Regulation of the promoters for the human bone morphogenetic protein 2 and4 genes

Citation
Lm. Helvering et al., Regulation of the promoters for the human bone morphogenetic protein 2 and4 genes, GENE, 256(1-2), 2000, pp. 123-138
Citations number
40
Categorie Soggetti
Molecular Biology & Genetics
Journal title
GENE
ISSN journal
03781119 → ACNP
Volume
256
Issue
1-2
Year of publication
2000
Pages
123 - 138
Database
ISI
SICI code
0378-1119(20001003)256:1-2<123:ROTPFT>2.0.ZU;2-N
Abstract
The bone morphogenetic proteins 2 and 4 are known to be important in bone f ormation and are expressed in both the developing and adult mammalian bone. Understanding the regulation of these genes in osteoblasts may yield metho ds by which we can control expression to induce bone formation. We have iso lated and characterized the human BMP-2 and BMP-4 promoters and report subs tantially more upstream sequence information than that which has been publi shed. Human osteoblasts were found to have a single transcript initiation s ite that is conserved across species, rather than multiple start sites, as has previously been reported (Feng, J.Q., Harris, M.A., Ghosh-Choudhury, N. , Feng, M., Mundy, G.R., Harris, S.E., 1994. Structure and sequence of mous e morphogenetic protein-2 gene (BMP-2): comparison of the structures and pr omoter regions of BMP-2 and BMP-4 genes. Biochim. Biophys. Acta 1218, 221-2 24; Helier, L.C., Li, Y., Abrams, K.L., Rogers, M.B., 1999. Transcriptional regulation of the Bmp2 gene. J. Biol. Chem. 274, 1394-1400; Sugiura, T., 1 999. Cloning and functional characterization of the 5'-flanking region of t he human bone morphogenetic protein-2 gene. Biochem. J. 338, 433-440). A se ries of promoter deletions for both human BMP-2 and BMP-4 fused to the luci ferase reporter gene were analyzed thoroughly in human and murine osteoblas tic cell lines. Several compounds and growth factors that stimulate general or osteogenic pathways were used to treat cells transfected with the promo ter constructs. Retinoic acid compounds and the phorbol ester, PMA were fou nd to stimulate BMP-2 and, to a lesser degree, BMP-I. The combination of al l trans-RA and PMA caused a synergistic increase in BMP-2 promoter activity and endogenous mRNA. The RA stimulation appears to be an indirect effect o n the BMP-2 promoter, as the most highly conserved RRE in the BMP-2 promote r was unable to functionally bind or compete for protein binding. Potential binding sites in both promoters for the bone-specific transcription factor , Cbfa-1, were found to specifically bind Cbfa-1 protein in osteoblast nucl ear extracts; however, deletion of these sites did not significantly affect transcriptional activity of the promoters in osteoblasts. These data thus present new sequence and regulatory information for the human BMP-2 and BMP -I promoters and clarify the human BMP-2 gene transcriptional start site in osteoblasts. (C) 2000 Elsevier Science B.V. All rights reserved.