Signal Transducers And Activators Of Transcription 6 Research Articles

Therapeutic action of Kushen recipe extractive and its inhibitory effect on eotaxin in mouse models with contact dermatitis.

Treatment of skin allergic diseases remains a challenging research topic. To investigate the effect of Kushen recipe extractive (KS) gel on contact dermatitis (CD) of mouse. Allergic contact dermatitis (ACD) model of mouse was established. Immunohistochemical method (ICH) and flow cytometry method (FCM) were used to detect CD4+ and CD8+ T lymphocytes and explore the regulation effect of KS on the immune status of the organism. The expression status of eotaxin tissue was evaluated by real-time polymerase chain reaction (RT-PCR), ICH, and western blotting method. The survival rates of HaCaT cell and Fibroblasts affected by KS were detected by methyl thiazolyl tetrazolium (MTT) method. The inhibitory effect of KS on eotaxin produced by HaCaT cell and FBs induced by TNF-α and interleukin (IL)-4 were evaluated using RT-PCR and enzyme-linked immunosorbent assay methods. The inhibitory effect of KS on nuclear factor-κB (NF-κB) and Signal transducers and activators of transcription 6 (STAT6) activation induced by TNF-α and IL-4 was detected by electrophoretic mobility shift assay and western blotting methods. We confirmed that KS shows favorable therapeutic effect on CD, which can obviously inhibit eotaxin expression and Eosinophils recruitment in allergic skin of mouse, as well as regulate the immune status of the organism. Furthermore, KS and its main effective components can inhibit TNF-α and IL-4 induced upregulation of eotaxin via the two signal transduction pathways, NF-κB and STAT6. The great importance of traditional Chinese recipe KS is evidenced by its therapeutic effect and mechanism in ACD of mouse.

The downstream network of STAT6 in promoting vascular smooth muscle cell phenotypic switch and neointimal formation.

Intimal thickening caused by the excessive multiplication of vascular smooth muscle cells (VSMCs) is the pathological process central to cardiovascular diseases, including restenosis. In response to vascular injury, VSMCs would undergo phenotypic switching from a fully differentiated, low proliferative rate phenotype to a more pro-proliferative, promigratory, and incompletely-differentiated state. The lack of a full understanding of the molecular pathways coupling the vascular injury stimuli to VSMCs phenotype switching largely limits the development of medical therapies for treating intima hyperplasia-related diseases. The role of signal transducers and activators of transcription 6 (STAT6) in modulating the proliferation and differentiation of various cell types, especially macrophage, has been well investigated, but little is known about its pathophysiological role and target genes in restenosis after vascular injury. In the present work, Stat6-/- mice were observed to exhibit less severe intimal hyperplasia compared with Stat6+/+ mice after carotid injury. The expression of STAT6 was upregulated in VSMCs located in the injured vascular walls. STAT6 deletion leads to decreased proliferation and migration of VSMCs while STAT6 overexpression enhances the proliferation and migration of VSMCs companies with reduced expression of VSMCs marker genes and organized stress fibers. The effect of STAT6 in mouse VSMCs was conserved in human aortic SMCs. RNA-deep-sequencing and experiments verification revealed LncRNA C7orf69/LOC100996318-miR-370-3p/FOXO1-ER stress signaling as the downstream network mediating the pro-dedifferentiation effect of STAT6 in VSMCs. These findings broaden our understanding of vascular pathological molecules and throw a beam of light on the therapy of a variety of proliferative vascular diseases.

STAT6 suppression prevents bleomycin-induced dermal fibrosis.

Fibrosis of the skin and internal organs is a hallmark of systemic sclerosis (SSc). Although the pathogenesis of SSc is poorly understood, increasing evidence suggests that interleukins (IL)-4 and - 13 contribute to the pathogenesis of skin fibrosis by promoting collagen production and myofibroblast differentiation. Signal transducers and activators of transcription 6 (STAT6) is one of the most important downstream transcription factors activated by both IL-4 and IL-13. However, it is not completely understood whether STAT6 plays a role during the pathogenesis of skin fibrosis in SSc. In this study, we observed increased STAT6 phosphorylation in fibrotic skin samples collected from SSc patients as well as bleomycin-injected murine mice. Knockout of Stat6 in mice significantly (1) suppressed the expression of fibrotic cytokines including Il13, Il17, Il22, Ccl2, and the alternatively activated macrophage marker Cd206; (2) reduced the production of collagen and fibronectin, and (3) attenuated late-stage skin fibrosis and inflammation induced by bleomycin. Consistently, mice treated with STAT6 inhibitor AS1517499 also attenuated skin fibrosis on day 28. In addition, a co-culture experiment demonstrated that skin epithelial cells with STAT6 knockdown had reduced cytokine expression in response to IL-4/IL-13, and subsequently attenuated fibrotic protein expression in skin fibroblasts. On the other side, STAT6 depletion in skin fibroblasts attenuated IL-4/IL-13-induced cytokine and fibrotic marker expression, and reduced CXCL2 expression in co-cultured keratinocytes. In summary, our study highlighted an important yet not fully understood role of STAT6 in skin fibrosis by driving innate inflammation and differentiation of alternatively activated macrophages in response to injury.

Brucella induces M1 to M2 polarization of macrophages through STAT6 signaling pathway to promote bacterial intracellular survival

Brucella are serious intracellular pathogens that parasitize macrophages and cause persistent infection in humans and animals. Although macrophages are an important bridge between natural and acquired immunity, their role in Brucella infection is not completely clear. Recently, studies have reported that Brucella can induce macrophage polarization, although the specific molecular mechanism involved is not known. Therefore, in the current study the replication ability of Brucella melitensis strain M5 (Brucella M5) was examined as well as its macrophage polarization and cytokine production, in a host. The role of Signal transducers and activators of transcription 6 (STAT6) in macrophage polarization induced by Brucella infection was also investigated. The results showed that Brucella M5 survived in vivo for a prolonged period of time and caused damage to the spleen and uterus tissues. The expression of type M2 cytokines was induced after Brucella M5 infection. Immunohistochemistry showed that STAT6 was upregulated in spleen and uterus tissues. At the cellular level, Brucella M5 induced macrophagetransformation from M1 to M2-type during the later stage of infection. When STAT6 was silenced, the polarization of M2-type was inhibited, and the intracellular survival rate of Brucella decreased significantly. In conclusion, these findings demonstrate that STAT6 is the key factor regulates M2 polarization of macrophages and promotes the intracellular survival of Brucella in the late stage of infection and provides an explanation of the mechanism responsible for persistent Brucella infection.

Tespa1 plays a role in the modulation of airway hyperreactivity through the IL-4/STAT6 pathway

BackgroundThymocyte-expressed, positive selection-associated 1 (Tespa1) is a critical signaling molecule in thymocyte development. This study aimed to investigate the regulatory effect of Tespa1 on mast cells in the pathogenesis of asthma and its relationship with the interleukin (IL)-4/signal transducers and activators of transcription 6 (STAT6) signaling pathway.MethodsTespa1 mRNA expression analysis and IgE levels were carried out using the induced sputum of 33 adults with stable asthma and 36 healthy controls. Tespa1-knockout mice (Tespa1−/−, KO) and C57BL/6 background (wild-type, WT) mice were sensitized and treated with ovalbumin (OVA) to establish an asthma model. Pathological changes, number and activity of mast cells, and changes in activation of the IL-4/STAT6 pathway in lung tissue were detected. The changes of tryptase expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The changes of enzyme expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The association between the Tespa1 and p-STAT6 was analyzed by co-immunoprecipitation method.ResultsCompared with the healthy controls, Tespa1 expression was decreased, and IgE levels were elevated in the sputum of asthmatic patients. Animal experiments showed that Tespa1−/− mice exhibited more severe inflammation, higher quantity of goblet cells and mast cells in the bronchium, and greater expression of mast cell tryptase, which is induced by ovalbumin, than WT mice. And IL-4, IL-13, phospho-Janus kinase 1, and p-STAT6 expressions presented a higher increase in the Tespa1−/− mouse model than in the WT mouse model. Further in vitro studies confirmed that IL-4 could more significantly promote tryptase and p-STAT6 activities in Tespa1−/− mast cells than their WT counterparts. Correlation analysis results showed a negative correlation between Tespa1 and p-STAT6. Co-immunoprecipitation results demonstrated an association between Tespa1 and p-STAT6.ConclusionsAltogether, our results indicate that Tespa1 can negatively regulate mast cell activity, and this event is related to the mast cell IL-4/STAT6 signaling pathway and could be therapeutically exploited to treat asthma attacks.

The roles of post-translational modifications and coactivators of STAT6 signaling in tumor growth and progression.

Signal transducers and activators of transcription 6 (STAT6) arehighly expressed in various tumors and associated with tumorigenesis, immunosuppression, proliferation, metastasisand poor prognosis in human cancers. In response to IL-4/13, STAT6 is phosphorylated, dimerizesand triggers transcriptional regulation after recruitment of coactivators to transcriptosome, such as CBP/p300, SRC-1, PARP-14 and PSF. Post-translational modifications, including phosphorylation, ubiquitination, ADP-ribosylation and acetylation, have been explored for molecular mechanisms of STAT6 in tumor development and management. STAT6 has been developed as a specific biomarker for distinguishing and diagnosing tumor phenotypes, although it is observed to be frequently mutated in metastatic tumors. In this article, we focus mainly on the structural characteristics ofSTAT6 and its rolein tumor growth and progression.

CD137 signaling induces macrophage M2 polarization in atherosclerosis through STAT6/PPARδ pathway

CD137 signaling plays an important role in the formation and development of atherosclerotic plaques. The purpose of the present study was to investigate the effects of CD137 signaling on macrophage polarization during atherosclerosis and to explore the underlying mechanisms. The effect of CD137 signaling on macrophage phenotype in atherosclerotic plaques was determined by intraperitoneal injection of agonist-CD137 recombinant protein in apolipoprotein E–deficient (ApoE−/−) mice, an established in vivo model of atherosclerosis. Murine peritoneal macrophages and RAW 264.7 cells were treated with AS1517499 and siPPARδ (peroxisome proliferator-activated receptor δ) to study the role of STAT6 (signal transducers and activators of transcription 6)/PPARδ signaling in CD137-induced M2 macrophage polarization in vitro. Results from both in vivo and in vitro experiments showed that CD137 signaling can transform macrophages into the M2 phenotype during the process of atherosclerotic plaque formation and regulate the angiogenic features of M2 macrophages. Furthermore, activation of the CD137 signaling pathway induces phosphorylation of STAT6 and enhances the expression of PPARδ. We further found that macrophage M2 polarization is reduced when the STAT6/PPARδ pathway is inhibited. Together, these data show a role for the STAT6/PPARδ signaling pathway in the CD137 signaling-induced M2 macrophage polarization pathway.

TNF-α mediated MEK-ERK signaling in invasion with putative network involving NF-κB and STAT-6: a new perspective in glioma.

Glioblastoma is the most common malignant primary brain tumor with poor prognosis. Invasion involves pro-inflammatory cytokines and major signaling hubs. Tumor necrosis factor-α (TNF-α) acts as a master switch in establishing an intricate link between inflammation and cancer. The present study attempted to explore the possible implication of MAPK extracellular signaling-regulated kinase kinase (MEK)-extracellular signaling-regulated kinase (ERK) signaling pathway and expression of nuclear factor-κB (NF-κB), signal transducers and activators of transcription-6 (STAT-6), ERK, and phosphorylated-ERK (p-ERK) signaling proteins in TNF-α microenvironment. U0126 and PD98059 were used to inhibit the MEK-ERK1/2 pathway. TNF-α stimulation enhanced invasion in U87MG, U251MG and patient-derived primary glioma cells, whereas cell viability was not altered. Matrix metalloproteinase-2 (MMP-2) activity was increased only in U251MG glioma cells. These data suggest that TNF-α microenvironment plays an important role in the invasion of U251MG, U87MG, and patient-derived primary glioma cells, without any cytotoxic effect. The MMP-2 activity is differentially regulated by TNF-α stimulation in these cells. TNF-α stimulation upregulated the protein expression of ERK-1, ERK-2 and also increased the level of p-ERK1/2. TNF-α stimulation further upregulated the expression of NF-κB1, STAT-6 in tandem with Ras-MEK signaling system in U87MG cells, which emphasized the possible involvement of these signaling hubs in the glioma microenvironment. MEK-ERK inhibitors significantly attenuated the invasion of U87MG cells mediated by the TNF-α stimulation, probably through their inhibitory impact on p-ERK1/2 and ERK-2. This study provides the possible rationale of invasion by glioma cells in a TNF-α-induced pro-inflammatory milieu, which involves direct role of MEK-ERK signaling, with possible implication of NF-κB and STAT-6.

Dietary Nondigestible Polysaccharides Ameliorate Colitis by Improving Gut Microbiota and CD4+ Differentiation, as Well as Facilitating M2 Macrophage Polarization.

The aim of this study was to investigate the therapeutic mechanism of a specific multifiber mix diet (MF) designed to match the fiber content of a healthy diet in interleukin-10 knockout (IL-10-/- ) mice with spontaneous chronic colitis displaying similar characteristics to those of human Crohn's disease (CD). Sixteen-week-old IL-10-/- mice were used for the experiments with MF diet for 4 weeks. Severity of colitis, the composition of the fecal microbiota, expression of Th1/Th17 cells, myeloperoxidase (MPO) concentrations, and inflammatory cytokines and chemokines (tumor necrosis factor-α [TNF-α], IL-6, macrophage inflammatory protein [MIP]-2, monocyte chemoattractant protein-1 [MCP-1], and MIP-1α), as well as arginase 1 (Arg1) and signal transducers and activators of transcription 6 (STAT6) proteins, were measured at the end of the experiment. In addition, the corresponding metabolites (short-chain fatty acids) of MF on CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) were also detected in vivo and in vitro. MF treatment significantly ameliorated colitis associated with decreased lamina propria frequency of Th1/Th17 cells, MPO concentrations, and inflammatory cytokines and chemokines (TNF-α, IL-6, MIP-2, MCP-1, and MIP-1α). An increase in gut microbial diversity was observed after MF treatment compared with IL-10-/- mice, including a significant increase in bacteria belonging to the Firmicutes phylum and a significant decrease in bacteria belonging to the Proteobacteria phylum. Moreover, MF treatment increased the differentiation of CD4+ CD25+ Foxp3+ Tregs mainly by microbial metabolites butyrate. In addition, Arg1 and STAT6 proteins were also significantly increased after MF treatment. These results shed light on the contribution of MF treatment to the CD mouse model and suggest that MF has potential as a therapeutic strategy for enhancing efficacy in inducing remission in patients with active CD.

Clarithromycin attenuates IL-13\u2013induced periostin production in human lung fibroblasts

BackgroundPeriostin is a biomarker indicating the presence of type 2 inflammation and submucosal fibrosis; serum periostin levels have been associated with asthma severity. Macrolides have immunomodulatory effects and are considered a potential therapy for patients with severe asthma. Therefore, we investigated whether macrolides can also modulate pulmonary periostin production.MethodsUsing quantitative PCR and ELISA, we measured periostin production in human lung fibroblasts stimulated by interleukin-13 (IL-13) in the presence of two 14-member–ring macrolides—clarithromycin or erythromycin—or a 16-member–ring macrolide, josamycin. Phosphorylation of signal transducers and activators of transcription 6 (STAT6), downstream of IL-13 signaling, was evaluated by Western blotting. Changes in global gene expression profile induced by IL-13 and/or clarithromycin were assessed by DNA microarray analysis.ResultsClarithromycin and erythromycin, but not josamycin, inhibited IL-13–stimulated periostin production. The inhibitory effects of clarithromycin were stronger than those of erythromycin. Clarithromycin significantly attenuated STAT6 phosphorylation induced by IL-13. Global gene expression analyses demonstrated that IL-13 increased mRNA expression of 454 genes more than 4-fold, while decreasing its expression in 390 of these genes (85.9%), mainly “extracellular,” “plasma membrane,” or “defense response” genes. On the other hand, clarithromycin suppressed 9.8% of the genes in the absence of IL-13. Clarithromycin primarily attenuated the gene expression of extracellular matrix protein, including periostin, especially after IL-13.ConclusionsClarithromycin suppressed IL-13–induced periostin production in human lung fibroblasts, in part by inhibiting STAT6 phosphorylation. This suggests a novel mechanism of the immunomodulatory effect of clarithromycin in asthmatic airway inflammation and fibrosis.

Abstract TP344: Stat6 Signaling in Microglia/macrophage is Critical for Functional Recovery After Intracerebral Hemorrhage

Objective: Intracerebral hemorrhage (ICH) is a devastating medical condition that remains a leading cause of death and long term disability in the US. Hematoma triggers a cascade of signaling events resulting in secondary brain injury and severe neurological deficits. Microglia and macrophage are activated within minutes after ICH, which may influence stroke outcome by clearing the hematoma and regulating neuro-inflammation. However, the molecular mechanism underlying the functional regulation of microglia and macrophages in ICH is poorly understood. The current study was designed to test the role of signal transducers and activators of transcription 6 (STAT6) in microglia/macrophage functions in a mouse model of ICH. Methods: Male adult WT and STAT6 KO mice were subjected to intrastriatal collagenase (0.075U) injection. Spatial and temporal profiles of STAT6 activation after ICH were determined by double-label immunostaining of phospho-STAT6 and cell markers. Neurobehavioral performances were assessed using rotarod, foot fault and water maze tests up to day 21 after ICH. Gray and white matter injury was examined quantitatively on sections stained with antibodies against MAP2/NeuN and MBP, respectively. Microglial phagocytosis of fluorescent labeled-red blood cells was evaluated in vitro by immunofluorescence and flow cytometry. Results: Robustly enhanced expression of phospho-STAT6 was found predominantly in Iba1 + cells, starting at day 1, peaking at day 5, and lasting for at least 14 d after ICH. STAT6 KO mice exhibited exacerbated sensorimotor and spatial learning and memory deficits compared to WT mice ( P <0.05). STAT6 KO mice also showed larger neuronal tissue loss and more severe white matter injury ( P <0.01). Hematoma resolving was slower in STAT6 KO mice than WT mice ( P =0.03). In cultures, primary microglia derived from STAT6 KO mice had 52.6% reduction of phagocytic activity compared to WT microglia ( P =0.006). Conclusions: STAT6 is activated in microglia/macrophage after ICH and plays a critical role in ameliorating gray and white matter injury and facilitating post-stroke neurological recovery. STAT6-afforded neuroprotection against ICH may be attributable, at least in part, to the enhanced hematoma clearance by phagocytes.

TSLP Induces Mast Cell Development and Aggravates Allergic Reactions through the Activation of MDM2 and STAT6

Thymic stromal lymphopoietin (TSLP) is known to promote T helper type 2 cell-associated inflammation. Mast cells are major effector cells in allergic inflammatory responses. We noted that the population and maturation of mast cells were reduced in TSLP-deficient mice (TSLP-/-). Thus, we hypothesized that TSLP might affect mast cell development. We found that TSLP induced the proliferation and differentiation of mast cells from bone marrow progenitors. TSLP-induced mast cell proliferation was abolished by depletion of mouse double minute 2 (MDM2) and signal transducers and activators of transcription 6 (STAT6), as an upstream activator of MDM2. TSLP-/-, in particular, had a considerable deficit in the expression of MDM2 and STAT6. Also, the TSLP deficiency attenuated mast cell-mediated allergic reactions through the downregulation of STAT6 and MDM2. In an antibody microarray chip analysis, MDM2 expression was increased in atopic dermatitis patients. These observations indicate that TSLP is a factor for mast cell development, and that it aggravates mast cell-mediated immune responses.

Regulation of recombinant Trichinella spiralis 53-kDa protein (rTsP53) on alternatively activated macrophages via STAT6 but not IL-4Rα in vitro

Classically activated macrophages (M1) or alternatively activated macrophages (M2) have different functions during helminth infections including Trichinella spiralis (T. spiralis). The excretory/secretory antigens (ESA) of T. spiralis can inhibit macrophage pro-inflammatory cytokines production. However, the specific molecules of ESA that regulate macrophages have not been identified. We previously reported that recombinant T. spiralis derived molecule 53-kDa protein (rTsP53) had protected mice from colitis. Furthermore, in the present study in vitro, we investigated rTsP53 showed anti-inflammatory function by inducing peritoneal macrophages to M2 with expressing M2 molecules of mannose receptor (MR), a novel mammalian lectin (Ym1), arginase-1 (Arg1), and interleukin (IL)-10. Next, we found the effect of rTsP53 on M2 independently of IL-4Rα. But rTsP53 can act dependently on signal transducers and activators of transcription 6 (STAT6). These results further imply that rTsP53 has potential as prospective immuno-therapeutics for inflammatory disorders.

A synthetic compound, 4-acetyl-3-methyl-6-(3,4,5-trimethoxyphenyl)pyrano[3,4-c]pyran-1,8-dione, ameliorates ovalbumin-induced asthma

Eosinophilia is one of the characteristic signs of allergic inflammation. Massive migration of eosinophils to the airways can cause epithelial tissue injury, contraction of airway smooth muscle and increased bronchial responsiveness. Previously, we discovered a new compound, 1H,8H-pyrano[3,4-c]pyran-1,8-dione (PPY), derived from the fruit of Vitex rotundifolia L. and evaluated its anti-inflammatory and anti-asthmatic properties. In this study, we synthesized a new modified compound, 4-acetyl-3-methyl-6-(3,4,5-trimethoxyphenyl) pyrano[3,4-c]pyran-1,8-dione (PPY-345), which was based on the PPY skeleton, and we evaluated its anti-asthmatic effects. To evaluate the anti-asthmatic effect of PPY-345 in vitro, A549 lung epithelial cells were stimulated with TNF-alpha, IL-4 and IL-1-beta to induce the expression of CCL11 (Eotaxin), a chemokine involved in eosinophil chemotaxis. To characterize the anti-asthmatic properties of PPY-345 in vivo, we examined the influence of PPY-345 in an ovalbumin (OVA)-induced asthma model. PPY-345 treatments significantly reduced CCL11 secretion. PPY-345 treatment did not inhibit the translocation of NF-κB into the nucleus but suppressed the phosphorylation of signal transducers and activators of transcription 6 (STAT6). PPY-345 treatment significantly reduced airway hyperreactivity as measured by whole-body plethysmography. PPY-345 further reduced total cells, including eosinophil, macrophage and lymphocytes, in the BAL fluid, goblet cell hyperplasia and myosin light chain 2 positive smooth muscle cell area in the lung tissue. Additionally, PPY-345 significantly suppressed the levels of OVA–IgE present in the serum. These results suggested that PPY-345 could improve asthma symptoms in OVA-sensitized mice.

A Decoy Oligodeoxynucleotides therapy for allergic skin diseases

Atopic dermatitis (AD) is a common, chronically relapsing skin disease. It is very difficult to treat severe type AD by steroid ointment. We therefore hypothesized that synthetic double-stranded DNA with a high affinity for Signal Transducers and Activators of Transcription 6 (STAT6) could be introduced in vivo as a decoy cis elements to bind the transcriptional factor and to block the gene activation of contributing the onset and progression of AD, thus providing effective therapy for AD. Treatment by the transfection of STAT6 decoy oligodeoxynucleotides (ODN), but not scrambled decoy ODN in the AD model mice, had a significantly inhibitory effect on not only STAT6 binding to nuclei but also on the third phase response. A histological analysis revealed that both edema and the infiltration of eosinophils and degranulated mast cells significantly decreased in STAT6 decoy ODN transfected mice. We herein report the first successful in vivo transfer of STAT6 decoy ODN to reduce the third phase reaction in AD model mice, thereby providing a new therapeutic strategy for atopic dermatitis. We also introduce another NF-kB Decoy Oligodeoxynucleotides therapy or STAT6siRNA therapy for allergic diseases.

Kaposi's Sarcoma-Associated Herpesvirus Inhibits Interleukin-4-Mediated STAT6 Phosphorylation To Regulate Apoptosis and Maintain Latency

Cytokine-mediated JAK/STAT signaling controls numerous important biologic responses like immune function, cellular growth, and differentiation. Inappropriate activation of this signaling pathway is associated with a range of malignancies. Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious viral agent associated with Kaposi's sarcoma and may also contribute to B-cell disorders, which include primary effusion lymphoma (PEL) and multicentric Castleman's disease. However, regulation of cytokine-mediated lymphocytic immune response by KSHV is not fully understood. In this report, we demonstrate that KSHV suppresses the interleukin-4 (IL-4)-stimulated immune response of B-lymphocyte activation and cell proliferation. Moreover, we show that the latency-associated nuclear antigen (LANA) encoded by KSHV is essential for viral blocking of IL-4-induced signaling. LANA reduces phosphorylation of the signal transducers and activators of transcription 6 (STAT6) on Y-641 and concomitantly its DNA binding ability. Importantly, knockdown of endogenous STAT6 dramatically increases the sensitivity of PEL cells to low-serum stress or chemical-mediated cellular apoptosis and reactivation of KSHV from latent replication. Thus, these findings suggest that the IL-4/STAT6 signaling network is precisely controlled by KSHV for survival, maintenance of latency, and suppression of the host cytokine immune response of the virus-infected cells.

Transforming growth factor‐β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor‐κB in airway smooth muscle cells

Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling. We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4. These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

Novel 7 H-pyrrolo[2,3- d]pyrimidine derivatives as potent and orally active STAT6 inhibitors

Signal transducers and activators of transcription 6 (STAT6) is an important transcription factor in interleukin (IL)-4 signaling pathway and a key regulator of the type 2 helper T (Th2) cell immune response. Therefore, STAT6 may be an excellent therapeutic target for allergic conditions, including asthma and atopic diseases. Previously, we reported 4-aminopyrimidine-5-carboxamide derivatives as STAT6 inhibitors. To search for novel STAT6 inhibitors, we synthesized fused bicyclic pyrimidine derivatives and identified a 7 H-pyrrolo[2,3- d]pyrimidine derivative as a STAT6 inhibitor. Optimization of the pyrrolopyrimidine derivatives led to identification of 2-[4-(4-{[7-(3,5-difluorobenzyl)-7 H-pyrrolo[2,3- d]pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetamide ( 24, AS1810722) which showed potent STAT6 inhibition and a good CYP3A4 inhibition profile. Compound 24 also inhibited in vitro Th2 differentiation without affecting type 1 helper T (Th1) cell differentiation and eosinophil infiltration in an antigen-induced mouse asthmatic model after oral administration.

Alcohol Primes the Airway for Increased Interleukin‐13 Signaling

Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol-mediated amplification of airway fibrosis is increased transforming growth factor beta-1(TGFbeta(1)) and alpha-smooth muscle actin expression. Other studies have shown that interleukin-13 (IL-13) modulates TGFbeta(1) signaling during experimentally-induced airway fibrosis. Therefore, we hypothesized that IL-13 is a component of alcohol-mediated amplification of pro-fibrotic mediators in the alcoholic lung. To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control- or alcohol-fed rats, alcohol-treated mouse lung fibroblasts, and human bronchial epithelial cells in vitro for expression of various components of the IL-13 signaling pathway. Signaling via the IL-13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription-6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control-fed and alcohol-fed donor rats and analyzed tracheal allografts for expression of components of the IL-13 signaling pathway by RT-PCR and immunocytochemical analyses. Interleukin-13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL-13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL-13 signaling receptor (IL-13R alpha(1)) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL-13 decoy receptor (IL-13R alpha(2)) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL-13 and lipopolysaccharide. Data from multiple cell types in the pulmonary system suggest that IL-13 and its receptors play a role in alcohol-mediated activation of pro-fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL-13 signaling and subsequent tissue remodeling upon injury such as transplantation.

Identification of 4-benzylamino-2-[(4-morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide derivatives as potent and orally bioavailable STAT6 inhibitors

Signal transducers and activators of transcription 6 (STAT6) is a key regulator of the type 2 helper T (Th2) cell immune response and a potential therapeutic target for allergic diseases such as asthma and atopic diseases. To search for potent and orally bioavailable STAT6 inhibitors, we synthesized a series of 4-benzylaminopyrimidine-5-carboxamide derivatives and evaluated their STAT6 inhibitory activities. Among these compounds, 2-[(4-morpholin-4-ylphenyl)amino]-4-[(2,3,6-trifluorobenzyl)amino]pyrimidine-5-carboxamide ( 25y, YM-341619, AS1617612) showed potent STAT6 inhibition with an IC 50 of 0.70 nM, and also inhibited Th2 differentiation in mouse spleen T cells induced by interleukin (IL)-4 with an IC 50 of 0.28 nM without affecting type 1 helper T (Th1) cell differentiation induced by IL-12. In addition, compound 25y showed an oral bioavailability of 25% in mouse.