Sutapa Ray

Requirement of histone deacetylase1 (HDAC1) in signal transducer and activator of transcription 3 (STAT3) nucleocytoplasmic distribution

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that plays a crucial role in interleukin-6 (IL-6) signaling, mediating the acute-phase induction of the human Angiotensinogen (hAGT) gene in hepatocytes. We showed earlier that IL-6 induces acetylation of the STAT3 NH2-terminus by the recruitment of the p300 coactivator. We had also observed a physical interaction of STAT3 and Histone Deacetylase1 (HDAC1) in an IL-6-dependent manner that leads to transcriptional repression. In this study, we sought to elucidate the mechanism by which HDAC1 controls STAT3 transcriptional activity. Here, we mapped the interacting domains of both STAT3 and HDAC1 and found that the COOH-terminal domain of HDAC1 is necessary for IL-6-induced STAT3 transcriptional repression, whereas the NH2-terminal acetylation domain of STAT3 is required for HDAC1 binding. Interestingly, over expression of HDAC1 in HepG2 cells leads to significantly reduced amounts of nuclear STAT3 after IL-6 induction, whereas silencing of HDAC1 resulted in accumulation of total and acetylated STAT3 in the nucleus. We have found that HDAC1 knockdown also interferes with the responsiveness of the STAT3-dependent MCP1 target gene expression to IL-6, as confirmed by real-time RT–PCR analysis. Together, our study reveals the novel functional consequences of IL-6-induced STAT3-HDAC1 interaction on nucleocytoplasmic distribution of STAT3.

The STAT3 NH2-terminal domain stabilizes enhanceosome assembly by interacting with the p300 bromodomain

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor mainly activated by the interleukin-6 cytokine family. Previous studies have shown that activated STAT3 recruits p300, a coactivator whose intrinsic histone acetyltransferase activity is essential for transcription. Here we investigated the function of the STAT3 NH2-terminal domain and how its interaction with p300 regulates STAT3 signal transduction. In STAT3-/- mouse embryonic fibroblasts, a stably expressed NH2 terminus-deficient STAT3 mutant (STAT3-ΔN) was unable to efficiently induce either STAT3-mediated reporter activity or endogenous mRNA expression. Chromatin immunoprecipitation assays were performed to determine whether the NH2-terminal domain regulates p300 recruitment or stabilizes enhanceosome assembly. Despite equivalent levels of STAT3 binding, cells expressing the STAT3-ΔN mutant were unable to recruit p300 and RNA polymerase II to the native socs3 promoter as efficiently as those expressing STAT3-full length. We previously reported that the STAT3 NH2-terminal domain is acetylated by p300 at Lys-49 and Lys-87. By introducing K49R/K87R mutations, here we found that the acetylation status of the STAT3 NH2-terminal domain regulates its interaction with p300. In addition, the STAT3 NH2-terminal binding site maps to the p300 bromodomain, a region spanning from amino acids 995 to 1255. Finally a p300 mutant lacking the bromodomain (p300-ΔB) exhibited a weaker binding to STAT3, and the enhanceosome formation on the socs3 promoter was inhibited when p300-ΔB was overexpressed. Taken together, our data suggest that the STAT3 NH2-terminal domain plays an important role in the interleukin-6 signaling pathway by interacting with the p300 bromodomain, thereby stabilizing enhanceosome assembly.

Roles of IL-6-gp130 Signaling in Vascular Inflammation

Interleukin-6 (IL-6) is a well-established, independent indicator of multiple distinct types of cardiovascular disease and all-cause mortality. In this review, we present current understanding of the multiple roles that IL-6 and its signaling pathways through glycoprotein 130 (gp130) play in cardiovascular homeostasis. IL-6 is highly inducible in vascular tissues through the actions of the angiotensin II (Ang II) peptide, where it acts in a paracrine manner to signal through two distinct mechanisms, the first being a classic membrane receptor initiated pathway and the second, a trans-signaling pathway, being able to induce responses even in tissues lacking the IL-6 receptor. Recent advances and new concepts in how its intracellular signaling pathways operate via the Janus kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) are described. IL-6 has diverse actions in multiple cell types of cardiovascular importance, including endothelial cells, monocytes, platelets, hepatocytes and adipocytes. We discuss central roles of IL-6 in endothelial dysfunction, cellular inflammation by affecting monocyte activation/differentiation, cellular cytoprotective functions from reactive oxygen species (ROS) stress, modulation of pro-coagulant state, myocardial growth control, and its implications in metabolic control and insulin resistance. These multiple actions indicate that IL-6 is not merely a passive biomarker, but actively modulates adaptive and pathological responses to cardiovascular stress. Summary: IL-6 is a multifunctional cytokine whose presence in the circulation is linked with diverse types of cardiovascular disease and is an independent risk factor for atherosclerosis. In this review, we examine the mechanisms by which IL-6 signals and its myriad effects in cardiovascular tissues that modulate the manifestations of vascular inflammation.

The Functional Role of an Interleukin 6-inducible CDK9·STAT3 Complex in Human γ-Fibrinogen Gene Expression

The signal transducer and activator of transcription 3 (STAT3) is an IL-6-inducible transcription factor that mediates the hepatic acute phase response (APR). Using γ-fibrinogen (FBG) as a model of the APR, we investigated the requirement of an IL-6-inducible complex of STAT3 with cyclin-dependent kinase 9 (CDK9) on γ-FBG expression in HepG2 hepatocarcinoma cells. IL-6 induces rapid nuclear translocation of Tyr-phosphorylated STAT3 that forms a nuclear complex with CDK9 in nondenaturing co-immunoprecipitation and confocal colocalization assays. To further understand this interaction, we found that CDK9-STAT3 binding is mediated via both STAT NH2-terminal modulatory and COOH-terminal transactivation domains. Both IL-6-inducible γ-FBG reporter gene and endogenous mRNA expression are significantly decreased after CDK9 inhibition using the potent CDK inhibitor, flavopiridol (FP), or specific CDK9 siRNA. Moreover, chromatin immunoprecipitation (ChIP) experiments revealed an IL-6-inducible STAT3 and CDK9 binding to the proximal γ-FBG promoter as well as increased loading of RNA Pol II and phospho-Ser2 CTD Pol II on the TATA box and coding regions. Finally, FP specifically and efficiently inhibits association of phospho-Ser2 CTD RNA Pol II on the γ-FBG promoter, indicating that CDK9 kinase activity mediates IL-6-inducible CTD phosphorylation. Our data indicate that IL-6 induces a STAT3·CDK9 complex mediated by bivalent STAT3 domains and CDK9 kinase activity is necessary for licensing Pol II to enter a transcriptional elongation mode. Therefore, disruption of IL-6 signaling by CDK9 inhibitors could be a potential therapeutic strategy for inflammatory disease.

Genomic Mechanisms of p210BCR-ABL Signaling INDUCTION OF HEAT SHOCK PROTEIN 70 THROUGH THE GATA RESPONSE ELEMENT CONFERS RESISTANCE TO PACLITAXEL-INDUCED APOPTOSIS

Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210BCR-ABL oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance. To further understand mechanisms mediating chemotherapy resistance, we identified 556 differentially regulated genes in HL-60 cells stably expressing p210BCR-ABL versus those expressing an empty vector using cDNA macro- and oligonucleotide microarrays. These BCR-ABL-regulated gene products play diverse roles in cellular function including apoptosis, cell cycle regulation, intracellular signaling, transcription, and cellular adhesion. In particular, we identified up-regulation of the inducible form of heat shock protein 70 (Hsp70), and further explored the mechanism for its up-regulation. In HL-60/BCR-ABL and K562 cells (expressing p210BCR-ABL), abundant cytoplasmic Hsp70 expression was detected by immunoblot analysis. Moreover, cells isolated from bone marrow aspirates of patients in different stages of CML (chronic, aggressive, and blast crisis) express Hsp70. Expression of p210BCR-ABL in BCR-ABL negative cells induced transcription of the proximal Hsp70 promoter. Mutational analysis mapped the major p210BCR-ABL responsive element to a high affinity 5′(A/T)GATA(A/G)-3′ “GATA” response element (GATA-RE) that binds GATA-1 in CML cells. The GATA-RE was sufficient to confer p210BCR-ABL- and p185BCR-ABL-mediated trans-activation to an inert promoter. Short interfering RNA mediated “knockdown” of Hsp70 expression in K562 cells induced marked sensitivity to paclitaxel-induced apoptosis. Together these findings indicate that BCR-ABL confers chemotherapeutic resistance through intracellular signaling to the GATA-RE element found in the promoter region of the anti-apoptotic Hsp70 protein. We suggest that down-regulation of the GATA-Hsp70 pathway may be useful in the treatment of chemotherapy-resistant CML.

The IL-6 trans-signaling-STAT3 pathway mediates ECM and cellular proliferation in fibroblasts from hypertrophic scar

The molecular mechanisms behind the pathogenesis of post-burn hypertrophic scar (HS) remain unclear. Here, we investigate the role of interleukin-6 (IL-6) trans-signaling-STAT3 pathway in HS fibroblasts (HSF) derived from burned-induced HS skin. HSF showed increased Tyr 705 STAT3 phosphorylation over normal fibroblast (NF) after IL-6•IL-6Rα stimulation by immunoassays. The endogenous STAT3 target gene, SOCS3, was upregulated in HSF and showed increased STAT3 binding on its promoter relative to NF in Chromatin Immunoprecipitation assay. We observed that the cell surface signaling transducer glycoprotein 130 is upregulated in HSF using Q-RT-PCR and flow cytometry. The production of excessive extracellular matrix (ECM), including the expression of alpha2 (1) procollagen (Col1A2) and fibronectin 1 (FN) were seen in HSFs. A STAT3 peptide inhibitor abrogated FN and Col1A2 gene expression in HSF indicating involvement of STAT3 in ECM production. The cellular proliferation markers Cyclin D1, Bcl-Xl and c-Myc were also upregulated in HSF and knockdown of STAT3 by siRNA attenuated c-Myc expression indicating the essential role of STAT3 in fibroblast proliferation. Taken together, our results suggest that the IL-6-trans-signaling-STAT3 pathway may play an integral role in HS pathogenesis and disruption of this pathway could be a potential therapeutic strategy for the treatment of burn-induced HS.

Bcr-Abl Regulates Protein Kinase Cι (PKCι) Transcription via an Elk1 Site in the PKCι Promoter

The protein kinase C (PKC) family of serine/threonine kinases plays an important role in numerous cancer signaling pathways, including those downstream of the bcr-abl oncogene. We demonstrated previously that atypical PKCι is required for Bcr-Abl-mediated resistance of human K562 chronic myelogenous leukemia (CML) cells to Taxol-induced apoptosis. Here, we report that the pattern of PKC isozyme expression characteristic of CML cells is regulated by Bcr-Abl. When Bcr-Abl was expressed in Bcr-Abl-negative HL-60 promyelocytic leukemia cells, expression of the PKCβI, PKCβII, and PKCι genes was induced, whereas expression of the PKCδ gene was reduced to levels similar to those found in CML cells. Given the importance of PKCι in Bcr-Abl-mediated transformation, we characterized the mechanism by which Bcr-Abl regulates PKCι expression. A 1200-bp PKCι promoter construct isolated from genomic DNA was highly active in Bcr-Abl-positive K562 cells and was activated when Bcr-Abl-negative cells were transfected with Bcr-Abl. Bcr-Abl-mediated induction of the PKCι promoter was dependent upon MEK1/2 activity, but not phosphatidylinositol 3-kinase or p38 MAPK activity. Mutational analysis of the PKCι promoter revealed a region between 97 and 114 bp upstream of the transcriptional start site that is responsible for Bcr-Abl-mediated regulation. Mutation of a consensus Elk1-binding site within this region abolished Bcr-Abl-mediated regulation. We conclude that Bcr-Abl regulates PKCι expression through the MEK-dependent activation of an Elk1 element within the proximal PKCι promoter. Our results indicate that Bcr-Abl-mediated transformation involves transcriptional activation of the PKCι gene, which in turn is required for Bcr-Abl-mediated chemoresistance.

Respiratory Syncytial Virus-Inducible BCL-3 Expression Antagonizes the STAT/IRF and NF-κB Signaling Pathways by Inducing Histone Deacetylase 1 Recruitment to the Interleukin-8 Promoter

ABSTRACT Respiratory syncytial virus (RSV) is a paramyxovirus that produces airway inflammation, in part by inducing interleukin-8 (IL-8) expression, a CXC-type chemokine, via the NF-κB/RelA and STAT/IRF signaling pathways. In RSV-infected A549 cells, IL-8 transcription attenuates after 24 h in spite of ongoing viral replication and persistence of nuclear RelA, suggesting a mechanism for transcriptional attenuation. RSV infection induces B-cell lymphoma protein -3 (Bcl-3) expression 6 to 12 h after viral infection, at times when IL-8 transcription is inhibited. By contrast, 293 cells, deficient in inducible Bcl-3 expression, show no attenuation of IL-8 transcription. We therefore examined Bcl-3s role in terminating virus-inducible IL-8 transcription. Transient expression of Bcl-3 potently inhibited virus-inducible IL-8 transcription by disrupting both the NF-κB and STAT/IRF pathways. Although previously Bcl-3 was thought to capture 50-kDa NF-κB1 isoforms in the cytoplasm, immunoprecipitation (IP) and electrophoretic mobility shift assays indicate that nuclear Bcl-3 associates with NF-κB1 without affecting DNA binding. Additionally, Bcl-3 potently inhibited the STAT/IRF pathway. Nondenaturing co-IP assays indicate that nuclear Bcl-3 associates with STAT-1 and histone deacetylase 1 (HDAC-1), increasing HDAC-1 recruitment to the IL-8 promoter. Treatment with the HDAC inhibitor trichostatin A blocks attenuation of IL-8 transcription. A nuclear targeting-deficient Bcl-3 is unable to enhance HDAC-1-mediated chemokine repression. Finally, small inhibitory RNA-mediated Bcl-3 “knockdown” resulted in enhanced RSV-induced chemokine expression in A549 cells. These data indicate that Bcl-3 is a virus-inducible inhibitor of chemokine transcription by interfering with the NF-κB and STAT/IRF signaling pathways by complexing with them and recruiting HDAC-1 to attenuate target promoter activity.

Interleukin-6–Signal Transducer and Activator of Transcription-3 Signaling Mediates Aortic Dissections Induced by Angiotensin II via the T-Helper Lymphocyte 17–Interleukin 17 Axis in C57BL/6 Mice

Objective—Dysregulated angiotensin II (Ang II) signaling induces local vascular interleukin-6 (IL-6) secretion, producing leukocyte infiltration and life-threatening aortic dissections. Precise mechanisms by which IL-6 signaling induces leukocyte recruitment remain unknown. T-helper 17 lymphocytes (Th17) have been implicated in vascular pathology, but their role in the development of aortic dissections is poorly understood. Here, we tested the relationship of IL-6–signal transducer and activator of transcription-3 signaling with Th17-induced inflammation in the formation of Ang II–induced dissections in C57BL/6 mice. Approach and Results—Ang II infusion induced aortic dissections and CD4+-interleukin 17A (IL-17A)–expressing Th17 cell accumulation in C57BL/6 mice. A blunted local Th17 activation, macrophage recruitment, and reduced incidence of aortic dissections were seen in IL-6−/− mice. To determine the pathological roles of Th17 lymphocytes, we treated Ang II–infused mice with IL-17A–neutralizing antibody or infused Ang II in genetically deficient IL-17A mice and found decreased aortic chemokine monocytic chemotactic protein-1 production and macrophage recruitment, leading to a reduction in aortic dissections. This effect was independent of blood pressure in IL-17A–neutralizing antibody experiment. Application of a cell-permeable signal transducer and activator of transcription-3 inhibitor to downregulate the IL-6 pathway decreased aortic dilation and Th17 cell recruitment. We also observed increased aortic Th17 infiltration and IL-17 mRNA expression in patients with thoracic aortic dissections. Finally, we found that Ang II–mediated aortic dissections occurred independent of blood pressure changes. Conclusions—Our results indicate that the IL-6–signal transducer and activator of transcription-3 signaling pathway converges on Th17 recruitment and IL-17A signaling upstream of macrophage recruitment, mediating aortic dissections.

Inducible STAT3 NH2 terminal mono-ubiquitination promotes BRD4 complex formation to regulate apoptosis

Signal Transducers and Activator of Transcription-3 (STAT3) are latent transcription factors that are regulated by post-translational modifications (PTMs) in response to cellular activation by the IL-6 superfamily of cytokines to regulate cell cycle progression and/or apoptosis. Here we observe that STAT3 is inducibly mono-ubiquitinated and investigate its consequences. Using domain mapping and highly specific selected reaction monitoring-mass spectrometric assays, we identify lysine (K) 97 in its NH2-terminal domain as the major mono-ubiquitin conjugation site. We constructed a mono-ubiquitinated mimic consisting of a deubiquitinase-resistant monomeric ubiquitin fused to the NH2 terminus of STAT3 (ubiquitinated-STAT3 FP). In complex assays of ectopically expressed ubi-STAT3-FP, we observed enhanced complex formation with bromodomain-containing protein 4 (BRD4), a component of the activated positive transcriptional elongation factor (P-TEFb) complex. Chromatin immunoprecipitation experiments in STAT3(+/-) and STAT3(-/-) MEFs showed BRD4 recruitment to STAT3-dependent suppressor of cytokine signaling-3 gene (SOCS3). The effect of a selective small molecule inhibitor of BRD4, JQ1, to inhibit SOCS3 expression demonstrated the functional role of BRD4 for STAT3-dependent transcription. Additionally, ectopic ubiquitinated-STAT3 FP expression upregulated BCL2, BCL2L1, APEX1, SOD2, CCND1 and MYC expression indicating the role of ubiquitinated STAT3 in anti-apoptosis and cellular proliferation. Finally we observed that ubiquitinated-STAT3 FP suppressed TNFα-induced apoptotic cell death, indicating the functional importance of mono-ubiquitinated STAT3 in antiapoptotic gene expression. We conclude that STAT3 mono-ubiquitination is a key trigger in BRD4-dependent antiapoptotic and pro-proliferative gene expression programs. Thus, inhibiting the STAT3 mono-ubiquitination-BRD4 pathway may be a novel therapeutic target for the treatment of STAT3-dependent proliferative diseases.