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 growt h factor-beta1 (TGFbeta1) has been connected to numerous diseases in h uman. TGFbeta1 expression is largely governed by three AP-1 binding si tes located in two different promoters of this gene. We have examined the ability of retinoid receptors to inhibit the activity of the two p romoters (especially the promoter 1) by cotransfection assays in the h epatocellular carcinoma cell line HepG2. When the TGFbeta1 promoter ac tivity is induced by 12-O-tetradecanoyl phorbol-13-acetate (an activat or of AP-1-controlled gene transcription), this activity can be strong ly repressed by retinoic acid receptor-alpha (RARalpha), RARbeta, or r etinoid X receptor-alpha (RXRalpha) as well as other members of the nu clear 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 antagonizin g of AP-1 activity. On further examining the anti-AP-1 activity of RXR alpha we observed that three different AP-1-controlled promoters (TGFb eta1, collagenase, and cFos) can be inhibited. Using gel shift assays, we demonstrated that RXRalpha inhibits Jun and Fos DNA binding and th at 9-cis RA enhances this inhibition, suggesting that a mechanism invo lving direct protein-protein interaction between RXR and AP-1 componen ts mediates the inhibitory effect observed in vivo. Transfection analy ses with RXRalpha point mutations revealed that residues L422, C432, a nd, to a lesser extent, residues L418 and L430, are involved in ligand -induced anti-AP1 activity of RXRalpha in vivo. Thus both types of ret inoid receptors can inhibit AP-1-activated promoters, including the TG Fbeta1 gene promoter, via a mechanism that involves protein-protein in teraction.