Mutation specific drug therapy for progressive familial or benign recurrent intrahepatic cholestasis: A new tool in a near future?
Article Outline
Abbreviations: PFIC, progressive familial intrahepatic cholestasis, BRIC, benign recurrent intrahepatic cholestasis, LT, liver transplantation, ER, endoplasmic reticulum, ERAD, endoplasmic reticulum associated degradation, 4-PBA, 4-phenylbutyrate, 6-ECDCA, 6-ethyl chenodeoxycholic acid
Keywords: BSEP, MDR3, FIC1, ABCB11, ABCB4, ATP8B1, PFIC, BRIC, Treatment, Nuclear receptor, Chaperone drug, Stop codon skipping
Commentary on: Folding defects in P-type ATP 8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate. van der Velden LM, Stapelbroek JM, Krieger E, van den Berghe PVE, Berger R, Verhulst PM, Holthuis JCM, Houwen RHJ, Klomp LWJ, van de Graaf SFJ. Hepatology 2010;51:286–296. Copyright 2010. Abstract reprinted with permission of John Wiley and Sons, Inc.
www.ncbi.nlm.nih.gov/pubmed/19918981
Abstract: Deficiency in P-type ATP8B1 is a severe and clinically highly variable hereditary disorder that is primarily characterized by intrahepatic cholestasis. It presents either as a progressive (progressive familial intrahepatic cholestasis type 1 [PFIC1]) or intermittent (benign recurrent intrahepatic cholestasis type 1 [BRIC1]) disease. ATP8B1 deficiency is caused by autosomal recessive mutations in the gene encoding ATP8B1, a putative aminophospholipid-translocating P-type adenosine triphosphatase. The exact pathogenesis of the disease is elusive, and no effective pharmacological therapy is currently available. Here, the molecular consequences of six distinct ATP8B1 missense mutations (p.L127P, p.G308V, p.D454G, p.D554N, p.I661T, and p.G1040R) and one nonsense mutation (p.R1164X) associated with PFIC1 and/or BRIC1 were systematically characterized. Except for the p.L127P mutation, all mutations resulted in markedly reduced ATP8B1 protein expression, whereas messenger RNA expression was unaffected. Five out of seven mutations resulted in (partial) retention of ATP8B1 in the endoplasmic reticulum. Reduced protein expression was partially restored by culturing the cells at 30
°C and by treatment with proteasomal inhibitors, which indicates protein misfolding and subsequent proteosomal degradation. Protein misfolding was corroborated by predicting the consequences of most mutations onto a homology model of ATP8B1. Treatment with 4-phenylbutyrate, a clinically approved pharmacological chaperone, partially restored defects in expression and localization of ATP8B1 substitutions G308V, D454G, D554N, and in particular I661T, which is the most frequently identified mutation in BRIC1. Conclusion: A surprisingly large proportion of ATP8B1 mutations resulted in aberrant folding and decreased expression at the plasma membrane. These effects were partially restored by treatment with 4-phenylbutyrate. We propose that treatment with pharmacological chaperones may represent an effective therapeutic strategy to ameliorate the recurrent attacks of cholestasis in patients with intermittent (BRIC1) disease.
Progressive familial intrahepatic cholestasis (PFIC) refers to a heterogeneous group of autosomal recessive liver disorders of childhood in which cholestasis of hepatocellular origin often presents in the neonatal period or first year of life and leads to death from liver failure at ages usually ranging from infancy to adolescence [1]. Recent molecular and genetic studies have shown that PFIC was related to severe mutations in canalicular transporter genes involved in bile formation [1]. PFIC1 is caused by mutation of the ATP8B1 gene encoding FIC1, a P-type ATPase which is a membrane aminophospholipid translocator expressed in several organs. In the liver it is essential for a proper composition of the canalicular membrane, and thus for normal bile flow [1]. PFIC2 is caused by a mutation in the ABCB11 gene encoding BSEP, the ATP-dependent canalicular bile salt export pump [1]. PFIC3 is caused by a mutation in the ABCB4 gene encoding MDR3, a P-glycoprotein of class III multidrug resistance proteins, involved in canalicular biliary phosphatidylcholine secretion [1]. Moderate deficiencies in FIC1 and BSEP may lead to a milder phenotype with episodes of cholestasis and intractable pruritus: benign recurrent intrahepatic cholestasis (BRIC1 and 2, respectively) [1].
Even though medical therapy such as ursodeoxycholic acid, rifampicin, cholestyramine, and/or surgical therapy such as biliary diversion might provide some symptomatic relief in PFIC1-3 patients, in the majority of cases liver transplantation (LT) is required because of severe cholestasis and unremitting pruritus, hepatic failure, or hepatocellular carcinoma [1], [2]. So far, transplantation of human hepatocytes has not proven efficient and gene therapy has not been initiated [1], [2]. While LT is curative in most cases, some severe complications may occur after LT. In PFIC1 patients, severe diarrhea and progressive fatty liver disease, as well as extradigestive symptoms, probably due to the extrahepatic expression of FIC1, have been described after LT [1], [2]. Moreover, in PFIC2 patients, recurrence of BSEP deficiency after LT has been recently described resulting from production of antibodies by the recipient in response to an allo-immunization against BSEP of the donor liver [1], [3], [4]. These specific complications of LT highlight the need for alternative therapies such as tailored pharmacological therapy in these diseases [1], [2].
As previously described, mutations in the canalicular transporters cause defective bile formation and retention of substances which are normally secreted into the bile. This may be due to decreased transporter expression at the canalicular membrane due to truncating or missense mutations that may lead to complete absence of protein expression, or to an abnormal intracellular trafficking with ER retention due to protein misfolding, respectively. Alternatively, some missense mutants, although correctly targeted at the canalicular membrane, may simply lead to a functional defect of the protein itself. Studies at the protein level have revealed that most ABCB11 missense mutations responsible for PFIC2 lead to trafficking impairment and increased proteasomal degradation of the resulting mutated BSEP [5], [6]. In PFIC3 it has been shown that one missense mutation leads to protein misfolding [7]. Such studies were lacking concerning PFIC1.
In a recent paper, van der Velden et al. studied protein expression in non-hepatocellular origin cell lines (HEK293T and U2OS cells) of human wild type FIC1 as well as 7 (6 missense, 1 nonsense) mutants known for being responsible for PFIC1 and/or BRIC1 [8]. A protein expression study was performed using transient transfection, and site-directed mutagenesis was performed in plasmid constructs containing the human ATP8B1 cDNA. The total expression level of FIC1 was decreased in all missense mutants but one. Plasma membrane expression was decreased in four of the missense mutants and completely absent in the nonsense mutant. An incubation with a proteasomal degradation inhibitor increased protein expression levels. Incubating at a reduced temperature, as well as with 4-phenylbutyrate (4-PBA), a pharmacological chaperone, increased protein expression and/or cell surface abundance of four of the FIC1 missense mutants. These results show that some mutations in ATP8B1 responsible for PFIC1 and/or BRIC1 lead to significant changes in FIC1 structure, which are responsible for protein misfolding and subsequent increased proteasomal degradation and/or protein mistrafficking/mislocalizing, eventually resulting in a reduction in the plasma membrane expression level of FIC1. The authors showed in vitro that proteasomal inhibitor and/or chaperone drugs such as 4-PBA can increase the cell surface abundance of some FIC1 mutants involved in PFIC1 and BRIC1. Even though no functional data about the consequence of the mutations before and after 4-PBA therapy are provided, these results support the concept of chaperone drug therapy in patients affected with PFIC1 or BRIC1. Such in vitro results are in line with previous observations showing that 4-PBA ameliorates cell surface abundance of a number of misfolded and mislocalized mutated membrane proteins involved in human liver diseases. These include some BSEP mutants identified in PFIC2 patients, cystic fibrosis transmembrane conductance regulator ΔF508 mutant, and some ATP7B mutants involved in Wilson disease [9], [10], [11].
Indeed, understanding the effects of selected mutations in these canalicular transporters at the protein level, and testing mutation specific drugs in vitro, constitute the basis for future in vivo personalized drug therapies for PFIC/BRIC (Fig. 1). Before translation into the clinical setting becomes feasible, confirmatory studies should be done, in vitro in human hepatocellular polarized cell lines, and/or in vivo in mutant liver models. The ultimate goal is to increase the canalicular expression level of a functional protein just enough to reverse the deficient phenotype. A partial reversion into a milder phenotype would also be of benefit. In case of a nonsense mutation, read through premature stop codons drugs (e.g. aminoglycosides, PTC124) could be used [9]. When a missense mutation is responsible for protein misfolding, pharmacological chaperones (e.g. 4-PBA, curcumin) could increase canalicular targeting whereas endoplasmic reticulum associated degradation (ERAD) inhibitors (e.g. MG132) could inhibit the subsequent proteasomal degradation [5], [8], [10], [11]. Lastly, agonists of nuclear receptors [e.g. 6-ethyl chenodeoxycholic acid (6-ECDCA), fibrates, statins] could be used to increase gene transcription of a missense mutant correctly addressed/targeted at the canalicular membrane and having residual activity [12]. These drugs could theoretically be used in combination to increase the expression level of functional protein at the canalicular membrane. Some of them, such as PTC124, 4-PBA, 6-ECDCA, or fenofibrate, are clinically approved and have already been tested, or are currently tested in human clinical trials [9], [12], [13]. The time to start clinical trials investigating mutation specific drug therapy in PFIC/BRIC patients is just around the corner. One should note that such treatment might also improve extrahepatic FIC1 related disease but might not abolish the risk of hepatocellular carcinoma associated with such chronic hepatocellular cholestasis, especially in BSEP deficiency, unless a high or full level of correction of protein dysfunction is achieved and no cell is already engaged in an irreversible malignant process.
Fig. 1.
Mutation specific drug therapy in PFIC/BRIC. Identification of the mutation class (nonsense, missense), assessment of the canalicular membrane expression in vivo of the mutant protein (immunohistochemistry), in vitro study of the protein trafficking/proteasomal degradation, of residual function of the mutated protein as well as of the effect of various drugs, constitute the rational to guide a mutation specific drug therapy strategy. Read through premature stop codon (mainly UGA) by drugs (e.g. aminoglycosides, PTC124), endoplasmic reticulum associated degradation (ERAD) inhibition by ERAD inhibitors (e.g. MG132), correction of protein misfolding by chaperone drugs (e.g. 4-PBA, curcumin), and increased gene transcription by nuclear receptor agonists (e.g. 6-ECDCA, fibrates, statins), are different approaches that could be used to render possible, sufficient expression of a functional protein at the canalicular membrane.
References
- . Progressive familial intrahepatic cholestasis. Orphanet J Rare Dis. 2009;4:1
- . Liver disease associated with canalicular transport defects: current and future therapies. J Hepatol. 2010;52:258–271
- Recurrence of bile salt export pump deficiency after liver transplantation. N Engl J Med. 2009;361:1359–1367
- . De novo bile salt transporter antibodies as a possible cause of recurrent graft failure after liver transplantation: a novel mechanism of cholestasis. Hepatology. 2009;50:510–517
- . Degradation of the bile salt export pump at endoplasmic reticulum in progressive familial intrahepatic cholestasis type II. Hepatology. 2008;48:1558–1569
- Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing. Hepatology. 2009;49:553–567
- A missense mutation in ABCB4 gene involved in progressive familial intrahepatic cholestasis type 3 leads to a folding defect that can be rescued by low temperature. Hepatology. 2009;49:1218–1227
- Folding defects in P-type ATP 8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate. Hepatology. 2010;51:286–296
- Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial. Lancet. 2008;372:719–727
- . 4-Phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps. Hepatology. 2007;45:1506–1516
- Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin. Hepatology. 2009;50:1783–1795
- . Nuclear receptors as therapeutic targets in cholestatic liver diseases. Br J Pharmacol. 2009;156:7–27
- . A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med. 1998;157:484–490
PII: S0168-8278(10)00379-X
doi:10.1016/j.jhep.2010.03.012
© 2010 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved.
