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Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures

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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.

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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

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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).

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Author notes

  1. Rachel Cavill

    Present address: Department of Toxicogenomics, Maastricht University, PO Box 616, 6200 MD, Maastricht, The Netherlands

Authors and Affiliations

  1. Department of Toxicology, Faculty of Medicine and Pharmacy, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussel, Belgium

    Mathieu Vinken, Michaël Maes, Tamara Vanhaecke & Vera Rogiers

  2. Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, UK

    Rachel Cavill, James K. Ellis, Timothy M. D. Ebbels & Hector C. Keun

  3. Vlaamse Instelling voor Technologisch Onderzoek, Boeretang 200, 2400, Mol, Belgium

    Dirk Valkenborg & Erwin Witters

  4. Department of Biology and Center for Proteomics, Universiteit Antwerpen, Groenenborgerlaan 171, 2020, Antwerpen, Belgium

    Dirk Valkenborg & Erwin Witters

  5. Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Agoralaan 1, 3590, Diepenbeek, Belgium

    Dirk Valkenborg

  6. Department of Basic Medical Sciences, Physiology Group, Faculty of Medicine and Health Sciences, Universiteit Gent, De Pintelaan 185, 9000, Ghent, Belgium

    Elke Decrock & Luc Leybaert

  7. Department of Medical Protein Research, Vlaams Instituut voor Biotechnologie, Albert Baertsoenkaai 3, 9000, Ghent, Belgium

    An Staes & Kris Gevaert

  8. Department of Biochemistry, Faculty of Medicine and Health Sciences, Universiteit Gent, Albert Baertsoenkaai 3, 9000, Ghent, Belgium

    An Staes & Kris Gevaert

  9. Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    André G. Oliveira & Gustavo B. Menezes

  10. Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, São Paulo, Brazil

    Bruno Cogliati & Maria Lúcia Zaidan Dagli

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  1. Mathieu Vinken
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Correspondence to Mathieu Vinken.

Electronic supplementary material

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204_2012_994_MOESM1_ESM.doc

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

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

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

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

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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Vinken, M., Maes, M., Cavill, R. et al. Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures. Arch Toxicol 87, 883–894 (2013). https://doi.org/10.1007/s00204-012-0994-0

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