Traore Kassim, Sharma Rajni, Thimmulappa Rajesh K, Watson Walter H, Biswal Shyam, Trush Michael A
Department of Environmental Health Sciences, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.
J Cell Physiol. 2008 Jul;216(1):276-85. doi: 10.1002/jcp.21403.
Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.
细胞外信号调节激酶（Erk）1/2的活性介导了由12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）诱导的髓样细胞分化。此前，我们报道TPA诱导的Erk1/2激活（磷酸化）需要活性氧（ROS）作为第二信使。在此，我们推测TPA刺激产生的ROS抑制了Erk1/2定向磷酸酶的活性，从而导致Erk1/2磷酸化增加，进而在ML - 1细胞中引发由p21（WAF1/Cip1）介导的生长停滞信号。用TPA孵育ML - 1细胞导致磷酸化Erk1/2显著积累，且这一过程发生在H2O2产生之后。有趣的是，用N - 乙酰半胱氨酸（NAC）对TPA处理后的细胞进行处理，会刺激Erk1/2发生显著且快速的去磷酸化，这表明NAC使Erk1/2定向磷酸酶活性得以恢复。基于以下观察结果，提示TPA在ML - 1细胞中诱导产生的ROS发生在线粒体电子传递链（METC）中：（i）未分化的ML - 1细胞不仅缺乏p67 - phox，而且p47 - phox（NADPH氧化酶酶活性所需的关键成分）表达水平较低；（ii）用NADH和NADPH依赖性酶的抑制剂二苯基碘（DPI）或复合物I的抑制剂大黄酸进行预处理，可阻断ROS的产生；（iii）对微阵列分析数据和蛋白质印迹分析数据的检测显示，响应TPA刺激，锰超氧化物歧化酶（MnSOD）在mRNA和蛋白质水平均有表达上调。MnSOD是线粒体防御系统中对抗线粒体来源超氧化物的关键成员。综上所述，本研究表明TPA刺激产生ROS作为第二信使，通过氧化还原介导的对Erk1/2定向磷酸酶的抑制作用来激活ML - 1细胞中的Erk1/2。
