Taylor B S, Liu S, Villavicencio R T, Ganster R W, Geller D A
Department of Surgery, University of Pittsburgh, Pittsburgh PA, USA.
Hepatology. 1999 Apr;29(4):1199-207. doi: 10.1002/hep.510290419.
Previously, we demonstrated that nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced hepatic inducible nitric oxide synthase (iNOS) expression. NF-kappaB activation is regulated by kinases and phosphatases whose function is only beginning to be understood. Therefore, experiments were performed to determine the role of protein phosphatases (PPase) in cytokine-induced iNOS expression. Hepatocytes were stimulated with cytokines in the presence or absence of tyrosine phosphatase inhibitors (pervanadate [PV], phenylarsine oxide [PAO]) and a serine-threonine phosphatase inhibitor (okadaic acid [OA]). Cytokines induced hepatocyte iNOS mRNA, protein, and NO2- production that was substantially decreased by the addition of the tyrosine phosphatase inhibitors (PAO and PV). The serine-threonine phosphatase inhibitor (OA) decreased NO release and protein levels in a concentration-dependent fashion; however, iNOS mRNA levels were not significantly reduced. Nuclear run-on experiments demonstrated that protein tyrosine phosphatases (PTPases) are required for iNOS transcription, while the serine-threonine phosphatase inhibitor (OA) had no effect on iNOS transcription. Electromobility shift assays (EMSAs) revealed that the tyrosine-phosphatase inhibitors blocked cytokine-induced NF-kappaB activation, while OA did not have a significant effect on NF-kappaB DNA binding activity. Therefore, tyrosine phosphatases are involved in the regulation of cytokine-induced activation of NF-kappaB, while serine-threonine phosphatases posttranscriptionally regulate iNOS translation. These results identify the regulatory role of specific protein phosphatases (PPases) in hepatic iNOS expression.
此前，我们证明核因子-κB（NF-κB）介导细胞因子诱导的肝诱导型一氧化氮合酶（iNOS）表达。NF-κB的激活受激酶和磷酸酶调节，其功能才刚刚开始被了解。因此，进行实验以确定蛋白磷酸酶（PPase）在细胞因子诱导的iNOS表达中的作用。在存在或不存在酪氨酸磷酸酶抑制剂（过氧钒酸盐[PV]、氧化苯胂[PAO]）和丝氨酸-苏氨酸磷酸酶抑制剂（冈田酸[OA]）的情况下，用细胞因子刺激肝细胞。细胞因子诱导肝细胞iNOS mRNA、蛋白表达及NO2-生成，添加酪氨酸磷酸酶抑制剂（PAO和PV）后其显著降低。丝氨酸-苏氨酸磷酸酶抑制剂（OA）以浓度依赖性方式降低NO释放和蛋白水平；然而，iNOS mRNA水平未显著降低。细胞核转录实验表明，蛋白酪氨酸磷酸酶（PTPases）是iNOS转录所必需的，而丝氨酸-苏氨酸磷酸酶抑制剂（OA）对iNOS转录无影响。凝胶迁移实验（EMSA）显示，酪氨酸磷酸酶抑制剂阻断细胞因子诱导的NF-κB激活，而OA对NF-κB DNA结合活性无显著影响。因此，酪氨酸磷酸酶参与细胞因子诱导的NF-κB激活的调节，而丝氨酸-苏氨酸磷酸酶在转录后调节iNOS翻译。这些结果确定了特定蛋白磷酸酶（PPases）在肝iNOS表达中的调节作用。
