Nuclear protein IκB-ζ inhibits the activity of STAT3

International Nuclear Information System (INIS)

Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

2009-01-01

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

IL-8 induces miR-424-5p expression and modulates SOCS2/STAT5 signaling pathway in oral squamous cell carcinoma.

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Peng, Hsuan-Yu; Jiang, Shih-Sheng; Hsiao, Jenn-Ren; Hsiao, Michael; Hsu, Yuan-Ming; Wu, Guan-Hsun; Chang, Wei-Min; Chang, Jang-Yang; Jin, Shiow-Lian Catherine; Shiah, Shine-Gwo

2016-06-01

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR-424-5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR-424-5p. The miR-424-5p-induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR-424-5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR-424-5p expression could be induced by the cytokine IL-8 primarily through enhancing STAT5 transcriptional activity rather than NF-κB signaling. Antagomir-mediated inactivation of miR-424-5p prevented the IL-8-induced cell migration and invasion, indicating that miR-424-5p is required for IL-8-induced cellular invasiveness. Taken together, these data indicate that STAT5-dependent expression of miR-424-5p plays an important role in mediating IL-8/STAT5/SOCS2 feedback loop, and scavenging miR-424-5p function using antagomir may have therapeutic potential for the treatment of OSCC. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

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Hansen, L. H.; Wang, X.; Kopchick, J J

1996-01-01

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

Reciprocal activation of α5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation.

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Zhang, Yao; Jia, Yanfei; Li, Ping; Li, Huanjie; Xiao, Dongjie; Wang, Yunshan; Ma, Xiaoli

2017-07-20

Cigarette smoking is the top environmental risk factor for lung cancer. Nicotine, the addictive component of cigarettes, induces lung cancer cell proliferation, invasion and migration via the activation of nicotinic acetylcholine receptors (nAChRs). Genome-wide association studies (GWAS) show that CHRNA5 gene encoding α5-nAChR is especially relevant to lung cancer. However, the mechanism of this subunit in lung cancer is not clear. In the present study, we demonstrate that the expression of α5-nAChR is correlated with phosphorylated STAT3 (pSTAT3) expression, smoking history and lower survival of non-small cell lung cancer (NSCLC) samples. Nicotine increased the levels of α5-nAChR mRNA and protein in NSCLC cell lines and activated the JAK2/STAT3 signaling cascade. Nicotine-induced activation of JAK2/STAT3 signaling was inhibited by the silencing of α5-nAChR. Characterization of the CHRNA5 promoter revealed four STAT3-response elements. ChIP assays confirmed that the CHRNA5 promoter contains STAT3 binding sites. By silencing STAT3 expression, nicotine-induced upregulation of α5-nAChR was suppressed. Downregulation of α5-nAChR and/or STAT3 expression inhibited nicotine-induced lung cancer cell proliferation. These results suggest that there is a feedback loop between α5-nAChR and STAT3 that contributes to the nicotine-induced tumor cell proliferation, which indicates that α5-nAChR is an important therapeutic target involved in tobacco-associated lung carcinogenesis. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

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Grange, Magali; Buferne, Michel; Verdeil, Grégory; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2012-01-01

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs. ©2011 AACR.

IGF-1 induces the epithelial-mesenchymal transition via Stat5 in hepatocellular carcinoma.

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Zhao, Chuanzong; Wang, Qian; Wang, Ben; Sun, Qi; He, Zhaobin; Hong, Jianguo; Kuehn, Florian; Liu, Enyu; Zhang, Zongli

2017-12-19

It has been reported that the epithelial-mesenchymal transition (EMT) plays an important role in hepatocellular carcinoma (HCC). However, the relationship between the insulin-like growth factor-1 (IGF-1) and EMT of HCC was not fully elucidated. In the present work, we found that the expression of N-cadherin, Vimentin, Snail1, Snail2, and Twist1 was positively associated with IGF-1R expression, while E-cadherin expression was negatively associated with IGF-1 expression in human HCC samples. Furthermore, we observed that IGF-1 up-regulated the expression of N-cadherin, Vimentin, Snail1, Snail2 and Twist1, and down-regulated the expression of E-cadherin. In addition, Stat5 was induced in IGF-1-treated HepG2 and Hep3B cells, and Stat5 inhibition or siRNA significantly affected IGF-1-induced EMT in HepG2 and Hep3B cells. In conclusion, IGF-1 induces EMT of HCC via Stat5 signaling pathway. Thus, IGF-1/Stat5 can be recommended as a potential and novel therapeutic strategy for HCC patients.

Cutaneous T cell lymphoma expresses immunosuppressive CD80 (B7-1) cell surface protein in a STAT5-dependent manner

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Zhang, Qian; Wang, Hong Yi; Wei, Fang

2014-01-01

as their joint ability to transcriptionally activate the CD80 gene. In IL-2-dependent CTCL cells, CD80 expression is induced by the cytokine in a Jak1/3- and STAT5a/b-dependent manner, whereas in the CTCL cells with constitutive STAT5 activation, CD80 expression is also STAT5a/b dependent but is independent......(+) and CD8(+) populations or the CD4(+) subset alone, transfected with CD152 mRNA, inhibits proliferation of normal T cells in a CD152- and CD80-dependent manner. These data identify a new mechanism of immune evasion in CTCL and suggest that the CD80-CD152 axis may become a therapeutic target in this type...

Ames dwarf (Prop1(df)/Prop1(df)) mice display increased sensitivity of the major GH-signaling pathways in liver and skeletal muscle.

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Miquet, Johanna G; Muñoz, Marina C; Giani, Jorge F; González, Lorena; Dominici, Fernando P; Bartke, Andrzej; Turyn, Daniel; Sotelo, Ana I

2010-04-01

Growth hormone (GH) is an anabolic hormone that regulates growth and metabolism. Ames dwarf mice are natural mutants for Prop1, with impaired development of anterior pituitary and undetectable levels of circulating GH, prolactin and TSH. They constitute an endocrine model of life-long GH-deficiency. The main signaling cascades activated by GH binding to its receptor are the JAK2/STATs, PI-3K/Akt and the MAPK Erk1/2 pathways. We have previously reported that GH-induced STAT5 activation was higher in Ames dwarf mice liver compared to non-dwarf controls. The aim of this study was to evaluate the principal components of the main GH-signaling pathways under GH-deficiency in liver and skeletal muscle, another GH-target tissue. Ames dwarf mice and their non-dwarf siblings were assessed. Animals were injected i.p. with GH or saline 15min before tissue removal. Protein content and phosphorylation of signaling mediators were determined by immunoblotting of tissue solubilizates. GH was able to induce STAT5 and STAT3 tyrosine phosphorylation in both liver and muscle, but the response was higher for Ames dwarf mice than for non-dwarf controls. When Erk1/2 activation was assessed in liver, only dwarf mice showed GH-induced phosphorylation, while in muscle no response to the hormone was found in either genotype. GH-induced Akt phosphorylation at Ser473 in liver was only detected in dwarf mice. In skeletal muscle, both normal and dwarf mice responded to a GH stimulus, although dwarf mice presented higher GH activation levels. The phosphorylation of GSK-3, a substrate of Akt, increased upon hormone stimulation only in dwarf mice in both tissues. In contrast, no differences in the phosphorylation of mTOR, another substrate of Akt, were observed after GH stimulus, either in normal or dwarf mice in liver, while we were unable to determine mTOR in muscle. Protein content of GH-receptor and of the signaling mediators studied did not vary between normal and dwarf animals in the assessed

ERK, Akt, and STAT5 are differentially activated by the two growth hormone receptors subtypes of a teleost fish (Oncorhynchus mykiss

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Jeffrey eKittilson

2011-09-01

Full Text Available Previously, we found that the teleost fish, rainbow trout, possesses two growth hormone receptor (GHR subtypes that display distinct ligand binding and agonist-induced regulation features. In this study, we used Chinese hamster ovary-K1 cells stably transfected individually with the two trout GHR subtypes, GHR1 and GHR2, to elucidate receptor-effector pathway linkages. Growth hormone (GH stimulated rapid (5-10 min phosphorylation of ERK, Akt, JAk2, and STAT5 in both GHR1- and GHR2-expressing cells; however; STAT5 was activated to a greater extent through GHR1 than through GHR2, whereas ERK and Akt were activated to a greater through GHR2 than through GHR1. Although blockade of the ERK pathway had no effect on the activation of Akt, inhibition of PI3k-Akt partially prevented activation of ERK, suggesting cross-talk between the ERK and PI3K-Akt pathways. JAK2 inhibition completely blocked activation of ERK, Akt, and STAT5, suggesting that all of these pathways link to GHR1 and GHR2 via JAK2. These findings establish important receptor-effector pathway linkages and suggest that the GHR subtypes of teleost fish may be functionally distinct.

Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma

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Dimberg Lina Y

2012-07-01

Full Text Available Abstract Background Multiple myeloma (MM is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM. Methods To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS. Results To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA, geldanamycin (17-AAG, doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered. Conclusion We conclude that Stat1 alters IL-6

Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma

International Nuclear Information System (INIS)

Dimberg, Lina Y; Nilsson, Kenneth; Öberg, Fredrik; Wiklund, Helena Jernberg; Dimberg, Anna; Ivarsson, Karolina; Fryknäs, Mårten; Rickardson, Linda; Tobin, Gerard; Ekman, Simon; Larsson, Rolf; Gullberg, Urban

2012-01-01

Multiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM. To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS). To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered. We conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

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Le Coquil Stéphanie

2011-04-01

Full Text Available Abstract Background The transcription factor STAT3 (signal transducer and activator of transcription 3 is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. Results The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. Conclusions The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is

STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-{alpha}2b peptide

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Blank, Viviana C.; Pena, Clara [Institute of Biochemistry and Biophysics (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956-C1113AAD Buenos Aires (Argentina); Roguin, Leonor P., E-mail: rvroguin@qb.ffyb.uba.ar [Institute of Biochemistry and Biophysics (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956-C1113AAD Buenos Aires (Argentina)

2010-02-15

In the search of mimetic peptides of the interferon-{alpha}2b molecule (IFN-{alpha}2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-{alpha}2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-{alpha}2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-α2b peptide

International Nuclear Information System (INIS)

Blank, Viviana C.; Pena, Clara; Roguin, Leonor P.

2010-01-01

In the search of mimetic peptides of the interferon-α2b molecule (IFN-α2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-α2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-α2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

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Sustarsic, Elahu G; Junnila, Riia K; Kopchick, John J.

2013-01-01

cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found...

Growth hormone (GH) differentially regulates NF-kB activity in preadipocytes and macrophages: implications for GH's role in adipose tissue homeostasis in obesity.

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Kumar, P Anil; Chitra, P Swathi; Lu, Chunxia; Sobhanaditya, J; Menon, Ram

2014-06-01

Adipose tissue remodeling in obesity involves macrophage infiltration and chronic inflammation. NF-kB-mediated chronic inflammation of the adipose tissue is directly implicated in obesity-associated insulin resistance. We have investigated the effect of growth hormone (GH) on NF-kB activity in preadipocytes (3T3-F442A) and macrophages (J774A.1). Our studies indicate that whereas GH increases NF-kB activity in preadipocytes, it decreases NF-kB activity in macrophages. This differential response of NF-kB activity to GH correlates with the GH-dependent expression of a cadre of NF-kB-activated cytokines in these two cell types. Activation of NF-kB by GH in preadipocytes heightens inflammatory response by stimulating production of multiple cytokines including TNF-α, IL-6, and MCP-1, the mediators of both local and systemic insulin resistance and chemokines that recruit macrophages. Our studies also suggest differential regulation of miR132 and SIRT1 expression as a mechanism underlying the observed variance in GH-dependent NF-kB activity and altered cytokine profile in preadipocytes and macrophages. These findings further our understanding of the complex actions of GH on adipocytes and insulin sensitivity.

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

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Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

2010-01-01

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

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Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Liver-specific deletion of the signal transducer and activator of transcription 5 gene aggravates fatty liver in response to a high-fat diet in mice.

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Baik, Myunggi; Nam, Yoon Seok; Piao, Min Yu; Kang, Hyeok Joong; Park, Seung Ju; Lee, Jae-Hyuk

2016-03-01

Growth hormone (GH) signal is mediated by signal transducer and activator of transcription 5 (STAT5), which controls hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) is clinically associated with a deficiency in GH. This study was performed to understand the role of local STAT5 signaling on hepatic lipid and glucose metabolism utilizing liver-specific STAT5 gene deletion (STAT5 LKO) mice under both normal diet and high-fat diet (HFD) feeding conditions. STAT5 LKO induced hepatic steatosis under HFD feeding, while this change was not observed in mice on normal diet. STAT5 LKO caused hyperglycemia, hyperinsulinemia, hyperleptinemia and elevated free fatty acid and cholesterol concentrations under HFD feeding but induced only hyperglycemia on normal diet. At the molecular level, STAT5 LKO up-regulated the expression of genes involved in lipid uptake (CD36), very low-density lipoprotein receptor (VLDLR), lipogenic stearoyl-CoA desaturase and adipogenic peroxisome proliferator-activated receptor gamma, in both diet groups. In response to HFD feeding, further increases in CD36 and VLDLR expression were found in STAT5 LKO mice. In conclusion, our study suggests that low STAT5 signaling on normal diet predisposes STAT5 LKO mice to early development of fatty liver by hyperglycemia and activation of lipid uptake and adipogenesis. A deficiency in STAT5 signaling under HFD feeding deregulates hepatic and body glucose and lipid metabolism, leading to the development of hepatic steatosis. Our study indicates that low STAT5 signaling, due to low GH secretion, may increase a chance for NAFLD development in elderly people. Copyright © 2015 Elsevier Inc. All rights reserved.

The JAK-STAT transcriptional regulator, STAT-5, activates the ATM DNA damage pathway to induce HPV 31 genome amplification upon epithelial differentiation.

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Shiyuan Hong

Full Text Available High-risk human papillomavirus (HPV must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.

Activated STAT5 Confers Resistance to Intestinal Injury by Increasing Intestinal Stem Cell Proliferation and Regeneration

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Shila Gilbert

2015-02-01

Full Text Available Intestinal epithelial stem cells (IESCs control the intestinal homeostatic response to inflammation and regeneration. The underlying mechanisms are unclear. Cytokine-STAT5 signaling regulates intestinal epithelial homeostasis and responses to injury. We link STAT5 signaling to IESC replenishment upon injury by depletion or activation of Stat5 transcription factor. We found that depletion of Stat5 led to deregulation of IESC marker expression and decreased LGR5+ IESC proliferation. STAT5-deficient mice exhibited worse intestinal histology and impaired crypt regeneration after γ-irradiation. We generated a transgenic mouse model with inducible expression of constitutively active Stat5. In contrast to Stat5 depletion, activation of STAT5 increased IESC proliferation, accelerated crypt regeneration, and conferred resistance to intestinal injury. Furthermore, ectopic activation of STAT5 in mouse or human stem cells promoted LGR5+ IESC self-renewal. Accordingly, STAT5 promotes IESC proliferation and regeneration to mitigate intestinal inflammation. STAT5 is a functional therapeutic target to improve the IESC regenerative response to gut injury.

Kaempferol attenuates COX-2 expression in IL-6-induced macrophages and carrageenan-induced mouse paw edema by targeting STAT3 and NF-kB

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Anandita Basu

2017-10-01

Full Text Available Dietary polyphenols are reported to possess varied pharmacological activities, viz. antioxidant, anti-inflammatory, anti-cancer, anti-allergic actions. Here, we report the efficacy of Kaempferol (kae to attenuate expression of IL-6 induced cycloxygenase-2 (COX-2, an inducible isoform of cycloxygenase enzyme family that catalyzes synthesis of inflammatory mediators, prostanoids and prostaglandins. IL-6 is a pleiotropic cytokine involved in both acute and chronic inflammation. Our results showed that kae attenuated COX-2 expression at both mRNA and protein level in IL-6-induced THP1 macrophages. This attenuation of COX-2 expression by kae involved dose-dependent inhibition of phosphorylation of STAT3 (Tyr 705 and NF-kB p65 (Ser 536 leading to their deactivation and reduced nuclear localization in THP-1 macrophages. Moreover, kae modulates COX-2 expression as well as STAT3 and NF-kB activation in carrageenan-induced mouse paw edema model. RT-PCR and western blot analysis from paw tissues were harvested after kae injection (i.p followed by carrageenan-treatment in sub-plantar region of right hind paw. Results showed that kae attenuated COX-2 expression and STAT3 and NF-kB activation in carrageenan-induced mouse paw edema, suggesting that inhibition of both IL-6-STAT3-COX-2 and IL-6-NFkB-COX-2 axes by kae might be stimulus-independent. To understand binding affinity of kae with NF-kB and STAT3, docking analysis was performed using Patchdock server. From our findings, we observed strong binding affinity and transient interaction in both NF-kB/kae and STAT3/kae complexes. We noticed negative atomic contact energy and greater interface area for both the complexes. Selected complexes obtained from Patchdock were refined using Firedock online server which also suggested similar negative binding energy profile. It is plausible that kae attenuates COX-2 expression by directly binding to both STAT3 and NF-kB proteins and inhibiting their activation and

STAT5 Activation in the Dermal Papilla Is Important for Hair Follicle Growth Phase Induction.

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Legrand, Julien M D; Roy, Edwige; Ellis, Jonathan J; Francois, Mathias; Brooks, Andrew J; Khosrotehrani, Kiarash

2016-09-01

Hair follicles are skin appendages that undergo periods of growth (anagen), regression (catagen), and rest (telogen) regulated by their mesenchymal component, the dermal papilla (DP). On the basis of the reports of its specific expression in the DP, we investigated signal transducer and activator of transcription (STAT5) activation during hair development and cycling. STAT5 activation in the DP began in late catagen, reaching a peak in early anagen before disappearing for the rest of the cycle. This was confirmed by the expression profile of suppressor of cytokine signaling 2, a STAT5 target in the DP. This pattern of expression starts after the first postnatal hair cycle. Quantification of hair cycling using the Flash canonical Wnt signaling in vivo bioluminescence reporter found that conditional knockout of STAT5A/B in the DP targeted through Cre-recombinase under the control of the Sox18 promoter resulted in delayed anagen entry compared with control. Microarray analysis of STAT5 deletion versus control revealed key changes in tumor necrosis factor-α, Wnt, and fibroblast growth factor ligands, known for their role in inducing anagen entry. We conclude that STAT5 activation acts as a mesenchymal switch to trigger natural anagen entry in postdevelopmental hair follicle cycling. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells

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Seyung S. Chung

2017-01-01

Full Text Available There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, activated colorectal cancer cells to be more invasive and stem-like. Combined treatment of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3, STAT1, and NF-κB physically interacted upon the cytokine stimulation. STAT3 was bound to the promoter region of human telomerase reverse transcriptase (hTERT. IL-6 and TNF-α stimulation further enhanced STAT3 binding affinity. Stem cell marker Oct-4 was upregulated in colorectal cancer cells upon IL-6 and TNF-α stimulation. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6- and TNF-α-induced cancer cell invasion and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3, STAT1, and NF-κB interactions. Oct-4 expression was also downregulated by withaferin A inhibition. The binding of STAT3 to the hTERT promoter region and telomerase activity showed reduction with withaferin A treatments. Proinflammatory cytokine-induced cancer cell invasiveness is mediated by a STAT3-regulated mechanism in colorectal cancer cells. Our data suggest that withaferin A could be a promising anticancer agent that effectively inhibits the progression of colorectal cancer.

GH signaling in skeletal muscle and adipose tissue in healthy human subjects

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Vestergaard, Poul Frølund; Vendelbo, Mikkel Holm; Pedersen, Steen Bønnelykke

2014-01-01

in women when compared with men (P=0.01). IGF1, SOCS1, SOCS2, SOCS3, and CISH mRNA expression increased significantly in muscle after 120 min in all subjects with no impact of age and gender. GH-induced pSTAT5b correlated inversely with lean body mass (LBM; r=-0.56, P=0.01) and positively with the CISH m...

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

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Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Growth hormone (GH)-releasing activity of chicken GH-releasing hormone (GHRH) in chickens.

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Harvey, S; Gineste, C; Gaylinn, B D

2014-08-01

Two peptides with sequence similarities to growth hormone releasing hormone (GHRH) have been identified by analysis of the chicken genome. One of these peptides, chicken (c) GHRH-LP (like peptide) was previously found to poorly bind to chicken pituitary membranes or to cloned and expressed chicken GHRH receptors and had little, if any, growth hormone (GH)-releasing activity in vivo or in vitro. In contrast, a second more recently discovered peptide, cGHRH, does bind to cloned and expressed cGHRH receptors and increases cAMP activity in transfected cells. The possibility that this peptide may have in vivo GH-releasing activity was therefore assessed. The intravenous (i.v.) administration of cGHRH to immature chickens, at doses of 3-100 μg/kg, significantly increased circulating GH concentrations within 10 min of injection and the plasma GH levels remained elevated for at least 30 min after the injection of maximally effective doses. The plasma GH responses to cGHRH were comparable with those induced by human (h) or porcine (p) GHRH preparations and to that induced by thyrotropin releasing hormone (TRH). In marked contrast, the i.v. injection of cGHRH-LP had no significant effect on circulating GH concentrations in immature chicks. GH release was also increased from slaughterhouse chicken pituitary glands perifused for 5 min with cGHRH at doses of 0.1 μg/ml or 1.0 μg/ml, comparable with GH responses to hGHRH1-44. In contrast, the perifusion of chicken pituitary glands with cGHRH-LP had no significant effect on GH release. In summary, these results demonstrate that cGHRH has GH-releasing activity in chickens and support the possibility that it is the endogenous ligand of the cGHRH receptor. Copyright © 2014 Elsevier Inc. All rights reserved.

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells.

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Schauwecker, Suzanne M; Kim, J Julie; Licht, Jonathan D; Clevenger, Charles V

2017-02-10

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Peroxiredoxin 5 Protects TGF-β Induced Fibrosis by Inhibiting Stat3 Activation in Rat Kidney Interstitial Fibroblast Cells.

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Hoon-In Choi

Full Text Available Renal fibrosis is a common final pathway of end-stage kidney disease which is induced by aberrant accumulation of myofibroblasts. This process is triggered by reactive oxygen species (ROS and proinflammatory cytokines generated by various source of injured kidney cells. Peroxiredoxin 5 (Prdx5 is a thiol-dependent peroxidase that reduces oxidative stress by catalyzing intramolecular disulfide bonds. Along with its antioxidant effects, expression level of Prdx5 also was involved in inflammatory regulation by immune stimuli. However, the physiological effects and the underlying mechanisms of Prdx5 in renal fibrosis have not been fully characterized. Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO for 1 or 7 days. For the in vitro model, NRK49F cells, a rat kidney interstitial fibroblast cell lines, were treated with transforming growth factor β (TGF-β for 0, 1, 3, or 5 days. To access the involvement of its peroxidase activity in TGF-β induced renal fibrosis, wild type Prdx5 (WT and double mutant Prdx5 (DM, converted two active site cysteines at Cys 48 and Cys 152 residue to serine, were transiently expressed in NRK49F cells. The protein expression of Prdx5 was reduced in UUO kidneys. Upregulation of fibrotic markers, such as fibronectin and alpha-smooth muscle actin (α-SMA, declined at 5 days in time point of higher Prdx5 expression in TGF-β treated NRK49F cells. The overexpression of wild type Prdx5 by transient transfection in NRK49F cells attenuated the TGF-β induced upregulation of fibronectin and α-SMA. On the other hand, the transient transfection of double mutant Prdx5 did not prevent the activation of fibrotic markers. Overexpression of Prdx5 also suppressed the TGF-β induced upregulation of Stat3 phosphorylation, while phosphorylation of Smad 2/3 was unchanged. In conclusion, Prdx5 protects TGF-β induced fibrosis in NRK49F cells by modulating Stat3 activation in a peroxidase activity dependent manner.

MicroRNA-20b-5p inhibits platelet-derived growth factor-induced proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3.

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Tang, Jin; Luo, Lingying

2018-06-01

Pediatric asthma is still a health threat to the pediatric population in recent years. The airway remodeling induced by abnormal airway smooth muscle (ASM) cell proliferation is an important cause of asthma. MicroRNAs (miRNAs) are important regulators of ASM cell proliferation. Numerous studies have reported that miR-20b-5p is a critical regulator for cell proliferation. However, whether miR-20b-5p is involved in regulating ASM cell proliferation remains unknown. In this study, we aimed to investigate the potential role of miR-20b-5p in regulating the proliferation of fetal ASM cell induced by platelet-derived growth factor (PDGF). Here, we showed that miR-20b-5p was significantly decreased in fetal ASM cells treated with PDGF. Biological experiments showed that the overexpression of miR-20b-5p inhibited the proliferation while miR-20b-5p inhibition markedly promoted the proliferation of fetal ASM cells. Bioinformatics analysis and luciferase reporter assay showed that miR-20b-5p directly targeted the 3'-UTR of signal transducer and activator of transcription 3 (STAT3). Further data showed that miR-20b-5p negatively regulated the expression of STAT3 in fetal ASM cells. Moreover, miR-20b-5p regulates the transcriptional activity of STAT3 in fetal ASM cells. Overexpression of STAT3 reversed the inhibitory effect of miR-20b-5p overexpression on fetal ASM cell proliferation while the knockdown of STAT3 abrogated the promoted effect of miR-20b-5p inhibition on fetal ASM cell proliferation. Overall, our results show that miR-20b-5p impedes PDGF-induced proliferation of fetal ASM cells through targeting STAT3. Our study suggests that miR-20b-5p may play an important role in airway remodeling during asthma and suggests that miR-20b-5p may serve as a potential therapeutic target for pediatric asthma. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

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Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

2016-11-15

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma.

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Tai, Wei-Tien; Chu, Pei-Yi; Shiau, Chung-Wai; Chen, Yao-Li; Li, Yong-Shi; Hung, Man-Hsin; Chen, Li-Ju; Chen, Pei-Lung; Su, Jung-Chen; Lin, Ping-Yi; Yu, Hui-Chuan; Chen, Kuen-Feng

2014-11-15

Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib ([Formula: see text] mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib. ©2014 American Association for Cancer Research.

Inhibition of STAT3 activity delays obesity-induced thyroid carcinogenesis in a mouse model

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Park, Jeong Won; Han, Cho Rong; Zhao, Li; Willingham, Mark C.; Cheng, Sheue-yann

2015-01-01

Compelling epidemiologic studies indicate that obesity is a risk factor for many human cancers, including thyroid cancer. In recent decades, the incidence of thyroid cancer has dramatically increased along with a marked rise in obesity prevalence. We previously demonstrated that a high fat diet (HFD) effectively induced the obese phenotype in a mouse model of thyroid cancer (ThrbPV/PVPten+/− mice). Moreover, HFD activates the STAT3 signal pathway to promote more aggressive tumor phenotypes. The aim of the present study was to evaluate the effect of S3I-201, a specific inhibitor of STAT3 activity, on HFD-induced aggressive cancer progression in the mouse model of thyroid cancer. Wild type and ThrbPV/PVPten+/− mice were treated with HFD together with S3I-201 or vehicle-only as controls. We assessed the effects of S3I-201 on HFD-induced thyroid cancer progression, the leptin-JAK2-STAT3 signaling pathway, and key regulators of epithelial-mesenchymal transition. S3I-201 effectively inhibited HFD-induced aberrant activation of STAT3 and its downstream targets to markedly inhibit thyroid tumor growth and to prolong survival. Decreased protein levels of cyclins D1 and B1, cyclin dependent kinase (CDK) 4, CDK 6, and phosphorylated retinoblastoma protein led to the inhibition of tumor cell proliferation in S3I-201-treated ThrbPV/PVPten+/− mice. Reduced occurrence of vascular invasion and blocking of anaplasia and lung metastasis in thyroid tumors of S3I-201-treated ThrbPV/PVPten+/− mice were mediated via decreased expression of vimentin and matrix metalloproteinases, two key effectors of epithelial-mesenchymal transition. The present findings suggest that inhibition of the STAT3 activity would be a novel treatment strategy for obesity-induced thyroid cancer. PMID:26552408

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma

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Kopp, Katharina L; Ralfkiaer, Ulrik; Gjerdrum, Lise Mette R

2013-01-01

show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells...... of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC.   In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data...

Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

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Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

2013-03-15

Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

Gastric Cancer Cell Proliferation and Survival Is Enabled by a Cyclophilin B/STAT3/miR-520d-5p Signaling Feedback Loop.

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Li, Ting; Guo, Hanqing; Zhao, Xiaodi; Jin, Jiang; Zhang, Lifeng; Li, Hong; Lu, Yuanyuan; Nie, Yongzhan; Wu, Kaichun; Shi, Yongquan; Fan, Daiming

2017-03-01

Molecular links between inflammation and cancer remain obscure despite their great pathogenic significance. The JAK2/STAT3 pathway activated by IL6 and other proinflammatory cytokines has garnered attention as a pivotal link in cancer pathogenesis, but the basis for its activation in cancer cells is not understood. Here we report that an IL6-triggered feedback loop involving STAT3-mediated suppression of miR-520d-5p and upregulation of its downstream target cyclophilin B (CypB) regulate the growth and survival of gastric cancer cells. In clinical specimens of gastric cancer, we documented increased expression of CypB and activation of STAT3. Mechanistic investigations identified miR-520d-5p as a regulator of CypB mRNA levels. This signaling axis regulated gastric cancer growth by modulating phosphorylation of STAT3. Furthermore, miR-520d-5p was identified as a direct STAT3 target and IL6-mediated inhibition of miR-520d-5p relied upon STAT3 activity. Our findings define a positive feedback loop that drives gastric carcinogenesis as influenced by H. pylori infections that involve proinflammatory IL6 stimulation. Cancer Res; 77(5); 1227-40. ©2016 AACR . ©2016 American Association for Cancer Research.

Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4+CD25+ regulatory T cells

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Jin, Yulan; Purohit, Sharad; Chen, Xueqin; Yi, Bing; She, Jin-Xiong

2012-01-01

Highlights: ► This is the first study to provide direct evidence of the role of Stat5b in NOD mice. ► Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. ► This protection may be mediated by the up-regulation of CD4 + CD25 + Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4 + T cells and especially CD8 + T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4 + and CD8 + T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-γ, TNF-α and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4 + CD25 + regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4 + CD25 + regulatory T cells.

GH signaling in human adipose and muscle tissue during 'feast and famine': amplification of exercise stimulation following fasting compared to glucose administration.

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Vendelbo, Mikkel H; Christensen, Britt; Grønbæk, Solbritt B; Høgild, Morten; Madsen, Michael; Pedersen, Steen B; Jørgensen, Jens O L; Jessen, Niels; Møller, Niels

2015-09-01

Fasting and exercise stimulates, whereas glucose suppresses GH secretion, but it is uncertain how these conditions impact GH signaling in peripheral tissues. To test the original 'feast and famine hypothesis' by Rabinowitz and Zierler, according to which the metabolic effects of GH are predominant during fasting, we specifically hypothesized that fasting and exercise act in synergy to increase STAT-5b target gene expression. Eight healthy men were studied on two occasions in relation to a 1 h exercise bout: i) with a concomitant i.v. glucose infusion ('feast') and ii) after a 36 h fast ('famine'). Muscle and fat biopsy specimens were obtained before, immediately after, and 30 min after exercise. GH increased during exercise on both examination days and this effect was amplified by fasting, and free fatty acid (FFA) levels increased after fasting. STAT-5b phosphorylation increased similarly following exercise on both occasions. In adipose tissue, suppressors of cytokine signaling 1 (SOCS1) and SOCS2 were increased after exercise on the fasting day and both fasting and exercise increased cytokine inducible SH2-containing protein (CISH). In muscle, SOCS2 and CISH mRNA were persistently increased after fasting. Muscle SOCS1, SOCS3, and CISH mRNA expression increased, whereas SOCS2 decreased after exercise on both examination days. This study demonstrates that fasting and exercise act in tandem to amplify STAT-5b target gene expression (SOCS and CISH) in adipose and muscle tissue in accordance with the 'feast and famine hypothesis'; the adipose tissue signaling responses, which hitherto have not been scrutinized, may play a particular role in promoting FFA mobilization. © 2015 European Society of Endocrinology.

Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

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Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

2009-09-01

DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway.

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Park, Ji-Hyun; Seo, Young Ho; Jang, Jung-Hee; Jeong, Chul-Ho; Lee, Sooyeun; Park, Byoungduck

2017-12-11

Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway. We used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways. METH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

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Takuji Oyama

Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

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Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

2013-01-01

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

STAT3 activation is associated with cerebrospinal fluid interleukin-10 (IL-10) in primary central nervous system diffuse large B cell lymphoma.

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Mizowaki, Takashi; Sasayama, Takashi; Tanaka, Kazuhiro; Mizukawa, Katsu; Takata, Kumi; Nakamizo, Satoshi; Tanaka, Hirotomo; Nagashima, Hiroaki; Nishihara, Masamitsu; Hirose, Takanori; Itoh, Tomoo; Kohmura, Eiji

2015-09-01

Signal transducers and activators of transcription 3 (STAT3) are activated by various cytokines and oncogenes; however, the activity and pathogenesis of STAT3 in diffuse large B cell lymphoma of the central nervous system have not been thoroughly elucidated. We investigated the phosphorylation levels of STAT3 in 40 specimens of primary central nervous system diffuse large B-cell lymphoma (PCNS DLBCL) and analyzed the association between phsopho-STAT3 (pSTAT3) expression and cerebrospinal fluid (CSF) concentration of interleukin-10 (IL-10) or IL-6. Immunohistochemistry and Western blot analysis revealed that most of the specimens in PCNS DLBCL expressed pSTST3 protein, and a strong phosphorylation levels of STAT3 was statistically associated with high CSF IL-10 levels, but not with CSF IL-6 levels. Next, we demonstrated that recombinant IL-10 and CSF containing IL-10 induced the phosphorylation of STAT3 in PCNS DLBCL cells. Furthermore, molecular subtype classified by Hans' algorithm was correlated with pSTAT3 expression levels and CSF IL-10 levels. These results suggest that the STAT3 activity is correlated with CSF IL-10 level, which is a useful marker for STAT3 activity in PCNS DLBCLs.

Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

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Jin, Yulan; Purohit, Sharad [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States); Chen, Xueqin; Yi, Bing [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); She, Jin-Xiong, E-mail: jshe@georgiahealth.edu [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States)

2012-08-10

Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.

ONC201 selectively induces apoptosis in cutaneous T-cell lymphoma cells via activating pro-apoptotic integrated stress response and inactivating JAK/STAT and NF-κB pathways.

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Ni, Xiao; Zhang, Xiang; Hu, Cheng-Hui; Langridge, Timothy; Tarapore, Rohinton S; Allen, Joshua E; Oster, Wolfgang; Duvic, Madeleine

2017-09-22

Cutaneous T-cell lymphomas (CTCLs) are extremely symptomatic and still incurable, and more effective and less toxic therapies are urgently needed. ONC201, an imipridone compound, has shown efficacy in pre-clinical studies in multiple advanced cancers. This study was to evaluate the anti-tumor activity of ONC201 on CTCL cells. The effect of ONC201 on the cell growth and apoptosis were evaluated in CTCL cell lines (n=8) and primary CD4 + malignant T cells isolated from CTCL patients (n=5). ONC201 showed a time-dependent cell growth inhibition in all treated cell lines with a concentration range of 1.25-10.0 μM. ONC201 also induced apoptosis in tested cells with a narrow concentration range of 2.5-10.0 μM, evidenced by increased Annexin V + cells, accompanied by accumulated sub-G1 portions. ONC201 only induced apoptosis in CD4 + malignant T cells, not in normal CD4 + T cells. The activating transcription factor 4 (ATF4), a hallmark of integrated stress response, was upregulated in response to ONC201 whereas Akt was downregulated. In addition, molecules in JAK/STAT and NF-κB pathways, as well as IL-32β, were downregulated following ONC201 treatment. Thus, ONC201 exerts a potent and selective anti-tumor effect on CTCL cells. Its efficacy may involve activating integrated stress response through ATF4 and inactivating JAK/STAT and NF-κB pathways.

Stat5 phosphorylation is responsible for the excessive potency of HB-EGF.

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Heo, Jeongyeon; Kim, Jae Geun; Kim, Sunghwan; Kang, Hara

2017-12-23

Heparin-binding EGF-like growth factor (HB-EGF) is a potent growth factor involved in wound healing and tumorigenesis. Despite the sequence similarity between HB-EGF and EGF, HB-EGF induces cellular proliferation and migration more potently than EGF. However, the differential regulation by HB-EGF and EGF has not been thoroughly elucidated. In this study, we compared signaling pathways activated by HB-EGF and EGF to understand the details of the molecular mechanism of the high potency induced by HB-EGF. HB-EGF specifically induced the phosphorylation of EGFR-Y1045 and activated Stat5, which is responsible for promoting cell proliferation, and migration. The competition of phosphorylated EGFR-Y1045 inhibited Stat5 activation and consequently lowered the effect of HB-EGF on cell proliferation, suggesting that the phosphorylation of EGFR-Y1045 is essential for the activation of Stat5. The phosphorylation of EGFR-Y1045 and Stat5 induced by HB-EGF was prevented by sequestering the heparin-binding domain, suggesting that the heparin-binding domain is critical for HB-EGF-mediated signaling and cellular responses. In conclusion, the heparin-binding domain of HB-EGF was responsible for EGFR-mediated Stat5 activation, resulting in a more potent cellular proliferation, and migration than that mediated by EGF. This molecular mechanism is useful for understanding ligand-specific EGFR signaling and developing biomedicines for wound healing or cancer therapy. © 2017 Wiley Periodicals, Inc.

Single nucleotide polymorphism in the STAT5b gene is associated with body weight and reproductive traits of the Jinghai Yellow chicken.

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Zhao, X H; Wang, J Y; Zhang, G X; Wei, Y; Gu, Y P; Yu, Y B

2012-04-01

In our research, signal transducer and activator of transcription 5b (STAT5b) gene was studied as candidate gene associated with body weight and reproductive traits of Jinghai Yellow chicken. Single nucleotide polymorphisms (SNPs) of STAT5b gene were examined in both Jinghai Yellow chicken and three reference chicken populations including the Bian, Youxi and Arbor Acre chickens. Two SNPs (C-1591T and G-250A) were detected in the 5' flanking region of STAT5b gene. Association indicated that the C-1591T mutation is significantly associated with age at fist egg, The G-250A mutation is significantly related with hatch weight and body weight at 300 days. Additionally four STAT5b haplotypes (H1, CG; H2, TG; H3, AC and H4, TA) and their frequency distributions were estimated using the phase program. Diplotype H3H4 is dominant for 8, 16 week-age-weight and body weight at first egg. Thus STAT5b gene may be served as a potential genetic marker for growth and reproduction traits evaluation of the Jinghai Yellow chicken. This study will provide valuable information for the protection and breeding of Jinghai Yellow chicken.

Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.

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Rong Wang

Full Text Available Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme.

An SH2 domain model of STAT5 in complex with phospho-peptides define "STAT5 Binding Signatures".

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Gianti, Eleonora; Zauhar, Randy J

2015-05-01

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

Science.gov (United States)

Gianti, Eleonora; Zauhar, Randy J.

2015-05-01

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

STAT6 silencing induces hepatocellular carcinoma-derived cell apoptosis and growth inhibition by decreasing the RANKL expression.

Science.gov (United States)

Qing, Tian; Yamin, Zhang; Guijie, Wang; Yan, Jin; Zhongyang, Shen

2017-08-01

Signal transducer and activator of transcription-6 (STAT6) is highly expressed in various human cancers and considered a regulator of multiple biological processes in cancers, including cell apoptosis. Evidence has indicated that STAT6 predicts a worse prognosis in hepatocellular carcinoma (HCC) patients. The objective of this study was to investigate the effects and mechanism of STAT6 in human HCC cells. We found that STAT6 silencing significantly inhibited HepG2 and Hep3B cell survival and proliferation. We observed that depletion of STAT6 increased HepG2 and Hep3B cell apoptosis by using a histone DNA ELISA detection kit. STAT6 silencing induced expression of apoptosis-associated genes Bax and caspase-3/7 and inhibited anti-apoptosis gene Bcl-2 levels. We also observed that STAT6 silencing downregulated the expression of receptor activator of NF-κB ligand (RANKL). Our results demonstrated that treatment with pcDNA3.1-RANKL abolished STAT6 depletion-induced HepG2 and Hep3B cell apoptosis and growth inhibition. Based on these findings, we believe that RANKL plays a major role in STAT6-induced HCC cell apoptosis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

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Matikainen, S; Ronni, T; Lehtonen, A; Sareneva, T; Melén, K; Nordling, S; Levy, D E; Julkunen, I

1997-06-01

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Gladiolus hybridus ABSCISIC ACID INSENSITIVE 5 (GhABI5) is an important transcription factor in ABA signaling that can enhance Gladiolus corm dormancy and Arabidopsis seed dormancy.

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Wu, Jian; Seng, Shanshan; Sui, Juanjuan; Vonapartis, Eliana; Luo, Xian; Gong, Benhe; Liu, Chen; Wu, Chenyu; Liu, Chao; Zhang, Fengqin; He, Junna; Yi, Mingfang

2015-01-01

The phytohormone abscisic acid (ABA) regulates plant development and is crucial for abiotic stress response. In this study, cold storage contributes to reducing endogenous ABA content, resulting in dormancy breaking of Gladiolus. The ABA inhibitor fluridone also promotes germination, suggesting that ABA is an important hormone that regulates corm dormancy. Here, we report the identification and functional characterization of the Gladiolus ABI5 homolog (GhABI5), which is a basic leucine zipper motif transcriptional factor (TF). GhABI5 is expressed in dormant vegetative organs (corm, cormel, and stolon) as well as in reproductive organs (stamen), and it is up-regulated by ABA or drought. Complementation analysis reveals that GhABI5 rescues the ABA insensitivity of abi5-3 during seed germination and induces the expression of downstream ABA response genes in Arabidopsis thaliana (EM1, EM6, and RD29B). Down-regulation of GhABI5 in dormant cormels via virus induced gene silence promotes sprouting and reduces the expression of downstream genes (GhLEA and GhRD29B). The results of this study reveal that GhABI5 regulates bud dormancy (vegetative organ) in Gladiolus in addition to its well-studied function in Arabidopsis seeds (reproductive organ).

Gladiolus hybridus ABSCISIC ACID INSENSITIVE 5 (GhABI5 is an important transcription factor in ABA signaling that can enhance Gladiolus corm dormancy and Arabidopsis seed dormancy.

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Jian eWu

2015-11-01

Full Text Available The phytohormone abscisic acid (ABA regulates plant development and is crucial for abiotic stress response. In this study, cold storage contributes to reducing endogenous ABA content, resulting in dormancy breaking of Gladiolus. The ABA inhibitor fluridone also promotes germination, suggesting that ABA is an important hormone that regulates corm dormancy. Here, we report the identification and functional characterization of the Gladiolus ABI5 homolog (GhABI5, which is a basic leucine zipper motif transcriptional factor (TF. GhABI5 is expressed in dormant vegetative organs (corm, cormel and stolon as well as in reproductive organs (stamen, and it is up-regulated by ABA or drought. Complementation analysis reveals that GhABI5 rescues the ABA insensitivity of abi5-3 during seed germination and induces the expression of downstream ABA response genes in Arabidopsis thaliana (EM1, EM6 and RD29B. Down-regulation of GhABI5 in dormant cormels via Virus Induced Gene Silence (VIGS promotes sprouting and reduces the expression of downstream genes (GhLEA and GhRD29B. The results of this study reveal that GhABI5 regulates bud dormancy (vegetative organ in Gladiolus in addition to its well-studied function in Arabidopsis seeds (reproductive organ.

Constitutive STAT5 Activation Correlates With Better Survival in Cervical Cancer Patients Treated With Radiation Therapy

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Chen, Helen H.W. [Department of Radiation Oncology, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Chou, Cheng-Yang [Department of Obstetrics and Gynecology, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Wu, Yuan-Hua; Hsueh, Wei-Ting; Hsu, Chiung-Hui [Department of Radiation Oncology, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Guo, How-Ran [Department of Environmental and Occupational Health, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Lee, Wen-Ying, E-mail: 7707@so-net.net.tw [Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan (China) and Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Su, Wu-Chou, E-mail: sunnysu@mail.ncku.edu.tw [Department of Internal Medicine, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China)

2012-02-01

Purpose: Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3, and STAT5, have been detected in a wide variety of human primary tumors and have been demonstrated to directly contribute to oncogenesis. However, the expression pattern of these STATs in cervical carcinoma is still unknown, as is whether or not they have prognostic significance. This study investigated the expression patterns of STAT1, STAT3, and STAT5 in cervical cancer and their associations with clinical outcomes in patients treated with radical radiation therapy. Methods and Materials: A total of 165 consecutive patients with International Federation of Gynecology and Obstetrics (FIGO) Stages IB to IVA cervical cancer underwent radical radiation therapy, including external beam and/or high-dose-rate brachytherapy between 1989 and 2002. Immunohistochemical studies of their formalin-fixed, paraffin-embedded tissues were performed. Univariate and multivariate analyses were performed to identify and to evaluate the effects of these factors affecting patient survival. Results: Constitutive activations of STAT1, STAT3, and STAT5 were observed in 11%, 22%, and 61% of the participants, respectively. While STAT5 activation was associated with significantly better metastasis-free survival (p < 0.01) and overall survival (p = 0.04), STAT1 and STAT3 activation were not. Multivariate analyses showed that STAT5 activation, bulky tumor ({>=}4 cm), advanced stage (FIGO Stages III and IV), and brachytherapy (yes vs. no) were independent prognostic factors for cause-specific overall survival. None of the STATs was associated with local relapse. STAT5 activation (odds ratio = 0.29, 95% confidence interval = 0.13-0.63) and advanced stage (odds ratio = 2.54; 95% confidence interval = 1.03-6.26) were independent predictors of distant metastasis. Conclusions: This is the first report to provide the overall expression patterns and prognostic significance of

Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-κB-STAT3-directed gene expression

International Nuclear Information System (INIS)

Ivanov, Vladimir N.; Ghandhi, Shanaz A.; Zhou, Hongning; Huang, Sarah X.; Chai, Yunfei; Amundson, Sally A.; Hei, Tom K.

2011-01-01

Mitochondrial DNA depleted (ρ 0 ) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ + HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ 0 cells compared to ρ + HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ + HSF, but this response was substantially decreased in ρ 0 HSF. Suppression of the IKK-NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ + HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ 0 HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6-JAK2-STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.

Ghrelin- and GH-induced insulin resistance

DEFF Research Database (Denmark)

Vestergaard, Esben Thyssen; Krag, Morten B; Poulsen, Morten M

2013-01-01

Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects.......Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects....

Screening for MPL mutations in essential thrombocythemia and primary myelofibrosis: normal Mpl expression and absence of constitutive STAT3 and STAT5 activation in MPLW515L-positive platelets.

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Glembotsky, Ana C; Korin, Laura; Lev, Paola R; Chazarreta, Carlos D; Marta, Rosana F; Molinas, Felisa C; Heller, Paula G

2010-05-01

To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO). Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA. MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced. The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.

Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides

DEFF Research Database (Denmark)

Dilokpimol, Adiphol

Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til oligosakkarider, som potentielt kan fungere som prebiotika ved at stimulere væksten af...... omfatter karakterisering af de producerede enzymer samt cDNA kloning af formodet GH61 endo Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til...

Molecular and biochemical analyses of the GH44 module of CbMan5B/Cel44A, a bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii.

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Ye, Libin; Su, Xiaoyun; Schmitz, George E; Moon, Young Hwan; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

2012-10-01

A large polypeptide encoded in the genome of the thermophilic bacterium Caldicellulosiruptor bescii was determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol. 78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases from C. bescii led to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization by C. bescii.

Growth hormone (GH)-independent dimerization of GH receptor by a leucine zipper results in constitutive activation

DEFF Research Database (Denmark)

Behncken, S N; Billestrup, Nils; Brown, R

2000-01-01

Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers of the gro......Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers...

Prognostic significance of signal transducer and activator of transcription 5 and 5b expression in Epstein-Barr virus-positive patients with chronic lymphocytic leukemia.

Science.gov (United States)

Diamantopoulos, Panagiotis T; Sofotasiou, Maria; Georgoussi, Zafiroula; Giannakopoulou, Nefeli; Papadopoulou, Vasiliki; Galanopoulos, Athanasios; Kontandreopoulou, Elina; Zervakis, Panagiotis; Pallaki, Paschalina; Kalala, Fani; Kyrtsonis, Marie-Christine; Dimitrakopoulou, Aglaia; Vassilakopoulos, Theodoros; Angelopoulou, Maria; Spanakis, Nikolaos; Viniou, Nora-Athina

2016-09-01

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein-Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real-time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA. Western blotting was performed for total STAT5 and STAT5b in protein extracts. STAT5b was only expressed in patients (not in healthy subjects) and STAT5 but particularly STAT5b expression was correlated with the presence of the virus (77.3% vs. 51.2%, P = 0.006 for STAT5b) and to the expression of LMP1 (58.3% vs. 21.6%, P = 0.011 for STAT5b). Moreover, the expression of STAT5b and the presence of EBV and LMP1 were strongly negatively correlated with the overall survival of the patients (log-rank test P = 0.011, 0.015, 0.006, respectively). Double positive (for EBV and STAT5b) patients had the lowest overall survival (log-rank test P = 0.013). This is the first report of a survival disadvantage of EBV+ patients with CLL, and the first time that STAT5b expression is correlated with survival. The correlation of STAT5 expression with the presence of the virus, along with our survival correlations defines a subgroup of patients with CLL that may benefit from anti-STAT agents. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

The Chemopreventive Phytochemical Moringin Isolated from Moringa oleifera Seeds Inhibits JAK/STAT Signaling.

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Carina Michl

Full Text Available Sulforaphane (SFN and moringin (GMG-ITC are edible isothiocyanates present as glucosinolate precursors in cruciferous vegetables and in the plant Moringa oleifera respectively, and recognized for their chemopreventive and medicinal properties. In contrast to the well-studied SFN, little is known about the molecular pathways targeted by GMG-ITC. We investigated the ability of GMG-ITC to inhibit essential signaling pathways that are frequently upregulated in cancer and immune disorders, such as JAK/STAT and NF-κB. We report for the first time that, similarly to SFN, GMG-ITC in the nanomolar range suppresses IL-3-induced expression of STAT5 target genes. GMG-ITC, like SFN, does not inhibit STAT5 phosphorylation, suggesting a downstream inhibitory event. Interestingly, treatment with GMG-ITC or SFN had a limited inhibitory effect on IFNα-induced STAT1 and STAT2 activity, indicating that both isothiocyanates differentially target JAK/STAT signaling pathways. Furthermore, we showed that GMG-ITC in the micromolar range is a more potent inhibitor of TNF-induced NF-κB activity than SFN. Finally, using a cellular system mimicking constitutive active STAT5-induced cell transformation, we demonstrated that SFN can reverse the survival and growth advantage mediated by oncogenic STAT5 and triggers cell death, therefore providing experimental evidence of a cancer chemopreventive activity of SFN. This work thus identified STAT5, and to a lesser extent STAT1/STAT2, as novel targets of moringin. It also contributes to a better understanding of the biological activities of the dietary isothiocyanates GMG-ITC and SFN and further supports their apparent beneficial role in the prevention of chronic illnesses such as cancer, inflammatory diseases and immune disorders.

Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts

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Tsang, Chi Man; Cheung, Yuk Chun; Lui, Vivian Wai-Yan; Yip, Yim Ling; Zhang, Guitao; Lin, Victor Weitao; Cheung, Kenneth Chat-Pan; Feng, Yibin; Tsao, Sai Wah

2013-01-01

Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC

STAT4 gene polymorphism and risk of chronic hepatitis B-induced hepatocellular carcinoma.

Science.gov (United States)

Zhao, Xiangqian; Jiang, Kai; Liang, Bin; Huang, Xiaoqiang

2015-01-01

STAT4 is a latent cytosolic factor that encodes a transcription factor transmitting signals stimulated by cytokines. Previous studies with different study designs in diverse ethnic populations have assessed the influence of STAT4 rs7574865 polymorphism on HBV-induced HCC risk. The aim of the current study was to investigate the effects in a larger sample. The individual reports published up to Dec. 30, 2013 were systematically identified by searching the PubMed and Embase databases. To combine the OR and corresponding 95% CI, we used the fixed effects model during meta-analysis. Based on eight independent populations with a total of 5,719 cases and 6,525 controls, we found a slightly reduced risk of HBV-induced HCC in individuals with the minor T allele compared with individuals with the common G allele (T versus G: OR = 0.87, 95% CI = 0.82-0.91, P(Het) = 0.974). Similar reductions were also indicated in all subgroups. The combined data indicate that STAT4 rs7574865 polymorphism may be associated with significantly reduced risk of HBV-induced HCC in Asian.

Growth hormone (GH) binding and effects of GH analogs in transgenic mice

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Bartke, A.; Steger, R.W. [Southern Illinois Univ., Carbondale, IL (United States); Turyn, D. [UBA-CONICET, Buenos Aires (Argentina)] [and others

1994-12-31

Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb{sub 2} cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo. 35 refs.

A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

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Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

1996-09-01

Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

Active Components with Inhibitory Activities on IFN-γ/STAT1 and IL-6/STAT3 Signaling Pathways from Caulis Trachelospermi

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Xiao-Ting Liu

2014-08-01

Full Text Available Initial investigation for new active herbal extract with inhibiting activity on JAK/STAT signaling pathway revealed that the extract of Caulis Trachelospermi, which was separated by 80% alcohol extraction and subsequent HP-20 macroporous resin column chromatography, was founded to strongly inhibit IFN-γ-induced STAT1-responsive luciferase activity (IFN-γ/STAT1 with IC50 value of 2.43 μg/mL as well as inhibiting IL-6-induced STAT3-responsive luciferase activity (IL-6/STAT3 with IC50 value of 1.38 μg/mL. Subsequent study on its active components led to the isolation and identification of two new dibenzylbutyrolactone lignans named 4-demethyltraxillaside (1 and nortrachelogenin 4-O-β-d-glucopyranoside (2, together with six known compounds. The lignan compounds 1–4 together with other lignan compounds isolated in previous study were tested the activities on IFN-γ/STAT1 and IL-6/STAT3 pathways. The following result showed that the main components trachelogenin and arctigenin had corresponding activities on IFN-γ/STAT1 pathway with IC50 values of 3.14 μM and 9.46 μM as well as trachelogenin, arctigenin and matairesinol strongly inhibiting IL-6/STAT3 pathway with IC50 values of 3.63 μM, 6.47 μM and 2.92 μM, respectively.

A novel small molecular STAT3 inhibitor, LY5, inhibits cell viability, cell migration, and angiogenesis in medulloblastoma cells.

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Xiao, Hui; Bid, Hemant Kumar; Jou, David; Wu, Xiaojuan; Yu, Wenying; Li, Chenglong; Houghton, Peter J; Lin, Jiayuh

2015-02-06

Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional diversity for biomass deconstruction in family 5 subfamily 5 (GH5_5) of fungal endo-β1,4-glucanases.

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Li, Bingyao; Walton, Jonathan D

2017-05-01

Endo-β1,4-glucanases in glycosyl hydrolase family 5 (GH5) are ubiquitous enzymes in the multicellular fungi and are common components of enzyme cocktails for biomass conversion. We recently showed that an endo-glucanase of subfamily 5 of GH5 (GH5_5) from Sporotrichum thermophile (StCel5A) was more effective at releasing glucose from pretreated corn stover, when part of an eight-component synthetic enzyme mixture, compared to its closely related counterpart from Trichoderma reesei, TrCel5A. StCel5A and TrCel5A belong to different clades of GH5_5 (GH5_5_1 and GH5_5_2, respectively). To test whether the superior activity of StCel5A was a general property of all enzymes in the GH5_5_2 clade, StCel5A, TrCel5A, and two additional members of each subfamily were expressed in a common host that had been engineered to suppress its native cellulases (T. reesei Δxyr1) and compared against each other alone on pure substrates, in synthetic mixtures on pure substrates, and against each other in synthetic mixtures on real biomass. The results indicated that superiority is a unique property of StCel5A and not of GH5_5_2 generally. The six Cel5A enzymes had significant differences in relative activities on different substrates, in specific activities, and in sensitivities to mannan inhibition. Importantly, the behavior of the six endo-glucanases on pure cellulose substrates did not predict their behavior in combination with other cellulolytic enzymes on a real lignocellulosic biomass substrate.

Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia.

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Tsunekawa, Taku; Banno, Ryoichi; Mizoguchi, Akira; Sugiyama, Mariko; Tominaga, Takashi; Onoue, Takeshi; Hagiwara, Daisuke; Ito, Yoshihiro; Iwama, Shintaro; Goto, Motomitsu; Suga, Hidetaka; Sugimura, Yoshihisa; Arima, Hiroshi

2017-02-01

Protein tyrosine phosphatase 1B (PTP1B) regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD), remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3) was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO) on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT). In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Differential binding activity of the transcription factor LIL-Stat in immature and differentiated normal and leukemic myeloid cells

NARCIS (Netherlands)

Tuyt, LML; Bregman, K; Lummen, C; Dokter, WHA; Vellenga, E

1998-01-01

Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients,

Lactococcus lactis KR-050L inhibit IL-6/STAT3 activation.

Science.gov (United States)

Hwang, J T; Jang, H-J; Kim, J H; Park, C S; Kim, Y; Lim, C-H; Lee, S W; Rho, M-C

2017-05-01

The purpose of this study was to investigate IL-6/STAT3 inhibitory activity using lactic acid bacteria (LABs) isolated from Gajuknamu kimchi. Six LABs were isolated from Gajuknamu kimchi and identified through 16S rRNA sequencing. Among them, the culture broth of Lactococcus lactis KR-050L inhibited IL-6-induced STAT3 luciferase activity. Fifteen compounds were isolated from the EtOAc extract of culture broth though column chromatography and preparative high-performance liquid chromatography, and they were identified as 2,5-diketopipperazine structures by spectroscopic analyses (MS, 1 H- and 13 C-NMR). They also showed inhibitory activities on IL-6-induced STAT3 activation, and showed the different in activity according to the presence of a phenylalanine residue, hydroxyl groups and isometric structure. The six new LABs isolated from Gajuknamu kimchi, and Lc. lactis KR-050L was selected as candidate IL-6/STAT3 inhibitors. The activity levels of 15 2,5-DKPs isolated from Lc. lactis KR-050L were verified. This study constitutes the first attempt to isolate various LABs from Gajuknamu kimchi and to discover IL-6/STAT3 inhibitors in the EtOAc extract of Lc. lactis KR-050L culture broth. Moreover, our data provide useful biochemical information regarding the commercialization of Lc. lactis isolated from Gajuknamu kimchi as an approach to use functional foods for the treatment of various diseases via IL-6/STAT3 activation. © 2017 The Society for Applied Microbiology.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

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S.C.F. Olinto

2012-11-01

Full Text Available The amino acid arginine (Arg is a recognized secretagogue of growth hormone (GH, and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO, which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g were removed, divided into two halves, pooled (three hemi-pituitaries and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM, the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM and a cyclic guanosine monophosphate (cGMP analogue (8-Br-cGMP, 1 mM increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS inhibitor, 55 mM abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

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Olinto, S.C.F. [Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Ituiutaba, MG (Brazil); Adrião, M.G. [Departamento de Morfologia e Fisiologia, Universidade Federal Rural de Pernambuco, Recife, PE (Brazil); Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)

2012-06-01

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

International Nuclear Information System (INIS)

Olinto, S.C.F.; Adrião, M.G.; Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T.

2012-01-01

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression

Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia

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Taku Tsunekawa

2017-02-01

Full Text Available Protein tyrosine phosphatase 1B (PTP1B regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD, remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3 was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT. In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions.

Direct targets of pSTAT5 signalling in erythropoiesis.

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Kevin R Gillinder

Full Text Available Erythropoietin (EPO acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25, epigenetic regulation (Suv420h2, and EpoR turnover (Clint1/EpsinR. Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.

Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia

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Rajasingh, Johnson; Raikwar, Himanshu P.; Muthian, Gladson; Johnson, Caroline; Bright, John J.

2006-01-01

Adult T cell leukemia is an aggressive and frequently fatal malignancy that expressess constitutively activated growth-signaling pathways in association with deregulated growth and resistance to apoptosis. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment, isolated from the rhizomes of the plant Curcuma longa that has traditionally been used in the treatment of injury and inflammation. But the effect and mechanism of action of curcumin on T cell leukemia is not known. To investigate the antitumor activity of curcumin in T cell leukemia, we examined its effect on constitutive phosphorylation of JAK and STAT proteins, proliferation, and apoptosis in HTLV-I-transformed T cell lines. HTLV-I-transformed T cell leukemia lines, MT-2, HuT-102, and SLB-1, express constitutively phosphorylated JAK3, TYK2, STAT3, and STAT5 signaling proteins. In vitro treatment with curcumin induced a dose-dependent decrease in JAK and STAT phosphorylation resulting in the induction of growth-arrest and apoptosis in T cell leukemia. The induction of growth-arrest and apoptosis in association with the blockade of constitutively active JAK-STAT pathway suggests this be a mechanism by which curcumin induces antitumor activity in T cell leukemia

Inhibition of interleukin-3- and interferon- α-induced JAK/STAT signaling by the synthetic α-X-2',3,4,4'-tetramethoxychalcones α-Br-TMC and α-CF3-TMC.

Science.gov (United States)

Jobst, Belinda; Weigl, Julia; Michl, Carina; Vivarelli, Fabio; Pinz, Sophia; Amslinger, Sabine; Rascle, Anne

2016-11-01

The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2',3,4,4'-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.

Effects of sexually dimorphic growth hormone secretory patterns on arachidonic acid metabolizing enzymes in rodent heart

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Zhang, Furong; Yu, Xuming [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); He, Chunyan [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Ouyang, Xiufang; Wu, Jinhua; Li, Jie; Zhang, Junjie; Duan, Xuejiao [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Wan, Yu [Department of Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Yue, Jiang, E-mail: yuejiang@whu.edu.cn [Department of Pharmacology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

2015-12-15

The arachidonic acid (AA) metabolizing enzymes are the potential therapeutic targets of cardiovascular diseases (CVDs). As sex differences have been shown in the risk and outcome of CVDs, we investigated the regulation of heart AA metabolizing enzymes (COXs, LOXs, and CYPs) by sex-dependent growth hormone (GH) secretory patterns. The pulsatile (masculine) GH secretion at a physiological concentration decreased CYP1A1 and CYP2J3 mRNA levels more efficiently in the H9c2 cells compared with the constant (feminine) GH secretion; however, CYP1B1 mRNA levels were higher following the pulsatile GH secretion. Sex differences in CYP1A1, CYP1B1, and CYP2J11 mRNA levels were observed in both the wild-type and GHR deficient mice. No sex differences in the mRNA levels of COXs, LOXs, or CYP2E1 were observed in the wild-type mice. The constant GH infusion induced heart CYP1A1 and CYP2J11, and decreased CYP1B1 in the male C57/B6 mice constantly infused with GH (0.4 μg/h, 7 days). The activity of rat Cyp2j3 promoter was inhibited by the STAT5B protein, but was activated by C/EBPα (CEBPA). Compared with the constant GH administration, the levels of the nuclear phosphorylated STAT5B protein and its binding to the rat Cyp2j3 promoter were higher following the pulsatile GH administration. The constant GH infusion decreased the binding of the nuclear phosphorylated STAT5B protein to the mouse Cyp2j11 promoter. The data suggest the sexually dimorphic transcription of heart AA metabolizing enzymes, which might alter the risk and outcome of CVDs. GHR-STAT5B signal transduction pathway may be involved in the sex difference in heart CYP2J levels. - Highlights: • The transcription of heart Cyp1a1, Cyp1b1 and Cyp2j genes is sexually dimorphic. • There are no sex differences in the mRNA levels of heart COXs, LOXs, or CYP2E1. • GHR-STAT5B pathway is involved in sexually dimorphic transcription of heart Cpy2j genes. • Heart CYPs-mediated metabolism pathway of arachidonic acid may be sex

Effects of sexually dimorphic growth hormone secretory patterns on arachidonic acid metabolizing enzymes in rodent heart

International Nuclear Information System (INIS)

Zhang, Furong; Yu, Xuming; He, Chunyan; Ouyang, Xiufang; Wu, Jinhua; Li, Jie; Zhang, Junjie; Duan, Xuejiao; Wan, Yu; Yue, Jiang

2015-01-01

The arachidonic acid (AA) metabolizing enzymes are the potential therapeutic targets of cardiovascular diseases (CVDs). As sex differences have been shown in the risk and outcome of CVDs, we investigated the regulation of heart AA metabolizing enzymes (COXs, LOXs, and CYPs) by sex-dependent growth hormone (GH) secretory patterns. The pulsatile (masculine) GH secretion at a physiological concentration decreased CYP1A1 and CYP2J3 mRNA levels more efficiently in the H9c2 cells compared with the constant (feminine) GH secretion; however, CYP1B1 mRNA levels were higher following the pulsatile GH secretion. Sex differences in CYP1A1, CYP1B1, and CYP2J11 mRNA levels were observed in both the wild-type and GHR deficient mice. No sex differences in the mRNA levels of COXs, LOXs, or CYP2E1 were observed in the wild-type mice. The constant GH infusion induced heart CYP1A1 and CYP2J11, and decreased CYP1B1 in the male C57/B6 mice constantly infused with GH (0.4 μg/h, 7 days). The activity of rat Cyp2j3 promoter was inhibited by the STAT5B protein, but was activated by C/EBPα (CEBPA). Compared with the constant GH administration, the levels of the nuclear phosphorylated STAT5B protein and its binding to the rat Cyp2j3 promoter were higher following the pulsatile GH administration. The constant GH infusion decreased the binding of the nuclear phosphorylated STAT5B protein to the mouse Cyp2j11 promoter. The data suggest the sexually dimorphic transcription of heart AA metabolizing enzymes, which might alter the risk and outcome of CVDs. GHR-STAT5B signal transduction pathway may be involved in the sex difference in heart CYP2J levels. - Highlights: • The transcription of heart Cyp1a1, Cyp1b1 and Cyp2j genes is sexually dimorphic. • There are no sex differences in the mRNA levels of heart COXs, LOXs, or CYP2E1. • GHR-STAT5B pathway is involved in sexually dimorphic transcription of heart Cpy2j genes. • Heart CYPs-mediated metabolism pathway of arachidonic acid may be sex

The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney

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O’Brown, Zach K.; Van Nostrand, Eric L.; Higgins, John P.; Kim, Stuart K.

2015-01-01

Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney. PMID:26678048

GH3::GUS reflects cell-specific developmental patterns and stress-induced changes in wood anatomy in the poplar stem.

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Teichmann, Thomas; Bolu-Arianto, Waode Hamsinah; Olbrich, Andrea; Langenfeld-Heyser, Rosemarie; Göbel, Cornelia; Grzeganek, Peter; Feussner, Ivo; Hänsch, Robert; Polle, Andrea

2008-09-01

GH3 genes related to the auxin-inducible Glycine max (L.) Merr. GmGH3 gene encode enzymes that conjugate amino acids to auxin. To investigate the role of GH3 enzymes in stress responses and normal wood development, Populus x canescens (Ait.) was transformed with the promoter-reporter construct GH3::GUS containing a GH3 promoter and the 5' UTR from soybean. beta-Glucuronidase (GUS) activity was present in the vascular tissues of leaves and in developing lateral roots and was inducible in silent tissues by external auxin application. A decrease in GUS activity from the stem apex to the bottom corresponded to decreases in auxin concentrations in these tissues. High auxin concentration and high GH3::GUS activity were present in the pith tissue, which may provide storage for auxin compounds. GH3 reporter was active in ray cells, paratracheal parenchyma cells, maturing vessels and in cells surrounding maturing phloem fibers but not in the cambium and immature phloem, despite high auxin concentrations in the latter tissues. However, the GH3 promoter in these tissues became active when the plants were exposed to abiotic stresses, like bending or salinity, causing changes in wood anatomy. We suggest that adjustment of the internal auxin balance in wood in response to environmental cues involves GH3 auxin conjugate synthases.

Psidium guajava extract inhibits thymus and activation-regulated chemokine (TARC/CCL17) production in human keratinocytes by inducing heme oxygenase-1 and blocking NF-κB and STAT1 activation.

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Han, Eun Hee; Hwang, Yong Pil; Choi, Jae Ho; Yang, Ji Hye; Seo, Jong Kwon; Chung, Young Chul; Jeong, Hye Gwang

2011-09-01

Psidium guajava (P. guajava) is a food and medicinal plant with antioxidant, anti-inflammatory, and anti-allergic activities that support its traditional uses. The aim of this study was to determine the effects of P. guajava ethyl acetate extract (PGEA) on atopic dermatitis and to investigate the possible mechanisms by which PGEA inhibits cytokine-induced Th2 chemokine expression in HaCaT human keratinocyte cells. We found that PGEA suppressed the IFN-γ/TNF-α-co-induced production of thymus and activation-regulated chemokine (TARC) protein and mRNA in HaCaT cells. Additionally, PGEA inhibited the TNF-α/IFN-γ-co-induced activation of NF-κB and STAT1 and increased the expression of heme oxygenase-1 (HO-1) protein and mRNA. HO-1 inhibitor enhanced the suppressive effects of PGEA on TNF-α/IFN-γ-co-induced TARC production and gene expression. Collectively, these data demonstrate that PGEA inhibits chemokine expression in keratinocytes by inducing HO-1 expression and it suggests a possible therapeutic application in atopic dermatitis and other inflammatory skin diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Perilla frutescens Extracts Protects against Dextran Sulfate Sodium-Induced Murine Colitis: NF-κB, STAT3, and Nrf2 as Putative Targets

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Deung Dae Park

2017-08-01

Full Text Available Perilla frutescens is a culinary and medicinal herb which has a strong anti-inflammatory and antioxidative effects. In the present study, we investigated the effects of Perilla frutescens extract (PE against dextran sulfate sodium (DSS-induced mouse colitis, an animal model that mimics human inflammatory bowel disease (IBD. Five-week-old male ICR mice were treated with a daily dose of PE (20 or 100 mg/kg, p.o. for 1 week, followed by administration of 3% DSS in double distilled drinking water and PE by gavage for another week. DSS-induced colitis was characterized by body weight loss, colon length shortening, diarrhea and bloody stool, and these symptoms were significantly ameliorated by PE treatment. PE administration suppressed DSS-induced expression of proinflammatory enzymes, including cyclooxygenase-2 and inducible nitric oxide synthase as well as cyclin D1, in a dose-dependent fashion. Nuclear factor-kappa B (NF-κB and signal transducer and activator of transcription 3 (STAT3 are major transcriptional regulators of inflammatory signaling. PE administration significantly inhibited the activation of both NF-κB and STAT3 induced by DSS, while it elevated the accumulation of Nrf2 and heme oxygenase-1 in the colon. In another experiment, treatment of CCD841CoN human normal colon epithelial cells with PE (10 mg/ml resulted in the attenuation of the tumor necrosis factor-α-induced expression/activation of mediators of proinflammatory signaling. The above results indicate that PE has a preventive potential for use in the management of IBD.

Perilla frutescens Extracts Protects against Dextran Sulfate Sodium-Induced Murine Colitis: NF-κB, STAT3, and Nrf2 as Putative Targets.

Science.gov (United States)

Dae Park, Deung; Yum, Hye-Won; Zhong, Xiancai; Kim, Seung Hyeon; Kim, Seong Hoon; Kim, Do-Hee; Kim, Su-Jung; Na, Hye-Kyung; Sato, Atsuya; Miura, Takehito; Surh, Young-Joon

2017-01-01

Perilla frutescens is a culinary and medicinal herb which has a strong anti-inflammatory and antioxidative effects. In the present study, we investigated the effects of Perilla frutescens extract (PE) against dextran sulfate sodium (DSS)-induced mouse colitis, an animal model that mimics human inflammatory bowel disease (IBD). Five-week-old male ICR mice were treated with a daily dose of PE (20 or 100 mg/kg, p.o. ) for 1 week, followed by administration of 3% DSS in double distilled drinking water and PE by gavage for another week. DSS-induced colitis was characterized by body weight loss, colon length shortening, diarrhea and bloody stool, and these symptoms were significantly ameliorated by PE treatment. PE administration suppressed DSS-induced expression of proinflammatory enzymes, including cyclooxygenase-2 and inducible nitric oxide synthase as well as cyclin D1, in a dose-dependent fashion. Nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) are major transcriptional regulators of inflammatory signaling. PE administration significantly inhibited the activation of both NF-κB and STAT3 induced by DSS, while it elevated the accumulation of Nrf2 and heme oxygenase-1 in the colon. In another experiment, treatment of CCD841CoN human normal colon epithelial cells with PE (10 mg/ml) resulted in the attenuation of the tumor necrosis factor-α-induced expression/activation of mediators of proinflammatory signaling. The above results indicate that PE has a preventive potential for use in the management of IBD.

Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

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Arzt, Lisa; Halbwedl, Iris; Gogg-Kamerer, Margit; Popper, Helmut H

2017-07-01

Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.

The characterization of six auxin-induced tomato GH3 genes uncovers a member, SlGH3.4, strongly responsive to arbuscular mycorrhizal symbiosis.

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Liao, Dehua; Chen, Xiao; Chen, Aiqun; Wang, Huimin; Liu, Jianjian; Liu, Junli; Gu, Mian; Sun, Shubin; Xu, Guohua

2015-04-01

In plants, the GH3 gene family is widely considered to be involved in a broad range of plant physiological processes, through modulation of hormonal homeostasis. Multiple GH3 genes have been functionally characterized in several plant species; however, to date, limited works to study the GH3 genes in tomato have been reported. Here, we characterize the expression and regulatory profiles of six tomato GH3 genes, SlGH3.2, SlGH3.3, SlGH3.4, SlGH3.7, SlGH3.9 and SlGH3.15, in response to different phytohormone applications and arbuscular mycorrhizal (AM) fungal colonization. All six GH3 genes showed inducible responses to external IAA, and three members were significantly up-regulated in response to AM symbiosis. In particular, SlGH3.4, the transcripts of which were barely detectable under normal growth conditions, was strongly activated in the IAA-treated and AM fungal-colonized roots. A comparison of the SlGH3.4 expression in wild-type plants and M161, a mutant with a defect in AM symbiosis, confirmed that SlGH3.4 expression is highly correlated to mycorrhizal colonization. Histochemical staining demonstrated that a 2,258 bp SlGH3.4 promoter fragment could drive β-glucuronidase (GUS) expression strongly in root tips, steles and cortical cells of IAA-treated roots, but predominantly in the fungal-colonized cells of mycorrhizal roots. A truncated 654 bp promoter failed to direct GUS expression in IAA-treated roots, but maintained the symbiosis-induced activity in mycorrhizal roots. In summary, our results suggest that a mycorrhizal signaling pathway that is at least partially independent of the auxin signaling pathway has evolved for the co-regulation of the auxin- and mycorrhiza-activated GH3 genes in plants. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

STAT3 and NF-κB are common targets for kaempferol-mediated attenuation of COX-2 expression in IL-6-induced macrophages and carrageenan-induced mouse paw edema

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Anandita Basu

2017-12-01

Full Text Available Cycloxygenase-2 (COX-2 is the inducible isoform of cycloxygenase enzyme family that catalyzes synthesis of inflammatory mediators, prostanoids and prostaglandins, and therefore, can be targeted by anti-inflammatory drugs. Here, we showed a plant polyphenol, kaempferol, attenuated IL-6-induced COX-2 expression in human monocytic THP-1 cells suggesting its beneficial role in chronic inflammation. Kaempferol deactivated and prevented nuclear localization of two major transcription factors STAT3 and NF-κB, mutually responsible for COX-2 induction in response to IL-6. Moreover, STAT3 and NF-κB were simultaneously deactivated by kaempferol in acute inflammation, as shown by carrageenan-induced mouse paw edema model. The concomitant reduction in COX-2 expression in paw tissues suggested kaempferol’s role in mitigation of inflammation by targeting STAT3 and NF-κB.

STAT3 activation in monocytes accelerates liver cancer progression

International Nuclear Information System (INIS)

Wu, Wen-Yong; Li, Jun; Wu, Zheng-Sheng; Zhang, Chang-Le; Meng, Xiang-Ling

2011-01-01

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor ubiquitously expressed in different cell types. STAT3 plays an essential role in cell survival, proliferation, and differentiation. Aberrantly hyper-activated STAT3 signaling in cancer cells and in the tumor microenvironment has been detected in a wide variety of human cancers and is considered an important factor for cancer initiation, development, and progression. However, the role of STAT3 activation in monocytes in the development of HCC has not been well understood. Immunohistochemical analysis of phosphorylated STAT3 was performed on tissue microarray from HCC patients. Using a co-culture system in vivo, HCC cell growth was determined by the MTT assay. In vivo experiments were conducted with mice given diethylinitrosamine (DEN), which induces HCC was used to investigate the role of STAT3 expression in monocytes on tumor growth. Real-time PCR was used to determine the expression of cell proliferation and cell arrest associated genes in the tumor and nontumor tissue from liver. Phosphorylated STAT3 was found in human hepatocellular carcinoma tissue samples and was expressed in tumor cells and also in monocytes. Phosphorylated STAT3 expression in monocyte was significantly correlated to advanced clinical stage of HCC and a poor prognosis. Using a co-culture system in vivo, monocytes promoted HCC cell growth via the IL-6/STAT3 signaling pathway. The STAT3 inhibitor, NSC 74859, significantly suppressed tumor growth in vivo in mice with diethylinitrosamine (DEN)-induced HCC. In this animal model, blockade of STAT3 with NSC 74859 induced tumor cell apoptosis, while inhibiting both tumor cells and monocytes proliferation. Furthermore, NSC 74859 treatment suppressed cancer associated inflammation in DEN-induce HCC. Our data suggest constitutively activated STAT3 monocytes promote liver tumorigenesis in clinical patients and animal experiments. Thus, STAT3 in tumor

Molecular engineering of fungal GH5 and GH26 beta-(1,4-mannanases toward improvement of enzyme activity.

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Marie Couturier

Full Text Available Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat /KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat /KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L that may confer more flexibility to the linker and another mutation (D416G located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM.

STAT5 induces miR-21 expression in cutaneous T cell lymphoma

DEFF Research Database (Denmark)

Lindahl, Lise M; Fredholm, Simon; Joseph, Claudine

2016-01-01

was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS...

Depletion of STAT5 blocks TEL–SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice

International Nuclear Information System (INIS)

Sprissler, C; Belenki, D; Maurer, H; Aumann, K; Pfeifer, D; Klein, C; Müller, T A; Kissel, S; Hülsdünker, J; Alexandrovski, J; Brummer, T; Jumaa, H; Duyster, J; Dierks, C

2014-01-01

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK wt , TEL–SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK wt enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK–SYK caused lethal T-cell lymphomas and the cytoplasmic TEL–SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL–SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL–SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL–SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases

5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization.

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Sandur, Santosh K; Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

2010-01-01

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

STAT proteins: from normal control of cellular events to tumorigenesis.

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Calò, Valentina; Migliavacca, Manuela; Bazan, Viviana; Macaluso, Marcella; Buscemi, Maria; Gebbia, Nicola; Russo, Antonio

2003-11-01

Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis.

JAK/STAT signaling pathway-mediated immune response in silkworm (Bombyx mori) challenged by Beauveria bassiana.

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Geng, Tao; Lv, Ding-Ding; Huang, Yu-Xia; Hou, Cheng-Xiang; Qin, Guang-Xing; Guo, Xi-Jie

2016-12-20

Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better

Cyclophilins contribute to Stat3 signaling and survival of multiple myeloma cells.

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Bauer, K; Kretzschmar, A K; Cvijic, H; Blumert, C; Löffler, D; Brocke-Heidrich, K; Schiene-Fischer, C; Fischer, G; Sinz, A; Clevenger, C V; Horn, F

2009-08-06

Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.

Multidimensional single cell based STAT phosphorylation profiling identifies a novel biosignature for evaluation of systemic lupus erythematosus activity.

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Xinfang Huang

Full Text Available INTRODUCTION: Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE, a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE. METHODS: Phospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA patients and 13 healthy donors (HDs. A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10 were also monitored. RESULTS: We observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF, and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells. CONCLUSIONS: The basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.

Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

International Nuclear Information System (INIS)

Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

2015-01-01

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl + K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5

Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

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Valsala Haridas

Full Text Available Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a the cross-talk that occurs between redox and phosphorylation processes, and (b the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3

DEFF Research Database (Denmark)

Sommer, V H; Clemmensen, O J; Nielsen, O

2004-01-01

in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible...... expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells...

Epstein-Barr virus-derived EBNA2 regulates STAT3 activation

International Nuclear Information System (INIS)

Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Togi, Sumihito; Kamitani, Shinya; Fujimuro, Masahiro; Harada, Shizuko; Oritani, Kenji; Matsuda, Tadashi

2009-01-01

The Epstein-Barr virus (EBV)-encoded latency protein EBNA2 is a nuclear transcriptional activator that is essential for EBV-induced cellular transformation. Here, we show that EBNA2 interacts with STAT3, a signal transducer for an interleukin-6 family cytokine, and enhances the transcriptional activity of STAT3 by influencing its DNA-binding activity. Furthermore, EBNA2 cooperatively acts on STAT3 activation with LMP1. These data demonstrate that EBNA2 acts as a transcriptional coactivator of STAT3.

GH mediates exercise-dependent activation of SVZ neural precursor cells in aged mice.

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Daniel G Blackmore

Full Text Available Here we demonstrate, both in vivo and in vitro, that growth hormone (GH mediates precursor cell activation in the subventricular zone (SVZ of the aged (12-month-old brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation.

GH Mediates Exercise-Dependent Activation of SVZ Neural Precursor Cells in Aged Mice

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Blackmore, Daniel G.; Vukovic, Jana; Waters, Michael J.; Bartlett, Perry F.

2012-01-01

Here we demonstrate, both in vivo and in vitro, that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise, and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast, no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury, we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely, infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation. PMID:23209615

Enterovirus 71 antagonizes the antiviral activity of host STAT3 and IL-6R with partial dependence on virus-induced miR-124.

Science.gov (United States)

Chang, Zhangmei; Wang, Yan; Bian, Liang; Liu, Qingqing; Long, Jian-Er

2017-12-01

Enterovirus 71 (EV71) has caused major outbreaks of hand, foot and mouth disease. EV71 infections increase the production of many host cytokines and pro-inflammatory factors, including interleukin (IL)-6, IL-10 and COX-2. Some of these molecules could stimulate the signal transducer and activator of transcription 3 (STAT3), which plays a key role in regulating host immune responses and several viral diseases. However, the role of STAT3 in EV71 infection remains unknown. This study found that the phosphorylation levels of STAT3 (p Y705 -STAT3) are closely related to EV71 infection. Further experiments revealed that STAT3 exerts an anti-EV71 activity. However, the antiviral activity of STAT3 is partially antagonized by EV71-induced miR-124, which directly targets STAT3 mRNA. Similarly, IL-6R, the α-subunit of the IL-6 receptor complex, exhibits anti-EV71 activity and is directly targeted by the virus-induced miR-124. These results indicate that EV71 can evade host IL-6R- and STAT3-mediated antiviral activities by EV71-induced miR-124. This suggests that controlling miR-124 and the downstream targets, IL-6R and STAT3, might benefit the antiviral treatment of EV71 infection.

Analysis of signal transducer and activator of transcription 3 (Stat 3) pathway in multiple myeloma: Stat 3 activation and cyclin D1 dysregulation are mutually exclusive events.

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Quintanilla-Martinez, Leticia; Kremer, Marcus; Specht, Katja; Calzada-Wack, Julia; Nathrath, Michaela; Schaich, Robert; Höfler, Heinz; Fend, Falko

2003-05-01

The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three

Different growth hormone (GH) response to GH-releasing peptide and GH-releasing hormone in hyperthyroidism.

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Ramos-Dias, J C; Pimentel-Filho, F; Reis, A F; Lengyel, A M

1996-04-01

Altered GH responses to several pharmacological stimuli, including GHRH, have been found in hyperthyroidism. The mechanisms underlying these disturbances have not been fully elucidated. GH-releasing peptide-6 (GHRP-6) is a synthetic hexapeptide that specifically stimulates GH release both in vitro and in vivo. The mechanism of action of GHRP-6 is unknown, but it probably acts by inhibiting the effects of somatostatin on GH release. The aim of this study was to evaluate the effects of GHRP-6 on GH secretion in patients with hyperthyroidism (n = 9) and in control subjects (n = 9). Each subject received GHRP-6 (1 microg/kg, iv), GHRH (100 microg, iv), and GHRP-6 plus GHRH on 3 separate days. GH peak values (mean +/- SE; micrograms per L) were significantly lower in hyperthyroid patients compared to those in control subjects after GHRH alone (9.0 +/- 1.3 vs. 27.0 +/- 5.2) and GHRP-6 plus GHRH (22.5 +/- 3.5 vs. 83.7 +/- 15.2); a lack of the normal synergistic effect of the association of both peptides was observed in thyrotoxicosis. However, a similar GH response was seen in both groups after isolated GHRP-6 injection (31.9 +/- 5.7 vs. 23.2 +/- 3.9). In summary, we have shown that hyperthyroid patients have a normal GH response to GHRP-6 together with a blunted GH responsiveness to GHRH. Our data suggest that thyroid hormones modulate GH release induced by these two peptides in a differential way.

Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells.

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Chiba, Takeshi; Maeda, Tomoji; Sanbe, Atsushi; Kudo, Kenzo

2016-04-22

Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Hippocalcin Is Required for Astrocytic Differentiation through Activation of Stat3 in Hippocampal Neural Precursor Cells.

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Min-Jeong Kang

2016-10-01

Full Text Available Hippocalcin (Hpca is a neuronal calcium sensor protein expressed in the mammalian brain. However, its function in neural stem/precursor cells has not yet been studied. Here, we clarify the function of Hpca in astrocytic differentiation in hippocampal neural precursor cells (HNPCs. When we overexpressed Hpca in HNPCs in the presence or absence of bFGF, expression levels of nerve-growth factors such as neurotrophin-3 (NT-3, neurotrophin-4/5 (NT-4/5 and brain-derived neurotrophic factor (BDNF, together with the proneural basic helix loop helix (bHLH transcription factors neuroD and neurogenin 1 (ngn1, increased significantly. In addition, there was an increase in the number of cells expressing glial fibrillary acidic protein (GFAP, an astrocyte marker, and in dendrite outgrowth, indicating astrocytic differentiation of the HNPCs. Downregulation of Hpca by transfection with Hpca siRNA reduced expression of NT-3, NT-4/5, BDNF, neuroD and ngn1 as well as levels of GFAP protein. Furthermore, overexpression of Hpca increased the phosphorylation of STAT3 (Ser727, and this effect was abolished by treatment with a STAT3 inhibitor (S3I-201, suggesting that STAT3 (Ser727 activation is involved in Hpca-mediated astrocytic differentiation. As expected, treatment with Stat3 siRNA or STAT3 inhibitor caused a complete inhibition of astrogliogenesis induced by Hpca overexpression. Taken together, this is the first report to show that Hpca, acting through Stat3, has an important role in the expression of neurotrophins and proneural bHLH transcription factors, and that it is an essential regulator of astrocytic differentiation and dendrite outgrowth in HNPCs.

Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts

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Mehrdad Sobhkhez

2014-01-01

Full Text Available Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar. Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD. Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

Loss of function mutations in PTPN6 promote STAT3 deregulation via JAK3 kinase in diffuse large B-cell lymphoma

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Demosthenous, Christos; Han, Jing Jing; Hu, Guangzhen; Stenson, Mary; Gupta, Mamta

2015-01-01

PTPN6 (SHP1) is a tyrosine phosphatase that negatively controls the activity of multiple signaling pathways including STAT signaling, however role of mutated PTPN6 is not much known. Here we investigated whether PTPN6 might also be a potential target for diffuse large B cell lymphoma (DLBCL) and performed Sanger sequencing of the PTPN6 gene. We have identified missense mutations within PTPN6 (N225K and A550V) in 5% (2/38) of DLBCL tumors. Site directed mutagenesis was performed to mutate wild type (WT) PTPN6 and stable cell lines were generated by lentiviral transduction of PTPN6WT, PTPN6N225K and PTPN6A550V constructs, and effects of WT or mutated PTPN6 on STAT3 signaling were analyzed. WT PTPN6 dephosphorylated STAT3, but had no effect on STAT1, STAT5 or STAT6 phosphorylation. Both PTPN6 mutants were unable to inhibit constitutive, as well as cytokines induced STAT3 activation. Both PTPN6 mutants also demonstrated reduced tyrosine phosphatase activity and exhibited enhanced STAT3 transactivation activity. Intriguingly, a lack of direct binding between STAT3 and WT or mutated PTPN6 was observed. However, compared to WT PTPN6, cells expressing PTPN6 mutants exhibited increased binding between JAK3 and PTPN6 suggesting a more dynamic interaction of PTPN6 with upstream regulators of STAT3. Consistent with this notion, both the mutants demonstrated increased resistance to JAK3 inhibitor, WHIP-154 relative to WT PTPN6. Overall, this is the first study, which demonstrates that N225K and A550V PTPN6 mutations cause loss-of-function leading to JAK3 mediated deregulation of STAT3 pathway and uncovers a mechanism that tumor cells can use to control PTPN6 substrate specificity. PMID:26565811

The Cytoplasmic Tail Domain of Epstein-Barr Virus gH Regulates Membrane Fusion Activity through Altering gH Binding to gp42 and Epithelial Cell Attachment

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Jia Chen

2016-11-01

Full Text Available Epstein-Barr virus (EBV is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD. In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706 of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain.

Synergistic Effect of Radiation and Interleukin-6 on Hepatitis B Virus Reactivation in Liver Through STAT3 Signaling Pathway

International Nuclear Information System (INIS)

Chou, C.H.; Chen, P.-J.; Jeng, Y.-M.; Cheng, A.-L.; Huang, L.-R.; Cheng, J.C.-H.

2009-01-01

Purpose: Hepatitis B virus (HBV) reactivation can occur after radiotherapy (RT) for hepatobiliary malignancies. Our previous in vitro culture study identified interleukin-6 (IL-6) as the main bystander mediator of RT-induced HBV replication. We attempted to examine the molecular mechanism in HBV-transgenic mice. Methods and materials: HBV transgenic mice were treated with whole liver RT (4 Gy daily for 5 days) with or without administration of IL-6 (400 ng twice daily for 15 days). The serum level of HBV DNA was measured using real-time polymerase chain reaction, and the IL-6 concentration was measured using enzyme-linked immunosorbent assay. The intensity of immunostaining with antibodies to HBV core protein and phosphorylated signal transducer and activator of transcription (STAT)3 in the mouse liver was qualitatively analyzed. HepG2.2.15 cells (a human hepatoblastoma cell line that persistently produces HBV DNA) were used to investigate the molecular role of IL-6 plus RT in HBV reactivation. Results: HBV reactivation was induced in vivo with IL-6 plus RT (5.58-fold) compared with RT alone (1.31-fold, p = .005), IL-6 alone (1.31-fold, p = .005), or sham treatment (1.22-fold, p = .004). HBV core protein staining confirmed augmentation of intrahepatic HBV replication. IL-6 plus RT-induced HBV DNA replication in HepG2.2.15 cells was suppressed by the STAT3 inhibitor AG490 and by transfection with dominant-negative STAT3 plasmid. Phosphorylated STAT3 staining was strongest in liver tissue from mice treated with IL-6 plus RT. The mobility shift assay demonstrated that reactivation was mediated through the interaction of phosphorylated STAT3/hepatocyte nuclear factor-3 complex with HBV enhancer 1. Conclusion: RT to the liver and longer sustained IL-6 induced HBV reactivation through the STAT3 signal transduction pathway.

The regulation effect of STAT 5 signaling pathway on the cell cycle progression of irradiated KG-1 cells

International Nuclear Information System (INIS)

Guo Dehuang; Dong Bo; Luo Qingliang; Wen Gengyun; Mao Bingzhi

2000-01-01

The author investigated the role of the JAK/STAT signaling pathway regulating cell cycle progression in the irradiated KG-1 cells. By permanent transfecting the cells with DN-STAT 5 cDNA to block the JAK/STAT signaling pathway and then transient transfecting with cyclin D 1 or cyclin B 1 cDNA, the effects of cyclin D 1 protein and cyclin B 1 protein on the cell cycle progression were examined. Results showed that after irradiation with 8Gy 60 Co rays, the irradiated KG-1 cells transfected with only DN-STAT 5 cDNA can not recover form the G 1 arrest, even though GM-CSF was added. Meanwhile, the cells transfected with both the DN-STAT 5 cDNA and cyclin D 1 cDNA or cyclin B 1 cDNA can recover from the G 1 arrest or the G 2 arrest to a great extent. Thus, it was proved indirectly that the JAK/STAT signaling pathway activated by GM-CSF regulated the cell cycle progression through cyclin D 1 and cyclin B 1 protein

Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

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Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

2015-03-19

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation.

Science.gov (United States)

Carruthers, Nicholas J; Stemmer, Paul M; Chen, Ben; Valeriote, Frederick; Gao, Xiaohua; Guatam, Subhash C; Shaw, Jiajiu

2017-09-15

UTL-5g is a novel small-molecule TNF-alpha modulator. It reduces cisplatin-induced side effects by protecting kidney, liver, and platelets, thereby increasing tolerance for cisplatin. UTL-5g also reduces radiation-induced acute liver toxicity. The mechanism of action for UTL-5g is not clear at the present time. A phosphoproteomic analysis to a depth of 4943 phosphopeptides and a luminescence-based transcription factor activity assay were used to provide complementary analyses of signaling events that were disrupted by UTL-5g in RAW 264.7 cells. Transcriptional activity downstream of the interferon gamma, IL-6, type 1 Interferon, TGF-β, PKC/Ca 2+ and the glucocorticoid receptor pathways were disrupted by UTL-5g. Phosphoproteomic analysis indicated that hyperphosphorylation of proteins involved in actin remodeling was suppressed by UTL-5g (gene set analysis, FDR 5g. This global characterization of UTL-5g activity in a macrophage cell line discovered that it disrupts selected aspects of LPS signaling including Stat3 activation and actin remodeling providing new insight on how UTL-5g acts to reduce cisplatin-induced side effects. Copyright © 2017 Elsevier B.V. All rights reserved.

The Role of the Interferon-Gamma-Jak/STAT Pathway in Rheumatoid Arthritis

Science.gov (United States)

2017-09-01

including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and...cytometry the T cell populations, the B cell populations and monocytes. Beginning Month 11, we will begin interrogating these cell populations for the... interrogate IFN-γ induced SAT1 activation along with the activation of STAT3 and STAT5 over a range of stimulation time- points. This approach is feasible

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

Science.gov (United States)

Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

Dynamic trafficking of STAT5 depends on an unconventional nuclear localization signal

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Shin, Ha Youn; Reich, Nancy C.

2013-01-01

Summary Signal transducer and activator of transcription 5 (STAT5) is crucial for physiological processes that include hematopoiesis, liver metabolism and mammary gland development. However, aberrant continual activity of STAT5 has been causally linked to human leukemias and solid tumor formation. As a regulated transcription factor, precise cellular localization of STAT5 is essential. Conventional nuclear localization signals consist of short stretches of basic amino acids. In this study, we provide evidence that STAT5 nuclear import is dependent on an unconventional nuclear localization signal that functions within the conformation of an extensive coiled-coil domain. Both in vitro binding and in vivo functional assays reveal that STAT5 nuclear import is mediated by the importin-α3/β1 system independently of STAT5 activation by tyrosine phosphorylation. The integrity of the coiled-coil domain is essential for STAT5 transcriptional induction of the β-casein gene following prolactin stimulation as well as its ability to synergize with the glucocorticoid receptor. The glucocorticoid receptor accumulates in the nucleus in response to prolactin and this nuclear import is dependent on STAT5 nuclear import. STAT5 continually shuttles in and out of the nucleus and live cell imaging demonstrates that STAT5 nuclear export is mediated by both chromosome region maintenance 1 (Crm1)-dependent and Crm1-independent pathways. A Crm1-dependent nuclear export signal was identified within the STAT5 N-terminus. These findings provide insight into the fundamental mechanisms that regulate STAT5 nuclear trafficking and cooperation with the glucocorticoid receptor and provide a basis for clinical intervention of STAT5 function in disease. PMID:23704351

A molecular model for the differential activation of STAT3 and STAT6 by the herpesviral oncoprotein tip.

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Eman Dey Mazumder

Full Text Available Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2 domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.

STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

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Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

2014-01-01

Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

Molecular integration of HoxB4 and STAT3 for self-renewal of hematopoietic stem cells: a model of molecular convergence for stemness.

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Hong, Sung-Hyun; Yang, Seung-Jip; Kim, Tae-Min; Shim, Jae-Seung; Lee, Ho-Sun; Lee, Ga-Young; Park, Bo-Bae; Nam, Suk Woo; Ryoo, Zae Young; Oh, Il-Hoan

2014-05-01

The upregulation of HoxB4 promotes self-renewal of hematopoietic stem cells (HSCs) without overriding the normal stem cell pool size. A similar enhancement of HSC self-renewal occurs when signal transducer and activator of transcription 3 (STAT3) is activated in HSCs. In this study, to gain insight into the functional organization of individual transcription factors (TFs) that have similar effects on HSCs, we investigated the molecular interplay between HoxB4 and STAT3 in the regulation of HSC self-renewal. We found that while STAT3-C or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both TFs did not have additive effects, indicating their functional redundancy in HSCs. In addition, activation of STAT3 did not cause changes in the expression levels of HoxB4. In contrast, the inhibition of STAT3 activity in HoxB4-overexpressing hematopoietic cells significantly abrogated the enhancing effects of HoxB4, and the upregulation of HoxB4 caused a ligand-independent Tyr-phosphorylation of STAT3. Microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis showed significant overlap in the candidate TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3. Interestingly, among these common TFs were the pluripotency-related genes Oct-4 and Nanog. These results indicate that tissue-specific TFs regulating HSC self-renewal are functionally organized to play an equivalent role in transcription and provide insights into the functional convergence of multiple entries of TFs toward a conserved transcription program for the stem cell state. © 2014 AlphaMed Press.

Activation of Stat3 in renal tumors.

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Guo, Charles; Yang, Guanyu; Khun, Kyle; Kong, Xiantian; Levy, David; Lee, Peng; Melamed, Jonathan

2009-02-28

Signal transducer and activator of transcription 3 (Stat3) plays a vital role in signal transduction pathways that mediate transformation and inhibit apoptosis. Oncogenic Stat3 is persistently activated in several human cancers and transformed cell lines. Previous studies indicate activation of Stat3 in renal cell carcinoma (RCC). However, the detailed characterization of the Stat3 expression pattern in different histologic types of RCC is lacking. We have analyzed the immunoprofile of activated or phosphorylated Stat3 (pStat3) in a tissue microarray of renal tumors of different histologic types, including 42 cases of conventional clear cell type, 24 chromophobe, and 7 papillary, 15 oncocytoma, 7 urothelial carcinoma and 21 normal kidney tissues using an anti-pStat3 antibody (recognizes only activated STAT3). pStat3 nuclear staining was observed in 25 of 42 conventional clear cell RCC (59.5 %), 8 of 24 chromophobe RCC (33.3%), 4 of 7 papillary RCC (57.1%). In the other tumor groups, 4 of 15 oncocytomas (26.7%) and 6 of 7 urothelial carcinomas (85.7%) showed positive nuclear staining. Weak nuclear immunoreactivity for pStat3 was seen in 4 of 21 cases of non-neoplastic kidney tissue (19.0%). The extent of Stat3 activation as determined by nuclear expression of its phosphorylated form is increased in histologic types of renal tumors with greater malignant potential, specifically conventional clear cell RCC, papillary RCC and urothelial carcinoma, only slightly increased in chromophobe RCC, and not increased in oncocytoma. These results suggest a role of Stat3 activation in different types of renal neoplasia, possibly serving as a prognostic marker or therapeutic target.

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

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Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.

2009-06-03

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

Identification of Novel STAT6-Regulated Proteins in Mouse B Cells by Comparative Transcriptome and Proteome Analysis.

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Mokada-Gopal, Lavanya; Boeser, Alexander; Lehmann, Christian H K; Drepper, Friedel; Dudziak, Diana; Warscheid, Bettina; Voehringer, David

2017-05-01

The transcription factor STAT6 plays a key role in mediating signaling downstream of the receptors for IL-4 and IL-13. In B cells, STAT6 is required for class switch recombination to IgE and for germinal center formation during type 2 immune responses directed against allergens or helminths. In this study, we compared the transcriptomes and proteomes of primary mouse B cells from wild-type and STAT6-deficient mice cultured for 4 d in the presence or absence of IL-4. Microarray analysis revealed that 214 mRNAs were upregulated and 149 were downregulated >3-fold by IL-4 in a STAT6-dependent manner. Across all samples, ∼5000 proteins were identified by label-free quantitative liquid chromatography/mass spectrometry. A total of 149 proteins was found to be differentially expressed >3-fold between IL-4-stimulated wild-type and STAT6 -/- B cells (75 upregulated and 74 downregulated). Comparative analysis of the proteome and transcriptome revealed that expression of these proteins was mainly regulated at the transcriptional level, which argues against a major role for posttranscriptional mechanisms that modulate the STAT6-dependent proteome. Nine proteins were selected for confirmation by flow cytometry or Western blot. We show that CD30, CD79b, SLP-76, DEC205, IL-5Rα, STAT5, and Thy1 are induced by IL-4 in a STAT6-dependent manner. In contrast, Syk and Fc receptor-like 1 were downregulated. This dataset provides a framework for further functional analysis of newly identified IL-4-regulated proteins in B cells that may contribute to germinal center formation and IgE switching in type 2 immunity. Copyright © 2017 by The American Association of Immunologists, Inc.

Novel mechanism for Fc epsilon RI-mediated signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and the selective influence of STAT5B over mast cell cytokine production

Czech Academy of Sciences Publication Activity Database

Pullen, N.A.; Barnstein, B.O.; Falanga, Y.T.; Wang, Z.Q.; Suzuki, R.; Tamang, T.D.L.; Khurana, M.C.; Harry, E.A.; Dráber, Petr; Bunting, K.D.; Mizuno, K.; Wilson, B.S.; Ryan, J.J.

2012-01-01

Roč. 287, č. 3 (2012), s. 2045-2054 ISSN 0021-9258 R&D Projects: GA MŠk 1M0506; GA ČR GAP302/10/1759; GA ČR GA301/09/1826 Grant - others:NIH(US) 1R01AI59638; NIH(US) R01DK059380; NIH(US) AI051575; NIH(US) GM065794; VCU(US) U19A1077435 Institutional support: RVO:68378050 Keywords : cytokine induction * STAT transcription factor * mast cell * Fyn kinase * STAT5 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.651, year: 2012

Dynamic mathematical modeling of IL13-induced signaling in Hodgkin and primary mediastinal B-cell lymphoma allows prediction of therapeutic targets.

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Raia, Valentina; Schilling, Marcel; Böhm, Martin; Hahn, Bettina; Kowarsch, Andreas; Raue, Andreas; Sticht, Carsten; Bohl, Sebastian; Saile, Maria; Möller, Peter; Gretz, Norbert; Timmer, Jens; Theis, Fabian; Lehmann, Wolf-Dieter; Lichter, Peter; Klingmüller, Ursula

2011-02-01

Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.

STAT3 inhibitor WP1066 as a novel therapeutic agent for bCCI neuropathic pain rats.

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Xue, Zhao-Jing; Shen, Le; Wang, Zhi-Yao; Hui, Shang-Yi; Huang, Yu-Guang; Ma, Chao

2014-10-02

Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in the development of chronic pain, but the complex regulation of STAT3-dependent pathway and the functional significance of inhibiting this pathway during the development of neuropathic pain remain elusive. To evaluate the contribution of the JAK2/STAT3 pathway to neuropathic pain and the potentiality of this pathway as a novel therapeutic target, we examined the effects of the STAT3 inhibitor WP1066 by intrathecal administration in a rat model of bilateral chronic constriction injury (bCCI). The pain behavior tests were performed before the surgery and on postoperative day 3, 7, 14 and 21. L4-L6 dorsal spinal cord were harvested at each time point. Both RT-PCR and Western blot were performed to evaluate the activation of JAK2/STAT3 pathway. To observe the influence of WP1066 on neuropathic pain and its molecular mechanism, WP1066 (10 μl, 10 mmol/L in DMSO) or the same capacity of DMSO as the control were applied through the intrathecal tube on the day before bCCI surgery, and on the postoperative day 3 and 5. Behavioral tests were performed to observe the therapeutic effect on mechanical, thermal and cold hyperalgesia. L4-L6 dorsal spinal cord was harvested on postoperative day fourteen, followed by RT-PCR and Western blot evaluation of the JAK2/STAT3 pathway activation. The mechanical, thermal and cold hyperalgesia of the bCCI rats were significantly decreased when compared with the Sham or the Naïve group at each postoperative time point (PbCCI rats, accompanied by SOCS3 mRNA with a similar tendency. Western blot analysis showed that JAK2 and phosphorylated STAT3 increased significantly since 3 days after bCCI. JAK2 peaked on postoperative day 14 while phosphorylated STAT3 peaked on postoperative day 7 and gradually decreased thereafter and SOCS3׳s peak level on postoperative day 3. When WP1066 were administered intrathecally, the pain behaviors of

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

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Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

Loss of cytokine-STAT5 signaling in the CNS and pituitary gland alters energy balance and leads to obesity.

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Ji-Yeon Lee

Full Text Available Signal transducers and activators of transcription (STATs are critical components of cytokine signaling pathways. STAT5A and STAT5B (STAT5, the most promiscuous members of this family, are highly expressed in specific populations of hypothalamic neurons in regions known to mediate the actions of cytokines in the regulation of energy balance. To test the hypothesis that STAT5 signaling is essential to energy homeostasis, we used Cre-mediated recombination to delete the Stat5 locus in the CNS. Mutant males and females developed severe obesity with hyperphagia, impaired thermal regulation in response to cold, hyperleptinemia and insulin resistance. Furthermore, central administration of GM-CSF mediated the nuclear accumulation of STAT5 in hypothalamic neurons and reduced food intake in control but not in mutant mice. These results demonstrate that STAT5 mediates energy homeostasis in response to endogenous cytokines such as GM-CSF.

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

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Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Turmeric (Curcuma longa) inhibits inflammatory nuclear factor (NF)-κB and NF-κB-regulated gene products and induces death receptors leading to suppressed proliferation, induced chemosensitization, and suppressed osteoclastogenesis.

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Kim, Ji H; Gupta, Subash C; Park, Byoungduck; Yadav, Vivek R; Aggarwal, Bharat B

2012-03-01

The incidence of cancer is significantly lower in regions where turmeric is heavily consumed. Whether lower cancer incidence is due to turmeric was investigated by examining its effects on tumor cell proliferation, on pro-inflammatory transcription factors NF-κB and STAT3, and on associated gene products. Cell proliferation and cell cytotoxicity were measured by the MTT method, NF-κB activity by EMSA, protein expression by Western blot analysis, ROS generation by FACS analysis, and osteoclastogenesis by TRAP assay. Turmeric inhibited NF-κB activation and down-regulated NF-κB-regulated gene products linked to survival (Bcl-2, cFLIP, XIAP, and cIAP1), proliferation (cyclin D1 and c-Myc), and metastasis (CXCR4) of cancer cells. The spice suppressed the activation of STAT3, and induced the death receptors (DR)4 and DR5. Turmeric enhanced the production of ROS, and suppressed the growth of tumor cell lines. Furthermore, turmeric sensitized the tumor cells to chemotherapeutic agents capecitabine and taxol. Turmeric was found to be more potent than pure curcumin for cell growth inhibition. Turmeric also inhibited NF-κB activation induced by RANKL that correlated with the suppression of osteoclastogenesis. Our results indicate that turmeric can effectively block the proliferation of tumor cells through the suppression of NF-κB and STAT3 pathways. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth.

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De Simone, V; Franzè, E; Ronchetti, G; Colantoni, A; Fantini, M C; Di Fusco, D; Sica, G S; Sileri, P; MacDonald, T T; Pallone, F; Monteleone, G; Stolfi, C

2015-07-01

Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of

Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

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Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

1994-12-31

In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

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Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Secoisolariciresinol diglucoside prevents the oxidative stress-induced apoptosis of myocardial cells through activation of the JAK2/STAT3 signaling pathway.

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Huang, Guiqiong; Huang, Xiaofang; Liu, Min; Hua, Yue; Deng, Bo; Jin, Wen; Yan, Wen; Tan, Zhangbin; Wu, Yifen; Liu, Bin; Zhou, Yingchun

2018-06-01

Myocardial cell apoptosis mediated by oxidative stress has previously been identified as a key process in ischemic heart disease. Secoisolariciresinol diglucoside (SDG), a polyphenolic plant lignan primarily found in flaxseed, has been demonstrated to effectively protect myocardial cells from apoptosis. In the present study, the role of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) was investigated in mediating the protective effect of SDG. Findings of the present study revealed that treatment with H2O2 reduced cell viability and induced apoptosis in H9C2 rat cardiomyocytes. However, SDG was able to reduce the effect of H2O2 in a dose‑dependent manner. H2O2 reduced the expression level of phosphorylated STAT3 and inhibited the levels of B‑cell lymphoma‑extra‑large and induced myeloid leukemia cell differentiation protein, which are the STAT3 target genes. Conversely, SDG rescued phosphorylation of STAT3 and increased the levels of STAT3 target genes. Treatment with SDG alone led to a dose‑dependent increased phosphorylation of JAK2 and STAT3, without activating Src. Furthermore, the anti‑apoptotic effects of SDG were partially abolished by a JAK2/STAT3 inhibitor. In addition, molecular docking revealed that SDG may bind to the protein kinase domain of JAK2, at a binding energy of ‑8.258 kcal/mol. Molecular dynamics simulations revealed that JAK2‑SDG binding was stable. In conclusion, activation of the JAK2/STAT3 signaling pathway contributed to the anti‑apoptotic activity of SDG, which may be a potential JAK2 activator.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

Directory of Open Access Journals (Sweden)

Deng D

2015-04-01

Full Text Available Dawei Deng,* Yang Li,* Jianpeng Xue, Jie Wang, Guanhua Ai, Xin Li, Yueqing GuDepartment of Biomedical Engineering, China Pharmaceutical University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: Messenger RNA (mRNA, a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP beacon containing a bare gold nanoparticle (AuNP as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.Keywords: molecular beacon, bioinformatics, gold nanoparticle, STAT5b mRNA, visual detection

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3 and KIT driven Leukemogenesis

Science.gov (United States)

Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H.; Waskow, Emily R.; Visconte, Valeria; Tiu, Ramon V.; Smith, Catherine C.; Shah, Neil; Bunting, Kevin D.; Boswell, H. Scott; Liu, Yan; Chan, Rebecca J.; Kapur, Reuben

2015-01-01

SUMMARY Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN) and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK), whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. PMID:25456130

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis

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Anindya Chatterjee

2014-11-01

Full Text Available Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML and myeloproliferative neoplasms (MPNs, and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.

Stat3 induces oncogenic Skp2 expression in human cervical carcinoma cells

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Huang, Hanhui [Shanghai Medical College of Fudan University, Shanghai 200032 (China); Zhao, Wenrong [Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011 (China); Yang, Dan, E-mail: yangdandr@gmail.com [Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University, Shanghai 200040 (China)

2012-02-03

Highlights: Black-Right-Pointing-Pointer Upregulation of Skp2 by IL-6 or Stat3 activation. Black-Right-Pointing-Pointer Stat3 activates Skp2 expression through bound to its promoter region. Black-Right-Pointing-Pointer Stat3 activates Skp2 expression through recruitment of P300. Black-Right-Pointing-Pointer Stat3 activation decreases the P27 stability. -- Abstract: Dysregulated Skp2 function promotes cell proliferation, which is consistent with observations of Skp2 over-expression in many types of human cancers, including cervical carcinoma (CC). However, the molecular mechanisms underlying elevated Skp2 expression have not been fully explored. Interleukin-6 (IL-6) induced Stat3 activation is viewed as crucial for multiple tumor growth and metastasis. Here, we demonstrate that Skp2 is a direct transcriptional target of Stat3 in the human cervical carcinoma cells. Our data show that IL-6 administration or transfection of a constitutively activated Stat3 in HeLa cells activates Skp2 mRNA transcription. Using luciferase reporter and ChIP assays, we show that Stat3 binds to the promoter region of Skp2 and promotes its activity through recruiting P300. As a result of the increase of Skp2 expression, endogenous p27 protein levels are markedly decreased. Thus, our results suggest a previously unknown Stat3-Skp2 molecular network controlling cervical carcinoma development.

Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes

DEFF Research Database (Denmark)

Nielsen, M; Svejgaard, A; Skov, S

1994-01-01

that stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL-2 ligation, whereas...... an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL-2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that IL-2...... induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84-kDa protein in antigen-specific human T cell lines....

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases

International Nuclear Information System (INIS)

Jeng, Yow-Jiun; Watson, Cheryl S

2009-01-01

Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH 3 /B 6 /F 10 , originally subcloned from GH 3 cells based on its ability to express high levels of the membrane estrogen receptor-α. We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities. Four phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH 3 /B 6 /F 10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens. Phytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary

Evaluation of in vivo [corrected] biological activity of new agmatine analogs of growth hormone-releasing hormone (GH-RH)

Science.gov (United States)

Bokser, L; Zarandi, M; Schally, A V

1990-01-01

The effects of agmatine analogs of growth hormone releasing hormone (GH-RH) were compared to GH-RH(1-29)-NH2 after intravenous (iv) and subcutaneous (sc) administration to pentobarbital-anesthetized male rats. After the iv injection, the analogs [desNH2-Tyr1,Ala15,Nle27] GH-RH(1-28)Agm (MZ-2-51); [desNH2-Tyr1,D-Lys12,Ala15,Nle27] GH-RH(1-28)Agm (MZ-2-57); [desNH2-Tyr1,Ala15,D-Lys21,Nle27] GH-RH(1-28)Agm (MZ-2-75) and [desNH2-Tyr1, D-Lys12,21, Ala15, Nle27] GH-RH(1-28)Agm (MZ-2-87) showed a potency equivalent to 4.4, 1.9, 1.07 and 1.03 times that of GH-RH (1-29)-NH2, respectively, at 5 min and 5.6, 1.8, 1.9 and 1.8 times higher, respectively, at 15 min. After sc administration, analogs MZ-2-51, MZ-2-57 and MZ-2-75 showed to be 34.3, 14.3 and 10.5 times more potent than the parent hormone at 15 min and 179.1, 88.9 and 45.0 times more active, respectively, at 30 min. In addition, MZ-2-51 had prolonged GH-releasing activity as compared to the standard. We also compared the activity of MZ-2-51 and MZ-2-57 with their homologous L-Arg and D-Arg analogs [desNH2-Tyr1,Ala15,Nle27] GH-RH(1-29)-NH2 (MZ-2-117), [des-NH2Tyr1,D-Lys12, Ala15, Nle27] GH-RH(1-29)NH2 (MZ-2-123) and [desNH2-Tyr1,D-Lys12,Ala15, Nle27,D-Arg29] GH-RH(1-29)NH2 (MZ-2-135) after intramuscular (im) injection. MZ-2-51 induced a somewhat greater GH release than MZ-2-117 at 15 min, both responses being larger than the controls (p less than 0.01) at 15 and 30 min. MZ-2-57, MZ-2-123 and MZ-2-135 given i.m. were able to stimulate GH release only at 15 minutes (p less than 0.05). Animals injected i.m. with MZ-2-51, but not with MZ-2-117, showed GH levels significantly higher than the control group (p less than 0.05) at 60 min. GH-RH(1-29)NH2 had low activity intramuscularly when tested at a dose of 2.5 micrograms. No toxic effects were observed after the iv administration of 1 mg/kg of Agm GH-RH analogs. These results indicate that our Agm analogs are active iv, sc and im and that the substitutions made in these

The STAT3 inhibitor pimozide impedes cell proliferation and induces ROS generation in human osteosarcoma by suppressing catalase expression.

Science.gov (United States)

Cai, Nan; Zhou, Wei; Ye, Lan-Lan; Chen, Jun; Liang, Qiu-Ni; Chang, Gang; Chen, Jia-Jie

2017-01-01

Currently, there is a considerable need to develop new treatments for osteosarcoma (OS), a very aggressive bone cancer. The activation of STAT3 signaling is positively associated with poor prognosis and aggressive progression in OS patients. Our previous study reported that the FDA-approved antipsychotic drug pimozide had anti-tumor activity against hepatocellular carcinoma and prostate cancer cells by suppressing STAT3 activity. Therefore, the aim of this study was to investigate the specific effect of pimozide on OS cells and the underlying molecular mechanism. Pimozide inhibited cell proliferation, colony formation, and sphere formation capacities of the OS cells in a dose-dependent manner, inducing G0/G1 phase cell cycle arrest. Pimozide reduced the percentage of side population cells representing cancer stem-like cells and enhanced the sensitivity of OS cells to 5-FU induced proliferative inhibition. In addition, pimozide induced apoptosis of U2OS cells, which showed increased expression of cleaved-PARP, a marker of programmed cell death. Moreover, pimozide suppressed Erk signaling in OS cells. Importantly, pimozide induced ROS generation by downregulating the expression of the antioxidant enzyme catalase (CAT). NAC treatment partially reversed the ROS generation and cytotoxic effects induced by pimozide. CAT treatment attenuated the pimozide-induced proliferation inhibition. The decrease of CAT expression induced by pimozide was potentially mediated through the suppression of cellular STAT3 activity in OS cells. Thus, pimozide may be a novel STAT3 inhibitor that suppresses cellular STAT3 activity to inhibit OS cells or stem-like cells and is a novel potential anti-cancer agent in OS treatment.

Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures

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Arumugam R. Jayakumar

2016-12-01

Full Text Available Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF. Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs, as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB, have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3, we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1 protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

International Nuclear Information System (INIS)

Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene; Raptis, Leda

2010-01-01

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac V12 ), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac V12 with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac V12 -expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac V12 is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac V12 expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac V12 . The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac V12 , as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

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Arulanandam, Rozanne; Geletu, Mulu [Departments of Microbiology and Immunology and Pathology and Molecular Medicine, and Queen' s University Cancer Institute, Queen' s University, Botterell Hall, Rm. 713, Kingston, Ontario, Canada K7L 3N6 (Canada); Feracci, Helene [Universite Bordeaux 1, Centre de Recherche Paul Pascal, CNRS UPR 8641, 33600 Pessac (France); Raptis, Leda, E-mail: raptisl@queensu.ca [Departments of Microbiology and Immunology and Pathology and Molecular Medicine, and Queen' s University Cancer Institute, Queen' s University, Botterell Hall, Rm. 713, Kingston, Ontario, Canada K7L 3N6 (Canada)

2010-03-10

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis.

Science.gov (United States)

Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H; Waskow, Emily R; Visconte, Valeria; Tiu, Ramon V; Smith, Catherine C; Shah, Neil; Bunting, Kevin D; Boswell, H Scott; Liu, Yan; Chan, Rebecca J; Kapur, Reuben

2014-11-20

Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The conserved Phe GH5 of importance for hemoglobin intersubunit contact is mutated in gadoid fish

Science.gov (United States)

2014-01-01

Background Functionality of the tetrameric hemoglobin molecule seems to be determined by a few amino acids located in key positions. Oxygen binding encompasses structural changes at the interfaces between the α1β2 and α2β1 dimers, but also subunit interactions are important for the oxygen binding affinity and stability. The latter packing contacts include the conserved Arg B12 interacting with Phe GH5, which is replaced by Leu and Tyr in the αA and αD chains, respectively, of birds and reptiles. Results Searching all known hemoglobins from a variety of gnathostome species (jawed vertebrates) revealed the almost invariant Arg B12 coded by the AGG triplet positioned at an exon-intron boundary. Rare substitutions of Arg B12 in the gnathostome β globins were found in pig, tree shrew and scaled reptiles. Phe GH5 is also highly conserved in the β globins, except for the Leu replacement in the β1 globin of five marine gadoid species, gilthead seabream and the Comoran coelacanth, while Cys and Ile were found in burbot and yellow croaker, respectively. Atlantic cod β1 globin showed a Leu/Met polymorphism at position GH5 dominated by the Met variant in northwest-Atlantic populations that was rarely found in northeast-Atlantic cod. Site-specific analyses identified six consensus codons under positive selection, including 122β(GH5), indicating that the amino acid changes identified at this position may offer an adaptive advantage. In fact, computational mutation analysis showed that the replacement of Phe GH5 with Leu or Cys decreased the number of van der Waals contacts essentially in the deoxy form that probably causes a slight increase in the oxygen binding affinity. Conclusions The almost invariant Arg B12 and the AGG codon seem to be important for the packing contacts and pre-mRNA processing, respectively, but the rare mutations identified might be beneficial. The Leu122β1(GH5)Met and Met55β1(D6)Val polymorphisms in Atlantic cod hemoglobin modify the

Suppression of STAT3 NH2 -terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21.

Science.gov (United States)

Ray, Sutapa; Coulter, Don W; Gray, Shawn D; Sughroue, Jason A; Roychoudhury, Shrabasti; McIntyre, Erin M; Chaturvedi, Nagendra K; Bhakat, Kishor K; Joshi, Shantaram S; McGuire, Timothy R; Sharp, John G

2018-04-01

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH 2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients. © 2017 Wiley Periodicals, Inc.

Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

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Xu Zheng

Full Text Available In response to interleukin 6 (IL-6 stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK, and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC/NF-κB

Clinical Implications of Phosphorylated STAT3 Expression in de novo Diffuse Large B-cell Lymphoma

DEFF Research Database (Denmark)

Ok, Chi Y; Chen, Jiayu; Xu-Monette, Ziju

2014-01-01

PURPOSE: Activated signal transducer and activator of transcription 3 (STAT3) regulates tumor growth, invasion, cell proliferation, angiogenesis, immune response, and survival. Data regarding expression of phosphorylated (activated) STAT3 in diffuse large B-cell lymphoma (DLBCL) and the impact of...

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

Energy Technology Data Exchange (ETDEWEB)

Sustarsic, Elahu G. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Junnila, Riia K. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Kopchick, John J., E-mail: kopchick@ohio.edu [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH (United States)

2013-11-08

Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

NFAT5 participates in seawater inhalation-induced acute lung injury via modulation of NF-κB activity

Science.gov (United States)

Li, Congcong; Liu, Manling; Bo, Liyan; Liu, Wei; Liu, Qingqing; Chen, Xiangjun; Xu, Dunquan; Li, Zhichao; Jin, Faguang

2016-01-01

Nuclear factor of activated T cells 5 (NFAT5) is a transcription factor that can be activated by extracellular tonicity. It has been reported that NFAT5 may increase the transcription of certain osmoprotective genes in the renal system, and the aim of the current study was to explore the role of NFAT5 in seawater inhalation-induced acute lung injury. Though establishing the model of seawater inhalation-induced acute lung injury, it was demonstrated that seawater inhalation enhanced the transcription and protein expression of NFAT5 (evaluated by reverse transcription-polymerase chain reaction, immunohistochemistry stain and western blotting) and activation of nuclear factor (NF)-κB (evaluated by western blotting and mRNA expression levels of three NF-κB-dependent genes) both in lung tissue and rat alveolar macrophage cells (NR8383 cells). When expression of NFAT5 was reduced in NR8383 cells using an siRNA targeted to NFAT5, the phosphorylation of NF-κB and transcription of NF-κB-dependent genes were significantly reduced. In addition, the elevated content of certain inflammatory cytokines [tumor necrosis factor α, interleukin (IL)-1 and IL-8] were markedly reduced. In conclusion, NFAT5 serves an important pathophysiological role in seawater inhalation-induced acute lung injury by modulating NF-κB activity, and these data suggest that NFAT5 may be a promising therapeutic target. PMID:27779669

Hyperglycaemia exacerbates choroidal neovascularisation in mice via the oxidative stress-induced activation of STAT3 signalling in RPE cells.

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Xia Li

Full Text Available Choroidal neovascularisation (CNV that occurs as a result of age-related macular degeneration (AMD causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3 and vascular endothelial growth factor (VEGF, in mice with CNV. The production of reactive oxygen species (ROS in retinal pigment epithelial (RPE cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV

Circulating interleukin-6 induces fever through a STAT3-linked activation of COX-2 in the brain.

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Rummel, Christoph; Sachot, Christelle; Poole, Stephen; Luheshi, Giamal N

2006-11-01

Interleukin (IL)-6 is an important humoral mediator of fever following infection and inflammation and satisfies a number of criteria for a circulating pyrogen. However, evidence supporting such a role is diminished by the moderate or even absent ability of the recombinant protein to induce fever and activate the cyclooxygenase-2 (COX-2) pathway in the brain, a prerequisite step in the initiation and maintenance of fever. In the present study, we investigated the role of endogenous circulating IL-6 in a rodent model of localized inflammation, by neutralizing its action using a specific antiserum (IL-6AS). Rats were injected with LPS (100 microg/kg) or saline into a preformed air pouch in combination with an intraperitoneal injection of either normal sheep serum or IL-6AS (1.8 ml/rat). LPS induced a febrile response, which was accompanied by a significant rise in plasma IL-6 and nuclear STAT3 translocation in endothelial cells throughout the brain 2 h after treatment, including areas surrounding the sensory circumventricular organs and the median preoptic area (MnPO), important regions in mediating fever. These responses were abolished in the presence of the IL-6AS, which also significantly inhibited the LPS-induced upregulation of mRNA expression or immunoreactivity (IR) of the inducible form of COX, the rate-limiting enzyme for PGE2-synthesis. Interestingly, nuclear signal transducer and activator of transcription (STAT)3-positive cells colocalized with COX-2-IR, signifying that IL-6-activated cells are directly involved in PGE2 production. These observations suggest that IL-6 is an important circulating pyrogen that activates the COX-2-pathway in cerebral microvasculature, most likely through a STAT3-dependent pathway.

Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway.

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Yao, Xiangyang; Zhu, Fenfen; Zhao, Zhihui; Liu, Chang; Luo, Lan; Yin, Zhimin

2011-10-01

Arctigenin is a dibenzylbutyrolactone lignan isolated from Bardanae fructus, Arctium lappa L, Saussureamedusa, Torreya nucifera, and Ipomea cairica. It has been reported to exhibit anti-inflammatory activities, which is mainly mediated through its inhibitory effect on nuclear transcription factor-kappaB (NF-κB). But the role of arctigenin in JAK-STAT3 signaling pathways is still unclear. In present study, we investigated the effect of arctigenin on signal transducer and activator of transcription 3 (STAT3) pathway and evaluated whether suppression of STAT3 activity by arctigenin could sensitize cancer cells to a chemotherapeutic drug cisplatin. Our results show that arctigenin significantly suppressed both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Inhibition of STAT3 tyrosine phosphorylation was found to be achieved through suppression of Src, JAK1, and JAK2, while suppression of STAT3 serine phosphorylation was mediated by inhibition of ERK activation. Pervanadate reversed the arctigenin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, arctigenin can obviously induce the expression of the PTP SHP-2. Furthermore, the constitutive activation level of STAT3 was found to be correlated to the resistance of cancer cells to cisplatin-induced apoptosis. Arctigenin dramatically promoted cisplatin-induced cell death in cancer cells, indicating that arctigenin enhanced the sensitivity of cancer cells to cisplatin mainly via STAT3 suppression. These observations suggest a novel anticancer function of arctigenin and a potential therapeutic strategy of using arctigenin in combination with chemotherapeutic agents for cancer treatment. Copyright © 2011 Wiley-Liss, Inc.

Stevia and stevioside protect against cisplatin nephrotoxicity through inhibition of ERK1/2, STAT3, and NF-κB activation.

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Potočnjak, Iva; Broznić, Dalibor; Kindl, Marija; Kropek, Matija; Vladimir-Knežević, Sanda; Domitrović, Robert

2017-09-01

We investigated the effect of natural sweetener Stevia rebaudiana and its constituent stevioside in cisplatin (CP)-induced kidney injury. Male BALB/cN mice were orally administered 10, 20, and 50 mg/kg body weight of Stevia rebaudiana ethanol extract (SE) or stevioside 50 mg/kg, 48 h after intraperitoneal administration of CP (13 mg/kg). Two days later, CP treatment resulted in histopathological changes showing kidney injury. Increased expression of 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and heme oxygenase-1 (HO-1) in mice kidneys suggested oxidative stress. CP treatment also increased renal expression of nuclear factor-kappaB (NF-κB) p65 subunit and phosphorylated inhibitor of NF-κB (IκBα), as well as expression of pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α). Induction of apoptosis and inhibition of the cell cycle in kidneys was evidenced by increased expression of p53, Bax, caspase-9, and p21, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), with concomitant suppression of Bcl-2 and cyclin D1 expression. The number of apoptotic cells in kidneys was also assessed. CP administration resulted in activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3). Both SE and stevioside attenuated CP nephrotoxicity by suppressing oxidative stress, inflammation, and apoptosis through mechanism involving ERK1/2, STAT3, and NF-κB suppression. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stat3 is involved in control of MASP2 gene expression

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Unterberger, Claudia; Hanson, Steven; Klingenhoff, Andreas; Oesterle, Daniela; Frankenberger, Marion; Endo, Yuichi; Matsushita, Misao; Fujita, Teizo; Schwaeble, Wilhelm; Weiss, Elisabeth H.; Ziegler-Heitbrock, Loems; Stover, Cordula

2007-01-01

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 to be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity

TC-PTP and PTP1B: Regulating JAK-STAT signaling, controlling lymphoid malignancies.

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Pike, Kelly A; Tremblay, Michel L

2016-06-01

Lymphoid malignancies are characterized by an accumulation of genetic lesions that act co-operatively to perturb signaling pathways and alter gene expression programs. The Janus kinases (JAK)-signal transducers and activators of transcription (STATs) pathway is one such pathway that is frequently mutated in leukemia and lymphoma. In response to cytokines and growth factors, a cascade of reversible tyrosine phosphorylation events propagates the JAK-STAT pathway from the cell surface to the nucleus. Activated STAT family members then play a fundamental role in establishing the transcriptional landscape of the cell. In leukemia and lymphoma, somatic mutations have been identified in JAK and STAT family members, as well as, negative regulators of the pathway. Most recently, inactivating mutations in the protein tyrosine phosphatase (PTP) genes PTPN1 (PTP1B) and PTPN2 (TC-PTP) were sequenced in B cell lymphoma and T cell acute lymphoblastic leukemia (T-ALL) respectively. The loss of PTP1B and TC-PTP phosphatase activity is associated with an increase in cytokine sensitivity, elevated JAK-STAT signaling, and changes in gene expression. As inactivation mutations in PTPN1 and PTPN2 are restricted to distinct subsets of leukemia and lymphoma, a future challenge will be to identify in which cellular contexts do they contributing to the initiation or maintenance of leukemogenesis or lymphomagenesis. As well, the molecular mechanisms by which PTP1B and TC-PTP loss co-operates with other genetic aberrations will need to be elucidated to design more effective therapeutic strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interleukin-6 induces S100A9 expression in colonic epithelial cells through STAT3 activation in experimental ulcerative colitis.

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Min Jeoung Lee

Full Text Available BACKGROUND: Intestinal epithelium is essential for maintaining normal intestinal homeostasis; its breakdown leads to chronic inflammatory pathologies, such as inflammatory bowel diseases (IBDs. Although high concentrations of S100A9 protein and interleukin-6 (IL-6 are found in patients with IBD, the expression mechanism of S100A9 in colonic epithelial cells (CECs remains elusive. We investigated the role of IL-6 in S100A9 expression in CECs using a colitis model. METHODS: IL-6 and S100A9 expression, signal transducer and activator of transcription 3 (STAT3 phosphorylation, and infiltration of immune cells were analyzed in mice with dextran sulfate sodium (DSS-induced colitis. The effects of soluble gp130-Fc protein (sgp130Fc and S100A9 small interfering (si RNA (si-S100A9 on DSS-induced colitis were evaluated. The molecular mechanism of S100A9 expression was investigated in an IL-6-treated Caco-2 cell line using chromatin immunoprecipitation assays. RESULTS: IL-6 concentrations increased significantly in the colon tissues of DSS-treated mice. sgp130Fc or si-S100A9 administration to DSS-treated mice reduced granulocyte infiltration in CECs and induced the down-regulation of S100A9 and colitis disease activity. Treatment with STAT3 inhibitors upon IL-6 stimulation in the Caco-2 cell line demonstrated that IL-6 mediated S100A9 expression through STAT3 activation. Moreover, we found that phospho-STAT3 binds directly to the S100A9 promoter. S100A9 may recruit immune cells into inflamed colon tissues. CONCLUSIONS: Elevated S100A9 expression in CECs mediated by an IL-6/STAT3 signaling cascade may play an important role in the development of colitis.

Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

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Nadine Wittkopf

Full Text Available Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function.

Distinct cytoplasmic domains of the growth hormone receptor are required for glucocorticoid- and phorbol ester-induced decreases in growth hormone (GH) binding. These domains are different from that reported for GH-induced receptor internalization

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King, A P; Tseng, M J; Logsdon, C D

1996-01-01

Glucocorticoids inhibit growth in children and antagonize the growth-promoting action of GH in peripheral tissues. Recently, they have been shown to decrease GH binding. In this study we examine the molecular mechanisms by which the glucocorticoid dexamethasone (DEX) and the phorbol ester phorbol...... of GH binding are also observed in a Chinese hamster ovary (CHO) cell line stably transfected with a rat liver GHR cDNA, further arguing that DEX and PMA act post-translationally on GHR. Using mutant GHRs stably expressed in CHO cells, amino acids 455-506 and tyrosines 333 and/or 338 of GHR were shown...... to be required for maximal DEX-induced inhibition of GH binding. DEX decreased GH binding to a GHR mutant F346A, which is reported to be deficient in ligand-induced internalization, suggesting that DEX decreases GH binding by a mechanism distinct from that of ligand-induced GHR internalization. PMA reduced GH...

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

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Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Gab2 promotes hematopoietic stem cell maintenance and self-renewal synergistically with STAT5.

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Geqiang Li

2010-02-01

Full Text Available Grb2-associated binding (Gab adapter proteins play major roles in coordinating signaling downstream of hematopoietic cytokine receptors. In hematopoietic cells, Gab2 can modulate phosphatidylinositol-3 kinase and mitogen associated protein kinase activities and regulate the long-term multilineage competitive repopulating activity of hematopoietic stem cells (HSCs. Gab2 may also act in a linear pathway upstream or downstream of signal transducer and activator of transcription-5 (STAT5, a major positive regulator of HSC function. Therefore, we aimed to determine whether Gab2 and STAT5 function in hematopoiesis in a redundant or non-redundant manner.To do this we generated Gab2 mutant mice with heterozygous and homozygous deletions of STAT5. In heterozygous STAT5 mutant mice, deficiencies in HSC/multipotent progenitors were reflected by decreased long-term repopulating activity. This reduction in repopulation function was mirrored in the reduced growth response to early-acting cytokines from sorted double mutant c-Kit(+Lin(-Sca-1(+ (KLS cells. Importantly, in non-ablated newborn mice, the host steady-state engraftment ability was impaired by loss of Gab2 in heterozygous STAT5 mutant background. Fetal liver cells isolated from homozygous STAT5 mutant mice lacking Gab2 showed significant reduction in HSC number (KLS CD150(+CD48(-, reduced HSC survival, and dramatic loss of self-renewal potential as measured by serial transplantation.These data demonstrate new functions for Gab2 in hematopoiesis in a manner that is non-redundant with STAT5. Furthermore, important synergy between STAT5 and Gab2 was observed in HSC self-renewal, which might be exploited to optimize stem cell-based therapeutics.

Malignant T cells exhibit CD45 resistant Stat 3 activation and proliferation in cutaneous T cell lymphoma

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Krejsgaard, T; Helvad, Rikke; Ralfkiær, Elisabeth

2010-01-01

CD45 is a protein tyrosine phosphatase, which is well-known for regulating antigen receptor signalling in T and B cells via its effect on Src kinases. It has recently been shown that CD45 can also dephosphorylate Janus kinases (Jaks) and thereby regulate Signal transducer and activator of transcr......CD45 is a protein tyrosine phosphatase, which is well-known for regulating antigen receptor signalling in T and B cells via its effect on Src kinases. It has recently been shown that CD45 can also dephosphorylate Janus kinases (Jaks) and thereby regulate Signal transducer and activator...... of transcription (Stat) activation and cytokine-induced proliferation in lymphocytes. Consequently, CD45 dysregulation could be implicated in aberrant Jak/Stat activation and proliferation in lymphoproliferative diseases. Despite high expression of the CD45 ligand, Galectin-1, in skin lesions from cutaneous T......-cell lymphoma (CTCL), the malignant T cells exhibit constitutive activation of the Jak3/Stat3 signalling pathway and uncontrolled proliferation. We show that CD45 expression is down-regulated on malignant T cells when compared to non-malignant T cells established from CTCL skin lesions. Moreover, CD45 cross...

Morbillivirus control of the interferon response: relevance of STAT2 and mda5 but not STAT1 for canine distemper virus virulence in ferrets.

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Svitek, Nicholas; Gerhauser, Ingo; Goncalves, Christophe; Grabski, Elena; Döring, Marius; Kalinke, Ulrich; Anderson, Danielle E; Cattaneo, Roberto; von Messling, Veronika

2014-03-01

The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that

Ovatodiolide of Anisomeles indica Exerts the Anticancer Potential on Pancreatic Cancer Cell Lines through STAT3 and NF-κB Regulation

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Ya-Ju Hsieh

2016-01-01

Full Text Available Pancreatic cancer is the eighth leading cause of cancer death worldwide. Patients with pancreatic cancer are normally diagnosed at an advanced stage and present poor survival rate. Ovatodiolide (OV, a bioactive macrocyclic diterpenoid isolated from Anisomeles indica, showed cytotoxicity effects in pancreatic cancer cells by inhibiting cell proliferation and inducing apoptosis. Moreover, not only were cell adhesion and invasion markedly suppressed in a dose-dependent manner, but the mRNA expression of matrix metalloproteinase-9 (MMP-9 and focal adhesion kinase (FAK was also significantly decreased. Western blot analysis indicated that OV potently suppressed the phosphorylation of STAT-3 and its upstream kinase including ERK1/2, P38, and AKT Ser473. Meanwhile, OV inactivated the nuclear factor kappa B (NF-κB by inhibiting IκB kinase (IKK α/β activation and the subsequent suppression of inhibitor of kappa B (IκB phosphorylation. These results demonstrated that OV could potentially inhibit Mia-PaCa2 cancer cells proliferation and induce apoptosis through modulation of NF-κB and STAT3 pathway. Moreover, OV suppressed cell invasiveness and interfered with cell-matrix adhesion in Mia-PaCa2 cancer cells by reducing MMP-9 and FAK transcription through suppressing NF-κB and STAT3 pathway. Taken together, our findings reveal a new therapeutic and antimetastatic potential of ovatodiolide for pancreatic cancer remedy.

JANEX-1, a JAK3 inhibitor, protects pancreatic islets from cytokine toxicity through downregulation of NF-{kappa}B activation and the JAK/STAT pathway

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Lv, Na; Kim, Eun-Kyung; Song, Mi-Young [Department of Biochemistry, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Choi, Ha-Na; Moon, Woo Sung [Department of Pathology, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Park, Sung-Joo [Department of Herbology, School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Park, Jin-Woo [Department of Biochemistry, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Kwon, Kang-Beom, E-mail: desson@wonkwang.ac.kr [Department of Physiology, School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Park, Byung-Hyun, E-mail: bhpark@chonbuk.ac.kr [Department of Biochemistry, Medical School and Diabetes Research Center, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of)

2009-07-15

JANEX-1/WHI-P131, a selective Janus kinase 3 (JAK3) inhibitor, has been shown to delay the onset of diabetes in the NOD mouse model. However, the molecular mechanism by which JANEX-1 protects pancreatic {beta}-cells is unknown. In the current study, we investigated the role of JANEX-1 on interleukin (IL)-1{beta} and interferon (IFN)-{gamma}-induced {beta}-cell damage using isolated islets. JANEX-1-pretreated islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced inducible form of NO synthase (iNOS) expression, and decreased islet destruction. The molecular mechanism by which JANEX-1 inhibits iNOS expression was mediated through suppression of the nuclear factor {kappa}B (NF-{kappa}B) and JAK/signal transducer and activator of transcription (STAT) pathways. Islets treated with the cytokines downregulated the protein levels of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, but pretreatment with JANEX-1 attenuated these decreases. Additionally, islets from JAK3{sup -/-} mice were more resistant to cytokine toxicity than islets from control mice. These results demonstrate that JANEX-1 protects {beta}-cells from cytokine toxicity through suppression of the NF-{kappa}B and JAK/STAT pathways and upregulation of SOCS proteins, suggesting that JANEX-1 may be used to preserve functional {beta}-cell mass.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

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Peter Y Yu

Full Text Available STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5.An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice.LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

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Yu, Peter Y; Gardner, Heather L; Roberts, Ryan; Cam, Hakan; Hariharan, Seethalakshmi; Ren, Ling; LeBlanc, Amy K; Xiao, Hui; Lin, Jiayuh; Guttridge, Denis C; Mo, Xiaokui; Bennett, Chad E; Coss, Christopher C; Ling, Yonghua; Phelps, Mitch A; Houghton, Peter; London, Cheryl A

2017-01-01

STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

Growth hormone action in hypothyroid infant rats.

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Humbert, J T; Bergad, P L; Masha, O; Stolz, A M; Kaul, S; Berry, S A

2000-02-01

In neonatal rats, expression of serine protease inhibitors 2.1 and 2.3 mRNA peaks on d 2 of life and declines shortly thereafter, coinciding with levels of circulating GH. To evaluate the role of GH in this increase and to test the hypothesis that GH is active in perinatal life, we studied GH action in a model of GH deficiency. Maternal/neonatal hypothyroidism with consequent GH deficiency was induced by methimazole administration to pregnant dams. The resultant hypothyroid neonates were treated at d 2 or 7 of age with GH or saline for 1 h before exsanguination. In d-7 neonates, but not at d 2, GH administration resulted in significant serine protease inhibitors 2.1 and 2.3 mRNA induction. This treatment did not result in increased production of either GH receptor or IGF-I mRNA at either age. There was a slight GH-independent increase in GH receptor and IGF-I mRNA expression by d 7. Electromobility shift assays using hepatic nuclear extracts from these neonates and the GH response element from the serine protease inhibitor 2.1 promoter showed signal transducer and activator of transcription 5 (Stat5) binding in response to GH in extracts from d-7 rats only. Immunoblots of these extracts showed twice as much Stat5 in the nuclei of d-7 treated neonates compared with d-2 treated neonates. We conclude that there is apparent insensitivity to GH treatment in d-2 neonates that remits by d 7 and that this remission correlates with increased abundance of GH receptor and Stat5.

[HSP90 inhibitor 17-AAG plays an important role in JAK3/STAT5 signaling pathways in HTLV-1 infection cell line HUT-102].

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Yang, Q Q; Tan, H; Fu, Z P; Ma, Q; Song, J L

2017-08-14

Objective: To analyze whether heat-shock protein 90 (HSP90) be involved in a permanently abnormal activated JAK/STAT signaling in ATL cells in vitro. Methods: The effect of 17-AAG on proliferation of ATL cell lines HUT-102 was assessed using CCK8 at different time points. Cell apoptosis was measured by flow cytometry. The specific proteins HSP90, STAT5, p-STAT5 and JAK3 were detected by Western blotting. Results: Overexpression of HSP90 in HUT-102 cell lines was disclosed ( P AAG led to reduced cell proliferation, but there was no significant change in terms of cell proliferation when the concentration of 17-AAG between 2 000-8 000 nmol/L ( P >0.05) . 17-AAG induced cell apoptosis in different time-points and concentrations. 17-AAG don't affect the expression of JAK3 gene. Conclusion: This study indicated that JAK3 as HSP90 client protein was aberrantly activated in HTLV-1-infected T-cell lines, leading to constitutive activation of p-STAT5 in JAK/STAT signal pathway, which demonstrated that HSP90-inhibitors 17-AAG inhibited the growth of HTLV-1-infected T-cell lines by reducing cell proliferation and inducing cell apoptosis.

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT genomic binding patterns discerns cell-specific cis-regulatory modules

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Kang Keunsoo

2013-01-01

Full Text Available Abstract Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM, which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

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Xu, Lei; Zhou, Xinying; Wang, Wenshi; Wang, Yijin; Yin, Yuebang; Laan, Luc J W van der; Sprengers, Dave; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

2016-10-01

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes. © FASEB.

Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

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Balart Luis A

2010-10-01

Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

Scoparone exerts anti-tumor activity against DU145 prostate cancer cells via inhibition of STAT3 activity.

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Jeong-Kook Kim

Full Text Available Scoparone, a natural compound isolated from Artemisia capillaris, has been used in Chinese herbal medicine to treat neonatal jaundice. Signal transducer and activator of transcription 3 (STAT3 contributes to the growth and survival of many human tumors. This study was undertaken to investigate the anti-tumor activity of scoparone against DU145 prostate cancer cells and to determine whether its effects are mediated by inhibition of STAT3 activity. Scoparone inhibited proliferation of DU145 cells via cell cycle arrest in G1 phase. Transient transfection assays showed that scoparone repressed both constitutive and IL-6-induced transcriptional activity of STAT3. Western blot and quantitative real-time PCR analyses demonstrated that scoparone suppressed the transcription of STAT3 target genes such as cyclin D1, c-Myc, survivin, Bcl-2, and Socs3. Consistent with this, scoparone decreased phosphorylation and nuclear accumulation of STAT3, but did not reduce phosphorylation of janus kinase 2 (JAK2 or Src, the major upstream kinases responsible for STAT3 activation. Moreover, transcriptional activity of a constitutively active mutant of STAT3 (STAT3C was inhibited by scoparone, but not by AG490, a JAK2 inhibitor. Furthermore, scoparone treatment suppressed anchorage-independent growth in soft agar and tumor growth of DU145 xenografts in nude mice, concomitant with a reduction in STAT3 phosphorylation. Computational modeling suggested that scoparone might bind the SH2 domain of STAT3. Our findings suggest that scoparone elicits an anti-tumor effect against DU145 prostate cancer cells in part through inhibition of STAT3 activity.

The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation

DEFF Research Database (Denmark)

Richter, H E; Albrektsen, T; Billestrup, Nils

2003-01-01

GH inhibits primary rat preadipocyte differentiation and expression of late genes required for terminal differentiation. Here we show that GH-mediated inhibition of fatty acid-binding protein aP2 gene expression correlates with the activation of the Janus kinase-2/signal transducer and activator ...

STAT3 Activities and Energy Metabolism: Dangerous Liaisons

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Camporeale, Annalisa, E-mail: annalisa.camporeale@unito.it [Molecular Biotechnology Center and Department of Molecular Biotechnology and Life Sciences, University of Turin, Via Nizza 52, Turin 10126 (Italy); Demaria, Marco [Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945 (United States); Monteleone, Emanuele [Molecular Biotechnology Center and Department of Molecular Biotechnology and Life Sciences, University of Turin, Via Nizza 52, Turin 10126 (Italy); Giorgi, Carlotta [Department of Experimental and Diagnostic Medicine, Section of General Pathology, Laboratory for Technologies of Advances Therapies (LTTA), University of Ferrara, Via Fossato di Mortara 70, Ferrara 44121 (Italy); Wieckowski, Mariusz R. [Nencki Institute of Experimental Biology, Department of Biochemistry, Pasteur Str. 3, Warsaw 02-093 (Poland); Pinton, Paolo [Department of Experimental and Diagnostic Medicine, Section of General Pathology, Laboratory for Technologies of Advances Therapies (LTTA), University of Ferrara, Via Fossato di Mortara 70, Ferrara 44121 (Italy); Poli, Valeria, E-mail: annalisa.camporeale@unito.it [Molecular Biotechnology Center and Department of Molecular Biotechnology and Life Sciences, University of Turin, Via Nizza 52, Turin 10126 (Italy)

2014-07-31

STAT3 mediates cytokine and growth factor receptor signalling, becoming transcriptionally active upon tyrosine 705 phosphorylation (Y-P). Constitutively Y-P STAT3 is observed in many tumors that become addicted to its activity, and STAT3 transcriptional activation is required for tumor transformation downstream of several oncogenes. We have recently demonstrated that constitutively active STAT3 drives a metabolic switch towards aerobic glycolysis through the transcriptional induction of Hif-1α and the down-regulation of mitochondrial activity, in both MEF cells expressing constitutively active STAT3 (Stat3{sup C/C}) and STAT3-addicted tumor cells. This novel metabolic function is likely involved in mediating pre-oncogenic features in the primary Stat3{sup C/C} MEFs such as resistance to apoptosis and senescence and rapid proliferation. Moreover, it strongly contributes to the ability of primary Stat3{sup C/C} MEFs to undergo malignant transformation upon spontaneous immortalization, a feature that may explain the well known causative link between STAT3 constitutive activity and tumor transformation under chronic inflammatory conditions. Taken together with the recently uncovered role of STAT3 in regulating energy metabolism from within the mitochondrion when phosphorylated on Ser 727, these data place STAT3 at the center of a hub regulating energy metabolism under different conditions, in most cases promoting cell survival, proliferation and malignant transformation even though with distinct mechanisms.

STAT3 Activities and Energy Metabolism: Dangerous Liaisons

International Nuclear Information System (INIS)

Camporeale, Annalisa; Demaria, Marco; Monteleone, Emanuele; Giorgi, Carlotta; Wieckowski, Mariusz R.; Pinton, Paolo; Poli, Valeria

2014-01-01

STAT3 mediates cytokine and growth factor receptor signalling, becoming transcriptionally active upon tyrosine 705 phosphorylation (Y-P). Constitutively Y-P STAT3 is observed in many tumors that become addicted to its activity, and STAT3 transcriptional activation is required for tumor transformation downstream of several oncogenes. We have recently demonstrated that constitutively active STAT3 drives a metabolic switch towards aerobic glycolysis through the transcriptional induction of Hif-1α and the down-regulation of mitochondrial activity, in both MEF cells expressing constitutively active STAT3 (Stat3 C/C ) and STAT3-addicted tumor cells. This novel metabolic function is likely involved in mediating pre-oncogenic features in the primary Stat3 C/C MEFs such as resistance to apoptosis and senescence and rapid proliferation. Moreover, it strongly contributes to the ability of primary Stat3 C/C MEFs to undergo malignant transformation upon spontaneous immortalization, a feature that may explain the well known causative link between STAT3 constitutive activity and tumor transformation under chronic inflammatory conditions. Taken together with the recently uncovered role of STAT3 in regulating energy metabolism from within the mitochondrion when phosphorylated on Ser 727, these data place STAT3 at the center of a hub regulating energy metabolism under different conditions, in most cases promoting cell survival, proliferation and malignant transformation even though with distinct mechanisms

Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma

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Lisette P. Yco

2014-01-01

Full Text Available Signal transducer and activator of transcription 3 (STAT3 is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM tumor cells, prevented interleukin-6 (IL-6–mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phosphotyrosine residue of the STAT3 Src homology 2 (SH2 domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM.

A novel STAT inhibitor, OPB-31121, has a significant antitumor effect on leukemia with STAT-addictive oncokinases

International Nuclear Information System (INIS)

Hayakawa, F; Sugimoto, K; Harada, Y; Hashimoto, N; Ohi, N; Kurahashi, S; Naoe, T

2013-01-01

Signal transduction and activator of transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate cell proliferation, apoptosis, differentiation, development and the immune response. Aberrant signals of STAT induce uncontrolled cell proliferation and apoptosis resistance and are strongly involved in cancer. STAT has been identified as a promising target for antitumor drugs, but to date most trials have not been successful. Here, we demonstrated that a novel STAT inhibitor, OPB-31121, strongly inhibited STAT3 and STAT5 phosphorylation without upstream kinase inhibition, and induced significant growth inhibition in various hematopoietic malignant cells. Investigation of various cell lines suggested that OPB-31121 is particularly effective against multiple myeloma, Burkitt lymphoma and leukemia harboring BCR–ABL, FLT3/ITD and JAK2 V617F, oncokinases with their oncogenicities dependent on STAT3/5. Using an immunodeficient mouse transplantation system, we showed the significant antitumor effect of OPB-31121 against primary human leukemia cells harboring these aberrant kinases and its safety for normal human cord blood cells. Finally, we demonstrated a model to overcome drug resistance to upstream kinase inhibitors with a STAT inhibitor. These results suggested that OPB-31121 is a promising antitumor drug. Phase I trials have been performed in Korea and Hong Kong, and a phase I/II trial is underway in Japan

Dual inhibition of STAT1 and STAT3 activation downregulates expression of PD-L1 in human breast cancer cells.

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Sasidharan Nair, Varun; Toor, Salman M; Ali, Bassam R; Elkord, Eyad

2018-05-02

Breast cancer is the most commonly diagnosed cancer, and it is a leading cause of cancer-related deaths in females worldwide. Triple-negative breast cancer (TNBC) constitutes 15% of breast cancer and shows distinct metastasis profiles with poor prognosis. Strong PD-L1 expression has been observed in some tumors, supporting their escape from immune surveillance. Targeting PD-L1 could be a promising therapeutic approach in breast cancer patients. We investigated potential molecular mechanisms for constitutive expression of PD-L1 by inhibiting upstream STAT1 and STAT3 signals. PD-L1 expression in three breast cancer cell lines was measured using quantitative PCR and western blotting. Activation of STAT1 and STAT3 was blocked using pharmacological inhibitors and siRNA. The mechanism underlying the constitutive expression of PD-L1 was investigated using ChIP and co-immunoprecipitation assays. We found that individual inhibition of STAT1 and STAT3 activation partially downregulated PD-L1, while combined inhibition completely downregulated PD-L1 expression. Moreover, our results suggest that pSTAT1-pSTAT3 dimerize in cytosol and translocate to the nucleus, where they bind to PD-L1 promoter and induce PD-L1 expression. These findings provide a rationale for combined targeting of STAT1 and STAT3 for the development of immune-based cancer therapies for down regulation of PD-L1 expression.

Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

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Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

2011-08-01

Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Chemokine CCL2–CCR2 Signaling Induces Neuronal Cell Death via STAT3 Activation and IL-1β Production after Status Epilepticus

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Tian, Dai-Shi; Feng, Li-Jie; Liu, Jun-Li

2017-01-01

Elevated levels of chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 have been reported in patients with temporal lobe epilepsy and in experimental seizures. However, the functional significance and molecular mechanism underlying CCL2–CCR2 signaling in epileptic brain remains largely unknown. In this study, we found that the upregulated CCL2 was mainly expressed in hippocampal neurons and activated microglia from mice 1 d after kainic acid (KA)-induced seizures. Taking advantage of CX3CR1GFP/+:CCR2RFP/+ double-transgenic mice, we demonstrated that CCL2–CCR2 signaling has a role in resident microglial activation and blood-derived monocyte infiltration. Moreover, seizure-induced degeneration of neurons in the hippocampal CA3 region was attenuated in mice lacking CCL2 or CCR2. We further showed that CCR2 activation induced STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1β production, which are critical for promoting neuronal cell death after status epilepticus. Consistently, pharmacological inhibition of STAT3 by WP1066 reduced seizure-induced IL-1β production and subsequent neuronal death. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. Together, we demonstrated that CCL2–CCR2 signaling contributes to neurodegeneration via STAT3 activation and IL-1β production after status epilepticus, providing potential therapeutic targets for the treatment of epilepsy. SIGNIFICANCE STATEMENT Epilepsy is a global concern and epileptic seizures occur in many neurological conditions. Neuroinflammation associated with microglial activation and monocyte infiltration are characteristic of epileptic brains. However, molecular mechanisms underlying neuroinflammation in neuronal death following epilepsy remain to be elucidated. Here we demonstrate that CCL2–CCR2 signaling is

Characterization of STAT3 activation and expression in canine and human osteosarcoma

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Li Pui-Kai

2009-03-01

Full Text Available Abstract Background Dysregulation of signal transducer and activator of transcription 3 (STAT3 has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor. Methods To examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656 or STAT3 (LLL3 inhibitors and cell proliferation (CyQUANT, caspase 3/7 activity (ELISA, apoptosis (Western blotting for PARP cleavage and/or viability (Wst-1 were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography. Results Our data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known

Characterization of STAT3 activation and expression in canine and human osteosarcoma

International Nuclear Information System (INIS)

Fossey, Stacey L; Liao, Albert T; McCleese, Jennifer K; Bear, Misty D; Lin, Jiayuh; Li, Pui-Kai; Kisseberth, William C; London, Cheryl A

2009-01-01

Dysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor. To examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography. Our data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3. These data

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

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Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-01-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB...

CRH promotes human colon cancer cell proliferation via IL-6/JAK2/STAT3 signaling pathway and VEGF-induced tumor angiogenesis.

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Fang, Xianjun; Hong, Yali; Dai, Li; Qian, Yuanyuan; Zhu, Chao; Wu, Biao; Li, Shengnan

2017-11-01

Corticotrophin-releasing hormone (CRH) has been demonstrated to participate in various diseases. Our previous study showed that its receptor CRHR1 mediated the development of colitis-associated cancer in mouse model. However, the detailed mechanisms remain unclear. In this study, we explored the oncogenetic role of CRH/CRHR1 signaling in colon cancer cells. Cell proliferation and colony formation assays revealed that CRH contributed to cell proliferation. Moreover, tube formation assay showed that CRH-treated colon cancer cell supernatant significantly promoted tube formation of human umbilical vein endothelial cells (HUVECs). And these effects could be reversed by the CRHR1 specific antagonist Antalarmin. Further investigation showed that CRH significantly upregulated the expressions of interlukin-6 (IL-6) and vascular endothelial growth factor (VEGF) through activating nuclear factor-kappa B (NF-κB). The CRH-induced IL-6 promoted phosphorylation of janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3). STAT3 inhibition by Stattic significantly inhibited the CRH-induced cell proliferation. In addition, silence of VEGF resulted in declined tube formation induced by CRH. Taken together, CRH/CRHR1 signaling promoted human colon cancer cell proliferation via NF-κB/IL-6/JAK2/STAT3 signaling pathway and tumor angiogenesis via NF-κB/VEGF signaling pathway. Our results provide evidence to support a critical role for the CRH/CRHR1 signaling in colon cancer progression and suggest its potential utility as a new therapeutic target for colon cancer. © 2017 Wiley Periodicals, Inc.

Inhibition of STAT3 reduces astrocytoma cell invasion and constitutive activation of STAT3 predicts poor prognosis in human astrocytoma.

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Qinchuan Liang

Full Text Available Astrocytoma cells characteristically possess high invasion potentials. Recent studies have revealed that knockdown of signal transducers and activators of transcription 3 (STAT3 expression by RNAi induces apoptosis in astrocytoma cell. Nevertheless, the distinct roles of STAT3 in astrocytoma's invasion and recurrence have not been elucidated. In this study, we silenced STAT3 using Small interfering RNAs in two human glioblastoma multiforme (GBM cell lines (U251 and U87, and investigated the effect on GBM cell adhesion and invasion. Our results demonstrate that disruption of STAT3 inhibits GBM cell's adhesion and invasion. Knockdown of STAT3 significantly increased E-cadherin but decreased N-cadherin, vascular endothelial growth factor, matrix metalloproteinase 2 and matrix metalloproteinase 9. Additionally, expression of pSTAT3(Tyr705 correlates with astrocytoma WHO classification, Karnofsky performance status scale score, tumor recurrence and survival. Furthermore, pSTAT3(Tyr705 is a significant prognostic factor in astrocytoma. In conclusion, STAT3 may affect astrocytoma invasion, expression of pSTAT3(Tyr705 is a significant prognostic factor in tumor recurrence and overall survival in astrocytoma patients. Therefore, STAT3 may provide a potential target for molecular therapy in human astrocytoma, and pSTAT3(Tyr705could be an important biomarker for astrocytoma prognosis.

Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways.

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Yang, Lichao; Guo, Han; Li, Ying; Meng, Xianglan; Yan, Lu; Dan Zhang; Wu, Sangang; Zhou, Hao; Peng, Lu; Xie, Qiang; Jin, Xin

2016-10-10

The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 μg/ml) in the presence or absence of OEA (10, 20 and 40 μM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases.

Hepatic expression of the GH/JAK/STAT/IGF pathway, acute-phase response signalling and complement system are affected in mouse offspring by prenatal and early postnatal exposure to maternal high-protein diet.

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Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Rehfeldt, Charlotte; Metges, Cornelia C

2011-12-01

Effects of pre- and early postnatal exposure to maternal high-protein diets are not well understood. Transcription profiling was performed in male mouse offspring exposed to maternal high-protein diet during pregnancy and/or lactation to identify affected hepatic molecular pathways. Dams were fed isoenergetic diets with control (20% w/w) or high protein levels (40%). The hepatic expression profiles were evaluated by differential microarray analysis 3 days (d3) and 3 weeks (d21) after birth. Offspring from three different high-protein dietary groups, HP (d3, high-protein diet during pregnancy), HPHP (d21, high-protein diet during pregnancy and lactation) and CHP (d21, control diet during pregnancy and high-protein diet during lactation), were compared with age-matched offspring from dams fed control diet. Offspring body and liver mass of all high-protein groups were decreased. Prenatal high-protein diet affected hepatic expression of genes mapping to the acute response/complement system and the GH/JAK/STAT/IGF signalling pathways. Maternal exposure to high-protein diet during lactation affected hepatic gene expression of the same pathways but additionally affected genes mapping to protein, fatty acid, hexose and pyruvate metabolism. (1) Genes of the acute response/complement system and GH/JAK/STAT/IGF pathways were down-regulated in offspring of dams exposed to high-protein diets during pregnancy and/or lactation. (2) Genes related to nutrient and energy metabolism, however, were only affected when high-protein diet was administered during lactation. (3) Modulation of the GH/JAK/STAT/IGF pathway might be responsible for reduced body and liver masses by maternal high-protein diet.

Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

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Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

2002-01-01

The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties. PMID:11909970

STAT3 can be activated through paracrine signaling in breast epithelial cells

International Nuclear Information System (INIS)

Lieblein, Jacqueline C; Ball, Sarah; Hutzen, Brian; Sasser, A Kate; Lin, Huey-Jen; Huang, Tim HM; Hall, Brett M; Lin, Jiayuh

2008-01-01

Many cancers, including breast cancer, have been identified with increased levels of phosphorylated or the active form of Signal Transducers and Activators of Transcription 3 (STAT3) protein. However, whether the tumor microenvironment plays a role in this activation is still poorly understood. Conditioned media, which contains soluble factors from MDA-MB-231 and MDA-MB-468 breast cancer cells and breast cancer associated fibroblasts, was added to MCF-10A breast epithelial and MDA-MB-453 breast cancer cells. The stimulation of phosphorylated STAT3 (p-STAT3) levels by conditioned media was assayed by Western blot in the presence or absence of neutralized IL-6 antibody, or a JAK/STAT3 inhibitor, JSI-124. The stimulation of cell proliferation in MCF-10A cells by conditioned media in the presence or absence of JSI-124 was subjected to MTT analysis. IL-6, IL-10, and VEGF levels were determined by ELISA analysis. Our results demonstrated that conditioned media from cell lines with constitutively active STAT3 are sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels. This signaling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and is persistent for at least 24 hours. ELISA analysis confirmed a correlation between elevated levels of IL-6 production and p-STAT3. Neutralization of the IL-6 ligand or gp130 was sufficient to block increased levels of p-STAT3 (Y705) in treated cells. Furthermore, soluble factors within the MDA-MB-231 conditioned media were also sufficient to stimulate an increase in IL-6 production from MCF-10A cells. These results demonstrate STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signaling through soluble factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The induction of STAT3 phosphorylation is through the IL-6/JAK pathway and appears to be associated with

Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.

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Ermelinda Porpiglia

2012-08-01

Full Text Available Erythropoietin (Epo-induced Stat5 phosphorylation (p-Stat5 is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a "knocked-in" EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the

Suppression of type I and type III IFN signalling by NSs protein of severe fever with thrombocytopenia syndrome virus through inhibition of STAT1 phosphorylation and activation.

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Chaudhary, Vidyanath; Zhang, Shuo; Yuen, Kit-San; Li, Chuan; Lui, Pak-Yin; Fung, Sin-Yee; Wang, Pei-Hui; Chan, Chi-Ping; Li, Dexin; Kok, Kin-Hang; Liang, Mifang; Jin, Dong-Yan

2015-11-01

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen causing significant morbidity and mortality in Asia. NSs protein of SFTSV is known to perturb type I IFN induction and signalling, but the mechanism remains to be fully understood. Here, we showed the suppression of both type I and type III IFN signalling by SFTSV NSs protein is mediated through inhibition of STAT1 phosphorylation and activation. Infection with live SFTSV or expression of NSs potently suppressed IFN-stimulated genes but not NFkB activation. NSs was capable of counteracting the activity of IFN-α1, IFN-β, IFN-λ1 and IFN-λ2. Mechanistically, NSs associated with STAT1 and STAT2, mitigated IFN-β-induced phosphorylation of STAT1 at S727, and reduced the expression and activity of STAT1 protein in IFN-β-treated cells, resulting in the inhibition of STAT1 and STAT2 recruitment to IFNstimulated promoters. Taken together, SFTSV NSs protein is an IFN antagonist that suppresses phosphorylation and activation of STAT1.

Vitamin D across growth hormone (GH) disorders: From GH deficiency to GH excess.

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Ciresi, A; Giordano, C

2017-04-01

The interplay between vitamin D and the growth hormone (GH)/insulin-like growth factor (IGF)-I system is very complex and to date it is not fully understood. GH directly regulates renal 1 alpha-hydroxylase activity, although the action of GH in modulating vitamin D metabolism may also be IGF-I mediated. On the other hand, vitamin D increases circulating IGF-I and the vitamin D deficiency should be normalized before measurement of IGF-I concentrations to obtain reliable and unbiased IGF-I values. Indeed, linear growth after treatment of nutritional vitamin D deficiency seems to be mediated through activation of the GH/IGF-I axis and it suggests an important role of vitamin D as a link between the proliferating cartilage cells of the growth plate and GH/IGF-I secretion. Vitamin D levels are commonly lower in patients with GH deficiency (GHD) than in controls, with a variable prevalence of insufficiency or deficiency, and this condition may worsen the already known cardiovascular and metabolic risk of GHD, although this finding is not common to all studies. In addition, data on the impact of GH treatment on vitamin D levels in GHD patients are quite conflicting. Conversely, in active acromegaly, a condition characterized by a chronic GH excess, both increased and decreased vitamin D levels have been highlighted, and the interplay between vitamin D and the GH/IGF-I axis becomes even more complicated when we consider the acromegaly treatment, both medical and surgical. The current review summarizes the available data on vitamin D in the main disorders of the GH/IGF-I axis, providing an overview of the current state of the art. Copyright © 2017 Elsevier Ltd. All rights reserved.

Study of the UV Light Conversion of Feruloyl Amides from Portulaca oleracea and Their Inhibitory Effect on IL-6-Induced STAT3 Activation.

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Hwang, Joo Tae; Kim, Yesol; Jang, Hyun-Jae; Oh, Hyun-Mee; Lim, Chi-Hwan; Lee, Seung Woong; Rho, Mun-Chual

2016-06-30

Two new feruloyl amides, N-cis-hibiscusamide (5) and (7'S)-N-cis-feruloylnormetanephrine (9), and eight known feruloyl amides were isolated from Portulaca oleracea L. and the geometric conversion of the ten isolated feruloyl amides by UV light was verified. The structures of the feruloyl amides were determined based on spectroscopic data and comparison with literature data. The NMR data revealed that the structures of the isolated compounds showed cis/trans-isomerization under normal laboratory light conditions. Therefore, cis and trans-isomers of feruloyl amides were evaluated for their convertibility and stability by UV light of a wavelength of 254 nm. After 96 h of UV light exposure, 23.2%-35.0% of the cis and trans-isomers were converted to trans-isomers. Long-term stability tests did not show any significant changes. Among all compounds and conversion mixtures collected, compound 6 exhibited the strongest inhibition of IL-6-induced STAT3 activation in Hep3B cells, with an IC50 value of 0.2 μM. This study is the first verification of the conversion rates and an equilibrium ratio of feruloyl amides. These results indicate that this natural material might provide useful information for the treatment of various diseases involving IL-6 and STAT3.

1,4-benzoquinone-induced STAT-3 hypomethylation in AHH-1 cells: Role of oxidative stress

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Jing Yang

2015-01-01

Full Text Available Benzene, a known occupational and environmental contaminant, is associated with increased risk of leukemia. The objectives of this study were to elucidate the regulatory mechanism of the hypomethylated STAT3 involved in benzene toxicity in vitro. As 1,4-benzoquinone (1,4-BQ is one of benzene’s major toxic metabolites, AHH-1 cells were treated by 1,4-BQ for 24 h with or without pretreatment of the antioxidant a-LA or the methyltransferase inhibitor, 5-aza-2′ deoxycytidine (5-aza. The cell viability was investigated using the 3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. ROS was determined via 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA flow cytometric assays. The level of oxidative stress marker 8-OHdG was measured by enzyme-linked immunosorbent assay. Methylation-specific PCR was used to detect the methylation status of STAT3. Results indicated the significantly increasing expression of ROS and 8-OHdG which accompanied with STAT3 hypomethylation in 1,4-BQ-treated AHH-1 cells. α-LA suppressed the expression of both ROS and 8-OHdG, simultaneously reversed 1,4-BQ-induced STAT3 hypomethylation. However, although the methylation inhibitor, 5-aza reduced the expression level of ROS and 8-OHdG, but had no obvious inhibiting effect on STAT3 methylation level. Taken together, oxidative stress are involved 1,4-BQ-induced STAT3 methylation expression.

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

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Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Leptin-induced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore

OpenAIRE

Smith, Christopher C. T.; Dixon, Richard A.; Wynne, Abigail M.; Theodorou, Louise; Ong, Sang-Ging; Subrayan, Sapna; Davidson, Sean M.; Hausenloy, Derek J.; Yellon, Derek M.

2010-01-01

Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the...

Single nucleotide polymorphisms in BMPR-IB and STAT5B genes and their association with growth and reproductive traits in chicken

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Shahram Niknafs

2014-04-01

Full Text Available The aim of the current study was to investigate the association of G4533815A SNP in STAT5B and A287G SNP in BMPR-IB genes with growth and reproduction related traits in chicken. A sample of 205 individuals from breeding station of Mazandaran native chicken population was selected randomly. All of the individuals were genotyped for both SNPs using PCR-RFLP technique. Marker-trait association analyses were performed using estimated breeding value of the traits as dependent variable in GLM procedure of SAS 9.1. Results suggested that breeding value least square means for genotypes of G4533815A SNP is significantly differed from each other for traits of body weight at 8 and 12 weeks (P<0.01. In the case of BMPR-IB gene, no significant difference was found. In conclusion, STAT5B gene may be associated with body growth in chicken and may be considered in Marker Assisted Selection program to improve chicken growth performance.

Heregulin Co-opts PR Transcriptional Action Via Stat3 Role As a Coregulator to Drive Cancer Growth.

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Proietti, Cecilia J; Izzo, Franco; Díaz Flaqué, María Celeste; Cordo Russo, Rosalía; Venturutti, Leandro; Mercogliano, María Florencia; De Martino, Mara; Pineda, Viviana; Muñoz, Sergio; Guzmán, Pablo; Roa, Juan C; Schillaci, Roxana; Elizalde, Patricia V

2015-10-01

Accumulated findings have demonstrated the presence of bidirectional interactions between progesterone receptor (PR) and the ErbB family of receptor tyrosine kinases signaling pathways in breast cancer. We previously revealed signal transducer and activator of transcription 3 (Stat3) as a nodal convergence point between said signaling pathways proving that Stat3 is activated by one of the ErbBs' ligands, heregulin (HRG)β1 via ErbB2 and through the co-option of PR as a signaling molecule. Here, we found that HRGβ1 induced Stat3 recruitment to the promoters of the progestin-regulated cell cycle modulators Bcl-XL and p21(CIP1) and also stimulated Stat3 binding to the mouse mammary tumor virus promoter, which carries consensus progesterone response elements. Interestingly, HRGβ1-activated Stat3 displayed differential functions on PR activity depending on the promoter bound. Indeed, Stat3 was required for PR binding in bcl-X, p21(CIP1), and c-myc promoters while exerting a PR coactivator function on the mouse mammary tumor virus promoter. Stat3 also proved to be necessary for HRGβ1-induced in vivo tumor growth. Our results endow Stat3 a novel function as a coregulator of HRGβ1-activated PR to promote breast cancer growth. These findings underscore the importance of understanding the complex interactions between PR and other regulatory factors, such as Stat3, that contribute to determine the context-dependent transcriptional actions of PR.

Heregulin Co-opts PR Transcriptional Action Via Stat3 Role As a Coregulator to Drive Cancer Growth

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Izzo, Franco; Díaz Flaqué, María Celeste; Cordo Russo, Rosalía; Venturutti, Leandro; Mercogliano, María Florencia; De Martino, Mara; Pineda, Viviana; Muñoz, Sergio; Guzmán, Pablo; Roa, Juan C.; Schillaci, Roxana

2015-01-01

Accumulated findings have demonstrated the presence of bidirectional interactions between progesterone receptor (PR) and the ErbB family of receptor tyrosine kinases signaling pathways in breast cancer. We previously revealed signal transducer and activator of transcription 3 (Stat3) as a nodal convergence point between said signaling pathways proving that Stat3 is activated by one of the ErbBs' ligands, heregulin (HRG)β1 via ErbB2 and through the co-option of PR as a signaling molecule. Here, we found that HRGβ1 induced Stat3 recruitment to the promoters of the progestin-regulated cell cycle modulators Bcl-XL and p21CIP1 and also stimulated Stat3 binding to the mouse mammary tumor virus promoter, which carries consensus progesterone response elements. Interestingly, HRGβ1-activated Stat3 displayed differential functions on PR activity depending on the promoter bound. Indeed, Stat3 was required for PR binding in bcl-X, p21CIP1, and c-myc promoters while exerting a PR coactivator function on the mouse mammary tumor virus promoter. Stat3 also proved to be necessary for HRGβ1-induced in vivo tumor growth. Our results endow Stat3 a novel function as a coregulator of HRGβ1-activated PR to promote breast cancer growth. These findings underscore the importance of understanding the complex interactions between PR and other regulatory factors, such as Stat3, that contribute to determine the context-dependent transcriptional actions of PR. PMID:26340407

Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases.

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Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

2015-01-01

Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

Ghrelin- and GH-induced insulin resistance: no association with retinol-binding protein-4

DEFF Research Database (Denmark)

Vestergaard, Esben Thyssen; Krag, Morten B; Poulsen, Morten M

2013-01-01

Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects....

Lung Adenocarcinomas and Lung Cancer Cell Lines Show Association of MMP-1 Expression With STAT3 Activation

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Alexander Schütz

2015-04-01

Full Text Available Signal transducer and activator of transcription 3 (STAT3 is constitutively activated in the majority of lung cancer. This study aims at defining connections between STAT3 function and the malignant properties of non–small cell lung carcinoma (NSCLC cells. To address possible mechanisms by which STAT3 influences invasiveness, the expression of matrix metalloproteinase-1 (MMP-1 was analyzed and correlated with the STAT3 activity status. Studies on both surgical biopsies and on lung cancer cell lines revealed a coincidence of STAT3 activation and strong expression of MMP-1. MMP-1 and tyrosine-phosphorylated activated STAT3 were found co-localized in cancer tissues, most pronounced in tumor fronts, and in particular in adenocarcinomas. STAT3 activity was constitutive, although to different degrees, in the lung cancer cell lines investigated. Three cell lines (BEN, KNS62, and A549 were identified in which STAT3 activitation was inducible by Interleukin-6 (IL-6. In A549 cells, STAT3 activity enhanced the level of MMP-1 mRNA and stimulated transcription from the MMP-1 promoter in IL-6–stimulated A549 cells. STAT3 specificity of this effect was confirmed by STAT3 knockdown through RNA interference. Our results link aberrant activity of STAT3 in lung cancer cells to malignant tumor progression through up-regulation of expression of invasiveness-associated MMPs.

Quantification of PRL/Stat5 signaling with a novel pGL4-CISH reporter.

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Fang, Feng; Antico, Giovanni; Zheng, Jiamao; Clevenger, Charles V

2008-02-06

Elevations of serum prolactin (PRL) are associated with an increased risk for breast cancer. PRL signaling through its prolactin receptor (PRLr) involves the Jak2/Stat5 pathway. Luciferase-based reporter assays have been widely used to evaluate the activity of this pathway. However, the existing reporters are often not sensitive enough to monitor the effect of PRL in this pathway. In this study, a new biologically relevant reporter, pGL4-CISH, was generated to study the PRL/Jak2/Stat5 signaling pathway. The sensitivity of pGL4-CISH to detect PRL was superior to that of several other commonly utilized Stat5-responsive reporters. Interestingly, the enhanced function pGL4-CISH was restricted to the estrogen receptor positive (ER+) human breast cancer cell lines T47D and MCF7, but not in the ER-MDA-231, BT-474, or MCF10A cell lines. Overexpression of Stat5 further enhanced the effect of PRL on pGL4-CISH. These studies demonstrate that pGL4-CISH is a novel and sensitive reporter for assessing the activity of the PRL/Stat5 signaling pathway in the ER+ human breast cancer cells.

Inhibitory effect of a tyrosine-fructose Maillard reaction product, 2,4-bis(p-hydroxyphenyl-2-butenal on amyloid-β generation and inflammatory reactions via inhibition of NF-κB and STAT3 activation in cultured astrocytes and microglial BV-2 cells

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Choi Im Seup

2011-10-01

Full Text Available Abstract Background Amyloidogenesis is linked to neuroinflammation. The tyrosine-fructose Maillard reaction product, 2,4-bis(p-hydroxyphenyl-2-butenal, possesses anti-inflammatory properties in cultured macrophages, and in an arthritis animal model. Because astrocytes and microglia are responsible for amyloidogenesis and inflammatory reactions in the brain, we investigated the anti-inflammatory and anti-amyloidogenic effects of 2,4-bis(p-hydroxyphenyl-2-butenal in lipopolysaccharide (LPS-stimulated astrocytes and microglial BV-2 cells. Methods Cultured astrocytes and microglial BV-2 cells were treated with LPS (1 μg/ml for 24 h, in the presence (1, 2, 5 μM or absence of 2,4-bis(p-hydroxyphenyl-2-butenal, and harvested. We performed molecular biological analyses to determine the levels of inflammatory and amyloid-related proteins and molecules, cytokines, Aβ, and secretases activity. Nuclear factor-kappa B (NF-κB DNA binding activity was determined using gel mobility shift assays. Results We found that 2,4-bis(p-hydroxyphenyl-2-butenal (1, 2, 5 μM suppresses the expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 as well as the production of nitric oxide (NO, reactive oxygen species (ROS, tumor necrosis factor-α (TNF-α, and interleukin-1β (IL-1β in LPS (1 μg/ml-stimulated astrocytes and microglial BV-2 cells. Further, 2,4-bis(p-hydroxyphenyl-2-butenal inhibited the transcriptional and DNA binding activity of NF-κB--a transcription factor that regulates genes involved in neuroinflammation and amyloidogenesis via inhibition of IκB degradation as well as nuclear translocation of p50 and p65. Consistent with the inhibitory effect on inflammatory reactions, 2,4-bis(p-hydroxyphenyl-2-butenal inhibited LPS-elevated Aβ42 levels through attenuation of β- and γ-secretase activities. Moreover, studies using signal transducer and activator of transcription 3 (STAT3 siRNA and a pharmacological inhibitor showed that 2

Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

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Buro Lauren J

2010-09-01

Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

STAT3 induces transcription of the DNA methyltransferase 1 gene (DNMT1) in malignant T lymphocytes

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Zhang, Qian; Wang, Hong Y; Woetmann, Anders

2006-01-01

In this study, we demonstrated that STAT3, a well-characterized transcription factor expressed in continuously activated oncogenic form in the large spectrum of cancer types, induces in malignant T lymphocytes the expression of DNMT1, the key effector of epigenetic gene silencing. STAT3 binds in ...

Short Stat5-interacting peptide derived from phospholipase C-β3 inhibits hematopoietic cell proliferation and myeloid differentiation.

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Hiroki Yasudo

Full Text Available Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN. Our recent study found that phospholipase C (PLC-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998 suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.

The ROS-mediated activation of IL-6/STAT3 signaling pathway is involved in the 27-hydroxycholesterol-induced cellular senescence in nerve cells.

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Liu, Jiao; Liu, Yun; Chen, Juan; Hu, Chunyan; Teng, Mengying; Jiao, Kailin; Shen, Zhaoxia; Zhu, Dongmei; Yue, Jia; Li, Zhong; Li, Yuan

2017-12-01

The oxysterol 27-hydroxycholesterol (27HC) is a selective estrogen receptor modulator (SERMs), which like endogenous estrogen 17β-estradiol (E 2 ) induces the proliferation of ER-positive breast cancer cells in vitro. Interestingly, the observation that 27HC induces adverse effects in neural system, distinguishing it from E 2 . It has been suggested that high levels of circulating cholesterol increase the entry of 27HC into the brain, which may induce learning and memory impairment. Based on this evidence, 27HC may be associated with neurodegenerative processes and interrupted cholesterol homeostasis in the brain. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that 27HC induced apparent cellular senescence in nerve cells. Senescence-associated β-galactosidase (SA-β-Gal) assay revealed that 27HC induced senescence in both BV2 cells and PC12 cells. Furthermore, we demonstrated that 27HC promoted the accumulation of cellular reactive oxygen species (ROS) in nerve cells and subsequently activation of IL-6/STAT3 signaling pathway. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly blocked 27HC-induced ROS production and activation of IL-6/STAT3 signaling pathway. Either blocking the generation of ROS or inhibition of IL-6/STAT3 both attenuated 27HC-induced cellular senescence. In sum, these findings not only suggested a mechanism whereby 27HC induced cellular senescence in nerve cells, but also helped to recognize the 27HC as a novel harmful factor in neurodegenerative diseases. Copyright © 2017. Published by Elsevier Ltd.

Changes in signal transducer and activator of transcription 3 (STAT3) dynamics induced by complexation with pharmacological inhibitors of Src homology 2 (SH2) domain dimerization.

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Resetca, Diana; Haftchenary, Sina; Gunning, Patrick T; Wilson, Derek J

2014-11-21

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A neuroprotective astrocyte state is induced by neuronal signal EphB1 but fails in ALS models.

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Tyzack, Giulia E; Hall, Claire E; Sibley, Christopher R; Cymes, Tomasz; Forostyak, Serhiy; Carlino, Giulia; Meyer, Ione F; Schiavo, Giampietro; Zhang, Su-Chun; Gibbons, George M; Newcombe, Jia; Patani, Rickie; Lakatos, András

2017-10-27

Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1-ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.

Human airway eosinophils exhibit preferential reduction in STAT signaling capacity and increased CISH expression.

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Burnham, Mandy E; Koziol-White, Cynthia J; Esnault, Stephane; Bates, Mary E; Evans, Michael D; Bertics, Paul J; Denlinger, Loren C

2013-09-15

Allergic asthma, a chronic respiratory disorder marked by inflammation and recurrent airflow obstruction, is associated with elevated levels of IL-5 family cytokines and elevated numbers of eosinophils (EOS). IL-5 family cytokines elongate peripheral blood EOS (EOS(PB)) viability, recruit EOS(PB) to the airways, and, at higher concentrations, induce degranulation and reactive oxygen species generation. Although airway EOS (EOS(A)) remain signal ready in that GM-CSF treatment induces degranulation, treatment of EOS(A) with IL-5 family cytokines no longer confers a survival advantage. Because the IL-5 family receptors have common signaling capacity, but are uncoupled from EOS(A) survival, whereas other IL-5 family induced endpoints remain functional, we tested the hypothesis that EOS(A) possess a JAK/STAT-specific regulatory mechanism (because JAK/STAT signaling is critical to EOS survival). We found that IL-5 family-induced STAT3 and STAT5 phosphorylation is attenuated in EOS(A) relative to blood EOS from airway allergen-challenged donors. However, IL-5 family-induced ERK1/2 phosphorylation is not altered between EOS(A) and EOS from airway allergen-challenged donors. These observations suggest EOS(A) possess a regulatory mechanism for suppressing STAT signaling distinct from ERK1/2 activation. Furthermore, we found, in EOS(PB), IL-5 family cytokines induce members of the suppressors of cytokine signaling (SOCS) genes, CISH and SOCS1. Additionally, following allergen challenge, EOS(A) express significantly more CISH and SOCS1 mRNA and CISH protein than EOS(PB) counterparts. In EOS(PB), long-term pretreatment with IL-5 family cytokines, to varying degrees, attenuates IL-5 family-induced STAT5 phosphorylation. These data support a model in which IL-5 family cytokines trigger a selective downregulation mechanism in EOS(A) for JAK/STAT pathways.

Chalepin: A Compound from Ruta angustifolia L. Pers Exhibits Cell Cycle Arrest at S phase, Suppresses Nuclear Factor-Kappa B (NF-κB) Pathway, Signal Transducer and Activation of Transcription 3 (STAT3) Phosphorylation and Extrinsic Apoptotic Pathway in Non-small Cell Lung Cancer Carcinoma (A549).

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Richardson, Jaime Stella Moses; Aminudin, Norhaniza; Abd Malek, Sri Nurestri

2017-10-01

Plants have been a major source of inspiration in developing novel drug compounds in the treatment of various diseases that afflict human beings worldwide. Ruta angustifolia L. Pers known locally as Garuda has been conventionally used for various medicinal purposes such as in the treatment of cancer. A dihydrofuranocoumarin named chalepin, which was isolated from the chloroform extract of the plant, was tested on its ability to inhibit molecular pathways of human lung carcinoma (A549) cells. Cell cycle analysis and caspase 8 activation were conducted using a flow cytometer, and protein expressions in molecular pathways were determined using Western blot technique. Cell cycle analysis showed that cell cycle was arrested at the S phase. Further studies using Western blotting technique showed that cell cycle-related proteins such as cyclins, cyclin-dependent kinases (CDKs), and inhibitors of CDKs correspond to a cell cycle arrest at the S phase. Chalepin also showed inhibition in the expression of inhibitors of apoptosis proteins. Nuclear factor-kappa B (NF-κB) pathway, signal transducer and activation of transcription 3 (STAT-3), cyclooxygenase-2, and c-myc were also downregulated upon treatment with chalepin. Chalepin was found to induce extrinsic apoptotic pathway. Death receptors 4 and 5 showed a dramatic upregulation at 24 h. Analysis of activation of caspase 8 with the flow cytometer showed an increase in activity in a dose- and time-dependent manner. Activation of caspase 8 induced cleavage of BH3-interacting domain death agonist, which initiated a mitochondrial-dependent or -independent apoptosis. Chalepin causes S phase cell cycle arrest, NF-κB pathway inhibition, and STAT-3 inhibition, induces extrinsic apoptotic pathway, and could be an excellent chemotherapeutic agent. This study reports the capacity of an isolated bioactive compound known as chalepin to suppress the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, signal

Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

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Hashimoto, Shoko; Yoshimura, Hiromi; Okada, Kazushi; Uramaru, Naoto; Sugihara, Kazumi; Kitamura, Shigeyuki; Imaoka, Susumu

2012-03-19

Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions. © 2011 American Chemical Society

The synthetic α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) inhibits the JAK/STAT signaling pathway.

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Pinz, Sophia; Unser, Samy; Brueggemann, Susanne; Besl, Elisabeth; Al-Rifai, Nafisah; Petkes, Hermina; Amslinger, Sabine; Rascle, Anne

2014-01-01

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.

Role of Stat in Skin Carcinogenesis: Insights Gained from Relevant Mouse Models

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Macias, E.; Rao, D.; DiGiovanni, J.; DiGiovanni, J.; DiGiovanni, J.

2013-01-01

Signal transducer and activator of transcription 3 (Stat) is a cytoplasmic protein that is activated in response to cytokines and growth factors and acts as a transcription factor. Stat plays critical roles in various biological activities including cell proliferation, migration, and survival. Studies using keratinocyte-specific Stat-deficient mice have revealed that Stat plays an important role in skin homeostasis including keratinocyte migration, wound healing, and hair follicle growth. Use of both constitutive and inducible keratinocyte-specific Stat-deficient mouse models has demonstrated that Stat is required for both the initiation and promotion stages of multistage skin carcinogenesis. Further studies using a transgenic mouse model with a gain of function mutant of Stat (Stat3C) expressed in the basal layer of the epidermis revealed a novel role for Stat in skin tumor progression. Studies using similar Stat-deficient and gain-of-function mouse models have indicated its similar roles in ultraviolet B (UVB) radiation-mediated skin carcinogenesis. This paper summarizes the use of these various mouse models for studying the role and underlying mechanisms for the function of Stat in skin carcinogenesis. Given its significant role throughout the skin carcinogenesis process, Stat is an attractive target for skin cancer prevention and treatment.

Immune function during GH treatment in GH-deficient adults

DEFF Research Database (Denmark)

Sneppen, S B; Mersebach, H; Ullum, H

2002-01-01

investigated were unaltered. CONCLUSIONS: GH deficiency was associated with changes in lymphocyte subsets and impaired unstimulated and stimulated natural killer cell activity, but these remained abnormal during 18 months of GH replacement therapy. Extra-pituitary GH gene expression in, e.g. lymphoid tissues...

SOCS3 Expression Correlates with Severity of Inflammation, Expression of Proinflammatory Cytokines, and Activation of STAT3 and p38 MAPK in LPS-Induced Inflammation In Vivo

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João Antônio Chaves de Souza

2013-01-01

Full Text Available SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway, which is relevant in inflammatory conditions. We used a model of LPS-induced periodontal disease in rats to correlate SOCS3 expression with the inflammatory status. In vitro we used a murine macrophage cell line to assess the physical interaction between SOCS3 and STAT3 by coimmunoprecipitation. 30 ug of LPS from Escherichia coli were injected in the gingival tissues on the palatal aspect of first molars of the animals 3x/week for up to 4 weeks. Control animals were injected with the vehicle (PBS. The rats were sacrificed at 7, 15, and 30 days. Inflammation and gene expression were assessed by stereometric analysis, immunohistochemistry, RT-qPCR, and western blot. LPS injections increased inflammation, paralleled by an upregulation of SOCS3, of the proinflammatory cytokines IL-1β, IL-6, and TNF-α and increased phosphorylation of STAT3 and p38 MAPK. SOCS3 expression accompanied the severity of inflammation and the expression of proinflammatory cytokines, as well as the activation status of STAT3 and p38 MAPK. LPS stimulation in a macrophage cell line in vitro induced transient STAT3 activation, which was inversely correlated with a dynamic physical interaction with SOCS3, suggesting that this may be a mechanism for SOCS3 regulatory function.

Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

International Nuclear Information System (INIS)

Wu Liguo; Hutt-Fletcher, Lindsey M.

2007-01-01

Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL

Quantification of PRL/Stat5 signaling with a novel pGL4-CISH reporter

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Zheng Jiamao

2008-02-01

Full Text Available Abstract Background Elevations of serum prolactin (PRL are associated with an increased risk for breast cancer. PRL signaling through its prolactin receptor (PRLr involves the Jak2/Stat5 pathway. Luciferase-based reporter assays have been widely used to evaluate the activity of this pathway. However, the existing reporters are often not sensitive enough to monitor the effect of PRL in this pathway. Results In this study, a new biologically relevant reporter, pGL4-CISH, was generated to study the PRL/Jak2/Stat5 signaling pathway. The sensitivity of pGL4-CISH to detect PRL was superior to that of several other commonly utilized Stat5-responsive reporters. Interestingly, the enhanced function pGL4-CISH was restricted to the estrogen receptor positive (ER+ human breast cancer cell lines T47D and MCF7, but not in the ER-MDA-231, BT-474, or MCF10A cell lines. Overexpression of Stat5 further enhanced the effect of PRL on pGL4-CISH. Conclusion These studies demonstrate that pGL4-CISH is a novel and sensitive reporter for assessing the activity of the PRL/Stat5 signaling pathway in the ER+ human breast cancer cells.

By Targeting Stat3 microRNA-17-5p Promotes Cardiomyocyte Apoptosis in Response to Ischemia Followed by Reperfusion

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Weijie Du

2014-08-01

Full Text Available Background: Several studies have confirmed the role of microRNAs in regulating ischemia/reperfusion-induced cardiac injury (I/R-I. MiR-17-5p has been regarded as an oncomiR in the development of cancer. However, its potential role in cardiomyocytes has not been exploited. The aim of this study is to investigate the role of miR-17-5p in I/R-I and the underlying mechanism through targeting Stat3, a key surviving factor in cardiomyocytes. Methods: MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide assay was used to detect the cell viability. ELISA and TUNEL were performed to measure apoptosis of neonatal rat ventricular cardiomyocytes (NRVCs. Infarct area was estimated by TTC (triphenyltetrazolium chloride and Evans blue staining. Western blot analysis was employed to detect the Stat3 and p-Stat3 levels and real-time RT-PCR was used to quantify miR-17-5p level. Results: The miR-17-5p level was significantly up-regulated in I/R-I mice and in NRVCs under oxidative stress. Overexpression of miR-17-5p aggravated cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death induced by H2O2, whereas inhibition of miR-17-5p by its antisense AMO-17-5p abrogated the deleterious changes. Moreover, the locked nucleic acid-modified antisense (LNA-anti-miR-17-5p markedly decreased the infarct area and apoptosis induced by I/R-I in mice. Furthermore, overexpression of miR-17-5p diminished the p-Stat3 level in response to H2O2. The results from Western blot analysis and luciferase reporter gene assay confirmed Stat3 as a target gene for miR-17-5p. Conclusion: Upregulation of miR-17-5p promotes apoptosis induced by oxidative stress via targeting Stat3, accounting partially for I/R-I.

The small molecule, LLL12, inhibits STAT3 phosphorylation and induces apoptosis in medulloblastoma and glioblastoma cells.

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Sarah Ball

Full Text Available Tumors of the central nervous system represent a major source of cancer-related deaths, with medulloblastoma and glioblastoma being the most common malignant brain tumors in children and adults respectively. While significant advances in treatment have been made, with the 5-year survival rate for medulloblastoma at 70-80%, treating patients under 3 years of age still poses a problem due to the deleterious effects of radiation on the developing brain, and the median survival for patients with glioblastoma is only 15 months. The transcription factor, STAT3, has been found constitutively activated in a wide variety of cancers and in recent years it has become an attractive therapeutic target. We designed a non-peptide small molecule STAT3 inhibitor, LLL12, using structure-based design. LLL12 was able to inhibit STAT3 phosphorylation, decrease cell viability and induce apoptosis in medulloblastoma and glioblastoma cell lines with elevated levels of p-STAT3 (Y705. IC(50 values for LLL12 were found to be between 1.07 µM and 5.98 µM in the five cell lines expressing phosphorylated STAT3. STAT3 target genes were found to be downregulated and a decrease in STAT3 DNA binding was observed following LLL12 treatment, indicating that LLL12 is an effective STAT3 inhibitor. LLL12 was also able to inhibit colony formation, wound healing and decreased IL-6 and LIF secretion. Our results suggest that LLL12 is a potent STAT3 inhibitor and that it may be a potential therapeutic treatment for medulloblastoma and glioblastoma.

Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia

International Nuclear Information System (INIS)

Al-Jamal, Hamid Ali Nagi; Mat Jusoh, Siti Asmaa; Hassan, Rosline; Johan, Muhammad Farid

2015-01-01

Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells. Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal–Wallis tests were used for statistical analysis. The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0

Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation

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Andersson, E I; Rajala, H L M; Eldfors, S; Ellonen, P; Olson, T; Jerez, A; Clemente, M J; Kallioniemi, O; Porkka, K; Heckman, C; Loughran, T P Jr; Maciejewski, J P; Mustjoki, S

2013-01-01

T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene

APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation.

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VanKlompenberg, Monica K; Leyden, Emily; Arnason, Anne H; Zhang, Jian-Ting; Stefanski, Casey D; Prosperi, Jenifer R

2017-11-28

Resistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus - Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT; Apc Min/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT; Apc Min/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT; Apc Min/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

Human Airway Eosinophils Exhibit Preferential Reduction in STAT Signaling Capacity and Increased CISH Expression1

Science.gov (United States)

Burnham, Mandy E.; Koziol-White, Cynthia J.; Esnault, Stephane; Bates, Mary E.; Evans, Michael D.; Bertics, Paul J.; Denlinger, Loren C.

2013-01-01

Allergic asthma, a chronic respiratory disorder marked by inflammation and recurrent airflow obstruction, is associated with elevated levels of Interleukin-5 (IL-5) family cytokines, and elevated numbers of eosinophils (EOS). IL-5 family cytokines elongate peripheral blood EOS (EOSPB) viability, recruit EOSPB to the airways, and at higher concentrations, induce degranulation and reactive oxygen species (ROS) generation. While, EOSA remain signal ready in that GM-CSF treatment induces degranulation, treatment of EOSA with IL-5 family cytokines no longer confers a survival advantage. Since the IL-5 family receptors have common signaling capacity, but are uncoupled from EOSA survival while other IL-5 family induced endpoints remain functional, we tested the hypothesis that EOSA possess a JAK/STAT specific regulatory mechanism (since JAK/STAT signaling is critical to EOS survival). We found that IL-5 family-induced STAT3 and STAT5 phosphorylation is attenuated in EOSA relative to blood EOS from airway allergen-challenged donors (EOSCPB). However, IL-5 family induced ERK1/2 phosphorylation is not altered between EOSA and EOSCPB. These observations suggest EOSA possess a regulatory mechanism for suppressing STAT signaling distinct from ERK1/2 activation. Furthermore, we found, in EOSPB, IL-5 family cytokines induce members of the suppressors of cytokine signaling (SOCS) genes, CISH and SOCS1. Additionally, following allergen challenge, EOSA express significantly more CISH and SOCS1 mRNA and CISH protein than EOSPB counterparts. In EOSPB, long-term pretreatment with IL-5 family cytokines, to varying degrees, attenuates IL-5 family induced STAT5 phosphorylation. These data support a model wherein IL-5 family cytokines trigger a selective down-regulation mechanism in EOSA for JAK/STAT pathways. PMID:23956426

Biologic activity of the novel small molecule STAT3 inhibitor LLL12 against canine osteosarcoma cell lines

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Couto Jason I

2012-12-01

Full Text Available Abstract Background STAT3 [1] has been shown to be dysregulated in nearly every major cancer, including osteosarcoma (OS. Constitutive activation of STAT3, via aberrant phosphorylation, leads to proliferation, cell survival and resistance to apoptosis. The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines. Results We evaluated the effects of LLL12 treatment on 4 canine OS cell lines and found that LLL12 inhibited proliferation, induced apoptosis, reduced STAT3 phosphorylation, and decreased the expression of several transcriptional targets of STAT3 in these cells. Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines. Conclusion LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.