Regular articleOxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation
Introduction
Reduction-oxidation (redox) reactions that generate reactive oxygen species (ROS), including H2O2, O2−, and OH−, have been identified as important chemical mediators that regulate signal transduction. Depending on its severity, oxidative stress can possibly lead to either cell necrosis or apoptosis [1]. It has been proposed that ROS in these two types of cell death function in fundamentally different ways: In necrosis, they serve as effectors, resulting in oxidative damage to lipids, nucleic acids, and proteins, whereas, in apoptosis, they may serve as signaling molecules via redox-sensitive pathways and cellular factors [2].
It has recently been shown that ROS stimulate intracellular signal events similar to those activated by growth factors, including stimulation of kinases and small G proteins such as c-Src, Ras, and ERK1/2 [3], [4]. Guyton et al. [5] have also shown H2O2-stimulated activation of ERK2 and a predominant pathway for ERK1/2 activation by H2O2 [6], [7]. However, the mechanism of how these signal transduction pathways are involved in ROS-mediated cell damage, especially apoptosis, remains to be clearly elucidated.
Recent studies with diverse systems have shown that both endogenously produced and exogenously added H2O2 can activate mitogen-activated protein kinases (MAPKs). MAPKs are serine/threonine protein kinases, which play pivotal roles in a variety of cell functions in many cell types [8], [9]. Families of MAPKs, consisting of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPK, have been identified to date in mammals. The regulatory network controlling ERK in mammalian cells is complex and consists of at least two pathways, which converge just upstream of MAP kinase at MAP kinase kinase (MEK). One pathway involves the c-raf proto-oncogene, while the other appears to be linked to serpentine receptors via G protein and protein kinase C (PKC) [10]. Stimulation of receptor tyrosine kinases often activates the Raf-1-MAPK/ERK kinase (MEK)-ERK cascade through Ras [11]. As for PKC-dependent ERK1/2 activation, it is well established that phorbol ester leads to a rapid and massive activation of ERK1/2 in most cell types [12].
It is generally accepted that the activation of the ERK cascade leads to cell proliferation [13]. The ERK1/2 pathway delivers a survival signal which counteracts proapoptotic effects elicited by JNK and the p38 activation [14]. However, investigations to unravel mechanisms, by which ERK mediates cell cycle arrest [15] and antiproliferation [16] as well as apoptotic [17] and nonapoptotic death [18], have only recently begun in a number of cells. The requirement of ERK1/2 in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells and ovarian cell lines [19] has also been demonstrated. Moreover, persistent activation of ERK1/2 contributes to glutamate-induced oxidative toxicity [20]. Although it is still unclear how the MAP kinase pathway affects cellular survival or death, earlier observations suggest that the MAP kinase cascade can be important for cellular responses to ROS.
In the present study, we, for the first time, observed that H2O2-mediated ERK1/2 activation, which was dependent on PKC (PKCδ) and tyrosine kinase phosphorylation (PDGFβ-receptor and c-Src)-dependent signaling, was responsible for the induction of apoptosis.
Section snippets
Chemicals and antibodies
MEK inhibitor, PD98059, JNK1/2 inhibitor, SP600125, GF109203X (broad range PKC inhibitors), safingol (PKCα inhibitor), Go6983 (PKCα and β inhibitor), rottlerin (PKCδ inhibitor), LY294002 (PI3 kinase inhibitor), AG490 (EGFR inhibitor), AG1296 (PDGF inhibitor), AG1478 (EGFR and PDGF inhibitor), genistein (EGFR inhibitor), and PP2 (Src inhibitor) were purchased from Calbiochem (La Jolla, CA). Anti-PKCα and PKCδ, anti-phospho-Akt, anti-Akt, anti-HA, anti-His, anti-Flag, anti-phosphotyrosine,
H2O2-induced apoptosis
As shown in Fig. 1A, 1 mM H2O2 induced DNA fragmentation of L929 cells in a time- and dose-dependent manner, and this was further confirmed by annexin V staining. Pretreatment of the cells with an antioxidant N-acetyl-l-cysteine (NAC) almost blocked H2O2-mediated DNA laddering (Fig. 1B).
H2O2 activated ERKs and JNKs
To further discern the effects of oxidative stress on L929 cells, we first examined whether H2O2 activated MAPKs in L929 cells. Thus, we exposed L929 cells to 1 mM H2O2 for 10 min and found that ERKs and JNKs
Discussion
In the present study, H2O2-mediated apoptosis in L929 cells was found to occur via ERK1/2 activation. In our L929 cell system, H2O2-induced ERK activation was dependent on both PKC-(PKCδ) and tyrosine kinase (PDGFβ-receptor, and c-Src)-dependent Raf-1-MAPK/ERK cascade through Ras.
Oxidative stress can trigger the activation of multiple signaling pathways that influence the cytotoxicity observed in affected cells, including the phosphorylation cascade leading to the activation of MAPKs. Although
Acknowledgements
We thank K.J. Kim for his excellent technical assistance. This work was supported by the Nuclear R&D Program from the Ministry of Science and Technology of Korea.
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