STAT3 Regulation by S-Nitrosylation: Implication for Inflammatory Disease

Abstract

Aims: S-nitrosylation and S-glutathionylation, redox-based modifications of protein thiols, are recently emerging as important signaling mechanisms. In this study, we assessed S-nitrosylation-based regulation of Janus-activated kinase 2/signal transducer and activator of transcription 3 ( JAK2/STAT3) pathway that plays critical roles in immune/inflammatory responses and tumorigenesis. Results: Our studies show that STAT3 in stimulated microglia underwent two distinct redox-dependent modifications, S-nitrosylation and S-glutathionylation. STAT3 S-nitrosylation was associated with inducible nitric oxide synthase (iNOS)-produced nitric oxide (NO) and S-nitrosoglutathione (GSNO), whereas S-glutathionylation of STAT3 was associated with cellular oxidative stress. NO produced by iNOS or treatment of microglia with exogenous GSNO inhibited STAT3 activation via inhibiting STAT3 phosphorylation (Tyr 705 ). Consequently, the interleukin-6 (IL-6)-induced microglial proliferation and associated gene expressions were also reduced. In cell-free kinase assay using purified JAK2 and STAT3, STAT3 phosphorylation was inhibited by its selective preincubation with GSNO, but not by preincubation of JAK2 with GSNO, indicating that GSNO-mediated mechanisms inhibit STAT3 phosphorylation through S-nitrosylation of STAT3 rather than JAK2. In this study, we identified that

S-nitrosoglutathione reductase (GSNOR) inhibitor as an immune modulator in experimental autoimmune encephalomyelitis

“…Similar observation was also made earlier by Yang et al [21] that S-nitroso-N-acetylpenicillamine (SNAP), another S-NO donor, selectively inhibited T H 17 without affecting T H 1 and T H 2. At present, the mechanism underlying GSNO and SNAP mediated inhibition of T H 17 is not well understood but we reproted that GSNO or S-nitroso-N-acetylcysteine (SNAC, a S-NO donor) inhibits IL-6-induced activation of STAT3 (phosphorylation on tyrosine 705 ) via S-nitrosylation of cysteine 259 of STAT3 [49, 60, 61]. Activated STAT3 by IL-6 and IL-23 plays a pivotal role in polarization and effector function of T H 17 cells [62], and thus documenting that GSNO mediated mechanism inhibit polarization and effector function of T H 17 via inactivation of STAT3.…”

“…Protein S-Nitrosylation was detected using the biotin-switch method with slight modification as described in our previous study [49]. Spleens were homogenized in 250 mM HEPES, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, 1% Nonidet P-40, 150 mM NaCl, 1 mM PMSF, 20mM methyl methanethiosulfonate (MMTS), 80 μM carmustine, protease inhibitor mixture (Sigma), and mixed with an equal volume of 25 mM HEPES, pH 7.7, 0.1 mM EDTA, 10 μM neocuproine, 5% SDS, 20 mM MMTS and incubated at 50°C for 20 min.…”

“…GSNO inhibited the IL-6-induced STAT3 activation (Tyr 705 phosphorylation) by S-nitrosylation of the STAT3 protein on Cys 259 [47] and also downregulated the IL-6 and TGF-β induced expression of RORγt, a T H 17 cell specific transcription factor [22]. Additionally, GSNO treatment also inhibited the IL-6/TGF-β and IL-23 induced T H 17 cell polarization and their effector function under in vitro cell culture conditions but without affecting T H 1 (IFN-γ) and T H 2 (IL-4) immune responses [22].…”

“…Additionally, GSNO treatment also inhibited the IL-6/TGF-β and IL-23 induced T H 17 cell polarization and their effector function under in vitro cell culture conditions but without affecting T H 1 (IFN-γ) and T H 2 (IL-4) immune responses [22]. Moreover, GSNO was reported to modulates the activities of proinflammatory transcription factors, such as NF-κB, AP-1, and STAT3 [35, 36, 48] and thus modulates gene expression for various proinflammatory effectors, such as ICAM-1, and VCAM-1 [35, 36, 43, 48, 49], as well as endothelial recruitment of peripheral immune cells for their CNS infiltration [36]. Therefore, it is important to understand the role of NO in immune and CNS disease process for MS and EAE.…”

The comparative proteomics analysis revealed the modulation of inducible nitric oxide on the immune response of scallop Chlamys farreri

“…The critical roles of NO in immune/inflammatory responses have been validated in vertebrates. NO can restrain the phosphorylation of STAT3 (Tyr 705 ) mediated by JAK2 to inhibit the transactivation of STAT3 and the expression of down-stream gene expressions in microglia inflammation via S-nitrosylation of STAT3 (Cys 259 ) [42]. In addition, Snitrosylation of p65 decreases the DNA binding of p50-p65 heterodimer to modulate NF-kB activity and to regulate gene expression in inflammation [43].…”