Chronic oxidative stress sensitizes hepatocytes to death from 4-hydroxynonenal by JNK/c-Jun overactivation

Abstract

Sustained activation of the c-Jun NH(2)-terminal kinase (JNK) signaling pathway mediates the development and progression of experimental diet-induced nonalcoholic fatty liver disease (NAFLD). Delineating the mechanism of JNK overactivation in the setting of a fatty liver is therefore essential to understanding the pathophysiology of NAFLD. Both human and experimental NAFLD are associated with oxidative stress and resultant lipid peroxidation, which have been proposed to mediate the progression of this disease from simple steatosis to steatohepatitis. The ability of oxidants and the lipid peroxidation product 4-hydroxynonenal (HNE) to activate JNK signaling suggested that these two factors may act synergistically to trigger JNK overactivation. The effect of HNE on hepatocyte injury and JNK activation was therefore examined in cells under chronic oxidant stress from overexpression of the prooxidant enzyme cytochrome P450 2E1 (CYP2E1), which occurs in NAFLD. CYP2E1-generated oxidant stress sensitized a rat hepatocyte cell line to death from normally nontoxic concentrations of HNE. CYP2E1-overexpressing cells underwent a more profound depletion of glutathione (GSH) in response to HNE secondary to decreased gamma-glutamylcysteine synthetase activity. GSH depletion led to overactivation of JNK/c-Jun signaling at the level of mitogen-activated protein kinase kinase 4 that induced cell death. Oxidant stress and the lipid peroxidation product HNE cause synergistic overactivation of the JNK/c-Jun signaling pathway in hepatocytes, demonstrating that HNE may not be just a passive biomarker of hepatic oxidant stress but rather an active mediator of hepatocellular injury through effects on JNK signaling.

Fig. 1.

Cytochrome P450 2E1 (CYP2E1) overexpression sensitizes to death from 4-hydroxynonenal (HNE). A: pCI-NEO vector cells (VEC) and S-CYP15 cells (S-CYP) were treated with the indicated micromolar concentrations of HNE, and the percentage of cell death was determined at 24 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (*P < 0.02; #P < 0.00001 compared with VEC cells treated with the same HNE concentration). B: VEC and S-CYP15 cells were left untreated (Con) or treated with 80 μM HNE for 8 or 12 h and costained with acridine orange and ethidium bromide, and the numbers of apoptotic (Apop) and necrotic (Nec) cells were determined by fluorescence microscopy as described in materials and methods (*P < 0.01; #P < 0.03 compared with the same type of cell death in VEC cells). C: percentage of cell death by MTT assay in S-CYP29 and S-CYP43 cells after 24 h of treatment with 80 or 85 μM HNE. Results are means + SE from 3 independent experiments each with duplicate data points.

Fig. 2.

S-CYP cells have constitutively increased reactive oxygen species production but normal levels of HNE. A: relative fluorescent intensity in untreated VEC and S-CYP15 (S-CYP) control cells (Con) and cells treated for 2 h with 85 μM HNE or 3.5 mM H₂O₂ (*P < 0.02; #P < 0.0001 compared with VEC cells with the same treatment). B: levels of HNE-histidine adducts in VEC and S-CYP cells untreated or treated for the indicated number of hours with 85 μM HNE (*P < 0.02 compared with VEC cells with the same treatment). Results are means + SE from 3 independent experiments each with duplicate data points.

Fig. 3.

S-CYP cells undergo marked glutathione (GSH) depletion with HNE treatment. GSH S-transferase activity (A) and cellular GSH content (B) were measured as described in materials and methods in VEC and S-CYP15 (S-CYP) cells untreated and treated with 80 μM HNE for the indicated times shown. Data are from 3 independent experiments performed in duplicate (*P < 0.04; #P < 0.0001 compared with VEC cells with the same treatment). C: percentage cell death in VEC cells treated with 80 μM HNE, diethyl maleate (DEM), or a combination of the two and in S-CYP15 cells treated with 80 μM HNE, 2 mM GSH ethyl ester, or both agents together. Results are means + SE from 3 independent experiments (*P < 0.03; #P < 0.0002 compared with cells treated with HNE alone).

Fig. 4.

S-CYP cells undergo increased GSH depletion from DEM and have decreased γ-glutamylcysteine synthetase (GCS) activity. A: GSH levels were determined in VEC and S-CYP15 (S-CYP) cells untreated and treated with DEM for the number of hours shown. Results are means + SE from 3 independent experiments (*P < 0.01 compared with VEC cells at the same time point). B: GCS activity in VEC and S-CYP15 cells untreated and treated for the indicated number of hours with 80 μM HNE. The data are means + SE from 4 independent experiments (*P < 0.003; #P < 0.001 compared with VEC cells at the same time point).

Fig. 5.

HNE treatment in the setting of CYP2E1 overexpression induces sustained c-Jun NH₂-terminal kinase (JNK)/c-Jun activation. A: protein was isolated from VEC and S-CYP15 (S-CYP) cells untreated or treated with 80 μM HNE for the indicated hours. Aliquots of protein were immunoblotted with antibodies for phospho-JNK (P-JNK) and total JNK (JNK), phospho-c-Jun (P-c-Jun) and total c-Jun (c-Jun), phospho-extracellular signal-regulated kinase (P-ERK1/2) and total ERK1/2 (ERK1/2), and phospho-MAPK kinase 4 (P-MKK4). B: identically treated cells were assayed for JNK activity by in vitro kinase assay. JNK activity is indicated by levels of P-c-Jun on immunoblots, and levels of total c-Jun (c-Jun) serve as a control for protein loading. C: relative levels of activator protein-1 (AP-1)-dependent luciferase activity in VEC and S-CYP15 cells untreated (Con) and treated with 80 μM HNE for 6 h (*P < 0.000001 compared with HNE-treated VEC cells). D: immunoblots with proteins from VEC, S-CYP15 (S15), S-CYP29 (S29), and S-CYP43 (S43) cells untreated or treated with 80 μM HNE for 4 h. The results in A, B, and D are representative of 3 independent experiments, and the data in C are means + SE from 3 independent experiments performed in duplicate. Numerical results under the immunoblots represent the relative mean signal intensity among samples from densitometry scanning of 3 experiments.

Fig. 6.

HNE toxicity is mediated by JNK/c-Jun overactivation resulting from GSH depletion. A: percentage cell death by MTT assay in S-CYP15 cells preinfected with Ad5LacZ or Ad5TAM and treated with the indicated micromolar concentrations of HNE for 24 h. Data are means + SE from 3 independent experiments performed in duplicate (*P < 0.001 compared with similarly treated Ad5LacZ-infected cells). B: protein was isolated from VEC and S-CYP15 cells that were untreated or treated with DEM for the number of hours indicated. Protein aliquots were immunoblotted with antibodies for P-JNK, total JNK, P-c-Jun, total c-Jun, P-ERK1/2, and total ERK1/2. C: protein was isolated from S-CYP15 cells that were untreated or treated with 80 μM HNE alone or together with GSH ethyl ester (GSH) for the indicated hours. Aliquots of protein were immunoblotted with the previous antibodies along with an antibody for P-MKK4. Results in B and C are each representative of 3 independent experiments with the numerical results representing the relative mean signal intensity from densitometry scanning of these experiments. D: total GSH levels in S-CYP15 cells preinfected with Ad5LacZ or Ad5TAM and left untreated or treated with 80 μM HNE for the indicated hours. Data are means + SE from 3 independent experiments performed in duplicate.

Fig. 7.

Inhibition of oxidant stress inhibits GSH depletion, JNK activation, and cell death. A: GSH levels in VEC and S-CYP15 cells in untreated control (Con) cells and cells treated for 1 h with HNE alone (HNE) or catalase and HNE (C/H). Data are means + SE from 3 independent experiments (*P < 0.01 compared with HNE-treated S-CYP cells). B: Western blots of proteins isolated from S-CYP15 cells treated with HNE alone or catalase and HNE (Cat + HNE) for the indicated number of hours and probed for the antibodies shown. C: percentage cell death in S-CYP15 cells treated with HNE alone or catalase and HNE for 24 h. Data are means + SE from 3 independent experiments (*P < 0.004 compared with HNE-treated cells).

Fig. 8.

S-CYP cells do not have a decreased rate of JNK dephosphorylation. A: VEC and S-CYP15 cells were heat shocked and treated with phosphorylation inhibitors as detailed in materials and methods. Total protein was isolated from untreated control cells (C) or cells at the indicated min after heat shock. Isolated protein was immunoblotted with antibodies to P-JNK, total JNK, P-c-Jun, and total c-Jun. Relative signal intensities from densitometric scanning of 3 independent experiments are indicated. B: rate of decline in JNK phosphorylation in VEC and S-CYP15 cells over time.

Fig. 9.

S-CYP cells are not sensitized to death from malondialdehyde (MDA). A: VEC and S-CYP15 cells were treated with the indicated micromolar concentrations of MDA, and the percentage cell death was determined at 24 h by MTT assay. Results are means + SE from 3 independent experiments performed in duplicate. B: protein was isolated from VEC and S-CYP15 cells at the indicated hours after treatment with 200 μM MDA and immunoblotted with antibodies for P-JNK, total JNK, P-c-Jun, and total c-Jun. The mean densitometric signal intensities from 3 independent experiments are shown.