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

The human alpha(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(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 Spl binding element located in the core region (positions -64 to +1) of the alpha(2)-integrin promoter plays an essential role in the alpha(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(2)-integrin promoter activity could also he achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha(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 mere 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 res (rasN17), confirming the importance of Sp1 and the ms pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha(2)-integrin promoter were observed in transient-cotransfection assays using alpha(2)-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha(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(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(2) beta(1)-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.