Tumor necrosis factor-alpha induces differentiation of human peripheral blood mononuclear cells into osteoclasts through the induction of p21(WAF1/Cip1)

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that mediates inflammation and induces bone loss caused by excessive bone resorption by osteoclasts. The interaction of TNF-alpha with its receptor activates several signal transduction pathways, including those of mitogen-activated protein (MAP) kinases (p38, JNK, and ERK) and NF-kappaB. Signaling from these molecules has been shown to play an important role in osteoclastogenesis. In the present study, we investigated the mechanism of TNF-alpha-induced osteoclast differentiation in human peripheral blood mononuclear cells (PBMCs). We found that TNF-alpha alone greatly induced differentiation of PBMCs into osteoclasts. The osteoclast differentiation induced by TNF-alpha was independent of RANKL binding to its receptor RANK on PBMCs. Furthermore, TNF-alpha potently activated p38 MAPK, JNK, and NF-kappaB. Western blotting analysis revealed that p21(WAF1/Cip1), a cyclin-dependent kinase (CDK) inhibitor, is significantly induced upon TNF-alpha stimulation. The induction of p21(WAF1/Cip1) during differentiation is responsible for arrest at G(0)/G(1) phase and associated with the JNK pathway. These results suggest that TNF-alpha regulates osteoclast differentiation through p21(WAF1/Cip1) expression and further shows that these events require JNK activity.     

Breast Cancer-derived Factors Stimulate Osteoclastogenesis through the Ca2+/Protein Kinase C and Transforming Growth Factor-��/MAPK Signaling Pathways

Breast cancer commonly metastasizes to bone where its growth depends on the action of bone-resorbing osteoclasts. We have previously shown that breast cancer cells secrete factors able to directly stimulate osteoclastogenesis from receptor activator of nuclear factor κB ligand (RANKL)-primed precursors and that transforming growth factor-β (TGFβ) plays a permissive role in this process. Now, we evaluate the signaling events triggered in osteoclast precursors by soluble factors produced by MDA-MB-231 human breast carcinoma cells. In mouse bone marrow cultures and RAW 264.7 murine monocytic cells, MDA-MB-231-derived factors increased osteoclast number, size, and nucleation. These factors failed to induce Smad2 phosphorylation, and short interfering RNAs against Smad4 did not affect their ability to induce osteoclastogenesis. In contrast, MDA-MB-231 factors induced phosphorylation of p38 and ERK1/2, and pharmacological inhibitors against p38 (SB203580) and MEK1/2 (PD98059) impeded the osteoclastogenic effects of cancer-derived factors. Neutralizing antibodies against TGFβ attenuated p38 activation, whereas activation of ERK1/2 was shortened in duration, but not decreased in amplitude. ERK1/2 phosphorylation induced by cancer-derived factors was blocked by MEK1/2 inhibitor, but not by Ras (manumycin A) or Raf (GW5074) inhibitors. Inhibition of protein kinase Cα using Gö6976 prevented both ERK1/2 phosphorylation and osteoclast formation in response to MDA-MB-231-derived factors. Using microspectrofluorimetry of fura-2-AM-loaded osteoclast precursors, we have found that cancer-derived factors, similar to RANKL, induced sustained oscillations in cytosolic free calcium. The calcium chelator BAPTA prevented calcium elevations and osteoclast formation in response to MDA-MB-231-derived factors. Thus, we have shown that breast cancer-derived factors induce osteoclastogenesis through the activation of calcium/protein kinase Cα and TGFβ-dependent ERK1/2 and p38 signaling pathways.     

Marrow stromal cells and osteoclast precursors differentially contribute to TNF-alpha-induced osteoclastogenesis in vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-kappaB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-alpha-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-alpha. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-alpha treatment or whether the recipient animals possess TNF-alpha-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-alpha requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-alpha increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-alpha is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-alpha depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-alpha may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.     

Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. In the present study, 3-isobutyl-1-methylxanthine (IBMX) and pentoxifylline, PDE inhibitors, induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. These inhibitors induced the expression of the osteoclast differentiation factor, TNF-related activation induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL), in calvarial osteoblasts. IBMX induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in osteoblasts. Induction of TRANCE expression by IBMX was partially suppressed by the inhibitors of protein kinase A (PKA), ERK, and p38 MAPK, suggesting that activation of ERK and p38 MAPK, as well as PKA, is involved in TRANCE expression by IBMX. Osteoblasts expressed PDE4, a PDE subtype, and rolipram, a selective inhibitor of PDE4, induced TRANCE expression. These results suggest that PDE4 is a key regulator of TRANCE expression in osteoblasts, which in turn controls osteoclast formation.     

Inflammatory cytokines activate p38 MAPK to induce osteoprotegerin synthesis by MG-63 cells

Inflammatory bone diseases are characterized by the presence of pro-inflammatory cytokines that regulate bone turnover. Osteoprotegerin (OPG) is a soluble osteoblast-derived protein that influences bone resorption by inhibiting osteoclast differentiation and activation. In the present study, we demonstrate that interleukin-1beta and tumor necrosis factor alpha induce OPG mRNA production and OPG secretion by osteoblast-like MG-63 cells. Maximum induction of OPG secretion by either cytokine requires activation of the p38 mitogen activated protein kinase (MAPK) pathway but neither the p42/p44 (ERK) nor the c-Jun N-terminal MAPK pathways. Induction of OPG mRNA by either cytokine is also p38 MAPK dependent. Taken together, these data indicate that cytokine-induced OPG gene expression and protein secretion are differentially regulated by specific MAP kinase signal transduction pathways.     

A novel PPARγ agonist,KR62776, suppresses RANKL-induced osteoclast differentiation and activity by inhibiting MAP kinase pathways

We investigated the effects of a novel peroxisome proliferator-activated receptor γ (PPARγ) agonist, KR62776, on osteoclast differentiation and function, and on the underlying signaling pathways. KR62776 markedly suppressed differentiation into osteoclasts in various osteoclast model systems, including bone marrow mononuclear (BMM) cells and a co-culture of calvarial osteoblasts and BMM cells. KR62776 suppressed the activation of tartrate-resistant acid phosphatase (TRAP) and the expression of genes associated with osteoclast differentiation, such as TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated receptor (OSCAR). Furthermore, KR62776 reduced resorption pit formation in osteoclasts, and down-regulated genes essential for osteoclast activity, such as Src and αvβ3 integrin. An analysis of a signaling pathway showed that KR62776 inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB). Together, these results demonstrate that KR62776 negatively affects osteoclast differentiation and activity by inhibiting the RANKL-induced activation of MAP kinases and NF-κB.     

Mechanism of stimulation of osteoclastic bone resorption through Gas6/Tyro 3, a receptor tyrosine kinase signaling, in mouse osteoclasts

The signaling through receptor tyrosine kinases expressed on mature osteoclasts has recently been suggested to be involved in osteoclastic bone resorption. This study investigated the mechanism and the possible physiological relevance of Gas6/Tyro 3, a receptor tyrosine kinase signaling pathway in osteoclasts in stimulating osteoclastic bone resorption using several mouse culture systems. Gas6, expressed ubiquitously in bone cells, did not affect the differentiation or the survival of osteoclasts, but stimulated osteoclast function to form resorbed pits on a dentine slice. The expression of its receptor, Tyro 3, was seen only in mature osteoclasts among bone cells. Gas6 up-regulated the phosphorylation of cellular proteins including p42/p44 mitogen-activated protein kinase (MAPK), but not p38 or c-Jun N-terminal kinase MAPK, and increased the kinase activity of immunoprecipitated Tyro 3 in isolated osteoclasts. The ability of Gas6 to stimulate pit formation resorbed by osteoclasts was abrogated by PD98059, a specific inhibitor of p42/p44 MAPK. In addition, the Gas6 mRNA level in bone marrow was up-regulated by ovariectomy and was reduced by estrogen replacement. These results strongly suggest that Gas6 acts directly on mature osteoclasts through activation of Tyro 3 and p42/p44 MAPK, possibly contributing to the bone loss by estrogen deficiency.     

Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.     

P38 mitogen-activated protein kinase inhibitor, FR167653, inhibits parathyroid hormone related protein-induced osteoclastogenesis and bone resorption

p38 mitogen-activated protein kinase (MAPK) acts downstream in the signaling pathway that includes receptor activator of NF-κB (RANK), a powerful inducer of osteoclast formation and activation. We investigated the role of p38 MAPK in parathyroid hormone related protein (PTHrP)-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo. The ability of FR167653 to inhibit osteoclast formation was evaluated by counting the number of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP-positive MNCs) in in vitro osteoclastgenesis assays. Its mechanisms were evaluated by detecting the expression level of c-Fos and nuclear factor of activated T cells c1 (NFATc1) in bone marrow macrophages (BMMs) stimulated with sRANKL and M-CSF, and by detecting the expression level of osteoprotegerin (OPG) and RANKL in bone marrow stromal cells stimulated with PTHrP in the presence of FR167653. The function of FR167653 on bone resorption was assessed by measuring the bone resorption area radiographically and by counting osteoclast number per unit bone tissue area in calvaria in a mouse model of bone resorption by injecting PTHrP subcutaneously onto calvaria. Whole blood ionized calcium levels were also recorded. FR167653 inhibited PTHrP-induced osteoclast formation and PTHrP-induced c-Fos and NFATc1 expression in bone marrow macrophages, but not the expression levels of RANKL and OPG in primary bone marrow stromal cells treated by PTHrP. Furthermore, bone resorption area and osteoclast number in vivo were significantly decreased by the treatment of FR167653. Systemic hypercalcemia was also partially inhibited. Inhibition of p38 MAPK by FR167653 blocks PTHrP-induced osteoclastogenesis in vitro and PTHrP-induced bone resorption in vivo, suggesting that the p38 MAPK signaling pathway plays a fundamental role in PTHrP-induced osteoclastic bone resorption.     

Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3 transgenic mice

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TL1A, induces osteoclast formation from monocyte, macrophage, and bone stromal marrow cells. However, the function of DcR3 on bone formation remains largely unknown. To understand the function of DcR3 in bone formation in vivo, transgenic mice overexpressing DcR3 were generated. Bone mineral density (BMD) and bone mineral content (BMC) of total body were significantly lower in DcR3 transgenic mice as compared with wild-type controls. The difference in BMD and BMC between DcR3 transgenic and control mice was confirmed by histomorphometric analysis, which showed a 35.7% decrease in trabecular bone volume in DcR3 transgenic mice in comparison with wild-type controls. The number of osteoclasts increased in DcR3 transgenic mice. In addition, local administration of DcR3 (30 microg/ml, 10 microl, once/day) into the metaphysis of the tibia via the implantation of a needle cannula significantly decreased the BMD, BMC, and bone volume of secondary spongiosa in tibia. Local injection of DcR3 also increased osteoclast numbers around trabecular bone in tibia. Furthermore, coadminstration of soluble tumor necrosis factor receptor inhibitor/Fc chimera (TNFRSF1A) but not osteoprotegerin inhibited the action of DcR3. In addition, in an assay of osteoclast activity on substrate plates, DcR3 significantly increased the resorption activity of mature osteoclasts. Treatment with higher concentrations of DcR3 slightly increased nodule formation and alkaline phosphatase activity of primary cultured osteoblasts. These results indicate that DcR3 may play an important role in osteoporosis or other bone diseases.     

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b⁺ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.     

Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis.     

Mitogen-activated protein kinases regulate Mycobacterium avium-induced tumor necrosis factor-alpha release from macrophages

Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines elicited by host macrophages upon challenge with pathogenic mycobacteria. Infection of human peripheral blood mononuclear cells or the murine macrophage cell line J774A-1 with Mycobacterium avium induced activation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and c-Jun N-terminal kinase. U0126, an MEK-specific inhibitor, abrogated M. avium-induced TNF-alpha secretion. Transfection of cells with dominant-negative MEK1 led to the suppression of TNF-alpha release in M. avium-challenged macrophages. M. avium activated p38 MAPK and use of the p38 MAPK inhibitor, SB203580, revealed that the p38 signaling pathway negatively regulates activation of ERK1/2 and release of TNF-alpha. Taken together, these results provide evidence that M. avium-induced TNF-alpha release from macrophages depends on an interplay between the ERK1/2 and the p38 MAPK signaling pathways.     

Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression

Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-beta (TGF-beta) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-beta in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-beta on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-beta was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-beta during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-beta was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-beta was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-beta, we examined RANK expression and p38 MAPK activation. We found the TGF-beta strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-beta to monocytes down-regulated RANK expression. The current results suggest that TGF-beta promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-beta abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.