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.Insulin-like growth factor-binding protein-5 (IGFBP-5) 1 belongs to a family of six structurally related proteins that are unrelated to the IGF receptors and that bind IGF-I and IGF-II with high affinity (1-3). IGFBPs modulate the mitogenic and metabolic activities of the IGFs in vitro, suggesting an important role in IGF physiology in vivo. Of the various IGFBPs, IGFBP-5 is unique in that it stimulates IGF-induced osteoblastic cell proliferation when added simultaneously with IGF I or II to serumfree osteoblast-like cell cultures (4 -7). Recent evidence suggests that IGFBP-5 may stimulate cell proliferation by an IGF-independent mechanism involving IGFBP-5 specific cell surface binding sites (7,8). In addition, IGFBP-5 binds with high affinity to hydroxyapatite and to various extracellular matrix proteins, properties that are consistent with a role for this binding protein in fixing IGFs to extracellular matrices including mineralized bone (7, 9, 10). IGFBP-5 may be a more potent enhancer of IGF activities when bound to extracellular matrix than in solution (10). Coding sequences and 5Ј-flanking regions of IGFBP-5 genes are highly conserved in human, rat, and mouse (4, 5, 11-13), and the mouse and human 5Ј-flanking regions have been found to impart basal promoter activity to reporter gene constructs in several cell types, including osteoblasts (12-16).IGFBP-5 is expressed by a number of cell types including fibroblasts, myoblasts, and osteoblasts, and production of IG-FBP-5 is regulated ...

The MN1 oncoprotein activates transcription of the IGFBP5 promoter through a CACCC-rich consensus sequence

“…When both elements were deleted, promoter activity was almost completely abolished. In the literature, the CA-rich element is described as a PR-sensitive element (Boonyaratanakornkit et al 1999). A mutant used by the authors of this article in which 3 Cs were exchanged with As was no longer able to confer PR activity to the IGFBP5 promoter.…”

“…The progesterone receptor (PR) does not stimulate the IGFBP5 promoter in Hep3B cells and U2 osteosarcoma cells (U2OS) cells, and the effect of MN1 is not affected by PR It has previously been shown by Boonyaratanakornkit et al (1999) that the PR can stimulate the IGFBP5 promoter in the presence of its ligand, progesterone. Although no canonical PR-responsive elements are present in the IGFBP5 promoter, Boonyaratanakornkit et al (1999) showed that induction by PR is mediated by the CACCC element in the promoter.…”

“…The mutants mPR, dCA, dGT, and dCAdGT were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The mPR mutant is as described by Boonyaratanakornkit et al (1999). The CA-rich element at positions K147 to K133 (CCCCACCCCCACCCC) is mutated in CCCCACCaaaACCCC.…”

“…Transcription factors that have been shown to affect the IGFBP5 promoter are the progesterone receptor (PR; Boonyaratanakornkit et al 1999), the retinoic acid receptor (RAR; Cesi et al 2004Cesi et al , 2005, the glucocorticoid receptor (Gabbitas et al 1996), activator protein-2 (AP-2) (Duan & Clemmons 1995) and Myb (Tanno et al 2002). Furthermore, prostaglandin E2, parathyroid hormone, and osteogenic protein-1 have been described to regulate the expression of IGFBP5 (Ji et al 1999, Yeh & Lee 2000, Erclik & Mitchell 2005.…”

Androgen, Estrogen and the Bone Marrow Microenvironment

“…In human bone and BM, IGFBP5 is expressed in chondrocytes, osteoblasts and osteocytes. These cell types express androgen receptors [26][27][28][29], however only androgen regulation of IGFBP 2, 3, 4 and not of IGFBP5 has been examined in-vitro [30].…”