Elevated hepatic multidrug resistance-associated protein 3/ATP-binding cassette subfamily C 3 expression in human obstructive cholestasis is mediated through tumor necrosis factor alpha and c-Jun NH2-terminal kinase/stress-activated protein kinase–signaling pathway †‡
Potential conflict of interest: Nothing to report.
This work was supported by the National Natural Science Foundation of China (30570842, 81070320) and the Natural Science Foundation of Chongqing (CQ CSTC 2007BA5030), and J.L.B. and S-Y.C. are supported by National Institutes of Health grants DK R37 25636 and DK P30 34989.
Multidrug resistance-associated protein 3 (MRP3, ABC subfamily C [ABCC]3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up-regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n = 22) with obstructive cholestasis caused by gallstone blockage of bile ducts and noncholestatic patient controls (n = 22). MRP3/ABCC3 messenger RNA (mRNA) and protein expression were significantly increased by 3.4- and 4.6-fold, respectively, in these cholestatic patients where elevated plasma tumor necrosis factor alpha (TNFα) (4.7-fold; P < 0.01) and hepatic specificity protein 1 transcription factor (SP1) and liver receptor homolog 1 expression (3.1- and 2.1-fold at mRNA level, 3.5- and 2.5-fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose- and time-dependent manner, where increased phosphorylation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor, SP600125. TNFα treatment enhanced HepG2 cell nuclear extract-binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125, as demonstrated by electrophoretic mobility shift assay (EMSA). An increase in nuclear protein-binding activity to the MRP3/ABCC3 promoter, consisting primarily of SP1, was also observed in liver samples from cholestatic patients, as assessed by supershift EMSA assays. Conclusions: Our findings indicate that up-regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activation of JNK/SAPK and SP1. (HEPATOLOGY 2012)
Multidrug resistance-associated protein 3 (MRP3/Mrp3, ABC subfamily C [ABCC]3/Abcc3) is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily. 1 It is expressed in many tissues, including liver, kidney, bladder, intestine, and adrenal gland, in humans and rodents. 1, 2 MRP3/Mrp3 is localized on the basolateral membrane of cells and excretes sulfated, glycine-, and taurine-conjugated bile salts, bilirubin glucuronides, 17α-glucuronosyl estradiol, leukotrienes, and a number of drugs. 1-3 The expression of hepatic MRP3/Mrp3 (ABCC3/Abcc3) is low under normal physiological conditions, but its expression is significantly up-regulated as an adaptive protective response in cholestasis, as demonstrated in rodents after bile duct ligation (BDL) or lipopolysaccharide/CCl4 treatment. 4-6 This up-regulation has also been observed in the liver of patients with advanced stages of primary biliary cirrhosis (PBC) or with extrahepatic cholestasis caused by a pancreatic malignancy, but not in patients with early stages of PBC or progressive familial intrahepatic cholestasis. 7-10 It is not known whether MRP3/ABCC3 expression is altered in cholestasis as a result of biliary obstruction from bile duct stones. Furthermore, it remains to be determined how MRP3/ABCC3 expression is up-regulated in cholestasis.
The nuclear receptor, liver receptor homolog 1 (LRH-1, NR5A2), also called cytochrome P450 (CYP)7A promoter-binding factor (CPF), is a positive regulator of MRP3/Mrp3 expression, where TNFα signaling is involved in this up-regulation. 5 However, details of this signaling pathway remain to be elucidated, particularly in human cholestatic patients.
We and others have also found that the transcription factor, specificity protein 1 transcription factor (SP1), can directly bind to the MRP3/ABCC3 promoter and stimulate its expression. 11-13 Whether tumor necrosis factor alpha (TNFα)-signaling plays a role in SP1-stimulated MRP3/ABCC3 expression is not known.
Recent studies indicate that TNFα-activated c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling plays an important role in pseudorabies virus–induced apoptosis in Vero cells and in RNA-dependent protein kinase (PKR)-deficient mice. 14, 15 In addition, inhibition of the JNK/SAPK-signaling pathway decreases transcription factor SP1 expression in natural killer (NK) cells and in PC-3 and PC-3N cells. 16, 17 Therefore, we hypothesized that up-regulation of hepatic MRP3/ABCC3 expression in cholestatic patients may be mediated by TNFα signaling, and that JNK/SAPK, SP1, and LRH-1 might be involved in this regulation.
To test this hypothesis, we assessed MRP3/ABCC3 expression in the livers of patients with obstructive cholestasis resulting from gallstone blockage of bile ducts. In this report, we describe that increased hepatic MRP3/ABCC3 expression is associated with elevated TNFα levels and enhanced SP1 and LRH-1 expression and binding activity to the MRP3/ABCC3 promoter, and speculate that JNK/SAPK signaling may mediate this up-regulation.
All liver samples were collected from Southwest Hospital (Chongqing, China). This study was approved by the hospital institutional ethics review board, and informed consent was obtained from all participants. Control liver samples were acquired by liver biopsy for exclusion of liver disease or staging of hematologic malignancy (n = 7) and were also obtained during resections for liver metastases without cholestasis (n = 15; 6 colorectal, 7 colonic, and 2 rectal metastases). Cholestatic liver samples (n = 22) were surgically resected from patients with obstructive cholestasis caused by biliary stones originating from the intrahepatic bile duct and common bile duct within 3 days of admission because of severe symptoms of biliary obstruction and jaundice. Neither ursodeoxycholic acid nor other preoperative therapy were administered. The isolated liver samples were immediately cut into small pieces and stored in liquid nitrogen. Biochemical characteristics of patients are listed in Table 1.
Table 1. Clinical Features of Patients
Obstructive Cholestasis Patients
Values are means ± SD.
Abbreviations: TBIL, total bilirubin; DBIL, direct bilirubin; IBIL, indirect bilirubin; TBA, total bile salts; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase.
Human hepatoma HepG2 cells were cultured as previously described. 5 Before chemical treatment, the cells were starved from serum overnight and then treated with indicated dose of chemicals for designated times. For JNK/SAPK-signaling inhibition experiments, HepG2 cells were pretreated with SP600125 (Sigma-Aldrich Chemical Co., St Louis, MO) for 2 hours before the addition of TNFα.
RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction.
Total RNA was extracted from tissues or cultured cells with Trizol reagent (Invitrogen; San Diego, CA). Total RNA was reverse transcribed into complementary DNA (cDNA) using a RevertAid first-strand cDNA synthesis kit (MBI Fermentas Inc., Burlington, Ontario, Canada). Real-time quantitative polymerase chain reaction (qPCR), using a SYBR premix Ex Taq II kit (Takara Biotechnology, Tokyo, Japan), was performed in a Bio-Rad CFX96 real-time system machine (Bio-Rad, Hercules, CA) to determine the mRNA levels of specific genes, whose primers are listed in Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin were used as references for normalizing data, and real-time PCR amplification efficiency of target genes was considered, when using CFX manager 2.0 for data analysis (Bio-Rad).
Table 2. Sense and Antisense Primers Used for Real-Time qPCR (SYBR Green)
Sense Primer (5′→3′)
Antisense Primer (5′→3′)
Primers were also used for semiquantitative real-time PCR, as described previously. 18
Total protein was extracted using radioimmunoprecipitation assay buffer (Sigma-Aldrich), containing protease and phosphatase inhibitors (Roche, Palo Alto, CA). Membrane protein and nuclear protein were prepared as described previously. 18 The dilution of primary antibodies were as follows: multidrug resistance protein (MDR)1 (1:2,000), MDR3 (1:2,000), MRP2 (1:1,300), MRP3 (1:1,300), bile salt export pump (BSEP; 1:1,000), sodium taurocholate cotransporting polypeptide (NTCP; 1:1,000), CYP7A1 (1:2,000), SP1 (1:1,600), JNK (1:2,000), LRH-1 (1:1,600) (Santa Cruz Biotechnology, Santa Cruz, CA), and p-JNK (1:2,500) (Cell Signaling, Danvers, MA). GAPDH and SH-PTP1 (Santa Cruz) were used as the loading reference for data analysis.
Electrophoretic Mobility Shift Assay.
Oligos containing the SP1 and LRH-1 response element in human MRP3/ABCC3 promoter or its mutant forms, as previously reported, 5, 11 were labeled with γ-32P-ATP (10 mCi/mL). The electrophoretic mobility shift assay (EMSA) and supershift EMSA assay were performed using nuclear extracts, as described previously. 11
Determination of Plasma Proinflammatory Cytokines TNFα, Interleukin-1β, and Interleukin-6.
Plasma samples from patients were collected before biopsy or liver resection (control patients, n = 22; obstructive cholestatic patients, n = 22) and stored at −80°C until analysis. Plasma cytokine TNFα, interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) levels were determined using a human TNFα, IL-1β, or IL-6 Quantikine enzyme-linked immunosorbent assay kit (BD Biosciences, San Jose, CA). Bilirubin and bile salt levels encountered in cholestatic plasma samples did not interfere with the quantification of TNFα, IL-1β, and IL-6.
Immunofluorescence and Immunohistochemistry Analysis.
Immunofluorescence (IF) microscopy, combining the antibodies with MRP3 (1:50), SP1 (1:100), p-JNK (1:400), and JNK (1:200), was performed as previously described. 18 Immunohistochemistry (IHC) was also performed using p-JNK (1:100) and JNK (1:200) antibodies, as described previously. 9
All data were analyzed using an independent-samples t test (two-tailed) and are expressed as the mean ± standard deviations (SDs). Linear regression analysis was also performed using SPSS software (PASW Statistics 18, IBM; SPSS, Inc., Chicago, IL). A value of P < 0.05 was considered significant.
MRP3/ABCC3 Expression Is Elevated in the Liver Samples of Patients With Obstructive Cholestasis.
Real-time qPCR revealed that liver MRP3/ABCC3 and MDR3/ABC subfamily B (ABCB)4 mRNA expression in obstructive cholestatic patients was significantly increased (3.4- and 1.5-fold, respectively), when compared to controls, whereas mRNA expression levels of MRP2/ABCC2, BSEP/ABCB11, and MDR1/ABCB1 were not significantly changed (Fig. 1A). In contrast, the mRNA expression of bile salt uptake transporter NTCP/SLC10A1 and bile acid synthetic enzyme CYP7A1 were significantly decreased to 57% and 33% of controls, respectively (Fig. 1A). Western blotting detected that MRP3 and MDR3 protein expression increased 4.6- and 3.0-fold, respectively (Fig. 1B,C). The increase in MRP3 protein expression was further confirmed by IF labeling of MRP3 in the tissues where MRP3 at the basolateral membrane of cholestatic hepatocytes was more predominant than in control livers (Fig. 1D). Reduced expression of NTCP and CYP7A1 protein was also observed in these obstructive cholestatic livers (65% and 50% of controls, respectively), whereas BSEP and MDR1 protein expression was not significantly altered (Fig. 1B,C).
Plasma TNFα Is Elevated in Patients With Obstructive Cholestasis.
Proinflammatory cytokine TNFα, IL-1β, and IL-6 levels were increased in cholestatic rodent models, where elevated hepatic Mrp3/Abcc3 expression was also observed. 5, 19 To verify whether increases in these cytokines also occurred in our obstructive cholestatic patients, we analyzed the plasma levels of these three cytokines. The level of TNFα was significantly increased by 4.7-fold in these cholestatic patients, as compared to the control group, whereas the levels of IL-1β and IL-6 were not significantly altered (Fig. 2A). To examine whether there was any correlation between TNFα levels and MRP3/ABCC3 induction, we analyzed the expression of these two genes by linear regression. Induction of hepatic MRP3/ABCC3 expression positively correlated with the level of plasma TNFα, but not IL-1β (Fig. 2B). Together, these results suggested that the increase in MRP3/ABCC3 expression could be induced by elevated TNFα in these patients, as in rodents. 5
Transcription Factors SP1 and LRH-1 Expression and DNA-Binding Activity to the MRP3/ABCC3 Promoter Are Increased in Human Obstructive Cholestasis.
Both SP1 and LRH-1 are positive regulators of MRP3/ABCC3 gene expression. 5, 11-13 To determine whether these two transcription factors would play any role in the up-regulation of MRP3/ABCC3 expression in our bile-duct–obstructed patients, we first detected the expression levels of these two transcription factors. SP1 and LRH-1 mRNA expression significantly increased by 3.1- and 2.1-fold, respectively, in the cholestatic patients (Fig. 3A). Western blotting detected increases in SP1 and LRH-1 nuclear protein levels by ∼3.5- and 2.5-fold, respectively (Fig. 3B). We observed the same results by IF labeling of SP1 and LRH-1 in the liver tissues of patients, where SP1 and LRH-1 staining in the nuclei of cholestatic hepatocytes was more predominant than in controls (data not shown). The increase in the expression of SP1 and LRH-1 prompted us to examine whether there were any changes in the binding of these transcription activators to the MRP3/ABCC3 promoter. To address this question, we performed EMSA using nuclear protein extract from the liver samples of patients with obstructive cholestasis and controls. Binding of SP1 and LRH-1 to the MRP3/ABCC3 promoter in liver samples from patients with obstructive cholestasis was increased by 5.1- and 4.8-fold, compared to control livers, respectively (Fig. 3C-E). This binding to the MRP3/ABCC3 promoter was specific, as confirmed by mutation and supershift EMSA experiments (Fig. 3C,D).
TNFα Increased SP1 Expression and DNA-Binding Activity to the MRP3/ABCC3 Promoter in HepG2 Cells.
It has been previously observed that TNFα stimulates MRP3/ABCC3 expression in cultured cells. 5 To test whether TNFα could induce SP1 expression and its nuclear localization, we treated HepG2 cells with TNFα. In this experiment, we found that TNFα increased SP1 mRNA and nuclear protein expression in a dose- and time-dependent manner (Fig. 4A,B), where MRP3/ABCC3 mRNA expression was also coordinately induced. 5 In addition, EMSA experiments, using nuclear extract from TNFα-treated HepG2 cells, demonstrated increased binding capacity to the MRP3/ABCC3 promoter, when using the SP1 response element (Fig. 4C). These results further confirm a role for TNFα-mediated SP1 expression and activity in the up-regulation of MRP3/ABCC3 expression.
JNK/SAPK-Signaling-Pathway–Mediated TNFα Stimulated SP1 Expression and Activity in HepG2 Cells.
Previous studies have shown that the JNK/SAPK-signaling pathway could regulate SP1 expression in NK cells and be activated by TNFα in Vero cells and choroidal neovascularization. 16, 17, 20 To investigate whether the JNK/SAPK-signaling pathway could also mediate TNFα-induced MRP3/ABCC3 expression, we treated HepG2 cells with TNFα (100 ng/mL). Phosphorylation of JNK (p-JNK) was increased in a time-dependent manner, whereas total JNK was not significantly changed (Fig. 5A). When HepG2 cells were pretreated with 30 μM of SP600125 (a JNK-specific inhibitor), the TNFα-induced p-JNK was abolished in association with reduced inductions of SP1, LRH-1, and MRP3/ABCC3 at mRNA and protein levels in these cells (Fig. 5B). In contrast, both MRP2/ABCC2 mRNA and protein levels were not significantly changed (Fig. 5B). Furthermore, we assessed SP1 and LRH-1 binding to the MRP3/ABCC3 promoter by TNFα treatment of HepG2 cells in the presence or absence of the JNK inhibitor, SP600125. It was demonstrated that SP600125 dramatically reduces TNFα-stimulated SP1 and LRH-1 binding to the MRP3/ABCC3 promoter (Fig. 5C,D). The specificity of SP1 and LRH-1 binding to the MRP3/ABCC3 promoter was further confirmed by competition, mutation, and supershift experiments (Fig. 5C,D). Together, these results indicate that TNFα-induced MRP3/ABCC3 expression may be mediated through the JNK/SAPK-signaling pathway and activation of SP1 and LRH-1.
JNK/SAPK-Signaling Pathway Is Also Activated in Liver Samples of Obstructive Cholestasis Patients.
To verify whether the JNK/SAPK-signaling pathway would also be activated in livers from human obstructive cholestasis, we assessed the p-JNK in both obstructive cholestatic patients and control patients. p-JNK was markedly induced in liver samples with obstructive cholestasis versus controls (4.3-fold), whereas total JNK was not changed significantly (Fig. 6). IF analysis also showed that p-JNK staining in the nucleus of patients' cholestatic hepatocytes was more prominent than in controls (Fig. 6B, a,b). JNK staining was also increased in the nucleus of cholestatic hepatocytes versus controls (Fig. 6B, c,d). IHC analysis also demonstrated that p-JNK and JNK expression were induced significantly in the nucleus of hepatocytes from cholestatic patients, when compared to the controls (Fig. 6B, e-h). All together, these findings suggest that the JNK/SAPK-signaling pathway was also activated in human obstructive cholestasis, where JNK is phosphorylated and translocated into the nucleus of hepatocytes under cholestatic conditions.
Elevated expression of MRP3/ABCC3 has been previously reported in cholestatic rodent models and some patients with PBC and obstructive cholestasis caused by pancreatic tumors. 4-6, 9, 10 The up-regulation of MRP3/ABCC3 is thought to be an adaptive protective response to cholestasis, although the detailed molecular mechanisms remain unclear. In this report, we assessed MRP3/ABCC3 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts. We demonstrate that MRP3/ABCC3 mRNA and protein expression were significantly increased by 3.4- and 4.6-fold, respectively. Interestingly, we found that hepatic MRP3/ABCC3 mRNA expression in these patients significantly and positively correlated with their serum levels of TNF-α, but not IL-1β. The correlation of TNF-α levels with hepatic MRP3/ABCC3 mRNA expression in these patients is consistent with our previous observations in cholestatic mice, 5 indicating that TNFα signaling modulates hepatic MRP3/Mrp3 expression in both humans and rodents.
SP1 and LRH-1 are two nuclear transcription activators in the human MRP3/ABCC3 promoter. 5, 11-13 Recent studies indicate that the JNK/SAPK-signaling pathway could regulate SP1 expression in human NK cells and in PC-3 and PC-3N cells. 16, 17 As mentioned previously, TNFα signaling can also activate JNK/SAPK in pseudorabies virus–induced Vero cell apoptosis and in PKR-deficient mice. 14, 15 To elucidate the molecular mechanism of TNFα-induced MRP3/ABCC3 expression, we found that (1) TNFα increased JNK phosphorylation in HepG2 cells (Fig. 5A), (2) TNFα also stimulated the expression of SP1 and LRH-1 in HepG2 cells in a time- and dose-dependent manner (Fig. 4A,B), 5 (3) increased SP1 and LRH-1 expression was also observed in livers from our cholestatic patients, but not from control subjects (Fig. 3A,B), (4) enhanced activities of SP1 and LRH-1 binding to the MRP3/ABCC3 promoter was detected, using nuclear extract from both cholestatic human liver and HepG2 cells treated with TNFα, by EMSA and supershift assays (Figs 3C-E and 4C), 5 (5) in HepG2 cells, the JNK specific inhibitor, SP600125, blocked TNFα-induced p-JNK, induction of transcription factors SP1 and LRH-1 expression, as well as MRP3/ABCC3 expression, and binding activities of SP1 and LRH-1 to the MRP3/ABCC3 promoter (Fig. 5B-D), and (6) increased p-JNK was also detected in hepatocytes from our cholestatic patients (Fig. 6). Therefore, we speculate that TNFα induces hepatic MRP3/ABCC3 expression through activation of the JNK/SAPK-signaling pathway, leading to an increase in SP1 and LRH-1 expression and function in human obstructive cholestasis.
Increases in TNFα could also explain the increased MRP3/ABCC3 expression that was reported in advanced stages of PBC, but not in patients with early stages of PBC. 7, 9 In severe cholestasis, liver inflammation may induce TNFα expression and result in up-regulation of MRP3/ABCC3 expression, whereas TNFα level may be normal in the early stages of PBC so that hepatic MRP3/ABCC3 expression does not change. In addition, induction of MRP3 by TNFα may also contribute to the jaundices and hyperbilirubinemia that is often observed in patients and animals with sepsis 21, 22 and otherwise usually attributed to down-regulation of MRP2. 23 An elevated level of plasma TNFα, but not IL-1β, was also observed in septic cats with hyperbilirubinemia, although it is not known whether hepatic Mrp3 expression was increased in these animals. 22 However, MRP3/Mrp3 is a bilirubin glucuronide transporter. In particular, Mrp3−/− mice with bile duct obstruction demonstrated lower serum bilirubin glucuronide than their wild-type control. 24 Furthermore, up-regulation of Mrp3 was associated with tienilic-acid–enhanced hyperbilirubinemia in Eisai hyperbilirubinuria rats. 25 Therefore, under certain conditions, MRP3/Mrp3 can play a key role in transporting bilirubin out of the hepatocyte back into the blood. During sepsis, serum level of TNFα may increase, which, in turn, induces hepatic MRP3 expression that further contributes to hyperbilirubinemia associated with sepsis.
Our findings indicate that both SP1 and LRH-1 play roles in TNFα-mediated induction of MRP3/ABCC3 expression. However, the relative contribution of each transcription factor remains to be determined. It is conceivable that with the loss of one of these transcription factors, the other may compensate to ensure up-regulation of MRP3/ABCC3 expression when TNFα level is elevated. In addition, activators of nuclear receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) and transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) induce MRP3/Mrp3 expression in vitro in human cells and in vivo in the liver of rodents. 26-28 However, Mrp3/Abcc3 basal expression and induction by Pxr or Car activators is retained in Pxr−/− and Car−/− knockout mice, indicating that Pxr and Car may not play a direct role in Mrp3/Abcc3 expression in the mouse. 29, 30 Interestingly, reduced Mrp3/Abcc3 expression was detected in the liver of Nrf2−/− knockout mice, suggesting that Nrf2 plays a role in regulating Mrp3/Abcc3 expression, although the detailed mechanism remains elusive. 31 Whether TNFα induction of MRP3/Mrp3 may also be mediated through an Nrf2 pathway needs further study.
Although this study focused on the mechanisms of the adaptive response of MRP3/ABCC3 in the human cholestatic liver, we also found altered expression of other genes involved in bile salt transport and synthesis in these obstructive cholestatic patients. These included down-regulation of NTCP/SLC10A1 and CYP7A1 and increases in expression of MDR3/ABCB4, MRP4/ABCC4, 18 and OSTα/β (data not shown), but no significant change in BSEP/ABCC11, MRP2/ABCC2, and MDR1/ABCB1 (Fig. 1). These changes in gene expression are consistent with previous reports and also contribute to the overall adaptive protective responses in cholestatic liver injury. 5, 9, 10
In summary, our current study demonstrates that up-regulation of MRP3/ABCC3 in obstructive cholestasis is positively correlated with serum levels of TNFα. JNK/SAPK activation and increased SP1 and LRH-1 expression and activity may mediate TNFα induction of MRP3/ABCC3 expression in human hepatocytes. These findings also suggest that the proinflammatory cytokine, TNFα, plays a cytoprotective role in cholestatic liver injury.