Chen Zhi Y, Shie Jue-Lon, Tseng Chi-Chuan
Section of Gastroenterology, Veterans Affairs Boston Healthcare System and Boston University School of Medicine, Boston, Massachusetts, 02118, USA.
J Biol Chem. 2002 Nov 29;277(48):46831-9. doi: 10.1074/jbc.M204816200. Epub 2002 Sep 23.
Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor that expresses in the gastrointestinal tract and mediates growth arrest of colonic epithelium. The molecular mechanisms governing its growth inhibitory effect have not been fully elucidated. In the present study, we showed that induction of GKLF mRNA and protein expression by interferon-gamma treatment was associated with reduction of ornithine decarboxylase (ODC) gene expression and enzyme activity in colon cancer HT-29 cells. Overexpression of GKLF in HT-29 cells significantly reduced ODC mRNA and protein levels as well as enzyme activity and resulted in growth arrest, indicating that ODC might be a downstream target of GKLF. This conclusion was further supported by data showing that GKLF mRNA and protein concentrations were the highest at the G(1)/S boundary of the cell cycle, where ODC mRNA and protein levels were the lowest and that overexpression of GKLF resulted in cell arrested at the G(1) phase. Reporter gene transfection studies and electrophoretic mobility gel shift assays demonstrated that GKLF repressed ODC promoter activity and that these effects appeared to be mediated through interaction with a GC box in the proximal portion of the promoter. Transfection studies using reporter constructs and chromatin immunoprecipitation assays also demonstrated that GKLF inhibited transactivation of the ODC gene by interfering with the binding of Sp1 to the ODC promoter. These results indicate that GKLF may function as a G(1)/S checkpoint regulator and exert its growth arrest effect through down-regulation of ODC gene expression. Furthermore, GKLF is a transcriptional repressor of the ODC gene, and these effects are mediated by interaction with the GC-rich region on the promoter.
肠道富集型锌指蛋白（GKLF，KLF4）是一种上皮特异性转录因子，在胃肠道中表达并介导结肠上皮细胞的生长停滞。其生长抑制作用的分子机制尚未完全阐明。在本研究中，我们发现γ干扰素处理诱导GKLF mRNA和蛋白表达与结肠癌HT-29细胞中鸟氨酸脱羧酶（ODC）基因表达及酶活性降低有关。HT-29细胞中GKLF的过表达显著降低了ODC mRNA和蛋白水平以及酶活性，并导致生长停滞，表明ODC可能是GKLF的下游靶点。细胞周期G(1)/S边界处GKLF mRNA和蛋白浓度最高，而此时ODC mRNA和蛋白水平最低，且GKLF过表达导致细胞停滞在G(1)期，这些数据进一步支持了这一结论。报告基因转染研究和电泳迁移率凝胶阻滞分析表明，GKLF抑制ODC启动子活性，且这些作用似乎是通过与启动子近端的一个GC盒相互作用介导的。使用报告基因构建体的转染研究和染色质免疫沉淀分析也表明，GKLF通过干扰Sp1与ODC启动子的结合来抑制ODC基因的反式激活。这些结果表明，GKLF可能作为一个G(1)/S检查点调节因子发挥作用，并通过下调ODC基因表达发挥其生长停滞作用。此外，GKLF是ODC基因的转录抑制因子，这些作用是通过与启动子上富含GC的区域相互作用介导的。
