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Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis

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Abstract

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl− channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.

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Data availability

The datasets generated during and/or analyzed during the current study are fully available upon contact with the corresponding author.

Abbreviations

AH:

Alcoholic hepatitis

AP:

Acute pancreatitis

CF:

Cystic fibrosis

CFLD:

CF-associated liver disease

CFTR:

Cystic fibrosis transmembrane conductance regulator

CPA:

Cyclopiazonic-acid

DMSO:

Dimethyl sulfoxide

EPI:

Exocrine pancreatic insufficiency

ER:

Endoplasmic reticulum

EtOH:

Ethanol

FAEE:

Fatty acid ethyl ester

HPO:

Human pancreatic organoids

IP3R:

Inositol triphosphate receptor

iPSC:

Induced pluripotent stem cells

ITGB1:

Integrin beta-1

KRT19:

Cytokeratin 19

MLO:

Mouse liver organoids

MPO:

Mouse pancreatic organoids

PA:

Palmitic acid

PMCA:

Plasma membrane calcium pump

POA:

Palmitoleic acid

SERCA:

Sarco/endoplasmic reticulum Ca2+-ATPase

SOCE:

Store-operated calcium entry

STIM1:

Stromal interaction molecule 1

ΔΨm:

Mitochondrial membrane potential

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Acknowledgements

The authors are grateful to Professor Ursula Seidler (Department of Gastroenterology, Hannover Medical School, Hanover, Germany) for providing us the Cftr KO mice. We also would like to thank Professor Gergely Lukács (Department of Physiology, McGill University, Montréal, Canada) for providing us the CFTR-3xHA plasmid.

Funding

The research was supported by funding from the Hungarian National Research, Development and Innovation Office (GINOP-2.3.2-15-2016-00048 to J.M., PD116553 to P.P.), the Ministry of Human Capacities (EFOP 3.6.2-16-2017-00006to J.M.), Bolyai Research Fellowship (BO/00569/17 to P.P.), the Hungarian Academy of Sciences (LP2017-18/2017 to J.M.), the National Excellence Programme (20391-3/2018/FEKUSTRAT,TUDFO/47138-1/2019/ITM, TKP2020 and TKP2021-EGA-28to J.M.), and the New National Excellence Program of the Ministry of Human Capacities (ÚNKP-21-4-SZTE-1116 to T.M. and ÚNKP-21-3-SZTE-68 to N.P). This work was also supported by the Albert Szent-Györgyi Research Grant (5S 470 (A202) to T.C.) by the Faculty of Medicine, University of Szeged. The project has also received funding from the EU’s Horizon 2020 research and innovation programme under grant agreement No. 739593. Current work was also supported by funding from the Hungarian Government (NTP-NFTÖ-B-0011 to Á.V., and NTP-NFTÖ-21-B-0205 to A.K and NTP-NFTÖ-21-B-0079 to V.Sz.)

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Authors and Affiliations

  1. Department of Medicine, University of Szeged, Szeged, 6720, Hungary

    Tamara Madácsy, Árpád Varga, Noémi Papp, Bálint Tél, Petra Pallagi, Viktória Szabó, Aletta Kiss, Júlia Fanczal & József Maléth

  2. HAS-USZ Momentum Epithelial Cell Signaling and Secretion Research Group, University of Szeged, Szeged, 6720, Hungary

    Tamara Madácsy, Árpád Varga, Noémi Papp, Bálint Tél, Petra Pallagi, Viktória Szabó, Aletta Kiss, Júlia Fanczal & József Maléth

  3. First Department of Pediatrics, Semmelweis University, Budapest, Hungary

    Bálint Tél

  4. Department of Pathophysiology, University of Szeged, Szeged, 6720, Hungary

    Zoltan Rakonczay Jr.

  5. Department of Pathology, University of Szeged, Szeged, Hungary

    László Tiszlavicz & Zsolt Rázga

  6. Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany

    Meike Hohwieler & Alexander Kleger

  7. Biosciences Institute, Newcastle University, Newcastle upon Tyne, England

    Mike Gray

  8. Institute for Translational Medicine, University of Pécs, Pécs, Hungary

    Péter Hegyi

  9. Centre for Translational Medicine and Division for Pancreatic Disorders, Semmelweis University, Budapest, Hungary

    Péter Hegyi

  10. HCEMM-USZ Molecular Gastroenterology Research Group, University of Szeged, Szeged, 6720, Hungary

    József Maléth

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  1. Tamara Madácsy
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  2. Árpád Varga
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Contributions

TM and JM: designed the research project; TM, ÁV, NP, VSZ, BD, BT, PP, JF, RZ, ZR, MH, AK, and TL: contributed to acquisition, analysis and interpretation of data for the work; TM and JM: drafted the work, PH, MG and ZR: revised the manuscript critically for important intellectual content. All the authors approved the final version of the manuscript, agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Corresponding author

Correspondence to József Maléth.

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None of the authors has conflict of interest to declare.

Ethics approval

Animals were used with adherence to the NIH guidelines and the EU directive 2010/63/EU. The study was approved by the National Scientific Ethical Committee on Animal Experimentation under license number XXI./2523/2018. The collection and use of human samples including cadaver donor pancreas and liver samples were executed in adherence with the EU standards and approved by the Regional Committee of Research Ethics of the Hungarian Medical Research Council under license number 37/2017-SZTE.

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Madácsy, T., Varga, Á., Papp, N. et al. Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis. Cell. Mol. Life Sci. 79, 265 (2022). https://doi.org/10.1007/s00018-022-04287-1

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