Transcriptional regulation of cyclooxygenase-2 gene expression by epidermal growth factor in A431 cells

• Main Authors: Lei-Chin Chen, 陳麗琴
• Other Authors: Wen-Chang Chang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/11908959658483444114

博士 === 國立成功大學 === 基礎醫學研究所 === 93 === 　Transcriptional activation of the cyclooxygenase-2 (COX-2) gene is responsible for high level of prostaglandin production during inflammation and carcinogenesis. In this study, the transcriptional regulation of COX-2 expression induced by epidermal growth factor (EGF) in human epidermoid carcinoma A431 cells was studied. EGF treatment induced the expression of COX-2 mRNA, protein, promoter and enzyme activity in a time-dependent manner. EGF-induced COX-2 promoter activity was inhibited by over-expression of the dominant-negative forms of Ras and ERK2. Induction of COX-2 and c-Jun by EGF was completely suppressed by MEK inhibitor combined with JNK inhibitor. Analysis of the EGF-responsive element on COX-2 promoter and COX-2 promoter binding proteins by reporter assay, gel mobility shift assay, DNA affinity precipitation assay and chromatin immunoprecipitation assay revealed that c-Jun and p300 binding to CRE/E-box site were responsible for the EGF-induced COX-2 gene transcription. Over-expression of p300 significantly enhanced COX-2 promoter activity in cells overexpressed of c-Jun or treated with EGF. EGF and c-Jun-induced transcription of COX-2 promoter was repressed by cotransfection of E1A in a dose-dependent manner. All together, these results indicated that the EGF-induced expression of COX-2 in A431 cells was mediated through the Ras-ERK/JNK signaling pathway, and subsequent induction of c-Jun following MAPK activation, in cooperation with coactivator p300, was required for the EGF response. In studying of the functional role of c-Jun in EGF-induced transcriptional activation of COX-2 in A431 cells, we found that SP600125, a pharmacological inhibitor of JNK, efficiently inhibited c-Jun N-terminal Ser-63 and Ser-73 phosphorylation, but failed to attenuate COX-2 expression induced by EGF. Over-expression of c-Jun N-terminal phosphorylation sites mutants had similar stimulatory effects on COX-2 promoter activity and protein expression as wild-type c-Jun. TAM-67, a mutant of c-Jun which lacks the N-terminal transactivation domain of c-Jun, also enhanced COX-2 promoter activity and protein expression in cells treated with EGF. In vitro DNA affinity precipitation assay and reporter assay revealed that unphosphorylated form of c-Jun C-terminus mutant enhanced c-Jun binding to COX-2 promoter and COX-2 promoter activity. Furthermore, in vitro DNA affinity precipitation assay, chromatin immunoprecipitation assay and GAL4 reporter assay systems demonstrated that c-Fos providing its transactivation function in Jun/Fos heterodimer was required for EGF-induced expression of COX-2. Moreover, cooperation of c-Jun with C/EBPdelta (NF-IL6beta) was also required for EGF-induced expression of COX-2. These results indicated that c-Jun N-terminal phosphorylation was not required for EGF-induced expression of COX-2. c-Jun acting as an anchor protein to recruit other transcription factors like c-Fos and C/EBPdelta which provided their transactivation activity was required for EGF-induced expression of COX-2 in A431 cells.