Sp1 binding plays a critical role in Erb-B2- and v-ras-mediated downregulation of alpha2-integrin expression in human mammary epithelial cells.
The human alpha2-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 alpha2-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 alpha2-integrin promoter plays an essential role in the alpha2-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 alpha2-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 alpha2-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 alpha2-integrin promoter were observed in transient-cotransfection assays using alpha2-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha2-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 alpha2-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.
Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
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J
Ye
R H
RH
Xu
J
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Taylor-Papadimitriou
P M
PM
Pitha
D015703:Antigens, CD / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D001940:Breast; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015536:Down-Regulation; D004848:Epithelium / Q000276:immunology / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D018734:Genes, erbB-2; D011905:Genes, ras; D005822:Genetic Vectors; D006801:Humans; D039421:Integrin alpha2; D009687:Nuclear Proteins / Q000378:metabolism; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis; D018739:Oncogene Proteins v-erbB / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014162:Transfection
D015703:Antigens, CD; D039421:Integrin alpha2; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D018739:Oncogene Proteins v-erbB; D000090063:Proto-Oncogene Mas; D016329:Sp1 Transcription Factor; D015689:Oncogene Protein p21(ras)