HLH TRANSCRIPTION FACTOR ACTIVITY IN OSTEOGENIC CELLS

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.