Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

PubMed

Marza, Esther; Taouji, Saïd; Barroso, Kim; Raymond, Anne-Aurélie; Guignard, Léo; Bonneu, Marc; Pallares-Lupon, Néstor; Dupuy, Jean-William; Fernandez-Zapico, Martin E; Rosenbaum, Jean; Palladino, Francesca; Dupuy, Denis; Chevet, Eric

2015-03-01

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes. © 2015 The Authors.

ER Stress-Mediated Signaling: Action Potential and Ca(2+) as Key Players.

PubMed

Bahar, Entaz; Kim, Hyongsuk; Yoon, Hyonok

2016-09-15

The proper functioning of the endoplasmic reticulum (ER) is crucial for multiple cellular activities and survival. Disturbances in the normal ER functions lead to the accumulation and aggregation of unfolded proteins, which initiates an adaptive response, the unfolded protein response (UPR), in order to regain normal ER functions. Failure to activate the adaptive response initiates the process of programmed cell death or apoptosis. Apoptosis plays an important role in cell elimination, which is essential for embryogenesis, development, and tissue homeostasis. Impaired apoptosis can lead to the development of various pathological conditions, such as neurodegenerative and autoimmune diseases, cancer, or acquired immune deficiency syndrome (AIDS). Calcium (Ca(2+)) is one of the key regulators of cell survival and it can induce ER stress-mediated apoptosis in response to various conditions. Ca(2+) regulates cell death both at the early and late stages of apoptosis. Severe Ca(2+) dysregulation can promote cell death through apoptosis. Action potential, an electrical signal transmitted along the neurons and muscle fibers, is important for conveying information to, from, and within the brain. Upon the initiation of the action potential, increased levels of cytosolic Ca(2+) (depolarization) lead to the activation of the ER stress response involved in the initiation of apoptosis. In this review, we discuss the involvement of Ca(2+) and action potential in ER stress-mediated apoptosis.

NHERF1, a novel GPER associated protein, increases stability and activation of GPER in ER-positive breast cancer

PubMed Central

Xiong, Ying; Wang, Yan; Zheng, Junfang; Zhao, Yuan; Tao, Tao; Wang, Qiqi; Liu, Hua; Wang, Songlin; Jiang, Wen G.; He, Junqi

2016-01-01

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC. PMID:27448983

NHERF1, a novel GPER associated protein, increases stability and activation of GPER in ER-positive breast cancer.

PubMed

Meng, Ran; Qin, Qiong; Xiong, Ying; Wang, Yan; Zheng, Junfang; Zhao, Yuan; Tao, Tao; Wang, Qiqi; Liu, Hua; Wang, Songlin; Jiang, Wen G; He, Junqi

2016-08-23

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC.

Transcription factor Brn-3α mRNA in cancers, relationship with AR, ER receptors and AKT/m-TOR pathway components

NASA Astrophysics Data System (ADS)

Spirina, L. V.; Gorbunov, A. K.; Chigevskaya, S. Y.; Usynin, Y. A.; Kondakova, I. V.; Slonimskaya, E. M.; Usynin, E. A.; Choinzonov, E. L.; Zaitseva, O. S.

2017-09-01

Transcription factors POU4F1 (neurogenic factor Brn-3α) play a pivotal role in cancers development. The aim of the study was to reveal the Brn-3α expression, AR, ER expression in cancers development, association with AKT/mTOR pathway activation. 30 patients with locally advanced prostate cancer, 20 patients with papillary thyroid cancer, T2-3N0-1M0 stages and 40 patients with renal cell cancer T2-3N0M0-1 were involved into the study. The expressions of Brn-3α, AR, ERα, components of AKT/m-TOR signaling pathway genes were performed by real-time PCR. The dependence of Brn-3α expression on mRNA levels of steroid hormone receptors and components of AKT/m-TOR signaling pathway in studied cancers were shown. High levels of mRNA of nuclear factor, steroid hormone receptors were found followed by the activation of this signaling pathway in prostate cancer tissue. The reduction of transcription factor Brn-3α was accompanied with tumor invasive growth with increasing rates of AR, ER and 4E-BP1 mRNA. Thyroid cancer development happened in a case of a Brn-3α and steroid hormone receptors decrease. The activation of AKT/m-TOR signaling pathway was established in the metastatic renal cancers, accompanied with the increase of ER mRNA. But there was no correlation between the steroid receptor and Brn-3α. One-direction changes of Brn-3α were observed in the development of prostate and thyroid cancer due to its effect on the steroid hormone receptors and the activation of AKT/m-TOR signaling pathway components. The influence of this factor on the development of the kidney cancer was mediated through m-TOR activity modifications, the key enzyme of oncogenesis.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

PubMed

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

Interleukin 17A exacerbates ER-stress-mediated inflammation of macrophages following ICH.

PubMed

Yang, Zhao; Liu, Qingjun; Shi, Hui; Jiang, Xuheng; Wang, Song; Lu, Yuanlan; Zhang, Ji; Huang, Xiaofei; Yu, Anyong

2018-05-30

IL-17A contributes to the initiation of inflammation following intracerebral hemorrhage (ICH). Endoplasmic reticulum (ER) stress acts on protein folding and contributes to inflammatory diseases. The role of IL-17A in the regulation of ER stress following ICH has not been well characterized. In this study, macrophages were stimulated with IL-17A, and then, ER stress and downstream pro-inflammatory factors were measured in vitro. In addition, brain edema and brain injury in ICH mice were assessed in vivo. We demonstrated that IL-17A induced ER stress in macrophages and thus promoted inflammation in vitro. Conversely, IL-17A inhibition attenuated ER stress and neuroinflammation. Furthermore, ERK 1/2 and p38 MAPK pathways mediated IL-17A-induced ER stress in macrophages. We also showed that IL-17A inhibition significantly attenuated ER stress and brain injury in ICH mice. In conclusion, our results demonstrate that IL 17A increases ER stress in macrophages and represents a novel mechanism in ICH. Copyright © 2018. Published by Elsevier Ltd.

O-GlcNAcylation of eIF2α regulates the phospho-eIF2α-mediated ER stress response.

PubMed

Jang, Insook; Kim, Han Byeol; Seo, Hojoong; Kim, Jin Young; Choi, Hyeonjin; Yoo, Jong Shin; Kim, Jae-woo; Cho, Jin Won

2015-08-01

O-GlcNAcylation is highly involved in cellular stress responses including the endoplasmic reticulum (ER) stress response. For example, glucosamine-induced flux through the hexosamine biosynthetic pathway can promote ER stress and ER stress inducers can change the total cellular level of O-GlcNAcylation. However, it is largely unknown which component(s) of the unfolded protein response (UPR) is directly regulated by O-GlcNAcylation. In this study, eukaryotic translation initiation factor 2α (eIF2α), a major branch of the UPR, was O-GlcNAcylated at Ser 219, Thr 239, and Thr 241. Upon ER stress, eIF2α is phosphorylated at Ser 51 by phosphorylated PKR-like ER kinase and this inhibits global translation initiation, except for that of specific mRNAs, including activating transcription factor 4, that induce stress-responsive genes such as C/EBP homologous protein (CHOP). Hyper-O-GlcNAcylation induced by O-GlcNAcase inhibitor (thiamet-G) treatment or O-GlcNAc transferase (OGT) overexpression hindered phosphorylation of eIF2α at Ser 51. The level of O-GlcNAcylation of eIF2α was changed by dithiothreitol treatment dependent on its phosphorylation at Ser 51. Point mutation of the O-GlcNAcylation sites of eIF2α increased its phosphorylation at Ser 51 and CHOP expression and resulted in increased apoptosis upon ER stress. These results suggest that O-GlcNAcylation of eIF2α affects its phosphorylation at Ser 51 and influences CHOP-mediated cell death. This O-GlcNAcylation of eIF2α was reproduced in thiamet-G-injected mouse liver. In conclusion, proper regulation of O-GlcNAcylation and phosphorylation of eIF2α is important to maintain cellular homeostasis upon ER stress. Copyright © 2015 Elsevier B.V. All rights reserved.

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

PubMed

Fragoso-Medina, Jorge; Rodriguez, Gabriela; Zarain-Herzberg, Angel

2018-05-01

The cardiac sarco/endoplasmic reticulum Ca 2+ -ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

Zinc deficiency mediates alcohol-induced apoptotic cell death in the liver of rats through activating ER and mitochondrial cell death pathways

PubMed Central

Sun, Qian; Zhong, Wei; Zhang, Wenliang; Li, Qiong; Sun, Xiuhua; Tan, Xiaobing; Sun, Xinguo; Dong, Daoyin

2015-01-01

Hepatic zinc deficiency has been well documented in alcoholic patients, but the mechanisms by which zinc deficiency mediates cell death have not been well defined. The objectives of this study were to determine whether alcohol perturbs subcellular zinc homeostasis and how organelle zinc depletion may link with cell death pathways. Wistar rats were pair-fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure significantly reduced zinc level in isolated hepatic endoplasmic reticulum (ER) and mitochondria. Among the detected zinc transporters, ER Zrt/Irt-like protein (ZIP)13 and mitochondrial ZIP8, which transport zinc from ER and mitochondria to cytosol, were significantly increased. Mitochondrial zinc transporter (ZnT) 4, which transports zinc from cytosol to mitochondria, was also increased. ER phosphorylated eukaryotic initiation factor 2α, activating transcription factor 4, and C/EBP homologous protein were significantly upregulated, and mitochondrial cytochrome c release and Bax insertion were detected in association with caspase-3 activation and apoptotic cell death. To define the role of zinc deficiency in ER and mitochondrial stress, H4IIEC3 cells were treated with 3 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine for 6 h with or without supplementation with zinc or N-acetylcysteine (NAC). The results demonstrated that zinc deprivation induced caspase-3 activation and apoptosis in association with ER and mitochondria dysfunction, which were inhibited by zinc as low as 10 μM but not by 2 mM NAC. These results suggest that chronic ethanol exposure induced in ER and mitochondrial zinc deficiency might activate intrinsic cell death signaling pathway, which could not be effectively rescued by antioxidant treatment. PMID:25767260

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription

PubMed Central

Marza, Esther; Taouji, Saïd; Barroso, Kim; Raymond, Anne-Aurélie; Guignard, Léo; Bonneu, Marc; Pallares-Lupon, Néstor; Dupuy, Jean-William; Fernandez-Zapico, Martin E; Rosenbaum, Jean; Palladino, Francesca; Dupuy, Denis; Chevet, Eric

2015-01-01

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPRER) to restore ER homeostasis. The AAA+ ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPRER genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA+ ATPase, as a novel repressor of a subset of UPRER genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPRER genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes. PMID:25652260

Curcumin induces ER stress-mediated apoptosis through selective generation of reactive oxygen species in cervical cancer cells.

PubMed

Kim, Boyun; Kim, Hee Seung; Jung, Eun-Ji; Lee, Jung Yun; K Tsang, Benjamin; Lim, Jeong Mook; Song, Yong Sang

2016-05-01

Prolonged accumulation of misfolded or unfolded proteins caused by cellular stress, including oxidative stress, induces endoplasmic reticulum stress, which then activates an unfolded protein response (UPR). ER stress is usually maintained at higher levels in cancer cells as compared to normal cells due to altered metabolism in cancer. Here, we investigated whether curcumin is ER stress-mediated apoptosis in cervical cancer cells, and ROS increased by curcumin are involved in the process as an upstream contributor. Curcumin inhibited proliferation of cervical cancer cells (C33A, CaSki, HeLa, and ME180) and induced apoptotic cell death. Curcumin activated ER-resident UPR sensors, such as PERK, IRE-1α, and ATF6, and their downstream-signaling proteins in cervical cancer cells, but not in normal epithelial cells and peripheral blood mononuclear cells (PBMCs). CHOP, a key factor involved in ER stress-mediated apoptosis, was also activated by curcumin. CHOP decreased the ratio of anti-apoptotic protein Bcl-2 to pro-apoptotic protein Bax expression, and subsequently increased the apoptotic population of cervical cancer cells. Furthermore, curcumin elevated levels of intracellular reactive oxygen species (ROS) in cervical cancer cells, but not in normal epithelial cells. Scavenging ROS resulted in inhibition of ER stress and partially restored cell viability in curcumin-treated cancer cells. Collectively, these observations show that curcumin promotes ER stress-mediated apoptosis in cervical cancer cells through increase of cell type-specific ROS generation. Therefore, modulation of these differential responses to curcumin between normal and cervical cancer cells could be an effective therapeutic strategy without adverse effects on normal cells. © 2015 Wiley Periodicals, Inc.

Human MI-ER1 Alpha and Beta Function as Transcriptional Repressors by Recruitment of Histone Deacetylase 1 to Their Conserved ELM2 Domain

PubMed Central

Ding, Zhihu; Gillespie, Laura L.; Paterno, Gary D.

2003-01-01

mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3′ end of hmi-er1 produces two major isoforms, hMI-ER1α and hMI-ER1β, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1α and hMI-ER1β in the regulation of transcription. Using fusion proteins of hMI-ER1α or hMI-ER1β tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1α and hMI-ER1β occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription. PMID:12482978

Human MI-ER1 alpha and beta function as transcriptional repressors by recruitment of histone deacetylase 1 to their conserved ELM2 domain.

PubMed

Ding, Zhihu; Gillespie, Laura L; Paterno, Gary D

2003-01-01

mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3' end of hmi-er1 produces two major isoforms, hMI-ER1alpha and hMI-ER1beta, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1alpha and hMI-ER1beta in the regulation of transcription. Using fusion proteins of hMI-ER1alpha or hMI-ER1beta tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1alpha and hMI-ER1beta occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription.

Estrogen promotes megakaryocyte polyploidization via estrogen receptor beta-mediated transcription of GATA1.

PubMed

Du, C; Xu, Y; Yang, K; Chen, S; Wang, X; Wang, S; Wang, C; Shen, M; Chen, F; Chen, M; Zeng, D; Li, F; Wang, T; Wang, F; Zhao, J; Ai, G; Cheng, T; Su, Y; Wang, J

2017-04-01

Estrogen is reported to be involved in thrombopoiesis and the disruption of its signaling may cause myeloproliferative disease, yet the underlying mechanisms remain largely unknown. GATA-binding factor 1 (GATA1) is a key regulator of megakaryocyte (MK) differentiation and its deficiency will lead to megakaryoblastic leukemia. Here we show that estrogen can dose-dependently promote MK polyploidization and maturation via activation of estrogen receptor beta (ERβ), accompanied by a significant upregulation of GATA1. Chromatin immunoprecipitation and a dual luciferase assay demonstrate that ERβ can directly bind the promoter region of GATA1 and activate its transcription. Steroid receptor coactivator 3 (SRC3) is involved in ERβ-mediated GATA1 transcription. The deficiency of ERβ or SRC3, similar to the inhibition of GATA1, leads to the impediment of estrogen-induced MK polyploidization and platelet production. Further investigations reveal that signal transducer and activator of transcription 1 signaling pathway downstream of GATA1 has a crucial role in estrogen-induced MK polyploidization, and ERβ-mediated GATA1 upregulation subsequently enhances nuclear factor erythroid-derived 2 expression, thereby promoting proplatelet formation and platelet release. Our study provides a deep insight into the molecular mechanisms of estrogen signaling in regulating thrombopoiesis and the pathogenesis of ER deficiency-related leukemia.

The Mediator complex and transcription regulation

PubMed Central

Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

PubMed

Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

2009-11-30

Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

PubMed Central

Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.

PubMed

Fang, Jingye; Liu, Ming; Zhang, Xuebao; Sakamoto, Takeshi; Taatjes, Douglas J; Jena, Bhanu P; Sun, Fei; Woods, James; Bryson, Tim; Kowluru, Anjaneyulu; Zhang, Kezhong; Chen, Xuequn

2015-08-01

Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.

Functional characterization of estrogen receptor subtypes, ER{alpha} and ER{beta}, mediating vitellogenin production in the liver of rainbow trout

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leanos-Castaneda, Olga; Kraak, Glen van der

2007-10-15

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ER{alpha} and ER{beta}, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ER{alpha} selective agonist, methyl-piperidino-pyrazole (MPP) an ER{alpha} selective antagonist, and diarylpropionitrile (DPN) an ER{beta} selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbowmore » trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [{sup 3}H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ER{alpha} could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ER{beta}. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ER{alpha}. On the other hand, once blocked ER{alpha} with MPP, the only manifestation of agonist activity of estradiol would be achieved via ER{beta}. In conclusion, the present results indicate that vitellogenin

Human leptin protein activates the growth of HepG2 cells by inhibiting PERK‑mediated ER stress and apoptosis.

PubMed

Xiong, Ying; Zhang, Jie; Liu, Man; An, Mingwei; Lei, Ling; Guo, Wuhua

2014-09-01

Current treatment modalities for various types of hepatic cancer, which has an increasing incidence rate, are inadequate and novel therapies are required. Therefore, identifying targets for liver cancer is becoming increasingly valuable to develop novel methods for therapy. The aim of the present study was to examine the growth activation mechanism of the leptin protein in the liver cancer cell line HepG2. The effects of the leptin protein on cell death were investigated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide analysis. DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis were also performed to detect cell apoptosis. The expression of leptin and three endoplasmic reticulum (ER) stress unfolded protein response (UPR) proteins, including activating transcription factor 6, phosphorylated‑PKR‑like ER kinase (p‑PERK) and inositol requiring protein 1, were investigated for the examination of ER stress. The mRNA UPR proteins were also detected by reverse transcription polymerase chain reaction. The apoptosis‑associated caspase 12 and C/EBP homologous protein (CHOP) was detected by western blot analysis. The expression of or incubation with the leptin protein was able to activate cell growth and inhibit cell death and apoptosis. In cells that expressed leptin or were incubated with leptin protein (pep-LPT), cisplatin‑induced ER stress‑associated mRNA transcription and protein activation were inhibited. Levels of the ER stress UPR pathway protein, PERK, increased significantly in leptin‑silenced cells when treated with cisplatin as compared with those in the leptin‑expressing or pep-LPT cells. Furthermore, caspase 12 activation was inhibited in ex‑LPT, pep‑LPT and HepG2 cells. In conclusion, human leptin protein is involved in promoting the proliferation of HepG2 cells through inhibiting the ER stress‑associated apoptotic pathway. The PERK UPR pathway and the apoptotic factor

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

PubMed

Petit, F G; Métivier, R; Valotaire, Y; Pakdel, F

1999-01-01

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

PubMed

Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

2016-07-01

The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

PubMed

Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

2016-06-07

Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

Insights on the involvement of (-)-epigallocatechin gallate in ER stress-mediated apoptosis in age-related macular degeneration.

PubMed

Karthikeyan, Bose; Harini, Lakshminarasimhan; Krishnakumar, Vaithilingam; Kannan, Velu Rajesh; Sundar, Krishnan; Kathiresan, Thandavarayan

2017-01-01

Endoplasmic reticulum (ER) stress-mediated apoptosis is a well-known factor in the pathogenesis of age-related macular degeneration (AMD). ER stress leads to accumulation of misfolded proteins, which in turn activates unfolded protein response (UPR) of the cell for its survival. The prolonged UPR of ER stress promotes cell death; however, the transition between adaptation and ER stress-induced apoptosis has not been clearly understood. Hence, the present study investigates the regulatory effect of (-)-epigallocatechin gallate (EGCG) on ER stress-induced by hydrogen peroxide (H 2 O 2 ) and disturbance of calcium homeostasis by thapsigargin (TG) in mouse retinal pigment epithelial (MRPE) cells. The oxidant molecules influenced MRPE cells showed an increased level of intracellular calcium [Ca 2+ ] i in ER and transferred to mitochondria through ER-mitochondrial tether site then increased ROS production. EGCG restores [Ca 2+ ] i homeostasis by decreasing ROS production through inhibition of prohibitin1 which regulate ER-mitochondrial tether site and inhibit apoptosis. Effect of EGCG on ER stress-mediated apoptosis was elucidated by exploring the UPR signalling pathways. EGCG downregulated GRP78, CHOP, PERK, ERO1α, IRE1α, cleaved PARP, cleaved caspase 3, caspase 12 and upregulated expression of calnexinin MRPE cells. In addition to this, inhibition of apoptosis by EGCG was also confirmed with expression of proteins Akt, PTEN and GSK3β. MRPE cells with EGCG upregulates phosphorylation of Akt at ser473 and phospho ser380 of PTEN, but phosphorylation at ser9 of GSK3β was inhibited. Further, constitutively active (myristoylated) CA-Akt transfected in MRPE cells had an increased Akt activity in EGCG influenced cells. These findings strongly suggest that antioxidant molecules inhibit cell death through the proper balancing of [Ca 2+ ] i and ROS production in order to maintain UPR of ER in MRPE cells. Thus, modulation of UPR signalling may provide a potential target for

The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

PubMed

Jeronimo, Célia; Robert, François

2017-10-01

Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lau, Wen Min; Doucet, Michele; Huang, David

2013-07-26

Highlights: •The effects of elevated CITED2 on ER function in breast cancer cells are examined. •CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. •CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found thatmore » CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a

Plant Mediator complex and its critical functions in transcription regulation.

PubMed

Yang, Yan; Li, Ling; Qu, Li-Jia

2016-02-01

The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted. © 2015 Institute of Botany, Chinese Academy of Sciences.

The SANT domain of human MI-ER1 interacts with Sp1 to interfere with GC box recognition and repress transcription from its own promoter.

PubMed

Ding, Zhihu; Gillespie, Laura L; Mercer, F Corinne; Paterno, Gary D

2004-07-02

To gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within-68/+144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-On-inducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative

Fungal mediator tail subunits contain classical transcriptional activation domains.

PubMed

Liu, Zhongle; Myers, Lawrence C

2015-04-01

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Malleable machines in transcription regulation: the mediator complex.

PubMed

Tóth-Petróczy, Agnes; Oldfield, Christopher J; Simon, István; Takagi, Yuichiro; Dunker, A Keith; Uversky, Vladimir N; Fuxreiter, Monika

2008-12-01

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the

Calcium and ER stress mediate hepatic apoptosis after burn injury

PubMed Central

Gauglitz, Gerd G.; Song, Juquan; Kulp, Gabriela A.; Finnerty, Celeste C.; Cox, Robert A.; Barral, José M.; Herndon, David N.; Boehning, Darren

2009-01-01

Abstract A hallmark of the disease state following severe burn injury is decreased liver function, which results in gross metabolic derangements that compromise patient survival. The underlying mechanisms leading to hepatocyte dysfunction after burn are essentially unknown. The aim of the present study was to determine the underlying mechanisms leading to hepatocyte dysfunction and apoptosis after burn. Rats were randomized to either control (no burn) or burn (60% total body surface area burn) and sacrificed at various time‐points. Liver was either perfused to isolate primary rat hepatocytes, which were used for in vitro calcium imaging, or liver was harvested and processed for immunohistology, transmission electron microscopy, mitochondrial isolation, mass spectroscopy or Western blotting to determine the hepatic response to burn injury in vivo. We found that thermal injury leads to severely depleted endoplasmic reticulum (ER) calcium stores and consequent elevated cytosolic calcium concentrations in primary hepatocytes in vitro. Burn‐induced ER calcium depletion caused depressed hepatocyte responsiveness to signalling molecules that regulate hepatic homeostasis, such as vasopressin and the purinergic agonist ATP. In vivo, thermal injury resulted in activation of the ER stress response and major alterations in mitochondrial structure and function – effects which may be mediated by increased calcium release by inositol 1,4,5‐trisphosphate receptors. Our results reveal that thermal injury leads to dramatic hepatic disturbances in calcium homeostasis and resultant ER stress leading to mitochondrial abnormalities contributing to hepatic dysfunction and apoptosis after burn injury. PMID:20141609

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

PubMed

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

ER stress and ER stress-induced apoptosis are activated in gastric SMCs in diabetic rats

PubMed Central

Chen, Xia; Fu, Xiang-Sheng; Li, Chang-Ping; Zhao, Hong-Xian

2014-01-01

AIM: To investigate the gastric muscle injury caused by endoplasmic reticulum (ER) stress in rats with diabetic gastroparesis. METHODS: Forty rats were randomly divided into two groups: a control group and a diabetic group. Diabetes was induced by intraperitoneal injection of 60 mg/kg of streptozotocin. Gastric emptying was determined at the 4th and 12th week. The ultrastructural changes in gastric smooth muscle cells (SMCs) were investigated by transmission electron microscopy. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis of SMCs. Expression of the ER stress marker, glucose-regulated protein 78 (GRP78), and the ER-specific apoptosis mediator, caspase-12 protein, was determined by immunohistochemistry. RESULTS: Gastric emptying was significantly lower in the diabetic rats than in the control rats at the 12th wk (40.71% ± 2.50%, control rats vs 54.65% ± 5.22%, diabetic rats; P < 0.05). Swollen and distended ER with an irregular shape was observed in gastric SMCs in diabetic rats. Apoptosis of gastric SMCs increased in the diabetic rats in addition to increased expression of GRP78 and caspase-12 proteins. CONCLUSION: ER stress and ER stress-mediated apoptosis are activated in gastric SMCs in diabetic rats with gastroparesis. PMID:25009401

TALE-mediated modulation of transcriptional enhancers in vivo.

PubMed

Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

PubMed

McInerney, E M; Ince, B A; Shapiro, D J; Katzenellenbogen, B S

1996-12-01

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable

HIV-1 Vpr triggers mitochondrial destruction by impairing Mfn2-mediated ER-mitochondria interaction.

PubMed

Huang, Chih-Yang; Chiang, Shu-Fen; Lin, Tze-Yi; Chiou, Shiow-Her; Chow, Kuan-Chih

2012-01-01

Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4(+) T lymphoblast cell line SupT1, or human primary CD4(+) T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1.

HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

PubMed Central

Huang, Chih-Yang; Chiang, Shu-Fen; Lin, Tze-Yi; Chiou, Shiow-Her; Chow, Kuan-Chih

2012-01-01

Human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM). The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4+ T lymphoblast cell line SupT1, or human primary CD4+ T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP) by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2) via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1) and increases bulging in mitochondria-associated membranes (MAM), the specific regions of endoplasmic reticulum (ER) which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1. PMID:22438978

Mediator-regulated transcription through the +1 nucleosome.

PubMed

Nock, Adam; Ascano, Janice M; Barrero, Maria J; Malik, Sohail

2012-12-28

Many genes are regulated at the level of a Pol II that is recruited to a nucleosome-free region upstream of the +1 nucleosome. How the Mediator coactivator complex, which functions at multiple steps, affects transcription through the promoter proximal region, including this nucleosome, remains largely unaddressed. We have established a fully defined in vitro assay system to delineate mechanisms for Pol II transit across the +1 nucleosome. Our results reveal cooperative functions of multiple cofactors, particularly of Mediator and elongation factor SII, in transcribing into this nucleosome. This is achieved, in part, through an unusual activity of SII that alters the intrinsic catalytic properties of promoter-proximal Pol II and, in concert with the Mediator, leads to enhancement in transcription of nucleosomal DNA. Our data provide additional mechanistic bases for Mediator function after recruitment of Pol II and, potentially, for regulation of genes controlled via nucleosome-mediated promoter-proximal pausing. Copyright © 2012 Elsevier Inc. All rights reserved.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

PubMed

Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

2015-06-01

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells

PubMed Central

JEONG, KWON; KIM, KIYOON; KIM, HUNSUNG; OH, YOOJUNG; KIM, SEONG-JIN; JO, YUNHEE; CHOE, WONCHAE

2015-01-01

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions. PMID:26137159

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP ▿

PubMed Central

Mita, Paolo; Savas, Jeffrey N.; Djouder, Nabil; Yates, John R.; Ha, Susan; Ruoff, Rachel; Schafler, Eric D.; Nwachukwu, Jerome C.; Tanese, Naoko; Cowan, Nicholas J.; Zavadil, Jiri; Garabedian, Michael J.; Logan, Susan K.

2011-01-01

Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes. PMID:21730289

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

The APP intracellular domain (AICD) potentiates ER stress-induced apoptosis.

PubMed

Kögel, Donat; Concannon, Caoimhín G; Müller, Thorsten; König, Hildegard; Bonner, Caroline; Poeschel, Simone; Chang, Steffi; Egensperger, Rupert; Prehn, Jochen H M

2012-09-01

Here we employed human SHEP neuroblastoma cells either stably or inducibly expressing the amyloid precursor protein (APP) intracellular domain (AICD) to investigate its ability to modulate stress-induced cell death. Analysis of effector caspase activation revealed that AICD overexpression was specifically associated with an increased sensitivity to apoptosis induced by the 2 endoplasmic reticulum (ER) stressors thapsigargin and tunicamycin, but not by staurosporine (STS). Basal and ER stress-induced expression of Bip/Grp78 and C/EBP-homologous protein/GADD153 were not altered by AICD implying that AICD potentiated cell death downstream or independent of the conserved unfolded protein response (UPR). Interestingly, quantitative polymerase chain reaction analysis and reporter gene assays revealed that AICD significantly downregulated messenger RNA levels of the Alzheimer's disease susceptibility gene ApoJ/clusterin, indicating transcriptional repression. Knockdown of ApoJ/clusterin mimicked the effect of AICD on ER stress-induced apoptosis, but had no discernible effect on staurosporine-induced cell death. Our data suggest that altered levels of AICD may abolish the prosurvival function of ApoJ/clusterin and increase the susceptibility of neurons to ER stress-mediated cell death, a pathway that may contribute to the pathogenesis of Alzheimer's disease. Copyright © 2012 Elsevier Inc. All rights reserved.

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito

2010-09-03

Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored.more » Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH{sub 2}-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with {gamma}-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.« less

Feedback regulation on PTEN/AKT pathway by the ER stress kinase PERK mediated by interaction with the Vault complex.

PubMed

Zhang, Wei; Neo, Suat Peng; Gunaratne, Jayantha; Poulsen, Anders; Boping, Liu; Ong, Esther Hongqian; Sangthongpitag, Kanda; Pendharkar, Vishal; Hill, Jeffrey; Cohen, Stephen M

2015-03-01

The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications for their potential use as therapeutic agents. Copyright © 2014. Published by Elsevier Inc.

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription

PubMed Central

Grierson, Patrick M.; Lillard, Kate; Behbehani, Gregory K.; Combs, Kelly A.; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-01-01

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. 3H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS. PMID:22106380

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.

PubMed

Grierson, Patrick M; Lillard, Kate; Behbehani, Gregory K; Combs, Kelly A; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-03-01

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling.

PubMed

Kang, Lianguo; Zhang, Xintian; Xie, Yan; Tu, Yaping; Wang, Dong; Liu, Zhenming; Wang, Zhao-Yi

2010-04-01

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

PubMed

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J; Jopling, Catherine L

2015-04-01

MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation.

PubMed

Batista, Marcelo Bueno; Wassem, Roseli; Pedrosa, Fábio de Oliveira; de Souza, Emanuel Maltempi; Dixon, Ray; Monteiro, Rose Adele

2015-05-07

Orthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation. The activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type. Fnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.

Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage.

PubMed

Layhadi, Janice A; Fountain, Samuel J

2017-06-03

Mechanisms controlling endoplasmic reticulum (ER) Ca 2+ homeostasis are important regulators of resting cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyto ) and receptor-mediated Ca 2+ signalling. Here we investigate channels responsible for ER Ca 2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca 2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca 2+ leak. Under these conditions ionomycin elevates [Ca 2+ ] cyto revealing a Ca 2+ leak response which is abolished by thapsigargin. IP 3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca 2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca 2+ ] cyto . In primary macrophage, translocon inhibition blocks Ca 2+ leak but does not influence resting [Ca 2+ ] cyto . We identify a role for translocon-mediated ER Ca 2+ leak in receptor-mediated Ca 2+ signalling in both model and primary human macrophage, whereby the Ca 2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca 2+ leak via the translocon in controlling resting cytoplasmic Ca 2+ in model macrophage and receptor-mediated Ca 2+ signalling in model macrophage and primary macrophage. Copyright © 2017 Elsevier Inc. All rights reserved.

Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

PubMed

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

2014-06-05

The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

Subunit architecture and functional modular rearrangements of the transcriptional Mediator complex

PubMed Central

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2014-01-01

SUMMARY The multisubunit Mediator comprising ~30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. PMID:24882805

BEND3 mediates transcriptional repression and heterochromatin organization

PubMed Central

Khan, Abid; Prasanth, Supriya G

2015-01-01

Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization. PMID:26507581

BEND3 mediates transcriptional repression and heterochromatin organization.

PubMed

Khan, Abid; Prasanth, Supriya G

2015-01-01

Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization.

Endoplasmic reticulum stress-mediated upregulation of miR-29a enhances sensitivity to neuronal apoptosis.

PubMed

Nolan, Katie; Walter, Franziska; Tuffy, Liam P; Poeschel, Simone; Gallagher, Ross; Haunsberger, Stefan; Bray, Isabella; Stallings, Raymond L; Concannon, Caoimhín G; Prehn, Jochen H M

2016-03-01

Disturbance of homeostasis within the endoplasmic reticulum (ER) lumen leads to the accumulation of unfolded and misfolded proteins. This results in the activation of an evolutionary conserved stress response termed ER stress that, if unresolved, induces apoptosis. Previously the Bcl-2 homology domain 3-Only Protein Puma was identified as a mediator of ER stress-induced apoptosis in neurons. In the search of alternative contributors to ER stress-induced apoptosis, a downregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 was noted during ER stress in both mouse cortical neurons and human SH-SY5Y neuroblastoma cells. Downregulation of Mcl-1 was associated with an upregulation of microRNA-29a (miR-29a) expression, and subsequent experiments showed that miR-29a targeted the 3'-untranslated region of the anti-apoptotic Bcl-2 family protein, Mcl-1. Inhibition of miR-29a expression using sequence-specific antagomirs or the overexpression of Mcl-1 decreased cell death following tunicamycin treatment, while gene silencing of Mcl-1 increased cell death. miR-29a did not alter the signalling branches of the ER stress response, rather its expression was controlled by the ER stress-induced transcription factor activating-transcription-factor-4 (ATF4). The current data demonstrate that the ATF4-mediated upregulation of miR-29a enhances the sensitivity of neurons to ER stress-induced apoptosis. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

The CDK inhibitor p21 is a novel target gene of ATF4 and contributes to cell survival under ER stress.

PubMed

Inoue, Yasumichi; Kawachi, Shiori; Ohkubo, Tsubasa; Nagasaka, Mai; Ito, Shogo; Fukuura, Keishi; Itoh, Yuka; Ohoka, Nobumichi; Morishita, Daisuke; Hayashi, Hidetoshi

2017-11-01

Activating transcription factor 4 (ATF4) is well known for its role in the endoplasmic reticulum (ER) stress response. ATF4 also transcriptionally induces multiple effectors that determine cell fate depending on cellular context. In addition, ATF4 can communicate both pro-apoptotic and pro-survival signals. How ATF4 mediates its prosurvival roles, however, requires further investigation. Here, we report that the CDK inhibitor p21 is a novel target gene of ATF4. We identified two ATF4-responsive elements, one of which directly binds ATF4, within the first intron of the p21 gene. Importantly, overexpression of p21 enhances cell survival following ER stress induction, while p21 knockdown increases cell death. These results suggest that p21 induction plays a vital role in the cellular response to ER stress and indicate that p21 is a prosurvival effector of ATF4. © 2017 Federation of European Biochemical Societies.

NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis.

PubMed

Widenmaier, Scott B; Snyder, Nicole A; Nguyen, Truc B; Arduini, Alessandro; Lee, Grace Y; Arruda, Ana Paula; Saksi, Jani; Bartelt, Alexander; Hotamisligil, Gökhan S

2017-11-16

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol. Copyright © 2017. Published by Elsevier Inc.

Mediator directs co-transcriptional heterochromatin assembly by RNA interference-dependent and -independent pathways.

PubMed

Oya, Eriko; Kato, Hiroaki; Chikashige, Yuji; Tsutsumi, Chihiro; Hiraoka, Yasushi; Murakami, Yota

2013-01-01

Heterochromatin at the pericentromeric repeats in fission yeast is assembled and spread by an RNAi-dependent mechanism, which is coupled with the transcription of non-coding RNA from the repeats by RNA polymerase II. In addition, Rrp6, a component of the nuclear exosome, also contributes to heterochromatin assembly and is coupled with non-coding RNA transcription. The multi-subunit complex Mediator, which directs initiation of RNA polymerase II-dependent transcription, has recently been suggested to function after initiation in processes such as elongation of transcription and splicing. However, the role of Mediator in the regulation of chromatin structure is not well understood. We investigated the role of Mediator in pericentromeric heterochromatin formation and found that deletion of specific subunits of the head domain of Mediator compromised heterochromatin structure. The Mediator head domain was required for Rrp6-dependent heterochromatin nucleation at the pericentromere and for RNAi-dependent spreading of heterochromatin into the neighboring region. In the latter process, Mediator appeared to contribute to efficient processing of siRNA from transcribed non-coding RNA, which was required for efficient spreading of heterochromatin. Furthermore, the head domain directed efficient transcription in heterochromatin. These results reveal a pivotal role for Mediator in multiple steps of transcription-coupled formation of pericentromeric heterochromatin. This observation further extends the role of Mediator to co-transcriptional chromatin regulation.

Extraction of nobiletin from Citrus Unshiu peels by supercritical fluid and its CRE-mediated transcriptional activity.

PubMed

Oba, Chisato; Ota, Masaki; Nomura, Koichiro; Fujiwara, Hironori; Takito, Jiro; Sato, Yoshiyuki; Ohizumi, Yasushi; Inomata, Hiroshi

2017-04-15

Polymethoxyflavone (PMF) is one of bioactive compounds in Citrus Unshiu and included mainly in the peels rather than the fruits, seeds and leaves. Supercritical CO 2 extraction is one candidate for selective extraction of polymethoxyflavone and in this study, supercritical CO 2 extraction with/without ethanol entrainer from Citrus Unshiu peels was examined at a temperature of 333K and a pressure of 30MPa. CRE (cyclic AMP response element)-mediated transcriptional assay was examined by using the extracts from supercritical fluid extraction. The results showed that extracts including nobiletin increased with increasing ethanol concentration in supercritical CO 2 and the elapsed extraction time. Extracts at ethanol concentration of 5 mol% showed high CRE-mediated transcription activity. This can be caused by activity of the extract including nobiletin in addition to the other methoxylated flavonoid species such as tangeretin. Extracts at ethanol concentration of 50% showed the highest CRE-mediated transcription activity, which can be attributed to flavonoid glycoside such as hesperidin. From our investigations, flavonoid glycoside can be one of promoters of CRE-mediated transcription activity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Differential effects of HTLV-1 Tax oncoprotein on the different estrogen-induced-ER α-mediated transcriptional activities

PubMed Central

Abou-Kandil, Ammar; Eisa, Nora; Jabareen, Azhar; Huleihel, Mahmoud

2016-01-01

ABSTRACT The activated estrogen (E2) receptor α (ERα) is a potent transcription factor that is involved in the activation of various genes by 2 different pathways; a classical and non-classical. In classical pathway, ERα binds directly to E2-responsive elements (EREs) located in the appropriate genes promoters and stimulates their transcription. However, in non-classical pathway, the ERα can indirectly bind with promoters and enhance their activity. For instance, ERα activates BRCA1 expression by interacting with jun/fos complex bound to the AP-1 site in BRCA1 promoter. Interference with the expression and/or functions of BRCA1, leads to high risk of breast or/and ovarian cancer. HTLV-1Tax was found to strongly inhibit BRCA1 expression by preventing the binding of E2–ERα complex to BRCA1 promoter. Here we examined Tax effect on ERα induced activation of genes by the classical pathway by testing its influence on E2-induced expression of ERE promoter-driven luciferase reporter (ERE-Luc). Our findings showed that E2 profoundly stimulated this reporter expression and that HTLV-1Tax significantly induced this stimulation. This result is highly interesting because in our previous study Tax was found to strongly block the E2-ERα-mediated activation of BRCA1 expression. ERα was found to produce a big complex by recruiting various cofactors in the nucleus before binding to the ERE region. We also found that only part of the reqruited cofactors are required for the transcriptional activity of ERα complex. Chip assay revealed that the binding of Tax to the ERα complex, did not interfere with its link to ERE region. PMID:27420286

Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

PubMed

Kim, Seong Keun; Oh, Eunhye; Yun, Mihee; Lee, Seong-Beom; Chae, Gue Tae

2015-07-16

Endoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells. RAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR. Palmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα. The findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

PubMed

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.

PubMed

Cho, Won-Ki; Spille, Jan-Hendrik; Hecht, Micca; Lee, Choongman; Li, Charles; Grube, Valentin; Cisse, Ibrahim I

2018-06-21

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. Here we used live cell super-resolution and light sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo. Copyright © 2018, American Association for the Advancement of Science.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

PubMed

Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Activating transcription factor 4 regulates stearate-induced vascular calcification.

PubMed

Masuda, Masashi; Ting, Tabitha C; Levi, Moshe; Saunders, Sommer J; Miyazaki-Anzai, Shinobu; Miyazaki, Makoto

2012-08-01

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate.

Activating transcription factor 4 regulates stearate-induced vascular calcification

PubMed Central

Masuda, Masashi; Ting, Tabitha C.; Levi, Moshe; Saunders, Sommer J.; Miyazaki-Anzai, Shinobu; Miyazaki, Makoto

2012-01-01

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate. PMID:22628618

The Mediator complex: a master coordinator of transcription and cell lineage development.

PubMed

Yin, Jing-wen; Wang, Gang

2014-03-01

Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells.

PubMed

Bernhart, Eva; Kogelnik, Nora; Prasch, Jürgen; Gottschalk, Benjamin; Goeritzer, Madeleine; Depaoli, Maria Rosa; Reicher, Helga; Nusshold, Christoph; Plastira, Ioanna; Hammer, Astrid; Fauler, Günter; Malli, Roland; Graier, Wolfgang F; Malle, Ernst; Sattler, Wolfgang

2018-05-01

Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H 2 O 2 -chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a 'clickable' alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)-6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP), a light for mammalian transcript analysis in low-input laboratories.

PubMed

Pandey, Mamta; Singh, Dheer; Onteru, Suneel K

2018-06-01

Transcript analysis is usually performed by costly, time-consuming, and expertise intensive methods, like real time-PCR, microarray, etc. However, they are not much feasible in low-input laboratories. Therefore, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a means of mammalian transcript analysis. Particularly, RT-LAMP was developed for buffalo aromatase cytochrome P450 (CYP19) transcript, to study its expression in 3D-cultured buffalo granulosa cells, which were exposed to lipopolysaccharide (LPS). The CYP19-RT-LAMP assay rapidly identified the LPS-induced downregulation of the CYP19 gene within 30 min at 63°C in a water bath. The assay was visualized via unaided eye by observing the change in turbidity and fluorescence, which were decreased by increasing the LPS exposure time to granulosa cells. Overall, the developed CYP19-RT-LAMP assay provided a hope on the application of RT-LAMP for mammalian transcript analysis in low-input laboratories. © 2017 Wiley Periodicals, Inc.

Lace plant ethylene receptors, AmERS1a and AmERS1c, regulate ethylene-induced programmed cell death during leaf morphogenesis.

PubMed

Rantong, Gaolathe; Evans, Rodger; Gunawardena, Arunika H L A N

2015-10-01

The lace plant, Aponogeton madagascariensis, is an aquatic monocot that forms perforations in its leaves as part of normal leaf development. Perforation formation occurs through developmentally regulated programmed cell death (PCD). The molecular basis of PCD regulation in the lace plant is unknown, however ethylene has been shown to play a significant role. In this study, we examined the role of ethylene receptors during perforation formation. We isolated three lace plant ethylene receptors AmERS1a, AmERS1b and AmERS1c. Using quantitative PCR, we examined their transcript levels at seven stages of leaf development. Through laser-capture microscopy, transcript levels were also determined in cells undergoing PCD and cells not undergoing PCD (NPCD cells). AmERS1a transcript levels were significantly lower in window stage leaves (in which perforation formation and PCD are occurring) as compared to all other leaf developmental stages. AmERS1a and AmERS1c (the most abundant among the three receptors) had the highest transcript levels in mature stage leaves, where PCD is not occurring. Their transcript levels decreased significantly during senescence-associated PCD. AmERS1c had significantly higher transcript levels in NPCD compared to PCD cells. Despite being significantly low in window stage leaves, AmERS1a transcripts were not differentially expressed between PCD and NPCD cells. The results suggested that ethylene receptors negatively regulate ethylene-controlled PCD in the lace plant. A combination of ethylene and receptor levels determines cell fate during perforation formation and leaf senescence. A new model for ethylene emission and receptor expression during lace plant perforation formation and senescence is proposed.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

PubMed Central

Rogers, Jason V.; Arlow, Tim; Inkellis, Elizabeth R.; Koo, Timothy S.; Rose, Mark D.

2013-01-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway. PMID:24152736

Structures of transcription pre-initiation complex with TFIIH and Mediator.

PubMed

Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

2017-11-09

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

Lack of TXNIP protects against mitochondria-mediated apoptosis but not against fatty acid-induced ER stress-mediated beta-cell death.

PubMed

Chen, Junqin; Fontes, Ghislaine; Saxena, Geetu; Poitout, Vincent; Shalev, Anath

2010-02-01

We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects beta-cells against lipoapoptosis. RESARCH DESIGN AND METHODS: To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis.

Loss of epigenetic Kruppel-like factor 4 histone deacetylase (KLF-4-HDAC)-mediated transcriptional suppression is crucial in increasing vascular endothelial growth factor (VEGF) expression in breast cancer.

PubMed

Ray, Alpana; Alalem, Mohamed; Ray, Bimal K

2013-09-20

Vascular endothelial growth factor (VEGF) is recognized as an important angiogenic factor that promotes angiogenesis in a series of pathological conditions, including cancer, inflammation, and ischemic disorders. We have recently shown that the inflammatory transcription factor SAF-1 is, at least in part, responsible for the marked increase of VEGF levels in breast cancer. Here, we show that SAF-1-mediated induction of VEGF is repressed by KLF-4 transcription factor. KLF-4 is abundantly present in normal breast epithelial cells, but its level is considerably reduced in breast cancer cells and clinical cancer tissues. In the human VEGF promoter, SAF-1- and KLF-4-binding elements are overlapping, whereas SAF-1 induces and KLF-4 suppresses VEGF expression. Ectopic overexpression of KLF-4 and RNAi-mediated inhibition of endogenous KLF-4 supported the role of KLF-4 as a transcriptional repressor of VEGF and an inhibitor of angiogenesis in breast cancer cells. We show that KLF-4 recruits histone deacetylases (HDACs) -2 and -3 at the VEGF promoter. Chronological ChIP assays demonstrated the occupancy of KLF-4, HDAC2, and HDAC3 in the VEGF promoter in normal MCF-10A cells but not in MDA-MB-231 cancer cells. Co-transfection of KLF-4 and HDAC expression plasmids in breast cancer cells results in synergistic repression of VEGF expression and inhibition of angiogenic potential of these carcinoma cells. Together these results identify a new mechanism of VEGF up-regulation in cancer that involves concomitant loss of KLF-4-HDAC-mediated transcriptional repression and active recruitment of SAF-1-mediated transcriptional activation.

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain.

PubMed

Cabukusta, Birol; Kol, Matthijs; Kneller, Laura; Hilderink, Angelika; Bickert, Andreas; Mina, John G M; Korneev, Sergei; Holthuis, Joost C M

2017-01-25

SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme's catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca 2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER.

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain

PubMed Central

Cabukusta, Birol; Kol, Matthijs; Kneller, Laura; Hilderink, Angelika; Bickert, Andreas; Mina, John G. M.; Korneev, Sergei; Holthuis, Joost C. M.

2017-01-01

SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme’s catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER. PMID:28120887

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

PubMed

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-12-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Activating transcription factor 4 underlies the pathogenesis of arsenic trioxide-mediated impairment of macrophage innate immune functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, Ritesh K.; Li, Changzhao

Chronic arsenic exposure to humans is considered immunosuppressive with augmented susceptibility to several infectious diseases. The exact molecular mechanisms, however, remain unknown. Earlier, we showed the involvement of unfolded protein response (UPR) signaling in arsenic-mediated impairment of macrophage functions. Here, we show that activating transcription factor 4 (ATF4), a UPR transcription factor, regulates arsenic trioxide (ATO)-mediated dysregulation of macrophage functions. In ATO-treated ATF4{sup +/+} wild-type mice, a significant down-regulation of CD11b expression was associated with the reduced phagocytic functions of peritoneal and lung macrophages. This severe immuno-toxicity phenotype was not observed in ATO-treated ATF4{sup +/−} heterozygous mice. To confirm thesemore » observations, we demonstrated in Raw 264.7 cells that ATF4 knock-down rescues ATO-mediated impairment of macrophage functions including cytokine production, bacterial engulfment and clearance of engulfed bacteria. Sustained activation of ATF4 by ATO in macrophages induces apoptosis, while diminution of ATF4 expression protects against ATO-induced apoptotic cell death. Raw 264.7 cells treated with ATO also manifest dysregulated Ca{sup ++} homeostasis. ATO induces Ca{sup ++}-dependent calpain-1 and caspase-12 expression which together regulated macrophage apoptosis. Additionally, apoptosis was also induced by mitochondria-regulated pathway. Restoring ATO-impaired Ca{sup ++} homeostasis in ER/mitochondria by treatments with the inhibitors of inositol 1,4,5-trisphosphate receptor (IP3R) and voltage-dependent anion channel (VDAC) attenuate innate immune functions of macrophages. These studies identify a novel role for ATF4 in underlying pathogenesis of macrophage dysregulation and immuno-toxicity of arsenic. - Highlights: • ATF4 regulates arsenic-mediated impairment in macrophage functions. • Arsenic-mediated alterations in pulmonary macrophage are diminished in ATF4{sup +/�

Morbillivirus Glycoprotein Expression Induces ER Stress, Alters Ca2+ Homeostasis and Results in the Release of Vasostatin

PubMed Central

Doucey, Marie-Agnès; Rosso, Lia; Curie, Thomas; Montagner, Alexandra; Wittek, Riccardo; Vandelvelde, Marc; Zurbriggen, Andreas; Hirling, Harald; Desvergne, Béatrice

2012-01-01

Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca2+ homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca2+ homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses. PMID:22403712

Endoplasmic reticulum turnover: ER-phagy and other flavors in selective and non-selective ER clearance.

PubMed

Fregno, Ilaria; Molinari, Maurizio

2018-01-01

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells. It is deputed to lipid and protein biosynthesis, calcium storage, and the detoxification of various exogenous and endogenous harmful compounds. ER activity and size must be adapted rapidly to environmental and developmental conditions or biosynthetic demand. This is achieved on induction of thoroughly studied transcriptional/translational programs defined as "unfolded protein responses" that increase the ER volume and the expression of ER-resident proteins regulating the numerous ER functions. Less understood are the lysosomal catabolic processes that maintain ER size at steady state, that prevent excessive ER expansion during ER stresses, or that ensure return to physiologic ER size during recovery from ER stresses. These catabolic processes may also be activated to remove ER subdomains where proteasome-resistant misfolded proteins or damaged lipids have been segregated. Insights into these catabolic mechanisms have only recently emerged with the identification of so-called ER-phagy receptors, which label specific ER subdomains for selective lysosomal delivery for clearance. Here, in eight chapters and one addendum, we comment on recent advances in ER turnover pathways induced by ER stress, nutrient deprivation, misfolded proteins, and live bacteria. We highlight the role of yeast (Atg39 and Atg40) and mammalian (FAM134B, SEC62, RTN3, and CCPG1) ER-phagy receptors and of autophagy genes in selective and non-selective catabolic processes that regulate cellular proteostasis by controlling ER size, turnover, and function.

Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression

PubMed Central

Gomez, Javier A; Rutkowski, D Thomas

2016-01-01

Endoplasmic reticulum (ER) stress is implicated in many chronic diseases, but very little is known about how the unfolded protein response (UPR) responds to persistent ER stress in vivo. Here, we experimentally reconstituted chronic ER stress in the mouse liver, using repeated injection of a low dose of the ER stressor tunicamycin. Paradoxically, this treatment led to feedback-mediated suppression of a select group of mRNAs, including those encoding the ER chaperones BiP and GRP94. This suppression was due to both silencing of the ATF6α pathway of UPR-dependent transcription and enhancement of mRNA degradation, possibly via regulated IRE1-dependent decay (RIDD). The suppression of mRNA encoding BiP was phenocopied by ectopic overexpression of BiP protein, and was also observed in obese mice. Our findings suggest that persistent cycles of UPR activation and deactivation create an altered, quasi-stable setpoint for UPR-dependent transcriptional regulation—an outcome that could be relevant to conditions such as metabolic syndrome. DOI: http://dx.doi.org/10.7554/eLife.20390.001 PMID:27938665

The Mediator complex: a central integrator of transcription

PubMed Central

Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment

PubMed Central

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-01-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3′ endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes. PMID:24298055

Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

PubMed Central

Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

1991-01-01

Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

Systemic effects of AGEs in ER stress induction in vivo.

PubMed

Adamopoulos, Christos; Mihailidou, Chrysovalantou; Grivaki, Christofora; Papavassiliou, Kostas A; Kiaris, Hippokratis; Piperi, Christina; Papavassiliou, Athanasios G

2016-08-01

Emerging evidence indicates that accumulation of advanced glycation end products (AGEs) in human tissues may contribute to cell injury, inflammation and apoptosis through induction of endoplasmic reticulum (ER) stress. Human metabolism relies on ER homeostasis for the coordinated response of all metabolic organs by controlling the synthesis and catabolism of various nutrients. In vitro studies have demonstrated AGE-induced enhancement of unfolded protein response (UPR) in different cell types including endothelial, neuronal, pancreatic cells and podocytes, suggesting this crosstalk as an underlying pathological mechanism that contributes to metabolic diseases. In this minireview, we describe in vivo studies undertaken by our group and others that demonstrate the diverse systemic effects of AGEs in ER stress induction in major metabolic tissues such as brain, kidney, liver and pancreas of normal mice. Administration of high-AGEs content diet to normal mice for the period of 4 weeks upergulates the mRNA and protein levels of ER chaperone Bip (GRP78) indicative of UPR initiation in all major metabolic organs and induces activation of the pivotal transcription factor XBP1 that regulates glucose and lipid metabolism. Furthermore, animals with genetic ablation of UPR-activated transcription factor C/EBP homologous protein CHOP allocated in high-AGEs diet, exhibited relative resistance to UPR induction (BiP levels) and XBP1 activation in major metabolic organs. Since CHOP presents a critical mediator that links accumulation and aggregation of unfolded proteins with induction of oxidative stress and ER stress-related apoptosis, it is revealed as an important molecular target for the management of metabolic diseases.

Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

PubMed

Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

2015-03-01

Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

ER stress triggers MCP-1 expression through SET7/9-induced histone methylation in the kidneys of db/db mice.

PubMed

Chen, Jigang; Guo, Yanhong; Zeng, Wei; Huang, Li; Pang, Qi; Nie, Ling; Mu, Jiao; Yuan, Fahuan; Feng, Bing

2014-04-15

Epigenetics plays a key role in the pathogenesis of diabetic nephropathy (DN), although the precise regulatory mechanism is still unclear. Here, we examined the role of endoplasmic reticulum (ER) stress in histone H3 lysine 4 (H3K4) methyltransferase SET7/9-induced monocyte chemoattractant protein-1 (MCP-1) expression in the kidneys of db/db mice. Our results indicate that the expression of MCP-1 significantly increased in the kidneys of db/db mice in a time-dependent manner. An increased chromatin mark associated with an active gene (H3K4me1) at MCP-1 promoters accompanied this change in expression. The expression of SET7/9 and the recruitment to these promoters were also elevated. SET7/9 gene silencing with small interfering (si) RNAs significantly attenuated the expression of H3K4me1 and MCP-1. Furthermore, expression of signaling regulator glucose-regulated protein 78 (GRP78), a monitor of ER stress, significantly increased in the kidneys of db/db mice. The expression of spliced X-box binding protein 1 (XBP1s), an ER stress-inducible transcription factor, and recruitment to the SET7/9 promoters were also increased. XBP1s gene silencing with siRNAs significantly attenuated the expression of SET7/9, H3K4me1, and MCP-1. The chaperone betaine not only effectively downregulated the GRP78 and XBP1s expression levels but also markedly decreased the SET7/9, H3K4me1, and MCP-1 levels. Luciferase reporter assay demonstrated that XBP1s participated in ER stress-induced SET7/9 transcription, Taken together, these results reveal that ER stress can trigger the expression of MCP-1, in part through the XBP1s-mediated induction of SET7/9.

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress.

PubMed

Kim, Min-Hyun; Aydemir, Tolunay B; Kim, Jinhee; Cousins, Robert J

2017-07-18

Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet-induced ER stress using Zip14 -/- (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders.

Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

PubMed

Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

2013-08-23

PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Restoration of Tamoxifen Sensitivity in Estrogen Receptor–Negative Breast Cancer Cells: Tamoxifen-Bound Reactivated ER Recruits Distinctive Corepressor Complexes

PubMed Central

Sharma, Dipali; Saxena, Neeraj K.; Davidson, Nancy E.; Vertino, Paula M.

2010-01-01

Breast tumors expressing estrogen receptor-α (ER) respond well to therapeutic strategies using selective ER modulators, such as tamoxifen. However, ~ 30% of invasive breast cancers are hormone independent because they lack ER expression due to hypermethylation of ER promoter. Treatment of ER-negative breast cancer cells with demethylating agents [5-aza-2′-deoxycytidine (5-aza-dC)] and histone deacetylase (HDAC) inhibitors (trichostatin A) leads to expression of ER mRNA and functional protein. Here, we examined whether epigenetically reactivated ER is a target for tamoxifen therapy. Following treatment with trichostatin A and 5-aza-dC, the formerly unresponsive ER-negative MDA-MB-231 breast cancer cells became responsive to tamoxifen. Tamoxifen-mediated inhibition of cell growth in these cells is mediated at least in part by the tamoxifen-bound ER. Tamoxifen-bound reactivated ER induces transcriptional repression at estrogen-responsive genes by ordered recruitment of multiple distinct chromatin-modifying complexes. Using chromatin immunoprecipitation, we show recruitment of two different corepressor complexes to ER-responsive promoters in a mutually exclusive and sequential manner: the nuclear receptor corepressor-HDAC3 complex followed by nucleosome remodeling and histone deacetylation complex. The mechanistic insight provided by this study might help in designing therapeutic strategies directed toward epigenetic mechanisms in the prevention or treatment of breast cancer. PMID:16778215

Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance.

PubMed

Giltnane, Jennifer M; Hutchinson, Katherine E; Stricker, Thomas P; Formisano, Luigi; Young, Christian D; Estrada, Monica V; Nixon, Mellissa J; Du, Liping; Sanchez, Violeta; Ericsson, Paula Gonzalez; Kuba, Maria G; Sanders, Melinda E; Mu, Xinmeng J; Van Allen, Eliezer M; Wagle, Nikhil; Mayer, Ingrid A; Abramson, Vandana; Gόmez, Henry; Rizzo, Monica; Toy, Weiyi; Chandarlapaty, Sarat; Mayer, Erica L; Christiansen, Jason; Murphy, Danielle; Fitzgerald, Kerry; Wang, Kai; Ross, Jeffrey S; Miller, Vincent A; Stephens, Phillip J; Yelensky, Roman; Garraway, Levi; Shyr, Yu; Meszoely, Ingrid; Balko, Justin M; Arteaga, Carlos L

2017-08-09

Inhibition of proliferation in estrogen receptor-positive (ER + ) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER + /human epidermal growth factor receptor 2-negative (HER2 - ) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including FGFR1 and CCND1 , respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER + tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER + FGFR1 / CCND1 coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal ESR1 fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Initiation and execution of lipotoxic ER stress in pancreatic β-cells

PubMed Central

Cunha, Daniel A.; Hekerman, Paul; Ladrière, Laurence; Bazarra-Castro, Angie; Ortis, Fernanda; Wakeham, Marion C.; Moore, Fabrice; Rasschaert, Joanne; Cardozo, Alessandra K.; Bellomo, Elisa; Overbergh, Lutgart; Mathieu, Chantal; Lupi, Roberto; Hai, Tsonwin; Herchuelz, Andre; Marchetti, Piero; Rutter, Guy A.; Eizirik, Décio L.; Cnop, Miriam

2013-01-01

Summary Free fatty acids (FFA) cause apoptosis of pancreatic β-cells and might contribute to β-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary β-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca2+ stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor β-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary β-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced β-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca2+ depletion. The IRE1 and resulting JNK activation contribute to β-cell apoptosis. PERK activation by palmitate also contributes to β-cell apoptosis via CHOP. PMID:18559892

Regulation of ERRα Gene Expression by Estrogen Receptor Agonists and Antagonists in SKBR3 Breast Cancer Cells: Differential Molecular Mechanisms Mediated by G Protein-Coupled Receptor GPR30/GPER-1

PubMed Central

Li, Yin; Birnbaumer, Lutz; Teng, Christina T.

2010-01-01

In selected tissues and cell lines, 17β-estradiol (E2) regulates the expression of estrogen-related receptor α (ERRα), a member of the orphan nuclear receptor family. This effect is thought to be mediated by the estrogen receptor α (ERα). However in the ERα- and ERβ-negative SKBR3 breast cancer cell line, physiological levels of E2 also stimulate ERRα expression. Here, we explored the molecular mechanism that mediates estrogen action in ER-negative breast cancer cells. We observed that E2, the ERα agonist, as well as the ERα antagonists ICI 182,780 and tamoxifen (TAM), a selective ER modulator, stimulate the transcriptional activity of the ERRα gene and increase the production of ERRα protein in SKBR3 cells. Moreover, the ERRα downstream target genes expression and cellular proliferation are also increased. We show further that the G protein-coupled receptor GPR30/GPER-1 (GPER-1) mediates these effects. The GPER-1 specific ligand G-1 mimics the actions of E2, ICI 182,780, and TAM on ERRα expression, and changing the levels of GPER-1 mRNA by overexpression or small interfering RNA knockdown affected the expression of ERRα accordingly. Utilizing inhibitors, we delineate a different downstream pathway for ER agonist and ER antagonist-triggered signaling through GPER-1. We also find differential histone acetylation and transcription factor recruitment at distinct nucleosomes of the ERRα promoter, depending on whether the cells are activated with E2 or with ER antagonists. These findings provide insight into the molecular mechanisms of GPER-1/ERRα-mediated signaling and may be relevant to what happens in breast cancer cells escaping inhibitory control by TAM. PMID:20211987

Endoplasmic reticulum stress is increased after spontaneous labor in human fetal membranes and myometrium where it regulates the expression of prolabor mediators.

PubMed

Liong, Stella; Lappas, Martha

2014-09-01

Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In nongestational tissues, several markers of the unfolded protein response (UPR) have been shown to regulate the inflammatory response. Thus, the aim of this study was to determine the effect of human labor on markers of ER stress in fetal membranes and myometrium. In addition, the effect of ER stress inhibition on the expression and secretion of proinflammatory and prolabor mediators was also assessed. The markers of ER stress, GRP78, IRE1, and spliced XBP1 (XBP1s), were significantly increased in fetal membranes and myometrium after term and preterm labor compared to nonlaboring samples. Given that inflammation is considered to be one of the leading causes of spontaneous preterm birth, here we used bacterial endotoxin lipopolysaccharide (LPS) as a model for infection-induced preterm birth. In term nonlabored fetal membranes and myometrium, LPS induced UPR activation as evidenced by a significant increase in the expression of GRP78, IRE1, and XBP1s in fetal membranes and myometrium. The use of the chemical chaperones 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA) alleviated ER stress induced by LPS. 4-PBA and TUDCA also ameliorated the increase in LPS-induced prolabor mediators. Our data suggest that the UPR may regulate the inflammatory responses associated with labor or infection in fetal membranes and myometrium of pregnant term and preterm women. Thus, the use of ER stress inhibitors, in particular 4-PBA or TUDCA, may be a potential therapeutic strategy for the prevention of infection-mediated spontaneous preterm birth. © 2014 by the Society for the Study of Reproduction, Inc.

Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum Stress and Attenuates Kidney Injury

PubMed Central

Mami, Iadh; Bouvier, Nicolas; El Karoui, Khalil; Gallazzini, Morgan; Rabant, Marion; Laurent-Puig, Pierre; Li, Shuping; Tharaux, Pierre-Louis; Beaune, Philippe; Thervet, Eric; Chevet, Eric; Hu, Guo-Fu

2016-01-01

Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang−/−) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism. PMID:26195817

Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

PubMed Central

Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

2011-01-01

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

Induced ER-chaperones regulate a novel receptor-like kinase to mediate a viral innate immune response

PubMed Central

Caplan, Jeffrey L.; Zhu, Xiaohong; Mamillapalli, Padmavathi; Marathe, Rajendra; Anandalakshmi, Radhamani; Dinesh-Kumar, S. P.

2009-01-01

Summary The plant innate immune response requires a rapid, global reprogramming of cellular processes. Here we employed two complementary proteomic methods, two-dimensional differential in-gel electrophoresis (2D-DIGE) and iTRAQ, to identify differentially regulated proteins early during a defense response. Besides defense-related proteins, the constituents of the largest category of up-regulated proteins were cytoplasmic- and endoplasmic reticulum (ER)-residing molecular chaperones. Silencing of ER-resident protein disulfide isomerases, NbERp57 and NbP5, and the calreticulins, NbCRT2 and NbCRT3, lead to a partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 are required for the expression of a novel induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for the N-mediated hypersensitive response programmed cell death (HR-PCD) and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane bound or secreted proteins that are necessary for innate immunity. PMID:19917500

Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matt Vaughn Greg Harrington Daniel R Bush

2002-08-06

This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with themore » loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 m

Histo-chemical and biochemical analysis reveals association of er1 mediated powdery mildew resistance and redox balance in pea.

PubMed

Mohapatra, Chinmayee; Chand, Ramesh; Navathe, Sudhir; Sharma, Sandeep

2016-09-01

Powdery mildew caused by Erysiphe pisi is one of the important diseases responsible for heavy yield losses in pea crop worldwide. The most effective method of controlling the disease is the use of resistant varieties. The resistance to powdery mildew in pea is recessive and governed by a single gene er1. The objective of present study is to investigate if er1 mediated powdery mildew resistance is associated with changes in the redox status of the pea plant. 16 pea genotypes were screened for powdery mildew resistance in field condition for two years and, also, analyzed for the presence/absence of er1 gene. Histochemical analysis with DAB and NBT staining indicates accumulation of reactive oxygen species (ROS) in surrounding area of powdery mildew infection which was higher in susceptible genotypes as compared to resistant genotypes. A biochemical study revealed that the activity of superoxide dismutase (SOD) and catalase, enzymes involved in scavenging ROS, was increased in, both, resistant and susceptible genotypes after powdery mildew infection. However, both enzymes level was always higher in resistant than susceptible genotypes throughout time course of infection. Moreover, irrespective of any treatment, the total phenol (TP) and malondialdehyde (MDA) content was significantly high and low in resistant genotypes, respectively. The powdery mildew infection elevated the MDA content but decreased the total phenol in pea genotypes. Statistical analysis showed a strong positive correlation between AUDPC and MDA; however, a negative correlation was observed between AUDPC and SOD, CAT and TP. Heritability of antioxidant was also high. The study identified few novel genotypes resistant to powdery mildew infection that carried the er1 gene and provided further clue that er1 mediated defense response utilizes antioxidant machinery to confer powdery mildew resistance in pea. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

PubMed

Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress

PubMed Central

Kim, Min-Hyun; Aydemir, Tolunay B.; Kim, Jinhee; Cousins, Robert J.

2017-01-01

Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet–induced ER stress using Zip14−/− (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders. PMID:28673968

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

PubMed

Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

2013-02-28

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

CRE-Mediated Transcription and COX-2 Expression in the Pilocarpine Model of Status Epilepticus

PubMed Central

Lee, Boyoung; Dziema, Heather; Lee, Kyu Hyun; Choi, Yun-Sik; Obrietan, Karl

2007-01-01

Status epilepticus (SE) triggers neuronal death, reactive gliosis and remodeling of synaptic circuitry, thus leading to profound pathological alterations in CNS physiology. These processes are, in part, regulated by the rapid upregulation of both cytotoxic and cytoprotective genes. One pathway that may couple SE to transcriptionally-dependent alterations in CNS physiology is the CREB (cAMP response element-binding protein)/CRE (cAMP response element) cascade. Here, we utilized the pilocarpine model of SE on a mouse strain transgenic for a CRE-reporter construct (β-galactosidase) to begin to characterize how seizure activity regulates the activation state of the CREB/CRE pathway in both glia and neurons of the hippocampus. SE triggered a rapid (4–8 hrs post SE) but transient increase in CRE-mediated gene expression in the neuronal sublayers. In contrast to neurons, SE induced a lasting increase (up to 20 days) in CRE-mediated transcription in both reactive astrocytes and microglia. CRE-mediated gene expression correlated with expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2). To examine the role of CREB in SE-induced COX-2 expression, we generated a transgenic mouse strain that expresses A-CREB, a potent repressor of CREB-dependent transcription. In these animals, the capacity of SE to stimulate COX-2 expression was markedly attenuated, indicating that CREB is a key intermediate in SE-induced COX-2 expression. Collectively these data show that SE triggers two waves of CREB-mediated gene expression, a transient wave in neurons and a long-lasting wave in reactive glial cells, and that CREB couples SE to COX-2 expression. PMID:17029965

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

PubMed

Lee, Ji Eun; Oney, McKenna; Frizzell, Kimberly; Phadnis, Nitin; Hollien, Julie

2015-02-13

Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells. Copyright © 2015 Lee et al.

Drosophila melanogaster Activating Transcription Factor 4 Regulates Glycolysis During Endoplasmic Reticulum Stress

PubMed Central

Lee, Ji Eun; Oney, McKenna; Frizzell, Kimberly; Phadnis, Nitin; Hollien, Julie

2015-01-01

Endoplasmic reticulum (ER) stress results from an imbalance between the load of proteins entering the secretory pathway and the ability of the ER to fold and process them. The response to ER stress is mediated by a collection of signaling pathways termed the unfolded protein response, which plays important roles in development and disease. Here we show that in Drosophila melanogaster S2 cells, ER stress induces a coordinated change in the expression of genes involved in carbon metabolism. Genes encoding enzymes that carry out glycolysis were up-regulated, whereas genes encoding proteins in the tricarboxylic acid cycle and respiratory chain complexes were down-regulated. The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh). Furthermore, Atf4 binding motifs in promoters for these genes could partially account for their regulation during ER stress. Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress. Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells. PMID:25681259

Stabilised DNA secondary structures with increasing transcription localise hypermutable bases for somatic hypermutation in IGHV3-23.

PubMed

Duvvuri, Bhargavi; Duvvuri, Venkata R; Wu, Jianhong; Wu, Gillian E

2012-07-01

Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER.

PubMed

Costello, Joseph L; Castro, Inês G; Hacker, Christian; Schrader, Tina A; Metz, Jeremy; Zeuschner, Dagmar; Azadi, Afsoon S; Godinho, Luis F; Costina, Victor; Findeisen, Peter; Manner, Andreas; Islinger, Markus; Schrader, Michael

2017-02-01

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering. © 2017 Costello et al.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

PubMed Central

Costello, Joseph L.; Hacker, Christian; Schrader, Tina A.; Zeuschner, Dagmar; Findeisen, Peter

2017-01-01

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering. PMID:28108524

Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

PubMed

Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

2016-04-26

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

An Actin-Dependent Step in Mitochondrial Fission Mediated by the ER-Associated Formin INF2

PubMed Central

Korobova, Farida; Ramabhadran, Vinay; Higgs, Henry N.

2013-01-01

Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state. PMID:23349293

A Lack of Ovarian Function Increases Neuroinflammation in Aged Mice

PubMed Central

Benedusi, Valeria; Meda, Clara; Della Torre, Sara; Monteleone, Giuseppina; Vegeto, Elisabetta

2012-01-01

Although several lines of evidence have indicated that menopause is associated with increased susceptibility to neurological disorders, the mechanisms involved in this phenomenon remain to be elucidated. Because neuroinflammation is a common feature of a number of brain diseases, we hypothesized that the cessation of ovarian functions and the consequent decrease in estrogen receptor (ER)-mediated antiinflammatory activity may represent a trigger for postmenopausal brain dysfunctions. The aim of the present study was to investigate the effects of aging and surgical menopause on the activity of ER in neuroinflammation. The present study shows that ER genes are expressed in the hippocampus, but ER transcriptional activity decreases significantly beginning at 12 months of age in intact and ovariectomized mice. With ovariectomy, we observe an age-dependent accumulation of mRNA encoding inflammatory mediators (e.g. TNFα, IL1β, and macrophage inflammatory protein-2) and changes in the morphology of astroglia and microglia. In addition, we show that aging itself is coupled with an exaggerated response to acute inflammatory stimuli with a major accumulation of TNFα, IL1β, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 mRNA in response to lipopolysaccharide administration. The response to acute inflammatory stimuli appears to be differentially modulated by the duration of hormone deprivation in 12-month-old mice. Taken together, the present results show that aging is associated with decreased ER activity, despite continuous ER synthesis, and that age-dependent neuroinflammation is strongly influenced by hormone deprivation. PMID:22492304

Microprocessor mediates transcriptional termination in long noncoding microRNA genes

PubMed Central

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J.; Jopling, Catherine L.

2015-01-01

MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with co-transcriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. While most miRNA are located within introns of protein coding genes, a substantial minority of miRNA originate from long non coding (lnc) RNA where transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) pathway, but instead use Microprocessor cleavage to terminate transcription. This Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells. PMID:25730776

Adenomatous polyposis coli protein (APC)-independent regulation of beta-catenin/Tcf-4 mediated transcription in intestinal cells.

PubMed Central

Baulida, J; Batlle, E; García De Herreros, A

1999-01-01

Alterations in the transcriptional activity of the beta-catenin-Tcf complex have been associated with the earlier stages of colonic transformation. We show here that the activation of protein kinase C by the phorbol ester PMA in several intestinal cell lines increases the levels of beta-catenin detected in the nucleus and augments the transcriptional activity mediated by beta-catenin. The response to PMA was not related to modifications in the cytosolic levels of beta-catenin and was observed not only in cells with wild-type adenomatous polyposis coli protein (APC) but also in APC-deficient cells. Binding assays in vitro revealed that PMA facilitates the interaction of the beta-catenin with the nuclear structure. Our results therefore show that beta-catenin-mediated transcription can be regulated independently of the presence of APC. PMID:10567241

Protein Kinase Cα Modulates Estrogen-Receptor-Dependent Transcription and Proliferation in Endometrial Cancer Cells

PubMed Central

Thorne, Alicia M.; Jackson, Twila A.; Willis, Van C.; Bradford, Andrew P.

2013-01-01

Endometrial cancer is the most common invasive gynecologic malignancy in developed countries. The most prevalent endometrioid tumors are linked to excessive estrogen exposure and hyperplasia. However, molecular mechanisms and signaling pathways underlying their etiology and pathophysiology remain poorly understood. We have shown that protein kinase Cα (PKCα) is aberrantly expressed in endometrioid tumors and is an important mediator of endometrial cancer cell survival, proliferation, and invasion. In this study, we demonstrate that expression of active, myristoylated PKCα conferred ligand-independent activation of estrogen-receptor- (ER-) dependent promoters and enhanced responses to estrogen. Conversely, knockdown of PKCα reduced ER-dependent gene expression and inhibited estrogen-induced proliferation of endometrial cancer cells. The ability of PKCα to potentiate estrogen activation of ER-dependent transcription was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K) and Akt. Evidence suggests that PKCα and estrogen signal transduction pathways functionally interact, to modulate ER-dependent growth and transcription. Thus, PKCα signaling, via PI3K/Akt, may be a critical element of the hyperestrogenic environment and activation of ER that is thought to underlie the development of estrogen-dependent endometrial hyperplasia and malignancy. PKCα-dependent pathways may provide much needed prognostic markers of aggressive disease and novel therapeutic targets in ER positive tumors. PMID:23843797

Protectin DX suppresses hepatic gluconeogenesis through AMPK-HO-1-mediated inhibition of ER stress.

PubMed

Jung, Tae Woo; Kim, Hyung-Chun; Abd El-Aty, A M; Jeong, Ji Hoon

2017-06-01

Several studies have shown that protectins, which are ω-3 fatty acid-derived proresolution mediators, may improve insulin resistance. Recently, protectin DX (PDX) was documented to attenuate insulin resistance by stimulating IL-6 expression in skeletal muscle, thereby regulating hepatic gluconeogenesis. These findings made us investigate the direct effects of PDX on hepatic glucose metabolism in the context of diabetes. In the current study, we show that PDX regulates hepatic gluconeogenesis in a manner distinct from its indirect glucoregulatory activity via IL-6. We found that PDX stimulated AMP-activated protein kinase (AMPK) phosphorylation, thereby inducing heme oxygenase 1 (HO-1) expression. This induction blocked hepatic gluconeogenesis by suppressing endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. These effects were significantly dampened by silencing AMPK or HO-1 expression with small interfering RNA (siRNA). We also demonstrated that administration of PDX to high fat diet (HFD)-fed mice resulted in increased hepatic AMPK phosphorylation and HO-1 expression, whereas hepatic ER stress was substantially attenuated. Furthermore, PDX treatment suppressed the expression of gluconeogenic genes, thereby decreasing blood glucose levels in HFD-fed mice. In conclusion, our findings suggest that PDX inhibits hepatic gluconeogenesis via AMPK-HO-1-dependent suppression of ER stress. Thus, PDX may be an effective therapeutic target for the treatment of insulin resistance and type 2 diabetes through the regulation of hepatic gluconeogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Apoptosis Induced by the UV Filter Benzophenone-3 in Mouse Neuronal Cells Is Mediated via Attenuation of Erα/Pparγ and Stimulation of Erβ/Gpr30 Signaling.

PubMed

Wnuk, A; Rzemieniec, J; Lasoń, W; Krzeptowski, W; Kajta, M

2018-03-01

Although benzophenone-3 (BP-3) has frequently been reported to play a role in endocrine disruption, there is insufficient data regarding the impact of BP-3 on the nervous system, including its possible adverse effects on the developing brain. Our study demonstrated that BP-3 caused neurotoxicity and activated apoptosis via an intrinsic pathway involving the loss of mitochondrial membrane potential and the activation of caspases-9 and -3 and kinases p38/MAPK and Gsk3β. These biochemical alterations were accompanied by ROS production, increased apoptotic body formation and impaired cell survival, and by an upregulation of the genes involved in apoptosis. The BP-3-induced effects were tissue-specific and age-dependent with the most pronounced effects observed in neocortical cells at 7 days in vitro. BP-3 changed the messenger RNA (mRNA) expression levels of Erα, Erβ, Gpr30, and Pparγ in a time-dependent manner. At 3 h of exposure, BP-3 downregulated estrogen receptor mRNAs but upregulated Pparγ mRNA. After prolonged exposures, BP-3 downregulated the receptor mRNAs except for Erβ mRNA that was upregulated. The BP-3-induced patterns of mRNA expression measured at 6 and 24 h of exposure reflected alterations in the protein levels of the receptors and paralleled their immunofluorescent labeling. Erα and Pparγ agonists diminished, but Erβ and Gpr30 agonists stimulated the BP-3-induced apoptotic and neurotoxic effects. Receptor antagonists caused the opposite effects, except for ICI 182,780. This is in line with a substantial reduction in the effects of BP-3 in cells with siRNA-silenced Erβ/Gpr30 and the maintenance of BP-3 effects in Erα- and Pparγ siRNA-transfected cells. We showed for the first time that BP-3-affected mRNA and protein expression levels of Erα, Erβ, Gpr30, and Pparγ, paralleled BP-3-induced apoptosis and neurotoxicity. Therefore, we suggest that BP-3-evoked apoptosis of neuronal cells is mediated via attenuation of Erα/Pparγ and

Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Payton-Stewart, Florastina; Tilghman, Syreeta L.; Williams, LaKeisha G.

Highlights: • The methyl-substituted benzimidazole was more effective at inhibiting growth in MDA-MB 231 cells. • The naphthyl-substituted benzimidazole was more effective at inhibiting growth in MCF-7 cells than ICI. • The benzimidazole molecules demonstrated a dose-dependent reduction in ERE transcriptional activity. • The benzimidazole molecules had binding mode in ERα and ERβ comparable to that of the co-crystallized ligand. - Abstract: Estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets formore » developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERβ receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERβ-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules

Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.

PubMed

Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K

2001-04-01

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

PubMed

Malik, Sohail; Roeder, Robert G

2010-11-01

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

PubMed

Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

2013-01-01

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice

PubMed Central

De Domenico, Ivana; Zhang, Tian Y.; Koening, Curry L.; Branch, Ryan W.; London, Nyall; Lo, Eric; Daynes, Raymond A.; Kushner, James P.; Li, Dean; Ward, Diane M.; Kaplan, Jerry

2010-01-01

Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses. PMID:20530874

Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters.

PubMed

Grünberg, Sebastian; Henikoff, Steven; Hahn, Steven; Zentner, Gabriel E

2016-11-15

Mediator is a conserved, essential transcriptional coactivator complex, but its in vivo functions have remained unclear due to conflicting data regarding its genome-wide binding pattern obtained by genome-wide ChIP Here, we used ChEC-seq, a method orthogonal to ChIP, to generate a high-resolution map of Mediator binding to the yeast genome. We find that Mediator associates with upstream activating sequences (UASs) rather than the core promoter or gene body under all conditions tested. Mediator occupancy is surprisingly correlated with transcription levels at only a small fraction of genes. Using the same approach to map TFIID, we find that TFIID is associated with both TFIID- and SAGA-dependent genes and that TFIID and Mediator occupancy is cooperative. Our results clarify Mediator recruitment and binding to the genome, showing that Mediator binding to UASs is widespread, partially uncoupled from transcription, and mediated in part by TFIID. © 2016 The Authors.

Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep

2010-04-25

Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNAmore » Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.« less

The Entangled ER-Mitochondrial axis as a potential therapeutic strategy in Neurodegeneration: A Tangled Duo Unchained

PubMed Central

Joshi, Amit U.; Kornfeld, Opher S.; Mochly-Rosen, Daria

2016-01-01

Endoplasmic reticulum (ER) and mitochondrial function have both been shown to be critical events in neurodegenerative diseases. The ER mediates protein folding, maturation, sorting as well acts as calcium storage. The unfolded protein response (UPR) is a stress response of the ER that is activated by the accumulation of misfolded proteins within the ER lumen. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Similarly, calcium-mediated mitochondrial function and dynamics not only contribute to ATP generation and calcium buffering but are also a linchpin in mediating cell fate. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintaining cellular homeostasis and determining cell fate under various pathophysiological conditions. Regulated Ca2+ transfer from the ER to the mitochondria is important in maintaining control of pro-survival/pro-death pathways. In this review, we summarize the latest therapeutic strategies that target these essential organelles in the context of neurodegenerative diseases. PMID:27212603

Light-mediated control of DNA transcription in yeast

PubMed Central

Hughes, Robert M.; Bolger, Steven; Tapadia, Hersh; Tucker, Chandra L.

2012-01-01

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light / cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems. PMID:22922268

Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca2+ homeostasis after ER stress.

PubMed

Kim, Jinhwan; Choi, Tae Gyu; Ding, Yan; Kim, Yeonghwan; Ha, Kwon Soo; Lee, Kyung Ho; Kang, Insug; Ha, Joohun; Kaufman, Randal J; Lee, Jinhwa; Choe, Wonchae; Kim, Sung Soo

2008-11-01

Prolonged accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in ER stress-mediated apoptosis. Cyclophilins are protein chaperones that accelerate the rate of protein folding through their peptidyl-prolyl cis-trans isomerase (PPIase) activity. In this study, we demonstrated that ER stress activates the expression of the ER-localized cyclophilin B (CypB) gene through a novel ER stress response element. Overexpression of wild-type CypB attenuated ER stress-induced cell death, whereas overexpression of an isomerase activity-defective mutant, CypB/R62A, not only increased Ca(2+) leakage from the ER and ROS generation, but also decreased mitochondrial membrane potential, resulting in cell death following exposure to ER stress-inducing agents. siRNA-mediated inhibition of CypB expression rendered cells more vulnerable to ER stress. Finally, CypB interacted with the ER stress-related chaperones, Bip and Grp94. Taken together, we concluded that CypB performs a crucial function in protecting cells against ER stress via its PPIase activity.

Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Mi-Sun; Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

Highlights: • FLII activates TGFβ-mediated expression of COL1A2 gene. • TGFβ induces the association of FLII with SMAD3 and BRG1 in A549 cells. • FLII is required for the recruitment of SWI/SNF complex and chromatin accessibility to COL1A2 promoter. - Abstract: Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signalingmore » pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2

Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

PubMed

D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

2016-06-23

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

Homo-trimerization is essential for the transcription factor function of Myrf for oligodendrocyte differentiation.

PubMed

Kim, Dongkyeong; Choi, Jin-Ok; Fan, Chuandong; Shearer, Randall S; Sharif, Mohamed; Busch, Patrick; Park, Yungki

2017-05-19

Myrf is a key transcription factor for oligodendrocyte differentiation and central nervous system myelination. We and others have previously shown that Myrf is generated as a membrane protein in the endoplasmic reticulum (ER), and that it undergoes auto-processing to release its N-terminal fragment from the ER, which enters the nucleus to work as a transcription factor. These previous studies allow a glimpse into the unusual complexity behind the biogenesis and function of the transcription factor domain of Myrf. Here, we report that Myrf N-terminal fragments assemble into stable homo-trimers before ER release. Consequently, Myrf N-terminal fragments are released from the ER only as homo-trimers. Our re-analysis of a previous genetic screening result in Caenorhabditis elegans shows that homo-trimerization is essential for the biological functions of Myrf N-terminal fragment, and that the region adjacent to the DNA-binding domain is pivotal to its homo-trimerization. Further, our computational analysis uncovered a novel homo-trimeric DNA motif that mediates the homo-trimeric DNA binding of Myrf N-terminal fragments. Importantly, we found that homo-trimerization defines the DNA binding specificity of Myrf N-terminal fragments. In sum, our study elucidates the molecular mechanism governing the biogenesis and function of Myrf N-terminal fragments and its physiological significance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

PubMed

Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

2015-05-01

Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

Resveratrol Induces Cancer Cell Apoptosis through MiR-326/PKM2-Mediated ER Stress and Mitochondrial Fission.

PubMed

Wu, Haili; Wang, Yingying; Wu, Changxin; Yang, Peng; Li, Hanqing; Li, Zhuoyu

2016-12-14

Resveratrol (Res), a natural phytoalexin found in a variety of plants, has significant antitumor activity. Pyruvate kinase M2 (PKM2) has abnormally high expression in various tumor cells, and it has been implicated in the survival of tumors. However, whether and how Res inhibits PKM2 expression is poorly understood. In the present study, we found that treatment with Res inhibited cell proliferation and induced cell apoptosis. The IC 50 values of Res against DLD1, HeLa, and MCF-7 cells were 75 ± 4.54, 50 ± 3.65, and 50 ± 3.32 μM, respectively. To elucidate mechanisms underlying its antitumor activities, serial experiments were performed. Results showed that reduction of PKM2 expression in tumor cells by Res treatment increased the expression of ER stress and mitochondrial fission proteins but reduced cell viability and the levels of fusion proteins. These phenomena were reversed by artificial overexpression of PKM2. Quantitative analyses showed that the expression of microRNA-326 (miR-326) was increased upon Res treatment. Treatment with the miR-326 mimic reduced PKM2 expression, promoting recovery from ER stress and mitochondrial fission. Overall, these results demonstrate that miR-326/PKM2-mediated ER stress and mitochondrial dysfunction participate in apoptosis induced by Res. These results provide novel insight into the molecular mechanisms by which Res suppresses tumors and further support for the use of Res as an antitumor drug.

Written Language Bursts Mediate the Relationship between Transcription Skills and Writing Performance

ERIC Educational Resources Information Center

Limpo, Teresa; Alves, Rui A.

2017-01-01

It is established that transcription skills (handwriting and spelling) constrain children's writing. Yet, little is known about the mechanism underlying this relationship. This study examined the mediating role of bursts and pauses on the link between transcription skills and writing fluency or text quality. For that, 174 second graders did the…

Transcription factor-mediated reprogramming toward hematopoietic stem cells

PubMed Central

Ebina, Wataru; Rossi, Derrick J

2015-01-01

De novo generation of human hematopoietic stem cells (HSCs) from renewable cell types has been a long sought-after but elusive goal in regenerative medicine. Paralleling efforts to guide pluripotent stem cell differentiation by manipulating developmental cues, substantial progress has been made recently toward HSC generation via combinatorial transcription factor (TF)-mediated fate conversion, a paradigm established by Yamanaka's induction of pluripotency in somatic cells by mere four TFs. This review will integrate the recently reported strategies to directly convert a variety of starting cell types toward HSCs in the context of hematopoietic transcriptional regulation and discuss how these findings could be further developed toward the ultimate generation of therapeutic human HSCs. PMID:25712209

A role for post-transcriptional control of endoplasmic reticulum dynamics and function in C. elegans germline stem cell maintenance.

PubMed

Maheshwari, Richa; Pushpa, Kumari; Subramaniam, Kuppuswamy

2016-09-01

Membrane-bound receptors, which are crucial for mediating several key developmental signals, are synthesized on endoplasmic reticulum (ER). The functional integrity of ER must therefore be important for the regulation of at least some developmental programs. However, the developmental control of ER function is not well understood. Here, we identify the C. elegans protein FARL-11, an ortholog of the mammalian STRIPAK complex component STRIP1/2 (FAM40A/B), as an ER protein. In the C. elegans embryo, we find that FARL-11 is essential for the cell cycle-dependent morphological changes of ER and for embryonic viability. In the germline, FARL-11 is required for normal ER morphology and for membrane localization of the GLP-1/Notch receptor involved in germline stem cell (GSC) maintenance. Furthermore, we provide evidence that PUF-8, a key translational regulator in the germline, promotes the translation of farl-11 mRNA. These findings reveal that ER form and function in the C. elegans germline are post-transcriptionally regulated and essential for the niche-GSC signaling mediated by GLP-1. © 2016. Published by The Company of Biologists Ltd.

Bilirubin Increases Insulin Sensitivity in Leptin-Receptor Deficient and Diet-Induced Obese Mice Through Suppression of ER Stress and Chronic Inflammation

PubMed Central

Dong, Huansheng; Huang, Hu; Yun, Xinxu; Kim, Do-sung; Yue, Yinan; Wu, Hongju; Sutter, Alton; Chavin, Kenneth D.; Otterbein, Leo E.; Adams, David B.; Kim, Young-Bum

2014-01-01

Obesity-induced endoplasmic reticulum (ER) stress causes chronic inflammation in adipose tissue and steatosis in the liver, and eventually leads to insulin resistance and type 2 diabetes (T2D). The goal of this study was to understand the mechanisms by which administration of bilirubin, a powerful antioxidant, reduces hyperglycemia and ameliorates obesity in leptin-receptor-deficient (db/db) and diet-induced obese (DIO) mouse models. db/db or DIO mice were injected with bilirubin or vehicle ip. Blood glucose and body weight were measured. Activation of insulin-signaling pathways, expression of inflammatory cytokines, and ER stress markers were measured in skeletal muscle, adipose tissue, and liver of mice. Bilirubin administration significantly reduced hyperglycemia and increased insulin sensitivity in db/db mice. Bilirubin treatment increased protein kinase B (PKB/Akt) phosphorylation in skeletal muscle and suppressed expression of ER stress markers, including the 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein (C/EBP) homologous protein, X box binding protein (XBP-1), and activating transcription factor 4 in db/db mice. In DIO mice, bilirubin treatment significantly reduced body weight and increased insulin sensitivity. Moreover, bilirubin suppressed macrophage infiltration and proinflammatory cytokine expression, including TNF-α, IL-1β, and monocyte chemoattractant protein-1, in adipose tissue. In liver and adipose tissue of DIO mice, bilirubin ameliorated hepatic steatosis and reduced expression of GRP78 and C/EBP homologous protein. These results demonstrate that bilirubin administration improves hyperglycemia and obesity by increasing insulin sensitivity in both genetically engineered and DIO mice models. Bilirubin or bilirubin-increasing drugs might be useful as an insulin sensitizer for the treatment of obesity-induced insulin resistance and type 2 diabetes based on its profound anti-ER stress and antiinflammatory properties. PMID

MYC2 Orchestrates a Hierarchical Transcriptional Cascade That Regulates Jasmonate-Mediated Plant Immunity in Tomato.

PubMed

Du, Minmin; Zhao, Jiuhai; Tzeng, David T W; Liu, Yuanyuan; Deng, Lei; Yang, Tianxia; Zhai, Qingzhe; Wu, Fangming; Huang, Zhuo; Zhou, Ming; Wang, Qiaomei; Chen, Qian; Zhong, Silin; Li, Chang-Bao; Li, Chuanyou

2017-08-01

The hormone jasmonate (JA), which functions in plant immunity, regulates resistance to pathogen infection and insect attack through triggering genome-wide transcriptional reprogramming in plants. We show that the basic helix-loop-helix transcription factor (TF) MYC2 in tomato ( Solanum lycopersicum ) acts downstream of the JA receptor to orchestrate JA-mediated activation of both the wounding and pathogen responses. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 655 MYC2-targeted JA-responsive genes. These genes are highly enriched in Gene Ontology categories related to TFs and the early response to JA, indicating that MYC2 functions at a high hierarchical level to regulate JA-mediated gene transcription. We also identified a group of MYC2-targeted TFs (MTFs) that may directly regulate the JA-induced transcription of late defense genes. Our findings suggest that MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output. As proof of concept, we showed that during plant resistance to the necrotrophic pathogen Botrytis cinerea , MYC2 and the MTF JA2-Like form a transcription module that preferentially regulates wounding-responsive genes, whereas MYC2 and the MTF ETHYLENE RESPONSE FACTOR.C3 form a transcription module that preferentially regulates pathogen-responsive genes. © 2017 American Society of Plant Biologists. All rights reserved.

Strategies to regulate transcription factor-mediated gene positioning and interchromosomal clustering at the nuclear periphery.

PubMed

Randise-Hinchliff, Carlo; Coukos, Robert; Sood, Varun; Sumner, Michael Chas; Zdraljevic, Stefan; Meldi Sholl, Lauren; Garvey Brickner, Donna; Ahmed, Sara; Watchmaker, Lauren; Brickner, Jason H

2016-03-14

In budding yeast, targeting of active genes to the nuclear pore complex (NPC) and interchromosomal clustering is mediated by transcription factor (TF) binding sites in the gene promoters. For example, the binding sites for the TFs Put3, Ste12, and Gcn4 are necessary and sufficient to promote positioning at the nuclear periphery and interchromosomal clustering. However, in all three cases, gene positioning and interchromosomal clustering are regulated. Under uninducing conditions, local recruitment of the Rpd3(L) histone deacetylase by transcriptional repressors blocks Put3 DNA binding. This is a general function of yeast repressors: 16 of 21 repressors blocked Put3-mediated subnuclear positioning; 11 of these required Rpd3. In contrast, Ste12-mediated gene positioning is regulated independently of DNA binding by mitogen-activated protein kinase phosphorylation of the Dig2 inhibitor, and Gcn4-dependent targeting is up-regulated by increasing Gcn4 protein levels. These different regulatory strategies provide either qualitative switch-like control or quantitative control of gene positioning over different time scales. © 2016 Randise-Hinchliff et al.

Leptin-induced ER-α-positive breast cancer cell viability and migration is mediated by suppressing CCN5-signaling via activating JAK/AKT/STAT-pathway.

PubMed

Haque, Inamul; Ghosh, Arnab; Acup, Seth; Banerjee, Snigdha; Dhar, Kakali; Ray, Amitabha; Sarkar, Sandipto; Kambhampati, Suman; Banerjee, Sushanta K

2018-01-25

In menopausal women, one of the critical risk factors for breast cancer is obesity/adiposity. It is evident from various studies that leptin, a 16 kDa protein hormone overproduced in obese people, plays the critical role in neovascularization and tumorigenesis in breast and other organs. However, the mechanisms by which obesity influences the breast carcinogenesis remained unclear. In this study, by analyzing different estrogen receptor-α (ER-α)-positive and ER-α-negative BC cell lines, we defined the role of CCN5 in the leptin-mediated regulation of growth and invasive capacity. We analyzed the effect of leptin on cell viability of ER-α-positive MCF-7 and ZR-75-1 cell lines and ER-α-negative MDA-MB-231 cell line. Additionally, we also determined the effect of leptin on the epithelial-mesenchymal transition (EMT) bio-markers, in vitro invasion and sphere-formation of MCF-7 and ZR-75-1 cell lines. To understand the mechanism, we determined the impact of leptin on CCN5 expression and the functional role of CCN5 in these cells by the treatment of human recombinant CCN5 protein(hrCCN5). Moreover, we also determined the role of JAK-STAT and AKT in the regulation of leptin-induced suppression of CCN5 in BC cells. Present studies demonstrate that leptin can induce cell viability, EMT, sphere-forming ability and migration of MCF-7 and ZR-75-1 cell lines. Furthermore, these studies found that leptin suppresses the expression of CCN5 at the transcriptional level. Although the CCN5 suppression has no impact on the constitutive proliferation of MCF-7 and ZR-75-1 cells, it is critical for leptin-induced viability and necessary for EMT, induction of in vitro migration and sphere formation, as the hrCCN5 treatment significantly inhibits the leptin-induced viability, EMT, migration and sphere-forming ability of these cells. Mechanistically, CCN5-suppression by leptin is mediated via activating JAK/AKT/STAT-signaling pathways. These studies suggest that CCN5 serves as a

Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

PubMed

Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

2005-03-01

Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

Toll-like Receptor 4-mediated Endoplasmic Reticulum Stress in Intestinal Crypts Induces Necrotizing Enterocolitis*

PubMed Central

Afrazi, Amin; Branca, Maria F.; Sodhi, Chhinder P.; Good, Misty; Yamaguchi, Yukihiro; Egan, Charlotte E.; Lu, Peng; Jia, Hongpeng; Shaffiey, Shahab; Lin, Joyce; Ma, Congrong; Vincent, Garrett; Prindle, Thomas; Weyandt, Samantha; Neal, Matthew D.; Ozolek, John A.; Wiersch, John; Tschurtschenthaler, Markus; Shiota, Chiyo; Gittes, George K.; Billiar, Timothy R.; Mollen, Kevin; Kaser, Arthur; Blumberg, Richard; Hackam, David J.

2014-01-01

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4ΔIEC-OVER mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4ΔIEC mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development. PMID:24519940

Toll-like receptor 4-mediated endoplasmic reticulum stress in intestinal crypts induces necrotizing enterocolitis.

PubMed

Afrazi, Amin; Branca, Maria F; Sodhi, Chhinder P; Good, Misty; Yamaguchi, Yukihiro; Egan, Charlotte E; Lu, Peng; Jia, Hongpeng; Shaffiey, Shahab; Lin, Joyce; Ma, Congrong; Vincent, Garrett; Prindle, Thomas; Weyandt, Samantha; Neal, Matthew D; Ozolek, John A; Wiersch, John; Tschurtschenthaler, Markus; Shiota, Chiyo; Gittes, George K; Billiar, Timothy R; Mollen, Kevin; Kaser, Arthur; Blumberg, Richard; Hackam, David J

2014-04-04

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Endoplasmic reticulum stress responses function in the HRT-mediated hypersensitive response in Nicotiana benthamiana.

PubMed

Moon, Ju Yeon; Lee, Jeong Hee; Oh, Chang-Sik; Kang, Hong-Gu; Park, Jeong Mee

2016-12-01

HRT is a plant coiled-coil, nucleotide-binding and leucine-rich repeat (CC-NB-LRR) disease resistance protein that triggers the hypersensitive response (HR) on recognition of Turnip crinkle virus (TCV) coat protein (CP). The molecular mechanism and significance of HR-mediated cell death for TCV resistance have not been fully elucidated. To identify the genes involved in HRT/TCV CP-mediated HR in Nicotiana benthamiana, we performed virus-induced gene silencing (VIGS) of 459 expressed sequence tags (ESTs) of pathogen-responsive Capsicum annuum genes. VIGS of CaBLP5, which encodes an endoplasmic reticulum (ER)-associated immunoglobulin-binding protein (BiP), silenced NbBiP4 and NbBiP5 and significantly reduced HRT-mediated HR. The induction of ER stress-responsive genes and the accumulation of ER-targeted BiPs in response to HRT-mediated HR suggest that ER is involved in HR in N. benthamiana. BiP4/5 silencing significantly down-regulated HRT at the mRNA and protein levels, and affected SGT1 and HSP90 expression. Co-expression of TCV CP in BiP4/5-silenced plants completely abolished HRT induction. Transient expression of TCV CP alone induced selected ER stress-responsive gene transcripts only in Tobacco rattle virus (TRV)-infected plants, and most of these genes were induced by HRT/TCV CP, except for bZIP60, which was induced specifically in response to HRT/TCV CP. TCV CP-mediated induction of ER stress-responsive genes still occurred in BiP4/5-silenced plants, but HRT/TCV CP-mediated induction of these genes was defective. Tunicamycin, a chemical that inhibits protein N-glycosylation, inhibited HRT-mediated HR, suggesting that ER has a role in HR regulation. These results indicate that BiP and ER, which modulate pattern recognition receptors in innate immunity, also regulate R protein-mediated resistance. © 2016 BSPP and John Wiley & Sons Ltd.

Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

PubMed

Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

2017-05-11

Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

The Transcription Elongation Complex Directs Activation-Induced Cytidine Deaminase-Mediated DNA Deamination†

PubMed Central

Besmer, Eva; Market, Eleonora; Papavasiliou, F. Nina

2006-01-01

Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter. PMID:16705187

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Munoz, Juan Pablo; Collao, Andres; Chiong, Mario

2009-10-09

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-inducedmore » MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.« less

ICI 182,780-regulated gene expression in DU145 prostate cancer cells is mediated by estrogen receptor-beta/NFkappaB crosstalk.

PubMed

Leung, Yuet-Kin; Gao, Ying; Lau, Kin-Mang; Zhang, Xiang; Ho, Shuk-Mei

2006-04-01

Estrogen receptor (ER)-beta is the predominant ER subtype in prostate cancer (PCa). We previously demonstrated that ICI 182,780 (ICI), but not estrogens, exerted dose-dependent growth inhibition on DU145 PCa cells by an ER-beta-mediated pathway. Transcriptional profiling detected a greater than three-fold upregulation of seven genes after a 12-hour exposure to 1 microM ICI. Semiquantitative reverse transcriptase polymerase chain reaction confirmed the upregulation of four genes by ICI: interleukin-12alpha chain, interleukin-8, embryonic growth/differentiation factor, and RYK tyrosine kinase. Treatment with an ER-beta antisense oligonucleotide reduced cellular ER-beta mRNA and induced loss of expression of these genes. Sequence analysis revealed the presence of consensus NFkappaB sites, but not estrogen-responsive elements, in promoters of all four genes. Reporter assay and chromatin immunoprecipitation experiments demonstrated that ICI-induced gene expression could be mediated by crosstalk between ER-beta and the NFkappaB signaling pathway, denoting a novel mechanism of ER-beta-mediated ICI action. Therefore, combined therapies targeting ER-beta and NFkappaB signaling may be synergistic as treatment for PCa.

The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth.

PubMed

Lozano-Durán, Rosa; Macho, Alberto P; Boutrot, Freddy; Segonzac, Cécile; Somssich, Imre E; Zipfel, Cyril

2013-12-31

The molecular mechanisms underlying the trade-off between plant innate immunity and steroid-mediated growth are controversial. Here, we report that activation of the transcription factor BZR1 is required and sufficient for suppression of immune signaling by brassinosteroids (BR). BZR1 induces the expression of several WRKY transcription factors that negatively control early immune responses. In addition, BZR1 associates with WRKY40 to mediate the antagonism between BR and immune signaling. We reveal that BZR1-mediated inhibition of immunity is particularly relevant when plant fast growth is required, such as during etiolation. Thus, BZR1 acts as an important regulator mediating the trade-off between growth and immunity upon integration of environmental cues. DOI: http://dx.doi.org/10.7554/eLife.00983.001.

Resveratrol triggers ER stress-mediated apoptosis by disrupting N-linked glycosylation of proteins in ovarian cancer cells.

PubMed

Gwak, HyeRan; Kim, Soochi; Dhanasekaran, Danny N; Song, Yong Sang

2016-02-28

Malignant tumors have a high glucose demand and alter cellular metabolism to survive. Herein, focusing on the utility of glucose metabolism as a therapeutic target, we found that resveratrol induced endoplasmic reticulum (ER) stress-mediated apoptosis by interrupting protein glycosylation in a cancer-specific manner. Our results indicated that resveratrol suppressed the hexosamine biosynthetic pathway and interrupted protein glycosylation through GSK3β activation. Application of either biochemical intermediates of the hexosamine pathway or small molecular inhibitors of GSK3β reversed the effects of resveratrol on the disruption of protein glycosylation. Additionally, an ER UDPase, ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), modulated protein glycosylation by Akt attenuation in response to resveratrol. By inhibition or overexpression of Akt functions, we confirmed that the glycosylation activities were dependent on ENTPD5 expression and regulated by the action of Akt in ovarian cancer cells. Resveratrol-mediated disruption of protein glycosylation induced cellular apoptosis as indicated by the up-regulation of GADD153, followed by the activation of ER-stress sensors (PERK and ATF6α). Thus, our results provide novel insight into cancer cell metabolism and protein glycosylation as a therapeutic target for cancers. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

PubMed

Pauli, Andrea; Montague, Tessa G; Lennox, Kim A; Behlke, Mark A; Schier, Alexander F

2015-01-01

Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

The role of proteosome-mediated proteolysis in modulating potentially harmful transcription factor activity in Saccharomyces cerevisiae

PubMed Central

Bonzanni, Nicola; Zhang, Nianshu; Oliver, Stephen G.; Fisher, Jasmin

2011-01-01

Motivation: The appropriate modulation of the stress response to variable environmental conditions is necessary to maintain sustained viability in Saccharomyces cerevisiae. Particularly, controlling the abundance of proteins that may have detrimental effects on cell growth is crucial for rapid recovery from stress-induced quiescence. Results: Prompted by qualitative modeling of the nutrient starvation response in yeast, we investigated in vivo the effect of proteolysis after nutrient starvation showing that, for the Gis1 transcription factor at least, proteasome-mediated control is crucial for a rapid return to growth. Additional bioinformatics analyses show that potentially toxic transcriptional regulators have a significantly lower protein half-life, a higher fraction of unstructured regions and more potential PEST motifs than the non-detrimental ones. Furthermore, inhibiting proteasome activity tends to increase the expression of genes induced during the Environmental Stress Response more than those in the rest of the genome. Our combined results suggest that proteasome-mediated proteolysis of potentially toxic transcription factors tightly modulates the stress response in yeast. Contact: jasmin.fisher@microsoft.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21685082

Proteins mediating DNA loops effectively block transcription.

PubMed

Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Integrative Analysis of Response to Tamoxifen Treatment in ER-Positive Breast Cancer Using GWAS Information and Transcription Profiling.

PubMed

Hicks, Chindo; Kumar, Ranjit; Pannuti, Antonio; Miele, Lucio

2012-01-01

Variable response and resistance to tamoxifen treatment in breast cancer patients remains a major clinical problem. To determine whether genes and biological pathways containing SNPs associated with risk for breast cancer are dysregulated in response to tamoxifen treatment, we performed analysis combining information from 43 genome-wide association studies with gene expression data from 298 ER(+) breast cancer patients treated with tamoxifen and 125 ER(+) controls. We identified 95 genes which distinguished tamoxifen treated patients from controls. Additionally, we identified 54 genes which stratified tamoxifen treated patients into two distinct groups. We identified biological pathways containing SNPs associated with risk for breast cancer, which were dysregulated in response to tamoxifen treatment. Key pathways identified included the apoptosis, P53, NFkB, DNA repair and cell cycle pathways. Combining GWAS with transcription profiling provides a unified approach for associating GWAS findings with response to drug treatment and identification of potential drug targets.

BDNF/TrkB Pathway Mediates the Antidepressant-Like Role of H2S in CUMS-Exposed Rats by Inhibition of Hippocampal ER Stress.

PubMed

Wei, Le; Kan, Li-Yuan; Zeng, Hai-Ying; Tang, Yi-Yun; Huang, Hong-Lin; Xie, Ming; Zou, Wei; Wang, Chun-Yan; Zhang, Ping; Tang, Xiao-Qing

2018-06-01

Our previous works have shown that hydrogen sulfide (H 2 S) significantly attenuates chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors and hippocampal endoplasmic reticulum (ER) stress. Brain-derived neurotrophic factor (BDNF) generates an antidepressant-like effect by its receptor tyrosine protein kinase B (TrkB). We have previously found that H 2 S upregulates the expressions of BDNF and p-TrkB in the hippocampus of CUMS-exposed rats. Therefore, the present work was to explore whether BDNF/TrkB pathway mediates the antidepressant-like role of H 2 S by blocking hippocampal ER stress. We found that treatment with K252a (an inhibitor of BDNF/TrkB pathway) significantly increased the immobility time in the forced swim test and tail suspension test and increased the latency to feed in the novelty-suppressed feeding test in the rats cotreated with sodium hydrosulfide (NaHS, a donor of H 2 S) and CUMS. Similarly, K252a reversed the protective effect of NaHS against CUMS-induced hippocampal ER stress, as evidenced by increases in the levels of ER stress-related proteins, glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12. Taken together, our results suggest that BDNF/TrkB pathway plays an important mediatory role in the antidepressant-like action of H 2 S in CUMS-exposed rats, which is by suppression of hippocampal ER stress. These data provide a novel mechanism underlying the protection of H 2 S against CUMS-induced depressive-like behaviors.

ICI 182,780-Regulated Gene Expression in DU145 Prostate Cancer Cells Is Mediated by Estrogen Receptor-β/NFκB Crosstalk1

PubMed Central

Leung, Yuet-Kin; Gao, Ying; Lau, Kin-Mang; Zhang, Xiang; Ho, Shuk-Mei

2006-01-01

Abstract Estrogen receptor (ER)-β is the predominant ER subtype in prostate cancer (PCa). We previously demonstrated that ICI 182,780 (ICI), but not estrogens, exerted dose-dependent growth inhibition on DU145 PCa cells by an ER-β-mediated pathway. Transcriptional profiling detected a greater than three-fold upregulation of seven genes after a 12-hour exposure to 1 µM ICI. Semi-quantitative reverse transcriptase polymerase chain reaction confirmed the upregulation of four genes by ICI: interleukin-12α chain, interleukin-8, embryonic growth/differentiation factor, and RYK tyrosine kinase. Treatment with an ER-β antisense oligonucleotide reduced cellular ER-β mRNA and induced loss of expression of these genes. Sequence analysis revealed the presence of consensus NFκB sites, but not estrogen-responsive elements, in promoters of all four genes. Reporter assay and chromatin immunoprecipitation experiments demonstrated that ICI-induced gene expression could be mediated by crosstalk between ER-α and the NFκB signaling pathway, denoting a novel mechanism of ER-β-mediated ICI action. Therefore, combined therapies targeting ER-β and NFκB signaling may be synergistic as treatment for PCa. PMID:16756716

MYC2 Orchestrates a Hierarchical Transcriptional Cascade That Regulates Jasmonate-Mediated Plant Immunity in Tomato[OPEN

PubMed Central

Liu, Yuanyuan; Deng, Lei; Wu, Fangming; Huang, Zhuo; Zhou, Ming; Chen, Qian; Zhong, Silin

2017-01-01

The hormone jasmonate (JA), which functions in plant immunity, regulates resistance to pathogen infection and insect attack through triggering genome-wide transcriptional reprogramming in plants. We show that the basic helix-loop-helix transcription factor (TF) MYC2 in tomato (Solanum lycopersicum) acts downstream of the JA receptor to orchestrate JA-mediated activation of both the wounding and pathogen responses. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 655 MYC2-targeted JA-responsive genes. These genes are highly enriched in Gene Ontology categories related to TFs and the early response to JA, indicating that MYC2 functions at a high hierarchical level to regulate JA-mediated gene transcription. We also identified a group of MYC2-targeted TFs (MTFs) that may directly regulate the JA-induced transcription of late defense genes. Our findings suggest that MYC2 and its downstream MTFs form a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output. As proof of concept, we showed that during plant resistance to the necrotrophic pathogen Botrytis cinerea, MYC2 and the MTF JA2-Like form a transcription module that preferentially regulates wounding-responsive genes, whereas MYC2 and the MTF ETHYLENE RESPONSE FACTOR.C3 form a transcription module that preferentially regulates pathogen-responsive genes. PMID:28733419

Humic Acid Increases Amyloid β-Induced Cytotoxicity by Induction of ER Stress in Human SK-N-MC Neuronal Cells

PubMed Central

Li, Hsin-Hua; Lu, Fung-Jou; Hung, Hui-Chih; Liu, Guang-Yaw; Lai, Te-Jen; Lin, Chih-Li

2015-01-01

Humic acid (HA) is a possible etiological factor associated with for several vascular diseases. It is known that vascular risk factors can directly increase the susceptibility to Alzheimer’s disease (AD), which is a neurodegenerative disorder due to accumulation of amyloid β (Aβ) peptide in the brain. However, the role that HA contributes to Aβ-induced cytotoxicity has not been demonstrated. In the present study, we demonstrate that HA exhibits a synergistic effect enhancing Aβ-induced cytotoxicity in cultured human SK-N-MC neuronal cells. Furthermore, this deterioration was mediated through the activation of endoplasmic reticulum (ER) stress by stimulating PERK and eIF2α phosphorylation. We also observed HA and Aβ-induced cytotoxicity is associated with mitochondrial dysfunction caused by down-regulation of the Sirt1/PGC1α pathway, while in contrast, treating the cells with the ER stress inhibitor Salubrinal, or over-expression of Sirt1 significantly reduced loss of cell viability by HA and Aβ. Our findings suggest a new mechanism by which HA can deteriorate Aβ-induced cytotoxicity through modulation of ER stress, which may provide significant insights into the pathogenesis of AD co-occurring with vascular injury. PMID:25961951

Characterization of the influence of mediator complex in HIV-1 transcription.

PubMed

Ruiz, Alba; Pauls, Eduardo; Badia, Roger; Riveira-Muñoz, Eva; Clotet, Bonaventura; Ballana, Ester; Esté, José A

2014-10-03

HIV-1 exploits multiple host proteins during infection. siRNA-based screenings have identified new proteins implicated in different pathways of the viral cycle that participate in a broad range of cellular functions. The human Mediator complex (MED) is composed of 28 elements and represents a fundamental component of the transcription machinery, interacting with the RNA polymerase II enzyme and regulating its ability to express genes. Here, we provide an evaluation of the MED activity on HIV replication. Knockdown of 9 out of 28 human MED proteins significantly impaired viral replication without affecting cell viability, including MED6, MED7, MED11, MED14, MED21, MED26, MED27, MED28, and MED30. Impairment of viral replication by MED subunits was at a post-integration step. Inhibition of early HIV transcripts was observed by siRNA-mediated knockdown of MED6, MED7, MED11, MED14, and MED28, specifically affecting the transcription of the nascent viral mRNA transactivation-responsive element. In addition, MED14 and MED30 were shown to have special relevance during the formation of unspliced viral transcripts (p < 0.0005). Knockdown of the selected MED factors compromised HIV transcription induced by Tat, with the strongest inhibitory effect shown by siMED6 and siMED14 cells. Co-immunoprecipitation experiments suggested physical interaction between MED14 and HIV-1 Tat protein. A better understanding of the mechanisms and factors controlling HIV-1 transcription is key to addressing the development of new strategies required to inhibit HIV replication or reactivate HIV-1 from the latent reservoirs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii.

PubMed

Kong, Hee Jeong; Lee, Ye Ji; Shin, Jihye; Cho, Hyun Kook; Kim, Woo-Jin; Kim, Hyung Soo; Cheong, Jaehun; Sohn, Young Chang; Lee, Sang-Jun; Kim, Bong-Seok

2012-09-01

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart. Copyright © 2012 Elsevier Inc. All rights reserved.

The Mediator subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination.

PubMed

Yao, Xiao; Tang, Zhanyun; Fu, Xing; Yin, Jingwen; Liang, Yan; Li, Chonghui; Li, Huayun; Tian, Qing; Roeder, Robert G; Wang, Gang

2015-12-02

The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. © 2015 The Authors.

INCREASED ENDOCRINE ACTIVITY OF XENOBIOTIC CHEMICALS AS MEDIATED BY METABOLIC ACTIVATION

EPA Science Inventory

This research is part of an effort to develop in vitro assays and QSARs applicable to untested chemicals on EPA inventories through study of estrogen receptor (ER) binding and estrogen mediated gene expression in fish. The current effort investigates metabolic activation of chemi...

Glucose Deprivation Induces ATF4-Mediated Apoptosis through TRAIL Death Receptors

PubMed Central

Iurlaro, Raffaella; Püschel, Franziska; León-Annicchiarico, Clara Lucía; O'Connor, Hazel; Martin, Seamus J.; Palou-Gramón, Daniel; Lucendo, Estefanía

2017-01-01

ABSTRACT Metabolic stress occurs frequently in tumors and in normal tissues undergoing transient ischemia. Nutrient deprivation triggers, among many potential cell death-inducing pathways, an endoplasmic reticulum (ER) stress response with the induction of the integrated stress response transcription factor ATF4. However, how this results in cell death remains unknown. Here we show that glucose deprivation triggered ER stress and induced the unfolded protein response transcription factors ATF4 and CHOP. This was associated with the nontranscriptional accumulation of TRAIL receptor 1 (TRAIL-R1) (DR4) and with the ATF4-mediated, CHOP-independent induction of TRAIL-R2 (DR5), suggesting that cell death in this context may involve death receptor signaling. Consistent with this, the ablation of TRAIL-R1, TRAIL-R2, FADD, Bid, and caspase-8 attenuated cell death, although the downregulation of TRAIL did not, suggesting ligand-independent activation of TRAIL receptors. These data indicate that stress triggered by glucose deprivation promotes the ATF4-dependent upregulation of TRAIL-R2/DR5 and TRAIL receptor-mediated cell death. PMID:28242652

Increased intracellular proteolysis reduces disease severity in an ER stress-associated dwarfism.

PubMed

Mullan, Lorna A; Mularczyk, Ewa J; Kung, Louise H; Forouhan, Mitra; Wragg, Jordan Ma; Goodacre, Royston; Bateman, John F; Swanton, Eileithyia; Briggs, Michael D; Boot-Handford, Raymond P

2017-10-02

The short-limbed dwarfism metaphyseal chondrodysplasia type Schmid (MCDS) is linked to mutations in type X collagen, which increase ER stress by inducing misfolding of the mutant protein and subsequently disrupting hypertrophic chondrocyte differentiation. Here, we show that carbamazepine (CBZ), an autophagy-stimulating drug that is clinically approved for the treatment of seizures and bipolar disease, reduced the ER stress induced by 4 different MCDS-causing mutant forms of collagen X in human cell culture. Depending on the nature of the mutation, CBZ application stimulated proteolysis of misfolded collagen X by either autophagy or proteasomal degradation, thereby reducing intracellular accumulation of mutant collagen. In MCDS mice expressing the Col10a1.pN617K mutation, CBZ reduced the MCDS-associated expansion of the growth plate hypertrophic zone, attenuated enhanced expression of ER stress markers such as Bip and Atf4, increased bone growth, and reduced skeletal dysplasia. CBZ produced these beneficial effects by reducing the MCDS-associated abnormalities in hypertrophic chondrocyte differentiation. Stimulation of intracellular proteolysis using CBZ treatment may therefore be a clinically viable way of treating the ER stress-associated dwarfism MCDS.

Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae.

PubMed

Shahi, Puja; Gulshan, Kailash; Näär, Anders M; Moye-Rowley, W Scott

2010-07-15

The multiprotein transcriptional Mediator complex provides a key link between RNA polymerase II and upstream transcriptional activator proteins. Previous work has established that the multidrug resistance transcription factors Pdr1 and Pdr3 interact with the Mediator component Med15/Gal11 to drive normal levels of expression of the ATP-binding cassette transporter-encoding gene PDR5 in Saccharomyces cerevisiae. PDR5 transcription is induced upon loss of the mitochondrial genome (rho(0) cells) and here we provide evidence that this rho(0) induction is Med15 independent. A search through other known Mediator components determined that Med12/Srb8, a member of the CDK8 Mediator submodule, is required for rho(0) activation of PDR5 transcription. The CDK8 submodule contains the cyclin C homologue (CycC/Srb11), cyclin-dependent kinase Cdk8/Srb10, and the large Med13/Srb9 protein. Loss of these other proteins did not lead to the same block in PDR5 induction. Chromatin immunoprecipitation analyses demonstrated that Med15 is associated with the PDR5 promoter in both rho(+) and rho(0), whereas Med12 recruitment to this target promoter is highly responsive to loss of the mitochondrial genome. Coimmunoprecipitation experiments revealed that association of Pdr3 with Med12 can only be detected in rho(0) cells. These experiments uncover the unique importance of Med12 in activated transcription of PDR5 seen in rho(0) cells.

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

PubMed

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2017-07-12

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

The fibroblast expression of RANKL in CoCrMo-particle-induced osteolysis is mediated by ER stress and XBP1s.

PubMed

Wang, Zhenheng; Huang, Zhen; Gan, Jingjing; Liu, Naicheng; Zhou, Gang; Shi, Tongguo; Wang, Zhenzhen; Wang, Rui; Bao, Nirong; Guo, Ting; Chen, Jiangning; Zhang, Junfeng; Dong, Lei; Zhao, Jianning

2015-09-01

Particle-induced osteolysis is a major cause of aseptic loosening, which is the most common reason for total hip arthroplasty (THA) failure and revision surgery. Although existing studies suggest that synovial fibroblasts present in the interfacial membrane are important targets of wear particles during bone resorption, the interaction mechanisms between the particles and fibroblasts remains elusive. In the present study, we investigated the effect of ER stress induced by CoCrMo particles (CoPs) in fibroblasts, calvarial resorption animal models and aseptic loosening clinical samples and its role in the stimulation of the RANKL expression. Our study further demonstrated that CoPs could induce significant ER stress in fibroblasts. Blocking ER stress with a specific inhibitor dramatically reduced the particle-induced expression of RANKL in vitro and in vivo. Furthermore, in fibroblasts, downregulation of the expression of XBP1s, a signaling molecule of ER stress, significantly reduced the expression of RANKL induced by wear particles. Moreover, inhibition of ER stress or XBP1s both ameliorated the CoPs-induced osteolysis in animal models. Collectively, these results suggested that in particle-induced osteolysis, CoPs could stimulate fibroblasts to secret RANKL through ER stress and the signaling molecule XBP1s. Therefore, downregulating ER stress or the signaling molecule XBP1s of fibroblasts represents a potential therapeutic approach for treating particle-induced peri-implant osteolysis. For the first time, our study demonstrated that ER stress mediated the induction of RANKL expression by CoPs in fibroblasts and promoted particle-induced osteolysis. Furthermore, the upregulation of RANKL by CoPs in fibroblasts was mediated by the ER stress signaling molecule XBP1s. Both blocking ER stress and inhibiting the protein XBP1s by specific inhibitors resulted in downregulation of the expression of RANKL and amelioration of osteolysis induced by the implanted particles

Hormone-mediated growth dynamics of the barley pericarp as revealed by magnetic resonance imaging and transcript profiling

PubMed Central

Pielot, Rainer; Kohl, Stefan; Manz, Bertram; Rutten, Twan; Weier, Diana; Tarkowská, Danuše; Rolčík, Jakub; Strnad, Miroslav; Volke, Frank; Weber, Hans

2015-01-01

The shape of the maternal pericarp affects cereal grain mass and yield. Pericarp growth was analysed by magnetic resonance imaging (MRI), revealing topological maps of mobile water in developing pericarp of barley (Hordeum vulgare) and displaying tissue regions actively elongating in specific temporal–spatial patterns. Correlation analysis of MRI signals and growth rates reveals that growth in length is mediated by dorsal and also lateral rather than ventral regions. Growth in thickness is related to ventral regions. Switching from dorsal to ventral growth is associated with differential expression of axial regulators of the HD-ZipIII and Kanadi/Ettin types, and NPH3 photoreceptors, suggesting light-mediated auxin re-distribution. Auxin increases with the highest levels in the basal pericarp at 6 days after fertilization (DAF), together with transcriptionally up-regulated auxin transport and signalling. Gibberellin biosynthesis is transcriptionally up-regulated only later, and levels of bioactive gibberellins increase from 7 to 13 DAF, with higher levels in ventral than dorsal regions. Differential gene expression related to cell expansion indicates genes related to apoplast acidification, wall relaxation, sugar cleavage, water transport, and cell wall biosynthesis. Candidate genes potentially involved in pericarp extension are distinguished by their temporal expression, representing potential isoforms responsible for dorsal-mediated early growth in length or ventral-mediated late growth in thickness. PMID:26276866

Serine 133 Phosphorylation Is Not Required for Hippocampal CREB-Mediated Transcription and Behavior

ERIC Educational Resources Information Center

Brian, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.; Blendy, Julie A.

2015-01-01

The cAMP response element (CRE)-binding protein, CREB, is a transcription factor whose activity in the brain is critical for long-term memory formation. Phosphorylation of Ser133 in the kinase-inducible domain (KID), that in turn leads to the recruitment of the transcriptional coactivator CREB-binding protein (CBP), is thought to mediate the…

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo

PubMed Central

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2017-01-01

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo. DOI: http://dx.doi.org/10.7554/eLife.28447.001 PMID:28699889

Transcription co-activator SAYP mediates the action of STAT activator

PubMed Central

Panov, Vladislav V.; Kuzmina, Julia L.; Doronin, Semen A.; Kopantseva, Marina R.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Vorobyeva, Nadezhda E.; Shidlovskii, Yulii V.

2012-01-01

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT. PMID:22123744

Transcription co-activator SAYP mediates the action of STAT activator.

PubMed

Panov, Vladislav V; Kuzmina, Julia L; Doronin, Semen A; Kopantseva, Marina R; Nabirochkina, Elena N; Georgieva, Sofia G; Vorobyeva, Nadezhda E; Shidlovskii, Yulii V

2012-03-01

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro.

PubMed

Gu, Wenwen; Dong, Nian; Wang, Peng; Shi, Changgen; Yang, Jun; Wang, Jian

2017-04-01

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER + ) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER + breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER + breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66 - /PR - /HER2 - ) and ER-α36 + /GPER1 + breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER + breast cancer.

Exocrine pancreas ER stress is differentially induced by different fatty acids.

PubMed

Danino, Hila; Ben-Dror, Karin; Birk, Ruth

2015-12-10

Exocrine pancreas acinar cells have a highly developed endoplasmic reticulum (ER), accommodating their high protein production rate. Overload of dietary fat (typical to obesity) is a recognized risk factor in pancreatitis and pancreatic cancer. Dietary fat, especially saturated fat, has been suggested by others and us to induce an acinar lipotoxic effect. The effect of different dietary fatty acids on the ER stress response is unknown. We studied the effect of acute (24h) challenge with different fatty acids (saturated, mono and poly-unsaturated) at different concentrations (between 200 and 500µM, typical to normal and obese states, respectively), testing fat accumulation, ER stress indicators, X-box binding protein 1 (Xbp1) splicing and nuclear translocation, as well as unfolded protein response (UPR) transcripts and protein levels using exocrine pancreas acinar AR42J and primary cells. Acute exposure of AR42J cells to different fatty acids caused increased accumulation of triglycerides, dependent on the type of fat. Different FAs had different effects on ER stress: most notably, saturated palmitic acid significantly affected the UPR response, as demonstrated by altered Xbp1 splicing, elevation in transcript levels of UPR (Xbp, CHOP, Bip) and immune factors (Tnfα, Tgfβ), and enhanced Xbp1 protein levels and Xbp1 time-dependent nuclear translocation. Poly-unsaturated FAs caused milder elevation of ER stress markers, while mono-unsaturated oleic acid attenuated the ER stress response. Thus, various fatty acids differentially affect acinar cell fat accumulation and, apart from oleic acid, induce ER stress. The differential effect of the various fatty acids could have potential nutritional and therapeutic implications. Copyright © 2015 Elsevier Inc. All rights reserved.

Signal transducers and activators of transcription: STATs-mediated mitochondrial neuroprotection.

PubMed

Lin, Hung Wen; Thompson, John W; Morris, Kahlilia C; Perez-Pinzon, Miguel A

2011-05-15

Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

PubMed

Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

2015-11-24

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

Understanding the origin of non-immune cell-mediated weakness in the idiopathic inflammatory myopathies - potential role of ER stress pathways.

PubMed

Lightfoot, Adam P; Nagaraju, Kanneboyina; McArdle, Anne; Cooper, Robert G

2015-11-01

Discussion of endoplasmic reticulum (ER) stress pathway activation in idiopathic inflammatory myopathies (IIM), and downstream mechanisms causative of muscle weakness. In IIM, ER stress is an important pathogenic process, but how it causes muscle dysfunction is unknown. We discuss relevant pathways modified in response to ER stress in IIM: reactive oxygen species (ROS) generation and mitochondrial dysfunction, and muscle cytokine (myokine) generation. First, ER stress pathway activation can induce changes in mitochondrial bioenergetics and ROS production. ROS can oxidize cellular components, causing muscle contractile dysfunction and energy deficits. Novel compounds targeting ROS generation and/or mitochondrial dysfunction can improve muscle function in several myopathologies. Second, recent research has demonstrated that skeletal muscle produces multiple myokines. It is suggested that these play a role in causing muscle weakness. Myokines are capable of immune cell recruitment, thus contributing to perturbed muscle function. A characterization of myokines in IIM would clarify their pathogenic role, and so identify new therapeutic targets. ER stress pathway activation is clearly of etiological relevance in IIM. Research to better understand mechanisms of weakness downstream of ER stress is now required, and which may discover new therapeutic targets for nonimmune cell-mediated weakness.

Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival

PubMed Central

Mori, Tomohisa; Hayashi, Teruo; Hayashi, Eri; Su, Tsung-Ping

2013-01-01

The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus. PMID:24204710

Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival.

PubMed

Mori, Tomohisa; Hayashi, Teruo; Hayashi, Eri; Su, Tsung-Ping

2013-01-01

The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.

hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis

PubMed Central

Park, Sun-Ji; Kim, Tae-Shin; Park, Choon-Keun; Lee, Sang-Hee; Kim, Jin-Man; Lee, Kyu-Sun; Lee, In-kyu; Park, Jeen-Woo; Lawson, Mark A; Lee, Dong-Seok

2014-01-01

Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells. PMID:23256993

Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor

PubMed Central

Omi, T; Tanimukai, H; Kanayama, D; Sakagami, Y; Tagami, S; Okochi, M; Morihara, T; Sato, M; Yanagida, K; Kitasyoji, A; Hara, H; Imaizumi, K; Maurice, T; Chevallier, N; Marchal, S; Takeda, M; Kudo, T

2014-01-01

We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell's responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress. PMID:25032855

WRKY23 is a component of the transcriptional network mediating auxin feedback on PIN polarity

PubMed Central

Vasileva, Mina; Sauer, Michael

2018-01-01

Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17- and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain- and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development. PMID:29377885

Bushen Zhuangjin decoction inhibits TM-induced chondrocyte apoptosis mediated by endoplasmic reticulum stress.

PubMed

Lin, Pingdong; Weng, Xiaping; Liu, Fayuan; Ma, Yuhuan; Chen, Houhuang; Shao, Xiang; Zheng, Wenwei; Liu, Xianxiang; Ye, Hongzhi; Li, Xihai

2015-12-01

Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type Ⅱ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)‑stimulated chondrocytes was detected using 4-phenylbutyric acid (4‑PBA). We found that 4‑PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM‑induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X‑box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP‑homologous protein (Chop), caspase‑9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase

Effects of environmental estrogenic chemicals on AP1 mediated transcription with estrogen receptors alpha and beta.

PubMed

Fujimoto, Nariaki; Honda, Hiroaki; Kitamura, Shigeyuki

2004-01-01

There has been much discussion concerning endocrine disrupting chemicals suspected of exerting adverse effects in both wildlife and humans. Since the majority of these compounds are estrogenic, a large number of in vitro tests for estrogenic characteristics have been developed for screening purpose. One reliable and widely used method is the reporter gene assay employing estrogen receptors (ERs) and a reporter gene with a cis-acting estrogen responsive element (ERE). Other elements such as AP1 also mediate estrogenic signals and the manner of response could be quite different from that of ERE. Since this has yet to be explored, the ER mediated AP1 activity in response to a series of environmental estrogens was investigated in comparison with ERE findings. All the compounds exhibited estrogenic properties with ERE-luc and their AP1 responses were quite similar. These was one exception, however, p,p'-DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane) did not exert any AP1-luc activity, while it appeared to be estrogenic at 10(-7) to 10(-5)M with the ERE action. None of the compounds demonstrated ER beta:AP1 activity. These data suggest that significant differences can occur in responses through the two estrogen pathways depending on environmental chemicals.

Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neff, Michael M.

This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

PubMed

Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

2017-08-31

Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator

PubMed Central

Grants, Jennifer M.; Goh, Grace Y. S.; Taubert, Stefan

2015-01-01

The Mediator multiprotein complex (‘Mediator’) is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. PMID:25634893

Signal Transducers and Activators of Transcription: STATs-Mediated Mitochondrial Neuroprotection

PubMed Central

Lin, Hung Wen; Thompson, John W.; Morris, Kahlilia C.

2011-01-01

Abstract Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia. Antioxid. Redox Signal. 14, 1853–1861. PMID:20712401

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

PubMed Central

Cabral, Wayne A.; Ishikawa, Masaki; Garten, Matthias; Makareeva, Elena N.; Sargent, Brandi M.; Weis, MaryAnn; Barnes, Aileen M.; Webb, Emma A.; Shaw, Nicholas J.; Ala-Kokko, Leena; Lacbawan, Felicitas L.; Högler, Wolfgang; Leikin, Sergey; Blank, Paul S.; Zimmerberg, Joshua; Eyre, David R.; Yamada, Yoshihiko; Marini, Joan C.

2016-01-01

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes. PMID:27441836

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

PubMed

Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

2015-02-27

The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/β-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

PubMed

Wang, Dongmao; Mohammad, Mardhiah; Wang, Yanyan; Tan, Rachel; Murray, Lydia S; Ricardo, Sharon; Dagher, Hayat; van Agtmael, Tom; Savige, Judy

2017-07-01

X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL 2 ) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 ( P  = 0.01 and P  = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels ( P  < 0.01), and reduced ER size ( P  < 0.01 by EM and P  < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P  < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only ( P  = 0.06). Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

Untangling the web: Mechanisms underlying ER network formation

PubMed Central

Goyal, Uma; Blackstone, Craig

2013-01-01

The ER is a continuous membrane system consisting of the nuclear envelope, flat sheets often studded with ribosomes, and a polygonal network of highly-curved tubules extending throughout the cell. Although protein and lipid biosynthesis, protein modification, vesicular transport, Ca2+dynamics, and protein quality control have been investigated in great detail, mechanisms that generate the distinctive architecture of the ER have been uncovered only recently. Several protein families including the reticulons and REEPs/DP1/Yop1p harbor hydrophobic hairpin domains that shape high-curvature ER tubules and mediate intramembrane protein interactions. Members of the atlastin/RHD3/Sey1p family of dynamin-related GTPases interact with the ER-shaping proteins and mediate the formation of three-way junctions responsible for the polygonal structure of the tubular ER network, with Lunapark proteins acting antagonistically. Additional classes of tubular ER proteins including some REEPs and the M1 spastin ATPase interact with the microtubule cytoskeleton. Flat ER sheets possess a different complement of proteins such as p180, CLIMP-63 and kinectin implicated in shaping, cisternal stacking and cytoskeletal interactions. The ER is also in constant motion, and numerous signaling pathways as well as interactions among cytoskeletal elements, the plasma membrane, and organelles cooperate to position and shape the ER dynamically. Finally, many proteins involved in shaping the ER network are mutated in the most common forms of hereditary spastic paraplegia, indicating a particular importance for proper ER morphology and distribution in large, highly-polarized cells such as neurons. PMID:23602970

The interaction between ER and NFκB in resistance to endocrine therapy

PubMed Central

2012-01-01

Endocrine therapy is a commonly used treatment for estrogen receptor (ER)-positive breast cancer. Although endocrine therapy has a favorable outcome in many patients, development of resistance is common. Recent studies have shown that NFκB, a transcription factor regulating a wide variety of cellular processes, might play a role in the development of endocrine resistance. The precise interaction between ER and NFκB and how this contributes to the attenuated responsiveness of ER-positive breast cancer cells to hormonal treatment remains unclear. This review provides an overview of the mechanisms of action for both transcription factors and focuses on the current knowledge explaining how ER and NFκB affect each other's activity and how this cross-talk might contribute to the development of an endocrine resistance phenotype in breast cancer cells. PMID:22963717

Cardiac hypertrophy induced by active Raf depends on Yorkie-mediated transcription

PubMed Central

Yu, Lin; Daniels, Joseph P.; Wu, Huihui; Wolf, Matthew J.

2015-01-01

Organ hypertrophy can result from enlargement of individual cells or from cell proliferation or both. Activating mutations in the serine-threonine kinase Raf cause cardiac hypertrophy and contribute to Noonan syndrome in humans. Cardiac-specific expression of activated Raf also causes hypertrophy in Drosophila melanogaster. We found that Yorkie (Yki), a transcriptional coactivator in the Hippo pathway that regulates organ size, is required for Raf-induced cardiac hypertrophy in flies. Although aberrant activation of Yki orthologs stimulates cardiac hyperplasia in mice, cardiac-specific expression of an activated mutant form of Yki in fruit flies caused cardiac hypertrophy without hyperplasia. Knockdown of Yki caused cardiac dilation without loss of cardiomyocytes and prevented Raf-induced cardiac hypertrophy. In flies, Yki-induced cardiac hypertrophy required the TEA domain–containing transcription factor Scalloped, and, in mammalian cells, expression of mouse RafL613V, an activated form of Raf with a Noonan syndrome mutation, increased Yki-induced Scalloped activity. Furthermore, overexpression of Tgi (a Tondu domain–containing Scalloped-binding corepressor) in the fly heart abrogated Yki- or Raf-induced cardiac hypertrophy. Thus, crosstalk between Raf and Yki occurs in the heart and can influence Raf-mediated cardiac hypertrophy. PMID:25650441

Reduced α-MSH Underlies Hypothalamic ER-Stress-Induced Hepatic Gluconeogenesis.

PubMed

Schneeberger, Marc; Gómez-Valadés, Alicia G; Altirriba, Jordi; Sebastián, David; Ramírez, Sara; Garcia, Ainhoa; Esteban, Yaiza; Drougard, Anne; Ferrés-Coy, Albert; Bortolozzi, Analía; Garcia-Roves, Pablo M; Jones, John G; Manadas, Bruno; Zorzano, Antonio; Gomis, Ramon; Claret, Marc

2015-07-21

Alterations in ER homeostasis have been implicated in the pathophysiology of obesity and type-2 diabetes (T2D). Acute ER stress induction in the hypothalamus produces glucose metabolism perturbations. However, the neurobiological basis linking hypothalamic ER stress with abnormal glucose metabolism remains unknown. Here, we report that genetic and induced models of hypothalamic ER stress are associated with alterations in systemic glucose homeostasis due to increased gluconeogenesis (GNG) independent of body weight changes. Defective alpha melanocyte-stimulating hormone (α-MSH) production underlies this metabolic phenotype, as pharmacological strategies aimed at rescuing hypothalamic α-MSH content reversed this phenotype at metabolic and molecular level. Collectively, our results posit defective α-MSH processing as a fundamental mediator of enhanced GNG in the context of hypothalamic ER stress and establish α-MSH deficiency in proopiomelanocortin (POMC) neurons as a potential contributor to the pathophysiology of T2D. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

HIV-1 Vpr Enhances PPARβ/δ-Mediated Transcription, Increases PDK4 Expression, and Reduces PDC Activity

PubMed Central

Shrivastav, Shashi; Zhang, Liyan; Okamoto, Koji; Lee, Hewang; Lagranha, Claudia; Abe, Yoshifusa; Balasubramanyam, Ashok; Lopaschuk, Gary D.; Kino, Tomoshige

2013-01-01

HIV infection and its therapy are associated with disorders of lipid metabolism and bioenergetics. Previous work has suggested that viral protein R (Vpr) may contribute to the development of lipodystrophy and insulin resistance observed in HIV-1–infected patients. In adipocytes, Vpr suppresses mRNA expression of peroxisomal proliferator-activating receptor-γ (PPARγ)-responsive genes and inhibits differentiation. We investigated whether Vpr might interact with PPARβ/δ and influence its transcriptional activity. In the presence of PPARβ/δ, Vpr induced a 3.3-fold increase in PPAR response element-driven transcriptional activity, a 1.9-fold increase in pyruvate dehydrogenase kinase 4 (PDK4) protein expression, and a 1.6-fold increase in the phosphorylated pyruvate dehydrogenase subunit E1α leading to a 47% decrease in the activity of the pyruvate dehydrogenase complex in HepG2 cells. PPARβ/δ knockdown attenuated Vpr-induced enhancement of endogenous PPARβ/δ-responsive PDK4 mRNA expression. Vpr induced a 1.3-fold increase in mRNA expression of both carnitine palmitoyltransferase I (CPT1) and acetyl-coenzyme A acyltransferase 2 (ACAA2) and doubled the activity of β-hydroxylacyl coenzyme A dehydrogenase (HADH). Vpr physically interacted with the ligand-binding domain of PPARβ/δ in vitro and in vivo. Consistent with a role in energy expenditure, Vpr increased state-3 respiration in isolated mitochondria (1.16-fold) and basal oxygen consumption rate in intact HepG2 cells (1.2-fold) in an etomoxir-sensitive manner, indicating that the oxygen consumption rate increase is β-oxidation–dependent. The effects of Vpr on PPAR response element activation, pyruvate dehydrogenase complex activity, and β-oxidation were reversed by specific PPARβ/δ antagonists. These results support the hypothesis that Vpr contributes to impaired energy metabolism and increased energy expenditure in HIV patients. PMID:23842279

Naltrexone ER/Bupropion ER: A Review in Obesity Management.

PubMed

Greig, Sarah L; Keating, Gillian M

2015-07-01

Oral naltrexone extended-release/bupropion extended-release (naltrexone ER/bupropion ER; Contrave(®), Mysimba(™)) is available as an adjunct to a reduced-calorie diet and increased physical activity in adults with an initial body mass index (BMI) of ≥ 30 kg/m(2) (i.e. obese) or a BMI of ≥ 27 kg/m(2) (i.e. overweight) in the presence of at least one bodyweight-related comorbidity, such as type 2 diabetes mellitus, hypertension or dyslipidaemia. In 56-week phase III trials in these patient populations, oral naltrexone ER/bupropion ER 32/360 mg/day was significantly more effective than placebo with regard to percentage bodyweight reductions from baseline and the proportion of patients who achieved bodyweight reductions of ≥ 5 and ≥ 10%. Significantly greater improvements in several cardiometabolic risk factors were also observed with naltrexone ER/bupropion ER versus placebo, as well as greater improvements in glycated haemoglobin levels in obese or overweight adults with type 2 diabetes. Naltrexone ER/bupropion ER was generally well tolerated in phase III trials, with nausea being the most common adverse event. Thus, naltrexone ER/bupropion ER 32/360 mg/day as an adjunct to a reduced-calorie diet and increased physical activity, is an effective and well tolerated option for chronic bodyweight management in obese adults or overweight adults with at least one bodyweight-related comorbidity.

Molecular mode of action of NKP-1339 - a clinically investigated ruthenium-based drug - involves ER- and ROS-related effects in colon carcinoma cell lines.

PubMed

Flocke, Lea S; Trondl, Robert; Jakupec, Michael A; Keppler, Bernhard K

2016-06-01

Sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (NKP-1339) is a clinically investigated ruthenium-based metal complex, which shows promising results in solid tumors, such as non-small cell lung cancer, colorectal carcinoma, and most distinctively in gastrointestinal neuroendocrine tumors. In previous studies, fast binding to albumin as well as transferrin could be shown. The enhanced permeability and retention (EPR) effect, which is diversely being exploited for tumor targeting, could therefore be applicable for NKP-1339. Here we studied the serum dependence of its biological activity in various methods, influencing its cellular accumulation, cytotoxicity as well as the generation of reactive oxygen species (ROS). ROS lead to Nrf2 activation, which is known to activate antioxidant response gene transcription. GRP78 down-regulation on the protein level suggests ER associated protein degradation (ERAD) as a mode of action, as RNA levels are only mildly affected. Another important part for the mode of action is endoplasmic reticulum (ER) stress, as different factors are highly upregulated on the protein level. For example PERK, a transmembrane receptor which is released by GRP78 when the ER is disturbed, is upregulated and phosphorylated. EIF2α is phosphorylated, which leads to an inhibition of CAP-dependent translation and other stress responses. The transcription factor CHOP (DDIT3), which promotes ER stress dependent apoptosis, is time and concentration dependently upregulated. Finally cytotoxicity tests could prove that inhibition of ER stress and ER stress-mediated apoptosis leads to decreased cytotoxic effects of NKP-1339, which highlights the involvement of this mechanism in the mode of action.

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

NASA Astrophysics Data System (ADS)

House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

2015-06-01

Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

PubMed Central

Zhang, Monica; Song, Lingyun; Lee, Bum-Kyu; Iyer, Vishwanath R.; Furey, Terrence S.; Crawford, Gregory E.; Yan, Hai; He, Yiping

2014-01-01

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes. PMID:25198066

WWOX sensitises ovarian cancer cells to paclitaxel via modulation of the ER stress response.

PubMed

Janczar, Szymon; Nautiyal, Jaya; Xiao, Yi; Curry, Edward; Sun, Mingjun; Zanini, Elisa; Paige, Adam Jw; Gabra, Hani

2017-07-27

There are clear gaps in our understanding of genes and pathways through which cancer cells facilitate survival strategies as they become chemoresistant. Paclitaxel is used in the treatment of many cancers, but development of drug resistance is common. Along with being an antimitotic agent paclitaxel also activates endoplasmic reticulum (ER) stress. Here, we examine the role of WWOX (WW domain containing oxidoreductase), a gene frequently lost in several cancers, in mediating paclitaxel response. We examine the ER stress-mediated apoptotic response to paclitaxel in WWOX-transfected epithelial ovarian cancer (EOC) cells and following siRNA knockdown of WWOX. We show that WWOX-induced apoptosis following exposure of EOC cells to paclitaxel is related to ER stress and independent of the antimitotic action of taxanes. The apoptotic response to ER stress induced by WWOX re-expression could be reversed by WWOX siRNA in EOC cells. We report that paclitaxel treatment activates both the IRE-1 and PERK kinases and that the increase in paclitaxel-mediated cell death through WWOX is dependent on active ER stress pathway. Log-rank analysis of overall survival (OS) and progression-free survival (PFS) in two prominent EOC microarray data sets (Tothill and The Cancer Genome Atlas), encompassing ~800 patients in total, confirmed clinical relevance to our findings. High WWOX mRNA expression predicted longer OS and PFS in patients treated with paclitaxel, but not in patients who were treated with only cisplatin. The association of WWOX and survival was dependent on the expression level of glucose-related protein 78 (GRP78), a key ER stress marker in paclitaxel-treated patients. We conclude that WWOX sensitises EOC to paclitaxel via ER stress-induced apoptosis, and predicts clinical outcome in patients. Thus, ER stress response mechanisms could be targeted to overcome chemoresistance in cancer.

c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

PubMed Central

Valentín-Acevedo, Aníbal; Sinquett, Frank L.; Covey, Lori R.

2011-01-01

LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death. PMID:21984918

Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj

2010-08-01

We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase weremore » enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.« less

Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane.

PubMed

Römisch, K

1999-12-01

Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.

Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization.

PubMed

Paterno, Gary D; Ding, Zhihu; Lew, Yuan-Y; Nash, Gord W; Mercer, F Corinne; Gillespie, Laura L

2002-07-24

mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1alpha and hMI-ER1beta. In all tissues except testis, transcripts encoding the beta isoform were predominant. hMI-ER1alpha lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1beta, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.

6-shogaol, a major compound in ginger, induces aryl hydrocarbon receptor-mediated transcriptional activity and gene expression.

PubMed

Yoshida, Kazutaka; Satsu, Hideo; Mikubo, Ayano; Ogiwara, Haru; Yakabe, Takafumi; Inakuma, Takahiro; Shimizu, Makoto

2014-06-18

Xenobiotics are usually detoxified by drug-metabolizing enzymes and excreted from the body. The expression of many of drug-metabolizing enzymes is regulated by the aryl hydrocarbon receptor (AHR). Some substances in vegetables have the potential to be AHR ligands. To search for vegetable components that exhibit AHR-mediated transcriptional activity, we assessed the activity of vegetable extracts and identified the active compounds using the previously established stable AHR-responsive HepG2 cell line. Among the hot water extracts of vegetables, the highest activity was found in ginger. The ethyl acetate fraction of the ginger hot water extract remarkably induced AHR-mediated transcriptional activity, and the major active compound was found to be 6-shogaol. Subsequently, the mRNA levels of AHR-targeting drug-metabolizing enzymes (CYP1A1, UGT1A1, and ABCG 2) and the protein level of CYP1A1 in HepG2 cells were shown to be increased by 6-shogaol. This is the first report that 6-shogaol can regulate the expression of detoxification enzymes by AHR activation.

Chronic Sleep Fragmentation During the Sleep Period Induces Hypothalamic Endoplasmic Reticulum Stress and PTP1b-Mediated Leptin Resistance in Male Mice

PubMed Central

Hakim, Fahed; Wang, Yang; Carreras, Alba; Hirotsu, Camila; Zhang, Jing; Peris, Eduard; Gozal, David

2015-01-01

Background: Sleep fragmentation (SF) is highly prevalent and may constitute an important contributing factor to excessive weight gain and the metabolic syndrome. Increased endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) leading to the attenuation of leptin receptor signaling in the hypothalamus leads to obesity and metabolic dysfunction. Methods: Mice were exposed to SF and sleep control (SC) for varying periods of time during which ingestive behaviors were monitored. UPR pathways and leptin receptor signaling were assessed in hypothalami. To further examine the mechanistic role of ER stress, changes in leptin receptor (ObR) signaling were also examined in wild-type mice treated with the ER chaperone tauroursodeoxycholic acid (TUDCA), as well as in CHOP −/+ transgenic mice. Results: Fragmented sleep in male mice induced increased food intake starting day 3 and thereafter, which was preceded by increases in ER stress and activation of all three UPR pathways in the hypothalamus. Although ObR expression was unchanged, signal transducer and activator of transcription 3 (STAT3) phosphorylation was decreased, suggesting reduced ObR signaling. Unchanged suppressor of cytokine signaling-3 (SOCS3) expression and increases in protein-tyrosine phosphatase 1B (PTP1B) expression and activity emerged with SF, along with reduced p-STAT3 responses to exogenous leptin. SF-induced effects were reversed following TUDCA treatment and were absent in CHOP −/+ mice. Conclusions: Sleep fragmentation (SF) induces hyperphagic behaviors and reduced leptin signaling in hypothalamus that are mediated by activation of endoplasmic reticulum (ER) stress, and ultimately lead to increased PTP1B activity. ER stress pathways are therefore potentially implicated in SF-induced weight gain and metabolic dysfunction, and may represent a viable therapeutic target. Citation: Hakim F, Wang Y, Carreras A, Hirotsu C, Zhang J, Peris E, Gozal D. Chronic sleep

Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics

PubMed Central

Jaipargas, Erica-Ashley; Barton, Kiah A.; Mathur, Neeta; Mathur, Jaideep

2015-01-01

Mitochondria are pleomorphic, double membrane-bound organelles involved in cellular energetics in all eukaryotes. Mitochondria in animal and yeast cells are typically tubular-reticulate structures and several micro-meters long but in green plants they are predominantly observed as 0.2–1.5 μm punctae. While fission and fusion, through the coordinated activity of several conserved proteins, shapes mitochondria, the endoplasmic reticulum (ER) has recently been identified as an additional player in this process in yeast and mammalian cells. The mitochondria-ER relationship in plant cells remains largely uncharacterized. Here, through live-imaging of the entire range of mitochondria pleomorphy we uncover the underlying basis for the predominantly punctate mitochondrial form in plants. We demonstrate that mitochondrial morphology changes in response to light and cytosolic sugar levels in an ER mediated manner. Whereas, large ER polygons and low dynamics under dark conditions favor mitochondrial fusion and elongation, small ER polygons result in increased fission and predominantly small mitochondria. Hypoxia also reduces ER dynamics and increases mitochondrial fusion to produce giant mitochondria. By observing elongated mitochondria in normal plants and fission-impaired Arabidopsis nmt1-2 and drp3a mutants we also establish that thin extensions called matrixules and a beads-on-a-string mitochondrial phenotype are direct consequences of mitochondria-ER interactions. PMID:26442089

Pancreatic and Duodenal Homeobox Protein 1 (Pdx-1) Maintains Endoplasmic Reticulum Calcium Levels through Transcriptional Regulation of Sarco-endoplasmic Reticulum Calcium ATPase 2b (SERCA2b) in the Islet β Cell*

PubMed Central

Johnson, Justin S.; Kono, Tatsuyoshi; Tong, Xin; Yamamoto, Wataru R.; Zarain-Herzberg, Angel; Merrins, Matthew J.; Satin, Leslie S.; Gilon, Patrick; Evans-Molina, Carmella

2014-01-01

Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/− mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca2+ regulation in Pdx-1-deficient states. PMID:25271154

β-Cell Dysfunction Due to Increased ER Stress in a Stem Cell Model of Wolfram Syndrome

PubMed Central

Shang, Linshan; Hua, Haiqing; Foo, Kylie; Martinez, Hector; Watanabe, Kazuhisa; Zimmer, Matthew; Kahler, David J.; Freeby, Matthew; Chung, Wendy; LeDuc, Charles; Goland, Robin; Leibel, Rudolph L.; Egli, Dieter

2014-01-01

Wolfram syndrome is an autosomal recessive disorder caused by mutations in WFS1 and is characterized by insulin-dependent diabetes mellitus, optic atrophy, and deafness. To investigate the cause of β-cell failure, we used induced pluripotent stem cells to create insulin-producing cells from individuals with Wolfram syndrome. WFS1-deficient β-cells showed increased levels of endoplasmic reticulum (ER) stress molecules and decreased insulin content. Upon exposure to experimental ER stress, Wolfram β-cells showed impaired insulin processing and failed to increase insulin secretion in response to glucose and other secretagogues. Importantly, 4-phenyl butyric acid, a chemical protein folding and trafficking chaperone, restored normal insulin synthesis and the ability to upregulate insulin secretion. These studies show that ER stress plays a central role in β-cell failure in Wolfram syndrome and indicate that chemical chaperones might have therapeutic relevance under conditions of ER stress in Wolfram syndrome and other forms of diabetes. PMID:24227685

The transcription factor DREAM represses A20 and mediates inflammation

PubMed Central

Tiruppathi, Chinnaswamy; Soni, Dheeraj; Wang, Dong-Mei; Xue, Jiaping; Singh, Vandana; Thippegowda, Prabhakar B.; Cheppudira, Bopaiah P.; Mishra, Rakesh K.; DebRoy, Auditi; Qian, Zhijian; Bachmaier, Kurt; Zhao, Youyang; Christman, John W.; Vogel, Stephen M.; Ma, Averil; Malik, Asrar B.

2014-01-01

Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/−) mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inflammatory stimuli. These studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy in diseases such as acute lung injury associated with unconstrained NF-κB activity. PMID:24487321

Curcumin abates hypoxia-induced oxidative stress based-ER stress-mediated cell death in mouse hippocampal cells (HT22) by controlling Prdx6 and NF-κB regulation

PubMed Central

Chhunchha, Bhavana; Fatma, Nigar; Kubo, Eri; Rai, Prerana; Singh, Sanjay P.

2013-01-01

Oxidative stress and endoplasmic reticulum (ER) stress are emerging as crucial events in the etiopathology of many neurodegenerative diseases. While the neuroprotective contributions of the dietary compound curcumin has been recognized, the molecular mechanisms underlying curcumin's neuroprotection under oxidative and ER stresses remains elusive. Herein, we show that curcumin protects HT22 from oxidative and ER stresses evoked by the hypoxia (1% O2 or CoCl2 treatment) by enhancing peroxiredoxin 6 (Prdx6) expression. Cells exposed to CoCl2 displayed reduced expression of Prdx6 with higher reactive oxygen species (ROS) expression and activation of NF-κB with IκB phosphorylation. When NF-κB activity was blocked by using SN50, an inhibitor of NF-κB, or cells treated with curcumin, the repression of Prdx6 expression was restored, suggesting the involvement of NF-κB in modulating Prdx6 expression. These cells were enriched with an accumulation of ER stress proteins, C/EBP homologous protein (CHOP), GRP/78, and calreticulin, and had activated states of caspases 12, 9, and 3. Reinforced expression of Prdx6 in HT22 cells by curcumin reestablished survival signaling by reducing propagation of ROS and blunting ER stress signaling. Intriguingly, knockdown of Prdx6 by antisense revealed that loss of Prdx6 contributed to cell death by sustaining enhanced levels of ER stress-responsive proapoptotic proteins, which was due to elevated ROS production, suggesting that Prdx6 deficiency is a cause of initiation of ROS-mediated ER stress-induced apoptosis. We propose that using curcumin to reinforce the naturally occurring Prdx6 expression and attenuate ROS-based ER stress and NF-κB-mediated aberrant signaling improves cell survival and may provide an avenue to treat and/or postpone diseases associated with ROS or ER stress. PMID:23364261

Diesel Exhaust Particulate Extracts Inhibit Transcription of Nuclear Respiratory Factor-1 and Cell Viability in Human Umbilical Vein Endothelial Cells

PubMed Central

Mattingly, Kathleen A.; Klinge, Carolyn M.

2011-01-01

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1 regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription and this suppression was not ablated by concomitant treatment with E2, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E2 increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. PMID:22105178

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

PubMed Central

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

2016-01-01

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

The Smad3 linker region contains a transcriptional activation domain

PubMed Central

2004-01-01

Transforming growth factor-β (TGF-β)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-β/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-β transcriptional activation responses, although it can be phosphorylated by the TGF-β receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-β. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control. PMID:15588252

The Smad3 linker region contains a transcriptional activation domain.

PubMed

Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

2005-02-15

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

PubMed Central

Posternak, Valeriya; Ung, Matthew H.; Cheng, Chao; Cole, Michael D.

2016-01-01

MYC is a pleiotropic transcription factor that activates and represses a wide range of target genes and is frequently deregulated in human tumors. While much is known about the role of MYC in transcriptional activation and repression, MYC can also regulate mRNA cap methylation through a mechanism that has remained poorly understood. Here it is reported that MYC enhances mRNA cap methylation of transcripts globally, specifically increasing mRNA cap methylation of genes involved in Wnt/β-catenin signaling. Elevated mRNA cap methylation of Wnt signaling transcripts in response to MYC leads to augmented translational capacity, elevated protein levels, and enhanced Wnt signaling activity. Mechanistic evidence indicates that MYC promotes recruitment of RNA methyltransferase (RNMT) to Wnt signaling gene promoters by enhancing phosphorylation of serine 5 on the RNA Polymerase II Carboxy-Terminal Domain, mediated in part through an interaction between the TIP60 acetyltransferase complex and TFIIH. Implications MYC enhances mRNA cap methylation above and beyond transcriptional induction. PMID:27899423

Anti-Fibrotic Effect of Losartan, an Angiotensin II Receptor Blocker, Is Mediated through Inhibition of ER Stress via Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin

PubMed Central

Kim, Hyosang; Baek, Chung Hee; Lee, Raymond Bok; Chang, Jai Won; Yang, Won Seok; Lee, Sang Koo

2017-01-01

Endoplasmic reticulum (ER) stress is increasingly identified as modulator of fibrosis. Losartan, an angiotensin II receptor blocker, has been widely used as the first choice of treatment in chronic renal diseases. We postulated that anti-fibrotic effect of losartan is mediated through inhibition of ER stress via SIRT1 (silent mating type information regulation 2 homolog 1) hemeoxygenase-1 (HO-1)/thioredoxin pathway. Renal tubular cells, tunicamycin (TM)-induced ER stress, and unilateral ureteral obstruction (UUO) mouse model were used. Expression of ER stress was assessed by Western blot analysis and immunohistochemical stain. ER stress was induced by chemical ER stress inducer, tunicamycin, and non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin. Losartan suppressed the TM-induced ER stress, as shown by inhibition of TM-induced expression of GRP78 (glucose related protein 78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α), through up-regulation of SIRT1 via HO-1 and thioredoxin. Losartan also suppressed the ER stress by non-chemical inducers. In both animal models, losartan reduced the tubular expression of GRP78, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor). Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis. These findings provide new insights into renoprotective effects of losartan and suggest that SIRT1, HO-1, and thioredoxin may be potential pharmacological targets in kidney diseases under excessive ER stress condition. PMID:28146117

Anti-Fibrotic Effect of Losartan, an Angiotensin II Receptor Blocker, Is Mediated through Inhibition of ER Stress via Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin.

PubMed

Kim, Hyosang; Baek, Chung Hee; Lee, Raymond Bok; Chang, Jai Won; Yang, Won Seok; Lee, Sang Koo

2017-01-31

Endoplasmic reticulum (ER) stress is increasingly identified as modulator of fibrosis. Losartan, an angiotensin II receptor blocker, has been widely used as the first choice of treatment in chronic renal diseases. We postulated that anti-fibrotic effect of losartan is mediated through inhibition of ER stress via SIRT1 (silent mating type information regulation 2 homolog 1) hemeoxygenase-1 (HO-1)/thioredoxin pathway. Renal tubular cells, tunicamycin (TM)-induced ER stress, and unilateral ureteral obstruction (UUO) mouse model were used. Expression of ER stress was assessed by Western blot analysis and immunohistochemical stain. ER stress was induced by chemical ER stress inducer, tunicamycin, and non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin. Losartan suppressed the TM-induced ER stress, as shown by inhibition of TM-induced expression of GRP78 (glucose related protein 78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α), through up-regulation of SIRT1 via HO-1 and thioredoxin. Losartan also suppressed the ER stress by non-chemical inducers. In both animal models, losartan reduced the tubular expression of GRP78, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor). Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis. These findings provide new insights into renoprotective effects of losartan and suggest that SIRT1, HO-1, and thioredoxin may be potential pharmacological targets in kidney diseases under excessive ER stress condition.

ER stress-mediated cell damage contributes to the release of EDA+ fibronectin from hepatocytes in nonalcoholic fatty liver disease.

PubMed

He, Lei; Yuan, Fa-Hu; Chen, Ting; Huang, Qiang; Wang, Yu; Liu, Zhi-Guo

2017-04-01

Fibronectin containing extra domain A (EDA + FN), a functional glycoprotein participating in several cellular processes, correlates with chronic liver disease. Herein, we aim to investigate the expression and secretion of EDA + FN from hepatocytes in nonalcoholic fatty liver disease (NAFLD) and the underlying mechanisms. Circulating levels of EDA + FN were determined by ELISA in clinical samples. Western blotting and flow cytometry were performed on L02 and HepG2 cell lines to analyze whether the levels of EDA + FN were associated with endoplasmic reticulum (ER) stress-related cell death. Circulating levels of EDA + FN in NAFLD patients were significantly higher than those in control subjects, and positively related with severity of ultrasonographic steatosis score. In cultured hepatocytes, palmitate up-regulated the expression of EDA + FN in a dose-dependent manner. Conversely, when the cells were pretreated with 4-phenylbutyrate, a specific inhibitor of ER stress, up-regulation of EDA + FN could be abrogated. Moreover, silencing CHOP by shRNA enhanced the release of EDA + FN from hepatocytes following palmitate treatment, which was involved in ER stress-related cell damage. These findings suggest that the up-regulated level of EDA + FN is associated with liver damage in NAFLD, and ER stress-mediated cell damage contributes to the release of EDA + FN from hepatocytes.

Ethylene and pollination decrease transcript abundance of an ethylene receptor gene in Dendrobium petals.

PubMed

Thongkum, Monthathip; Burns, Parichart; Bhunchoth, Anjana; Warin, Nuchnard; Chatchawankanphanich, Orawan; van Doorn, Wouter G

2015-03-15

We studied the expression of a gene encoding an ethylene receptor, called Ethylene Response Sensor 1 (Den-ERS1), in the petals of Dendrobium orchid flowers. Transcripts accumulated during the young floral bud stage and declined by the time the flowers had been open for several days. Pollination or exposure to exogenous ethylene resulted in earlier flower senescence, an increase in ethylene production and a lower Den-ERS1 transcript abundance. Treatment with 1-methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, decreased ethylene production and resulted in high transcript abundance. The literature indicates two kinds of ethylene receptor genes with regard to the effects of ethylene. One group shows ethylene-induced down-regulated transcription, while the other has ethylene-induced up-regulation. The present gene is an example of the first group. The 5' flanking region showed binding sites for Myb and myb-like, homeodomain, MADS domain, NAC, TCP, bHLH and EIN3-like transcription factors. The binding site for the EIN3-like factor might explain the ethylene effect on transcription. A few other transcription factors (RAV1 and NAC) seem also related to ethylene effects. Copyright © 2015 Elsevier GmbH. All rights reserved.

Transcriptional Regulation of CYP2D6 Expression

PubMed Central

Pan, Xian; Ning, Miaoran

2017-01-01

CYP2D6-mediated drug metabolism exhibits large interindividual variability. Although genetic variations in the CYP2D6 gene are well known contributors to the variability, the sources of CYP2D6 variability in individuals of the same genotype remain unexplained. Accumulating data indicate that transcriptional regulation of CYP2D6 may account for part of CYP2D6 variability. Yet, our understanding of factors governing transcriptional regulation of CYP2D6 is limited. Recently, mechanistic studies of increased CYP2D6-mediated drug metabolism in pregnancy revealed two transcription factors, small heterodimer partner (SHP) and Krüppel-like factor 9, as a transcriptional repressor and an activator, respectively, of CYP2D6. Chemicals that increase SHP expression (e.g., retinoids and activators of farnesoid X receptor) were shown to downregulate CYP2D6 expression in the humanized mice as well as in human hepatocytes. This review summarizes the series of studies on the transcriptional regulation of CYP2D6 expression, potentially providing a basis to better understand the large interindividual variability in CYP2D6-mediated drug metabolism. PMID:27698228

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

PubMed

Penno, Christophe; Sharma, Virag; Coakley, Arthur; O'Connell Motherway, Mary; van Sinderen, Douwe; Lubkowska, Lucyna; Kireeva, Maria L; Kashlev, Mikhail; Baranov, Pavel V; Atkins, John F

2015-04-21

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji

2014-01-17

Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains amore » highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.« less

Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway.

PubMed

Larios, Jorge A; Jausoro, Ignacio; Benitez, Maria-Luisa; Bronfman, Francisca C; Marzolo, Maria-Paz

2014-09-19

ApoER2 and the neurotrophin receptors Trk and p75(NTR) are expressed in the CNS and regulate key functional aspects of neurons, including development, survival, and neuronal function. It is known that both ApoER2 and p75(NTR) are processed by metalloproteinases, followed by regulated intramembrane proteolysis. TrkA activation by nerve growth factor (NGF) increases the proteolytic processing of p75(NTR) mediated by ADAM17. Reelin induces the sheeding of ApoER2 ectodomain depending on metalloproteinase activity. However, it is not known if there is a common regulation mechanism for processing these receptors. We found that TrkA activation by NGF in PC12 cells induced ApoER2 processing, which was dependent on TrkA activation and metalloproteinases. NGF-induced ApoER2 proteolysis was independent of mitogen activated protein kinase activity and of phosphatidylinositol-3 kinase activity. In contrast, the basal proteolysis of ApoER2 increased when both kinases were pharmacologically inhibited. The ApoER2 ligand reelin regulated the proteolytic processing of its own receptor but not of p75(NTR). Finally, in primary cortical neurons, which express both ApoER2 and TrkB, we found that the proteolysis of ApoER2 was also regulated by brain-derived growth factor (BDNF). Our results highlight a novel relationship between neurotrophins and the reelin-ApoER2 system, suggesting that these two pathways might be linked to regulate brain development, neuronal survival, and some pathological conditions.

FOXK2 transcription factor suppresses ERα-positive breast cancer cell growth through down-regulating the stability of ERα via mechanism involving BRCA1/BARD1.

PubMed

Liu, Ying; Ao, Xiang; Jia, Zhaojun; Bai, Xiao-Yan; Xu, Zhaowei; Hu, Gaolei; Jiang, Xiao; Chen, Min; Wu, Huijian

2015-03-05

Estrogen receptors (ERs) are critical regulators of breast cancer development. Identification of molecules that regulate the function of ERs may facilitate the development of more effective breast cancer treatment strategies. In this study, we showed that the forkhead transcription factor FOXK2 interacted with ERα, and inhibited ERα-regulated transcriptional activities by enhancing the ubiquitin-mediated degradation of ERα. This process involved the interaction between FOXK2 and BRCA1/BARD1, the E3 ubiquitin ligase of ERα. FOXK2 interacted with BARD1 and acted as a scaffold protein for BRCA1/BARD1 and ERα, leading to enhanced degradation of ERα, which eventually accounted for its decreased transcriptional activity. Consistent with these observations, overexpression of FOXK2 inhibited the transcriptional activity of ERα, decreased the transcription of ERα target genes, and suppressed the proliferation of ERα-positive breast cancer cells. In contract, knockdown of FOXK2 in MCF-7 cells promoted cell proliferation. However, when ERα was also knocked down, knockdown of FOXK2 had no effect on cell proliferation. These findings suggested that FOXK2 might act as a negative regulator of ERα, and its association with both ERα and BRCA1/BARD1 could lead to the down-regulation of ERα transcriptional activity, effectively regulating the function of ERα.

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection.

PubMed

Inácio, Patricia; Zuzarte-Luís, Vanessa; Ruivo, Margarida T G; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

2015-08-01

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s--the active form of the UPR mediator XBP1--and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. © 2015 The Authors.

STIM1L traps and gates Orai1 channels without remodeling the cortical ER

PubMed Central

Saüc, Sophie; Bulla, Monica; Nunes, Paula; Orci, Lelio; Marchetti, Anna; Antigny, Fabrice; Bernheim, Laurent; Cosson, Pierre; Frieden, Maud; Demaurex, Nicolas

2015-01-01

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides. PMID:25736291

Repairing RNA Transcripts that Mediate Breast Cancer Susceptibility

DTIC Science & Technology

2005-08-01

is actually the yield of TES product plus the yield of cryptic is in contrast to hammerhead and hairpin ribozymes , which products. This increases the...therapeutics. To this end, we have developed a novel biomolecule (a ribozyme ) that can specifically excise regions from RNA transcripts. In this work, we...designed a ribozyme that excises an insertion mutation that is linked to breast cancer predisposition from a short mimic of the p53 transcript in a

Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

PubMed

Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

2013-06-01

To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner

Endoplasmic reticulum stress inhibits expression of genes involved in thyroid hormone synthesis and their key transcriptional regulators in FRTL-5 thyrocytes

PubMed Central

Wen, Gaiping; Eder, Klaus

2017-01-01

Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins due to an impairment of ER quality control pathways leading to the activation of a defense system, called unfolded protein response (UPR). While thyrocytes are supposed to be highly susceptible to environmental conditions that cause ER stress due to the synthesis of large amounts of secretory proteins required for thyroid hormone synthesis, systematic investigations on the effect of ER stress on expression of key genes of thyroid hormone synthesis and their transcriptional regulators are lacking. Since the aim of the ER stress-induced UPR is to restore ER homeostasis and to facilitate cell survival through transient shutdown of ribosomal protein translation, we hypothesized that the expression of genes involved in thyroid hormone synthesis and their transcriptional regulators, all of which are not essential for cell survival, are down-regulated in thyrocytes during ER stress, while sterol regulatory element-binding proteins (SREBPs) are activated during ER stress in thyrocytes. Treatment of FRTL-5 thyrocytes with the ER stress inducer tunicamycin (TM) dose-dependently increased the mRNA and/or protein levels of known UPR target genes, stimulated phosphorylation of the ER stress sensor protein kinase RNA-like ER kinase (PERK) and of the PERK target protein eukaryotic initiation factor 2α (eIF2α) and caused splicing of the ER stress-sensitive transcription factor X-box binding protein (XBP-1) (P < 0.05). The mRNA levels and/or protein levels of genes involved in thyroid hormone synthesis, sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), their transcriptional regulators and thyrotropin (TSH) receptor and the uptake of Na125I were reduced at the highest concentration of TM tested (0.1 μg/mL; P < 0.05). Proteolytic activation of the SREBP-1c pathway was not observed in FRTL-5 cells treated with TM, whereas TM reduced proteolytic activation of

Endoplasmic reticulum stress inhibits expression of genes involved in thyroid hormone synthesis and their key transcriptional regulators in FRTL-5 thyrocytes.

PubMed

Wen, Gaiping; Ringseis, Robert; Eder, Klaus

2017-01-01

Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins due to an impairment of ER quality control pathways leading to the activation of a defense system, called unfolded protein response (UPR). While thyrocytes are supposed to be highly susceptible to environmental conditions that cause ER stress due to the synthesis of large amounts of secretory proteins required for thyroid hormone synthesis, systematic investigations on the effect of ER stress on expression of key genes of thyroid hormone synthesis and their transcriptional regulators are lacking. Since the aim of the ER stress-induced UPR is to restore ER homeostasis and to facilitate cell survival through transient shutdown of ribosomal protein translation, we hypothesized that the expression of genes involved in thyroid hormone synthesis and their transcriptional regulators, all of which are not essential for cell survival, are down-regulated in thyrocytes during ER stress, while sterol regulatory element-binding proteins (SREBPs) are activated during ER stress in thyrocytes. Treatment of FRTL-5 thyrocytes with the ER stress inducer tunicamycin (TM) dose-dependently increased the mRNA and/or protein levels of known UPR target genes, stimulated phosphorylation of the ER stress sensor protein kinase RNA-like ER kinase (PERK) and of the PERK target protein eukaryotic initiation factor 2α (eIF2α) and caused splicing of the ER stress-sensitive transcription factor X-box binding protein (XBP-1) (P < 0.05). The mRNA levels and/or protein levels of genes involved in thyroid hormone synthesis, sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), their transcriptional regulators and thyrotropin (TSH) receptor and the uptake of Na125I were reduced at the highest concentration of TM tested (0.1 μg/mL; P < 0.05). Proteolytic activation of the SREBP-1c pathway was not observed in FRTL-5 cells treated with TM, whereas TM reduced proteolytic activation of

Pulpal temperature increase with high-speed handpiece, Er:YAG laser and ultrasound tips.

PubMed

Mollica, Fernanda Brandão; Camargo, Fernanda Pelogia; Zamboni, Sandra Costa; Pereira, Sarina Maciel Braga; Teixeira, Symone Cristina; Nogueira, Lafayette

2008-01-01

The aim of this study was to compare intrapulpal temperature increase produced by high-speed handpiece, Er:YAG laser and CVDentus ultrasound tips during cavity preparation. Thirty bovine mandibular incisors with an enamel/dentin thickness of 4 mm at buccal surface had their roots amputated and were allocated to the following groups (n=10): Group I- high-speed handpiece; Group II- noncontact Er:YAG laser (250 mJ/4 Hz); and Group III- CVDentus ultrasouns tips. All devices were used with water cooling. Class V cavities were prepared to a depth of 3.5 mm, measured with a periodontal probe. A type T thermocouple was placed inside the pulp chamber to determine the temperature increase (degrees C), which was recorded by a data acquisition system ADS 2000 IP (Lynx Technology) linked to a notebook computer. Data were analyzed statistically by one-way ANOVA and Tukey's test (p=0.05). The mean temperature rises were: 1.10 degrees C (+/-0.56) for Group I, 0.84 degrees C (+/-0.55) for Group II, and 3.00 degrees C (+/- 1.34) for Group III. There were no statistically significant differences (p>0.05) between Groups I and II, but both of them differed significantly from Group III (p<0.05). In conclusion, the use of Er:YAG laser and high-speed handpiece for cavity preparation resulted in similar temperature increase. Although ultrasound tips generated significantly higher intrapulpal temperature increase, it remained below the critical value of 5.5 degrees C and may be considered safe for use.

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

NASA Technical Reports Server (NTRS)

Cardoza, V.; Stewart, C. N.

2003-01-01

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

SON and its alternatively spliced isoforms control MLL complex-mediated H3K4me3 and transcription of leukemia-associated genes

PubMed Central

Kim, Jung-Hyun; Baddoo, Melody C.; Park, Eun Young; Stone, Joshua K.; Park, Hyeonsoo; Butler, Thomas W.; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M.; Tan, Ming; Flemington, Erik K.; Lim, Ssang-Taek; Erin Ahn, Eun-Young

2016-01-01

SUMMARY Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy, but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

YY1 and HDAC9c transcriptionally regulate p38-mediated mesenchymal stem cell differentiation into osteoblasts

PubMed Central

Chen, Ya-Huey; Chung, Chiao-Chen; Liu, Yu-Chia; Lai, Wei-Chen; Lin, Zong-Shin; Chen, Tsung-Ming; Li, Long-Yuan; Hung, Mien-Chie

2018-01-01

Mesenchymal stem cells (MSCs) have a high self-renewal potential and can differentiate into various types of cells, including adipocytes, osteoblasts, and chondrocytes. Previously, we reported that the enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, and HDAC9c mediate the osteogenesis and adipogenesis of MSCs. In the current study, we identify the role of p38 in osteogenic differentiation from a MAPK antibody array screen and investigate the mechanisms underlying its transcriptional regulation. Our data show that YY1, a ubiquitously expressed transcription factor, and HDAC9c coordinate p38 transcriptional activity to promote its expression to facilitate the osteogenic potential of MSCs. Our results show that p38 mediates osteogenic differentiation, and this has significant implications in bone-related diseases, bone tissue engineering, and regenerative medicine. PMID:29637005

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

PubMed Central

Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

2015-01-01

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

Cooperative Dynamics of AR and ER Activity in Breast Cancer

PubMed Central

D’Amato, Nicholas C.; Gordon, Michael A.; Babbs, Beatrice L.; Spoelstra, Nicole S.; Carson Butterfield, Kiel T.; Torkko, Kathleen C.; Phan, Vernon T.; Barton, Valerie N.; Rogers, Thomas J.; Sartorius, Carol A; Elias, Anthony D.; Gertz, Jason; Jacobsen, Britta M.; Richer, Jennifer K.

2016-01-01

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha positive (ER+) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared to dihydrotestosterone (DHT). Estradiol-induced AR binding sites were enriched for estrogen response elements and had significant overlap with ER binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER+/AR+ breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER+/AR+ patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Lastly, in a comparison of ER+/AR+ primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide pre-clinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single agent efficacy may be possible in tumors resistant to traditional endocrine therapy, since clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. Implications This study suggests that AR plays a previously-unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers. PMID:27565181

Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

PubMed

Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

2015-11-01

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

PubMed

Menezes, Regina Andrade; Pimentel, Catarina; Silva, Ana Rita Courelas; Amaral, Catarina; Merhej, Jawad; Devaux, Frédéric; Rodrigues-Pousada, Claudina

2017-04-01

Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes. Copyright © 2017. Published by Elsevier B.V.

Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

PubMed

Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

2010-05-21

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

TRIM45 negatively regulates NF-{kappa}B-mediated transcription and suppresses cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shibata, Mio; Sato, Tomonobu; Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638

2012-06-22

Highlights: Black-Right-Pointing-Pointer NF-{kappa}B plays an important role in cell survival and carcinogenesis. Black-Right-Pointing-Pointer TRIM45 negatively regulates TNF{alpha}-induced NF-{kappa}B-mediated transcription. Black-Right-Pointing-Pointer TRIM45 overexpression suppresses cell growth. Black-Right-Pointing-Pointer TRIM45 acts as a repressor for the NF-{kappa}B signal and regulates cell growth. -- Abstract: The NF-{kappa}B signaling pathway plays an important role in cell survival, immunity, inflammation, carcinogenesis, and organogenesis. Activation of NF-{kappa}B is regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. The NF-{kappa}B signaling pathway is activated by two distinct signaling mechanisms and is strictly modulated by the ubiquitin-proteasome system. It has been reported that overexpression of TRIM45, one ofmore » the TRIM family ubiquitin ligases, suppresses transcriptional activities of Elk-1 and AP-1, which are targets of the MAPK signaling pathway. In this study, we showed that TRIM45 also negatively regulates TNF{alpha}-induced NF-{kappa}B-mediated transcription by a luciferase reporter assay and that TRIM45 lacking a RING domain also has an activity to inhibit the NF-{kappa}B signal. Moreover, we found that TRIM45 overexpression suppresses cell growth. These findings suggest that TRIM45 acts as a repressor for the NF-{kappa}B signal and regulates cell growth.« less

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

PubMed

Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

2015-12-17

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Post-transcriptional regulation mediated by specific neurofilament introns in vivo.

PubMed

Wang, Chen; Szaro, Ben G

2016-04-01

Neurons regulate genes post-transcriptionally to coordinate the supply of cytoskeletal proteins, such as the medium neurofilament (NEFM), with demand for structural materials in response to extracellular cues encountered by developing axons. By using a method for evaluating functionality of cis-regulatory gene elements in vivo through plasmid injection into Xenopus embryos, we discovered that splicing of a specific nefm intron was required for robust transgene expression, regardless of promoter or cell type. Transgenes utilizing the nefm 3'-UTR but substituting other nefm introns expressed little or no protein owing to defects in handling of the messenger (m)RNA as opposed to transcription or splicing. Post-transcriptional events at multiple steps, but mainly during nucleocytoplasmic export, contributed to these varied levels of protein expression. An intron of the β-globin gene was also able to promote expression in a manner identical to that of the nefm intron, implying a more general preference for certain introns in controlling nefm expression. These results expand our knowledge of intron-mediated gene expression to encompass neurofilaments, indicating an additional layer of complexity in the control of a cytoskeletal gene needed for developing and maintaining healthy axons. © 2016. Published by The Company of Biologists Ltd.

RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation

PubMed Central

Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E.; Liau, Brian B.; Mack, Stephen C.; Yang, Kailin; Factor, Daniel C.; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E.; Bao, Shideng; Rich, Jeremy N.

2016-01-01

Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor–initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH. PMID:27322055

RANK-c attenuates aggressive properties of ER-negative breast cancer by inhibiting NF-κB activation and EGFR signaling.

PubMed

Sirinian, Chaido; Papanastasiou, Anastasios D; Schizas, Michail; Spella, Magda; Stathopoulos, Georgios T; Repanti, Maria; Zarkadis, Ioannis K; King, Tari A; Kalofonos, Haralabos P

2018-05-29

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2007-05-01

The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection

PubMed Central

Inácio, Patricia; Zuzarte-Luís, Vanessa; Ruivo, Margarida TG; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

2015-01-01

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s—the active form of the UPR mediator XBP1—and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. PMID:26113366

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

PubMed

Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

2016-04-20

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Role of ERRF, a Novel ER-Related Nuclear Factor, in the Growth Control of ER-Positive Human Breast Cancer Cells

PubMed Central

Su, Dan; Fu, Xiaoying; Fan, Songqing; Wu, Xiao; Wang, Xin-Xin; Fu, Liya; Dong, Xue-Yuan; Ni, Jianping Jenny; Fu, Li; Zhu, Zhengmao; Dong, Jin-Tang

2012-01-01

Whereas estrogen–estrogen receptor α (ER) signaling plays an important role in breast cancer growth, it is also necessary for the differentiation of normal breast epithelial cells. How this functional conversion occurs, however, remains unknown. Based on a genome-wide sequencing study that identified mutations in several breast cancer genes, we examined some of the genes for mutations, expression levels, and functional effects on cell proliferation and tumorigenesis. We present the data for C1orf64 or ER-related factor (ERRF) from 31 cell lines and 367 primary breast cancer tumors. Whereas mutation of ERRF was infrequent (1 of 79 or 1.3%), its expression was up-regulated in breast cancer, and the up-regulation was more common in lower-stage tumors. In addition, increased ERRF expression was significantly associated with ER and/or progesterone receptor (PR) positivity, which was still valid in human epidermal growth factor receptor 2 (HER2)–negative tumors. In ER-positive tumors, ERRF expression was inversely correlated with HER2 status. Furthermore, higher ERRF protein expression was significantly associated with better disease-free survival and overall survival, particularly in ER- and/or PR-positive and HER2-negative tumors (luminal A subtype). Functionally, knockdown of ERRF in two ER-positive breast cancer cell lines, T-47D and MDA-MB-361, suppressed cell growth in vitro and tumorigenesis in xenograft models. These results suggest that ERRF plays a role in estrogen-ER–mediated growth of breast cancer cells and could, thus, be a potential therapeutic target. PMID:22341523

SR/ER-mitochondrial local communication: Calcium and ROS

PubMed Central

Csordás, György; Hajnóczky, György

2009-01-01

Mitochondria form junctions with the sarco/endoplasmic reticulum (SR/ER), which support signal transduction and biosynthetic pathways and affect organellar distribution. Recently, these junctions have received attention because of their pivotal role in mediating calcium signal propagation to the mitochondria, which is important for both ATP production and mitochondrial cell death. Many of the SR/ER-mitochondrial calcium transporters and signaling proteins are sensitive to redox regulation and are directly exposed to the reactive oxygen species (ROS) produced in the mitochondria and SR/ER. Although ROS has been emerging as a novel signaling entity, the redox signaling of the SR/ER-mitochondrial interface is yet to be elucidated. We describe here possible mechanisms of the mutual interaction between local Ca2+ and ROS signaling in the control of SR/ER-mitochondrial function. PMID:19527680

Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells

PubMed Central

Beck, Daniela; Niessner, Heike; Smalley, Keiran S.M.; Flaherty, Keith; Paraiso, Kim H.T.; Busch, Christian; Sinnberg, Tobias; Vasseur, Sophie; Iovanna, Juan Lucio; Drießen, Stefan; Stork, Björn; Wesselborg, Sebastian; Schaller, Martin; Biedermann, Tilo; Bauer, Jürgen; Lasithiotakis, Konstantinos; Weide, Benjamin; Eberle, Jürgen; Schittek, Birgit; Schadendorf, Dirk; Garbe, Claus; Kulms, Dagmar; Meier, Friedegund

2013-01-01

The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase (MAPK) signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of anti-apoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein GRP78, increased the abundance of the spliced isoform of the transcription factor X-box protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein ATF4 significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance. PMID:23362240

Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription.

PubMed

Stavreva, Diana A; Wiench, Malgorzata; John, Sam; Conway-Campbell, Becky L; McKenna, Mervyn A; Pooley, John R; Johnson, Thomas A; Voss, Ty C; Lightman, Stafford L; Hager, Gordon L

2009-09-01

Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation.

A self-defeating anabolic program leads to β-cell apoptosis in endoplasmic reticulum stress-induced diabetes via regulation of amino acid flux.

PubMed

Krokowski, Dawid; Han, Jaeseok; Saikia, Mridusmita; Majumder, Mithu; Yuan, Celvie L; Guan, Bo-Jhih; Bevilacqua, Elena; Bussolati, Ovidio; Bröer, Stefan; Arvan, Peter; Tchórzewski, Marek; Snider, Martin D; Puchowicz, Michelle; Croniger, Colleen M; Kimball, Scot R; Pan, Tao; Koromilas, Antonis E; Kaufman, Randal J; Hatzoglou, Maria

2013-06-14

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Binet, Francois; Chiasson, Sonia; Girard, Denis, E-mail: denis.girard@iaf.inrs.ca

2010-01-01

Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenicmore » trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2{alpha} are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.« less

AAV-mediated delivery of the transcription factor XBP1s into the striatum reduces mutant Huntingtin aggregation in a mouse model of Huntington's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zuleta, Amparo; Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago; Vidal, Rene L.

2012-04-13

Highlights: Black-Right-Pointing-Pointer The contribution of ER stress to HD has not been directly addressed. Black-Right-Pointing-Pointer Expression of XBP1s using AAVs decreases Huntingtin aggregation in vivo. Black-Right-Pointing-Pointer We describe a new in vivo model of HD based on the expression of a large fragment of mHtt-RFP. -- Abstract: Huntington's disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptationmore » to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588{sup Q95}-mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588{sup Q95}-mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.« less

Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

PubMed

Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

2016-03-07

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. © 2016 Thomas-Claudepierre et al.

Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation.

PubMed

Nayebosadri, Arman; Ji, Julie Y

2013-08-01

The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm(2)) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction.

Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation

PubMed Central

Nayebosadri, Arman

2013-01-01

The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm2) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction. PMID:23703529

Transforming growth factor β-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

PubMed

Stephen, Tom L; Rutkowski, Melanie R; Allegrezza, Michael J; Perales-Puchalt, Alfredo; Tesone, Amelia J; Svoronos, Nikolaos; Nguyen, Jenny M; Sarmin, Fahmida; Borowsky, Mark E; Tchou, Julia; Conejo-Garcia, Jose R

2014-09-18

Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation. Copyright © 2014 Elsevier Inc. All rights reserved.

COPA mutations impair ER-Golgi transport causing hereditary autoimmune-mediated lung disease and arthritis

PubMed Central

Watkin, Levi B.; Jessen, Birthe; Wiszniewski, Wojciech; Vece, Timothy; Jan, Max; Sha, Youbao; Thamsen, Maike; Santos-Cortez, Regie L. P.; Lee, Kwanghyuk; Gambin, Tomasz; Forbes, Lisa; Law, Christopher S.; Stray-Petersen, Asbjørg; Cheng, Mickie H.; Mace, Emily M.; Anderson, Mark S.; Liu, Dongfang; Tang, Ling Fung; Nicholas, Sarah K.; Nahmod, Karen; Makedonas, George; Canter, Debra; Kwok, Pui-Yan; Hicks, John; Jones, Kirk D.; Penney, Samantha; Jhangiani, Shalini N.; Rosenblum, Michael D.; Dell, Sharon D.; Waterfield, Michael R.; Papa, Feroz R.; Muzny, Donna M.; Zaitlen, Noah; Leal, Suzanne M.; Gonzaga-Jauregui, Claudia; Boerwinkle, Eric; Eissa, N. Tony; Gibbs, Richard A.; Lupski, James R.; Orange, Jordan S.; Shum, Anthony K.

2015-01-01

Advances in genomics have allowed unbiased genetic studies of human disease with unexpected insights into the molecular mechanisms of cellular immunity and autoimmunity1. We performed whole exome sequencing (WES) and targeted sequencing in patients with an apparent Mendelian syndrome of autoimmune disease characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease (ILD). In five families, we identified four unique deleterious variants in the Coatomer subunit alpha (COPA) gene all located within the same functional domain. We hypothesized that mutant COPA leads to a defect in intracellular transport mediated by coat protein complex I (COPI)2–4. We show that COPA variants impair binding of proteins targeted for retrograde Golgi to ER transport and demonstrate that expression of mutant COPA leads to ER stress and the upregulation of Th17 priming cytokines. Consistent with this pattern of cytokine expression, patients demonstrated a significant skewing of CD4+ T cells toward a T helper 17 (Th17) phenotype, an effector T cell population implicated in autoimmunity5,6. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease. These findings provide a unique opportunity to understand how alterations in cellular homeostasis caused by a defect in the intracellular trafficking pathway leads to the generation of human autoimmune disease. PMID:25894502

The Saccharomyces cerevisiae Cdk8 Mediator Represses AQY1 Transcription by Inhibiting Set1p-Dependent Histone Methylation.

PubMed

Law, Michael J; Finger, Michael A

2017-03-10

In the budding yeast Saccharomyces cerevisiae , nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus. Copyright © 2017 Law and Finger.

A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis[W][OPEN

PubMed Central

Ng, Sophia; Ivanova, Aneta; Duncan, Owen; Law, Simon R.; Van Aken, Olivier; De Clercq, Inge; Wang, Yan; Carrie, Chris; Xu, Lin; Kmiec, Beata; Walker, Hayden; Van Breusegem, Frank; Whelan, James; Giraud, Estelle

2013-01-01

Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ∼33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions. PMID:24045017

REF4 and RFR1, Subunits of the Transcriptional Coregulatory Complex Mediator, Are Required for Phenylpropanoid Homeostasis in Arabidopsis*

PubMed Central

Bonawitz, Nicholas D.; Soltau, Whitney L.; Blatchley, Michael R.; Powers, Brendan L.; Hurlock, Anna K.; Seals, Leslie A.; Weng, Jing-Ke; Stout, Jake; Chapple, Clint

2012-01-01

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs. PMID:22167189

REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis.

PubMed

Bonawitz, Nicholas D; Soltau, Whitney L; Blatchley, Michael R; Powers, Brendan L; Hurlock, Anna K; Seals, Leslie A; Weng, Jing-Ke; Stout, Jake; Chapple, Clint

2012-02-17

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.

Basic leucine zipper transcription factor SlbZIP1 mediates salt and drought stress tolerance in tomato.

PubMed

Zhu, Mingku; Meng, Xiaoqing; Cai, Jing; Li, Ge; Dong, Tingting; Li, Zongyun

2018-05-08

Basic region/leucine zipper (bZIP) transcription factors perform as crucial regulators in ABA-mediated stress response in plants. Nevertheless, the functions for most bZIP family members in tomato remain to be deciphered. Here we examined the functional characterization of SlbZIP1 under salt and drought stresses in tomato. Silencing of SlbZIP1 in tomato resulted in reduced expression of multiple ABA biosynthesis- and signal transduction-related genes in transgenic plants. In stress assays, SlbZIP1-RNAi transgenic plants exhibited reduced tolerance to salt and drought stresses compared with WT plants, as are evaluated by multiple physiological parameters associated with stress responses, such as decreased ABA, chlorophyll contents and CAT activity, and increased MDA content. In addition, RNA-seq analysis of transgenic plants revealed that the transcription levels of multiple genes encoding defense proteins related to responses to abiotic stress (e.g. endochitinase, peroxidases, and lipid transfer proteins) and biotic stress (e.g. pathogenesis-related proteins) were downregulated in SlbZIP1-RNAi plants, suggesting that SlbZIP1 plays a role in regulating the genes related to biotic and abiotic stress response. Collectively, the data suggest that SlbZIP1 exerts an essential role in salt and drought stress tolerance through modulating an ABA-mediated pathway, and SlbZIP1 may hold potential applications in the engineering of salt- and drought-tolerant tomato cultivars.

Seven-day cumulative effects of air pollutants increase respiratory ER visits up to threefold.

PubMed

Schvartsman, Cláudio; Pereira, Luiz Alberto Amador; Braga, Alfésio Luiz Ferreira; Farhat, Sylvia Costa Lima

2017-02-01

Children are especially vulnerable to respiratory injury induced by exposure to air pollutants. In the present study, we investigate periods of up to 7 days, and evaluate the lagged effects of exposure to air pollutants on the daily number of children and adolescents visiting the emergency room (ER) for the treatment of lower respiratory obstructive diseases (LROD), in the city of São Paulo, Brazil. Daily records of LROD-related ER visits by children and adolescents under the age of 19, from January 2000 to December 2007 (2,922 days) were included in the study. Time-series regression models (generalized linear Poisson) were used to control for short- and long-term trends, as well as for temperature and relative humidity. Third-degree polynomial lag models were used to estimate both lag structures and the cumulative effects of air pollutants. Effects of air pollutants were expressed as the percentage increase in LROD-related ER visits. We observed an acute effect at the same day of exposure to air pollutants; however, the cumulative effects of air pollutants on the number of LROD-related ER visits was almost threefold greater than the one observed at the same day of exposure to PM 10 , SO 2 , and NO 2 mainly in children aged 5 years and under. The 7-day cumulative effect of SO 2 reached 11.0% (95% CI: 5.0-16.7) increase in visits. Conclusion and Relevance: This study highlights the effects of intermediate-term exposure to air pollutants on LROD in children. Pediatr Pulmonol. 2017;52:205-212. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Regulation of Memory Formation by the Transcription Factor XBP1.

PubMed

Martínez, Gabriela; Vidal, René L; Mardones, Pablo; Serrano, Felipe G; Ardiles, Alvaro O; Wirth, Craig; Valdés, Pamela; Thielen, Peter; Schneider, Bernard L; Kerr, Bredford; Valdés, Jose L; Palacios, Adrian G; Inestrosa, Nibaldo C; Glimcher, Laurie H; Hetz, Claudio

2016-02-16

Contextual memory formation relies on the induction of new genes in the hippocampus. A polymorphism in the promoter of the transcription factor XBP1 was identified as a risk factor for Alzheimer's disease and bipolar disorders. XBP1 is a major regulator of the unfolded protein response (UPR), mediating adaptation to endoplasmic reticulum (ER) stress. Using a phenotypic screen, we uncovered an unexpected function of XBP1 in cognition and behavior. Mice lacking XBP1 in the nervous system showed specific impairment of contextual memory formation and long-term potentiation (LTP), whereas neuronal XBP1s overexpression improved performance in memory tasks. Gene expression analysis revealed that XBP1 regulates a group of memory-related genes, highlighting brain-derived neurotrophic factor (BDNF), a key component in memory consolidation. Overexpression of BDNF in the hippocampus reversed the XBP1-deficient phenotype. Our study revealed an unanticipated function of XBP1 in cognitive processes that is apparently unrelated to its role in ER stress. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Milk: an epigenetic amplifier of FTO-mediated transcription? Implications for Western diseases.

PubMed

Melnik, Bodo C

2015-12-21

Single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity-associated) gene are associated with enhanced FTO expression, increased body weight, obesity and type 2 diabetes mellitus (T2DM). The N (6) -methyladenosine (m(6)A) demethylase FTO plays a pivotal regulatory role for postnatal growth and energy expenditure. The purpose of this review is to provide translational evidence that links milk signaling with FTO-activated transcription of the milk recipient. FTO-dependent demethylation of m(6)A regulates mRNA splicing required for adipogenesis, increases the stability of mRNAs, and affects microRNA (miRNA) expression and miRNA biosynthesis. FTO senses branched-chain amino acids (BCAAs) and activates the nutrient sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), which plays a key role in translation. Milk provides abundant BCAAs and glutamine, critical components increasing FTO expression. CpG hypomethylation in the first intron of FTO has recently been associated with T2DM. CpG methylation is generally associated with gene silencing. In contrast, CpG demethylation generally increases transcription. DNA de novo methylation of CpG sites is facilitated by DNA methyltransferases (DNMT) 3A and 3B, whereas DNA maintenance methylation is controlled by DNMT1. MiRNA-29s target all DNMTs and thus reduce DNA CpG methylation. Cow´s milk provides substantial amounts of exosomal miRNA-29s that reach the systemic circulation and target mRNAs of the milk recipient. Via DNMT suppression, milk exosomal miRNA-29s may reduce the magnitude of FTO methylation, thereby epigenetically increasing FTO expression in the milk consumer. High lactation performance with increased milk yield has recently been associated with excessive miRNA-29 expression of dairy cow mammary epithelial cells (DCMECs). Notably, the galactopoietic hormone prolactin upregulates the transcription factor STAT3, which induces miRNA-29 expression. In a retrovirus-like manner

Roles of Arenavirus Z Protein in Mediating Virion Budding, Viral Transcription-Inhibition and Interferon-Beta Suppression.

PubMed

Shao, Junjie; Liang, Yuying; Ly, Hinh

2018-01-01

The smallest arenaviral protein is the zinc-finger protein (Z) that belongs to the RING finger protein family. Z serves as a main component required for virus budding from the membrane of the infected cells through self-oligomerization, a process that can be aided by the viral nucleoprotein (NP) to form the viral matrix of progeny virus particles. Z has also been shown to be essential for mediating viral transcriptional repression activity by locking the L polymerase onto the viral promoter in a catalytically inactive state, thus limiting viral replication. The Z protein has also recently been shown to inhibit the type I interferon-induction pathway by directly binding to the intracellular pathogen-sensor proteins RIG-I and MDA5, and thus inhibiting their normal functions. This chapter describes several assays used to examine the important roles of the arenaviral Z protein in mediating virus budding (i.e., either Z self-budding or NP-Z budding activities), viral transcriptional inhibition in a viral minigenome (MG) assay, and type I IFN suppression in an IFN-β promoter-mediated luciferase reporter assay.

Chronic sleep fragmentation during the sleep period induces hypothalamic endoplasmic reticulum stress and PTP1b-mediated leptin resistance in male mice.

PubMed

Hakim, Fahed; Wang, Yang; Carreras, Alba; Hirotsu, Camila; Zhang, Jing; Peris, Eduard; Gozal, David

2015-01-01

Sleep fragmentation (SF) is highly prevalent and may constitute an important contributing factor to excessive weight gain and the metabolic syndrome. Increased endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) leading to the attenuation of leptin receptor signaling in the hypothalamus leads to obesity and metabolic dysfunction. Mice were exposed to SF and sleep control (SC) for varying periods of time during which ingestive behaviors were monitored. UPR pathways and leptin receptor signaling were assessed in hypothalami. To further examine the mechanistic role of ER stress, changes in leptin receptor (ObR) signaling were also examined in wild-type mice treated with the ER chaperone tauroursodeoxycholic acid (TUDCA), as well as in CHOP-/+ transgenic mice. Fragmented sleep in male mice induced increased food intake starting day 3 and thereafter, which was preceded by increases in ER stress and activation of all three UPR pathways in the hypothalamus. Although ObR expression was unchanged, signal transducer and activator of transcription 3 (STAT3) phosphorylation was decreased, suggesting reduced ObR signaling. Unchanged suppressor of cytokine signaling-3 (SOCS3) expression and increases in protein-tyrosine phosphatase 1B (PTP1B) expression and activity emerged with SF, along with reduced p-STAT3 responses to exogenous leptin. SF-induced effects were reversed following TUDCA treatment and were absent in CHOP -/+ mice. SF induces hyperphagic behaviors and reduced leptin signaling in hypothalamus that are mediated by activation of ER stress, and ultimately lead to increased PTP1B activity. ER stress pathways are therefore potentially implicated in SF-induced weight gain and metabolic dysfunction, and may represent a viable therapeutic target. © 2014 Associated Professional Sleep Societies, LLC.

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

A crosstalk between p21 and UPR-induced transcription factor C/EBP homologous protein (CHOP) linked to type 2 diabetes.

PubMed

Mihailidou, Chrysovalantou; Papavassiliou, Athanasios G; Kiaris, Hippokratis

2014-04-01

Type 2 diabetes (T2D) is a disease that is characterized by raised levels of glucose in the blood combined with insulin resistance and relative insulin deficiency. The pathogenesis of type 2 diabetes is associated with the induction of the unfolded protein response (UPR). While UPR aims to restore tissue homeostasis following stress of the endoplasmic reticulum (ER), prolonged ER stress triggers apoptosis at least in part through the unfolded protein response (UPR)-activated transcription factor C/EBP (CCAAT/enhancer binding protein) homologous protein (CHOP). CHOP has elevated as a critical mediator connecting accumulation and aggregation of unfolded proteins in the ER and oxidative stress and also contributes to the induction of apoptosis in β-cell (beta-cell) - cells under conditions of increased insulin demand. p21 is a cell cycle regulator that is implicated in the regulation of the UPR by various mechanisms involving inhibition of apoptosis and facilitation of the regeneration capacity of the β cells. In this review we summarize the role of ER stress in the pathogenesis of type 2 diabetes which is associated with the induction of the unfolded protein response (UPR). We also review recent evidence associating p21 activity with β cell health and regenerative capacity by mechanisms that may interfere with the effects of p21 in the UPR or operate independently of ER stress. Most likely understanding the molecular details of the pathogenesis of type 2 diabetes will be beneficial for the management of the disease. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation

DOE PAGES

Wu, Rui; Sanishvili, Ruslan; Belitsky, Boris R.; ...

2017-03-27

Here, the Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of gamma-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic.-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoicmore » acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.« less

PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Rui; Sanishvili, Ruslan; Belitsky, Boris R.

Here, the Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of gamma-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic.-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoicmore » acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.« less

Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

PubMed Central

Dalbiès-Tran, Rozenn; Stigger-Rosser, Evelyn; Dotson, Travis; Sample, Clare E.

2001-01-01

Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes. PMID:11119577

Cadmium-induced teratogenicity: Association with ROS-mediated endoplasmic reticulum stress in placenta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhen; Wang, Hua; Xu, Zhong Mei

The placenta is essential for sustaining the growth of the fetus. An increased endoplasmic reticulum (ER) stress has been associated with the impaired placental and fetal development. Cadmium (Cd) is a potent teratogen that caused fetal malformation and growth restriction. The present study investigated the effects of maternal Cd exposure on placental and fetal development. The pregnant mice were intraperitoneally injected with CdCl{sub 2} (4.5 mg/kg) on gestational day 9. As expected, maternal Cd exposure during early limb development significantly increased the incidences of forelimb ectrodactyly in fetuses. An obvious impairment in the labyrinth, a highly developed tissue of bloodmore » vessels, was observed in placenta of mice treated with CdCl{sub 2}. In addition, maternal Cd exposure markedly repressed cell proliferation and increased apoptosis in placenta. An additional experiment showed that maternal Cd exposure significantly upregulated the expression of GRP78, an ER chaperone. Moreover, maternal Cd exposure induced the phosphorylation of placental eIF2α, a downstream molecule of PERK signaling. In addition, maternal Cd exposure significantly increased the level of placental CHOP, another target of PERK signaling, indicating that the unfolded protein response (UPR) signaling was activated in placenta of mice treated with CdCl{sub 2}. Interestingly, alpha-phenyl-N-t-butylnitrone, a free radical spin-trapping agent, significantly alleviated Cd-induced placental ER stress and UPR. Taken together, these results suggest that reactive oxygen species (ROS)-mediated ER stress might be involved in Cd-induced impairment on placental and fetal development. Antioxidants may be used as pharmacological agents to protect against Cd-induced fetal malformation and growth restriction. -- Highlights: ► Cd induces fetal malformation and growth restriction. ► Cd induced placental ER stress and UPR. ► PBN alleviates Cd-induced ER stress and UPR in placenta. ► ROS-mediated

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C.; Loros, Jennifer J.; Kück, Ulrich

2013-01-01

Summary The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41–EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41–EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi. PMID:17501918

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation

PubMed Central

Zhao, Yingxin; Sun, Hong; Zhang, Yueqing; Yang, Jun; Brasier, Allan R.

2016-01-01

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases. PMID:27793799

Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection

NASA Astrophysics Data System (ADS)

Xu, Yun; Zhang, Wenri; Klaus, Judith; Young, Jennifer; Koerner, Ines; Sheldahl, Laird C.; Hurn, Patricia D.; Martínez-Murillo, Francisco; Alkayed, Nabil J.

2006-09-01

Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases. ischemia | stroke | estrogen

High-intensity training reduces intermittent hypoxia-induced ER stress and myocardial infarct size.

PubMed

Bourdier, Guillaume; Flore, Patrice; Sanchez, Hervé; Pepin, Jean-Louis; Belaidi, Elise; Arnaud, Claire

2016-01-15

Chronic intermittent hypoxia (IH) is described as the major detrimental factor leading to cardiovascular morbimortality in obstructive sleep apnea (OSA) patients. OSA patients exhibit increased infarct size after a myocardial event, and previous animal studies have shown that chronic IH could be the main mechanism. Endoplasmic reticulum (ER) stress plays a major role in the pathophysiology of cardiovascular disease. High-intensity training (HIT) exerts beneficial effects on the cardiovascular system. Thus, we hypothesized that HIT could prevent IH-induced ER stress and the increase in infarct size. Male Wistar rats were exposed to 21 days of IH (21-5% fraction of inspired O2, 60-s cycle, 8 h/day) or normoxia. After 1 wk of IH alone, rats were submitted daily to both IH and HIT (2 × 24 min, 15-30m/min). Rat hearts were either rapidly frozen to evaluate ER stress by Western blot analysis or submitted to an ischemia-reperfusion protocol ex vivo (30 min of global ischemia/120 min of reperfusion). IH induced cardiac proapoptotic ER stress, characterized by increased expression of glucose-regulated protein kinase 78, phosphorylated protein kinase-like ER kinase, activating transcription factor 4, and C/EBP homologous protein. IH-induced myocardial apoptosis was confirmed by increased expression of cleaved caspase-3. These IH-associated proapoptotic alterations were associated with a significant increase in infarct size (35.4 ± 3.2% vs. 22.7 ± 1.7% of ventricles in IH + sedenary and normoxia + sedentary groups, respectively, P < 0.05). HIT prevented both the IH-induced proapoptotic ER stress and increased myocardial infarct size (28.8 ± 3.9% and 21.0 ± 5.1% in IH + HIT and normoxia + HIT groups, respectively, P = 0.28). In conclusion, these findings suggest that HIT could represent a preventive strategy to limit IH-induced myocardial ischemia-reperfusion damages in OSA patients. Copyright © 2016 the American Physiological Society.

Estrogen receptor alpha and nuclear factor Y coordinately regulate the transcription of the SUMO-conjugating UBC9 gene in MCF-7 breast cancer cells.

PubMed

Ying, Shibo; Dünnebier, Thomas; Si, Jing; Hamann, Ute

2013-01-01

UBC9 encodes a protein that conjugates small ubiquitin-related modifier (SUMO) to target proteins thereby changing their functions. Recently, it was noted that UBC9 expression and activity play a role in breast tumorigenesis and response to anticancer drugs. However, the underlying mechanism is poorly understood. To investigate the transcriptional regulation of the UBC9 gene, we identified and characterized its promoter and cis-elements. Promoter activity was tested using luciferase reporter assays. The binding of transcription factors to the promoter was detected by chromatin immunoprecipitation (ChIP), and their functional role was confirmed by siRNA knockdown. UBC9 mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of UBC9 mRNA and protein was found in MCF-7 breast cancer cells treated with 17β-estradiol (E2). Analysis of various deletion mutants revealed a 137 bp fragment upstream of the transcription initiation site to be sufficient for reporter gene transcription. Mutations of putative estrogen receptor α (ER-α) (one imperfect estrogen response element, ERE) and/or nuclear factor Y (NF-Y) binding sites (two CCAAT boxes) markedly reduced promoter activity. Similar results were obtained in ER-negative MDA-MB-231 cells except that the ERE mutation did not affect promoter activity. Additionally, promoter activity was stimulated upon E2 treatment and overexpression of ER-α or NF-YA in MCF-7 cells. ChIP confirmed direct binding of both transcription factors to the UBC9 promoter in vivo. Furthermore, UBC9 expression was diminished by ER-α and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal UBC9 promoter and provided evidence that ER-α and NF-Y regulate UBC9 expression on the transcriptional level in response to E2 in MCF-7 cells. These findings may contribute to a better understanding of the regulation of UBC9 in ER

Progression of Mouse Skin Carcinogenesis Is Associated with Increased Erα Levels and Is Repressed by a Dominant Negative Form of Erα

PubMed Central

Michalopoulos, Ioannis; Sideridou, Maria; Tsimaratou, Katerina; Christodoulou, Ioannis; Pyrillou, Katerina; Gorgoulis, Vassilis; Vlahopoulos, Spiros; Zoumpourlis, Vassilis

2012-01-01

Estrogen receptors (ER), namely ERα and ERβ, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ERα in skin cancer, using chemically-induced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ERα is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ERβ levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ERα gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ERβ levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ERα (dnERα) resulted in reduced ERα levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ERα promoter through which dnERα decreased endogenous ERα levels. The dnERα-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ERα in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnERα. PMID:22870269

Characterization of a new ARID family transcription factor (Brightlike/ARID3C) that co-activates Bright/ARID3A-mediated immunoglobulin gene transcription

PubMed Central

Tidwell, Josephine A.; Schmidt, Christian; Heaton, Phillip; Wilson, Van; Tucker, Philip W.

2011-01-01

Two members, Bright/ARID3A and Bdp/ARID3B, of the ARID (AT-Rich Interaction Domain) transcription family are distinguished by their ability to specifically bind to DNA and to self-associate via a second domain, REKLES. Bright and Bdp positively regulate immunoglobulin heavy chain gene (IgH) transcription by binding to AT-rich motifs within Matrix Associating Regions (MARs) residing within a subset of VH promoters and the Eµ intronic enhancer. In addition, REKLES provides Bright nuclear export function, and a small pool of Bright is directed to plasma membrane sub-domains/lipid rafts where it associates with and modulates signaling of the B cell antigen receptor (BCR). Here, we characterize a third, highly conserved, physically condensed ARID3 locus, Brightlike/ARID3C. Brightlike encodes two alternatively spliced, SUMO-I-modified isoforms that include or exclude (Δ6) the REKLES-encoding exon 6. Brightlike transcripts and proteins are expressed preferentially within B lineage lymphocytes and coordinate with highest Bright expression--in activated follicular B cells. Brightlike, but not BrightlikeΔ6, undergoes nuclear-cytoplasmic shuttling with a fraction localizing within lipid rafts following BCR stimulation. Brightlike, but not BrightlikeΔ6, associates with Bright in solution, at common DNA binding sites in vitro, and is enriched at Bright binding sites in chromatin. Although possessing little transactivation capacity of its own, Brightlike significantly co-activates Bright-dependent IgH transcription with maximal activity mediated by the unsumoylated form. In sum, this report introduces Brightlike as an additional functional member of the family of ARID proteins, which should be considered in regulatory circuits, previously ascribed to be mediated by Bright. PMID:21955986

Intrapulpal Temperature Increase During Er:YAG Laser-Aided Debonding of Ceramic Brackets.

PubMed

Yilanci, Hilal; Yildirim, Zeynep Beyza; Ramoglu, Sabri Ilhan

2017-04-01

The purpose of this study was to evaluate the temperature changes in the pulp chamber while using a newly introduced application of Er:YAG laser to debond ceramic brackets in a study model with a pulpal circulation with and without thermocycled samples. An esthetic alternative to stainless steel brackets, ceramic brackets have been proposed. However, because of their low fracture resistance and high bond strengths, ceramic brackets can cause a problem when they are being removed using conventional techniques. Experimental Groups A and B were established for samples with or without thermocycling. The same 20 maxillary central incisor and 20 premolar teeth were used in both groups. Pulpal blood microcirculation was simulated using an apparatus described in a previous study. Monocrystalline brackets were bonded by using Transbond XT. In Group A, brackets were debonded using the Er:YAG laser (600 mJ, 2 Hz, long pulse, and no air or water spray) after being stored in distilled water for 24 h. In Group B, brackets were debonded using the same laser system as that used in Group A after being stored in distilled water for 24 h and then thermocycled for a total of 5000 cycles between 5°C and 55°C. The laser irradiation duration and intrapulpal temperature changes were measured. In Group B, the intrapulpal temperature increase of the central incisors was significantly higher than that of the premolar teeth. In the central incisor and premolar teeth groups, there were no statistically significant difference between Groups A and B (p > 0.05). A positive correlation was found between laser irradiation duration and temperature increase (p < 0.01). The use of Er:YAG laser is an effective method for debonding the monocrystalline ceramic brackets. This method can be used safely under the consideration of intrapulpal temperature changes.

Overcoming Endocrine Resistance by Targeting ER/FoxA1/IL 8 Axis

DTIC Science & Technology

2016-10-01

INTRODUCTION Approximately 75% of breast cancers express the hormone estrogen receptor α (ER). As a critical determinant in estrogen response and oncogenic...factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we...positive tumors. FOXA1 | estrogen receptor | breast cancer | transcriptional reprogramming | endocrine resistance About 75% of breast cancers express

Increased mitochondrial-encoded gene transcription in immortal DF-1 cells.

PubMed

Kim, H; You, S; Kim, I J; Farris, J; Foster, L K; Foster, D N

2001-05-01

We have established, in continuous cell culture, a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (DF-1) as well as several other immortal CEF cell lines. The immortal DF-1 cells divided more rapidly than primary and other immortal CEF cells. To identify the genes involved in rapidly dividing DF-1 cells, we have used differential display RT-PCR. Of the numerous genes analyzed, three mitochondrial-encoded genes (ATPase 8/6, 16S rRNA, and cytochrome b) were shown to express at higher levels in DF-1 cells compared to primary and other immortal CEF cells. The inhibition of mitochondrial translation by treatment with chloramphenicol markedly decreased ATP production and cell proliferation in DF-1 cells, while not affecting growth in either primary or other immortal CEF cells. This result suggests a correlation between rapid cell proliferation and the increased mitochondrial respiratory functions. We also determined that the increased transcription of mitochondrial-encoded genes in DF-1 cells is due to increased de novo transcript synthesis as shown by mitochondrial run-on assays, and not the result of either increased mitochondrial biogenesis or mitochondrial transcript half-lives. Together, the present studies suggest that the transcriptional activation of mitochondrial-encoded genes and the elevated respiratory function should be one of the characteristics of rapidly dividing immortal cells. Copyright 2001 Academic Press.

3' Untranslated regions mediate transcriptional interference between convergent genes both locally and ectopically in Saccharomyces cerevisiae.

PubMed

Wang, Luwen; Jiang, Ning; Wang, Lin; Fang, Ou; Leach, Lindsey J; Hu, Xiaohua; Luo, Zewei

2014-01-01

Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3'-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3'-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision

Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komoike, Yuta, E-mail: komoike@research.twmu.ac.jp

Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level ofmore » c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway. - Highlights: • Exposure of SH-SY5Y cells to AA activates the eIF2α–ATF4 pathway of the UPR. • Exposure of SH-SY5Y cells to AA induces the CHOP expression and apoptosis. • Exposure of zebrafish to AA induces the chop expression and apoptosis in the brain. • AA possibly induces apoptotic neuronal cell death through the

Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice

PubMed Central

Ai, Ding; Baez, Juan M.; Jiang, Hongfeng; Conlon, Donna M.; Hernandez-Ono, Antonio; Frank-Kamenetsky, Maria; Milstein, Stuart; Fitzgerald, Kevin; Murphy, Andrew J.; Woo, Connie W.; Strong, Alanna; Ginsberg, Henry N.; Tabas, Ira; Rader, Daniel J.; Tall, Alan R.

2012-01-01

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease. PMID:22466652

Auxin-Mediated Transcriptional System with a Minimal Set of Components Is Critical for Morphogenesis through the Life Cycle in Marchantia polymorpha

PubMed Central

Kato, Hirotaka; Ishizaki, Kimitsune; Kouno, Masaru; Shirakawa, Makoto; Bowman, John L.; Nishihama, Ryuichi; Kohchi, Takayuki

2015-01-01

The plant hormone auxin regulates many aspects of plant growth and development. Recent progress in Arabidopsis provided a scheme that auxin receptors, TIR1/AFBs, target transcriptional co-repressors, AUX/IAAs, for degradation, allowing ARFs to regulate transcription of auxin responsive genes. The mechanism of auxin-mediated transcriptional regulation is considered to have evolved around the time plants adapted to land. However, little is known about the role of auxin-mediated transcription in basal land plant lineages. We focused on the liverwort Marchantia polymorpha, which belongs to the earliest diverging lineage of land plants. M. polymorpha has only a single TIR1/AFB (MpTIR1), a single AUX/IAA (MpIAA), and three ARFs (MpARF1, MpARF2, and MpARF3) in the genome. Expression of a dominant allele of MpIAA with mutations in its putative degron sequence conferred an auxin resistant phenotype and repressed auxin-dependent expression of the auxin response reporter proGH3:GUS. We next established a system for DEX-inducible auxin-response repression by expressing the putatively stabilized MpIAA protein fused with the glucocorticoid receptor domain (MpIAAmDII-GR). Repression of auxin responses in proMpIAA:MpIAAmDII-GR plants caused severe defects in various developmental processes, including gemmaling development, dorsiventrality, organogenesis, and tropic responses. Transient transactivation assays showed that the three MpARFs had different transcriptional activities, each corresponding to their phylogenetic classifications. Moreover, MpIAA and MpARF proteins interacted with each other with different affinities. This study provides evidence that pleiotropic auxin responses can be achieved by a minimal set of auxin signaling factors and suggests that the transcriptional regulation mediated by TIR1/AFB, AUX/IAA, and three types of ARFs might have been a key invention to establish body plans of land plants. We propose that M. polymorpha is a good model to investigate the

Auxin-Mediated Transcriptional System with a Minimal Set of Components Is Critical for Morphogenesis through the Life Cycle in Marchantia polymorpha.

PubMed

Kato, Hirotaka; Ishizaki, Kimitsune; Kouno, Masaru; Shirakawa, Makoto; Bowman, John L; Nishihama, Ryuichi; Kohchi, Takayuki

2015-05-01

The plant hormone auxin regulates many aspects of plant growth and development. Recent progress in Arabidopsis provided a scheme that auxin receptors, TIR1/AFBs, target transcriptional co-repressors, AUX/IAAs, for degradation, allowing ARFs to regulate transcription of auxin responsive genes. The mechanism of auxin-mediated transcriptional regulation is considered to have evolved around the time plants adapted to land. However, little is known about the role of auxin-mediated transcription in basal land plant lineages. We focused on the liverwort Marchantia polymorpha, which belongs to the earliest diverging lineage of land plants. M. polymorpha has only a single TIR1/AFB (MpTIR1), a single AUX/IAA (MpIAA), and three ARFs (MpARF1, MpARF2, and MpARF3) in the genome. Expression of a dominant allele of MpIAA with mutations in its putative degron sequence conferred an auxin resistant phenotype and repressed auxin-dependent expression of the auxin response reporter proGH3:GUS. We next established a system for DEX-inducible auxin-response repression by expressing the putatively stabilized MpIAA protein fused with the glucocorticoid receptor domain (MpIAA(mDII)-GR). Repression of auxin responses in (pro)MpIAA:MpIAA(mDII)-GR plants caused severe defects in various developmental processes, including gemmaling development, dorsiventrality, organogenesis, and tropic responses. Transient transactivation assays showed that the three MpARFs had different transcriptional activities, each corresponding to their phylogenetic classifications. Moreover, MpIAA and MpARF proteins interacted with each other with different affinities. This study provides evidence that pleiotropic auxin responses can be achieved by a minimal set of auxin signaling factors and suggests that the transcriptional regulation mediated by TIR1/AFB, AUX/IAA, and three types of ARFs might have been a key invention to establish body plans of land plants. We propose that M. polymorpha is a good model to investigate

Increased global transcription activity as a mechanism of replication stress in cancer

PubMed Central

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M.; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-01-01

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. PMID:27725641

Increased global transcription activity as a mechanism of replication stress in cancer.

PubMed

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-10-11

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRAS V12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRAS V12 , elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs).

PubMed

Wang, Li; Zhang, Yi; Wang, Weiguo; Zhu, Yunjie; Chen, Yang; Tian, Bole

2017-01-01

-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs. Gemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs)

PubMed Central

Wang, Weiguo; Zhu, Yunjie; Chen, Yang

2017-01-01

interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs. Conclusion Gemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed. PMID:28854261

Arabidopsis CRY2 and ZTL mediate blue-light regulation of the transcription factor CIB1 by distinct mechanisms

PubMed Central

Liu, Hongtao; Wang, Qin; Liu, Yawen; Zhao, Xiaoying; Imaizumi, Takato; Somers, David E.; Tobin, Elaine M.; Lin, Chentao

2013-01-01

Plants possess multiple photoreceptors to mediate light regulation of growth and development, but it is not well understood how different photoreceptors coordinate their actions to jointly regulate developmental responses, such as flowering time. In Arabidopsis, the photoexcited cryptochrome 2 interacts with the transcription factor CRYPTOCHROME-INTERACTING basic helix–loop–helix 1 (CIB1) to activate transcription and floral initiation. We show that the CIB1 protein expression is regulated by blue light; CIB1 is highly expressed in plants exposed to blue light, but levels of the CIB1 protein decreases in the absence of blue light. We demonstrate that CIB1 is degraded by the 26S proteasome and that blue light suppresses CIB1 degradation. Surprisingly, although cryptochrome 2 physically interacts with CIB1 in response to blue light, it is not the photoreceptor mediating blue-light suppression of CIB1 degradation. Instead, two of the three light–oxygen–voltage (LOV)-domain photoreceptors, ZEITLUPE and LOV KELCH PROTEIN 2, but not FLAVIN-BINDING KELCH REPEAT 1, are required for the function and blue-light suppression of degradation of CIB1. These results support the hypothesis that the evolutionarily unrelated blue-light receptors, cryptochrome and LOV-domain F-box proteins, mediate blue-light regulation of the same transcription factor by distinct mechanisms. PMID:24101505

VirF-Independent Regulation of Shigella virB Transcription is Mediated by the Small RNA RyhB

PubMed Central

Broach, William H.; Egan, Nicholas; Wing, Helen J.; Payne, Shelley M.; Murphy, Erin R.

2012-01-01

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression. PMID:22701677

Lipocalin 2, a new GADD153 target gene, as an apoptosis inducer of endoplasmic reticulum stress in lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsin, I-Lun; Hsiao, Yueh-Chieh; Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan

2012-09-15

Endoplasmic reticulum (ER) stress is activated under severe cellular conditions. GADD153, a member of the C/EBP family, is an unfolded protein response (UPR) responsive transcription factor. Increased levels of lipocalin 2, an acute phase protein, have been found in several epithelial cancers. The aim of this study is to investigate the function of lipocalin 2 in lung cancer cells under ER stress. Treatment with thapsigargin, an ER stress activator, led to increases in cytotoxicity, ER stress, apoptosis, and lipocalin 2 expression in A549 cells. GADD153 silencing decreased lipocalin 2 expression in A549 cells. On chromatin immunoprecipitation assay, ER stress increasedmore » GADD153 DNA binding to lipocalin 2 promoter. Furthermore, silencing of lipocalin 2 mitigated ER stress-mediated apoptosis in A549 cells. Our findings demonstrated that lipocalin 2 is a new GADD153 target gene that mediates ER stress-induced apoptosis. Highlights: ► We demonstrate that Lipocalin 2 is a new GADD153 target gene. ► Lipocalin 2 mediates ER stress-induced apoptosis. ► ER stress-induced lipocalin 2 expression is calcium-independent in A549 cells. ► Lipocalin 2 dose not play a major role in ER stress-induced autophagy.« less

Dioxin Toxicity In Vivo Results from an Increase in the Dioxin-Independent Transcriptional Activity of the Aryl Hydrocarbon Receptor

PubMed Central

Céspedes, Miguel Angel; Galindo, Maximo Ibo; Couso, Juan Pablo

2010-01-01

The Aryl hydrocarbon receptor (Ahr) is the nuclear receptor mediating the toxicity of dioxins -widespread and persistent pollutants whose toxic effects include tumor promotion, teratogenesis, wasting syndrome and chloracne. Elimination of Ahr in mice eliminates dioxin toxicity but also produces adverse effects, some seemingly unrelated to dioxin. Thus the relationship between the toxic and dioxin-independent functions of Ahr is not clear, which hampers understanding and treatment of dioxin toxicity. Here we develop a Drosophila model to show that dioxin actually increases the in vivo dioxin-independent activity of Ahr. This hyperactivation resembles the effects caused by an increase in the amount of its dimerisation partner Ahr nuclear translocator (Arnt) and entails an increased transcriptional potency of Ahr, in addition to the previously described effect on nuclear translocation. Thus the two apparently different functions of Ahr, dioxin-mediated and dioxin-independent, are in fact two different levels (hyperactivated and basal, respectively) of a single function. PMID:21079739

All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation.

PubMed

Fontán-Gabás, Lorena; Oliemuller, Erik; Martínez-Irujo, Juan José; de Miguel, Carlos; Rouzaut, Ana

2007-01-01

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.

Hypothalamic AMPK-ER Stress-JNK1 Axis Mediates the Central Actions of Thyroid Hormones on Energy Balance.

PubMed

Martínez-Sánchez, Noelia; Seoane-Collazo, Patricia; Contreras, Cristina; Varela, Luis; Villarroya, Joan; Rial-Pensado, Eva; Buqué, Xabier; Aurrekoetxea, Igor; Delgado, Teresa C; Vázquez-Martínez, Rafael; González-García, Ismael; Roa, Juan; Whittle, Andrew J; Gomez-Santos, Beatriz; Velagapudi, Vidya; Tung, Y C Loraine; Morgan, Donald A; Voshol, Peter J; Martínez de Morentin, Pablo B; López-González, Tania; Liñares-Pose, Laura; Gonzalez, Francisco; Chatterjee, Krishna; Sobrino, Tomás; Medina-Gómez, Gema; Davis, Roger J; Casals, Núria; Orešič, Matej; Coll, Anthony P; Vidal-Puig, Antonio; Mittag, Jens; Tena-Sempere, Manuel; Malagón, María M; Diéguez, Carlos; Martínez-Chantar, María Luz; Aspichueta, Patricia; Rahmouni, Kamal; Nogueiras, Rubén; Sabio, Guadalupe; Villarroya, Francesc; López, Miguel

2017-07-05

Thyroid hormones (THs) act in the brain to modulate energy balance. We show that central triiodothyronine (T3) regulates de novo lipogenesis in liver and lipid oxidation in brown adipose tissue (BAT) through the parasympathetic (PSNS) and sympathetic nervous system (SNS), respectively. Central T3 promotes hepatic lipogenesis with parallel stimulation of the thermogenic program in BAT. The action of T3 depends on AMP-activated protein kinase (AMPK)-induced regulation of two signaling pathways in the ventromedial nucleus of the hypothalamus (VMH): decreased ceramide-induced endoplasmic reticulum (ER) stress, which promotes BAT thermogenesis, and increased c-Jun N-terminal kinase (JNK) activation, which controls hepatic lipid metabolism. Of note, ablation of AMPKα1 in steroidogenic factor 1 (SF1) neurons of the VMH fully recapitulated the effect of central T3, pointing to this population in mediating the effect of central THs on metabolism. Overall, these findings uncover the underlying pathways through which central T3 modulates peripheral metabolism. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Nfatc1 Is a Functional Transcriptional Factor Mediating Nell-1-Induced Runx3 Upregulation in Chondrocytes.

PubMed

Li, Chenshuang; Zheng, Zhong; Zhang, Xinli; Asatrian, Greg; Chen, Eric; Song, Richard; Culiat, Cymbeline; Ting, Kang; Soo, Chia

2018-01-06

Neural EGFL like 1 (Nell-1) is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1's pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3)-mediated Indian hedgehog (Ihh) signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1) as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the -833--810 region of the Runx3 -promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

A Self-defeating Anabolic Program Leads to β-Cell Apoptosis in Endoplasmic Reticulum Stress-induced Diabetes via Regulation of Amino Acid Flux*

PubMed Central

Krokowski, Dawid; Han, Jaeseok; Saikia, Mridusmita; Majumder, Mithu; Yuan, Celvie L.; Guan, Bo-Jhih; Bevilacqua, Elena; Bussolati, Ovidio; Bröer, Stefan; Arvan, Peter; Tchórzewski, Marek; Snider, Martin D.; Puchowicz, Michelle; Croniger, Colleen M.; Kimball, Scot R.; Pan, Tao; Koromilas, Antonis E.; Kaufman, Randal J.; Hatzoglou, Maria

2013-01-01

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes. PMID:23645676

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

PubMed

Gallagher, Thomas L; Tietz, Kiel T; Morrow, Zachary T; McCammon, Jasmine M; Goldrich, Michael L; Derr, Nicolas L; Amacher, Sharon L

2017-09-01

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased anxiety and synaptic plasticity in estrogen receptor -deficient mice

NASA Astrophysics Data System (ADS)

Krel, Wojciech; Dupont, Sonia; Krust, Andrée; Chambon, Pierre; Chapman, Paul F.

2001-10-01

Estrogens are powerful modulators of neuronal physiology and in humans may affect a broad range of functions, including reproductive, emotional, and cognitive behaviors. We studied the contribution of estrogen receptors (ERs) in modulation of emotional processes and analyzed the effects of deleting ER or ER in mice. Behavior consistent with increased anxiety was observed principally in ER mutant females and was associated with a reduced threshold for the induction of synaptic plasticity in the basolateral amygdala. Local increase of 5-hydroxytryptamine 1a receptor expression inmedial amygdala may contribute to these changes. Our data show that, particularly in females, there is an important role for ERβ-mediated estrogen signaling in the processing of emotional behavior.

Cellular Insulin Resistance Disrupts Leptin-Mediated Control of Neuronal Signaling and Transcription

PubMed Central

Nazarians-Armavil, Anaies; Menchella, Jonathan A.

2013-01-01

Central resistance to the actions of insulin and leptin is associated with the onset of obesity and type 2 diabetes mellitus, whereas leptin and insulin signaling is essential for both glucose and energy homeostasis. Although it is known that leptin resistance can lead to attenuated insulin signaling, whether insulin resistance can lead to or exacerbate leptin resistance is unknown. To investigate the molecular events underlying crosstalk between these signaling pathways, immortalized hypothalamic neuronal models, rHypoE-19 and mHypoA-2/10, were used. Prolonged insulin exposure was used to induce cellular insulin resistance, and thereafter leptin-mediated regulation of signal transduction and gene expression was assessed. Leptin directly repressed agouti-related peptide mRNA levels but induced urocortin-2, insulin receptor substrate (IRS)-1, IRS2, and IR transcription, through leptin-mediated phosphatidylinositol 3-kinase/Akt activation. Neuronal insulin resistance, as assessed by attenuated Akt phosphorylation, blocked leptin-mediated signal transduction and agouti-related peptide, urocortin-2, IRS1, IRS2, and insulin receptor synthesis. Insulin resistance caused a substantial decrease in insulin receptor protein levels, forkhead box protein 1 phosphorylation, and an increase in suppressor of cytokine signaling 3 protein levels. Cellular insulin resistance may cause or exacerbate neuronal leptin resistance and, by extension, obesity. It is essential to unravel the effects of neuronal insulin resistance given that both peripheral, as well as the less widely studied central insulin resistance, may contribute to the development of metabolic, reproductive, and cardiovascular disorders. This study provides improved understanding of the complex cellular crosstalk between insulin-leptin signal transduction that is disrupted during neuronal insulin resistance. PMID:23579487

Intestinal IRE1 Is Required for Increased Triglyceride Metabolism and Longer Lifespan under Dietary Restriction.

PubMed

Luis, Nuno Miguel; Wang, Lifen; Ortega, Mauricio; Deng, Hansong; Katewa, Subhash D; Li, Patrick Wai-Lun; Karpac, Jason; Jasper, Heinrich; Kapahi, Pankaj

2016-10-25

Dietary restriction (DR) is one of the most robust lifespan-extending interventions in animals. The beneficial effects of DR involve a metabolic adaptation toward increased triglyceride usage. The regulatory mechanism and the tissue specificity of this metabolic switch remain unclear. Here, we show that the IRE1/XBP1 endoplasmic reticulum (ER) stress signaling module mediates metabolic adaptation upon DR in flies by promoting triglyceride synthesis and accumulation in enterocytes (ECs) of the Drosophila midgut. Consistently, IRE1/XBP1 function in ECs is required for increased longevity upon DR. We further identify sugarbabe, a Gli-like zinc-finger transcription factor, as a key mediator of the IRE1/XBP1-regulated induction of de novo lipogenesis in ECs. Overexpression of sugarbabe rescues metabolic and lifespan phenotypes of IRE1 loss-of-function conditions. Our study highlights the critical role of metabolic adaptation of the intestinal epithelium for DR-induced lifespan extension and explores the IRE1/XBP1 signaling pathway regulating this adaptation and influencing lifespan. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Quantifying the Role of Circulating Unconjugated Estradiol in Mediating the Body Mass Index-Breast Cancer Association.

PubMed

Schairer, Catherine; Fuhrman, Barbara J; Boyd-Morin, Jennifer; Genkinger, Jeanine M; Gail, Mitchell H; Hoover, Robert N; Ziegler, Regina G

2016-01-01

Higher body mass index (BMI) and circulating estrogen levels each increase postmenopausal breast cancer risk, particularly estrogen receptor-positive (ER(+)) tumors. Higher BMI also increases estrogen production. We estimated the proportion of the BMI-ER(+) breast cancer association mediated through estrogen in a case-control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Participants included 143 women with invasive ER(+) breast cancer and 268 matched controls, all postmenopausal and never having used hormone therapy at baseline. We used liquid chromatography-tandem mass spectrometry to measure 15 estrogens and estrogen metabolites in baseline serum. We calculated BMI from self-reported height and weight at baseline. We estimated the mediating effect of unconjugated estradiol on the BMI-ER(+) breast cancer association using Aalen additive hazards and Cox regression models. All estrogens and estrogen metabolites were statistically significantly correlated with BMI, with unconjugated estradiol most strongly correlated [Pearson correlation (r) = 0.45]. Approximately 7% to 10% of the effect of overweight, 12% to 15% of the effect of obesity, and 19% to 20% of the effect of a 5 kg/m(2) BMI increase on ER(+) breast cancer risk was mediated through unconjugated estradiol. The BMI-breast cancer association, once adjusted for unconjugated estradiol, was not modified by further adjustment for two metabolic ratios statistically significantly associated with both breast cancer and BMI. Circulating unconjugated estradiol levels partially mediate the BMI-breast cancer association, but other potentially important estrogen mediators (e.g., bioavailable estradiol) were not evaluated. Further research is required to identify mechanisms underlying the BMI-breast cancer association. ©2015 American Association for Cancer Research.

AP-1 mediated transcriptional repression of matrix metalloproteinase-9 by recruitment of histone deacetylase 1 in response to interferon β.

PubMed

Mittelstadt, Megan L; Patel, Rekha C

2012-01-01

Matrix metalloproteinase-9 (MMP-9) is a 92 kDa zinc-dependant endopeptidase that degrades components of the extracellular matrix. Increased expression of MMP-9 is implicated in many pathological conditions including metastatic cancer, multiple sclerosis, and atherosclerosis. Although it has been widely noted that interferon-β (IFNβ) downregulates both the basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression at the transcriptional level, the molecular mechanism of this repression is poorly understood. In the present study we identify a novel mechanism for repression of MMP-9 transcription by IFNβ in HT1080 fibrosarcoma cells. Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site. Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment. ChIP analysis shows that IFNβ induced HDAC1 recruitment to the MMP-9 promoter and IFNβ mediated transcriptional repression is lost when the AP-1 binding site is inactivated by a point mutation. Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

PubMed

Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

2013-03-01

Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Long non-coding RNA MIAT is estrogen-responsive and promotes estrogen-induced proliferation in ER-positive breast cancer cells.

PubMed

Li, Yuehua; Jiang, Baohong; Wu, Xiaoping; Huang, Qin; Chen, Wenqi; Zhu, Hongbo; Qu, Xiaofei; Xie, Liming; Ma, Xin; Huang, Guo

2018-05-21

Estrogen drives the development and progression of estrogen receptor (ER)-positive breast cancer. However, the detailed mechanism underlying ER-driven carcinogenesis remains unclear despite extensive studies. Previously reports indicated higher expression of long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) in ER-positive breast cancer tissues than in ER-negative tissues. However, the functional relevance of MIAT in ER-positive breast cancer tumorigenesis was poorly understood. Here, we investigated the role of lncRNA MIAT in ER-positive breast cancer cells. MIAT was over-expressed in ER-positive breast cancer tissues and ER-positive breast cancer cell line MCF-7. Activating estrogen signaling by diethylstilbestrol (DES) led to a dose- and time-dependent up-regulation of MIAT in MCF-7 cells that was dependent on ERα, as evidenced by ERα silencing and pharmacological inhibition using ER antagonist ICI 182780. Silencing MIAT decreased DES-induced MCF-7 cell proliferation while overexpressing MIAT increased MCF-7 cell proliferation. Further mechanistic study identified that MIAT was critical for G1 to S phase cell cycle transition. Taken together, these results suggest that lncRNA MIAT is an estrogen-inducible lncRNA and a key regulator in ER-positive breast cancer cell growth. MIAT could serve as a potential biomarker and promising therapeutic target for ER-positive breast cancer. Copyright © 2018. Published by Elsevier Inc.

Icaritin enhances mESC self-renewal through upregulating core pluripotency transcription factors mediated by ERα

PubMed Central

Tsang, Wing Pui; Zhang, Fengjie; He, Qiling; Cai, Waijiao; Huang, Jianhua; Chan, Wai Yee; Shen, Ziyin; Wan, Chao

2017-01-01

Utilization of small molecules in modulation of stem cell self-renewal is a promising approach to expand stem cells for regenerative therapy. Here, we identify Icaritin, a phytoestrogen molecule enhances self-renewal of mouse embryonic stem cells (mESCs). Icaritin increases mESCs proliferation while maintains their self-renewal capacity in vitro and pluripotency in vivo. This coincides with upregulation of key pluripotency transcription factors OCT4, NANOG, KLF4 and SOX2. The enhancement of mESCs self-renewal is characterized by increased population in S-phase of cell cycle, elevation of Cylin E and Cyclin-dependent kinase 2 (CDK2) and downregulation of p21, p27 and p57. PCR array screening reveals that caudal-related homeobox 2 (Cdx2) and Rbl2/p130 are remarkably suppressed in mESCs treated with Icaritin. siRNA knockdown of Cdx2 or Rbl2/p130 upregulates the expression of Cyclin E, OCT4 and SOX2, and subsequently increases cell proliferation and colony forming efficiency of mESCs. We then demonstrate that Icaritin co-localizes with estrogen receptor alpha (ERα) and activates its nuclear translocation in mESCs. The promotive effect of Icaritin on cell cycle and pluripotency regulators are eliminated by siRNA knockdown of ERα in mESCs. The results suggest that Icaritin enhances mESCs self-renewal by regulating cell cycle machinery and core pluripotency transcription factors mediated by ERα. PMID:28091581

Icaritin enhances mESC self-renewal through upregulating core pluripotency transcription factors mediated by ERα.

PubMed

Tsang, Wing Pui; Zhang, Fengjie; He, Qiling; Cai, Waijiao; Huang, Jianhua; Chan, Wai Yee; Shen, Ziyin; Wan, Chao

2017-01-16

Utilization of small molecules in modulation of stem cell self-renewal is a promising approach to expand stem cells for regenerative therapy. Here, we identify Icaritin, a phytoestrogen molecule enhances self-renewal of mouse embryonic stem cells (mESCs). Icaritin increases mESCs proliferation while maintains their self-renewal capacity in vitro and pluripotency in vivo. This coincides with upregulation of key pluripotency transcription factors OCT4, NANOG, KLF4 and SOX2. The enhancement of mESCs self-renewal is characterized by increased population in S-phase of cell cycle, elevation of Cylin E and Cyclin-dependent kinase 2 (CDK2) and downregulation of p21, p27 and p57. PCR array screening reveals that caudal-related homeobox 2 (Cdx2) and Rbl2/p130 are remarkably suppressed in mESCs treated with Icaritin. siRNA knockdown of Cdx2 or Rbl2/p130 upregulates the expression of Cyclin E, OCT4 and SOX2, and subsequently increases cell proliferation and colony forming efficiency of mESCs. We then demonstrate that Icaritin co-localizes with estrogen receptor alpha (ERα) and activates its nuclear translocation in mESCs. The promotive effect of Icaritin on cell cycle and pluripotency regulators are eliminated by siRNA knockdown of ERα in mESCs. The results suggest that Icaritin enhances mESCs self-renewal by regulating cell cycle machinery and core pluripotency transcription factors mediated by ERα.

Mouse Stbd1 is N-myristoylated and affects ER-mitochondria association and mitochondrial morphology.

PubMed

Demetriadou, Anthi; Morales-Sanfrutos, Julia; Nearchou, Marianna; Baba, Otto; Kyriacou, Kyriacos; Tate, Edward W; Drousiotou, Anthi; Petrou, Petros P

2017-03-01

Starch binding domain-containing protein 1 (Stbd1) is a carbohydrate-binding protein that has been proposed to be a selective autophagy receptor for glycogen. Here, we show that mouse Stbd1 is a transmembrane endoplasmic reticulum (ER)-resident protein with the capacity to induce the formation of organized ER structures in HeLa cells. In addition to bulk ER, Stbd1 was found to localize to mitochondria-associated membranes (MAMs), which represent regions of close apposition between the ER and mitochondria. We demonstrate that N -myristoylation and binding of Stbd1 to glycogen act as major determinants of its subcellular targeting. Moreover, overexpression of non-myristoylated Stbd1 enhanced the association between ER and mitochondria, and further induced prominent mitochondrial fragmentation and clustering. Conversely, shRNA-mediated Stbd1 silencing resulted in an increase in the spacing between ER and mitochondria, and an altered morphology of the mitochondrial network, suggesting elevated fusion and interconnectivity of mitochondria. Our data unravel the molecular mechanism underlying Stbd1 subcellular targeting, support and expand its proposed function as a selective autophagy receptor for glycogen and uncover a new role for the protein in the physical association between ER and mitochondria. © 2017. Published by The Company of Biologists Ltd.

Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

PubMed

Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

2016-04-01

Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stars and Symbiosis: MicroRNA- and MicroRNA*-Mediated Transcript Cleavage Involved in Arbuscular Mycorrhizal Symbiosis1[W][OA

PubMed Central

Devers, Emanuel A.; Branscheid, Anja; May, Patrick; Krajinski, Franziska

2011-01-01

The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out high-throughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development. PMID:21571671

3′ Untranslated Regions Mediate Transcriptional Interference between Convergent Genes Both Locally and Ectopically in Saccharomyces cerevisiae

PubMed Central

Wang, Luwen; Jiang, Ning; Wang, Lin; Fang, Ou; Leach, Lindsey J.; Hu, Xiaohua; Luo, Zewei

2014-01-01

Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3′-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3′-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision

Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

PubMed

Lewis, Brian A

2010-01-15

The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

Mediator of DNA damage checkpoint 1 (MDC1) contributes to high NaCl-induced activation of the osmoprotective transcription factor TonEBP/OREBP.

PubMed

Kunin, Margarita; Dmitrieva, Natalia I; Gallazzini, Morgan; Shen, Rong-Fong; Wang, Guanghui; Burg, Maurice B; Ferraris, Joan D

2010-08-11

Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.

The association of TIF-IA and polymerase I mediates promoter recruitment and regulation of ribosomal RNA transcription in Acanthamoeba castellanii.

PubMed

Gogain, Joseph C; Paule, Marvin R

2005-01-01

Large amounts of energy are expended for the construction of the ribosome during both transcription and processing, so it is of utmost importance for the cell to efficiently regulate ribosome production. Understanding how this regulation occurs will provide important insights into cellular growth control and into the coordination of gene expression mediated by all three transcription systems. Ribosomal RNA (rRNA) transcription rates closely parallel the need for protein synthesis; as a cell approaches stationary phase or encounters conditions that negatively affect either growth rate or protein synthesis, rRNA transcription is decreased. In eukaryotes, the interaction of RNA polymerase I (pol I) with the essential transcription initiation factor IA (TIF-IA) has been implicated in this downregulation of transcription. In agreement with the first observation that rRNA transcription is regulated by altering recruitment of pol I to the promoter in Acanthamoeba castellanii, we show here that pol I and an 80-kDa homologue of TIF-IA are found tightly associated in pol I fractions competent for specific transcription. Disruption of the pol I-TIF-IA complex is mediated by a specific dephosphorylation of either pol I or TIF-IA. Phosphatase treatment of TIF-IA-containing A. castellanii pol I fractions results in a downregulation of both transcriptional activity and promoter binding, reminiscent of the inactive pol I fractions purified from encysted cells. The fraction of pol I competent for promoter recruitment is enriched in TIF-IA relative to that not bound by immobilized promoter DNA. This downregulation coincides with an altered electrophoretic mobility of TIF-IA, suggesting at least it is phosphorylated.

TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis.

PubMed

Wu, Haibo; Wang, Yongsheng; Zhang, Yan; Yang, Mingqi; Lv, Jiaxing; Liu, Jun; Zhang, Yong

2015-03-31

Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of Mycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein-Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis.

Thermotolerance induced at a mild temperature of 40°C alleviates heat shock-induced ER stress and apoptosis in HeLa cells.

PubMed

Bettaieb, Ahmed; Averill-Bates, Diana A

2015-01-01

Hyperthermia (39-45°C) has emerged as an alternate prospect for cancer therapy in combination with radiation and chemotherapy. Despite promising progress in the clinic, molecular mechanisms involved in hyperthermia-induced cell death are not clear. Hyperthermia causes protein denaturation/aggregation, which results in cell death by apoptosis and/or necrosis. Hyperthermia also induces thermotolerance, which renders cells resistant to subsequent exposure to lethal heat shock. This study investigates the role of both lethal (42-43°C) and mild (40°C) hyperthermia in regulating ER stress and ER stress-induced apoptosis in HeLa cells. The ability of mild thermotolerance induced at 40°C to alleviate either or both of these processes is also determined. Hyperthermia (42-43°C) induced ER stress, revealed by phosphorylation of PERK, eIF2α and IRE1α, cleavage of ATF6 and increased expression of BiP and sXBP1. Real-time PCR revealed that mRNA levels of ATF6, ATF4, BiP, sXBP1 and CHOP increased in cells exposed to hyperthermia. Moreover, hyperthermia caused disruption of calcium homeostasis and activated the calpain-calpastatin proteolytic system and ER resident caspase 4. Pre-exposure to mild hyperthermia (40°C) alleviated the induction of cytotoxicity and ER stress by hyperthermia (42-43°C) and protected cells against ER stress-induced apoptosis. ShRNA-mediated depletion of Hsp72 abrogated protective effects of mild thermotolerance (40°C) against heat-shock induced ER stress and sensitized cells to ER stress-mediated apoptosis. Our findings show that Hsp72 contributes to the protective effects of mild hyperthermia (40°C) against hyperthermia-induced ER stress and apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development.

PubMed

Al-Shanti, Nasser; Stewart, Claire E

2009-11-01

The loss of muscle mass with age and disuse has a significant impact on the physiological and social well-being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross-sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age-related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin-like growth factor I (IGF-I) and Ca(2+)/calmodulin-dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF-I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca(2+)/calmodulin-dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T-cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca(2+) sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca(2+)/calmodulin-dependent kinases

Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation.

PubMed

Frabutt, Dylan A; Wang, Bin; Riaz, Sana; Schwartz, Richard C; Zheng, Yong-Hui

2018-01-01

Innate immunity provides an immediate defense against infection after host cells sense danger signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I α-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as "nonself" or misfolded protein and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication. IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used IAV to investigate how host innate immunity is activated. We found that IAV HA glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I α-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins

Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways.

PubMed

Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

2015-01-01

Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia.

Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

PubMed Central

Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

2015-01-01

Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia. PMID:26157452

Arctigenin alleviates ER stress via activating AMPK

PubMed Central

Gu, Yuan; Sun, Xiao-xiao; Ye, Ji-ming; He, Li; Yan, Shou-sheng; Zhang, Hao-hao; Hu, Li-hong; Yuan, Jun-ying; Yu, Qiang

2012-01-01

Aim: To investigate the protective effects of arctigenin (ATG), a phenylpropanoid dibenzylbutyrolactone lignan from Arctium lappa L (Compositae), against ER stress in vitro and the underlying mechanisms. Methods: A cell-based screening assay for ER stress regulators was established. Cell viability was measured using MTT assay. PCR and Western blotting were used to analyze gene and protein expression. Silencing of the CaMKKβ, LKB1, and AMPKα1 genes was achieved by RNA interference (RNAi). An ATP bioluminescent assay kit was employed to measure the intracellular ATP levels. Results: ATG (2.5, 5 and 10 μmol/L) inhibited cell death and unfolded protein response (UPR) in a concentration-dependent manner in cells treated with the ER stress inducer brefeldin A (100 nmol/L). ATG (1, 5 and 10 μmol/L) significantly attenuated protein synthesis in cells through inhibiting mTOR-p70S6K signaling and eEF2 activity, which were partially reversed by silencing AMPKα1 with RNAi. ATG (1-50 μmol/L) reduced intracellular ATP level and activated AMPK through inhibiting complex I-mediated respiration. Pretreatment of cells with the AMPK inhibitor compound C (25 μmol/L) rescued the inhibitory effects of ATG on ER stress. Furthermore, ATG (2.5 and 5 μmol/L) efficiently activated AMPK and reduced the ER stress and cell death induced by palmitate (2 mmol/L) in INS-1 β cells. Conclusion: ATG is an effective ER stress alleviator, which protects cells against ER stress through activating AMPK, thus attenuating protein translation and reducing ER load. PMID:22705729

The role of Nb in intensity increase of Er ion upconversion luminescence in zirconia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smits, K., E-mail: smits@cfi.lu.lv; Sarakovskis, A.; Grigorjeva, L.

2014-06-07

It is found that Nb co-doping increases the luminescence and upconversion luminescence intensity in rare earth doped zirconia. Er and Yb-doped nanocrystalline samples with or without Nb co-doping were prepared by sol-gel method and thermally annealed to check for the impact of phase transition on luminescence properties. Phase composition and grain sizes were examined by X-ray diffraction; the morphology was checked by scanning- and high-resolution transmission electron microscopes. Both steady-state and time-resolved luminescence were studied. Comparison of samples with different oxygen vacancy concentrations and different Nb concentrations confirmed the known assumption that oxygen vacancies are the main agents for tetragonalmore » or cubic phase stabilization. The oxygen vacancies quench the upconversion luminescence; however, they also prevent agglomeration of rare-earth ions and/or displacement of rare-earth ions to grain surfaces. It is found that co-doping with Nb ions significantly (>20 times) increases upconversion luminescence intensity. Hence, ZrO{sub 2}:Er:Yb:Nb nanocrystals may show promise for upconversion applications.« less

ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

PubMed

Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis

2017-06-01

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.

Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

PubMed Central

MacVicar, Thomas D. B.; Mannack, Lilith V. J. C.; Lees, Robert M.; Lane, Jon D.

2015-01-01

Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis. PMID:26110381

The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation.

PubMed

Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

2013-07-01

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells.

Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

PubMed Central

Rojas, Cristina; Pan-Castillo, Belén; Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Arola, Lluis; Mulero, Miquel

2014-01-01

Background Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines. Principal Findings We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity. Conclusions Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.

PubMed

Dejeans, Nicolas; Tajeddine, Nicolas; Beck, Raphaël; Verrax, Julien; Taper, Henryk; Gailly, Philippe; Calderon, Pedro Buc

2010-05-01

Increase in cytosolic calcium concentration ([Ca2+](c)), release of endoplasmic reticulum (ER) calcium ([Ca2+](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca2+](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca2+](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca2+](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca2+](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca2+](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells. 2009 Elsevier Inc. All rights reserved.

Sensitizing properties of luminescence centers on the emission of Er3+ in Si-rich SiO2 film

NASA Astrophysics Data System (ADS)

Fu, Qianyu; Gao, Yuhan; Li, Dongsheng; Yang, Deren

2016-05-01

In this paper, we report on the luminescence-center (LC)-mediated excitation of Er3+ as a function of annealing temperature in Er-doped Si-rich SiO2 (SRO) films fabricated by electron beam evaporation. It is found that the annealing temperature has significant effects on the emission of Er3+ and the specific optical-active point-defects called LCs within Er-doped SRO films. Different luminescence centers generated by the evolution of microstructures during annealing process act as efficient sensitizers for Er3+ in the films when the annealing temperature is below 1100 °C. Moreover, the temperature dependence of the energy coupling between LCs and Er3+ demonstrates the effective phonon-mediated energy transfer process. In addition, when the annealing temperature reaches 1100 °C, the decreased density of activable erbium ions induced by the aggregation of Er will bring detrimental effects on the emission of Er3+. It is demonstrated that an appropriate annealing process can be designed to achieve efficiently enhanced emissions from Er3+ ions by optimizing the density of LCs and the coupling between Er3+ and LCs.

TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis

PubMed Central

Wu, Haibo; Wang, Yongsheng; Zhang, Yan; Yang, Mingqi; Lv, Jiaxing; Liu, Jun; Zhang, Yong

2015-01-01

Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of Mycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein–Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis. PMID:25733846

ER-plasma membrane junctions: Why and how do we study them?

PubMed

Chang, Chi-Lun; Chen, Yu-Ju; Liou, Jen

2017-09-01

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are membrane microdomains important for communication between the ER and the PM. ER-PM junctions were first reported in muscle cells in 1957, but mostly ignored in non-excitable cells due to their scarcity and lack of functional significance. In 2005, the discovery of stromal interaction molecule 1 (STIM1) mediating a universal Ca 2+ feedback mechanism at ER-PM junctions in mammalian cells led to a resurgence of research interests toward ER-PM junctions. In the past decade, several major advancements have been made in this emerging topic in cell biology, including the generation of tools for labeling ER-PM junctions and the unraveling of mechanisms underlying regulation and functions of ER-PM junctions. This review summarizes early studies, recently developed tools, and current advances in the characterization and understanding of ER-PM junctions. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcriptional activation of transposable elements in mouse zygotes is independent of Tet3-mediated 5-methylcytosine oxidation.

PubMed

Inoue, Azusa; Matoba, Shogo; Zhang, Yi

2012-12-01

The methylation state of the paternal genome is rapidly reprogrammed shortly after fertilization. Recent studies have revealed that loss of 5-methylcytosine (5mC) in zygotes correlates with appearance of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). This process is mediated by Tet3 and the 5mC oxidation products generated in zygotes are gradually lost during preimplantation development through a replication-dependent dilution process. Despite these findings, the biological significance of Tet3-mediated oxidation of 5mC to 5hmC/5fC/5caC in zygotes is unknown. DNA methylation plays an important role in silencing gene expression including the repression of transposable elements (TEs). Given that the activation of TEs during preimplantation development correlates with loss of DNA methylation, it is believed that paternal DNA demethylation may have an important role in TE activation. Here we examined this hypothesis and found that Tet3-mediated 5mC oxidation does not have a significant contribution to TE activation. We show that the expression of LINE-1 (long interspersed nucleotide element 1) and ERVL (endogenous retroviruses class III) are activated from both paternal and maternal genomes in zygotes. Inhibition of 5mC oxidation by siRNA-mediated depletion of Tet3 affected neither TE activation, nor global transcription in zygotes. Thus, our study provides the first evidence demonstrating that activation of both TEs and global transcription in zygotes are independent of Tet3-mediated 5mC oxidation.

Mediator MED23 Links Pigmentation and DNA Repair through the Transcription Factor MITF.

PubMed

Xia, Min; Chen, Kun; Yao, Xiao; Xu, Yichi; Yao, Jiaying; Yan, Jun; Shao, Zhen; Wang, Gang

2017-08-22

DNA repair is related to many physiological and pathological processes, including pigmentation. Little is known about the role of the transcriptional cofactor Mediator complex in DNA repair and pigmentation. Here, we demonstrate that Mediator MED23 plays an important role in coupling UV-induced DNA repair to pigmentation. The loss of Med23 specifically impairs the pigmentation process in melanocyte-lineage cells and in zebrafish. Med23 deficiency leads to enhanced nucleotide excision repair (NER) and less DNA damage following UV radiation because of the enhanced expression and recruitment of NER factors to chromatin for genomic stability. Integrative analyses of melanoma cells reveal that MED23 controls the expression of a melanocyte master regulator, Mitf, by modulating its distal enhancer activity, leading to opposing effects on pigmentation and DNA repair. Collectively, the Mediator MED23/MITF axis connects DNA repair to pigmentation, thus providing molecular insights into the DNA damage response and skin-related diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Ripk3 regulates cardiac microvascular reperfusion injury: The role of IP3R-dependent calcium overload, XO-mediated oxidative stress and F-action/filopodia-based cellular migration.

PubMed

Zhou, Hao; Wang, Jin; Zhu, Pingjun; Hu, Shunying; Ren, Jun

2018-05-01

Ripk3-mediated cellular apoptosis is a major contributor to the pathogenesis of myocardial ischemia reperfusion (IR) injury. However, the mechanisms by which Ripk3 influences microvascular homeostasis and endothelial apoptosis are not completely understood. In this study, loss of Ripk3 inhibited endothelial apoptosis, alleviated luminal swelling, maintained microvasculature patency, reduced the expression of adhesion molecules and limited the myocardial inflammatory response. In vitro, Ripk3 deficiency protected endothelial cells from apoptosis and migratory arrest induced by HR injury. Mechanistically, Ripk3 had the ability to migrate onto the endoplasmic reticulum (ER), leading to ER damage, as evidenced by increased IP3R and XO expression. The higher IP3R content was associated with cellular calcium overload, and increased XO expression was involved in cellular oxidative injury. Furthermore, IP3R-mediated calcium overload and XO-dependent oxidative damage were able to initiate cellular apoptosis. More importantly, IP3R and XO also caused F-actin degradation into G-actin via post-transcriptional modification of cofilin, impairing the formation of the filopodia and limiting the migratory response of endothelial cells. Altogether, our data confirmed that Ripk3 was involved in microvascular IR injury via regulation of IP3R-mediated calcium overload, XO-dependent oxidative damage and filopodia-related cellular migration, ultimately leading to endothelial apoptosis and migratory inhibition. These findings provide a potential target for treating cardiac microcirculatory IR injury. Copyright © 2018 Elsevier Inc. All rights reserved.

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES).

PubMed

Han, Eun Hee; Gorman, Amanda A; Singh, Puja; Chi, Young-In

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Doping porous silicon with erbium: pores filling as a method to limit the Er-clustering effects and increasing its light emission.

PubMed

Mula, Guido; Printemps, Tony; Licitra, Christophe; Sogne, Elisa; D'Acapito, Francesco; Gambacorti, Narciso; Sestu, Nicola; Saba, Michele; Pinna, Elisa; Chiriu, Daniele; Ricci, Pier Carlo; Casu, Alberto; Quochi, Francesco; Mura, Andrea; Bongiovanni, Giovanni; Falqui, Andrea

2017-07-20

Er clustering plays a major role in hindering sufficient optical gain in Er-doped Si materials. For porous Si, the long-standing failure to govern the clustering has been attributed to insufficient knowledge of the several, concomitant and complex processes occurring during the electrochemical Er-doping. We propose here an alternative road to solve the issue: instead of looking for an equilibrium between Er content and light emission using 1-2% Er, we propose to significantly increase the electrochemical doping level to reach the filling the porous silicon pores with luminescent Er-rich material. To better understand the intricate and superposing phenomena of this process, we exploit an original approach based on needle electron tomography, EXAFS and photoluminescence. Needle electron tomography surprisingly shows a heterogeneous distribution of Er content in the silicon thin pores that until now couldn't be revealed by the sole use of scanning electron microscopy compositional mapping. Besides, while showing that pore filling leads to enhanced photoluminescence emission, we demonstrate that the latter is originated from both erbium oxide and silicate. These results give a much deeper understanding of the photoluminescence origin down to nanoscale and could lead to novel approaches focused on noteworthy enhancement of Er-related photoluminescence in porous silicon.

The multitalented Mediator complex.

PubMed

Carlsten, Jonas O P; Zhu, Xuefeng; Gustafsson, Claes M

2013-11-01

The Mediator complex is needed for regulated transcription of RNA polymerase II (Pol II)-dependent genes. Initially, Mediator was only seen as a protein bridge that conveyed regulatory information from enhancers to the promoter. Later studies have added many other functions to the Mediator repertoire. Indeed, recent findings show that Mediator influences nearly all stages of transcription and coordinates these events with concomitant changes in chromatin organization. We review the multitude of activities associated with Mediator and discuss how this complex coordinates transcription with other cellular events. We also discuss the inherent difficulties associated with in vivo characterization of a coactivator complex that can indirectly affect diverse cellular processes via changes in gene transcription. Copyright © 2013 Elsevier Ltd. All rights reserved.