Evidence that Retinoid X Receptors Mediate Retinoid-dependent Transcriptional Activation of the Retinoic Acid Receptor β Gene in S91 Melanoma Cells

S91 melanoma cells are growth arrested and differentiate when treated with retinoids. These processes correlate with expression of the retinoic acid receptor (RAR) beta gene, which is induced through a retinoic acid response element (beta RARE). We wished to determine which endogenous retinoid receptors (RARs and retinoid X receptors, RXRs) mediate induction of the RAR beta gene. We show that RXR alpha and RXR beta are constitutively expressed. Electrophoretic mobility shift assays with nuclear extracts show specific binding to the beta RARE (Complex I) in untreated cells, which can be supershifted by antibodies against RXRs but not by anti-RAR antibodies. After 48 h of treatment with retinoic acid, Complex I is replaced by a faster migrating Complex II, which can be supershifted by anti-RAR beta and anti-RXR alpha antibodies. This suggests that induction of the RAR beta gene is largely mediated by RXRs only. Accordingly, we also find that 9-cis RA, which activates both RAR and RXR, is a more potent inducer of the RAR beta gene than RA, which only activates RAR. After 48 h, all RXRs appear to be titrated by the newly synthesized RAR beta into an RAR beta.RXR heterodimer complex. Thus, it appears that the beta RARE is sequentially occupied by RXR dimers and RAR-RXR heterodimers.