Yong-Joon Chwae

STAT3 transcriptional factor activated by reactive oxygen species induces IL6 in starvation-induced autophagy of cancer cells.

Autophagy is one of the survival processes of cancer cells, especially in stressful conditions such as starvation, hypoxia and chemotherapeutic agents. However, its roles in tumor survival have not yet been fully elucidated. Here, we found for the first time that JAK2/STAT3 was activated in HeLa cells when they were starved or treated with rapamycin. STAT3 activation was associated with autophagic processes, because it was completely inhibited by 3-methyladenine, partially inhibited by knockdown of molecules associated with autophagic processes and blocked by antioxidants, DPI, a Nox inhibitor and knockdown of p22 phox, indicating that ROS generated by Nox that was activated during autophagic processes activated JAK2/STAT3 pathway. Activated STAT3 directly bound to IL6 promoter and increased IL6 mRNA and protein secretion. Finally, the conditioned media, which included IL6, from starved HeLa cells promoted cancer cell survival in both normal and starved conditions, confirmed by clonogenic, proliferation and cell death assays. These data together indicate that the autophagic process in cancer cells can contribute to their survival by JAk2/STAT3 activation and subsequent secretion of growth factors.

Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication

ABSTRACT Phosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However, in vivo phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [32P]orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication. IMPORTANCE Threonine 162, within the carboxyl-terminal end of the hepatitis B virus (HBV adw) core (C) protein, has long been ignored as a phosphoacceptor, even though it is highly conserved among mammalian hepadnaviruses and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication.

Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor γ, controls hepatitis B virus replication

In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adiponectin, liver X receptor α (LXRα), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPARγ and C/EBPα. Conversely, HBV replication was upregulated by adiponectin and PPARγ agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.

Neutrophil autophagy and extracellular DNA traps contribute to airway inflammation in severe asthma.

Autophagy and neutrophil extracellular DNA traps (NETs) are implicated in asthma; however, their roles in asthma pathogenesis have not been elucidated.

The role of autophagy in allergic inflammation: a new target for severe asthma

Autophagy has been investigated for its involvement in inflammatory diseases, but its role in asthma has been little studied. This study aimed to explore the possible role of autophagy and its therapeutic potential in severe allergic asthma. BALB/c mice were sensitized with ovalbumin (OVA) on days 0 and 14, followed by primary OVA challenge on days 28–30. The mice received a secondary 1 or 2% OVA challenge on days 44–46. After the final OVA challenge, the mice were assessed for airway responsiveness (AHR), cell composition and cytokine levels in bronchoalveolar lavage fluid (BALF). LC3 expression in lung tissue was measured by western blot and immunofluorescence staining. Autophagosomes were detected by electron microscopy. 3-Methyladenine (3-MA) treatment and Atg5 knockdown were applied to investigate the potential role of autophagy in allergic asthma mice. AHR, inflammation in BALF and LC3 expression in lung tissue were significantly increased in the 2% OVA-challenged mice compared with the 1% OVA-challenged mice (P<0.05). In addition, eosinophils showed prominent formation of autophagosomes and increased LC3 expression compared with other inflammatory cells in BALF and lung tissue. After autophagy was inhibited by 3-MA and Atg5 shRNA treatment, AHR, eosinophilia, interleukin (IL)-5 levels in BALF and histological inflammatory findings were much improved. Finally, treatment with an anti-IL-5 antibody considerably reduced LC3 II expression in lung homogenates. Our findings suggest that autophagy is closely correlated with the severity of asthma through eosinophilic inflammation, and its modulation may provide novel therapeutic approaches for severe allergic asthma.

Down-regulation of interleukin-2 production by CD4(+) T cells expressing TIM-3 through suppression of NFAT dephosphorylation and AP-1 transcription.

TIM-3 is expressed by TH1 cells and negatively regulates cytokine production by these cells. The aim of the present study was to explore the mechanisms by which IL-2 production is suppressed in TIM-3-expressing T cells. First, the activity of two transcription factors that bind to the IL-2 promoter was examined in Jurkat T cells expressing TIM-3. Both AP-1 and NFAT activity were reduced in TIM-3-expressing cells stimulated with a phorbol ester and a calcium ionophore. At the same time, expression of the AP-1 components, c-Fos and c-Jun, was induced to a lesser extent in stimulated human primary CD4(+) T cells expressing high levels of TIM-3 than in those expressing low levels of TIM-3. Furthermore, TIM-3-expression inhibited the stimulation-induced dephosphorylation and nuclear translocation of NFAT in Jurkat T cells and primary CD4(+) T cells. Finally, the cytoplasmic tail of TIM-3 was required for the suppression of IL-2 production and for AP-1 and NFAT activation. Taken together, these results suggest that IL-2 production by T cells may be downregulated by TIM-3-mediated signals, leading to suppression of NFAT dephosphorylation and AP-1 transcription.

Activation of mitogen activated protein kinase-Erk kinase (MEK) increases T cell immunoglobulin mucin domain-3 (TIM-3) transcription in human T lymphocytes and a human mast cell line

The immune regulatory molecule T cell immunoglobulin mucin domain (TIM-3) is expressed in activated T cells and in mast cells treated with transforming growth factor (TGF)-β, but underlying mechanisms for induction of TIM-3 transcription have not been well-explored. We studied the role of mitogen-activated protein kinase (MAPK) in TIM-3 transcription on the basis of the involvement of MAPK in T cell activation and TGF-β signaling. Inhibitors of MAPK-Erk kinase (MEK) as well as p38 suppressed TIM-3 transcription in phorbol myristic acid (PMA)-stimulated T cells, but inhibitors of c-Jun NH2-terminal kinase (JNK) did not. MEK over-expression enhanced TIM-3 transcription in PMA-stimulated T cells. Furthermore, -1.5kb TIM-3 promoter was activated by PMA stimulation and repressed by MEK inhibitors in Jurkat T cells. Similarly, MEK activation enhanced TIM-3 transcription in TGF-β-stimulated HMC-1 human mast cells, although MEK seemed not directly activated by TGF-β. Concordantly, -1.5kb TIM-3 promoter activity was reduced by MEK inhibitors, but was not responsive to TGF-β stimulation in HMC-1 cells. These results suggest the regulatory role of MEK in TIM-3 transcription by human CD4+ T cells and mast cells.

Vaccination with Lentiviral Vector Expressing the nfa1 Gene Confers a Protective Immune Response to Mice Infected with Naegleria fowleri

ABSTRACT Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Phosphatidylinositol 4-phosphate 5-kinase α is induced in ganglioside-stimulated brain astrocytes and contributes to inflammatory responses

In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kα is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kα mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kα knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1β and TNFα. PIP5Kα knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-κB and the degradation of IκB-α, indicating that PIP5Kα knockdown interfered with the ganglioside-activated NF-κB signaling. Together, these results suggest that PIP5Kα is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-κB activation in ganglioside-stimulated astrocytes.