Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation

Abstract

NO, produced from l-arginine in a reaction catalyzed by NO synthase, is an endogenous free radical with multiple functions in mammalian cells. Here, we demonstrate that endogenously produced NO can suppress c-Jun N-terminal kinase (JNK) activation in intact cells. Treatment of BV-2 murine microglial cells with IFN-gamma induced endogenous NO production, concomitantly suppressing JNK1 activation. Similarly, IFN-gamma induced suppression of JNK1 activation in RAW264.7 murine macrophage cells and rat alveolar macrophages. The IFN-gamma-induced suppression of JNK1 activation in BV-2, RAW264.7, or rat alveolar macrophage cells was completely prevented by N(G)-nitro-l-arginine, a NO synthase inhibitor. Interestingly, the IFN-gamma-induced suppression of JNK1 activation was not affected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase. 8-Bromo-cGMP, a membrane-permeant analogue of cGMP, did not change JNK1 activation in intact cells either. In contrast, S-nitro-N-acetyl-dl-penicillamine (SNAP), a NO donor, inhibited JNK1 activity in vitro. Furthermore, a thiol reducing agent, DTT, reversed not only the in vitro inhibition of JNK1 activity by SNAP but also the in vivo suppression of JNK1 activity by IFN-gamma. Substitution of serine for cysteine-116 in JNK1 abolished the inhibitory effect of IFN-gamma or SNAP on JNK1 activity in vivo or in vitro, respectively. Moreover, IFN-gamma enhanced endogenous S-nitrosylation of JNK1 in RAW264.7 cells. Collectively, our data suggest that endogenous NO mediates the IFN-gamma-induced suppression of JNK1 activation in macrophage cells by means of a thiol-redox mechanism.

Figure 1

IFN-γ suppresses JNK1 activation by means of the induction of NO generation in microglial and macrophage cells. (A) BV2 murine microglial cells were pretreated with IFN-γ (100 units/ml) for the indicated times and then exposed to 10 μM anisomycin for 15 min at 37°C. Enzymatic activities of JNK1 in the BV2 cells were measured by immunocomplex kinase assay using anti-JNK1 antibody. For NO measurement, 100 μl of culture medium in each plate was collected and analyzed for NO release by the Griess method. (B) BV2, RAW264.7, and rat primary alveolar macrophage cells were pretreated with IFN-γ (100 units/ml) for 16 h, and then incubated with 2 mM l-NNA for 30 min, where indicated. After the chemical treatments, the cells were exposed to UV light (60 J/m²) and further incubated for 30 min at 37°C. JNK1 activities in the cells were determined by immunocomplex kinase assay using anti-JNK1 antibody.

Figure 2

IFN-γ does not affect either SEK1 or MEKK1 in RAW264.7 cells. RAW264.7 cells were treated with IFN-γ (100 units/ml) for 16 h. Where indicated, the cells were exposed to UV light (60 J/m²) and further incubated for 30 min at 37°C. Cell lysates were subjected to immunoprecipitation using anti-SEK1 or anti-MEKK1 antibody. The immunopellets were examined for SEK1 or MEKK1 activity by immunocomplex kinase assay using GST-SAPKβ(K55R) or GST-SEK1(K129R) as substrate.

Figure 3

cGMP does not mediate NO-induced suppression of JNK activation in intact cells. (A) RAW264.7 cells were pretreated with 100 μM 8-bromo-cGMP (8-Br-cGMP) for 30 min, and then irradiated with 60 J/m² UV light, followed by an additional 1-h incubation at 37°C. (B) RAW264.7 cells were pretreated with IFN-γ (100 units/ml) for 16 h, and then treated with 100 μM ODQ for 30 min. After the chemical treatments, the cells were exposed to 60 J/m² UV light, then incubated further for 1 h at 37°C. (A and B) The cells were harvested, lysed, and analyzed for JNK1 activity by immunocomplex kinase assay using anti-JNK1 antibody.

Figure 4

In vitro effect of SNAP on JNK1, SEK1, or MEKK1 activity. HEK293 cells were irradiated with 60 J/m² UV light and then incubated for 1 h at 37°C. The cell lysates were subjected to immunoprecipitation using anti-MEKK1, anti-SEK1, or anti-JNK1 antibody, as indicated. In A, the resultant immunopellets were treated with 100 μM SNAP for 20 min on ice, and then examined for the indicated protein kinase activities by immunocomplex kinase assay. In B, the resultant immunopellets were exposed to 100 μM SNAP for 20 min on ice, and then incubated with 10 mM DTT for 20 min on ice. JNK1 activity in the immunopellets was determined by immunocomplex kinase assay.

Figure 5

Cys¹¹⁶ of JNK1 is critical for the NO-mediated thiol-redox regulation of JNK1. HEK293 cells were transfected with a plasmid vector expressing HA-JNK1(C79S), HA-JNK1(C116S), or HA-JNK1(C137S). After 50 h of transfection, the cells were exposed to UV light (60 J/m²) and then incubated further for 1 h at 37°C. The cell lysates were subjected to immunoprecipitation using mouse monoclonal anti-HA antibody. The immunopellets were treated with 100 μM SNAP for 20 min on ice, washed twice with PBS solution, and then examined for JNK1 activity by immunocomplex kinase assay.

Figure 6

IFN-γ suppresses the c-Jun-mediated luciferase reporter activity through an induction of NO production. RAW264.7 cells were transiently transfected with luciferase reporter gene, c-Jun, ΔMEKK1, and SEK1(K129R) constructs, as indicated. pcDNA3-β-galactosidase was also included in all transfections. After 24 h of transfection, the cells were exposed to 100 units/ml IFN-γ for 16 h at 37°C, then incubated with 2 mM l-NNA for 6 h. The cells were harvested and assayed for luciferase activity. Luciferase activity was normalized to β-galactosidase activity.

Figure 7

INF-γ enhances in vivo S-nitrosylation of JNK1. (A) RAW264.7 cells were pretreated with 100 units/ml of IFN-γ for 16 h, and then treated with 10 μg/ml anisomycin for 15 min at 37°C. The cell lysates were subjected to immunoprecipitation using mouse monoclonal anti-JNK1 antibody. The immunopellets were incubated either with 10 mM DTT for 20 min on ice or with 100 μM HgCl₂ for 30 min at room temperature, where indicated. JNK1 activities in the immunopellets were determined by immunocomplex kinase assay. (B) RAW264.7 cells were transiently transfected with pcDNA3 expressing wild-type JNK1 or JNK1(C116) mutant. After 30 h of transfection, the cells were pretreated with 100 units/ml IFN-γ for 16 h, and then treated with 10 μg/ml anisomycin for 15 min at 37°C. The cell lysates were determined for JNK1 activity by immunocomplex kinase assay using anti-JNK1 antibody. (C) RAW264.7 cells were pretreated with 100 units/ml of IFN-γ for 16 h, and then treated with 10 μg/ml anisomycin for 15 min at 37°C. The cell lysates were subjected to immunoprecipitation using mouse monoclonal anti-JNK1 antibody. The JNK1 immunoprecipitates were extensively washed with washing buffer (20 mM Hepes, pH 7.4) (the purity of JNK1 in the immunoprecipitates: >95%). The JNK1 immunoprecipitates were then reacted with 100 μM HgCl₂ and 100 μM DAN for 30 min at room temperature, followed by addition of 1 M NaOH. NO released from S-nitrosylated JNK1 was quantified by measuring the fluorescence intensity of the reaction product using a fluorimeter with an excitation wavelength of 375 nm and an emission wavelength of 450 nm.