Nuclear receptors RXRalpha:RARalpha are repressors for human MRP3 expression

Multidrug resistance-associated protein MRP3/Mrp3 (ABCC3) is upregulated in cholestasis, an adaptive response that may protect the liver from accumulation of toxic compounds, such as bile salts and bilirubin conjugates. However, the mechanism of this upregulation is poorly understood. We and others have previously reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression. In searching for additional regulatory elements in the human MRP3 promoter, we have now identified nuclear receptor retinoic X receptor-alpha:retinoic acid receptor-alpha (RXRalpha:RARalpha) as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that cotransfection of transcription factor Sp1 stimulates the MRP3 promoter activity and that additions of RXRalpha:RARalpha abrogated this activation in a dose-dependent manner. Site mutations and gel shift assays have identified a Sp1 binding GC box motif at -113 to -108 nts upstream from the MRP3 translation start site, where RXRalpha:RARalpha specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXRalpha:RARalpha as a repressor of MRP3 expression was further confirmed by RARalpha small-interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXRalpha:RARalpha act in concert to regulate MRP3 expression. Since RXRalpha:RARalpha expression is diminished by cholestatic liver injury, loss of RXRalpha:RARalpha may lead to upregulation of MRP3/Mrp3 expression in these disorders.