The long term objective of this application is to elucidate the role of gelsolin and the associated signalling complex in podosome assembly/disassembly and osteoclast function. Significant progress has been made in the understanding of the cell biology of osteoclast organization associated with bone resorption. Osteoclasts are highly motile cells, and they utilize unique types of cell/matrix interaction, the podosome, to yield high rates of cell motility. Podosomes ligate to the matrix by the alphavbeta3 integrin and have specialized cytoskeletal association. Integrin alphavbeta3 ligand-mediated signaling for stimulation of bone resorption involves activation of a gelsolin based signalling complex in the podosome which includes c-src, PI3-kinase, and its product, phosphatidylinositol trisphosphate (PtdIns P3). Gelsolin is an actin-binding protein with multiple functions, including filament severing and gelsolin barbed-end capping. Gelsolin uncapping of actin oligomers results in filament polymerization. We have demonstrated that PtdIns P3 produced by PI3-kinase activation participates in osteopontin/alphavbeta3 stimulated actin filament formation and podosome assembly/disassembly. Transgenic mice null for gelsolin failed to express podosomes in their osteoclasts and are hypomotile. The result is a significant decrease in the rates of bone resorption.
The specific aims of the proposal are to: 1) analyze the mechanisms of podosome assembly/disassembly; and 2) determine the mechanism of gelsolin associated signalling complex formation. Studies in specific aim 1 will focus on the role of protein tyrosine kinases in the assembly of the gelsolin-based signalling complex. Additionally, various gelsolin constructs will be used in an attempt to rescue the gelsolin null phenotype and stimulate podosome formation. Studies in specific aim 2 will analyze the role of phosphoinositides in the recognition of SH2 domains of the signalling molecules associated with gelsolin. The binding sites of phosphoinositides to short peptides of SH2 domains will be analyzed. These studies should delineate the structures necessary for the development of short peptide reagents capable of regulating osteoclasts function. Peptide based manipulations of SH2 domain/PtdIns P3/gelsolin organized signalling has the potential for pharmacological manipulation. There remains a tremendous need for therapeutic agents designed for the control of bone cell function in numerous diseases, especially osteoporosis, and thus these studies are extremely significant. This project should provide fundamental insights into the mechanisms of bone resorption and yield a potential for their pharmacologic control.
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