Elsevier

Journal of Hepatology

Volume 35, Issue 4, October 2001, Pages 436-443
Journal of Hepatology

FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte

https://doi.org/10.1016/S0168-8278(01)00158-1Get rights and content

Abstract

Background/Aims: FIC1 (familial intrahepatic cholestasis 1) is affected in two clinically distinct forms of hereditary cholestasis, namely progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis. Here we examined the subcellular localization of this protein within the liver.

Methods: Antibodies raised against different epitopes of human FIC1 were used for immunoblot analysis and immunohistochemical detection of FIC1.

Results: Immunoblot analysis of intestine and liver tissue extracts from human, rat and mouse origin indicated that the antibodies raised against FIC1 specifically detected FIC1 as a 140-kDa protein. In the liver homogenate of a PFIC1 patient, FIC1 could not be detected. Analysis of isolated rat liver membrane vesicles indicated that this protein is predominantly present in the canalicular membrane fraction. Immunohistochemical detection of the protein in liver sections confirmed that FIC1 was present in the canalicular membrane, whereas no staining was observed in the PFIC1 patients liver. Double label immunofluorescence of murine liver revealed that FIC1 colocalized with cytokeratin 7 in cholangiocytes.

Conclusions: The localization of FIC1 in the canalicular membrane and cholangiocytes suggests that it may directly or indirectly play a role in bile formation since mutations in FIC1 are associated with severe symptoms of cholestasis.

Introduction

Bile flow is one of the critical functions of the liver for excretion of drugs and lipids as well as endogenous and exogenous waste products [1], [2]. To comply with the demands of an ongoing bile flow, the hepatocytes and intrahepatic bile duct epithelial cells (cholangiocytes) express polarized transport systems at the basolateral and apical plasma membrane domains [3], [4]. Defective bile secretion results in cholestasis with accumulation of bile salts and other cholephiles within hepatocytes and blood plasma. Cholestasis may occur because of hereditary or acquired impairment of selective canalicular bile secretory processes or as a secondary consequence of chronic hepatocyte or bile duct damage [5]. A number of inheritable human disorders leading to cholestasis are associated with mutations in genes that encode hepatocyte transport proteins [6]. Progressive familial intrahepatic cholestasis type 1 (PFIC1; Ref. [7], [8]) and benign recurrent intrahepatic cholestasis (BRIC; Ref. [9], [10], [11]) are two such autosomal recessive inherited disorders, which are characterized by an impaired bile secretion [12]. These disorders initially manifest with similar clinical and biochemical features but they greatly differ in severity. Both are characterized by normal serum γ-glutamyltransferase activity and cholesterol levels, high concentrations of serum primary bile acids and low concentrations of biliary bile salts (especially chenodeoxycholic acid). BRIC is characterized by recurrent episodes of intrahepatic cholestasis, lasting weeks to months and starting in childhood or adulthood. The cholestatic attacks resolve spontaneously without leaving detectable damage in the liver. A distinguishing feature of PFIC1 is the progressive cholestasis starting in early childhood and leading to end-stage liver disease before reaching adulthood. The molecular basis of these forms of intrahepatic cholestasis was determined by positional cloning [13], [14]. PFIC1 and BRIC are caused by mutations in a single gene at chromosome 18q21, FIC1 [14], now officially renamed ATP8B1. Recently, we established that a third syndrome characterized by intrahepatic cholestasis, Greenland familial cholestasis, is also associated with a mutation in FIC1 [15]. FIC1 is the first characterized human member of the P4 subfamily of P-type ATPases; the proteins within this subfamily were suggested to function as aminophospholipid translocases [14], [16], [17], [18]. However, the aminophospholipid translocase activity of one of the other members of this subfamily (Drs2p from yeast) was questioned [19]. Thus, the molecular function of FIC1 is presently unclear. Besides its physiological role the subcellular localization within the liver also remains elusive. FIC1 mRNA is expressed in many epithelial tissues, including small intestine, pancreas, kidney and to a lesser extent in the liver [14]. The aim of the present study was to localize the FIC1 protein within the human and rodent liver to provide a basis for subsequent studies on the function of this protein.

Section snippets

Antibodies

cDNA fragments encoding different FIC1 cytoplasmic domains were individually subcloned into expression vector pQE30 (Qiagen, Düsseldorf, Germany) to create His6-tagged fusions. The sequences of the FIC1-C31, FIC1-C32 and FIC1-C6 fusion peptides correspond to the amino acid residues 539-748, 468–748 and 1164–1251, respectively of FIC1. These FIC1 sequences shared no significant sequence homology to other known liver membrane proteins. These His6-tagged fusion proteins were purified using Ni2+

Generation and characterization of FIC1 antibodies

To determine the subcellular localization of human FIC1 protein in the liver, polyclonal antibodies were raised against two distinct cytoplasmic domains of this protein. cDNA fragments encoding these domains were subcloned in a His6-affinity tag expression vector. To perform immunization of the rabbits, the His6-tagged fusion peptides FIC1-C31, FIC1-C32 and FIC1-C6 were overexpressed and purified from an Escherichia coli expression strain (Fig. 1a). After immunization, the raised antisera

Discussion

The intrahepatic cholestasis observed in the two clinically distinct disorders PFIC1 and BRIC is caused by mutations in a single gene, FIC1. The function of the FIC1-encoded protein is presently unclear. So far, the question of the localization of FIC1 in the liver has only been addressed in preliminary reports. However, these reports were in conflict with each other since either a localization in the cholangiocytes [28] or in the canalicular membrane [29] was suggested. Here, polyclonal

Acknowledgements

We express our appreciation to Carin Nibbering for stimulating discussions and to Dr R.J. Thompson for providing the liver biopsy material from the PFIC1 patient. We thank Titia Rolf and Ellen van Beurden for excellent technical assistance. EFE is supported by a grant from the Anton Meelmeyer Foundation (Academic Medical Center Amsterdam). SWCvM is supported by grant WS98-12 of the Dutch Digestive Disease Foundation; LWJK is the recipient of Ster grant SW006 of the University Medical Center

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