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Mature osteoclasts are multinuclear, macrophage-like cells derived from hematopoietic stem cells in the bone marrow. Several transcription factors regulating osteoclast differentiation have been identified. However, the molecular basis of transcriptional regulation in osteoclasts at epigenetic levels is largely unknown. In fact, no osteoclast-specific transcriptional co-regulators have been characterized. Recently, selective ablation of estrogen receptor alpha (ERalpha) in mature osteoclasts derived from female mice (ERalpha(Deltaoc/Deltaoc)) exhibited trabecular bone loss due to induced apoptosis via upregulated expression of Fas ligand mRNA. In general, the component composition of the ERalpha-associated co-activator complex and its expression levels are distinct among tissues. However, ERalpha transcriptional co-regulators in mature osteoclasts remain unclear. In the present study, we achieved large-scale cultivation of mature, multinucleated osteoclasts and established a purification system for ERalpha-associated proteins. In addition to co-regulators previously found in other ERalpha target cells, several unexpected factors were found such as CAP-H. The mRNA expression level of CAP-H was high during osteoclast differentiation. These results demonstrate the existence of osteoclast-specific transcriptional co-regulators supporting ERalpha function.

Osteoblasts and adipocytes differentiate from common pleiotropic mesenchymal stem cells under transcriptional controls by numerous factors and multiple intracellular signalings. However, cellular signaling factors that determine cell fates of mensenchymal stem cells in bone marrow remain to be largely uncovered, though peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is well established as a prime inducer of adipogenesis. Here, we describe two signaling pathways that induce the cell fate decision into osteoblasts from adipocytes. One signaling is a TAK1/TAB1/NIK cascade activated by TNF-alpha and IL-1, and the activated NF-kappaB blocked the DNA binding of PPAR-gamma, attenuating the activated PPAR-mediated adipogenesis. The second signaling is the noncanonical Wnt pathway through CaMKII-TAK1/TAB2-NLK. Activated NLK by a noncanonical Wnt ligand (Wnt-5a) transrepresses PPAR transactivation through a histone methyltransferase, SETDB1. Wnt-5a induces phosphorylation of NLK, leading to the formation of a corepressor complex that inactivates PPAR function through histone H3-K9 methylation. Thus, two signaling pathways lead to an osteoblastic cell lineage decision from mesenchymal stem cells through two distinct modes of PPAR transrepression.

Estrogens play a key role in regulation of bone mass and strength by controlling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. Cellular effects of estrogens are mediated predominantly by the action of estrogen receptor alpha (ERalpha). In earlier studies, ablation of the ERalpha gene in mice did not result in osteoporotic phenotypes due to systemic endocrine disturbance and compensatory effects of elevated levels of testosterone. Despite the relatively well-established effects in osteoblasts, little is known about the direct action of estrogen in osteoclasts. Development in the last decade of more sophisticated genetic manipulation approaches opened new possibilities to explore cell-specific roles of nuclear receptors in bone tissue. Recently, we have generated osteoclast-specific ERalpha gene knockout mice and shown that in vivo estrogens directly regulate the life span of mature osteoclasts by inducing the expression of pro-apoptotic Fas ligand (FasL). Inhibitory effects of estrogens on osteoclast function were further studied in vitro. We observed sufficiently detectable ERalpha expression in osteoclasts differentiating from primary bone marrow cells or RAW264 cells, although levels of ERalpha were decreasing during progression of the differentiation into mature osteoclasts. Treatment with estrogens led to reduction in expression of osteoclast-specific genes controlling bone resorption activity. However, estrogens did not affect the size of multinucleated osteoclasts or number of nuclei in a mature osteoclast. In conclusion, in osteoclasts, estrogens function to inhibit bone resorption activity and vitality rather than differentiation.