EXPRESSION AND REGULATION OF RETINOID-X-RECEPTORS IN B16 MELANOMA-CELLS

Recently, a new subfamily of nuclear retinoid receptors that is distinct from that of RARs has been identified and named Retinoid X receptors (RXRs). These receptors specifically bind 9-cis-retinoic acid (9cisRA), but not all-trans-retinoic acid (ATRA). We determined which RXR subtypes were expressed in B16 mouse melanoma cells and then studied the effect of ATRA, 8-bromo-cyclic AMP (8BrcA), and phorbol dibutyrate (PDB) on RXR mRNA levels. ATRA induces differentiation in these cells while 8BrcA and PDB antagonize the RA-induced differentiation of B16 melanoma cells. Northern analysis demonstrated the expression of RXR alpha and RXR beta mRNA in B16 cells, but RXR gamma was not detectable. Further analysis using RT-PCR also failed to detect RXR gamma in these cells. Longterm RA treatment decreased the expression of RXR alpha, but not RXR beta mRNAs. PDB did not alter the expression of either RXR mRNAs, however, 8BrcA treatment resulted in a time dependent decrease in the amount of RXR beta, but not RXR alpha mRNA. Inhibition of protein synthesis by cycloheximide resulted in a large increase in RXR alpha and RXR beta mRNA levels. This effect of cycloheximide was time and concentration dependent with maximal stimulation of RXR alpha and RXR beta mRNAs occurring at 4 h of treatment. Inhibition of transcription with actinomycin D completely abolished the cycloheximide-induced increase of RXR beta. In contrast to its effect on other genes, such as immediate response genes, cycloheximide treatment did not increase the half-life of RXR beta mRNA. Nuclear run-on assays showed that cycloheximide treatment of intact B16 melanoma cells stimulated the transcription rate of RXRP, but not RXR alpha. These results suggest the presence of an unstable transcription factor that negatively regulates the expression of RXRP in B16 melanoma cells. In addition, since RXR beta is the predominant isotype in B16 cells, 8BrcA may, at least partially, inhibit RA-induced differentiation through down-regulation of this RXR. (C) 1995 Wiley-Liss, Inc.