在一些與細胞過度增生有關的皮膚疾病 (例如牛皮癬) 中，角質細胞中的角質素十六 (keratin 16) 呈現大量表現。因此，將keratin 16 歸類為與疾病形成相關的角質素。根據之前的研究報告顯示，表皮生長因子 (EGF) 或甲型轉形生長因子 (TGF-α)會促進keratin 16 的啟動區活性，並證實其EGF 反應區間是位於啟動區上-212 至-192bp。而除了此EGF 反應區間外，位於啟動區上-127 至-121 bp 之Sp1 結合序列，對於keratin 16 啟動區之基本轉錄活性亦為必須的。本研究目的為更進一步探討在人類角質細胞株HaCaT 中，EGF 如何調控keratin 16 之基因表現，且有哪些轉錄因子參與中。首先證實在HaCaT 細胞中，keratin 16 mRNA 及蛋白質表現會隨著EGF 處理時間增加而增加。利用不同5′端段切長度的keratin 16 啟動區之報告基因來做分析，我們發現EGF 誘導keratin 16 基因表現之反應區間是位於啟動區上-162 至-114 bp，此結果與先前學者所發表的區間位置並不相同。進一步將此區間內Sp1 結合序列 (-127 至-122 bp) 及AP1 結合序列 (-148 至-142 bp) 作雙點突變修飾，結果顯示EGF 所誘導的keratin 16 啟動區活性會明顯地受到抑制。另外透過Ras 蛋白質的活化所誘導keratin 16 基因表現的反應位置與上述之EGF 反應位置相同，由此結果得知在EGF 誘導keratin 16 基因表現之訊息傳遞路徑中，Ras 的活化參與著重要的角色。接著利用DNA親和力沉澱分析法 (DNA affinity precipitation assay) 分析啟動區上核蛋白質之結合情形。我們發現在HaCaT 細胞及果蠅SL2 細胞中，Sp1 核蛋白質會結合於此Sp1 結合序列上，而c-Jun 核蛋白質會透過與Sp1 核蛋白質進行交互作用，進而間接地結合於此Sp1 結合序列上。我們進一步以cotransfection assay 分析其功能上的角色，實驗證明此二轉錄因子c-Jun 及Sp1 對於keratin 16 基因表現呈現協同性的增加。另外分別送入不同劑量的coactivators (p300 或CBP)，由結果顯示p300/CBP 皆會對c-Jun/Sp1 complex 誘導keratin 16 的基因表現呈現加成性的增加，且EGF 誘導keratin 16 啟動區活性會被已知可阻斷p300 作用之病毒蛋白質E1A 所抑制，證實p300/CBP 確實參與在EGF 誘導keratin 16 之基因調控路徑中。由以上結果得知，keratin 16 啟動區之-162至-114 bp 區間中，Sp1 結合序列及AP1 結合序列為EGF 誘導keratin 16 基因表現之最重要位置，且Sp1 協同c-Jun 及p300/CBP 共同調控EGF 誘導keratin 16 的基因表現。接著我們想要進一步釐清，EGF 誘導keratin 16 基因表現需經由哪些訊息傳遞路徑，且所參與的核蛋白質是否需透過轉譯後修飾機轉 (post-translational modification)來調控此系統。由結果顯示，在HaCaT 細胞中，EGF 會活化ERK 及JNK，進而促使c-Jun 表現增加。而EGF 誘導keratin 16 基因的表現主要是透過Ras-MEK-ERK 訊息傳遞路徑，只有少部分經由JNK 路徑。另一方面，在HaCaT 細胞中，c-Jun 核蛋白質NH2 端上的Ser63 及Ser73 位置會受到EGF 磷酸化。然而，利用一系列c-Jun NH2端的突變質體做功能上的分析，我們發現c-Jun 的NH2 端對於EGF 誘導keratin 16 基因表現是不需要的。接著將c-Jun COOH 端之三個可能受乙醯化的lysines 位置突變成arginines 的質體myc-c-JunK3R 送入細胞中，我們發現此突變質體無法增加EGF 所誘導的keratin 16 基因表現，由結果顯示，c-Jun COOH 端之乙醯化修飾作用在此調控系統中至少參與著一部份的角色。有趣的是，藉由染色質免疫沉澱法(Chromatin Immunoprecipitation) 和DNA 親和力沉澱分析法，我們發現EGF 會透過ERK 訊息傳遞路徑而促使p300 聚集至keratin 16 啟動區。綜合以上實驗結果得知，經由ERK 及JNK 活化所增加的c-Jun 表現量，對於調控EGF 誘導keratin 16 基因表現是需要的。另外透過EGF 刺激ERK 的活化，會增加p300 聚集至keratin 16 啟動區，並進而調控keratin 16 基因表現，且此調控機制有一部份可能是透過增加c-Jun COOH 端之乙醯化所致。

　　Overexpression of keratin 16 has been observed in keratinocytes in those skin diseases characterized by hyperproliferation such as psoriasis. Therefore, keratin 16 is usually referred to as a disease-associated keratin. In studies of gene regulation of keratin 16, it has been reported previously that the hyperproliferation-inducing agents, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), induce keratin 16 expression in normal human epidermal keratinocytes and found that an EGF-responsiveelement (EGF-RE), located at the promoter ranged from -212 to -192 bp, is required for EGF and TGF-α responses. Furthermore, in the study of basal expression of keratin 16 in keratinocytes, in addition to EGF-RE, an Sp1 binding site located in the promoter range from -127 to -121 bp is also essential. The aim of the present work is to further characterize the EGF response region in the human keratin 16 gene promoter in a spontaneously immortalized keratinocyte (HaCaT) cell line. We found that EGF increased the expression of keratin 16 mRNA and protein synthesis in a time-dependent manner in HaCaT cells. Reporter assays revealed that the EGF response region was in the range of -162 to -114 bp, which was distinct from EGF-RE reported previously by other investigators. Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the keratin 16 promoter by site-directed mutagenesis significantly inhibited keratin 16 promoter activity induced by EGF. Furthermore, keratin 16 gene expression induced by Ras activation was also regulated in the same manner as the EGF response. By using the DNA affinity precipitation assay in HaCaT and SL2 cells, Sp1 directly interacted with the Sp1 site of the promoter, and c-Jun and c-Fos precipitated with the Sp1 oligonucleotide was attributable to the interaction between the Sp1 and AP1 proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activate keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of keratin 16 promoter, and EGF-induced promoter activation was blocked by the viral oncoprotein E1A. Taken together, these results suggest that Sp1 and AP1 sites in the essential promoter region are critical for EGF response, and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of EGF-induced keratin 16 gene expression. In addition, we further investigated the signaling networks relaying EGF stimulation of keratin 16 gene expression and the underlying regulatory mechanism, such as post-translational modification, of those nuclear factors involved in the transcriptional control of keratin 16 upon EGF treatment. Evidences obtained from the present study showed that treatment of HaCaT cells with EGF resulted in a rapid phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), and subsequent induction of c-Jun. The stimulated expression of keratin 16 by EGF was mediated mainly through the Ras-MEK-ERK signaling pathway, but partly through the JNK cascade. On the other hand, Ser63 and Ser73 on c-Jun NH2-terminal transactivation domain could be phosphorylated in HaCaT cells treated with EGF. Nevertheless, we found surprisingly that the c-Jun NH2-terminus was not required for EGF-induced expression of keratin 16. The activation of keratin 16 promoter by EGF treatment could not be enhanced by overexpression of myc-c-JunK3R, in which three putative acetylation lysine residues on the c-Jun COOH-terminus were all mutated into arginines, indicating that c-Jun acetylation might partially play a functional role in this system. Interestingly, by using a chromatin immunoprecipitation assay and a DNA affinity precipitation assay, EGF treatment was found to up-regulate p300 recruitment through ERK signaling to the promoter region in regulating keratin 16 transcriptional activity. In conclusion, these results strongly suggest that c-Jun biosynthesis, stimulated through ERK and JNK activation, plays an essential role in regulating keratin 16 gene expression by EGF. Recruitment of p300 to the keratin 16 gene promoter was due to EGF-activation of the ERK signaling pathway, and p300 mediated and regulated EGF-induced keratin 16 gene expression at least in part through multiple mechanisms including a selective acetylation of c-Jun COOH-terminus.

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