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Michael L. Freeman - One of the best experts on this subject based on the ideXlab platform.

Tsonwin Hai - One of the best experts on this subject based on the ideXlab platform.

  • Activating Transcription Factor 3 regulates canonical TGFβ signalling in systemic sclerosis
    Annals of the rheumatic diseases, 2015
    Co-Authors: Tatjana Mallano, Tsonwin Hai, Katrin Palumbo-zerr, Pawel Zerr, Andreas Ramming, Barbara Zeller, Christian Beyer, Clara Dees, Jingang Huang, Oliver Distler
    Abstract:

    Background Activating Transcription Factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of Transcription Factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc). Methods ATF3 was analysed by reverse Transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation. Results Transforming growth Factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the Transcriptional activity of Smad3. Conclusions We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.

  • Immunohistochemical Detection of Activating Transcription Factor 3, a Hub of the Cellular Adaptive–Response Network
    Methods in enzymology, 2011
    Co-Authors: Tsonwin Hai, Swati P. Jalgaonkar, Christopher C. Wolford, Xin Yin
    Abstract:

    Activating Transcription Factor 3 (ATF3) gene encodes a member of the ATF family of Transcription Factors and is induced by various stress signals, including many of those that induce the unfolded protein response (UPR). Emerging evidence suggests that ATF3 is a hub of the cellular adaptive–response network and studies using various mouse models indicate that ATF3 plays a role in the pathogenesis of various diseases. One way to investigate the potential relevance of ATF3 to human diseases is to determine its expression in patient samples and test whether it correlates with disease progression or clinical outcomes. Due to the scarcity and preciousness of patient samples, methods that can detect ATF3 on archival tissue sections would greatly facilitate this research. In this chapter, we briefly review the roles of ATF3 in cellular adaptive–response and UPR, and then describe the detailed steps and tips that we developed based on general immunohistochemistry (IHC) protocols to detect ATF3 on paraffin embedded sections.

  • Activating Transcription Factor 3 Confers Protection against Ventilator-induced Lung Injury
    American journal of respiratory and critical care medicine, 2010
    Co-Authors: Ali Akram, Tsonwin Hai, Bing Han, Hussain Masoom, Claudia Peng, Emily Lam, Michael L. Litvack, Xiao-hui Bai, Yuexin Shan, Jane Batt
    Abstract:

    Rationale: Ventilator-induced lung injury (VILI) significantly contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury. Understanding the molecular basis for response to cyclic stretch (CS) and its derangement during high-volume ventilation is of high priority. Objectives: To identify specific molecular regulators involved in the development of VILI. Methods: We undertook a comparative examination of cis-regulatory sequences involved in the coordinated expression of CS-responsive genes using microarray analysis. Analysis of stretched versus nonstretched cells identified significant enrichment for genes containing putative binding sites for the Transcription Factor activating Transcription Factor 3 (ATF3). To determine the role of ATF3 in vivo, we compared the response of ATF3 gene–deficient mice to wild-type mice in an in vivo model of VILI. Measurements and Main Results: ATF3 protein expression and nuclear translocation is increased in the lung after mechanical ventilation in wild-type mice. ATF3-deficient mice have greater sensitivity to mechanical ventilation alone or in conjunction with inhaled endotoxin, as demonstrated by increased cell infiltration and proinflammatory cytokines in the lung and bronchoalveolar lavage, and increased pulmonary edema and indices of tissue injury. The expression of stretch-responsive genes containing putative ATF3 cis-regulatory regions was significantly altered in ATF3-deficient mice. Conclusions: ATF3 deficiency confers increased sensitivity to mechanical ventilation alone or in combination with inhaled endotoxin. We propose ATF3 acts to counterbalance CS and high volume–induced inflammation, dampening its ability to cause injury and consequently protecting animals from injurious CS.

  • negative regulation of tlr signaling pathways by activating Transcription Factor 3
    Journal of Immunology, 2007
    Co-Authors: Mark M Whitmore, Amaya Iparraguirre, Lindsey Kubelka, Wolfgang Weninger, Tsonwin Hai, Bryan R G Williams
    Abstract:

    Activating Transcription Factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory Transcription Factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.

  • activating Transcription Factor 3 atf3 represses the expression of ccl4 in murine macrophages
    Molecular Immunology, 2007
    Co-Authors: Ciera H Khuu, Tsonwin Hai, Roberto M Barrozo, Steven L Weinstein
    Abstract:

    Abstract Acute expression of macrophage inflammatory protein-1 beta (also known as CCL4) promotes beneficial leukocyte recruitment to infected tissues, but chronic expression of this chemokine contributes to inflammatory disease. CCL4 expression is controlled largely at the Transcriptional level and an ATF/CRE sequence located in the promoter (−104 to −97 bp, relative to the Transcriptional start site) has been identified as a critical cis -acting element. The trans -acting binding proteins that influence CCL4 Transcription via this site are largely unknown. We investigated whether activating Transcription Factor 3 (ATF3), a member of the ATF/CREB family of Transcription Factors, binds to the CCL4 ATF/CRE site in macrophages. Using the electrophoretic mobility shift assay and the chromatin immunoprecipitation assay, we found that ATF3 binds to the ATF/CRE site within the CCL4 promoter in untreated and lipopolysaccharide (LPS)-stimulated macrophages. Quantitative RT-PCR analysis showed that CCL4 mRNA levels in elicited peritoneal macrophages from ATF3 −/− mice are significantly higher than in congenic ATF3 +/+ macrophages under both unstimulated and LPS-stimulated conditions, suggesting that ATF3 represses Transcription of the CCL4 gene. Consistent with the higher gene expression, ATF3-deficient macrophages secreted more CCL4 protein than ATF3 +/+ macrophages. Similar results were obtained in bone-marrow-derived macrophages treated with Toll-like receptor 2, 3, 4 and 5 agonists. Thus, we conclude that ATF3 constitutively binds to the ATF/CRE site in the CCL4 promoter where it represses basal and pathogen-associated molecular pattern (PAMP)-stimulated Transcription. Consequently, ATF3 appears to be part of a control mechanism that limits the amount of CCL4 released by macrophages, preventing excessive inflammation.

Chunhong Yan - One of the best experts on this subject based on the ideXlab platform.

  • the activating Transcription Factor 3 protein suppresses the oncogenic function of mutant p53 proteins
    Journal of Biological Chemistry, 2014
    Co-Authors: Saisai Wei, Hongbo Wang, Chunhong Yan, Sarah Malmut, Jianqiao Zhang, Shumei Ren, Wei Wang, Dale D Tang
    Abstract:

    Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with Transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating Transcription Factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the Transcription Factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

  • MDM2 mediates ubiquitination and degradation of activating Transcription Factor 3
    The Journal of biological chemistry, 2010
    Co-Authors: Hongbo Wang, Douglas D Boyd, Chunhong Yan
    Abstract:

    Activating Transcription Factor 3 (ATF3) is a common stress sensor, and its rapid induction by cellular stresses (e.g. DNA damage) is crucial for cells to mount appropriate responses (e.g. activating the tumor suppressor p53) and maintain homeostasis. Although emerging evidence suggests that dysregulation of ATF3 contributes to occurrences of human diseases including cancer, the mechanism(s) by which ATF3 expression is regulated is largely unknown. Here, we demonstrate that mouse double minute 2 (MDM2) is a bona fide E3 ubiquitin ligase for ATF3 and regulates ATF3 expression by promoting its degradation. MDM2 via its C-terminal RING finger can bind to the Basic region of ATF3 and mediate the addition of ubiquitin moieties to the ATF3 leucine zipper domain. As a consequence, ATF3, but not a mutant deficient in MDM2 binding (Δ80–100), is degraded by MDM2-mediated proteolysis. Consistent with these results, ablation of MDM2 in cells not only increases basal ATF3 levels, but results in stabilization of ATF3 in late stages of DNA damage responses. Because ATF3 was recently identified as a p53 activator, these results suggest that MDM2 could inactivate p53 through an additional feedback mechanism involving ATF3. Therefore, we provide the first evidence demonstrating that ATF3 is regulated by a posttranslational mechanism.

  • activating Transcription Factor 3 activates p53 by preventing e6 associated protein from binding to e6
    Journal of Biological Chemistry, 2010
    Co-Authors: Hongbo Wang, Shumei Ren, Chunhong Yan
    Abstract:

    Genomic integration of human papillomavirus (HPV) DNA accounts for more than 90% of cervical cancers. High-risk genital HPVs encode E6 proteins that can interact with a cellular ubiquitin ligase E6-associated protein (E6AP) and target the tumor suppressor p53 for ubiquitin-mediated proteolysis. Currently, how this critical event is regulated is largely unknown. Here we report that activating Transcription Factor 3 (ATF3), a broad DNA damage sensor whose expression is frequently downregulated in cervical cancer, interacted with E6 and prevented p53 from ubiquitination and degradation mediated by the viral protein. Consistent with its role as a potent E6 antagonist, ATF3 expressed enforcedly in HPV-positive SiHa cells activated p53, leading to expression of p53-target genes (e.g. p21 and PUMA), cell cycle arrest and apoptotic cell death. The leucine zipper domain of ATF3 appears indispensable for these effects as an ATF3 mutant lacking this domain failed to interact with E6 and activate p53 in the cervical cancer cells. The prevention of p53 degradation was unlikely caused by binding of ATF3 to the tumor suppressor, but rather was a consequence of disruption of the E6-E6AP interaction by ATF3. These results indicate that ATF3 plays a key role in a mechanism defending against HPV-induced carcinogenesis, and could serve as a novel therapeutic target for HPV-positive cancers.

Stephan L. Brown - One of the best experts on this subject based on the ideXlab platform.

Hidenori Shiraha - One of the best experts on this subject based on the ideXlab platform.

  • Loss of runt-related Transcription Factor 3 induces gemcitabine resistance in pancreatic cancer.
    Molecular oncology, 2013
    Co-Authors: Shigeru Horiguchi, Hidenori Shiraha, Shin Ichi Nishina, Minoru Matsubara, Masaya Iwamuro, Teruya Nagahara, Jyunnro Kataoka, Hironari Kato, Akinobu Takaki, Kazuhiro Nouso
    Abstract:

    Abstract Background & Aim Runt-related Transcription Factor 3 (RUNX3) is a tumor suppressor gene that is expressed in gastric and other cancers including pancreatic cancer. However, the precise function of RUNX3 in pancreatic cancer has not been fully elucidated. In this study, we aimed to determine the effect of decreased RUNX3 expression in pancreatic cancer. Methods This study included 36 patients with primary pancreatic cancer, who had undergone pancreaticoduodenectomy. All patients were treated with 1000 mg/m2 gemcitabine after the surgery. The pancreatic cancer cell lines PANC-1, MIAPaCa-2, BxPC-3, SUIT-2, and KLM-1 were used for immunoblotting analysis of RUNX3 and multidrug resistance protein (MRP) expressions. Ectopic RUNX3 expression was achieved by cDNA transfection of the cells, and small interfering RNA (siRNA) against RUNX3 was used to knock down endogenous RUNX3. Cell growth in the presence of gemcitabine was assessed using the MTT assay. Results Patients with RUNX3-positive and RUNX3-negative pancreatic cancer had a median survival of 1006 and 643 days, respectively. Exogenous RUNX3 expression reduced the expression of MRP1, MRP2, and MRP5 in endogenous RUNX3-negative cells, whereas RUNX3 siRNA increased the expressions of these genes in endogenous RUNX3-positive cells. Exogenous RUNX3 expression decreased gemcitabine IC50 in RUNX3-negative cells. Conclusion Loss of RUNX3 expression contributes to gemcitabine resistance by inducing MRP expression, thereby resulting in poor patient survival.

  • Runt-related Transcription Factor 3 reverses epithelial-mesenchymal transition in hepatocellular carcinoma.
    International journal of cancer, 2012
    Co-Authors: Shigetomi Tanaka, Hidenori Shiraha, Shin Ichi Nishina, Yutaka Nakanishi, Minoru Matsubara, Shigeru Horiguchi, Masaya Iwamuro, Nobuyuki Takaoka, Junro Kataoka, Kenji Kuwaki
    Abstract:

    Loss or decreased expression of runt-related Transcription Factor 3 (RUNX3), a tumor suppressor gene involved in gastric and other cancers, has been frequently observed in hepatocellular carcinoma (HCC). The objective of this study was to identify the regulatory mechanism of the epithelialmesenchymal transition (EMT) by RUNX3 in HCC. Human HCC cell lines, Hep3B, Huh7, HLF and SK-Hep1, were divided into low- and high-EMT lines, based on their expression of TWIST1 and SNAI2, and were used in this in vitro study. Ectopic RUNX3 expression had an anti-EMT effect in low-EMT HCC cell lines characterized by increased E-cadherin expression and decreased N-cadherin and vimentin expression. RUNX3 expression has previously been reported to reduce jagged-1 (JAG1) expression; therefore, JAG1 ligand peptide was used to reinduce EMT in RUNX3-expressing low-EMT HCC cells. Immunohistochemical analyses were performed for RUNX3, E-cadherin, N-cadherin and TWIST1 in 33 human HCC tissues, also divided into low- and high-EMT HCC, based on TWIST1 expression. E-cadherin expression was correlated positively and N-cadherin expression was correlated negatively with RUNX3 expression in low-EMT HCC tissues. Correlations between EMT markers and RUNX3 mRNA expression were analyzed using Oncomine datasets. Similarly, mRNA expression of E-cadherin was also significantly correlated with that of RUNX3 in low-EMT HCC, while mRNA expression of JAG1 was negatively correlated with that of RUNX3. These results suggest a novel mechanism by which loss or decreased expression of RUNX3 induces EMT via induction of JAG1 expression in low-EMT HCC.

  • Loss of runt-related Transcription Factor 3 causes development and progression of hepatocellular carcinoma.
    Journal of cellular biochemistry, 2011
    Co-Authors: Hidenori Shiraha, Shin Ichi Nishina, Kazuhide Yamamoto
    Abstract:

    Runt-related Transcription Factor 3 (RUNX3) is reported as a tumor suppressor gene for gastric cancer, and may be important in the development of hepatocellular carcinoma (HCC). RUNX3 expression is frequently lost or decreased by hemizygous deletion or hypermethylation of its promoter lesion in HCC. The significance of decreased expression of RUNX3 in HCC has not been fully elucidated, but is likely related to dysfunction of cell cycle regulation, decrement of apoptosis, enhancement of angiogenesis, and development of epithelial–mesenchymal transition. RUNX3 is a promising candidate as a tumor suppressor gene for HCC. J. Cell. Biochem. 112: 745–749, 2011. © 2010 Wiley-Liss, Inc.

  • Loss of runt-related Transcription Factor 3 expression leads hepatocellular carcinoma cells to escape apoptosis
    BMC cancer, 2011
    Co-Authors: Yutaka Nakanishi, Hidenori Shiraha, Shin Ichi Nishina, Shigetomi Tanaka, Minoru Matsubara, Shigeru Horiguchi, Masaya Iwamuro, Nobuyuki Takaoka, Masayuki Uemura, Kenji Kuwaki
    Abstract:

    Runt-related Transcription Factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC). RUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis. RUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 ± 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 ± 4% and 4 ± 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation. RUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis.