Partitioning of IGFBP-5 actions in myogenesis: IGF-independent anti-apoptotic function.
Igfbp5 is upregulated during the differentiation of several key cell lineages and in some tumours; the function of IGFBP-5 in these physiological and pathological situations is unknown. Since IGFBP-5 contains sequence motifs consistent with IGF-independent actions, the aim of these studies was to distinguish between IGF-dependent and -independent actions of IGFBP-5. Myc-tagged wild-type (termed wtIGFBP-5) and non-IGF binding mouse Igfbp5 (termed mutIGFBP-5) cDNAs were generated and used to transfect C2 myoblasts, a cell line that undergoes differentiation to myotubes in an IGF- and IGFBP-5-regulated manner. WtIGFBP-5, but not mutIGFBP-5, inhibited myogenesis, as assessed by cell morphology, MHC immunocytochemistry and caveolin 3 expression. However, both wt- and mutIGFBP-5 increased cell survival and decreased apoptosis, as indicated by decreased caspase-3 activity and cell surface annexin V binding. Further examination of apoptotic pathways revealed that wt- and mutIGFBP-5 ameliorated the increase in caspase-9 but not the modest increase in caspase-8 during myogenesis, suggesting that IGFBP-5 increased cell survival via inhibition of intrinsic cell death pathways in an IGF-independent manner. The relationship between IGF-II and IGFBP-5 was examined further by cotransfecting C2 myoblasts with antisense Igf2 (previously established to induce increased cell death) and Igfbp5; both wt- and mutIGFBP-5 conferred equivalent protection against the decreased cell survival and increased apoptosis. In conclusion, we have partitioned IGFBP-5 action in myogenesis into IGF-dependent inhibition of differentiation and IGF-independent cell survival. Our findings suggest that, by regulation of cell survival, IGFBP-5 has an autonomous role in the regulation of cell fate in development and in tumourigenesis.
Cobb
Laura J
LJ
Signalling Programme, The Babraham Institute, Cambridge CB2 4AT, UK.
Salih
Dervis A M
DA
Gonzalez
Ivelisse
I
Tripathi
Gyanendra
G
Carter
Emma J
EJ
Lovett
Fiona
F
Holding
Cathy
C
Pell
Jennifer M
JM
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D002460:Cell Line; D004789:Enzyme Activation; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D024510:Muscle Development; D018995:Myosin Heavy Chains / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D061945:Caspase Inhibitors; D018975:Insulin-Like Growth Factor Binding Protein 5; D012333:RNA, Messenger; D007335:Insulin-Like Growth Factor II; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D018995:Myosin Heavy Chains