Xanthine oxidoreductase mediates genotoxic drug-induced autophagy and apoptosis resistance by uric acid accumulation and TGF-ß-activated kinase 1 (TAK1) activation.

Autophagy is a highly conserved cellular process that profoundly impacts the efficacy of genotoxic chemotherapeutic drugs. TGF-ß-activated kinase 1 (TAK1) is a serine/threonine kinase that activates several signaling pathways involved in inducing autophagy and suppressing cell death. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid and hydrogen peroxide in the purine catabolism pathway. Recent studies showed that uric acid can bind to TAK1 and prolong its activation. We hypothesized that genotoxic drugs may induce autophagy and apoptosis resistance by activating TAK1 through XOR-generated uric acid. Here, we report that gemcitabine and 5-fluorouracil (5-FU), two genotoxic drugs, induced autophagy in HeLa and HT-29 cells by activating TAK1 and its two downstream kinases, AMP-activated kinase (AMPK) and c-Jun terminal kinase (JNK). XOR knockdown and the XOR inhibitor allopurinol blocked gemcitabine-induced TAK1, JNK, AMPK, and Unc51-like kinase 1 (ULK1)S555 phosphorylation and gemcitabine-induced autophagy. Inhibition of the ATM-Chk pathway, which inhibits genotoxic drug-induced uric acid production, blocked gemcitabine-induced autophagy by inhibiting TAK1 activation. Exogenous uric acid in its salt form, monosodium urate (MSU), induced autophagy by activating TAK1 and its downstream kinases JNK and AMPK. Gene knockdown or the inhibitors of these kinases blocked gemcitabine- and MSU-induced autophagy. Inhibition of autophagy by allopurinol, chloroquine, and 5Z-7-oxozeaenol (5Z), a TAK1-specific inhibitor, enhanced gemcitabine-induced apoptosis. Our study uncovers a previously unrecognized role of XOR in regulating genotoxic drug-induced autophagy and apoptosis and has implications for designing novel therapeutic strategies for cancer treatment.

Leflunomide alleviates obesity via activation of the TAK1-AMPK pathway and induction of lipophagy.

Lipophagy is a subset of selective autophagy that specifically degrades lipid droplets and plays an important role in obesity. Leflunomide treatment in rheumatoid arthritis (RA) patients has been associated with weight loss and decreased blood glucose levels, which cannot be attributed to its known side effects. Our prior studies showed that A77 1726, the active metabolite of leflunomide, acts as an inhibitor of S6K1 to sensitize the insulin receptor and control hyperglycemia. Whether the anti-obesity effect of leflunomide is mediated by targeting S6K1 and its underlying mechanisms remain unclear. Here, we report that A77 1726 induced LC3 lipidation and increased the formation of autophagosomes and lipoautolysosomes in 3T3-L1 adipocytes by activating TGF-ß-activated kinase 1 (TAK1), AMP-activated kinase (AMPK), and Unc-51 like autophagy-activated kinase 1 (ULK1). A77 1726 reduced the content of lipid droplets in 3T3-L1 adipocytes, which was blocked by bafilomycin or by beclin-1 knockdown. Similar observations were made in murine adipocytes differentiated from S6K1-/- embryonic fibroblasts (MEFs). Leflunomide treatment restricted bodyweight gains in ob/ob mice and reduced the visceral fat deposit and the size of adipocytes. Leflunomide treatment induced autophagy in adipose and liver tissues and reduced hepatic lipid contents. Consistently, S6K1 knockout increased the levels of LC3 lipidation in the liver, muscle, and fat of S6K-/- mice. Leflunomide treatment and S6K1 deficiency both induced TAK1, AMPK, and ULK1 phosphorylation in these tissues. These observations collectively suggest that leflunomide controls obesity in part by activating AMPK and inducing lipophagy. Our study provides insights into the mechanisms of leflunomide-mediated anti-obesity activity.

Virological evaluation of natural and modified attapulgite against porcine epidemic diarrhoea virus.

BACKGROUND: The Porcine Epidemic Diarrhea Virus (PEDV) has caused significant economic losses in the global swine industry. As a potential drug for treating diarrhea, the antiviral properties of attapulgite deserve further study. METHODS: In this study, various methods such as RT-qPCR, Western blot, viral titer assay, Cytopathic Effect, immunofluorescence analysis and transmission electron microscopy were used to detect the antiviral activity of attapulgite and to assess its inhibitory effect on PEDV. RESULTS: When exposed to the same amount of virus, there was a significant decrease in the expression of the S protein, resulting in a viral titer reduction from 10-5.613 TCID50/mL to 10-2.90 TCID50/mL, which represents a decrease of approximately 102.6 folds. Results of cytopathic effect and indirect immunofluorescence also indicate a notable decrease in viral infectivity after attapulgite treatment. Additionally, it was observed that modified materials after acidification had weaker antiviral efficacy compared to powdered samples that underwent ultrasonic disintegration, which showed the strongest antiviral effects. CONCLUSION: As a result, Attapulgite powders can trap and adsorb viruses to inhibit PEDV in vitro, leading to loss of viral infectivity. This study provides new materials for the development of novel disinfectants and antiviral additives.

XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity.

XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

A77 1726, the active metabolite of the anti-rheumatoid arthritis drug leflunomide, inhibits influenza A virus replication in vitro and in vivo by inhibiting the activity of Janus kinases.

The newly reassorted IAV subtypes from zoonotic reservoirs respond poorly to current vaccines and antiviral therapy. There is an unmet need in developing novel antiviral drugs for better control of IAV infection. The cellular factors that are crucial for virus replication have been sought as novel molecular targets for antiviral therapy. Recent studies have shown that Janus kinases (JAK), JAK1, and JAK2, play an important role in IAV replication. Leflunomide is an anti-inflammatory drug primarily used for treating rheumatoid arthritis (RA). Prior studies suggest that A77 1726, the active metabolite of leflunomide, inhibits the activity of JAK1 and JAK3. Our current study aims to determine if A77 1726 can function as a JAK inhibitor to control IAV infection. Here, we report that A77 1726 inhibited the replication of three IAV subtypes（H5N1, H1N1, H9N2）in three cell types (chicken embryonic fibroblasts, A549, and MDCK). A77 1726 inhibited JAK1, JAK2, and STAT3 tyrosine phosphorylation. Similar observations were made with Ruxolitinib (Rux), a JAK-specific inhibitor. JAK2 overexpression enhanced H5N1 virus replication and compromised the antiviral activity of A77 1726. Leflunomide inhibited virus replication in the lungs of IAV-infected mice, alleviated their body weight loss, and prolonged their survival. Our study demonstrates for the first time the ability of A77 1726 to inhibit JAK2 activity and suggests that inhibition of JAK activity contributes to its antiviral activity.

The Gli1-Snail axis contributes to Salmonella Typhimurium-induced disruption of intercellular junctions of intestinal epithelial cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that damages gastrointestinal tissue and causes severe diarrhoea. The mechanisms by which Salmonella disrupts epithelial barrier and increases the paracellular permeability are incompletely understood. Our present study aims to determine the role of Gli1, a transcription factor activated in the sonic hedgehog (Shh) pathway, in decreasing the levels of apical junction proteins in a Salmonella-infected human colonic epithelial cancer cell line, Caco-2, and in the intestinal tissue of Salmonella-infected mice. Here, we report that S. Typhimurium increased the mRNA and protein levels of Gli1 and Snail, a downstream transcription factor that plays an important role in the epithelial-to-mesenchymal transition (EMT). S. Typhimurium also decreased the levels of E-cadherin and three tight junction proteins (ZO-1, claudin-1, and occludin). Gli1 siRNA and GANT61, a Gli1-specific inhibitor, blocked S. Typhimurium-induced Snail expression, restored the levels of E-cadherin and tight junction proteins, and prevented S. Typhimurium-increased paracellular permeability. Further study showed that Gli1 was cross-activated by the MAP and PI-3 kinase pathways. S. Typhimurium devoid of sopB, an effector of the Type 3 secretion system (T3SS) responsible for AKT activation, was unable to induce Snail expression and to decrease the expression of apical junction proteins. Our study uncovered a novel role of Gli1 in mediating the Salmonella-induced disruption of the intestinal epithelial barrier.

Toll-like receptor signalling cross-activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages.

It has been long recognised that activation of toll-like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR-induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium-induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium-induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP-activated protein kinase (AMPK) through activation of the TGF-ß-activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc-51-like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium-infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR-mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow-derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4-deficient macrophages. TAK1 and AMPK-specific inhibitors blocked S. Typhimurium-induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1-AMPK axis through TLRs is essential for S. Typhimurium-induced autophagy and that TLR signalling cross-activates the autophagic pathway to clear intracellular bacteria.

Astaxanthin Inhibits p70 S6 Kinase 1 Activity to Sensitize Insulin Signaling.

Astaxanthin (AST) is a carotenoid with therapeutic values on hyperglycemia and diabetic complications. The mechanisms of action of AST remain incompletely understood. p70 S6 kinase 1 (S6K1) is a serine/threonine kinase that phosphorylates insulin receptor substrate 1 (IRS-1)S1101 and desensitizes the insulin receptor (IR). Our present study aims to determine if AST improves glucose metabolisms by targeting S6K1. Western blot analysis revealed that AST inhibited the phosphorylation of two S6K1 substrates, S6S235/236 and IRS-1S1101, but enhanced the phosphorylation of AKTT308, AKTS473, and S6K1T389 by feedback activation of the phosphatidylinositol-3 (PI-3) kinase in 3T3-L1 adipocytes and L6 myotubes. In vitro kinase assays revealed that AST inhibited S6K1 activity with an IC50 value of approximately 13.8 µM. AST increased insulin-induced IR tyrosine phosphorylation and IRS-1 binding to the p85 subunit of PI-3 kinase. Confocal microscopy revealed that AST increased the translocation of the glucose transporter 4 (GLUT4) to the plasma membrane in L6 cells. Glucose uptake assays using a fluorescent dye, 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), revealed that AST increased glucose uptake in 3T3-L1 adipocytes and L6 myotubes under insulin resistance conditions. Our study identifies S6K1 as a previously unrecognized molecular target of AST and provides novel insights into the mechanisms of action of AST on IR sensitization.

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses.

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 103.0EID50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway.

Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA-, and a plasmid-mediated complementary strain, S.E-AvrA-/pAvrA+ (S.E-AvrA+), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA+ than in cells infected with S.E-AvrA- Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA+ strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA- strain led to enhanced bacterial invasion, both in vitro and in vivo Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases.

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. RESULTS: A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 106.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. CONCLUSIONS: These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Immunogenicity and protective efficacy of a Salmonella Enteritidis sptP mutant as a live attenuated vaccine candidate.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) is a highly adaptive pathogen in both humans and animals. As a Salmonella Type III secretion system (T3SS) effector, Salmonella protein tyrosine phosphatase (SptP) is critical for virulence in this genus. To investigate the feasibility of using C50336ΔsptP as a live attenuated oral vaccine in mice, we generated the sptP gene deletion mutant C50336ΔsptP in S. Enteritidis strain C50336 by λ-Red mediated recombination and evaluated the protective ability of the S. Enteritidis sptP mutant strain C50336ΔsptP against mice salmonellosis. RESULTS: We found that C50336ΔsptP was a highly immunogenic, effective, and safe vaccine in mice. Compared to wild-type C50336, C50336ΔsptP showed reduced virulence as confirmed by the 50% lethal dose (LD50) in orally infected mice. C50336ΔsptP also showed decreased bacterial colonization both in vivo and in vitro. Immunization with C50336ΔsptP had no significant effect on body weight and did not result in obvious clinical symptoms relative to control animals treated with phosphate-buffered saline (PBS), but induced humoral and cellular immune responses at 12 and 26 days post inoculation. Immunization with 1 × 108 colony-forming units (CFU) C50336ΔsptP per mouse provided 100% protection against subsequent challenge with the wild-type C50336 strain, and immunized mice showed mild and temporary clinical symptoms as compared to those of control group. CONCLUSIONS: These results demonstrate that C50336ΔsptP can be a live attenuated oral vaccine for salmonellosis.

Vemurafenib activates the sonic hedgehog pathway and promotes thyroid cancer stem cell self-renewal.

B-Raf kinase inhibitors such as vemurafenib (PLX4032) and dabrafenib have limited therapeutic efficacy on BRAF-mutated thyroid cancer. Cancer stem cells (CSCs) play important roles in tumor recurrence, drug resistance, and metastasis. Whether CSCs play a role in dampening the antitumor activity of B-Raf kinase inhibitors remains unknown. Here, we report that vemurafenib (PLX4032) induced the expression of several stemness-related genes including Gli1, Snail, BMI1, and SOX2 in two anaplastic thyroid cancer cell lines, SW1736 and 8505C, but decreased the expression of these genes in A375 cells, a human melanoma cell line. PLX4032 promoted thyroid cancer stem cell self-renewal, as evidenced by increased numbers of aldehyde dehydrogenase-positive cells and thyrospheres. Mechanistically, PLX4032 activates the PI-3 and mitogen-activated protein kinase pathways through HER3 to cross-activate Gli1, a transcription factor of the sonic hedgehog (Shh) pathway. GANT61, a specific inhibitor of Gli1, blocked the expression of the stemness-related genes in PLX4032-treated thyroid cancer cells in vitro and in vivo in two thyroid cancer xenograft models. GANT61 treatment alone weakly inhibited SW1736 tumor growth but enhanced the antitumor activity of PLX4032 when used in combination. Our study provides mechanistic insights into how thyroid cancer poorly responds to B-Raf kinase inhibitors and suggests that targeting B-Raf and the Shh pathway in combination may overcome thyroid cancer drug resistance.

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K.

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P â€‹+ â€‹M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P â€‹+ â€‹M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.

Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication.

Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.

Uric acid accumulation in DNA-damaged tumor cells induces NKG2D ligand expression and antitumor immunity by activating TGF-ß-activated kinase 1.

DNA damage by genotoxic drugs such as gemcitabine and 5-fluorouracil (5-FU) activates the ataxia telangiectasia, mutated (ATM)-Chk pathway and induces the expression of NKG2D ligands such as the MHC class I-related chain A and B (MICA/B). The mechanisms underlying this remain incompletely understood. Here we report that xanthine oxidoreductase (XOR), a rate-limiting enzyme that produces uric acid in the purine catabolism pathway, promotes DNA damage-induced MICA/B expression. Inhibition of the ATM-Chk pathway blocks genotoxic drug-induced uric acid production, TGF-ß-activated kinase 1 (TAK1) activation, ERK phosphorylation, and MICA/B expression. Inhibition of uric acid production by the XOR inhibitor allopurinol blocks DNA damage-induced TAK1 activation and MICA/B expression in genotoxic drug-treated cells. Exogenous uric acid activates TAK1, NF-κB, and the MAP kinase pathway. TAK1 inhibition blocks gemcitabine- and uric acid-induced MAP kinase activation and MICA/B expression. Exogenous uric acid in its salt form, monosodium urate (MSU), induces MICA/B expression and sensitizes tumor cells to NK cell killing. MSU immunization with irradiated murine breast cancer cell line RCAS-Neu retards breast cancer growth in syngeneic breast cancer models and delays breast cancer development in a somatic breast cancer model. Our study suggests that uric acid accumulation plays an important role in activating TAK1, inducing DNA damage-induced MICA/B expression, and enhancing antitumor immunity.

H5N1 infection impairs the alveolar epithelial barrier through intercellular junction proteins via Itch-mediated proteasomal degradation.

The H5N1 subtype of the avian influenza virus causes sporadic but fatal infections in humans. H5N1 virus infection leads to the disruption of the alveolar epithelial barrier, a pathologic change that often progresses into acute respiratory distress syndrome (ARDS) and pneumonia. The mechanisms underlying this remain poorly understood. Here we report that H5N1 viruses downregulate the expression of intercellular junction proteins (E-cadherin, occludin, claudin-1, and ZO-1) in several cell lines and the lungs of H5N1 virus-infected mice. H5N1 virus infection activates TGF-ß-activated kinase 1 (TAK1), which then activates p38 and ERK to induce E3 ubiquitin ligase Itch expression and to promote occludin ubiquitination and degradation. Inhibition of the TAK1-Itch pathway restores the intercellular junction structure and function in vitro and in the lungs of H5N1 virus-infected mice. Our study suggests that H5N1 virus infection impairs the alveolar epithelial barrier by downregulating the expression of intercellular junction proteins at the posttranslational level.

Major histocompatibility complex class I-related chain A/B (MICA/B) expression in tumor tissue and serum of pancreatic cancer: role of uric acid accumulation in gemcitabine-induced MICA/B expression.

BACKGROUND: Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind the immunoreceptor NKG2D and play an important role in mediating the cyotoxicity of NK and T cells. In this study, we sought to study MICA/B expression in pancreatic cancer and to determine whether and how genotoxic drugs such as gemcitabine can affect MICA/B expression and natural killer cytotoxity. METHODS: Seven pancreatic cancer cell lines were analyzed for MICA/B expression by flow cytometry and for their sensitivity to NK-92 cell killing by a 51Cr release assay. MICA/B expression in tumor tissues and sera of pancreatic cancer was analyzed by immunohistochemical staining (IHC) and ELISA, respectively. RESULTS: Two MICA/B-positive cell lines were sensitive to the cytotoxic activity of NK-92 cells. Other two MICA/B-positive cell lines and three MICA/B-negative cell lines were resistant to NK-92 cell killing. MICA/B expression was positive in 17 of 25 (68%) pancreatic ductal adenocarcinomas but not in normal pancreatic ductal epithelial cells. Serum MICA/B levels were significantly elevated in patients with pancreatic adenocarcinomas but did not correlate with the stage of pancreatic cancer and patient survival. Gemcitabine therapy led to increased serum MICA levels in 6 of 10 patients with detectable serum MICA. Allopurinol, an inhibitor of xanthine oxidoreductase that converts xanthine to uric acid, blocked uric acid production, MICA/B expression, and sensitivity to NK-92 cell killing toward a PANC-1 cancer cell line exposed to radiation and two genotoxic drugs, gemcitabine and 5-fluorouracil. CONCLUSIONS: The levels of MICA/B expression in serum and tissue of pancreatic cancer are elevated. DNA damage-induced MICA/B expression is mediated through increased uric acid production.