Cellular and Molecular Life Sciences

, Volume 67, Issue 6, pp 907–918 | Cite as

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

  • Mathieu Vinken
  • Elke Decrock
  • Elke De Vuyst
  • Marijke De Bock
  • Roosmarijn E. Vandenbroucke
  • Bruno G. De Geest
  • Joseph Demeester
  • Niek N. Sanders
  • Tamara Vanhaecke
  • Luc Leybaert
  • Vera Rogiers
Research 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.

Keywords

Apoptosis Primary hepatocyte Connexin32 Gap junction Hemichannel 

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

Notes

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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Copyright information

© Birkhäuser Verlag, Basel/Switzerland 2009

Authors and Affiliations

  • Mathieu Vinken
    • 1
  • Elke Decrock
    • 2
  • Elke De Vuyst
    • 2
  • Marijke De Bock
    • 2
  • Roosmarijn E. Vandenbroucke
    • 3
  • Bruno G. De Geest
    • 4
    • 5
  • Joseph Demeester
    • 4
  • Niek N. Sanders
    • 4
    • 6
  • Tamara Vanhaecke
    • 1
  • Luc Leybaert
    • 2
  • Vera Rogiers
    • 1
  1. 1.Department of Toxicology, Faculty of Medicine and PharmacyVrije Universiteit BrusselBrusselsBelgium
  2. 2.Physiology Group, Department of Basic Medical Sciences, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium
  3. 3.Department for Molecular Biomedical ResearchGhent University-VIBGhentBelgium
  4. 4.Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical SciencesGhent UniversityGhentBelgium
  5. 5.Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical SciencesGhent UniversityGhentBelgium
  6. 6.Laboratory of Gene Therapy, Faculty of Veterinary MedicineGhent UniversityMerelbekeBelgium

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