Güller Meryem, Toualbi-Abed Kahina, Legrand Agnès, Michel Laurence, Mauviel Alain, Bernuau Dominique, Daniel Fanny
Institut National de la Sante et de la Recherche Medicale U773, Hôpital Saint-Louis, and Université Paris 7 Denis Diderot, Centre de Recherche Bichat Beaujon CRB3, BP 416, Paris F-75018, France.
World J Gastroenterol. 2008 Nov 7;14(41):6339-46. doi: 10.3748/wjg.14.6339.
To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation.
IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3beta and epidermal growth factor receptor (EGF-R) were assayed by Western blotting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments.
Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpressing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3beta, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein compared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyrosine kinase inhibitor, prevented the phosphorylation of GSK-3beta induced by serum stimulation and decreased Cyclin D1 stability in the nucleus.
Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.
研究稳定过表达c-Fos对永生化人肝细胞（IHH）增殖的影响。
将稳定转染c-Fos（IHH-Fos）或空载体（IHH-C）的IHH细胞在补充有1%血清的培养基中培养或用10%血清刺激。通过细胞计数、3H-胸腺嘧啶核苷摄取和流式细胞术分析评估细胞增殖。通过蛋白质印迹法检测细胞周期调节蛋白（细胞周期蛋白D1、E、A）、细胞周期蛋白依赖性激酶（cdk）cdk2、cdk4、cdk6及其抑制剂p15、p16、p21、p27、总及磷酸化的糖原合成酶激酶-3β（GSK-3β）和表皮生长因子受体（EGF-R）的水平。通过逆转录-聚合酶链反应和实时聚合酶链反应（PCR）分析进行细胞周期蛋白D1 mRNA水平的分析。通过放线菌酮阻断实验研究细胞周期蛋白D1的稳定性。
稳定过表达c-Fos在低血清条件下增加细胞增殖，并导致血清添加后[3H]-胸腺嘧啶核苷掺入增加两倍。流式细胞术细胞周期分析表明，c-Fos加速了细胞周期动力学。血清刺激后，细胞周期蛋白D1在过表达c-Fos的细胞中表达更为丰富。细胞周期蛋白D1的积累不是由于转录激活增加，而是由于核内稳定。与IHH-C相比，在血清饥饿和血清刺激的细胞中，c-Fos的过表达与核内无活性磷酸化GSK-3β水平升高相关，GSK-3β是一种参与细胞周期蛋白D1降解的激酶，且EGF-R mRNA和EGF-R蛋白水平升高。用特异性EGF-R酪氨酸激酶抑制剂AG1478处理IHH-Fos中的EGF-R信号通路，可防止血清刺激诱导的GSK-3β磷酸化，并降低细胞核中细胞周期蛋白D1的稳定性。
我们的结果清楚地表明c-Fos在肝细胞细胞周期调节中具有积极作用。重要的是，我们阐明了一种新机制，通过该机制c-Fos可通过稳定细胞核内的细胞周期蛋白D1促进肝癌发生，为c-Fos在肝细胞癌中的作用引入了一个新特征。
