Identification of the functional domain of osteoclast inhibitory peptide-1/hSca.

Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide-1/human Sca [OIP-1/hSca]). OIP-1/hSca is a glycosylphosphatidylinositol (GPI)-linked membrane protein (16 kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression of OIP-1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms responsible for the inhibitory effects of OIP-1/hSca on OCL formation are unknown. Therefore, we expressed deletion mutants of OIP-1 in 293 cells and tested their effects on OCL formation. These studies indicated that the carboxy-terminal peptide (c-peptide) region is critical for OIP-1/hSca activity. A 33 amino acid OIP-1 c-peptide (10-100 ng/ml) significantly inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced OCL formation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore, the c-peptide (10-100 ng/ml) significantly inhibited early human OCL precursor (granulocyte-macrophage colony-forming unit [GM-CFU]) colony formation in methylcellulose cultures. The polyclonal antibody against the OIP-1 c-peptide neutralized the inhibitory effect of OIP-1 c-peptide on OCL formation in mouse bone marrow cultures in vitro. These results show that the OIP-1 c-peptide is the functional domain of OIP-1 and that availability of neutralizing antibody specific to the OIP-1 c-peptide should provide important mechanistic insights into OIP-1/hSca inhibition of osteoclastogenesis in the bone microenvironment.     

Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment.

Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-kappaB (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors.     

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH)2D3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-kappaB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10(-10) M 1,25-(OH)2D3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH)2D3. ECF-L-Fc by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH)2D3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.     

c-myc is required for osteoclast differentiation.

The role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-a tumor necrosis factor (TNF)-related cytokine-in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.     

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.     

Stromal cell-derived factor-1 (SDF-1) recruits osteoclast precursors by inducing chemotaxis, matrix metalloproteinase-9 (MMP-9) activity, and collagen transmigration.

Signals targeting OCs to bone and resorption sites are not well characterized. A chemoattractant receptor (CXCR4), highly expressed in murine OC precursors, mediated their chemokine (SDF-1)-induced chemoattraction, collagen transmigration, and MMP-9 expression. Thus, bone vascular and stromal SDF-1 may direct OC precursors into bone and marrow sites for development and bone resorption. INTRODUCTION: Although chemokines are essential for trafficking and homing of circulating hematopoietic cells under normal and pathological conditions, their potential roles in osteoclast (OC) recruitment or function are generally unknown. CXCR4 and its unique ligand, stromal cell-derived factor-1 (SDF-1), critically control the matrix metalloproteinase (MMP)-dependent targeting of hematopoietic cells into bone and within the marrow microenvironment. Therefore, SDF-1/CXCR4 may regulate OC precursor recruitment to sites for development and activation. METHODS: Chemokine receptor mRNA expression was analyzed during OC formation induced by RANKL in murine RAW 264.7 cells. SDF-1 versus RANKL effects on chemotaxis, transcollagen migration, MMP-9 expression and activity, OC development, and bone resorption were evaluated in RAW cells or RAW-OCs. RESULTS: CXCR4 was highly expressed in RAW cells and downregulated during their RANKL development into bone-resorptive RAW-OCs. SDF-1, but not RANKL, elicited RAW cell chemotaxis. Conversely, RANKL, but not SDF-1, promoted RAW-OC development, TRAP activity, cathepsin K expression, and bone pit resorption, and SDF-1 did not modify these RANKL responses. Both SDF-1 and RANKL increased MMP-9, a matrix-degrading enzyme essential for OC precursor migration into developing bone marrow cavities, and increased transcollagen migration of RAW cells in a MMP-dependent manner. SDF-1 also upregulated MMP-9 in various primary murine OC precursor cells. Because RANKL induced a higher, more sustained expression of MMP-9 in RAW cells than did SDF-1, MMP-9 may have an additional role in mature OCs. Consistent with this, MMP-9 upregulation during RANKL-induced RAW-OC development was necessary for initiation of bone pit resorption. CONCLUSIONS: SDF-1, a chemokine highly expressed by bone vascular endothelial and marrow stromal cells, may be a key signal for the selective attraction of circulating OC precursors into bone and their migration within marrow to appropriate perivascular stromal sites for RANKL differentiation into resorptive OCs. Thus, SDF-1 and RANKL likely serve complementary physiological functions, partly mediated through increases in MMP-9, to coordinate stages of OC precursor recruitment, development, and function.     

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.     

Eosinophil chemotactic factor-L (ECF-L): a novel osteoclast stimulating factor.

Screening a cDNA library enriched for genes expressed in OCLs identified ECF-L. ECF-L enhanced OCL formation without increasing RANKL levels. Anti-ECF-L inhibited RANKL-induced OCL formation. These results support a potent role of ECF-L in osteoclastogenesis. INTRODUCTION: To investigate the molecular mechanisms that control osteoclastogenesis, we developed an immortalized osteoclast (OCL) precursor cell line that forms mature OCLs in the absence of stromal cells and used it to form pure populations of OCLs. MATERIALS AND METHODS: Polymerase chain reaction (PCR) selective cDNA subtraction was used to identify genes that are highly expressed in mature OCLs compared with OCL precursors employing OCL and OCL precursors derived from this cell line. RESULTS: Eosinophil chemotactic factor-L (ECF-L), a previously described chemotactic factor for eosinophils, was one of the genes identified. Conditioned media from 293 cells transfected with mECF-L cDNA, or purified ECF-L Fc protein, increased OCL formation in a dose-dependent manner in mouse bone marrow cultures treated with 10(-10) M 1,25(OH)2D3. OCLs derived from marrow cultures treated with ECF-L conditioned media formed increased pit numbers and resorption area per dentin slice compared with OCLs induced by 1,25(OH)2D3 (p < 0.01). Addition of an antisense S-oligonucleotide to mECF-L inhibited OCL formation in murine bone marrow cultures treated only with 10(-9) M 1,25(OH)2D3 compared with the sense S-oligonucleotide control. Time course studies demonstrated that ECF-L acted at the later stages of OCL formation, and chemotactic assays showed that mECF-L increased migration of OCL precursors. mECF-L mRNA was detectable in mononuclear and multinucleated cells by in situ hybridization. Interestingly, a neutralizing antibody to ECF-L blocked RANKL or 10(-9) M 1,25(OH)2D3-induced OCL formation in mouse bone marrow cultures, although ECF-L did not induce RANKL expression. CONCLUSIONS: These data show ECF-L is a previously unknown factor that is a potent mediator of OCL formation, which acts at the later stages of OCL formation and enhances the effects of RANKL.     

低分子量褐藻糖胶对破骨细胞凋亡的影响

目的探讨低分子量褐藻糖胶(LMWF)对小鼠单核细胞RAW264.7诱导成熟破骨细胞凋亡的影响。方法通过100ng/m L RANKL诱导RAW264.7细胞株分化为破骨细胞,经TRAP特异性染色和骨吸收陷窝对诱导后的细胞进行鉴定。鉴定成功后,用100 ng/m L RANKL诱导RAW264.7细胞株5 d后,使用含有LMWF的培养基继续培养3 d,通过对TRAP阳性细胞计数和分析骨吸收面积来观察低分子量褐藻糖胶对破骨细胞的抑制和骨吸收功能情况;采用流式细胞术检测LMWF对破骨细胞凋亡的影响,capsase-3活性测试试剂盒检测LMWF对capsase-3活性进行测定;RT-PCR检测LMWF对成熟破骨细胞BAX与BCL-2基因表达的影响。结果单纯采用100 ng/m L的RANKL可成功诱导成熟的、有功能的破骨细胞。LMWF可以明显抑制RANKL诱导成熟破骨细胞的形成以及成熟破骨细胞的骨吸收功能;流式细胞术显示LMWF可增加成熟破骨细胞的早期凋亡率;并且能升高capsase-3的活性;PCR显示LMWF可明显下调破骨细胞凋亡相关的BCL-2和上调BAX基因mRNA表达,降低BCL-2/BAX的比值。结论低分子量褐藻糖胶可抑制破骨细胞的活性与骨吸收能力,促进破骨细胞凋亡,其主要机制是通过下调BCL-2和上调BAX mRNA基因表达实现的。     

Annexin II stimulates RANKL expression through MAPK.

We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. INTRODUCTION: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. MATERIALS AND METHODS: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. RESULTS AND CONCLUSION: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.     

Prostate-specific antigen induces apoptosis of osteoclast precursors: potential role in osteoblastic bone metastases of prostate cancer

BACKGROUND: Prostate cancer is frequently associated with bone metastases with marked osteoblastic changes and low osteoclastic activity but its mechanism is not well understood. We previously reported that prostate-specific antigen (PSA) stimulated the proliferation and the activation of osteoblasts. In this study, we investigated the effect of PSA on osteoclastogenesis. METHODS: Two human prostate cancer cell lines and PSA were directly injected into human adult bone (HAB) implanted into NOD/SCID mice, followed by morphological analysis. RAW 264.7 cells, murine osteoclast precursor, were treated with PSA. RESULTS: PSA-producing LNCaP and PSA caused a significant decrease of osteoclast precursors and osteoclasts in HAB accompanied by osteoblast proliferation and new bone formation, while PSA-nonproducing PC3 showed increasing osteoclasts with osteolysis. PSA induced apoptosis of RAW 264.7 cells in vitro. PSA-induced apoptosis was dependent of enzymatic activity of PSA and was specific to immature tartrate-resistant acid phosphatase-negative mononuclear RAW 264.7 cells. CONCLUSIONS: PSA plays a crucial role for osteoblastic bone metastasis by promoting both osteoblasts proliferation and apoptosis of osteoclast precursors.     

Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation.

We determined that sFRP-1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP-1 neutralizing antibodies and siRNA complementary to sFRP-1 coding sequence enhanced, while recombinant sFRP-1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP-1, we found that sFRP-1 could bind recombinant RANKL. These results suggest potential cross-talk between Wnt and RANKL pathways. INTRODUCTION: Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage-colony-stimulating factor (M-CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled-related protein-1 (sFRP-1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast-derived factor that impinges on osteoclast formation and activity. MATERIALS AND METHODS: Differential display of mRNA from osteoblast lineage cell lines established sFRP-1 to be highly expressed in an osteoclast supporting cell line. sFRP-1 expression in bone was determined by in situ hybridization, and the effects of sFRP-1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP-1 and recombinant protein. RESULTS: In situ hybridization revealed sFRP-1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP-1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2 (PGE2) or interleukin (IL)-11, whereas many other osteotropic agents (e.g., IL-1, IL-6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP-1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP-1 enhanced TRACP+ mononuclear and multinuclear osteoclast formation (3- and 2-fold, respectively) in co-cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP-1 coding sequence significantly enhanced osteoclast formation in co-cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2 and 1,25(OH)2 vitamin D3. Recombinant sFRP-1 dose-dependently inhibited osteoclast formation in osteoblast/spleen co-cultures, RANKL + M-CSF-treated splenic cultures, and RANKL-treated RAW264.7 cell cultures, indicating a direct action of sFRP-1 on hematopoietic cells. Consistent with this, sFRP-1 was found to bind to RANKL in ELISAs. CONCLUSION: sFRP-1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP-1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP-1.     

The surface antigen CD45R identifies a population of estrogen-regulated murine marrow cells that contain osteoclast precursors

We examined the osteoclastogenic potential of murine bone marrow cells that were fractionated according to their expression of the surface antigen CD45R. Osteoclast-like cells (OCL) with many authentic osteoclast characteristics readily formed in purified CD45R⁺ murine bone marrow cell cultures after treatment with receptor activator of nuclear factor κB ligand (RANKL) and M-CSF. Ovariectomy (Ovx) caused a 1.5- to 2-fold increase in OCL number in unfractionated and CD45R⁺ murine bone marrow cell cultures without affecting OCL formation in CD45R⁻ marrow cells. Limiting dilution assays confirmed that Ovx caused an increase in osteoclast precursor cell number in CD45R⁺ but not CD45R⁻ cells. Mice deficient in the type 1 IL-1 receptor (IL-1R1 KO) do not lose bone mass after Ovx. We found that unfractionated, CD45R⁺, and CD45R⁻ bone marrow cells from IL-1R1 KO mice showed no increase in OCL formation in vitro after Ovx. In both the wild-type (WT) and the IL-1R1 KO mice Ovx was associated with a 2-fold increase in pre-B-lymphocytes. About 1.3–3.5% of murine marrow cells expressed surface RANK (the receptor for RANKL) while about 11.9–15% of murine bone marrow cells expressed c-Fms (the receptor for M-CSF). There was little effect of Ovx on cells expressing either RANK or c-Fms. These results demonstrate that CD45R expression identifies a subset of murine bone marrow cells whose ability to form OCL in vivo is regulated by estrogen in WT but not IL-1R1 KO cells. The effects of estrogen on bone mass may be related to these responses.     

CCR1 chemokines promote the chemotactic recruitment, RANKL development, and motility of osteoclasts and are induced by inflammatory cytokines in osteoblasts.

Chemoattractants that recruit OC precursors to locally inflamed sites of resorption are not well known. A chemokine receptor, CCR1, was expressed in OC precursors and elevated in mature OCs, and its ligands promoted OC precursor recruitment, RANKL development, and OC motility. Cytokines induced OB release of such chemokines, which may therefore significantly contribute to inflammatory bone loss. INTRODUCTION: Chemokines, primarily of two major (CXC, CC) families, are essential signals for the trafficking and localization of circulating hematopoietic cells into tissues. However, little is known about their potential roles in osteoclast (OC) recruitment, development, or function. Previously, we analyzed CXC receptors in murine OC precursors and found high expression of CXCR4 that mediated their stromal-derived factor-1(SDF-1)-induced chemotaxis and collagen invasion. Here, we investigated if CC receptors and ligands, which are elevated in inflammatory and other osteolytic diseases, also play important roles in the recruitment, formation, or activity of murine bone-resorptive OCs. MATERIALS AND METHODS: CC chemokine receptor (CCR) mRNA expression was analyzed during OC formation induced by RANKL in murine RAW 264.7 cells and primary marrow cells. Corresponding CC chemokines were tested for their ability to elicit precursor chemotaxis or OC development, or to influence motility, bone resorption, adhesion, or survival in RANKL-differentiated OCs. Constitutive and inflammatory cytokine-induced release of the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and regulated on activation, normal T-cell expressed and secreted (RANTES) was measured by ELISA for OCs, osteoblasts (OBs), and their precursor cells. RESULTS: CCR1 was expressed in murine marrow cells, the most prominent CCR in RAW cells, and upregulated by RANKL in marrow or RAW cells. Chemokines that bind CCR1 (MIP-1alpha, RANTES, and monocyte chemoattractant protein-3 [MCP-3]) were produced to varying degrees by murine OCs, OBs, and their precursors, and markedly increased by interleukin (IL)-1alpha and TNFalpha in differentiating OBs. RANTES, and especially MIP-1alpha, increased mature OC motility, but did not alter OC resorption activity, adhesion, or survival. All three chemokines stimulated chemotaxis of marrow or RAW cell precursors, leading to the greater formation of OCs (in number and size) after RANKL development of such chemoattracted marrow cells. All three chemokines also directly and dramatically enhanced OC formation in marrow cultures, through a pathway dependent on the presence of RANKL but without altering RANK expression. CONCLUSIONS: Pathological increases in secretion of these chemokines from activated OBs or other cells may potently stimulate the chemotactic recruitment and RANKL formation of bone-resorptive OCs, thereby exacerbating local osteolysis in multiple skeletal diseases.     

Inhibition of RANKL-induced osteoclast formation in mouse bone marrow cells by IL-12: involvement of IFN-gamma possibly induced from non-T cell population

IL-12 was shown to have the potential to inhibit osteoclast formation in mouse bone marrow cells treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). When bone marrow macrophages (BMM) were used as osteoclast precursors, IL-12 failed to inhibit M-CSF/RANKL-induced osteoclast formation from BMM. In coculture experiments using transwells, IL-12 did inhibit osteoclast formation from BMM cocultured with whole bone marrow cells. These results indicated that IL-12 indirectly affected M-CSF/RANKL-induced osteoclastogenesis in bone marrow cells and that the inhibition of IL-12 on osteoclast formation was caused by a humoral factor from bone marrow cells treated with IL-12. Experiments with anti-interferon (IFN)-gamma antibody and bone marrow cells from IFN-gamma receptor knockout mice revealed that IFN-gamma might be involved in the inhibition of osteoclast formation in this system. The expression of osteoprotegerin mRNA in bone marrow cells was not affected by treatment with IL-12. The inhibitory effect of IL-12 on osteoclast formation was also seen in the T cell-depleted bone marrow cells of normal mice and the whole bone marrow cells of athymic nude mice, while the inhibitory effect of IL-12 was partially suppressed in the B cell-depleted bone marrow cells. The inhibitory effect of IL-12 on M-CSF/RANKL-induced osteoclastogenesis was not accompanied with cell death, in contrast with our previous finding that the inhibitory effect of IL-12 on M-CSF/TNF-alpha-induced osteoclastogenesis is attributable to Fas and FasL-mediated apoptosis.     

ADAM8: a novel osteoclast stimulating factor.

We used polymerase chain reaction (PCR)-selective complementary DNA (cDNA) subtraction hybridization with an immortalized murine osteoclast (OCL) precursor cell line to identify genes that are highly expressed in OCLs compared with OCL precursors and which may be involved in the OCL differentiation process. ADAM8 was one of the 50 genes identified. ADAM (a disintegrin and metalloproteinase) peptides are membrane-bound proteins that can act as cell-to-cell and cell-to-matrix adhesion molecules, degrade the extracellular matrix, and play a role in tissue morphogenesis. Addition of antisense (AS) S-oligonucleotides for ADAM8 (1-10 nM) to mouse bone marrow cultures treated with 10(-9) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibited OCL formation compared with treatment with the control S-oligonucleotide. Furthermore, conditioned media from 293 cells transiently transfected with a secretable form of the ADAM8 cDNA increased OCL formation in a dose-dependent manner. In addition, treatment of OCLs with soluble ADAM8 conditioned media significantly increased pit formation per dentin slice compared with control OCLs. Time course studies indicated that ADAM8 increased OCL formation only when it was present during days 4-7 of the 7-day culture period. Structural analysis, using truncated constructs of ADAM8, showed that the cysteine-rich/disintegrin domain was responsible for its OCL stimulatory activity. Western blot analysis confirmed that the soluble form of ADAM8 is present in normal marrow cultures. These data suggest that ADAM8 plays an important role in OCL formation and acts primarily at the later stages of OCL differentiation.     

不同浓度地西他滨对破骨细胞分化的影响

目的：探讨不同浓度梯度地西他滨对破骨细胞形成、活性及吸收功能的影响。方法不同浓度地西他滨（0、0.1、0.25和0.5μmol/L）处理单核巨噬（RAW264.7）细胞。通过4’,6?联眯?2?苯基吲哚（4,6?Diamidino?2?phenylindole dihydrochloride, DAPI）染色和微丝绿色荧光探针（F?actin?Trakcer Green）染色后观察F?actin环的形成即破骨细胞轮廓;抗酒石酸酸性磷酸酶检测试剂盒检测细胞上清中的抗酒石酸酸性磷酸酶活性,骨板吸收实验检测破骨细胞的骨吸收能力;Q?PCR实验检测破骨细胞标志基因抗酒石酸酸性磷酸酶（tartrate?resistant acid phosphatase, TRAP）、组织蛋白酶K（cathepsin K, CK）和脊髓基质金属蛋白酶?9（matrix metalloproteinase?9, MMP?9）的mRNA表达。结果不同浓度地西他滨抑制核因子NF?κB配体激活因子（receptor activator of NF?κB ligand, RANKL）诱导RAW264.7细胞形成F?actin环,降低了破骨细胞的TRAP酶活性,抑制了破骨细胞的骨吸收能力,同时也下调了破骨细胞标志基因TRAP、CK和MMP?9的mRNA表达。且随着药物浓度的增高,上述的抑制作用越明显。结论地西他滨抑制破骨细胞形成、活性和骨吸收能力,且这种抑制作用随着药物浓度的增加而逐渐增强。     

RANKL-stimulated osteoclast-like cell formation in vitro is partially dependent on endogenous interleukin-1 production

Receptor activator of NF-kappaB ligand (RANKL) and interleukin-1 (IL-1) individually plays a critical role in the differentiation and activation of osteoclasts in bone. In addition, both RANKL and IL-1 activate similar signal transduction pathways including p38 MAP kinase and c-Jun NH(2) terminal kinase (JNK). We examined if endogenously produced IL-1 influenced osteoclast-like cell (OCL) formation in murine bone marrow and bone marrow monocyte (BMM) cultures that were stimulated with M-CSF and RANKL. RANKL stimulated OCL formation in a dose-dependent manner in bone marrow cultures, and this response was significantly inhibited by IL-1 RA (100 ng/ml), a specific IL-1 antagonist. Interleukin-1 further increased OCL formation in BMM cultures that were treated with M-CSF (30 ng/ml) and RANKL (1, 3, 10 and 30 ng/ml). In addition, BMM cultures from IL-1 type I receptor-deficient mice, which do not respond to IL-1, demonstrated significantly less OCL formation compared to wild-type BMM cultures. We examined the time course and dose response of IL-1alpha protein expression by ELISA in BMM cultures that were treated with or without M-CSF and RANKL. RANKL dose dependently stimulated IL-1alpha protein significantly (up to 46%) in 6-day cultures. The interaction of RANKL and IL-1 on osteoclastogenesis did not appear significantly dependent on prostaglandin synthesis since PGE(2) expression in the conditioned medium of BMM cultures was nearly undetectable and the PGHS-2 specific inhibitor, NS-398, was without effect. We also investigated the effect of IL-1 on p38 MAP kinase and JNK in BMM cultures. The combination of RANKL and IL-1 had additive effects on JNK but not p38 MAP kinase compared to results in cultures treated with RANKL or IL-1 alone. In addition, SP600125, a specific JNK inhibitor, markedly reduced OCL formation in BMM cultures that were treated with RANKL or the combination of RANKL and IL-1. These findings demonstrate that endogenously produced IL-1 augments the response of bone marrow cells to RANKL, and this effect appears mediated by mechanisms that are associated with enhancement of JNK activity.