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Connexin32 hemichannels contribute to the apoptotic-to-necrotic transition during Fas-mediated hepatocyte cell death

Cellular and Molecular Life Sciences volume 67, pages 907–918 (2010)Cite this article

Abstract

The present study was set up to investigate the fate of connexin32 and its channels in hepatocellular apoptosis. Primary hepatocyte cultures were exposed to Fas ligand and cycloheximide, and modifications in connexin32 expression and localization, and gap junction functionality were studied. We found that gap junction functionality rapidly declined upon progression of cell death, which was associated with a decay of the gap junctional connexin32 protein pool. Simultaneously, levels of newly synthesized connexin32 protein increased and gathered in a hemichannel configuration. This became particularly evident towards the end stages of the cell death process and was not reflected at the transcriptional level. We next either silenced connexin32 expression or inhibited connexin32 hemichannel activity prior to cell death induction. Both approaches resulted in a delayed termination of the cell death response. We conclude that connexin32 hemichannels facilitate the apoptotic-to-necrotic transition, which typically occurs in the final stage of hepatocellular apoptosis.

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Abbreviations

Ac-DEVD-AFC:

Acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin

ATP:

Adenosine triphosphate

CHX:

Cycloheximide

Cx:

Connexin

FasL:

Fas ligand

FRAP:

Fluorescence recovery after photobleaching

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GJIC:

Gap junctional intercellular communication

HBSS–Hepes:

Hank’s balanced salt solution supplemented with Hepes

LDH:

Lactate dehydrogenase

PbAE1:

1,4-Butanediol diacrylate-based poly-beta-aminoester

PBS:

Phosphate-buffered saline solution

PBSD+ :

Divalent ion-supplemented PBS

qRT-PCR:

Quantitative real-time reverse transcriptase-polymerase chain reaction

siRNA:

Small interfering RNA

TBS:

Tris-buffered saline solution

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Acknowledgments

The authors wish to thank Mr. Bart Degreef, Mr. Roel Fiey and Miss Sofie Wijthouck for their excellent technical assistance. This work was supported by grants from the Fund for Scientific Research Flanders (FWO-Vlaanderen), the Interuniversity Attraction Poles Program (Belgian Science Policy), the Research Council of the Vrije Universiteit Brussel (OZR-VUB) and the European Union (FP6 projects CARCINOGENOMICS and LIINTOP).

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Affiliations

  1. Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium

    Mathieu Vinken, Tamara Vanhaecke & Vera Rogiers

  2. Physiology Group, Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium

    Elke Decrock, Elke De Vuyst, Marijke De Bock & Luc Leybaert

  3. Department for Molecular Biomedical Research, Ghent University-VIB, Technologiepark 927, 9052, Ghent, Belgium

    Roosmarijn E. Vandenbroucke

  4. Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000, Ghent, Belgium

    Bruno G. De Geest, Joseph Demeester & Niek N. Sanders

  5. Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000, Ghent, Belgium

    Bruno G. De Geest

  6. Laboratory of Gene Therapy, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820, Merelbeke, Belgium

    Niek N. Sanders

Authors
  1. Mathieu Vinken
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  2. Elke Decrock
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  3. Elke De Vuyst
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  6. Bruno G. De Geest
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  9. Tamara Vanhaecke
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  10. Luc Leybaert
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  11. Vera Rogiers
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Correspondence to Mathieu Vinken.

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Vinken, M., Decrock, E., De Vuyst, E. et al. Connexin32 hemichannels contribute to the apoptotic-to-necrotic transition during Fas-mediated hepatocyte cell death. Cell. Mol. Life Sci. 67, 907–918 (2010). https://doi.org/10.1007/s00018-009-0220-2

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  • DOI: https://doi.org/10.1007/s00018-009-0220-2

Keywords

  • Apoptosis
  • Primary hepatocyte
  • Connexin32
  • Gap junction
  • Hemichannel