Choi Hee-Jung, Chung Tae-Wook, Kim Seok-Jo, Cho Soo-Young, Lee Young-Seek, Lee Young-Choon, Ko Jeong-Heon, Kim Cheorl-Ho
Department of Biological Science, Molecular and Cellular Glycobiology Unit, SungKyunKwan University, 300 Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Korea.
Glycobiology. 2008 May;18(5):395-407. doi: 10.1093/glycob/cwn016. Epub 2008 Mar 3.
Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2alpha-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking region of the PTEN gene showed that the region between -1175 and -1077 from the translational initiation site, which contains the AP-2alpha binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2alpha is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2alpha diminished the enhancement of AP-2alpha and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2alpha also results in the induction of PTEN transcription in AP-2alpha-negative colon cancer cells. Additionally, GM3 induced AP-2alpha-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene.
神经节苷脂GM3通过调节细胞信号通路抑制多种癌细胞的生长并诱导细胞周期停滞。我们之前的研究结果表明，GM3可抑制肿瘤抑制因子PTEN介导的癌细胞增殖。然而，GM3诱导PTEN基因转录调控的确切分子机制仍不清楚。在此，我们首次表明，GM3可诱导转录因子AP-2α介导结肠癌细胞中PTEN的表达。GM3在HCT116和p53基因缺失的HCT116细胞中增强PTEN的表达，这与p53功能无关。因此，为了进一步确定GM3调控PTEN基因表达的机制，我们对PTEN基因的启动子区域进行了表征。对PTEN基因5'侧翼区域的启动子分析表明，从翻译起始位点起-1175至-1077之间的区域，包含AP-2α结合位点，在结肠癌细胞中作为GM3诱导型启动子发挥作用。此外，凝胶迁移实验、定点诱变和染色质免疫沉淀实验明显表明，AP-2α对于GM3刺激的结肠癌细胞中PTEN的表达至关重要。此外，针对AP-2α的小干扰RNA减少了GM3诱导的结肠癌细胞中AP-2α和PTEN表达的增强。AP-2α的瞬时表达也导致AP-2α阴性结肠癌细胞中PTEN转录的诱导。此外，GM3通过抑制自分泌配体介导的EGFR激活诱导AP-2α介导的PTEN表达。这些结果表明，转录因子AP-2α是神经节苷脂GM3刺激的PTEN基因转录调控所必需的。
