title: Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells.
abstract: 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 beta-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 p38alpha-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCdelta (protein kinase Cdelta), 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-1beta and interferon-gamma, 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.
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
authors.forename: Natalia
authors.initials: N
authors.lastname: Makeeva
authors.forename: Jason W
authors.initials: JW
authors.lastname: Myers
authors.forename: Nils
authors.initials: N
authors.lastname: Welsh
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D016207:Cytokines / Q000494:pharmacology*; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D048371:MAP Kinase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D048669:MAP Kinase Kinase 6 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D048056:Mitogen-Activated Protein Kinase 9 / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; D007328:Insulin; D007527:Isoenzymes; D009569:Nitric Oxide; D048056:Mitogen-Activated Protein Kinase 9; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6