Abstract
Ethanol metabolism plays a central role in activating the mitogen-activated protein kinase (MAPK) cascade leading to inflammation and apoptosis. Sustained activation of c-Jun N-terminal kinase (JNK), one of the MAPKs, has been shown to induce apoptosis in hepatocytes. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate MAPKs in several cells. The aim of the study is to evaluate the role of MKP-1 in sustained JNK activation as a mechanism to explain ethanol-induced hepatocyte apoptosis. VL-17A cells (HepG2 cells overexpressing alcohol dehydrogenase and cytochrome P450-2E1) were exposed to ethanol for different time periods. Western blots were performed for MKP-1, phospho-JNK, phosphotyrosine, and protein kinase Cdelta (PKCdelta). Electrophoretic mobility shift assays for AP-1 were performed. Apoptosis was measured by caspase-3 activity assay, TUNEL, and 4',6-diamidino-2-phenylindole staining. Reactive oxygen species were neutralized by overexpressing both superoxide dismutase-3 and catalase genes using lentiviral vectors in VL-17A cells. Ethanol incubation markedly decreased the MKP-1 protein levels to 15% of control levels and was associated with sustained phosphorylation of p46 JNK and p54 JNK, as well as increased apoptosis. VL-17A cells overexpressing superoxide dismutase-3 and catalase, treatment with a tyrosine kinase inhibitor, or incubation of the cells with PKCdelta small interference RNAs significantly inhibited the ethanol-induced MKP-1 degradation and apoptosis. Ethanol-induced oxidative stress enhanced the tyrosine phosphorylation of PKCdelta, which in turn caused the proteasomal degradation of MKP-1, leading to sustained JNK activation and increased apoptosis in VL-17A cells.
Highlights
Alcoholic liver damage is one of the major causes of cirrhosis
Transient activation of Jun N-terminal kinase (JNK) is necessary for liver regeneration [3], whereas sustained activation is involved in apoptosis [4, 5], and ethanol had been shown to induce the sustained activation of both isoforms of JNK (p54 JNK and p46 JNK) in hepatocytes [3, 5,6,7]
Incubation of VL-17A cells with ethanol caused an increase in the phosphorylation of both p46 JNK and p54 JNK at 30 min that remained increased up to 4 h (Fig. 1A), and this increase was associated with an enhancement in the nuclear translocation of AP-1 (Fig. 1B)
Summary
Alcoholic liver damage is one of the major causes of cirrhosis. Recent evidence suggests that many cellular responses of ethanol are mediated by the modulation of mitogen activated protein kinases (MAPK) signaling [1, 2]. MAPKs are activated by dual phosphorylation on Ser/Thr and Tyr residues, whereas 11 dual-specificity phosphatases or MAPK phosphatases (MKPs) terminate such activation [8] These phosphatases exhibit differential specificity toward MAPK substrates, show distinct subcellular localizations, and have different modes of regulation. Several PKC isoforms have been shown to have important roles in the regulation of cell survival and apoptosis [16, 17], and a series of studies suggests that PKC␦ is involved in apoptosis by acting as a proapoptotic signal [18, 19]. A selective inhibitor of PKC␦, rottlerin, and a dominant-negative mutant of PKC␦, attenuate apoptosis induced by phorbol ester [20], hydrogen peroxide (H2O2) [21], UV radiation [22], or etoposide [23] These studies demonstrate that PKC␦ is one of the prime regulators of apoptosis in several cell types. Cent protein; LV, lentiviral vector; EMSA, electrophoretic mobility shift assay; ROS, reactive oxygen species; SOD3, superoxide dismutase-3; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; RT, reverse transcription; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; DAPI, 4Ј,6-diamidino-2-phenylindole; siRNA, small interference RNA; E100, 100 mM ethanol; cat, catalase; DCF, 2Ј,7Ј-dichlorofluorescein; H2DCFDA, 2Ј,7Ј-dichlorodihydrofluorescein diacetate
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