Choudhury G G, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, Abboud H E
Department of Medicine, University of Texas Health Science Center, San Antonio, USA.
Am J Physiol. 1997 Dec;273(6):F931-8. doi: 10.1152/ajprenal.1997.273.6.F931.
Proliferation and migration are important biological responses of mesangial cells to injury. Platelet-derived growth factor (PDGF) is a prime candidate to mediate these responses in glomerular disease. PDGF and its receptor (PDGFR) are upregulated in the mesangium during glomerular injury. We have recently shown that PDGF activates phosphatidylinositol 3-kinase (PI-3-kinase) in cultured mesangial cells. The role of this enzyme and other more distal signaling pathways in regulating migration and proliferation of mesangial cells has not yet been addressed. In this study, we used two inhibitors of PI-3-kinase, wortmannin (WMN) and LY-294002, to investigate the role of this enzyme in these processes. Pretreatment of mesangial cells with WMN and LY-294002 dose-dependently inhibited PDGF-induced PI-3-kinase activity assayed in antiphosphotyrosine immunoprecipitates. WMN pretreatment also inhibited the PI-3-kinase activity associated with anti-PDGFR beta immunoprecipitates prepared from mesangial cells treated with PDGF. Pretreatment of the cells with different concentrations of WMN resulted in a dose-dependent inhibition of PDGF-induced DNA synthesis. Both WMN and LY-294002 inhibited PDGF-stimulated migration of mesangial cells in a dose-dependent manner. It has recently been shown that PI-3-kinase physically interacts with Ras protein. Because Ras is an upstream regulator of the kinase cascade leading to the activation of mitogen-activated protein kinase (MAPK), we determined whether activation of PI-3-kinase is necessary for activation of MAPK. Pretreatment of mesangial cells with WMN and LY-294002 significantly inhibited PDGF-induced MAPK activity as measured by immune complex kinase assay of MAPK immunoprecipitates. Furthermore, PD-098059, an inhibitor of MAPK-activating kinase inhibited PDGF-induced MAPK activity and resulted in significant reduction of mesangial cell migration in response to PDGF. These data indicate that MAPK is a downstream target of PI-3-kinase and that both these enzymes are involved in regulating proliferation and migration of mesangial cells.
增殖和迁移是系膜细胞对损伤的重要生物学反应。血小板衍生生长因子（PDGF）是介导肾小球疾病中这些反应的主要候选因子。在肾小球损伤期间，系膜中PDGF及其受体（PDGFR）表达上调。我们最近发现，PDGF可激活培养的系膜细胞中的磷脂酰肌醇3激酶（PI-3激酶）。该酶以及其他更远端信号通路在调节系膜细胞迁移和增殖中的作用尚未得到研究。在本研究中，我们使用两种PI-3激酶抑制剂渥曼青霉素（WMN）和LY-294002来研究该酶在这些过程中的作用。用WMN和LY-294002对系膜细胞进行预处理，以剂量依赖的方式抑制了在抗磷酸酪氨酸免疫沉淀中检测到的PDGF诱导的PI-3激酶活性。WMN预处理还抑制了与从用PDGF处理的系膜细胞制备的抗PDGFRβ免疫沉淀相关的PI-3激酶活性。用不同浓度的WMN对细胞进行预处理导致对PDGF诱导的DNA合成的剂量依赖性抑制。WMN和LY-294002均以剂量依赖的方式抑制PDGF刺激的系膜细胞迁移。最近发现PI-3激酶与Ras蛋白发生物理相互作用。由于Ras是导致丝裂原活化蛋白激酶（MAPK）激活的激酶级联的上游调节因子，我们确定PI-3激酶的激活对于MAPK的激活是否必要。用WMN和LY-294002对系膜细胞进行预处理，通过MAPK免疫沉淀的免疫复合物激酶测定法测量，显著抑制了PDGF诱导的MAPK活性。此外，MAPK激活激酶的抑制剂PD-098059抑制了PDGF诱导的MAPK活性，并导致系膜细胞对PDGF的迁移显著减少。这些数据表明MAPK是PI-3激酶的下游靶点，并且这两种酶都参与调节系膜细胞的增殖和迁移。
