The receptor activator of NF-B (RANKL) is the essential signal required for full osteoclast (OC) development, activation, and survival. RANKL is highly expressed in areas of trabecular bone remodeling and inflammatory bone loss, is increased on marrow stromal cells or osteoblasts by osteotropic hormones or cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy receptor also crucial for preventing arterial calcification. Vascular endothelial cells (VEC) are critically involved in bone development and remodeling and influence OC recruitment, formation, and activity. Although OCs develop and function in close association with bone VEC and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-␣ and interleukin-1␣ elevate RANKL and OPG expression 5-40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the surface of tumor necrosis factor-␣-activated HMVEC. Cytokine-activated HMVEC promoted the formation, fusion, and bone resorption of OCs formed in co-cultures with circulating human monocytic precursors via a RANKLmediated mechanism fully antagonized by exogenous OPG. Furthermore, paraffin sections of human osteoporotic fractured bone exhibited increased RANKL immunostaining in vivo on VEC located near resorbing OCs in regions undergoing active bone turnover. Therefore, cytokine-activated VEC may contribute to inflammatorymediated bone loss via regulated production of RANKL and OPG. VEC-derived OPG may also serve as an autocrine signal to inhibit blood vessel calcification.The receptor activator of NF-B ligand (RANKL), 1 also known as osteoprotegerin ligand (OPGL), osteoclast differentiation factor, or TNF-related activation-induced cytokine (TRANCE), is a recently discovered transmembrane molecule of the tumor necrosis factor (TNF) ligand superfamily that is highly expressed in lymphoid tissues and trabecular bone, particularly in areas associated with active bone remodeling or inflammatory osteolysis (1-4). RANKL is the essential and final common signal required both in vitro and in vivo for full osteoclastic (OC) differentiation from multipotential hematopoietic precursor cells into mature multinucleated bone-resorptive OCs in the presence of the permissive factor macrophage colony-stimulating factor (M-CSF) (1-7). RANKL expressed on the surface of osteoblasts (OB) or bone marrow stromal cells (BMSC) interacts with a cell surface receptor, RANK, present on pre-OC (induced by M-CSF) and mature OC to stimulate their fusion, development, bone resorption, and cell survival (5-9). RANKL expression increases during early OB development and is up-regulated in OB and BMSC by various proresorptive stimuli such as parathyroid hormone (PTH), 1,25-dihydroxyvitamin D 3 (VD 3 ), dexamethasone (Dex), prostaglandin E 2 , or interleukin-11 (IL-11) (6, 10 -12). Recently, the pro-reso...
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.
We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NOgenerating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.Bone-remodeling disorders such as osteoporosis, osteoarthritis, and periodontal disease are frequently associated with perturbations in the interplay between local and systemic bone-remodeling regulatory pathways. Inflammatory cytokines and arachidonic acid derivatives have been implicated as intercellular messengers involved in humoral-mediated and local osteopenia (1, 2). Postmenopausal bone loss associated with diminished estrogen levels is correlated with increased levels of interleukin 1 and stromal cell-derived interleukin 6, cytokines known to stimulate osteoclast activity and development (3, 4). Estrogen also directly inhibits osteoclast-mediated resorption (5,6) Howard (13) reported that NO-generating compounds may increase cGMP levels in isolated chicken osteoclasts. Furthermore, sodium nitroprusside (SNP) has been shown to inhibit the parathyroid hormone or 1,25-(OH)2-vitamin D3 stimulation of resorption in the 19-day fetal rat limb resorption assay system, with concomitant increases in cGMP (14).The current study was designed to investigate the role of NO in both an isolated in vitro avian osteoclast system and an in vivo rat osteoporosis model system using a NOgenerating agent and selective NOS inhibitors. These findings demonstrate that NO regulates osteoclast bone-resorption activity in vitro and in vivo and that similar effects are seen in birds and mammals.MATERIALS AND METHODS Animals. Three-month-old female Sprague-Dawley rats (250-300 g) from Charles River Breeding Laboratories were used in all in vivo experim...
Although in vivo effects of 17f3-estradiol (estrogen) on bone turnover have been shown to occur mainly through influences on osteoclast-mediated bone resorption, the mechanism by which estrogen reduces bone resorption is unclear. To approach this question, we have examined authentic osteoclasts for evidence of a direct osteoclast response to estrogen in vitro. Highly purified (>90%) viable avian osteoclasts from birds maintained on a low calcium diet were obtained using an osteoclast-specific monoclonal antibody coupled to magnetic beads. Isolated cells were either analyzed directly for estrogen receptor (ER) levels or cultured to assess the biological effects of estrogen. Northern blot analysis revealed a 5.2-kilobase mRNA that hybridized with a cDNA to human ER mRNA in the osteoclasts. An anti-human ER antibody recognized proteins of 66 kDa and 140 kDa in osteoclast extracts by Western blot analysis. The 66-kDa size is in close agreement with the reported size of the human ER. Nuclear binding of estrogen to intact viable osteoclasts was steroid-specific and saturable, with 5662 ± 1420 molecules bound per nucleus (mean ± SEM). In vitro estrogen responses in osteoclasts included a dose-dependent decrease in resorption as well as an increase in nuclear protooncogene mRNA levels. These observations indicate that osteoclasts are capable of directly responding to estrogen in vivo.Postmenopausal osteoporosis is a major health problem in the United States resulting in 1.5 million fractures and costing $7 to $10 million each year (1). 17,6-Estradiol (estrogen) deficiency has long been recognized as a cause of postmenopausal osteoporosis and estrogen replacement therapy is an effective treatment for the prevention of bone loss (2-6). Formerly, it was believed that the effects of estrogen on bone were indirect since early studies were unable to identify estrogen receptors (ERs) in bone tissue. Recent reports, however, have demonstrated that cells of the osteoblast lineage contain ERs and respond physiologically to estrogen (7,8). These findings were paradoxical because the primary biological effect of estrogen on bone in vivo is to decrease bone resorption (2-6) and resulted in the hypothesis that estrogen effects on osteoclast activity may be regulated through the osteoblast.On the basis of immunohistochemical localization and radioimmunoassay studies of bone samples from four children, Pensler et al. (9) have suggested that human osteoclasts may contain ERs. However, these studies were not conclusive and, to date, the direct effects of estrogen on osteoclasts have yet to be demonstrated. This report describes the use of an immunomagnetic separation method (10) for the isolation of highly purified viable avian osteoclasts to assess the potential for a direct estrogen effect on osteoclasts in vitro. The results presented show unequivocally that not only are functional ERs and their mRNAs present in these osteoclasts, but also estrogen rapidly modulates steady-state mRNA levels of osteoclast nuclear protooncogene...
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