Chi Yun Wang

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ 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-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-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-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.

Anesthetic Propofol Reduces Endotoxic Inflammation by Inhibiting Reactive Oxygen Species-regulated Akt/IKKβ/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 IKKβ (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/IKKβ/NF-κB signaling pathways.

Glycogen Synthase Kinase-3β Facilitates IFN-γ-Induced STAT1 Activation by Regulating Src Homology-2 Domain-Containing Phosphatase 2

Glycogen synthase kinase-3β (GSK-3β)-modulated IFN-γ-induced inflammation has been reported; however, the mechanism that activates GSK-3β and the effects of activation remain unclear. Inhibiting GSK-3β decreased IFN-γ-induced inflammation. IFN-γ treatment rapidly activated GSK-3β via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephosphorylation at Ser9, and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr216. 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-3β was potentially regulated by IFN-γ receptor 2-associated Jak2, but it was independent of IFN-γ receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A2 positively regulated neutral sphingomyelinase. Inhibiting GSK-3β activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-γ stimulation. All these results showed that activated GSK-3β synergistically affected IFN-γ-induced STAT1 activation by inhibiting SHP2.

IFN‐γ synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase‐3‐inhibited IL‐10

Interferon‐γ (IFN‐γ) 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‐γ increased iNOS expression and NO production in a time‐dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor‐α 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‐γ/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‐γ/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‐3β, caused inhibition on IFN‐γ/LPS‐induced GSK‐3β 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‐γ synergized with LPS. J. Cell. Biochem. 105: 746–755, 2008.

Annexin A2 silencing induces G2 arrest of non-small cell lung cancer cells through p53-dependent and -independent mechanisms

Background: Aberrant expression of annexin A2 has been found in several cancers; however, its role in tumorigenesis is unknown. Results: Silencing annexin A2 partly caused p53-regulated cell cycle arrest at the G2 phase by inactivating JNK/c-Jun signaling. Conclusion: Annexin A2 promotes the cell cycle and cell proliferation in lung cancer in part by inhibiting p53. Significance: This study provides new insight into the tumorigenic role of annexin A2. Annexin A2 (ANXA2) overexpression is required for cancer cell proliferation; however, the molecular mechanisms underlying ANXA2-mediated regulation of the cell cycle are still unknown. ANXA2 is highly expressed in non-small cell lung cancer (NSCLC) and is positively correlated with a poor prognosis. NSCLC A549 cells lacking ANXA2 exhibited defects in tumor growth in vivo and in cell proliferation in vitro without cytotoxicity. ANXA2 knockdown induced cell cycle arrest at G2 phase. Unexpectedly, ANXA2 silencing increased the expression of p53 and its downstream genes, which resulted in p53-dependent and -independent G2 arrest. Aberrant JNK inactivation, which was observed in ANXA2-deficient cells, inhibited cell proliferation following G2 arrest. A lack of ANXA2 caused a loss of JNK-regulated c-Jun expression, resulting in an increase in p53 transcription. These results demonstrate a novel role for ANXA2 in NSCLC cell proliferation by facilitating the cell cycle partly through the regulation of p53 via JNK/c-Jun.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis

Inactivation of glycogen synthase kinase (GSK)‐3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK‐3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr‐Abl tyrosine kinase. In Bcr‐Abl signaling, the role of GSK‐3 is not well defined. Here, we report that enforced expression of constitutively active GSK‐3 reduced proliferation and increased Bcr‐Abl inhibition‐induced apoptosis by nearly 1‐fold. Bcr‐Abl inhibition activated GSK‐3 and GSK‐3‐dependent apoptosis. Inactivation of GSK‐3 by Bcr‐Abl activity is, therefore, confirmed. To reactivate GSK‐3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor‐suppressor sphingo‐lipid and a potent GSK‐3 activator. We found that either PDMP or silence of GCS increased Bcr‐Abl inhibition‐induced GSK‐3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug‐resistant CML T315I mutant to Bcr‐Abl inhibitor GNF‐2‐, imatinib‐, or nilotinib‐induced apoptosis by >5‐fold. Combining PDMP and GNF‐2 eliminated transplanted‐CML‐T315I‐mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF‐2‐induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr‐Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK‐3‐mediated apoptosis. This study suggests a feasible novel anti‐CML strategy by accumulating endogenous ceramide to reactivate GSK‐3 and abrogate drug resistance.—Huang, W.‐C., Tsai, C.‐C., Chen, C.‐L., Chen, T.‐Y., Chen, Y.‐P., Lin, Y.‐S., Lu, P.‐J., Lin, C.‐M., Wang, S.‐W., Tsao, C.‐W., Wang, C.‐Y., Cheng, Y.‐L., Hsieh, C.‐Y., Tseng, P.‐C., Lin, C.‐F. Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr‐Abl inhibitor and cooperatively induces glycogen synthase kinase‐3‐regulated apoptosis. FASEB J. 25, 3661–3673 (2011). www.fasebj.org

Increased galectin-3 facilitates leukemia cell survival from apoptotic stimuli

Galectin-3 is regulated for cancer cell survival and apoptosis depending upon the cell type and stimulus. We investigated a glycogen synthase kinase (GSK)-3β/galectin-3-regulated mechanism used by leukemia cells to escape from apoptotic stimuli. Galectin-3 expression was time- and transcription-dependently deregulated in K562 chronic myeloid leukemia cells stimulated for apoptosis by cisplatin (a platinum-based chemotherapy drug), sphingolipid ceramide analog C(2)-ceramide, and LY294002 (a phosphatidylinositol 3-kinase inhibitor). Notably, galectin-3 was upregulated in survival cells. Forced galectin-3 expression caused resistance to apoptosis, whereas knockdown galectin-3 expression increased susceptibility to apoptosis. Sub-cellular distribution of inducible galectin-3 was mitochondria-specific. Apoptotic stimuli decreased pro-survival Bcl-2 family protein expression (especially Mcl-1), whereas galectin-3 overexpression reversed but it was enhanced by a galectin-3 expression knockdown. Under apoptotic stimulation, GSK-3β was activated after Akt was inactivated and GSK-3β was inhibited-either pharmacologically or using short hairpin RNA to abolish galectin-3, increase apoptosis, and inhibit colony formation-which suggests a pro-survival role for GSK-3β. We found that GSK-3β upregulated galectin-3 and stabilized anti-apoptotic Bcl-2 family proteins, which is important for the escape of leukemia cells from apoptotic stimuli.

Anesthetic Propofol Causes Glycogen Synthase Kinase-3β-regulated Lysosomal/Mitochondrial Apoptosis in Macrophages

Background: Overdose propofol treatment with a prolong time causes injury to multiple cell types; however, its molecular mechanisms remain unclear. Activation of glycogen synthase kinase (GSK)-3&bgr; is proapoptotic under death stimuli. The authors therefore hypothesize that propofol overdose induces macrophage apoptosis through GSK-3&bgr;. Methods: Phagocytic analysis by uptake of Staphylococcus aureus showed the effects of propofol overdose on murine macrophages RAW264.7 and BV2 and primary human neutrophils in vitro. The authors further investigated cell apoptosis in vitro and in vivo, lysosomal membrane permeabilization, and the loss of mitochondrial transmembrane potential (MTP) by propidium iodide, annexin V, acridine orange, and rhodamine 123 staining, respectively. Protein analysis identified activation of apoptotic signals, and pharmacologic inhibition and genetic knockdown using lentiviral-based short hairpin RNA were further used to clarify their roles. Results: A high dose of propofol caused phagocytic inhibition and apoptosis in vitro for 24 h (25 &mgr;g/ml, in triplicate) and in vivo for 6 h (10 mg/kg/h, n = 5 for each group). Propofol induced lysosomal membrane permeabilization and MTP loss while stabilizing MTP and inhibiting caspase protected cells from mitochondrial apoptosis. Lysosomal cathepsin B was required for propofol-induced lysosomal membrane permeabilization, MTP loss, and apoptosis. Propofol decreased antiapoptotic Bcl-2 family proteins and then caused proapoptotic Bcl-2-associated X protein (Bax) activation. Propofol-activated GSK-3&bgr; and inhibiting GSK-3&bgr; prevented Mcl-1 destabilization, MTP loss, and lysosomal/mitochondrial apoptosis. Forced expression of Mcl-1 prevented the apoptotic effects of propofol. Decreased Akt was important for GSK-3&bgr; activation caused by propofol. Conclusions: These results suggest an essential role of GSK-3&bgr; in propofol-induced lysosomal/mitochondrial apoptosis.

Interferon-γ stimulates p11-dependent surface expression of annexin A2 in lung epithelial cells to enhance phagocytosis

Annexin A2 (p36) is usually present together with its natural ligand p11 as a heterotetramer complex, which has multiple biological functions depending on its cellular localization. However, the detailed mechanism of annexin A2 translocation and its physiological role in inflammation remain unclear. Here, we show that IFN‐γ stimulation enhances surface translocation of annexin A2 on lung epithelial cells. While total annexin A2 protein remains unchanged, the expression of p11 is upregulated via the IFN‐γ‐activated JAK2/STAT1 signal pathway. Notably, IFN‐γ‐induced p11 expression is required for annexin A2 translocation to the cell surface. Since annexin A2 lacks a signal peptide for surface translocation by the classical endoplasmic reticulum‐Golgi route, its mode of trafficking remains unclear. We observed that p11‐dependent surface translocation of annexin A2 is associated with the exosomal secretion pathway. The IFN‐γ‐induced increase of annexin A2 in the exosomes is blocked in p11‐silenced cells. Furthermore, IFN‐γ‐induced surface expression of annexin A2 mediates phagocytosis of apoptotic cells by lung epithelial cells. These findings provide insights into the surface translocation mechanism of annexin A2 and illustrate a pivotal function of surface annexin A2 in the phagocytic response to IFN‐γ. J. Cell. Physiol. 227: 2775–2787, 2012.

Autophagy facilitates an IFN-γ response and signal transduction

Autophagy, that is directly triggered by invaded pathogens and indirectly triggered by IFN-γ, acts as a defense by mediating intracellular microbial recognition and clearance. In addition, autophagy contributes to inflammation by facilitating an IFN-γ response and signal transduction. For immune escape, downregulated autophagy may be a strategy used by microbes.