Sp1 binding plays a critical role in Erb-B2- and v-ras-mediated downregulation of alpha sub(2)-integrin expression in human mammary epithelial cells

• Published in: Molecular and cellular biology Vol. 16; no. 11; pp. 6178 - 6189
• Main Authors: Ye, Jianping, Xu, Ruo Hui, Taylor-Papadimitriou, J, Pitha, P M
• Format: Journal Article
• Language: English
• Published: 01.01.1996
• ISSN: 0270-7306

The human alpha sub(2)-integrin gene is transcriptionally downregulated in a nontumorigenic human mammary epithelial cell line, MTSV1-7, and its clonal variant HB2, overexpressing the Erb-B2 oncogene. In this study, we have used deletion mutations within the alpha sub(2)-integrin promoter inserted 5' of the chloramphenicol acetyltransferase or luciferase reporter genes to identify the element that is responsible for the Erb-B2-mediated downregulation. The results of the transient-transfection assay showed that the Sp1 binding element located in the core region (positions -64 to +1) of the alpha sub(2)-integrin promoter plays an essential role in the alpha sub(2)-integrin promoter activity and its downregulation by Erb-B2. By gel shift assay, we have demonstrated that this element binds with a high degree of affinity not only to Sp1, but also to Sp3. The downregulation of the alpha sub(2)-integrin promoter activity could also be achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha sub(2)-integrin gene expression, may proceed through the ras pathway. Both the Erb-B2- and the v-ras-overexpressing cells exhibited a Sp1 DNA binding activity lower than that of the parental line, while the relative levels of Sp1 protein in these cells were not altered. The Erb-B2- and v-ras-mediated downregulation could be reversed by the overexpression of Sp1 and by a dominant negative variant of ras (rasN17), confirming the importance of Sp1 and the ras pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha sub(2)-integrin promoter were observed in transient-cotransfection assays using alpha sub(2)-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha sub(2)-integrin reporter gene construct was transfected into MTSV1-7 or HB2 cells permanently overexpressing Erb-B2 or v-ras. The effects of Erb-B2 or v-ras on the transcriptional activity of the alpha sub(2)-integrin promoter were observed in nontumorigenic luminal epithelial cell lines (MTSV1-7 and HB2) as well as in the breast cancer cell line T47D. These data suggest that in luminal epithelial cells and the breast cancers which develop from them, the Erb-B2 proto-oncogene signaling leads to inhibition of alpha sub(2) beta sub(1)-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.