GELSOLIN BASED SIGNALING IN OSTEOCLAST FUNCTION

本申请的长期目标是阐明凝溶胶蛋白和相关信号传导复合物在足体组装/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已经取得了重大进展。 破骨细胞是高度运动的细胞，并且它们利用独特类型的细胞/基质相互作用，即podosome，来产生高速率的细胞运动性。 足状体通过α v β 3整联蛋白与基质连接，并具有专门的细胞骨架缔合。 用于刺激骨吸收的整联蛋白α v β 3配体介导的信号传导涉及在足体中基于凝溶胶蛋白的信号传导复合物的活化，所述信号传导复合物包括c-src、PI 3-激酶及其产物三磷酸磷脂酰肌醇（PtdIns P3）。 凝溶胶蛋白是一种肌动蛋白结合蛋白，具有多种功能，包括切断纤维和凝溶胶蛋白倒刺端帽。 肌动蛋白寡聚体的凝溶胶蛋白脱帽导致细丝聚合。 我们已经证明，由PI 3-激酶激活产生的PtdIns P3参与骨桥蛋白/α v β 3刺激的肌动蛋白丝的形成和podosome装配/拆卸。 无凝溶胶蛋白的转基因小鼠在其破骨细胞中不能表达足体，并且运动迟缓。 结果是骨吸收率显著降低。 该提案的具体目的是：1）分析podosome组装/拆卸的机制; 2）确定凝溶胶蛋白相关信号复合物形成的机制。 具体目标1中的研究将集中于蛋白酪氨酸激酶在基于凝溶胶的信号传导复合物的组装中的作用。 此外，将使用各种凝溶胶蛋白构建体来试图拯救凝溶胶蛋白无效表型并刺激足体形成。 具体目标2中的研究将分析磷酸肌醇在识别与凝溶胶蛋白相关的信号分子的SH 2结构域中的作用。将分析磷酸肌醇与SH 2结构域的短肽的结合位点。 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。 基于肽的操纵SH 2结构域/PtdIns P3/凝溶胶蛋白有组织的信号传导具有药理学操纵的潜力。仍然存在对设计用于控制许多疾病，特别是骨质疏松症中的骨细胞功能的治疗剂的巨大需求，因此这些研究是极其重要的。 该项目应提供骨吸收机制的基本见解，并产生潜在的药理学控制。

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.