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

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. 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.

Glycogen synthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation by regulating Src homology-2 domain-containing phosphatase 2.

Glycogen synthase kinase-3beta (GSK-3beta)-modulated IFN-gamma-induced inflammation has been reported; however, the mechanism that activates GSK-3beta and the effects of activation remain unclear. Inhibiting GSK-3beta decreased IFN-gamma-induced inflammation. IFN-gamma treatment rapidly activated GSK-3beta via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephosphorylation at Ser(9), and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr(216). Pyk2 was activated through phosphatidylcholine-specific phospholipase C (PC-PLC)-, protein kinase C (PKC)-, and Src-regulated pathways. The activation of PC-PLC, Pyk2, and GSK-3beta was potentially regulated by IFN-gamma receptor 2-associated Jak2, but it was independent of IFN-gamma receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A(2) positively regulated neutral sphingomyelinase. Inhibiting GSK-3beta activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-gamma stimulation. All these results showed that activated GSK-3beta synergistically affected IFN-gamma-induced STAT1 activation by inhibiting SHP2.

Glycogen synthase kinase-3ß indirectly facilitates interferon-γ-induced nuclear factor-κB activation and nitric oxide biosynthesis.

Either glycogen synthase kinase (GSK)-3ß or nuclear factor (NF)-κB regulates interferon (IFN)-γ-induced nitric oxide (NO) biosynthesis; however, the inter-regulation between GSK-3ß and NF-κB is unknown. We have previously shown that IFN-γ-activated GSK-3ß negatively regulates Src homology-2 domain-containing phosphatase (SHP) 2 to facilitate Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 1 activation. Because Jaks-IFN-inducible dsRNA-activated serine-threonine protein kinase (PKR) axis signaling is essential for IFN-γ-activation of NF-κB, in this study we investigate the potential mechanism for GSK-3ß-facilitated NF-κB activation in IFN-γ-stimulated RAW264.7 murine macrophages. Pharmacological inhibitors of GSK-3ß or NF-κB signaling, such as the inhibitor of κB (IκB) kinase ß (IKKß) and IκBα, inhibited IFN-γ-induced inducible NO synthase (iNOS) and thus NO biosynthesis. Inhibiting GSK-3ß decreased IFN-γ-induced NF-κB phosphorylation (Ser536) and activation. The upstream regulators for GSK-3ß activation, including okadaic acid-sensitive protein phosphatase and proline-rich tyrosine kinase 2, were also important for IFN-γ-induced IκBα phosphorylation (Ser32) and degradation. Under IFN-γ stimulation, Jak2-PKR axis signaling induced IκBα inactivation as well as iNOS/NO biosynthesis. It is notable that inhibiting GSK-3ß caused SHP2-mediated dephosphorylation of PKR (Thr446), IKKß (Ser180), and NF-κB (Ser536). Taken together, we provide the first evidence to demonstrate that GSK-3ß indirectly facilitates IFN-γ-induced NF-κB activation by inhibiting SHP2, in turn activating the PKR-IKKß-IκBα axis signaling pathway.

IFN-gamma synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase-3-inhibited IL-10.

Interferon-gamma (IFN-gamma) plays a crucial role in innate immunity and inflammation. It causes the synergistic effect on endotoxin lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS)/NO biosynthesis; however, the mechanism remains unclear. In the present study, we investigated the effects of glycogen synthase kinase-3 (GSK-3)-mediated inhibition of anti-inflammatory interleukin-10 (IL-10). We found, in LPS-stimulated macrophages, that IFN-gamma increased iNOS expression and NO production in a time-dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor-alpha and regulated on activation, normal T expressed and secreted, and the downregulation of IL-10. RT-PCR further showed changes in the IL-10 mRNA level as well. Treating cells with recombinant IL-10 showed a decrease in IFN-gamma/LPS-induced iNOS/NO biosynthesis, whereas anti-IL-10 neutralizing antibodies enhanced this effect, suggesting that IL-10 acts in an anti-inflammatory role. GSK-3-inhibitor treatment blocked IFN-gamma/LPS-induced iNOS/NO biosynthesis but upregulated IL-10 production. Inhibiting GSK-3 using short-interference RNA showed similar results. Additionally, treating cells with anti-IL-10 neutralizing antibodies blocked these effects. We further showed that inhibiting GSK-3 increased phosphorylation of transcription factor cyclic AMP response element binding protein. Inhibiting protein tyrosine kinase Pyk2, an upstream regulator of GSK-3beta, caused inhibition on IFN-gamma/LPS-induced GSK-3beta phosphorylation at tyrosine 216 and iNOS/NO biosynthesis. Taken together, these findings reveal the involvement of GSK-3-inhibited IL-10 on the induction of iNOS/NO biosynthesis by IFN-gamma synergized with LPS.

Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKß/NF-κB signaling.

BACKGROUND: Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKß (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages. CONCLUSIONS/SIGNIFICANCE: These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKß/NF-κB signaling pathways.