Katabami Kouji, Kato Takumi, Sano Rikio, Ogura Masaharu, Mizuno Hiromi, Itoh Saotomo, Tsuji Tsutomu
Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
J Cell Biochem. 2006 Feb 15;97(3):530-43. doi: 10.1002/jcb.20663.
The alpha3beta1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for alpha3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5'-flanking region of the mouse alpha3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cell lines. However, we found that these cell lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for alpha3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of alpha3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate alpha3 integrin gene expression and that the contribution of each element depends on the type of tumor cells.
α3β1整合素是包括层粘连蛋白异构体在内的细胞外基质蛋白的黏附受体，在上皮组织和内皮组织的组织形成中发挥关键作用。这种黏附分子在肿瘤细胞上的异常表达与其侵袭和转移潜能相关。在本研究中，我们通过荧光素酶测定分析了不同组织来源的各种肿瘤细胞系中α3整合素基因表达所必需的元件。小鼠α3整合素基因5'-侧翼区的一个约0.3 kb片段（相对于主要转录起始位点为-260 / + 84）在所有六个检测的肿瘤细胞系中均显示出强启动子活性。然而，我们发现这些细胞系可根据对位于-133处的假定Ets转录因子结合基序的依赖程度分为两组。该基序先前已被证明对MKN1胃癌细胞中α3整合素的表达至关重要。一组细胞系中的基因表达主要由Ets基序上调，而另一组中的基因表达对Ets基序的依赖性较小。然后我们推测还有其他调节元件负责α3整合素的表达，并发现-69处富含GC的基序是另一个重要元件。使用特异性抗体的电泳迁移率变动分析和核蛋白的蛋白质印迹分析表明，Sp3转录因子与这个富含GC的基序结合。这些结果表明，Sp3和Ets转录因子协同调节α3整合素基因表达，并且每个元件的作用取决于肿瘤细胞的类型。
