目前已知EGF可經由c-Jun/Sp1此複合體調控與癌症密切相關的基因如12(S)-lipoxygenase 和p21WAF1/CIP1的表現，在此c-Jun透過跟Sp1交互作用而結合到沒有AP1的結合序列的啟動區上，進而去調控該基因的表現。然而在EGF訊息傳遞路徑下，是否尚有其他未知的因子可以經由這樣的機制來調控基因的表現呢？於是我們利用蛋白質體學的技術，分析在EGF處理下與Sp1結合情況有明顯改變的蛋白質，發現ARNT此一屬於hypoxia inducible factor-1的轉錄因子在EGF處理下與Sp1結合有明顯的增加。這可能暗示著ARNT可能在EGF的訊息傳遞路徑下扮演了某些角色，基於此，我們便試著去探討ARNT是否有可能會參與在EGF所調控的基因表現過程中。首先從免疫沉澱的實驗中得知EGF可促進 ARNT與c-Jun、Sp1 的交互作用，而在DNA親和免疫沈澱分析中得知ARNT/c-Jun/Sp1複合體可以結合到12(S)-lipoxygenase啟動區上。將細胞中的Sp1 knock down後發現ARNT與12(S)-lipoxygenase啟動區結合有抑制的情形，且 overexpression ARNT並無法活化Sp1 binding site突變的12(S)-lipoxygenase啟動區，顯示ARNT調控12(S)-lipoxygenase的基因表現需透過Sp1。使用ARNT siRNA將ARNT knock down後發現EGF所induce的12(S)-lipoxygenase啟動區的活性及mRNA都有抑制的現象，而大量表現ARNT則會活化該啟動區活性，證實了ARNT可透過transcriptional level來調控該基因的表現。而在ARNT/c-Jun/Sp1調控12(S)-lipoxygenase的基因表現中，c-Jun為主要的轉錄活化因子負責轉錄活化基因的表現，ARNT對基因的轉錄活化作用在此並不是那麼重要。在免疫沉澱的實驗中，我們發現c-Jun與Sp1的交互作用會因ARNT knock down而減少，因此ARNT很有可能經由影響c-Jun與Sp1的交互作用而參與在12(S)-lipoxygenase的基因調控中。除了12(S)-lipoxygenase 外，其他由c-Jun/Sp1所調控的基因，如p21WAF1/CIP1也有可能受到相同的調控機制，此結果顯示ARNT可以在normoxia的情形下參與c-Jun/Sp1所調控的基因表現，此研究也為hypoxia inducible factor促進腫瘤生長的機制提供了一個新的解釋。

It is well documented that EGF regulates the gene expression including 12(S)-lipoxygenase and p21WAF1/CIP1 through the transcription factor c-Jun and Sp1. However, the signaling molecules involved in EGF-responsive gene expression are not totally calified. Here we used the immunoprecipitation and followed by MALDI-TOF to analyze Sp1-associated proteins. We found that ARNT, a hypoxia related transcription factor, co-immunoprecipitated with Sp1. This result suggested that ARNT may play a role in EGF signaling event under physicial condiction. We found that EGF stimulated the formation of c-Jun/ARNT/Sp1 complexs as well as the binding of the transcription factor complex to the gene promoter in a time-dependent manner. ARNT small interfering RNA (siRNA) inhibited the promoter activity and the mRNA level of 12(S)-lipoxygenase in cells treated with EGF. Furthermore, overexpression of ARNT increased the 12(S)-lipoxygenase promoter activity. We also identified that Sp1 binding site of 12(S)-lipoxygenase promoter was responsible for overexpressing ARNT activation. In addition, ARNT apparently acted via c-Jun because expression of c-Jun mutant nullified the action of ARNT. Contrary to the transcriptional activity of c-Jun, ARNT mediated the interaction between c-Jun and Sp1 in EGF-treated cells. Furthermore, ARNT siRNA also inhibited EGF-induced expression of p21WAF1/CIP1. These results revealed that ARNT mediated c-Jun/Sp1-regulated gene expression under normoxic condition and provided an additional mechanism in considering the HIF factor as regulator of tumor growth.

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