PHOSPHORYLATION IN RENAL CELL INJURY

描述(摘自申请者摘要)：血小板源性生长 因子受体b(Pdgfr)在受损肾小球和活化的肾小球中表达。 培养的系膜细胞。PDGFR激活刺激系膜细胞 增殖和迁移，表型在许多肾小球疾病中表现出来 包括系膜增生性肾炎(GN)。我们最近 证实PDGFR刺激的磷脂酰肌醇-3-激酶(PI-3K) 活性是系膜细胞增殖和迁移所必需的 细胞。丝氨酸苏氨酸激酶，Akt，已被确定为下游 PI 3K的目标。我们的假设是Akt调节系膜细胞的激活 包括肾小球过程中这些细胞的增殖和迁移 受伤。我们建议描述Akt在培养细胞中的作用途径。 肾小球系膜细胞和体内抗Thy-1诱导的肾小球肾炎模型。AKT 在肾小球肾炎进展过程中将测定其活性。Akt的角色 肾小球系膜细胞激活中的激酶将通过检测其作用来确定 这种蛋白质的显性负性和结构性活性版本 系膜细胞的增殖和迁移。调节Akt激酶的蛋白质 该酶的活性或代表底物将使用 酵母双杂交蛋白质-蛋白质相互作用策略。的开放阅读框架 相互作用的蛋白质将通过核苷酸测序来确定。 这些蛋白质的特性将通过提高抗肽来实现。 和GST融合蛋白抗体。这些基因对Akt活性的调节 AKT相关蛋白将在体外和培养的系膜细胞中进行研究 细胞。这些蛋白在Akt信号转导通路中的作用及其调控 将测定肾小球系膜细胞的增殖和迁移。 我们的初步数据表明，PDGFR酪氨酸激酶和PDGFR之间的串扰 骨形态发生蛋白受体丝氨酸苏氨酸激酶存在于 系膜细胞。我们最近证明了受体的激活 骨形态发生蛋白2(BMP-2)激活的丝氨酸苏氨酸激酶 TGFb超家族在缺乏基质的情况下抑制PDGF诱导的DNA合成 扩张。这种抑制是由于抑制了PDGF诱导的ERK1/2型 丝裂原活化蛋白激酶(MAPK)。在我们的第二个具体目标中，我们将 骨形态发生蛋白-2在治疗大鼠系膜增生性肾炎中的应用 将构建表达BMP-2的腺病毒载体。腺病毒载体 基因转移和工程系膜细胞载体将用于表达 BMP-2体内对肾小球肾炎系膜细胞增殖的抑制作用 细胞外基质扩张。PI3K、MAPK和Akt的活性将 在以媒介为靶标的动物的肾小球裂解物中确定。这些 研究将确定涉及肾小球的重要信号机制 病理和帮助建立有效的治疗方式 增殖型肾小球肾炎。

DESCRIPTION (Adapted from the Applicant's Abstract): Platelet-derived growth factor receptor b (PDGFR) is expressed in injured glomeruli and in activated cultured mesangial cells. Activation of PDGFR stimulates mesangial cell proliferation and migration, phenotypes manifest in many glomerular diseases including mesangioproliferative glomerulonephritis (GN). We recently demonstrated that PDGFR-stimulated phosphatidylinositol 3 kinase (PI 3K) activity is necessary for proliferation and migration of cultured mesangial cells. A serine threonine kinase, Akt, has been identified as a downstream target of PI 3K. Our hypothesis is that Akt regulates mesangial cell activation which includes proliferation and migration of these cells during glomerular injury. We propose to characterize pathways by which Akt functions in cultured mesangial cells and in vivo in a model of anti-Thy-1-induced GN in rats. Akt kinase activity will be determined during progression of GN. The role of Akt kinase in mesangial cell activation will be determined by examining the effect of dominant negative and constitutively active versions of this protein on mesangial cell proliferation and migration. Proteins that regulate Akt kinase activity or represent substrates for this enzyme will be identified using a yeast two-hybrid protein-protein interaction strategy. Open reading frames of interacting proteins will be determined by nucleotide sequencing. Characterization of these proteins will be carried out by raising antipeptide and GST-fusion protein antibodies. Regulation of the Akt activity by these Akt-associated proteins will be studied in vitro and in cultured mesangial cells. The role of these proteins in pathways involving Akt and regulating mesangial cell proliferation and migration will be determined. Our preliminary data indicate that cross-talk between PDGFR tyrosine kinase and bone morphogenetic protein receptor serine threonine kinases exists in mesangial cells. We have recently demonstrated that activation of receptor serine threonine kinase by bone morphogenetic protein 2 (BMP-2), a member of TGFb superfamily, inhibits PDGF-induced DNA synthesis in the absence of matrix expansion. This inhibition is due to inhibition of PDGF-induced Erk1/2 type of MAPK (mitogen-activated protein kinase). In our second specific aim, we will use BMP-2 in a therapeutic approach to treat mesangioproliferative GN in rats. An adenovirus vector expressing BMP-2 will be constructed. Adenovirus-mediated gene transfer and engineered mesangial cell vectors will be used to express BMP-2 in vivo to inhibit mesangial cell proliferation in GN, without inducing extracellular matrix expansion. Activities of PI 3 kinase, MAPK and Akt will be determined in the glomerular lysates from vector-targeted animals. These studies will identify important signaling mechanisms involved in glomerular pathology and help to establish effective therapeutic modalities for treatment of proliferative forms of GN.