Pharmacological Premature Termination Codon Readthrough of ABCB11 in Bile Salt Export Pump Deficiency: An In Vitro Study.

BACKGROUND AND AIMS: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X, and p.E1302X) identified in patients with PFIC2. APPROACH AND RESULTS: The ability of G418, gentamicin, and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full-length protein was studied in human embryonic kidney 293 (HEK293), HepG2, and Can 10 cells using immunodetection assays. The function of the gentamicin-induced full-length protein was studied by measuring the [3 H]-taurocholate transcellular transport in stable Madin-Darby canine kidney clones co-expressing Na+-taurocholate co-transporting polypeptide (Ntcp). Combinations of gentamicin and chaperone drugs (ursodeoxycholic acid, 4-phenylbutyrate [4-PB]) were investigated. In NIH3T3, aminoglycosides significantly increased the readthrough level of all mutations studied, while PTC124 only slightly increased the readthrough of p.E1302X. Gentamicin induced a readthrough of p.R415X, p.R470X, p.R1057X, and p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for BsepR1090X , which was also detected at the plasma membrane of human embryonic kidney HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and ursodeoxycholic acid significantly increased the canalicular proportion of full-length BsepR1090X protein in Can 10 cells. In Madin-Darby canine kidney clones, gentamicin induced a 40% increase of the BsepR1090X [3 H]-taurocholate transport, which was further increased with additional 4-PB treatment. CONCLUSION: This study constitutes a proof of concept for readthrough therapy in selected patients with PFIC2 with nonsense mutations.

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Functional rescue of an ABCB11 mutant by ivacaftor: A new targeted pharmacotherapy approach in bile salt export pump deficiency.

BACKGROUND & AIM: The canalicular bile salt export pump (BSEP/ABCB11) of hepatocytes is the main adenosine triphosphate (ATP)-binding cassette (ABC) transporter responsible for bile acid secretion. Mutations in ABCB11 cause several cholestatic diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) often lethal in absence of liver transplantation. We investigated in vitro the effect and potential rescue of a BSEP mutation by ivacaftor, a clinically approved cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) potentiator. METHODS: The p.T463I mutation, identified in a PFIC2 patient and located in a highly conserved ABC transporter motif, was studied by 3D structure modelling. The mutation was reproduced in a plasmid encoding a rat Bsep-green fluorescent protein. After transfection, mutant expression was studied in Can 10 cells. Taurocholate transport activity and ivacaftor effect were studied in Madin-Darby canine kidney (MDCK) clones co-expressing the rat sodium-taurocholate co-transporting polypeptide (Ntcp/Slc10A1). RESULTS: As the wild-type protein, BsepT463I was normally targeted to the canalicular membrane of Can 10 cells. As predicted by 3D structure modelling, taurocholate transport activity was dramatically low in MDCK clones expressing BsepT463I . Ivacaftor treatment increased by 1.7-fold taurocholate transport activity of BsepT463I (P < .0001), reaching 95% of Bsepwt activity. These data suggest that the p.T463I mutation impairs ATP-binding, resulting in Bsep dysfunction that can be rescued by ivacaftor. CONCLUSION: These results provide experimental evidence of ivacaftor therapeutic potential for selected patients with PFIC2 caused by ABCB11 missense mutations affecting BSEP function. This could represent a significant step forward for the care of patients with BSEP deficiency.

Successful Treatment with Rituximab and Immunoadsorption for an Auto-Antibody Induced Bile Salt Export Pump Deficiency in a Liver Transplanted Patient.

We present an 8 years old girl who was diagnosed at 6 months of age of Progressive Familial Intrahepatic Cholestasis type 2. Although liver transplantation (LT) was classically considered curative for these patients, cholestasis recurrence with normal gamma-glutamyl transpeptidase (GGT), mediated by anti-bile salt export pump (BSEP) antibodies after LT (auto-antibody Induced BSEP Deficiency, AIBD) has been recently reported. Our patient underwent LT at 14 months. During her evolution, patient presented three episodes of acute rejection. Seven years after the LT, the patient presented pruritus with cholestasis and elevation of liver enzymes with persistent normal GGT. Liver biopsy showed intrahepatic cholestasis and giant-cell transformation with very low BSEP activity. Auto-antibodies against BSEP were detected therefore an AIBD was diagnosed. She was treated with Rituximab and immunoadsorption with resolution of the AIBD. As a complication of the treatment she developed a pneumocystis infection successfully treated with corticoids, cotrimoxazol and anidulafungin.

Characterization of the effect of the mitochondrial protein Hint2 on intracellular Ca(2+) dynamics.

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca(2+) handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca(2+) dynamics in this cell type. We found that in hepatocytes isolated from Hint2(-/-) mice, the frequency of Ca(2+) oscillations induced by 1 µM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca(2+) pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2(-/-) mice; we found that Hint2 accelerates Ca(2+) pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca(2+) in suspensions of mitochondria. This prediction was then confirmed experimentally.

Differential redistribution of Ca2+-handling proteins during polarisation of MDCK cells: Effects on Ca2+ signalling.

The spatial organisation of the Ca(2+) signal in microdomains enables the regulation of various processes in specific regions of the cell and is essential for the versatility of cell responses to various stimuli. Ca(2+) signals can be independently regulated in the cytoplasm and in the nucleoplasm. Increases in the concentration of Ca(2+) in the nucleus can have specific effects different from those due to increases of Ca(2+) in the cytoplasm. We investigated the influence of cell polarity on the subcellular distribution of molecules responsible for intracellular Ca(2+) homeostasis (Ca(2+) release channels, Ca(2+) pumps and Ca(2+) binding proteins) and its influence on the intracellular Ca(2+) signal in MDCK cells with respect to its cytoplasmic or nucleoplasmic localisation. The intracellular Ca(2+) store was largely reorganised during cell polarisation, with a differential redistribution of IP3R, Ca(2+)-binding proteins and SERCA between the nuclear envelope and the periphery of the cell. This was accompanied by morphological changes in cell shape, which condense the cytoplasm around the nucleus, and in the shape of the nucleus, resulting in invaginations of the nuclear envelope. This facilitates Ca(2+) exchanges between the cytoplasm and the nucleoplasm, and preserves the ability to generate nucleoplasmic Ca(2+) transients in agonist-stimulated polarised MDCK cells.

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIbeta in the Golgi apparatus in cerebellar granule cells.

BACKGROUND INFORMATION: Interconnections between the Ca2+ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase-anchoring proteins). In many cell types, the activation of InsP3R (inositol 1,4,5-trisphosphate receptor), an endoplasmic reticulum Ca2+ channel, is a key event of Ca2+ signalling. The phosphorylation of InsP3R1 by PKA stimulates Ca2+ mobilization. This control is thought to be tight, involving the association of PKA with InsP3R1. The InsP3R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP3R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. RESULTS: Immunoprecipitation experiments showed that InsP3R1 associated with PKA type IIbeta and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co-purification of AKAP450 with InsP3R1 on heparin-agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP3R1 and RIIbeta (regulatory subunit of PKA IIbeta) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP3R1 was detected in this organelle only in granule cells. CONCLUSIONS: Taken together these results suggest that InsP3R1 forms a complex with AKAP450 and PKAIIbeta, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP3R1 with PKA in Purkinje cells would require a different macromolecular complex.