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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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 %
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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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DOI: https://doi.org/10.1007/s00204-012-0994-0