Molecular Mechanism Study On The Expression Of Hepatic Multidrug Resistance-associated Protein 2 Regulated By Interleukin-18

Cholestasis is a common syndrome in clinic, which is caused by obstacles in the synthesis and excretion of bile, result in the accumulation of bile in hepatocytes and blood. Liver is the most important organ for the metabolism of bile, for its elaborate system for taking in and out of bile components. This system consists of various translocators, such as organic anion transporting polypeptides(OATPs), bile salt export pump(BSEP), sodium/taurocholate cotransporting polypeptide(NTCP), multidrug resistance-associated proteins(MRPs) and so on. These transporters are located in different parts of liver cell membrane, working in cooperation to maintain the normal bile metabolism. The changes of bile transporters is considered to be the important molecular mechanisms of cholestasis.Multidrug resistance-associated protein 2(MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. Dysfunction of MRP2 im human may lead to accumulation of bilirubin in blood, which is called Dubin-Johnson syndrome. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. It is reported that serum level of interleukin-18(IL-18) in patients with bile duct obstruction was elevated obviously, and returned to normal when obstruction was relieved. It is also reported that there was a negative correlation between IL-18 and hepatic MRP2 in necrotic enteritis in rodents. These data indicate that IL-18 may participate in regulating expression of MRP2. In this study, we expected to reveal how IL-18 influences the expression of MRP2 and to discuss the molecular mechanism.Main methods and results1. IL-18 down-regulates MRP2 expression via NF-κB/FXR/YY1 pathway in HepG2 cells.We treated human hepatoma HepG2 cells with IL-18 and measured the expression of MRP2, nuclear factor kappa B(NF-κB), farnesoid X receptor(FXR), and the transcription factor Yin Yang 1(YY1) by quantitative real-time quantitative polymerase chain reaction(PCR) and western blotting. Then we transfected HepG2 cells with p65 ShRNA or YY1 ShRNA to observe whether these gene interferences affect the changes induced by IL-18.1.1 MRP2 and FXR were repressed and YY1 was enhanced by IL-18 in HepG2 cells at both the mRNA and protein level, in a dose-dependent manner.1.2 IL-18 activation of the NF-κB signaling pathway was indicated by nuclear p65 and total phosphorylated p65 enhancement.1.3 Changes in MRP2, FXR, and YY1 expression induced by IL-18 which were attenuated by p65 knockdown.1.4 Attenuation of the IL-18-induced decreases in MRP2 and FXR expression by YY1 knockdown.2. Elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression were observed in bile duct-ligated Sprague Dawley rat livers.Ten male Sprague Dawley(SD) rats were randomly divided into two groups: the bile duct-ligated(BDL) group(n=5) and the sham operation group(n=5). 7 days post-surgery, their livers were collected for RNA and protein extraction and detection.2.1 Elevated expression of IL-18, p65, and YY1 and decreased expression of FXR and MRP2 were observed in the BDL rats.2.2 Decreased amount of FXR bound to the MRP2 promoter was detected in the BDL rats livers by chromatin immunoprecipitation(ChIP) assay.DiscussionMRP2 is a key transporter for the balance of bile acid metabolism. High expression of MRP2 is critical for the maintenance of enterohepatic bile acid circulation, which may explain why most known molecular regulations of MRP2 are positive. Previous studies have reported that MRP2 expression was down-regulated at both the mRNA and protein level in some pathological states, mainly in cholestasis; however, the mechanism for this remains unclear. Many studies have supported the hypothesis that cytokines are essential in the regulation of transporter expression in cholestasis. For example, TNF-α up-regulates MRP3 expression at both the mRNA and protein level. IL-18 was reported to be significantly elevated in patients with obstructive jaundice and other diseases associated with cholestasis, such as primary biliary cirrhosis and intrahepatic cholestasis of pregnancy. Furthermore, IL-18 was associated with the depression of hepatic MRP2 in necrotic enteritis in rodents. These findings indicate that the increased IL-18 maybe not only be a bodily response to cholestasis, but may also affect the development of cholestasis, specifically via the suppression of MRP2 expression in the liver. Our study is the first to confirm this supposition. We found MRP2 expression was decreased by IL-18 treatment at both the mRNA and protein level and in a dose- and time-dependent manner. In BDL rats, we also detected a significantly elevated IL-18 mRNA expression and repressed MRP2 mRNA expression, which were confirmed to be significantly related by linear regression analysis.So the next question is which transcription factor mediates this down-regulation of the MRP2 gene. It is already known that MRP2 is directly regulated by several nuclear receptors such as FXR, PXR, CAR, and RAR. All these nuclear receptors enable positive regulation to promote the expression of MRP2. No transcription factor has yet been found to suppress MRP2 expression. So the most likely explanation is that IL-18 causes a reduction of a certain nuclear receptor that promotes MRP2 expression and thus, leads to the down-regulation of MRP2. FXR is a nuclear receptor that plays a key role in maintaining cholesterol and bile acid homeostasis, and is also a major transcriptional factor that regulates lipid and glucose metabolism in the liver. FXR has been proven to be an important nuclear factor that promotes the expression of MRP2. We detected the expression of several nuclear receptors and found that the expression of FXR was significantly repressed in HepG2 cells treated with IL-18. Decreased expression of FXR was also observed in BDL rats. This is an interesting finding, because the bile acids chenodeoxycholic acid, lithocholic acid, and deoxycholic acid are ligands for FXR. Therefore, FXR activity should be up-regulated in cholestasis because of the increased bile acids. However, our data indicates that FXR expression is down-regulated in cholestasis at both the mRNA and protein level, which may explain why MRP2 is down-regulated in cholestasis while FXR activity is likely up-regulated. Meanwhile, Cyp7a1 expression was shown to be elevated by IL-18 in HepG2 cells(S2 Fig), which further confirmed that the target genes of FXR could be regulated not only by the activation of FXR, which occurs mainly via ligand binding, but also by the expression level of FXR. ChIP assays further verified that FXR bound to the promoter region in the MRP2 gene was significantly less in liver extracts from the BDL rats than the sham-operated rats. This result further confirms that the reduction of MRP2 in the BDL rats is very likely to be associated with the reduction of FXR. Furthermore, these findings indicate that there must be other inhibitive factors which repress FXR expression. One candidate is the transcription factor YY1.YY1 is a ubiquitous and multifunctional zinc-finger transcription factor of the polycomb group protein family, which can act as a transcriptional repressor, activator, or initiator element-binding protein. A previous study demonstrated that YY1 can suppress FXR expression by binding to the YY1 responsive element in intron 1 of the FXR gene. We detected the expression of YY1 in HepG2 cells with IL-18 treatment and found that YY1 expression was elevated by IL-18 in a dose-dependent manner at both the mRNA and protein level. In the BDL rats, YY1 expression was also observed to be elevated and closely related to IL-18 expression. To investigate whether down-regulation of FXR and MRP2 were induced by YY1, we transfected HepG2 cells with YY1 shRNA and then treated the cells with IL-18. We discovered that FXR and MRP2 expression were still reduced, but the fold reduction was significantly lower than in normal HepG2 cells. This suggested that the IL-18-mediated decrease of MRP2 and FXR was attenuated by YY1 interference. In the BDL rats, we observed that YY1 mRNA expression was significantly negatively correlated with FXR mRNA expression. These data demonstrate that YY1 expression is enhanced in HepG2 cells with IL-18 treatment, and FXR expression is inhibited by YY1, which then leads to the down-regulation of MRP2.We then attempted to demonstrate the mechanism by which IL-18 induces YY1 expression. NF-κB is a primary signaling pathway conducted by IL-18, and has been shown in hepatocytes to be activated by IL-18. Upon binding to the IL-18 receptor(IL-18R), IL-18 initiates a signaling cascade that results in the activation of NF-κB. Furthermore, YY1 expression has been shown to be enhanced by NF-κB, which directly binds to the YY1 promoter using its p50/p65 heterodimer. Blockade of NF-κB results in the inhibition of YY1 expression. In our study, NF-κB was confirmed to be activated by IL-18 in HepG2 cells. In the BDL rats, NF-κB was also activated.To study whether IL-18 repression of MRP2 expression is mediated by the NF-κB pathway, we transfected HepG2 cells with p65 shRNA and then treated the p65 knockdown HepG2 cells with IL-18. We observed that the elevated YY1 expression and repressed MRP2 and FXR levels(i.e., conditions induced by IL-18) were all impaired in the p65 knockdown HepG2 cells. These results reveal that IL-18 can enhance YY1 expression and inhibit FXR and MRP2 expression, and that this regulation is partially mediated by activation of the NF-κB pathway.ConclusionsWe demonstrated that IL-18 can repress MRP2 expression through FXR. Furthermore, we observed that the transcription factor YY1 was enhanced by IL-18 via the NF-κB signaling pathway, and that the decreased expression of FXR was associated with enhanced YY1. Animal experiments verified these results, and furtherly proved that the reduced FXR lead to the expression of MRP2. Our findings represent a novel negative regulation of MRP2, which may contribute to increased understanding of the decreased expression of MRP2 in cholestasis and may also provide new directions to explore in the development of novel therapeutic strategies for clinical treatment of cholestasis.