RANKL Expression In Osteoblasts Research Articles

Regulatory mechanism of osteoclastogenesis by Wnt signaling

Bone-resorbing osteoclasts develop from monocyte-macrophage lineage cells under the regulation of bone-forming osteoblasts. Osteoblasts express two cytokines essential for osteoclastogenesis, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Osteoblasts also produce osteoprotegerin (OPG), a decoy receptor for RANKL, which inhibits the interaction between RANKL and RANK, a receptor of RANKL. Wnt proteins (Wnts) play a central role in the development of organs and tissues. There are two pathways of Wnt signaling. β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. The discovery that loss-of-function mutations in low density lipoprotein receptor-related protein 5 (LRP5), a Wnt co-receptor, led to low bone mass in humans revealed the possible role of Wnt signaling in bone formation: Wnts act on osteoblast precursor cells and promote their differentiation into osteoblasts through the β-catenin-dependent canonical pathway. In addition, Wnts suppress bone resorption by the up-regulation of OPG expression and the down-regulation of RANKL expression in osteoblasts through the same pathway. In contrast, the activation of the β-catenin-independent noncanonical pathway enhances the RANKL-induced osteoclastogenesis. Recent studies have shown that the β-catenin independent noncanonical pathway is also involved in bone resorption induced by arthritis. This review summarizes the regulatory mechanism of bone resorption by Wnt signals.

OA/Gpnmb Mutation Impairs Osteoblast and Osteoclast Differentiation and Function

Osteoactivin/Gpnmb (OA/Gpnmb) is a glycoprotein identified in bone, and later found to be strongly localized to bone‐forming osteoblasts. There exists in mice a naturally occurring mutation for OA/Gpnmb, which is attributable to premature stop codon resulting in a truncated 150 amino acid OA/Gpnmb protein. Micro‐CT analysis of aged mice show decreased bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb. Th.) in OA/Gpnmb mutant mice when compared to wild‐type, implying that OA/Gpnmb mutant mice are more osteoporotic. Immunoflourescent analysis has shown OA/Gpnmb protein in mutant osteoblasts (OB) is retained in the peri‐nuclear/cis‐Golgi area. Electron microscopy analysis of osteoblasts showed no morphological differences in mutant osteoblasts versus control, suggesting OA/Gpnmb retention is not cytotoxic. qPCR mRNA and western blot analyses showed increased RANKL expression in OB from OA/Gpnmb mutant. Co‐culture of bone marrow cells showed marked increase in osteoclast (OC) numbers and size in mutant cultures when compared to WT. Treatment of WT and Mutant osteoblasts with recombinant OA/Gpnmb protein resulted in enhanced osteoclast number and size in vitro in WT and but not in mutant OB, suggesting an inherent defect to in mutant compared to WT OC. Collectively, these data suggest OA/Gpnmb acts as a regulator of osteoblast‐osteoclast differentiation and function.Grant Funding Source: NIAMS

Trichostatin A inhibits osteoclastogenesis and bone resorption by suppressing the induction of c-Fos by RANKL

Histone deacetylases are enzymes involved in the remodeling of chromatin structure, in the regulation of transcriptional activity, and in epigenetic integrity. Histone deacetylase inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) have emerged as potent anticancer drugs that have proved useful in preclinical and early clinical trials. The role of histone deacetylase inhibitors in regulating osteoclast differentiation, however, is not well established. In this study, we analyzed the effects of TSA on osteoclast differentiation induced by the differentiation factor RANKL (receptor activator of NF-κB ligand). TSA strongly inhibited osteoclast formation in coculture of bone marrow cells and osteoblasts without reducing RANKL expression in osteoblasts. Furthermore, TSA suppressed RANKL-induced osteoclast formation from primary bone marrow-derived macrophages. TSA was only effective when present during the early stage of osteoclast differentiation. This effect was accompanied by a significant decrease in the RANKL-stimulated induction of c-Fos and NFATc1, which are key transcription factors during early osteoclastogenesis. The ectopic introduction of c-Fos and a constitutively active form of NFATc1 reversed the TSA-induced antiosteoclastogenic effect. Consistent with the in vitro results, TSA inhibited lipopolysaccharide- and interleukin-1-induced bone resorption and osteoclast formation in an in vivo model. Taken together, our findings suggest a novel action of TSA: inhibiting RANKL-induced osteoclast formation by suppressing the induction of the osteoclastogenic transcription factor c-Fos. Also, the inhibitory effect of TSA on bone destruction in vivo suggests that histone deacetylase inhibitors may be novel therapeutics for treating typical bone diseases.

Bisphosphonates in Patients with Glucocorticoids: Time for Implementation

Use of glucocorticoids (GC) may have devastating effects on bone quality1. From earlier histomorphometric data, we know that bone formation is inhibited by direct suppression of osteoblast function during the use of GC, while bone resorption is unchanged or even elevated, at least partly related to decreased intestinal calcium absorption, elevated urinary calcium excretion, and a tendency to secondary hyperparathyroidism2,3. However, exciting new data provide additional insights into the pathogenesis of Glucocorticoid Induced OsteoPorosis (GIOP)2,3: (1) upregulation of the Wnt inhibitor Dickkopf-1, leading to inhibition of bone formation; (2) increased apoptosis of osteoblasts and osteocytes; (3) differentiation of mesenchymal stem cells into the direction of adipocytes, instead of into osteoblasts; (4) elevated lifespan of osteoclasts, due to decreased apoptosis; and (5) upregulation of expression of RANKL in osteoblasts (and downregulation of osteoprotegerin). Nevertheless, it is important to realize that the (negative) direct and indirect effects of GC on bone are counteracted by the immunosuppressive effects of GC on the underlying disease2,3. Another complicating factor is that use of GC and the underlying disease may both be associated with muscular weakness and, subsequently, increased risk of falling. Use of GC is associated with bone loss, particularly in the early phase of treatment, when the dosage of GC is usually high and the underlying disease for which the GC are prescribed is active. In contrast, in the chronic phase, bone loss may be mild in GC-treated patients, related to the use of lower dosages of GC and to low or preferably absent disease activity. In addition, use of GC is also associated with elevated risk of fracture: In a retrospective cohort study with 244,235 GC-treated patients and the same number of age and sex matched controls, the fracture … Address correspondence to Prof. Lems. E-mail: wf.lems{at}vumc.nl

A single application of hydrogen sulphide induces a transient osteoclast differentiation with RANKL expression in the rat model

Objective Oral malodor is mainly attributed to volatile sulphur compounds (VSCs) such as hydrogen sulphide (H 2S), methyl mercaptan and dimethyl sulphide. VSC accelerate periodontal soft tissue destruction. However, there is little information about the potential role of H 2S in alveolar bone loss. The purpose of this animal study was to examine the effects of sodium hydrogen sulphide (NaHS), H 2S donor drug, on osteoclast differentiation in rat periodontal tissue. Design Twenty-four male Wistar rats (8 weeks old) were divided into four groups: a control group and three experimental groups, which were examined at 3 h, 1 day, and 3 days after topical application of 3 μl NaHS (l M in physiological saline) into the gingival sulcus of rat first molar. Expression of tumour necrosis factor (TNF)-α, RANKL, NF-κB and tartrate-resistant acid phosphatase (TRAP) was evaluated in the periodontal tissue. Results Three hours after NaHS application, TNF-α expression increased in the periodontal ligament. The numbers of RANKL-positive osteoblasts and TRAP-positive osteoclasts significantly increased progressively with time and reached a maximum level after 1 day. Significant up-regulation of RANKL and NF-κB mRNA was observed at 3 h after NaHS application. Conclusions H 2S application caused a transient increase of osteoclast differentiation with up-regulation of RANKL expression in osteoblasts. H 2S, which is primarily responsible for halitosis, may also contribute to alveolar bone resorption through RANKL expression.

Role of Prostaglandin E in Receptor Activator of Nuclear Factor-.KAPPA.B Ligand (RANKL) Expression in Osteoblasts Induced by Cell Adhesion to Bone Marrow B-lymphocytes

Estrogen deficiency caused by ovariectomy (OVX) induces bone loss and increased B-lymphopoiesis in bone marrow. In OVX mice, the production of prostaglandin E (PGE) and the expression of receptor activator of nuclear factor-κB ligand (RANKL) were elevated in osteoblasts, and the cell adhesion of B cells induced RANKL expression in osteoblasts. However, the roles of PGE in RANKL expression and bone resorption are not clear. To examine the relationship between B-lymphopoiesis and PGE production by osteoblasts, B cells were purified from bone marrow, fixed, and co-cultured with mouse osteoblasts. Most of the fixed B cells adhered to cell surface of osteoblasts, and the cell-cell interaction markedly elevated the expression of cyclooxygenase (COX)-2 and membrane-bound PGE synthase (mPGES)-1 mRNAs, and PGE2 production in osteoblasts. Adding B cells also induced the expression of RANKL mRNA in osteoblasts, and the RANKL expression was suppressed by indomethacin, COX-2 inhibitor (NS398) and selective antagonist for PGE receptor EP4, suggesting that PGE production and EP4 signals are involved in RANKL-dependent bone resorption induced by cell-cell contact between B cells and osteoblasts. Therefore, the increased B-lymphopoiesis and PGE production by osteoblasts may contribute to the pathogenesis of osteoporosis due to estrogen deficiency.

Phosphodiesterase 3 and 4 Negatively Regulate Receptor Activator of Nuclear Factor-.KAPPA.B Ligand-Mediated Osteoclast Formation by Prostaglandin E2

Prostaglandin E2 (PGE2) stimulates osteoclast formation by increasing receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) mRNA expression via cAMP-protein kinase A (PKA) pathways in osteoblasts. Since phosphodiesterases (PDEs) balance the concentration of intracellular cAMP stimulated by PGE2, we investigated the role of PDEs in PGE2-mediated osteoclast formation using various cAMP-specific PDEs inhibitors. In the presence of PGE(2), PDE3 and 4 inhibitors were shown to dose-dependently increase the osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. In agreement with this finding, they stimulated PGE2-induced cAMP production followed by increased RANKL mRNA expression in osteoblasts, suggesting that PDE3 and 4 negatively regulate PGE2-mediated RANKL expression in osteoblasts. RT-PCR analysis revealed that PDE3A, 3B, 4A, 4B and 4D are expressed in osteoblasts. The PDE8 inhibitor did not increase osteoclast formation, although it stimulated PGE2-induced RANKL mRNA expression in osteoblasts. The four subtypes of PGE receptors are designated EP1, EP2, EP3, and EP4. PDE3 and 4 inhibitors were found to increase EP1/3, EP4 and/or EP2 agonist-stimulated RANKL expression, indicating that PDE3 and PDE4 negatively regulate PGE2-induced RANKL mRNA expression through four EPs. Taken together, these data suggest that PDE3 and PDE4 could have important pharmacological and clinical implications in bone-related diseases.

Treatment With Anti-γ-Glutamyl Transpeptidase Antibody Attenuates Osteolysis in Collagen-Induced Arthritis Mice

The effectiveness of a new antibody treatment on arthritis-associated osteolysis was studied by using CIA mice. GGT, a newly identified bone-resorbing factor, was upregulated in arthritic joints. We generated monoclonal antibodies against GGT and injected them into CIA mice. Mice treated with antibodies showed a reduction in osteoclast number and bone erosion. Gamma-glutamyl transpeptidase (GGT) acts as a bone-resorbing factor that stimulates osteoclast formation. GGT expression has been detected in active lymphocytes that accumulate at inflammation sites, such as rheumatoid arthritis (RA). We hypothesize that GGT is an effective target for suppression of arthritis-related osteoclastogenesis and joint destruction. Here, we describe the therapeutic effect of neutralizing antibodies against GGT on joint destruction using a collagen-induced arthritis (CIA) mouse model. GGT expression in the synovium of RA patients and CIA mice was determined by immunohistochemistry and RT-PCR. Monoclonal antibodies were generated against recombinant human GGT (GGT-mAbs) using BALB/c mice. Antibody treatment was performed by intraperitoneal injections of GGT-mAbs into CIA mice. Effects of antibody treatment on arthritis and bone erosion were evaluated by incidence score, arthritis score, and histopathological observations. The role of GGT in osteoclast development was examined by using the established osteoclastogenic culture system. GGT expression was significantly upregulated in inflamed synovium. Immunohistochemistry revealed that GGT was present in lymphocytes, plasma cells, and macrophages, as well as capillaries. Injection of GGT-mAbs significantly decreased the number of osteoclasts and attenuated the severity of joint destruction in CIA mice. In vitro examination showed that GGT enhanced RANKL-dependent osteoclast formation. GGT stimulated the expression of RANKL in osteoblasts and its receptor RANK in osteoclast precursors, respectively. This study indicates that inflamed synovial tissue-derived GGT acts as a risk factor for joint destruction and that the antibody-mediated inhibition of GGT significantly decreases osteoclast number and bone erosion in CIA mice. GGT antagonists might be novel therapeutic agents for attenuating joint destruction in RA patients.

Long chain polyunsaturated fatty acids alter membrane-bound RANK-L expression and osteoprotegerin secretion by MC3T3-E1 osteoblast-like cells

Inflammation triggers an increase in osteoclast (bone resorbing cell) number and activity. Osteoclastogenesis is largely controlled by a triad of proteins consisting of a receptor (RANK), a ligand (RANK-L) and a decoy receptor (osteoprotegerin, OPG). Whilst RANK is expressed by osteoclasts, RANK-L and OPG are expressed by osteoblasts. The long chain polyunsaturated fatty acid (LCPUFA) arachidonic acid (AA, 20:4n-6) and its metabolite prostaglandin E2 (PGE2), are pro-inflammatory and PGE2 is a potent stimulator of RANKL expression. Various LCPUFAs such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) have anti-inflammatory activity. We aimed to determine if AA itself can stimulate RANKL expression and whether EPA, DHA and GLA inhibit RANKL expression in osteoblasts. MC3T3-E1/4 osteoblast-like cells were cultured under standard conditions with each of the LCPUFAs (5 μg/ml) for 48 h. Membrane-bound RANKL expression was measured by flow cytometry and OPG secretion measured by ELISA. In a second experiment, RANKL expression in MC3T3-E1/4 cells was stimulated by PGE2 treatment and the effect of EPA, DHA and GLA on membrane-bound RANKL expression and OPG secretion determined. The percentage of RANKL-positive cells was higher ( p < 0.05) than controls following treatment with AA or GLA but not after co-treatment with the cyclooxygenase inhibitor, indomethacin. DHA and EPA had no effect on membrane-bound RANKL expression under standard cell culture conditions. Secretion of OPG was lower ( p < 0.05) in AA-treated cells but not significantly different from controls in GLA, EPA or DHA treated cells. Treatment with prostaglandin E2 (PGE2) resulted in an increase ( p < 0.05) in the percentage of RANK-L positive cells and a decrease ( p < 0.05) in mean OPG secretion. The percentage of RANKL positive cells was significantly lower following co-treatment with PGE2 and either DHA or EPA compared to treatment with PGE2 alone. Mean OPG secretion remained lower than controls in cells treated with PGE2 regardless of co-treatment with EPA or DHA. Results from this study suggest COX products of GLA and AA induce membrane-bound RANKL expression in MC3T3-E1/4 cells. EPA and DHA have no effect on membrane-bound RANKL expression in cells cultured under standard conditions however both EPA and DHA inhibit the PGE2-induced increase in RANKL expression in MC3T3-E1/4 cells.

Bacterial components and bone resorption

Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage under the control of bone-forming osteoblasts. Bone-resorbing factors such as 1α,25(OH)2D3 and IL-1 stimulated osteoclast formation in cocultures of mouse osteoblasts and hemopoietic cells through the induction of RANKL expression in osteoblasts. We examined the mechanism of action of LPS and MDP in osteoclastic bone resorption using the coculture system. LPS stimulated osteoclast formation through the enhancement of RANKL expression in osteoblasts. LPS also stimulated the survival of mature osteoclasts. Using MyD88-deficient mice, we showed that MyD88 was involved in both RANKL expression in osteoblasts and the survival of osteoclasts induced by LPS. On the other hand, MDP alone could not induce osteoclast formation in the coculture, but enhanced it induced LPS but not 1α,25(OH)2D3. LPS stimulated Nod2 expression in osteoblasts, but 1α,25(OH)2D3 did not. MDP enhanced RANKL expression induced by LPS in osteoblasts. The depletion of intracellular Nod2 by siRNA blocked the MDP-induced upregulation of RANKL mRNA in osteoblasts. These results suggest that bacterial components stimulate osteoclastic bone resorption through several signaling pathways including TLR/MyD88- and Nod-mediated ones.

Effects of muramyl dipeptide on osteoclast formation induced by LPS, IL-1 and TNF-α in mouse culture system

Muramyl dipeptide (MDP), the essential structure responsible for the immunoadjuvant activity of peptidoglycan, exists in Gram-positive and -negative bacterial walls. MDP synergistically enhances osteoclast formation induced by LPS, IL-1 and TNF-α through RANKL expression in osteoblasts. Nod2-mediated signals appear to be involved in the MDP-induced RANKL expression in osteoblasts.

Muramyl Dipeptide Enhances Osteoclast Formation Induced by Lipopolysaccharide, IL-1α, and TNF-α through Nucleotide-Binding Oligomerization Domain 2-Mediated Signaling in Osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and PGE2, LPS and IL-1alpha stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-kappaB ligand (RANKL) or TNF-alpha. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-alpha but not 1alpha,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-alpha in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-alpha-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1alpha, and TNF-alpha through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.

SC-19220, Antagonist of Prostaglandin E2 Receptor EP1, Inhibits Osteoclastogenesis by RANKL

We examined the direct effect of SC-19220, an EP1 prostaglandin (PG) E2 receptor antagonist, on osteoclastogenesis induced by RANK/RANKL signaling in mouse cell cultures. We found that SC-19220 inhibited RANKL-induced osteoclastogenesis by suppression of the RANK/RANKL signaling pathway in osteoclast precursors. Bone growth is accomplished by a dynamic equilibrium between formation by osteoblasts and resorption by osteoclasts, which are regulated by many systemic and local osteotropic factors that induce osteoclast formation from hematopoietic precursors through RANK/RANKL signaling. There are four subtypes of prostaglandin E (PGE) receptors, EP1, EP2, EP3, and EP4, and PGE2 facilitates bone resorption by a mechanism mediated by EP2/EP4. It is well known that SC-19220 is an EP1-specific antagonist. We previously found that SC-19220 inhibited osteoclastogenesis induced by osteotropic factors, including PGE2; however, the inhibitory mechanism is not clear. In this study, we investigated the inhibitory effects of SC-19220 on osteoclastogenesis induced by RANK/RANKL signaling in mouse cell cultures and analyzed the mechanism involved. A bone marrow culture system and bone marrow macrophages were used to examine the effects of SC-19220 on PGE2-, 11-deoxy-PGE1-, and RANKL-induced osteoclastogenesis. We analyzed RANKL expression in osteoblasts induced by PGE2 using RT-PCR. We also examined the effects of SC-19220 on the macrophage-colony-stimulating factor (M-CSF) receptor (c-Fms) and RANK expression in osteoclast precursors as well as RANK/RANKL signaling using RT-PCR and Western blotting analyses. SC-19220 dose-dependently inhibited osteoclast formation induced by PGE2, 11-deoxy-PGE1, and RANKL in the mouse culture system; however, it had no influence on RANKL expression in osteoblasts induced by PGE2. Furthermore, the expression of RANK and c-Fms in osteoclast precursors was decreased by SC-19220 at the mRNA and protein levels. In RANK signaling networks, SC-19220 inhibited c-Src and NFAT2 expression. Our findings indicated that SC-19220 inhibits RANKL-induced osteoclastogenesis through the suppression of RANK, c-Fms, c-Src, and NFAT2, suggesting that this EP1-specific antagonist inhibits osteoclast formation induced by RANKL from the early stage of osteoclastogenesis.

11 骨芽細胞でのRANKL発現におけるPKC-δの関与(第491回大阪歯科学会例会)

11 骨芽細胞でのRANKL発現におけるPKC-δの関与(第491回大阪歯科学会例会)

Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway.

Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-kappaB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-kappaB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production.

Involvement of PKC-δ in RANKL expression in osteoblasts

Involvement of PKC-δ in RANKL expression in osteoblasts

B-Lymphocytes are Elevated in Mouse Bone Marrow by Estrogen Deficiency, and Induce Receptor Activator of Nuclear Factor .KAPPA.B Ligand (RANKL) Expression in Osteoblasts via Cell Adhesion

Loss of estrogen caused by ovariectomy (OVX) stimulates bone resorption and bone marrow B-lymphopoiesis, resulting in a marked bone loss and an accumulation of B cells in mouse bone marrow. In OVX mice, the expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA was elevated in trabecular bone and bone marrow compared with sham mice. To examine the roles of B-lymphocytes in bone resorption, B cells were purified from bone marrow, fixed, and co-cultured with mouse osteoblasts. Most of the fixed B cells adhered to cell surface of osteoblasts. The expression of RANKL mRNA in osteoblasts was markedly elevated by the contact with the fixed B cells, and the induction rate of RANKL was correlated with the number of B cells added. Treatment with inhibitors of ERK 1/2 and p38 MAP kinases suppressed the B cell-induced RANKL expression in osteoblasts, suggesting the involvement of these kinases in the signals via the cell-to-cell contact. These findings emphasize the roles of B-lymphocytes in RANKL-induced osteoclastogenesis and in pathogenesis of bone loss due to estrogen deficiency.

P38 MAPK-Mediated Signals Are Required for Inducing Osteoclast Differentiation But Not for Osteoclast Function

Receptor activator of nuclear factor-κB ligand (RANKL)-induced signals play critical roles in osteoclast differentiation and function. SB203580, an inhibitor of p38 MAPK, blocked osteoclast formation induced by 1α,25-dihydroxyvitamin D3 and prostaglandin E2 in cocultures of mouse osteoblasts and bone marrow cells. Nevertheless, SB203580 showed no inhibitory effect on RANKL expression in osteoblasts treated with 1α,25-dihydroxyvitamin D3 and prostaglandin E2. 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 TNFα all stimulated the survival of osteoclasts, which was not inhibited by SB203580. Phosphorylation of p38 MAPK was induced by RANKL, IL-1, TNFα, 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 IκB 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.

Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts.

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.