title: 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.
abstract: 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(-8) 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.
authors.affiliation: J. L. Pettis Veterans Affairs Medical Center, Loma Linda University, Loma Linda, California 92357, USA.
authors.forename: V
authors.initials: V
authors.lastname: Boonyaratanakornkit
authors.forename: D D
authors.initials: DD
authors.lastname: Strong
authors.forename: S
authors.initials: S
authors.lastname: Mohan
authors.forename: D J
authors.initials: DJ
authors.lastname: Baylink
authors.forename: C A
authors.initials: CA
authors.lastname: Beck
authors.forename: T A
authors.initials: TA
authors.lastname: Linkhart
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics*; D015735:Mifepristone / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D011374:Progesterone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D006727:Hormone Antagonists; D018975:Insulin-Like Growth Factor Binding Protein 5; D009687:Nuclear Proteins; D015735:Mifepristone; D011374:Progesterone