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Expression and regulation of retinoid X receptors in B16 melanoma cells
1Department of Biochemistry and Molecular Biology, Marshall University School of Medicine, Huntington, West Virginia 25755-9310, USA.
Abstract:
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. Long-term 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 RXR beta, but not RXR alpha. These results suggest the presence of an unstable transcription factor that negatively regulates the expression of RXR beta 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.
Insights
Retinoid X receptors (RXRs) alpha and beta are present in B16 melanoma cells. 8-bromo-cyclic AMP (8BrcA) reduces RXR beta mRNA, potentially inhibiting all-trans-retinoic acid (ATRA)-induced differentiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Retinoid X receptors (RXRs) are a distinct subfamily of nuclear retinoid receptors that bind 9-cis-retinoic acid (9cisRA).
- B16 mouse melanoma cells are a model system for studying retinoid signaling and differentiation.
- All-trans-retinoic acid (ATRA) induces differentiation in B16 melanoma cells, while 8-bromo-cyclic AMP (8BrcA) and phorbol dibutyrate (PDB) antagonize this effect.
Purpose of the Study:
- To determine which RXR subtypes are expressed in B16 mouse melanoma cells.
- To investigate the effects of ATRA, 8BrcA, and PDB on RXR mRNA levels in these cells.
- To elucidate the regulatory mechanisms controlling RXR expression in melanoma.
Main Methods:
- Northern blot analysis to detect RXR alpha, beta, and gamma mRNA expression.
- Reverse transcription-polymerase chain reaction (RT-PCR) for RXR gamma detection.
- Treatment with ATRA, 8BrcA, PDB, cycloheximide (protein synthesis inhibitor), and actinomycin D (transcription inhibitor).
- Nuclear run-on assays to assess transcription rates.
Main Results:
- RXR alpha and RXR beta mRNA were detected in B16 cells, while RXR gamma was not.
- Long-term ATRA treatment decreased RXR alpha mRNA but not RXR beta mRNA.
- 8BrcA treatment caused a time-dependent decrease in RXR beta mRNA, but not RXR alpha mRNA.
- PDB did not affect RXR mRNA levels.
- Cycloheximide increased RXR alpha and RXR beta mRNA levels in a time- and concentration-dependent manner, primarily by stimulating RXR beta transcription.
- Actinomycin D abolished the cycloheximide-induced increase in RXR beta mRNA, indicating the involvement of a labile transcription factor.
Conclusions:
- B16 melanoma cells express RXR alpha and RXR beta, with RXR beta being the predominant isotype.
- An unstable transcription factor negatively regulates RXR beta expression in B16 melanoma cells.
- 8BrcA may inhibit ATRA-induced differentiation, at least partially, by down-regulating RXR beta.