Eberhardt W, Huwiler A, Beck K F, Walpen S, Pfeilschifter J
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe Universität, Frankfurt am Main, Germany.
J Immunol. 2000 Nov 15;165(10):5788-97. doi: 10.4049/jimmunol.165.10.5788.
The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1beta-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2, 3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1beta-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5'-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1beta-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-kappaB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1beta. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1beta-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-kappaB.
自由基对细胞信号传导的调节作用在炎症性疾病的发病机制中具有重要意义。最近，我们发现一氧化氮（NO）可降低白细胞介素-1β（IL-1β）诱导的肾小球系膜细胞（MC）中基质金属蛋白酶（MMP-9）的表达。在此我们报告，由次黄嘌呤/黄嘌呤氧化酶系统（HXXO）或氧化还原循环剂2,3-二甲氧基-1,4-萘醌产生的外源性超氧化物，可导致IL-1β引发的、稳定状态下MMP-9信使核糖核酸（mRNA）水平显著升高，并使条件培养基中的明胶酶活性增强。单独使用超氧化物生成剂则无效。HXXO对细胞因子诱导的MMP-9内源性抑制剂金属蛋白酶组织抑制因子-1（TIMP-1）的稳定状态mRNA水平也有类似影响。用大鼠MMP-9基因5'侧翼区的0.6千碱基对进行大鼠系膜细胞的瞬时转染，证实超氧化物对MMP-9表达具有转录调控作用。HXXO增强了IL-1β触发的p65和c-Jun的核转位，同时增加了核因子κB（NF-κB）和激活蛋白-1（AP-1）的DNA结合活性。任一反应元件的突变均可完全阻止IL-1β对MMP-9启动子的激活。此外，经典的细胞外信号调节激酶（ERK）途径和p38丝裂原活化蛋白激酶（MAPK）级联反应的特异性抑制剂，可部分逆转HXXO对MMP-9 mRNA水平的影响，从而证明ERK和p38 MAPK参与了MMP-9的表达。此外，超氧化物可显著增强IL-1β引发的包括p38-MAPK、c-Jun氨基末端激酶和ERK在内的所有三种MAPK的磷酸化。我们的数据表明，超氧化物作为一种共刺激因子，通过干扰导致AP-1和NF-κB激活的信号级联反应，放大细胞因子诱导的MMP-9表达。
