Sharma Chandan, Pradeep Anamika, Pestell Richard G, Rana Basabi
Division of Molecular Cardiology, The Texas A&M University System Health Science Center, College of Medicine, Temple, Texas 76504, USA.
J Biol Chem. 2004 Apr 23;279(17):16927-38. doi: 10.1074/jbc.M309045200. Epub 2004 Feb 5.
Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) following exposure to PPARgamma-specific ligands resulted in growth inhibition in various carcinoma cell lines. Our aim was to elucidate the pathway of PPARgamma2 activation-mediated modulation of cyclin D1 transcription in mouse hepatocytes. To address this we utilized stable control and PPARgamma hepatocyte cell lines created via retroviral overexpression utilizing AML-12 hepatocytes. Addition of PPARgamma ligand troglitazone (TZD) activated PPARgamma2 in proliferating hepatocytes and resulted in growth arrest accompanied by a down-regulation of proliferating cell nuclear antigen, cyclin D1, and beta-catenin expression. Furthermore activation of PPARgamma2 attenuated cyclin D1 promoter activity indicating a transcriptional regulation of cyclin D1. Since beta-catenin plays a pivotal role in regulating cyclin D1 transcription, we studied whether PPARgamma2-mediated inhibition of cyclin D1 transcription involved beta-catenin. Interestingly overexpression of either wild-type or S37A mutant beta-catenin was unable to rescue PPARgamma2-mediated suppression of cyclin D1 transcription, whereas overexpression of cAMP-response element-binding protein (CREB) was capable of antagonizing this inhibitory effect of PPARgamma2. Additionally pretreatment with okadaic acid antagonized PPARgamma2-mediated inhibition of cyclin D1 transcription without any effect on beta-catenin expression. These studies also showed a TZD-mediated inhibition of total and phospho-CREB(Ser133) levels, CREB promoter activity, and cAMP-response element-mediated transcription in PPARgamma hepatocytes. Pretreatment of PPARgamma hepatocytes with okadaic acid, however, maintained higher total and phospho-CREB(Ser133) levels in the presence of TZD. These results indicated that PPARgamma2 activation inhibited cyclin D1 transcription in hepatocytes via CREB-dependent and beta-catenin-independent pathways.
暴露于过氧化物酶体增殖物激活受体γ（PPARγ）特异性配体后，PPARγ的激活导致多种癌细胞系生长受到抑制。我们的目的是阐明PPARγ2激活介导的小鼠肝细胞中细胞周期蛋白D1转录调控的途径。为解决这一问题，我们利用逆转录病毒过表达技术，以AML-12肝细胞为基础创建了稳定的对照和PPARγ肝细胞系。添加PPARγ配体曲格列酮（TZD）可激活增殖肝细胞中的PPARγ2，并导致生长停滞，同时增殖细胞核抗原、细胞周期蛋白D1和β-连环蛋白表达下调。此外，PPARγ2的激活减弱了细胞周期蛋白D1启动子活性，表明细胞周期蛋白D1存在转录调控。由于β-连环蛋白在调节细胞周期蛋白D1转录中起关键作用，我们研究了PPARγ2介导的细胞周期蛋白D1转录抑制是否涉及β-连环蛋白。有趣的是，野生型或S37A突变型β-连环蛋白的过表达均无法挽救PPARγ2介导的细胞周期蛋白D1转录抑制，而环磷酸腺苷反应元件结合蛋白（CREB）的过表达能够拮抗PPARγ2的这种抑制作用。此外，用冈田酸预处理可拮抗PPARγ2介导的细胞周期蛋白D1转录抑制，而对β-连环蛋白表达无任何影响。这些研究还表明，TZD介导了PPARγ肝细胞中总CREB和磷酸化CREB（Ser133）水平、CREB启动子活性以及环磷酸腺苷反应元件介导的转录的抑制。然而，在TZD存在的情况下，用冈田酸预处理PPARγ肝细胞可维持较高的总CREB和磷酸化CREB（Ser133）水平。这些结果表明，PPARγ2激活通过CREB依赖性和β-连环蛋白非依赖性途径抑制肝细胞中细胞周期蛋白D1的转录。
