RETINOIC ACID RECEPTORS AND RETINOID-X RECEPTOR-ALPHA DOWN-REGULATE THE TRANSFORMING GROWTH FACTOR-BETA(1) PROMOTER BY ANTAGONIZING AP-1 ACTIVITY

Overexpression of the multifunctional growth factor transforming growth factor-beta1 (TGFbeta1) has been connected to numerous diseases in human. TGFbeta1 expression is largely governed by three AP-1 binding sites located in two different promoters of this gene. We have examined the ability of retinoid receptors to inhibit the activity of the two promoters (especially the promoter 1) by cotransfection assays in the hepatocellular carcinoma cell line HepG2. When the TGFbeta1 promoter activity is induced by 12-O-tetradecanoyl phorbol-13-acetate (an activator of AP-1-controlled gene transcription), this activity can be strongly repressed by retinoic acid receptor-alpha (RARalpha), RARbeta, or retinoid X receptor-alpha (RXRalpha) as well as other members of the nuclear receptor family. Repression was hormone dependent and a function of receptor concentration. Heterodimerization of RARa or RARbeta with RXRalpha did not modify the inhibition activities of these receptors, indicating that heterodimer formation is not required for antagonizing of AP-1 activity. On further examining the anti-AP-1 activity of RXRalpha we observed that three different AP-1-controlled promoters (TGFbeta1, collagenase, and cFos) can be inhibited. Using gel shift assays, we demonstrated that RXRalpha inhibits Jun and Fos DNA binding and that 9-cis RA enhances this inhibition, suggesting that a mechanism involving direct protein-protein interaction between RXR and AP-1 components mediates the inhibitory effect observed in vivo. Transfection analyses with RXRalpha point mutations revealed that residues L422, C432, and, to a lesser extent, residues L418 and L430, are involved in ligand-induced anti-AP1 activity of RXRalpha in vivo. Thus both types of retinoid receptors can inhibit AP-1-activated promoters, including the TGFbeta1 gene promoter, via a mechanism that involves protein-protein interaction.