Identification of Non-Genomic Mechanisms for Estrogen Effects on Monocyte Responses to Inflammatory and Osteoclastogenic Stimuli.

In previous studies we found that estradiol can act directly on primary human monocytes to inhibit RANKL stimulated osteoclastic differentiation, a potential mechanism for anti-osteoporotic effects of estrogen. Estrogen effects were rapid, suggesting involvement of non-genomic mechanisms in the inhibition of osteoclastogenic signals. Direct effects of estradiol on monocytic cells have also been implicated in anti-inflammatory effects of estrogen. It was recently shown that in murine monocytic cells, estrogen could acutely inhibit signaling events downstream from the lipopolysaccharide receptor, Toll-like receptor (TLR) 4 (Ghisletti et al. Mol Cell Biol 25 (2005) 2957). A critical component of TLR effects is the activation of the transcription factor NF-kB, resulting in increased expression of cytokines and chemokines that initiate the inflammatory response to LPS and other stimuli. NF-kB is also a critical mediator of the effects of TNF family members including RANKL. RANKL differs from most TNF family members in relying primarily on a signaling pathway through Traf6 for activation of NF-kB. Traf6 is also a key component of the pathway from TLR to NF-kB, and signaling events downstream from Traf6 appear to be common to RANKL and TLR4 signaling. We used CD14-selected primary human monocytes to examine the role of estrogen effects on NF-kB in its inhibition of monocyte response to RANKL and to determine whether common molecular mechanisms underlie estrogen effects on inflammation and osteoclastogenesis. Estradiol was added to CD14 cell cultures that, after 5 minutes, were treated with RANKL or lipopolysaccharide. After 30 minutes, cells were fixed and NF-kB localization determined by immunofluorescence microscopy using antibodies to p65. The nuclear translocation of NF-kB, evident in parallel cultures treated with RANKL or LPS but not estrogen, was markedly inhibited when cells were cotreated with estradiol. (Estrogen alone showed no effect on NF-kB localization.) Alternatively, after treatment with or without estrogen, LPS or RANKL, nuclear and cytosolic extracts were prepared and evaluated by SDS-PAGE and DNA binding, to confirm estrogen inhibition of the nuclear translocation of NF-kB. Nuclear localization of NF-kB follows the phosphorylation and degradation of IkB. Cells stimulated with RANKL or LPS for 5 or 20 minutes showed inhibition of IkB degradation if cells were cotreated with estradiol. This suggested involvement of the kinase cascade initiated by a signaling protein complex including Traf6, which is the most proximal signaling protein common to the TLR and RANKL pathways. We investigated the possibility that estrogen inhibited TLR and RANKL signal transduction through effects on the Traf6 protein complex. After 5 minutes stimulation with ligands, coimmunoprecipitation studies revealed the association of Traf6 with estrogen receptor alpha (ER) when cells were cotreated with estradiol. Coimmunoprecipitation further revealed the association of the scaffolding protein BCAR1 (p130 Cas), a protein previously implicated in non-genomic estrogen effects in breast cancer cells. We evaluated the role of BCAR1 using siRNA to inhibit its expression. In cells lacking BCAR1, the inhibitory effect of estradiol on NF-kB nuclear translocation was abrogated. The results overall suggest that estradiol may inhibit both TLR and RANKL signaling through ER protein interactions, potentially mediated by BCAR1, that disrupt the Traf6 signaling complex critical for activation of NF-kB.