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Alcohol could compromise the anti-hepatitis C virus (HCV) function of interferon-alpha (IFN-α). However, little information is available about the effect of alcohol on interferon-lambda (IFN-λ, type III IFN), a novel candidate for development of therapy for HCV infection. Huh7 cells were infected with HCV JFH-1 virus, then treated with alcohol, and/or IFN-λ1. RT-PCR and Western blot were used to detect the levels of HCV and key cellular factors. Overexpression or silencing expression was performed to verify the role of key factors in alcohol-attenuated anti-HCV function of IFN-λ1. Alcohol treatment compromised anti-HCV effect of IFN-λ1 in HCV JFH-1-infected Huh7 cells, evidenced by the significantly increased levels of HCV RNA, and HCV core protein in alcohol-/IFN-λ1-treated cells compared to cells with IFN-λ1 treatment alone. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol enhanced the expression of protein inhibitor of activated STAT (PIASy). Overexpression of PIASy compromised anti-HCV ability of IFN-λ1, whereas silencing expression of PIASy partly restored the alcohol-attenuated anti-HCV effect of IFN-λ1. More importantly, overexpression of PIASy significantly down-regulated the level of IFN-λ1-indcued phosphorylation of STAT1 (p-STAT1), an important adaptor in IFN-λ pathway, as well as reduced the expression of IFN-λ1-induced IFN-stimulated genes 56 (ISG56), and myxovirus resistance 1 (Mx1), two antiviral effectors in in IFN-λ pathway. These findings indicate that alcohol, through inducing the expression of negative regulator in IFN-λ pathway, inhibits IFN-λ-mediated anti-HCV action in human hepatic cells, which may lead to the poor efficacy of IFN-λ-based therapy against HCV infection.

The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres.

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development.

Originally identified as cytokine inhibitors, protein inhibitors of activated STAT (PIAS) are shown to regulate activities of a plethora of proteins and influence diverse processes such as immune response, cancer formation, and cell cycle progression. However, the roles of PIAS during vertebrate embryogenesis are less understood. In this study, we report isolation and initial characterization of all four PIAS genes from Xenopus laevis. The Xenopus PIAS genes are expressed throughout early development and have overlapping and distinct expression patterns, with, for example, high levels of PIAS2 in the notochord and strong expression of PIAS4 in the neural and neural crest derivatives. Overexpression of PIAS disrupts mesoderm induction and impairs body axis formation. PIAS proteins have differential ability to regulate signals from the growth factors activin, bone morphogenetic protein 4 (BMP4), and Wnt8. Our data suggest that Xenopus PIAS play important roles in mesodermal induction and patterning during early frog development.

The small ubiquitin-related modifier (SUMO) system of higher eukaryotes plays important roles in normal cell division, especially in chromosome segregation. However, only a few mitotic SUMO substrates have been identified in mammals. Here, we show that the mitotic kinase Aurora-B can be modified by SUMO. The E3 SUMO-protein ligase PIAS3 [protein inhibitor of activated STAT (signal transducer and activator of transcription)] dramatically enhanced poly-SUMO-2/3 conjugation of Aurora-B, whereas the SUMO-specific isopeptidase SENP2 (Sentrin/SUMO-specific protease) specifically deconjugated SUMO from Aurora-B. The Lys-202 residue on human Aurora-B was preferentially modified by SUMO, and enhancement of SUMOylation in cells facilitated Aurora-B autophosphorylation, which is essential for its activation. Conversely, SENP2-mediated deSUMOylation of Aurora-B down-regulated its autophosphorylation in cells and also impaired its re-activation in Aurora inhibitor VX-680-treated mitotic cells. Poly-SUMO-2 conjugation of Aurora-B occurred during the M phase of the cell cycle, and both SUMO-2 and PIAS3 were localized adjacent to Aurora-B in the kinetochores in early mitosis. Based on these results, we propose that Aurora-B is a novel mitotic SUMO substrate and that its kinase activity is fine-tuned by the SUMO system.

The induction of oxidative stress and inflammation has been closely linked in traumatic brain injury (TBI). Transcriptional factors of signal transducers and activators of transcription (STAT) proteins are redox sensitive and participate in the regulation of cytokine signaling. Previous studies demonstrated that melatonin protects neurons through its antioxidative and anti-inflammatory effects in various neuropathological conditions. However, the effect of melatonin on STAT activity after TBI has not yet been explored. In this study, we used a controlled weight-drop TBI model and found that brain contusion induced oxidative stress (a decreased level of total glutathione and an increased ratio of oxidized glutathione to total glutathione), a reduction in STAT1 DNA-binding activity, and consequently neuronal loss in a contusion depth-dependent manner. A significant increased mRNA expression of suppressor of cytokine signaling (SOCS3), inducible nitric oxide synthetase (iNOS), and interleukine-6 (IL-6), but a decreased protein expression of protein inhibitor of activated STAT (PIAS1), was found 24 hr after brain contusion. SOCS3 and PIAS1 are endogenous negative regulators of STAT1. Moreover, the combination of intraperitoneal and local (presoaked in gelfoam and placed on the traumatic cortex) administration of melatonin had the most pronounced influence in inhibiting all effects except the PIAS1 downregulation induced by brain contusion. The results suggest that SOCS-3 upregulation and oxidative stress may contribute to the STAT1 inactivation after TBI. Melatonin protects neurons from TBI by reducing oxidative stress, STAT1 inactivation, and upregulation of SOCS-3 and pro-inflammatory cytokines.

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is one of the main signaling pathways in eukaryotic cells. This pathway is used during diverse growth and developmental processes in multiple tissues to control cell proliferation, differentiation, survival, and apoptosis. In addition to its role during development, the JAK/STAT pathway has also been implicated in tumorigenesis. Drosophila melanogaster is a powerful genetic tool, and its eyes have been used extensively as a platform to study signaling pathways. Many reports have demonstrated that the JAK/STAT pathway plays pleiotropic roles in Drosophila eye development. Its functions and activation are decided by its interplay with other signal pathways and the epigenetic status. In this review, we focus on the functions and regulation of the JAK/STAT pathway during eye development and provide some insights into the study of this pathway in tumorigenesis. Developmental Dynamics 239:2522–2533, 2010. © 2010 Wiley-Liss, Inc.

The family of STAT proteins consists of seven members that mediate highly specific functions in cytokine signaling. STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. In this report, we show that the coacitivator p100 protein can interact with STAT6 through its SN domain both in vivo and in vitro, resulting in enhancement of STAT6-mediated gene transcriptional acitivation. Consistent with our previous reports, we identified intracellular localization of p100 and STAT-6 by confocal microscopy examined in response to IL-4. Moreover, in consideration of STAT molecules sharing significant homology in structure and function, we detected whether p100 can associate with STAT-1. In conclusion, this study found no evidence that p100 functions as a transcriptional coactivator for STAT1-dependent gene regulation. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1’s corepressor activity.Structured summaryMINT-6787569:PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with HDAC3 (uniprotkb:O15379) by anti tag coimmunoprecipitation (MI:0007)MINT-6787767:PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with SUMO-1 (uniprotkb:P63165) by anti tag coimmunoprecipitation (MI:0007)

STAT transcription factors have been implicated in many biological processes, particularly host immune defense and development. Here we characterize a STAT orthologue from the nematode, C. elegans. We show that this protein, termed STA-1, is structurally and functionally related to other vertebrate and invertebrate STAT proteins, recognizing a cis DNA element conserved through phylogeny. Unexpectedly, STA-1 lacks the conserved amino-terminal oligomerization domain found in vertebrate and other invertebrate STAT proteins, a feature also lacking in orthologues from a distantly related nematode species and from the slime mold, Dictyostelium discoideum. This absence suggests that a primordial STAT protein lacked this domain, which was accreted later in evolution to provide further regulatory control of STAT signaling. Derivation of null mutants demonstrated that STA-1 is not required for nematode viability, despite its widespread expression in multiple tissues of the worm. However, mutant STA-1 proteins that lack functional coiled-coil and DNA binding domains could still be activated and accumulated in the nucleus, suggesting that DNA binding is not a necessary prerequisite for nuclear retention of activated STAT proteins. Our results shed new light on the evolution and function of the STAT signaling pathway and on the structural requirements for STAT activation.

Cytokine signaling is essential for intercellular communication and affects cell proliferation, differentiation, and survival. In the immune system, cytokines coordinate the activities of many cell types ultimately leading to both innate and adaptive immune responses. Dysregulation of these processes can result in a wide spectrum of diseases ranging from defective host responses to invading pathogens to autoimmunity. Most cytokines signal through the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway initiated by the cytokine binding to its cell surface receptor, which leads to the activation of STAT proteins, their binding to response elements near target promoters ultimately changing the transcription of STAT-responsive genes. STAT proteins do not exist in isolation but act in concert with other transcription factors and cofactors which can either stimulate or inhibit their activity. One such factor is a ligand-dependent transcriptional regulator termed the glucocorticoid (GC) receptor (GR), which transduces the information conveyed by GC hormones and their synthetic analogs. GR is known for its anti-inflammatory and immunosuppressive properties; GC-like molecules have been used as drugs for inflammatory, autoimmune and lymphoproliferative diseases since the 1950s. In contrast, cytokines frequently promote activation of the immune system. In last several years, functional interactions have been described between virtually every member of the STAT family and GR or its cofactors. This review focuses on the recent literature on the modes and levels of interactions between these seemingly unrelated regulators and potential biological implications of STAT : GR cross-talk.

PIAS3, a member of the protein inhibitor of activated STAT family, was found to interact in vivo and in vitro with TIF2, a previously described coactivator for nuclear receptors. The interaction is mediated by two distinct non-contiguous regions of TIF2. We found that TIF2–PIAS3 interaction occurs through a unique domain of PIAS3, very rich in acidic residues and conserved throughout the PIAS family. PIAS3 modulates the ability of TIF2 to mediate ligand-enhanced transcription activation positively or negatively, for different steroid receptors. Taken together, our results indicate a potential role of PIAS3 as transcriptional modulator of TIF2-mediated signalling.

This review will discuss how STAT (Signal Transducers and Activators of Transcription) proteins, a group of transcription factors that transmit signals from the extracellular surface of cells to the nucleus, are involved in growth control. I will discuss the anatomy of a STAT protein, how it works as a transcription factor, the molecules that regulate its “activity”, the phenotypes of mice that lack individual STAT proteins and their involvement in growth, differentiation, apoptosis, and transformation. Finally, a number of examples will be presented of how dysregulated STAT signaling may be involved in the pathogenesis of cancer. BioEssays 23:161–169, 2001. © 2001 John Wiley & Sons, Inc.

Nerve injury leads to the release of a number of cytokines which have been shown to play an important role in cellular activation after peripheral nerve injury. The members of the signal transducer and activator of transcription (STAT) gene family are the main mediators in the signal transduction pathway of cytokines. After phosphorylation, STAT proteins are transported into the nucleus and exhibit transcriptional activity. Following axotomy in rat regenerating facial and hypoglossal neurons, a transient increase of mRNA for JAK2, JAK3, STAT1, STAT3 and STAT5 was detected using in situ hybridization and semi-quantitative polymerase chain reaction (PCR). Of the investigated STAT molecules, only STAT3 protein was significantly increased. In addition, activation of STAT3 by phosphorylation on position Tyr705 and enhanced nuclear translocation was found within 3 h in neurons and after 1 day in astrocytes. Unexpectedly, STAT3 tyrosine phosphorylation was obvious for more than 3 months. In contrast, none of these changes was found in response to axotomy of non-regenerating Clarke's nucleus neurons, although all the investigated models express c-Jun and growth-associated protein-43 (GAP-43) in response to axonal injury. Increased expression of Janus kinase (JAK) and STAT molecules after peripheral nerve transection suggests changes in the responsiveness of the neurons to signalling molecules. STAT3 as a transcription factor, which is expressed early and is activated persistently until the time of reinnervation, might be involved in the switch from the physiological gene expression to an 'alternative program' activated only after peripheral nerve injury.

A new insect member of the STAT family of transcription factors (Ag-STAT) has been cloned from the human malaria vector Anopheles gambiae. The domain involved in DNA interaction and the SH2 domain are well conserved. Ag-STAT is most similar to Drosophila D-STAT and to vertebrate STATs 5 and 6, constituting a proposed ancient class A of the STAT family. The mRNA is expressed at all developmental stages, and the protein is present in hemocytes, pericardial cells, midgut, skeletal muscle and fat body cells. There is no evidence of transcriptional activation following bacterial challenge. However, bacterial challenge results in nuclear translocation of Ag-STAT protein in fat body cells and induction of DNA-binding activity that recognizes a STAT target site. In vitro treatment with pervanadate (vanadate and H2O2) translocates Ag-STAT to the nucleus in midgut epithelial cells. This is the first evidence of direct participation of the STAT pathway in immune responses in insects.

Background The recently discovered Jak/STAT signal transduction pathway is associated with cytokine or growth factor receptors; whether members of the G protein-coupled receptor superfamily also activate this pathway is not yet clear. As a first member, the angiotensin (AT)1A receptor has been demonstrated to phosphorylate Jak and STAT proteins. Bombesin, a neurotransmitter and growth factor in many cells and tissues, activates its G protein-coupled receptor and in addition phosphorylates proteins that might be members of the Jak/STAT family. This study investigated whether bombesin- or angiotensin-mediated growth effects are associated with STAT protein activation. Methods Functional receptors were characterized using ligand-binding studies, second-messenger activation and determination of ligand-mediated growth effects. STAT protein activation was analysed by electrophoretic mobility shift assay (EMSA) using labelled DNA response elements recognizing all known STAT proteins. Results Functional bombesin receptors mediating mitogenic effects were demonstrated on Swiss 3T3 fibroblasts, human melanoma cells (A375-6) and primary human lung fibroblasts; however, bombesin-related STAT protein activation was not observed by EMSA. Interferon-α typically activated a STAT1-STAT2-p48 heterotrimer, as well as STAT1-3 hetero- and homodimers in human melanoma cells and significantly inhibited growth of this cell line in vitro. Functional AT1A receptors on primary rat cardiac fibroblasts mediated angiotensin-stimulated growth effects but, in contrast to recently published data, did not activate any known STAT protein. Conclusion Interferon α-stimulated growth inhibition is mediated by activation of the Jak/STAT pathway, whereas bombesin or AT1A receptor-mediated effects on cellular proliferation do not involve phosphorylation of STAT proteins.

The JAK-STAT pathway is important in cytokine-mediated immune responses. Research in the JAK/STAT field has elucidated its roles in various cellular processes such as proliferation, apoptosis and migration, and has found frequent dysregulation of the JAK/STAT pathway in diverse types of cancer. In recent years, JAK and STAT have also been implicated in a noncanonical mode of action: the regulation of genomic stability independent of their functions in cytokine signalling. Research in Drosophila has shown that nuclear, unphosphorylated STAT physically interacts with heterochromatin protein 1 (HP1) and stabilises its binding to heterochromatin. A similar interaction occurs in human cells, where unphosphorylated STAT5A interacts with HP1α and acts as a tumour suppressor. Nuclear JAK2, however, functions as a histone tyrosine kinase, displacing HP1α from chromatin. These data have important implications for human cancer: They suggest new drug therapies, which could target the noncanonical functions of JAK and STAT. In this article, we discuss how the canonical arm of the JAK-STAT pathway functions in tumourigenesis, focussing on JAK and on STAT1, STAT3 and STAT5. We then discuss recent findings regarding epigenetic silencing of JAK/STAT pathway components, and the direct involvement of JAK and STAT in regulating heterochromatin integrity. Key Concepts: The tyrosine kinase JAK and its downstream target STAT respond to cytokine signalling in cells. In response to cytokines, JAK itself is phosphorylated, leading to its activation. The activated JAK kinase then phosphorylates specific STATs. STAT proteins dimerise and translocate into the nucleus upon phosphorylation by JAK, where they bind to DNA and regulate transcription. Overactivation of the JAK-STAT pathway can cause cancer by bypassing apoptosis and cell cycle checkpoints. Unphosphorylated STAT is also found in the nuclei and mitochondria of cells that are not stimulated by cytokines. Mitochondrial STAT upregulates cellular respiration and can promote oncogenic transformation. Unphosphorylated nuclear STAT binds to HP1α and stabilises heterochromatin. STAT5A in colon cancer cells acts as a tumour suppressor via this mechanism. Nuclear JAK2 is a histone tyrosine kinase. Phosphorylation of histone 3 tyrosine 41 displaces HP1α/CBX5 from chromatin and contributes to tumourigenicity. In some cancers, DNA methylation suppresses the expression of inhibitory SOCS proteins, resulting in uncontrolled JAK/STAT pathway activation. Keywords: JAK; STAT; cancer; epigenetic; heterochromatin; metastasis; angiogenesis; HP1; CpG methylation

The activation of Janus protein tyrosine kinases (Jak) and STAT (signal transducer and activator of transcription) proteins has recently been linked to the signal transduction mechanism of several cytokines. IL-7 was observed to induce a rapid and dose-dependent tyrosine phosphorylation of Jak 1 and Jak 3 and concomitantly, the tyrosine phosphorylation and DNA binding activity of multiple STAT proteins. The STAT proteins utilized by IL-7 were identical to those induced by IL-2 and could be identified as various STAT 5 isoforms. Moreover, the induction of both Jak 1 and 3, and STAT 5 activity strongly correlated with the growth-promoting effects of IL-7, suggesting that this signal transduction mechanism may play a key role in IL-7-induced proliferation.

The Janus Kinase (JAK)/signal transducer and achivator of transcription (STAT) pathway is activated not only by various cytokines such as interferon and interleukin, but also by many growth factors. This pathway plays a crucial role in the signal transduction of these factors. Keywords: Cytokine; JAK; STAT; SOCS; PIAS; PTP