REGULATION OF MESANGIAL CELL PROLIFERATION BY SPARC

肾小球系膜细胞增殖与肾小球系膜细胞扩张 肾小球系膜细胞外基质似乎是许多 肾小球疾病的类型。此应用程序试图确定如何 肾小球系膜细胞在SPARC介导的信号背景下生长 分泌的蛋白质，调节内皮细胞的行为和 成纤维细胞。我们提出了三个目标来确定这种Ca+2-和Cu2+- 结合蛋白控制系膜细胞的形态和增殖。 SPARC的具体活动被分配到不同的领域 通过合成肽和结构域特异性研究该分子 抗体。这些多肽研究的一个重要扩展是使用 重组突变以确定SPARC的活性位点。它是 我们的假设是SPARC是系膜细胞生长所必需的，而且 一个内部的阳离子铜离子结合结构域包含生物活性元素 系膜细胞增殖所必需的。我们将通过以下方式验证我们的假设 对特定突变体的生物活性的评估。突变者 将通过体外寡核苷酸引导的突变进行工程改造， 在大肠杆菌中表达，并通过镍-螯合亲和纯化 层析法。重组SPARC、突变体和多肽片段将 检测它们结合铜+2的能力和影响表型的能力 和系膜细胞周期的进展。扩散将是 通过将[~3H]-胸腺嘧啶核苷掺入小鼠细胞DNA来测量 大鼠肾小球系膜细胞。蜂窝传播将是 由四舍五入指数量化。 我们还假设SPARC可以影响形态和 系膜细胞通过依赖钙离子的活动附着。我们 提出了两个钙离子结合区负责抗肿瘤的机制。 SPARC的黏附活性。我们将通过以下方法来验证这一假设 缺乏其中一种钙离子结合的突变体的构建 结构域和这些突变体在多大程度上 结合钙离子，影响细胞形态和细胞铺展。 通过这些目标，我们希望获得定义这一点的有用见解 SPARC是一种胞外蛋白，具有潜在的 在肾小球系膜细胞增殖调控中发挥动态作用 以及细胞与细胞外基质之间的相互作用。 从这个项目中得到的见解将被应用于对 项目2中SPARC的肾脏病理生理学。

The proliferation of glomerular mesangial cells and expansion of mesangial extracellular matrix appear to be common denominators in many types of glomerular disease. This application seeks to determine how mesangial cells grow in the context of signals mediated by SPARC, a secreted protein that modulates the behavior of endothelial cells and fibroblasts. We propose three Aims to determine how this Ca+2-and Cu+2- binding protein controls the shape an proliferation of mesangial cells. Specific activities of SPARC have been assigned to distinct domains of the molecule through studies with synthetic peptides and domain specific antibodies. An important extension of these peptide studies is the use of recombinant mutations to identify the active sites of SPARC. It is our hypothesis that SPARC is required for mesangial cell growth and that an internal, cationic Cu+2-binding domain contains bioactive elements necessary for mesangial proliferation. We will test our hypothesis by the assessment of the biological activities of specific mutants. Mutants will be engineered by in vitro oligonucleotide-directed mutagenesis, expressed in E. coli, and purified by nickel-chelate affinity chromatography. Recombinant SPARC, mutants and peptide fragments will be assayed for their ability to bind Cu+2 and to influence the phenotype and progression of the mesangial cell cycle. Proliferation will be measured by the incorporation of [3H]-thymidine into the cellular DNA of mesangial cells from rat glomeruli. Cellular spreading will be quantified by a Rounding index. We also hypothesize that SPARC can influence the morphology and attachment of mesangial cells through Ca+2-dependent activities. We propose that two Ca+2-binding regions are responsible for the anti- adhesive activity of SPARC. We will test this hypothesis by the construction of mutants that lack one or the other of these Ca+2-binding domains and by the determination of the extent to which these mutants bind Ca+2 and affect cell shape and cellular spreading. Through these Aims, we hope to acquire useful insights that define that active sites of SPARC, an extracellular protein with the potential to play a dynamic role in the regulations of mesangial cell proliferation and in the interaction between cells and their extracellular matrix. Insights derived from this project will be applied to studies of the renal pathophysiology of SPARC in project #2.