STAT3-responsive Genes Research Articles

Increased IL-6 receptor expression and signaling in ageing cartilage can be explained by loss of TGF-β-mediated IL-6 receptor suppression

Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-β (TGF-β) signaling in cartilage. Recently, we showed that TGF-β suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-β signaling results in increased IL-6R expression and signaling in ageing cartilage. Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-β1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR. Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-β to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-β-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed. Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-β signaling, resulting in loss of TGF-β-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.

Interleukin-6 requires JAK to stimulate inner cell mass expansion in bovine embryos.

Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.

TGF-β dampens IL-6 signaling in articular chondrocytes by decreasing IL-6 receptor expression

Transforming growth factor-β (TGF-β) is an important homeostatic regulator of cartilage. In contrast, interleukin-6 (IL-6) is a pro-inflammatory cytokine implicated in cartilage degeneration. Cross-talk between TGF-β and IL-6 is reported in tissues other than articular cartilage. Here, we investigated regulation of IL-6 signaling by TGF-β in articular chondrocytes. Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with TGF-β1 or interleukin-1β (IL-1β). Expression of IL-6 and IL-6 receptor (IL-6R) was determined on mRNA and protein level. TGF-β regulation of IL-6 signaling via phosho-STAT3 (p-STAT3) was determined using Western blot, in presence of inhibitors for IL-6R, and Janus kinase(JAK)- and activin receptor-like kinase ALK)5 kinase activity. Furthermore, induction of STAT3-responsive genes was used as a read-out for IL-6 induced gene expression. TGF-β1 increased IL-6 mRNA and protein expression in both G6 and primary chondrocytes. Moreover, TGF-β1 stimulation clearly induced p-STAT3), which was abolished by inhibition of either IL-6R, JAK- or ALK5 kinase activity. However, TGF-β1 did not increase expression of the STAT3-responsive gene SOCS3 and pre-treatment with TGF-β1 even inhibited induction of p-STAT3 and SOCS3 by rhIL-6. Interestingly, TGF-β1 potently decreased IL-6R expression. In contrast, IL-1β did increase IL-6 levels, but did not affect IL-6R expression. Finally, addition of recombinant IL-6R abolished the inhibitory effect of TGF-β1 on IL-6-induced p-STAT3 and downstream SOCS3, BCL3, SAA1 and MMP1 expression. In this study we show that TGF-β decreases IL-6R expression, thereby dampening IL-6 signaling in chondrocytes. This reveals a novel effect of TGF-β, possibly important to restrict pro-inflammatory IL-6 effects to preserve cartilage homeostasis.

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

The replication and transcription activator of murine gammaherpesvirus 68 cooperatively enhances cytokine-activated, STAT3-mediated gene expression

Gammaherpesviruses (γHVs) have a dynamic strategy for lifelong persistence, involving productive infection, latency, and intermittent reactivation. In latency reservoirs, such as B lymphocytes, γHVs exist as viral episomes and express few viral genes. Although the ability of γHV to reactivate from latency and re-enter the lytic phase is challenging to investigate and control, it is known that the γHV replication and transcription activator (RTA) can promote lytic reactivation. In this study, we provide first evidence that RTA of murine γΗV68 (MHV68) selectively binds and enhances the activity of tyrosine-phosphorylated host STAT3. STAT3 is a transcription factor classically activated by specific tyrosine 705 phosphorylation (pTyr705-STAT3) in response to cytokine stimulation. pTyr705-STAT3 forms a dimer that avidly binds a consensus target site in the promoters of regulated genes, and our results indicate that RTA cooperatively enhances the ability of pTyr705-STAT3 to induce expression of a STAT3-responsive reporter gene. As indicated by coimmunoprecipitation, in latently infected B cells that are stimulated to reactivate MHV68, RTA bound specifically to endogenous pTyr705-STAT3. An in vitro binding assay confirmed that RTA selectively recognizes pTyr705-STAT3 and indicated that the C-terminal transactivation domain of RTA was required for enhancing STAT3-directed gene expression. The cooperation of these transcription factors may influence both viral and host genes. During MHV68 de novo infection, pTyr705-STAT3 promoted the temporal expression of ORF59, a viral replication protein. Our results demonstrate that MHV68 RTA specifically recognizes and recruits activated pTyr705-STAT3 during the lytic phase of infection.

Abstract 4101: Targeting the EGFR/STAT3 axis in NSCLC with resistance to EGFR tyrosine kinase inhibitors using an oligonucleotide-based decoy

Abstract EGFR (Epidermal Growth Factor Receptor) is a major target for the treatment of Non-Small Cell Lung Cancer (NSCLC), but patients lacking EGFR mutation are insensitive to EGFR inhibition. Patients with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs), but eventually develop acquired resistance as a result of secondary mutations in the EGFR tyrosine kinase domain. Upregulation of Signal Transducer and Activator of Transcription (STAT3), a key oncogenic molecule in NSCLC that is downstream of EGFR signaling, contributes to intrinsic and acquired resistance to EGFR TKIs. In the clinic, agents targeting STAT3 have not been satisfactory to date. To address the limitations of available STAT3 inhibitors, we are currently testing a cyclic oligonucleotide molecule with a novel mechanism that acts as a STAT3 “decoy” (Cyclic STAT3 decoy, CS3D). CS3D mimics the DNA consensus sequence in the promoter region of STAT3-responsive genes, causing the binding of STAT3 dimers and preventing transcriptional regulation of STAT3-target genes. The effects of CS3D are compared to mutant cyclic STAT3 decoy (CS3M), which differs from CS3D by one base-pair. We have tested the ability of CS3D to produce anti-tumor effects in NSCLC cells that are EGFR WT (201T) and in EGFR mutant cells that also carry the T790M resistant mutation (H1975). Initial in vitro studies showed that CS3D caused a 50% inhibition in cell proliferation in 201T and H1975 relative to CS3M using MTS assays. Flow cytometry studies also demonstrated that CS3D caused a 2-fold increase in the percent of apoptotic cells as compared to CS3M. CS3D also caused a 2-fold reduction in expression of STAT3-target genes c-Myc, Bcl-xL and IL-6. Compared to CS3M, CS3D inhibited colony formation by 70%. Using an in vivo mouse xenograft model of 201T and H1975, CS3D caused a 96.5% reduction in tumor growth in 201T (P<0.007) compared to CS3M, while an 81.7% inhibition was seen in H1975 (P<.0001). Utilizing IHC, analysis of residual tumors also illustrated that CS3D induced more caspase3 cleavage relative to CS3M. Additionally, western blot analysis showed 70% reduction in c-Myc protein level in response to CS3D. These results suggest CS3D can be effective as a single therapeutic agent. Combining CS3D with EGFR inhibitors such as afatinib in vitro significantly suppressed cell viability by 85.2% and 80% in 201T and H1975 respectively, as compared to single treatment (CS3D alone reduced cell viability by 53%, and 51.5%, and afatinib alone reduced viability by 31.4% and 34.03% in 201T and H1975, respectively). The combination of CS3D and afatinib warrants further testing in vivo. These data suggest that CS3D alone or in combination with EGFR tyrosine kinase inhibitors produces anti-tumor effects in NSCLC with intrinsic and acquired resistance to EGFR TKIs. Supported by funding to the Masonic Cancer Center from the Minnesota 5th Order of Eagles. Citation Format: Christian Njatcha, Mariya Farooqui, Jennifer R. Grandis, Jill M. Siegfried. Targeting the EGFR/STAT3 axis in NSCLC with resistance to EGFR tyrosine kinase inhibitors using an oligonucleotide-based decoy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2017-4101

Abstract 4170: Oncogenic activation of LMO2 by EGFR signaling regulates STAT3 phosphorylation and transcriptional activity in human glioma

Abstract Glioblastoma multiforme (GBM) is a highly heterogeneous disease characterized by multiple genetic abnormalities. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene represent signature genetic abnormalities occurred in over 60% of GBM. The successful application of EGFR-targeted therapy for the treatment of GBM is thus dependent on a thorough understanding of the intricate signal pathways cross-linkage that underlies the pathobiology of this disease. The LIM-domain only factor 2 (LMO2) gene is a crucial oncogene involved in hematopoietic development and leukemogenesis. Herein, we showed, from the TCGA GBM data, that LMO2 is highly expressed in EGFR-aberrant GBM samples and might serve as a downstream effector of EGFR signaling in GBM. Depletion of LMO2 expression impairs the maintenance of glioma cell proliferation in vitro and in vivo. In addition, EGFR signaling induces LMO2 nuclear translocation and enables LMO2 to form a triplex with EGFR and Signal transducer and activator of transcription 3 (STAT3). More importantly, LMO2 serves as a bridging molecule to promote the formation of EGFR/STAT3 complex and to enhance EGFR-mediated STAT3 Y705 phosphorylation upon EGF stimulation, which in turn up-regulates the expression of STAT3-responsive genes involved in the regulation of cell proliferation and tumor development. In conclusion, these findings reveal that LMO2 might serve as a critical effector of EGFR signaling in human glioma and highlight the essential function of LMO2 in EGF-stimulated STAT3 activation and tumorigenesis. Citation Format: Jun Fu, Dimpy Koul, Shaofang Wu, Siyuan Zheng, Roel G. Verhaak, W.K Yung. Oncogenic activation of LMO2 by EGFR signaling regulates STAT3 phosphorylation and transcriptional activity in human glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2014-4170

Angoline: A selective IL-6/STAT3 signaling pathway inhibitor isolated from Zanthoxylum nitidum

STAT3 signaling pathway is an important target for human cancer therapy. Thus, the identification of small-molecules that target STAT3 signaling will be of great interests in the development of anticancer agents. The aim of this study was to identify novel inhibitors of STAT3 pathway from the roots of Zanthoxylum nitidum (Roxb.) DC. The bioassay-guided fractionation of MeOH extract of Z. nitidum using a STAT3-responsive gene reporter assay led to the isolation of angoline (1) as a potent and selective inhibitor of the STAT3 signaling pathway (IC50=11.56μM). Angoline inhibited STAT3 phosphorylation and its target gene expression and consequently induced growth inhibition of human cancer cells with constitutively activated STAT3 (IC50=3.14–4.72μM). This work provided a novel lead for the development of anti-cancer agents targeting the STAT3 signaling pathway.

P330 ACTIVATION OF INTERFERON AND NF-κB PATHWAYS IN EARLY HUMAN ALCOHOLIC LIVER DISEASE DEVELOPMENT

Background and aims : Animal data suggest a pathogenic role for TLR4 with subsequent activation of NFkB in alcohol liver injury. Here, we assessed cytokines, TLR and TLR-dependent pathways expressions in human ALD. Methods : Liver biopsies from 41 actively drinking ALD patients were compared with normal liver tissue (n=6) and with liver biopsies from ALD patients after 2 weeks of alcohol abstinence (n=27). Quantitative PCR and Western blotting were used for mRNA and protein analysis, respectively, immunohistochemistry for cellular localization of inflammatory processes. The study was approved by our local ethics committee. Results : Compared with normal livers, already early ALD (with minimal fibrosis) showed a significant activation of NFkB with increased total and phosphorylated p65 protein expression and up-regulation of NFkB responsive genes mRNA levels TNFalphaand IL-1beta. P65 immunohistochemistry evidenced positive staining mainly in inflammatory cells in ALD patients. Interestingly, TLR4 mRNA expression remained unchanged whereas CD14 and TNF receptor 1 mRNA were down-regulated in active ALD. Only IL-1beta returned to control levels after abstinence. STAT3 phosphorylation was also significantly induced in early ALD patients. Intriguingly, STAT3 mRNA expression as well as STAT3 responsive genes SOCS3, IL-6 and MCP-1 were significantly downregulated in ALD patients suggesting an overall inhibition of the STAT3 pathway. STAT3 inhibition did not recover following 2 weeks of alcohol abstinence. The interferon pathway was strongly activated in early ALD with significant increase in total and phosphorylated interferon regulatory factor-3, up-regulation of interferon-beta and interferon responsive genes ISG6-16 and OAS-1 mRNA levels. Interferon-beta levels significantly correlated with up-regulation of TLR3 and 7 mRNA. Activation of the interferon pathway was not reversed after abstinence. CD68 mRNA was significantly increased and both CD68 and CD163 immunohistochemistry revealed enlarged, aggregated Kupffer cells and small inflammatory cells in early ALD compared to scattered Kupffer cells in normal liver. Conclusions: Besides NFkB, early activation of the interferon pathway could play an important role in human ALD pathogenesis. Inhibition of the STAT3 pathway could contribute to impaired regenerative capacity in ALD.

Monoallelic loss of tumor suppressor GRIM-19 promotes tumorigenesis in mice

Gene-associated with retinoid-interferon induced mortality-19 (GRIM-19), a STAT3-inhibitory protein, was isolated as a growth-suppressive gene product using a genome-wide expression knockdown screen. We and others have shown a loss of expression and occurrence of mutations in the GRIM-19 gene in a variety of primary human cancers, indicating its potential role as tumor suppressor. To help investigate its role in tumor development in vivo, we generated a genetically modified mouse in which Grim-19 can be conditionally inactivated. Deletion of Grim-19 in the skin significantly increased the susceptibility of mice to chemical carcinogenesis, resulting in development of squamous cell carcinomas. These tumors had high Stat3 activity and an increased expression of Stat3-responsive genes. Loss of Grim-19 also caused mitochondrial electron transport dysfunction resulting from failure to assemble electron transport chain complexes and altered the expression of several cellular genes involved in glycolysis. Surprisingly, the deletion of a single copy of the Grim-19 gene was sufficient to promote carcinogenesis and formation of invasive squamous cell carcinomas. These observations highlight the critical role of GRIM-19 as a tumor suppressor.

125 : The Sin3a repressor complex regulates the balance between the activities of different STAT proteins

The activity of Signal Transducer and Activator of Transcription (STAT) proteins is regulated by a multitude of posttranslational modifications, which control different aspects of signal transduction. In particular, lysine acetylation and deacetylation has emerged as a critical modification, regulating the activity of all seven STAT members. Unlike tyrosine phosphorylation/dephosphorylation, which mainly acts as an on/off switch of STAT activity, the control exerted by acetylation appears multifaceted and by far more complex, exerting distinct and even opposed regulation to different STAT members. We observed that type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide siRNA screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as a novel regulator of STAT activity. Sin3a directly interacts with STAT3 and controls its acetylation status, its nuclear accumulation and DNA binding, strongly influencing the transcription of a subset of STAT3-responsive genes. Interfering with Sin3a expression restores, at least partially, the STAT3 transcriptional blockade observed upon IFN treatment. Strikingly, Sin3a exerts opposed regulation on ISGF3 activity, as it is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. We conclude that Sin3a contributes to the regulation of the balance between the activities of different STAT members, and interfering with its activity or expression may re-direct cytokine signals, favoring the onset of pathological conditions, such as autoimmune syndromes, inflammation and cancer.

Gadd45a Suppresses Tumor Angiogenesis via Inhibition of the mTOR/STAT3 Protein Pathway*

Gadd45a, a p53-regulated and DNA damage-inducible gene, is implicated in protection against tumor malignancy, although the underlying mechanism remains to be defined further. Here we demonstrate that Gadd45a plays an important role in suppression of tumor angiogenesis. Gadd45a deletion significantly increases microvessel density in tumors and stimulates an angiogenic response in a chicken embryo chorioallantoic membrane assay. Disruption of endogenous Gadd45a promotes tube formation and migration of endothelial cells. We further show that Gadd45a deletion increases phosphorylation of STAT3 at Ser-727 and, in turn, elevates the STAT3 transcriptional activity. This process substantially induces both expression and secretion of VEGFa, a STAT3 responsive gene, and promotes tumor angiogenesis. Interestingly, Gadd45a is able to physically associate with mammalian target of rapamycin (mTOR), a kinase that mediates Ser-727 phosphorylation of STAT3. The interaction of Gadd45a with mTOR suppresses STAT3 phosphorylation at Ser-727 and leads to down-regulated expression of VEGFa. Further analysis reveals that Gadd45a overexpression attenuates the association between mTOR and STAT3, whereas Gadd45a disruption strengthens this interaction, indicating that Gadd45a suppression of STAT3 phosphorylation is mainly through the dissociation of mTOR with STAT3. Taken together, these findings provide the first evidence that Gadd45a inhibits tumor angiogenesis via blocking of the mTOR/STAT3 pathway.

Early Activation of Rat Skeletal Muscle IL-6/STAT1/STAT3 Dependent Gene Expression in Resistance Exercise Linked to Hypertrophy

Cytokine interleukin-6 (IL-6) is an essential regulator of satellite cell-mediated hypertrophic muscle growth through the transcription factor signal transducer and activator of transcription 3 (STAT3). The importance of this pathway linked to the modulation of myogenic regulatory factors expression in rat skeletal muscle undergoing hypertrophy following resistance exercise, has not been investigated. In this study, the phosphorylation and nuclear localization of STAT3, together with IL-6/STAT3-responsive gene expression, were measured after both a single bout of resistance exercise and 10 weeks of training. Flexor Digitorum Profundus muscle samples from Wistar rats were obtained 2 and 6 hours after a single bout of resistance exercise and 72 h after the last bout of either 2, 4, or 10 weeks of resistance training. We observed an increase in IL-6 and SOCS3 mRNAs concomitant with phosphorylation of STAT1 and STAT3 after 2 and 6 hours of a single bout of exercise (p<0.05). STAT3-dependent early responsive genes such as CyclinD1 and cMyc were also upregulated whereas MyoD and Myf5 mRNAs were downregulated (p<0.05). BrdU-positive satellite cells increased at 2 and 6 hours after exercise (p<0.05). Muscle fiber hypertrophy reached up to 100% after 10 weeks of training and the mRNA expression of Myf5, c-Myc and Cyclin-D1 decreased, whereas IL-6 mRNA remained upregulated. We conclude that the IL-6/STAT1/STAT3 signaling pathway and its responsive genes after a single bout of resistance exercise are an important event regulating the SC pool and behavior involved in muscle hypertrophy after ten weeks of training in rat skeletal muscle.

Prohibitin Attenuates Colitis-Associated Tumorigenesis in Mice by Modulating p53 and STAT3 Apoptotic Responses

Although inflammatory bowel disease is associated with higher risk of colorectal cancer, the precise pathogenic mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicated in the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In this study, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type and transgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classical two-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell models were used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHB protein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in a manner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2 expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, and caspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3 activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated with phospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to show interaction between PHB and STAT3 in vivo. In summary, our findings suggest that PHB protects against colitis-associated cancer by modulating p53- and STAT3-mediated apoptosis. Modulation of PHB expression in intestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis.

P131 STAT transcriptional activity is controlled by regulated deacetylation

Tyrosine phosphorylation is a hallmark for activation of Signal Transducer and Activator of Transcription (STAT) proteins, but their transcriptional activity also depends on other secondary modifications. Type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Simultaneous treatment with IFNa2 and Trichostatin A, as well as combined HDAC1/HDAC2 silencing, restores STAT3-dependent reporter gene and endogenous genes expression, strongly suggesting that HDAC1 and HDAC2 are directly involved in repressing IFNa-activated STAT3. We used the IFN-dependent STAT3 transcriptional blockade as read-out for a genome-wide RNAi repressor screen. Quite significantly, we identified multiple partners of the Sin3a/HDAC transcriptional repressor complex as negative regulators of STAT3 transcriptional activity, regardless to the STAT3 activating stimulus. Sin3a directly interacts with STAT3 and controls its acetylation status, its nuclear accumulation and DNA binding, strongly influencing the transcription of a subset of STAT3-responsive genes. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.

Conserved Intergenic Elements and DNA Methylation Cooperate to Regulate Transcription at the il17 Locus

Naive CD4(+) T cells can differentiate into distinct lineages with unique immune functions. The cytokines TGFβ and IL-6 promote the development of Th17 cells that produce IL-17, an inflammatory cytokine not expressed by other T helper lineages. To further understand how IL-17 production is controlled, we studied an ~120-kb genomic region containing the murine il17a and il17f genes and seven evolutionarily conserved, intergenic noncoding sequences. We show that the +28-kb noncoding sequence cooperates with STAT3, RORγt, and Runx1 to enhance transcription from both il17a and il17f promoters. This enhancer and both promoters exhibited Th17 lineage-specific DNA demethylation, accompanied by demethylation of lysine 27 of histone H3 (H3K27) and increased H3K4 methylation. Loss of DNA methylation tended to occur at STAT3 consensus elements, and we show that methylation of one of these elements in the il17a promoter directly inhibits STAT3 binding and transcriptional activity. These results demonstrate that TGFβ and IL-6 synergize to epigenetically poise the il17 loci for expression in Th17 cells, and suggest a general mechanism by which active STAT3 may be epigenetically excluded from STAT3-responsive genes in non-Th17 lineages.

AP-1 Transcription Factors, Mucin-Type Molecules and MMPs Regulate the IL-11 Mediated Invasiveness of JEG-3 and HTR-8/SVneo Trophoblastic Cells

This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo cells.

Preclinical Evaluation of Local JAK1 and JAK2 Inhibition in Cutaneous Inflammation

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100 nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

Suppression of FoxO1 Activity by Long-Chain Fatty Acyl Analogs

OBJECTIVEOveractivity of the Forkhead transcription factor FoxO1 promotes diabetic hyperglycemia, dyslipidemia, and acute-phase response, whereas suppression of FoxO1 activity by insulin may alleviate diabetes. The reported efficacy of long-chain fatty acyl (LCFA) analogs of the MEDICA series in activating AMP-activated protein kinase (AMPK) and in treating animal models of diabesity may indicate suppression of FoxO1 activity.RESEARCH DESIGN AND METHODSThe insulin-sensitizing and anti-inflammatory efficacy of a MEDICA analog has been verified in guinea pig and in human C-reactive protein (hCRP) transgenic mice, respectively. Suppression of FoxO1 transcriptional activity has been verified in the context of FoxO1- and STAT3-responsive genes and compared with suppression of FoxO1 activity by insulin and metformin.RESULTSTreatment with MEDICA analog resulted in total body sensitization to insulin, suppression of lipopolysaccharide-induced hCRP and interleukin-6–induced acute phase reactants and robust decrease in FoxO1 transcriptional activity and in coactivation of STAT3. Suppression of FoxO1 activity was accounted for by its nuclear export by MEDICA-activated AMPK, complemented by inhibition of nuclear FoxO1 transcriptional activity by MEDICA-induced C/EBPβ isoforms. Similarly, insulin treatment resulted in nuclear exclusion of FoxO1 and further suppression of its nuclear activity by insulin-induced C/EBPβ isoforms. In contrast, FoxO1 suppression by metformin was essentially accounted for by its nuclear export by metformin-activated AMPK.CONCLUSIONSSuppression of FoxO1 activity by MEDICA analogs may partly account for their antidiabetic anti-inflammatory efficacy. FoxO1 suppression by LCFA analogs may provide a molecular rational for the beneficial efficacy of carbohydrate-restricted ketogenic diets in treating diabetes.

Chronic IL-11 Promotes Proliferation, Bmp1 Signalling and Activation of the Mucosal Alarmin IL-33 in the Colon

Background and AimsIL-33 is an IL-1 related cytokine and like IL-1a has been suggested to amplify the immune response during tissue injury. IL-33 is expressed in endothelium and mucosal epithelium and is a ligand for the receptor ST2. Recently IL-33 has been shown to be a crucial amplifier of immunity that can modulate the response in a number of models of innate-type mucosal immunity. Significantly IL-33 is involved in the development of DSS induced colitis. Additionally we have demonstrated that IL-11, a member of the IL-6 family of cytokines, is also important in DSS induced colitis and others have demonstrated an antiinflammatory role for IL-11 in intestinal inflammation. Here we examine the effects of IL11 on the normal colon and establish that IL-11 is upstream of Bmp1 and IL-33, and that it regulates mucosal proliferation via Reg3β in a STAT3-dependent manner. MethodsNormal mice were treated with 5 ug of recombinant human IL-11 (rhIL-11) every 6 hours and culled either 6 hours, 24 hours of 5 days after the initial dose. Colons were removed for mRNA and protein analysis. ResultsIL-11 is a ligand for gp130 and should initiate both the Ras/MAPK/ERK and STAT1/STAT3 signalling pathways. Chronic IL-11 treatment of normal mice activates STAT3 (Tyr705) but not ERK1/2 (Thr202/Tyr204), and acute IL-11 caused a drop in ERK1/2 activation. This is a very similar to the signalling outcome downstream of IL-11 in the gastric mucosa, where signalling emanating from gp130 activation by IL-11 is skewed towards STAT3. Expression of SOCS3, a bona fide STAT3 responsive gene, is also increased in the colon following IL-11 treatment. In the gastric antrum IL-11 treatment is pro-proliferative. The same is true of the colonic response to IL-11 with a 1.6 fold increase in the expression of the proliferation marker PCNA by immunoblot. IL-11 also caused an increase in the expression of the pro-proliferative gene Reg3β and the mRNAs involved in BMP signalling Bmp1 and Bmp1rb. However, most significantly IL-11 treatment caused a large and significant increase in the expression of IL-33 and other mediators of the innate immune response in the absence of any significant damage to the mucosa. ConclusionsColonic tissue responds to IL-11 largely by activation of STAT3 signalling. There are three major outcomes resulting from this signalling; 1) increased proliferation and expression of pro-proliferative genes, 2) increased expression of components of BMP signalling and 3) activation of IL-33 and the innate immune system.