ATP potentiates interleukin-1β-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR
ATP potentiates interleukin-1β-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR
复制标题
DOI：
10.1074/jbc.m305722200
复制
发表时间：
2003-12-19
影响因子：
4.8
通讯作者：
Eberhardt, W
中科院分区：
文献类型：
--
作者：
Huwiler, A;Akool, ES;Eberhardt, W
肾小球系膜细胞在炎性细胞因子如白介素1β的作用下，高水平表达基质金属蛋白酶9。我们证明了稳定的三磷酸腺苷类似物5‘-O-(硫代三磷酸)(ATPGammaS)能有效地增强细胞因子诱导的系膜细胞明胶分解的含量，主要是通过增加基质金属蛋白酶-9的稳态mRNA水平来实现的。含有1.3kb的基质金属蛋白酶-9 5‘-启动子区域的荧光素酶报告基因对ATPGammaS的反应较弱，但在基质金属蛋白酶-9的3’-非翻译区的额外控制下，荧光素酶活性强烈地依赖于ATP。通过体外降解实验和放线菌素D实验，我们发现ATPGammaS有效地延缓了基质金属蛋白酶-9mRNA的衰退。凝胶移位和超移位分析表明，存在于基质金属蛋白酶-9 3‘-非翻译区的三个富含AU的元件(ARs)与含有mRNA稳定因子Hur的复合体结合。ATPGammaS可显著提高这些复合体的RNA结合力。每个ARE元素的突变都会严重损害含有HUR的复合体的RNA结合。报告基因分析表明，其中一个ARE的突变不影响ATPGammaS的刺激作用，但这三个ARE的突变都会导致ATP依赖的荧光素酶活性的丧失，而不影响IL-1β的诱导性。在共聚焦显微镜下，我们证明了ATPGammaS增加了HUR的核质穿梭，并引起了细胞分裂实验所显示的胞质HUR水平的增加。综上所述，我们的结果表明，细胞外ATP对基质金属蛋白酶-9表达的放大是通过可能涉及到Hur依赖的基质金属蛋白酶-9 mRNA稳定性上升的机制来触发的。
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATPgammaS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATPgammaS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPgammaS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPgammaS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPgammaS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1beta-inducibility. By confocal microscopy we demonstrate that ATPgammaS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.