The Role Of Protein S-nitrosylation In Inhibitory Effects Of NO On CREB And STAT-1 Activities In RSC-364 Cells

Nitric oxide (NO), as a gaseous messenger, regulates cell function in a variety of physiologic and pathophysiologic states. NO may act on target site of protein directly, in a membrane receptor-independent way, and plays multiple biological roles. Previous studies on direct action of NO on protein were mainly focused on the metal ion of the target protein. For example, NO can bind to heme iron of soluble guanylyl cyclase (sGC), produce the second messenger 3′-5′guanosine monophosphate (cGMP) and plays important roles in relaxation of vasculum and inhibition of platelets aggregation. Recently, NO-dependent cysteine thiols (Cys-SH) oxidative modification was paid attention to. In 1994, Stamler proposed the concept of protein S-nitrosylation: the coupling of a NO moiety to a reactive cysteine thiol and formation of an S-nitrosothiol (SNO) or S-nitrosylated protein (PSNO) can regulate conformation and function of protein. Protein S-nitrosylation, as a reversible cysteine redox modification, may regulate protein function directly or indirectly through formation of other NO-dependent thiols redox modifications including formation of the mixed disulfide or glutathiolation et al. Protein S-nitrosylation is tightly linked to cell type, cellular rodox status, hydrophobic environment around the target thiols, the colocalization of NOS with target protein, the concentration of NO and the characteristic of reactive oxygen species (ROS) et al. Protein S-nitrosylation has been recognized gradually as an ubiquitous regulatory mechanism in cell signal transduction comparable to phosphorylation.Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synoviocytes proliferation and synovium inflammation. Fibroblast-like synoviocytes (FLS) isolated from synovium of RA play a major role in the pathogenesis of RA by uncontrolled proliferation and secreting effector molecules including cytokines, chemokines, and proteolytic enzymes, which promote inflammation and joint destruction. NO is elevated in the synovial fluids of patients with RA, may inhibit FLS proliferation and induce FLS apoptosis. These studies indicate that NO exists in the micro-environment around FLS and may regulate function of FLS in pathogenesis of RA.Marshall et al. showed that protein S-nitrosylation may inhibit nuclear factor-kappa B (NF-κB) activities, Nikitovic et al. found that NO inhibited activating protein-1(AP-1) DNA binding activities by protein S-nitrosylation. These studies suggested that protein S-nitrosylation of transcription factors may serve as one of the mechanisms by which NO regulates cell homeostasis. Takeba et al. confirmed that cAMP response element-binding protein (CREB) is involved in the FLS proliferation in patients with RA. Analogous to CREB,the transcription factor signal transducer and activator of transcription-1(STAT-1) is activated in RA synovium by increased phosphorylation level. STAT-1 mainly distributes in FLS, infiltrated T cell et al. and plays a pivotal role in the expression of inflammatory gene products involved in RA. Both CREB and STAT-1 may play important roles in the pathogenesis of RA. It has been confirmed that NO may modulate CREB and STAT-1 activities. A number of researches demonstrated that most of transcription factors contained cysteine residues are sensitive to NO-dependent thiols redox modification. There are several conserved cysteine residues in kinase induced domain of CREB and DNA binding domain of STAT-1, whether NO may result in protein S-nitrosylation in FLS; whether protein S-nitrosylation can modulate CREB or STAT-1 activities by thiols redox modification; whether protein S-nitrosylation may regulate phosphorylation levels and downstream genes transcription of CREB or STAT-1 are all still unclear.Nitrosothiols are exceptionally labile as a result of reactivity with reducing agents and degradation in light, so it is difficult to detect proteins contained nitrosothiols. Recently, Jaffrey et al. developed the biotin switch method to specifically transform S-nitrosylated thiols in biotinylated thilos. The expression levels of S-nitrosylated proteins were evaluated by detection the expression levels of biotinylated proteins. Biotin switch method becomes a new method for research protein S-nitrosylation.To explore the role of protein S-nitrosylation and to reveal the fast regulatory effect of NO in pathogenesis of RA, we assaied the expression levels of S-nitrosylated proteins using the biotin switch method and Western blotting, investigated the effect of protein S-nitrosylation on DNA binding activities of CREB or STAT-1 using electrophoresis mobility shift assay (EMSA)and detected the effect of protein S-nitrosylation on phosphorylation levels and downstream genes transcription of CREB or STAT-1 in RSC-364(a kind of murine FLS).1 The effect of NO on protein S-nitrosylation in RSC-364 cells.1.1 The effect of NO on protein S-nitrosylation in RSC-364 cells extracts.To investigate the effect of NO on protein S-nitrosylation in RSC-364 cells extracts, RSC-364 cell extracts were prepared and were divided randomly into four groups:①control group;②glutathione (GSH) group;③S-nitroso-glutathione (GSNO) group;④GSNO+dithiothreitol (DTT) group. After S-nitrosylation reaction in vitro, extracts were precipitated with acetone to remove the GSNO or GSH. The resulting S-nitrosylated proteins were subjected to the biotin switch assay and some samples were treated with DMSO vehicle solution in non-biotin added groups as a control. After biotin switch assay, the expression of S-nitrosylated proteins was assayed by Western blotting. Results: in samples that were treated with biotin-HPDP, there was a wide range of biotinylated proteins and theoretically, S-nitrosylated proteins, in GSNO group, but none were present in unstimulated control group. After incubated with GSNO, addition of DTT as a reducing agent results in ablation of all signal. In samples that were not treated with biotin-HPDP, the endogenous biotinylated proteins are detectable. In conclusion, the present study confirmed that treating RSC-364 cell extracts with GSNO lead to protein S-nitrosylation . 1.2 The effect of NO on protein S-nitrosylation in RSC-364 cells.Above experiments showed that treating RSC-364 cell extracts with GSNO lead to protein S-nitrosylation, which indicated that there are a wide range of S-nitrosylation targets in RSC-364 cells. The purpose of the present study was to observe the effect of NO on protein S-nitrosylation in RSC-364 cells. The cultured RSC-364 cells were divided randomly into nine groups treated with different agents:①control group I;②GSH group;③GSNO group;④GSNO+DTTgroup;⑤control group II;⑥interferon-γ(IFN-γ)+ interleukin-1(IL-1β) group;⑦iNOS inhibitor aminoguanidine (AG) group;⑧IFN-γ+IL-1β+AG group;⑨IFN-γ+IL-1β+DTT group. The cell extracts from each group were prepared and were subjected to the biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. The contents of NO in RSC-364 cells culture supernatant in⑤-⑧groups were measured according protocols. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. The results were as follows: (1) GSNO obviously increased the expression of S-nitrosylated proteins in RSC-364 cells, which was reversed by DTT. Administration of GSH alone has no effect on the expression of nitrosylated proteins in cell. In samples that were not treated with biotin-HPDP, the endogenous biotinylated proteins were detectable. (2) In samples that were treated with biotin-HPDP, there was a wide range of biotinylated proteins in IFN-γ+IL-1βgroup, but none were present in control group and AG group. Interestingly, the pattern of IFN-γ+IL-1β+AG group was not different from that noted in control group. (3) Compared with control group, NO content in culture supernatant in IFN-γ+IL-1βgroup increased significantly (4.34±2.82μmol/L vs. 30.93±4.8μmol/L, p<0.01). Co-incubation with AG and cytokines mixture (IFN-γand IL-1β) resulted in a significant decrease of NO content in culture supernatant (4.7±2.63μmol/L, p<0.01). AG alone did not affect NO production (5.4±2.47μmol/L, p>0.05).These data confirmed that: 1 GSNO resulted in protein S-nitrosylation in RSC-364 cells. 2 This combination stimulation of cytokines (IFN-γand IL-1β) lead to protein S-nitrosylation in RSC-364 cells by increasing NO production.2 The effect of protein S-nitrosylation on CREB activities in RSC-364 cells2.1 The effect of protein S-nitrosylation in vitro on CREB activities in RSC-364 cellsAbove study showed that NO lead to protein S-nitrosylation by reaction with directly RSC-364 cell extracts. Whether protein S-nitrosylation is involved in regulation of CREB activity by NO has remained unclear, the aim of the present study was to investigate the effect of protein S-nitrosylation in vitro on CREB activies in RSC-364 cells. The nuclear extracts from RSC-364 cells treated with vehicle (NS) or IL-1βfor 2h were prepared and were incubated with GSNO in the absence or presence of DTT for 15 min at room temperature. After S-nitrosylation reaction in vitro, the nuclear extracts in each groups were subjected to electropehoresis mobility shift assay (EMSA) to explore the CREB binding activity. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: The CREB binding activity was significantly higher in RSC-364 cells stimulated with IL-1βin comparison with unstimulated cells (p<0.01). Treating the nuclear extracts from RSC-364 cells stimulated with IL-1βwith GSNO decreased CREB activation. If DTT was included in the nuclear extracts after GSNO exposure, the effect of GSNO on CREB activation was reversed. DTT treatment alone had no effect on the CREB binding activity (p>0.05). The binding specificity was confirmed by using homologous (CREB) and nonhomologous (AP-2) oligonucleotides as competitors. These results showed that, NO resulted in protein S-nitrosylation in vitro, which could inhibit CREB activity by thiols oxidative modification.2.2 The effect of protein S-nitrosylation on CREB activity in RSC-364 cellsAbove experiments showed that NO could result in protein S-nitrosylation in RSC-364 cell extracts and inhibit CREB activity by thiols oxidative modification in vitro. It is remained unclear that the effect of protein S-nitrosylation in RSC-364 cells on CREB/DNA binding activity and on CREB phosphorylation. In the present study, the cultured RSC-364 cells were divided randomly into ten groups after treatment with different agents:①control group;②IL-1βgroup;③GSNO group;④GSNO+IL-1βgroup;⑤the thiol-alkylating agent N-ethylmaleimide (NEM) group;⑥NEM+GSNO+ IL-1βgroup;⑦GSNO+IL-1β+DTT group;⑧IL-1β+IFN-γgroup;⑨AG+ IL-1β+IFN-γgroup;⑩AG group. The CREB binding activity was analyzed by EMSA; the CREB and phosphorylated CREB levels in①-⑥groups were detected by western blotting. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: (1) The CREB binding activity was significantly higher in RSC-364 cells stimulated with IL-1βin comparison with unstimulated cells (p<0.01). Pre-treatment RSC-364 cells with GSNO decreased IL-1β-induced CREB activation (p<0.01). If DTT was added in the nuclear extracts from the RSC-364 cells incubated with GSNO and IL-1β, the effect of GSNO on CREB activation was reversed (p<0.01). If RSC-364 cells were treated with NEM for 30 min followed by stimulation with GSNO and IL-1β, the inhibition effect of GSNO on CREB activation was also reversed (p<0.01). Pre-treatment with NEM alone had no effect on the CREB binding activity (p>0.05). The binding specificity was confirmed by using homologous (CREB) and nonhomologous (AP-2) oligonucleotides as competitors.(2) The CREB binding activity was significantly higher in RSC-364 cells stimulated with IL-1βin comparison with unstimulated cells (p<0.01). The combination stimulation of cytokines (IFN-γand IL-1β) for 12h resulted in a obvious decrease in CREB activation induced by IL-1β(p<0.01), but co-incubation with AG and cytokines increased CREB activation (p<0.01). AG alone had no significant effect on CREB activation (p>0.05). The binding specificity was confirmed by using homologous (CREB) and nonhomologous (AP-2) oligonucleotides as competitors.(3) The phosphorylated CREB level in RSC-364 cells was markedly increased 2h after incubation with IL-1βin comparison with the control group (p<0.01). Pre-treatment with GSNO had no obvious effect on the phosphorylated CREB level in RSC-364 cells in comparison with the IL-1βgroup (p>0.05), pre-treatment with NEM had no significant effect on the phosphorylated CREB level in RSC-364 cells in comparison with the GSNO+IL-1βgroup (p>0.05). Treatment with GSNO or NEM alone had no effect on the phosphorylated CREB level in comparison with the control group (both p>0.05). NO significant changes in CREB level were observed in all groups (all p>0.05). These results showed that: (1) NO resulted in protein S-nitrosylation in RSC-364 cell, which might CREB inhibit activity by thiols oxidative modification. (2) Protein S-nitrosylation induced by NO had no significant effect on the phosphorylated CREB level. (3) This combination stimulation of cytokines (IFN-γand IL-1β) might inhibit CREB activity by increasing S-nitrosylation level.3 The effect of protein S-nitrosylation on STAT-1 activities in RSC-364 cells3.1 The effect of protein S-nitrosylation in vitro on STAT-1 activities in RSC-364 cellsWhether protein S-nitrosylation is involved in regulation of STAT-1 activity by NO has remained unproved, the aim of the present study was to investigate the effect of protein S-nitrosylation in vitro on STAT-1 activies in RSC-364 cells. The nuclear extracts from RSC-364 cells treated with vehicle (NS) or IFN-γfor 30min were prepared and were incubated with GSNO in the absence or presence of DTT for 15 min at room temperature. After S-nitrosylation reaction in vitro, the nuclear extracts in each groups were subjected to EMSA to assay the STAT-1 binding activity. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: The STAT-1 binding activity was significantly higher in RSC-364 cells stimulated with IFN-γin comparison with control group (p<0.01). Treating the nuclear extracts from RSC-364 cells stimulated with IFN-γwith GSNO decreased STAT-1 activation induced by IFN-γ(p<0.01). If DTT was included in the the nuclear extracts after GSNO exposure, the effect of NO on STAT-1 activation was reversed (p<0.01). Treatment with DTT alone had no effect on the STAT-1 binding activity (p>0.05). The binding specificity was confirmed by using homologous (STAT-1) and nonhomologous (AP-2) oligonucleotides as competitors. These results showed that, NO resulted in protein S-nitrosylation in vitro, which might inhibit STAT-1 activity by thiols oxidative modification.3.2 The effect of protein S-nitrosylation in cells on STAT-1 activity in RSC-364 cellsAbove experiments showed that NO could result in protein S-nitrosylation in cell extracts from RSC-364 cells and inhibit STAT-1 activity by cysteine oxidative modification, which suggested that STAT-1 may be sensitive to thiols oxidative modification. It is unclear that the effect of protein S-nitrosylation in cell on STAT-1/DNA binding activity and STAT-1 phosphorylation. To elucidate the effect of protein S-nitrosylation in cells on STAT-1/DNA binding activity and STAT-1 phosphorylation, the cultured RSC-364 cells were divided randomly into ten groups treated with different agents:①control group;②IFN-γgroup;③GSNO group;④GSNO+IFN-γgroup;⑤NEM group;⑥NEM+GSNO+IFN-γgroup;⑦GSNO+IFN-γ+DTT group;⑧IL-1β+IFN-γgroup;⑨AG+IL-1β+ IFN-γgroup;⑩AG group. The STAT-1 binding activity was analyzed by EMSA; the STAT-1 and phosphorylated STAT-1 levels in①-⑥groups were detected by western blotting. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: (1) The STAT-1 binding activity was significantly higher in RSC-364 cells stimulated with IFN-γin comparison with unstimulated cells (p<0.01). Pre-treatment with GSNO decreased IFN-γ-induced STAT-1 activation (p<0.01). If DTT was added in the nuclear extracts from the RSC-364 cells incubated with GSNO and IFN-γ, the effect of GSNO on STAT-1 activation was reversed (p<0.01). Pre-treatment with NEM also reversed the effect of GSNO on STAT-1 activation (p<0.01). Treatment with NEM alone had no effect on the STAT-1 binding activity (p>0.05).The binding specificity was confirmed by using homologous (STAT-1) and nonhomologous (AP-2) oligonucleotides as competitors. (2) The STAT-1 binding activity was significantly higher in RSC-364 cells stimulated with IFN-γin comparison with unstimulated cells (p<0.01), the combination stimulation of cytokines (IFN-γand IL-1β) for 12h resulted in a obvious decrease in STAT-1 activation induced by IFN-γ(p<0.01), while co-incubation with AG and cytokines increased STAT-1 activation (p<0.01). AG alone had no significant effect on STAT-1 activation (p>0.05). The binding specificity was confirmed by using homologous (STAT-1) and nonhomologous (AP-2) oligonucleotides as competitors.(3)The phosphorylated STAT-1 level in RSC-364 cells was markedly increased 30min after incubation with IFN-γin comparison with the control group (p<0.01).Pre-treatment with GSNO had no obvious effect on the phosphorylated STAT-1 level in RSC-364 cells in comparison with the IFN-γgroup (p>0.05), pre-treatment with NEM had also no significant effect on the phosphorylated STAT-1 level in RSC-364 cells in comparison with the GSNO+IFN-γgroup (p>0.05). GSNO or NEM alone had no effect on the phosphorylated STAT-1 level in comparison with the control group (both p>0.05). NO significant changes in STAT-1 level were observed in all groups (all p>0.05).These results shown that: 1 NO resulted in protein S-nitrosylation in cells, which might inhibit STAT-1 activity by thiols oxidative modification. 2 protein S-nitrosylation had no effect on phosphorylated STAT-1. 3 The combination stimulation of cytokines (IFN-γand IL-1β) inhibited STAT-1 activity by increasing protein S-nitrosylation level.4 The effects of protein S-nitrosylation on interleukin-6 (IL-6) and IFN-γinducible protein of 10kDa (IP-10) protein expression in RSC-364 cells4.1 The effect of protein S-nitrosylation on the expression of IL-6 in RSC-364 cellsAbove experiments showed that NO might inhibit CREB activity by thiols oxidative modification. Because IL-6 gene transcription was mainly mediated by CREB, the present study was undertaken to determine the effect of CREB thiols oxidative modification on the protein expression of IL-6 induced by IL-1β. The cultured RSC-364 cells were divided randomly into six groups treated with different agents:①control group;②IL-1βgroup;③GSNO group;④GSNO+IL-1βgroup;⑤NEM group;⑥NEM+GSNO+IL-1βgroup. The IL-6 protein expression level was detected by ELISA. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: Stimulating RSC-364 cells with IL-1βfor 24h resulted in an obvious increase in IL-6 production in supernatant in comparison with control group (431.69±14.15pg/ml vs. 66.92±8.2 pg/ml, p<0.01). Compared with IL-1βgroup, the IL-6 content in supernatant in GSNO+IL-1βgroup decreased (143.96±14.7pg/ml, p<0.01). But the IL-6 content in supernatant NEM+ GSNO+IL-1βgroup significantly increased in comparison with GSNO+IL-1βgroup(433.27±14.4pg/ml,p<0.01). NEM alone had no effect on the IL-6 level (70.06±10.21 pg/ml, p>0.05). These results confirmed that CREB thiols oxidative modification inhibited the expression level of IL-6 induced by IL-1β.4.2 The effect of protein S-nitrosylation on the expression of IP-10 in RSC-364 cellsAbove experiments showed that NO resulted in protein S-nitrosylation in vivo, which might inhibit STAT-1 activity by thiols oxidative modification. Because IP-10 gene transcription was mainly mediated by STAT-1, the present study was undertaken to determine the effect of cysteine thiols modification of STAT-1 on the protein expression of IP-10 induced by IFN-γ, The cultured RSC-364 cells were divided randomly into six groups after treated with different agents:①control group;②IFN-γgroup;③GSNO group;④GSNO+IFN-γgroup;⑤NEM group;⑥NEM+GSNO+IFN-γgroup. The expression of IP-10 was detected by Western blotting. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: The IP-10 level in RSC-364 cells was markedly increased 3h after incubation with IFN-γin comparison with the control group (p<0.01). Pre-treatment with GSNO resulted in a decrease in the IP-10 level in RSC-364 cells in comparison with the IFN-γgroup (p<0.01). But pre-treatment with NEM reversed the effect of GSNO (p<0.01), NEM alone had no effect on the IP-10 level (p>0.05). These results demonstrated that thiols oxidative modification of STAT-1 inhibited the expression level of IP-10 induced by IFN-γ. CONCLUSIONSIn the present study, we investigated the modulatory effects of protein S-nitrosylation on phosphorylation levels of CREB and STAT-1, the DNA/binding activities of these two transcription factors and the downstream gene expression, the conclusions were as follows:1 NO could lead to CREB and STAT-1 thiols oxidative modification by protein S-nitrosylation, and as a result the DNA/binding activities of CREB and STAT-1 were inhibited, and the downstream gene expression were reduced. It is an important mechanism for NO to regulate FLS homeostasis and inhibit RA progression.2 As a gaseous messenger, the production and disappearance of NO is rapid. NO can freely enter into cells without the existence of receptors or channels. The present study demonstrated that NO could regulate the DNA/binding activities of transcription factors, which suggested that protein S-nitrosylation is a fast regulatory mechanism. Compared with other regulation mechanism such as neuroendocrine pathway, protein S-nitrosylation may be the most fast stress reaction.3 GSNO, as the conservator and transport agent of NO, distributes in many varieties of tissues and organs including hematological system. The present study showed that NO released by GSNO could enter into cells and regulate the DNA/binding activities of transcription factors, which suggested that NO played biological roles not only in autocrine or paracrine way but also in endocrine way.