To examine possible mechanisms underlying osteoblast differentiation f
rom mesenchymal stem cells, we investigated bHLH functional activity i
n cell lines representing different stages of osteoblast maturation. I
nteraction of nuclear proteins with oligonucleotides corresponding to
various bHLH binding sequences (known as E-boxes) was determined in mo
bility shift assays. Both ADD-1 oligonucleotide, a binding site for tr
anscription factor ADD-1, and OCE-1, an E-box from osteocalcin promote
r, produced retarded bands after incubation with nuclear extracts from
osteogenic cells. Cells at different stages of osteogenic maturation
demonstrated similar patterns and intensity of binding, as did cells t
reated with different osteogenic inducers. Binding to ADD-1 and OCE-1
was not tissue-specific as it was also observed in fibroblastic 10T1/2
cells. MEF-1 oligonucleotide, the E-box sequence from the muscle crea
tine kinase enhancer, demonstrated no changes in binding with nuclear
extracts from moderately differentiated (W-20) or relatively mature (R
OS 17/2.8) cells under any conditions tested. However, in poorly diffe
rentiated RI-2J cells, which do not express osteogenic markers unless
treated with dexamethasone, induction of differentiation was reflected
in transient inhibition of binding to MEF-1. Inhibition of binding wa
s not seen under differentiation-restrictive conditions. Promoter-repo
rter studies also demonstrated inhibition of MEF-1 driven CAT expressi
on by dexamethasone under differentiation-permissive conditions in RI-
2J cells. These data suggest that bHLH gene expression is not required
for the early steps of osteogenesis; moreover, inhibition of bHLH pro
tein binding to a MEF1-type E box might be an integral part of osteoge
nic commitment. (C) 1997 Wiley-Liss, Inc.