Makeeva Natalia, Myers Jason W, Welsh Nils
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Biochem J. 2006 Jan 1;393(Pt 1):129-39. doi: 10.1042/BJ20050814.
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.
本研究的目的是进一步阐明p38丝裂原活化蛋白激酶（MAPK）在一氧化氮和细胞因子诱导的β细胞死亡中的重要性。为此，将分离的人胰岛用d-小干扰RNA（diced small interfering RNA）处理，然后暴露于一氧化氮供体DETA/ NONOate [2,2'-（羟基亚硝基肼基）双乙胺]。我们观察到，用p38α特异性d-小干扰RNA处理的细胞，而不是用靶向GL3（萤火虫荧光素酶小干扰RNA质粒）或PKCδ（蛋白激酶Cδ）的d-小干扰RNA处理的细胞，对一氧化氮诱导的死亡具有抗性。这与Bcl-XL（B细胞白血病/淋巴瘤-X长链）水平的升高相平行。为了深入研究p38激活的机制，MKK3（MAPK激酶3）、MKK6及其显性负突变体在产生胰岛素的RIN-5AH细胞中过表达。在瞬时转染中，MKK3过表达导致p38磷酸化增加，而在稳定过表达MKK3的RIN-5AH克隆中，p38和JNK（c-Jun N端激酶）的蛋白水平降低，导致磷酸化p38水平未受影响。此外，长期MKK3过表达不影响细胞对细胞因子白细胞介素-1β和干扰素-γ的死亡率，而短期MKK3表达导致细胞因子诱导的RIN-5AH细胞死亡增加。一氧化氮合酶抑制剂消除了MKK3对细胞因子诱导的细胞死亡的增强作用，磷酸酶抑制剂增强了MKK3刺激的p38磷酸化。最后，由于MKK3的显性负突变体不影响细胞因子诱导的p38磷酸化，并且由于野生型MKK3不影响p38自身磷酸化，可能是p38在响应细胞因子和一氧化氮时通过MKK3/6非依赖性途径被激活。此外，p38活性的长期增加可能被p38、JNK和p42基因表达的降低以及p38去磷酸化的增加所抵消。
