Osteoclastic Differentiation of Monocytes Is Inhibited by Non-Transcriptional Effects of Estrogen Mediated by BCAR1.

Abstract Estrogen is a major positive regulator of bone mass with important inhibitory effects on bone resorption. However, knowledge of the mechanisms underlying estrogen effects remains incomplete. We investigated whether estrogen altered osteoclastic differentiation of human monocytes in response to Receptor Activator of NF-kB (RANK) Ligand (RANKL), a critical stimulus for osteoclast formation. For these studies, human monocytic cells were isolated from peripheral blood by positive selection for CD14 expression (“CD14 cells”) and maintained in charcoal stripped medium with monocyte colony-stimulating factor. Compared to RANKL treatment alone, co-treatment with 17-β -estradiol reduced osteoclast formation, as shown by decreased development of tartrate resistant acid phosphatase (TRAP) activity, multinucleation, and resorption pit formation on bone. A possible mechanism for the estrogen effects was interference with RANKL signal transduction. We examined estrogen effects on a key event in the RANKL signaling: nuclear translocation of NF-kB following phosphorylation and degradation of IkB. Estrogen cotreatment of RANKL-stimulated CD14 cells inhibited the nuclear translocation of NF-kB observed with RANKL alone. This effect was evident after only 30 minutes of treatment. Cotreatment with estrogen also inhibited RANKL-stimulated IkB phosphorylation; this was evident within 5 minutes after treatment. The time course of the estrogen effects argued strongly for a non-transcriptional mechanism. We therefore investigated the involvement of proteins previously implicated in rapid estrogen effects, including the breast cancer anti-estrogen resistance protein 1(BCAR1, also called p130Cas), a scaffolding/adaptor protein recently reported to interact with estrogen receptors in breast cancer cells. Co-immunoprecipitation from ligand treated CD14 cells revealed an estrogen-dependent association of estrogen receptor-α (ER) with BCAR1 that was enhanced by cotreatment with RANKL. The coordinate regulation of this complex by both RANKL and estrogen suggested that BCAR1 might represent a link between the two signaling systems. In addition, in cells cotreated with estrogen and RANKL, co-immunoprecipitation revealed a further association of ER with Traf6, a critical proximal signaling intermediate for RANKL. These protein interactions were also rapid, evident 5 minutes after ligand addition. To test the functional significance of BCAR1 in estrogen effects, its expression was inhibited by transduction with BCAR1 siRNA. In cells deficient in BCAR1, estrogen no longer inhibited RANKL-stimulated NF-kB nuclear translocation. Unexpectedly, suppression of BCAR1 itself partially blocked RANKL stimulated NF-kB translocation, suggesting a role for BCAR1 even in the absence of estrogen. Furthermore, when BCAR1 deficient CD14 cells were treated with RANKL, osteoclastic differentiation, as assessed by formation of TRAP-positive multinucleated cells, was reduced compared to RANKL-treated cells transduced with control siRNA. The results suggest that BCAR1 is involved in the RANK-NF-kB pathway that mediates osteoclast differentiation signals in monocytes. In addition, BCAR1 appears necessary for estrogen inhibition of RANKL signaling. The estrogen dependent association of ER with BCAR1 may, like BCAR1 deficiency, block BCAR1 actions in the RANKL-NF-kB pathway, and thus inhibit the osteoclastic differentiation of monocytes.