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 Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 hours.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...

Granulocyte colony-stimulating factor (G-CSF), the major cytokine regulator of neutrophilic granulopoiesis, stimulates both the proliferation and differentiation of myeloid precursors. A variety of signaling proteins have been identified as mediators of G-CSF signaling, but understanding of their specific interactions and organization into signaling pathways for particular cellular effects is incomplete. The present study examined the role of the scaffolding protein Grb2-associated binding protein-2 (Gab2) in G-CSF signaling. We found that a chemical inhibitor of Janus kinases inhibited G-CSF-stimulated Gab2 phosphorylation. Transfection with Jak2 antisense and dominant negative constructs also inhibited Gab2 phosphorylation in response to G-CSF. In addition, G-CSF enhanced the association of Jak2 with Gab2. In vitro, activated Jak2 directly phosphorylated specific Gab2 tyrosine residues. Mutagenesis studies revealed that Gab2 tyrosine 643 (Y643) was a major target of Jak2 in vitro, and a key residue for Jak2-dependent phosphorylation in intact cells. Mutation of Gab2 Y643 inhibited G-CSF-stimulated Erk1/2 activation and Shp2 binding to Gab2. Loss of Y643 also inhibited Gab2-mediated G-CSF-stimulated cell proliferation. Together, these results identify a novel signaling pathway involving Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation in response to G-CSF.

The Drosophila kinesin-family protein Costal 2 (Cos2) and its mammalian ortholog Kif7 play dual roles in Hedgehog (Hh) signaling. In the absence of Hh, Cos2 and Kif7 contribute to proteolytic processing and silencing of the Hh-regulated transcription factors, Drosophila Cubitus interruptus (Ci) and mammalian Gli proteins. Cos2 and Kif7 are also necessary for full activation of full-length Ci-155 and Gli transcription factors in response to Hh proteins. Here, we use classical fused alleles and transgenic Cos2 products deficient for Fused (Fu) association to show that Cos2 must bind to Fu to support efficient Ci-155 processing. Residual Ci-155 processing in the absence of Cos2-Fu interaction did not require Suppressor of Fused, which has been implicated in processing mammalian Gli proteins. We also provide evidence that Cos2 binding to the CORD domain of Ci-155 contributes to both Ci-155 processing and Ci-155 silencing in the absence of Hh. In the presence of Hh, Ci-155 processing is blocked and Cos2 now promotes activation of Ci-155, which requires Fu kinase activity. Here, we show that normal Ci-155 activation by Hh requires Cos2 binding to Fu, supporting the hypothesis that Cos2 mediates the apposition of Fu molecules suitable for cross-phosphorylation and consequent full activation of Fu kinase. We also find that phosphorylation of Cos2 by Fu at two previously mapped sites, S572 and S931, which is thought to mediate Ci-155 activation, is not required for normal activation of Ci-155 by Hh or by activated Fu.

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