The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced β-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2′-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38α-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCδ (protein kinase Cδ), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1β and interferon-γ, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.
Research Article| December 12 2005
Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells
Jason W. Myers;
Nils Welsh 1
*Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
1To whom correspondence should be addressed, at Department of Cell Biology, Uppsala University, Biomedicum, P.O. Box 571, S-751 23 Uppsala, Sweden (email Nils.Welsh@medcellbiol.uu.se).
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Natalia Makeeva, Jason W. Myers, Nils Welsh; Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells. Biochem J 1 January 2006; 393 (1): 129–139. doi: https://doi.org/10.1042/BJ20050814
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