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.