Follicle-stimulating hormone does not impact male bone mass in vivo or human male osteoclasts in vitro.

Bone loss in the elderly is mainly caused by osteoclast-induced bone resorption thought to be causally linked to the decline in estrogen and testosterone levels in females and males. Recently, involvement of follicle stimulating-hormone (FSH) in this process has been suggested to explain in part the etiology of the disease in females, whereas its role in males has never been examined. In this study, the direct impact of FSH on bone mass of 16-week-old C57BL/6J male mice by either daily intermittent application of 6 or 60 mug/kg of FSH or continuous delivery via miniosmotic pump of a dose of 6 mug/kg over the course of a month was assessed. Femoral peripheral quantitative computed tomographic and microcomputed tomographic analyses at 0, 2, and 4 weeks of FSH-treated mice did not reveal any differences in cancellous and cortical bone compared to sham-treated mice. FSH functionality was verified by demonstrating cAMP induction and activation of a cAMP-response element-containing reporter cell line by FSH. Furthermore, osteoclastogenesis from human mononuclear cell precursors and from RAW 264.7 cells was not affected by FSH (3, 10, 30 ng/mL) compared to control. No direct effect of FSH on gene regulation was observed by Affymetrix Gene Array on RAW 264.7 cells. Lastly, no expression of FSH receptor (FSHR) mRNA or FSHR was observed by quantitative polymerase chain reaction and Western blot in either human male osteoclasts or RAW 264.7 cells. These data show that FSH does not appear to modulate male bone mass regulation in vivo and does not act directly on osteoclastogenesis in vitro.

Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts.

The proliferation inhibitor of the macrolide class, everolimus, is a drug shown to be effective in the prevention of organ transplant rejection and to have a potential in the treatment of rheumatoid arthritis and certain cancers. As these diseases or their current treatments are associated with bone loss, we examined the effect of everolimus on mouse and human bone cells in vitro and on bone in an ovariectomized (OVX) rat model. Everolimus potently inhibited primary mouse and human osteoclast activity in the pit assay (IC50 values of 0.6-4.0 nM), as well as osteoclast formation, measured as the number of tartrate-resistant acid phosphatase (TRAP) multinucleated cells (IC50 values of 7.7-10.5 nM). Inhibition of osteoblastic differentiation was also observed (IC50 value of 13.5 nM). As expected, everolimus inhibited proliferation of osteoclast precursors and stimulated apoptosis, albeit with insufficient potency and efficacy to explain inhibition of osteoclast activity. Thus, everolimus appeared to directly inhibit bone resorption, which is in accord with the detected inhibition of mRNA and protein expression of cathepsin K; the main collagen-degrading protease in osteoclasts. Despite the in vitro antiproliferative activity of everolimus and the observed inhibition of osteoblast differentiation, no detrimental effects were detected at different skeletal sites in mature OVX rats at doses up to 3 mg/kg/day. This everolimus dose also prevented the OVX-induced loss of cancellous bone by 60%, an effect predominantly associated with decreased osteoclast-mediated bone resorption, resulting in a partial preservation of the cancellous bone network. Everolimus inhibited S6 kinase 1 activity in rat blood cells, skin, and bone, at doses equivalent to those used for efficacy experiments in the OVX rat model, which demonstrated in vivo targeting of the expected molecular pathway. In conclusion, everolimus directly inhibits bone resorption by osteoclasts and thus could at least be neutral or protective for bone in vivo, which would favor its use in disease indications associated with bone loss.

Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay.

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase) and activity (resorbed area and collagen fragments released from bone slices) in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay.

Transcriptional program of mouse osteoclast differentiation governed by the macrophage colony-stimulating factor and the ligand for the receptor activator of NFkappa B.

Cytokines macrophage colony stimulating factor (M-CSF) and the receptor activator of NFkappaB ligand (RANKL) induce differentiation of bone marrow hematopoietic precursor cells into bone-resorbing osteoclasts without the requirement for stromal cells of mesenchymal origin. We used this recently described mouse cell system and oligonucleotide microarrays representing about 9,400 different genes to analyze gene expression in hematopoietic cells undergoing differentiation to osteoclasts. The ability of microarrays to detect the genes of interest was validated by showing expression and expected regulation of several osteoclast marker genes. In total 750 known transcripts were up-regulated by > or =2-fold, and 91% of them at an early time in culture, suggesting that almost the whole differentiation program is defined already in pre-osteoclasts. As expected, M-CSF alone induced the receptor for RANKL (RANK), but also, unexpectedly, other RANK/NFkappaB pathway components (TRAF2A, PI3-kinase, MEKK3, RIPK1), providing a molecular explanation for the synergy of M-CSF and RANKL. Furthermore, interleukins, interferons, and their receptors (IL-1alpha, IL-18, IFN-beta, IL-11Ralpha2, IL-6/11R gp130, IFNgammaR) were induced by M-CSF. Although interleukins are thought to regulate osteoclasts via modulation of M-CSF and RANKL expression in stromal cells, we showed that a mix of IL-1, IL-6, and IL-11 directly increased the activity of osteoclasts by 8.5-fold. RANKL induced about 70 novel target genes, including chemokines and growth factors (RANTES (regulated on activation, normal T cell expressed and secreted), PDGFalpha, IGF1), histamine, and alpha1A-adrenergic receptors, and three waves of distinct receptors, transcription factors, and signaling molecules. In conclusion, M-CSF induced genes necessary for a direct response to RANKL and interleukins, while RANKL directed a three-stage differentiation program and induced genes for interaction with osteoblasts and immune and nerve cells. Thus, global gene expression suggests a more dynamic role of osteoclasts in bone physiology than previously anticipated.