Mitochondria-specific transgenic overexpression of connexin-43 simulates preconditioning-induced cytoprotection of stem cells.
OBJECTIVE
We previously reported that preconditioning of stem cells with insulin-like growth factor-1 (IGF-1) translocated connexin-43 (Cx-43) into mitochondria, causing cytoprotection. We posit that these preconditioning effects could be simulated by mitochondria-specific overexpression of Cx-43.


RESULTS
During IGF-1-induced preconditioning of C57black/6 mouse bone marrow stem cell antigen-1(+) (Sca-1(+)) cells, Cx-43 was mainly translocated onto the mitochondrial inner membrane, which was abrogated by an extracellular signal-regulated kinases 1 and 2 (ERK1/2) blocker PD98059. To investigate the role of mitochondrial Cx-43, we successfully designed a vector coding for full-length mouse Cx-43 with a mitochondria-targeting sequence (mito-Cx-43) and cloned into a shuttle vector (pShuttle-IRES-hrGFP-1) for mitochondria-specific overexpression of Cx-43 (mito-Cx-43). Sca-1(+) cells with mito-Cx-43 reduced cytosolic accumulation of cytochrome c, lowered caspase-3 activity, and improved survival during index oxygen-glucose deprivation as determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling and lactate dehydrogenase assays. Computational analysis revealed a B-cell lymphoma-2 (Bcl-2) homology domain-3 (BH3) motif in Cx-43 with a conserved pattern of amino acids consistent with the Bcl-2 family that regulated cytochrome c release. Moreover, computational secondary structure prediction indicated an extended α-helix in this region, a known condition for BH3-driven protein-protein interactions.


CONCLUSIONS
Cx-43 translocation into mitochondria during preconditioning was ERK1/2-dependent. Expression of mito-Cx-43 simulated the cytoprotective effects of preconditioning in stem cells. Structural features of Cx-43 were shared with the Bcl-2 family as determined by computational analysis.
Lu
Gang
G
Department of Pathology and Lab Medicine, University of Cincinnati, Cincinnati, OH 45220, USA.
Haider
Husnain Kh
HKh
Porollo
Aleksey
A
Ashraf
Muhammad
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D045304:Cytochromes c / Q000378:metabolism; D019610:Cytoprotection; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051336:Mitochondrial Membranes / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
D000950:Antigens, Ly; D018031:Connexin 43; C543076:GJA1 protein, mouse; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D045304:Cytochromes c; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3