Dynamic control of gene expression by ISGF3 and IRF1 during IFNß and IFNγ signaling.

Type I interferons (IFN-I, including IFNß) and IFNγ produce overlapping, yet clearly distinct immunological activities. Recent data show that the distinctness of global transcriptional responses to the two IFN types is not apparent when comparing their immediate effects. By analyzing nascent transcripts induced by IFN-I or IFNγ over a period of 48 h, we now show that the distinctiveness of the transcriptomes emerges over time and is based on differential employment of the ISGF3 complex as well as of the second-tier transcription factor IRF1. The distinct transcriptional properties of ISGF3 and IRF1 correspond with a largely diverse nuclear protein interactome. Mechanistically, we describe the specific input of ISGF3 and IRF1 into enhancer activation and the regulation of chromatin accessibility at interferon-stimulated genes (ISG). We further report differences between the IFN types in altering RNA polymerase II pausing at ISG 5' ends. Our data provide insight how transcriptional regulators create immunological identities of IFN-I and IFNγ.

How cancer cells make and respond to interferon-I.

Acute exposure of cancer cells to high concentrations of type I interferon (IFN-I) drives growth arrest and apoptosis, whereas chronic exposure to low concentrations provides important prosurvival advantages. Tyrosine-phosphorylated IFN-stimulated gene (ISG) factor 3 (ISGF3) drives acute deleterious responses to IFN-I, whereas unphosphorylated (U-)ISGF3, lacking tyrosine phosphorylation, drives essential constitutive prosurvival mechanisms. Surprisingly, programmed cell death-ligand 1 (PD-L1), often expressed on the surfaces of tumor cells and well recognized for its importance in inactivating cytotoxic T cells, also has important cell-intrinsic protumor activities, including dampening acute responses to cytotoxic high levels of IFN-I and sustaining the expression of the low levels that benefit tumors. More thorough understanding of the newly recognized complex roles of IFN-I in cancer may lead to the identification of novel therapeutic strategies.

Restoration of HBV-specific CD8+ T-cell responses by sequential low-dose IL-2 treatment in non-responder patients after IFN-α therapy.

Patients with chronic hepatitis B (CHB) undergoing interferon (IFN)-α-based therapies often exhibit a poor HBeAg serological response. Thus, there is an unmet need for new therapies aimed at CHB. This study comprised two clinical trials, including 130 CHB patients, who were treatment-naïve; in the first, 92 patients were systematically analyzed ex vivo for interleukin-2 receptor (IL-2R) expression and inhibitory molecules expression after receiving Peg-IFN-α-2b therapy. In our second clinical trial, 38 non-responder patients, in whom IFN-α therapy had failed, were treated with or without low-dose IL-2 for 24 weeks. We then examined the hepatitis B virus (HBV)-specific CD8+ T-cell response and the clinical outcome, in these patients. Although the majority of the participants undergoing Peg-IFN-α-2b therapy were non-responders, we observed a decrease in CD25 expression on their CD4+ T cells, suggesting that IFN-α therapy may provide a rationale for sequential IL-2 treatment without increasing regulatory T cells (Tregs). Following sequential therapy with IL-2, we demonstrated that the non-responders experienced a decrease in the numbers of Tregs and programmed cell death protein 1 (PD-1) expression. In addition, sequential IL-2 administration rescued effective immune function, involving signal transducer and activator of transcription 1 (STAT1) activation. Importantly, IL-2 therapy significantly increased the frequency and function of HBV-specific CD8+ T cells, which translated into improved clinical outcomes, including HBeAg seroconversion, among the non-responder CHB patients. Our findings suggest that sequential IL-2 therapy shows efficacy in rescuing immune function in non-responder patients with refractory CHB.

STAT1 Isoforms Differentially Regulate NK Cell Maturation and Anti-tumor Activity.

Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1ß isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1ß (Stat1ß/ß ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1ß/ß mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1ß/ß mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1ß/ß mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.

Regulation of interferon stimulated gene expression levels at homeostasis.

Interferon stimulated genes (ISGs), a collection of genes important in the early innate immune response, are upregulated in response to stimulation by extracellular type I interferons. The regulation of ISGs has been extensively studied in cells exposed to significant interferon stimulation, but less is known about ISG regulation in homeostatic regimes in which extracellular interferon levels are low. Using a collection of pre-existing, publicly available microarray datasets, we investigated ISG regulation at homeostasis in CD4, pulmonary epithelial, fibroblast and macrophage cells. We used a linear regression model to predict ISG expression levels from regulator expression levels. Our results suggest significant regulation of ISG expression at homeostasis, both through the ISGF3 molecule and through IRF7 and IRF8 associated pathways. We find that roughly 50% of ISGs have expression levels significantly correlated with ISGF3 expression levels at homeostasis, supporting previous results suggesting that homeostatic IFN levels have broad functional consequences. We find that ISG expression levels varied in their correlation with ISGF3, with epithelial and macrophage cells showing more correlation than CD4 and fibroblast cells. Our analysis provides a novel approach for decomposing and quantifying ISG regulation.

A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.

Mitochondrial DNA Stress Signalling Protects the Nuclear Genome.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that instability and cytoplasmic release of mtDNA activates the cGAS-STING-TBK1 pathway resulting in interferon stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs, that includes Parp9, remains activated by the unphosphorylated form of ISGF3 (U-ISGF3) that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS-STING-dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam +/- mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting mtDNA is a genotoxic stress sentinel.

A Positive Feedback Amplifier Circuit That Regulates Interferon (IFN)-Stimulated Gene Expression and Controls Type I and Type II IFN Responses.

Interferon (IFN)-I and IFN-II both induce IFN-stimulated gene (ISG) expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2. STAT1 homodimers, known as γ-activated factor (GAF), activate transcription in response to all types of IFNs by direct binding to IFN-II activation site (γ-activated sequence)-containing genes. Association of interferon regulatory factor (IRF) 9 with STAT1-STAT2 heterodimers [known as interferon-stimulated gene factor 3 (ISGF3)] or with STAT2 homodimers (STAT2/IRF9) in response to IFN-I, redirects these complexes to a distinct group of target genes harboring the interferon-stimulated response element (ISRE). Similarly, IRF1 regulates expression of ISGs in response to IFN-I and IFN-II by directly binding the ISRE or IRF-responsive element. In addition, evidence is accumulating for an IFN-independent and -dependent role of unphosphorylated STAT1 and STAT2, with or without IRF9, and IRF1 in basal as well as long-term ISG expression. This review provides insight into the existence of an intracellular amplifier circuit regulating ISG expression and controlling long-term cellular responsiveness to IFN-I and IFN-II. The exact timely steps that take place during IFN-activated feedback regulation and the control of ISG transcription and long-term cellular responsiveness to IFN-I and IFN-II is currently not clear. Based on existing literature and our novel data, we predict the existence of a multifaceted intracellular amplifier circuit that depends on unphosphorylated and phosphorylated ISGF3 and GAF complexes and IRF1. In a combinatorial and timely fashion, these complexes mediate prolonged ISG expression and control cellular responsiveness to IFN-I and IFN-II. This proposed intracellular amplifier circuit also provides a molecular explanation for the existing overlap between IFN-I and IFN-II activated ISG expression.

STAT1ß enhances STAT1 function by protecting STAT1α from degradation in esophageal squamous cell carcinoma.

STAT1, which carries tumor suppressor functions in several models, consists of two isoforms, namely STAT1α and STAT1ß. The biological function and significance of STAT1ß has never been examined in human cancer. We examined STAT1ß function in esophageal squamous cell carcinoma (ESCC) by transfecting a STAT1ß gene into various ESCC cell lines. The interaction between STAT1α and STAT1ß was examined by using co-immunoprecipitation and confocal microscopy. The prognostic significance of STAT1ß expression, detectable by immunohistochemistry and western blot, was evaluated in a large cohort of ESCC patients. Enforced expression of STAT1ß induced and prolonged the expression and phosphorylation of STAT1α in ESCC cells, and these effects were amplified by gamma-interferon (IFN-γ). We also found that STAT1ß interacts with STAT1α and decreases STAT1α degradation by the proteasome. Moreover, STAT1ß substantially increased the DNA binding and transcription activity of STAT1. STAT1ß also sensitized ESCC cells to chemotherapeutic agents, including cisplatin and 5-flurouracil. Using western blot and immunohistochemistry, we found that STAT1ß was frequently decreased in esophageal cancer, as compared to their adjacent benign esophageal epithelial tissue. Loss of STAT1ß significantly correlated with lymph node metastasis, invasion and shorter overall survival in ESCC patients. Therefore, STAT1ß plays a key role in enhancing the tumor suppressor function of STAT1α, in ESCC, in a manner that can be amplified by IFN-γ.

Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells.

LINE-1 (L1) retrotransposons can mobilize (retrotranspose) within the human genome, and mutagenic de novo L1 insertions can lead to human diseases, including cancers. As a result, cells are actively engaged in preventing L1 retrotransposition. This work reveals that the human Condensin II complex restricts L1 retrotransposition in both non-transformed and transformed cell lines through inhibition of L1 transcription and translation. Condensin II subunits, CAP-D3 and CAP-H2, interact with members of the Gamma-Interferon Activated Inhibitor of Translation (GAIT) complex including the glutamyl-prolyl-tRNA synthetase (EPRS), the ribosomal protein L13a, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NS1 associated protein 1 (NSAP1). GAIT has been shown to inhibit translation of mRNAs encoding inflammatory proteins in myeloid cells by preventing the binding of the translation initiation complex, in response to Interferon gamma (IFN-γ). Excitingly, our data show that Condensin II promotes complexation of GAIT subunits. Furthermore, RNA-Immunoprecipitation experiments in epithelial cells demonstrate that Condensin II and GAIT subunits associate with L1 RNA in a co-dependent manner, independent of IFN-γ. These findings suggest that cooperation between the Condensin II and GAIT complexes may facilitate a novel mechanism of L1 repression, thus contributing to the maintenance of genome stability in somatic cells.

IRF9-Stat2 Fusion Protein as an Innate Immune Inducer to Activate Mx and Interferon-Stimulated Gene Expression in Zebrafish Larvae.

Virus infection often causes large amounts of mortality during teleost larvae stage. Strong induction of innate immunity to increase survival rates of teleost larvae has been less reported. In this study, we present a zebrafish IRF9-Stat2 fusion protein (zIRF9-S2C) as a strong innate immunity inducer and characterized induction of interferon-stimulated genes (ISGs) in zebrafish larvae. zIRF9-S2C could mimic IFN-stimulated gene factor 3 (ISGF3) complex to constitutively activate transcription of Mx promoter through IFN-stimulatory element (ISRE) sites. Mutation of two ISRE sites on Mx promoter reduced transactivation activities of Mx promoter induced by zIRF9-S2C. An electrophoretic mobility shift assay experiment shows that zIRF9-S2C could directly bind to two ISRE sites of Mx promoter. Induction of transactivation of Mx promoter by zIRF9-S2C shows significantly higher activity than by zebrafish IFN1 (zIFN1), IFNγ (zIFNγ), and Tetraodon IRF9-S2C (TnIRF9-S2C). zIRF9-S2C raises transcription of Mxa, Mxb, Mxc, Ifnφ1, Ifnφ2, and Ifnφ3 in zebrafish liver ((ZFL) cell line) cells and zebrafish larvae. Collectively, we suggest that IRF9-S2C could activate transcription of ISGs with species-specific recognition and could be an innate immunity inducer in teleost larvae.

Unphosphorylated ISGF3 drives constitutive expression of interferon-stimulated genes to protect against viral infections.

Interferon (IFN)-stimulated genes (ISGs) are antiviral effectors that are induced by IFNs through the formation of a tripartite transcription factor ISGF3, which is composed of IRF9 and phosphorylated forms of STAT1 and STAT2. However, we found that IFN-independent ISG expression was detectable in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. The constitutive expression of ISGs was mediated by the unphosphorylated ISGF3 (U-ISGF3) complex, consisting of IRF9 together with unphosphorylated STAT1 and STAT2. Under homeostatic conditions, STAT1, STAT2, and IRF9 were found in the nucleus. Analysis of a chromatin immunoprecipitation sequencing data set revealed that STAT1 specifically bound to the promoters of ISGs even in the absence of IFNs. Knockdown of STAT1, STAT2, or IRF9 by RNA interference led to the decreased expression of various ISGs in Huh7.5 human liver cells, which was confirmed in mouse embryonic fibroblasts (MEFs) from STAT1-/-, STAT2-/-, or IRF9-/- mice. Furthermore, decreased ISG expression was accompanied by increased replication of hepatitis C virus and hepatitis E virus. Conversely, simultaneous overexpression of all ISGF3 components, but not any single factor, induced the expression of ISGs and inhibited viral replication; however, no phosphorylated STAT1 and STAT2 were detected. A phosphorylation-deficient STAT1 mutant was comparable to the wild-type protein in mediating the IFN-independent expression of ISGs and antiviral activity, suggesting that ISGF3 works in a phosphorylation-independent manner. These data suggest that the U-ISGF3 complex is both necessary and sufficient for constitutive ISG expression and antiviral immunity under homeostatic conditions.

Type I interferon-regulated gene expression and signaling in murine mixed glial cells lacking signal transducers and activators of transcription 1 or 2 or interferon regulatory factor 9.

Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-α-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-α was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-α exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.

Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma.

BACKGROUND: In spite of modern therapies for non-small-cell lung cancer (NSCLC), prognosis for many patients is still poor and survival rates are low. Immunotherapy is the possibility to improve the lung immune response surrounding the tumour. However, this approach requires detailed understanding of the local immune-responses of NSCLC patients. METHODS: We analysed samples from three different regions within the lungs of NSCLC patients, whereas we distinguished between patients suffering from adenocarcinoma and squamous cell carcinoma. Expression of type 1 T helper (Th1)/type 1 cytotoxic (Tc1) factors was assessed by quantitative real-time PCR, western blot analyses or immunohistochemistry. Cytotoxic cell activity of CD8(+) T cells was determined via co-culture with autologous tumour cells and apoptosis assay. RESULTS: We found decreased levels of the transcription factor T-box expressed in T cells (T-bet or Tbx21) and of the downstream activated IFN-γ-dependent pSTAT1α isoform in the lung tumour areas of patients with NSCLC as compared with tumour-free control regions. In these patients, reduced T-bet and pSTAT1α levels were found associated with increased immunosuppressive markers like cytotoxic T lymphocyte-associated protein 4, programmed cell death 1 and with a suppression of the Th1 cell cytokine production and Tc1 cell activity. CONCLUSIONS: These findings confirm a central role of T-bet in targeted immunotherapy for patients with NSCLC.

Cooperative Transcriptional Activation of Antimicrobial Genes by STAT and NF-κB Pathways by Concerted Recruitment of the Mediator Complex.

The transcriptional response to infection with the bacterium Listeria monocytogenes (Lm) requires cooperative signals of the type I interferon (IFN-I)-stimulated JAK-STAT and proinflammatory NF-κB pathways. Using ChIP-seq analysis, we define genes induced in Lm-infected macrophages through synergistic transcriptional activation by NF-κB and the IFN-I-activated transcription factor ISGF3. Using the Nos2 and IL6 genes as prime examples of this group, we show that NF-κB functions to recruit enzymes that establish histone marks of transcriptionally active genes. In addition, NF-κB regulates transcriptional elongation by employing the mediator kinase module for the recruitment of the pTEFb complex. ISGF3 has a major role in associating the core mediator with the transcription start as a prerequisite for TFIID and RNA polymerase II (Pol II) binding. Our data suggest that the functional cooperation between two major antimicrobial pathways is based on promoter priming by NF-κB and the engagement of the core mediator for Pol II binding by ISGF3.

Antiviral activity of Rheum palmatum methanol extract and chrysophanol against Japanese encephalitis virus.

Rheum palmatum, Chinese traditional herb, exhibits a great variety of anti-cancer and anti-viruses properties. This study rates antiviral activity of R. palmatum extracts and its components against Japanese encephalitis virus (JEV) in vitro. Methanol extract of R. palmatum contained higher levels of aloe emodin, chrysophanol, rhein, emodin and physcion than water extract. Methanol extract (IC50 = 15.04 µg/ml) exhibited more potent inhibitory effects on JEV plaque reduction than water extract (IC50 = 51.41 µg/ml). Meanwhile, IC50 values determined by plaque reduction assay were 15.82 µg/ml for chrysophanol and 17.39 µg/ml for aloe-emodin, respectively. Virucidal activity of agents correlated with anti-JEV activity, while virucidal IC50 values were 7.58 µg/ml for methanol extract, 17.36 µg/ml for water extract, 0.75 µg/ml for chrysophanol and 0.46 µg/ml for aloe-emodin, respectively. In addition, 10 µg/ml of extract, chrysophanol or aloe emodin caused 90 % inhibition of JEV yields in cells and significantly activated gamma activated sequence-driven promoters. Hence, methanol extract of R. palmatum and chrysophanol with high therapeutic index might be useful for development of antiviral agents against JEV.

Recent advances in understanding viral evasion of type I interferon.

The type I interferon (IFN) system mediates a wide variety of antiviral effects and represents an important first barrier to virus infection. Consequently, viruses have developed an impressive diversity of tactics to circumvent IFN responses. Evasion strategies can involve preventing initial virus detection, via the disruption of the Toll-like receptors or the retinoic acid inducible gene I (RIG-I) -like receptors, or by avoiding the initial production of the ligands recognized by these receptors. An alternative approach is to preclude IFN production by disarming or degrading the transcription factors involved in the expression of IFN, such as interferon regulatory factor 3 (IRF3)/IRF7, nuclear factor-κB (NF-κB), or ATF-2/c-jun, or by inducing a general block on host cell transcription. Viruses also oppose IFN signalling, both by disturbing the type I IFN receptor and by impeding JAK/STAT signal transduction upon IFN receptor engagement. In addition, the global expression of IFN-stimulated genes (ISGs) can be obstructed via interference with epigenetic signalling, and specific ISGs can also be selectively targeted for inhibition. Finally, some viruses disrupt IFN responses by co-opting negative regulatory systems, whereas others use antiviral mechanisms to their own advantage. Here, we review recent developments in this field.

Immunostimulatory effect of laminarin on RAW 264.7 mouse macrophages.

This study investigated the immunostimulatory effects of laminarin with respect to inflammatory mediators such as hydrogen peroxide, calcium, nitric oxide, various cytokines, transcription factors, and immune response gene in RAW 264.7 mouse macrophages. Laminarin did not reduce the cell proliferation of RAW 264.7 mouse macrophages at concentrations up to 500 µg/mL. Laminarin significantly increased the release of hydrogen peroxide, calcium, nitric oxide, monocyte chemotactic protein-1, vascular endothelial growth factor, leukemia inhibitory factor, and granulocyte-colony stimulating factor with enhancing expression of Signal Transducer and Activator of Transcription 1 (STAT1), STAT3, c-Jun, c-Fos, and cyclooxygenase-2 mRNA in RAW 264.7 cells. The results suggest that laminarin has immunostimulatory properties, strengthening immune reactions via the transcription factor pathway in macrophages.

Differential effects of LY294002 and wortmannin on inducible nitric oxide synthase expression in glomerular mesangial cells.

Nitric oxide (NO) that is produced by inducible nitric oxide synthase (iNOS) is associated with the pathophysiology of glomerulonephritis. Numerous studies have focused on the regulation of NO production by iNOS to reduce NO-mediated cytotoxicity. In the present study, we demonstrated the differential effects of two phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, on lipopolysaccharide- (LPS) and interferon (IFN)-γ-induced NO production in a glomerular mesangial cell line, MES-13 cells. At dosages without affecting cell viability of MES-13 cells, 5µM LY294002 showed a more-significant inhibitory effect on LPS/IFN-γ-induced NO production, and iNOS protein and gene expressions than did 1µM wortmannin. Akt phosphorylation in MES-13 cells declined upon the addition of wortmannin, but not upon treatment with LY294002. Suppression of PI3K expression by small interfering (si)RNA exhibited no effect on LPS/IFN-γ-stimulated NO production or iNOS protein expression in MES-13 cells. Neither LY294002 nor wortmannin reduced IFN-γ-induced STAT-1α phosphorylation. LY294002 exhibited a more-significant inhibitory effect on NF-κB luciferase activities than wortmannin in LPS/IFN-γ-stimulated MES-13 cells. Moreover, LY294002, but not wortmannin, accelerated iNOS protein degradation and reduced the iNOS dimer/monomer ratio in MES-13 cells. Although both LY294002 and wortmannin are known as PI3K inhibitors, their differential effects on iNOS expression in MES-13 cells indicate that the effects of LY294002 on inhibiting NF-κB activation and accelerating iNOS protein degradation are through a mechanism independent of PI3K.