Nuclear receptors RXRalpha:RARalpha are repressors for human MRP3 expression - PubMed (original) (raw)

Nuclear receptors RXRalpha:RARalpha are repressors for human MRP3 expression

Wensheng Chen et al. Am J Physiol Gastrointest Liver Physiol. 2007 May.

Abstract

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.

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Figures

Fig. 1

Luciferase reporter assays demonstrate that the first 234-bp sequence of the human multidrug resistance-associated protein MRP3 promoter contain a retinoic X receptor-α:retinoic acid receptor-α (RARα:RXRα) response element. Cotransfection of 75 ng RARα and/or 75 ng RXRα expression plasmid (s) with 300 ng of either of 3 human MRP3 promoter region constructs (p-200Luc, p-500Luc, or p-1 kbLuc) into HepG2 cells significantly inhibit MRP3 promoter activity when compared with empty vector control (Ctrl) (N = 3, *P < 0.01). Firefly luciferase activity was normalized to Renilla luciferase activity. RLU, relative light units.

Fig. 2

Endogenous MRP3 is upregulated in RARα small-interfering RNA (siRNA) transfected HepG2 cells at 48 h. Left: Western blot and densitometry demonstrate that endogenous RARα expression is knocked down by its specific siRNA, #P < 0.01, N = 3. There was no change in the nuclear protein SH-PTP1. Middle: quantitative PCR demonstrates that MRP3 mRNA is upregulated when RARα is knocked down, #P < 0.01. Right: Western blot and densitometry demonstrate an increase in expression of MRP3 protein when RARα is knocked down, *P < 0.05. There was no change in β-actin.

Fig. 3

The 234-bp DNA sequence of the MRP3 gene 5′-flank region in p-200Luc construct. Fetoprotein transcription factor (FTF)/cholesterol 7α hydroxylase promoter factor (CPF)/liver receptor homolog-1 (Lrh-1) and putative Sp1 binding GC box motifs are indicated.

Fig. 4

Luciferase reporter assays demonstrate that RXRα:RARα and Sp1 coregulate the MRP3 promoter. Left: Sp1 specifically activates the MRP3 promoter in a dose-dependent manner in transfected HEK293 cells. p-200Luc (200 ng) was cotransfected with the indicated amount of either the Sp1 vector, the Sp3 vector, or the empty vector. Middle: RARα:RXRα abrogated Sp1 activation on MRP3 promoter in a dose-dependent manner in transfected S2 cells. p-200Luc (200 ng) was cotransfected with the indicated amount of Sp1, RAR, and RXR. Right: mutations of the Sp1 binding GC box motif reduced MRP3 promoter activity. p-200Luc (200 ng) wild-type and mutants were cotransfected with or without 100 ng Sp1 and 50 ng RARα and 50 ng RXRα expression vectors in HepG2 cells. N = 3, *P < 0.01. Firefly luciferase activity was normalized to total cell protein.

Fig. 5

EMSA indicates that RXRα:RARα and Sp1 are in the complex of MRP3 promoter. Sp1 (left) and nuclear extract from HepG2 cell (middle) bind to the GC box motif, and mutation on the GC box abrogates these bindings. Right: recombinant RXRα and RARα specifically reduced Sp1 binding to the GC box motif. NE, nuclear extract. WT, wild type; GCB, both guanine and cytosine box are mutated; GC5, 5′ end of guanine and cytosine box are mutated; GST, glutathione-_S_-transferase.

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Nuclear receptors RXRalpha:RARalpha are repressors for human MRP3 expression - PubMed (original) (raw)