title: Gap junctional communication promotes apoptosis in a connexin-type-dependent manner.
abstract: Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to α-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca(2+) signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca(2+) concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca(2+) or a Ca(2+)-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca(2+) increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells.
authors.affiliation: Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München and Munich University Hospital, Munich, Germany. Kameritsch@lmu.de
authors.forename: P
authors.initials: P
authors.lastname: Kameritsch
authors.forename: N
authors.initials: N
authors.lastname: Khandoga
authors.forename: U
authors.initials: U
authors.lastname: Pohl
authors.forename: K
authors.initials: K
authors.lastname: Pogoda
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D053200:Fas-Associated Death Domain Protein / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013308:Streptonigrin / Q000494:pharmacology; D000097006:Gap Junction alpha-4 Protein
chemical_list: D002118:Calcium; D053179:Caspase 7; D018031:Connexin 43; D017630:Connexins; D053200:Fas-Associated Death Domain Protein; D049452:Green Fluorescent Proteins; D011065:Poly(ADP-ribose) Polymerases; D013308:Streptonigrin; D000097006:Gap Junction alpha-4 Protein; C484032:GJD4 protein, human