Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice.

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.

[Current Topics on Vitamin D. Evolution of animals and vitamin D].

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.

The paired-box homeodomain transcription factor Pax6 binds to the upstream region of the TRAP gene promoter and suppresses receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-ß (IFN-ß), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Vitamin D3 Promotes Not Motor Coordination but Motor Skill Learning without Influence on Muscle Function.

Although motor coordination or motor skill learning are improved by taking vitamin D in the animal experiment, muscle function have not been estimated. Here we examined the effect of vitamin D3 administration on motor coordination and motor skill learning, muscle strength, and muscle volume in mice fed a vitamin D deficient diet. In mice fed a vitamin D deficient diet, serum calcium and 25(OH)D3 concentrations were measured. We then conducted Rotarod test, beam walking assay, micro-CT analysis, and forelimb grip strength test. Administration of vitamin D3 elongated the retention time in the Rotarod test in a time dependent manner. In contrast, the time to reach a beam goal box in beam walking assay was not changed in mice administered with vitamin D3, compared to the control. Oral administration of vitamin D3 did not affect muscle strength nor muscle volume. Oral administration of vitamin D3 promotes not motor coordination but motor skill learning and does not affect muscle function.

Bone effects of vitamin D - Discrepancies between in vivo and in vitro studies.

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. In 1952, Carlsson reported that administration of vitamin D(3) to rats fed a vitamin D-deficient, low calcium diet raised serum calcium levels. Since the diet did not contain appreciable amounts of calcium, the rise in serum calcium was considered to be derived from bone. Since then, this assay has been used as a standard bioassay for vitamin D compounds. Osteoclasts, the cells responsible for bone resorption, develop from hematopoietic cells of the monocyte-macrophage lineage. Several lines of evidence have shown that the active form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is one of the most potent inducers of receptor activator of NF-κB ligand (RANKL), a key molecule for osteoclastogenesis, in vitro. In fact, 1α,25(OH)(2)D(3) strongly induced osteoclast formation and bone resorption in vitro. Nevertheless, 1α,25(OH)(2)D(3) and its prodrug, Alfacalcidol (1α-hydroxyvitamin D(3)) have been used as therapeutic agents for osteoporosis since 1983, because they increase bone mineral density and reduce the incidence of bone fracture in vivo. Furthermore, a new vitamin D analog, Eldecalcitol [2ß-(3-hydroxypropoxy)-1α,25(OH)(2)D(3)], has been approved as a new drug for osteoporosis in Japan in January 2011. Interestingly, these beneficial effects of in vivo administration of vitamin D compounds are caused by the suppression of osteoclastic bone resorption. The present review article describes the mechanism of the discrepancy of vitamin D compounds in osteoclastic bone resorption between in vivo and in vitro.

Regulation of osteoclast function.

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.

[A history of the research on bone metabolism for the past 50 years in Japan and a memory of Dr. Hirotoshi Morii].

It has been 20 years since the monthly Journal "CLINICAL CALCIUM" was published in November, 1991. This review Journal edited by Drs. Hirotoshi Morii and Yoshio Yazaki has contributed a lot to the understanding of the importance of calcium in bone and cardiovascular systems. This article describes a history of the research on bone metabolism for the past 50 years in Japan. Also, a memory of my dear friend, Hirotoshi Morii, is retraced.

Generation of 1,25-dihydroxyvitamin D3 in Cyp27b1 knockout mice by treatment with 25-hydroxyvitamin D3 rescued their rachitic phenotypes.

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.

Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment.

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.

miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation.

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.

Contributions to osteoclast biology from Japan.

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.

Mechanisms involved in bone resorption regulated by vitamin D.

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).

Generation of osteoclasts in vitro, and assay of osteoclast activity.

Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte-macrophage lineage. The authors have developed a mouse marrow culture system and a coculture system of mouse osteoblasts and hemopoietic cells, in which osteoclasts are formed in response to various osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, prostaglandin E2, and interleukin-11. Recent studies have revealed that osteoblasts express two cytokines essential for osteoclastogenesis: receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Using RANKL and M-CSF, we can induce osteoclasts from monocyte-macrophage lineage cells even in the absence of osteoblasts. This chapter describes the methods for osteoclast formation in vitro in the presence and absence of osteoblasts, and for pit-formation assay using dentine slices and osteoclasts formed in vitro. These culture systems have made it possible to investigate each step of osteoclast development and function separately.

VDR in Osteoblast-Lineage Cells Primarily Mediates Vitamin D Treatment-Induced Increase in Bone Mass by Suppressing Bone Resorption.

Long-term treatment with active vitamin D [1α,25(OH)2 D3 ] and its derivatives is effective for increasing bone mass in patients with primary and secondary osteoporosis. Derivatives of 1α,25(OH)2 D3 , including eldecalcitol (ELD), exert their actions through the vitamin D receptor (VDR). ELD is more resistant to metabolic degradation than 1α,25(OH)2 D3 . 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.

Mechanisms for the reduction of 24,25-dihydroxyvitamin D3 levels and bone mass in 24-hydroxylase transgenic rats.

24-Hydroxylase (CYP24) is an enzyme distributed in the target tissues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Two functions for this enzyme have been reported: One is production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and the other is inactivation of 1alpha,25(OH)2D3. To elucidate other physiologic roles of CYP24 in vivo, we previously generated rats that constitutively express the CYP24 gene. These transgenic (Tg) rats developed unexpected phenotypes, such as low plasma levels of 24,25(OH)2D3, lipidemia, and albuminuria. In this study, we elucidated the mechanisms for inducing low plasma 24,25(OH)2D3 levels and bone loss. Tg rats excreted massive amounts of vitamin D binding protein (DBP), which coincided with the loss of albumin. In Tg rats, the renal expression pattern of megalin, which serves as an endocytotic receptor responsible for the reuptake of urinary proteins such as DBP and albumin, was identical to that of the wild-type rats. Excreted albumin appeared to compete for the binding and reabsorption of the DBP-25-hydroxyvitamin D3 [25(OH)D3] complex with megalin, resulting in a loss of 25(OH)D3 into the urine and subsequent reduction of plasma 24,25(OH)2D3. In this prominent rat model of nephritis, supplementation of 25(OH)D3 was effective in preventing bone loss in an early stage of renal insufficiency.

Physiological functions of vitamin D: what we have learned from global and conditional VDR knockout mouse studies.

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.

Donepezil prevents RANK-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase.

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.

Effects of geranylgeranoic acid in bone: induction of osteoblast differentiation and inhibition of osteoclast formation.

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.

p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function.

Receptor activator of nuclear factor-kappaB ligand (RANKL)-induced signals play critical roles in osteoclast differentiation and function. SB203580, an inhibitor of p38 MAPK, blocked osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2) in cocultures of mouse osteoblasts and bone marrow cells. Nevertheless, SB203580 showed no inhibitory effect on RANKL expression in osteoblasts treated with 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2). RANKL-induced osteoclastogenesis in bone marrow cultures was inhibited by SB203580, suggesting a direct effect of SB203580 on osteoclast precursors, but not on osteoblasts, in osteoclast differentiation. However, SB203580 inhibited neither the survival nor dentine-resorption activity of osteoclasts induced by RANKL. Lipopolysaccharide (LPS), IL-1, and TNFalpha all stimulated the survival of osteoclasts, which was not inhibited by SB203580. Phosphorylation of p38 MAPK was induced by RANKL, IL-1, TNFalpha, and LPS in osteoclast precursors but not in osteoclasts. LPS stimulated phosphorylation of MAPK kinase 3/6 and ATF2, upstream and downstream signals of p38 MAPK, respectively, in osteoclast precursors but not in osteoclasts. Nevertheless, LPS induced degradation of IkappaB and phosphorylation of ERK in osteoclasts as well as in osteoclast precursors. These results suggest that osteoclast function is induced through a mechanism independent of p38 MAPK-mediated signaling.