Morphological aspects of the biological function of the ... - Core

Oral Science International 9 (2012) 1–8

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Review

Morphological aspects of the biological function of the osteocytic lacunar canalicular system and of osteocyte-derived factors Muneteru Sasaki a , Hiromi Hongo a , Tomoka Hasegawa a , Reiko Suzuki a , Liu Zhusheng a , Paulo Henrique Luiz de Freitas b , Tamaki Yamada c , Kimimitsu Oda d , Tsuneyuki Yamamoto a , Minqi Li a,e , Yasunori Totsuka c , Norio Amizuka a,∗ a

Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan Department of Oral and Maxillofacial Surgery, Dr. Mário Gatti Municipal Hospital, Campinas, Brazil c Department of Oral and Maxillofacial Surgery, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan d Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan e Shandong Provincial Key Laboratory of Oral Biomedicine, The School of Stomatology, Shandong University, Jinan, China b

a r t i c l e

i n f o

Article history: Received 15 December 2011 Received in revised form 11 January 2012 Accepted 18 January 2012 Keywords: Osteocyte OLCS Sclerostin FGF23 Bone remodeling

a b s t r a c t Osteocytes are organized in functional syncytia collectively referred to as the osteocytic lacunar–canalicular system (OLCS). The osteocytes are interconnected through gap junctions between their cytoplasmic processes, which pass through narrow passageways referred to as osteocytic canaliculi. There are two possible ways molecules can be transported throughout the OLCS: via the cytoplasmic processes and their gap junctions, and via the pericellular space in the osteocytic canaliculi. Transport of minerals and small molecules through a spatially well-organized OLCS is vital for bone mineral homeostasis, mechanosensing, and bone remodeling control. Recently, osteocyte-derived molecules – sclerostin, dentin matrix protein-1, fibroblast growth factor 23 (FGF23) – have been put in evidence as they may be related to osteocytic functions such as mechanosensing, regulation of bone remodeling, and so forth. FGF23 regulates serum phosphate concentration by affecting renal function, while sclerostin can inhibit osteoblastic activities. In our observations, FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS. This review will introduce our recent morphological studies on the regularity of OLCS and the synthesis of osteocyte-derived FGF23 and sclerostin. © 2012 Japanese Stomatological Society. Published by Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Putative function of osteocytic osteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible role of osteocytes in mineral transport and as transducers of mechanical strains into biochemical signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of bone minerals by mediating osteocytes-derived factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Role of osteocyte-derived sclerostin in bone remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Distribution of FGF23 in the OLCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Disrupted osteocytic function in klotho-deficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

∗ Corresponding author at: Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo, 060-8586, Japan. Tel.: +81 11 706 4223; fax: +81 11 706 4226. E-mail address: [email protected] (N. Amizuka).

Osteocytes, the most abundant cells in bone, are at the center of bone turnover control as they establish 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. Osteocytes are embedded in the bone matrix, and are derived from osteoblasts that got

1348-8643/$ – see front matter © 2012 Japanese Stomatological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S1348-8643(12)00009-2

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Fig. 1. The ultrastructure of osteocytes depending on the site and on the interval since embedding into the bone matrix. Panel A shows a recently embedded osteocyte (ocy) connected to an osteoblast (ob) by its cytoplasmic processes (arrows). This osteocyte has abundant rough endoplasmic reticulum (rER) and shares ultrastructural similarities with osteoblasts. In panel B, osteocytes close to the bone surface but embedded into mineralized matrix tend to have rER and are surrounded by the rough walls of the osteocytic lacuna. As shown in the right-hand panel, there are collagen fibrils in the pericellular space in the osteocytic lacuna. In contrast, as shown in panels C and D, osteocytes embedded in the deeper portions of the matrix or those that have been buried for a long time show fewer cell organelles, and the nucleus becomes more significant. Please note the nucleus of osteocyte in D was much more condensed than that seen in C. The electron dense lines on the wall of lacunae, the lamina limitans (arrowheads in C and D), indicate the absence of bone deposition. Bar: A–D, 1 ␮m.

“trapped” within the bone matrix. Depending on the site and on the interval since the embedding, osteocytic ultrastructure can vary significantly (Fig. 1). Recently embedded osteocytes have abundant rough endoplasmic reticulum (rER) and Golgi apparatus, indicating some residual bone formation capacity. Osteocytes close to the bone surfaces have rER, and their osteocytic lacunae have rough walls, suggesting that osteocytes embedded in the superficial bone layers may still be capable of synthesizing bone matrix. Aarden et al. termed osteocytes located in osteoid and those embedded in recently mineralized matrix as osteoid osteocytes and young osteocytes, respectively [1]. In contrast, osteocytes embedded in the deeper portion of matrix or those that have been buried for a long time (mature osteocytes) show fewer cell organelles with the nucleus becoming more prominent. The process of osteocyte embedding in bone is not at all random. Osteocytes act as a functional group, since their cytoplasmic processes are connected through gap junctions [2–4]. Such a network of cytoplasmic processes permits the passage of small cytoplasmic molecules from one osteocyte to the next. In addition, the pericellular space (annular space) in the osteocytic canaliculi may serve as an alternative transport path (Fig. 2). The diffusion coefficient of fluorescein in the pericellular space has been shown to be similar to diffusion coefficients measured for comparably sized molecules in cartilage matrix [5]. Through these possible paths,

embedded osteocytes communicate and establish the osteocytic lacunar–canalicular system (OLCS) [1,6,7]. The three-dimensional OLCS has been examined in vivo [8], and our group has recently demonstrated that, in mice, the OLCS becomes progressively more regular as the individual grows [9]. For the OLCS to function properly, its anatomic arrangement has to be correct. In mature, cortical bone, osteocytic bodies parallel the bone surface and extend their cytoplasmic processes perpendicularly to it (Fig. 3) [10]. This regularity may relate with the direction of the collagen bundles: while the longitudinal axis of the osteocytes parallels the direction of the collagen fibrils, their cytoplasmic processes are perpendicular to them. The regular OLCS may enable the osteocytes to sense mechanical loading and efficiently transport small molecules via their cytoplasmic processes and through the pericellular space of their canaliculi. Bone disease, on the other hand, may significantly affect the arrangement of the OLCS. In human osteomalacia, haphazardly connected, non-regular OLCS are seen, and in the late stage of osteoporosis, a remarkable compromise of connectivity and regularity of that system is present [7]. In this review, we will introduce and elaborate on morphological aspects of osteocytic function, especially the biological function of the regularly arranged OLCS and the pivotal roles of osteocytederived factors in bone mineralization.

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Fig. 2. Cellular network of osteocytes and the pericellular space of osteocytic canaliculi. (A–C) Images of scanning electron microscopic observation on the cellular network of osteocytes (ocy) after elimination of extracellular bone matrices. As shown in (A), flattened osteocytes in the endosteal region of cortical bone lies parallel to the bone surface, extending their cytoplasmic processes perpendicularly to it. However, the periosteal region of the cortical bone (B) reveals randomly oriented osteocytes extending their processes in all directions. Please note the bifurcation of osteocytic processes (arrows) when observed at a higher magnification (C). Panels D and E show the transmission electron microscopic images of pericellular space, or annular space in the osteocytic canaliculi (D is an image obtained from mineralized bone, while E shows demineralized osteocytic canaliculi). Please note that there are two possible pathways for molecular transport, one formed by the cytoplasmic processes, and the other being the pericellular space in the osteocytic canaliculi. Bar: A, 10 ␮m; B, 5 ␮m; C, 2 ␮m; D and E, 0.5 ␮m. cp; cytoplasmic process, BM; bone marrow.

2. Putative function of osteocytic osteolysis Can an osteocyte regulate the mineralization of the bone matrix that encloses it? It is possible that osteocytes respond to parathyroid hormone (PTH) or to a low calcium diet by altering serum calcium levels. This phenomenon was reported a hundred years ago by Recklinghausen [11] and by Kind [12], and in the 1960s, Bélanger proposed a new notion of “osteocytic osteolysis” [13]. Six hours after injection of human PTH (1–34) into the jugular vein of mice, we observed enlarged osteocytic lacunae in the cortical bone with regular OLCS. One may question wonder whether osteoclasts can create such enlargement by secreting acids and proteolytic enzymes, which may erode the lacunar matrix. In order to address

the issue at hand, it seems necessary to verify the occurrence of enlarged lacunae in mice lacking osteoclasts. In addition, some researchers may attribute the acute elevation of serum calcium levels after PTH injection to osteocytic osteolysis, while others may question whether such a raise may result from stimulated renal calcium reabsorption. Some findings suggest possible mechanisms for osteocytic osteolysis, e.g., synthesis of tartrate-resistant acid phosphates by osteocytes [14], enlarged osteocytic lacunae and acid phosphates activity in osteocytes after continuous infusion of PTH for 4 weeks [15], as well as synthesis of matrix metalloproteinase-2 by osteocytes [16]. More recently, the groups of Teti and Bonewald reported osteocytic remodeling of the perilacunar and pericanalicular matrix [17,18].

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Fig. 3. Histological imaging of osteocytic lacunar–canalicular system using paraffin sections. Panels A, B and C, D are silver impregnation modified of Bodian’s method and phase contrast images of the distribution of osteocytes (ocy) and their canaliculi. In the cortical bone (A and C), osteocytes’ bodies were parallel to the bone surface, but they extended the cytoplasmic processes perpendicularly to it. However, as shown in B and D, the distribution of osteocytes and their canaliculi was similar in the metaphyseal secondary trabeculae. Bar: A–D, 30 ␮m.

3. Possible role of osteocytes in mineral transport and as transducers of mechanical strains into biochemical signals While the true function of the osteocytic osteolysis is still under discussion, the fact that minerals and other small molecules are carried through the OLCS serves as the basis for assuming that osteocytes might be intimately involved in chemical transduction [5,19,20]. Regarding mineral transport, ablation of osteocytes in transgenic mice expressing osteocyte-specific receptor for diphtheria toxin, strongly indicated that osteocytes control mineral traffic in bone [21]. After diphtheria toxin injection, transgenic

mice showed many empty lacunae, indicating osteocyte death. Curiously, trabeculae were shortened after toxin injection, and the cortical bones started to show broad demineralized areas in the periphery of empty osteocytic lacunae. It seems that disruption of osteocytic function leads to some type of bone mineral shortage. Another 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 [6,19,22,23]. Bone remodeling appears to have three different aspects: (1) balance of essential serum minerals, (2) skeletal adaptation to the environment and (3) repairing

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of load-related microdamage [24]. While the first aspect does not need site-dependent remodeling, the other two do require that specificity. This concept is named “targeted bone remodeling” [25]. The proposed cellular mechanism of targeted bone remodeling regards osteocytes as sensors for microdamage in bone [26–28]. Once damage is detected, osteocytes would undergo apoptosis, triggering the resorption of the damaged region [29–31]. There are many reports on osteocytic apoptosis and accumulated microdamage as important factors for initiating new remodeling sites [26,27,32–34]. It seems likely that osteocytic apoptosis and microdamage disturb the signals carried throughout the OLCS, leading to signaling misinterpretation by osteocytes and osteoblasts and initiation of targeted bone remodeling. An intact OLCS seems crucial for molecular transport among osteocytes, and may be important for mechanosensing and bone remodeling control. We have shown that osteocytes embedded in remodeled bone were flat and extended their cytoplasmic processes perpendicularly to the longitudinal axis of trabecular and cortical bones (Fig. 3). Using biomechanical simulation analyses, McCreadie et al. [35] demonstrated that strains were higher in elongated cells compared to less elongated ones, when load parallels the long axis of the lacuna. Also, finite element models showed that longer, thinner cells have higher maximum strains [36]. Flattened osteocytes are found among 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 osteocytic processes, when geometrically harmonized with the surrounding collagenous architecture, may be very effective in recognizing mechanical loading. In addition, a regular OLCS may efficiently transport small molecules from one osteocyte to others, and to osteoblasts as well [6,19,20,37]. Irregularly distributed osteocytic processes, on the other hand, 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 [24,25] supports our idea that osteocytes develop a well-organized OLCS as normal bone remodeling progresses. 4. Regulation of bone minerals by mediating osteocytes-derived factors Osteocyte-derived molecules were recently highlighted because these molecules may reflect osteocytic function responding to mechanosensing, regulation of bone remodeling and so forth. Dentin matrix protein-1 (DMP-1) was shown to be a bone matrix protein expressed in osteocytes (Fig. 4A–C), and has been assumed to play a pivotal role in bone mineral homeostasis, due to its high calcium-binding affinity [38,39]. Another osteocytederived factor, sclerostin, is a glycoprotein encoded by the SOST gene [40], and was reported to bind the LRP5/6 receptor, thereby antagonizing Wnt signaling and increasing ␤-catenin degradation [41,42]. Sclerostin has been reported as a negative regulator of osteoblastic bone formation [40,43–45]: Sclerostin secreted by osteocytes may pass through the osteocytic canaliculi and inhibit bone-lining osteoblasts. Fibroblast growth factor (FGF) 23, which is also an osteocyte-derived factor (Fig. 4D–F), modulates serum phosphate concentration, by co-operating in kidney [46]. Thus, recently highlighted factors such as FGF23, sclerostin, and DMP-1 would provide clues for better understanding for osteocytic function. 4.1. Role of osteocyte-derived sclerostin in bone remodeling Sclerostin, which is secreted by osteocytes, passes through osteocytic canaliculi and inhibits osteoblasts, and therefore is

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characterized as a factor that regulates bone formation. We have examined the distribution of sclerostin and OLCS in long bones of mice lacking osteoprotegerin (OPG) [47], a decoy receptor for the receptor activator of the nuclear factor ␬B ligand (RANKL). OPG has been characterized as an inhibitor of osteoclastogenesis [48]. OPG-deficient mice showed accelerated bone remodeling and an irregular OLCS. While DMP-1 was found in all osteocytes in the OPG-deficient bone, sclerostin reactivity was significantly diminished in OPG-deficient epiphyses and cortical bone. Sclerostin appears to be synthesized specifically once osteocytes possess a regular OLCS, for example in normal, mature cortical bone. It has been suggested that PTH administration inhibits sclerostin synthesis by osteocytes, thereby allowing for active bone remodeling [44]. By examining the mesial region of the mandibular interradicular septum in ovariectomized rats, we attempted to elucidate whether estrogen deficiency would affect the synthesis of sclerostin by means of accelerating bone resorption or through a more direct effect on the estrogen receptors in osteocytes (unpublished data). The mesial region of ovariectomized interradicular septa showed an increased number of osteoclasts with intense RANKL labeling, as well as widespread absence of sclerostin-positive osteocytes. Accelerated bone remodeling induced by estrogen deficiency, rather than having a direct effect through the estrogen receptors in osteocytes, appeared to inhibit sclerostin expression by osteocytes in the mesial region of interradicular septa. These findings imply that bone remodeling or its participating cells, osteoblasts and osteoclasts, would primarily affect sclerostin synthesis. Recently, two reports demonstrated that osteocytes express high amounts of RANKL and by doing so support osteoclastic differentiation; meanwhile, the authors believe that RANKL produced by osteoblasts seems not to contribute to adult bone remodeling [49,50]. This is a radical shift from the presently prevailing knowledge of osteoclastogenesis. It seems necessary to examine in detail whether osteoclasts differentiate only after triggered by osteocytederived RANKL. Also, one may consider how matrix-embedded osteocytes could affect osteoclast precursors that lie distant from bone surfaces. Yet, examining the involvement of osteocytes in osteoclastogenesis is crucial for new breakthroughs in bone biology, and these two reports may provide a new horizon in the field. 4.2. Distribution of FGF23 in the OLCS FGF23 was originally reported as a phosphaturic factor in autosomal dominant hypophosphatemic rickets [51], tumor-induced osteomalacia [52], McCune-Albright syndrome/fibrous dysplasia [53], familial tumoral calcinosis [54], and in X-linked hypophosphatemic rickets [55]. Although FGF23 mRNA is found in several tissues [51,52,54], this molecule is most abundantly expressed in bone [56]. FGF23 serves as a phosphaturic agent that inhibits 1,25(OH)2D3 production and the function of sodium/phosphate cotransporter II (NaPiII), which inhibits reabsorption of phosphates in the proximal renal tubules [57–59]. Investigations on the biological functions of FGF23 have broadened the understanding of the systemic regulation of phosphate homeostasis, as well as the knowledge about maintaining mineralization in the bone matrix [60]. We have observed FGF23-immunopositive osteocytes in disease-free secondary trabeculae and cortical bone with regularly oriented OLCS: FGF23 appears to be synthesized mainly by osteocytes forming regularly distributed OLCS in mature bone that had been remodeled (Fig. 4D–F) [10]. Alternatively, intense immunopositivity for FGF23 in metaphyseal primary trabeculae with irregular OLCS could not be verified. Mature bone could, therefore, serve as an organ regulating serum phosphorus levels. Although preliminary, one of our recent experiments hinted

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Fig. 4. Distribution of dentin matrix protein (DMP-1) and fibroblast growth factor (FGF) 23 in the epiphysis (epi) and metaphysis (meta). Panel A shows the immunolocalization of DMP-1 (brown color) throughout the femoral epiphysis and metaphysis. DMP-1 positivity is found in osteocytes (ocy) and their lacunae and canaliculi of both the primary (B) and secondary (C) metaphyseal trabeculae. In contrast, as shown in panel D, while FGF23 positive osteocytes (red) are prominent in the epiphysis, hardly any positivity is seen in the metaphysis. When observing at a higher magnification, osteocytes (ocy) do not show FGF23-positivity in the metaphyseal primary trabeculae, while osteocytes in the secondary trabecules tend to exhibit relatively intense FGF23 immunoreactivity. Modified from Ubaidus et al. [10]. Bar: A and D, 100 ␮m; B, C, E, and F, 30 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

on enhancement of FGF23 synthesis by osteocytes after 2 weeks of PTH administration. However, PTH administration also accelerated bone remodeling and formation of an irregular OLCS. PTH may induce FGF23 synthesis in osteocytes even when the OLCS is malformed; also, PTH could directly attenuate phosphate reabsorption, but also by inhibiting NaPiII in the proximal renal tubules. These preliminary findings further support an important role of the osteocytes in phosphate homeostasis. 4.3. Disrupted osteocytic function in klotho-deficient mice The klotho gene is involved in multiple aging phenotypes and age-related disorders. Klotho-deficient mice develop normally until 3 weeks of age, then become less active and ultimately die by 8–9 weeks of age [61]. The defect in klotho gene causes osteoporosis, skin atrophy, ectopic mineralization (vascular calcification), pulmonary emphysema, gonadal dysplasia, and defective hearing in mice – all of which also appear in human aging. Recently, two klotho proteins have been recognized: transmembrane klotho and circulating klotho. The transmembrane klotho is a main co-receptor for the FGF23 signaling pathways [46,62,63]. Circulating klotho, on the other hand, regulates the activity of multiple glycoproteins on the cell surface, including ion channels and insulin growth factor1 receptor [64]. Since klotho was shown to regulate phosphate homeostasis as an obligate co-receptor for FGF23, its deficiency

could lead to defective FGF23 signaling, consequently accelerating phosphate reabsorption in kidney [46,57–59,62]: Klotho-deficient mice feature high serum calcium, phosphorus [61,65], and osteoprotegerin [66,67] levels. The abnormal histology found in klotho-deficient mice has been attributed to a disrupted FGF23-klotho axis. We, however, postulated that klotho deficiency might also affect bone in a way not linked to the FGF23-klotho axis. We have observed that klotho-deficient primary metaphyseal trabeculae were excessively mineralized, probably due to markedly increased concentrations of calcium and phosphate, while the secondary metaphyseal trabeculae showed defective mineralization [68,69]. Using electron probe microanalysis, we demonstrated higher and lower contents of calcium and phosphorus in primary and secondary metaphyseal trabeculae of klotho-deficient mice, respectively. More recently, we found matrix proteins – matrix Gla proteins (MGP), DMP-1, and osteocalcin with a high affinity to hydroxyapatite – localized in the excessively mineralized osteocytic lacunae of klotho-deficient bone, indicative of dying osteocytes [70]. These findings suggest that osteocalcin and MGP are ectopically synthesized in klotho-deficient osteocytes, which also synthesize a large amount of DMP-1. Consequently, the osteocytic lacunae are filled with mineralized crystals and osteocytic function is disrupted. It is feasible, therefore, that there is some degree of malfunction

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in klotho-deficient osteocytes in a manner independent from the FGF23-klotho axis. 5. Conclusion The regularly arranged OLCS is a functional syncytia where FGF23 and sclerostin expression is observed. The regularity of the OLCS seems to be established by physiological bone remodeling, which secures bone maturation. Acknowledgment This study was partially supported by grants from the Japanese Society for the Promotion of Science (Amizuka N, Suzuki R, Li M). References [1] Aarden EM, Burger EH, Nijweide PJ. Function of osteocytes in bone. J Cell Biochem 1994;55:287–99. [2] Doty SB. Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 1981;33:509–12. [3] Shapiro F. Variable conformation of GAP junctions linking bone cells: a transmission electron microscopic study of linear stacked linear, curvilinear, oval, and annular junctions. Calcif Tissue Int 1997;61:285–93. [4] Donahue HJ. Gap junctions and biophysical regulation of bone cell differentiation. Bone 2000;26:417–22. [5] Wang L, Wang Y, Han Y, et al. In situ measurement of solute transport in the bone lacunar–canalicular system. Proc Natl Acad Sci USA 2005;102:11911–6. [6] Burger EH, Klein-Nulend J. Mechanotransduction in bone role of the lacunocanalicular network. FASEB J 1999;13:101–12. [7] Knothe Tate ML, Adamson JR, Tami AE, et al. The osteocyte. Int J Biochem Cell Biol 2004;36:1–8. [8] Kamioka H, Honjo T, Takano-Yamamoto T. A three dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 2001;28:145–9. [9] Hirose S, Li M, Kojima T, et al. A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining. J Bone Miner Metab 2007;25:374–82. [10] Ubaidus S, Li M, Sultana S, et al. FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling. J Electron Microsc 2009;58:381–92. [11] Recklinghausen Fv. Untersuchungen über Rachitis und Osteomalacia. Jena: Gustav Fischer; 1910. [12] Kind H. “Periosteocytäre osteolyse” Studien zur Frage der Osteolyse. Beitr Path Anat 1951;111:283–312. [13] Bélanger LF. Osteocytic osteolysis. Calcif Tissue Res 1969;4:1–12. [14] Nakano Y, Toyosawa S, Takano Y. Eccentric localization of osteocytes expressing enzymatic activities, protein, and mRNA signals for type 5 tartrate-resistant acid phosphatase (TRAP). J Histochem Cytochem 2004;52:1475–82. [15] Tazawa K, Hoshi K, Kawamoto S. Osteocytic osteolysis observed in rats to which parathyroid hormone was continuously administered. J Bone Miner Metab 2004;22:524–9. [16] Inoue K, Mikuni-Takagaki Y, Oikawa K, et al. A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism. J Biol Chem 2006;281:33814–24. [17] Teti A, Zallone A. Do osteocytes contribute to bone mineral homeostasis? Osteocytic osteolysis revisited. Bone 2008;44:11–6. [18] Qing H, Bonewald LF. Osteocyte remodeling of the perilacunar and pericanalicular matrix. Int J Oral Sci 2009;1:59–65. [19] Klein-Nulend J, van der Plas A, Semeins CM, et al. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 1995;9:441–5. [20] Johnson DL, McAllister TN, Frangos JA. Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts. Am J Physiol 1996;271:E205–8. [21] Tatsumi S, Ishii K, Amizuka N, et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab 2007;5:464–75. [22] Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 1994;27:339–60. [23] Burger EH, Klein-Nulend J, van der Plas A, et al. Function of osteocytes in bone—their role in mechanotransduction. J Nutr 1995;125:2020S–3S. [24] Burr DB. Targeted and nontargeted remodeling. Bone 2002;30:2–4. [25] Frost HM. Presence of microscopic cracks in vivo in bone. Bull Henry Ford Hosp 1960;8:25–35. [26] Mori S, Burr DB. Increased intracortical remodeling following fatigue damage. Bone 1993;14:103–9. [27] Hazenberg JG, Freeley M, Foran E, et al. Microdamage: a cell transducing mechanism based on ruptured osteocyte processes. J Biomech 2006;39:2096–103. [28] Burr DB, Forwood MR, Fyhrie DP, et al. Bone microdamage and skeletal fragility in osteoporotic and stress fractures. J Bone Miner Res 1997;12:6–15.

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