GELSOLIN BASED SIGNALING IN OSTEOCLAST FUNCTION

这项应用的长期目标是阐明凝胶和相关信号复合物在足小体组装/拆卸和破骨细胞功能中的作用。在理解与骨吸收相关的破骨细胞组织的细胞生物学方面取得了重大进展。破骨细胞是高度运动性的细胞，它们利用独特类型的细胞/基质相互作用，即足质体，产生高速率的细胞运动性。足小体通过α - β 3整合素连接到基质上，并具有特殊的细胞骨架关联。整合素- α - β 3配体介导的刺激骨吸收的信号通路涉及激活足小体中基于凝胶的信号复合物，该复合物包括c-src、pi3激酶及其产物磷脂酰肌醇三磷酸（PtdIns P3）。Gelsolin是一种具有多种功能的肌动蛋白结合蛋白，包括切断细丝和Gelsolin倒钩端盖。肌动蛋白低聚物的凝胶脱帽导致长丝聚合。我们已经证明，由pi3激酶激活产生的PtdIns P3参与骨桥蛋白/ α β 3刺激的肌动蛋白丝的形成和足小体的组装/拆卸。gelsolin基因缺失的转基因小鼠在破骨细胞中不能表达足小体，运动能力低下。结果是骨吸收率显著降低。该提案的具体目的是：1)分析足体组装/拆卸的机制；2)确定凝胶相关信号复合物形成的机制。具体目标1的研究将集中在蛋白酪氨酸激酶在凝胶基信号复合物组装中的作用。此外，各种凝胶结构将被用于尝试挽救凝胶无表型和刺激podosome的形成。具体目标2的研究将分析磷酸肌苷在识别与凝胶相关的信号分子的SH2结构域中的作用。磷酸肌苷与SH2结构域短肽的结合位点将被分析。这些研究应该描绘出开发能够调节破骨细胞功能的短肽试剂所必需的结构。SH2结构域/PtdIns P3/gelsolin组织信号的肽基操作具有药理操作的潜力。在许多疾病中，特别是骨质疏松症，仍然需要设计用于控制骨细胞功能的治疗药物，因此这些研究非常重要。该项目将为骨吸收的机制提供基本的见解，并为其药理学控制提供潜力。

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.