title: IL-17 intensifies IFN-γ-induced NOS2 upregulation in RAW 264.7 cells by further activating STAT1 and NF-κB.
abstract: Interleukin-17 (IL-17) is a signature cytokine of Th17 cells. Previous research has indicated that IL-17 plays a proinflammatory role by exacerbating interferon-γ (IFN-γ)-induced inflammation. However, prior to this study, it was not known whether inducible nitric oxide synthase (iNOS or NOS2), a signature molecule of inflammation, could be intensified by IL-17 when combined with IFN-γ. Thus, we explored the roles and underlying mechanisms of IL-17 and IFN-γ in the regulation of NOS2 expression in RAW 264.7 cells using qPCR, western blot analysis, colorimetric analysis, ChIP assay and statistical analysis. Although IL-17 alone did not induce NOS2 expression or nitric oxide (NO) production, as shown by western blot analysis and colorimetric analysis, it intensified IFN-γ-induced NOS2 upregulation and NO production in RAW 264.7 cells. The alteration of relevant transcription factors demonstrated that a combination of IFN-γ and IL-17 enhanced Tyr701-phosphorylated signal transducer and activator of transcription 1 [p-STAT1(Y701)] and nuclear factor-κB (NF-κB) activation, nuclear translocations and their binding to the NOS2 promoter, compared with IFN-γ alone, as illustrated by the results of the western blot analysis and ChIP assay. Also, using the corresponding inhibitors of STAT1 and NF-κB, we noted downregulation of the expression of NOS2 induced by IFN-γ alone or in combination with IL-17, respectively. In addition, IFN-γ increased phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK), and accelerated the activation of the NF-κB pathway and the expression of NOS2, but phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was reduced by treatment with IFN-γ and IL-17. IL-17 intensified the activation of the NF-κB pathway and NOS2 upregulation induced by IFN-γ by increasing the phosphorylation of p38 MAPK and limiting the phosphorylation of ERK1/2. Taken together, these results suggest that IL-17 intensified IFN-γ-induced NOS2 upregulation and NO production by increasing the transcription activity of p-STAT1(Y701) and NF-κB in RAW 264.7 cells. Further activation of the NF-κB pathway induced by IL-17 relied on enhanced phosphorylation of p38 MAPK and decreased phosphorylation of ERK1/2. The mechanism suggested in this study provides novel information which may be used for anti-inflammatory therapy with IL-17.
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Qiyue
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wen
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Weiping
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Ning
authors.initials: N
authors.lastname: Guo
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Zuyi
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D000818:Animals; D015971:Gene Expression Regulation, Enzymologic; D007249:Inflammation / Q000235:genetics* / Q000378:metabolism; D007371:Interferon-gamma / Q000008:administration & dosage* / Q000378:metabolism; D020381:Interleukin-17 / Q000008:administration & dosage* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000378:metabolism; D010766:Phosphorylation; D000067996:RAW 264.7 Cells; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics
chemical_list: D020381:Interleukin-17; D016328:NF-kappa B; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases