Larsen L, Størling J, Darville M, Eizirik D L, Bonny C, Billestrup N, Mandrup-Poulsen T
Steno Diabetes Center, 2 Niels Steensens Vej, 2820, Gentofte, Denmark.
Diabetologia. 2005 Dec;48(12):2582-90. doi: 10.1007/s00125-005-0039-9. Epub 2005 Nov 11.
AIMS/HYPOTHESIS: The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NFkappaB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1beta-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFkappaB activation in beta cells.
The MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1beta-induced iNOS production and kappa B inhibitor protein (IkappaB) degradation were examined by western blotting. NFkappaB DNA binding was investigated by electrophoretic mobility shift assay, while NFkappaB-induced gene transcription was evaluated by gene reporter assays.
Inhibition of the MAPKs did not affect IkappaB degradation or NFkappaB DNA binding. However, inhibition of ERK reduced NFkappaB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFkappaB complex seemed critical, as evaluated by amino acid mutation analysis.
CONCLUSIONS/INTERPRETATION: ERK activity is required for NFkappaB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFkappaB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.
目的/假设：1型糖尿病的特征是β细胞破坏和胰岛素缺乏，这部分是由细胞因子（如IL-1β）以及一氧化氮（NO）依赖性和非依赖性效应机制介导的。IL-1β激活丝裂原活化蛋白激酶（MAPK），包括细胞外信号调节激酶（ERK）、p38和c-Jun氨基末端激酶（JNK），以及核因子κB（NFκB）途径。这两条途径对于编码诱导型一氧化氮合酶（iNOS）的基因表达以及IL-1β介导的β细胞死亡都是必需的。目前尚不清楚这两条途径调节β细胞Nos2表达的分子机制。因此，本研究的目的是阐明β细胞中MAPK和NFκB激活之间的假定相互作用。
通过SB203580、PD98059或Tat-JNK结合结构域或过表达JNK结合结构域的细胞抑制MAPK ERK、p38和JNK。通过蛋白质印迹法检测MAPK抑制对IL-1β诱导的iNOS产生和κB抑制蛋白（IkappaB）降解的影响。通过电泳迁移率变动分析研究NFκB DNA结合，而通过基因报告分析评估NFκB诱导的基因转录。
MAPK的抑制不影响IkappaB降解或NFκB DNA结合。然而，ERK的抑制降低了NFκB介导的Nos2表达；通过氨基酸突变分析评估，NFκB复合物p65亚基的丝氨酸276磷酸化似乎至关重要。
结论/解读：在产生胰岛素的INS-1E细胞中，ERK活性是NFκB介导的Nos2转录所必需的，这表明ERK通过增加NFκB的反式激活能力来调节Nos2表达。这可能涉及一种尚未确定的激酶对p65上Ser276的磷酸化。
