Pratt Phillip F, Bokemeyer Dirk, Foschi Marco, Sorokin Andrey, Dunn Michael J
Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
J Biol Chem. 2003 Dec 19;278(51):51928-36. doi: 10.1074/jbc.M309256200. Epub 2003 Oct 6.
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.
内皮素-1（ET-1）是一种强效血管收缩肽，具有与酪氨酸激酶信号通路激活相关的促有丝分裂作用。ET-1诱导环氧化酶-2（COX-2），该酶可将花生四烯酸转化为促炎类二十烷酸。已证实，三种主要的丝裂原活化蛋白激酶（MAPK）途径，即细胞外信号调节激酶1/2（ERK1/2）、应激活化蛋白激酶/应激激活蛋白激酶（JNK/SAPK）和p38 MAPK（p38）中的每一种途径的激活，均可增强COX-2的表达。MAPK的负调控可能通过一类称为丝裂原活化蛋白激酶磷酸酶（MKP）的双特异性磷酸酶家族发生。本研究的目的是检验以下假设：野生型MKP-1通过抑制培养的肾小球系膜细胞（GMC）中p38的激活来调节ET-1诱导的COX-2表达。构建了表达野生型和催化失活突变体MKP-1（MKP-1/CS）的腺病毒，以研究培养的GMC中ET-1调节的MAPK信号传导和COX-2表达。ET-1刺激ERK和p38α MAPK的磷酸化，并诱导COX-2的表达。COX-2的表达被MEK抑制剂U0126和p38 MAPK抑制剂SB 203580部分阻断。MKP-1/CS的腺病毒表达增强了基础和ET-1诱导的p38α MAPK的磷酸化，对ERK1/2磷酸化的影响较小。野生型MKP-1的异位表达阻断了ET-1对p38α MAPK的磷酸化，但增加了p38γ MAPK的磷酸化。共沉淀研究表明MKP-1与p38α MAPK和ERK1/2相关。免疫荧光图像分析表明，MKP-1/CS/绿色荧光蛋白将磷酸化的p38 MAPK捕获在细胞质中。与感染LacZ或绿色荧光蛋白的对照细胞相比，MKP-1/CS中ET-1刺激的COX-2表达增加。这些结果表明，在GMC中，MKP-1对p38α MAPK相对于p38γ MAPK表现出相对选择性，并且可能间接调节COX-2的表达。
