The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ~1 nM reduced RANKLdependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 hours. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (~5 minute) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKLsignaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 minutes of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα. KeywordsEstrogen receptor-α; Breast cancer antiestrogen resistance-1; p130Cas; Nuclear factor-κB; TNFreceptor associated factorsOsteoclasts are multinucleated bone-degrading cells that differentiate from monocytic precursors. They are critical to bone modeling and maintenance, and mediate the release of skeletal mineral for calcium and pH homeostasis. Consequently, osteoclast differentiation and activity are regulated by several systems. Because bone loss occurs when estrogen declines at the menopause, the modulation of osteoclast formation and activity by estrogen is an important issue. The mechanisms by which estrogen regulates bone turnover remain unclear [1]. *Corresponding author: Lisa J. Robinson, 707 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261, Tel: +1 412 647-0365, Fax: +1 412 647-4008, robinsonlj@msx.upmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptUnresolved issues include whether estrogen causes apoptosis of osteoclasts or osteoclast precursors, and whether estrogen suppresses osteoclast formation directly or whether effects are mediated via oth...

We previously reported that a soluble form of the TNF-family receptor death receptor-3 (DR3) is expressed in osteoblasts. DR3 regulates death or differentiation in other tissues, and DR3 ligands occur in bone, but the function of DR3 in the osteoblast was unknown. We studied the expression of DR3 and the effects crosslinking antibodies to DR3 or of natural DR3 ligands in human osteoblasts. Western analysis showed that nontransformed osteoblasts and the MG63 osteosarcoma cell line produce both soluble decoy receptor and transmembrane isoforms of DR3. Cell surface labeling showed that low and high DR3-expressing osteoblast populations occur. Verification of by cloning showed a point mutation in DR3 from MG63 cells. Activation of DR3 by antibody crosslinking or with DR3 ligands caused apoptosis in osteoblasts and in MG63 cells, but only in low-density cell cultures. In dense cultures apoptosis did not occur, but nuclear factor-kappaB nuclear translocation was observed under some conditions. Crosslinking of DR3 in high-density MG63 cultures blocked expression of bone matrix elements. DR3 activation in high-density nontransformed osteoblasts had only minor effects on cell maturation. We conclude that DR3 activation can mediate apoptosis in osteoblasts. Its activity is, however, highly restricted by its soluble ligand-binding isoform and possibly also by alternate survival signals. In the presence of survival signals, DR3 may affect cell maturation although effects on differentiation were clearly seen only in the MG63 transformed cell line.

We studied the effects of serum growth factors and of TNF-family proteins on osteoblast gap-junction connectivity. Serum starvation of human MG63 osteosarcoma cells or nontransformed osteoblasts decreased connexin43 protein. TNFα or TRAIL reduced connexin43 further. Serum starvation redistributed gap junctions but did not reduce intercellular diffusion. In contrast, TNFα or TRAIL reduced gap junctions on cell processes and decreased intercellular diffusion. Effects of TNFs on connexin43 were mediated by lysosomal proteolysis. Activating analogs of cAMP increased connexin43 protein, but did not block effects of serum starvation, TNFα, or TRAIL on connexin43 protein. Connexin43 and connectivity recovered overnight if stimuli were withdrawn. Surprisingly, connexin43 mRNA increased in serum starvation and with TNFα or TRAIL. Since β-catenin is a binding partner of connexin43, when connexin43 is degraded, β-catenin activation may contribute to a reflexive increase in connexin43 transcription. We conclude that osteoblast connectivity is regulated by a multifactorial system that maintains intercellular connections. Serum starvation, TNFα and TRAIL augmented connexin43 degradation and connexin43 transcription. Cell-cell communication was maintained in serum starvation, which may model response to acute injury, but was sensitive to TNFs. These inflammatory agents mediated selective, reversible removal of connexin43 from cell processes.