Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse.

The development of osteoclastic cell lines would greatly facilitate analysis of the cellular and molecular biology of bone resorption. Several cell lines have previously been reported to be capable of osteoclastic differentiation. However, such cell lines form at best only occasional excavations, suggesting that osteoclastic differentiation is either incomplete or that osteoclasts represent a very small proportion of the cells present. We have used the recently developed H-2KbtsA58 transgenic mouse, in which the interferon-inducible major mouse histocompatibility complex H-2Kb promoter drives the temperature-sensitive (ts) immortalizing gene of simian virus 40 (tsA58), to develop cell lines from bone marrow with high efficiency. Bone marrow cells were incubated with gamma interferon at 33 degrees C, then cloned, and expanded. The cell lines were characterized at 39.5 degrees C in the absence of gamma interferon. First, stromal cell lines were established that induced osteclast formation (resorption of bone slices) when cocultured with hemopoietic spleen cells. Some of the stromal cell lines so generated were able to resorb approximately 30 mm2/cm2 of bone surface. We then established cell lines of hemopoietic origin, several of which possess osteoclastic potential. When these osteoclast-precursor cell lines were cocultured with stromal cell lines, extensive bone resorption was observed. Osteoclast formation did not occur if the precursor cell lines were incubated on bone slices without stromal cells; osteoclast formation was also dependent upon the presence of 1 alpha,25-dihydroxyvitamin D3. These cell lines represent a model for osteoclast formation and a valuable resource for identification of the mechanisms and factors that regulate osteoclast differentiation and function.     

Cells of the osteoclast lineage as mediators of the anabolic actions of parathyroid hormone in bone

PTH is an anabolic agent used to treat osteoporosis, but its mechanisms of action are unclear. This study elucidated target cells and mechanisms for anabolic actions of PTH in mice during bone growth. Mice with c-fos ablation are osteopetrotic and lack an anabolic response to PTH. In this study, there were no alterations in PTH-regulated osteoblast differentiation or proliferation in vitro in cells from c-fos -/- mice compared with +/+; hence, the impact of osteoclastic cells was further investigated. A novel transplant model was used to rescue the osteopetrotic defect of c-fos ablation. Vertebral bodies (vossicles) from c-fos -/- and +/+ mice were implanted into athymic hosts, and the c-fos -/- osteoclast defect was rescued. PTH treatment to vossicle-bearing mice increased 5-bromo-2'-deoxyuridine (BrdU) positivity in the bone marrow and increased bone area regardless of the vossicle genotype. To inhibit recruitment of osteoclast precursors to wild-type vossicles, stromal derived factor-1 signaling was blocked, which blunted the PTH anabolic response. Treating mice with osteoprotegerin to inhibit osteoclast differentiation also blocked the anabolic action of PTH. In contrast, using c-src mutant mice with a late osteoclast differentiation defect did not hinder the anabolic action, suggesting key target cells reside in the intermediately differentiated osteoclast population in the bone marrow. These results indicate that c-fos in osteoblasts is not critical for PTH action but that cells of the osteoclast lineage are intermediate targets for the anabolic action of PTH.     

Differential effects of glucocorticoid on recruitment and activity of osteoclasts induced by normal and osteocalcin-deficient bone implanted in rats.

Prolonged glucocorticoid excess is associated with bone loss. Among the contributory factors are glucocorticoids' suppression of bone formation and stimulation of bone resorption. In this study, the effects of glucocorticoids on bone resorption were evaluated in a rodent model. Subcutaneous implants of devitalized mineralized bone particles (BPs) elicit the recruitment of progenitor cells and their differentiation to osteoclasts which resorb the BPs. The effects of glucocorticoids on both the recruitment and the activity of cells induced by normal BPs were distinguished based upon when treatment was initiated. When treatment with hydrocortisone or dexamethasone was initiated at the time of BP implantation, the recruitment of bone-resorbing cells was impaired and a subsequent decrease in BP resorption was found. On the other hand, when treatment was initiated on day 7, glucocorticoids increased osteoclastic resorption and tartrate-resistant acid phosphatase activity. We also tested hydrocortisone's effect to stimulate the activity of cells associated with osteocalcin-deficient BPs. As previously reported, BPs deficient in osteocalcin were poorly resorbed as a result of decreased formation and activity of osteoclasts. Hydrocortisone had an even more pronounced effect in stimulating the low level resorption of the osteocalcin-deficient BP implants than of the normal BP implants. These findings show differential effects of glucocorticoids on two aspects of bone resorption: they inhibit the recruitment and/or differentiation of bone-resorbing cells, but they stimulate the activity of existing osteoclastic cells. The ability of glucocorticoids to increase resorption of normal bone and to overcome resistance to resorption of osteocalcin-deficient bone suggests an important regulatory effect of glucocorticoids in the activation of osteoclasts to increase bone resorption.     

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.     

Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells

Osteoclasts are derived from hematopoietic precursor cells belonging to the monocyte/macrophage lineage. Osteoclast development has been reported to be regulated by several molecules such as macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), and a decoy receptor of RANKL, osteoprotegerin (OPG). Recently, it was demonstrated that the Notch signaling pathway regulates myeloid differentiation and antagonizes cell fate determination, however, the effect of Notch signaling on the osteoclast lineage has not been reported. In this study, we examined the effect of signaling via Notch receptors on the differentiation into osteoclasts by using cells from the bone marrow, spleen, and peritoneal cavity, and a cloned macrophagelike cell line. Osteoclastogenesis was inhibited by an immobilized Notch ligand, Delta-1. The dish-adherent bone marrow cells precultured with M-CSF expressed both Mac-1 and M-CSF receptors, c-Fms; osteoclastogenesis of these cells was efficiently inhibited. The immobilized Delta-1 also down-regulated the surface c-Fms expression, while the c-Fms gene expression was not changed. Genes for Notch receptors and Notch ligands are expressed in not only hematopoietic cells but also stromal cells that support osteoclast development. Constitutively active Notch1-transfected stromal cells showed increased expression of RANKL and OPG genes, and strong inhibition of M-CSF gene expression, resulting in reduction of their ability to support osteoclast development. Taken together, these findings indicate that Notch signaling affects both osteoclast precursors and stromal cells and thereby negatively regulates osteoclastogenesis.     

Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture

Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K(1) treatment. These cells had the ability to mineralize in the presence of alpha-glycerophosphate. In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively. Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.     

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.     

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.     

Inhibitor of DASH proteases affects expression of adhesion molecules in osteoclasts and reduces myeloma growth and bone disease

Dipeptidyl peptidase (DPP) IV activity and/or structure homologues (DASH) are serine proteases implicated in tumourigenesis. We previously found that a DASH protease, fibroblast activation protein (FAP), was involved in osteoclast-induced myeloma growth. Here we further demonstrated expression of various adhesion molecules in osteoclasts cultured alone or cocultured with myeloma cells, and tested the effects of DASH inhibitor, PT-100, on myeloma cell growth, bone disease, osteoclast differentiation and activity, and expression of adhesion molecules in osteoclasts. PT-100 had no direct effects on viability of myeloma cells or mature osteoclasts, but significantly reduced survival of myeloma cells cocultured with osteoclasts. Real-time PCR array for 85 adhesion molecules revealed upregulation of 17 genes in osteoclasts after coculture with myeloma cells. Treatment of myeloma/osteoclast cocultures with PT-100 significantly downregulated 18 of 85 tested genes in osteoclasts, some of which are known to play roles in tumourigenesis and osteoclastogenesis. PT-100 also inhibited osteoclast differentiation and subsequent pit formation. Resorption activity of mature osteoclasts and differentiation of osteoblasts were not affected by PT-100. In primary myelomatous severe combined immunodeficient (SCID)-hu mice PT-100 reduced osteoclast activity, bone resorption and tumour burden. These data demonstrated that DASH proteases are involved in myeloma bone disease and tumour growth.     

Minireview: the OPG/RANKL/RANK system.

The identification of the OPG/RANKL/RANK system as the dominant, final mediator of osteoclastogenesis represents a major advance in bone biology. It ended a long-standing search for the specific factor produced by preosteoblastic/stromal cells that was both necessary and sufficient for osteoclast development. The initial cloning and characterization of OPG as a soluble, decoy receptor belonging to the TNF receptor superfamily was the first step that eventually led to an unraveling of this system. Soon thereafter, the molecule blocked by OPG, initially called OPG-ligand/osteoclast differentiating factor (ODF) and subsequently RANKL, was identified as the key mediator of osteoclastogenesis in both a membrane-bound form expressed on preosteoblastic/stromal cells as well as a soluble form. RANKL, in turn, was shown to bind its receptor, RANK, on osteoclast lineage cells. The decisive role played by these factors in regulating bone metabolism was demonstrated by the findings of extremes of skeletal phenotypes (osteoporosis vs. osteopetrosis) in mice with altered expression of these molecules. Over the past several years, work has focused on identifying the factors regulating this system, the signaling mechanisms involved in the RANKL/RANK pathway, and finally, potential alterations in this system in metabolic bone disorders, from the extremely common (i.e. postmenopausal osteoporosis) to the rare (i.e. familial expansile osteolysis).     

Direct stimulation of osteoclastogenesis by MIP-1alpha: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.     

Overexpression of gamma-glutamyltransferase in transgenic mice accelerates bone resorption and causes osteoporosis

We previously identified gamma-glutamyltransferase (GGT) by expression cloning as a factor inducing osteoclast formation in vitro. To examine its pathogenic role in vivo, we generated transgenic mice that overexpressed GGT in a tissue-specific manner utilizing the Cre-loxP recombination system. Systemic as well as local production of GGT accelerated osteoclast development and bone resorption in vivo by increasing the sensitivity of bone marrow macrophages to receptor activator of nuclear factor-kappaB ligand, an essential cytokine for osteoclastogenesis. Mutated GGT devoid of enzyme activity was as potent as the wild-type molecule in inducing osteoclast formation, suggesting that GGT acts not as an enzyme but as a cytokine. Recombinant GGT protein increased receptor activator of nuclear factor-kappaB ligand expression in marrow stromal cells and also stimulated osteoclastogenesis from bone marrow macrophages at lower concentrations. Thus, GGT is implicated as being involved in diseases characterized by accelerated osteoclast development and bone destruction and provides a new target for therapeutic intervention.     

Cell biology of the osteoclast.

The osteoclast is a hematopoietic cell derived from CFU-GM and branches from the monocyte-macrophage lineage early during the differentiation process. The marrow microenvironment appears critical for osteoclast formation due to production of RANK ligand, a recently described osteoclast differentiation factor, by marrow stromal cells in response to a variety of osteotropic factors. In addition, factors such as osteoprotegerin, a newly described inhibitor of osteoclast formation, as well as secretory products produced by the osteoclast itself and other cells in the marrow enhance or inhibit osteoclast formation. The identification of the role of oncogenes such as c-fos and pp60 c-src in osteoclast differentiation and bone resorption have provided important insights in the regulation of normal osteoclast activity. Current research is beginning to delineate the signaling pathways involved in osteoclastic bone resorption and osteoclast formation in response to cytokines and hormones. The recent development of osteoclast cell lines may make it possible for major advances to our understanding of the biology of the osteoclast to be realized in the near future.     

Mechanisms involved in bone resorption

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product ofosteo blasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-κ Bligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-β super family members, such as bone morphogenetic proteins(BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cross-talk between the interleukin-6 and prostaglandin E(2) signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-{kappa}B (RANK) ligand/RANK system

The osteoprotegerin (OPG)/receptor activator of nuclear factor-kappaB ligand (RANKL)/receptor activator of nuclear factor-kappaB (RANK) system is the dominant and final mediator of osteoclastogenesis. Abnormalities of this system have been implicated in the pathogenesis of many skeletal diseases. Cyclooxygenase (COX)-2 and prostaglandin (PG)E(2), a major eicosanoid product of the COX-2-catalyzed pathway, play key roles in normal bone tissue remodeling. PGE(2) exerts its actions by binding and activating the E series of prostaglandin (EP) receptor. Activation of EP(2) and EP(4) receptors is associated with PGE(2)-induced osteoclast differentiation. IL-6, a major proinflammatory cytokine, has also been reported to induce osteoclast differentiation. Although interactions between the COX-2/PGE(2) and IL-6 systems have been described in bone cells, the mechanisms underlying these cooperative signaling pathways and the possible involvement of the OPG/RANKL/RANK system have not been fully elucidated. We demonstrate that COX-2, PGE(2), and IL-6 stimulate osteoblast growth and osteoclast differentiation. Effects on osteoclast differentiation, particularly with IL-6, were most marked when osteoclast precursor cells were grown in coculture with osteoblasts, indicating a possible role of the RANK/RANKL/OPG system. COX-2 and PGE(2) stimulated osteoclastogenesis through inhibition of OPG secretion, stimulation of RANKL production by osteoblasts, and up-regulation of RANK expression in osteoclasts. PGE(2) stimulated IL-6 secretion by bone cells, whereas COX-2 inhibitors decreased this same parameter. IL-6, in turn, increased PGE(2) secretion, COX-2, and EP receptor subtype expression in bone cells. Finally, IL-6 was the mediator of PGE(2)-induced suppression of OPG production by osteoblasts. These findings provide evidence for cross-talk between the PGE(2) and IL-6 signaling enhance osteoclast differentiation via effects on the OPG/RANKL/RANK system in bone cells.     

Human mesenchymal stem cells promote human osteoclast differentiation from CD34+ bone marrow hematopoietic progenitors.

Interactions between osteoclast progenitors and stromal cells derived from mesenchymal stem cells (MSCs) within the bone marrow are important for osteoclast differentiation. In vitro models of osteoclastogenesis are well established in animal species; however, such assays do not necessarily reflect human osteoclastogenesis. We sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human marrow-derived MSCs (hMSCs) and CD34+ hematopoietic stem cells (HSCs). After 3 weeks, coculture of hMSCs and HSCs resulted in an increase in hematopoietic cell number with formation of multinucleated osteoclast-like cells (Ocls). Coculture of hMSCs with HSCs, transduced with a retroviral vector that expresses enhanced green fluorescent protein, produced enhanced green fluorescent protein+ Ocls, further demonstrating that Ocls arise from HSCs. These Ocls express calcitonin and vitronectin receptors and tartrate-resistant acid phosphatase and possess the ability to resorb bone. Ocl formation in this assay is cell contact dependent and is independent of added exogenous factors. Conditioned medium from the coculture contained high levels of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), and macrophage-colony stimulating factor. IL-6 and LIF were present at low levels in cultures of hMSCs but undetectable in cultures of HSCs alone. These data suggest that coculture with HSCs induce hMSCs to secrete cytokines involved in Ocl formation. Addition of neutralizing anti-IL-6, IL-11, LIF, or macrophage-colony stimulating factor antibodies to the coculture inhibited Ocl formation. hMSCs seem to support Ocl formation as undifferentiated progenitor cells, because treatment of hMSCs with dexamethasone, ascorbic acid, and beta-glycerophosphate (to induce osteogenic differentiation) actually inhibited osteoclastogenesis in this coculture model. In conclusion, we have developed a simple and reproducible assay using culture-expanded hMSCs and purified HSCs with which to study the mechanisms of human osteoclastogenesis.     

Inhibition of inflammatory bone erosion by constitutively active STAT-6 through blockade of JNK and NF-kappaB activation

OBJECTIVE: NF-kappaB and JNK signaling pathways play key roles in the pathogenesis of inflammatory arthritis. Both factors are also activated in response to osteoclastogenic factors, such as RANKL and tumor necrosis factor alpha. Inflammatory arthritis and bone erosion subside in the presence of antiinflammatory cytokines such as interleukin-4 (IL-4). We have previously shown that IL-4 inhibits osteoclastogenesis in vitro through inhibition of NF-kappaB and JNK activation in a STAT-6-dependent manner. This study was undertaken to investigate the potential of constitutively active STAT-6 to arrest the activation of NF-kappaB and JNK and to subsequently ameliorate the bone erosion associated with inflammatory arthritis in mice. METHODS: Inflammatory arthritis was induced in wild-type and STAT-6-null mice by intraperitoneal injection of arthritis-eliciting serum derived from K/BxN mice. Bone erosion was assessed in the joints by histologic and immunostaining techniques. Cell-permeable Tat-STAT-6 fusion proteins were administered intraperitoneally. Cells were isolated from bone marrow and from joints for the JNK assay, the DNA-binding assays (electrophoretic mobility shift assays), and for in vitro osteoclastogenesis. RESULTS: Activation of NF-kappaB and JNK in vivo was increased in extracts of cells retrieved from the joints of arthritic mice. Cell-permeable, constitutively active STAT-6 (i.e., STAT-6-VT) was effective in blocking NF-kappaB and JNK activation in RANKL-treated osteoclast progenitors. More importantly, STAT-6-VT protein significantly inhibited the in vivo activation of NF-kappaB and JNK, attenuated osteoclast recruitment in the inflamed joints, and decreased bone destruction. CONCLUSION: Our findings indicate that the administration of STAT-6-VT presents a novel approach to the alleviation of bone erosion in inflammatory arthritis.     

Osteopenia in X-linked hyper-IgM syndrome reveals a regulatory role for CD40 ligand in osteoclastogenesis

We report that osteopenia is a prominent and previously unappreciated clinical feature of patients with X-linked hyper-IgM syndrome, an inherited immune deficiency disorder caused by mutations in the gene encoding CD40 ligand (CD40L). We therefore conducted studies to determine the relationship between CD40L and osteoclastogenesis. Recognizing that activated T cells express surface receptor activator of NF-kappaB ligand (RANKL) and can induce osteoclast differentiation of myeloid cells expressing RANK, we assessed the capacity of wild-type T cells and CD40L(-/-) T cells to induce osteoclastogenesis in vitro. Relative to wild-type T cells, activated CD40L(-/-) T cells from both humans and mice promoted robust osteoclast differentiation of myeloid cells. Whereas activated CD40L(-/-) T cells had normal expression of RANKL, they were deficient in IFN-gamma production. In subsequent studies, we cultured activated CD40L(-/-) T cells in the presence of IFN-gamma, and we found that the osteoclastic capacity of CD40L(-/-) T cells could be greatly diminished. These results show that CD40L can influence RANKL signaling through T cell priming, and thus they demonstrate a regulatory role for CD40L in bone mineralization that is absent in patients with X-linked hyper-IgM syndrome.