Archives of Toxicology

, Volume 87, Issue 5, pp 883–894 | Cite as

Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures

  • Mathieu Vinken
  • Michaël Maes
  • Rachel Cavill
  • Dirk Valkenborg
  • James K. Ellis
  • Elke Decrock
  • Luc Leybaert
  • An Staes
  • Kris Gevaert
  • André G. Oliveira
  • Gustavo B. Menezes
  • Bruno Cogliati
  • Maria Lúcia Zaidan Dagli
  • Timothy M. D. Ebbels
  • Erwin Witters
  • Hector C. Keun
  • Tamara Vanhaecke
  • Vera Rogiers
In vitro systems

Abstract

Freshly established cultures of primary hepatocytes progressively adopt a foetal-like phenotype and display increased production of connexin43. The latter is a multifaceted cellular entity with variable subcellular locations, including the mitochondrial compartment. Cx43 forms hemichannels and gap junctions that are involved in a plethora of physiological and pathological processes, such as apoptosis. The present study was conducted with the goal of shedding more light onto the role of connexin43 in primary hepatocyte cultures. Connexin43 expression was suppressed by means of RNA interference technology, and the overall outcome of this treatment on the hepatocellular proteome and metabolome was investigated using tandem mass tag-based differential protein profiling and 1H NMR spectroscopy, respectively. Global protein profiling revealed a number of targets of the connexin43 knock-down procedure, including mitochondrial proteins (heat shock protein 60, glucose-regulated protein 75, thiosulphate sulphurtransferase and adenosine triphosphate synthase) and detoxifying enzymes (glutathione S-transferase μ 2 and cytochrome P450 2C70). At the metabolomic level, connexin43 silencing caused no overt changes, though there was some evidence for a subtle increase in intracellular glycine quantities. Collectively, these data could further substantiate the established existence of a mitochondrial connexin pool and could be reconciled with the previously reported involvement of connexin43 signalling in spontaneously occurring apoptosis in primary hepatocyte cultures.

Keywords

Primary hepatocyte Connexin43 Proteomics Metabolomics 

Abbreviations

ATP

Adenosine triphosphate

Cx

Connexin

DDA

Data-dependent acquisition

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GO

Gene ontology

GRP75

Glucose-regulated protein 75

HSP60

Heat shock protein 60

IPI

International protein index

LC/MS

Liquid chromatography/mass spectrometry

LOWESS

Locally weighted scatterplot smoothing

LTQ

Linear ion trap

NMR

Nuclear magnetic resonance

OT

Orbitrap

PbAE2

1,6-Hexanediol diacrylate-based poly-beta-aminoester

PBS

Phosphate-buffered saline solution

RF

Relative frequence

SDS–PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

siRNA

Small interfering RNA

TBS

Tris-buffered saline solution

TMT

Tandem mass tags

TSP

Trimethylsilyltetradeuteropropionic acid

Notes

Acknowledgments

The authors wish to thank Mr. P. Claes and Mrs. K. Schildermans for their excellent technical assistance. This work was supported by grants from the Research Council of the Vrije Universiteit Brussel-Belgium (OZR-VUB), the Fund for Scientific Research Flanders-Belgium (FWO-Vlaanderen) and the European Union (FP6 project carcinoGENOMICS and FP7/Cosmetics Europe projects HeMiBio and DETECTIVE).

Supplementary material

204_2012_994_MOESM1_ESM.doc (24 kb)
Supplementary material 1 (DOC 23 kb): Xcorr thresholds as a function of the charge state. Representation of the minimal Xcorr value for the different charge states (z) of the ions for confident (P0.05) and highly confident (P0.01) peptide-based homology protein identifications
204_2012_994_MOESM2_ESM.doc (30 kb)
Supplementary material 2 (DOC 29 kb): Presentation of the hepatocyte GO profile used as compared to the total rat GO profile. Numbers are relative abundances (%) of the top 25 GO identifiers found in the total set of identified proteins for the hepatocytes used and compared with the corresponding set of IPI rat GO identifiers. GO identifiers that differ more than 2-fold are presented in bold
204_2012_994_MOESM3_ESM.doc (28 kb)
Supplementary material 3 (DOC 27 kb): Presentation of the affected GO profile of the hepatocytes upon Cx43 suppression. All GO identifiers that occurred at least 3 times were added to the list. The relative frequence (RF) of each identifier was used to rank the GO terms
204_2012_994_MOESM4_ESM.docx (12 kb)
Supplementary material 4 (DOCX 11 kb): Proteins changed in the Cx43 siRNA condition versus the control condition. The 27 selected proteins have an abundance variability of less than 30 %
204_2012_994_MOESM5_ESM.docx (11 kb)
Supplementary material 5 (DOCX 11 kb): Proteins changed in the non-targeting siRNA condition versus the control condition. The 25 selected proteins have an abundance variability of less than 30 %

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Mathieu Vinken
    • 1
  • Michaël Maes
    • 1
  • Rachel Cavill
    • 2
    • 11
  • Dirk Valkenborg
    • 3
    • 4
    • 5
  • James K. Ellis
    • 2
  • Elke Decrock
    • 6
  • Luc Leybaert
    • 6
  • An Staes
    • 7
    • 8
  • Kris Gevaert
    • 7
    • 8
  • André G. Oliveira
    • 9
  • Gustavo B. Menezes
    • 9
  • Bruno Cogliati
    • 10
  • Maria Lúcia Zaidan Dagli
    • 10
  • Timothy M. D. Ebbels
    • 2
  • Erwin Witters
    • 3
    • 4
  • Hector C. Keun
    • 2
  • Tamara Vanhaecke
    • 1
  • Vera Rogiers
    • 1
  1. 1.Department of Toxicology, Faculty of Medicine and Pharmacy, Center for Pharmaceutical ResearchVrije Universiteit BrusselBrusselBelgium
  2. 2.Biomolecular Medicine, Department of Surgery and Cancer, Faculty of MedicineImperial College LondonLondonUK
  3. 3.Vlaamse Instelling voor Technologisch OnderzoekMolBelgium
  4. 4.Department of Biology and Center for ProteomicsUniversiteit AntwerpenAntwerpenBelgium
  5. 5.Interuniversity Institute for Biostatistics and Statistical BioinformaticsHasselt UniversityDiepenbeekBelgium
  6. 6.Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine and Health SciencesUniversiteit GentGhentBelgium
  7. 7.Department of Medical Protein ResearchVlaams Instituut voor BiotechnologieGhentBelgium
  8. 8.Department of Biochemistry, Faculty of Medicine and Health SciencesUniversiteit GentGhentBelgium
  9. 9.Department of Morphology, Institute of Biological SciencesUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  10. 10.Department of Pathology, School of Veterinary Medicine and Animal ScienceUniversity of Sao PauloSão PauloBrazil
  11. 11.Department of ToxicogenomicsMaastricht UniversityMaastrichtThe Netherlands

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