Title: Mitochondria-specific transgenic overexpression of connexin-43 simulates preconditioning-induced cytoprotection of stem cells.

Authors: Lu, Gang; Haider, Husnain Kh; Porollo, Aleksey; Ashraf, Muhammad

Published In Cardiovasc Res, (2010 Nov 01)

Abstract: 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.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.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.

PubMed ID: 20833648

MeSH Terms: Amino Acid Motifs; Amino Acid Sequence; Animals; Antigens, Ly/metabolism; Apoptosis*/drug effects; Cells, Cultured; Connexin 43/chemistry; Connexin 43/genetics; Connexin 43/metabolism*; Conserved Sequence; Cytochromes c/metabolism; Cytoprotection; Insulin-Like Growth Factor I/metabolism; Male; Membrane Proteins/metabolism; Mice; Mice, Inbred C57BL; Mitochondria/drug effects; Mitochondria/metabolism*; Mitochondria/pathology; Mitochondrial Membranes/metabolism; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors; Mitogen-Activated Protein Kinase 1/metabolism; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors; Mitogen-Activated Protein Kinase 3/metabolism; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Kinase Inhibitors/pharmacology; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins c-bcl-2/chemistry; Proto-Oncogene Proteins c-bcl-2/metabolism; Recombinant Fusion Proteins/metabolism; Stem Cells/drug effects; Stem Cells/immunology; Stem Cells/metabolism*; Stem Cells/pathology; Structure-Activity Relationship; Time Factors; Transfection; Up-Regulation