GELSOLIN BASED SIGNALING IN OSTEOCLAST FUNCTION

破骨细胞功能中基于凝溶胶蛋白的信号传导

• 批准号: 6532990
• 负责人: MEENAKSHI A CHELLAIAH
• 金额: $ 20.69万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6532990
• 关键词: actin binding protein; binding sites; biological signal transduction; cell cell interaction; cell motility; chickens; extracellular matrix; fluorescence microscopy; gelsolin; genetically modified animals; immunocytochemistry; immunoprecipitation; laboratory mouse; microinjections; osteoclasts; pathologic bone resorption; phosphatidylinositols; polymerase chain reaction; protein biosynthesis; protein degradation; protein signal sequence; protein tyrosine kinase; tissue /cell culture; western blottings

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.

该应用的长期目标是阐明凝溶胶蛋白和相关信号复合物在足体组装/拆卸和破骨细胞功能中的作用。对与骨吸收相关的破骨细胞组织的细胞生物学的理解已经取得了重大进展。 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（足体）来产生高细胞运动率。 足体通过 alphavbeta3 整合素连接至基质，并具有专门的细胞骨架关联。 用于刺激骨吸收的整合素 αvβ3 配体介导的信号传导涉及足体中基于凝溶胶蛋白的信号复合物的激活，其中包括 c-src、PI3-激酶及其产物磷脂酰肌醇三磷酸 (PtdIns P3)。 凝溶胶蛋白是一种具有多种功能的肌动蛋白结合蛋白，包括细丝切断和凝溶胶蛋白倒刺末端加帽。 肌动蛋白寡聚物的凝溶胶蛋白脱帽导致丝聚合。 我们已经证明，由 PI3 激酶激活产生的 PtdIns P3 参与骨桥蛋白/alphavbeta3 刺激的肌动蛋白丝形成和足小体组装/拆卸。 凝溶胶蛋白无效的转基因小鼠不能在其破骨细胞中表达足小体并且运动能力低下。 结果是骨吸收率显着降低。 该提案的具体目标是：1）分析足体组装/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 的研究将重点关注蛋白酪氨酸激酶在基于凝溶胶蛋白的信号复合物组装中的作用。 此外，将使用各种凝溶胶蛋白构建体来尝试拯救凝溶胶蛋白无效表型并刺激足小体形成。 具体目标 2 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 SH2 结构域中的作用。将分析磷酸肌醇与 SH2 结构域短肽的结合位点。 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需的结构。 基于肽的 SH2 结构域/PtdIns P3/凝溶胶蛋白组织信号传导的操作具有药理学操作的潜力。对于许多疾病，特别是骨质疏松症，对于控制骨细胞功能的治疗剂仍然存在巨大的需求，因此这些研究极其重要。 该项目应为骨吸收机制提供基本见解，并为其药理学控制提供潜力。