Submitted on May 4, 2006
Accepted on January 30, 2007
NUCLEAR RECEPTORS RXR
:RAR ARE REPRESSORS FOR HUMAN MRP3 EXPRESSION
Wensheng Chen1, SHI-YING CAI2, SHUHUA XU2, Lee Denson3, Carol Soroka2, and James L. Boyer4*
1 Department of Gastroenterology, Third Military Medical University, Chongqing, Chongqing, China
2 Internal Medicine, Yale University, New Haven, Connecticut, United States
3 Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
4 School of Medicine, Liver Center, Yale University, New Haven, Connecticut, United States; Internal Medicine, Yale University, New Haven, Connecticut, United States
* To whom correspondence should be addressed. E-mail: james.boyer{at}yale.edu.
MRP3/Mrp3 (ABCC3) is up-regulated 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 up-regulation is poorly understood. We and others have previously reported that FTF/Lrh-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 RXR
:RAR as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that co-transfection of transcription factor Sp1 stimulates the MRP3 promoter activity, and that additions of RXR:RAR abrogated this activation in a dose dependant manner. Site mutations and gel shift assays have identified a Sp1 binding GC-box motif at -113~-108 nts upstream from the MRP3 translation start site, where RXR:RAR specifically reduced Sp1 binding to this site. Mutation of the GC-box also reduced MRP3 promoter activity. The functional role of RXR:RAR as a repressor of MRP3 expression was further confirmed by RAR siRNA knockdown in HepG2 cells which up-regulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXR:RAR act in concert to regulate MRP3 expression. Since RXR:RAR expression is diminished by cholestatic liver injury, loss of RXR:RAR may lead to up-regulation of MRP3/Mrp3 expression in these disorders.
