在發炎組織以及癌化的過程中，環氧酵素二型(cyclooxygenase-2, COX-2)可被誘導表現增加，進而代謝花生四烯酸(Arachidonic acid)產生的前列腺素(prostaglandins)扮演重要的角色。在本研究中，我們針對表皮生長因子(Epidermal Growth Factor, EGF)誘導COX-2表現的轉錄調控機制做深入的探討。A431細胞在EGF處理之下，可隨著EGF處理時間的增加，可誘導COX-2 mRNA、蛋白質、promoter以及酵素活性增加；當細胞送入dominant negative mutant型態的Ras以及ERK2，可將EGF誘導的COX-2 promoter活性抑制下來，而且當細胞同時處理MEK以及JNK抑制劑可以將EGF誘導的COX-2以及c-Jun表現完全抑制。接著我們利用reporter assay、凝膠電泳位移測定(Electrophoretic mobility shift assay)、DNA親和免疫沈澱分析(DNA affinity precipitation assay)以及染色質免疫沈澱分析方法，得知EGF誘導COX-2基因轉錄機制中需要c-Jun以及p300結合到COX-2 promoter上CRE/E-box element。同時當細胞過度表現p300，可以增加COX-2 promoter活性，而且此現象可被EGF處理或同時過度表現c-Jun更加增強。另外，隨著細胞送入E1A量的增加，可以抑制EGF以及c-Jun誘導的COX-2 promoter活性。由以上實驗結果得知在A431細胞中，EGF主要經由Ras-ERK/JNK訊息傳遞路徑誘導COX-2表現增加。而MAPK活化誘導產生的c-Jun與p300的共同合作參與在EGF誘導COX-2轉錄機制之中。分析c-Jun在EGF誘導COX-2轉錄活化的功能角色的研究中，我們發現JNK抑制劑(SP600125)可以有效的抑制c-Jun N端Ser-63以及Ser-73的磷酸化，但卻無法抑制EGF誘導COX-2的表現。若將細胞過度表現c-Jun N端磷酸化位置突變質體則可與wild type c-Jun一樣，對COX-2 promoter以及蛋白質表現依然具有誘導作用，而且當細胞送入不具N端transactivation domain的c-Jun突變質體-TAM-67，於EGF處理之下也可以誘導COX-2 promoter以及蛋白質表現增加。由in vitro DNA親和免疫沈澱分析以及reporter assay實驗結果顯示c-Jun C端無法磷酸化的突變質體比wild type c-Jun具有更好的COX-2 promoter活性誘導能力。另外由DNA親和免疫沈澱分析、染色質免疫沈澱分析以及GAL4報告者分析系統等實驗的結果証明細胞在EGF處理之下c-Fos提供了本身的transactivation功能給c-Jun/c-Fos異質雙體，進而促使COX-2表現增加。同時我們也由實驗證實c-Jun與C/EBPdelta (NF-IL6beta)的共同合作，也參與在EGF誘導COX-2表現機制之中，由以上實驗結果顯示EGF誘導COX-2表現的機制中，並不需要c-Jun N端的磷酸化，而c-Jun扮演著anchor蛋白質的角色，幫助recruit其他提供transactivation活性的轉錄因子，例如c-Fos以及C/EBPdelta進而促使COX-2表現增加。

　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.

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