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J Biol Chem, Vol. 274, Issue 37, 26431-26438, September 10, 1999

Progesterone Stimulation of Human Insulin-like Growth Factor-binding Protein-5 Gene Transcription in Human Osteoblasts Is Mediated by a CACCC Sequence in the Proximal Promoter

Viroj Boonyaratanakornkit, Donna D. Strong^¶, Suburraman Mohan^**, David J. Baylink, Candice A. Beck, and Thomas A. Linkhart^§§

From the J. L. Pettis Veterans Affairs Medical Center and the Departments of  Biochemistry, ^¶ Microbiology and Molecular Genetics, ^§§ Pediatrics, ^** Physiology, and  Medicine, Loma Linda University, Loma Linda, California 92357 and the  University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262

Insulin-like growth factor-binding protein-5 (IGFBP-5) is produced by osteoblasts and potentiates insulin-like growth factor mitogenic stimulation in osteoblast cell cultures. Progesterone (PG) increased IGFBP-5 expression in normal human osteoblasts and increased IGFBP-5 transcription in U2 human osteosarcoma cells. We developed a chloramphenicol acetyltransferase reporter construct containing the human IGFBP-5 proximal promoter sequence, which includes TATA and CAAT boxes, and five putative PG response element half-sites. 10⁸ M PG increased promoter activity of this construct in U2 cells co-transfected with a PG receptor isoform A (PR_A) expression vector. Analysis of 5' deletion constructs indicates that PG transactivation of IGFBP-5 promoter activity does not require the PG response element half-sites but does require the region 162 to 124 containing two tandem CACCC box sequences. Mutation of the proximal CACCC box at 139 eliminated PG transactivation. Gel shift assays using a 162 to 124 DNA fragment, U2 cell nuclear extracts, and purified PR_A protein indicate that nuclear factors bind to a CACCC sequence at 139 and that PR_A alters the pattern of transcription factor interaction with the CACCC sequence. Using a luciferase reporter construct containing base pairs 252 to +24 of the IGFBP-5 promoter, we found that both PR_A and PR_B isoforms mediated PG stimulation of promoter activity. These results suggest that PG may stimulate IGFBP-5 gene transcription via a novel mechanism involving PR and CACCC-binding factors.

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