Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways.one is a dynamic tissue that continuously remodels and can undergo regeneration throughout life. This continuous remodeling occurs through a dynamic process of breakdown by osteoclasts and rebuilding by osteoblasts. Bone mass in adults is maintained by a local balance between osteoclastic bone resorption and osteoblastic activities that are mediated via various factors such as hormones, growth factors, cytokines, and matrix proteins. Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblastmediated bone formation are tightly coupled. We now have clear evidence of how osteoblasts direct osteoclast differentiation through RANK and RANKL as well as other pathways (1). Questions remain as to how osteoblasts are recruited to the site after the resorption phase and how the amount of bone produced is controlled. This has led to consideration of how osteoclasts and/or their activity could promote bone formation.Mouse models and humans in whom osteoclastogenesis is perturbed have provided important insights into the role of b...

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFκB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IκB, and NFκB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFκB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFκB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclX L expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFκB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival.

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains solved. A recently developed technique for the isolation of avian osteoclasts has been modified to obtain highly purified multinucleated activity remained unresolved. To determine if there is an estrogen effect on osteoclast activity in humans, we have examined multinucleated cells from human osteoclast-like giant cell tumors of the bone for evidence of ERs and responses to 17,3-estradiol (173-E2) treatment. These cells have been reported to exhibit all documented characteristics of authentic osteoclasts, including the ability to form pits when cultured on slices of cortical bone and are therefore appropriate osteoclast surrogates (5). An efficient technique for the isolation of highly purified avian osteoclasts was adapted to separate multinucleated cells from moftonuclear cells in the human piant cell tumors (6). These multinucleated cells were examined for evidence ofestrogen responsiveness. pelleted by centrifugation at 900 rpm for 10 min and an avian osteoclast-directed monoclonal antibody (121; a gift from P. Osdoby, Washington University, St. Louis) coupled to immunomagnetic beads (Dynal, Great Neck, NY) was used to obtain antigen 121-positive (121+) cell populations that contain at least 90% pure multinucleated and 10%6 or less unidentified mononuclear cells. Avian osteoclasts purified by this method exhibit all of the phenotypic attributes of osteoclasts including multinucleation, ruffled border formation when cultured with bone particles, and the ability to form resorption pits when cultured on slices of cortical bone (6). The number of total cells, multinucleated cells (more than three nuclei), and 121+ (bead-binding cells) were scored. The antigen 121-negative (121-)

For many years it has been recognized that sex steroids have profound effects on bone metabolism. The current perception is that estrogen decreases bone resorption and androgen increases bone deposition. To investigate the potential for androgens to directly modulate bone resorption, we have examined avian osteoclast and human and mouse osteoclast-like cells for androgen responsiveness. There was a dose-dependent decrease in resorption activity in response to ␣-dihydrotestosterone (␣-DHT), ␤-DHT, testosterone, or the synthetic androgen RU1881. This decrease was blocked by cotreatment with the specific androgen antagonist hydroxyf lutamide. Further examination of avian osteoclasts revealed that the cells exhibited specific and saturable nuclear binding of tritiated RU1881 and that ␣-DHT stimulated the activity of the androgen response element as measured by using a chloramphenicol acetyltransferase reporter plasmid. In addition, avian osteoclasts responded to androgen treatment with elevated production and secretion of transforming growth factor ␤, a well documented response to androgen exposure in other cell systems. Treatment with either ␣-DHT or ␤-DHT for 24 hours resulted in a significant dosedependent decrease in secretion of cathepsin B and tartrateresistant acid phosphatase. This response to ␤-DHT, a stereoisomer of ␣-DHT that is inactive in other androgen receptor-dependent systems, supports the hypothesis that the osteoclast androgen receptor has unusual ligand-binding properties. Taken together, these results confirm the presence of functional androgen receptors in these cells and support the conclusion that osteoclasts are able to respond directly to androgens in vitro and thus are potential androgen target cells in vivo.Androgens have long been recognized to play an important role in the normal development and physiology of bone and have profound influences on bone development and metabolism (1). The evidence published to date suggests that androgens exert their effects on bone metabolism through stimulation of bone formation by both indirect and direct effects on bone-forming osteoblasts derived from both males and females (2-4). Although the incidence of hip fractures in elderly men is less than in elderly women (17% in men versus 32% in women), osteoporosis in men is still a significant health problem with an aging population (5). Decreased androgen levels have been linked to lower bone density in men, yet there has been no definitive proof that there is a link between lower androgen levels and the increase in the incidence of hip fracture (6). There is, however, a strong correlation between hypogonadism in elderly men and hip fracture and spinal osteoporosis (5, 6). Although one study of males matched for age, race, and lifestyle has shown a direct correlation between hip fracture incidences and lower gonadal steroid levels (7), it has proven difficult in other studies to correlate serum androgen levels and risk for osteoporosis in elderly men, and it is likely that there are many other fa...