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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-X_Lprotein, 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.

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Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine produced mainly by monocytes and macrophages in response to bacterial infection and other harmful stimuli. The binding of IL-1 to its type 1 receptor leads to activation of several intracellular signalling molecules, including the three major Mitogen Activated Protein kinases (MAPKs): p42 MAPK, p38 MAPK and p54 MAPK, which is also known as Jun N-terminal kinase (JNK) or Stress Activated Protein kinase (SAPK). Strong activation of JNK/SAPK was observed in rabbit liver upon systemic administration of IL-1. This tissue was therefore used as a source to look for an IL-1-regulated activator of this protein kinase. A single activator was identified and, although it could not be purified by sequential column chromatography, it was identified using an antiserum of MAPK kinase 7 (MKK7), a newly identified activator of JNK/SAPK. It was not recognised by an antiserum to MKK4, the only other known activator of this MAPK. Activation of p38 MAPK in rabbit liver was not regulated by IL-1. Instead, a constitutively active p38 MAPK kinase was identified. Mutants of recombinant p38 MAPK were constructed to show that the activator phosphorylated the threonine and tyrosine residues of p38 MAPK, revealing it to be a dual-specificity kinase, like the other known MKKs. An antiserum which recognised MKK3 and MKK6 strongly precipitated the activator. Assays were developed so that the three MAPKs could be immunoprecipitated from various tissues and their activities measured. In most cell lines, IL-1 activates JNK/SAPK and p38 MAPK and in some it activates p42 MAPK. In five tissues JNK/SAPK was strongly regulated by IL-1 whilst p38 and p42 MAPKs were not. Antisera which recognise distinct JNK/SAPKs were also tested and revealed that the 54 and 46 kDa forms of JNK2/SAPKα were the main JNK/SAPK forms activated in the tissues.

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