Transcriptional activation of insulin-like growth factor-binding protein-4 by 17β-estradiol in MCF-7 cells:: Role of estrogen receptor-Sp1 complexes

Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17 beta-estradiol (E-2) resulted in induction of IGFBP-4 gene expression (>3-fold) and protein secretion (>6-fold). To identify genomic sequences associated with E-2 responsiveness, the 5'-promoter region (-1214 to + 18) of the IGFBP-4 gene was cloned into a vector upstream from the firefly luciferase reporter gene, and E-2 induced a 10-fold increase in luciferase activity in MCF-7 cells transiently transfected with this construct. Deletion analysis of this region of the IGFBP-4 gene promoter identified two GC-rich sequences at -559 to -553 and -72 to -64 that were important for E-2-induced trans-activation. Gel mobility shift assays using P-32-labeled -569 to -540 and -83 to -54 oligonucleotides from the IGFBP-4 gene promoter showed that Sp1 protein bound these oligonucleotides to form a retarded band, and the intensity of the band was competitively decreased after coincubation with unlabeled IGFBP-4-derived and consensus Sp1 oligonucleotides. Mutation of the GC-rich sites within these sequences resulted in loss of the retarded band formation. Wildtype human estrogen receptor did not bind directly to the IGFBP-4 oligonucleotides; however, human estrogen receptor enhanced Sp1-DNA binding in a concentration-dependent manner. The results of this study demonstrate that at least two GC-rich sequences at -559 to -553 and -72 to -64 are required for induction of IGFBP-4 gene expression by E-2 in MCF-7 cells.