intestinal damage to minor mismatch GVHD. EXPERIMENTAL DESIGN: Lethally irradiated wild-type (WT), MLCK-/-, and constitutively active (CA)-MLCK B6 recipients received a BMT of 5x106 bone marrow cells and 30x106 splenocytes from 129 donors. Sublethally irradiated B6/Rag1-/- recipients received an adoptive transfer (AT) of 30x106 129 splenocytes. Preliminary studies showed that BMT alone is insufficient to cause GVHD but is required to rescue recipients from lethal irradiation. Data are represented as mean ± SEM. RESULTS: 129 BMT into lethally irradiated B6 WT recipients resulted in weight loss with clinical symptoms and histopathological features of GVHD. Similar disease developed following AT of 129 splenocytes (without BMT) into sublethally irradiated B6/Rag1-/- mice. In contrast, 129 AT into B6/Rag1-/- mice without irradiation conditioning did not cause GVHD. This was not due to graft rejection, as circulating 129 donor T cells were present 35 days after AT. To further assess the effects of irradiation in driving minor mismatch GVHD, irradiated and non-irradiated B6/Rag1-/- mice were compared after 129 AT. At day 7 after AT, serum IFNγ, TNF, and IL-6 were significantly greater in mice that received irradiation and AT (compared to AT alone, P<0.05). To determine whether alloreactive donor cells were more effectively primed after irradiation, we assessed In Vivo killing of labeled B6 targets. After irradiation and AT, killing efficiency was 66±8%, while killing efficiency after AT only was 23±6%. To determine if intestinal damage, as occurs with irradiation, contributes to GVHD, colonic injury was induced with dextran sulfate sodium (DSS) prior to 129 AT. 129 AT into DSS-treated mice caused systemic GVHD and increased In Vivo killing of B6 targets (40±4%). To assess the role of TNF-induced tight junction dysregulation in GVHD perpetuation, MLCK-/- and CA-MLCK B6 mice were given a 129 BMT after lethal irradiation. Barrier loss was reduced in MLCK-/- and enhanced in CA-MLCK mice, and this was associated with less and more severe GVHD, respectively, relative to WT mice. CONCLUSIONS: Intestinal damage and barrier dysfunction, initiated by the pretransplant conditioning regimen and perpetuated by epithelial MLCK activation, are vital to minor mismatch GVHD pathogenesis. Both intestinal protection and MLCK inhibition represent potential novel therapeutic approaches for treatment of systemic GVHD.
Hyaluronic Acid Induces Radioprotection in the Intestine by a TLR-4 and COX-2 Dependent Mechanism Terrence Riehl, William F. Stenson Background: Hyaluronic acid (HA), a glycosaminoglycan that forms a part of the extracellular matrix, affects inflammation and wound repair through binding to its receptors CD44, TLR2 and TLR4. HA expression is increased in dextran sodium sulfate (DSS) colitis and exogenous HA is protective in DSS colitis through a mechanism that was both MyD88 and COX-2 dependent. PGE2 produced by COX-1 and COX-2 is radioprotective in the intestine. Here we sought to determine if radiation induces HA expression and if exogenous HA induces radioprotection in the intestine. Methods: All experiments were done in wild type, MyD88-/, TLR4-/-, and COX-2-/- mice on a C57Bl/6 background. To determine if radiation induces HA expression, mice were irradiated (12Gy), six hours later they were sacrificed. Small intestinal HA expression was assessed by immunohistochemistry. Plasma HA was assessed by ELISA. To determine if exogenous HA is radioprotective, mice were given either vehicle or HA (30mg/kg) intraperitoneally and were irradiated (12Gy). Six hours later the mice were sacrificed and radiation-induced apoptosis was assessed by histology. Mice were given vehicle or HA and irradiated, 84 hours later they were sacrificed and crypt survival was assessed. Using immunohistochemistry for surface markers we identified a population of constitutively COX-2 positive lamina propria mononuclear cells as mesenchymal stem cells (MSCs). We followed the effects of HA on the number and distribution of the MSCs. Results: Radiation increased HA expression in the small intestine with all the expression in the lamina propria. Plasma HA levels six hours after radiation increased six fold (p<.001). Administration of HA prior to irradiation induced a 60% decrease in radiation induced apoptosis at positions 4-12 (p<.01). Crypt survival was 27 crypts per cross section in the mice receiving HA compared with 15 crypts per cross section in mice receiving vehicle (p<.001). The effects of HA on radiation induced apoptosis and crypt survival were MyD88, TLR4, and COX-2dependent. The total number of intestinal MSCs in Wild Type mice changed little after HA treatment, but the distribution changed from 20% in crypts and 80% in villi in controls to 40% in crypts and 60% in villi at 8 hours after HA treatment. In TLR4-/- mice, the total number of cells COX-2 expressing decreased after HA treatment, but the distribution remained unchanged with 32% in crypts and 68% in villi. Conclusion: Radiation induces HA expression in the small intestine and increases plasma HA. Intraperitoneal HA induces radioprotection in the small intestine through a COX-2, MyD88, and TLR4 dependent pathway. Intraperitoneal HA induces the migration of COX-2 positive MSCs from the lamina propria in the villi to the lamina propria near the crypt epithelial cells. This migration may relate to the radioprotective effects of HA.
1025 Increased MicroRNA-29b Inhibits CDK2 mRNA Translation and Intestinal Mucosal Growth After Polyamine Depletion Lan Xiao, Yu-hong Cui, Jaladanki N. Rao, Tontong Zou, Lan Liu, Myriam Gorospe, JianYing Wang Maintenance of intestinal epithelial integrity requires polyamines that regulate expression of various genes involved in cell proliferation and apoptosis, but the exact mechanism underlying polyamines remains unclear. MicroRNAs (miRNAs) generally base-pair to sequences in the 3'-untranslated regions (UTR) of labile mRNAs and regulate the stability and translation of target transcripts. miRNAs are implicated in many aspects of biological processes, and their expression is in tissue-specific manner and is highly regulated by numerous factors. This study determined if polyamines regulate intestinal mucosal growth by altering miRNA profiling and also defined the involvement of polyamine-regulated miR29b in the control of cyclin-dependent kinase 2 (CDK2) expression. Methods: Studies were conducted in mice and IEC-6 cells derived from rat small intestinal crypts. miRNAs profiles were examined by miRNA-microarray and real-time PCR (Q-PCR) analyses. Interaction of miR-29b with the CDK2 mRNA was examined by pmir-Glo reporter system and biotin labeled miR-29b pull-down assays. CDK2 translation was measured by chimeric CDK2 3'UTR luciferease reporter and newly synthesized CDK2 protein assays. Polyamine levels were depleted by DFMO (a specific inhibitor of polyamine biosynthesis) but increased by ODC gene overexpression. The functions of miRNA-29b were examined by miR-29b-directed siRNA and ectopic overexpression of its precursor. Results: Comparison of the miRNA expression profiles in the small intestinal mucosa in control mice relative to mice exposed to DFMO for 4 days or fasted for 48 h revealed several miRNAs that increased during mucosal growth inhibition, including miR-29b, miR-222, miR-140, and miR-195, while other miRNAs like miR-503 decreased. To keep the focus, changes in miR-29b levels were confirmed by Q-PCR analysis and increased by >2.5-fold after polyamine depletion. In cultured IECs, the levels of primary and mature miR-29b were also increased by polyamine depletion (by ~2-fold) but they were decreased by increasing the levels of cellular polyamines. miR-29b bound the CDK2 mRNA via its 3'-UTR rather than coding region; this [miR-29b/ CDK2 mRNA] association increased after polyamine depletion (by ~3-fold), which was associated with inhibited CDK2 expression (by ~75%). miR-29b silencing decreased [miR29b/CDK2 mRNA] complex and enhanced CDK2 translation in normal and polyaminedeficient cells, whereas increased miR-29b induced the associations and repressed CDK2 translation. miR-29b overexpression also inhibited IEC proliferation and resulted in G1 phase growth arrest. Conclusions: These results indicate that 1) miRNA expression profiling is regulated by cellular polyamines and 2) polyamine depletion-induced miR-29b represses CDK2 translation, thus inhibiting IEC proliferation.
1023 Oral Glutamine Supplementation Improves Intestinal Permeability Dysfunction in a Murine Acute Graft Versus Host Disease Model Rainer Noth, Robert Haesler, Julia Lange-Grumfeld, Jochen Hampe, Stefan Schreiber, Alexander Arlt Background Hematopoietic stem cell transplantation is increasingly performed . One of the severe side effects of the procedure are graft versus host reactions leading among other complications to gastrointestinal symptoms including diarrhea, nausea, vomiting, abdominal cramping, anorexia and gastrointestinal bleeding. Up to now little is known about the pathophysiological mechanism evoking these GI-symptoms, especially the manifestation in the small intestine. The influence of different nutritional components like omega-3 polyunsaturated fatty acids and zinc for disorders characterized by intestinal inflammation are under investigation. Barrier-protective and immune-modulatory effects have also been described for the amino acid glutamine. Aim The aim of the study was to evaluate the influence of a oral glutamine supplementation on the intestinal permeability, TNF-a expression, expression of the tight-junction proteins occludin and ZO-1 and on the mucosal architecture in a murine GvHD model. Methods To induce acute semi allogenic graft versus host disease prepared donor lymphocytes from C57BL/6 mice were transferred to 8-14-week-old irradiated B6D2F1 mice (n=40) of the same sex by intraperitoneal injection.Half of the GvHD animals (n=20) received oral glutamine supplementation for six days started at the time of lymphocyte transfer. Six days after induction of the semi allogenic GvHD jejunum specimens were prepared. The expression of the proinflammatory cytokin TNF-α, and the tight-junction proteins occludin and ZO-1 was investigated by PCR. Histological changes were evaluated and intestinal permeability was assessed by the differential uptake of lactulose and mannitol. Results As compared to control group, animals with GvHD have a significant impaired epithelial paracellular barrier function. The GvHD leads to a raised expression of TNF-α and reduced expression of the tight junction protein occludin. The ZO-1 expression was unchanged in GvHD-animals. The GvHD leads to mucosal atrophy and crypt hyperplasia. The oral supplementation of glutamine improves impaired intestinal permeability in GvHDanimals. Glutamin treated mice showed a reduced expression of TNF-α, an increased occludin expression and less macroscopic disease symptoms in the jejunum. Conclusions Oral glutamine supplementation leads to an improved intestinal permeability, a reduction of the increased TNF-α levels and a restoration of the tight junctions. Furthermore glutamine reduces histological changes like villous atrophy and crypt hyperplasia. Oral glutamine supplementation seems to have different beneficial effects on the severity of inflammatory changes in the course of the murine semiallogenic graft versus host disease.
1027 Intestinal Epithelial Cell Responses to Total Parenteral Nutrition (TPN) Are Under Complex Regulation by ADAM17 Yongjia Feng, Yu-Hwai Tsai, Daniel H. Teitelbaum, Peter J. Dempsey Background: ADAM17 (TACE) participates in the shedding of a variety of substrates, including TNFa and ErbB ligand/receptor families. Although ADAM17 is abundant in the small bowel mucosa, its precise function is unknown. Removal of enteral nutrition using TPN, results in decreased epithelial cell (EC) proliferation and increased EC apoptosis. Using a mouse TPN model, EGF, and its receptor EGFR decreased in small bowel ECs, whereas TNFa and TNFR1 increased. Because of these unique changes, we used intestine-specific ADAM17 KO mice to investigate the role of ADAM17 in the modulation of these signaling paths. Methods: Intestine-specific ADAM17 KO mice (Vil-ADAM17 KO) were generated by breeding ADAM17loxP/loxP mice to Villin-Cre mice. Vil-ADAM17 KO mice were viable and did not show an overt intestinal phenotype. 10 week wild-type (WT) or Vil-ADAM17 KO male mice received enteral nutrition (control) or TPN for 7 days. EC proliferation was assessed with BrdU incorporation and PCNA expression. Other samples were quantified with RT-PCR or immunoblotting. Results: TPN significantly up-regulated ADAM17 abundance and
1024 Intestinal Damage and Myosin Light Chain Kinase (MLCK)-Dependent Epithelial Barrier Dysfunction Are Vital to Minor Mismatch Graft-Versus-Host Disease Sam C. Nalle, Nora E. Joseph, Peter A. Savage, Jerrold R. Turner BACKGROUND: GVHD is a serious complication of allogeneic bone marrow transplantation (BMT) that targets the intestine, liver, skin, and lung. Previous studies suggest that the intestine may also be a site of GVHD initiation. AIM: To characterize the contribution of
its inhibitor TIMP3 in WT mice, with an associated decline in EC proliferation (Table). Loss of proliferation was partially prevented in Vil-ADAM17 KO mice. TPN also led to decreased in EGFR expression in WT mice, which was also partially prevented in Vil-ADAM17 KO mice. Glucagon-like peptide-2 can also mediate EC proliferation. Interestingly, GLP2 receptor (GLP2R) expression decreased in the jejunum of WT TPN mice, and markedly increased in Vil-ADAM17 KO TPN mice. ADAM17 can also modulate TNFα signaling. In WT TPN mice, TNFa and its receptor TNFR1 increased, whereas TNFR2 was unchanged. TNFa increased 2-fold in WT TPN mice and 4-fold in Vil-ADAM17 KO TPN mice compared to control mice. No difference in TNFR1 expression was found between WT and Vil-ADAM17 KO TPN mice. WT TPN mice showed increased EC apoptosis, whereas active Caspase3 staining was significantly reduced in Vil-ADAM17 KO mice. Downstream, phosphorylation of MAPK p38 (PP38) expression increased 2-fold in both WT and Vil-ADAM17 KO TPN mice. Bcl-2 protein expression declined 2.7-fold in TPN WT mice, and was totally prevented in Vil-ADAM17 KO TPN mice; whereas Bax protein increased 2.3-fold in WT TPN mice, and did not change in Vil-ADAM17 KO TPN mice. Summary: TPN led to increased ADAM17, which may promote activation of TNFa and TNFR1, and a down-regulation of EGFR and its ligands. Removal of ADAM17 in intestinal EC partly protected against these changes and resulted in preservation of EC proliferation and prevention of EC apoptosis. Conclusions: These changes provide In Vivo evidence of the functional aspects of ADAM17 signaling in the small intestinal mucosa, and a potential mechanism for the development of mucosal atrophy associated with TPN.
tumor sizes were measured with vernier calipers and tumor volume determined using the LxWxW/2 formula. In resulting tumors, expression and activation of pathways downstream of ErbB4 were determined by immunohistochemical staining of fixed sections and immunoblot of tumor homogenates. To validate pathways identified in this screen, pharmacological inhibitors were used in soft agar colony forming assays. RESULTS: ErbB4 expression promoted cellular transformation as shown by acquisition of soft agar colony formation ability in YAMC cells. Furthermore, ErbB4 enhanced the colony formation ability of IMCERas cells by 2.2-fold (p < 0.01). In nude mouse xenograft injections, ErbB4 alone was not sufficient to induce tumor establishment of non-transformed YAMC cells; however, ErbB4 over-expression in IMCE-Ras cells resulted in a 2.1-fold increase in xenograft tumor size after 2 weeks (p < 0.02). Immunohistochemical and immunoblot analysis of tumors showed increased Akt phosphorylation and COX-2 expression but decreased p38 phosphorylation in ErbB4-expressing versus vector-expressing tumors. Pharmacological inhibitors to PI 3kinase (LY294002, 5μM) and COX-2 (celecoxib, 1μM) suppressed soft agar colony formation of ErbB4-expressing IMCE-Ras cells In Vitro, suggesting that increased activity of these cascades in the tumors is physiologically relevant. CONCLUSIONS: ErbB4 independently induces hallmarks of transformation in colonocytes In Vitro, and also co-operates with Apcmin and oncogenic Ras to promote colon epithelial cell tumorigenicity In Vivo. Chronic ErbB4 over-expression in the context of inflammation may contribute to colorectal carcinogenesis, and tumors with high levels of the receptor are likely to have enhanced cell survival signaling through PI 3-kinase and COX-2. There results suggest ErbB4 as a novel therapeutic target for colorectal cancer therapy. 1030 Glucagon-Like Peptide-2 (GLP-2) and Insulin-Like Growth Factor-I (IGF-I) Induce Differential Intestinal Growth in IGF Binding Protein (Igfbp)-3 and -5 Double Knock out Mice Sangita G. Murali, Patrick Solverson, Wing Pun, Adam S. Brinkman, John E. Pintar, Denise M. Ney
** p<0.001, *P<0.01, compared to control ## P<0.001, #p<0.01, & p<0.05,compared to WT TPN
Glucagon-like peptide-2(GLP-2) and insulin-like growth factor-I(IGF-I) are potent intestinal growth factors. The intestinotrophic actions of GLP-2 have been shown to be dependent on the presence of IGF-I. IGF binding proteins(IGFBP) modulate IGF-I action and IGFBP5 potentiates the intestinotrophic action of IGF-I. We previously reported that the intestinotrophic actions of exogenous IGF-I are not diminished in IGFBP-5 knock out (KO) mice, although IGFBP-3 expression was increased possibly compensating for the lack of IGFBP5. Our objective was to assess if stimulation of intestinal growth due to GLP-2 or IGF-I was dependent on expression of IGFBP-3 and -5. Three groups of wild type (WT) mice and three groups of double IGFBP-3 and -5 KO mice were maintained with a standard casein diet and given twice daily IP injections of GLP-2 (0.5 μg/g body wt), IGF-I (4 μg/g body wt) or vehicle for 7d. The mass of small Intestine was similar in vehicle-treated KO and WT mice. GLP-2 treated mice showed a 2-fold increase in jejunal mucosal mass, protein and DNA, as well as increased villus height and crypt depth, with no increase in plasma IGF-I compared with vehicle-treated mice. In contrast, growth of jejunal muscularis in response to GLP-2 was impaired in KO compared with WT mice. This suggests that GLP2 action in jejunal mucosa is not dependent on IGFBP-3 and -5; whereas the response in muscularis is dependent on expression of IGFBP-3 and -5. IGF-I treated mice showed a 1.4-fold increase in plasma IGF-I levels and greater gain in body weight compared to vehicletreated mice. IGF-I did not stimulate growth of either mucosa or muscularis in KO compared with WT mice. This suggests that IGF-I stimulated intestinal growth is dependent on expression of IGFBP-3 and -5. In summary, expression of IGFBP-3 and -5 has differential effects on the ability of exogenous GLP-2 or IGF-I to stimulate intestinal growth in mice. Whereas the ability of IGF-I to stimulate growth of both mucosa and muscularis is dependent on IGFBP-3 and -5, GLP-2 stimulation of mucosa growth is independent of IGFBP-3 and -5. These findings suggest interactions between GLP-2 and IGFBPs for muscularis growth and support the role of downstream mediators other than IGFBPs for the mucosal growth induced by GLP-2.
1028 IL-13 Induces Colon Epithelial Cell Apoptosis and Barrier Dysfunction in a STAT6-Dependent Manner Michael J. Rosen, Lindsay A. Kuhnhein, Scott S. Coggeshall, Rupesh Chaturvedi, Mark R. Frey, Poojitha Matta, D Brent Polk, Keith T. Wilson Background & Aims: The Th2 cytokine Interleukin 13 (IL-13) is upregulated in ulcerative colitis (UC) and increases permeability of colon epithelial monolayers by inducing apoptosis and expression of the pore-forming tight junction protein claudin-2. IL-13 binding to its receptor leads to activation of signal transducer and activator of transcription 6 (STAT6) and our lab has demonstrated increased phosphorylated STAT6 in patients with ulcerative colitis. The role of STAT6 in IL-13-induced apoptosis and barrier dysfunction has not been established. Therefore, the aim of this study was to determine if IL-13-induced apoptosis, claudin-2 expression, and resultant barrier dysfunction are STAT6-dependent. Methods: Human HT-29 colon epithelial cells were transfected with STAT6 or nontargeting (NT) small interfering RNA (siRNA) prior to treatment with IL-13 at 10 ng/ml for 48 hours. Apoptosis was assessed by flow cytometry of Annexin V stained cells, and confirmed by Western blot analysis for cleaved caspase-3. Claudin-2 expression was determined by Western blot analysis. Human T84 cell monolayers were grown on membrane inserts and treated with IL-13 at 10 ng/ml for 48 hours in the presence of suberolyanilide hydroxamic acid (SAHA), a known STAT6 inhibitor. Transepithelial resistance (TER) across each monolayer was assessed with a voltmeter. Results: STAT6 siRNA transfection resulted in a 72±5% reduction in STAT6 protein expression (P<0.001) and a 95±1% reduction in IL-13-induced phosphorylated STAT6 (P<0.001). In cells transfected with NT siRNA, IL-13 exposure increased Annexin V+ cells from 13.6±0.7% to 20.4±0.9% (P<0.01), but had no effect on apoptosis in cells transfected with STAT6 siRNA. In NT siRNA-transfected cells treated with IL-13, 20.4±0.9% were Annexin V+, compared to 13.5±1.3% in STAT6 siRNA-transfected cells (P<0.01). Similarly, IL-13 induced a 3-fold increase in caspase-3 cleavage in NT siRNA transfected cells (P<0.01), but did not induce caspase-3 cleavage in STAT6 siRNA-transfected cells. As expected, IL-13 induced a 1.4-fold increase in claudin-2 expression in NT siRNAtransfected cells (P<0.01). In contrast, in STAT6 siRNA-transfected cells, IL-13 caused a nonstatistically significant 30% decrease in claudin-2 expression. Furthermore, IL-13 exposure for 12, 24, and 48 hours decreased TER by 43±4%, 46±7%, and 65±4%, respectively, compared to 5±5%, 19±6%, and 57±4%, respectively, in the presence of SAHA (P<0.001, P<0.01, and P<0.05 respectively for IL-13 + SAHA vs. IL-13 alone). Conclusions: IL-13-induced apoptosis and claudin-2 expression are STAT6 dependent, and SAHA, a known STAT6 inhibitor, abrogates IL-13-induced epithelial barrier dysfunction in a cell culture model of colon epithelium. The potential role of epithelial STAT6 inhibition in the treatment of Th-2 mediated colitis warrants further investigation.
1031 Intestinal Epithelial Insulin-Like Growth Factor-1 Receptor Mediates Chronic Glucagon-Like Peptide-2-Induced Epithelial Proliferation and Associated Growth of the Crypt-Villus Unit Katherine J. Rowland, Daiyoon Lee, Ken Wan, Patricia L. Brubaker Glucagon-like peptide-2 (GLP-2) is an intestinal-specific mitogenic peptide. In Vivo studies have previously demonstrated that GLP-2 requires insulin-like growth factor-1 (IGF-1) to induce its intestinotropic effects. Moreover, we have confirmed that expression of the IGF1 receptor specifically in intestinal epithelial cells (IEC) is required for the proliferative effects of GLP-2 during the transition from fasting to re-feeding, and for GLP-2-induced crypt cell β-catenin signaling. AIM: To determine the mechanism by which GLP-2 exerts its chronic effects on intestinal growth. METHODS: Villin-Cre-ERT2+/0 and Igf1rflox/flox mice were bred to achieve homozygous deletion of IGF-1 receptor in the IEC upon induction with tamoxifen. IE-igf1rKO and control (Igf1rflox/flox and villin-Cre-ERT2+/0) mice were treated with tamoxifen for 5 d followed by GLP-2 or saline treatment for 10 d. Expression of growth factors and their receptors known to be involved in epithelial growth were assessed by qRT-PCR. Intestinal growth parameters included weight, mucosal cross-sectional area, crypt-villus height and expression of the proliferative marker, Ki67, as determined by immunohistochemistry. RESULTS: IE-igf1rKO mice displayed decreased mRNA expression levels of IGF-1R (p<0.05) and proglucagon (p<0.05) in jejunal mucosa relative to controls. However, no significant differences were observed in mRNA transcript levels of IGF-1, GLP2R, the ErbB ligands epiregulin, amphiregulin and HB-EGF, and the ErbB receptors ErbB1 and ErbB2, in jejunal mucosa of control and IE-igf1rKO mice10 d after tamoxifen treatment. Chronic administration of GLP-2 for 10 d to villin-Cre-ERT2+/0 control mice significantly increased small intestinal weight (p<0.01), crypt-villus height (p<0.01), and crypt cell proliferation (p<0.05) as compared to saline controls, in both tamoxifen- and vehicle-pretreated mice, indicating no effects of either Cre-recombinase or tamoxifen on GLP-2-induced gut growth. No significant differences were detected between the body weights and mucosal
1029 ErbB4 Promotes Colon Epithelial Cell Survival Signals and Tumorigenicity Christopher S. Williams, Jessica K. Bernard, D Brent Polk, Mark R. Frey The ErbB4 receptor tyrosine kinase, which is expressed at high levels in the intestinal mucosa of mice with experimental colitis and human IBD patients, promotes colon epithelial cell survival via a phosphatidylinositol 3-kinase (PI 3-kinase) and cyclooxygenase-2 (COX-2) dependent pathway. Elevated PI 3-kinase and COX-2 activities are associated with colorectal tumorigenesis. Thus, in this study, we used coordinated In Vitro and In Vivo assays to ask whether ErbB4's anti-apoptotic signaling could promote transformation of colon epithelial cells. METHODS: To assess ErbB4's effects on colonocyte transformation In Vitro, wild-type mouse colon epithelial cells (non-transformed YAMC cells) or cells harboring Apcmin and oncogenic Ras mutations (IMCE-Ras cells) were infected with retroviral expression vectors encoding ErbB4 and subjected to soft agar colony forming assays. In Vivo tumorigenicity was tested by injecting 2x106 cells subcutaneously into the flank of athymic nude mice;