Osteoclasts utilize the integrin alphabeta3 to mediate attachment to bone, the initial step in athe resorptive process. We find, in a murine system of in vitro osteoclastogenesis, precursor cells initially express and utilize the integrin alphabeta5 and not alphabeta3 and that, during formation of bone-resorbing polykaryons, alphabeta5 and alphabeta3 levels fall and rise respectively. This reciprocal change in levels of two functional integrins can be replicated by treating osteoclast precursors with GM-CSF, a cytokine involved in osteoclast differentiation. Levels of beta5 and beta3 mRNA fall alphabeta5 and alphabeta3 regulation is mediated through GM-CSF-dependent alterations in beta5 and beta3 gene expression. GM-CSF regulates gene expression by phosphorylation of STAT5, a cytosol- residing transcription factor, two isoforms of which have been cloned in mouse. The activated STAT5 translocates to the nucleus, where it binds to specific sequences in target genes, resulting in new protein synthesis. Most importantly as regards this proposal, the promoter region of the murine beta3 contains multiple STAT binding sequences. Based on our findings, and athe current model of GM-CSF action, we hypothesize that 1) specific STAT5 isoforms bind to and mediate GM-CSF expression of the murine beta3 gene and 2) GM-CSF inhibits murine beta5 gene expression through a cis-acting DNA sequence. Thus, our specific aims are; 1. CHARACTERIZE THE STAT5 ISOFORMS AND ASSOCIATED DNA BINDING DOMAINS WHICH MEDIATE GM-CSF REGULATION OF THE MURINE BETA3 GENE; 2. IDENTIFY THE DNA SEQUENCE BY WHICH GM-CSF INHIBITS EXPRESSION OF THE MURINE BETA5 GENE.
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