| Popis: |
Mutation of the N-terminal IGF-binding site of IGFBP-5 and its transfection into myoblasts revealed that the functions of IGFBP-5 in myogenesis can be divided in to IGF-dependent inhibition of myogenesis, and an IGF-independent anti-apoptotic role, assessed by decreased caspase-3 and PARP cleavage, caspase-3 and -9 activities, and decreased annexin V staining of cell populations overexpressing wild type (wtIGFBP-5) and non-IGF binding IGFBP-5 (mutIGFBP-5). Further analysis revealed the decreased apoptosis stimulated by wt and mutIGFBP-5 correlates with increased Bcl-XL protein, and Bad phosphorylation levels. Transfection of myoblasts with wtIGFBP-5 resulted in decreased Akt phosphorylation, increased p38 activation and slightly increased p21 protein levels. Closer examination of cells overexpressing mutIGFBP-5 revealed enhanced myogenesis, increased p38 activation, and slightly, but consistently, increased p21 levels. Intriguingly, increased Akt phosphorylation was also observed in mutIGFBP-5 overexpressing cells which occurred independently of type I receptor activation. This suggests that mutIGFBP-5 was activating an alternative pathway to enhance the myogenic programme. To study the influence of the secretory pathway on the functions of IGFBP-5, the signal peptide was removed (nsIGFBP-5). The overexpression of nsIGFBP-5 in C2 cells had little effect on the rate or extent of myogenesis, Akt phosphorylation or p38 activation when compared with control cells, but increased p21 levels to a greater extent than did wtIGFBP-5. However, more dead cells were visible. When apoptosis was examined in these cell populations, nsIGFBP-5 no longer had a protective effect, as assessed by annexin V staining and caspase-3. Indeed, cells overexpressing nsIGFBP-5 appeared to have slightly elevated apoptosis, and an intriguing 4-fold increase in caspase-9 activation, which was not translated fully in to an increase in caspase-3 activity. The slightly elevated caspase-3 activity interestingly appeared to correlate with an approximate 50% reduction in Bcl-2 protein levels. |
