Nitric oxide represses inhibitory kappaB kinase through S-nitrosylation

Abstract

Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-kappaB. A commonly proposed mode of action for inhibition of NF-kappaBbyNO involves interference with NF-kappaB binding to DNA. Because activation of inhibitory kappaB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-kappaB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-kappaB activation. We demonstrate here that S-nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKbeta, the catalytic subunit required for NF-kappaB activation, was a direct target for S-nitrosylation. A mutant version of IKKbeta containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKbeta, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor alpha to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-kappaB activation by cytokines. Collectively, our findings demonstrate that IKKbeta is an important target for the redox regulation of NF-kappaB by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.

Fig. 1.

Inactivation of IKK but not JNK by SNAP in vitro. (A) C10 cells were exposed to 10 ng/ml TNFα for 5 min to induce IKK activity. Immunoprecipiated IKK was then exposed to indicated concentrations of SNAP for 15 min, and an in vitro kinase assay was performed by using GST-IκB as a substrate. IB, Western blotting for IKKγ.(B) C10 cells were treated with 10 ng/ml TNFα for 15 min to activate JNK, and JNK1 was immunoprecipitated from lysates. After a 15-min exposure to the indicated concentrations of SNAP or GSNO, an in vitro kinase assay was performed by using GST-c-Jun as a substrate. IB, Western blotting for JNK1.

Fig. 2.

Repression of IKK activity in intact cells by exposure to SNO. (A Left) C10 cells were treated with 1 mM SNAP in presence or absence of 1 mM l-cys for 15 min before exposure to 10 ng/ml TNFα for 5 min, and an in vitro kinase assay was performed. IB, Western blot of IKKβ. (Right) Jurkat T cells were treated with 500 μM l-CSNO for 30 min and subsequently with 10 ng/ml TNFα for 10 min, and an in vitro kinase assay was performed. (B) C10 cells were treated with 250 μM l-or d-CSNO, or 500 μM GSNO in presence of 500 μMof l-or d-cys for 15 min, before stimulation with 10 ng/ml TNFα for 5 min. The activity of IKK was assessed in an in vitro kinase assay. IB, Western blot of IKKβ.(C) C10 cells were treated with indicated concentrations of l-CSNO for 15 min and subsequently with 10 ng/ml TNFα for 5 min. The activity of IKK was assessed in an in vitro kinase assay. Results were quantified by phospho-image analysis and expressed as the percent kinase activity compared with TNFα-only-treated cells. (D) C10 cells were incubated with 2.5 μM clasto lactacystin β-lactone for 30 min to block proteasomal degradation of proteins and then exposed to l-CSNO for 15 min, followed by a 5-min incubation with 10 ng/ml TNFα. The amount of phosphorylated IκBα (p-IκBα, Upper) and total IκBα (IκBα, Lower) was assessed by Western blotting. (E) C10 cells were treated with 250 μM l-CSNO for 15 min and subsequently with 10 ng/ml TNFα for 5 min. IKKβ was immunoprecipitated from lysates, and phosphoserine content was assessed by Western blot by using a phosphoserine antibody. IB, Western blot of IKKβ.

Fig. 3.

S-nitrosylation of IKKβ. (A) Jurkat T cells were treated with 1 mM l-CSNO for 30 min and subsequently with TNFα for 10 min. IKKβ was immunoprecipitated from lysates containing 2.2 mg of protein by using an IKKβ antibody. A control immunoprecipitation was performed by using an isotype-matched Ig (IgG). After immunoprecipitation, selected samples were treated with HgCl₂, and all samples were treated with sulfanilamide to ensure specificity. S-nitrosylation of IKKβ was assessed by chemiluminescence. *, P < 0.05 (Student's t test) compared with NO signal obtained in the TNFα + l-CSNO subjected to immunoprecipitation with the IgG control antibody. (B) IKKβ was immunoprecipitated from untreated Jurkat T cells, samples boiled in sample buffer, separated on SDS/PAGE gel, and silver stained. The location of IKKβ is indicated. HC, antibody heavy chain; LC, antibody light chain. (C) Cells were treated as in A, and lysates were subjected to biotin derivatization. Biotinylation of IKKβ was detected after immunoprecipitation of the IKKβ-containing complex and Western blotting using streptavidin–horseradish peroxidase. In control samples, reduction by ascorbate (–vitC) was omitted. IB, anti-IKKβ immunoblot; ns, nonspecific reactivity.

Fig. 4.

Cys-179 of IKKβ is target for S-nitrosylation. (A) C10 cells were transfected with wt or C179A HA-IKKβ, treated with 1 mM GSNO/l-cys or l-CSNO for 15 min, before exposure to 10 ng/ml TNFα for 5 min. IKK activity was assessed in an in vitro kinase assay, after immunoprecipitation with an HA antibody. IB, anti-HA immunoblot. (Bottom) Quantitation by phosphoimage analysis. Results are expressed as percentage of IKK activity compared with TNFα-only-treated cells. (B Left)wt(Upper) or C179A HA-IKKβ-transfected C10 cells (Lower) were treated with 1 mM GSNO/l-cys for 15 min before exposure to 10 ng/ml TNFα for 5 min. S-nitrosylated proteins were immunoprecipitated, by using an S-nitrosocysteine antibody (IP SNO) and IKKβ detected by detection of HA by Western blotting. (Lower) HA Western blots on total cell lysates. (B Right) Assessment of specificity of the S-nitrosocysteine antibody. wt (Upper) or C179A HA-IKKβ transfected cells (Lower) were left untreated (left lane), treated with 1 mM l-CSNO for 15 min (middle lane) or treated with 1 mM l-CSNO for 15 min followed by incubation with HgCl₂ (right lane) before immunoprecipitation. S-nitrosylated proteins were then immunoprecipitated by using an S-nitrosocysteine antibody (IP: SNO) and IKKβ detected by Western blotting for HA; (Lower) HA Western blots on total cell lysates.

Fig. 5.

Repression of IKK activity in intact cells by endogenous NOS activity. Jurkat T cells were treated with 1 mM l-NMMA for 4 h followed by stimulation with 10 ng/ml TNFα for 10 min or mock manipulations. Selected dishes were treated with TNFα alone. (A) IKK activity was assessed in an in vitro kinase assay. IB, anti-IKKβ immunoblot. (B) Lysates were subjected to biotin derivatization, and biotinylated IKKβ was detected after immunoprecipitation of IKKβ and Western blotting by using streptavidin–horseradish peroxidase. In control samples, reduction by ascorbate (–vit C) or labeling with N-(3-malemidylpropionyl)biocytin (–biotin) was omitted. IB, anti-IKKβ immunoblot.