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

Recently, a new subfamily of nuclear retinoid receptors that is distin ct from that of RARs has been identified and named Retinoid X receptor s (RXRs). These receptors specifically bind 9-cis-retinoic acid (9cisR A), but not all-trans-retinoic acid (ATRA). We determined which RXR su btypes were expressed in B16 mouse melanoma cells and then studied the effect of ATRA, 8-bromo-cyclic AMP (8BrcA), and phorbol dibutyrate (P DB) on RXR mRNA levels. ATRA induces differentiation in these cells wh ile 8BrcA and PDB antagonize the RA-induced differentiation of B16 mel anoma cells. Northern analysis demonstrated the expression of RXR alph a and RXR beta mRNA in B16 cells, but RXR gamma was not detectable. Fu rther analysis using RT-PCR also failed to detect RXR gamma in these c ells. Longterm RA treatment decreased the expression of RXR alpha, but not RXR beta mRNAs. PDB did not alter the expression of either RXR mR NAs, 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 co ncentration dependent with maximal stimulation of RXR alpha and RXR be ta mRNAs occurring at 4 h of treatment. Inhibition of transcription wi th actinomycin D completely abolished the cycloheximide-induced increa se of RXR beta. In contrast to its effect on other genes, such as imme diate response genes, cycloheximide treatment did not increase the hal f-life of RXR beta mRNA. Nuclear run-on assays showed that cycloheximi de treatment of intact B16 melanoma cells stimulated the transcription rate of RXRP, but not RXR alpha. These results suggest the presence o f an unstable transcription factor that negatively regulates the expre ssion of RXRP in B16 melanoma cells. In addition, since RXR beta is th e predominant isotype in B16 cells, 8BrcA may, at least partially, inh ibit RA-induced differentiation through down-regulation of this RXR. ( C) 1995 Wiley-Liss, Inc.