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. 2010 Sep 10;285(37):28715-22.
doi: 10.1074/jbc.M110.133355. Epub 2010 Jun 30.

"VSports" Autophagy facilitates IFN-gamma-induced Jak2-STAT1 activation and cellular inflammation

Yu-Ping Chang  1 , Cheng-Chieh Tsai, Wei-Ching Huang, Chi-Yun Wang, Chia-Ling Chen, Yee-Shin Lin, Jui-In Kai, Chia-Yuan Hsieh, Yi-Lin Cheng, Pui-Ching Choi, Shun-Hua Chen, Shih-Ping Chang, Hsiao-Sheng Liu, Chiou-Feng Lin
Affiliations

Autophagy facilitates IFN-gamma-induced Jak2-STAT1 activation and cellular inflammation

Yu-Ping Chang et al. J Biol Chem. .

Abstract

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects VSports手机版. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. .

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Figures

FIGURE 1.
FIGURE 1.
IFN-γ-induced autophagy was critical for cellular inflammation. A, Western blotting was used to determine LC3 conversion in IFN-γ (10 ng/ml)-treated WT and Atg5−/− MEFs. β-Actin was the internal control. Data are representative of three individual experiments. B, confocal fluorescence microscopic observation of EGFP-LC3 punctate formation 6 h after IFN-γ (10 ng/ml) treatment in WT and Atg5−/− MEFs is shown. Scale bars, 20 μm. C, luciferase reporter assay of IRF1 promoter transactivation 1 h after IFN-γ (10 ng/ml) treatment in WT and Atg5−/− MEFs is shown. D, Griess reagent was used to detect the generation of nitrite 48 h after IFN-γ (10 ng/ml) treatment. E and F, ELISA was used to measure RANTES and IP-10 production 24 h after IFN-γ (10 ng/ml) treatment. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05.
FIGURE 2.
FIGURE 2.
Inhibiting autophagy reduced IFN-γ-induced inflammation. Griess reagent was used to detect nitrite generation 48 h after IFN-γ (10 ng/ml) treatment in WT MEFs (A) and RAW264.7 cells (B) pretreated for 0.5 h with (+) and without (−) 3-MA (1 mm). ELISA was used to measure RANTES and TNF-α 24 h after treatment. Dimethyl sulfoxide (DMSO) was the control. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05.
FIGURE 3.
FIGURE 3.
Effects of autophagy on the expression of IFN-γ receptors and the activation of Jak2 signaling. A, flow cytometry was used to detect the expression of IFNGR1 and IFNGR2 using specific antibodies in WT and Atg5−/− MEFs. The percentages of positive cells are shown. B, after 0.25 h of IFN-γ (10 ng/ml) treatment, co-immunoprecipitation (IP) was used to detect Jak2 interaction with IFNGR2. Western blotting (IB) was used to determine the expression of Jak2 and IFNGR2. H, heavy chain; L, light chain; NB, nonspecific binding. C, after IFN-γ (10 ng/ml) treatment, Western blotting was used to determine the time kinetic phosphorylation of Jak2 (Tyr1007/Tyr1008) and Jak1 (Tyr1022/Tyr1023). β-Actin was the internal control. Data are representative of three individual experiments.
FIGURE 4.
FIGURE 4.
Autophagy was important for IFN-γ-activated STAT1. A, after IFN-γ (10 ng/ml) treatment, Western blotting was used to determine the time kinetic phosphorylation of STAT1α/β (Tyr701) as well as IRF1 and SOCS1 expression in WT and Atg5−/− MEFs. B, HMEC-1 cells were pretreated with or without lentivirus-based short hairpin Atg5 RNA (shAtg5) or control luciferase-shRNA (shLuc) transfection and then treated with IFN-γ (10 ng/ml) for the indicated times. Western blotting was used to determine the expression of phosphorylation of STAT1α/β (Tyr701) and Atg5. β-Actin was the internal control. Data are representative of three individual experiments.
FIGURE 5.
FIGURE 5.
Autophagy was required for IFN-γ-activated Jak2-STAT1 and inflammation. Western blotting was used to determine LC3 conversion (A) and the time kinetic phosphorylation of Jak2 (Tyr1007/Tyr1008) and STAT1α/β (Tyr701) (B) in IFN-γ (10 ng/ml)-treated WT and Atg7−/− MEFs. β-Actin was the internal control. Data are representative of three individual experiments. C, Griess reagent was used to detect nitrite generation 48 h after IFN-γ (10 ng/ml) treatment. D, ELISA was used to measure IP-10 production 24 h after IFN-γ (10 ng/ml) treatment. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05.
FIGURE 6.
FIGURE 6.
ROS generation was deregulated in the absence of autophagy and negatively regulated IFN-γ-induced STAT1 activation. A, confocal fluorescence microscopic observation of the co-localization (Merge, yellow) of EGFP-LC3 punctate formation (green) and MitoSOX Red staining (red) in WT MEFs with or without IFN-γ (10 ng/ml) treatment for 3 h. 3D, three-dimensional. Scale bars, 20 μm. B, Western blotting, PCR, and flow cytometry, with COX IV antibodies, COX I primer, and MitoTracker Green staining, respectively, were used to detect mitochondria expression in WT and Atg5−/− MEFs. The ratios of mitochondrial DNA COX I to the total genomic DNA GAPDH were calculated based on the intensities of their PCR product in agarose gel. MFI, mean fluorescence intensity. C, CM-H2DCFDA staining and a microplate reader were used to measure ROS generation. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05. D, Western blotting was used to determine IFN-γ (10 ng/ml)-induced phosphorylation of STAT1α/β (Tyr701) in WT MEFs 0.25 h after they had been pretreated with (+) and without (−) caffeic acid phenethyl ester (CAPE, 25 μm) or H2O2 (10 mm) for 0.5 h. β-Actin was the internal control. Data are representative of three individual experiments. E, with (+) and without (−) H2O2 pretreatment for 0.5 h, generation of nitrite in IFN-γ (10 ng/ml)-treated WT MEFs was detected using Griess reagent at 48 h. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05.
FIGURE 7.
FIGURE 7.
ROS-regulated SHP2 inhibited IFN-γ-activated STAT1 in the absence of autophagy. A, Western blotting was used to determine IFN-γ (10 ng/ml)-induced phosphorylation of STAT1α/β (Tyr701) in WT MEFs 0.25 h after they had been pretreated with (+) and without (−) SHP2-shRNA clone 2 (shSHP2–2) or control luciferase-shRNA (shLuc) transfection and then with H2O2 (10 mm) for 0.5 h. B, after IFN-γ (10 ng/ml) treatment with (+) and without (−) SHP2-shRNAs (shSHP2) or control (shLuc) transfection, Western blotting was used to determine the phosphorylation of STAT1α/β (Tyr701) in Atg5−/− MEFs. shSHP2-1, inefficiently silenced; shSHP2-2, efficiently silenced. β-Actin was the internal control. Data are representative of three individual experiments.
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