PHOSPHORYLATION IN RENAL CELL INJURY

描述（改编自申请人摘要）：血小板源性生长 因子受体B（PDGFR）在受损的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，表型表现在许多肾小球疾病 包括系膜增生性肾小球肾炎（GN）。我们最近 证明PDGFR刺激的磷脂酰肌醇3激酶（PI 3 K） 活性对于培养的系膜细胞的增殖和迁移是必需的 细胞丝氨酸苏氨酸激酶Akt已被鉴定为下游激酶。 PI 3 K的目标我们的假设是Akt调节系膜细胞活化 这包括这些细胞的增殖和迁移， 损伤我们建议表征Akt在培养细胞中发挥作用的途径， 系膜细胞和体内抗Thy-1诱导的大鼠GN模型。Akt 在GN进展期间测定激酶活性。Akt的作用 将通过检查激酶在系膜细胞活化中的作用来确定 这种蛋白质的显性负性和组成性活性版本在 系膜细胞增殖和迁移。调节Akt激酶的蛋白 活性或该酶的代表底物将使用 酵母双杂交蛋白质相互作用策略。打开的阅读帧 相互作用的蛋白质将通过核苷酸测序来确定。 这些蛋白质的表征将通过提高抗肽 和GST融合蛋白抗体。Akt活性的调节， Akt相关蛋白将在体外和培养的系膜中进行研究 细胞这些蛋白质在涉及Akt和调节Akt的途径中的作用， 将测定系膜细胞增殖和迁移。 我们的初步数据表明，PDGFR酪氨酸激酶和 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）的丝氨酸苏氨酸激酶， TGF β超家族，在无基质存在下抑制PDGF诱导的DNA合成 扩张.这种抑制作用是由于抑制PDGF诱导的Erk 1/2类型的 MAPK（丝裂原活化蛋白激酶）。在第二个具体目标中，我们将 使用BMP-2在治疗方法中治疗大鼠系膜增生性GN。 将构建表达BMP-2的腺病毒载体。腺病毒 基因转移和工程系膜细胞载体将用于表达 BMP-2在体内抑制GN系膜细胞增殖，而不诱导 细胞外基质扩张PI 3激酶，MAPK和Akt的活性将被检测到。 在来自载体靶向动物的肾小球裂解物中测定。这些 研究将确定参与肾小球疾病的重要信号机制， 病理学，并帮助建立有效的治疗模式， 增生性肾小球肾炎的症状

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.