Choudhury G G, Marra F, Kiyomoto H, Abboud H E
Department of Medicine, University of Texas, Health Science Center at San Antonio, USA.
Kidney Int. 1996 Jan;49(1):19-25. doi: 10.1038/ki.1996.3.
Platelet-derived growth factor (PDGF) exerts multiple effects in glomerular mesangial cells, including transcription of genes that mediate its biological activity. We have partially characterized PDGF-mediated early mitogenic signal transduction pathways that include activation of protein kinase C alpha and phosphatidylinositol 3 kinase. However, the precise mechanism of PDGF-induced gene transcription is not yet clear. A family of cytoplasmic transcription factors referred to as signal transducers and activators of transcription (STAT) has recently been identified. This group of transcription factors is activated by different cytokines via tyrosine phosphorylation. We studied the effect of PDGF on STATs in human mesangial cells. Using a gel retardation assay, nuclear and cytoplasmic extracts from PDGF-stimulated mesangial cells contained protein factors that bind to a DNA sequence representing the sis-inducible element (SIE) present in the c-fos gene promoter. These protein factors also bind to the enhancer element present in interferon-gamma responsive genes, suggesting the involvement of STAT proteins. The addition of monoclonal antibody that recognizes STAT 1 results in "supershift" of the DNA-protein complex stimulated by PDGF indicating the presence of STAT 1. Immunoblotting experiments with a monoclonal STAT 1 antibody revealed the presence of STAT1 alpha and STAT1 beta in mesangial cells. Since certain cytokines activate STATs via tyrosine phosphorylation mediated by JAK family of tyrosine kinases, we studied the effect of PDGF on JAK kinases. Antiphosphotyrosine immunoblotting of JAK 1 immunoprecipitates from PDGF-stimulated mesangial cell lysate showed increased tyrosine phosphorylation of this tyrosine kinase. In vitro immune complex kinase assay of JAK 1 immunoprecipitates from PDGF-stimulated mesangial cell lysate revealed activation of this tyrosine kinase. Taken together, these data demonstrate that PDGF activates the transcription factor STAT 1 in mesangial cells. The data also provide the first evidence that PDGF stimulates tyrosine phosphorylation of JAK 1, the cytoplasmic tyrosine kinase stimulated by many other cytokines to activate transcription via STATs. These observations indicate that JAK 1 is a downstream tyrosine kinase in PDGF receptor signaling and is a candidate for activation of STAT 1.
血小板衍生生长因子（PDGF）在肾小球系膜细胞中发挥多种作用，包括介导其生物活性的基因转录。我们已部分阐明了PDGF介导的早期促有丝分裂信号转导途径，其中包括蛋白激酶Cα和磷脂酰肌醇3激酶的激活。然而，PDGF诱导基因转录的确切机制尚不清楚。最近发现了一类被称为信号转导子和转录激活子（STAT）的细胞质转录因子家族。这组转录因子通过酪氨酸磷酸化被不同的细胞因子激活。我们研究了PDGF对人系膜细胞中STAT的影响。使用凝胶阻滞试验，PDGF刺激的系膜细胞的核提取物和细胞质提取物中含有与代表c-fos基因启动子中sis诱导元件（SIE）的DNA序列结合的蛋白质因子。这些蛋白质因子也与干扰素-γ反应基因中的增强子元件结合，提示有STAT蛋白参与。加入识别STAT 1的单克隆抗体导致PDGF刺激的DNA-蛋白质复合物出现“超迁移”，表明存在STAT 1。用单克隆STAT 1抗体进行的免疫印迹实验显示系膜细胞中存在STAT1α和STAT1β。由于某些细胞因子通过JAK家族酪氨酸激酶介导的酪氨酸磷酸化激活STAT，我们研究了PDGF对JAK激酶的影响。对PDGF刺激的系膜细胞裂解物中JAK 1免疫沉淀物进行抗磷酸酪氨酸免疫印迹显示，该酪氨酸激酶的酪氨酸磷酸化增加。对PDGF刺激的系膜细胞裂解物中JAK 1免疫沉淀物进行体外免疫复合物激酶测定显示该酪氨酸激酶被激活。综上所述，这些数据表明PDGF在系膜细胞中激活转录因子STAT 1。这些数据还首次证明PDGF刺激JAK 1的酪氨酸磷酸化，JAK 1是一种细胞质酪氨酸激酶，被许多其他细胞因子刺激以通过STAT激活转录。这些观察结果表明JAK 1是PDGF受体信号传导中的下游酪氨酸激酶，是激活STAT 1的候选激酶。
