Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. We previously reported that E297G and D482G BSEP, which are frequently found mutations in European patients, result in impaired membrane trafficking, whereas both mutants retain their transport function. The dysfunctional localization is probably attributable to the retention of BSEP in endoplasmic reticulum (ER) followed by proteasomal degradation. Because sodium 4-phenylbutyrate (4PBA) has been shown to restore the reduced cell surface expression of mutated plasma membrane proteins, in the current study, we investigated the effect of 4PBA treatment on E297G and D482G BSEP.
Pravastatin is a well known 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. Cumulative studies have shown that pravastatin is taken up into hepatocytes by the organic anion transporting polypeptide family transporters and excreted into the bile as an intact form by multidrug resistance-associated protein 2 (MRP2). It is generally accepted that the bile salt export pump (BSEP/ABCB11) mainly transports bile acids and plays an indispensable role in their biliary excretion. Interestingly, we found that BSEP could accept pravastatin as a substrate. Significant ATP-dependent uptake of pravastatin by human BSEP (hBSEP)-and rat BSEP (rBsep)-expressing membrane vesicles was observed, and the ratio of the uptake activity of pravastatin to that of taurocholic acid (TCA) by hBSEP was 3.3-fold higher than that by rBsep. The K m value of pravastatin for hBSEP was 124 M. A mutual inhibition study between TCA and pravastatin revealed that they competitively interact with hBSEP. Several statins inhibited the hBSEP-and rBsep-mediated uptake of TCA; however, the specific uptake of other statins (cerivastatin, fluvastatin, and pitavastatin) by hBSEP and rBSEP was not detected. The inhibitory effects of hydrophilic statins (pravastatin and rosuvastatin) on the uptake of TCA by BSEP were relatively lower than those of lipophilic statins. These data suggest that BSEP may be partly involved in the biliary excretion of pravastatin in both rats and humans.
ABSTRACT:Vectorial transport of bile acids across hepatocytes is a major driving force for bile flow, and bile acid retention in the liver causes hepatotoxicity. The basolateral and apical transporters for bile acids are thought to be targets of drugs that induce cholestasis. Previously, we constructed polarized LLC-PK1 cells that express both a major bile acid uptake transporter human Na
Fexofenadine (FEX) is mainly eliminated from the liver into bile in unchanged form. We demonstrated previously that organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are involved in the hepatic uptake of FEX. However, little is known about the mechanisms controlling the hepatic efflux of FEX from the liver to bile and blood. In the present study, the involvement of hepatic efflux transporters in the pharmacokinetics of FEX was investigated in both in vitro and in vivo studies. Vectorial transport of FEX was observed in OATP1B3/ human bile salt export pump (hBSEP) double transfectants but not in OATP1B3/human breast cancer resistance protein double transfectants, which indicates the possible contribution of hBSEP to the biliary excretion of FEX in humans. In multidrug resistance-associated protein 2 (Mrp2) Ϫ/Ϫ mice, the biliary excretion clearance based on the plasma concentration and the liver-to-plasma concentration ratio significantly decreased, whereas the biliary excretion clearance based on the liver concentration decreased only with 20%, suggesting the minimum contribution of Mrp2 to its biliary excretion. ATP-dependent transport of FEX was observed in hMRP3-enriched membrane vesicles but not hMRP4. In Mrp3 Ϫ/Ϫ mice, the biliary excretion clearance based on both the plasma and liver concentration and the liver-to-plasma concentration ratio increased, suggesting the significant contribution of Mrp3 to its sinusoidal efflux and the up-regulation of its biliary excretion in Mrp3 Ϫ/Ϫ mice. On the other hand, pharmacokinetics of FEX remained unchanged in Mrp4 Ϫ/Ϫ mice. This information provides a novel insight into the transporters important for FEX disposition.
The reduced expression of the bile salt export pump (BSEP/ ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We demonstrated previously that 4-phenylbutyrate (4PBA) treatment, a U.S. Food and Drug Administration-approved drug for the treatment of urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. On the other hand, BSEP mutations, E297G and D482G, found in progressive familial intrahepatic cholestasis type 2 (PFIC2), reduced it by shortening the degradation rate of cell-surfaceresident BSEP. Therefore, to help the development of the medical treatment of cholestasis, we investigated the underlying mechanism by which 4PBA and PFIC2-type mutations affect the BSEP degradation from cell surface, focusing on shortchain ubiquitination. In Madin-Darby canine kidney II (MDCK II) cells expressing BSEP and rat canalicular membrane vesicles, the molecular mass of the mature form of BSEP/Bsep shifted from 170 to 190 kDa after ubiquitin modification (molecular mass, 8 kDa). Ubiquitination susceptibility of BSEP/Bsep was reduced in vitro and in vivo by 4PBA treatment and, conversely, was enhanced by BSEP mutations E297G and D482G. Moreover, biotin-labeling studies using MDCK II cells demonstrated that the degradation of cell-surface-resident chimeric protein fusing ubiquitin to BSEP was faster than that of BSEP itself. In conclusion, BSEP/Bsep is modified with two to three ubiquitins, and its ubiquitination is modulated by 4PBA treatment and PFIC2-type mutations. Modulation of short-chain ubiquitination can regulate the change in the degradation rate of cell-surfaceresident BSEP by 4PBA treatment and PFIC2-type mutations.
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