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)β gene, which is induced through a retinoic acid response element (βRARE). We wished to determine which endogenous retinoid receptors (RARs and retinoid X receptors, RXRs) mediate induction of the RARβ gene. We show that RXRα and RXRβ are constitutively expressed. Electrophoretic mobility shift assays with nuclear extracts show specific binding to the βRARE (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-RARβ and anti-RXRα antibodies. This suggests that induction of the RARβ 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 RARβ gene than RA, which only activates RAR. After 48 h, all RXRs appear to be titrated by the newly synthesized RARβ into an RARβ-RXR heterodimer complex. Thus, it appears that the βRARE is sequentially occupied by RXR dimers and RAR-RXR heterodimers.