Sp1 and AP-1 Regulate Expression of the Human Gene VIL2 in Esophageal Carcinoma Cells
Sp1 and AP-1 Regulate Expression of the Human Gene VIL2 in Esophageal Carcinoma Cells
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DOI：
10.1074/jbc.m809734200
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发表时间：
2009-03-20
影响因子：
4.8
通讯作者：
Xu, Li-Yan
中科院分区：
文献类型：
--
作者：
Gao, Shu-Ying;Li, En-Min;Xu, Li-Yan
由VIL 2编码的埃兹蛋白是一种膜-细胞骨架连接蛋白，已被认为参与肿瘤发生。Ezrin在食管鳞状细胞癌（esophageal squamous cell carcinoma，ESCC）中的表达是近年来发现的，但其表达调控的分子机制及其临床意义尚不清楚。因此，我们回顾性评估ezrin表达的免疫组织化学在组织芯片代表193 ESCC。在193个肿瘤中的90个（46.6%）中Ezrin过表达与较差的存活率相关（p = 0.048）。然后，我们通过将基因的5 '侧翼区的缺失或定点突变体融合到荧光素酶报告基因来评估人VIL 2的转录调控区，探索了ESCC中ezrin表达受控制的机制。我们发现，在ESCC来源的食管癌细胞（EC 109）中，包含共有Sp1（-75/-69）和AP-1（-64/-58）结合位点的-87/-32区域是VIL 2启动子活性的关键。AP-1由c-Jun和c-Fos组成。电泳迁移率变化和染色质免疫沉淀实验表明，Sp1和c-Jun结合特异性的VIL 2启动子内的各自的结合位点。此外，Sp1，c-Jun或c-Fos的瞬时表达增加了ezrin表达和VIL 2启动子活性。使用选择性抑制剂显示VIL 2反式激活需要MEK 1/2信号转导通路，而不是JNK或p38 MAPK。综上所述，我们提出了一种可能的信号转导途径，MEK 1/2磷酸化ERK 1/2，磷酸化Sp1和AP-1，进而结合到各自的结合位点，以调节人VIL 2在ESCC细胞中的表达。
Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.