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Osteoclasts are multinucleated cells responsible for bone resorption. The differentiation of osteoclasts from bone marrow macrophages (BMMs) is induced by receptor activator of NF-κB ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor of RANKL, inhibits osteoclastogenesis by blocking RANKL signaling. Here we investigated the degradation of OPG in vitro. Osteoclasts, but not BMMs, secreted OPG-degrading enzymes. Using mass spectrometry and RNA-sequencing analysis, we identified high-temperature requirement A serine peptidase 1 (HtrA1) as an OPG-degrading enzyme. HtrA1 did not degrade OPG pre-reduced by dithiothreitol, suggesting that HtrA1 recognizes the three-dimensional structure of OPG. HtrA1 initially cleaved the amide bond between leucine 90 and glutamine 91 of OPG, then degraded OPG into small fragments. Inhibitory activity of OPG on RANKL-induced osteoclastogenesis was suppressed by adding HtrA1 in RAW 264.7 cell cultures. These results suggest that osteoclasts potentially prepare a microenvironment suitable for osteoclastogenesis. HtrA1 may be a novel drug target for osteoporosis., Nagahiro Ochiai et al. report that osteoclasts, but not bone marrow macrophages, secrete a serine protease HtrA1 that can degrade osteoprotegerin in the bone microenvironment. Their results suggest HtrA1 recognizes the three-dimensional structure of osteoprotegerin and may function to prepare the microenvironment for osteoclastogenesis.

We have reported that 25-hydroxyvitamin D3 [25(OH)D3] binds to vitamin D receptor and exhibits several biological functions directly in vitro. To evaluate the direct effect of 25(OH)D3 in vivo, we used Cyp27b1 knockout (KO) mice, which had no detectable plasma 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] when fed a diet containing normal Ca and vitamin D. Daily treatment with 25(OH)D3 at 250 μg kg-1 day-1 rescued rachitic phenotypes in the Cyp27b1 KO mice. Bone mineral density, female sexual cycles, and plasma levels of Ca, P, and PTH were all normalized following 25(OH)D3 administration. An elevated Cyp24a1 mRNA expression was observed in the kidneys, and plasma concentrations of Cyp24a1-dependent metabolites of 25(OH)D3 were increased. To our surprise, 1,25(OH)2D3 was detected at a normal level in the plasma of Cyp27b1 KO mice. The F1 to F4 generations of Cyp27b1 KO mice fed 25(OH)D3 showed normal growth, normal plasma levels of Ca, P, and parathyroid hormone, and normal bone mineral density. The curative effect of 25(OH)D3 was considered to depend on the de novo synthesis of 1,25(OH)2D3 in the Cyp27b1 KO mice. This suggests that another enzyme than Cyp27b1 is present for the 1,25(OH)2D3 synthesis. Interestingly, the liver mitochondrial fraction prepared from Cyp27b1 KO mice converted 25(OH)D3 to 1,25(OH)2D3. The most probable candidate is Cyp27a1. Our findings suggest that 25(OH)D3 may be useful for the treatment and prevention of osteoporosis for patients with chronic kidney disease.

Active forms of vitamin D enhance osteoclastogenesis in vitro and in vivo through the vitamin D receptor (VDR) in osteoblast-lineage cells consisting of osteoblasts and osteocytes. This pro-resorptive activity was evident basically with higher concentrations of active vitamin D than those expected in physiological conditions. Nevertheless, vitamin D compounds have been used in Japan for treating osteoporosis to increase bone mineral density (BMD). Of note, the increase in BMD by long-term treatment with pharmacological (=near-physiological) doses of vitamin D compounds was caused by the suppression of bone resorption. Therefore, whether vitamin D expresses pro-resorptive or anti-resorptive properties seems to be dependent on the treatment protocols. We established osteoblast lineage-specific and osteoclast-specific VDR conditional knockout (cKO) mice using Osterix-Cre transgenic mice and Cathepsin K-Cre knock-in mice, respectively. According to our observation using these cKO mouse lines, neither VDR in osteoblast-lineage cells nor that in osteoclasts played important roles for osteoclastogenesis and bone resorption at homeostasis. However, using our cKO lines, we observed that VDR in osteoblast-lineage cells, but not osteoclasts, was involved in the anti-resorptive properties of pharmacological doses of vitamin D compounds in vivo. Two different osteoblast-lineage VDR cKO mouse lines were reported. One is a VDR cKO mouse line using alpha 1, type I collagen (Col1a1)-Cre transgenic mice (here we call Col1a1-VDR-cKO mice) and the other is that using dentin matrix protein 1 (Dmp1)-Cre transgenic mice (Dmp1-VDR-cKO mice). Col1a1-VDR-cKO mice exhibited slightly increased bone mass due to lowered bone resorption. In contrast, Dmp1-VDR-cKO mice exhibited no difference in BMD in agreement with our results regarding Ob-VDR-cKO mice. Here we discuss contradictory results and multiple modes of actions of vitamin D in bone resorption in detail. (279 words).

Long-term treatment with active vitamin D [1α,25(OH)2D3] and its derivatives is effective for increasing bone mass in patients with primary and secondary osteoporosis. Derivatives of 1α,25(OH)2D3, including eldecalcitol (ELD), exert their actions through the vitamin D receptor (VDR). ELD is more resistant to metabolic degradation than 1α,25(OH)2D3. It is reported that ELD treatment causes a net increase in bone mass by suppressing bone resorption rather than by increasing bone formation in animals and humans. VDR in bone and extraskeletal tissues regulates bone mass and secretion of osteotropic hormones. Therefore, it is unclear what types of cells expressing VDR preferentially regulate the vitamin D–induced increase in bone mass. Here, we examined the effects of 4-week treatment with ELD (50 ng/kg/day) on bone using osteoblast lineage-specific VDR conditional knockout (Ob-VDR-cKO) and osteoclast-specific VDR cKO (Ocl-VDR-cKO) male mice aged 10 weeks. Immunohistochemically, VDR in bone was detected preferentially in osteoblasts and osteocytes. Ob-VDR-cKO mice showed normal bone phenotypes, despite no appreciable immunostaining of VDR in bone. Ob-VDR-cKO mice failed to increase bone mass in response to ELD treatment. Ocl-VDR-cKO mice also exhibited normal bone phenotypes, but normally responded to ELD. ELD-induced FGF23 production in bone was regulated by VDR in osteoblast-lineage cells. These findings suggest that the vitamin D treatment-induced increase in bone mass is mediated by suppressing bone resorption through VDR in osteoblast-lineage cells. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

We have reported that 25-hydroxyvitamin D

Vitamin D compounds, 1,25(OH)2D3 and its analogs, enhance osteoclastogenesis in vitro and in vivo through the vitamin D receptor (VDR) in osteoblastic cells. This proresorptive activity was detected only with much higher concentrations of vitamin D compounds than those expected in physiological conditions. On the other hand, these compounds have been used as therapeutic drugs in Japan for treating osteoporosis to increase bone mineral density (BMD). Notably, the increase in BMD by long-term treatment with pharmacological doses of vitamin D compounds is caused by the suppression of bone resorption. Therefore, whether vitamin D expresses proresorptive or antiresorptive properties seemingly depends on the treatment protocols. We established osteoblastic cell-specific and osteoclast-specific VDR conditional knockout mice. Neither VDR in osteoblastic cells nor that in osteoclasts plays important roles for osteoclastogenesis at homeostasis. However, VDR in osteoblastic cells, but not osteoclasts, was involved in the antiresorptive properties of pharmacological doses of vitamin D compounds in vivo. Multiple modes of action of vitamin D in bone resorption are discussed in detail in this chapter.

The physiological role of vitamin D depends on calcium supply and calcium balance. When the calcium balance is normal, the major target of vitamin D is intestine. Vitamin D stimulates mainly active intestinal calcium transport mechanism. During a negative calcium balance, bone effects of vitamin D become dominant. Thus, the role of vitamin D in maintaining normocalcemia appears to have priority over skeletal integrity in these situations.

Long-term treatment with active vitamin D [1α,25(OH)

Osteoclasts are terminally differentiated multinucleated cells that are the principal resorptive cells of bone, playing a central role in the formation of the skeleton and regulation of its mass. The molecular events involved in the differentiation and function of osteoclasts had not been clarified for a long time. Over the past two decades, several novel approaches have been developed and adopted to investigate osteoclast biology. In the present review, we would like to update recent progress in the elucidation of the molecular mechanism of osteoclast activation and function.

Eldecalcitol (ED-71) is a new vitamin D₃ derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED-71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED-71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED-71 (50 ng/kg body weight) to 8-week-old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED-71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle-arrested receptor activator of NF-κB (RANK)-positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED-71 affected neither the number of RANK-positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED-71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL-positive cell surface around the trabecular bone was significantly reduced in ED-71-treated mice than in the control mice. ED-71 administration also increased BMD in 12-week-old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED-71 increases BMD by suppressing RANKL expression in trabecular bone in vivo.

Chronic immune responses and inflammatory reactions in rheumatoid arthritis (RA) often cause severe destruction of cartilage and bone, but its mechanism is still a matter of controversy. We reported that interleukin-6 (IL-6) alone does not induce osteoclast formation, but soluble interleukin-6 receptors (sIL-6R) triggered the formation in the presence of IL-6 in cocultures of murine osteoblastic cells and bone marrow cells. In this study, we examined the involvement of sIL-6R and IL-6 in joint destruction in patients with RA. Although the frequency of patients having osteoclast-like multinucleated cells in synovium derived from the knee joint was not significantly different between RA (65%) and osteoarthritis (OA) patients (43%), the number of osteoclast-like cells found in the synovium was greater in the former than in the latter. Multinucleated cells obtained from RA synovium expressed the osteoclast-specific phenotype such as tartrate-resistant acid phosphatase, carbonic anhydrase II, vacuolar proton-ATPase and vitronectin receptors at similar levels to those from a human giant cell tumor of bone. The concentration of both IL-6 and sIL-6R was significantly higher in the synovial fluids from patients with RA than with OA. The concentration of IL-6 and sIL-6R correlated well with the roentgenologic grades of joint destruction. Dose-response curves for human IL-6 and human sIL-6R in inducing osteoclast-like cell formation in cocultures indicated that the RA synovial fluids contained sufficient IL-6 and sIL-6R to induce osteoclastogenesis. When synovial fluids from RA and OA patients were added to the cocultures, some of the RA synovial fluids containing high levels of IL-6 and sIL-6R stimulated osteoclast-like cell formation, which was strikingly inhibited by adding anti-IL-6R antibody simultaneously. These results suggest that IL-6 in the RA synovial fluids is at least in part responsible for joint destruction in the presence of sIL-6R through osteoclastogenesis.

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.

We have reported that 1-alpha,25-dihydroxyvitamin D3 [1-alpha,25-(OH)2D3] directly induces fusion of mouse alveolar macrophages at a very high rate (circa 70-80%) by a mechanism involving protein synthesis (Proc. Natl. Acad. Sci. USA 80:5583, 1983; FEBS Letters 174:61, 1984). While examining further the mechanism of the 1-alpha,25-(OH)2D3-induced fusion of macrophages, we found that polyamines are involved in this mechanism. Mouse alveolar macrophages incubated with 12 nM 1-alpha,25-(OH)2D3 began to fuse at 36 h and the fusion rate increased linearly up to 60 h. Addition of as much as 0.05-5 mM alpha-difluoromethylornithine (alpha-DFMO), a specific inhibitor of ornithine decarboxylase, did not inhibit fusion appreciably, but addition of 0.05-5 microM methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, strikingly inhibited fusion. When macrophages were treated with both 12 nM 1-alpha,25-(OH)2D3 and 5 microM MGBG for the first 12 h and incubated further for 60 h in fresh medium containing 1-alpha,25-(OH)2D3, fusion was significantly inhibited, suggesting that the 1-alpha,25-(OH)2D3-induced synthesis of polyamines precedes fusion. The inhibition by MGBG of the 1-alpha,25-(OH)2D3-induced fusion was restored completely by adding 1 microM spermidine or spermine or 100 microM putrescine. None of the polyamines alone induced fusion. MGBG suppressed the 1-alpha,25-(OH)2D3-induced incorporation of [3H]-leucine into the trichloroacetic acid-insoluble fraction in macrophages, but its inhibitory effect was restored completely by adding 1 microM spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously reported a procedure to obtain a preparation containing a large number of mouse osteoclast (OCL)-like multinucleated cells (MNCs) formed in cocultures of mouse osteoblastic and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3]. The MNCs satisfied major criteria of OCLs, such as tartrate-resistant acid phosphatase (TRAP) activity, acid production, calcitonin (CT) receptors, and the ability to form resorption pits on bone slices. In this report, we describe a simple resorption assay system using MNC preparations. After culturing MNC preparations or disaggregated rat OCL preparations on dentin slices, they were stained with Mayer's hematoxylin. The stained area corresponded exactly with the resorption pits visualized by scanning electron microscopy and were measured using an image analysis system attached to a light microscope. Pit formation by MNCs was gradually enhanced by reducing the medium pH (pH 7.5 < 7.2 < 6.9). The plan area resorbed by MNCs increased linearly for up to 72 h. These results are very similar to those obtained with OCL preparations. In multiple standard assays with MNC preparations, more than 250 MNCs could be placed on a dentin slice, and the total area resorbed to a level of up to 9% of the whole surface within 48 h. In contrast, in multiple assays with OCL preparations, it was not easy to place more than 50 OCLs on a slice and the resorbed area was only 0.7% of the surface. The resorbing activity of the MNC preparation expressed by the resorbed area per a TRAP-positive MNC (4.4 ± 0.4 × 10−3 μm2) was greater than that of the OCL preparation (1.9 ± 0.4 × 10−3 μm2). Pit formation by MNCs was dose dependently inhibited by salmon and human CT (ED50: salmon CT, 10−14 M; human CT, 3 × 10−13 M). Bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase, also inhibited pit formation by MNCs at concentrations of 10−9 M and above. In contrast, estrogen at 10−12–10−6 M had no significant inhibitory effect on pit formation in this assay system. The assay system presented here is sensitive and reproducible and will be useful for examining the effects of various natural factors and synthetic compounds on osteoclastic bone resorption. We have previously established a culture system for examining MNC formation. Therefore, it is now possible to examine the effects of various test compounds on the recruitment of OCLs and pit formation by the same OCLs in a series of experiments.

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures (Ref. 12). In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor β (TGF-β). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.

1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] directly induces both fusion and cytotoxicity in murine alveolar macrophages. Unlike 1 alpha,25(OH)2D3, retinoic acid per se did not induce fusion of alveolar macrophages, but it greatly enhanced the 1 alpha,25(OH)2D3-induced fusion every time the macrophages were treated simultaneously with the two vitamins. The giant cells induced by the two vitamins were much larger than those induced by 1 alpha,25(OH)2D3 alone. The macrophages treated with 1 alpha,25(OH)2D3 started to fuse 36 h after the addition of the vitamin, whereas the macrophages pretreated with retinoic acid for 24 h began to fuse immediately after 1 alpha,25(OH)2D3 was added. 1 alpha,25(OH)2D3 and retinoic acid activated alveolar macrophages similarly, measured by the enhancement of glucose consumption and the induction of cytotoxicity against tumor cells, though 1 alpha,25(OH)2D3 was 100 times more potent than retinoic acid on a molar basis. Simultaneous treatment with physiological concentrations of 1 alpha,25(OH)2D3 (0.12 nM) and retinoic acid (10 nM) induced cytotoxicity additively. Morphological examinations revealed that the treated cells were enlarged and flattened with numerous filopodia. These results clearly indicate that both 1 alpha,25(OH)2D3 and retinoic acid similarly activate alveolar macrophages, and the activated state is prerequisite to the fusion of macrophages induced by 1 alpha,25(OH)2D3.

The effects of parathyroid hormone (1-34) (PTH (1-34) on osteoblast differentiation were investigated using primary osteoblast-like cells isolated from newborn mouse calvaria. The osteoblast-like cells cultured at low cell densities, in which the cells remained in a subconfluent state at the end of culture, were exposed for 7 days to PTH. This stimulated alkaline phosphatase (ALP) activity in a dose-dependent manner. In contrast, PTH dose-dependently inhibited both ALP activity and osteocalcin production in cells inoculated at high cell densities, in which they had reached a confluent state before the end of culture. The changes of ALP activity by PTH were accompanied with the expression of ALP messenger RNA. PTH induced no changes of the hydroxyproline content in the cell layer when the cells were exposed to the hormone at a subconfluent state, but reduced the content at a postconfluent state. The stimulation of ALP activity by PTH at a preconfluent state was retained even after the removal of PTH from the culture media. The opposite effect of PTH, observed between the preconfluent and the postconfluent state, was reproduced by adding dibutyryl cyclic adenosine monophosphate (cAMP) or forskolin, but not by adding phorbol myristate acetate. In a colony-forming unit fibroblastic (CFU-F) assay, using bone marrow cells isolated from tibiae of 10-week-old mice, PTH induced no changes in the total number of CFU-Fs, but increased the proportion of ALP-positive colonies. These results indicate that PTH exerts opposite effects on the phenotypic expression of osteoblasts, depending on their differentiation stages of osteoblasts. PTH may preferentially stimulate osteoblast differentiation in immature osteoblasts but inhibit it in more mature cells.

Effects of various colony-stimulating factors (CSFs) [interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), and granulocyte CSF (G-CSF)] on osteoclast-like cell formation were examined in two different culture systems: the one-step mouse marrow culture system and the two-step coculture system of mouse primary osteoblastic cells with the bone marrow cells collected from the colonies that formed in the methylcellulose in the presence of the CSFs. In the one-step mouse marrow cultures, none of the CSFs stimulated the formation of tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts)-positive multinucleated cells (MNCs). Furthermore, the CSFs other than G-CSF inhibited in a dose-dependent manner the TRAP-positive MNC formation induced by 1 alpha-25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. In contrast, when marrow cells were first cultured in semisolid methylcellulose in the presence of a CSF and the recovered marrow cells from the semisolid cultures were subsequently cocultured with primary osteoblastic cells in the presence of 1 alpha,25-(OH)2D3, numerous TRAP-positive MNCs were formed. [125I]salmon calcitonin specifically bound to TRAP-positive cells formed in this two-step culture system. Over 90% of the TRAP-positive mononuclear cells and MNCs accumulated [125I]calcitonin. M-CSF was the most potent in inducing TRAP-positive MNCs, followed by GM-CSF, IL-3, and G-CSF in that order. No TRAP-positive cells were formed in the absence of either osteoblastic cells or 1 alpha,25-(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

The multinucleation process of isolated alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] was examined using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). At the beginning of culture, most of the macrophages were spherical in shape. During incubation with 1.2 X 10(-8) M 1 alpha,25(OH)2D3, spreading macrophages appeared among the spherical macrophages, and they increased in number. Spreading macrophages extended many cytoplasmic processes toward adjacent macrophages, and interdigitations of these processes between those of neighboring cells were often seen. Two types of cell contact have been observed in the 1 alpha,25(OH)2D3-treated cells. In some, cytoplasmic processes were put into the cytoplasm of the adjacent cells, where clathrinlike structures were observed at the inner membrane of the concave portion. In others, spreading macrophages occasionally came in contact with adjacent cells by a peripheral rim of their cytoplasm with gap junctions. Cytoplasmic continuity was rarely observed at the boundaries between the closely associated cells. The two types of cell contact were also found, though not frequently, in the untreated cells. These results indicate that 1 alpha,25(OH)2D3 promotes multinucleation of alveolar macrophages through spreading forms with the formation of gap junctions and the coated membrane invagination.

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

Bone is a dynamic tissue, in which bone formation by osteoblasts and bone resorption by osteoclasts continue throughout life. In 1998, we molecularly cloned osteoclast differentiation factor (ODF), a long-thought factor responsible for osteoclast formation. This review article describes how Japanese scientists contributed to osteoclast biology before and after the discovery of ODF. This review article is based on the Louis V. Avioli Memorial Lecture of the American Society for Bone and Mineral Research (ASBMR) held in Honolulu in September, 2007.

Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation.

Interleukin (IL)-4 and IL-13 are closely related cytokines known to inhibit osteoclast formation by targeting osteoblasts to produce an inhibitor, osteoprotegerin (OPG), as well as by directly targeting osteoclast precursors. However, whether their inhibitory actions are the same remains unclear. The inhibitory effect of IL-4 was stronger than that of IL-13 in an osteoclast-differentiation culture system containing mouse osteoblasts and osteoclast precursors. Both cytokines induced OPG production by osteoblasts in similar time- and dose-dependent manners. However, IL-4 was stronger in direct inhibition that targeted osteoclast precursors. Furthermore, IL-4 induced phosphorylation of signal transducer and activator of transcription-6 (STAT6) at lower concentrations than those of IL-13 in osteoclast precursors. IL-4 but not IL-13 strongly inhibited the expression of nuclear factor of activated T-cells, cytoplasmic 1 (nuclear factor-ATc1), a key factor of osteoclast differentiation, by those precursors. Thus, the activities of IL-4 and IL-13 toward osteoclast precursors were shown to be different in regards to inhibition of osteoclast differentiation, whereas those toward osteoblasts for inducing OPG expression were equivalent.

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.

Objective Donepezil, an inhibitor of acetylcholinesterase (AChE) targeting the brain, is a common medication for Alzheimer's disease. Interestingly, a recent clinical study found that administration of this agent is associated with lower risk of hip fracture independently of falling, suggesting its direct effect on bone tissues as well. AChE has been reported to be involved in osteoblast function, but the role of AChE on osteoclastogenesis still remains unclear. We analyzed the effect of AChE and donepezil on osteoclastogenesis in vivo and in vitro. Methods Cell-based assays were conducted using osteoclasts generated in cultures of murine bone marrow macrophages (BMMs) with receptor activator of nuclear factor-kappa B ligand (RANKL). The effect of donepezil was also determined in vivo using a mouse model of RANKL-induced bone loss. Results Recombinant AChE in BMMs cultured with RANKL further promoted RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation. RANKL also upregulated AChE expression in BMMs. RNA interference-mediated knockdown of AChE significantly inhibited RANKL-induced osteoclast differentiation and suppressed gene expression specific for osteoclasts. AChE upregulated expression of RANK, the receptor of RANKL, in BMMs. Donepezil decreased cathepsin K expression in BMMs and the resorptive function of osteoclasts on dentine slices. Donepezil decreased RANK expression in BMMs, resulting in the inhibition of osteoclast differentiation with downregulation of c-Fos and upregulation of Id2. Moreover, administration of donepezil prevented RANKL-induced bone loss in vivo, which was associated with the inhibition of bone resorption by osteoclasts. Conclusions AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

Metabolism of 25-hydroxyvitamin D3 (25-OH-D3) was examined in chicks supplemented with vitamin D3. Kidney homogenates metabolized in vitro [3H]-25-OH-D3 to 3 new metabolites (peaks A, C and E) by way of 24,25-dihydroxyvitamin D3. The enzymes responsible for the synthesis of these metabolites appeared to be induced by 1 alpha,25-dihydroxyvitamin D3. Production of these metabolites was increased in parallel with the increase of the supplemented levels of vitamin D3, while recovery of the radioactivity in the chloroform phase was sharply decreased. The production of peak C was considered to be closely related to the transfer of the radioactive metabolites to the water-soluble phase. These results may indicate that 24-hydroxylation is a degradation step in the 25-OH-D3 metabolism.

Vitamin D3 is already found in the early evolution of life, but essentially as inactive products of the photochemical reaction of 7-dehydrocholesterol. The full vitamin D endocrine system characterized by the specific vitamin D transport protein (DBP) , specific vitamin D-metabolizing CYP P450 enzymes, active vitamin D metabolites, 1α,25 (OH) 2D3, specific vitamin D nuclear receptor (VDR), and fibroblast growth factor 23 (FGF23) became essential for maintaining calcium and bone homeostasis in terrestrial animals cope with the challenging of higher gravity and calcium-poor environment. The present review describes the story about the evolution of animals and vitamin D.

Myogenic differentiation is suppressed in vitro by unknown factors present in fetal bovine serum (FBS). We found that specific inhibitors of bone morphogenetic proteins (BMPs) stimulated myogenic differentiation even in the presence of 20% FBS, which in turn activated specific BMP signaling. Moreover, these specific BMP inhibitors blocked maturation of osteoblastic cells induced by FBS, indicating that BMP-like factor(s) in serum regulate both myogenic and osteoblastic differentiation. The factor identified had an apparent molecular weight (Mw) of over 100 kDa on a Superdex 200 column for molecular sieving HPLC, but an apparent Mw of 33 kDa on SDS–PAGE under non-reducing conditions. Analysis of a purified preparation from FBS (5 L) by liquid chromatography-tandem mass spectrometry revealed the presence of an amino acid sequence conserved between mature human and murine BMP-4. This is the first study to show that BMP-4 is present in FBS as a large complex.

In the clinical field, cell-based therapies are used to treat bone defects. Adipose tissues contain many osteoblast progenitors, among other cell types. We separated mouse adipose tissue-derived stromal cells (ATSCs) according to their cell adhesive properties. Cells in a fraction adherent to the culture dishes 0.5 h after inoculation (AF-0.5) had a potent ability to differentiate into both osteoblasts and adipocytes in vitro. Their differentiation pathways depended on the culture conditions. In these cells, the expression of marker genes for osteoblast differentiation was induced in osteogenic medium. Moreover, the AF-0.5 cells, which were induced to differentiate into osteoblasts in vitro, formed abundant bone tissues in vivo. These results suggest that the AF-0.5 cells have been enriched with bi-potential progenitor cells destined for either osteoblasts or adipocytes. This simple and efficient method for preparing osteoblast progenitor cells from ATSCs may be utilized for bone defect treatment clinically.

Vitamin D was discovered as an anti-rachitic agent, but even at present, there is no direct evidence to support the concept that vitamin D directly stimulates osteoblastic bone formation and mineralization. It appears to be paradoxical, but vitamin D functions in the process of osteoclastic bone resorption. Osteoclasts, the only cells responsible for bone resorption, develop from hematopoietic cells of the monocyte-macrophage lineage. In 1992, we hypothesized that a membrane-bound factor, designated as "osteoclast differentiation factor (ODF)", is expressed on the plasma membrane of osteoblasts/stromal cells in response to osteotropic factors including the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. Recently, four research groups including ours independently identified three key molecules (RANKL, RANK, and OPG) responsible for osteoclastogenesis. A long-sought-after ligand, ODF, was identical to RANKL. RANKL was a member of the membrane-associated TNF ligand family, which induced differentiation of spleen cells (osteoclast progenitors) into osteoclasts in the presence of M-CSF. RANK, a member of the TNF receptor family, was a signaling receptor essential for the RANKL-mediated osteoclastogenesis. OPG, a secreted member of the TNF receptor family, was a decoy receptor for RANKL. The discovery of RANKL, RANK and OPG opens a new era in the study of bone biology and the therapy of several metabolic bone diseases such as osteoporosis, rheumatoid arthritis, and periodontal diseases. (Communicated by Tadamitsu KISHIMOTO, M.J.A.)

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)2D3 was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 μg/kg of 1,25(OH)2D3 inhibited the PTH-induced RANKL mRNA expression, but 0.5 μg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)2D3 dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)2D3 suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)2D3 stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling. J. Cell. Biochem. 90: 267–277, 2003. © 2003 Wiley-Liss, Inc.

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.

Background: Bone morphogenetic protein-2 (BMP-2) stimulates osteoblast differentiation, but inhibits myogenic differentiation in C2C12 myoblasts. BMP-2 induces transcription of Id1, an inhibitor for myogenesis, within 1 h in the cells. To examine the molecular mechanism of the action of BMP-2, we analysed a BMP-2-responsive element (BRE) in the 5′ flanking region of the human Id1 gene. Results: A GC-rich region between −985 bp and −957 bp of the human Id1 gene was identified as a BRE. The BRE containing promoter activity was stimulated by BMP-2 or by constitutively active BMP receptors (BMPR-IA and BMPR-IB). The stimulation was blocked by co-transfecting with dominant negative BMPR-IA or Smad7. A unique DNA–protein complex was induced in response to BMP-2 on the BRE. The complex induced by BMP-2 contained Smad1 and Smad4, possibly as a complex of both Smads. BMP-2 failed to stimulate the expression of Id1 mRNA in Smad4-deficient cells. Over-expression of Smad4, but not Smad1, stimulated the Id1 reporter activity and the expression of endogenous Id1 mRNA in Smad4-deficient cells. Conclusion: Signalling of BMP-2 to stimulate the expression of Id1 would be transduced by BMPR-IA and mediated by Smad1 and Smad4, both of which form a complex on the 29 bp GC-rich element.

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3-E1 cells, but also up-regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1alpha,25(OH)2D3- or prostaglandin E2 (PGE2)-induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor kappaB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence-accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin-4 inhibits osteoclast differentiation in vitro and in vivo. Co-cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin-4, restored in vitro osteoclast differentiation. Administration of netrin-4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin-4.

Vitamin D was discovered as an anti-rachitic agent preventing a failure in bone mineralization, but it is now established that the active form of vitamin D3 (1α,25(OH)2D3) induces bone resorption. Discovery of the receptor activator of nuclear factor -κB ligand (RANKL) uncovered the molecular mechanism by which 1α,25(OH)2D3 stimulates bone resorption. Treating osteoblastic cells with 1α,25(OH)2D3 stimulates RANKL expression, which in turn induces osteoclastogenesis. Nevertheless, active vitamin D compounds such as calcitriol (1α,25(OH)2D3), alfacalcidol (1α(OH)D3) and eldecalcitol (1α,25-dihydroxy-2β-(3-hydroxypropoxy) vitamin D3) have been used as therapeutic drugs for osteoporosis, as they increase bone mineral density (BMD) in osteoporotic patients. Paradoxically, the increase in BMD is caused by the suppression of bone resorption. Several studies have been performed to elucidate the mechanism by which active vitamin D compounds suppress bone resorption in vivo. Our study showed that daily administration of eldecalcitol to mice suppressed neither the number of osteoclast precursors in the bone marrow nor the number of osteoclasts formed in ex vivo cultures. Eldecalcitol administration suppressed RANKL expression in osteoblasts. This review discusses how the difference between in vitro and in vivo effects of active vitamin D compounds on bone resorption is induced.

Vitamin D3 is already found early in the evolution of life but essentially as inactive end products of the photochemical reaction of 7-dehydrocholestol with ultraviolet light B. A full vitamin D (refers to vitamin D2 and D3) endocrine system, characterized by a specific VDR (vitamin D receptor, member of the nuclear receptor family), specific vitamin D metabolizing CYP450 enzymes regulated by calciotropic hormones and a dedicated plasma transport-protein is only found in vertebrates. In the earliest vertebrates (lamprey), vitamin D metabolism and VDR may well have originated from a duplication of a common PRX/VDR ancestor gene as part of a xenobiotic detoxification pathway. The vitamin D endocrine system, however, subsequently became an important regulator of calcium supply for an extensive calcified skeleton. Vitamin D is essential for normal calcium and bone homeostasis as shown by rickets in vitamin D-deficient growing amphibians, reptiles, birds and mammals. From amphibians onward, bone is gradually more dynamic with regulated bone resorption, mainly by combined action of PTH and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the generation and function of multinucleated osteoclasts. Therefore, bone functions as a large internal calcium reservoir, under the control of osteoclasts. Osteocytes also display a remarkable spectrum of activities, including mechanical sensing and regulating mineral homeostasis, but also have an important role in global nutritional and energy homeostasis. Mineralization from reptiles onward is under the control of well-regulated SIBLING proteins and associated enzymes, nearly all under the control of 1,25(OH)2D3. The vitamin D story thus started as inert molecule but gained an essential role for calcium and bone homeostasis in terrestrial animals to cope with the challenge of higher gravity and calcium-poor environment.

Osteocalcin is a noncollagenous protein that is abundant in mineralized bone matrix. Mice have a gene cluster of osteocalcin that consists of OG1, OG2, and ORG. We established a new method to directly analyze the expression levels of OG1, OG2, and ORG mRNAs relative to total osteocalcin mRNA. They were amplified as 371-bp fragments by reverse transcription-polymerase chain reaction (RT-PCR) at the same time using common primers, digested with ApaLI, and separated in a polyacrylamide gel. ApaLI digestion did not affect the mobility of the OG1-derived 371-bp fragment, whereas both 371-bp fragments, derived from OG2 and ORG, were digested into 350 bp. Total RNA prepared from mouse bone was then subjected to RT-PCR followed by ApaLI digestion. OG1 and OG2 mRNAs were found to be expressed at ratios of 80%-86% and 14%-20%, respectively, to the total osteocalcin mRNA in mouse bone. The ratios were almost constant in various bones in vivo, independent of the animal's genetic background, age, or gender, or different parts of bone. RT-PCR using specific primers revealed that mouse bone tissues strongly expressed osteocalcin mRNA derived from OG1 and OG2, but not ORG. In contrast, cells cultured in vitro showed different expression ratios of osteocalcin mRNA: 53%-65% for OG1 and 35%-47% for OG2 to the total osteocalcin mRNA in the osteoblast cell line and primary osteoblasts in culture even though they formed many mineralized bone nodules. Similar results were obtained in both KS483 osteoblasts and C2C12 myoblasts, when they were cultured with bone morphogenetic protein-2 (BMP-2) to induce osteocalcin mRNA. Taken together, these findings indicate that OG1 is the predominant transcript among the three osteocalcin genes in mouse bone in vivo. It is also suggested that the expression of OG1 and OG2 is regulated differently in bone tissues and osteoblast cultures.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

Objective To elucidate the direct role of human T cells in the induction of osteoclastogenesis in rheumatoid arthritis (RA), by studying human monocytes and the pathogenetic roles of receptor activator of nuclear factor κB ligand (RANKL), RANK, and osteoprotegerin (OPG). Methods Synovial tissue obtained at total knee replacement was stained immunohistologically using anti-RANKL, CD3, and CD4 antibodies. Synovial fluid was obtained from patients with RA, osteoarthritis (OA), gout, or trauma. Concentrations of the soluble form of RANKL (sRANKL) and OPG in the synovial fluid were measured by enzyme-linked immunosorbent assay. Activated T cells from peripheral blood mononuclear cells (PBMC) of healthy volunteers were cultured with human monocytes from PBMC. Results Immunostaining of the synovial tissue of RA patients demonstrated that RANKL-positive cells were detected in a subset of fibroblast-like synoviocytes and infiltrating mononuclear cells. Double immunostaining revealed that RANKL-positive cells were detected in a subset of CD3+ cells and CD4+ cells. An increased concentration of sRANKL and a decreased concentration of OPG were detected in synovial fluid from RA patients. The ratio of the concentration of sRANKL to that of OPG was significantly higher in synovial fluid of RA patients than in synovial fluid of patients with OA or gout. The activated T cells expressing RANKL induced osteoclastogenesis from autologous peripheral monocytes. The role of RANKL in this osteoclastogenetic process was confirmed by dose-dependent inhibition by OPG. Conclusion The present study is the first to demonstrate osteoclastogenesis using human-derived T cells and monocytes. In addition, the present findings suggest that excess production of RANKL by activated T cells increases the level of sRANKL in synovial fluid and may contribute to osteoclastic bone resorption in RA patients.

Recently, three new family members of the tumor necrosis factor (TNF) ligand and receptor signaling system that play a critical role in the regulation of bone resorption have been identified and cloned. These also have been shown to play an important role in regulating the immune system. A proliferation of synonyms for these molecules has led to miscommunication and redundancy. To resolve this, the President of the American Society for Bone and Mineral Research (ASBMR) appointed a special committee to recommend a standard nomenclature. After considerable deliberation and after vetting by workers in the field, the Committee recommends the names of receptor activator of NF-κB (RANKL) for the membrane receptor, RANK ligand (RANK) for the ligand, and osteoprotegerin (OPG) for the decoy receptor.

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.

Osteoblasts arise from common progenitors with chondrocytes, muscle and adipocytes, and various hormones and local factors regulate their differentiation. We review here regulation of osteoblast differentiation mediated by the local factors such as bone morphogenetic proteins (BMPs) and hedgehogs and the transcription factor, core-binding factor α-1 (Cbfa1). BMPs are the most potent regulators of osteoblast differentiation among the local factors. Sonic and Indian hedgehogs are involved in osteoblast differentiation by interacting with BMPs. Cbfa1, a member of the runt domain gene family, plays a major role in the processes of a determination of osteoblast cell lineage and maturation of osteoblasts. Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation, because Cbfa1-deficient mice completely lacked bone formation due to maturation arrest of osteoblasts. Although the regulatory mechanism of Cbfa1 expression has not been fully clarified, BMPs are an important local factor that up-regulates Cbfa1 expression. Thus, the intimate interaction between local factors such as BMPs and hedgehogs and the transcription factor, Cbfa1, is important to osteoblast differentiation and bone formation.

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.