Aryl hydrocarbon receptor and NF-E2-related factor 2 are key regulators of human MRP4 expression

Abstract

Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids in hepatocytes and renal proximal tubule epithelium. MRP4/Mrp4 undergoes adaptive upregulation in response to oxidative and cholestatic liver injury in human and animal models of cholestasis. However, the molecular mechanism of this regulation remains to be determined. The aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) play important roles in protecting cells from oxidative stress. Here we examine the role of these two nuclear factors in the regulation of the expression of human MRP4. HepG2 cells and human hepatocytes were treated with the AhR and Nrf2 activators, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), or oltipraz and other nuclear receptor agonists. TCDD, 3-MC, and oltipraz significantly increased MRP4 expression at mRNA and protein levels. Computer program analysis revealed three Xenobiotic response element (XRE) and one Maf response element sites within the first 500 bp of the MRP4 proximal promoter. Luciferase reporter assay detected strong promoter activity (53-fold higher than vector control) in this region. TCDD and 3-MC also induced promoter activity in the reporter assays. Mutation of any of these XRE sites significantly decreased MRP4 promoter activity in reporter assays, although XRE2 demonstrated the strongest effects on both basal and TCDD-inducible activity. EMSA and chromatin immunoprecipitation assays further confirmed that both AhR and Nrf2 bind to the proximal promoter of MRP4. Our findings indicate that AhR and Nrf2 play important roles in regulating MRP4 expression and suggest that agents that activate their activity may be of therapeutic benefit for cholestasis.

Fig. 1.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and oltipraz (OPZ) induce the expression of multidrug resistance-associated protein 4 (MRP4) mRNA in HepG2 cells and primary human hepatocytes. A: dose-dependent induction by TCDD in HepG2 cells after 24-h treatment. *P < 0.05, n = 3. B: OPZ upregulated MRP4 mRNA levels in a dose-dependent manner in HepG2 cells (48-h treatment). C: OPZ induction of MRP4 mRNA is time dependent. HepG2 cells were treated with 50 μM OPZ for different time points. All data were normalized to GAPDH level. (n = 3). D: TCDD induced MRP4 mRNA expression in primary human hepatocytes. Human hepatocytes from 2 individuals were exposed to DMSO or 5 nM TCDD for indicated times. The data presented are averages from triplicate measurements.

Fig. 2.

TCDD and OPZ upregulate MRP4 protein expression. Western blots analysis of total protein expression (A) and cell surface protein expression (B) in HepG2 cells 48 h after 50 μM OPZ treatment. C: immunofluorescence staining of MRP4 in primary human hepatocytes. Human hepatocytes were treated with DMSO, OPZ (50 μM), 3-methylcholanthrene (3-MC; 1 μM), or 4-PBA (1 mM) for 48 h. Bar represents 10 μm. Arrows point to the increase in plasma membrane staining of MRP4.

Fig. 3.

Endogenous MRP4 expression is decreased in aryl hydrocarbon receptor (AhR) knockdown HepG2 cells. Reduced AhR expression (A) led to diminished MRP4 mRNA (B) and protein (C) expression. CTL, control; siRNA, small interfering RNA. *P < 0.01, n = 3.

Fig. 4.

A: sequence of the proximal promoter of the MRP4 gene. The numbered positions refer to the translational initiation codon (ATG). →, Transcription start site at −119 bp. Potential XRE and MARE-like regulatory elements are underlined. B: reporter assays demonstrate that the highest promoter activity is within the first 700-bp 5′-flank region of MRP4 gene. The regulatory DNA elements are indicated by the symbols: triangle, MARE; oval, XRE. The promoter activity of each reporter constructs is presented as fold to pGL3-basic vector.

Fig. 5.

A: 3-MC or TCDD increased MRP4 promoter activity in reporter assays. The pGL3–761/-72Luc reporter construct was transfected in HepG2 cells and treated for 24 h; *P < 0.05, n = 3. B: mutations of XREs reduced MRP4 promoter activity in reporter assays. Ovals on the left of the bars represent XREs. *P < 0.005, #P < 0.05, n = 3. C: the XRE2 mutation prevents TCDD induction of the MRP4 promoter activity in reporter assay. HepG2 cells were transfected for 24 h and treated with 10 nM TCDD for additional 24 h. Data were normalized to Renilla luciferase activity. *P < 0.05, n = 3. n. s., Not significant.

Fig. 6.

A: cotransfection of NF-E2-related factor 2 (Nrf2) increases MRP4 promoter activity and deletion of MARE core sequence reduces the promoter activity in reporter assay. HepG2 cells were cotransfected with MRP4 promoter constructs of either the wild-type or the deletion of MARE core motifs for 48 h; n = 3 experiments. *P < 0.05, pGL3–761/-72Luc plus Nrf2 expression plasmid DNA vs. pGL3–761/-72Luc plus empty vector, **P < 0.05, the wild-type vs. the deletion of MARE, n = 3. B: mutation of XRE3 prevented the induction by Nrf2. HepG2 cells were cotransfected with pGL3–761/-72Luc wild type, XRE2 mutant or XRE3 mutant construct with either empty vector or Nrf2 expression plasmid (25 ng). The results are represented as fold induction compared with its control vector. *P < 0.05, n = 3.

Fig. 7.

A–C: EMSA demonstrate XRE1, XRE2, or XRE3 forms a complex (retarded band) with extracts from HepG2 cells. wt, Wild type; m, mutated; NE, nuclear extract. D: the specificity of MARE complex (retarded band) was confirmed by competition with an unlabeled oligo (wt) or the unlabeled deleted oligo (del). Loss of the complex occurred when the reaction mix was incubated with the labeled 4-bp deleted oligos. E: chromatin immunoprecipitation assay (ChIP) confirms that AhR and Nrf2 bind to the proximal promoter region of MRP4. MK, 100-bp DNA marker.