[Estrogen actions on osteocytes].

Estrogen is well-known to play essential roles for maintenance of bone mass. Although its osteoprotective actions are mediated mainly through suppressing bone resorption, several reports has suggested that ERα/estrogen signal in osteocytes has some osteoprotective effects. For example, ERαis reported to be involved in adaptive response to loading which is thought to be sensed by osteocytes. Estrogen is also thought to be a viable factor for osteocytes. In addition, our group recently found that osteocytic ERαexhibited decreased bone mass, suggesting that osteocytic ERαexerts osteoprotective function. Also, functions of osteocyte itself have been characterized recently. These findings may lead to an understanding of estrogen actions on osteocytes and to a comprehensive understanding of estrogen actions on bone.

[Mechanotransduction via estrogen receptors in bone].

Bone mass and strength is mediated by appropriate weight bearing, and it is clear that bone tissue senses and adopts mechanical stresses. However, the precise molecular mechanisms in mechanotransduction remain elusive. Recently, several studies clarified that estrogen receptor alpha (ERalpha) plays important role in mechanotransduction. For example, the lifespan of osteocytes is prolonged by mechanical loading through ERalpha action, which is produced by the plentiful expression of ERalpha in osteocytes. Although these recent findings clearly show the important role of ERalpha in mechanotransduction, the intracellular function of ERalpha is still largely unknown. In this point of view, it will be a great interest to elucidate how ERs functionally associate with regulators involved with mechanotransductions in bone cells.

Estrogen receptor α in osteocytes regulates trabecular bone formation in female mice.

Estrogens are well known steroid hormones necessary to maintain bone health. In addition, mechanical loading, in which estrogen signaling may intersect with the Wnt/ß-catenin pathway, is essential for bone maintenance. As osteocytes are known as the major mechanosensory cells embedded in mineralized bone matrix, osteocyte ERα deletion mice (ERα(ΔOcy/ΔOcy)) were generated by mating ERα floxed mice with Dmp1-Cre mice to determine the role of ERα in osteocytes. Trabecular bone mineral density of female, but not male ERα(ΔOcy/ΔOcy) mice was significantly decreased. Bone formation parameters in ERα(ΔOcy/ΔOcy) were significantly decreased while osteoclast parameters were unchanged. This suggests that ERα in osteocytes exerts osteoprotective function by positively controlling bone formation. To identify potential targets of ERα, gene array analysis of Dmp1-GFP osteocytes sorted by FACS from ERα(ΔOcy/ΔOcy) and control mice was performed. Gene expression microarray followed by gene ontology analyses revealed that osteocytes from ERα(ΔOcy/ΔOcy) highly expressed genes categorized in 'Secreted' when compared to control osteocytes. Among them, expression of Mdk and Sostdc1, both of which are Wnt inhibitors, was significantly increased without alteration of expression of the mature osteocyte markers such as Sost and ß-catenin. Moreover, hindlimb suspension experiments showed that trabecular bone loss due to unloading was greater in ERα(ΔOcy/ΔOcy) mice without cortical bone loss. These data suggest that ERα in osteocytes has osteoprotective functions in trabecular bone formation through regulating expression of Wnt antagonists, but conversely plays a negative role in cortical bone loss due to unloading.

Minireview: osteoprotective action of estrogens is mediated by osteoclastic estrogen receptor-alpha.

The osteoprotective action of estrogen in women has drawn considerable attention because estrogen deficiency-induced osteoporosis became one of the most widely spread diseases in developed countries. In men, the significance of estrogen action for bone health maintenance is also apparent from the osteoporotic phenotype seen in male patients with genetically impaired estrogen signaling. Severe bone loss and high bone turnover, including typical osteofeatures seen in postmenopausal women, can also be recapitulated in rodents after ovariectomy. However, the expected osteoporotic phenotype is not observed in female mice deficient in estrogen receptor (ER)-alpha or -beta or both, even though the degenerative defects are clearly seen in other estrogen target tissues together with up-regulated levels of circulating testosterone. It has also been reported that estrogens may attenuate bone remodeling by cell autonomous suppressive effects on osteoblastogenesis and osteoclastogenesis. Hence, the effects of estrogens in bone appear to be complex, and the molecular role of bone estrogen receptors in osteoprotective estrogen action remains unclear. Instead, it has been proposed that estrogens indirectly control bone remodeling. For example, the enhanced production of cytokines under estrogen deficiency induces bone resorption through stimulation of osteoclastogenesis. However, the osteoporotic phenotype without systemic defects has been recapitulated in female (but not in male) mice by osteoclast-specific ablation of the ERalpha, proving that bone cells represent direct targets for estrogen action. An aberrant accumulation of mature osteoclasts in these female mutants indicates that in females, the inhibitory action of estrogens on bone resorption is mediated by the osteoclastic ERalpha through the shortened lifespan of osteoclasts.

Mechanical induction of PGE2 in osteocytes blocks glucocorticoid-induced apoptosis through both the ß-catenin and PKA pathways.

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid-flow shear stress blocks dexamethasone-induced apoptosis of osteocyte-like cells (MLO-Y4). Prostaglandin E(2) (PGE(2) ), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone-induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3-kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of ß-catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/ß. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid-induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase-2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce ß-catenin protein expression. ß-catenin siRNA knockdown experiments abrogated the protective effects of PGE(2), confirming the central role of ß-catenin in mediating the protection against dexamethasone-induced cell death. Our data support a central role for PGE(2) acting through the cAMP/PKA and ß-catenin signaling pathways in the protection of osteocyte apoptosis by fluid-flow shear stress.

Regulation of bone metabolism by nuclear receptors.

Bone tissue protects and supports soft organs and maintains calcium homeostasis. Steroid sex hormones and fat-soluble vitamins play a pivotal role in regulation of bone homeostasis, turnover and remodeling. These molecules act as ligands of nuclear receptors, through which they control gene expression in bone cells, namely bone-forming osteoblasts, bone-resorptive osteoclasts and osteocytes. Significant advances in our understanding of nuclear receptor physiology have been achieved due to development of novel genetic manipulation approaches and generation of experimental animal models in which nuclear receptor genes were mutated in specific cell types. In this review, we summarized some aspects of recent progress in studies on molecular mechanisms of cell-specific action of nuclear hormone receptors in bone tissue.

Estrogens maintain bone mass by regulating expression of genes controlling function and life span in mature osteoclasts.

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.