Abstract
Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP3 receptors or stimulating IP3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP3 permeability. However, IP3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.
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Abbreviations
- AI:
-
apoptotic index
- AUC:
-
area under the curve
- BH4-Bcl-2:
-
BH4 domain of Bcl-2
- BIND:
-
a mutant BH4-Bcl-2 peptide in which the surface-accessible residues have been altered
- Cbx:
-
carbenoxolone
- Cx:
-
connexin
- CytC:
-
cytochrome C
- [Ca2+]i:
-
cytoplasmic Ca2+ concentration
- DAPI:
-
2′,6′-diamidino-2-phenylindole
- DMEM:
-
Dulbecco's modified Eagle's medium
- DTR:
-
dextran Texas Red
- ER:
-
endoplasmic reticulum
- EV:
-
empty vector
- GJ:
-
gap junction channel
- HBSS-Hepes:
-
Hanks’ balanced salt solution buffered with Hepes
- IDP:
-
IP3R-derived peptide
- IP3:
-
inositol 1,4,5-trisphosphate
- IP3R:
-
inositol 1,4,5-trisphosphate receptor
- NALC:
-
N-acetyl L-cysteine
- PBSD+:
-
PBS supplemented with Ca2+ and Mg2+
- PFA:
-
paraformaldehyde
- PI:
-
propidium iodide
- PLC:
-
phospholipase C
- PTP:
-
permeability transition pore
- ROS:
-
reactive oxygen species
- SCR:
-
scrambled version of BH4-Bcl-2
- SLDT:
-
scrape loading and dye transfer
- WT:
-
wild-type
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Acknowledgements
This work was impossible without the technical assistance of Ms Kirsten Leurs, Ms Anneleen Decock, Ms Marlies Bekaert, Ms Liesbeth Heyndrickx and Mr Eric Tack. We also gratefully acknowledge Mr Timothy Voorspoels for developing analysis tools and software. We thank Dr. D Boehning (Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA) for providing the IP3RCYT peptide. Our work is supported by: the Fund for Scientific Research Flanders (FWO-Vlaanderen), Belgium (G.0140.08, G.0134.09, G.0298.11N and WO.005.10N) and the Interuniversity Attraction Poles Program (Belgian Science Policy, project P6/31) assigned to LL; FWO (G.072810) assigned to DVK; a Methusalem grant (BOF09/01M00709) from the Flemish Government assigned to PV; FWO (G.0788.11N), the Research Council of the KU Leuven via OT START-1 (SRT/10/044) and the Interuniversity Attraction Poles Program (P6/28) assigned to GB; and the Interuniversity Attraction Poles Program (P6/28) assigned to CE.
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Decrock, E., Krysko, D., Vinken, M. et al. Transfer of IP3 through gap junctions is critical, but not sufficient, for the spread of apoptosis. Cell Death Differ 19, 947–957 (2012). https://doi.org/10.1038/cdd.2011.176
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DOI: https://doi.org/10.1038/cdd.2011.176
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