Nitric Oxide Protects Cultured Rat Hepatocytes from Tumor Necrosis Factor-α-induced Apoptosis by Inducing Heat Shock Protein 70 Expression

Young-Myeong Kim,Michael E De Vera,Simon C Watkins,Timothy R Billiar

doi:10.1074/jbc.272.2.1402

Young-Myeong Kim, Michael E De Vera + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.2.1402

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Abstract

Nitric oxide (NO) and tumor necrosis factor-alpha (TNFalpha) play important roles in the pathogenesis of liver disease during acute inflammation. The present study was designed to elucidate the effect of NO pre-exposure on TNFalpha-induced hepatotoxicity. Pretreatment of primary cultures of rat hepatocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of heat shock protein 70 (HSP70) mRNA and protein, which was associated with thermotolerance and cytoprotection from TNFalpha+actinomycin D-induced hepatotoxicity and apoptosis. SNAP transiently changed the intracellular redox state by inducing glutathione (GSH) oxidation associated with the formation of S-nitrosoglutathione (GSNO). HSP70 mRNA was also induced by the GSH-oxidizing agent diamide and the GSH-conjugating agent N-ethylmaleimide, suggesting that NO induces HSP70 expression through GSH oxidation. The protective effect of SNAP pretreatment on TNFalpha-induced apoptosis correlated with the level of HSP70 expression. SNAP pretreatment inhibited reactive oxygen intermediate generation and lipid peroxidation effects that were reversed by blocking HSP70 expression using an antisense oligonucleotide to HSP70. Finally, endogenous NO formation, induced in hepatocytes stimulated with interferon-gamma and interleukin-1beta, led to the formation of GSNO and GSSG, induced HSP70, and attenuated TNFalpha-mediated cytotoxicity. These findings demonstrated that NO can induce resistance to TNFalpha-induced hepatotoxicity, possibly through the stimulation of HSP70 expression.

Highlights

The protective effect of SNAP pretreatment on tumor necrosis factor-␣ (TNF␣)induced apoptosis correlated with the level of heat shock protein 70 (HSP70) expression
To determine if Nitric oxide (NO) pretreatment protects against TNF␣-induced toxicity, freshly isolated rat hepatocytes were exposed to 750 ␮M SNAP as an NO donor, and 18 h later the cells were washed, TNF␣ was added, and viability was determined by crystal violet staining (Fig. 1)
SNAP Pretreatment Protects from TNF␣ϩActD-induced Apoptosis—Since TNF␣ϩActD toxicity in hepatocytes is associated with the induction of apoptosis, we examined the effect of SNAP pretreatment on TNF␣ϩActD-induced DNA fragmentation

Summary

EXPERIMENTAL PROCEDURES

Materials—Williams medium E, penicillin, streptomycin, L-glutamine, and HEPES were purchased from Life Technologies, Inc. Harvested hepatocytes (5 ϫ 106 cells) were lysed in 100 ␮l of 20 mM Tris-HCl buffer, pH 7.4, containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 5 ␮g/ml aprotinin, 5 ␮g/ml pepstatin A, and 1 ␮g/ml cymostatin) by three freeze-thaw cycles, and the cytosolic fraction was obtained by centrifugation at 13,000 ϫ g for 20 min at 4 °C. Nuclear extracts (10 ␮g of protein) were incubated with ϳ40,000 cpm (ϳ0.5 ng) of 32P-labeled oligonucleotide for min at room temperature in a buffer containing 2 ␮g of poly(dI-dC), 4.2 mM HEPES, pH 7.4, 2.5% glycerol, 4.2 mM KCl, 1 mM MgCl2, 0.02 mM EDTA, 2% Ficoll, and mM dithiothreitol (final volume of 30 ␮l). Hepatocytes (2.5 ϫ 105 cells/well in 12-well plates) were pretreated with 750 ␮M SNAP for 16 h, washed with serum-free medium twice, and incubated with 125I-labeled human TNF␣ (0.2–1.6 nM, 780 Ci/mmol) in the presence or absence of excess unlabeled TNF␣ (200-fold). Differences were considered significant when the p value was equal to or less than 0.05

RESULTS

Catalase Superoxide dismutase GSH peroxidase

DISCUSSION