Cebpd調控GSK3β-Cyclin D1參與在methyl methanesulophonate 所造成細胞毒性之機轉探討

Cebpd regulates MMS-mediated cytotoxicity through GSK3β-Cyclin D1 pathways

王聖文(Sheng-Wen Wang)

指導教授 ： 黃阿梅

高雄醫學大學/醫學院/生物化學研究所/碩士(2010年)

摘要

C/EBP轉錄調控因子(CCAAT/Enhancer Binding Protein)家族具有basic leucine zipper 以及與DNA結合部位，可以分別形成homo-或hetero-dimer和DNA結合，進而進行轉錄功能。C/EBP家族有Cebpa、Cebpb、Cebpg、Cebpd、Cebpe以及Cebpz六個成員。Cebpd首先在發炎反應中被發現，往後研究發現Cebpd在許多不同種類的細胞中都會經由外來刺激而表現，具有調控細胞複製、分化、代謝、發炎反應以及維持染色體穩定等角色。在先前的研究發現，缺乏cebpd的老鼠與正常老鼠外型上沒有很大的差異，然而在缺乏cebpd的老鼠胚胎纖維母細胞中發現有染色體不穩定的現象。Cyclin D1是cyclin- dependent kinase 4 and 6 (CDK4/6)的異位調控者，當Cyclin D1的T286磷酸化時，會結合CDK4/6促使G1到S的細胞週期進行，此磷酸化主要是由glycogen synthase kinase 3 beta (GSK3β)所調控。另外有研究指出在老鼠乳腺細胞中發現Cebpd的表現與Cyclin D1表現量下降的情形有密切的關係。因此本研究提出假設推論Cebpd可作為抑制腫瘤的功能可能主要是透過GSK3β進而調控Cyclin D1的降解。
Methyl methanesulophonate (MMS)是一種DNA烷化劑，會在DNA上加上甲基進而形成DNA adducts對細胞產生損害。本研究利用老鼠胚胎纖維母細胞(MEFs)探討Cebpd參與在MMS對細胞毒性作用的分子機轉。首先發現Cebpd KO MEFs (NKO)相較於WT MEFs (NWT)對MMS有較高的抗性。NKO在MMS刺激下會降低GSK3β的活性及Cyclin D1的降解速率，所以從中可以推測Cebpd可參與GSK3β調控Cyclin D1的降解過程。另外也發現MMS的刺激在NWT都可誘導Cebpd、phospho-Ser-15 p53以及p21蛋白質量增加；有趣的是在NKO中phospho-Ser-15 p53 並沒有受到MMS刺激下而被誘導出來，但p21蛋白質仍可被有效的誘導出。MMS刺激之下不管Cebpd的存在與否都可誘導出磷酸化p38，所以推測Cebpd可能參與p38與p53的交互作用進而調控細胞的行為。
綜合以上實驗結果，顯示Cebpd在MEFs受到基因致毒物時對於調控細胞凋亡、DNA修復和細胞週期扮演重要角色。未來可以更加深入探討在MMS所誘發的細胞毒性相關分子機制，以釐清Cebpd在細胞中所扮演的角色與功能。

關鍵字

Cebpd調控GSK3β-Cyclin D1

並列摘要

C/EBP (CCAAT/enhancer binding protein) proteins are a family of basic-leucine zipper (bZIP) transcription factors that includes six members: Cebpa, Cebpb, Cebpg, Cebpd, Cebpe and Cebpz. Cebpd was first characterized as an acute phase inflammatory response gene. Cebpd is expressed in various tissues and cell types and involved in the control of cellular proliferation, differentiation, metabolism, inflammation and maintenance of genomic stability. Cebpd null mice are fertile and have no overt phenotypes. However, Cebpd knockout mouse embryonic fibroblasts (MEFs) showed severe chromosomal rearrangement in vitro. Cyclin D1 is an allosteric regulator for cyclin-dependent kinase 4 and 6 (CDK4/6) and promotes G1/S transition through phosphorylation at T286 by several kinases including GSK3β. Cebpd induction during involution correlated with repression of Cyclin D1 expression in mouse mammary glands. Thus, we hypothesizes that Cebpd executes its tumor suppressor functions by down-regulating Cyclin D1 through GSK3β.
In this study, an alkylating agent methyl methanesulophonate (MMS) which mainly causes DNA adducts by adding methyl groups to DNA, was used to treat mouse embryonic fibroblasts (MEFs). Cebpd knockout MEFs (KO) showed higher resistance to MMS when compared to wild type (WT). Cebpd, phospho-Ser-15 p53 was induced by MMS in WT but diminished in KO MEFs. However, p21 protein and p38 phosphorylation can be induced by MMS regardless of p53 activation and Cebpd expression level. In addition, MMS reduced GSK3β activation and delayed Cyclin D1 degradation was observed in Cebpd KO cells. These data demonstrates that Cebpd may play an important role in regulating GSK3β-dependent degradation of cyclin D1 and responsible for MMS-mediated cytotoxicity.
In conclusion, these results indicate that Cebpd plays an important role in regulating apoptosis, DNA repair and cell cycle in MEFs under genotoxic stresses. The underlying molecular mechanisms by which Cebpd might involve in MMS- induced cytotoxicity responses need to be further explored.

並列關鍵字

Cebpd

參考文獻

參考文獻

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