Choudhury G G
Department of Medicine, University of Texas Health Science Center at San Antonio, 78229-3900, USA.
J Biol Chem. 2001 Sep 21;276(38):35636-43. doi: 10.1074/jbc.M100946200. Epub 2001 Jul 26.
Proliferation of mesangial cells requires platelet-derived growth factor receptor beta (PDGFR)-mediated signal transduction. We have previously shown that activation of phosphatidylinositol (PI) 3-kinase is necessary for PDGFR-induced DNA synthesis in these cells. The mechanism by which PI 3-kinase stimulates DNA synthesis is not known. One target of PI 3-kinase, Akt serine threonine kinase, regulates survival of many cells by inhibiting the actions of certain proapoptotic proteins. In this study, we investigated the role of Akt in PDGF-induced DNA synthesis in mesangial cells. PDGF increased Akt serine threonine kinase activity in a time- and PI 3-kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced activation of endogenous Akt in mesangial cells, resulting in complete inhibition of DNA synthesis. On the other hand, inhibition of MAPK attenuated PDGF-induced DNA synthesis only partially. Inhibition of Akt also attenuated PDGF-induced c-fos gene transcription, with concomitant inhibition of Elk-1-dependent transcription, indicating positive regulation of this early response gene by Akt. To further determine the role of Akt in PDGF-induced DNA synthesis, we investigated its effect on cyclin-dependent kinase 2 (CDK2). PDGF stimulated CDK2 activity in mesangial cells and decreased the level of p27(kip1) cyclin kinase inhibitor protein. Expression of dominant negative Akt increased p27(kip1) protein and resulted in inhibition of CDK2 activity. The increase in p27(kip1) expression in response to Akt kinase inhibition was due to increased transcription of the p27(kip1) gene. p27(kip1) transcription similarly was decreased by expression of constitutively active Akt kinase in mesangial cells. These data provide the first evidence that Akt kinase regulates PDGF-induced DNA synthesis by regulating CDK2 activity and define Akt-mediated inhibition of transcription of p27(kip1) as one of the mechanisms for PDGF-induced DNA synthesis in mesangial cells.
系膜细胞的增殖需要血小板衍生生长因子受体β（PDGFR）介导的信号转导。我们先前已表明，磷脂酰肌醇（PI）3激酶的激活对于这些细胞中PDGFR诱导的DNA合成是必需的。PI 3激酶刺激DNA合成的机制尚不清楚。PI 3激酶的一个靶点Akt丝氨酸苏氨酸激酶，通过抑制某些促凋亡蛋白的作用来调节许多细胞的存活。在本研究中，我们调查了Akt在血小板衍生生长因子（PDGF）诱导的系膜细胞DNA合成中的作用。PDGF以时间和PI 3激酶依赖性方式增加Akt丝氨酸苏氨酸激酶活性。通过腺病毒介导的基因转移表达显性负性Akt可阻断PDGF诱导的系膜细胞内源性Akt的激活，从而完全抑制DNA合成。另一方面，抑制丝裂原活化蛋白激酶（MAPK）仅部分减弱PDGF诱导的DNA合成。抑制Akt也减弱了PDGF诱导的c-fos基因转录，并同时抑制了Elk-1依赖性转录，表明Akt对该早期反应基因有正向调节作用。为了进一步确定Akt在PDGF诱导的DNA合成中的作用，我们研究了其对细胞周期蛋白依赖性激酶2（CDK2）的影响。PDGF刺激系膜细胞中的CDK2活性并降低p27（kip1）细胞周期蛋白激酶抑制蛋白的水平。显性负性Akt的表达增加了p27（kip1）蛋白并导致CDK2活性受到抑制。响应Akt激酶抑制而导致的p27（kip1）表达增加是由于p27（kip1）基因转录增加所致。在系膜细胞中组成型活性Akt激酶的表达同样降低了p27（kip1）转录。这些数据首次证明Akt激酶通过调节CDK2活性来调控PDGF诱导的DNA合成，并将Akt介导的对p27（kip1）转录的抑制定义为系膜细胞中PDGF诱导的DNA合成机制之一。
