The distribution of osteocytic lacunar–canalicular system, and immunolocalization of FGF23 and sclerostin in osteocytes

The distribution of osteocytic lacunar–canalicular system, and immunolocalization of FGF23 and sclerostin in osteocytes

Journal of Oral Biosciences 54 (2012) 37–42 Contents lists available at SciVerse ScienceDirect Journal of Oral Biosciences journal homepage: www.els...

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Journal of Oral Biosciences 54 (2012) 37–42

Contents lists available at SciVerse ScienceDirect

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The distribution of osteocytic lacunar–canalicular system, and immunolocalization of FGF23 and sclerostin in osteocytes Norio Amizuka a,n, Hiromi Hongo a, Muneteru Sasaki a, Tomoka Hasegawa a, Reiko Suzuki a, Chihiro Tabata a, Ubaidus Sobhan b, Hideo Masuki a, Guo Ying a, Paulo Henrique Luiz de Freitas c, Kimimitsu Oda d, Minqi Li a a

Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan High-tech Research Center-8, Tokyo Dental College, Chiba, Tokyo, Japan c ´rio Gatti Municipal Hospital, Campinas, Brazil Department of Oral and Maxillofacial Surgery, Dr. Ma d Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan b

a r t i c l e i n f o

abstract

Article history: Received 16 June 2011 Received in revised form 26 June 2011 Accepted 27 June 2011 Available online 15 February 2012

Osteocytes build up functional syncytia, i.e., the osteocytic lacunar–canalicular system (OLCS). The sites of active bone remodeling revealed the irregularly arranged OLCS, while those of slow remodeling featured well arranged OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of OLCS. Recently, osteocytes were shown to regulate phosphorus serum levels and osteoblastic activity through the expression of fibroblast growth factor (FGF) 23 and sclerostin. In our observations, FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS: both proteins were consistently expressed by osteocytes in epiphyses and cortical bones showing regularly arranged OLCS. In contrast, osteoprotegerin-deficient (OPG  /  ) mice revealed rapid bone remodeling with irregular OLCS. Sclerostin-immunoreactivity was markedly diminished in OPG  /  bones. However, sclerostin expression seemed to be a function of osteoclastic and osteoblastic activities rather than being influenced by the regularity of OLCS. This review will introduce our recent studies on the regularity of OLCS and the synthesis of osteocyte-derived FGF23 and sclerostin. & 2012 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Keywords: Osteocyte OLCS Sclerostin FGF23 Bone remodeling

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The OLCS becomes spatially regular as trabecular bone matures Regional differences of OLCS distribution in long bones . . . . . . . Putative function of OLCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunolocalization of FGF23 in OLCS . . . . . . . . . . . . . . . . . . . . . Immunolocalization of sclerostin in OLCS. . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

1. Introduction Osteocytes are the most abundant cells in bone. These cells are at the center of bone turnover’s mainframe, since they establish

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Corresponding author. Tel.: þ81 11 706 4223; fax: þ81 11 706 4226. E-mail address: [email protected] (N. Amizuka).

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the network through which osteoblasts and bone lining cells communicate. All osteocytes lie within osteocytic lacunae and connect to other osteocytes and to osteoblasts on the bone surface via thin cytoplasmic processes that pass through narrow channels named osteocytic canaliculi. Osteocytes can build functional syncytia where osteocytic processes interconnect by means of gap junctions [1–3]. In these syncytia, embedded osteocytes and osteoblasts on the bone surface communicate, forming the

1349-0079/$ - see front matter & 2012 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.job.2011.06.002

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N. Amizuka et al. / Journal of Oral Biosciences 54 (2012) 37–42

osteocytic lacunar–canalicular system (OLCS) [4–6]. The threedimensional network of OLCS has been examined in vivo [7], and our group has recently demonstrated that OLCS become progressively more regular as an individual mouse grows [8]. Knothe Tate et al. [7] documented that human osteomalacia features highly connected, non-regular OLCS, while late stage osteoporosis revealed remarkably decreased connectivity and regularity of that system. It has been postulated that the OLCS would serve as a conduit for bone minerals and other chemicals, and would take part in mechanosensing and bone turnover regulation. The canaliculi network guarantees nutrition to distant osteocytes, and also allows the transit of small molecules and minerals originated from the extracellular fluid as evidenced by tracer experiments [9–11]. Bone remodeling appears to have three different aspects: (1) balance of essential minerals in serum, (2) skeletal adaptation to its environment, and (3) repairing of load-related microdamage [12]. While the first aspect does not need site-dependent remodeling, the other two do require that specificity. This concept is named targeted remodeling [13]. There are many reports that osteocytic apoptosis and accumulated microdamage are important factors in initiating new remodeling sites [14–19]. It seems likely that osteocytic apoptosis and microdamage may disturb the signals carried throughout the lacunar–canalicular system, leading to signaling misinterpretation by osteocytes and osteoblasts, thereby initiating the targeted bone remodeling. Thus, the OLCS may be crucial for molecular transporting among osteocytes, and may also be important in interpreting mechanical load and in bone remodeling regulation. In addition, recent reports suggest active participation of osteocytes in bone metabolism by means of specific molecules—fibroblast growth factor (FGF) 23 and sclerostin. In this review, we will introduce our recent reports on the distribution of the osteocytic lacunar–

canalicular system and the immunolocalization of FGF23 and sclerostin in osteocytes.

2. The OLCS becomes spatially regular as trabecular bone matures Osteocytic lacunar–canalicular system, OLCS is a functional syncytia formed by osteocytes, and its geometrical arrangement is an important parameter in bone biology. First, we examined chronological alterations of the geometry of OLCS. With the use of Schoen’s silver staining, irregularly oriented metaphyseal trabeculae and thin cortices were identified in femora of neonatal mice [8]. Osteocytic canaliculi were randomly spread, and osteocytes were spatially very close. This feature was also identified in trabecular bones of 2- and 3-weeks-old mice. In 4-weeks-old mice bones, some osteocytes (especially those at the terminal region of the trabeculae) displayed cell processes that were perpendicular to the bone surfaces. At 8 weeks of age, terminal regions of trabecular bone had undergone remodeling, and OLCS were well arranged. At 12 weeks of age, the geometrical regularity of OLCS was more evident than that in 8-weeks-old samples. Most osteocytic canaliculi were perpendicular to the bone surfaces, which were covered by flattened osteoblasts. These findings indicate that, as bone matures, the geometrical distribution of OLCS becomes increasingly regular.

3. Regional differences of OLCS distribution in long bones Round-shaped osteocytes embedded in primary metaphyseal trabeculae extended their cytoplasmic processes in multiple

epi

GP

meta primary trabecule

secondary trabecule

ocy

cortical bone

ocy

ocy cartilaginous cores

ocy ocy

ocy

Fig. 1. Triple staining for silver impregnation, ALPase and TRAPase. (A) Alkaline phosphatase (ALPase)-positive osteoblasts (brown), tartrate-resistant acid phosphatase (TRAPase)-positive osteoclasts (red) and the silver impregnated traces representative of the OLCS (black) are depicted in the femoral epiphysis (epi) and metaphysis (meta). (B) Primary metaphyseal trabeculae displaying intensely ALPase-positive osteoblasts (brown) and several TRAPase-reactive osteoclasts (red) cover the trabecule. The cytoplasmic processes of ovoid osteocytes (ocy) spread in multiple directions, but the connectivity among these processes is interrupted by the presence of inner cartilaginous cores in the primary trabeculae. (C) In the secondary trabeculae, OLCS are regularly arranged and osteocytes (ocy) are flattened and distributed parallel to the bone surface. Osteocytic canaliculi run perpendicular to the bone surface. (D) In cortical bones, all the osteocytes (ocy) localized their cell bodies parallel to the bone surface, and had their processes extended perpendicularly to the bone surface. Bar: (A) 100 mm and (B–D) 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Modified from Ref. [20].

N. Amizuka et al. / Journal of Oral Biosciences 54 (2012) 37–42

metaphysis

metaphysis

middle region

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diaphysis

ocy ocy ocy

A

diaphysis

D

C

B

Fig. 2. Bone deposition and the regularity of OLCS. (A) The endosteal surface of the cortical bone permits only bone deposition but not resorption, and the distance between the two calcein labels (fluorescent green) gradually narrows from the metaphyseal towards the diaphyseal regions. (B) In the metaphyseal cortical bone, ALPasepositive cuboidal osteoblasts (brown) and ovoid osteocytes (ocy) with irregularly spread canaliculi are discernible. (C) In the middle region of cortical bone, osteocytes (ocy) become slender and are more regularly distributed. (D) In the diaphyseal region, osteocytes (ocy) are flattened and extend their cytoplasmic process perpendicular to the bone surface. Additionally, the layer of ALPase-positive osteoblasts becomes less pronounced towards the diaphysis. Bar: (A) 100 mm and (B,C) 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Modified from Ref. [20].

DMP1

FGF23 epi

epi

meta

meta

A

D

primary trabecule

secondary trabecule

primary trabecule

secondary trabecule

ocy ocy ocy

B

ocy

C

E

F

Fig. 3. Distribution of DMP1 and FGF23 immunopositivity in the metaphyseal trabeculae. (A) Dentin matrix protein (DMP)1 immunopositivity (brown) is seen throughout the femoral epiphysis (epi) and metaphysic (meta). (B,C) DMP1-positivity is found in the osteocytic lacunae and canaliculi of both the primary (B) and secondary (C) metaphyseal trabeculae. Note the similar intensity of immunoreaction between the primary and secondary trabeculae. (D) While FGF23-positive osteocytes (red) are ubiquitous in the epiphysis (epi), hardly any positivity is seen in the metaphysis (meta). (E) When observing at a higher magnification, osteocytes (ocy) do not show FGF23-positivity in the metaphyseal primary trabeculae, where the OLCS is irregularly distributed. (F) In the region of the secondary trabeculae featuring regular OLCS, osteocytes (ocy) exhibit relatively intense FGF23 immunoreactivity. Bar: (A,B) 100 mm and (B–F) 25 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Modified from Ref. [20].

directions. However, the inner cartilaginous cores of primary trabeculae seemed to interrupt the connections among osteocytic processes. Therefore, not only the regularity of OLCS, but also the connectivity of osteocytes seemed disrupted. Such finding suggests that the osteocytic functional syncytia could not be functioning optimally in the primary trabeculae (Fig. 1) [20]. In contrast, in remodeled secondary trabeculae, OLCS was regularly arranged and osteocytes were flat. Osteocytes’ bodies paralleled the bone surfaces, while osteocytic canaliculi ran perpendicular to them. It seems that bone remodeling is the driving force of OLCS regularization. We showed in previous reports that, as observations

progressed from the metaphysis towards the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling with a related increase in OLCS regularity (Fig. 2) [20]. It is possible that bone deposition rate during bone remodeling affects the OLCS regularity.

4. Putative function of OLCS While the true function of the osteocyte is still under discussion, the fact that transport of minerals and small molecules is carried

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N. Amizuka et al. / Journal of Oral Biosciences 54 (2012) 37–42

wild type epiphysis

OPG-/epiphysis

cortical bone

cortical bone

E Fig. 4. Immunolocalization of sclerostin in the wild type (A,C,E) and OPG  /  (B,D,F) epiphyses and diaphyses. (A,B) Triple staining for ALPase (blue), TRAPase (red) and sclerostin (brown) demonstrated a thick layer of ALPase-positive osteoblasts accompanied with many TRAPase-reactive osteoclasts in the OPG  /  epiphysis (B) compared with the wild-type ones (A). Many sclerostin-positive (brown) osteocytes were present in the wild-type epiphyses (A), while few sclerostin-positive osteocytes can be seen in the OPG  /  epiphyses (B). (C,D) The endosteal surface of wild-type cortical bone was covered with thin ALPase-positive flattened cells accompanied with few TRAPasepositive osteoclasts (C). However, OPG  /  cortical bone had many ALPase-positive osteoblastic cells (blue) and TRAPase-positive osteoclasts (red) localized on its surface and also in the inner regions (D). (E,F) At a higher magnification, compared with the wild-type cortical bone (E), plump osteoblasts (blue) adjacent to the pore in the inner region of the OPG  /  cortical bone can be seen (F). However, note that osteocytes (arrows), which are distant from the thick osteoblastic layer on the bone surface and/or adjacent to the pore in the inner regions, reveal sclerostin (brown). Bar: (A,B) 50 mm, (C,D) 100 mm and (E,F) 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Modified from Ref. [53].

through their OLCS serves as a fundamental for assuming that this cell type might be intimately involved in chemical transduction [21–23]. The chemical transduction may ultimately regulate bone remodeling [24–26] and mineral metabolism [27–29]. Consistently, ablation of osteocytes in transgenic mice expressing osteocytespecific HB-EGF, a receptor for diphteria toxin, strongly indicated that osteocytes control mineral traffic in bone and might as well regulate osteoclastic and osteoblastic activities occurring at the bone surface [27]. These putative functions imply that OLCS must be finely arranged, and that such arrangement could be altered by physical or chemical imbalances. The most accepted theory for osteocytic function places these cells as transducers of mechanical strains into biochemical signals that affect communication among

osteocytes and between osteocytes and osteoblasts [5,21,30,31]. An osteocytic response to mechanical load has been suggested [32–34], and it has been proposed that osteocytes may detect microdamage in bone [14,19,35] and undergo apoptosis, thus signaling for resorption of the damaged region [16,36,37]. Thus, the OLCS seems to be an appropriate network for transferring exogenous and endogenous signals, both mechanically and chemically. We have previously shown that osteocytes embedded in remodeled bone were flat and extended their cytoplasmic processes perpendicularly to the longitudinal axis of trabecular and cortical bones. Using biomechanical simulation analyses, McCreadie et al. [38] demonstrated that strains were higher in an elongated cell compared to a less anisotropic one, when load parallels the long axis

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of the lacuna. Also, finite element models showed that longer, thinner cells have higher maximum strains [39]. Flattened osteocytes can be found between collagen bundles, which run parallel to each other, and therefore may not disturb the seam of collagen bundles in compact bones. Orderly distributed osteocytes and their cytoplasmic processes, which geometrically harmonize with collagenous architecture, may easily recognize mechanical loading. In addition, regular OLCS may efficiently transport small molecules from one osteocyte to the others and to osteoblasts [5,11,21,22]. Alternatively, irregularly distributed osteocytic processes may not be so efficient when it comes to recognizing the direction and the intensity of mechanical loading. The notion that bone remodeling occurs as the skeleton adapts itself to its mechanical environment [12,13] supports our idea that osteocytes develop a well organized osteocytic lacunar–canalicular system as normal bone remodeling progresses.

5. Immunolocalization of FGF23 in OLCS Fibroblast growth factor 23, FGF 23 was originally reported as a phosphaturic factor in autosomal dominant hypophosphatemic rickets [40], tumor-induced osteomalacia [41], McCune-Albright syndrome/fibrous dysplasia [42], familial tumoral calcinosis [43] and possibly in X-linked hypophosphatemic rickets [44]. Although FGF23 mRNA is found in several tissues [40,41,45], this molecule is most abundantly expressed in bone [46]. Consistently, we observed FGF23-immunopositive osteocytes in secondary trabeculae and cortical bone with regularly oriented OLCS. In contrast, in our observations, dentin matrix protein1, DMP1, another hallmark of osteocytes, was broadly expressed in osteocytes (Fig. 3) [20]. Unlike DMP1, however, FGF23 appears to be synthesized principally by osteocytes in regularly distributed OLCS that have been established after bone remodeling. A circulating factor synthesized by osteocytes, FGF23 serves as a systemic, phosphaturic agent that inhibits 1,25(OH)2D3 renal production and maintains the balance between phosphate homeostasis and skeletal mineralization [47]. Considering that mature bone possesses regular OLCS, and that FGF23 synthesis occurs consistently in well-arranged OLCS, it seems likely that mature bone could serve as an organ regulating serum phosphorus levels. Thus, investigations on the biological functions of FGF23 have broadened the understanding of the systemic regulation of phosphate homeostasis, as well as the knowledge of maintenance of proper mineralization in the bone matrix [47].

6. Immunolocalization of sclerostin in OLCS Osteocytes secrete sclerostin, a product of the SOST gene [49] which binds the LRP5/6 receptor, thus antagonizing Wnt signaling and increasing b-catenin degradation [49,50]. Sclerostin has been highlighted as a negative regulator of osteoblastic bone formation [25,26,48,50–52], and is also regarded as an important mediator of mechanical loading in bone [52]. We recently demonstrated the extremely reduced sclerostin immunopositivity in osteoprotegerin-deficient (OPG  /  ) epiphyses and cortices, while wild-type mice revealed intense sclerostin staining (Fig. 4) [53]. OLCS in OPG  /  bones was markedly disturbed and, therefore, one may think that sclerostin, like FGF23, would be present only in a well-arranged OLCS. However, OPG  /  cortical bones, especially at the endosteal portion, did not show irregular OLCS; also, osteocytes close to active osteoblasts and bone-resorbing osteoclasts did not reveal sclerostin immunoreactivity. This indicates that, rather than OLCS regularity, osteoclastic and osteoblastic activities may influence

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sclerostin synthesis in osteocytes; it also implies the existence of some sort of intercellular signaling from osteoblasts to osteocytes.

7. Conclusion The geometrically regular osteocyte lacunar–canalicular system (OLCS) is a functional syncytia where FGF23 and sclerostin expression can be observed. The regularity of OLCS seems to be established by the physiological bone remodeling that secures bone maturation.

Conflict of interest No potential conflicts of interest are disclosed.

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