GELSOLIN BASED SIGNALING IN OSTEOCLAST FUNCTION

这一应用的长期目标是阐明明胶蛋白及其相关的信号复合体在足小体组装/拆解和破骨细胞功能中的作用。在了解破骨细胞组织与骨吸收相关的细胞生物学方面取得了重大进展。破骨细胞是高度运动的细胞，它们利用独特类型的细胞/基质相互作用，即足体，产生高运动率的细胞。足体通过αvbeta3整合素连接到基质上，并具有特殊的细胞骨架结合。整合素Alphavbeta3配体介导的刺激骨吸收的信号涉及激活足体中基于明胶蛋白的信号复合体，该复合体包括c-src、PI3-激酶及其产物磷脂酰肌醇三磷酸(PtdIns P3)。明胶蛋白是一种肌动蛋白结合蛋白，具有多种功能，包括细丝切断和明胶蛋白带刺末端封顶。肌动蛋白低聚体的明胶解帽可导致细丝聚合。我们已经证明，由PI3-激酶激活产生的PtdIns P3参与了骨桥蛋白/α-β3刺激的肌动蛋白细丝的形成和足体的组装/拆卸。明胶蛋白缺失的转基因小鼠无法在破骨细胞中表达足体，运动能力低下。其结果是骨吸收的比率显著降低。该提案的具体目的是：1)分析足小体组装/拆解的机制；2)确定明胶结合的信号复合体的形成机制。具体目标1的研究将集中在蛋白酪氨酸激酶在基于明胶的信号复合体组装中的作用。此外，将使用各种明胶蛋白构建物来尝试挽救明胶蛋白缺失的表型并刺激足体的形成。具体目标2的研究将分析肌醇磷脂在识别与明胶蛋白相关的信号分子的SH2结构域中的作用。将分析磷脂酰肌醇与SH2结构域短肽的结合部位。这些研究应该勾勒出开发能够调节破骨细胞功能的短肽试剂所需的结构。基于多肽的SH2结构域/PtdIns P3/Gelsolin组织信号的操纵具有潜在的药理学操纵作用。对于许多疾病，特别是骨质疏松症，设计用于控制骨细胞功能的治疗药物仍然是非常需要的，因此这些研究是非常重要的。该项目将提供对骨吸收机制的基本见解，并为其药物控制提供潜在的可能性。

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.