9-cis-retinoic acid elevates MRP3 expression by inhibiting sumoylation of RXRα to alleviate cholestatic liver injury.

AIMS: Vitamin A and its metabolites has been found to be protective against cholestatic liver injury, but the exact underlying mechanisms involved in cholestatic liver injury remain unclear. The objective of this study was to determine the function and mechanisms of 9-cis-retinoic acid, the metabolite of vitamin A, in cholestatic liver injury. METHODS: The bile duct ligated (BDL) mice were treated with 9-cis-retinoic acid by intravenous injection through the tail for 10 days. The liver function and histology were assessed in the matched group and experimental group. The expression of MRP3 in liver tissue was tested by qRT-PCR, Western blotting, and IHC. Effect of RXRα sumoylation on MRP3 expression was investigated at a cellular level. Influence of 9-cis-retinoic acid on RXRα sumoylation was also tested in cells. RESULTS: Our findings showed that 9-cis-retinoic acid significantly decreases the serum ALT and AST level, alleviates hepatic necrosis of the BDL-mice. We also identified MRP3, an important protective hepatobiliary transporter in cholestasis, was elevated by 9-cis-retinoic acid in vivo and in vitro. 9-cis-retinoic acid weakened the sumoylation of RXRα, which promotes the cytoplasmic location of RXRα and lightens the interaction of RXRα and RARα. Inhibition of RXRα and RARα interaction increased MRP3 expression. CONCLUSIONS: 9-cis-retinoic acid alleviates cholestatic liver injury by elevating MRP3 expression through its mechanism of inhibiting sumoylation of RXRα.

Identification of novel MRP3 inhibitors based on computational models and validation using an in vitro membrane vesicle assay.

INTRODUCTION: Multidrug resistance-associated protein 3 (MRP3), an efflux transporter on the hepatic basolateral membrane, may function as a compensatory mechanism to prevent the accumulation of anionic substrates (e.g., bile acids) in hepatocytes. Inhibition of MRP3 may disrupt bile acid homeostasis and is one hypothesized risk factor for the development of drug-induced liver injury (DILI). Therefore, identifying potential MRP3 inhibitors could help mitigate the occurrence of DILI. METHODS: Bayesian models were developed using MRP3 transporter inhibition data for 86 structurally diverse drugs. The compounds were split into training and test sets of 57 and 29 compounds, respectively, and six models were generated based on distinct inhibition thresholds and molecular fingerprint methods. The six Bayesian models were validated against the test set and the model with the highest accuracy was utilized for a virtual screen of 1470 FDA-approved drugs from DrugBank. Compounds that were predicted to be inhibitors were selected for in vitro validation. The ability of these compounds to inhibit MRP3 transport at a concentration of 100µM was measured in membrane vesicles derived from stably transfected MRP3-over-expressing HEK-293 cells with [3H]-estradiol-17ß-d-glucuronide (E217G; 10µM; 5min uptake) as the probe substrate. RESULTS: A predictive Bayesian model was developed with a sensitivity of 73% and specificity of 71% against the test set used to evaluate the six models. The area under the Receiver Operating Characteristic (ROC) curve was 0.710 against the test set. The final selected model was based on compounds that inhibited substrate transport by at least 50% compared to the negative control, and functional-class fingerprints (FCFP) with a circular diameter of six atoms, in addition to one-dimensional physicochemical properties. The in vitro screening of predicted inhibitors and non-inhibitors resulted in similar model performance with a sensitivity of 64% and specificity of 70%. The strongest inhibitors of MRP3-mediated E217G transport were fidaxomicin, suramin, and dronedarone. Kinetic assessment revealed that fidaxomicin was the most potent of these inhibitors (IC50=1.83±0.46µM). Suramin and dronedarone exhibited IC50 values of 3.33±0.41 and 47.44±4.41µM, respectively. CONCLUSION: Bayesian models are a useful screening approach to identify potential inhibitors of transport proteins. Novel MRP3 inhibitors were identified by virtual screening using the selected Bayesian model, and MRP3 inhibition was confirmed by an in vitro transporter inhibition assay. Information generated using this modeling approach may be valuable in predicting the potential for DILI and/or MRP3-mediated drug-drug interactions.

Farnesoid X receptor, the bile acid sensing nuclear receptor, in liver regeneration.

The liver is unique in regenerative potential, which could recover the lost mass and function after injury from ischemia and resection. The underlying molecular mechanisms of liver regeneration have been extensively studied in the past using the partial hepatectomy (PH) model in rodents, where 2/3 PH is carried out by removing two lobes. The whole process of liver regeneration is complicated, orchestrated event involving a network of connected interactions, which still remain fully elusive. Bile acids (BAs) are ligands of farnesoid X receptor (FXR), a nuclear receptor of ligand-activated transcription factor. FXR has been shown to be highly involved in liver regeneration. BAs and FXR not only interact with each other but also regulate various downstream targets independently during liver regeneration. Moreover, recent findings suggest that tissue-specific FXR also contributes to liver regeneration significantly. These novel findings suggest that FXR has much broader role than regulating BA, cholesterol, lipid and glucose metabolism. Therefore, these researches highlight FXR as an important pharmaceutical target for potential use of FXR ligands to regulate liver regeneration in clinic. This review focuses on the roles of BAs and FXR in liver regeneration and the current underlying molecular mechanisms which contribute to liver regeneration.

Protective effect of heme oxygenase induction in ethinylestradiol-induced cholestasis.

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 µmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis.

Restoring multidrug resistance-associated protein 3 attenuates cell proliferation in the polycystic kidney.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by abnormal proliferation of renal tubular epithelial cells, resulting in the loss of renal function. Despite identification of the genes responsible for ADPKD, few effective drugs are currently available for the disease. Thus finding additional effective drug targets is necessary. The functions of multidrug- resistance-associated protein 3 (MRP3) have been reported only in the field of drug resistance, and the renal functions of MRP3 are mostly unknown. In this study, we found that MRP3 was significantly downregulated in kidneys of human patients with ADPKD and polycystic kidney disease (PKD) mouse models. Our results suggest that downregulated MRP3 stimulated renal epithelial cell proliferation through the B-Raf/MEK/ERK signaling pathway. In contrast, we found that restoring MRP3 reduced cell proliferation and cystogenesis in vitro. These results suggest that the renal function of MRP3 is related to renal cell proliferation and cyst formation and that restoring MRP3 may be an effective therapeutic approach for PKD.

Multidrug resistance-associated protein 3 confers resistance to chemoradiotherapy for rectal cancer by regulating reactive oxygen species and caspase-3-dependent apoptotic pathway.

This study aimed to clarify the role of multidrug resistance-associated protein 3 (MRP3) in resistance to neoadjuvant chemoradiotherapy and long-term prognosis of advanced rectal cancer. Immunohistochemistry was used to measure MRP3 expression in biopsy specimens of 144 stage II-III rectal cancer patients who received preoperative chemoradiotherapy. The effect of MRP3 expression on short-term pathological response and postoperative long-term prognosis were assessed using the Cox proportional hazards model. Short interfering RNAs targeting MRP3 were synthesized and used to transfect human colorectal carcinoma cell lines. The effect of MRP3 down-regulation on cell proliferation and apoptosis in response to 5-fluorouracil and/or irradiation were examined in vitro and in xenograft mouse models, respectively. The content of intracellular reactive oxygen species and the activity of caspase-3-dependent apoptotic pathway in response to irradiation were further evaluated. High expression (immunoreactive score > 6) of MRP3 significantly predicted poor pathological response to chemoradiotherapy (tumor regression grade ≤ 2 vs. ≥3, p = 0.002) in univariate analysis and unfavorable long-term prognosis (5-year overall survival: HR = 1.612, 95% CI, 1.094-2.375, p = 0.016; 5-year disease-free survival: HR = 1.513, 95% CI, 1.041-2.200, p = 0.030) in multivariate Cox analysis. MRP3 down-regulation significantly increased 5-fluorouracil or irradiation-induced cell apoptosis and attenuated tumor growth following irradiation in animal models. MRP3 inhibition significantly reduced intracellular reactive oxygen species exporting from cells following irradiation, and increased expression of cleaved poly ADP-ribose polymerase and caspase-3. Aberrant expression of MRP3 in rectal cancer confers chemo-radioresistance. MRP3 might be a predictive factor and an attractive target in treating advanced rectal cancer.