Transcriptional regulation by transforming growth factor beta of the expression of retinoic acid and retinoid X receptor genes in osteoblastic cells is mediated through AP-1

We now report that transforming growth factor pi (TGF-PI), a potent regulatory cytokine of bone remodeling, is a powerful stimulator for gene expression of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in osteoblastic MC3T3-E1 cells. TGF-beta 1 transcriptionally stimulated the expression of RAR alpha, RAR gamma, and RXR alpha genes, but did not do so for RAR beta, RXR beta, and RXR gamma genes. We also observed that AP-1, a transcriptional factor, plays an important role in the signal pathway for expression of RAR alpha, RAR gamma, and RXR alpha genes stimulated by TGF-beta 1 because stimulation of the expression of these genes in the cytokine-treated cells was markedly inhibited by a mixture of antisense c-fos and c-jun. A gel mobility shift assay demonstrated that TGF-beta 1 is able to increase, in a dose-dependent manner, the binding of nuclear proteins to direct repeat 5, a consensus sequence with high affinity for RAR-RXR heterodimers. The mobility shift assay, using specific antibody for each receptor, showed that direct repeat B-binding proteins may be RAR and RXR isoforms. The stimulated binding to direct repeat 5 was inhibited strongly by H-7, an inhibitor of serine/threonine kinase, and by curcumin, an inhibitor of AP-1, The present study suggests a novel pathway for TGF-beta 1 action in osteoblastic cells via stimulation of RAR-RXR transcriptional activity in a ligand-dependent fashion.