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Endocrinology Vol. 140, No. 6 2501-2508
Copyright © 1999 by The Endocrine Society

ARTICLES

Transcriptional Activation of Insulin-Like Growth Factor-Binding Protein-4 by 17ß-Estradiol in MCF-7 Cells: Role of Estrogen Receptor-Sp1 Complexes1

Chunhua Qin, Pomila Singh and Stephen Safe

Department of Veterinary Physiology and Pharmacology, Texas A&M University (C.Q., S.S.), College Station, Texas 77843-4466; and the Department of Anatomy and Neurosciences (P.S.), University of Texas Medical Branch, Galveston, Texas 77555

Address all correspondence and requests for reprints to: Dr. Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu

Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17ß-estradiol (E2) resulted in induction of IGFBP-4 gene expression (>3-fold) and protein secretion (>6-fold). To identify genomic sequences associated with E2 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 E2 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 E2-induced trans-activation. Gel mobility shift assays using 32P-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. Wild-type 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 E2 in MCF-7 cells.




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