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

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. 10−8m PG increased promoter activity of this construct in U2 cells co-transfected with a PG receptor isoform A (PRA) 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 PRA protein indicate that nuclear factors bind to a CACCC sequence at −139 and that PRA 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 PRA and PRB 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.