Nuclear receptors RXRα:RARα are repressors for human MRP3 expression

Multidrug resistanceassociated 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 ( RXR alpha: RAR alpha) 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 RXR alpha: RAR alpha 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 RXR alpha: RAR alpha specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXR alpha: RAR alpha as a repressor of MRP3 expression was further confirmed by RAR alpha small- interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXR alpha: RAR alpha act in concert to regulate MRP3 expression. Since RXR alpha: RAR alpha expression is diminished by cholestatic liver injury, loss of RXR alpha: RAR alpha may lead to upregulation of MRP3/ Mrp3 expression in these disorders.