conference.book Page 70 Wednesday, February 7, 2001 8:44 PM
Abstracts - Oral Presentations
IBMS/ECTS 2001 - First Joint Meeting
A NOVEL OSTEOBLAST-DERIVED MOLECULE THAT INHIBITS OSTEOCLAST FORMATION
INTERLEUKIN-1 REGULATES CYTOSKELETAL ORGANIZATION IN OSTEOCLASTS VIA TRAF6/C-SRC COMPLEX
H. Zhou, V. Kartsogiannis, Y. S. Hu, J. Elliott, J. M. W. Quinn, W. J. McKinstry, M. T. Gillespie*, K. W. Ng St. Vincent's Inst Med. Res. and Dept Med. Uni. Melbourne, Melbourne, Australia A novel mRNA species was identified from UMR 201 rat preosteoblast cells ± retinoic acid treatment, using a differential display PCR strategy. The full length murine homolog of 1kb was isolated and termed OCIL (Osteoclast Inhibitory Lectin). mOCIL was predicted to be a type II membrane-bound molecule of 21 kDa. Antisense oligonucleotides to mOCIL in murine in vitro osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells increased TRAP+ve mononucleate cell formation up to four fold: sense or scrambled oligonucleotides had no effect. These data suggested that OCIL was inhibiting the formation of mononuclear osteoclast precursors. The extracellular domain of mOCIL was expressed in E. coli and used in in vitro osteoclast formation assays. In murine osteoblast with spleen cell cocultures, as well as in spleen cell cultures treated with RANKL and M-CSF, recombinant mOCIL dose-dependently inhibited multinucleate osteoclast formation. mOCIL acted directly on macrophage/ monocyte cells as evidenced by its inhibitory action on adherent cell cultures, which were depleted of stromal and lymphocytic cells. Further, recombinant mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation (days 0-3) and resulted in 70% inhibition during the differentiation phase (days 4-7). mOCIL mRNA levels in primary murine calvarial osteoblasts was enhanced by PTH, calcitriol, IL-1 alpha and IL-11 (each >10 fold). These osteotropic factors have been shown to enhance RANKL mRNA expression in osteoblasts. The concordant regulation of mOCIL with that for RANKL suggests that OCIL may be a tonic inhibitor of the actions of RANKL. Further support for this notion results from Northern blot, in situ hybridization and immunohistochemistry analyses of rodent tissues that established that the expression of OCIL mRNA and protein was concordant with the distribution for RANKL. OCIL was expressed by osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues such as brain, heart, kidney, spleen, skeletal muscle and skin. The overlapping wide tissue distribution of OCIL mRNA and protein with that for RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.
I. N. Nakamura1,2*, Y. Kadono1, H. Takayanagi1, E. Jimi3, T. Miyazaki1, H. Oda1, K. Nakamura1, S. Tanaka1, G. A. Rodan2, L. T.Duong2 1 Univ of Tokyo, Tokyo, Japan 2 Merck Res Labs, West Point, USA 3Yale Univ, New Haven, USA Targeted disruption of either c-Src or TRAF6 in mice results in osteopetrotic phenotype due to osteoclast dysfunction, suggesting that both molecules play important roles in osteoclastic bone resorption. We previously demonstrated that interleukin-1 (IL-1) induces actin ring formation, leading to osteoclast activation. In this study, the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways was examined in osteoclast-like cells (pre-fusion cells: pOCs and multinucleated cells: OCLs) formed in the murine co-culture system. In pOCs, IL-1 induced actin ring formation and tyrosine phosphorylation of Cas, a known substrate of c-Src. However, in Src-deficient pOCs, Cas was not tyrosinephosphorylated with IL-1 treatment. In normal pOCs treated with IL-1, antiTRAF6 antibodies co-precipitated three tyrosine-phosphorylated proteins (p130, p120 and p60) which were identified to be Cas, PYK2 and c-Src, respectively. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is a key molecule for the complex formation of TRAF6, Cas and PYK2. In addition, in OCLs, anti-TRAF6 and anti-c-Src antibodies pulled down c-Src and TRAF6, respectively. Moreover, an immunocytochemical analysis revealed that in OCLs, IL-1 induced redistribution of TRAF6 to actin ring structure formed at cell periphery of OCLs where TRAF6 also co-localized with c-Src. In summary, IL-1 induces the formation of TRAF6-Src molecular complex including Cas and PYK2, leading to actin ring formation in osteoclasts. Taken together, these data suggest that IL-1 might cross-regulate the tyrosine kinase pathway, leading to cytoskeletal reorganization essential for osteoclast activation.
OR4 EXPRESSION AND FUNCTION OF SMALL GTPASES OF RAB FAMILY IN BONE-RESORBING OSTEOCLASTS H. B. Zhao*, O. Ettala, H. K. Väänänen Department of Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland Extensive intracellular vesicular trafficking is essential for the polarization and bone resorption activities of osteoclasts. Small GTPases of rab family have emerged as key regulators of intracellular membrane trafficking pathways along the biosynthetic/secretory and endocytic pathways in mammalian cells. The expression array of members of rab family in osteoclasts was investigated by a PT-PCR based cloning strategy. Seventeen different partial cDNA sequences of small GTPases including rab proteins were isolated. Full-length cDNAs of 6 rat rabs were obtained by RACE-PCR. Subcellular distribution of rab proteins in osteoclasts was then examined by confocal laser scanning microscopy. Non-resorbing osteoclasts have a large number of rab7, a late endosomal rab protein, positive vesicles while in boneresorbing osteoclasts the number of these vesicles decreased and rab7 is predominantly localized at the periphery of the ruffled border (RB). Another late endosomal rab protein, rab9, is found in perinuclear vesicles in osteoclasts. Interestingly, a fraction of rab9 is also seen at the central RB complementary to rab7 localization. Rab3 which is associated with secretory granules in other types of cells was localized in cytoplasmic vesicles in non-resorbing osteoclasts while in bone-resorbing osteoclasts it was restricted to the membrane structures corresponding to the trans Golgi network. Rab11 was found to associate with the perinuclear recycling endosomes in osteoclasts. Since the antibody against rab5C is not available, we studied the subcellular localization of rab5 effector, EEA1, in osteoclasts. A punctate vesicular labelling of EEA1 was seen at the peripheral areas of osteoclasts corresponding to the early endosomes. No EEA1 was found at RB in resorbing osteoclasts. After added to the culture medium for 5 to 15 minutes, transferrin, an endosomal tracer, was found to colocalize with EEA1 in the peripheral vesicles in osteoclasts. After 30 minutes transferrin was seen to mainly colocalize with rab11 in perinuclear vesicles and at the same time point a large amount of transferrin was also localized at RB and in the resorption lacuna. Further functional study showed that the antisense oligonucleotides against rab7 decreased the number of resorbing osteoclasts and significantly inhibited the osteoclastic bone resorption in vitro. These results indicate the participation of late endocytic pathway in the formation and maintenance of RB and may be a useful target for the intervention of osteoclast function.
OR2 CBL NEGATIVELY REGULATES SRC KINASE ACTIVITY AND VITRONECTIN RECEPTOR-MEDIATED CELL ADHESION A. Sanjay*, A. Houghton, E. DiDomenico, W. Horne, R. Baron Dept.of Orthopaedics, Yale University, USA c-Cbl, a substrate of Src in osteoclasts, is required for in vitro bone resorption. Vitronectin receptor (VnR)-mediated cell adhesion induces the association of c-Cbl with Src and the Src-dependent tyrosine phosphorylation of Cbl. We have previously reported that the Src SH3 domain binds to c-Cbl via proline-rich sequences that are absent from the truncated oncogene product, v-Cbl. We also noted an interaction between Src and v-Cbl (weaker than the SH3-mediated binding of Src to c-Cbl) that we speculated might involve the phosphotyrosinebinding (PTB) domain of Cbl and phosphorylated Src Tyr416, Src’s autophosphorylation site. To characterize the Cbl PTB/Src pY416 interaction and its functional consequences, we reconstituted this signaling system in VnRexpressing HEK 293 cells (293-VnR). Mutation of either the v-Cbl PTB domain (G306E) or Src Y416 (Y416F) abolished the v-Cbl/Src interaction but did not affect the association of Src with c-Cbl. v-Cbl did not bind to mutants of Src that do not autophosphorylate Y416. Thus, the Cbl PTB binding requires Src autophosphorylation. We then studied the functional consequences of the PTB/Src interaction. Overexpressing isoforms of Cbl with an intact PTB domain inhibited Src kinase activity and reduced the adhesion of these cells to vitronectin (but not to fibronectin). Mutation of the v-Cbl PTB domain (v-Cbl G306E) abolished the inhibition of Src kinase. Overexpression of c-Cbl with the same mutation led to a marked and significant increase in cell adhesion (144% p<0.001), suggesting that endogenous c-Cbl (which would be displaced by the mutant protein) downregulates cell adhesion. We conclude that Cbl regulates Src kinase activity and VnRdependent cell adhesion via the interaction of its PTB domain with the Src kinase domain. Thus, a Src/Cbl complex appears to regulate the VnR in an inside-out manner. These results may explain the decreased motility of osteoclasts from Src or Cbl knockout mice.
conference.book Page 71 Wednesday, February 7, 2001 8:44 PM
IBMS/ECTS 2001 - First Joint Meeting
Abstracts - Oral Presentations
CONNEXIN43 DEFICIENCY LEADS TO IMPAIRED OSTEOCLASTOGENESIS
INTERLEUKIN-17 IS A CATABOLIC CYTOKINE FOR BONE AND CARTILAGE IN PATHOLOGICAL CONDITIONS CHARACTERIZED BY INFLAMMATION
F. Lecanda1*, F. Furlan2, L. Weitzmann2, S. Sheikh2, R. Civitelli2 1University of Navarra, Pamplona, Spain 2Washington University, St. Louis, USA Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). Cx43 is also present in cells of the monocyte-macrophage lineage and in osteoclasts. We have recently shown that genetic deficiency of Cx43 leads to developmental defects, including craniofacial abnormalities and delayed ossification of the axial and appendicular skeleton. To analyze the role of this protein for osteoclast differentiation, we have used Cx43-null and wild type (wt) littermates to prepare co-cultures of collagenase-digested cells from calvaria (CV) which can support osteoclastogenesis - and spleen monocytes as a source of osteoclast precursors. After two weeks postconfluence, the abundance of osteocalcin and collagen type I mRNA is severely reduced in Cx43-null compared to wt cells, two hallmarks of osteoblast dysfunction. Co-cultures of CV cells of different Cx43 genotypes with wt mouse spleen monocytes yielded TRAP-positive multinucleated cells in all co-cultures in the presence of 1a,25(OH)D3 but their number was significantly lower in co-culture with Cx43-null than either heterozygous or wt osteoblasts (p<0.01). Monocytes heterozygous for Cx43 were able to undergo osteoclastic differentiation in a similar fashion as wt monocytes, when supported by calvaria cells, implying that the presence of Cx43 in the osteoblastic-stromal compartment, more than in the hematopoietic compartment, is critical for osteoclastogenesis. To investigate this issue further, we induced osteoclast differentiation in monocytes of different Cx43 genotypes by exposure to M-CSF and RANKL. At variance with the co-culture system, single allele deletion of Cx43 resulted in greatly reduced formation of TRAP+ cells, whereas Cx43-null monocytes were unable to undergo differentiation, indicating that an hematopoietic defect is also present in Cx43 deficiency. By semi-quantitative RT-PCR, RANKL mRNA abundance was ~30% lower in Cx43-null than in wt CV. Conversely, basal levels of OPG were 3-fold higher in mutant relative to wt cells, whereas M-CSF abundance was very similar in different Cx43 genotypes. Thus, osteoblast support of osteoclastogenesis requires functional Cx43 gap junctions. Although loss of Cx43 leads to a reduced RANKL/OPG ratio, calvaria cells provide additional stimulatory mechanisms or survival factors that support monocyte differentiation, other than M-CSF and RANKL, in condition of Cx43 deficiency. Gap junctional communication appears to be involved in both arms of bone remodeling.
R. L. van Bezooijen*, L. van der Wee-Pals, B. Oudshoorn, S. E. Papapoulos, C. W. G. M. Löwik Leiden University Medical Center, Leiden, The Netherlands IL-17 is a cytokine exclusively produced by activated T-cells. Like other cytokines produced by inflammatory cells, IL-17 may affect bone and cartilage in pathological conditions characterized by the presence of activated T-cells. In the synovial fluid of patients with rheumatoid arthritis (RA), for example, elevated levels of IL-17 have been found. We have recently shown that IL-17 stimulated osteoclastic resorption largely independent of RANK/RANKL and NF-kappaB signaling in fetal mouse metatarsals in vitro. In this study, we examined whether IL-17 affected the cartilage of these metatarsals and whether it affected osteoblastogenesis. IL-17 alone, up to 100 ng/ml, had no effect on the morphology of the bone explants. IL-17 (1 ng/ml and higher), however, synergized with TNF-alpha (10 ng/ ml and higher) to induce cartilage degradation. Histological analysis showed that IL-17 + TNF-alpha induced degradation of the proliferating and hypertrophic cartilage zones and decreased alcian blue staining, a marker of proteoglycans. Nitric oxide (NO) production induced by the cytokine combination was not involved, as abolishment of NO production by the NO synthesis inhibitor L-NMMA (100 microM) or the NF-kappaB inhibitor PDTC (50 microM) did not prevent cartilage degradation. Cartilage degradation was associated with matrix metalloproteinase (MMP) activity, as treatment with the MMP inhibitor marimastat (0.1 microM) prevented loss of proliferating and hypertrophic cartilage zones. Marimastat, however, did not prevent the decrease in alcian blue staining. While IL-17 needed co-treatment with TNF-alpha to stimulate osteoclastic resorption and cartilage degradation, IL-17 alone (1 ng/ml and higher) inhibited osteoblastic differentiation of KS483 cells; measured as a decrease in alkaline phosphatase activity. KS483 cells can differentiate into mature osteoblasts capable of forming mineralized bone nodules. TNF-alpha alone also inhibited osteoblastic differentiation of these cells. Induction of NO production by KS483 cells, however, only occurred when IL-17 and TNF-alpha were given together. Both cytokines alone did not induce the production of NO. In pathological conditions characterized by the presence of activated T-cells, such as rheumatoid arthritis and loosing of bone implants, IL-17 may contribute to the catabolic effects observed on cartilage and bone by stimulating osteoclastic bone resorption and cartilage degradation and by inhibiting bone formation.
OR6 INTERLEUKIN-12 ALONE, AND IN SYNERGY WITH INTERLEUKIN-18, INHIBITS OSTEOCLAST FORMATION IN VITRO
OR8 TUMOR NECROSIS FACTOR-ALPHA INDUCES OSTEOCLASTOGENESIS BY DIRECT STIMULATION OF MACROPHAGES EXPOSED TO PERMISSIVE LEVELS OF RANK LIGAND J. Lam1*, S. Takeshita1, J. E. Barker2, O. Kanagawa1, F. P. Ross1, S. L. Teitelbaum1 1 Washington University School of Medicine, Saint Louis, MO, USA 2The Jackson Laboratory, Bar Harbor, ME, USA While tumor necrosis factor-alpha (TNF-alpha) is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-alpha alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NFkB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-alpha and RANKL markedly potentiate NFkB and SAPK/JNK activity, two signaling pathways essential for osteoclastogenesis. TNF-alpha stimulates osteoclast formation in co-cultures of TNF receptor (TNFR)-deficient marrow stromal cells with TNFR-heterozygous macrophages, without addition of exogenous RANKL. These data suggest that while TNF-alpha alone cannot prompt osteoclastogenesis, it may do so when stromal/osteoblastic RANKL is present at basal, non-stimulated levels. To test this hypothesis in vivo, we generated chimeric animals by bone marrow transplantation in which beta-galactosidase positive, TNF-responsive macrophages develop within a TNFR-deficient stromal environment. In vivo administration of TNF-alpha prompts robust osteoclast formation in these chimeric animals, in which TNFresponsive macrophages develop within a TNF-non-responsive stromal environment. Thus, while TNF-alpha alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages exposed to permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-alpha to promote osteoclastogenesis, pharmacological blockade of RANKL will be successful only if this molecule is virtually eliminated. TNF-alpha may therefore be a more convenient clinical target than RANKL in arresting inflammatory osteolysis.
N. J. Horwood, J. Elliott, T. J. Martin, M. T. Gillespie* St. Vincent's Inst. Med. Res., Melbourne, Australia IL-12 and IL-18 are expressed by cells present in the bone microenvironment and in rheumatoid arthritis joints and promote the production of the osteoclast inhibitors, IFN gamma and GM-CSF. We therefore examined the potential of IL-12, and IL-12 in combination with IL-18, to effect osteoclast formation. IL-12, like IL-18, potently inhibited osteoclast formation in cocultures of murine osteoblast and spleen cells treated with 1,25(OH)2 vitamin D3, as well as in adult spleen cell cultures treated with M-CSF and RANKL. The inhibitory actions of IL-12, like that of IL-18, occurred during the first three days of the culture. Furthermore IL-12, like IL-18, was found to act via T cells, since 1) depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12, 2) IL-12 was without action in cultures established from Rag1-/- mice, and 3) addition of T cells from C57/BL6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW 264.7 cells, demonstrating that each agent was unable to act directly on osteoclastic precursors. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at a concentration 20- to 1000-fold less, respectively, than when added individually. Since IL-12 and IL-18 are reported to stimulate IFN-gamma and GM-CSF production, the involvement of IFN-gamma and GM-CSF in osteoclast inhibition was determined in osteoblasts/spleen cell cocultures from either GM-CSF R-/mice or IFN-gamma R-/- mice. Both IL-12 and IL-12 with IL-18 were able to inhibit osteoclast formation in cocultures established from these mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition by IL-12 alone or in combination with IL-18. Subsequently the T cell-derived inhibitor was identified as either a secreted or shed factor since conditioned media of T cells treated with either IL-12 or IL-12/IL-18 was able to inhibit osteoclast formation of RANKL-stimulated RAW264.7 cells. In addition, these agents were able to inhibit osteoclast formation in cultures where T cells were physically separated from RAW264.7. Neutralizing antibodies to either IL-4, IL-10 or IL-13 were unable to rescue IL-12- or IL-12/IL-18-induced inhibition suggesting that these agents induce the production of an as yet unknown T cell-derived inhibitor of osteoclast formation.
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Abstracts - Oral Presentations
IBMS/ECTS 2001 - First Joint Meeting
META-ANALYSIS OF COL1A1 SP1 POLYMORPHISM IN RELATION TO BONE MINERAL DENSITY AND OSTEOPOROTIC FRACTURE
GENETIC EFFECTS ON FALLS RISK MAY HELP EXPLAIN WHY FRACTURES RUN IN FAMILIES: A TWIN STUDY J. D. Wark1*, K. Hill2, A-M. Cassano1,2, N. El Haber1, R. MacInnis1 of Melbourne, Parkville, Australia 2 National Ageing Research Institute, Parkville, Australia Falls in older women are a major global cause of injury, disability and death. Many risk factors for falls are known also to be associated with increased risk of osteoporotic fractures, in particular at the hip. Theoretical modelling (after Hopper and Carlin, 1992) suggested that the familial clustering of hip fractures was explained only in part by the heritability of bone mineral density and of hip axis length. Therefore, we performed a classical twin study to ask whether genetic or shared environmental effects on falls risk contributed to the familial aggregation of fractures. We recorded historical risk factors for falls and performed laboratory tests reflecting gait and balance function, and tested cognitive function and vision in 35 pairs of monozygotic (MZ) and 39 pairs of dizygotic (DZ) female twins who were peri- or postmenopausal and aged 46 - 82 years. Test results in the Table showed highly-significant within-MZ pair correlations on multiple outcome measures; within-DZ pair correlations tended strongly to be lower, indicating the likelihood of moderate to strong additive genetic effects on these falls predictors. MZ pairs were not more concordant than DZ pairs for other falls risk factors (e.g., relevant medication use, impaired vision, reported arthritis). This ongoing study provides important new information on the genetic epidemiology of osteoporosis, suggesting that heritability of gait and balance function contributes to the familial clustering of osteoporotic fractures.
V. Mann*, S. H. Ralston University of Aberdeen, Aberdeen, UK We have previously reported an association between a polymorphic Sp1 binding site in the COL1A1 gene, bone mineral density (BMD) and osteoporotic fracture. Since these findings have been replicated in some populations but not in others we conducted a meta-analysis of sixteen eligible studies which have looked at the COL1A1 Sp1 polymorphism in relation to BMD or fracture. Standardised mean differences between the genotype groups were calculated for continuous variables and odds ratios for categorical variables using Revman 4.1 software. The studies included a total of 5041 subjects with 3287 (65.2%) "SS"; 1507 (29.9%) "Ss" and 247 (4.9%) "ss". No evidence of publication bias was found using a funnel plot (p=0.51). The main results are shown in the table below. BMD values were significantly lower in the "Ss" and "ss" genotype groups when compared with "SS" at the lumbar spine (LS-BMD) and femoral neck (FN-BMD) with evidence of a gene-dose effect. The odds ratio of fracture was increased to +1.57 in the "Ss" group and +2.26 in the "ss" group when compared with "SS" homozygotes. We also found a small but significant reduction in body mass index (BMI) in "Ss" and "ss" subjects but no significant difference between genotypes in age, menopausal age, smoking or calcium intake. We conclude that the COL1A1 Sp1 polymorphism is a genetic marker for BMD, BMI and osteoporotic fracture. The odds ratio for fracture is too great to be explained by the difference in BMD and BMI values between the genotype groups, which at most should account for an increased risk of +1.15 per copy of the "s" allele. This supports the view that COL1A1 alleles increase the risk of fracture, by as yet undefined mechanisms, that are independent of bone mass. Comparison SS vs Ss
SS vs ss
LS-BMD FN- BMD Fracture BMI
<0.00001 0.0002 <0.00001 0.001
-0.19 [0.05-0.33] -0.26 [0.12- 0.40] +2.26 [1.53- 3.35] - 0.16 [0.01-0.31]
0.007 0.0002 0.0009 0.04
-0.15 [0.09- 0.22] -0.12 [0.06-0.18] +1.57 [1.31- 1.88] -0.11 [0.04-0.17]
TESTS OF GAIT AND BALANCE FUNCTION IN FEMALE TWINS (WITHIN-PAIR CORRELATIONS) TEST
Adjusted activity score 0.63 0.29 Muscle strength (R ankle dorsiflexion) 0.86 0.69 Stride velocity 0.62 0.20 Double support duration 0.76 0.25 Lord's balance test 0.51 0.22 Chattecx balance testing 1 0.54 0.02 Chattecx balance testing 2 0.48 0.18 Step test 0.62 0.42 rMZ, rDZ: Pearson's r; Lord's test: eyes closed, foam surface; Chattecx testing: anteroposterior perturbation,1 - no distraction, 2 with distraction.
OR10 EVIDENCE FOR A LOCUS OF A MAJOR OSTEOPOROSIS GENE IN AN ICELANDIC LINKAGE STUDY U. Styrkarsdottir1*, K. Jonasson1, V. D. Johannsdottir1, K. H. Gudjonsdottir1, H. S. Erlingsdottir1, S. Oscarson2, J. Gulcher1, K. Stefansson1, G. Sigurdsson3 1 Decode Genetics, Reykjavik, Iceland 2Clinical Research Center, Reykjavik, Iceland 3University Hospital, Reykjavik, Iceland We have conducted a multipoint linkage analysis, based on a genome wide scan with 860 microsatellite markers in 190 osteoporotic Icelandic families. A total of 1200 people from these families participated in the study. We used the Allegro linkage program in an affected-only type of approach. Using a new “combined” phenotype definition we identified a likely location of an osteoporosis gene with a LOD score of 5. The primary aim is to find genes that are involved in osteoporosis, not only at identifying genes controlling BMD. We therefore include in the analysis biphosphonate users and low impact fracture cases, as well as subjects with low BMD. For identifying families suitable for linkage analysis we used the Decode Genetics Genealogy Database and a list of people with osteopenia/osteoporosis, generated from over 9600 BMD measurements made at Reykjavik Hospital’s osteoporosis clinic. We identified families with at least two low BMD members (< -1 age matched SD) connecting within 5 meioses in the database. All 1200 participants had a complete clinical record, and DEXA-BMD measurements, both total hip and lumbar spine. The BMD values were corrected for sex, age, weight and hormone replacement therapy. We analysed hip and spine separately for identifying genes controlling BMD and osteoporosis at these different skeletal sites. For identifying genes that control both these sites, we used a “combined” phenotype, which is based on the sum of the corrected hip and spine measurements, plus bisphosphonate users and fracture cases. In the combined analysis we identified two chromosomal regions with significant evidence of linkage (LOD>3.6). For one of these regions, we maximized the LOD score by varying the threshold used as a cut-off for affected, giving a maximum when the lower 10th percentile was used as threshold. Additional 25 markers resulted in a LOD score of 5.05, and a much narrower LOD peak (6 cM one-LOD-drop). A LOD of 4 was reached using quantitative trait analysis approach with the Allegro program at the same location. Apart from the previous regions, the hip analysis identified another chromosomal region suggestive of linkage (LOD>3.0).
OR12 CHROMOSOMAL LOCATION OF GENES THAT CONTRIBUTE TO VERTEBRAL TRABECULAR BONE DENSITY AND MICROARCHITECTURE IN MICE M. L. Bouxsein1*, T. Uchiyama1, J. Mytar2, W. G. Beamer2, L. R. Donahue2, C. J. Rosen2, C. H. Turner3, R. Mueller1 1Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA 2The Jackson Laboratory, Bar Harbor, ME, USA 3Indiana University, Indianapolis, IN, USA Bone mineral density (BMD) is a complex trait influenced by both environmental and genetic factors, with heritability explaining much of the variance in BMD. In addition to BMD, other phenotypic traits that affect bone strength, such as trabecular architecture, may be genetically determined. To identify heritable determinants of trabecular architecture, we evaluated mice from the F2 intercross of C57BL/6J (B6) and C3H/HeJ (C3H) progenitor strains. Microcomputed tomography was used to assess trabecular bone volume (BV/TV), thickness (Tb.Th), separation (Tb.Sp), and number (Tb.N) in the 5th lumbar vertebral body of the progenitors (n=8 / strain) and B6C3H-F2 progeny (n=914). B6 had greater vertebral trabecular BV/TV (+114%), TbN (+68%), and lower TbSp (-42%) compared to C3H (p<0.001 for all). These traits were normally distributed in the F2 population. Genomic DNA from F2 progeny was screened for 107 PCRbased markers that discriminate B6 and C3H alleles on all 19 autosomes. Regression analysis using MapmanagerQT was used to identify quantitative trait loci (QTLs) contributing to these vertebral trabecular bone traits. As expected, these traits were found to be highly polygenic quantitative traits. Heritability estimates (H2) ranged from 28% for Tb.Sp to 88% for BV/TV. The number of QTLs underlying these traits ranged from 6 to 10, with LOD scores ranging from 3.1 to 13.2 (Table). Two loci were common for all the vertebral trabecular traits: one on Chr 4 and another on Chr 8. BV/TV, Tb.N, and Tb.Sp also shared common QTLs on Chr 1, 9, 12, and 13. Other unique QTLs were identified on Chr 7, 14, and 18 for TbTh; and on Chr 6 for BV/TV. In summary, we identified several QTLs that may contribute to vertebral trabecular architecture in mice. We previously identified Chr 1, 4, and 18 as major regulators of femoral and vertebral BMD. However, many of the QTLs that we identified here appear to be unique to vertebral trabecular bone architecture and may be independent of bone density.
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Abstracts - Oral Presentations We found that a substantively increased risk of further fracturing existed across different sites, with increases of 2 to 3 times the expected rate in the general population. This indicates that fracture history is a strong risk factor for new fractures.
OR15 REDUCED EXPRESSION OF eNOS IN THE FRACTURED FEMORAL NECK N. Loveridge1*, S. Fletcher2, J. Power1, J. Reeve1, 1University of Cambridge, Cambridge, UK 2Royal Veterinary College, London, UK
Nitric oxide (NO) is important in the control of bone turnover. Endothelial NOS (eNOS) is the predominant isoform expressed in osteoblasts and osteocytes and appears responsible for the increases in NO in response to load. While NO donors prevent bone loss in ovarietomised rats, there is no direct evidence supporting a role of eNOS in human osteoporotic fractures. We have analysed the presence of eNOS in cortical osteocytes in complete femoral neck biopsies of cases of intracapsular hip fracture (n=5, F, 70-87y) and similar control material (n=4, F, 68-87y). eNOS expression in osteocytes was assessed by immunocytochemistry; osteocyte density by propidium iodide (PI). Confocal (NOS) or normal (PI) images were obtained from the inferior (I) and superior (S) regions of the femoral neck and the proportion of eNOS+ve osteocytes calculated. The distance of each osteocyte or eNOS+ve osteocyte from the centre of the nearest haversian canal was also measured. In control biopsies, the proportion of eNOS+ve osteocytes was higher in the inferior region (42±13.4%; normally loaded in compression) than the superior region (14.4±6.5%, tension). There were no differences between cases and controls in the superior region (cases: 7.5±1.6%) but the cases had a reduced proportion of eNOS+ve osteocytes (12.6±2.9%, p=0.04) in the inferior region. The mean distance between the centre of the nearest haversian canal and ecNOS+ve osteocytes (122.7±6µm) was significantly greater than that for all osteocytes (103.4±2.7µm, p=0.006). The mean difference between eNOS+ve and all osteocytes was higher (p=0.019) in the cases (24±9.7µm) than the controls (13.5±5µm). This pilot study suggests the following conclusions: 1) The 50% reduction in eNOS+ve osteocytes in the inferior region of the cases suggests that differences in the perception of loading are a contributing cause to the pattern of cortical bone loss. 2) Strain mode may influence eNOS expression in osteocytes. 3) As eNOS expression is highest in osteocytes distant from the haversian canal, NO may act as a limiter of bone resorption. If so, the reduction in eNOS expressing osteocytes may be involved in the formation of the giant canals (diameter >385 µm) which are a predominant feature of the inferior half of the fractured femoral neck.
ASSOCIATION BETWEEN STATIN-USE, BONE MASS AND FRACTURE RISK IN AUSTRALIAN WOMEN: GEELONG OSTEOPOROSIS STUDY J. A. Pasco, M. A. Kotowicz*, M. J. Henry, K. M. Sanders, G. C. Nicholson The University of Melbourne, Department of Clinical and Biomedical Sciences, Barwon Health, Geelong, Victoria, Australia Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) stimulate bone formation in vitro and in rodents [Science 1999;286:1946]. Recent data suggest that statins, used in the treatment of hypercholesterolaemia, decrease fracture risk [Lancet 2000:355:2185; JAMA 2000;283:3205 and 3211] and increase bone mineral density (BMD) [Lancet 2000;355:2218]. We aimed to determine whether statins decrease the risk of fracture and evaluate the association between statin use and BMD in Australian women. Current statin use was evaluated in 580 women (50-95 yr) with incident fractures occurring 1994-6 and in a random sample of 804 women (50-94 yr) without incident fractures. BMD (Lunar DPX-L) was determined for all subjects, with 83% of the fracture group being assessed within 4 months of sustaining the fracture. Both groups were drawn concurrently from the Barwon Statistical Division. Excluding pathological fractures, there were 16 statin-users in the fracture group and 53 in the non-fracture group. The proportion of women using HRT, glucocorticoids or calcium/vitamin D supplements did not differ between statinusers and non-users (p>0.05). Unadjusted odds ratio (OR) for fracture associated with statin use was 0.40 (95% CI 0.23, 0.71); adjusting for age and weight had no effect on the OR. Adjusting for BMD at the femoral neck, spine and whole body increased the OR to 0.45 (95% CI 0.25, 0.80), 0.42 (95% CI 0.23, 0.75) and 0.43 (95% CI 0.24, 0.78), respectively. Statin use was associated with a 3% greater ageand weight-adjusted BMD at the femoral neck (p=0.08), with non-significant increases at the spine and whole body. Our results suggest that increases in BMD associated with statins are too small to account for the observed 60% reduction in fracture risk. Unless confounded by unrecognised factors, statins confer substantial protection against fracture, but the mechanisms of action remain unclear.
BV/TV * *** * ** ** ** * * Tb.Th *** * * * * Tb.Sp ** ** ** ** ** * Tb.N ** ** ** ** ** * LOD score of *3-6, **6-10, *** >10; ^ = two distinct QTLs on each chromosome
OR16 VITAMIN D DEFICIENCY, SERUM IGF-1 AND MARKERS OF BONE TURNOVER PREDICT FRACTURES IN THE LONGITUDINAL AGING STUDY AMSTERDAM
P. Lips1,2*, S. M. F. Pluijm2, C. Popp-Snijders1, J. H. Smit3 1Endocrinology, Academic Hospital, Vrije Universiteit, Amsterdam,
T. P. van Staa1,2,3*, H. G. M. Leufkens1, C. Cooper2 1Department of Pharmacoepidemiology and Pharmacotherapy, University of Utrecht, Sorbonnelaan 16, Utrecht, the Netherlands 2 Medical Research Council Environmental Epidemiology Unit, Southampton University Hospital, Southampton, UK 3Procter & Gamble Pharmaceuticals, Lovett House, Lovett Road, Staines, UK The purpose of this report was to estimate the risk of further fracturing at other skeletal sites for a broad group of fracture types. Information was obtained from the General Practice Research database in the UK, which contains medical records of general practitioners. The study population consisted of all patients aged 20 years or older with a fracture during 1988 to 1998. There were 222 369 subjects, 119 317 women and 103 052 men, who had sustained at least 1 fracture during follow-up. There was an increased risk of almost every other type of fracture following an initial fracture, with 2-3 times higher than expected rates of subsequent fracture at different skeletal sites. A patient with a radius / ulna fracture had a standardised incidence ratio (SIR) of 3.0 (95% confidence interval 2.9-3.1) for fractures at a different skeletal site. For vertebral fractures cases, this ratio was 2.9 (2.8-3.1) and for femur/hip fractures cases, this was 2.6 (2.5-2.7). The SIRs were generally higher among men than women. Men aged 65 to 74 years with a radius / ulna fracture or vertebral fracture had substantially higher rates of subsequent femur / hip fractures than expected. The respective SIRs were 6.0 (3.4-9.9) and 13.4 (7.3-22.5). The corresponding SIRs among women of similar age were 3.3 (2.8-3.9) and 5.8 (4.1-8.1), respectively. Men and women with a vertebral fracture had a 5-year risk of femur / hip fracture of 6.7% and 13.3%, respectively. Stable SIRs over time were generally also found for the relationship between other fracture types.
The Netherlands 2Institute for Research in Extramural Medicine (EMGO-Institute), Vrije Universiteit, Amsterdam, The Netherlands 3Department of Sociology and Social Gerontology, Vrije Universiteit, Amsterdam, The Netherlands Metabolic diseases may cause osteoporosis and fractures by increasing bone resorption and decreasing bone formation. The role of vitamin D deficiency, parathyroid hormone, sex hormone binding globulin, insulin-like growth factor 1 (IGF-1) and markers of bone turnover as predictors of osteoporotic fractures was investigated in a cohort of 1350 community-dwelling women and men older than 65 years from the Longitudinal Aging Study Amsterdam. Fasting blood samples were obtained in 1995-96 and prospective follow-up for fractures was done during 3 years. Serum 25(OH)D, PTH, SHBG, IGF-1, osteocalcin and urinary DPD were measured by (radio)immunochemical methods. Serum 25(OH)D (mean±SD) was 58±24 nmol/l. When serum 25(OH)D was < 30, 30-50 or >50 nmol/l, serum PTH was 4.9±4.2, 3.8±2.2 and 3.1±1.4 pmol/l respectively. Serum IGF-1 was 14±5 nmol/l (range 2-37). Serum osteocalcin was 2.2±1.1 nmol/l. Urinary DPD/ creatinine was 5.7±2.7 nmol/mmol. During 3 years of follow-up 88 patients reported 108 fractures, i.e. 23 hip, 26 wrist and 59 other fractures. The following variables were associated with an increased risk for fractures: serum 25(OH)D < 30 nmol/l (vs > 30 nmol/l) RR 1.65 (CI: 1.02-2.68); a high serum osteocalcin in men RR 1.32 per SD increase (CI: 1.04-1.67); a high urinary DPD/creat RR 1.30 per SD increase (CI: 1.04-1.63). A high serum IGF-1 was protective RR 0.78 per SD increase (CI: 0.62-0.98). We conclude that a low serum 25(OH)D (<50 nmol/l) is associated with increased serum PTH. Vitamin D deficiency, a high serum osteocalcin and a high urinary DPD excretion are risk factors for fractures in community-dwelling elderly, while a high serum IGF–1 is protective.
DOES A FRACTURE AT ONE SITE PREDICT LATER FRACTURES AT ANY OTHER SITE?
conference.book Page 74 Wednesday, February 7, 2001 8:44 PM
Abstracts - Oral Presentations
IBMS/ECTS 2001 - First Joint Meeting
ISOCALORIC PROTEIN UNDERNUTRITION IS ASSOCIATED WITH ALTERATION OF SOMATOTROPIC AND GONADOTROPIC AXES RESULTING IN OSTEOPOROSIS IN MALE AND FEMALE ADULT RATS
PROTEASOME INHIBITORS STIMULATE OSTEOBLAST DIFFERENTIATION AND BONE FORMATION BY INHIBITING GLI3 DEGRADATION AND ENHANCING BMP-2 EXPRESSION
D. Chen1,2*, I. R. Garrett1,2, M. Qiao2, G. Rossini2, M. Zhao1, Z. Mi1, C. M. Crews3, S. Ishii4, G. R. Mundy1,2 1University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 2OsteoScreen Inc., San Antonio, Texas, USA 3Yale University, New Haven, Connecticut, USA 4 RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan We have found that structurally unrelated proteasome inhibitors stimulate osteoblast differentiation and bone formation in vitro and in vivo. Since we have previously shown that BMP-2 is an autocrine factor in osteoblast differentiation, and regulators of BMP-2 expression such as statins stimulate bone formation in vitro and in vivo, we examined the effects of proteasome inhibitors such as epoxomicin and proteasome inhibitor-1(PSI) on BMP-2 gene expression. We found that these proteasome inhibitors stimulated BMP-2 promoter activity and BMP-2 mRNA expression in osteoblast precursor 2T3 cells, and enhanced BMP-2 protein production by MG-63 human osteoblastic cells. When bone organ cultures were treated with noggin, the endogenous inhibitor of a number of BMPs including BMP-2, the increase in bone formation caused by epoxomicin and PSI was abrogated. To determine the molecular mechanism by which proteasome inhibition increased BMP-2 transcription and bone formation, we examined the effects of proteasome inhibitors on potential modulators of BMP-2 expression. Since the degradation product of the drosophila transcription factor cubitus interruptus (Ci) suppresses the expression of dpp, the mammalian homologue of the BMP-2/4 genes, and enzymatic processing of Ci is regulated by the ubiquitin-proteasome pathway, we determined the effect of Gli3, the mammalian counterpart of Ci, on BMP-2 gene expression and the effects of proteasome inhibitors on Gli3 degradation. We detected Gli3 protein expression after transfection of full-length Gli3 cDNA in osteoblast progenitor C3H10T1/2 cells. Slimb, a specific E3 ubiquitin ligase upon which Ci degradation depends, induced Gli3 degradation to the N-terminal truncated short form of Gli3 (sGli3). Transfection of sGli3 cDNA in the same cells significantly inhibited BMP-2 promoter activity and mRNA expression. The structurally-unrelated proteasome inhibitors, epoxomicin (20 nM) and PSI (50 nM), inhibited Slimb-induced Gli3 degradation. These results indicate that proteasome inhibitors stimulate BMP-2 gene expression by inhibiting Gli3 degradation, and suggest that the osteoblast proteasome regulates Gli3 degradation, which in turn controls BMP-2 expression, osteoblast differentiation and subsequent bone formation.
P. M. L. J. P. S. R. 1Division of Bone Diseases, Department of Internal Medicine 2 Division of Pediatric Endocrinology and Diabetology, Department of Pediatrics, University of Geneva, Switzerland Growth Hormone (GH), Insulin-like Growth Factor-I (IGF-I), sex hormones, as well as nutritional intakes are essential for the maintenance of metabolic function, bone mass/density, and muscle mass during aging. To investigate the mechanisms involved in the decrease of metabolic functions during aging, we developed an animal model of frailty in which either male or female adult rats are fed an isocaloric low protein diet, and studied the somatotropic and gonadotropic axes. Six-month old Sprague-Dawley rats were fed isocaloric diets containing 15 or 2.5% protein (LPD). In females, LPD caused a rapid decrease in IGF-I levels, which was significant already by 2 weeks. After 16 weeks on LPD, IGF-I was 271±50* vs 411±28ng/ml (mean±SE; *p<0.05). In males, a marked decrease in IGF-I was observed after 12 weeks on LPD (349±29* vs 675±29ng/ml). Pulsatile GH secretion was not altered after 20 days of LPD in either gender despite a decrease in IGF-I levels. No proestrus/estrus signs were detected in vaginal smear in rats fed the LPD. Testosterone levels were decreased in males after 24 weeks: 494±65* vs 1306±25pg/ml, as was seminal vesicle weight: 199±14* vs 309±13mg. In both genders, bone mineral density (BMD) and strength were decreased by 10-25% in skeletal sites containing cancellous or cortical bone. This was associated with early depressed bone formation in male and female as evaluated by histomorphometry, and subsequent increased bone resorption, which was detected however earlier in female than in male. High doses of IGF-I-IGF-I binding protein complex failed to influence bone or muscle in animal fed a LPD, indicating a target organ resistance. Similarly, there was a partial resistance to GH administration at the level of the liver, bone and muscle on LPD. In conclusion, protein undernutrition with normal calorie intake reduced IGF-I levels independently of GH secretion. A resistance to IGF-I or GH at the level of the target organs were also detected. This was associated with an early depressed bone formation. A progressive inhibition of sex steroid secretion in both male and female rats was observed. This could explain in part the increased bone resorption, which was detectable earlier in female than in male. These mechanisms might be relevant to understand the progressive bone loss and increased bone fragility observed in elderly.
REGULATION OF OSTEOBLAST DIFFERENTIATION IN VITRO BY THE DELTAFOSB FAMILY OF TRANSCRIPTION FACTORS
FRACTURES IN PATIENTS WITH ANOREXIA NERVOSA, BULIMIA, AND OTHER EATING DISORDERS
G. Sabatakos1*, M. Wu1, G. Rawadi2, S. Roman-Roman2, R. Baron1 1Yale University, USA 2 Aventis, France The AP1 family of transcription factors consists of Fos- and Jun-related basicleucine zipper proteins that modulate gene transcription via targeting of homo- and heterodimers to specific DNA sequences. Studies using transgenic animal models have implicated AP1 family members as important regulators of bone remodelling and skeletal development. We have shown previously that the overexpression of DeltaFosB, an alternative splice product of the fosB transcript, in transgenic mice leads to increased bone formation throughout the skeleton and a continuous postdevelopmental increase in bone mass resulting in progressive and profound osteosclerosis. The present study was aimed at elucidating the molecular mechanisms by which expression of the DeltaFosB protein up-regulates bone formation. For this purpose we have generated stable transfectants of DeltaFosB, as well as of DeltaFosB mutants expressing only the complete DeltaFosB, or solely the N-terminally truncated isoform Delta2-DeltaFosB (D2DFosB) in a variety of osteoblastic and bipotential cell lines (C2C12, 10T1/2 and ST2). The effects of stable expression of DeltaFosB and its two mutants on osteoblast differentiation and in response to osteogenic agents such as BMP2 have then been studied. In all cell types analysed, we observed a decrease in proliferation that was correlated with an early increase in the expression of several markers of osteoblast differentiation (osteopontin, osteocalcin, type I collagen), increased alkaline phosphatase activity, and nodule formation only in cells expressing the truncated D2DFosB isoform. Transactivation studies using the osteocalcin promoter demonstrated that D2DFosB-expressing cells exhibited higher levels of activation as well as a progressive increase in AP1 binding activity. Furthermore, we determined the ability of these isoforms to transduce signals through Smad family members. Interestingly, only overexpression of D2DFosB resulted in an early increase of Smad1 mRNA levels and increased translocation of phospho-Smad1 to the nucleus whereas there was no significant change in the levels of Smad4, Smad8 as well as of the BMP-receptors studied. It is therefore concluded, that the D2DFosB is a potent regulator of osteoblast differentiation in vitro by activating the transcription of several osteoblast markers. In addition, D2DFosB up-regulates the expression of Smad1, a signalling molecule downstream of BMP2 receptors, possibly favoring the osteogenic activity of BMP2.
P. Vestergaard1*, C. Emborg2, R. Støving3, C. Hagen3, L. Mosekilde1, K. Brixen3 1Department of Endocrinology and Metabolism C, Aarhus University Hospital, Aarhus, Denmark 2 Department of Psychiatric Demography, Aarhus University Hospital, Aarhus, Denmark 3Department of Endocrinology, Odense University Hospital, Odense, Denmark Aim: To evaluate if fracture risk was increased before or after a diagnosis of either anorexia nervosa, bulimia nervosa, or other eating disorders. Design: Cohort study. Main outcome variable: Fractures. Material and methods. All subjects diagnosed with anorexia nervosa (n=2,149), bulimia nervosa (n=1,294), or any other eating disorder (n=942) between January 1 1977 and December 31 1998 in Denmark. Each patient was compared with three age and gender matched control subjects randomly drawn from the background population. Results: Mean age at diagnosis was 21.2±9.2 years in anorexia nervosa, 23.0±6.5 years in bulimia nervosa, and 24.6±11.8 years among those with other types of eating disorders (2p less than 0.01). Overall fracture risk was increased in anorexia nervosa after the diagnosis had been made (RR=1.98, 95% CI: 1.60-2.44), but not before the diagnosis (RR=1.20, 95% CI: 0.98-1.47). The increased fracture risk persisted more than ten years after the diagnosis of anorexia nervosa. In bulimia nervosa there was a borderline significant increase in overall fracture risk before diagnosis (RR=1.31, 95% CI: 1.04-1.64), that disappeared after diagnosis (RR=1.44, 95% CI: 0.93-2.22). Patients with other types of eating disorders had a similar increase in fracture risk before (RR=1.39, 95% CI: 1.06-1.81) and after diagnosis (RR=1.77, 95% CI: 1.25-2.51). Conclusion: In anorexia nervosa an increased fracture risk persists many years after the diagnosis. Bulimia nervosa is associated with a borderline significant increase in fracture risk before, but not after diagnosis. In patients with other eating disorders, the fracture risk was the same before and after diagnosis.
conference.book Page 75 Wednesday, February 7, 2001 8:44 PM
IBMS/ECTS 2001 - First Joint Meeting
Abstracts - Oral Presentations
RETROVIRAL BMP4 STIMULATES OSTEOGENIC DIFFERENTIATION OF MUSCLE-DERIVED STEM CELLS AND INDUCES DE NOVO BONE FORMATION: POTENTIAL FOR BONE GENE THERAPY
INACTIVATION OF GLUCOCORTICOIDS IN OSTEOBLASTS BY TARGETED OVEREXPRESSION OF 11ß-HYDROXYSTEROID DEHYDROGENASE TYPE 2 IN TRANSGENIC MICE H. W. Woitge1,2*, I. Kalajzic3, S. H. Clark3, G. Gronowicz4, Z. Krozowski5, J. R. Harrison2, B. E. Kream2 1Department of Medicine I, University of Heidelberg, Heidelberg, Germany 2 Department of Medicine, University of Connecticut Health Center, Farmington, USA 3Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, USA 4Department of Orthopedic Surgery, University of Connecticut Health Center, Farmington, USA 5 Laboratory of Molecular Hypertension, Baker Medical Research Institute, Prahran, Australia Glucocorticoid action in some target cells can be regulated by a pre-receptor mechanism involving several enzymes of the 11ß-hydroxysteroid dehydrogenase family. The NAD-dependent enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites. The goal of the present study was to generate a transgenic mouse model that utilizes osteoblast-directed overexpression of 11ßHSD2 as a novel means of disrupting glucocorticoid signaling in osteoblasts, which normally express only low levels of the enzyme. A rat 11ßHSD2 cDNA was cloned downstream of a 2.3 kb rat Col1a1 promoter fragment and microinjected into the pronucleus of fertilized CD-1 mouse embryos. Four transgenic Col2.311ßHSD2 founder lines were generated producing viable litters with the expected Mendelian ratio of wild-type and transgenic offspring. Col2.3-11ßHSD2 mice showed no gross phenotypic abnormalities, including no signs of infertility or premature death. As demonstrated by Northern blot analysis and immunohistochemical staining, transgenic 11ßHSD2 mRNA and protein were highly expressed in bone and tendon with little or no expression in skin, liver, lung, kidney, brain, and reproductive organs. In calvarial cultures from 7-day old Col2.311ßHSD2 mice, rates of conversion of [3H]corticosterone to [3H]dehydrocorticosterone were higher than in calvarial cultures from wild-type littermates. Neonatal Col2.3-11ßHSD2 and wild-type mice were injected twice with vehicle or 30 mg/kg cortisol during a 24 h period and sacrificed. Calvariae were incubated for 2 h with [3H]proline to measure percent collagen synthesis (PCS). In wild-type calvariae, cortisol treatment decreased PCS, whereas in Col2.3-11ßHSD2 calvariae, cortisol treatment did not affect PCS. In summary, we have generated transgenic mouse lines expressing 11ßHSD2 under the control of a 2.3 kb Col1a1 promoter fragment that is selective for cells of the osteoblast lineage. Our strategy provides a novel loss-of-function model to study the role of glucocorticoid signaling in bone.
V. Wright, H. Peng*, A. Usas, J. Huard University of Pittsburgh Department of Orthopaedic Surgery, Pittsburgh, PA, USA BMP4 plays a pivotal role in embryonic skeletal development and is an early mediator of bone healing. The aim of this study is to test the in vitro and in vivo effects of retroviral BMP4 transduced muscle-derived stem cells and their potential application in promoting bone healing or regeneration. Muscle-derived stem cells (MC13) were transduced with retroviral vector expressing human BMP4. This retroviral vector contained a BMP2/4 hybrid construct that was shown, in our previous work, to markedly enhance the level of BMP4 secretion. The efficiency of transduction was higher than 80% as assessed by immunochemical staining for BMP4 expression. The level of BMP4 secretion was estimated by bioasssay based on C2C12 cells with rhBMP4 as standards. One week after transduction, MC13 cells transduced with retroviral BMP4 secreted BMP4 at 250 ng/106 cells/24hr. This level increased to 612 ng/106cells/24 hr three weeks post transduction. The ostogenic potential of transduced BMP4 expressing cells was determined by the expression of alkaline phosphotase (ALP) in transduced cells. Two weeks after transduction, 96-98% of retroviral BMP4 transduced MC13 cells became ALP positive, compared to 10% of the control cells transduced with a retroviral vector expressing LacZ. This was correlated to a 67-fold increase in the overall ALP activity in BMP4 expressing cells. To determined the potential of de novo bone formation of transduced MC13 cells expressing BMP4, 3 x 105 cells werBL mice, untransduced cells were injected as negative controls in the contralateral limb. De novo bone formation occurred within 2 weeks following injection in both SCID and C57BL mice. H&E and von Kossa staining revealed mineralized bone surrounding trabecular space and abutting muscle. No bone formation was detected in muscle injected with untransduced cells. Transduced MC13 cells expressing BMP4 was also found within the osteoid suggesting their differentiation into osteocytes. In summary, this study demonstrated that muscle-derived stem cells can be efficiently transduced by retroviral vector, express high level of BMP4, capable of osteogenic differentiation, and more importantly, induce de novo bone formation in immune competent animals. These cell and vectors system should have great potential in promoter bone healing in a variety of orthopeadic conditions.
OR22 STATINS MEDIATE BONE FORMATION BY ENHANCING BMP2 EXPRESSION Garrett1,2*,
I. R. D. M. G. A. D. J. Esparza1, G. R. Mundy1,2 1 OsteoScreen Inc., San Antonio, Texas, USA 2University of Texas Health Science Center at San Antonio, Texas, USA Inhibitors of the HMG-CoA reductase enzyme (statins), used to lower serum cholesterol in patients, enhance bone formation in vitro and in vivo. To determine if the statin effects on bone are due to their capacity to inhibit HMG-CoA reductase activity, we examined the effects of the immediate downstream metabolites mevalonate (100uM) and GGPP (10uM) on bone organ cultures and found that they inhibited both bone formation and BMP-2 transcription stimulated by statins. Statin bone effects are thus dependent on HMG-CoA reductase inhibition in bone cells. Although statins increase BMP2 expression, it is not clear that this is the mechanism by which these compounds cause increased bone formation. We found that statins stimulate BMP2 promoter activity and increase BMP2 (but not BMP4) mRNA and protein expression in osteoblastic cells, and showed statins increased the expression of BMP2 mRNA in rat tibia in vivo following a single oral dose of statin. Since statins increase BMP-2 transcription in bone cells, we next determined if this was necessary and required for statin effects on bone formation. First, we used bone cells stably transfected with mutant receptors to BMP-2 that make them unresponsive to BMP-2- we found that these cells were also unresponsive to statins. Further, we examined the effect of the BMP antagonist noggin (2ug/ml) in bone organ cultures treated with either lovastatin or simvastatin. Although noggin inhibited bone formation stimulated by these statins and BMP2, it did not affect FGF1-stimulated bone formation. Finally, we found that statin effects were impaired in vivo in transgenic mice in which the transgene comprised the dominant-negative truncated BMP receptor 1B linked to the type 1 collagen promoter. We injected lovastatin (5mg/kg/day), rBMP-2 (20ug/kg/day) and FGF1 20ug/kg/day subcutaneously for 5 days over the calvaria of truncated BMP receptor-1B homozygous transgenic mice and wild-type littermates. Mice were sacrificed at Day14 and histomorphometric analysis indicated both lovastatin and rBMP2 induced woven bone in wild-type mice but not in transgenic mice, in contrast to FGF1. We conclude that statins stimulate bone by inhibiting the HMG-CoA reductase enzyme, which increases BMP2 transcription and protein production resulting in enhanced osteoblast differentiation and bone formation.
THE NMDA-TYPE GLUTAMATE RECEPTOR ANTAGONIST MK801 REGULATES DIFFERENTIATION OF NON-ADHERENT RAT BONE MARROW OSTEOPROGENITORS.
K. R. Dobson*, T. M. Skerry University of York, York, UK We have demonstrated the expression of NMDA-type glutamate receptors and associated signalling apparatus in osteoblasts, suggesting a novel communication mechanism involved in bone remodelling. We have previously shown that MK801, a non-competitive ion channel blocker of NMDA receptors has significant effects on the differentiation of bone marrow osteoprogenitors, whereby osteoblast differentiation was inhibited and increased adipogenic capacity was measured. We now describe the effects of MK801 on the osteoblastic differentiation of nonadherent bone marrow osteoprogenitors. After an overnight incubation of total bone marrow cells, the non-adherent cells were transferred to fresh culture plates and treated. In both the cfu-f assay and the nodule formation assay, MK801 treatment resulted in a dose dependent (1-50 micromolar) decrease in osteoblastic phenotype as measured by ALP activity and mineralisation. Proliferation was not significantly effected in either culture method. As previously observed in cultures of total bone marrow cells, MK801 treatment caused a change in cellular morphology and cells became more rounded and contained perinuclear granules. When MK801 treated cells were further cultured in adipogenic permissive culture conditions cells containing Oil Red O positive lipid droplets were formed. RT-PCR analysis showed that both total bone marrow cells and the non-adherent bone marrow cells were positive for the osteoblast lineage marker Cbfa-1 and NMDAreceptor subunits. These results indicate that the non-adherent bone marrow cells are able to form mineralised colonies in the cfu-f and nodule formation assays similarly to total bone marrow cells. The presence of cbfa-1 mRNA in the nonadherent fraction confirms that osteoprogenitors reside in this fraction of bone marrow cells. The similar response to MK801 in cultures from both the nonadherent fraction of bone marrow cells and total bone marrow cells suggests that the target cells for MK801 reside in both these fractions. These observations therefore indicate that blocking signalling through NMDA receptors in bone marrow cells results in a reduced differentiation of osteoprogenitors. These data suggest that glutamate signalling within the bone marrow microenvironment plays a critical role in osteoprogenitor differentiation and plays an important role in maintenance of bone formation and therefore bone remodelling.
conference.book Page 76 Wednesday, February 7, 2001 8:44 PM
Abstracts - Oral Presentations
IBMS/ECTS 2001 - First Joint Meeting
CORTICAL BONE RESORPTION IS DRAMATICALLY INCREASED IN ADULT MICE OVEREXPRESSING OSF2/CBFA1 IN OSTEOBLASTS UNDER THE CONTROL OF COLLAGEN TYPE I
HORMONAL REPLACEMENT THERAPY REDUCES FOREARM FRACTURE INCIDENCE IN RECENT POSTMENOPAUSAL WOMEN - RESULTS OF THE DANISH OSTEOPOROSIS PREVENTION STUDY
M. Kneissel1*, A. Boyde2, B. Fournier1, P. Matthias3, V. Geoffroy3 1Novartis Pharma AG, Basle, Switzerland 2 University College London, London, UK 3Friedrich Miescher Institute, Basle, Switzerland The transcription factor Cbfa1 is required for bone formation during development. However its role in the adult skeleton is not fully understood yet. To address this question we have evaluated the bone phenotype of adult mice overexpressing Osf2/Cbfa1 under the control of the collagen type I promoter, which is expressed early during osteoblast commitment. As reported previously, we obtained 6 founder lines all presenting surprisingly with osteopenia (1). Expression of the transgene is tissue specific in these mice and the level of transgene expression correlates with the severity of the osteopenic phenotype. Multiple fractures occur predominantly in the hindlimbs and the tail vertebrae of these transgenics. We found now that osteopenia and fracture incidence increase continuously from the age of 2 to 16 months as determined by double energy x-ray absorptiometry, peripheral quantitative computed tomography, and radiographs. Interestingly, the bone phenotype is much more distinct in the cortical bone compartment than in cancellous bone. Both the amount of cortical bone and its volumetric density are dramatically reduced. Histomorphometric analysis of bones of 4-month-old mice, who have reached their peak bone mass, revealed that cortical bone modelling is severely disturbed. Bone resorption is dramatically increased at the endocortical bone envelope, resulting in expansion of the marrow cavity, while bone formation is enhanced at the subperiosteal bone envelope. The extremely reduced cortical thickness indicates that resorption exceeds formation. The observed subperiosteal increase in bone formation is only related to a rise in the number of bone forming cells and not in their performance rate. Bone mineralization as determined by back scattered electron imaging is lowered in transgenic animals. Whether the decreased matrix mineralization is due to the high bone turnover in these mice or whether itself triggers elevation in bone modeling rates cannot be decided from investigations at the tissue level and has to be determined in in vitro experiments. The observed dramatic increase in cortical bone resorption is unexpected. Our findings suggest that an increase of Cbfa1 at an early stage of osteoblast commitment enhances indirectly bone resorption in vivo. (1) Geoffroy et al. 2000 Calcif Tiss Int 66: O24
L. Mosekilde1*, H. Beck-Nielsen2, O. H. Sørensen3, S. P. Nielsen4, P. Charles1, P. Vestergaard1 1 Department of Endocrinology and Metabolism C, Aarhus University Hospital, Aarhus, Denmark 2Department of Endocrinology M, Odense University Hospital, Odense, Denmark 3 Department of Medicine, Hvidovre Hospital, Hvidovre, Denmark 4Department of Clinical Physiology, Hillerød Hospital, Hillerød, Denmark Objectives: To study the fracture reducing potential of hormonal replacement therapy (HRT) in recent postmenopausal women in a primary preventive scenario. Methods: Prospective controlled comprehensive cohort trial. 2,016 healthy women aged 45-58 years, from three to 24 month past last menstrual bleeding were recruited from a random sample of the background population. Mean age was 50.8±2.8 years, and the number of person years followed was 9,335.3. There were two main study arms: a randomised arm (randomised to HRT [n=502] or not [n=504]) and a non-randomised arm (on HRT [n=221] or not [n=789] by own choice). First line HRT was oral sequential oestradiol/norethisterone in women with intact uterus and oral continuous oestradiol in hysterectomised women. Results: After five years, a total of 156 fractures were sustained by 140 women. There were 51 forearm fractures in 51 women. By intention-to-treat analysis (n=2,016), overall fracture risk was borderline statistically significantly reduced (RR=0.73, 95% CI: 0.50-1.05), and forearm fracture risk was significantly reduced (RR=0.45, 95% CI: 0.22-0.90) with HRT. Restricting the analysis to women who had adhered to their initial allocation of either HRT (n=395) or no HRT (n=977) showed a significant reduction in both the overall fracture risk (RR=0.61, 95% CI: 0.39-0.97) and the risk of forearm fractures (RR=0.24, 95% CI: 0.09-0.69). Compliance with HRT was 65% after five years. Conclusions: It is possible to reduce the number of forearm fractures and possibly the total number of fractures in recent postmenopausal women by use of HRT as primary prevention.
OR28 SIDE EFFECT SEVERITY AND DISCONTINUATION RATE AMONG WOMEN INITIATING HORMONE REPLACEMENT THERAPY, RALOXIFENE, OR ALENDRONATE FOLLOWING BMD TESTING.
B. Ettinger1*, A. N. A. Tosteson2, M. Grove2, M. Moncur2, A. R. Pressman1, G. T. Ray1, G. M. Hebert1, C. Hammond2 1Division of Research, Kaiser Permanente Medical Care Program, Oakland, USA 2Clinical Research Section, Darmouth-Hitchcock Medical Center, Lebanon, USA Many women initiating osteoporosis treatment discontinue within one year. To characterize treatment side effects and their association with discontinuation we studied Kaiser Foundation Health Plan in Northern California women with bone mineral density (BMD) t-scores below -1.0 who began treatment with HRT, raloxifene, or alendronate within 6 months after BMD testing. We excluded those using any osteoporosis therapies prior to BMD. In structured telephone interviews, reasons for initiation, side effect experiences, discontinuation and reasons for discontinuation were documented. To quantify the bothersomeness of side effects, women who reported side effects were asked how many days in the next month, if any, they would give-up to avoid their side effects (i.e., disutility). Thus far, among 213 HRT, 110 raloxifene, and 253 alendronate users interviewed an average of 7 months after starting treatment, more than 90% initiated treatment due to osteoporosis concern. Side effects were reported for 40% of HRT, 30% of raloxifene, and 27% of alendronate users and were very or extremely bothersome for 22% of HRT, 8% of raloxifene (chi-squared p=0.002 vs. HRT), and 13% of alendronate (chi-squared p=0.01 vs. HRT) users. Among women with side effects, disutility differed across treatments with 29% for HRT, 6% for raloxifene (p=0.01 vs. HRT) and 21% of alendronate (p=0.31 vs. HRT) users reporting any disutility. The proportion of women discontinuing differed by treatment and was 27% for HRT, 14% for raloxifene (p=0.01 vs. HRT), and 21% for alendronate (p=0.17 vs. HRT). Side effects were a reason for discontinuation among 60-76% of those stopping. Across treatments, there were no significant differences in continuation by BMD score (osteopenic vs. osteoporotic). We conclude that osteoporosis is a primary reason for treatment initiation among women with low BMD and that among these 3 treatments, raloxifene had the lowest rate of troublesome side effects and the highest rate of continuation. Differences between raloxifene and alendronate were not statistically significant, however our analyses are currently limited due to small sample sizes. To effectively prevent osteoporotic fractures through enhanced long-term treatment continuation, it is critical that the most tolerable treatments be identified; raloxifene appears most promising in this regard.
IDENTIFICATION OF A UNIQUE POPULATION OF BONE MORPHOGENETIC PROTEIN-6-EXPRESSING CELLS DURING OSTEOGENESIS IN VIVO A. Plant*, J. H. Tobias Rheumatology Unit, Division of Medicine, University of Bristol, Bristol, UK It is well recognised that administration of high-dose estrogen induces osteosclerosis in the long bones of female mice. To explore the mechanisms which mediate this response, we examined the changes in mRNA expression of candidate growth factors which occur within the long bones of female mice following highdose estrogen administration. Total and polyA+ RNA was isolated from whole femurs prior to, or 1, 2, 4, 8, 12 and 16 days following treatment with 17betaestradiol (E2) 0.5mg/animal/week (10 animals per time-point). RNA was pooled for each treatment group, and resultant Northern blots hybridised with cDNAs for TGF-beta 1, PDGF-A, PDGF-B, IGF-1, BMP-2 and BMP-6. Results, which were corrected for beta-actin expression, were combined from three independent experiments. Interestingly, BMP-6 showed a rapid and selective increase in expression, which reached significance following eight days of treatment (p<0.05 by ANOVA). We then investigated changes in BMP-6 at the protein level by indirect immunofluorescence, as assessed in longitudinal sections of the distal femur from female mice treated with estrogen as above. In untreated animals, BMP-6 protein was found to be expressed by growth plate chondrocytes and a sparse population of marrow cells dispersed uniformly through the marrow cavity, but no expression was observed by osteoblasts on trabecular, endosteal or periosteal surfaces. As early as four days after commencing treatment with E2, an additional population of BMP-6-expressing cells was observed, forming discrete clusters within bone marrow. Analysis of sections from later time-points revealed clusters of BMP-6 positive cells adjacent to trabecular surfaces at sites of new bone formation. Double immunolabelling demonstrated that these BMP-6 positive cells express neither leukocyte nor erythrocyte markers (i.e. CD45 and TER-119 respectively), consistent with a stromal origin. We conclude, therefore, that estrogen-induced osteogenesis is associated with the appearance within bone marrow of a unique population of BMP-6 expressing stromal cells. The rapidity with which these cells appear following E2 administration raises the possibility that they play a major role in mediating the osteogenic response to high-dose estrogen, and possibly other anabolic stimuli.
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Time, Lumbar Spine, Lumbar Spine, Femoral Neck, Months placebo hPTH 1-34 Placebo 6 mo 12 mo 18 mo 24 mo
0.7% 9.2% * 0.2% 0.1% 0.7% 20.5% * 0.0% 4.0% ** O.6% 23.6% * 0.0% 7.3% * 0.9% 29.2% * 0.2% 11.0% * Pbo DC PTH DC Pbo DC PTH DC 30 mo 0.8% 27.5% * 0.2% 12.0% * 36 mo 0.9% 25.1% * 0.2% 11.0% * *p<0.0001, **p<0.002 Of those randomized to PTH who had DXA at 24 months, 89% had increases of lumbar spine BMD greater than the equivalent of 2 population SD. Trabecular bone density in L1, L2 as measured with QCT, increased in the PTH group a median of 74% from baseline at 24 months (p=0.027) and decreased 2.1% in the placebo group. During the observational phase of the study BMD decreased slightly but significantly at the spine but did not change significantly at the femoral neck. These findings indicate that hPTH 1-34 and estrogen therapy is effective in producing large relatively durable increases in bone density at the lumbar spine and femoral neck in postmenopausqal women with osteoporosis.
G. Goemaere1*, J-M. Kaufman1, O. Wang2, B. H. Mitlak2 University Hospital, Ghent, Belgium 2 Eli Lilly and Company, Indianapolis, IN, USA We evaluated the relationship between radiographic vertebral fracture grade and clinical sequelae of fracture (height loss and back pain) in a double-blind randomized, placebo-controlled study of LY333334 [recombinant human parathyroid hormone (rhPTH(1-34)] treatment of postmenopausal osteoporosis. In this study 1637 women with one or more prevalent vertebral fracture were assigned to 1 of 3 study arms: placebo, 20 (PTH20) or 40 (PTH40) microgram rhPTH(1-34) administered once-daily by self-injection. All patients received daily calcium and vitamin D supplements. The median follow-up was 21 months. Baseline and endpoint lateral spine radiographs were assessed centrally for incident vertebral fractures using a semi-quantitative grading score (J Bone Miner Res 1993;8:1137). A new fracture was reported when the score for any vertebra increased from 0 at baseline to 1, 2, or 3; a moderate or severe fracture was reported when the grade increased from 0 to 2 or 3. One or more incident vertebral fractures occurred in 105 women (7.9% of those evaluable). Relative to women receiving placebo, those receiving PTH20 or PTH40 had relative risks for vertebral fracture of 0.35 [95% confidence interval (CI), 0.2 to 0.6] and 0.31 (0.2 to 0.5), respectively. For the 55 women with one or more moderate or severe fracture, the risk of fracture was 0.10 (0.04 to 0.3) and 0.22 (0.1 to 0.4) for the PTH20 and PTH40 groups compared with placebo, respectively. Among patients with one or more new vertebral fracture, the proportion with moderate or severe fractures was significantly less in the PTH groups compared with placebo (chi square, p<0.001). Among patients with new vertebral fractures, reports of new or worsening back pain and changes in height were compared across treatment groups (table). Treatment with rhPTH(1-34) strikingly decreased the incidence and severity of vertebral fractures and the clinical sequelae of fracture. *for the PTH Fracture Prevention Study Investigators
N (%) of patients with 64 (14%) 22 (5%)* one or more new vertebral fracture % reporting back pain 45% 23%# Mean height loss (cm) 1.1 0.2* *p<0.001, **p<0.05 compared with placebo #p=0.015 for pooled PTH doses compared with placebo
TREATMENT WITH RECOMBINANT HUMAN PARATHYROID HORMONE (1-34) DECREASES THE SEVERITY AND SEQUELAE OF VERTEBRAL FRACTURES
Femoral Neck, hPTH 1-34
OR31 EFFECTS OF THE SELECTIVE ESTROGEN RECEPTOR MODULATOR RALOXIFENE ON BONE METABOLISM AND PITUITARY-GONADAL AXIS IN HEALTHY MIDDLE-AGED MEN B. Uebelhart1*, J. P. Bonjour1, M. Draper2, I. Pavo3, R. Rizzoli1 1Division of Bone Diseases, University Hospital, Geneva, Switzerland 2 Eli Lilly and Company, Indianapolis, Indiana, USA 3Lilly Area Medical Center, Vienna, Austria Raloxifene (RLX) is a Selective Estrogen Receptor Modulator (SERM) which acts in bone as an estrogen-agonist, since it has been shown to decrease bone turnover, increase bone mineral density, and prevent vertebral fractures in postmenopausal women (JAMA, 282:637-47, 1999). There is a great interest to study the influence on bone metabolism and pituitary-gonadal axis of RLX in men, since testosterone action on bone in men may be partly mediated by estradiol. We conducted a randomized RLX vs placebo (PL) two-sequence crossover study in 43 healthy adult men with normal gonadal function (mean age: 56 years, range 49-70). The subjects were randomly assigned to receive either RLX 120 mg/day (RLX/PL, N=22) or placebo (PL/RLX, N=21) for 6 weeks, followed by a 8-week wash-out period. Then, all subjects were crossed-over for the same schedule. Blood and urine were collected at the end of each treatment period, before and after an acute oral calcium load. RLX decreased serum osteocalcin (17.8vs19.8µg/l, p<0.001) without affecting fasting urinary Ca-to-creatinine ratio or urinary free deoxypyridinoline, suggesting that net bone resorption was not changed. RLX decreased serum phosphate (Pi) (1.06vs1.10mmol/l, p=0.03), an effect still detectable after the acute oral Ca load. The maximal tubular Pi transport was reduced (1.25vs1.33mmol/l GFR, p=0.01). Increases in urinary Ca-to-creatinine ratio before and after an oral Ca load were lower in RLX-treated men (p=0.04), suggesting a decrease in intestinal Ca absorption on RLX. Calcitriol was decreased (119vs131pmol/l, p=0.02), but PTH was not modified. RLX caused a 29% increase in FSH (p<0.001), a 18% increase in LH (p=0.03), a 13% increase in total testosterone (p=0.002), a 8% increase in SHBG (p=0.03), a 22% increase in free prostate specific antigene (p=0.002), and a 18% increase in cortisol (p<0.001) compared to PL. RLX treatment caused a 10% decrease in prolactin (p=0.01) and a 14% decrease in IGFI (p<0.001). Serum lipids and renal function were not affected. RLX was well tolerated, and devoid of side effects. In summary, in healthy middle-aged men, RLX has significant influences on the pituitary-gonadal axis and on bone metabolism, with a decrease in biochemical markers of bone formation, of IGF-I, calcitriol, Pi, and of renal tubular Pi transport.
PTH40 19 (4%)* 21%# 0.3**
OR30 TWO YEARS OF PARATHYROID HORMONE 1-34 AND ESTROGEN PRODUCE DRAMATIC BONE DENSITY INCREASES IN POSTMENOPAUSAL OSTEOPOROTIC WOMEN THAT DISSIPATE ONLY SLIGHTLY DURING A THIRD YEAR OF TREATMENT WITH ESTROGEN ALONE:- RESULTS FROM A PLACEBO-CONTROLLED RANDOMIZED TRIAL C. D. Arnaud1*, E. B. Roe2, M. S. Sanchez1, P. Bacchetti1, D. M. Black1, C. E. Cann1 1 University of California, San Francisco, USA 2University of Manitoba, USA We determined the bone-building efficacy of daily, self administered, subcutaneous hPTH 1-34 (400 IU/day) plus oral estrogen in a 2 - year, randomized, double blind, placebo controlled trial. Eligible women were at least 5-years postmenopausal, had osteoporosis [lumbar spine or hip bone mineral density (BMD) more than 2.5 SD below the mean for young normal women] and were required to have taken hormone replacement therapy (HRT) for at least one year immediately prior to entry into the study. Seventy four women were randomized to receive either hPTH or placebo. In addition, the participants received Premarin 0.625 mg daily, medroxyprogesterone if needed, vitamin D 800 IU daily, and calcium supplements to ensure a total daily calcium intake of 1500 mg. At 2 years, hPTH 1-34 and placebo injections were discontinued, the blind was broken, and participants were followed for an additional year. During the entire 2 year treatment and one year observational phases of the study lumbar spine, hip and forearm BMD were measured by dual energy absorptiometry (DXA) and lumbar spine and hip BMD by quantitative computed tomography (QCT). Secondary endpoints included biochemical markers of bone metabolism and spine x-rays. A total of 60 subjects (81%) completed the treatment phase and 56 (76%) completed the observational phase of the trial. The median bone density changes as measured by DXA are shown in the following table:
OR32 THE LEVEL OF ENDOGENOUS ESTRADIOL DETERMINES THE THERAPEUTIC RESPONSE TO A SELECTIVE ESTROGEN RECEPTOR MODULATOR IN ELDERLY MEN
P. M. Doran*, B. L. Riggs, E. J. Atkinson, W. M. O'Fallon, S. Khosla Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA Several lines of evidence implicate estrogen deficiency as a cause of bone loss in elderly men. Thus, we assessed the effect on bone turnover of six months of treatment with raloxifene (60 mg/day), a selective estrogen receptor modulator (SERM) that has an agonist effect on bone but is not feminizing, versus placebo in 50 elderly men (mean age±SD 69.1±6.0 years). The mean change in urinary N-telopeptide of type I collagen (NTx) excretion with treatment did not differ between the groups. However, the change in NTx following treatment was directly related to the baseline serum estradiol level in the raloxifene (r=0.57, P=0.004) but not in the placebo-treated (r=0.15, P=0.485) men (P=0.015 for the difference between the groups). Moreover, the men in whom NTx excretion decreased following raloxifene treatment had significantly lower baseline estradiol levels (mean±SEM, 22±2 pg/mL) than the men in whom urinary NTx excretion did not change or increased following raloxifene therapy (30±3 pg/mL,
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P=0.03), and no such difference was found in the placebo group. Consistent with this, regression analysis indicated that approximately 26 pg/mL defined a baseline estradiol level above which raloxifene tended to increase urine NTx excretion. Thus, the effect of raloxifene on bone resorption in elderly men may be dependent on endogenous estradiol levels. These studies also define a level of skeletal estrogen sufficiency in elderly men (approximately 26 pg/mL) above which raloxifene increases bone resorption. Interestingly, we have also found in population studies that elderly men with estradiol levels below this value have higher rates of bone loss and bone resorption than men with estradiol levels above this value (JBMR (Suppl) 15:S159, 2000). We speculate that above this threshold raloxifene may compete with the more potent estradiol for binding to the estrogen receptor, whereas below this level it may bind to unoccupied estrogen receptor sites. Our findings also suggest that future studies assessing the effects of SERMs on bone in elderly men should focus on men with estradiol levels below this value.
Type n Ht Z start Ht Z 12 mo. n GMFM Start %
GMFM 12 mo. %
I 21 -1.8±1.0 III 29 -6.9±1.9 IV 50 -3.4±2.1 V 8 -2.2±1.4 ? 13 -4.3±3.6 (* p<0.05)
91.2±3.3* 42.2±23.1* 69.9±20.4* 82.2±26.8 52.6±28.7*
-1.5±1.0* -6.0±1.9* -3.3±2.13 -1.9±1.1 -3.9±3.4
5 9 8 4 5
78.4±1.4 24.2±24.7 52.5±28.8 78.5±26.6 33.0±38.3
OR35 MALE MICE LACKING ESTROGEN RECEPTOR-BETA LOSE CANCELLOUS AND CORTICAL BONE FOLLOWING ORCHIDECTOMY
H. Z. Ke*, T. A. Brown, K. L. Chidsey-Frink, H. Qi, D. T. Crawford, H. A. Simmons, D. N. Petersen, M. R. Allen, J. D. McNeish, D. D.Thompson Global Research and Development, Groton Laboratories, Pfizer Inc., Groton, Connecticut, USA The effects of androgens on the male skeletons may be through the direct stimulation of the androgen receptor and/or through aromatization of androgens into estrogens, and then indirectly, through stimulation of estrogen receptors (ERs), ER-alpha and ER-beta. However, the relative importance of ER-alpha and ER-beta in skeletal growth, development and maintenance of males is not clearly understood. The purpose of this study was to evaluate the effects of androgen deficiency induced by ORX on bone and seminal vesicle in ER-beta knockout (BERKO) male mice. BERKO male mice and wild-type (WT) littermates were ORX or sham-operated (sham) at 4.5 months of age. All mice were necropsied 8 weeks post-surgery. Compared with WT-sham, BERKO-sham mice had significantly lower seminal vesicle weight. There was no significant difference between WT-sham and BERKO-sham in body weight, PQCT parameters of the distal femoral metaphysis (DFM) and tibial shafts, and histomorphometric parameters of the DFM. Following ORX, body weight gain decreased significantly in both WT and BERKO mice as compared with their sham controls. Seminal vesicle wet weight decreased significantly in WT-ORX and BERKO-ORX compared with their sham controls. In the DFM, ORX induced significant decreases (from -11% to -40%) in volumetric mineral content and volumetric mineral density of total, trabecular and cortical bone compartments in both the WT and BERKO mice. There was no significant difference in these parameters between WT-ORX compared with BERKO-ORX. The changes in PQCT parameters of the tibial shaft were similar to those of the DFM. Histomorphometric analysis of the DFM showed that ORX induced a decrease in trabecular bone volume and trabecular number, and an increase in trabecular separation, osteoclast number, osteoclast perimeter, and bone formation rate-bone volume referent in both WT and BERKO mice. These data indicate that androgen deficiency stimulates bone turnover and induces bone loss in both WT and BERKO mice. In conclusion, we have demonstrated that ORX induced significant loss in bone mass and seminal vesicle weight in male mice lacking ER-beta. Thus, the mechanisms of loss in bone and seminal vesicle weight following androgen deficiency in male mice are independent from ER-beta.
CAN THREE DROPS OF VITAMIN D PREVENT HIP FRACTURES? M. Billsten1*, I. Rodine1, E. Ornstein1, O. Johnell2, I. Sernbo2 of Orthopaedics Hassleholm-Kristianstad hospital, S-29185 Kristianstad, Sweden 2 Department of Orthopaedics, Malmo University Hospital, S-205 02 Malmo, Sweden At present there are some studies with the combination of Vitamin D and calcium which show fracture reduction. However for Vitamin D alone the results are not conclusive Method. In a prospective control clinical trial, we investigated the effects of oral vitamin D supplements on hip fractures and other non-vertebral fractures. We included 2404 women 50 years of age and older (mean age 85.8±6.3) living in 174 nursing homes in southern part of Sweden. The patients were followed for three years. One district was selected as treatment area and the surrounding districts as control areas.1594 women (mean age 85.7±6.5) were assigned to receive 21,000 IU vitamin-D3 once per month. The dose were distributed by nurse as three drops of ergocalciferol (294,000IU/ml) per month. In the control group we included 854 women (mean age 85.9±5.9). The average daily dietary calcium intake was 650 mg. Results. Hip fracture: After 12 months there had been 120 fractures, after 24 months 186 and after 36 months 220. For all participants the odds ratio during the first 12 months was 0.66 (0.46-0.93), 24 months 0.79 (0.59-1.6) and during 36 months 0.85 (0.65-1.11) However during the trial, which was a real life trial, some patients were treated with calcium or bisphosphonates. That group included 320 women. With these excluded the significant levels changed somewhat, after 12 months the odds ratio was 0.48 (0.32-0.73), after 24 months 0.68 (0.49-0.94), 36 months 0.77 (0.58-1.04). Other fractures: After 12 months there had been 124 fractures, after 24 months 203 and after 36 months 227. The corresponding odds ratios for these were 0.60 (0.42-0.86), 0.70 (0.55-0.95) and 0.78 (0.60-1.01). If those who had been taking calcium or bisphosphonates during the trial were excluded the corresponding RR was 0.49 (0.33-0.75), 0.65 (0.48-0.88) and 0.72 (0.54-0.95), respectively. Conclusion. Women living in nursing homes with short life expectancy have a greater chance to die without a hip fracture if they take three drops of Vitamin D per month.
ESTROGEN INHIBITS ADIPOCYTIC DIFFERENTIATION OF BONE MARROW STROMAL CELLS THROUGH EITHER ESTROGEN RECEPTOR ALPHA OR BETA
PAMIDRONATE TREATMENT IMPROVES GROSS MOTOR FUNCTION AND GROWTH IN CHILDREN WITH OSTEOGENESIS IMPERFECTA
R. Okazaki1*, D. Inoue2, M. Shibata1, M. Saika2, S. Kido2, Y. Sakamoto1, T. Matsumoto2 1 Teikyo University, Ichihara, Japan 2 Tokushima University, Tokushima, Japan Protective effects of estrogen (E) on bone have been mainly attributed to its inhibitory effects on osteoclast differentiation/activation through suppression of bone-resorptive cytokines. However, evidence suggests that increased lipid accumulation in the bone marrow may be associated with osteporosis in the aged women. Since both osteoblasts and adipocytes stem from bone marrow stromal cells (BMSCs) of mesenchymal origin, E may be involved in the regulation of BMSC differentiation toward the osteoblast and adipocyte lineages. In order to test this hypothesis, we examined effects of E on differentiation of wild-type ST-2, a mouse BMSC line, as well as stable cell lines overexpressing human E receptor (ER) alpha (ST2ERalpha) or ERbeta (ST2ERbeta), developed in our laboratories. Overexpression of functional ER in these stable cell lines was confirmed by enhancement of transcription via E response element using luciferase assay. All the cell lines expressed BMP receptor (BMPR) 1A but not BMPR1B. Addition of BMP-2 for 6 days, at a dose of 100-500 ng/ml significantly increased alkaline phosphatase (ALP) activity as well as the number of oil red-O positive adipocytes, indicating that BMP-2 has positive effects on both adipogenesis and osteoblastogenesis in these bipotential cells. As we previously demonstrated, 17beta-estradiol (E2) enhanced induction of ALP activity as well as expression of ALP mRNA by BMP-2. In contrast, 1nM of E2 inhibited basal as well as BMP-2-induced adipocyte formation by 30-50% in both ST2ERalpha and ST2ERbeta cells. These effects of E2 were completely reversed by the addition of 100-fold excess of type 2 E antagonist. E inhibition of adipogenic differentiation was accompanied by a substantial decrease in the mRNA level of peroxisome proliferator activated receptor gamma2 (PPARgamma2), an early marker of
H. Plotkin*, J. Gibis, F. H. Glorieux Genetics Unit, Shriners Hospital for Children, and the Departments of Surgery and Pediatrics, McGill University, Montréal, Québec, Canada Osteogenesis imperfecta (OI) is a disorder characterized by osteopenia and bone fragility. Bisphosphonates have proven to be effective to increase bone density, and to decrease pain and fracture rate in children with OI. We present here the results of the changes in height and gross motor function (GMF) in 121 (50 boys) patients with OI treated with pamidronate IV for one year. Age at start ranged from 0.1 to 21 years of age (mean 7.7±4.9). Twenty-one patients had type I OI, 29 type III, 50 type IV, 8 type V and 13 patients could not be labeled as per the current classification. All patients received pamidronate at a dose of 3 mg/kg/cycle (3 days) every 4 months (>3 years of age), or 1.5 mg/kg/cycle every 2 months (<3 years of age), and physiotherapy (pt) assessment and stimulation. GMF was evaluated in 31 patients over 5 years of age using the GMFM method, quantifying GMF in 5 dimensions (lying and rolling, sitting, crawling and kneeling, standing, and walking, running and jumping). Normal subjects over 5 years of age should score 100%. An annual improvement of 7% is clinically significant for children with disabilities. Height was measured with a Harpender stadiometer. Mean height Z-score for the patients as a group was –3.7±2.6 at baseline, and increased to –3.4±2.5 after 12 months of treatment (p<0.001 by Wilcoxon Signed Rank Test) (n=121). Height Z-score increased significantly in type I and type III patients, and did not change in patients with OI type IV, V and unclassified (?). Patients showed an overall 14% improvement in GMFM scores. There were no negative effects of therapy on growth. Data in fact suggest catchup growth in patients with types I and IV. GMF improves significantly under interdisciplinary intervention including pamidronate treatment and a rehabilitation program.
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adipocytes, but not that of PPARgamma1. E2 also inhibited induction of adipocytes by a PPAR-ligand, troglitazone (1 microM), suggesting that E may affect the activity of PPARgamma itself or events after induction of PPARgamma. Collectively, these results suggest that E inhibits adipogenesis and, at the same time, stimulates osteoblastogensis from BMSCs via either ERalpha or ERbeta. Therefore, E may directly promote commitment of the BMSC into the osteoblast in preference to the adipocyte, thereby stimulating bone formation.
REGULATION AND FUNCTIONS OF VITAMIN D RECEPTOR SHUTTLING
OR37 ESTROGEN DEFICIENCY CAUSES BONE LOSS THROUGH UPREGULATED T CELL PRODUCTION OF TNF ALPHA C. Roggia*, M. N. Weitzmann, S. Cenci, G. Toraldo, J. Kindle, R. Pacifici Division of Bone and Mineral Diseases Washington University School of Medicine, St. Louis MO, USA A key mechanism by which estrogen (E2) deficiency induces bone loss is by increasing osteoclast (OC) formation through upregulated production of osteoclastogenic cytokines. However, the contribution of specific cells and cytokines remains controversial. We found that T cell deficient athymic nude mice were completely protected against the increase in bone resorption (as measured by pyridinium excretion) and the resulting bone loss (as measured in vivo by pQCT) induced by ovariectomy (ovx). Bone loss was reinduced by transplant into nude mice of T cells harvested from wild type ovx mice. In contrast, no bone loss took place after transplant into nude mice of T cells harvested from TNF deficient ovx mice. These findings demonstrate that T cell produced TNF plays a key role as a cause of ovx induced bone loss. Ovx increased by 3 fold T cell proliferation, by 2 fold the number of T cells in the bone marrow and by 4 fold the T cell production of TNF alpha. The stimulatory effects of ovx on T cell proliferation and TNF alpha production were not reversed by in vitro E2 treatment, thus suggesting mature T cells are not directly regulated by E2. T cells (or T cell conditioned media) from ovx mice increased M-CSF and RANKL-induced differentiation of purified monocytes into OC by 3 fold. In contrast T cells (or T cell media) from E2 replete mice had no effect. The osteoclastogenic activity released by T cells was completely neutralized by anti TNF alpha antibody, confirming that T cells upregulate OC formation via a TNF dependent mechanism. T cells from wild type ovx mice failed to augment OC differentiation of monocytes lacking the TNF receptor p55, while they promoted the differentiation of monocytes from mice lacking the p75 receptor. Furthermore, p55 deficient mice (but not WT and p75 KO mice) were completely protected against ovx induced bone, thus establishing that T cell derived TNF upregulates osteoclastogenesis and causes bone loss via p55. The finding that T cell produced TNF alpha mediates the catabolic effects of E2 deficiency on bone provides a novel paradigm for T cells as an essential target of E2 in bone.
J. Barsony*, K. Prufer, C. Segovis Laboratory of Cell Biochemistry and Biology, NIDDK, NIH, USA We showed recently that hormone-induced changes in vitamin D receptor (VDR) cytoplasmic/nuclear distribution are important for controlling transcriptional activity. To explore the mechanisms, regulation, and functional significance of this receptor translocation, we tagged VDR and RXR with multicolor fluorescent protein variants and tagged calcitriol, the ligand for VDR, with a red-BODIPY label (red calcitriol). Photobleaching experiments on multinucleated COS-7 cells demonstrated that both VDR and RXR shuttle rapidly across the nuclear membrane. GFP-VDR export from the nucleus was blocked by point mutations in the ligand- and DNAbinding region previously known to be important for heterodimerization. Nuclear import of GFP-VDR was blocked unexpectedly by point mutations in the ligand binding Helix-3 domain of GFP-VDR (nls2GFP-VDR). To explore the influence of heterodimerization on the subcellular distribution of receptors, we introduced RXR-BFP into cells that stably express GFP-VDR. Fluorescence resonance energy transfer microscopy (FRET) demonstrated that the unliganded GFP-VDR and RXR-BFP form heterodimers. RXR-BFP coexpression promoted hormone-independent nuclear accumulation of GFP-VDR and nls2GFPVDR by influencing both nuclear import and retention. The increase in nuclear heterodimer content correlated with an increase in basal transcriptional activity. RXR-BFP also promoted hormone-dependent nuclear accumulation of receptors that harbor mutations in the known nuclear localization signal within the DNAbinding region (nls1GFP-VDR). Coexpression of RXR-BFP with nls1GFP-VDR rescued calcitriol-dependent transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nls1GFP-VDR distribution or activity. Simultaneous recordings of red calcitriol and GFP-VDR import into the nucleus indicated that hormone and receptor translocation coincide. Nuclear distribution of hormone and receptor was also similar. Hormone-bound receptors were visualized by reverse FRET microscopy. Interaction between the green GFP-VDR (donor) and the red calcitriol (acceptor) was indicated by the increase in donor emission after photodestruction of the acceptor. These FRET experiments were used to monitor the shuttling of liganded wild type and import/export mutant receptors. The results revealed a role for the receptor to carry the hormone into the nucleus. Combined, our data demonstrate that VDR shuttles between the cytoplasm and the nucleus, show that heterodimerization with RXR influences this shuttling, and strongly support the model that shuttling is essential for ligand-dependent transcriptional activity.
VITAMIN D IS A NEGATIVE REGULATOR OF BONE FORMATION
THE TRABECULAR BONE MINERAL DENSITY IS PRESERVED BOTH BY AN ACTIVATION OF THE ANDROGEN RECEPTOR AND BY AN ACTIVATION OF THE ESTROGEN RECEPTOR ALPHA IN MALE MICE
H. Tanaka1*, K. Kinuta1, N. Inoue1, M. Shinohara1, S. Kato2, Y. Seino1 1Okayama University Medical School, Okayama, Japan 2 University of Tokyo, Tokyo, Japan Vitamin D is an important hormone in bone metabolism, but there is a little evidence that vitamin D actively participates in this process. Vitamin D receptor (VDR) null mice have provided new insights in vitamin D metabolism and its role in vivo. However, like other nuclear receptor gene knockout, calcium supplementation experiments aimed at establishing physiological direct functions of VDR in many organs including bone have been inconclusive owing to essential roles of mineral in biological function. Although calcium supplementation showed apparent cure of rickets, we could not exclude the compensatory mechanisms such as hyperparathyroidism in this process. To evaluate the direct function of vitamin D in bone, we analyzed ectopically transplanted VDRKO bone in wild mice. 2 weeks old VDRKO bone is indistinguishable from wild bone in terms of the histology and the biochemistry. Therefore, each wild mouse or VDRKO was received 2 weeks old VDRKO bone and wild one in each side of the back muscle. After 2, 3, 4 weeks, the bones were retrieved and analyzed by micro focus X-ray, Micro CT and histological method. As expected, wild bone in VDRKO showed marked bone resorption because of hyperparathyroidism and high 1,25(OH)2D. However, the VDRKO bone in wild mouse (KOW) showed dramatic increase in bone density compared with wild bone in wild mouse (WW). Bone volume was 0.175 mm3 in KOW and 0.105 mm3 in WW at 4 weeks after transplantation. Similarly, bone thickness of calvaria was 180 microm, which was 2.7 fold compared to WW. To explore this mechanism, we next examined mRNA expression of the genes, which may play key roles in bone metabolism, by RT-PCR in the transplanted bone. The results demonstrated increased expression of Cbfa1, BMP4 and OCIF/OPG in KO bone. From these results, we conclude that vitamin D may play a important role in normal bone by protecting it from excessive bone formation by suppressing the expression of Cbfa1, BMP4 and OCIF/OPG.
S. Movérare1*, M. K. Lindberg1, S. Skrtic1, J. Å. Gustafsson2, C. Ohlsson1 1Department of Internal Medicine, Division of Endocrionology, Sahlgrenska University Hospital, Göteborg, Sweden 2Department of Medical Nuitrition, Karolinska Institute, Novum, Huddinge, Sweden Androgens may regulate the male skeleton either directly via a stimulation of androgen receptor (AR) or indirectly via aromatisation of androgens into estrogen and, thereafter, via stimulation of estrogen receptors (ERs). There are two known estrogen receptors, ERalpha and ERbeta. The aim of this study was to investigate the mechanism of action for the effect of androgens on trabecular BMD in male mice. Furthermore, the effect of estrogen on orchidectomized male mice, lacking one or both of the two estrogen receptors, was investigated. Seven months old male mice, lacking ERalpha (ERKO), ERbeta (BERKO) or both receptors (DERKO), were orchidectomized and treated for three weeks with 0.7 mg/mouse/day of 17beta-estradiol or the vehicle. Before orchidectomy, the trabecular BMD did not differ between WT, ERKO, BERKO and DERKO mice, demonstrating that neither ERalpha nor ERbeta is required for the maintenance of a normal trabecular BMD in male mice. After orchidectomy, there was a similar decrease in trabecular BMD, resulting in equal levels of trabecular BMD in all groups. This reduction was completely reversed in WT and BERKO mice treated with estrogen, while no significant effect of estrogen was found in ERKO and DERKO mice. In summary, the trabecular BMD is preserved both by an activation of the androgen receptor and by an activation of ERalpha, indicating that the AR and ERalpha are redundant in the maintenance of a normal trabecular BMD in male mice. In contrast, ERbeta is of no importance for the regulation of trabecular BMD in male mice.
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induced by renal ischemia. The cellular basis for this protection requires further study. These studies suggest that HGF may have therapeutic potential in preventing ischemia-induced renal failure.
MECHANISMS OF BIPHASIC ANABOLIC AND CATABOLIC EFFECTS OF PARATHYROID HORMONE (PTH) ON BONE CELLS
Mouse Type Creat. 24 Hrs BUN 24 Hrs Creat 48 Hrs BUN 48 Hrs
R. M. Locklin*, S. Khosla, B. L. Riggs Endocrine Research Unit, Mayo Clinic and Mayo Foundation, Rochester, MN, USA Continuous treatment with PTH is catabolic whereas intermittent treatment is anabolic and is a promising formation-stimulating regimen for treating osteoporosis. However, the mechanism(s) mediating these divergent responses is unclear. RANK ligand (RANKL) and osteoprotegerin (OPG) are recently discovered members of the TNF and TNF receptor superfamily, are produced by stromal-osteoblastic cells, and are the final regulators of osteoclast formation (OCF). OCF is stimulated when RANKL binds to its receptor RANK on osteoclast lineage cells, but is inhibited when it binds to the soluble decoy receptor, OPG. We investigated whether the different effects of the two PTH regimens could be explained by differences in gene expression of IGF-I and RANKL/OPG. Marrow aspirates from C57 BL/6 mice were plated at 1 x 106/cm2 and cultured for 5 days in alpha-MEM with 10% heat-inactivated FBS. PTH(1-34) (10 nM) was then administered in culture medium for an additional 4 days, either continuously (CONT) or intermittently for the first 6 hours of each 48-hour period (INT). Changes in steady-state mRNA levels of osteoblast differentiation genes (alkaline phosphatase [AP], type I collagen [COL1], and cbfa-1) and IGF-I, and in OPG and RANKL were assessed by real-time quantitative RT-PCR. Results are given as mean (±SEM) compared to vehicle controls (C, set at 1.0). OCF (assessed by multinucleated TRAP+ cells counted in 10 fields) was nonexistent in controls, very low with INT treatment but frequent with CONT treatment. There were increases or trends for increases in expression of osteoblast differentiation genes and IGF-I with INT, but not CONT, treatment. Conversely, there were increases in OCF with CONT, but not INT, treatment that was associated with a 12-fold increase in the RANKL/OPG ratio. Thus, selective activation of IGF-I by INT and of RANKL by CONT treatment regimens explains the respective anabolic and catabolic responses to PTH treatment regimens. RANKL (C=1)
RANKL/ OCF OPG (no. (C=1) cells)
5.14±1.74 0.24±0.03 0.06±0.01 51.2±7.8 2.67±1.67
1.41±0.40 0.94±0.19 0.73±0.12 6.5±2.9
Control 1.3±0.3 105±22 PTHrP 1.0±0.2 64±39 HGF 0.7±0.2 64±14 units in mg/dl. ** = p<0.01; *** = p<0.05;
THE SKELETAL EFFICACY OF STATINS DO NOT COMPARE WITH LOW-DOSE PARATHYROID HORMONE M. Sato*, A. Schmidt, H. Cole, S. Smith, E. Rowley, L. Ma Lilly Research Laboratories, Indianapolis IN, USA Statins were shown previously to increase cancellous bone volume in ovariectomized rats (Mundy et al. 1999, Science 286:1946). Therefore, we attempted to characterize the biomechanical effects of cerivastatin and simvastatin on cancellous and cortical bone from osteopenic, ovariectomized rats. Specifically, mature Sprague-Dawley rats (6 months old) were ovariectomized 1 month prior to initiation of treatment for the following 3 months with maximally tolerated doses of statins and were compared to parathyroid hormone (PTH) as a positive control. Quantitative computed tomography (QCT) showed a 20% to 25% reduction in the volumetric bone mineral density (BMD) of cancellous bone sites in ovariectomized controls (Ovx) compared to sham-ovariectomy controls (Sham) by 1 month postsurgery. Therefore, rats were significantly osteopenic before treatment with compounds. No differences in body weight were observed between groups; however, 8/16 of the animals administered 1 mg/kg cerivastatin were euthanized before study termination. Significant reductions in serum cholesterol and triglyceride levels were observed for 0.01-1 mg/kg cerivastatin and 0.1-10 mg/kg simvastatin, confirming biological activity in vivo. Dual energy x-ray absorptiometry analysis of whole body composition showed no effect of statins on whole body bone mineral content (BMC) or lean mass; however, 0.1 mg/kg cerivastatin and 3-10 mg/kg simvastatin reduced fat mass by 49% to 60% as compared to Ovx. QCT and biomechanical analyses showed no significant effects of statins on the proximal tibial metaphysis, lumbar vertebra, or the femoral midshaft, compared to Ovx. By contrast, PTH increased whole body BMC, and restored bone mass, architecture, and strength at both cancellous and cortical bone sites in a dose-dependent manner to levels greater than Ovx, Sham, and baseline controls. Dose response analyses showed a minimally efficacious dose of 0.3 microg/kg/day for PTH in the proximal tibial metaphysis of this model. PTH had no effect on cholesterol levels, but was observed to slightly elevate triglyceride levels. These data clearly showed no skeletal effects of either cerivastatin or simvastatin in osteopenic ovariectomized rats. Unlike PTH, no evidence was generated to suggest that cerivastatin or simvastatin stimulates new bone formation in the proximal tibial metaphysis, vertebra, or femoral midshaft.
2.28±0.58 0.94±0.17 0.45±0.17 0.008
93±26 99±30 21±4***
12.53±1.84 5.19±0.86 1.75±0.81 0.91±0.13 0.006
2.7±0.5 2.4±0.9 0.4±0.03**
OR42 PREVENTION OF ACUTE ISCHEMIC RENAL FAILURE BY TARGETED DELIVERY OF GROWTH FACTORS TO THE PROXIMAL TUBULE IN TRANSGENIC MICE: THE ROLES OF HEPATOCYTE GROWTH FACTOR AND PTH-RELATED PROTEIN. N. M. Fiaschi-Taesch1*, S. Santos1, S. Van Why2, P. Esbrit3, J. J. Orloff4, A. F. Stewart1, A. Garcia-Ocana1 1Division of Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 2 Division of Pediatric Nephrology, Yale University School of Medicine, New Haven, CT, USA 3Fundacion Jimenez Diaz, Madrid, Spain 4Merck and Co, Rahway, NJ, USA Renal ischemia leads to renal tubular damage, impaired glomerular filtration, and acute renal failure. Treatment of renal failure would be enhanced by identification of factors that accelerate renal recovery from injury. Parathyroid hormone-related protein (PTHrP) and hepatocyte growth factor (HGF) stimulate proliferation in cells derived from the proximal nephron. To study the pathophysiological roles and therapeutic potential of these two factors in renal failure, we have developed transgenic mice overexpressing PTHrP or HGF in the proximal tubule under the direction of the gamma-glutamyl transpeptidase (GGT) promoter I. Baseline evaluation of GGT-PTHrP and GGT-HGF mice revealed the following characteristics: a) abundant expression of the respective transgenes in the kidney at the mRNA and protein levels, assessed by immunohistochemical staining and renal extracts; b) PTH/PTHrP type I receptor mRNA levels similar in GGT-PTHrP and normal littermates; c) normal renal morphology; and, d) normal plasma BUN and creatinine concentrations. Interestingly, GGT-PTHrP mice display significant hypophosphatemia (6.10±0.6 mg/dl) as compared to control siblings (7.97±0.5 mg/ dl, p=0.037), providing biological confirmation of overexpression of PTHrP in the proximal nephron. Acute ischemic renal injury was induced in transgenic and control mice by bilateral clamping (30 min) of the renal pedicle followed by re-perfusion. The Table shows the effects of renal ischemia on plasma creatinine and blood urea nitrogen (BUN) in normal and transgenic mice overexpressing the two transgenes as compared to controls, at 24 and 48 hours following renal ischemia. Results are mean±SEM: Overexpression of PTHrP in the proximal nephron of mice results in hypophosphatemia but no protection against ischemia-induced renal injury. In contrast, HGF overexpression results in dramatic protection from acute renal failure
OR44 NOVEL CGRP/AMYLIN RECEPTOR FUNCTIONS OF THE HUMAN CALCITONIN RECEPTOR ISOTYPE 2 MODULATED BY ACCESSORY RECEPTOR-ACTIVITY-MODIFYING PROTEINS (RAMP) K. Leuthäuser1*, R. Gujer1, A. Aldecoa1, R. A. McKinney2, R. Muff1, J. A. Fischer1, W. Born1 1 Research Laboratory for Calcium Metabolism, University of Zurich, Klinik Balgrist, Zurich, Switzerland 2Brain Research Institute, University of Zurich, Zurich, Switzerland RAMP are single transmembrane-domain accessory proteins required for CGRP and adrenomedullin receptor function of a calcitonin (CT) receptor-like receptor (CRLR) that exhibits 60% amino acid sequence homology with the human CT receptor isotype 2 (hCTR2). The hCTR2 predominantly interacts with CT in the absence of RAMP, but its coexpression with human RAMP1 revealed mixed-type CGRP/amylin receptors. In the presence of hRAMP3 the hCTR2 is specific for amylin alone. Cell surface protein cross-linking, co-immunoprecipitation and confocal microscopy have identified [125I]CGRP- and [125I]amylin-binding hCTR2/hRAMP1 complexes. [125I]CGRP binding was inhibited by human alphaCGRP, human amylin and hCT with IC50 of 15, 11 and 193 nM, respectively. Inhibition of [125I]amylin binding was observed with IC50 of 36, 8 and 731 nM for human alphaCGRP, human amylin and hCT, respectively. In COS-7 cells co-expressing hCTR2 and hRAMP1 maximal cAMP accumulation by human alphaCGRP was 17-fold (EC50 = 0.77±0.37 nM (n=5)). The response was attenuated by sCT(8-32) with a Ki of 2.9±1.4 nM (n=5), but was not affected by 100 nM human alphaCGRP(8-37). This defined hCTR2/RAMP1 as a CGRP receptor subtype distinct from a CRLR/ RAMP1 CGRP receptor isotype antagonized by CGRP(8-37) with a Ki of 49±22 nM (n=4). In conclusion, RAMP1 and RAMP3 co-expressed with hCTR2 define novel CGRP/amylin and amylin receptors, respectively. They are comparable to the well characterized CGRP/amylin and CGRP receptors in the nucleus accumbens and the hypothalamus.
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Abstracts - Oral Presentations
INTERACTIONS BETWEEN MULTIPLE MYELOMA AND BONE CELLS FORMING A VICIOUS CYCLE
IN VIVO OSTEOCLASTOGENIC EFFECTS OF THE C-C CHEMOKINE MACROPHAGE INFLAMMATORY PROTEIN-1 ALPHA
M. Abe, K. Hiura, J. Wilde, S. Kido, S. Hashimoto, S. Ozaki, K. Moriyama, D. Inoue, T. Matsumoto* Tokushima University, Tokushima, Japan Multiple myeloma (MM) develops and expands in the bone marrow, and causes a devastating bone destruction by enhancing osteoclastic resorption in their close vicinity. Therefore, close interactions between MM and bone cells including osteoclasts may be important to the development of MM bone lesions. Consistent with this idea, we found that co-cultures of MM cells and human PBMC resulted in increased osteoclast (OC) formation as well as enhanced survival of MM cells in vitro. Osteoclastogenic C-C chemokines macrophage inflammatory protein (MIP)-1 alpha and beta were secreted by most MM cell lines and primary cells, and OC induction by these cells in co-cultures was largely blocked by anti-MIP-1alpha and beta neutralizing antibodies. MIP-1 induced stromal cell expression of RANK ligand, which was also expressed by MM itself. Moreover, MIP-1 was able to enhance adhesion of MM cells to VCAM-1-coated plates as well as VCAM-1positive stromal cells in a manner dependent on VLA-4, a VCAM-1 receptor abundantly expressed on MM cell surface. MIP-1 also enhanced chemotaxis of preOC cell lines. We also found that PBMC-derived OCs enhanced survival and growth of human MM cells. These effects of OCs were only partially inhibited by an anti-human IL-6 neutralizing antibody, although we observed increased production of IL-6 by OCs or OC progenitors in co-cultures with MM cells. In addition, human MM cell growth was enhanced even in co-cultures with a mouse or rabbit OCs whose IL-6 cannot act on human cells. Furthermore, prevention of cellular contact by membrane filters completely abolished the OC effect. Therefore, OCs may enhance MM cell growth through a close cell-cell interaction by elaborating unknown factor(s) other than IL-6. In conclusion, MIP-1 may play a central role in establishing a microenvironment in which MM, stromal cells and OC progenitors interact and efficiently induce osteoclastogenesis. Osteclasts may in turn augment growth and activity of MM, thereby forming a vicious cycle that leads to extensive bone destruction and MM cell expansion.
B. O. Oyajobi1*, P. J. Williams1, D. Pulkrabek1, G. Franchin2, B. Sherry2, G. R. Mundy1 1 Medicine/Endocrinology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 2 The Picower Institute for Medical Research, Manhasset, NY, USA Multiple myeloma (MM) is characterized by extensive bone destruction due to excessive osteoclast (Ocl) activity induced by as yet unidentified factor(s) produced by the tumor cells. The C-C chemokine MIP-1alpha is produced by myeloma cells and its levels are reportedly elevated in bone marrow plasma of MM patients compared to other hematological malignancies and normal controls. MIP-1alpha, which is chemotactic for human and rodent osteoclast precursors, stimulates formation of TRAP+ Ocl-like cells in vitro. However, if important in the development of myeloma bone disease, MIP-1alpha should cause typical myeloma osteolytic lesions in vivo. To test this, we investigated the ability of MIP-1alpha to stimulate osteoclastogenesis and bone resorption in vivo. First, conditioned media (CM) harvested from clonal Chinese hamster ovary (CHO) cells stably transfected with full-length human MIP-1alpha cDNA (CHO/MIP-1alpha) which secrete human MIP-1alpha, stimulated bone resorption assessed by 45Ca release from radiolabeled fetal rat long bones. In contrast, CM from empty vector-transfected CHOneo cells had no effect in this assay. Next, when intramuscular tumors were induced in athymic mice by inoculation of either CHO clone into left thigh muscles, CHO/MIP-1alpha mice developed significantly greater numbers of osteolytic lesions, which occurred earlier and were larger in area as assessed by blinded image analysis of radiographs. Importantly, lytic lesions in the right hind limbs (contralateral side) were significantly more and larger in CHO/MIP-1alpha mice. To further examine the relevance of locally produced MIP-1alpha in bone marrow to typical myeloma bone lesions, we utilized a model in which tumors develop within 3 weeks in the medullary cavities of long bones of the limbs in nude mice following intracardiac inoculation of CHO cells. Compared to CHOneo tumors, CHO/MIP-1alpha tumors were associated with significantly more TRAP+ Ocl evident at both tumor-bone and tumor-marrow interfaces. Finally, we found that recombinant MIP-1alpha induced OC formation and bone resorption in vivo following daily injections of MIP-1alpha into the subcutaneous tissue overlying calvariae of normal mice. Together, these data show that MIP-1alpha increases Ocl formation and bone resorption in vivo when expressed adjacent to bone surfaces, consistent with the notion that the chemokine plays an important role in the pathogenesis of MM-associated bone destruction.
OR46 THE TRANSCRIPTION FACTOR NF-KB PLAYS A CRITICAL ROLE IN IGF-PROMOTED BONE METASTASIS OF BREAST CANCER
OR48 OSTEOPROTEGERIN INHIBITS THE DEVELOPMENT OF OSTEOLYTIC BONE DISEASE IN MULTIPLE MYELOMA P. I. Croucher1*, C. M. Shipman1, M. Perry2, J. Lippitt1, K. Asosingh3, E. J. R. van Beek4, B. Van Camp3, R. G. G. Russell1, C. Dunstan5, K.Vanderkerken3 1University of Sheffield Medical School, Sheffield, UK 2 University of Bristol, Bristol, UK 3Free University, Brussels, Belgium 4Royal Hallamshire Hospital, Sheffield, UK 5 Amgen Inc., Thousand Oaks, CA, USA Multiple myeloma (MM) is often associated with the development of osteolytic bone disease, although, the mechanisms that lead to this aspect of the disease are unclear. However, with improvements in our understanding of the mechanism of bone loss, novel therapeutic targets may be identified. Recent studies have shown that binding of the ligand for receptor activator of NF-kB (RANKL) to RANK, on osteoclast precursors, is essential for osteoclast formation and this process can be inhibited by the soluble decoy receptor, osteoprotegerin (OPG). Myeloma cells also express RANKL raising the possibility that targeting this system may have therapeutic potential. The aim of this study therefore was to determine whether an OPG fusion protein (Fc-OPG) could inhibit the development of osteolytic bone disease in a murine model of MM. 5T2MM murine myeloma cells were injected intravenously into C57BL/KaLwRij mice and the development of myeloma monitored by measuring serum paraprotein. After 8 weeks all animals had a detectable paraprotein and were treated with Fc-OPG (25mg/kg, iv, 3 times/week) or vehicle for a further 4 weeks. All animals injected with 5T2MM cells developed bone disease characterised by radiological evidence of osteolytic lesions in the femora, tibiae and lumbar vertebrae. Histomorphometric analyses demonstrated that this was associated with a significant decrease in bone volume (BV/TV) in the proximal tibial and distal femoral metaphyses and a significant increase in osteoclast number. DXA analyses demonstrated a decrease in bone mineral density (BMD) at each of these sites. Treatment of 5T2MM-bearing mice with Fc-OPG almost completely prevented the development of lytic bone lesions in the femora and tibiae. Treatment was associated with a partial preservation of BV/TV in the femoral and tibial metaphyses and the absence of osteoclasts. Fc-OPG also promoted an increase in femoral, tibial and vertebral BMD. Fc-OPG had no effect on paraprotein levels or tumour volume. These data demonstrate that Fc-OPG inhibits the development of lytic bone disease in a model of established MM and may represent a new approach to the treatment of myeloma bone disease.
T. Hiraga1*, A. Myoui1, P. J. Williams2, G. R. Mundy2, T. Yoneda1,2 Biochemistry and Orthopedics, Osaka Univ., Osaka, Japan 2Div. Endocrinol., Univ. TX Hlth. Sci. Ctr., San Antonio, TX, USA Evidence is accumulating that colonization of breast cancer in bone is under the influence of growth factors (GFs) released from bone as a consequence of bone resorption. However, the mechanism by which these bone-derived GFs affect bone metastases is poorly understood. Here, we studied the role of IGF, which is the most abundant GF in bone, and its signaling pathways in bone metastasis of breast cancer. The MDA-MB-231 human breast cancer cells were overexpressed with a cDNA of IGF type I receptors (MDA-231/IGFIR) and examined for the capacity to develop bone metastases using an animal model of bone metastasis. Radiographic and histomorphometric examination showed bone metastases were promoted in MDA-231/IGFIR compared with empty vector-transfected MDA-231 cells (MDA231/EV). Consistent with these in vivo results, a neutralizing antibody against IGFIR but not TGF-beta, FGF or PDGF decreased the proliferation of MDA-231 cells in culture that was stimulated by the resorbed-bone conditioned medium. These results suggest bone-derived IGF plays a critical role in bone metastasis in MDA-231 breast cancer. We subsequently unraveled signaling events that mediated the effects of IGF in MDA-231/IGFIR cells. Upon treatment with IGF-I, the transcription factor NF-kB (p50/p65) was activated in conjunction with an activation of IGFIR, IRS-I, PI-3 kinase and Akt but not Shc and ERK. NF-kB activation was blocked by a PI-3 kinase inhibitor wortmannin. Since NF-kB is implicated in both the malignant behavior of breast cancer and responsivity to IGF, we next examined the role of NF-kB in bone metastasis in MDA-231 breast cancer. To approach this, MDA-231 cells were stably transfected with a N-terminustruncated dominant-negative IkBalpha that was shown to inhibit NF-kB activation (MDA-231/IkBalpha-deltaN). MDA-231/IkBalpha-deltaN cells exhibited reduced bone metastases with decreased number of osteoclasts and increased apoptosis in metastatic cancer cells compared with MDA-231/EV cells. Taken together, these results suggest bone-derived IGF stimulates proliferation and prevents apoptosis in metastatic breast cancer cells in bone through activation of IGFIR, IRS-1, Akt and NF-kB, leading to a promotion of bone metastases. They also suggest NF-kB is a potential molecular target for design of therapeutic agents for bone metastases in breast cancer.
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apoptosis by chemotherapy. Our results indicate that chemotherapy and TRAIL act synergistically to kill cancer cells but not normal cells, which has exciting implications for the future management of osteosarcoma.
TRANSFORMING GROWTH FACTOR-BETA (TGF-BETA) STIMULATES TUMOR PARATHYROID HORMONE-RELATED PROTEIN (PTHrP) PRODUCTION VIA MITOGEN ACTIVATED PROTEIN (MAP) KINASE PATHWAYS IN A CELL-SPECIFIC MANNER
OR51 INACTIVATION OF MENIN, A SMAD3-INTERACTING PROTEIN, BLOCKS TGFBETA SIGNALING
S. Kakonen1*, M. Neal1, J. M. Chirgwin1, K. S. Selander2, B. G. Grubbs1, J. J. Yin1, T. A. Guise1 1Medicine/Endocrinology, University of Texas Health Science Center, San Antonio, USA 2University of Alabama, Birmingham, USA PTHrP mediates local osteolysis by metastatic breast cancer. TGF-beta, stored in bone matrix and released during osteoclastic resorption, stimulates tumor PTHrP production, but the responsible signaling pathways are unclear. Previous data indicate that TGF-beta stimulation of PTHrP by MDA-MB-231 breast cancer is both Smad-dependent and –independent. The p38 MAP kinase pathway is a major component of this Smad-independent signaling since the combination of p38 inhibition and dominant-negative Smad overexpression completely blocked the TGF-beta-induced PTHrP production compared with either modality alone. MEK (MAP kinase kinase of the Erk pathway) inhibition reduced the basal, but not TGFbeta-stimulated, PTHrP production in MD-MB-231 cells. To determine if the MAP kinase pathway is an important mediator of the effects of TGF-beta to stimulate PTHrP in other cancer cell types associated with bone metastases, we tested the effect of MEK and p38 inhibitors on PTHrP production in 7 cancer cell lines. Breast cancer (MDA-MB-231, Hs578t, MDA-MB-435S, BT549), prostate cancer (PC-3, DU145) and lung cancer (RWGT2) cell lines were treated with either MEK inhibitor, p38 inhibitor, or both, ±TGF-beta. All cell lines secreted PTHrP in the basal state and this was significantly increased by TGF-beta. The cell lines which secreted the most PTHrP in response to TGF-beta (RWGT2, PC-3 and MDA-MB231) caused the most osteolysis when inoculated into the left cardiac ventricle of nude mice. The TGF-beta-induced PTHrP production was significantly reduced by the p38 inhibitor in all cell lines except DU145. Complete blockade was observed in PC-3 and Hs578t cancer cells. The p38 inhibitor also reduced the basal PTHrP production by MDA-MB-231, Hs578t, and MDA- MB-435s. In contrast, the MEK inhibitor significantly reduced the basal, but not TGF-beta-stimulated, PTHrP production by MDA-MB-231 and Hs578t breast cancer lines and had no effects on the other cell lines. These data indicate a significant contribution of the p38 MAP kinase signaling pathway in mediating the effects of TGF-beta to stimulate PTHrP production in a variety of cancer cell lines associated with bone metastases. The relative contribution of the p38 MAP kinase pathway in TGF-beta signaling appears to be cell-specific and represents new molecular targets for anti-osteolytic therapy.
L. Canaff1*, H. Kaji1, J-J. Lebrun1, D. Goltzman1,2, G. N. Hendy1,2,3 of Medicine, McGill University, Montreal, Qc, Canada 2Department of Physiology, McGill University, Montreal, Qc, Canada 3Department of Human Genetics, McGill University, Montreal, Qc, Canada Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers, inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. To explore the function of menin, GH4C1 cells, a rat anterior pituitary cell-line, were stably transfected with an antisense menin cDNA. The antisense menin mRNA effectively reduced endogenous menin expression. TGFbeta inhibited proliferation of GH4C1 cells stably-transfected with empty vector. However, antisense menin antagonized the inhibitory growth effects of TGFbeta in cell number, [3H] thymidine uptake and MTT assays. HepG2 cells were transfected with the TGFbeta -responsive, plasminogen activator inhibitor 1 (PAI-I) promoter containing, 3TP-Lux construct. TGFbeta greatly increased luciferase activity, however antisense menin cotransfection markedly blocked TGFbeta -induced transcriptional activity and this was fully reversible by co-transfection of a sense menin construct. TGFbeta exerts growth inhibitory and transcriptional responses through the two receptor-regulated Smads, Smad2 and Smad3, which upon association with the common partner Smad4 translocate to the nucleus. To identify the target through which antisense menin blocks the TGFbeta signal pathway we examined whether menin would interact with the Smad proteins. COS cells were transiently transfected with DNA coding for myc-tagged Smad2 or 3, or flag-tagged Smad4 in the presence or absence of the menin cDNA. Menin specifically co-immunoprecipitated with Smad3 but not with Smad2 or Smad4. This indicated that menin interacts physically with Smad3. Smad3 and Smad4 are TGFbeta -inducible DNA binding proteins and the Smad3/4 complex recognizes a specific binding site within the PAI-1 promoter in 3TP-Lux. In an electrophoretic mobility shift assay (EMSA) with a probe derived from the PAI-1 promoter the levels of the Smad3/4-DNA complex were significantly decreased in nuclear extracts from cells in which antisense menin was co-expressed with Smad3 and Smad4. Thus, menin seems to interact with the TGFbeta pathway in the nucleus through Smad3 and inactivation of menin interrupts Smad3 binding to DNA thereby blocking TGFbeta signaling.
CHEMOTHERAPEUTIC DRUGS SENSITISE OSTEOGENIC SARCOMA CELLS BUT NOT NORMAL HUMAN BONE CELLS TO TRAIL-MEDIATED APOPTOSIS
A GENOME-WIDE SEARCH IN FAMILIAL PAGET'S DISEASE OF BONE
G. J. Atkins1*, A. Evdokiou1, S. Bouralexis1, F. Chai2, S. Hay1, M. T. Clayer2, M. Findlay1 1Dept Orthopaedics, Royal Adelaide Hospital, The University of Adelaide,
L. J. Hocking1*, C. A. Herbert2, R. K. Nicholls2, S. T. Bennett2, T. Cundy3, G. C. Nicholson4, W. van Hul5, W. Wuyts5, S. H. Ralston1 1 Department of Medicine and Therapeutics, University of Aberdeen, UK 2Oxagen, Abingdon, UK 3University of Auckland, New Zealand 4University of Melbourne, Australia 5University of Antwerp, Belgium Paget’s Disease of Bone (PDB) is a common disorder characterised by increased bone turnover on a focal or multifocal basis throughout the skeleton. Genetic factors are important in the pathogenesis of Paget's disease and previous linkage studies have identified candidate loci for the disease on chromosomes 6p (PDB1) and 18q21-22 (PDB2). Positional cloning studies mapped the TNFRSF11A gene encoding RANK to the PDB2 locus and mutation screening of this gene identified insertion mutations in the signal peptide as the cause of Familial Expansile Osteolysis. However mutation screening of RANK, coupled with linkage studies in several kindreds with familial Paget's have excluded the involvement of RANK in the vast majority of cases. In order to define other genes involved in the pathogenesis of PDB, we conducted a genome-wide search for Paget's disease in 327 individuals (166 affected) from 65 kindreds with familial PDB who were predominantly of UK descent. Genotyping was carried out by semi-automated PCR based methods using 389 fluorescent microsatellite markers located at approximately 10cM intervals throughout the genome. Alleles were called manually using ABI Genotyper software and binned using Linkage Designer. Linkage analysis was carried out using Genehunter version 1.3 assuming an autosomal dominant mode of inheritance with penetrance set at 90% for individuals aged 55 and over. The disease gene frequency was set at 0.00465 and phenocopy rate 0.02635. Linkage analysis in 22 of the 65 families studied so far has identified one region with definite evidence of linkage on chromosome 5 (LOD 3.5) and two others on chromosomes 1 (LOD 2.2) and 3 (LOD 3.0) with suggestive evidence of linkage. Some families were observed with negative lodscores in the regions of interest however, suggesting that the disease may be genetically heterogeneous. These data support the importance of genetic factors in familial Paget's disease and indicate that several candidate loci for the pathogenesis of the disease may exist with evidence of a strong susceptibility locus on chromosome 5.
Adelaide, Australia 2Dept Medicine, Dept Orthopaedics, The Queen Elizabeth Hospital, The University
of Adelaide, Woodville, Australia TRAIL/Apo2L is a tumour necrosis factor (TNF) family member that induces death of cancer cells but not of normal cells. Its apoptotic activity is mediated through cell surface death domain containing receptors, DR4 and DR5. TRAIL also binds to three “decoy” receptors, DcR1, DcR2 and osteoprotegerin (OPG), which lack functional death domains. The aim of this study was to investigate the cytotoxic activity of TRAIL alone or in combination with anti-cancer drugs, such as doxorubicin, cisplatin, methotrexate, cyclophosphamide and etoposide, on human osteogenic sarcoma cell lines and on primary normal human bone cells (NHBC). Of six cell lines tested, TRAIL induced apoptosis (>80%) only in one (BTK-143). However, TRAIL-resistant lines became sensitive to apoptosis with combinations of TRAIL and doxorubicin, cisplatin and etoposide, but not with TRAIL and methotrexate or cyclophosphamide. Importantly, neither TRAIL alone, nor in combination with any of these drugs, affected NHBC. The broad caspase inhibitor zVAD-fmk, or the caspase 3-specific inhibitor zDEVD-fmk, prevented apoptosis in response to TRAIL when used alone or in combination with drug treatment. The pattern of basal TRAIL receptor mRNA expression or endogenous expression of the caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with sensitivity to TRAIL–induced apoptosis. However, we show that augmentation of TRAIL effects by chemotherapy was the result of the druginduced up-regulation of DR4 and DR5. Although, we found no apparent correlation between the expression of OPG mRNA or protein and susceptibility to TRAIL-induced apoptosis, the addition of recombinant OPG to TRAIL-sensitive BTK-143 cells blocked apoptosis in response to TRAIL, suggesting a functional role for OPG in regulating the activity of TRAIL. We have further shown that stable overexpression of a dominant negative form of the Fas-associated death domain protein, (FADD) in TRAIL-sensitive BTK-143 cells was able to block caspase 8 and caspase 3 activation and completely inhibit TRAIL-induced death. However, dominant negative FADD could not inhibit the augmentation of TRAIL-induced
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collagen fibrils results in biomechanical alterations in the joints of the BGN-FM double-deficient mice, and are the common origin of the gait impairment, ectopic ossification and premature osteoarthritis noted in these mice.
OR55 LEPTIN AS A SKELETAL GROWTH FACTOR
Y. Yamanaka1*, H. Tanaka1, M. Koike1, R. Nishimura2, Y. Seino1 1 Okayama University, Okayama, Japan 2Osaka University, Osaka, Japan Constitutively activated mutations in the FGFR3 gene are responsible for the pathogenesis of achondroplasia (ACH) and related disorders. The activated FGFR3 has been thought to suppress the proliferation of chondrocytes, resulting in disturbing the growth of long bones. Recent studies have suggested that Stat1 is constitutively activated in chondrocytes of a severe type of ACH, thanatophoric dysplasia (TD), and implicated in inhibition of proliferation caused by this mutation in FGFR3. Little is, however, known about the precise role of Stat1 in chondrocytes of these skeletal disorders. Therefore, we investigated the functional role of Stat1 in FGFR3 signaling pathway by using chondrogenic cell lines, ATDC5 cells which were transfected several FGFR3 mutants, including G380R (ACH), K650E (TD type II: TDII). The ACH or TDII mutant receptor exhibited 20- or 100fold greater autophosphorylation than wild-type (WT) FGFR3, determined by in vitro kinase assay. The TDII mutant receptors exhibited 61-fold greater phosphorylation of Stat1 than WT receptor, determined by immunoblot with antiphospho-Stat1 antibody. In ACH cells, Stat1 was modestly phosphorylated. To further assess the state of Stat1 in the cells expressing these FGFR3 mutants, we determined the subcellular localization of Stat1 using chimera construct fused Stat1 to GFP (green fluorescent protein). Stat1 was specifically localized in nuclei in ACH or TDII cells, whereas Stat1 remained in cytoplasm of WT cells. [3H]thymidine incorporation assay showed that DNA synthesis was decreased by 60% in TDII cells, compared to WT cells. Importantly, introduction of dominantnegative (DN)-Stat1 into TDII cells restored DNA synthesis by 85%, while DNStat5a or DN-Stat5b had no effect. Consistently, constitutively active mutant of Stat1 decreased DNA synthesis of ATDC5 cells. In conclusion, an excessive activation of Stat1 induced by the FGFR3 mutants inhibits proliferation of chondrocytes. Thus, our data suggest that Stat1 may account for the disturbance of endochondral bone formation in achondroplasia and its related disorders.
G. Maor1*, M. Ben-Eliezer1, Y. Segev3, M. Phillip2 1 Technion, Haifa, Israel 2Schneider Children's Medical Center, Petach Tikva, Israel. 3Ben-Gurion University of the Negev, Beer-Sheva Obese children frequently show a concomitant increase in height velocity, with acceleration of epiphyseal maturation in spite of very low levels of serum Growth Hormone (GH) levels. Indeed, serum IGF1 levels are normal, but since it is well documented that it is the local (GH-dependent) IGF1 which accounts, we assumed that a circulating factor-other than GH, is responsible for skeletal growth in obese children. Leptin-a 16 kDa protein, which is secreted from adipocytes, circulates in high levels in obese children. We could show that chondrocytes population of skeletal growth centers in two models for endochondral ossification: the mandibular condyle and the humerus growth plate, do contain specific binding sites for leptin. Using an in vitro organ culture of murine derived mandibular condyle, we also show that 0.5-1.5µg/ml leptin stimulates, in a dose dependent manner, the chondroprigenitor zone (up to 80%) and the overall condylar length (up to 90%). Increase in BrdU incorporation into DNA, and in (acidic) alcian blue staining of the cartilaginous matrix accompanied with an increase in the expression of cartilagespecific proteoglycans, confirm that leptin induces both proliferation and differentiation activities in the mandibular condyle. Leptin also increases the immunohistochemically positive staining of IGF1 and of IGF1 receptors within the chondrocytes and the progenitor cells respectively. IGF1 activity seems to be pivotal for the leptin-induced increased skeletal growth, since immunoinhibition of IGF1 abolished the inductive effect of leptin. Our results indicate that leptin might be considered as skeletal growth factor that is probably mediated by increasing production of and sensitivity to local IGF1. We assume that high circulating levels of leptin might contribute to normal height velocity in obese children.
AN EXCESSIVE ACTIVATION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1 (STAT1) INHIBITS PROLIFERATION OF CHONDROCYTES IN FGFR3-RELATED SKELETAL DISORDERS
OR56 SIGNALING VIA THE PTH/PTHrP RECEPTOR NEGATIVELY REGULATES THE FORMATION OF HEMATOPOIETIC MARROW
L. Ameye1*, D. Aria1, G. Miller2, X-D. Chen1, P. Gehron Robey1, S. Chakravarti3, A. Oldberg4, T. Xu1, M. F. Young1 1 CSDB, NIDCR, NIH, Bethesda, USA 2Veterinary Resources Program, NIH, Bethesda, USA 3Dept. of Medecine, John Hopkins University, Baltimore, USA 4 Dept of Cell and Molecular Biology, University of Lund, Lund, USA Biglycan (BGN) and fibromodulin (FM) belong to the family of the small leucine-rich proteoglycans that are secreted into extracellular matrices. Mice deficient in each of these genes exhibit abnormal collagen fibril morphology. In order to investigate possible interactions between BGN and FM in vivo, doubleknockout mice (DKO) were generated by interbreeding the BGN and FB single knockout mice. DKO mice were fertile and viable, but were leaner on average than the wild-type (WT) and single knockouts. Compared to age-matched WT and single knockouts, one-month old DKO exhibited a decreased flexibility of their knee and ankle joints, thereby impairing their gait. Radiographs of these joints revealed ectopic radiolucent areas in the three knockout mice at the age of three months. Histological analysis demonstrated that these radiolucent areas were sesamoid bones forming within tendons through ossification of fibrocartilage. Radiographs of nine month-old DKO also showed ectopic ossification in the tendons of the elbow, wrist and hip joints. Semiquantitative scores based on radiographs were developed in order to assess the severity of the ectopic ossification in the ankle and knee joints in the four populations of mice at the age of three, six and nine months. Statistical analyses performed on these scores showed that the ectopic ossification increased with age and was more severe in the DKO than in the WT and single knockouts. Histological analysis also revealed osteoarthritis in the medial compartment of the knee starting at three months of age in the knee joints of the FMKO and DKO, with the DKO being the most profoundly affected. Between 6 and 9 months of age, bony cysts and severe cartilage loss with exposure of the underlying bone were observed in the DKO knee joints. The phenotype of the BGN-FM DKO showed striking similarities with the phenotype of the STR/ORT mice, a strain that develops spontaneous ectopic ossification and osteoarthritis in the knee joints. We theorize that abnormal
P. Bianco1*, S. A. Kuznetsov2, M. Riminucci3, N. Ziran2, L. Calvi4, H. M. Kronenberg4, P. G. Robey2, E. Schipani4 1Universita "La Sapienza", Rome, Italy 2 CSDB, NIDCR, NIH, Bethesda, MD, USA 3Universita dell'Aquila, L'Aquila, Italy 4Endocrine Unit, Massachuesetts General Hospital, Harvard Medical School, Boston, MA, USA Mice expressing a constitutively active PTH/PTHrP receptor (H223R) in bone develop a dramatic increase in trabecular bone mass. A detailed histological and micro-CT analysis of long bones of mutant mice showed that excess primary bone obliterates the prospective marrow cavity at 2 weeks. Subsequent multiple remodeling cycles of the excess bone leads to a distinct histological pattern characterized by irregular bone trabeculae and a fibrotic marrow, resembling human fibrous dysplasia and hyperparathyroidism. Hematopoiesis was initially absent from the abnormal bone/bone marrow organ of the mutant mice, but a marrow cavity with hematopoiesis was partially restored with increasing age perhaps due to decreased activity of the bone-specific type I collagen promoter driving expression of the transgene. We postulated that constitutive activation of the PTH/PTHrP signaling pathway during bone modeling resulted in disruption of normal myelogenesis, whereby a marrow cavity is established in bone prior to seeding with hematopoietic cells. This effect might reflect the impact of the transgene on the marrow stromal progenitor cell population, which normally comprises precursors for both the osteogenic cells involved in formation of the spongiosa and stromal cells that instead support hematopoiesis in marrow spaces. To test this hypothesis, clonogenic marrow stromal cells were isolated from normal and mutant mice, expanded in culture, and transplanted subcutaneously into immunocompromised mice using an HA/TCP carrier in duplicate experiments. Normal stromal cells generated a complete ossicle (bone and marrow). In contrast, no marrow was detectable at 4-8 weeks post-transplantation of mutant stromal cells. The amount of bone formed by normal and mutant cells was nearly identical, indicating that failure to establish an ectopic marrow was not due a paucity of bone. Thus, stromal cells expressing the consitutively active PTH/PTHrP receptor form bone, but fail to support hematopoiesis. When matched to the impaired formation of a marrow cavity in the mutant mice, these data suggest that the normal transition from hematopoiesis-free primary spongiosa to hematopoietic marrow may require stromal cells without active PTH/PTHrP signaling, which is made impossible by a constitutively active receptor. This indicates a major role for PTH/PTHrP signaling in regulation of normal marrow ontogeny.
BIGLYCAN AND FIBROMODULIN DOUBLE KNOCKOUT MICE EXHIBIT GAIT IMPAIRMENT AND DEVELOP ECTOPIC SESAMOID BONES AND PREMATURE OSTEOARTHRITIS IN THEIR JOINTS.
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DEVELOPMENT AND CHARACTERIZATION OF COL1A1-CRE TRANSGENIC MICE FOR OSTEOBLAST-DIRECTED GENOMIC RECOMBINATION
CAMURATI-ENGELMANN DISEASE IS CAUSED BY MUTATIONS IN THE TGFB1 GENE K. Janssens1*, R. Gershoni-Baruch2, N. Guañabens3, N. Migone4, S. H. Ralston5, H. MacPherson5, M. Bonduelle6, W. Lissens6, L. Van Maldergem7, F. Vanhoenacker8, L. Verbruggen9, W. Van Hul1 1Dept. of Medical Genetics, University of Antwerp, Antwerp, Belgium 2Institute of Human Genetics, Rambam Medical Center, Haifa, Israel 3Dept. of Rheumatology, Hospital Clínic., Barcelona, Spain 4Dept. of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy 5Dept. of Medicine and Therapeutics, University of Aberdeen Medical School, Aberdeen, UK 6 Dept. of Medical Genetics, University Hospital of Brussels, Belgium 7Center of Human Genetics, Institute of Pathology and Genetics, Loverval, Belgium 8Dept. of Radiology, University Hospital of Antwerp, Belgium 9 Dept. of Rheumatology, University Hospital of Brussels, Belgium Camurati-Engelmann disease (CED; MIM 131300) or progressive diaphyseal dysplasia is a rare, sclerosing bone dysplasia inherited in an autosomal dominant manner. It is manifested by severe pain in the legs, muscular weakness and a waddling gait. Other clinical symptoms include easy fatigability, reduced muscle mass, general weakness, exophthalmos, facial paralysis, hearing difficulties and loss of vision. Radiologically, CED is characterised by fusiform thickening of the cortex in the diaphyseal portions of the long bones causing a narrowing of the medullary canals; in some cases, the metaphyses are also affected. The distribution of the lesions in the skeleton is symmetrical. Sclerotic changes can also be found at the base of the skull. Recently, we and others localised the gene causing CED to chromosome 19q13. This region contains the gene encoding transforming growth factor-beta1 (TGFB1), known to be involved in bone remodelling. We evaluated TGFB1 as a candidate gene and found in ten families of European origin five different missense mutations and one small in frame duplication in the signal peptide at the N-terminal part of the peptide. All missense mutations are located in the latency associated peptide (LAP) of the gene. TGF-beta1 is synthesised as a large precursor molecule that is processed in the Golgi apparatus and cleaved into mature TGF-beta1 and the LAP, which remains non-covalently linked to the mature TGF-beta1, rendering it inactive. The effects of TGF-beta1 on bone have been investigated thoroughly, illustrating that TGF-beta1 has a keyrole in the coupling process between bone formation and resorption. However, the underlying mechanisms are far from understood and data are even sometimes contradictory. Since all missense mutations are located in the LAP, we hypothesise that they impair the ability of the LAP to keep the mature TGF-beta1 inactive. Ongoing functional analysis of the effect of the mutations might lead to a better understanding of the mechanisms by which TGF-beta1 regulates bone remodelling.
H. W. Woitge1,2*, I. Kalajzic3, M. S. Kronenberg3, S. H. Clark3, G. Gronowicz4, A. C. Lichtler3, B. E. Kream2 1Department of Medicine I, University of Heidelberg, Heidelberg, Germany 2 Department of Medicine, University of Connecticut Health Center, Farmington, USA 3Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, USA 4Department of Orthopedic Surgery, University of Connecticut Health Center, Farmington, USA To develop a model of osteoblast-directed gene targeting using Cre/loxP technology, Cre cDNA was cloned downstream of either a 2.3 kb (Col2.3-Cre) or a 3.6 kb (Col3.6-Cre) rat Col1a1 promoter fragment and transgenic mice were produced by embryo microinjection. In calvarial osteoblast and bone marrow stromal cultures, Col3.6 is expressed earlier and more broadly during osteoblast differentiation than Col2.3, which is expressed primarily by mature osteoblasts within mineralizing bone nodules By Northern blotting, 6-week old - Col2.3-Cre and Col3.6-Cre mice showed strong transgene mRNA expression in bone, whereas Col3.6-Cre mice also showed transgene mRNA expression in tendon and skin. To determine the temporal and spatial pattern of Col-Cre transgene expression, ColCre mice were bred with the Cre deletor strain GtROSA26tmSor (R26R). In these mice, excision of a floxed cassette by Cre allows ß-galactosidase to be expressed. ßgalactosidase activity was detectable in bones and skin of R26R/Col2.3-Cre mice and in bones, skin, muscles and reproductive organs of R26R/Col3.6-Cre mice. ßgalactosidase activity was detectable from embryonic day 10 onwards as demonstrated by whole-mount X-Gal stained R26R/Col2.3-Cre and R26R/Col3.6Cre embryos. In summary, we have developed Col2.3-Cre and Col3.6-Cre transgenic mouse lines for osteoblast-directed Cre-mediated deletion of any loxPflanked genomic sequence.
OR58 INCREASED BONE MASS IN MEGAKARYOCYTE DEFICIENT MICE WITH A TARGETED GENE DELETION OF THE THROMBOPOIETIN RECEPTOR M. J. Perry*, R. L. Gibson, J. H. Tobias University of Bristol, Bristol, UK Increased production of megakaryocytes (MKs) has been reported to be associated with excess bone formation. To determine whether MKs play a physiological role in regulating the cellular processes responsible for skeletal integrity, we characterised the skeletal phenotype of mice rendered deficient in MKs through a null mutation of the thrombopoietin receptor, c-Mpl (mpl KO mice) (WS Alexander, Blood 87:2162). Skeletal indices were compared between male and female mpl KO mice, and male and female wild-types (WTs) matched for age and genetic background. Bone mineral density (BMD; mg/cm2) as measured by Lunar PIXImus was significantly increased in mpl KO mice at all measurement sites (p<0.0005 by two-way ANOVA; results show mean±SEM). Cancellous and cortical histomorphometric indices were subsequently analysed at the tibial metaphysis and diaphysis respectively. Mpl KO mice showed a significant gain in both cancellous and cortical bone, as reflected by cancellous bone volume and medullary area (p<0.05 by two-way ANOVA). Indices of cancellous and cortical bone formation were also increased in mpl KO mice, as indicated by cancellous double-labelled surface and endocortical mineral apposition rate (p<0.02 by two-way ANOVA). Further studies were performed to analyse the mechanisms by which osteoblast function is elevated in mpl KO mice. Ex-vivo CFUf cultures revealed that bone marrow from mpl KO mice contains a similar number of osteoprogenitor cells compared to WT animals. The proportion of apoptotic cells in distal femurs were compared using TUNEL. No differences were observed in the proportion of apoptotic osteoblasts or bone lining cells between mpl KO and WT mice, although in the former, a significant increase in proportion of apoptotic cells was noted within bone marrow. We conclude that mpl KO mice show an abnormal bone phenotype characterised by increased bone mass secondary to enhanced osteoblast function, the exact basis of which remains unclear. Site Total body Whole femur Distal femur
OR60 RETINOIC ACID REGULATES OSTEOCLAST AND CHONDROCYTE DIFFERENTIATION IN DEER ANTLERS WHICH EXPRESS RETINOIC ACID RECEPTORS IN VIVO S. P. Allen1*, M. Maden2, J. S. Price1 1University College, London, UK 2Kings College, London, UK Retinoic acid (RA) has multiple functions during limb development and at sites of endochondral ossification in embryonic bones chondrogenic cells are a critical RA target. RA also regulates key events during limb regeneration in lower vertebrates. RA binds to two families of receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs) that form dimers when activated by ligand. This study explores the hypothesis that RA also plays a functional role when bone regenerates in an adult animal. We use the deer antler as a model since antlers are the only mammalian appendages capable of true regeneration; these complex bony structures are cast and regrow annually by an endochondral process. Our specific objectives were to determine sites of RA receptor expression in the distal growing tip of the antler and to determine the effect of exogenous RA on osteoclast (OC) and chondrocyte differentiation. Three receptors (alpha, beta, gamma) for both the RAR and RXR families were cloned from antler tissue by RT-PCR and their expression mapped by in situ hybridisation. RARalpha and RXRbeta were expressed in skin and the underlying perichondium. In antler cartilage, RXRbeta was specifically expressed in chondrocytes and RARalpha in the perivascular cells of the osteoblast lineage (these cells also express type I collagen, and alkaline phosphatase). Thus these receptors may be involved in osteoblast and chondrocyte differentiation respectively. In micromass cultures of antler cartilage cells, which also express RARalpha and RXRbeta mRNAs, all trans RA (10-7-10-8M) significantly stimulated the formation of OC-like cells after 10 days, which was preceded by an increase in RANKL mRNA expression. RA (10-6-10-8M) also dose-dependently inhibited glycosaminoglycan synthesis in micromass cultures of chondrocytes and in explant cultures of antler cartilage. In conclusion, these results show that RA receptors are expressed in deer antlers and that their in vitro activation regulates chondrocyte and osteoclast differentiation. The demonstration that RA may be an important molecular signal in
73.4±1.5 54.9±1.9 104.5±5.3
82.1±1.2 62.7±1.3 118.8±4.3
74.9±1.6 52.1±0.9 98.3±3.1
85.0±2.1 63.4±1.4 126.6±5.6
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the antler is further evidence that embryonic signalling pathways are recapitualted during mammalian bone regeneration. This study also shows for the first time that the stimulation of OC formation by RA may be mediated by RANKL.
T. P. van Staa1,2,3*, H. G. M. Leufkens1, C. Cooper2 1Department of Pharmacoepidemiology and Pharmacotherapy, University of Utrecht, Sorbonnelaan 16, Utrecht, the Netherlands 2 Medical Research Council Environmental Epidemiology Unit, Southampton University Hospital, Southampton, United Kingdom 3 Procter & Gamble Pharmaceuticals, Lovett House, Lovett Road, Staines, United Kingdom The objective of this study was to evaluate the performance of risk factors predicting fractures at various sites. Information was obtained from the General Practice Research database in the UK, which contains medical records of general practitioners. The study population consisted of all patients aged 65 years or older with a fracture. Each case was matched by age, sex and medical practice to a control patient. There were 77 828 fracture cases in our study population. The majority of the fracture cases were women (79.0%) and the mean age was 78 years. The following diseases and drugs were associated with an increased risk of fractures: history of anaemia (odds ratio [OR] of 1.5; 95% confidence interval 1.4-1.6), dementia (OR 1.6; 1.5-1.7), cerebrovascular disease (OR 1.4; 1.3-1.4), and chronic obstructive pulmonary disease (OR 1.4; 1.3-1.4) and recent use of oral corticosteroids (OR 1.5; 1.4-1.6), anticonvulsants (OR 1.9; 1.8-2.0), NSAIDs (OR 1.4; 1.3-1.4), antiarrythmics (OR 1.6; 1.4-1.8), antipsychotics (OR 1.4; 1.3-1.5), antidepressants (OR 1.5; 1.5-1.6), and anti-Parkinson drugs (OR 1.8; 1.6-1.9). Patients with combinations of these risk factors showed substantially higher odds ratios for fractures compared to those without. The highest odds ratios for femur / hip fractures were found among patient with dementia and recent use of antipsychotics (OR 3.8; 3.3-4.5). It was found that vertebral, pelvis and femur/hip fractures were among the fracture types predicted more accurately by the risk factors. The area under the curve of receiver-operator characteristic curves was 61% for any fracture, 81% for vertebral, and 67% for femur/hip fractures. The positive predictive value over a 5-year period was 10.9% for women aged 65 years and 4.6% for men of similar age. The positive predictive value was 22.9% for women and 10.4% for men, aged 85 years or older. In conclusion, risk factors did not predict the occurrence of fracture in an individual patient with any certainty. However, the risk factor information allowed identification of groups of patients with a substantively increased risk of fracture. Men and women showed similar screeeing characteristics and relative rates of fractures with the risk factors.
DISEASES AND DRUGS ASSOCIATED WITH OSTEOPOROSIS PREDICT FRACTURES AT DIFFERENT SITES
C. H. Turner1*, Q. Sun1, M. L. Bouxsein2, C. J. Rosen3, L. R. Donahue3, K. L. Shultz3, W. G. Beamer3 1 Indiana University, Indianapolis, USA 2Beth Israel/Deaconess Medical Center, Boston, USA 3The Jackson Laboratory, Bar Harbor, USA. The inbred mice strains, C57BL/6J (B6) and C3H/HeJ (C3H), differ significantly in femoral bone mineral density as assessed by pQCT. Genetic analyses of femoral BMD from 1000 B6C3H-F2 females identified 10 quantitative trait loci (QTLs) linked to BMD on chromosomes 1, 2, 4, 6, 11, 12, 13, 14, 16 and 18. Congenic strains of mice were developed for 6 of these QTLs by transferring each QTL region of the chromosome from C3H onto the B6 mice by selective backcrossing. After 6 cycles of backcrossing, the effect of each QTL from C3H chromosomes 1, 4 (two segments), 6, 13, and 18 on BMD were confirmed in mice with a B6 background. Excised femora from these congenic strains of mice were analyzed biomechanically to test the effect of the donated C3H QTLs on bending force to fracture (F), stiffness (S) and work to failure (U) of the mid-diaphyseal region. The C3H QTLs significantly influenced the mechanical properties of the femoral midshaft (Table 1). The greatest genetic influence was observed at Chr 4T where the QTL improved femoral F by 21%, S by 35%, and U by 29%, compared to B6. QTLs at Chrs 4A and 18 also improved some biomechanical properties. These results indicate that biomechanical properties of the femoral diaphysis are influenced by genes on several chromosomes, with the QTL region on Chr 4T having the most influence. Table 1. Biomechanical properties for congenic strains (N=9-16) B6 C3H Chr1 Chr4A Chr4T Chr6 Chr13 Chr18 F (N) 23.0 40.4* 23.5 26.5* 27.8* 20.9 S (N/mm) 98 132* 108 125* 132* 98 U (mJ) 4.2 8.6* 4.1 4.7 5.4* 3.7 Asterisks indicate a significant difference from B6.
23.0 107 4.2
23.6 112* 4.3
IMPROVED BONE STRENGTH IN LOW BONE DENSITY C57BL/6J MICE CARRYING DONATED QTLS FROM C3H/HEJ MICE
EARLY ONSET AND SUSTAINED EFFICACY OF RALOXIFENE ON INCIDENT VERTEBRAL FRACTURES IN POSTMENOPAUSAL WOMEN WITH OSTEOPOROSIS: 4-YEAR RESULTS FROM THE MORE TRIAL
POTENTAL ROLES FOR AP-1/INTERLEUKIN-11 PATHWAYS IN MECHANICAL STRESS-INDUCED BONE FORMATION
H. Pols1*, R. Eastell2, P. Delmas3, J. Adachi4, K. Ensrud5, K. Harper6, S. Sarkar6, C. Gennari7, J. Y. Reginster8, R. Recker9, S. Harris10, W. Wu6, D. Black10, H. Genant10 for the MORE investigators 1Erasmus University, Rotterdam, The Netherlands 2University of Sheffield, UK 3 INSERM, Lyon, France 4 McMaster University, Hamilton, Canada 5 VA Medical Center, Minneapolis, USA 6 Eli Lilly and Company, Indianapolis, USA 7University of Siena, Siena, Italy 8CHU Centre-Ville, Liège, Belgium 9Creighton University, Omaha, USA 10UCSF, San Francisco, USA Raloxifene (RLX) therapy decreased the incidence of new vertebral fractures (VF) over a 3-year period in the MORE (Multiple Outcomes of Raloxifene Evaluation) trial, which studied 7705 postmenopausal women (<80 years) with osteoporosis randomized to placebo or RLX (60 or 120 mg/day) [JAMA 282(1999): 637]. The present analyses evaluate whether the decreased risk of incident VF persists into the 4th year of RLX therapy, and if RLX has early onset of effects on clinical VF. New semiquantitative/morphometric VF were assessed annually after year 2 using spinal radiographs. Primary VF endpoints were determined cumulatively from baseline to year 3, with an extension through year 4. Clinical VF were confirmed radiographically after women presented with signs or symptoms suggestive of new VF at any post-baseline visit. A post-hoc analysis examined whether the reduced risk of new VF was sustained in the 4th year. At 4 years, the cumulative relative risk (RR) of new VF was 0.61 (95% confidence interval 0.51, 0.73) with the pooled RLX doses. In the first year, RLX60 significantly decreased the incidence of new clinical VF in total study population (RR 0.32), and among women with prevalent VF (RR 0.34). In the total MORE population, the RR for new VF with RLX60 was 0.65 (95%CI 0.53, 0.79) from baseline to year 3; this was sustained during the 4th year [RR 0.61 (95%CI 0.43, 0.88)]. In women with prevalent VF, RLX60 significantly reduced the incidence of new VF from baseline to year 3 (RR 0.70), which was sustained in the 4th year (RR 0.62), and corresponded to 8.1% and 3.0% reductions in absolute risk. RLX60 also significantly decreased the incidence of new VF in women without prevalent VF from baseline to year 3 (RR 0.45), which was sustained in the 4th year (RR 0.50),
S. Kido, D. Inoue*, E. Tohjima, T. Matsumoto Tokushima University, Tokushima, Japan Critical roles for mechanical stress in maintaining bone mass and strength is evidenced by severe bone loss in human subjects during space flight as well as immobilization. We have shown that fos family transcription factors including delta-fosB, a C-terminally truncated splicing variant of fosB, fra-1 and c-fos are rapidly induced in mice reloaded in rotating cages after tail suspension as well as in calvarial osteoblasts stimulated by fluid shear stress in vitro. Recent reports that over-expression of either delta-fosB or fra-1 in transgenic mice results in osteosclerosis due to increased bone formation support their important roles in osteogenesis. However, signaling pathways inducing these fos family members as well as their target genes in osteoblasts remain to be elucidated. Searching for the mechanism of aging-associated bone loss, we found that antiadipogenic cytokine, IL-11, is expressed in osteoblastic cells in a manner dependent on AP-1 transcription factors composed of Fos and Jun. Impaired bone formation with aging was associated with reduction in AP-1 activity and IL-11 expression in marrow stromal cells. Notably, transgenic mice over-expressing IL-11 have been shown to exhibit progressively increasing bone mass with age due to accelerated bone formation. We therefore tested a hypothesis that IL-11 acts downstream of AP-1 in mechanical stress-induced osteogenic signaling pathways and analyzed signaling components required for induction of delta-fosB and IL-11. Consistent with our hypothesis, we found that induction of fos family members by either mechanical reloading in vivo or fluid shear stress in vitro was followed by IL-11 induction. Since rise in intracellular calcium is considered to be essential to initiation of mechanical stress signaling pathways, we next examined effects of a calcium ionophore, A23187, on calvarial osteoblasts in vitro. The results indicated that A23187 induced activation of MAP kinases, expression of delta-fosB and subsequently IL-11 as well. Moreover, we demonstrated that U0126, a specific inhibitor of Erk1/2, but not SB203580, an inhibitor of p38 kinase, almost completely blocked the induction of both delta-fosB and IL-11 by fluid shear stress in calvarial osteoblasts. Taken together, these results suggest critical roles for Ca/ Erk/fos/IL-11 cascades in mechanical stress-induced osteogenic signaling pathways.
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and corresponded to absolute risk reductions of 2.0% and 1.1%. RLX significantly decreased the cumulative risks of incident VF for up to 4 years in postmenopausal women with or without prevalent VF, rapidly decreased the clinical VF incidence in the 1st year, and most importantly, sustained the risk reduction of new VF in the 4th year of MORE.
biomechanical strength. The greater periosteal distribution of cortical bone, reflected in the moments of inertia, may contribute to the reduction in nonvertebral fractures that is not explained by changes in DXA bone mineral density alone. Proximal radius parameters
*Torsional strength index (mg*mm) **Flexural strength index (mg*mm)
A PROSPECTIVE POPULATION-BASED POST-FRACTURE OSTEOPOROSIS INTERVENTION
*Polar moment of inertia (mm-4) *Axial moment of inertia (mm-4) *Periosteal circumference (mm)
M. T. Cuddihy1*, S. E. Gabriel1, P. C. Amadio1, S. Khosla1, R. Kurland2, W. M. O'Fallon1, L. J. Melton Iii1 1Mayo Clinic and Mayo Foundation, Rochester, MN, USA 2Olmsted Medical Center, Rochester, MN, USA Objective: Little attention has been given to osteoporosis intervention after fractures, despite known benefits. We previously reported that osteoporosis interventions were made in only 16% of postmenopausal women with a distal forearm fracture (DFF) in this community (1993-1997), a group known to be at high risk for osteoporosis. Consequently, we developed a practice intervention offering both men and women over age 45 an osteoporosis evaluation as part of their post-DFF care. Methods: Using the resources of the Rochester Epidemiology Project, Olmsted County residents over age 45 who sustained a DFF due to minor trauma (i.e. fall from standing height or less) between 1/1999 and 8/2000, were identified. Participants were offered femur bone densitometry (BD), and follow up with a physician to address osteoporosis management. We assessed compliance with BD, acceptance of osteoporosis medication, quality of life and osteoporosis health beliefs. Results: Of the 105 cases identified (80% women), 58 agreed to participate, while 38 declined and 9 were ineligible because of dementia. There were 51(88%) women with a mean age of 66.3 years. BD assessment was completed in 45 (78%), 30 of which were new evaluations; of the 15 women with established osteoporosis, 7 agreed to repeat BD. Altogether, 28 (62%) had a T score below –1.5 and 8 had previously diagnosed osteoporosis. Twenty-four (47%) women received pharmacologic treatment for osteoporosis: 13 continued or altered existing regimens and 11 initiated new osteoporosis pharmacologic treatment. Of the 25 women with T score < -1.5, only 10 (40%) accepted medication advice; none of the three men with T score < -1.5 accepted therapy. Of the 33 participants in whom 12 month follow-up was complete to date, 15 (94%) were still on their osteoporosis medication. Discussion: A targeted program such as ours increased BD measurement from 5% to 78% and osteoporosis intervention from 16% to 47%; compared to our previously reported rates of osteoporosis evaluation and management after low impact DFF in the same population between 1993-1997. These data indicate that practice interventions improve assessment, but there remain barriers to acceptance of osteoporosis interventions known to reduce future fracture risk, particularly among men.
**Cortical density (mg/cm-3) **Cortical area (mm-2) Cortical bone mineral content (mg) (SD) *p<0.05 by trend analysis **p<0.10 by trend analysis
1948 (457) 2153 (525) 2292 (623) 662 (205) 695 (178) 751 (222) 2032.3 (505.2) 2223.0 (536.6) 2459.1 (606.2) 691.2 (234.5)
38.6 (2.9) 961.8 (71.1)
39.2 (2.9) 969.4 (53.3)
40.6 (2.6) 930.3 (70.4)
OR67 BMP-6 RESTORES LOST BONE M. Basic-Koretic1*, V. Kusec2, J. Buljan-Culej1, G. Grahovac1, A. Grskovic1, M. Habek1, M. Ljubic1, I. Vukoja1, K. Vukovic1, S.Vukicevic1 1Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia 2 Central Laboratory, University Hospital Zagreb, Zagreb, Croatia As current therapy for osteoporosis is based only on antiresorptive drugs, there is a great need for agents that could restore lost bone and prevent fractures. Good candidates are bone morphogenetic proteins (BMPs), which can, when applied locally, induce formation of new tissues like bone, cartilage and ligament. We have demonstrated that BMP-6, when administered systematically in a rat tail vein, induces new bone formation at a site of subcutaneously or intramuscularly implanted demineralized bone matrix. However, there is no evidence that a human recombinant BMP, given systematically, can restore bone in an osteoporotic animal model. Wistar female rats were ovariectomized at 14 weeks of age and therapy started four months later. Bone mineral density (BMD) of total body, lumbar spine and hind limbs was measured monthly. Animals were divided into five groups (n=12): (1)sham, (2)ovariectomized (OVX), (3)OVX+17beta-estradiol (E2)(3x/week, 50+50+75 microg. sc), (4)OVX+BMP-6 (BMP-6)(3x/week, 10 microg/kg iv), (5)OVX+17beta-estradiol+BMP-6 (E2BMP-6). Within four months of treatment, in BMP-6 treated rats, BMD values of total body, lumbar spine and hind limbs reached the values of sham rats. Four months following treatment, ex vivo BMD was measured on excised femur, tibia and lumbar spine, and the values were similar to in vivo results. BMP-6 in combination with estrogen synergistically increased the proximal femur bone volume. Histology of uteri showed significant thickening of endometrium in estradiol treated animals, while BMP-6 had no effect. Histomorphometrically determined bone volume of femora and vertebrae confirmed the BMD measurements. The bone volume in proximal tibiae of E2BMP-6 treated animals was significantly higher even when compared to sham controls (P<0.002). Both cortical and trabecular bone volume were increased in BMP-6 treated rats. These results suggest, for the first time, an anabolic effect of systemically administered BMP-6 on bone volume in osteoporotic rats.
OR66 EFFECTS OF LY333334 [RECOMBINANT PARATHYROID HORMONE (1-34)] ON CORTICAL BONE STRENGTH INDICES AS ASSESSED BY PERIPHERAL QUANTITATIVE COMPUTED TOMOGRAPHY. J. R. Zanchetta1*, C. Bogado1, J. L. Ferretti1, O. Wang2, M. Sato2, G. A. Gaich2 1 Instituto de Investigaciones Metabolicas and USAL University School of Medicine, Buenos Aires, AR 2 Eli Lilly and Company, Indianapolis, IN, USA Treatment of postmenopausal women with LY333334 [recombinant parathyroid hormone (1-34), rhPTH(1-34)] significantly reduces the risk of both vertebral and nonvertebral fractures (Neer et al. Endocrine Society, 2000). Although the greatest increase in bone mineral density occurs in the predominantly trabecular regions such as the lumbar vertebrae, cortical regions, as typified by the midshaft radius, do not appear to respond as well. Structural properties of the cortical bone of the proximal radius, as assessed by peripheral quantitative computed tomography (pQCT), may detect skeletal effects of rhPTH(1-34) that are not reflected in measurements of integral bone mineral density by dual energy x-ray absorptiometry (DXA). In primate models, PTH stimulated both modeling and intracortical remodeling, resulting in thicker cortical bones with greater cortical area. A cross-sectional pilot study was conducted to assess this effect in humans as part of a large prospective, multicenter study of the effect of rhPTH(1-34) on fracture risk and bone mineral density. Women were randomly assigned either placebo, 20 (PTH20) or 40 (PTH40) microgram rhPTH(1-34) administered once daily by self-injection for up to 2 years. pQCT was performed with a Stratec XCT960. Results at study closeout (median 21 months) are shown in the table (mean±standard deviation (SD)). Compared with placebo-treated patients, the PTH-treated patients in this study showed greater periosteal circumference and cortical area, similar bone mineral content, and lower bone density, all consistent with treatment effects of rhPTH in monkey studies. These differences resulted in greater polar and axial moments of inertia and torsional bone strength index, which are predictive of increased
OR68 INTRAVENOUS IBANDRONATE IN THE TREATMENT OF CORTICOID-INDUCED OSTEOPOROSIS J. D. Ringe*, A. Dorst, H. Faber, K. Ibach, K. Mammes Med. Klinikum 4, University of Cologne, Leverkusen, Germany Chronically ill patients with corticoid-induced osteoporosis (CIO) often take multiple medications. Intravenous ibandronate may be a new attractive alternative to oral bisphosphonates (BPs) as it is offered as an intermittent bolus injection. In this controlled, prospective, comparative two-arm study 75 patients with established CIO (average age 65 years) were treated with either 2 mg ibandronate every 3 months iv bolus (group A) or with a daily dose of 1ug alfacalcidol orally (group B). All patients received 500 mg calcium per day. The patients had been on corticosteroids for an average of 8 years. The baseline mean BMD values were very low (T-score lumbar spine: -3.7 SD, femoral neck: -3.0 SD). 88% of the patients had 1 or more prevalent vertebral fractures. During the 24 months of observation there was a progressive reduction in back pain in both groups and no relevant adverse events were recorded. Mean BMD at the lumbar spine increased by +11.8% in the ibandronate treated group and +1.8% in the 1-alpha group (p< 0.0001). The corresponding changes after 2 years for the femoral neck were +4.4% and +1.1% (p=0.0005), respectively. Five new vertebral fractures (in 5 patients) were observed in group A and 10 (in 9 patients) in group B (ns). We conclude that ibandronate may be superior to alfacalcidol in the treatment of CIO. These data confirm that BPs are highly effective in the treatment of CIO. The very good acceptance and excellent compliance with the 3 monthly ibandronate
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bolus therapy, the scarcity of adverse events and the avoidance of poor intestinal absorption make iv ibandronate a very interesting and promising alternative to oral BP treatment.
LOW CONCENTRATIONS OF MEVASTATIN INHIBIT BONE RESORPTION IN VITRO
LONG-TERM POTENTIATION IN BONE: A CELLULAR BASIS OF MEMORY IN OSTEOBLASTS?
K. J. Armour*, K. E. Armour, R. J. van't Hof, C. E. Clarkin, M. H. Helfrich, S. H. Ralston, M. J. Rogers University of Aberdeen, Aberdeen, UK We have recently reported that 1-100microM mevastatin causes osteoclast apoptosis and inhibits bone resorption in vitro, whereas other studies have suggested that lower concentrations (0.06-1microM) of statins have anabolic effects on bone in vitro, possibly due to upregulation of endothelial nitric oxide synthase (eNOS). Since the anabolic vs anti-resorptive effects of statins and the involvement of eNOS remain unclear, we examined the effects of low concentrations of mevastatin on osteoclasts and bone resorption using calvarial organ cultures from C57 BL6 (WT) and eNOS knockout (-/-) mice, and osteoblast/ bone marrow co-cultures. 2-day old WT mice were injected with 45calcium chloride and, 4 days later, calvariae were excised and cultured with mevastatin for 3 days. When compared with controls, 0.01, 0.1 or 1.0microM mevastatin significantly inhibited the release of 45Ca in a concentration-dependent manner by 11%, 32% and 45% respectively (n=10; all p<0.01). A similar, concentration-dependent inhibition of resorption was observed in eNOS -/- calvariae. Furthermore, inhibition of NO synthesis in WT calvariae by co-treatment with 1mM L-NMMA did not alter the response to mevastatin. We then used histological analysis to study the effect of mevastatin on WT calvarial organ cultures in vitro. Treatment with 1microM mevastatin for 10 days caused a marked increase (67.3%; p<0.05) in the area of remodelling foci within the calvariae and an increase of 74.4% (p<0.05) in average calvarial width. Mevastatin also caused reductions in osteoclast number/bone surface (1.46 ± 0.28x109 vs 0.83 ± 0.11x109; n=6, p<0.05) and total resorption surface (7.82 ± 1.88 vs 4.26 ± 0.46; n=6, p<0.05) compared with untreated calvariae. In osteoblast/bone marrow co-cultures on ivory discs, treatment with 1microM mevastatin for the final 3 days of culture reduced the number of osteoclasts/disc (411.6 ± 54.2 vs 234.8 ± 42.0; n=5; p<0.05) and inhibited osteoclastic resorption (3.29 ± 0.34x106 µm2/slice vs 0.93 ± 0.23x106 µm2/slice; n=5; p<0.01) compared with control cultures. These results demonstrate that the bone anabolic effect of low concentrations of statins in vitro may be due, at least in part, to inhibition of osteoclastic bone resorption. Furthermore, the inhibitory effect of mevastatin on bone resorption does not appear to involve the eNOS pathway.
G. J. Spencer*, T. S. Grewal, P. G. Genever, T. M. Skerry Univesity of York, York, UK In the central nervous system (CNS), glutamate signalling directs long-lasting neuronal functions, such as learning and memory formation, through a process known as long-term potentiation (LTP). The mechanism of LTP is not fully understood, but involves use-dependent modulation of signalling, by phosphorylation of the glutamate receptor C-terminus and autophosphorylation of activated CAM kinase II (CaMKII). Here we show that LTP may be implicated in long-lasting responses of osteoblasts to brief stimuli including those induced by brief periods of mechanical loading. At central synapses, glutamate receptors are clustered through specific interactions between C-termini of receptor subunits and PDZ (PSD-95/Dlg/ZO-1) domains of multivalent clustering proteins, which also have important roles in the assembly of downstream signalling complexes. Osteoblasts express prominent pericellular immunoreactivity for these PDZ domain-containing proteins. We have demonstrated (by RT-PCR, immunolocalisation, Western and Northern blot analyses) that osteoblasts also express all of the essential molecules required for initiating LTP at central glutamatergic synapses. By co-immunoprecipitation, we show that these proteins associate physically with the C-termini of osteoblastic NMDAR2A/B receptor subunits, two of which, the supposedly neurospecific CaMKII isoforms, -alpha and -beta are known to play fundamental roles in LTP. In primary rat osteoblasts, application of either a depolarising concentration of KCl (60mM), or glutamate (100microM) produced significant increases in CaMKIIalpha and beta autophosphorylation resulting in a 35% and 47% increase in calcium-independent CaMKII activity. Significantly, inhibition of CaMKII has marked effects on primary rat osteoblast differentiation. The CAMK inhibitor KN62 (50nM-10microM) caused a time- and dose-dependent reduction in alkaline phosphatase activity and bone nodule formation without affecting cell proliferation suggesting that distinct phases of CaMKII activity are required for normal osteoblast function. Finally, mechanical loading also stimulated CaMKII autophosphorylation and calcium-independent activity. In ROS 17/2.8 osteosarcoma cells, cyclical loading for 10 minutes at a frequency of 1Hz (peak strain 0.0035) caused a rapid transient increase in both CaMKII-alpha and -beta autophosphorylation. This activation peaked at responses of 350% (CaMKII-alpha) and 50% (CaMKII-beta). These data suggest that in bone, a mechanism similar or identical to LTP in the CNS regulates long-term osteogenic responses that result from brief periods of mechanical loading.
S. B. Rodan*, M. E. Duggan, L. T. Duong, J. E. Fisher, M. A. Gentile, G. D. Hartman, N. C. Ihle, D. Kimmel, C-T. Leu, R. Nagy, J. G. Seedor, G. Wesolowski, A. E. Zartman, G. A. Rodan Merck Research Laboratories, West Point, Pennsylvania, USA The alpha v beta 3 integrin was shown to play a major role in bone resorption in vitro and in vivo. Alpha v beta 3 binds to extracellular matrix proteins containing the RGD sequence, and is highly expressed in osteoclasts. We report here the pharmacological properties of a low molecular weight RGD peptidomimetic, L770874, and its in vivo activity in rats and in ovariectomized cynomolgus macaques. In binding assays, which utilize purified human, rhesus or rat alpha v beta 3 and a radiolabeled RGD peptidomimetic ligand, L-770874 displaces binding with IC50s of about 0.1-0.3 nM. In attachment assays utilizing HEK 293 cells transfected with either alpha v beta 3 or alpha v beta 5 integrin, L-770874 inhibits alpha v beta 3 and alpha v beta 5-mediated attachment to vitronectin with IC50s of 0.5 and 9.5 nM, respectively. In an assay dependent on the platelet integrin alpha IIb beta 3, L-770874 inhibits human platelet aggregation with an IC50 of 156 nM. L-770874 is also a potent inhibitor of murine osteoclast formation and of rabbit osteoclast bone resorption in vitro, with IC50s of 1.5 and 4.2 nM, respectively. L770874 infused s.c. to thyroparathyroidectomized rats at 0.67 mg/kg/hr, completely blocked the parathyroid hormone-induced increase in serum calcium, 50% inhibition observed at 0.134 mg/kg/hr, at serum levels of about 50 nM. To evaluate the efficacy of L-770874 in ovariectomized cynomolgus macaques, it was administered s.c. at 10 mg/kg/day for 21 days. Urinary NTx levels were reduced starting 1 day after administration of L-770874 through day 21. In conclusion, L770874 is a potent alpha v beta 3 integrin antagonist which inhibits bone resorption in vitro and in vivo.
E. Bonnelye*, L. Meerdad, J. E. Aubin University of Toronto, Ontario, Canada The orphan estrogen related receptor, ERRa, is known to be expressed by osteoblastic cells and to transactivate at least one osteoblast-associated gene, osteopontin. Its relationship to the estrogen receptors, ERa and ERb, makes it an interesting and potentially important regulator of bone formation and maintenance of bone mass. We found that ERRa is expressed in rat calvaria (RC) in vivo and in RC cells in vitro. Moreover, by global amplification PCR of individual bone nodules, we found ERRa mRNA to be expressed throughout all osteoblast differentiation stages in vitro, from early osteoprogenitors to mature osteoblasts. Immunocytochemistry of cultured RC cells and tissue sections showed that ERa is co-expressed in vitro and in vivo in osteoblastic cells expressing ERRa, while ERb was less widely detectable. Comparison of the mRNA expression levels of ERRa, ERa and ERb by RT-PCR in RC cell populations or single developing bone colonies showed that ERRa is much more highly expressed than either ERa or ERb. We overexpressed or inhibited ERRa by transient transfection or phosphorothioatemodified antisense oligonucleotides respectively in differentiating RC cell cultures. After transfection, we found that mineralized bone nodule formation was increased significantly by 15%, consistent with the approximately 10-15% transfection efficiency. Concomitantly, we detected an increase in type I collagen, alkaline phosphatase and osteocalcin mRNA. Blocking ERRa expression with antisense oligonucleotides resulted in a striking dose-dependent decrease in bone nodule formation, i.e., 70% decrease at 0.5mM and 100% at 1 mM and 2mM. Control/ sense oligonucleotides had only a small dose-independent effect on nodule numbers. Inhibition of ERRa had effects during both the proliferation and differentiation developmental time windows, with concomitant reduction in expression of osteoblast-associated markers. Our findings show that ERRa is more highly expressed in differentiating osteoblastic cells than either ERa or ERb. They also indicate a critical role for ERRa in bone formation, with both up- and down-regulation of bone nodule formation concomitant with up- and down-regulation of ERRa in vitro. Our data suggest that ERRa plays a widespread and physiologically-relevant role in bone formation and predict specific ERRa target genes at different osteoblast developmental time windows.
A POTENT NON-PEPTIDE ANTAGONIST OF ALPHA V BETA 3 INTEGRIN INHIBITS CALCIUM MOBILIZATION IN THYROPARATHYROIDECTOMIZED RATS AND INHIBITS BONE RESORPTION IN OVARIECTOMIZED CYNOMOLGUS MACAQUES
THE ORPHAN RECEPTOR, ESTROGEN RELATED RECEPTOR ERRA, IS HIGHLY EXPRESSED IN OSTEOBLASTS AND IS REQUIRED FOR BONE FORMATION
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PTH1Rc internalization. Accordingly, exposure of human osteosarcoma (SaOS-2) cells to Bpa1-PTHrP-(1-36) induces a sustained activation of adenylyl cyclase compared to PTH and PTHrP. We have now investigated a series of PTH- and PTHrP-derived agonists modified in position 1 with regard to ß-arrestin2 recruitment, PTH1Rc endocytosis, and cAMP signaling. Our results suggest that the amino acid in position 1 of PTH and PTHrP is required to stimulate ß-arrestin2 mobilization and PTH1Rc internalization, but not necessarily to regulate PTH1Rcmediated cAMP signaling. Based on our previous observations of a contact point between Bpa1 in the ligand and Met425 in PTH1Rc’s transmembrane domain 6 (TM 6), we have generated mutated PTH1Rcs carrying single alanine substitutions in TM 6. Thus, using fluorescence microscopy, we have identified a PTH1Rc site of interaction with Bpa1-PTHrP-(1-36) involved in ß-arrestin2 mobilization, receptor internalization, and regulation of cAMP signaling. Coupled with results from NMR spectroscopy and computer simulation, we are developing the first experimentallybased molecular model of the conformation of ligand-associated PTH1Rc responsible for the interaction with ß-arrestin2. In conclusion, we have initiated the identification of the molecular and structural determinants of both ligand and receptor involved in the regulation of cellular trafficking and signaling in the PTH/ PTHrP system.
THE DIRECT ACTIONS OF LEPTIN ON BONE CELLS INCREASE BONE STRENGTH IN VIVO - AN EXPLANATION OF LOW FRACTURE RATES IN OBESITY J. Cornish*, K. E. Callon, U. Bava, Q. X. Lin, D. Naot, B. L. Hill, N. D. Broom, I. R. Reid University of Auckland, Auckland, New Zealand Fat mass is one of the principal determinants of bone density but the mechanism of this remains controversial. Leptin has been identified as a circulating peptide originating from the adipocyte, raising the possibility that it may influence bone cell function. This hypothesis is supported by recent evidence that administration of leptin into the cerebral ventricles of mice is associated with profound bone loss (suggesting that bone mass is centrally regulated). However, these results would suggest that obesity is associated with low bone mass, the opposite of what is actually found. Since leptin originates in the periphery, we examined the effect of its peripheral administration on bone is necessary to address this major discrepancy. Leptin increased proliferation of isolated fetal rat osteoblasts at concentrations of 10-10M and greater and the magnitude of the maximal effect was comparable to IGF-1. In mouse bone marrow cultures, leptin (10-11M - 10-9M) inhibited osteoclastogenesis, but it had no effect on bone resorption in two assays of mature osteoclast activity. Systemic administration of leptin to sexually mature male mice (20 injections of 40 micrograms over 4 weeks) increased bone strength by greater than 20% (displacement, control vs leptin: 0.44±0.02 vs 0.53±0.03 mm, p=0.004; and work to failure: 5.5±0.3 vs 7.0±0.4 Nmm, p=0.02). It is concluded that leptin's direct effects on bone tend to increase bone strength which may contribute to the high bone mass and low fracture rates of obesity. These results indicate that when leptin is administered systemically, its direct effects on bone outweigh its CNS-mediated effects on the skeleton, the latter of which are likely to be mediated by leptin's actions on insulin sensitivity via the autonomic nervous system.
OR76 THE CARBOXY-TERMINAL, "OSTEOSTATIN", REGION OF PTHRELATED PROTEIN IS ESSENTIAL FOR INDUCTION OF CELLULAR PROLIFERATION FOLLOWING NUCLEAR ENTRY. N. M. Fiaschi-Taesch1*, F. deMiguel1, J-C. Lopez-Talavera1, K. K. Takane1, T. Massfelder2, J-J. Helwig2, A. F. Stewart1 1 Divisionof Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 2Université Louis Pasteur, Strasbourg, France In the arterial wall, PTHrP is upregulated by vascular injury, by vasodistention, and by vasoconstrictors, and acts as a vascular smooth muscle (VSM) relaxant. These effects of PTHrP are believed to be principally paracrine and/or autocrine. PTHrP also has a nuclear/nucleolar localization signal (NLS) in the 96-107 region. Full-length PTHrP has been shown to powerfully influence proliferation in VSM cells. Interestingly, PTHrP with an intact NLS drives VSM cell proliferation, whereas PTHrP lacking an NLS markedly inhibits proliferation in VSM cells. The NLS previously has been demonstrated to be required for both nuclear entry as well as activation of proliferation in VSM. In the current study, we wanted to determine what other regions of the PTHrP molecule in addition to the NLS are required for this "intracrine" stimulation of VSM proliferation. Twelve PTHrP deletion mutant constructs lacking the signal peptide, the NLS, the (1-37), (38-86), (108-139) regions, or combinations of the above, were stably expressed in A-10 rat VSM cells. Proliferation rates were examined over 12 days in culture. Control (A-10) and vector-alone A-10 cells proliferated at the same rate. In contrast, A-10 cells with NLS deletions, as described previously, proliferated at a reduced rate, approximately 50% of control. In contrast, A-10 cell overexpressing any mutant of PTHrP with both an intact NLS and carboxy-terminal (108-139) region, regardless of signal peptide, 1-36 or 38-86 status, proliferated at rates 3-4 times faster than controls. This indicates that (1-37), (38-86) regions are not required for proliferation. Surprisingly, and in marked contrast to carboxyterminally intact constructs, A-10 cells expressing PTHrP in which the carboxyterminal (108-139) was deleted proliferated at a normal rate, 25-30% that of wildtype PTHrP VSM. These findings indicate that the carboxy-terminal region of PTHrP, which contains the so-called "osteostatin" region, together with a functional NLS, are essential requirements for PTHrP to activate cell proliferation in VSM. Surprisingly, this is the region of PTHrP which is least conserved. Further studies are required to more finely map the 107-139 region with respect to activation of proliferation, and to define the nuclear partners of the carboxy-terminal region which mediate the proliferation.
OR74 LEPTIN REGULATES BONE MASS IN RATS A. F. Schilling1*, T. Holzmann1, J. M. Rueger1, G. Karsenty2, M. Amling1 Hamburg University 2 Baylor College, Houston Leptin is a polypeptide hormone secreted by the adipocyte that has been identified as a key regulator of body weight. Leptin controls starvation and adiposity following its binding to a specific receptor located in the hypothalamus. Recently it has been demonstrated that mice deficient in leptin signaling have a high bone mass phenotype due to an increase in bone formation. This result identified on a genetic basis leptin as a major regulator of bone formation, in mice. To determine whether this was a more general feature of vertebrate skeleton biology, we studied the skeleton of the zucker or fa/fa rats that have an inactivating mutation in the gene encoding the leptin-receptor. Here we show that these mutant rats have an increased bone formation rate and a high bone mass phenotype similar to the one observed in ob/ob and db/db mice. Unlike what is the case in the ob/ob and db/db mice the bone phenotype of the fa/fa rats is not restricted to trabecular bone as these animals have also an increased cortical thickness. In fa/fa rats both trabecular bone volume and cortical thickness are significantly increased. Threepoint bending tests further confirmed that the absence of leptin signaling results in a significant increase in biomechanical strength of fa/fa long bones. That the bone phenotype is present in heterozygous animals that are not obese, confirms that the increase in bone mass is not secondary to obesity but dominant to the fat phenotype. Thus, this study extends the importance of leptin signaling in regulating bone remodeling.
OR75 LIGAND-RECEPTOR INTERACTIONS REGULATING CELLULAR TRAFFICKING AND SIGNALING OF THE HUMAN PARATHYROID HORMONE/ PARATHYROID HORMONERELATED PROTEIN RECEPTOR
OR77 MUTATIONS IN SOST, A NOVEL SECRETED PROTEIN, CAUSE INCREASED BONE FORMATION IN SCLEROSTEOSIS
S. L. Ferrari1,2*, L. Monticelli3, D. Mierke3, A. Bisello1,4 Israel Deaconess Medical Center and Harvard Medical School, Boston, USA 2 University Hospital, Geneva, Switzerland 3Brown University, RI, USA 4University of Pittsburgh Medical Center, PA, USA Desensitization of G protein-coupled receptors is a fundamental mechanism regulating the cellular responsiveness to agonists. Until recently, the cellular and molecular mechanisms responsible for the desensitization of the human parathyroid hormone (PTH)/ parathyroid hormone-related protein (PTHrP) receptor (PTH1Rc) were mostly unknown. Using fluorescent PTH- and PTHrP-derived ligands and a green fluorescence protein (GFP)-PTH1Rc conjugate, we have recently demonstrated that stimulation of PTH1Rc by its cognate agonists triggers the rapid internalization of ligand-receptor complexes in living cells. This phenomenon is dependent on the interaction between the activated receptor and a cytoplasmic adaptor molecule, ß-arrestin2. In turn, this interaction has been shown to be responsible for the rapid desensitization of PTH1Rc-mediated cAMP signaling. We have further described a PTHrP-derived, Gs alpha-signaling selective agonist, Bpa1-PTHrP-(1-36), which does not stimulate mobilization of ß-arrestin2 nor
W. Balemans1*, M. Ebeling2, N. Patel3, E. Van Hul1, P. Olson3, M. Dioszegi3, C. Lacza3, W. Wuyts1, J. Van Den Ende1, P. Willems4, A. F. Paes-Alves5, S. Hill6, M. Bueno7, F. J. Ramos7, P. Tacconi8, F. G. Dikkers9, K. Lindpaintner10, B. Vickery3, D. Foernzler10, W. Van Hul1 1 Dept. Medical Genetics, Antwerp, Belgium 2F. Hoffmann-La Roche Ltd, Basel, Switzeland 3Roche Bioscience, Palo Alto, USA 4Dept. Clinical Genetics, Rotterdam, The Netherlands 5 Departamento de Ginecologica, Salvador, Brazil 6National Institutes of Health, Bethesda, USA 7Universidad de Zaragoza, Spain 8 Instituto di Neurologia, Cagliari, Italy 9University Hospital Groningen, Groningen, The Netherlands 10Roche Genetics, Basle, Switzerland Sclerosteosis (MIM269500) is an autosomal recessive disorder classified among the craniotubular hyperostoses. The condition is characterized by a generalized osteosclerosis and hyperostosis of the skeleton, most pronounced in skull and
conference.book Page 89 Wednesday, February 7, 2001 8:48 PM
IBMS/ECTS 2001 - First Joint Meeting
Abstracts - Oral Presentations We performed an extensive literature search and meta-analysis to determine the relationship between statin use and hip and non-spine fracture risk. Using abstracts from major meetings, published studies and one unpublished study (Rotterdam), we found 8 observational studies that reported statin use and prospectively assessed fracture risk. Studies of men and women were included. We used the relative hazard or odds ratio and 95% confidence intervals (CI), adjusted for potential confounders, when available, or calculated the unadjusted risk from raw data. Studies were combined using a random effects model, and summary estimates are reported as relative risk (RR). For studies with multiple published results, we tested the effect of each of the differing results on our summary estimate. The overall results are shown in the Table. The hip fracture results were heterogeneous (test of homogeneity p=0.03) when data from the Women's Health Initiative (WHI) were included, but the summary relative risk was similar when that study was omitted (RR=0.39, CI: 0.27, 0.58). Conflicting results from the General Practice Research Database (GPRD) had no appreciable effect on the overall results. Our meta-analysis of these 8 observational studies support a protective effect of statins on hip and non-spine fractures. Additional controlled trials of these agents among individuals at high risk of fracture are needed. Fracture
# Studies # Subjects # Statin Users # Fractures
Hip 8 Non-spine 6
mandible. Sclerosteosis is clinically and radiologically very similar to van Buchem disease (MIM239100), mainly differentiated by hand malformations and gigantism appearing in sclerosteosis. Linkage analysis in one extended van Buchem family and two consanguineous sclerosteosis families assigned both genes to the same chromosomal region 17q12q21, supporting the hypothesis that both conditions are caused by mutations in the same gene. We now performed further linkage analysis in the Dutch van Buchem family and were able to reduce the previously delineated region of 0.7 cM to about 1 Mb flanked by D17S1326 (proximal) and D17S1860 (distal). To identify the disease causing gene(s) we simultaneously performed mutation analysis on known genes and ESTs from this interval and screened three genomic clones from the linkage region with exon prediction programs. Mutation analysis finally revealed sequence variations in sclerosteosis patients in one putative exon located in genomic sequence HPRC905N1. This exon belongs to a two exon gene, which we called SOST, with an ORF of 642 bp. We were able to detect two different nonsense mutations in two consanguineous sclerosteosis families and one splice site mutation in an isolated sclerosteosis case from Senegal. Extensive mutation analysis of the SOST gene did not reveal a mutation in a Spanish sclerosteosis patient, nor in the Dutch van Buchem patients. The SOST gene encodes a 213 amino acid propeptide containing a signal sequence for secretion. Screening for functional domains and motifs produced a weak but significant match to the cystine-knot motif, which is known to participate in dimerization and receptor binding. SOST has a restricted expression pattern, with highest expression in kidney tissue and interestingly also expression in osteoblasts. The three disease causing mutations lead to a loss-of-function of the SOST protein resulting in the excess of normally structured bone tissue. Therefore, the physiological role of SOST is probably to suppress bone formation, making this gene an interesting target for the development of anabolic agents against osteoporosis.
Summary RR (CI) 0.43 (0.25, 0.75) 0.66 (0.55, 0.88)
OR80 A SINGLE ANNUAL INJECTION OF THE BISPHOSPHONATE, ZOLEDRONIC ACID, STABLY REDUCES BONE TURNOVER AND INCREASES BONE DENSITY IN POSTMENOPAUSAL OSTEOPOROSIS
I. R. Reid1*, J. Brown2, P. Burckhardt3, Z. Horowitz4, P. Richardson4, U. Trechsel4, for the Zoledronic Acid Study Group5 1 Medicine, University of Auckland, New Zealand 2Universitaire de Quebec-Le Centre Hospitalier, Sainte-Foy, PQ, Canada 3CHUV University Hospital, Lausanne, Switzerland. 4 Novartis 5(25 centers in 10 countries) Current bisphosphonate (BP) therapies are effective treatments of osteoporosis. Poor bioavailability and relative lack of potency require frequent (usually daily) administration away from food. Poor GI tolerability limits maximal dosing, and prescription in patients with GI disorders, as well as compliance, especially if osteoporosis is asymptomatic. Intravenous BPs have been widely used in oncology to avoid these problems. Intermittent (3-monhtly) treatments have also been explored in osteoporosis, but the effect of increasing the inter-dose interval has not been systematically explored. In this study we explore whether the use of zoledronic acid, which in preclinical studies appears the most potent BP identified to date, allows very infrequent administration, and results in a sustained suppression of bone turnover, whilst avoiding the limitations of regular oral dosing. In this 1-year, randomized controlled trial including 351 postmenopausal women with T-scores < -2, zoledronic acid was given intravenously in doses of 0.25mg, 0.5mg and 1mg at 3 month intervals. In addition, the total annual dose of 4mg was also given as one dose of 4mg, or two 6-monthly doses of 2mg. BMD results (ITT analysis) are shown in the table below as mean changes from baseline (differences from placebo). After 1 year, mean BSAP and serum CTX were about 40%, and median urine NTX about 50% below baseline. Increases in BMD and decreases in markers were significantly different from placebo in all groups. There were no other betweengroups differences. Myalgia and pyrexia occurred more commonly in the zoledronic acid groups, but the treatment was generally well tolerated. We conclude that zoledronic acid given at intervals of up to 1 year between injections produces effects on bone turnover and bone density as great as those seen with daily oral dosing of agents of proven anti-fracture efficacy, such as alendronate and risedronate. This result suggests that an annual iv injection of zoledronic acid might be an effective treatment for postmenopausal osteoporosis.
FGF23 AS A CAUSATIVE FACTOR OF HYPOPHOSPHATEMIC DISEASES T. Shimada1, S. Mizutani1, T. Muto1, T. Yoneya1, R. Hino1, Y. Takeuchi2, T. Fujita2, S. Fukumoto3, T. Yamashita1* 1KIRIN Pharmaceutical Research Lab., Takasaki, Japan 2 University of Tokyo School of Medicine, Tokyo, Japan 3University of Tokyo Branch Hospital, Tokyo, Japan Tumor-induced osteomalacia (TIO) is one of paraneoplastic diseases characterized by hypophosphatemia due to renal phosphate wasting. Because removal of the responsible tumors rapidly normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. In order to identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO, and constructed tumor-specific cDNA contigs. Based on the novel sequence of one major contig, we cloned 2270 bp cDNA, which turned out to encode fibroblast growth factor (FGF)23. Administration of recombinant FGF23 decreased serum phosphate in mice within 12 hours. When CHO cells stably expressing FGF23 were subcutaneously implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced biochemical, clinical and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by CHO cells indicated proteolytic cleavage of FGF23 at RXXR motif. Administration of full length or cleaved FGF23 separated by ion exchange chromatography revealed that full length FGF23 has hypophosphatemic activity. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets (ADHR), another hypophosphatemic disease with similar features to TIO. To investigate the significance of missense mutations in ADHR patients, we generated mutant FGF23 with double missense mutations (R176Q and R179Q) in the RXXR motif. The mutant FGF23 produced in CHO cells was resistant to the proteolytic processing that was observed in wild type FGF23. This protein also showed the ability to induce hypophosphatemia in vivo. We conclude that overproduction of FGF23 causes TIO, while mutations in FGF23 gene result in ADHR by preventing proteolytic cleavage and enhancing biological activity of FGF23.
Spine BMD Hip BMD
4x0.25 mg (every 3 months) 4x0.5 mg (every 3 months) 4x1.0 mg (every 3 months) 2x2.0 mg (at start and 6 months) 1x4.0 mg (at start only)
+5.1% +4.9% +4.3% +4.3% +4.6%
+3.1 +3.1% +3.2% +3.6% +3.3%
STATIN USE AND FRACTURE: A META-ANALYSIS OF 8 OBSERVATIONAL STUDIES
D. C. Bauer1*, D. M. Black1, M. van der Klift2 1University of California, San Francisco, USA 2 Erasmus University, Rotterdam, Netherlands Several, but not all, recent observational studies have suggested a reduction in hip and non-spine fractures among individuals taking HMG-CoA reductase inhibitors (statins). Two placebo-controlled cardiovascular trials (4S and LIPID) found no effect of statins on fracture rates, but fractures were not confirmed and few hip fractures were reported.