Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor α through its interaction with Sp1

• Published in: Journal of molecular endocrinology Vol. 36; no. 1; pp. 91 - 105
• Main Authors: Panno, M L, Mauro, L, Marsico, S, Bellizzi, D, Rizza, P, Morelli, C, Salerno, M, Giordano, F, Ando’, S
• Format: Journal Article
• Language: English
• Published: England BioScientifica 01.02.2006
• Subjects: Animals; Base Sequence; Blotting, Western; Cell Line, Tumor; CHO Cells; Cricetinae; DNA Primers; Electrophoretic Mobility Shift Assay; Estrogen Receptor alpha - metabolism; Humans; Insulin Receptor Substrate Proteins; Mice; Mutagenesis, Site-Directed; Phosphoproteins - genetics; Promoter Regions, Genetic; Protein Binding; Regular papers; Sp1 Transcription Factor - metabolism
• ISSN: 0952-5041; 1479-6813
• DOI: 10.1677/jme.1.01848

In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3′ different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt −1420 to −160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position −1500 to −1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt −1500 to −1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERα and Sp1.