The Studies Of The Molecular Mechanism Of Hepatic Adaptive Response And MRP2/ABCC2 Post-transcriptional Down-regulation In Human Obstructive Cholestasis

Background & ObjectiveChronic cholestasis resulting from gallstone obstruction, hepatitis, biliary atresia, pancreas tumors and drug toxicity leads to fibrosis, cirrhosis, liver failure and death [1-4] The expression levels of hepatic membrane transporters and bile acid metabolic enzymes have been shown to be significantly altered in cholestasis [1-5]. Most of these changes are thought to be an adaptive response against cholestatic liver injury by inhibiting of bile acids synthesis (e.g. the reduction of CYP7A1) (Phase Ⅰ), reducing its hepatotoxicity (e.g. the induction of Cyp3a11, Ugt2b and Sult2al) (Phase Ⅱ) and enhancing its efflux (e.g. the induced expression of MRP3, MRP4 and OSTα/β) (Phase Ⅲ)[1-11]. For example, bile acid efflux transporter multidrug resistance-associated protein 4 (Mrp4) deficiency worsened liver injury and decreased plasma bile acid levels in bile duct-ligated mice. However, the expression of MRP4/Mrp4 at the basolateral membrane of cholestatic hepatocytes was markedly induced in human and rodent cholestasis [1-4,6-8]. Inhibition of bile acid synthetic enzymes, Cyp7al and Cyp7b1, and induction of detoxification enzymes, Cyp3all, Ugt2b and Sult2al, that contribute to decrease bile acid synthesis and toxicity have been observed in cholestatic rodents [1-5,9] However, only CYP7A1 repression was detected in human obstructive cholestasis and primary biliary cirrhosis (PBC)[7,10,11].In contrast, the induced expression of CYP8B1 was observed in extrahepatic cholestasis caused by pancreatic tumors[7].These data suggested that the molecular mechanism of adaptive response may vary depending on the organism and type of cholestasis. Whether a similar adaptive response is coordinately regulated in human obstructive cholestasis originating from gallstone blockage of bile ducts remains unclear.In particularly, multidrug resistance-associated protein 2 (MRP2; ABCC2) is expressed at the canalicular membrane of hepatocytes, secreting conjugated organic anions including bilirubin and bile acids and is the main determinant of bile acid-independent bile flow [2, 12, 13]. Dysfunction of MRP2/Mrp2 leads to jaundice and hyperbilirubinemia in patients with Dubin-Johnson syndrome (DJS) [13]. Jaundice is also seen in patients with obstructive cholestasis. Hepatic MRP2 mRNA expression was not changed in these patients [7,10]but how its protein expression is regulated remains uncertain. Previous studies in cholestatic rodent models have found reduced hepatic Mrp2 protein expression without changes in mRNA expression[14,15], indicating that post-transcriptional regulation most likely plays an important role in regulating Mrp2 function. Indeed loss of Mrp2 from bile canalicular membranes without altering its mRNA expression in Radixin-deficient mice and Radixin knockdown rat hepatocytes emphasized the importance of ’Radixin in the expression and function of rodent Mrp2 at post-transcriptional levels[17,18] Radixin is a member of the ERM protein family that consists of two more closely related proteins, i.e. Ezrin and Moesin [19]. Studies from Caco2 cells and rat intestine tissue indicate that Ezrin plays a role in regulating MRP2/Mrp2 membrane expression and function [20,21]. When the intestine was treated with the PKCa specific activator Thymeleatoxin, Ezrin Thr567 phosphorylation and Mrp2 brush-border membrane expression and efflUx activity were reduced although the mechanism remained unexplained [21].In contrast, activation of PKCa stimulated Ezrin phosphorylation in human breast carcinoma cells (MCF-7 cells)[23]. Elevated PKC activity, including PKCa,δ, and ε, was also reported in cholestatic rodent liver [14,24]. Thus, we hypothesize that elevated expression of PKCa during cholestasis leads to Ezrin Thr567 phosphorylation which may then result in MRP2 internalization and degradation, mediating by ubiquitin ligase E3 gp78.To address the molecular mechanism of hepatic adaptive response and MRP2 post-transcriptional down-regulation in human cholestasis, we analyzed the collected liver samples of patients with and without obstructive cholestasis by using Real time qPCR, Western-blot, stably transfected cell lines, immunohistochemistry (IHC), immunofluorescence (IF), co-immunoprecipitation(CO-IP), etc.Methods1. We collected liver samples from patients with or without obstructive cholestasis. All liver samples were immediately cut into small pieces and fixed in 4% paraformaldehyde or stored in liquid nitrogen until used.2. The mRNA levels of bile acids synthetic enzymes (e.g. CYP7B1, CY8B1, and CYP27A1), hepatic detoxification enzymes (e.g. UGT2B4/7, SULT2A1, GSTA1-4, and GSTM1-4), hepatic basolateral membrane transporters (e.g. OSTα/β and OCT1), canalicular membrane transporters (e.g. ABCG2 and ABCG5/8), and the relative nuclear receptors (e.g. VDR, PPARa, HNFla, HNF4a, RXRa, RARa, LXR, FXR and SHP) and transcriptional factors (e.g. HNF3β, NRF2 and AHR) in human obstructive cholestatic livers were measured by Real time qPCR (SYBR).3. The protein levels of these bile acids metabolic genes in human obstructive cholestatic livers and control livers were detected by Western-blot analysis.4. The protein expression and localization of hepatic transporters (OSTα/β, OCT1, ABCG2 and ABCG8) and nuclear receptors (VDR, HNF4a, RARa and FXR) in livers of patients with and without obstructive cholestasis were analyzed by immunofluorescence.5. We detected the mRNA expressions of hepatic cytoskeleton-linker proteins (Ezrin and Radixin), protein kinase Cs (PKCα, PKCδ and PKCε), and ubiquitin ligase E3s (gp78, TEB4 and HRD1) in human obstructive cholestatic livers and control livers by using Real time qPCR (SYBR).6. The protein expressions of these genes mentioned above were analyzed by Western blotting. Moreover, we also detected the phosphorylation of Ezrin Thr567 in human obstructive cholestasis by Western-blot.7. The protein expression and localization of MRP2, Ezrin, and p-Ezrin Thr567 in human hepatocytes of livers with and without obstructive cholestasis were measured by immunofluorescence and immunohistochemistry.8. The protein-protein interactions among Ezrin, p-Ezrin Thr567, Radixin, PKCs (PKCα, PKCδ and PKCε), and MRP2 were analyzed by co-immunoprecipitation.9. HepG2-Ezrin WT, T567A and T567D stably transfected cell lines and PKCa WT, DN, CAT stably transfected cell lines were used to detect MRP2 mRNA, total protein, and membrane protein by Real time qPCR, Western-blot, etc.Results1. The hepatic classic bile acids synthetic pathway (CYP7A1) was significantly repressed while the alternative synthetic pathway (CYP7B1 and CYP8B1) was activated in human obstructive cholestasis.2. The hepatic expression of detoxification enzymes, including UGT2B4/7, SULT2A1, GSTA1-4 and GSTM1-5 were markedly reduced in human obstructive cholestatic livers, compared to control livers.3. The hepatic membrane transporters OSTβ and OCT1 expression were significantly increased while OSTα and ABCG2/8 expression were markedly decreased in human obstructive cholestatic livers.4. The liver expression of nuclear receptor FXR was dramatically decreased while VDR, RARa and HNF4a expression were significantly increased in livers of patients with obstructive cholestasis, when compared to controls.5. There were significantly positive correlations between FXR mRNA reduction and detoxification enzymes UGT2B4/7, SULT2A1, or GSTA1 mRNA reduction in human obstructive cholestatic livers.6. Hepatic MRP2 protein and canalicular localization were dramatically reduced in human obstructive cholestatic livers, compared with control livers.7. The hepatic mRNA and protein levels of Ezrin and Radixin were unchanged in human obstructive cholestasis. However, Ezrin Thr567 phosphorylation was markedly increased in obstructive cholestatic livers, where enhanced the protein-protein interaction of Ezrin with MRP2. The stimulation of Ezrin Thr567 phosphorylation reduces membrane MRP2 expression in stably transfected HepG2 cell lines.8. The expressions of PKCs (PKCα, PKCδ and PKCε) at mRNA and protein levels were significantly increased in livers of patients with obstructive cholestasis. In HepG2 stably transfected cell lines, PKCa can stimulate Ezrin Thr567 phosphorylation and membrane MRP2 protein expression.9. Gp78 expression at mRNA and protein levels and MRP2 ubiquitination were significantly increased in human obstructive cholestatic livers, compared to control livers. Knockdown of gp78 increased the expression of MRP2 protein in HepG2 cells.ConclusionIn the current studies, we observed CYP7B1 and CYP8B1 up-regulation during cholestasis indicating that the alternative pathway of bile acid synthesis was activated, directing the bile acid synthesis towards to cholic acid (CA), but not chenodeoxycholic acid (CDCA). In particularly, dramatic detoxification enzymes reduction, including UGT2B4/7, SULT2A1, GSTA1-4 and GSTM1-4, suggests that hepatic detoxification ability may be greatly impaired in human obstructive cholestatic patients. Moreover, there were significantly positive correlations between the reduced FXR mRNA level and the decreased detoxification enzymes UGT2B4/7, SULT2A1, and GSTA1, which can explain why FXR agonists reduce liver injury (e.g. UDCA and INT747) in human cholestasis.In addition, we observed that hepatic MRP2 protein and canalicular membrane localization were dramatically reduced in human obstructive cholestasis while MRP2 mRNA levels were unchanged. We provided a mechanistic explanation for MRP2 post-transcriptional down-regulation in patients with obstructive cholestasis. Increased PKCa expression and activity phosphorylate Ezrin Thr567. The phosphorylated Ezrin enhances its binding to canalicular membrane MRP2 and retrieves MRP2 from canalicular membrane into cytosol. The internalized MRP2 is polyubiquitinated by ubiquitin ligase E3 gp78, and then is degraded by protease degradation (ERAD) pathway. Understanding the molecular regulation of hepatic adaptive response and MRP2 down-regulation may lead to new strategies for therapeutic interventions.