Iskar, Murat; Campillos, Monica; Kuhn, Michael;
further newly identified drug-induced differential regulation of Lanosterol 14-alpha demethylase, Endoplasmin, DNA topoisomerase 2-alpha and Calmodulin 1. The feedback regulation in these and other targets is likely to be relevant for the success or failure of the molecular intervention....
Jacki E. Heraud-Farlow
Full Text Available RNA-binding proteins play crucial roles in directing RNA translation to neuronal synapses. Staufen2 (Stau2 has been implicated in both dendritic RNA localization and synaptic plasticity in mammalian neurons. Here, we report the identification of functionally relevant Stau2 target mRNAs in neurons. The majority of Stau2-copurifying mRNAs expressed in the hippocampus are present in neuronal processes, further implicating Stau2 in dendritic mRNA regulation. Stau2 targets are enriched for secondary structures similar to those identified in the 3′ UTRs of Drosophila Staufen targets. Next, we show that Stau2 regulates steady-state levels of many neuronal RNAs and that its targets are predominantly downregulated in Stau2-deficient neurons. Detailed analysis confirms that Stau2 stabilizes the expression of one synaptic signaling component, the regulator of G protein signaling 4 (Rgs4 mRNA, via its 3′ UTR. This study defines the global impact of Stau2 on mRNAs in neurons, revealing a role in stabilization of the levels of synaptic targets.
The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.
Martin-Sanchez, Diego; Poveda, Jonay; Fontecha-Barriuso, Miguel; Ruiz-Andres, Olga; Sanchez-Niño, María Dolores; Ruiz-Ortega, Marta; Ortiz, Alberto; Sanz, Ana Belén
The term acute tubular necrosis was thought to represent a misnomer derived from morphological studies of human necropsies and necrosis was thought to represent an unregulated passive form of cell death which was not amenable to therapeutic manipulation. Recent advances have improved our understanding of cell death in acute kidney injury. First, apoptosis results in cell loss, but does not trigger an inflammatory response. However, clumsy attempts at interfering with apoptosis (e.g. certain caspase inhibitors) may trigger necrosis and, thus, inflammation-mediated kidney injury. Second, and most revolutionary, the concept of regulated necrosis emerged. Several modalities of regulated necrosis were described, such as necroptosis, ferroptosis, pyroptosis and mitochondria permeability transition regulated necrosis. Similar to apoptosis, regulated necrosis is modulated by specific molecules that behave as therapeutic targets. Contrary to apoptosis, regulated necrosis may be extremely pro-inflammatory and, importantly for kidney transplantation, immunogenic. Furthermore, regulated necrosis may trigger synchronized necrosis, in which all cells within a given tubule die in a synchronized manner. We now review the different modalities of regulated necrosis, the evidence for a role in diverse forms of kidney injury and the new opportunities for therapeutic intervention. Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.
Piatek, Agnieszka Anna
Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group
Full Text Available Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia, and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219, pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638 as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.
Aleem, Eiman; Arceci, Robert J
Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.
Full Text Available Kegiatan pembangunan yang diwarnai oleh market driven akan menyebabkan kerusakan lingkungan. Apabila terjadi secara terus-menerus, bisa mengakibatkan penurunan kualitas lingkungan hidup. Oleh karena itu penataan ruang (spatial planning menjadi aspek yang amat penting agar ruang yang terbatas dapat digunakan secara efisien dengan tetap memelihara kelestarian dan daya dukung lingkungan hidup. Proses invasi dan urban sprawl sebagai akibat dari keterbatasan ruang perkotaan telah merembet ke sebagian daerah di Kabupaten Sleman, terutama di wilayah Kecamatan Mlati. Perkembangan wilayah di daerah ini perlu mendapatkan perhatian khusus agar di kemudian hari tidak menjadi unmanaged growth. Untuk menjaga kelestarian alam dan mengurangi dampak kerusakan lingkungan yang bisa berakibat terhadap terganggunya sistem ekologi wilayah sekitar Kabupaten Sleman, Badan Pengendalian Pertanahan Daerah (BPPD Kabupaten Sleman melakukan pengendalian atas perubahan penggunaan tanah melalui sosialisasi atas kebijakan pertanahan yang telah ditetapkan Pemerintah Kabupaten Sleman. BPPD memasang/membuat papan informasi/baliho pada tempat strategis, sehingga dapat dibaca dengan mudah oleh masyarakat. Salah satu baliho dipasang di kawasan pertanian di Jalan Sendari-Gombang, Tirtoadi, Kecamatan Mlati. Berkenaan dengan hal di atas, penelitian ini bertujuan untuk menilai sikap target grup terhadap zoning regulations dengan studi kasus di Jalan Sendari Gombang Tirtoadi, Kecamatan Mlati. Penelitian juga ingin mengungkap faktor-faktor yang memengaruhi dukungan target grup terhadap implementasi zoning regulations di Kecamatan Mlati, terutama di Jalan Sendari-Gombang, Tirtoadi. Penelitian menggunakan pendekatan deskriptif naturalistik dengan metode studi kasus.
Huang, Yi; Marton, Laurence J; Woster, Patrick M; Casero, Robert A
Over the past three decades the metabolism and functions of the polyamines have been actively pursued as targets for antineoplastic therapy. Interactions between cationic polyamines and negatively charged nucleic acids play a pivotal role in DNA stabilization and RNA processing that may affect gene expression, translation and protein activity. Our growing understanding of the unique roles that the polyamines play in chromatin regulation, and the discovery of novel proteins homologous with specific regulatory enzymes in polyamine metabolism, have led to our interest in exploring chromatin remodelling enzymes as potential therapeutic targets for specific polyamine analogues. One of our initial efforts focused on utilizing the strong affinity that the polyamines have for chromatin to create a backbone structure, which could be combined with active-site-directed inhibitor moieties of HDACs (histone deacetylases). Specific PAHAs (polyaminohydroxamic acids) and PABAs (polyaminobenzamides) polyamine analogues have demonstrated potent inhibition of the HDACs, re-expression of p21 and significant inhibition of tumour growth. A second means of targeting the chromatin-remodelling enzymes with polyamine analogues was facilitated by the recent identification of flavin-dependent LSD1 (lysine-specific demethylase 1). The existence of this enzyme demonstrated that histone lysine methylation is a dynamic process similar to other histone post-translational modifications. LSD1 specifically catalyses demethylation of mono- and di-methyl Lys4 of histone 3, key positive chromatin marks associated with transcriptional activation. Structural and catalytic similarities between LSD1 and polyamine oxidases facilitated the identification of biguanide, bisguanidine and oligoamine polyamine analogues that are potent inhibitors of LSD1. Cellular inhibition of LSD1 by these unique compounds led to the re-activation of multiple epigenetically silenced genes important in tumorigenesis. The use of
Despite having been available for decades, target benefit pension plans (TBPs) will continue to be resisted by federally regulated employers unless a legal flaw is fixed, according to a report from the C.D. Howe Institute. In “Target Benefit Plans: Improving Access for Federally Regulated Employees,” author Randy Bauslaugh finds that TBPs are rarely adopted by federally regulated private-sector employers because federal pension law casts doubt over the ability of employers to limit their fina...
Full Text Available Abstract Hypoxia is an important microenvironmental factor that induces cancer metastasis. Hypoxia/hypoxia-inducible factor-1α (HIF-1α regulates many important steps of the metastatic processes, especially epithelial-mesenchymal transition (EMT that is one of the crucial mechanisms to cause early stage of tumor metastasis. To have a better understanding of the mechanism of hypoxia-regulated metastasis, various hypoxia/HIF-1α-regulated target genes are categorized into different classes including transcription factors, histone modifiers, enzymes, receptors, kinases, small GTPases, transporters, adhesion molecules, surface molecules, membrane proteins, and microRNAs. Different roles of these target genes are described with regards to their relationship to hypoxia-induced metastasis. We hope that this review will provide a framework for further exploration of hypoxia/HIF-1α-regulated target genes and a comprehensive view of the metastatic picture induced by hypoxia.
Makarevich, Grigory; Leroy, Olivier; Akinci, Umut; Schubert, Daniel; Clarenz, Oliver; Goodrich, Justin; Grossniklaus, Ueli; Köhler, Claudia
Polycomb group (PcG) proteins convey epigenetic inheritance of repressed transcriptional states. Although the mechanism of the action of PcG is not completely understood, methylation of histone H3 lysine 27 (H3K27) is important in establishing PcG-mediated transcriptional repression. We show that the plant PcG target gene PHERES1 is regulated by histone trimethylation on H3K27 residues mediated by at least two different PcG complexes in plants, containing the SET domain proteins MEDEA or CURLY LEAF/SWINGER. Furthermore, we identify FUSCA3 as a potential PcG target gene and show that FUSCA3 is regulated by MEDEA and CURLY LEAF/SWINGER. We propose that different PcG complexes regulate a common set of target genes during the different stages of plant development.
Full Text Available MicroRNAs (miRs are key post-transcriptional regulators that silence gene expression by direct base pairing to target sites of RNAs. They have a wide variety of tissue expression patterns and are differentially expressed during development and disease. Their activity and abundance is subject to various levels of control ranging from transcription and biogenesis to miR response elements on RNAs, target cellular levels and miR turnover. This review summarizes and discusses current knowledge on the regulation of miR activity and concludes with novel non-canonical functions that have recently emerged.
Dove, Simon L; Darst, Seth A; Hochschild, Ann
Bacterial sigma factors play a key role in promoter recognition, making direct contact with conserved promoter elements. Most sigma factors belong to the sigma70 family, named for the primary sigma factor in Escherichia coli. Members of the sigma70 family typically share four conserved regions and, here, we focus on region 4, which is directly involved in promoter recognition and serves as a target for a variety of regulators of transcription initiation. We review recent advances in the understanding of the mechanism of action of regulators that target region 4 of sigma.
Tremblay, Luc; Crainic, Valentin A; de Grosbois, John; Bhattacharjee, Arindam; Kennedy, Andrew; Hansen, Steve; Welsh, Timothy N
The utilization of visual information for the control of ongoing voluntary limb movements has been investigated for more than a century. Recently, online sensorimotor processes for the control of upper-limb reaches were hypothesized to include a distinct process related to the comparison of limb and target positions (i.e., limb-target regulation processes: Elliott et al. in Psychol Bull 136:1023-1044. doi: 10.1037/a0020958 , 2010). In the current study, this hypothesis was tested by presenting participants with brief windows of vision (20 ms) when the real-time velocity of the reaching limb rose above selected velocity criteria. One experiment tested the perceptual judgments of endpoint bias (i.e., under- vs. over-shoot), and another experiment tested the shifts in endpoint distributions following an imperceptible target jump. Both experiments revealed that limb-target regulation processes take place at an optimal velocity or "sweet spot" between movement onset and peak limb velocity (i.e., 1.0 m/s with the employed movement amplitude and duration). In contrast with pseudo-continuous models of online control (e.g., Elliott et al. in Hum Mov Sci 10:393-418. doi: 10.1016/0167-9457(91)90013-N , 1991), humans likely optimize online limb-target regulation processes by gathering visual information at a rather limited period of time, well in advance of peak limb velocity.
Full Text Available Abstract Background In Drosophila, the transport regulator Klar displays tissue-specific localization: In photoreceptors, it is abundant on the nuclear envelope; in early embryos, it is absent from nuclei, but instead present on lipid droplets. Differential targeting of Klar appears to be due to isoform variation. Droplet targeting, in particular, has been suggested to occur via a variant C-terminal region, the LD domain. Although the LD domain is necessary and sufficient for droplet targeting in cultured cells, lack of specific reagents had made it previously impossible to analyze its role in vivo. Results Here we describe a new mutant allele of klar with a lesion specifically in the LD domain; this lesion abolishes both droplet localization of Klar and the ability of Klar to regulate droplet motion. It does not disrupt Klar's function for nuclear migration in photoreceptors. Using a GFP-LD fusion, we show that the LD domain is not only necessary but also sufficient for droplet targeting in vivo; it mediates droplet targeting in embryos, in ovaries, and in a number of somatic tissues. Conclusions Our analysis demonstrates that droplet targeting of Klar occurs via a cis-acting sequence and generates a new tool for monitoring lipid droplets in living tissues of Drosophila.
Full Text Available Through combinatorial regulation, regulators partner with each other to control common targets and this allows a small number of regulators to govern many targets. One interesting question is that given this combinatorial regulation, how does the number of regulators scale with the number of targets? Here, we address this question by building and analyzing co-regulation (co-transcription and co-phosphorylation networks that describe partnerships between regulators controlling common genes. We carry out analyses across five diverse species: Escherichia coli to human. These reveal many properties of partnership networks, such as the absence of a classical power-law degree distribution despite the existence of nodes with many partners. We also find that the number of co-regulatory partnerships follows an exponential saturation curve in relation to the number of targets. (For E. coli and Bacillus subtilis, only the beginning linear part of this curve is evident due to arrangement of genes into operons. To gain intuition into the saturation process, we relate the biological regulation to more commonplace social contexts where a small number of individuals can form an intricate web of connections on the internet. Indeed, we find that the size of partnership networks saturates even as the complexity of their output increases. We also present a variety of models to account for the saturation phenomenon. In particular, we develop a simple analytical model to show how new partnerships are acquired with an increasing number of target genes; with certain assumptions, it reproduces the observed saturation. Then, we build a more general simulation of network growth and find agreement with a wide range of real networks. Finally, we perform various down-sampling calculations on the observed data to illustrate the robustness of our conclusions.
Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in American males today. Novel and effective treatment such as gene therapy is greatly desired. The early viral based gene therapy uses tissue-nonspecific promoters, which causes unintended toxicity to other normal tissues. In this chapter, we will review the transcriptionally regulated gene therapy strategy for prostate cancer treatment. We will describe the development of transcriptionally regulated prostate cancer gene therapy in the following areas: (1) Comparison of different routes for best viral delivery to the prostate; (2) Study of transcriptionally regulated, prostate-targeted viral vectors: specificity and activity of the transgene under several different prostate-specific promoters were compared in vitro and in vivo; (3) Selection of therapeutic transgenes and strategies for prostate cancer gene therapy (4) Oncolytic virotherapy for prostate cancer. In addition, the current challenges and future directions in this field are also discussed.
Li, Binghua; Fan, Junkai; Liu, Xinran; Qi, Rong; Bo, Linan; Gu, Jinfa; Qian, Cheng; Liu, Xinyuan
A major goal in cancer gene therapy is to develop efficient gene transfer protocols that allow tissue-specific and tightly regulated expression of therapeutic genes. The ideal vector should efficiently transduce cancer cells with minimal toxicity on normal tissues and persistently express foreign genes. One of the most promising regulatory systems is the mifepristone/RU486-regulated system, which has much lower basal transcriptional activity and high inducibility. In this work, we modified this system by incorporating a cancer-specific promoter, the human telomerase reverse transcriptase (hTERT) promoter. By utilizing hTERT promoter to control the regulator, RU486 could specifically induce the expression of foreign genes in cancer cells but not in normal cells. In the context of this system, a dominant negative mutant of survivin (surDN) was controllably expressed in colorectal tumor cells. The surDN expression induced by RU486 showed a dosage- and time-dependent pattern. Regulated expression of surDN caused caspase-dependent apoptosis in colorectal tumor cells but had little effect on normal cells. Analysis of cell viability showed that RU486-induced expression of surDN suppressed colorectal tumor cell growth and had synergic effect in combination with chemotherapeutic agents. The potential of this system in cancer therapy was evaluated in experimental animals. Tumor xenograft models were established in nude mice with colorectal tumor cells, and RU486 was intraperitoneally administered. The results showed that conditional expression of surDN efficiently inhibited tumor growth in vivo and prolonged the life of tumor-burdened mice. Synergized with the chemotherapeutic drug cisplatin, regulated surDN expression completely suppressed tumor growth. These results indicated that this modified RU486-regulated system could be useful in cancer-targeting therapy.
Full Text Available Demands for plant growth regulators (chemicals that control plant growth are increasing globally, especially in developing countries. Both positive and negative plant growth regulators are widely used to enhance crop production and to suppress unwanted shoot growth, respectively. Strigolactones (SLs are multifunctional molecules that function as phytohormones, inhibiting shoot branching and also functioning in the rhizospheric communication with symbiotic fungi and parasitic weeds. Therefore, it is anticipated that chemicals that regulate the functions of SLs will be widely used in agricultural applications. Although the SL biosynthetic pathway is not fully understood, it has been demonstrated that beta-carotene isomerases, carotenoid cleavage dioxygenases (CCDs, and a cytochrome P450 monooxygenase are involved in strigolactone biosynthesis. A CCD inhibitor, abamine, which is also an inhibitor of abscisic acid biosynthesis, reduces the levels of SL in several plant species and reduces the germination rate of Orobanche minor seeds grown with tobacco. On the basis of the structure of abamine, several chemicals have been designed to specifically inhibit CCDs during SL synthesis. Cytochrome P450 monooxygenase is another target enzyme in the development of SL biosynthesis inhibitors, and the triazole-derived TIS series of chemicals is known to include SL biosynthesis inhibitors, although their target enzyme has not been identified. Recently, DWARF14 (D14 has been shown to be a receptor for SLs, and the D-ring moiety of SL is essential for its recognition by D14. A variety of SL agonists are currently under development and most agonists commonly contain the D-ring or a D-ring-like moiety. Several research groups have also resolved the crystal structure of D14 in the last two years. It is expected that this information on the D14 structure will be invaluable not only for developing SL agonists with novel structures but also in the design of inhibitors
Sander, Jeffry D; Joung, J Keith
Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
Chang, Aeson [Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052 (Australia); Kim-Fuchs, Corina [Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052 (Australia); Department of Visceral Surgery and Medicine, University Hospital Bern, Bern 3010 (Switzerland); Le, Caroline P. [Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052 (Australia); Hollande, Frédéric [Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052 (Australia); Department of Pathology, University of Melbourne, Parkville 3010 (Australia); Sloan, Erica K., E-mail: email@example.com [Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052 (Australia); Cousins Center for PNI, UCLA Semel Institute, Jonsson Comprehensive Cancer Center, and UCLA AIDS Institute, University of California Los Angeles, Los Angeles, CA 90095 (United States); Peter MacCallum Cancer Centre, Division of Cancer Surgery, East Melbourne, Victoria 3002 (Australia)
The tumor microenvironment is known to play a pivotal role in driving cancer progression and governing response to therapy. This is of significance in pancreatic cancer where the unique pancreatic tumor microenvironment, characterized by its pronounced desmoplasia and fibrosis, drives early stages of tumor progression and dissemination, and contributes to its associated low survival rates. Several molecular factors that regulate interactions between pancreatic tumors and their surrounding stroma are beginning to be identified. Yet broader physiological factors that influence these interactions remain unclear. Here, we discuss a series of preclinical and mechanistic studies that highlight the important role chronic stress plays as a physiological regulator of neural-tumor interactions in driving the progression of pancreatic cancer. These studies propose several approaches to target stress signaling via the β-adrenergic signaling pathway in order to slow pancreatic tumor growth and metastasis. They also provide evidence to support the use of β-blockers as a novel therapeutic intervention to complement current clinical strategies to improve cancer outcome in patients with pancreatic cancer.
Jung, Chang Hwa; Kim, Heemun; Ahn, Jiyun; Jeon, Tae-Il; Lee, Dae-Hee; Ha, Tae-Youl
Fisetin, a flavonol present in vegetables and fruits, possesses antioxidative and anti-inflammatory properties. In this study, we have demonstrated that fisetin prevents diet-induced obesity through regulation of the signaling of mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth, cellular proliferation and lipid biosynthesis. To evaluate whether fisetin regulates mTORC1 signaling, we investigated the phosphorylation and kinase activity of the 70-kDa ribosomal protein S6 kinase 1 (S6K1) and mTORC1 in 3T3-L1 preadipocytes. Fisetin treatment of preadipocytes reduced the phosphorylation of S6K1 and mTORC1 in a time- and concentration-dependent manner. To further our understanding of how fisetin negatively regulates mTORC1 signaling, we analyzed the phosphorylation of S6K1, mTOR and Akt in fisetin-treated TSC2-knockdown cells. The results suggested that fisetin treatment inhibits mTORC1 activity in an Akt-dependent manner. Recent studies have shown that adipocyte differentiation is dependent on mTORC1 activity. Fisetin treatment inhibited adipocyte differentiation, consistent with the negative effect of fisetin on mTOR. The inhibitory effect of fisetin on adipogenesis is dependent of mTOR activity, suggesting that fisetin inhibits adipogenesis and the accumulation of intracellular triglycerides during adipocyte differentiation by targeting mTORC1 signaling. Fisetin supplementation in mice fed a high-fat diet (HFD) significantly attenuated HFD-induced increases in body weight and white adipose tissue. We also observed that fisetin efficiently suppressed the phosphorylation of Akt, S6K1 and mTORC1 in adipose tissue. Collectively, these results suggest that inhibition of mTORC1 signaling by fisetin prevents adipocyte differentiation of 3T3-L1 preadipocytes and obesity in HFD-fed mice. Therefore, fisetin may be a useful phytochemical agent for attenuating diet-induced obesity. Copyright © 2013 Elsevier Inc. All rights reserved.
Reactive stromal cells are an integral part of tumor microenvironment (TME) and interact with cancer cells to regulate their growth. Although targeting stromal cells could be a viable therapy to regulate the communication between TME and cancer cells, identification of stromal targets that make canc...
Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.
Nathan, N; Sperandio, M; Erdmann, W; Westerkamp, B; Van Dijk, G; Scherpereel, P; Feiss, P
Physi Flex is the first commercially available apparatus capable for quantitative, or self-regulating target controlled inhalational anaesthesia, with a totally closed circuit, in adults and children. The fresh gas supply to the circuit is intermittent, automatically regulated by continuous monitoring of the volume and composition of the gas mixture in the breathing circuit. The circle system includes, instead of the two conventional one way valves, a blower creating a continuous unidirectional flow at 70 L.min-1. In addition to the CO2-absorber it contains an absorber with carbon, absorbing the anaesthetic vapour when switched into the circuit. The ventilator consists of four ventilating chambers, each one with a membrane separating the patient and the motor compartments. The displacement of the membranes generates and measures the tidal volume. Automatic ventilation is achieved by electric valves and motor gas, and manual ventilation using a bag. Spontaneous ventilation is also possible. The machine is operated via a computer with selects the number of ventilating chambers (one, two or four), and the tidal volume between 50 and 2,000 mL, depending on age, gender and weight of the patient. The computer maintains the gas volume and the gas and vapour concentrations at their preset values. The O2-flow and consumption, the N2O flow and uptake, FICO2 and FETCO2, FI and FET of the volatile anaesthetic, all other important data are displayed in a numerical and graphical form on a color screen and registered for a delayed analysis. The end tidal concentration of the volatile anaesthetic drives a stepmotor with a syringe containing the selected volatile anaesthetic agent with is directly injected into the breathing circuit where it is vaporized. Therefore the concentration of the anaesthetic vapour can be instantaneously increased with this injector at induction and lowered at end of anaesthesia with the carbon absorber, and the fresh gas consumption is significantly
Yoon, Young Sil; Ryu, Dongryeol; Lee, Min Woo; Hong, Sungpyo; Koo, Seung Hoi
During fasting periods, hepatic glucose production is enhanced by glucagon to provide fuels for other organs. This process is mediated via cAMP-dependent induction of the CREB regulated transcriptional coactivator (CRTC) 2, a critical transcriptional activator for hepatic gluconeogenesis. We have previously shown that CRTC2 activity is regulated by AMP activated protein kinase (AMPK) family members. Here we show that adiponectin and thiazolidinedione directly regulate AMPK to modulate CRTC2 activity in hepatocytes. Adiponectin or thiazolidinedione lowered glucose production from primary hepatocytes. Treatment of both reagents reduced gluconeogenic gene expression as well as cAMP-mediated induction of CRE reporter, suggesting that these reagents directly affect CREB/CRTC2- dependent transcription. Furthermore, adiponectin or thiazolidinedione mediated repression of CRE activity is largely blunted by co-expression of phosphorylation defective mutant CRTC2, underscoring the importance of serine 171 residue of this factor. Taken together, we propose that adiponectin and thiazolidinedione promote the modulation of AMPK-dependent CRTC2 activity to influence hepatic gluconeogenesis.
Parry, Martin A J; Andralojc, P John; Scales, Joanna C; Salvucci, Michael E; Carmo-Silva, A Elizabete; Alonso, Hernan; Whitney, Spencer M
Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO(2) and NH(3) and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to 'supercharge' photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO(2) can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco's inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.
Cooke, Amy; Prigge, Andrew; Opperman, Laura; Wickens, Marvin
Regulatory complexes formed on mRNAs control translation, stability, and localization. These complexes possess two activities: one that binds RNA and another--the effector--that elicits a biological function. The Pumilio and FBF (PUF) protein family of RNA binding proteins provides a versatile scaffold to design and select proteins with new specificities. Here, the PUF scaffold is used to target translational activation and repression of specific mRNAs, and to induce specific poly(A) addition and removal. To do so, we linked PUF scaffold proteins to a translational activator, GLD2, or a translational repressor, CAF1. The chimeric proteins activate or repress the targeted mRNAs in Xenopus oocytes, and elicit poly(A) addition or removal. The magnitude of translational control relates directly to the affinity of the RNA-protein complex over a 100-fold range of K(d). The chimeric proteins act on both reporter and endogenous mRNAs: an mRNA that normally is deadenylated during oocyte maturation instead receives poly(A) in the presence of an appropriate chimera. The PUF-effector strategy enables the design of proteins that affect translation and stability of specific mRNAs in vivo.
Wallace, Sean W.; Magalhaes, Ana; Hall, Alan
Rho GTPases regulate multiple signaling pathways to control a number of cellular processes during epithelial morphogenesis. To investigate the downstream pathways through which Rho regulates epithelial apical junction formation, we screened a small interfering RNA (siRNA) library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C-related kinase 2, also called PKN2). Depletion of...
Qinglin Du, Honghai Wang, Jianping Xie
Full Text Available Drug resistance of pathogens has necessitated the identification of novel targets for antibiotics. Thiamin (vitamin B1 is an essential cofactor for all organisms in its active form thiamin diphosphate (ThDP. Therefore, its metabolic pathways might be one largely untapped source of antibiotics targets. This review describes bacterial thiamin biosynthetic, salvage, and transport pathways. Essential thiamin synthetic enzymes such as Dxs and ThiE are proposed as promising drug targets. The regulation mechanism of thiamin biosynthesis by ThDP riboswitch is also discussed. As drug targets of existing antimicrobial compound pyrithiamin, the ThDP riboswitch might serves as alternative targets for more antibiotics.
Full Text Available Abstract Background Precise connections of neural circuits can be specified by genetic programming. In the Drosophila olfactory system, projection neurons (PNs send dendrites to single glomeruli in the antenna lobe (AL based upon lineage and birth order and send axons with stereotyped terminations to higher olfactory centers. These decisions are likely specified by a PN-intrinsic transcriptional code that regulates the expression of cell-surface molecules to instruct wiring specificity. Results We find that the loss of longitudinals lacking (lola, which encodes a BTB-Zn-finger transcription factor with 20 predicted splice isoforms, results in wiring defects in both axons and dendrites of all lineages of PNs. RNA in situ hybridization and quantitative RT-PCR suggest that most if not all lola isoforms are expressed in all PNs, but different isoforms are expressed at widely varying levels. Overexpression of individual lola isoforms fails to rescue the lola null phenotypes and causes additional phenotypes. Loss of lola also results in ectopic expression of Gal4 drivers in multiple cell types and in the loss of transcription factor gene lim1 expression in ventral PNs. Conclusion Our results indicate that lola is required for wiring of axons and dendrites of most PN classes, and suggest a need for its molecular diversity. Expression pattern changes of Gal4 drivers in lola-/- clones imply that lola normally represses the expression of these regulatory elements in a subset of the cells surrounding the AL. We propose that Lola functions as a general transcription factor that regulates the expression of multiple genes ultimately controlling PN identity and wiring specificity.
Claro, Anthony; Boulanger, Marie-Michelle; Shaw, Steven R.
The paper examined the effectiveness of an in-school intervention for adolescents designed to target emotional regulation skills related to risky behaviors. The Cognitive Emotion Regulation Intended for Youth (CERTIFY) program was delivered to at-risk adolescents in Montreal, Canada. Participants were drawn from an alternative high school and a…
Medvid Viktoriia Yu.
Full Text Available The article studies theoretical features, target orientation and functional content of economic regulation of regional development. It identifies the content of the “economic regulation of regional development” notion with respect to the region as a territory, economic system and economic space. It establishes that economic regulation of regional development is a system impact on certain parameters, revision of which would ensure positive dynamics in revision of basic characteristics of regions such as properties, features and boundaries. It identifies the main goal and complex of tasks (complexity, target character, variability, resource provision, addressness and manageability of economic regulation of regional development. It shows directions of increase of efficiency of the system of economic regulation of regional development.
Gevaert, Olivier; Plevritis, Sylvia
Vast amounts of molecular data characterizing the genome, epigenome and transcriptome are becoming available for a variety of cancers. The current challenge is to integrate these diverse layers of molecular biology information to create a more comprehensive view of key biological processes underlying cancer. We developed a biocomputational algorithm that integrates copy number, DNA methylation, and gene expression data to study master regulators of cancer and identify their targets. Our algorithm starts by generating a list of candidate driver genes based on the rationale that genes that are driven by multiple genomic events in a subset of samples are unlikely to be randomly deregulated. We then select the master regulators from the candidate driver and identify their targets by inferring the underlying regulatory network of gene expression. We applied our biocomputational algorithm to identify master regulators and their targets in glioblastoma multiforme (GBM) and serous ovarian cancer. Our results suggest that the expression of candidate drivers is more likely to be influenced by copy number variations than DNA methylation. Next, we selected the master regulators and identified their downstream targets using module networks analysis. As a proof-of-concept, we show that the GBM and ovarian cancer module networks recapitulate known processes in these cancers. In addition, we identify master regulators that have not been previously reported and suggest their likely role. In summary, focusing on genes whose expression can be explained by their genomic and epigenomic aberrations is a promising strategy to identify master regulators of cancer.
Fujisawa, Masaki; Shima, Yoko; Higuchi, Naoki; Nakano, Toshitsugu; Koyama, Yoshiyuki; Kasumi, Takafumi; Ito, Yasuhiro
The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.
Beck, Michael W.; Derrick, Jeffrey S.; Kerr, Richard A.; Oh, Shin Bi; Cho, Woo Jong; Lee, Shin Jung C.; Ji, Yonghwan; Han, Jiyeon; Tehrani, Zahra Aliakbar; Suh, Nayoung; Kim, Sujeong; Larsen, Scott D.; Kim, Kwang S.; Lee, Joo-Yong; Ruotolo, Brandon T.; Lim, Mi Hee
The absence of effective therapeutics against Alzheimer's disease (AD) is a result of the limited understanding of its multifaceted aetiology. Because of the lack of chemical tools to identify pathological factors, investigations into AD pathogenesis have also been insubstantial. Here we report chemical regulators that demonstrate distinct specificity towards targets linked to AD pathology, including metals, amyloid-β (Aβ), metal-Aβ, reactive oxygen species, and free organic radicals. We obtained these chemical regulators through a rational structure-mechanism-based design strategy. We performed structural variations of small molecules for fine-tuning their electronic properties, such as ionization potentials and mechanistic pathways for reactivity towards different targets. We established in vitro and/or in vivo efficacies of the regulators for modulating their targets' reactivities, ameliorating toxicity, reducing amyloid pathology, and improving cognitive deficits. Our chemical tools show promise for deciphering AD pathogenesis and discovering effective drugs.
Beck, Michael W; Derrick, Jeffrey S; Kerr, Richard A; Oh, Shin Bi; Cho, Woo Jong; Lee, Shin Jung C; Ji, Yonghwan; Han, Jiyeon; Tehrani, Zahra Aliakbar; Suh, Nayoung; Kim, Sujeong; Larsen, Scott D; Kim, Kwang S; Lee, Joo-Yong; Ruotolo, Brandon T; Lim, Mi Hee
The absence of effective therapeutics against Alzheimer's disease (AD) is a result of the limited understanding of its multifaceted aetiology. Because of the lack of chemical tools to identify pathological factors, investigations into AD pathogenesis have also been insubstantial. Here we report chemical regulators that demonstrate distinct specificity towards targets linked to AD pathology, including metals, amyloid-β (Aβ), metal-Aβ, reactive oxygen species, and free organic radicals. We obtained these chemical regulators through a rational structure-mechanism-based design strategy. We performed structural variations of small molecules for fine-tuning their electronic properties, such as ionization potentials and mechanistic pathways for reactivity towards different targets. We established in vitro and/or in vivo efficacies of the regulators for modulating their targets' reactivities, ameliorating toxicity, reducing amyloid pathology, and improving cognitive deficits. Our chemical tools show promise for deciphering AD pathogenesis and discovering effective drugs.
Iwatsubo, Kousaku; Tsunematsu, Takashi; Ishikawa, Yoshihiro
Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoform dependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.
Kristensen, Dorte Enggaard; Albers, Peter Hjorth; Prats, Clara
are expressed in a fibre type-dependent manner and that fibre type-specific activation of AMPK and downstream targets is dependent on exercise intensity. Pools of type I and II fibres were prepared from biopsies of m. vastus lateralis from healthy men before and after two exercise trials; A) continuous cycling......AMP-activated protein kinase (AMPK) is a regulator of energy homeostasis during exercise. Studies suggest muscle fibre type-specific AMPK expression. However, fibre type-specific regulation of AMPK and downstream targets during exercise has not been proven. We hypothesized that AMPK subunits...
Singh, Noopur; Sharma, Ashok
microRNA is known to play an important role in growth and development of the plants and also in environmental stress. Ocimum basilicum (Basil) is a well known herb for its medicinal properties. In this study, we used in-silico approaches to identify miRNAs and their targets regulating different functions in O. basilicum using EST approach. Additionally, functional annotation, gene ontology and pathway analysis of identified target transcripts were also done. Seven miRNA families were identified. Meaningful regulations of target transcript by identified miRNAs were computationally evaluated. Four miRNA families have been reported by us for the first time from the Lamiaceae. Our results further confirmed that uracil was the predominant base in the first positions of identified mature miRNA sequence, while adenine and uracil were predominant in pre-miRNA sequences. Phylogenetic analysis was carried out to determine the relation between O. basilicum and other plant pre-miRNAs. Thirteen potential targets were evaluated for 4 miRNA families. Majority of the identified target transcripts regulated by miRNAs showed response to stress. miRNA 5021 was also indicated for playing an important role in the amino acid metabolism and co-factor metabolism in this plant. To the best of our knowledge this is the first in silico study describing miRNAs and their regulation in different metabolic pathways of O. basilicum.
Barnea, Eytan R; Hayrabedyan, Soren; Todorova, Krassimira; Almogi-Hazan, Osnat; Or, Reuven; Guingab, Joy; McElhinney, James; Fernandez, Nelson; Barder, Timothy
Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders. Copyright © 2016 Elsevier GmbH. All rights reserved.
Slavov, Nikolai; Carey, Jannette; Linse, Sara
Diverse physiological processes are regulated differentially by Ca(2+) oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca(2+) binding affinities, and target exchange rates that depend on both target identity and Ca(2+) occupancy. The binding dynamics among Ca(2+), Mg(2+), calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca(2+)-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca(2+) transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin.
Full Text Available The characteristics of formation of the inversion type of the stock market and its contradictions were investigated, the necessity of transition to a functional-target regulation of the stock market was proved the ways of optimization of the institutional system by integrating the functions of regulatory authorities were proposed.
Gu, Yuanzheng; Barry, Joshua; McDougel, Robert; Terman, David; Gu, Chen
Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.
Van De Werken, C.; Avo Santos, M.; Laven, J. S E; Eleveld, C.; Fauser, B. C J M; Lens, S. M A; Baart, E. B.
STUDY QUESTION Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? SUMMARY ANSWER Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal
Van De Werken, C.; Avo Santos, M.; Laven, J. S E; Eleveld, C.; Fauser, B. C J M; Lens, S. M A; Baart, E. B.
STUDY QUESTION Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? SUMMARY ANSWER Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal
Full Text Available Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1 target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2 serve as a resource for identifying new molecular targets for treatment.
Full Text Available miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.
Polioudakis, Damon; Abell, Nathan S; Iyer, Vishwanath R
miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191's regulation of primary human fibroblast proliferation.
Full Text Available Cytokine and growth factor signaling pathways involving STAT3 are frequently constitutively activated in many human primary tumors, and are known for the transcriptional role they play in controlling cell growth and cell cycle progression. However, the extent of STAT3's reach on transcriptional control of the genome as a whole remains an important question. We predicted that this persistent STAT3 signaling affects a wide variety of cellular functions, many of which still remain to be characterized. We took a broad approach to identify novel STAT3 regulated genes by examining changes in the genome-wide gene expression profile by microarray, using cells expressing constitutively-activated STAT3. Using computational analysis, we were able to define the gene expression profiles of cells containing activated STAT3 and identify candidate target genes with a wide range of biological functions. Among these genes we identified Necdin, a negative growth regulator, as a novel STAT3 target gene, whose expression is down-regulated at the mRNA and protein levels when STAT3 is constitutively active. This repression is STAT3 dependent, since inhibition of STAT3 using siRNA restores Necdin expression. A STAT3 DNA-binding site was identified in the Necdin promoter and both EMSA and chromatin immunoprecipitation confirm binding of STAT3 to this region. Necdin expression has previously been shown to be down-regulated in a melanoma and a drug-resistant ovarian cancer cell line. Further analysis of Necdin expression demonstrated repression in a STAT3-dependent manner in human melanoma, prostate and breast cancer cell lines. These results suggest that STAT3 coordinates expression of genes involved in multiple metabolic and biosynthetic pathways, integrating signals that lead to global transcriptional changes and oncogenesis. STAT3 may exert its oncogenic effect by up-regulating transcription of genes involved in promoting growth and proliferation, but also by down-regulating
Ariel, Federico; Brault-Hernandez, Marianne; Laffont, Carole; Huault, Emeline; Brault, Mathias; Plet, Julie; Moison, Michael; Blanchet, Sandrine; Ichanté, Jean Laurent; Chabaud, Mireille; Carrere, Sébastien; Crespi, Martin; Chan, Raquel L.; Frugier, Florian
Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues. PMID:23023168
Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi
This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon
Full Text Available MicroRNAs (miRNAs have been reported to have diverse biological roles in regulating many biological processes, including osteogenic differentiation. In the present study, we identified that miR-24 was a critical regulator during osteogenic differentiation. We found that overexpression of miR-24 significantly inhibited osteogenic differentiation, which decreased alkaline phosphatase activity, matrix mineralization and the expression of osteogenic differentiation markers. In contrast, inhibition of miR-24 exhibited an opposite effect. Furthermore, we delineated that miR-24 regulates post-transcriptionals of T-cell factor-1 (Tcf-1 via targeting the 3'-untranslated region (UTR of Tcf-1 mRNA. MiR-24 was further found to regulate the protein expression of Tcf-1 in the murine osteoprogenitors cells and bone mesenchymal stem cells. Additionally, the positive effect of miR-24 suppression on osteoblast differentiation was apparently abrogated by Tcf-1 silencing. Taken together, our data suggest that miR-24 participates in osteogenic differentiation by targeting and regulating Tcf-1 expression in osteoblastic cells.
Parks, Scott K; Pouysségur, Jacques
Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pHi) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pHi regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pHi in the continued presence of external acidification (pHe). Considerable experimentation has revealed targets that successfully disrupt tumour pHi regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na(+)/H(+) exchangers (NHEs), carbonic anhydrases (CAs), Na(+)/HCO3(-) co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pHi when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives.
Abstract Background microRNAs (miRNAs) represent a class of small (typically 22 nucleotides in length) non-coding RNAs that can degrade their target mRNAs or block their translation. Recent disease research showed the exposure to some environmental chemicals (ECs) can regulate the expression patterns of miRNAs, which raises the intriguing question of how miRNAs and their targets cope with the exposure to ECs throughout the genome. Results In this study, we comprehensively analyzed the propert...
Sreekanth, Sreekumaran; Rasheed, Vazhanthodi A; Soundararajan, Lalitha; Antony, Jayesh; Saikia, Minakshi; Sivakumar, Krishnankutty Chandrika; Das, Ani V
Retinal histogenesis requires coordinated and temporal functioning of factors by which different cell types are generated from multipotent progenitors. Development of rod photoreceptors is regulated by multiple transcription factors, and Nrl is one of the major factors involved in their fate specification. Presence or absence of Nrl at the postnatal stages decides the generation of cone photoreceptors or other later retinal cells. This suggests the need for regulated expression of Nrl in order to accelerate the generation of other cell types during retinal development. We found that miR cluster 143/145, comprising miR-143 and miR-145, targets and imparts a posttranscriptional inhibition of Nrl. Expression of both miRNAs was differentially regulated during retinal development and showed least expression at PN1 stage in which most of the rod photoreceptors are generated. Downregulation of rod photoreceptor regulators and markers upon miR cluster 143/145 overexpression demonstrated that this cluster indeed negatively regulates rod photoreceptors. Further, we prove that Nrl positively regulates miR cluster 143/145, thus establishing a feedback loop regulatory mechanism. This may be one possible mechanism by which Nrl is posttranscriptionally regulated to facilitate the generation of other cell types in retina.
Gratz, Kim L.; Weiss, Nicole H.; Tull, Matthew T.
This paper reviews the extant literature on emotion regulation (ER) in psychological interventions. First, we review current conceptualizations of ER, highlighting a model with established clinical utility (particularly with regard to the development of new interventions and modification of existing interventions). Next, we review the literature on the effects of psychological interventions on ER, from traditional cognitive-behavioral and acceptance-based behavioral interventions that do not target ER directly to treatments that directly target ER as one component of a larger or more comprehensive treatment, as well as the preliminary research examining ER as a mechanism of change in these treatments. Finally, extant data on three treatments developed specifically to address ER are reviewed, with an emphasis on the ER-specific treatment with the most empirical support to date (emotion regulation group therapy). PMID:25859561
Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M
In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role of the prot......In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of genome instability, which is suggested to drive tumorigenesis and possibly aging, our data will facilitate future functional studies in the fields of DNA metabolism and cancer biology....
Sun, Xiang; Lou, Longhua; Zhong, Kezhao; Wan, Lijuan
Renal cell carcinoma (RCC) is a malignant tumor, which severely threatens human's life, moreover, the multi-drug resistance (MDR) under RCC undoubtedly strengthen the difficulties in the treatment. MiR-451 has been considered to play an important role in regulation of MDR in several cancers, but the role of it in MDR of RCC has not been explored. This study aims to explore the mechanism of miR-451 as a target to regulate chemotherapy resistance, which is crucial for further exploring novel therapy for RCC. Two human cell lines (ACHN and GRC-1) were performed in this study and adriamycin (ADM) was used to construct MDR cell lines. qRT-PCR was used to determine the mRNA expression of miR-451 and ATF-2. Weston blot was used to determine protein expression. MTT assay and flow cytometry were used for assessing cell viability and apoptosis, individually. Luciferase reporter assay was used to detect the targeting of miR-451 and ATF-2. Results presented that the expression of miR-451 was higher in low MDR cell line (ACHN) comparing with the high MDR cell line (GRC-1), while the expression of ATF-2 revealed an opposite results. MiR-451 targeted ATF-2 and regulated its expression. Overexpression of miR-451 strengthened drug resistance, decreased cell viability, and increased cell apoptosis of GRC-1 pretreated by ADM, while overexpressed ATF-2 reversed the effect induced by miR-451 overexpression. Then miR-451 knockdown improved drug susceptibility, decreased cell apoptosis, and increased cell viability of ACHN induced by ADM, however, ATF-2 suppression reversed the low rate of cell apoptosis and high rate of cell viability induced by miR-451 knockdown. Our results revealed that miR-451 regulates the drug resistance of RCC by targeting ATF-2 gene, which might be critical for overcoming MDR in RCC patients. Impact statement This is the first study to emphasize the expression of miR-451 on regulating multi-drug resistance (MDR) in renal cell carcinoma (RCC). Our study found
Li, Shi-Jie; Liu, Hong-Lin; Tang, Shi-Lei; Li, Xiao-Juan; Wang, Xiao-yin
Warburg effect, characterized by enhanced glycolysis and lactate production, even under aerobic conditions, is one of the hallmarks of cancer cells. However, the mechanism underlying this phenomenon remains poorly understood. Previous studies have shown that microRNA-150 (miR-150) is significantly up-regulated in various malignancies and represents a putative onco-miRNA in human cancers. In the present study, we aim to investigate the functional significance and molecular target of miR-150 in...
Chaudhuri, Aadel A; So, Alex Yick-Lun; Mehta, Arnav; Minisandram, Aarathi; Sinha, Nikita; Jonsson, Vanessa D; Rao, Dinesh S; O'Connell, Ryan M; Baltimore, David
MicroRNA-125b (miR-125b) is up-regulated in patients with leukemia. Overexpression of miR-125b alone in mice causes a very aggressive, transplantable myeloid leukemia. Before leukemia, these mice do not display elevation of white blood cells in the spleen or bone marrow; rather, the hematopoietic compartment shows lineage-skewing, with myeloid cell numbers dramatically increased and B-cell numbers severely diminished. miR-125b exerts this effect by up-regulating the number of common myeloid progenitors while inhibiting development of pre-B cells. We applied a miR-125b sponge loss of function system in vivo to show that miR-125b physiologically regulates hematopoietic development. Investigating the mechanism by which miR-125b regulates hematopoiesis, we found that, among a panel of candidate targets, the mRNA for Lin28A, an induced pluripotent stem cell gene, was most repressed by miR-125b in mouse hematopoietic stem and progenitor cells. Overexpressing Lin28A in the mouse hematopoietic system mimicked the phenotype observed on inhibiting miR-125b function, leading to a decrease in hematopoietic output. Relevant to the miR-125b overexpression phenotype, we also found that knockdown of Lin28A led to hematopoietic lineage-skewing, with increased myeloid and decreased B-cell numbers. Thus, the miR-125b target Lin28A is an important regulator of hematopoiesis and a primary target of miR-125b in the hematopoietic system.
Mao, Langyong; Zhang, Yan; Deng, Xiaolong; Mo, Wenjuan; Yu, Yao; Lu, Hong
The deregulation of microRNAs has been demonstrated in various tumor processes. Here, we report that microRNA-544 (miR-544) is decreased in cervical cancer tissues compared with normal cervical tissues. To identify the mechanisms involved in miR-544 deregulation, we studied the regulation of miR-544 expression at the transcriptional level. We first identified the transcriptional start site of miR-544 by 5' rapid amplification of cDNA ends and subsequently determined the miR-544 promoter. We discovered that the transcription factor Krueppel-like factor 4 (KLF4) is involved in the transcriptional regulation of miR-544 through interaction with the miR-544 promoter. In addition, we found that miR-544 directly targets the YWHAZ oncogene and functions as a tumor suppressor in cervical cancer cells. miR-544 is involved in cell cycle regulation and suppresses cervical cancer cell proliferation, colony formation, migration and invasion in a manner associated with YWHAZ downregulation. In summary, our findings demonstrate that KLF4 upregulates miR-544 transcription by activating the miR-544 promoter and that miR-544 functions as a tumor suppressor by targeting YWHAZ. Therefore, miR-544 may be a potential novel therapeutic target and prognostic marker for cervical cancer.
Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H
Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.
Block, Gregory J; Eskiw, Christopher H; Dellaire, Graham; Bazett-Jones, David P
Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.
Budanov, Andrei V
The tumor suppressor p53 protects organisms from most types of cancer through multiple mechanisms. The p53 gene encodes a stress-activated transcriptional factor that transcriptionally regulates a large set of genes with versatile functions. These p53-activated genes mitigate consequences of stress regulating cell viability, growth, proliferation, repair, and metabolism. Recently, we described a novel antioxidant function of p53, which is important for its tumor suppressor activity. Among the many antioxidant genes activated by p53, Sestrins (Sesns) are critical for suppression of reactive oxygen species (ROS) and protection from oxidative stress, transformation, and genomic instability. Sestrins can regulate ROS through their direct effect on antioxidant peroxiredoxin proteins and through the AMP-activated protein kinase-target of rapamycin signaling pathway. The AMP-activated protein kinase-target of rapamycin axis is critical for regulation of metabolism and autophagy, two processes associated with ROS production, and deregulation of this pathway increases vulnerability of the organism to stress, aging, and age-related diseases, including cancer. Recently, we have shown that inactivation of Sestrin in fly causes accumulation of age-associated damage. Hence, Sestrins can link p53 with aging and age-related diseases.
Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel
UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known...... to have wide-ranging and substantial effects on cellular function, both as part of signalling network modulation and more directly by modifying the function of key proteins. In this study, we show that PTM regulation is differentially targeted at different areas of the proteome, and that cytoskeletal...... proteins involved in neuronal process extension and maintenance are both more heavily modified and more frequently regulated at a PTM level. This suggests a clear role not only for PTMs in these processes, but possibly also for heavy protein modification in general. BIOLOGICAL SIGNIFICANCE: This study...
Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of small non-coding single-stranded RNA molecules that inhibit gene expression at post-transcriptional level. Gadd45g (growth arrest and DNA-damage-inducible 45 gamma is a stress-response protein, which has been implicated in several biological processes, including DNA repair, the cell cycle and cell differentiation. RESULTS: In this work, we found that miR-383 is a negative regulator of Gadd45g. Forced expression of miR-383 decreased the expression of Gadd45g through binding to the 3' untranslated region (3'-UTR, whereas inhibition of miR-383 increased Gadd45g expression. The presence of miR-383 increased the cellular sensitivity to DNA damage in breast cancer cells, which was rescued by ectopic expression of Gadd45g without the 3'-UTR. miR-383 also regulates the expression of Gadd45g in embryonic stem (ES cells, but not their apoptosis under genotoxic stress. miR-383 was further showed to negatively regulate ES cell differentiation via targeting Gadd45g, which subsequently modulates the pluripotency-associated genes. Taken together, our study demonstrates that miR-383 is a negative regulator of Gadd45g in both tumor cells and ES cells, however, has distinct function in regulating cell apoptosis. miR-383 may be used as antineoplastic agents in cancer chemotherapy. CONCLUSION: We demonstrate for the first time that miR-383 can specifically regulates the expression of Gadd45g by directly targeting to the 3-UTR region of Gadd45g mRNA, a regulatory process conserved in human tumor cells and mouse embryonic stem cells. These two compotents can be potentially used as antineoplastic agents in cancer chemotherapy.
O'Rourke John P
Full Text Available Abstract Background The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. Methods We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. Results By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Conclusions Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer
WU De-gang; WANG Xi-rui; YOU Yong-ping; LIU Ning; WANG Ying-yi; FAN Li-gang; LUO Hui; HAN Bin; SUN Li-hua; WANG Xie-feng; ZHANG Jun-xia; CAO Lei
Background Invasion growth is the most characteristic biological phenotype of glioblastoma,but the molecular mechanism in glioma cell invasion is poorly understood.Recent data have showed that microRNA plays an essential role in tumor invasion.Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion.Methods Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells.Luciferase reporter assay was used to determine focal adhesion kinase (FAK) as a target of miR-7.The levels of miR-7,matrix metalloproteinases (MMP)-2 and MMP-9 mRNA were detected by PCR assay,and the levels of FAK,MMP-2,MMP-9,total and phosphorylation serine/threonine kinase (AKT),and extracellular signal-regulated kinase (ERK) 1/2 were measured by Western blotting analysis.Results Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells.And up-regulation of miR-7 reduced FAK protein expression,Further,luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3'UTR of FAK mRNA.In addition,miR-7 repressed p-ERK1/2 and p-AKT level,MMP-2 and MMP-9 expression.Finally,the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues.Conclusion To our knowledge,these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.
Davudian, Sadaf; Mansoori, Behzad; Shajari, Neda; Mohammadi, Ali; Baradaran, Behzad
BACH1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1) is a transcriptional factor and a member of cap 'n' collar (CNC) and basic region leucine zipper factor family. In contrast to other bZIP family members, BACH1 appeared as a comparatively specific transcription factor. It acts as transcription regulator and is recognized as a recently hypoxia regulator and functions as an inducible repressor for the HO-1 gene in many human cell types in response to stress oxidative. In regard to studies lately, although, BACH1 has been related to the regulation of oxidative stress and heme oxidation, it has never been linked to invasion and metastasis. Recent studies have showed that BACH1 is involved in bone metastasis of breast cancer by up-regulating vital metastatic genes like CXCR4 and MMP1. This newly discovered aspect of BACH1 gene provides new insight into cancer progression study and stands on its master regulator role in metastasis process, raising the possibility of considering it as a potential target for cancer therapy.
Rosa H Jimenez
Full Text Available BACKGROUND: We investigated mTOR regulation of gene expression by studying rapamycin effect in two hepatic cell lines, the non-tumorigenic WB-F344 cells and the tumorigenic WB311 cells. The latter are resistant to the growth inhibitory effects of rapamycin, thus providing us with an opportunity to study the gene expression effects of rapamycin without confounding effects on cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: The hepatic cells were exposed to rapamycin for 24 hr. Microarray analysis on total RNA preparations identified genes that were affected by rapamycin in both cell lines and, therefore, modulated independent of growth arrest. Further studies showed that the promoter regions of these genes included E-box-containing transcription factor binding sites at higher than expected rates. Based on this, we tested the hypothesis that c-Myc is involved in regulation of gene expression by mTOR by comparing genes altered by rapamycin in the hepatic cells and by c-Myc induction in fibroblasts engineered to express c-myc in an inducible manner. Results showed enrichment for c-Myc targets among rapamycin sensitive genes in both hepatic cell lines. However, microarray analyses on wild type and c-myc null fibroblasts showed similar rapamycin effect, with the set of rapamycin-sensitive genes being enriched for c-Myc targets in both cases. CONCLUSIONS/SIGNIFICANCE: There is considerable overlap in the regulation of gene expression by mTOR and c-Myc. However, regulation of gene expression through mTOR is c-Myc-independent and cannot be attributed to the involvement of specific transcription factors regulated by the rapamycin-sensitive mTOR Complex 1.
Howick, Ken; Griffin, Brendan T.; Cryan, John F.; Schellekens, Harriët
Ghrelin is the only known peripherally-derived orexigenic hormone, increasing appetite and subsequent food intake. The ghrelinergic system has therefore received considerable attention as a therapeutic target to reduce appetite in obesity as well as to stimulate food intake in conditions of anorexia, malnutrition and cachexia. As the therapeutic potential of targeting this hormone becomes clearer, it is apparent that its pleiotropic actions span both the central nervous system and peripheral organs. Despite a wealth of research, a therapeutic compound specifically targeting the ghrelin system for appetite modulation remains elusive although some promising effects on metabolic function are emerging. This is due to many factors, ranging from the complexity of the ghrelin receptor (Growth Hormone Secretagogue Receptor, GHSR-1a) internalisation and heterodimerization, to biased ligand interactions and compensatory neuroendocrine outputs. Not least is the ubiquitous expression of the GHSR-1a, which makes it impossible to modulate centrally-mediated appetite regulation without encroaching on the various peripheral functions attributable to ghrelin. It is becoming clear that ghrelin’s central signalling is critical for its effects on appetite, body weight regulation and incentive salience of food. Improving the ability of ghrelin ligands to penetrate the blood brain barrier would enhance central delivery to GHSR-1a expressing brain regions, particularly within the mesolimbic reward circuitry. PMID:28134808
Barry, Joshua; Gu, Yuanzheng; Gu, Chen
Proper axonal and dendritic bundling is essential for the establishment of neuronal connections and the synchronization of synaptic inputs, respectively. Cell adhesion molecules of the L1-CAM (L1-cell adhesion molecule) family regulate axon guidance and fasciculation, neuron migration, dendrite morphology, and synaptic plasticity. It remains unclear how these molecules play so many different roles. Here we show that polarized axon-dendrite targeting of an avian L1-CAM protein, NgCAM (neuron-glia cell adhesion molecule), can regulate the switch of bundling of the two major compartments of rat hippocampal neurons. Using a new in-vitro model for studying neurite-neurite interactions, we found that expressed axonal NgCAM induced robust axonal bundling via the trans-homophilic interaction of immunoglobulin domains. Interestingly, dendritic bundling was induced by the dendritic targeting of NgCAM, caused by either deleting its fibronectin repeats or blocking activities of protein kinases. Consistent with the NgCAM results, expression of mouse L1-CAM also induced axonal bundling and blocking kinase activities disrupted its axonal targeting. Furthermore, the trans-homophilic interaction stabilized the bundle formation, probably through recruiting NgCAM proteins to contact sites and promoting guided axon outgrowth. Taken together, our results suggest that precise localization of L1-CAM is important for establishing proper cell-cell contacts in neural circuits.
Full Text Available Ghrelin is the only known peripherally-derived orexigenic hormone, increasing appetite and subsequent food intake. The ghrelinergic system has therefore received considerable attention as a therapeutic target to reduce appetite in obesity as well as to stimulate food intake in conditions of anorexia, malnutrition and cachexia. As the therapeutic potential of targeting this hormone becomes clearer, it is apparent that its pleiotropic actions span both the central nervous system and peripheral organs. Despite a wealth of research, a therapeutic compound specifically targeting the ghrelin system for appetite modulation remains elusive although some promising effects on metabolic function are emerging. This is due to many factors, ranging from the complexity of the ghrelin receptor (Growth Hormone Secretagogue Receptor, GHSR-1a internalisation and heterodimerization, to biased ligand interactions and compensatory neuroendocrine outputs. Not least is the ubiquitous expression of the GHSR-1a, which makes it impossible to modulate centrallymediated appetite regulation without encroaching on the various peripheral functions attributable to ghrelin. It is becoming clear that ghrelin’s central signalling is critical for its effects on appetite, body weight regulation and incentive salience of food. Improving the ability of ghrelin ligands to penetrate the blood brain barrier would enhance central delivery to GHSR-1a expressing brain regions, particularly within the mesolimbic reward circuitry.
Howick, Ken; Griffin, Brendan T; Cryan, John F; Schellekens, Harriët
Ghrelin is the only known peripherally-derived orexigenic hormone, increasing appetite and subsequent food intake. The ghrelinergic system has therefore received considerable attention as a therapeutic target to reduce appetite in obesity as well as to stimulate food intake in conditions of anorexia, malnutrition and cachexia. As the therapeutic potential of targeting this hormone becomes clearer, it is apparent that its pleiotropic actions span both the central nervous system and peripheral organs. Despite a wealth of research, a therapeutic compound specifically targeting the ghrelin system for appetite modulation remains elusive although some promising effects on metabolic function are emerging. This is due to many factors, ranging from the complexity of the ghrelin receptor (Growth Hormone Secretagogue Receptor, GHSR-1a) internalisation and heterodimerization, to biased ligand interactions and compensatory neuroendocrine outputs. Not least is the ubiquitous expression of the GHSR-1a, which makes it impossible to modulate centrallymediated appetite regulation without encroaching on the various peripheral functions attributable to ghrelin. It is becoming clear that ghrelin's central signalling is critical for its effects on appetite, body weight regulation and incentive salience of food. Improving the ability of ghrelin ligands to penetrate the blood brain barrier would enhance central delivery to GHSR-1a expressing brain regions, particularly within the mesolimbic reward circuitry.
Wang, Hui; Gui, Hongxing; Rallo, Michael S; Xu, Zhiyan; Matise, Michael P
The Notch signaling pathway controls cell fate decision, proliferation and other biological functions in both vertebrates and invertebrates. Precise regulation of the canonical Notch pathway ensures robustness of the signal throughout development and adult tissue homeostasis. Aberrant Notch signaling results in profound developmental defects and is linked to many human diseases. In this study, we identified the Atrophin family protein RERE (also called Atro2) as a positive regulator of Notch target Hes genes in the developing vertebrate spinal cord. Prior studies have shown that during early embryogenesis in mouse and zebrafish, deficit of RERE causes various patterning defects in multiple organs including the neural tube. Here, we detected the expression of RERE in the developing chick spinal cord, and found that normal RERE activity is needed for proper neural progenitor proliferation and neuronal differentiation possibly by affecting Notch mediated Hes expression. In mammalian cells, RERE co-immunoprecipitates with CBF1 and Notch intracellular domain (NICD), and is recruited to nuclear foci formed by overexpressed NICD1. RERE is also necessary for NICD to activate the expression of Notch target genes. Our findings suggest that RERE stimulates Notch target gene expression by preventing degradation of NICD protein, thereby facilitating the assembly of a transcriptional activating complex containing NICD, CSL and other coactivators. This article is protected by copyright. All rights reserved.
Broaddus Russell R
Full Text Available Abstract In eukaryotic cells, control mechanisms have developed that restrain cell-cycle transitions in response to stress. These regulatory pathways are termed cell-cycle checkpoints. The G2/M checkpoint prevents cells from entering mitosis when DNA is damaged in order to afford these cells an opportunity to repair the damaged DNA before propagating genetic defects to the daughter cells. If the damage is irreparable, checkpoint signaling might activate pathways that lead to apoptosis. Since alteration of cell-cycle control is a hallmark of tumorigenesis, cell-cycle regulators represent potential targets for therapy. The centrosome has recently come into focus as a critical cellular organelle that integrates G2/M checkpoint control and repairs signals in response to DNA damage. A growing number of G2/M checkpoint regulators have been found in the centrosome, suggesting that centrosome has an important role in G2/M checkpoint function. In this review, we discuss centrosome-associated regulators of the G2/M checkpoint, the dysregulation of this checkpoint in cancer, and potential candidate targets for cancer therapy.
Ortells, M Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R; López-Rodríguez, Cristina; Aramburu, Jose
Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses.
Choi, Sunga; Joo, Hee Kyoung; Jeon, Byeong Hwa
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.
Liu, Zhenyang; Wang, Junyu; Li, Yunjun; Fan, Juan; Chen, Lihua; Xu, Ruxiang
Glioblastoma is the most aggressive manifestation of malignant gliomas and considered to be among the deadliest forms of human cancers. MicroRNAs are found to tightly regulate diverse biological processes and considered to play important roles in cancer etiology. In this study, we found that microRNA-153 was significantly downregulated in glioblastoma tissues compared to matched non-tumor tissues and in glioblastoma cell lines. To investigate the potential function of microRNA-153 in glioblastoma, we transfected glioblastoma cell line U87MG as well as U373MG with synthetic microRNA-153 oligos and observed decreased cell proliferation and increased apoptosis. We further found that microRNA-153 restrained glutamine utilization and glutamate generation. Bioinformatics analysis revealed that glutaminase, which catalyzed the formation of glutamate from glutamine, is the potential target of microRNA-153. Indeed, microRNA-153 cannot further reduce glutamine utilization when glutaminase was knocked down. Overexpression of glutaminase abrogates the effect of microRNA-153 on glutamine utilization. Furthermore, the relative expression of microRNA-153 and glutaminase in glioblastoma versus matched non-tumor tissues showed a reverse correlation, further indicating that microRNA-153 may negatively regulate glutaminase in vivo. These results demonstrate an unexpected role of microRNA-153 in regulating glutamine metabolism and strengthen the role of microRNA-153 as a therapeutic target in glioblastoma.
Full Text Available Objective(s: Vascular calcification is one the major characteristics in patients with various types of chronic inflammatory disorders. MiRNAs have been shown to be involved in many normal biological functions as well as diseases; however, their role in vascular calcification has not received much attention. Materials and Methods: In the current study, we built a vascular calcification rat model using vitamin D3 plus nicotine and analyzed miRNA expression profile by miRNA chip assay. Potential target of one selected miRNA with sharpest variation in expression were predicted by both PicTar and TargetScan. The impact of the selected miRNA on the expression of the potential target on both mRNA and protein levels were measured by RT-PCR and Western blot, respectively. Results: Our results identified 16 dysregulated miRNAs, among which miR-297a showed the sharpest variation. Further analysis focusing on miR-297a revealed that fibroblast growth factor 23 (FGF23 was a potential target of miR297a. Measurement of FGF23 and its regulator Klotho on both mRNA and protein levels demonstrated that FGF23 was significantly increased while Klotho was decreased in rats with vascular calcification. Conclusion: Our results indicated that FGF23 was target of miR-297a and decreased miR-297a in vascular calcification lead to the increase of FGF23, which together with Klotho might enhance vascular calcification. The findings of this study could provide valuable information for the understanding of mechanisms underlying miR-dependent vascular calcification as well as potential treatment target for the disease.
Zou, Yuhai; Liu, Wenting; Zhang, Jinxia; Xiang, Dingcheng
MicroRNAs (miRs) are a class of important regulators, which are involved in the regulation of apoptosis. Oxidative stress‑induced apoptosis is the predominant factor accounting for cardiac ischemia‑reperfusion injury. miR‑153 has been previously shown to have an antitumor effect in cancer. However, whether miR‑153 is involved in oxidative stress‑induced apoptosis in the heart remains to be elucidated. To this end, the present study used reverse transcription‑quantitative polymerase chain reaction to detect miR-153 levels upon oxidative stress, and evaluated apoptosis, autophagy and expression of critical genes by western blotting. A luciferase assay was also used to confirm the potential target gene. In the present study, it was found that the expression of miR‑153 was significantly increased upon H2O2 stimulation, and the inhibition of endogenous miR‑153 decreased apoptosis. To further identify the mechanism underlying the pro‑apoptotic effect of miR‑153, the present study analyzed the 3'untranslated region of myeloid cell leukemia‑1 (Mcl‑1), and found that Mcl‑1 was potentially targeted by miR‑153. The forced expression of miR‑153 inhibited the expression of Mcl‑1 and luciferase activity, which was reversed by its antisense inhibitor. Furthermore, it was shown that the inhibition of miR‑153 induced autophagy during oxidative stress, and that its effects of autophagy induction and apoptosis inhibition were efficiently abrogated by Mcl‑1 small interfering RNA. In conclusion, the results of the present study elucidated a novel mechanism by which miR‑153 regulates the survival of cardimyocytes during oxidative stress through the modulation of apoptosis and autophagy. These effects may be mediated directly by targeting Mcl‑1. These finding revealed the potential clinical value of miR‑153 in the treatment of cardiovascular disease.
Wang, Yunxia; Zhu, Kongxi; Yu, Weihua; Wang, Hongjuan; Liu, Lan; Wu, Qiong; Li, Shuai; Guo, Jianqiang
Non-alcoholic fatty liver diseases (NAFLD) is one of the leading cause of chronic liver diseases in the world. However, the pathogenesis of NAFLD is still unclear. Emerging studies have demonstrated that microRNAs (miRs) are profoundly involved in NAFLD and related metabolic diseases. Here, we investigated the mechanisms by which miR-181b influences NAFLD via direct targeting SIRT1. The expression of miR181b was up-regulated while SIRT1 was down-regulated in both human NAFLD patients and high fat diet (HFD) induced NAFDL mice model. And palmitic acid (PA) treatment increased the miR-181b expression while decreased SIRT1 expression in HepG2 cells. Further, we identified that SIRT1 is a direct downstream target of miR-181b. Ectopic expression of miR-181b significantly repressed the 3'-UTR reporter activities of SIRT1 in a dose-dependent manner, while the effect of miR-181b was interrupted when the binding site of miR-181b within the SIRT1 3'-UTR was mutated. And overexpression of miR-181b reduced both the mRNA and protein levels of SIRT1 in HepG2 cells. We also found that inhibition of miR-181b expression alleviates hepatic steatosis both in vitro and in vivo. And the effect of miR-181b on steatosis was blocked by SIRT1 overexpression. Taken together, our data indicated that increased expression of miR-181b potentially contributes to altered lipid metabolism in NAFLD. Downregulation of miR-34a may be a therapeutic strategy against NAFLD by regulating its target SIRT1. Copyright © 2017. Published by Elsevier Inc.
Li, Jiehan; Papadopoulos, Vassilios
As a major regulator in obesity and its associated metabolic complications, the proper functioning of adipocytes is crucial for health maintenance, thus serving as an important target for the development of anti-obese and anti-diabetic therapies. There is increasing evidence that mitochondrial malfunction is a pivotal event in disturbing adipocyte cell homeostasis. Among major mitochondrial structure components, the high-affinity drug- and cholesterol-binding outer mitochondrial membrane translocator protein (18 kDa; TSPO) has shown importance across a broad spectrum of mitochondrial functions. Recent studies demonstrated the presence of TSPO in white adipocyte mitochondria of mice, and administration of TSPO drug ligands to obese mice reduced weight gain and lowered glucose level. Therefore, it is of great interest to assess whether TSPO in adipocytes could serve as a drug target to regulate adipocyte activities with potential influence on weight control and glucose metabolism. Two structurally distinct TSPO drug ligands, PK 11195 and FGIN-1-27, improved the intracellular dynamics of 3T3-L1 adipocytes, such as the production and release of adipokines, glucose uptake, and adipogenesis. TSPO knockdown in either differentiated adipocytes or preadipocytes impaired these functions. Findings from 3T3-L1 cells were related to human primary cells, where TSPO expression was tightly associated with the metabolic state of primary adipocytes and the differentiation of primary preadipocytes. These results suggest that TSPO expression is essential to safeguard healthy adipocyte functions, and that TSPO activation in adipocytes improves their metabolic status in regulating glucose homeostasis. Adipocyte TSPO may serve as a pharmacologic target for the treatment of obesity and diabetes.
Fair, K; Anderson, M; Bulanova, E; Mi, H; Tropschug, M; Diaz, M O
The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.
Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.
Zhang, Honghao; Guo, Yujie; Mishra, Amarjit; Gou, Deming; Chintagari, Narendranath Reddy; Liu, Lin
Lung surfactant secretion is a highly regulated process. Our previous studies have shown that VAMP-2 is essential for surfactant secretion. In the present study we investigated the role of miR-206 in surfactant secretion through VAMP-2. VAMP-2 was confirmed to be a target of miR-206 by 3'-untranslational region (3'-UTR) luciferase assay. Mutations in the predicated miR-206 binding sites reduced the binding of miR-206 to the 3'-UTR of VAMP-2. miR-206 decreased the expression of VAMP-2 protein and decreased the lung surfactant secretion in alveolar type II cells. In conclusion, miR-206 regulates lung surfactant secretion by limiting the availability of VAMP-2 protein.
Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan
Most microalgae abundantly accumulate lipid droplets (LDs) containing triacylglycerols (TAGs) under several stress conditions, but the underlying molecular mechanism of this accumulation remains unclear. In a recent study, we found that inhibition of TOR (target of rapamycin), a highly conserved protein kinase of eukaryotes, by rapamycin resulted in TAG accumulation in microalgae, indicating that TOR negatively regulates TAG accumulation. Here, we show that formation of intracellular LDs and TAG accumulation were also induced in the unicellular green alga Chlamydomonas reinhardtii after exposure to Torin1 or AZD8055, which are novel TOR inhibitors that inhibit TOR activity in a manner different from rapamycin. These results supported quite well our previous conclusion that TOR is a central regulator of TAG accumulation in microalgae.
Kondratyeva, L G; Vinogradova, T V; Chernov, I P; Sverdlov, E D
The transformation of normal precursors into cancer cells is an intricately regulated, multistep process. The master regulatory genes that play a crucial role in the process of organism development may also play a key role in carcinogenesis. From such a point of view, cancer is not simply a genetic disease that is due to a progressive accumulation of mutation--it is also a disorder of the developmental system of the tissue in which cancer emerges. Master regulators and their genes disturb stem cell differentiation upon mutation and thus may serve as targets for cancer therapy, in addition to the classic oncogenes and suppressors of tumor formation. This review is an attempt to give a modern concept of master genes and their functions in adult stem cells of the organism and in carcinogenesis, with pancreatic cancer as an example.
Full Text Available The central nervous system monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3'-untranslated regions (3'UTR of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3’UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3'UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p and mir-488. We observed that mir-383, mir-384-3p and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus.
Derghal, Adel; Djelloul, Mehdi; Airault, Coraline; Pierre, Clément; Dallaporta, Michel; Troadec, Jean-Denis; Tillement, Vanessa; Tardivel, Catherine; Bariohay, Bruno; Trouslard, Jérôme; Mounien, Lourdes
The central nervous system (CNS) monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC) neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs) are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3′-untranslated regions (3′UTR) of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3′UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3′UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p, and mir-488. We observed that mir-383, mir-384-3p, and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p, and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p, and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus. PMID:25999818
Brackertz, B; Conrad, H.; Daniel, J.; Kast, B; Krönig, H; Busch, D.H.; Adamski, J.; C Peschel; Bernhard, H
The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD+ myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. A...
Full Text Available CD69 has been identified as an early activation marker of lymphocytes. However, recent work has indicated that CD69 plays an essential role for the regulation of inflammatory processes. Particularly, CD69 is highly expressed by lymphocytes at mucosal sites being constantly exposed to the intestinal microflora (one of the nature’s most complex and most densely populated microbial habitats and food antigens, while only a small number of circulating leukocytes express this molecule. In this review we will discuss the role of CD69 in mucosal tissue and consider CD69 as a potential target for the development of novel treatments of intestinal inflammation.
Wan, S M; Peng, P; Guan, T
Ovarian cancer is the second most common gynaecological cancer worldwide, and its molecular mechanism has not been completely understood. Ets-1 is a member of the Ets transcription family and can play important roles in the regulation of extracellular matrix remodelling, invasion, angiogenesis and drug resistance in several malignancies, including ovarian cancer. In the current study, we downloaded two datasets from Gene Expression Omnibus database and sought to explore the regulation mechanism of Ets-1 in ovarian cancer by computational analysis of gene expression profiles. Microarray analysis identified a total of 548 genes that were regulated by Ets-1 in ovarian cancer. Functional annotation of these genes revealed that Ets-1 may be involved in several biological processes, both physiological and pathological, such as system development, response to stimulus, vascular endothelial growth factor (VEGF) production, morphogenesis, cell proliferation, cell adhesion and signal transduction. Further, DNA methylation analysis of the DEGs found that 26.5% (145) of them were differentially methylated genes in ovarian cancer. Our results provide insight into the mechanism of Ets-1 regulating the transcription of its target genes in the complex and multistep process of ovarian cancer progression.
The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.
Li, Ran; Zhang, Jin; Li, Jiancai; Zhou, Guoxin; Wang, Qi; Bian, Wenbo; Erb, Matthias; Lou, Yonggen
Plants generally respond to herbivore attack by increasing resistance and decreasing growth. This prioritization is achieved through the regulation of phytohormonal signaling networks. However, it remains unknown how this prioritization affects resistance against non-target herbivores. In this study, we identify WRKY70 as a specific herbivore-induced, mitogen-activated protein kinase-regulated rice transcription factor that physically interacts with W-box motifs and prioritizes defence over growth by positively regulating jasmonic acid (JA) and negatively regulating gibberellin (GA) biosynthesis upon attack by the chewing herbivore Chilo suppressalis. WRKY70-dependent JA biosynthesis is required for proteinase inhibitor activation and resistance against C. suppressalis. In contrast, WRKY70 induction increases plant susceptibility against the rice brown planthopper Nilaparvata lugens. Experiments with GA-deficient rice lines identify WRKY70-dependent GA signaling as the causal factor in N. lugens susceptibility. Our study shows that prioritizing defence over growth leads to a significant resistance trade-off with important implications for the evolution and agricultural exploitation of plant immunity. DOI: http://dx.doi.org/10.7554/eLife.04805.001 PMID:26083713
Li, Ran; Zhang, Jin; Li, Jiancai; Zhou, Guoxin; Wang, Qi; Bian, Wenbo; Erb, Matthias; Lou, Yonggen
Plants generally respond to herbivore attack by increasing resistance and decreasing growth. This prioritization is achieved through the regulation of phytohormonal signaling networks. However, it remains unknown how this prioritization affects resistance against non-target herbivores. In this study, we identify WRKY70 as a specific herbivore-induced, mitogen-activated protein kinase-regulated rice transcription factor that physically interacts with W-box motives and prioritizes defence over growth by positively regulating jasmonic acid (JA) and negatively regulating gibberellin (GA) biosynthesis upon attack by the chewing herbivore Chilo suppressalis. WRKY70-dependent JA biosynthesis is required for proteinase inhibitor activation and resistance against C. suppressalis. In contrast, WRKY70 induction increases plant susceptibility against the rice brown planthopper Nilaparvata lugens. Experiments with GA-deficient rice lines identify WRKY70-dependent GA signaling as the causal factor in N. lugens susceptibility. Our study shows that prioritizing defence over growth leads to a significant resistance trade-off with important implications for the evolution and agricultural exploitation of plant immunity.
Tamimi, Yahya; Skarie, Jonathan M; Footz, Tim; Berry, Fred B; Link, Brian A; Walter, Michael A
The forkhead C1 (FOXC1) transcription factor is involved in the development and regulation of several organs, including the eye, where FOXC1 alterations cause iris, trabecular meshwork and corneal anomalies. Using nickel agarose chromatin enrichment with human anterior segment cells, we previously identified the fibroblast growth factor 19 (FGF19) locus as a gene potentially regulated by FOXC1. Here, we demonstrate that FGF19 is a direct target of FOXC1 in the eye. FOXC1 positively regulates FGF19 expression in corneal and periocular mesenchymal cells in cell culture and in zebrafish embryos. Through the FGFR4 tyrosine kinase, FGF19 promotes MAPK phosphorylation in the developing and mature cornea. During development, loss of either FOXC1 or FGF19 results in complementary, but distinct, anterior segment dysgeneses. This study reveals an important role for FOXC1 in the direct regulation of the FGF19-FGFR4-MAPK pathway to promote both the development and maintenance of anterior segment structures within the eye.
Guo, Yunxue; Chen, Yaosheng; Zhang, Yun; Zhang, Yue; Chen, Luxi; Mo, Delin
Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.
Brohée, Laura; Demine, Stéphane; Willems, Jérome; Arnould, Thierry; Colige, Alain C; Deroanne, Christophe F
Lipogenesis inhibition was reported to induce apoptosis and repress proliferation of cancer cells while barely affecting normal cells. Lipins exhibit dual function as enzymes catalyzing the dephosphorylation of phosphatidic acid to diacylglycerol and as co-transcriptional regulators. Thus, they are able to regulate lipid homeostasis at several nodal points. Here, we show that lipin-1 is up-regulated in several cancer cell lines and overexpressed in 50 % of high grade prostate cancers. The proliferation of prostate and breast cancer cells, but not of non-tumorigenic cells, was repressed upon lipin-1 knock-down. Lipin-1 depletion also decreased cancer cell migration through RhoA activation. Lipin-1 silencing did not significantly affect global lipid synthesis but enhanced the cellular concentration of phosphatidic acid. In parallel, autophagy was induced while AKT and ribosomal protein S6 phosphorylation were repressed. We also observed a compensatory regulation between lipin-1 and lipin-2 and demonstrated that their co-silencing aggravates the phenotype induced by lipin-1 silencing alone. Most interestingly, lipin-1 depletion or lipins inhibition with propranolol sensitized cancer cells to rapamycin. These data indicate that lipin-1 controls main cellular processes involved in cancer progression and that its targeting, alone or in combination with other treatments, could open new avenues in anticancer therapy.
Li, Yao; Li, Shengjie; Jin, Ping; Chen, Liming; Ma, Fei
MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with the following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'-untranslated region in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis. Copyright © 2017 the American Physiological Society.
Wallace, Sean W; Magalhaes, Ana; Hall, Alan
Rho GTPases regulate multiple signaling pathways to control a number of cellular processes during epithelial morphogenesis. To investigate the downstream pathways through which Rho regulates epithelial apical junction formation, we screened a small interfering RNA (siRNA) library targeting 28 known Rho target proteins in 16HBE human bronchial epithelial cells. This led to the identification of the serine-threonine kinase PRK2 (protein kinase C-related kinase 2, also called PKN2). Depletion of PRK2 does not block the initial formation of primordial junctions at nascent cell-cell contacts but does prevent their maturation into apical junctions. PRK2 is recruited to primordial junctions, and this localization depends on its C2-like domain. Rho binding is essential for PRK2 function and also facilitates PRK2 recruitment to junctions. Kinase-dead PRK2 acts as a dominant-negative mutant and prevents apical junction formation. We conclude that PRK2 is recruited to nascent cell-cell contacts through its C2-like and Rho-binding domains and promotes junctional maturation through a kinase-dependent pathway.
Full Text Available Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.
Verschoor, Meghan L; Singh, Gurmit
Ovarian cancer is characterized by high rates of metastasis and therapeutic resistance. Many chemotherapeutic agents rely on the induction of oxidative stress to cause cancer cell death, thus targeting redox regulation is a promising strategy to overcome drug resistance. We have used a tetracycline-inducible Ets-1 overexpression model derived from 2008 ovarian cancer cells in the present study. To examine the role of Ets-1 in glutathione regulation we have measured intracellular reactive oxygen species and glutathione levels, as well as glutathione peroxidase enzyme activity. Glutathione synthesis was limited using transsulfuration or Sx(c)- pathway blocking agents, and glutamate release was measured to confirm Sx(c)- blockade. Cell viability following drug treatment was assessed via crystal violet assay. Oxidative stress was induced through glucose oxidase treatment, which produces hydrogen peroxide by glucose oxidation. The protein expressions of redox-related factors were measured through western blotting. Overexpression of Ets-1 was associated with decreased intracellular ROS, concomitantly with increased intracellular GSH, GPX antioxidant activity, and Sx(c)- transporter activity. Under basal conditions, inhibition of the transsulfuration pathway resulted in decreased GSH levels and GPX activity in all cell lines, whereas inhibition of Sx(c)- by sulfasalazine decreased GPX activity in Ets-1-expressing cells only. However, under oxidative stress the intracellular GSH levels decreased significantly in correlation with increased Ets-1 expression following sulfasalazine treatment. In this study we have identified a role for proto-oncogene Ets-1 in the regulation of intracellular glutathione levels, and examined the effects of the anti-inflammatory drug sulfasalazine on glutathione depletion using an ovarian cancer cell model. The findings from this study show that Ets-1 mediates enhanced Sx(c)- activity to increase glutathione levels under oxidative stress
Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.
Ribeiro, Carolina Werner; Baldacci-Cresp, Fabien; Pierre, Olivier; Larousse, Marie; Benyamina, Sofiane; Lambert, Annie; Hopkins, Julie; Castella, Claude; Cazareth, Julie; Alloing, Geneviève; Boncompagni, Eric; Couturier, Jérémy; Mergaert, Peter; Gamas, Pascal; Rouhier, Nicolas; Montrichard, Françoise; Frendo, Pierre
Legumes associate with rhizobia to form nitrogen (N2)-fixing nodules, which is important for plant fitness [1, 2]. Medicago truncatula controls the terminal differentiation of Sinorhizobium meliloti into N2-fixing bacteroids by producing defensin-like nodule-specific cysteine-rich peptides (NCRs) [3, 4]. The redox state of NCRs influences some biological activities in free-living bacteria, but the relevance of redox regulation of NCRs in planta is unknown [5, 6], although redox regulation plays a crucial role in symbiotic nitrogen fixation [7, 8]. Two thioredoxins (Trx), Trx s1 and s2, define a new type of Trx and are expressed principally in nodules . Here, we show that there are four Trx s genes, two of which, Trx s1 and s3, are induced in the nodule infection zone where bacterial differentiation occurs. Trx s1 is targeted to the symbiosomes, the N2-fixing organelles. Trx s1 interacted with NCR247 and NCR335 and increased the cytotoxic effect of NCR335 in S. meliloti. We show that Trx s silencing impairs bacteroid growth and endoreduplication, two features of terminal bacteroid differentiation, and that the ectopic expression of Trx s1 in S. meliloti partially complements the silencing phenotype. Thus, our findings show that Trx s1 is targeted to the bacterial endosymbiont, where it controls NCR activity and bacteroid terminal differentiation. Similarly, Trxs are critical for the activation of defensins produced against infectious microbes in mammalian hosts. Therefore, our results suggest the Trx-mediated regulation of host peptides as a conserved mechanism among symbiotic and pathogenic interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.
Romagnolo, Donato F; Zempleni, Janos; Selmin, Ornella I
Posttranslational modifications of histones, alterations in the recruitment and functions of non-histone proteins, DNA methylation, and changes in expression of noncoding RNAs contribute to current models of epigenetic regulation. Nuclear receptors (NRs) are a group of transcription factors that, through ligand-binding, act as sensors to changes in nutritional, environmental, developmental, pathophysiologic, and endocrine conditions and drive adaptive responses via gene regulation. One mechanism through which NRs direct gene expression is the assembly of transcription complexes with cofactors and coregulators that possess chromatin-modifying properties. Chromatin modifications can be transient or become part of the cellular "memory" and contribute to genomic imprinting. Because many food components bind to NRs, they can ultimately influence transcription of genes associated with biologic processes, such as inflammation, proliferation, apoptosis, and hormonal response, and alter the susceptibility to chronic diseases (e.g., cancer, diabetes, obesity). The objective of this review is to highlight how NRs influence epigenetic regulation and the relevance of dietary compound-NR interactions in human nutrition and for disease prevention and treatment. Identifying gene targets of unliganded and bound NRs may assist in the development of epigenetic maps for food components and dietary patterns. Progress in these areas may lead to the formulation of disease-prevention models based on epigenetic control by individual or associations of food ligands of NRs.
Osterwalder, Marco; Speziale, Dario; Shoukry, Malak; Mohan, Rajiv; Ivanek, Robert; Kohler, Manuel; Beisel, Christian; Wen, Xiaohui; Scales, Suzie J.; Christoffels, Vincent M.; Visel, Axel; Lopez-Rios, Javier; Zeller, Rolf
Summary The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatio-temporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then, we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting on Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. Our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling. PMID:25453830
Full Text Available Abstract Background The tetracycline-controlled transactivator system is a powerful tool to control gene expression in vitro and to generate consistent and conditional transgenic in vivo model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator. Findings In this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results. Conclusions The purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time.
Bhattacharjee, Saikat; Halane, Morgan K; Kim, Sang Hee; Gassmann, Walter
Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.
Fuglseth, Bente Beckstroem
This report has explored changes in two regulations targeting energy performance of buildings in Sweden, energy requirements and certification of buildings. The objective has been to investigate the effect of the implementation of the EU directive on energy performance of buildings (EPBD) on these two regulations and to what degree the directive can explain the regulative changes. The analytical framework has also included domestic factors; the influence of the national government and the organizational field. The analysis revealed that whereas the EPBD has acted only as facilitator in connection with the changes in energy requirements, it has been the sole driver of some of the changes in Sweden's new certification system. Several of the changes during the period studied can however be traced to the national government and the organizational field. But the EPBD has also worked as a facilitator of the changes promoted by domestic actors. The directive has been used to legitimize radical changes that would have been difficult to implement in other ways. (Author). 40 refs., 2 tabs
Li, Yingxian; Wang, Xiaogang; Li, Qi; Lv, Ke; Wan, Yumin; Li, Yinghui; Bai, Yanqiang
Background: MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR) of mRNAs, resulting in translational repression. Growing evidence shows that microRNAs (miRNAs) regu-late various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; How-ever, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast dif-ferentiation remains elusive. Methods: To study the potential involvement of miRNAs in osteoblast differentiation under stimulated microgravity, we analyzed the expression of 20 bone relative miRNAs using real time PCR platform to find particularly miRNAs whose expression is altered during osteoblast differentiation. TargetScan, miRBase and Miranda were used to predict the target gene of candidate miRNA. To investigate whether ATF4 can be directly targeted by miR-214, we engineered luciferase reporters that have either the wild-type 3'UTRs of these genes, or the mutant UTRs with a 6 base pair (bp) deletion in the target sites. Lastly, to address the in vivo role of miR-214 in bone formation, tail suspension mice model was used to simulate the change of osteoblast function and bone loss. Results: Recent studies have sug-gested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify miR-214 in MC3T3-E1 cells, which is a primary mouse osteoblasts cell line, to promote osteoblast differentiation by repressing Activating Transcription Factor4 (ATF4) ex-pression at the posttranscriptional level. What is more, miR-214 was found to be transcribed in C2C12 cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-214 attenuated BMP2-induced osteoblastogenesis
Liu, Ning-Ai; Araki, Takako; Cuevas-Ramos, Daniel; Hong, Jiang; Ben-Shlomo, Anat; Tone, Yukiko; Tone, Masahide
Context: Cushing disease, due to pituitary corticotroph tumor ACTH hypersecretion, drives excess adrenal cortisol production with adverse morbidity and mortality. Loss of glucocorticoid negative feedback on the hypothalamic-pituitary-adrenal axis leads to autonomous transcription of the corticotroph precursor hormone proopiomelanocortin (POMC), consequent ACTH overproduction, and adrenal hypercortisolism. We previously reported that R-roscovitine (CYC202, seliciclib), a 2,6,9-trisubstituted purine analog, suppresses cyclin-dependent-kinase 2/cyclin E and inhibits ACTH in mice and zebrafish. We hypothesized that intrapituitary cyclin E signaling regulates corticotroph tumor POMC transcription independently of cell cycle progression. The aim was to investigate whether R-roscovitine inhibits human ACTH in corticotroph tumors by targeting the cyclin-dependent kinase 2/cyclin E signaling pathway. Methods: Primary cell cultures of surgically resected human corticotroph tumors were treated with or without R-roscovitine, ACTH measured by RIA and quantitative PCR, and/or Western blot analysis performed to investigate ACTH and lineage-specific transcription factors. Cyclin E and E2F transcription factor 1 (E2F1) small interfering RNA (siRNA) transfection was performed in murine corticotroph tumor AtT20 cells to elucidate mechanisms for drug action. POMC gene promoter activity in response to R-roscovitine treatment was analyzed using luciferase reporter and chromatin immunoprecipitation assays. Results: R-roscovitine inhibits human corticotroph tumor POMC and Tpit/Tbx19 transcription with decreased ACTH expression. Cyclin E and E2F1 exhibit reciprocal positive regulation in corticotroph tumors. R-roscovitine disrupts E2F1 binding to the POMC gene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. Conclusion: R-roscovitine inhibits human pituitary corticotroph tumor ACTH by targeting the
James M Acton
By 2003. as military planners had become worried that the country's long-range conventional weapons, such as cruise missiles, might be too slow to reach hypothetical distant targets that needed to be struck urgently...
Ren, B; Maniatis, T
The stage-specific expression of the Drosophila alcohol dehydrogenase (Adh) gene is achieved through the alternate activation of two tandem promoters. The proximal promoter is active primarily during late embryonic development and early larval stages, while the distal promoter is active in late third instar larvae and adults. Here, we provide evidence that this Adh promoter switch is regulated by a zinc finger repressor protein (AEF-1) that is expressed predominantly in adult flies and targets the initiator region of the proximal promoter. We propose that AEF-1 plays a critical role in Adh promoter switching by blocking interactions between a component of the general transcription machinery and the initiator region of the proximal promoter. PMID:9463385
Hébert, J M; Rosenquist, T; Götz, J; Martin, G R
Fibroblast growth factor 5 (FGF5) is a secreted signaling protein. Mice homozygous for a predicted null allele of the Fgf5 gene, fgf5neo, produced by gene targeting in embryonic stem cells, have abnormally long hair. This phenotype appears identical to that of mice homozygous for the spontaneous mutation angora (go). The fgf5neo and go mutations fail to complement one another, and exon 1 of Fgf5 is deleted in DNA from go homozygotes, demonstrating that go is a mutant allele of Fgf5. Expression of Fgf5 is detected in hair follicles from wild-type mice and is localized to the outer root sheath during the anagen VI phase of the hair growth cycle. These findings provide evidence that FGF5 functions as an inhibitor of hair elongation, thus identifying a molecule whose normal function is apparently to regulate one step in the progression of the follicle through the hair growth cycle.
Yang, Hui; Wu, Xiao-Li; Wu, Kai-Hua; Zhang, Rong; Ju, Li-Li; Ji, Ying; Zhang, Yan-Wei; Xue, Song-Lin; Zhang, Ye-Xin; Yang, Yong-Feng; Yu, Min-Min
Cervical cancer is one of the most lethal malignancies amongst women, partially because it is unresponsive to many chemotherapeutic drugs. The mechanism underlying cisplatin (DDP) resistance in cervical cancer remains largely elusive. In this study, by detecting the 12 most reported down-regulated miRNAs in chemotherapy-sensitive and -resistant cervical cancer cells, we found that miR-497 was significantly reduced in chemotherapy-resistant HeLa/DDP cells and contributed to DDP chemosensitivity. Transketolase (TKT), a thiamine-dependent enzyme that plays a role in the channeling of excess glucose phosphates to glycolysis in the pentose phosphate pathway, was identified as a direct target of miR-497. TKT expression in clinical specimens was characterized by immunohistochemistry and the result showed that TKT was highly expressed in 81.1% (60/74) of samples examined. Data from Oncomine databases revealed that TKT was significantly up-regulated in cervical cancer tissues compared to normal controls. Gain-of-function and loss-of-function studies showed that the miR-497/TKT axis was a critical modulator in DDP chemosensitivity as demonstrated by cell viability and apoptosis assays. Mechanistically, DDP chemosensitivity induced by the miR-497/TKT axis was associated with glutathione (GSH) depletion and reactive oxygen species (ROS) generation, and GSH treatment effectively abrogated miR-497/TKT-mediated chemosensitivity. In conclusion, these findings suggest that a deregulated miR-497/TKT axis has important implications in the cervical cancer cellular response to DDP, and thus targeting this axis may be a promising way to improve chemosensitivity in cervical cancer. PMID:27904781
Turkington, R C; Longley, D B; Allen, W L; Stevenson, L; McLaughlin, K; Dunne, P D; Blayney, J K; Salto-Tellez, M; Van Schaeybroeck, S; Johnston, P G
The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.
Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela
Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ(-/-) mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper-tyrosine-phosphorylated in the PTPσ(-/-) mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ(-/-) mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.
Wang, Shizhi; Ma, Gaoxiang; Zhu, Haixia; Lv, Chunye; Chu, Haiyan; Tong, Na; Wu, Dongmei; Qiang, Fulin; Gong, Weida; Zhao, Qinghong; Tao, Guoquan; Zhou, Jianwei; Zhang, Zhengdong; Wang, Meilin
Our previous genome-wide miRNA microarray study revealed that miR-107 was upregulated in gastric cancer (GC). In this study we aimed to explore its biological role in the pathogenesis of GC. Integrating in silico prediction algorithms with western blotting assays revealed that miR-107 inhibition enhanced NF1 (neurofibromin 1) mRNA and protein levels, suggesting that NF1 is one of miR-107 targets in GC. Luciferase reporter assay revealed that miR-107 suppressed NF1 expression by binding to the first potential binding site within the 3'-UTR of NF1 mRNA. mRNA stable assay indicated this binding could result in NF1 mRNA instability, which might contribute to its abnormal protein expression. Functional analyses such as cell growth, transwell migration and invasion assays were used to investigate the role of interaction between miR-107 and its target on GC development and progression. Moreover, We investigated the association between the clinical phenotype and the status of miR-107 expression in 55 GC tissues, and found the high expression contributed to the tumor size and depth of invasion. The results exhibited that down regulation of miR-107 opposed cell growth, migration, and invasion, whereas NF1 repression promoted these phenotypes. Our findings provide a mechanism by which miR-107 regulates NF1 in GC, as well as highlight the importance of interaction between miR-107 and NF1 in GC development and progression.
Tsutsuki, Hiroyasu; Jung, Minkyung; Zhang, Tianli; Ono, Katsuhiko; Ida, Tomoaki; Kunieda, Kohei; Ihara, Hideshi; Akaike, Takaaki; Sawa, Tomohiro
8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections.
Jones, M D; Ademi, I; Yin, X; Gong, Y; Zamble, D B
Nickel is an essential transition metal for the survival of Helicobacter pylori in the acidic human stomach. The nickel-responsive transcriptional regulator HpNikR is important for maintaining healthy cytosolic nickel concentrations through the regulation of multiple genes, but its complete regulon and role in nickel homeostasis are not well understood. To investigate potential gene targets of HpNikR, ChIP sequencing was performed using H. pylori grown at neutral pH in nickel-supplemented media and this experiment identified HPG27_866 (frpB2) and HPG27_1499 (ceuE). These two genes are annotated to encode a putative iron transporter and a nickel-binding, periplasmic component of an ABC transporter, respectively. In vitro DNA-binding assays revealed that HpNikR binds both gene promoter sequences in a nickel-responsive manner with affinities on the order of ∼10(-7) M. The recognition sites of HpNikR were identified and loosely correlate with the HpNikR pseudo-consensus sequence (TATTATT-N11-AATAATA). Quantitative PCR experiments revealed that HPG27_866 and HPG27_1499 are transcriptionally repressed following growth of H. pylori G27 in nickel-supplemented media, and that this response is dependent on HpNikR. In contrast, iron supplementation results in activation of HPG27_1499, but no impact on the expression of HPG27_866 was observed. Metal analysis of the Δ866 strain revealed that HPG27_866 has an impact on nickel accumulation. These studies demonstrate that HPG27_866 and HPG27_1499 are both direct targets of HpNikR and that HPG27_866 influences nickel uptake in H. pylori.
Jiang, Lulu; Hindmarch, Charles C. T.; Rogers, Mark; Campbell, Colin; Waterfall, Christy; Coghill, Jane; Mathieson, Peter W.; Welsh, Gavin I.
Glucocorticoids are steroids that reduce inflammation and are used as immunosuppressive drugs for many diseases. They are also the mainstay for the treatment of minimal change nephropathy (MCN), which is characterised by an absence of inflammation. Their mechanisms of action remain elusive. Evidence suggests that immunomodulatory drugs can directly act on glomerular epithelial cells or ‘podocytes’, the cell type which is the main target of injury in MCN. To understand the nature of glucocorticoid effects on non-immune cell functions, we generated RNA sequencing data from human podocyte cell lines and identified the genes that are significantly regulated in dexamethasone-treated podocytes compared to vehicle-treated cells. The upregulated genes are of functional relevance to cytoskeleton-related processes, whereas the downregulated genes mostly encode pro-inflammatory cytokines and growth factors. We observed a tendency for dexamethasone-upregulated genes to be downregulated in MCN patients. Integrative analysis revealed gene networks composed of critical signaling pathways that are likely targeted by dexamethasone in podocytes. PMID:27774996
Full Text Available Cystic fibrosis (CF is caused by genetic mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR protein. These mutations can impact the synthesis and transfer of the CFTR protein to the apical membrane of epithelial cells, as well as influencing the gating or conductance of chloride and bicarbonate ions through the channel. CFTR dysfunction results in ionic imbalance of epithelial secretions in several organ systems, such as the pancreas, gastrointestinal tract, liver and the respiratory system. Since discovery of the CFTR gene in 1989, research has focussed on targeting the underlying genetic defect to identify a disease-modifying treatment for CF. Investigated management strategies have included gene therapy and the development of small molecules that target CFTR mutations, known as CFTR modulators. CFTR modulators are typically identified by high-throughput screening assays, followed by preclinical validation using cell culture systems. Recently, one such modulator, the CFTR potentiator ivacaftor, was approved as an oral therapy for CF patients with the G551D-CFTR mutation. The clinical development of ivacaftor not only represents a breakthrough in CF care but also serves as a noteworthy example of personalised medicine.
Ho, Gary P H; Selvakumar, Balakrishnan; Mukai, Jun; Hester, Lynda D; Wang, Yuxuan; Gogos, Joseph A; Snyder, Solomon H
PSD-95, a principal scaffolding component of the postsynaptic density, is targeted to synapses by palmitoylation, where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA-mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction. Copyright © 2011 Elsevier Inc. All rights reserved.
Brackertz, B; Conrad, H; Daniel, J; Kast, B; Krönig, H; Busch, D H; Adamski, J; Peschel, C; Bernhard, H
The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD(+) myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT(+) myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin.
Ho, Gary P. H.; Selvakumar, Balakrishnan; Mukai, Jun; Hester, Lynda D.; Wang, Yuxuan; Gogos, Joseph A.; Snyder, Solomon H.
PSD-95, a principal scaffolding component of the post-synaptic density, is targeted to synapses by palmitoylation where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction. PMID:21745643
Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.
Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010
Full Text Available Kaposi's sarcoma herpesvirus (KSHV encodes a cluster of twelve micro (miRNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3, a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.
Beningo, Karen A.; Wang, Yu-li
Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.
Dmitriev, Petr; Stankevicins, Luiza; Ansseau, Eugenie; Petrov, Andrei; Barat, Ana; Dessen, Philippe; Robert, Thomas; Turki, Ahmed; Lazar, Vladimir; Labourer, Emmanuel; Belayew, Alexandra; Carnac, Gilles; Laoudj-Chenivesse, Dalila; Lipinski, Marc; Vassetzky, Yegor S.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder linked to the deletion of an integral number of 3.3-kb-long macrosatellite repeats (D4Z4) within the subtelomeric region of chromosome 4q. Most genes identified in this region are overexpressed in FSHD myoblasts, including the double homeobox genes DUX4 and DUX4c. We have carried out a simultaneous miRNome/transcriptome analysis of FSHD and control primary myoblasts. Of 365 microRNAs (miRNAs) analyzed in this study, 29 were found to be differentially expressed between FSHD and normal myoblasts. Twenty-one microRNAs (miR-1, miR-7, miR-15a, miR-22, miR-30e, miR-32, miR-107, miR-133a, miR-133b, miR-139, miR-152, miR-206, miR-223, miR-302b, miR-331, miR-362, miR-365, miR-382, miR-496, miR-532, miR-654, and miR-660) were up-regulated, and eight were down-regulated (miR-15b, miR-20b, miR-21, miR-25, miR-100, miR-155, miR-345, and miR-594). Twelve of the miRNAs up-regulated in FHSD were also up-regulated in the cells ectopically expressing DUX4c, suggesting that this gene could regulate miRNA gene transcription. The myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206 were highly expressed in FSHD myoblasts, which nonetheless did not prematurely enter myogenic differentiation. This could be accounted for by the fact that in FSHD myoblasts, functionally important target genes, including cell cycle, DNA damage, and ubiquitination-related genes, escape myogenic microRNA-induced repression. PMID:24145033
Ren, Maozhi; Venglat, Prakash; Qiu, Shuqing; Feng, Li; Cao, Yongguo; Wang, Edwin; Xiang, Daoquan; Wang, Jinghe; Alexander, Danny; Chalivendra, Subbaiah; Logan, David; Mattoo, Autar; Selvaraj, Gopalan; Datla, Raju
Target of Rapamycin (TOR) is a major nutrition and energy sensor that regulates growth and life span in yeast and animals. In plants, growth and life span are intertwined not only with nutrient acquisition from the soil and nutrition generation via photosynthesis but also with their unique modes of development and differentiation. How TOR functions in these processes has not yet been determined. To gain further insights, rapamycin-sensitive transgenic Arabidopsis thaliana lines (BP12) expressing yeast FK506 Binding Protein12 were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf, and shoot growth and development leading to poor nutrient uptake and light energy utilization. Experimental limitation of nutrient availability and light energy supply in wild-type Arabidopsis produced phenotypes observed with TOR knockdown plants, indicating a link between TOR signaling and nutrition/light energy status. Genetic and physiological studies together with RNA sequencing and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and life span in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, and rRNA and protein synthesis. Gain- and loss-of-function Ribosomal Protein S6 (RPS6) mutants additionally show that TOR function involves RPS6-mediated nutrition and light-dependent growth and life span in Arabidopsis.
Beisang, Daniel; Reilly, Cavan; Bohjanen, Paul R.
Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation PMID:25123787
Full Text Available Extracellular acidification occurs not only in pathologicalconditions such as inflammation and brain ischemia, but alsoin normal physiological conditions such as synaptic transmission.Acid-sensing ion channels (ASICs can detect a broadrange of physiological pH changes during pathological andsynaptic cellular activities. ASICs are voltage-independent,proton-gated cation channels widely expressed throughout thecentral and peripheral nervous system. Activation of ASICs isinvolved in pain perception, synaptic plasticity, learning andmemory, fear, ischemic neuronal injury, seizure termination,neuronal degeneration, and mechanosensation. Therefore,ASICs emerge as potential therapeutic targets for manipulatingpain and neurological diseases. The activity of these channelscan be regulated by many factors such as lactate, Zn2+, andPhe-Met-Arg-Phe amide (FMRFamide-like neuropeptides byinteracting with the channel’s large extracellular loop. ASICsare also modulated by G protein-coupled receptors such asCB1 cannabinoid receptors and 5-HT2. This review focuses onthe physiological roles of ASICs and the molecularmechanisms by which these channels are regulated. [BMBReports 2013; 46(6: 295-304
Annadurai, Narendran; Agrawal, Khushboo; Džubák, Petr; Hajdúch, Marián; Das, Viswanath
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects normal functions of the brain. Currently, AD is one of the leading causes of death in developed countries and the only one of the top ten diseases without a means to prevent, cure, or significantly slow down its progression. Therefore, newer therapeutic concepts are urgently needed to improve survival and the quality of life of AD patients. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neurons. However, their role in hyperphosphorylation of tau makes them potential druggable target for AD therapy. Despite the relevance of MARKs in AD pathogenesis, only a few small molecules are known to have anti-MARK activity and not much has been done to progress these compounds into therapeutic candidates. But given the diverse role of MARKs, the specificity of novel inhibitors is imperative for their successful translation from bench to bedside. In this regard, a recent co-crystal structure of MARK4 in association with a pyrazolopyrimidine-based inhibitor offers a potential scaffold for the development of more specific MARK inhibitors. In this manuscript, we review the biological role of MARKs in health and disease, and draw attention to the largely unexplored area of MARK inhibitors for AD.
Li, Maolan; Chen, Wei; Zhang, Hongchen; Zhang, Yong; Ke, Fayong; Wu, Xiangsong; Zhang, Yijian; Weng, Mingzhe; Liu, Yingbin; Gong, Wei
Gallbladder cancer (GBC) is a malignant tumor highly resistant to chemotherapy. MicroRNAs (miRNAs) are found extensively involved in modulation of carcinogenesis and chemoresistance. This study aimed to investigate cisplatin (DDP)-susceptibility regulated by expression of the miRNAs and underlying pathways in GBC. The microRNA-31 (miR-31) was selected by microarray due to the biggest fold change between DDP-resistant and parental cells. Ectopic overexpression of miR-31 decreased cell proliferation, viability and invasion capacity, but promoted apoptosis in DDP-resistant cells and in xenograft tumor models. Cell apoptosis and DDP-chemosensitivity was remarkably increased by knockdown of Src proto-oncogene (Src) expression, which was subsequently reversed by rescue of Src expression in miR-31-expressing cells. The microarray was used to select the candidate miRNA in two DDP-resistant GBC cell lines. The effect of regulated expression of the miRNA on cell migration, invasion, proliferation and apoptosis was examined by wound healing, transwell assays, CCK-8 assays, colony formation and flow cytometry assays, respectively. Xenograft tumor models were used to validate the function of the downstream target. Our results demonstrated that miR-31reduced significantly in GBC cells rendering resistance to cisplatin, and upregulated expression of miR-31 augmented chemosensitivity, presenting a therapeutic potential to overcome drug resistance in GBC.
Nelson S. Yee
Histone deacetylases (HDACs and RNA polymerase III (POLR3 play vital roles in fundamental cellular processes, and deregulation of these enzymes has been implicated in malignant transformation. Hdacs and Polr3 are required for exocrine pancreatic epithelial proliferation during morphogenesis in zebrafish. We aim to test the hypothesis that Hdacs and Polr3 cooperatively control exocrine pancreatic growth, and combined inhibition of HDACs and POLR3 produces enhanced growth suppression in pancreatic cancer. In zebrafish larvae, combination of a Hdac inhibitor (Trichostatin A and an inhibitor of Polr3 (ML-60218 synergistically prohibited the expansion of exocrine pancreas. In human pancreatic adenocarcinoma cells, combination of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA and ML-60218 produced augmented suppression of colony formation and proliferation, and induction of cell cycle arrest and apoptotic cell death. The enhanced cytotoxicity was associated with supra-additive upregulation of the pro-apoptotic regulator BAX and the cyclin-dependent kinase inhibitor p21CDKN1A. tRNAs have been shown to have pro-proliferative and anti-apoptotic roles, and SAHA-stimulated expression of tRNAs was reversed by ML-60218. These findings demonstrate that chemically targeting developmental regulators of exocrine pancreas can be translated into an approach with potential impact on therapeutic response in pancreatic cancer, and suggest that counteracting the pro-malignant side effect of HDAC inhibitors can enhance their anti-tumor activity.
Doan, Khanh V.; Kinyua, Ann W.; Yang, Dong Joo; Ko, Chang Mann; Moh, Sang Hyun; Shong, Ko Eun; Kim, Hail; Park, Sang-Kyu; Kim, Dong-Hoon; Kim, Inki; Paik, Ji-Hye; DePinho, Ronald A.; Yoon, Seul Gi; Kim, Il Yong; Seong, Je Kyung; Choi, Yun-Hee; Kim, Ki Woo
Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis. PMID:27681312
Condello, S; Morgan, C A; Nagdas, S; Cao, L; Turek, J; Hurley, T D; Matei, D
Cancer cells form three-dimensional (3D) multicellular aggregates (or spheroids) under non-adherent culture conditions. In ovarian cancer (OC), spheroids serve as a vehicle for cancer cell dissemination in the peritoneal cavity, protecting cells from environmental stress-induced anoikis. To identify new targetable molecules in OC spheroids, we investigated gene expression profiles and networks upregulated in 3D vs traditional monolayer culture conditions. We identified ALDH1A1, a cancer stem cell marker as being overexpressed in OC spheroids and directly connected to key elements of the β-catenin pathway. β-Catenin function and ALDH1A1 expression were increased in OC spheroids vs monolayers and in successive spheroid generations, suggesting that 3D aggregates are enriched in cells with stem cell characteristics. β-Catenin knockdown decreased ALDH1A1 expression levels and β-catenin co-immunoprecipitated with the ALDH1A1 promoter, suggesting that ALDH1A1 is a direct β-catenin target. Both short interfering RNA-mediated β-catenin knockdown and A37 ((ethyl-2-((4-oxo-3-(3-(pryrrolidin-1-yl)propyl)-3,4-dihydrobenzo [4,5]thioeno [3,2-d]pyrimidin-2-yl)thio)acetate)), a novel ALDH1A1 small-molecule enzymatic inhibitor described here for the first time, disrupted OC spheroid formation and cell viability (Pmodel. These data strongly support the role of β-catenin-regulated ALDH1A1 in the maintenance of OC spheroids and propose new ALDH1A1 inhibitors targeting this cell population.
Zambrano, Joelle N; Neely, Benjamin A; Yeh, Elizabeth S
Hormonally up-regulated neu-associated Kinase (Hunk) is a protein kinase that was originally identified in the murine mammary gland and has been shown to be highly expressed in Human Epidermal Growth Factor Receptor 2 positive (HER2(+)/ErbB2(+)) breast cancer cell lines as well as MMTV-neu derived mammary tumor cell lines. However, the physiological role of Hunk has been largely elusive since its identification. Though Hunk is predicted to be a Serine/Threonine (Ser/Thr) protein kinase with homology to the SNF1/AMPK family of protein kinases, there are no known Hunk substrates that have been identified to date. Recent work demonstrates a role for Hunk in HER2(+)/ErbB2(+) breast cancer progression, including drug resistance to HER2/ErbB2 inhibitors, with Hunk potentially acting downstream of HER2/ErbB2 and the PI3K/Akt pathway. These studies have collectively shown that Hunk plays a vital role in promoting mammary tumorigenesis, as Hunk knockdown via shRNA in xenograft tumor models or crossing MMTV-neu or Pten-deficient genetically engineered mouse models into a Hunk knockout (Hunk-/-) background impairs mammary tumor growth in vivo. Because the majority of HER2(+)/ErbB2(+) breast cancer patients acquire drug resistance to HER2/ErbB2 inhibitors, the characterization of novel drug targets like Hunk that have the potential to simultaneously suppress tumorigenesis and potentially enhance efficacy of current therapeutics is an important facet of drug development. Therefore, work aimed at uncovering specific regulatory functions for Hunk that could contribute to this protein kinase's role in both tumorigenesis and drug resistance will be informative. This review focuses on what is currently known about this under-studied protein kinase, and how targeting Hunk may prove to be a potential therapeutic target for the treatment of breast cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.
Fang Wang; Jia Yu; Gui-Hua Yang; Xiao-Shuang Wang; Jun-Wu Zhang
Lineage differentiation is a continuous process during which fated progenitor cells execute specific programs to produce mature counterparts. This lineage-restricted pathway can be controlled by particular regulators, which are usually exclusively expressed in certain cell types or at specific differentiation stages. Here we report that miR376a participates in the regulation of the early stages of human erythropoiesis by targeting cyclin-dependent kinase 2 (CDK2) and Argonaute 2 (Ago2). Among various human leukemia cell lines, miR-376a was only detected in K562 cells which originated from a progenitor common to the erythroid and megakaryotic lineages. Enforced expression of miR-376a or silencing of CDK2 and Ago2 by RNAi inhibits erythroid differentiation of K562 cells. Hematopoietic progenitor cells transduced with miR-376a showed a significant reduction of their erythroid clonogenic capacity. MiR-376a is relatively abundant in erythroid progenitor cells, where it reduces expression of CDK2 and maintains a low level of differentiation due to cell cycle arrest and decreased cell growth. Following erythroid induction, miR376a is significantly down-regulated and CDK2 is released from miR-376a inhibition, thereby facilitating the escape of progenitor cells from the quiescent state into erythroid differentiation. Moreover, our results establish a functional link between miR-376a and Ago2, a key factor in miRNA biogenesis and silencing pathways with novel roles in human hematopoiesis.
Regulation and function of protein-coding genes are increasingly well-understood, but no comparable evidence exists for non-coding RNA (ncRNA) genes, which appear to be more numerous than protein-coding genes. We developed a novel machine-learning model to distinguish promoters of long ncRNA (lncRNA) genes from those of protein-coding genes. This represents the first attempt to make this distinction based on properties of the associated gene promoters. From our analyses, several transcription factors (TFs), which are known to be regulated by lncRNAs, also emerged as potential global regulators of lncRNAs, suggesting that lncRNAs and TFs may participate in bidirectional feedback regulatory network. Our results also raise the possibility that, due to the historical dependence on protein-coding gene in defining the chromatin states of active promoters, an adjustment of these chromatin signature profiles to incorporate lncRNAs is warranted in the future. Secondly, we developed a novel method to infer functions for lncRNA and microRNA (miRNA) transcripts based on their transcriptional regulatory networks in 119 tissues and 177 primary cells of human. This method for the first time combines information of cell/tissueVspecific expression of a transcript and the TFs and transcription coVfactors (TcoFs) that control activation of that transcript. Transcripts were annotated using statistically enriched GO terms, pathways and diseases across cells/tissues and associated knowledgebase (FARNA) is developed. FARNA, having the most comprehensive function annotation of considered ncRNAs across the widest spectrum of cells/tissues, has a potential to contribute to our understanding of ncRNA roles and their regulatory mechanisms in human. Thirdly, we developed a novel machine-learning model to identify LD motif (a protein interaction motif) of paxillin, a ncRNA target that is involved in cell motility and cancer metastasis. Our recognition model identified new proteins not
Noble, Daniel C; Aoki, Scott T; Ortiz, Marco A; Kim, Kyung Won; Verheyden, Jamie M; Kimble, Judith
Germ cell specification as sperm or oocyte is an ancient cell fate decision, but its molecular regulation is poorly understood. In Caenorhabditis elegans, the FOG-1 and FOG-3 proteins behave genetically as terminal regulators of sperm fate specification. Both are homologous to well-established RNA regulators, suggesting that FOG-1 and FOG-3 specify the sperm fate post-transcriptionally. We predicted that FOG-1 and FOG-3, as terminal regulators of the sperm fate, might regulate a battery of gamete-specific differentiation genes. Here we test that prediction by exploring on a genomic scale the messenger RNAs (mRNAs) associated with FOG-1 and FOG-3. Immunoprecipitation of the proteins and their associated mRNAs from spermatogenic germlines identifies 81 FOG-1 and 722 FOG-3 putative targets. Importantly, almost all FOG-1 targets are also FOG-3 targets, and these common targets are strongly biased for oogenic mRNAs. The discovery of common target mRNAs suggested that FOG-1 and FOG-3 work together. Consistent with that idea, we find that FOG-1 and FOG-3 proteins co-immunoprecipitate from both intact nematodes and mammalian tissue culture cells and that they colocalize in germ cells. Taking our results together, we propose a model in which FOG-1 and FOG-3 work in a complex to repress oogenic transcripts and thereby promote the sperm fate.
Di, Jia; Jiang, Lei; Zhou, Yang; Cao, Hongdi; Fang, Li; Wen, Ping; Li, Xiurong; Dai, Chunsun; Yang, Junwei
Fibroblast activation is one of the most important mechanisms for Angiotensin II (Ang II) in promoting renal fibrosis. Transcription factor Ets-1 is recognized to play a key role in kidney diseases. However, the role and mechanisms of Ets-1 in Ang-II induced fibroblast activation and kidney fibrosis are not fully understood. Mice were treated with Ang II via osmotic mini-pumps or Ang II expression plasmid (pAng II). Cultured normal rat kidney interstitial fibroblast (NRK-49F) cells were incubated with Ang II. Role of Ets-1 in renal fibrosis and fibroblast activation were assessed by Western blot, Immunohistochemical staining'MTT, Boyden chamber and Immunofluorescence staining. Effects of miR-221 on Ets-1 and fibroblast activation were investigated by MTT, Boyden chamber, Western blot and Q-PCR. We found that Ets-1 was up-regulated in fibrotic kidneys. Similarly, Ang II could activate NRK-49F cells as demonstrated by up-regulated α-SMA and fibronectin(FN) expression and enhanced cell proliferation and migration. Ang II also induced Ets-1 expression in NRK-49F cells in a dose and time dependent manner. Knock-down of Ets-1 by RNA interference attenuated Ang II-induced activation of NRK-49F cells. Ets-1 was previously reported as a target of microRNA-221 (miR-221). In Ang II-induced fibrotic kidney, miR-221 was down-regulated. Similar results were observed in Ang II treated NRK-49F cells. Ectopic expression of miR-221 mimic attenuated the up-regulation of Ets-1 by Ang II in NRK-49F cells, which further prevented the activation of NRK-49F cells. However, the inhibitor of miR-221 aggravated Ang II induced Ets-1 expression and NRK-49F cells activation. Our study suggests that miR-221/Ets-1 axis takes an important role in mediating AngII induced interstitial fibroblast activation and renal fibrosis. © 2014 S. Karger AG, Basel.
Charmandari, Evangelia; Chrousos, George P.; Lambrou, George I.; Pavlaki, Aikaterini; Koide, Hisashi; Ng, Sinnie Sin Man; Kino, Tomoshige
Context and Objective Circulating cortisol fluctuates diurnally under the control of the “master” circadian CLOCK, while the peripheral “slave” counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. Design and Participants We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. Results GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. Conclusions Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night. PMID:21980503
Chun, Kyung-Soo; Surh, Young-Joon
Expression of cyclooxygenase-2 (COX-2) has been reported to be elevated in human colorectal adenocarcinoma and other tumors, including those of breast, cervical, prostate, and lung. Genetic knock-out or pharmacological inhibition of COX-2 has been shown to protect against experimentally-induced carcinogenesis. Results from epidemiological and laboratory studies indicate that regular intake of selective COX-2 inhibitors reduces the risk of several forms of human malignancies. Thus, it is conceivable that targeted inhibition of abnormally or improperly elevated COX-2 provides one of the most effective and promising strategies for cancer chemoprevention. The COX-2 promoter contains a TATA box and binding sites for several transcription factors including nuclear factor-kappaB (NF-kappaB), nuclear factor for interleukin-6/CCAAT enhancer-binding protein (NF-IL6/C/EBP) and cyclic AMP response element (CRE) binding protein. Upregulation of COX-2 is mediated by a variety of stimuli including tumor promoters, oncogenes, and growth factors. Stimulation of either protein kinase C (PKC) or Ras signaling enhances mitogen-activated protein kinase (MAPK) activity, which, in turn, activates transcription of cox-2. Celecoxib, the first US FDA approved selective COX-2 inhibitor, initially developed for the treatment of adult rheumatoid arthritis and osteoarthritis, has been reported to reduce the formation of polyps in patients with familial adenomatous polyposis. This COX-2 specific inhibitor also protects against experimentally-induced carcinogenesis, but the underlying molecular mechanisms are poorly understood. The present review covers the signal transduction pathways responsible for regulating COX-2 expression as novel molecular targets of chemopreventive agents with celecoxib as a specific example.
Ginestier, Christophe; Monville, Florence; Wicinski, Julien; Cabaud, Olivier; Cervera, Nathalie; Josselin, Emmanuelle; Finetti, Pascal; Guille, Arnaud; Larderet, Gaelle; Viens, Patrice; Sebti, Said; Bertucci, François; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle
There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.
Pfluger, Paul T; Castañeda, Tamara R; Heppner, Kristy M; Strassburg, Sabine; Kruthaupt, Traci; Chaudhary, Nilika; Halem, Heather; Culler, Michael D; Datta, Rakesh; Burget, Lukas; Tschöp, M H; Nogueiras, Ruben; Perez-Tilve, Diego
Recent studies suggest that spontaneous physical activity (SPA) may be under the non-conscious control of neuroendocrine circuits that are known to control food intake. To further elucidate endocrine gut-brain communication as a component of such circuitry, we here analyzed long-term and acute effects of the gastrointestinal hormones ghrelin and PYY 3-36 as well as their hypothalamic neuropeptide targets NPY, AgRP and POMC (alpha-MSH), on locomotor activity and home cage behaviors in rats. For the analysis of SPA, we used an automated infrared beam break activity measuring system, combined with a novel automated video-based behavior analysis system (HomeCageScan (HCS)). Chronic (one-month) peripheral infusion of ghrelin potently increased body weight and fat mass in rats. Such positive energy balance was intriguingly not due to an overall increased caloric ingestion, but was predominantly associated with a decrease in SPA. Chronic intracerebroventricular infusion (7 days) of ghrelin corroborated the decrease in SPA and suggested a centrally mediated mechanism. Central administration of AgRP and NPY increased food intake as expected. AgRP administration led to a delayed decrease in SPA, while NPY acutely (but transiently) increased SPA. Behavioral dissection using HCS corroborated the observed acute and transient increases of food intake and SPA by central NPY infusion. Acute central administration of alpha-MSH rapidly decreased food intake but did not change SPA. Central administration of the NPY receptor agonist PYY 3-36 transiently increased SPA. Our data suggest that the control of spontaneous physical activity by gut hormones or their neuropeptide targets may represent an important mechanistic component of energy balance regulation. Copyright © 2011 Elsevier Inc. All rights reserved.
Full Text Available CONTEXT AND OBJECTIVE: Circulating cortisol fluctuates diurnally under the control of the "master" circadian CLOCK, while the peripheral "slave" counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. DESIGN AND PARTICIPANTS: We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs as non-synchronized controls. RESULTS: GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. CONCLUSIONS: Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.
Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D
This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (qhypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the hypothalamus as a response to HFD, mechanisms mediating a sustained 'perpetual cycle' of appetite enhancement were observed. The GlyR protein may constitute a novel treatment target for the reduction of central orexigenic signals in obesity.
Danos, Arpad M; Osmanovic, Senad; Brady, Matthew J
PTG and G(L) are hepatic protein phosphatase-1 (PP1) glycogen-targeting subunits, which direct PP1 activity against glycogen synthase (GS) and/or phosphorylase (GP). The C-terminal 16 amino residues of G(L) comprise a high affinity binding site for GP that regulates bound PP1 activity against GS. In this study, a truncated G(L) construct lacking the GP-binding site (G(L)tr) and a chimeric PTG molecule containing the C-terminal site (PTG-G(L)) were generated. As expected, GP binding to glutathione S-transferase (GST)-G(L)tr was reduced, whereas GP binding to GST-PTG-G(L) was increased 2- to 3-fold versus GST-PTG. In contrast, PP1 binding to all proteins was equivalent. Primary mouse hepatocytes were infected with adenoviral constructs for each subunit, and their effects on glycogen metabolism were investigated. G(L)tr expression was more effective at promoting GP inactivation, GS activation, and glycogen accumulation than G(L). Removal of the regulatory GP-binding site from G(L)tr completely blocked the inactivation of GS seen in G(L)-expressing cells following a drop in extracellular glucose. As a result, G(L)tr expression prevented glycogen mobilization under 5 mm glucose conditions. In contrast, equivalent overexpression of PTG or PTG-G(L) caused a similar increase in glycogen-targeted PP1 levels and GS dephosphorylation. Surprisingly, GP dephosphorylation was significantly reduced in PTG-G(L)-overexpressing cells. As a result, PTG-G(L) expression permitted glycogenolysis under 5 mm glucose conditions that was prevented in PTG-expressing cells. Thus, expression of constructs that contained the high affinity GP-binding site (G(L) and PTG-G(L)) displayed reduced glycogen accumulation and enhanced glycogenolysis compared with their respective controls, albeit via different mechanisms.
Full Text Available Abstract Background As nuclear mediators of wnt/β-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/β-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants. Results We identified cold-inducible RNA binding protein (CIRP as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3. Conclusion Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.
Gabriela A Ferreyra
Full Text Available BACKGROUND: Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB challenge was investigated in six tissues. RESULTS: The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC, spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. CONCLUSION: Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes.
Shin, Hee Soon; Shon, Dong-Hwa
An immune hypersensitivity disorder called allergy is caused by diverse allergens entering the body via skin contact, injection, ingestion, and/or inhalation. These allergic responses may develop into allergic disorders, including inflammations such as atopic dermatitis, asthma, anaphylaxis, food allergies, and allergic rhinitis. Several drugs have been developed to treat these allergic disorders; however, long-term intake of these drugs could have adverse effects. As an alternative to these medicines, food and natural materials that ameliorate allergic disorder symptoms without producing any side effects can be consumed. Food and natural materials can effectively regulate successive allergic responses in an allergic chain-reaction mechanism in the following ways:  Inhibition of allergen permeation via paracellular diffusion into epithelial cells,  suppression of type 2 T-helper (Th) cell-related cytokine production by regulating Th1/Th2 balance,  inhibition of pathogenic effector CD4(+) T cell differentiation by inducing regulatory T cells (Treg), and  inhibition of degranulation in mast cells. The immunomodulatory effects of food and natural materials on each target mechanism were scientifically verified and shown to alleviate allergic disorder symptoms. Furthermore, consumption of certain food and natural materials such as fenugreek, skullcap, chitin/chitosan, and cheonggukjang as anti-allergics have merits such as safety (no adverse side effects), multiple suppressive effects (as a mixture would contain various components that are active against allergic responses), and ease of consumption when required. These merits and anti-allergic properties of food and natural materials help control various allergic disorders.
Full Text Available The innate immune response is a host defense mechanism against infection by viruses and bacteria. Type I interferons (IFNα/β play a crucial role in innate immunity. If not tightly regulated under normal conditions and during immune responses, IFN production can become aberrant, leading to inflammatory and autoimmune diseases. In this study, we identified TRIM11 (tripartite motif containing 11 as a novel negative regulator of IFNβ production. Ectopic expression of TRIM11 decreased IFNβ promoter activity induced by poly (I:C stimulation or overexpression of RIG-I (retinoic acid-inducible gene-I signaling cascade components RIG-IN (constitutively active form of RIG-I, MAVS (mitochondrial antiviral signaling protein, or TBK1 (TANK-binding kinase-1. Conversely, TRIM11 knockdown enhanced IFNβ promoter activity induced by these stimuli. Moreover, TRIM11 overexpression inhibited the phosphorylation and dimerization of IRF3 and expression of IFNβ mRNA. By contrast, TRIM11 knockdown increased the IRF3 phosphorylation and IFNβ mRNA expression. We also found that TRIM11 and TBK1, a key kinase that phosphorylates IRF3 in the RIG-I pathway, interacted with each other through CC and CC2 domain, respectively. This interaction was enhanced in the presence of the TBK1 adaptor proteins, NAP1 (NF-κB activating kinase-associated protein-1, SINTBAD (similar to NAP1 TBK1 adaptor or TANK (TRAF family member-associated NF-κB activator. Consistent with its inhibitory role in RIG-I-mediated IFNβ signaling, TRIM11 overexpression enhanced viral infectivity, whereas TRIM11 knockdown produced the opposite effect. Collectively, our results suggest that TRIM11 inhibits RIG-I-mediated IFNβ production by targeting the TBK1 signaling complex.
Full Text Available Topoisomerase II (TOP2 targeting drugs like doxorubicin and etoposide are frontline chemotherapeutics for a wide variety of solid and hematological malignancies, including breast and ovarian adenocarcinomas, lung cancers, soft tissue sarcomas, leukemias and lymphomas. These agents cause a block in DNA replication leading to a pronounced DNA damage response and initiation of apoptotic programs. Resistance to these agents is common, however, and elucidation of the mechanisms causing resistance to therapy could shed light on strategies to reduce the frequency of ineffective treatments. To explore these mechanisms, we utilized an unbiased shRNA screen to identify genes that regulate cell death in response to doxorubicin treatment. We identified the Filamin A interacting protein 1-like (FILIP1L gene as a crucial mediator of apoptosis triggered by doxorubicin. FILIP1L shares significant similarity with bacterial SbcC, an ATPase involved in DNA repair. FILIP1L was originally described as DOC1, or "down-regulated in ovarian cancer" and has since been shown to be downregulated in a wide variety of human tumors. FILIP1L levels increase markedly through transcriptional mechanisms following treatment with doxorubicin and other TOP2 poisons, including etoposide and mitoxantrone, but not by the TOP2 catalytic inhibitors merbarone or dexrazoxane (ICRF187, or by UV irradiation. This induction requires the action of the OCT1 transcription factor, which relocalizes to the FILIP1L promoter and facilitates its expression following doxorubicin treatment. Our findings suggest that the FILIP1L expression status in tumors may influence the response to anti-TOP2 chemotherapeutics.
Ma, Changle; Hagstrom, Danielle; Polley, Soumi Guha; Subramani, Suresh
In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.
Full Text Available Synaptic clustering on dendritic branches enhances plasticity, input integration and neuronal firing. However, the mechanisms guiding axons to cluster synapses at appropriate sites along dendritic branches are poorly understood. We searched for such a mechanism by investigating the structural overlap between dendritic branches and axons in a simplified model of neuronal networks--the hippocampal cell culture. Using newly developed software, we converted images of meshes of overlapping axonal and dendrites into topological maps of intersections, enabling quantitative study of overlapping neuritic geometry at the resolution of single dendritic branch-to-branch and axon-to-branch crossings. Among dendro-dendritic crossing configurations, it was revealed that the orientations through which dendritic branches cross is a regulated attribute. While crossing angle distribution among branches thinner than 1 µm appeared to be random, dendritic branches 1 µm or wider showed a preference for crossing each other at angle ranges of either 50°-70° or 80°-90°. It was then found that the dendro-dendritic crossings themselves, as well as their selective angles, both affected the path of axonal growth. Axons displayed 4 fold stronger tendency to traverse within 2 µm of dendro-dendritic intersections than at farther distances, probably to minimize wiring length. Moreover, almost 70% of the 50°-70° dendro-denritic crossings were traversed by axons from the obtuse angle's zone, whereas only 15% traversed through the acute angle's zone. By contrast, axons showed no orientation restriction when traversing 80°-90° crossings. When such traverse behavior was repeated by many axons, they converged in the vicinity of dendro-dendritic intersections, thereby clustering their synaptic connections. Thus, the vicinity of dendritic branch-to-branch crossings appears to be a regulated structure used by axons as a target for efficient wiring and as a preferred site for
Yang, Zi-hui; Wu, Bao-lei; Ye, Chen; Jia, Sen; Yang, Xin-jie; Hou, Rui; Lei, De-lin; Wang, Lei
Distraction osteogenesis (DO) is a widely used self-tissue engineering. However, complications and discomfort due to the long treatment period are still the bottleneck of DO. Novel strategies to accelerate bone formation in DO are still needed. P38 is capable of regulating the osteogenic differentiation of both mesenchymal stem cells (MSCs) and osteoblasts, which are crucial to bone regeneration. However, it is not clear whether targeting p38 could regulate bony formation in DO. The purpose of the current work was to investigate the effects of local application of either p38 agonist anisomycin or p38 inhibitor SB203580 in a rat model of DO. 30 adult rats were randomly divided into 3 groups: (A) rats injected with DMSO served as the control group; (B) rats injected with p38 agonist anisomycin; (C) rats injected with p38 inhibitor SB203580. All the rats were subjected to mandibular distraction and the injection was performed daily during this period. The distracted mandibles were harvested on days 15 and 30 after surgery and subjected to the following analysis. Micro-computed tomography and histological evaluation results showed that local application of p38 agonist anisomycin increased new bone formation in DO, whereas p38 inhibitor SB203580 decreased it. Immunohistochemical analysis suggested that anisomycin promoted MSC recruitment in the distraction gap. In conclusion, this study demonstrated that local application of p38 agonist anisomycin can increase new bone formation during DO. This study may lead to a novel cell-based strategy for the improvement of bone regeneration. PMID:27766028
Ai, Zhihong; Lu, Yang; Qiu, Songbo; Fan, Zhen
Cisplatin is currently one of the most effective chemotherapeutic drugs used for treating ovarian cancer; however, resistance to cisplatin is common. In this study, we explored an experimental strategy for overcoming cisplatin resistance of human ovarian cancer from the new perspective of cancer cell metabolism. By using two pairs of genetically matched cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines, we tested the hypothesis that downregulating hypoxia-inducible factor-1 (HIF-1), which regulates metabolic enzymes involved in glycolysis, is a promising strategy for overcoming cisplatin resistance of human ovarian cancer cells. We found that cisplatin downregulated the level of the regulatable α subunit of HIF-1, HIF-1α, in cisplatin-sensitive ovarian cancer cells through enhancing HIF-1α degradation but did not downregulate HIF-1α in their cisplatin-resistant counterparts. Overexpression of a degradation-resistant HIF-1α (HIF-1α ΔODD) reduced cisplatin-induced apoptosis in cisplatin-sensitive cells, whereas genetic knockdown of HIF-1α or pharmacological promotion of HIF-1α degradation enhanced response to cisplatin in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. We further demonstrated that knockdown of HIF-1α improved the response of cisplatin-resistant ovarian cancer cells to cisplatin by redirecting the aerobic glycolysis in the resistant cancer cells toward mitochondrial oxidative phosphorylation, leading to cell death through overproduction of reactive oxygen species. Our findings suggest that the HIF-1α-regulated cancer metabolism pathway could be a novel target for overcoming cisplatin resistance in ovarian cancer.
Munabi, Naikhoba C.O.; England, Ryan W.; Edwards, Andrew K.; Kitajewski, Alison A.; Tan, Qian Kun; Weinstein, Andrew; Kung, Justin E.; Wilcox, Maya; Kitajewski, Jan K.; Shawber, Carrie J.
Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs and the downstream pathways in mediating propranolol’s effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of hemangiomas (IHs). IHs are the most common vascular tumor in children and have been proposed to arise from a hemangioma stem cell (HemSC). Propranolol, a nonselective β-adrenergic receptor (βAR) antagonist, has proven efficacy; however, understanding of its mechanism of action on HemSCs is limited. The presented data demonstrate that propranolol, via βAR perturbation, dose dependently suppresses cAMP levels and activated extracellular signal-regulated kinase 1/2. Furthermore, propranolol acts via perturbation of β2AR, and not β1AR, although both receptors are expressed in HemSCs. These results provide important insight into propranolol’s action in IHs and can be used to guide the development of more targeted therapy. PMID:26574555
Boursereau, Raphaël; Abou-Samra, Michel; Lecompte, Sophie; Noel, Laurence; Brichard, Sonia M
Muscle inflammation worsens metabolic disorders as well as devastating myopathies. The hormone adiponectin (ApN) has emerged has a master regulator of inflammation/immunity in several tissues including the skeletal muscle. In this work, we explore whether microRNAs regulated by ApN may represent novel mechanisms for controlling muscle inflammation. By screening arrays, we found miR-711 as a strong candidate for mediating ApN action. Thus, ApN-knockout mice showed decreased muscular expression of miR-711 together with enhanced inflammation/oxidative stress markers, while mice overexpressing ApN showed increased miR-711 levels. Likewise, electrotransfer of the ApN gene in muscle of ApN-knockout mice upregulated miR-711 while reducing inflammation and oxidative stress. Similar data were obtained in murine C2C12 cells or in human primary myotubes treated with ApN. MiR-711 overexpression downregulated several components of the Toll-like receptor-4 (TLR4) pathway, which led to repression of NF-κB activity and downstream pro-inflammatory cytokines. MiR-711 blockade had opposite effects. Moreover, muscle electrotransfer of pre-miR-711 recapitulated in vivo the anti-inflammatory effects observed in vitro. Thus, miR-711, which is upregulated by ApN represses TLR4 signaling, acting therefore as a major mediator of the anti-inflammatory action of ApN. This novel miRNA and its related target genes may open new therapeutic perspectives for controlling muscle inflammation.
Chung, Woo Young; Park, Hyun Woo; Han, Jung Woo; Lee, Min Goo; Kim, Joo Young
WNK4, a serine/threonine kinase, plays a critical role in the expression of membrane proteins in the cell surface; however, the underlying mechanism of WNK4 is not clear. Here, we demonstrate that WNK4 inhibits the fusion of plasma membrane delivering vesicle with sorting/recycling endosome through disrupting SNARE formation of syntaxin13, an endosomal t-SNARE and VAMP2, the v-SNARE in plasma membrane delivering vesicle. Their interaction and co-localization were enhanced by hyperosmotic stimulation which is known for WNK4 activation. The kinase domain of WNK4 interacts with the transmembrane domain (TM) of syntaxin13 and this interaction was abolished when the TM was replaced with that of syntaxin16. Interestingly, cell fractionation using sucrose gradients revealed that WNK4 inhibited the formation of the syntaxin13/VAMP2 SNARE complex in the endosomal compartment, but not syntaxin16/VAMP2 or syntaxin13/VAMP7. Syntaxin13 was not phosphorylated by WNK4 and WNK4KI also showed the same binding strength and similar inhibitory regulation on SNARE formation of syntaxin13. Physiological relevance of this mechanism was proved with the expression of NCC (Na(+) C1(-) co-transporter) in the cell surface. The inhibiting activity of WNK4 on surface expression of NCC was abolished by syntaxin13 siRNA transfection. These results suggest that WNK4 attenuates PM targeting of NCC proteins through regulation of syntaxin13 SNARE complex formation with VAMP2 in recycling and sorting endosome. © 2013.
Full Text Available Abstract Background During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Results Using immunoprecipitated (IPed DNA fragments recovered by chromatin immunoprecipitation (ChIP with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. Conclusions The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes
Binger, Katrina J.; Côrte-Real, Beatriz F.; Kleinewietfeld, Markus
Interleukin-17-producing T helper (Th17) cells are critical for the host defense of bacterial and fungal pathogens and also play a major role in driving pathogenic autoimmune responses. Recent studies have indicated that the generation of Th17 cells from naïve CD4+ T cells is coupled with massive cellular metabolic adaptations, necessary to cope with different energy and metabolite requirements associated with switching from a resting to proliferative state. Furthermore, Th17 cells have to secure these metabolic adaptations when facing nutrient-limiting environments, such as at the sites of inflammation. Accumulating data indicates that this metabolic reprogramming is significantly linked to the differentiation of T helper cells and, particularly, that the metabolic changes of Th17 cells and anti-inflammatory Forkhead box P3+ regulatory T cells are tightly and reciprocally regulated. Thus, a better understanding of these processes could offer potential new targets for therapeutic interventions for autoimmune diseases. In this mini-review, we will highlight some of the recent advances and discoveries in the field, with a particular focus on metabolic demands of Th17 cells and their implications for autoimmunity.
Li, Yanyan; Wang, Xingguo; Yu, Jianfeng; Shao, Fang; Zhang, Yanping; Lu, Xiangyun; Gu, Zhiliang
As the most abundant microRNA (miRNA) in the liver, miR-122 plays important roles in the growth and development of liver, lipid metabolism, and liver diseases. Vanin 1 (VNN1) plays an important role in hepatic lipid metabolism, and VNN1 may serve as a potential therapeutic target for the treatment of metabolic diseases caused by overactivated gluconeogenesis. In our previous RNA-seq study, we found the expression of VNN1 increased significantly when the expression of miR-122 (gga-miR-122-5p) was knocked down in primary chicken hepatocytes. In this study, we verified this result by real-time qRT-PCR, and we also found that the chicken VNN1 was highly expressed in the liver. By bioinformatics analyses, we found the 3'UTR of VNN1 contained sequences completely complementary to the nucleotides 1 to 8 of miR-122. Co-transfection and dual-luciferase reporter assays showed that overexpression of miR-122 decreased the expression of luciferase reporter gene linked to the 3'UTR of chicken VNN1 in the Chinese hamster ovary cells (Pchicken hepatocytes. Overall, this study suggests that miR-122 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of the VNN1 gene.
Jonathan M. Goodwin
Full Text Available Iron is vital for many homeostatic processes, and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4 are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening, we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34, and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron-depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover, but its deletion leads to TAX1BP1-dependent activation of TBK1 that regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an amyotrophic lateral sclerosis (ALS-associated TBK1 allele are incapable of degrading ferritin suggesting a molecular mechanism that explains the presence of iron deposits in patient brain biopsies.
Silva, Dillian Adelaine Cesar da; Cunha, Antonio Carlos Rodrigues da; Cunha, Thiago Rocha da; Rosaneli, Caroline Filla
When it comes to food marketing, children are one of the major targets. Regulatory actions can play a strategic role in health protection. The objective of this research was to characterize the ethical perspective in the discourse against state regulatory actions on food marketing directed at children, aiming to understand the context of the discourse's production and how it creates meaning. The methodology adopted was qualitative, with documentary analysis and use of concepts and procedures from Discourse Analysis. The work of Hans Jonas, specifically his Responsibility Principle, and Garrafa and Port's Intervention Bioethics oriented the analysis. The self-regulation discourse analysis showed an ethical perspective in which relations of consumption predominate over the children´s vulnerability. The rhetorical excess is constant, as well as the use of resources like naturalization, untruthfulness, ideological dissimulation and euphemism. An erasure of social conflicts takes place, and an ahistorical perspective is present. The discourse does not align with Jonas´ Responsibility Principle, nor those of Intervention Bioethics. Lastly, the ethical perspective of the discourse represents a double paradox, because it is a business discourse that hides its competitive roots and metamorphoses into an ethical one.
The peroxisome proliferator activated receptor alpha (PPARα) is a ligand activated tran- scription factor involved in the regulation of a variety of processes, ranging from inflam- mation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic
Full Text Available BACKGROUND: microRNAs (miRNAs are small noncoding RNAs that regulate cognate mRNAs at the post-transcriptional stage. Several studies have shown that miRNAs modulate gene expression in mammalian cells by base pairing to complementary sites in the 3'-untranslated region (3'-UTR of the target mRNAs. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, miR-24 was found to target fas associated factor 1(FAF1 by binding to its amino acid coding sequence (CDS region, thereby regulating apoptosis in DU-145 cells. This result supports an augmented model whereby animal miRNAs can exercise their effects through binding to the CDS region of the target mRNA. Transfection of miR-24 antisense oligonucleotide (miR-24-ASO also induced apoptosis in HGC-27, MGC-803 and HeLa cells. CONCLUSIONS/SIGNIFICANCE: We found that miR-24 regulates apoptosis by targeting FAF1 in cancer cells. These findings suggest that miR-24 could be an effective drug target for treatment of hormone-insensitive prostate cancer or other types of cancers. Future work may further develop miR-24 for therapeutic applications in cancer biology.
Buch, Susanne; Melcher, Christoph; Bauer, Matthias; Katzenberger, Joerg; Pankratz, Michael J
Specific neurosecretory cells of the Drosophila brain express insulin-like peptides (dilps), which regulate growth, glucose homeostasis, and aging. Through microarray analysis of flies in which the insulin-producing cells (IPCs) were ablated, we identified a target gene, target of brain insulin (tobi), that encodes an evolutionarily conserved alpha-glucosidase. Flies with lowered tobi levels are viable, whereas tobi overexpression causes severe growth defects and a decrease in body glycogen. Interestingly, tobi expression is increased by dietary protein and decreased by dietary sugar. This pattern is reminiscent of mammalian glucagon secretion, which is increased by protein intake and decreased by sugar intake, suggesting that tobi is regulated by a glucagon analog. tobi expression is also eliminated upon ablation of neuroendocrine cells that produce adipokinetic hormone (AKH), an analog of glucagon. tobi is thus a target of the insulin- and glucagon-like signaling system that responds oppositely to dietary protein and sugar.
Bartlett, Danielle; Boyle, Glen M; Ziman, Mel; Medic, Sandra
Melanoma is a highly aggressive and drug resistant form of skin cancer. It arises from melanocytes, the pigment producing cells of the skin. The formation of these melanocytes is driven by the transcription factor PAX3 early during embryonic development. As a result of alternative splicing, the PAX3 gene gives rise to eight different transcripts which encode isoforms that have different structures and activate different downstream target genes involved in pathways of cell proliferation, migration, differentiation and survival. Furthermore, post-translational modifications have also been shown to alter the functions of PAX3. We previously identified PAX3 downstream target genes in melanocytes and melanoma cells. Here we assessed the effects of PAX3 down-regulation on this panel of target genes in primary melanocytes versus melanoma cells. We show that PAX3 differentially regulates various downstream target genes involved in cell proliferation in melanoma cells compared to melanocytes. To determine mechanisms behind this differential downstream target gene regulation, we performed immunoprecipitation to assess post-translational modifications of the PAX3 protein as well as RNAseq to determine PAX3 transcript expression profiles in melanocytes compared to melanoma cells. Although PAX3 was found to be post-translationally modified, there was no qualitative difference in phosphorylation and ubiquitination between melanocytes and melanoma cells, while acetylation of PAX3 was reduced in melanoma cells. Additionally, there were differences in PAX3 transcript expression profiles between melanocytes and melanoma cells. In particular the PAX3E transcript, responsible for reducing melanocyte proliferation and increasing apoptosis, was found to be down-regulated in melanoma cells compared to melanocytes. These results suggest that alternate transcript expression profiles activate different downstream target genes leading to the melanoma phenotype.
Oglesby, Irene K
MicroRNAs (miRNAs) have emerged as a class of regulatory RNAs with immense significance in numerous biological processes. When aberrantly expressed miRNAs have been shown to play a role in the pathogenesis of several disease states. Extensive research has explored miRNA involvement in the development and fate of immune cells and in both the innate and adaptive immune responses whereby strong evidence links miRNA expression to signalling pathways and receptors with critical roles in the inflammatory response such as NF-κB and the toll-like receptors, respectively. Recent studies have revealed that unique miRNA expression profiles exist in inflammatory lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and lung cancer. Evaluation of the global expression of miRNAs provides a unique opportunity to identify important target gene sets regulating susceptibility and response to infection and treatment, and control of inflammation in chronic airway disorders. Over 800 human miRNAs have been discovered to date, however the biological function of the majority remains to be uncovered. Understanding the role that miRNAs play in the modulation of gene expression leading to sustained chronic pulmonary inflammation is important for the development of new therapies which focus on prevention of disease progression rather than symptom relief. Here we discuss the current understanding of miRNA involvement in innate immunity, specifically in LPS\\/TLR4 signalling and in the progression of the chronic inflammatory lung diseases cystic fibrosis, COPD and asthma. miRNA in lung cancer and IPF are also reviewed.
Oglesby, Irene K
Abstract MicroRNAs (miRNAs) have emerged as a class of regulatory RNAs with immense significance in numerous biological processes. When aberrantly expressed miRNAs have been shown to play a role in the pathogenesis of several disease states. Extensive research has explored miRNA involvement in the development and fate of immune cells and in both the innate and adaptive immune responses whereby strong evidence links miRNA expression to signalling pathways and receptors with critical roles in the inflammatory response such as NF-κB and the toll-like receptors, respectively. Recent studies have revealed that unique miRNA expression profiles exist in inflammatory lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and lung cancer. Evaluation of the global expression of miRNAs provides a unique opportunity to identify important target gene sets regulating susceptibility and response to infection and treatment, and control of inflammation in chronic airway disorders. Over 800 human miRNAs have been discovered to date, however the biological function of the majority remains to be uncovered. Understanding the role that miRNAs play in the modulation of gene expression leading to sustained chronic pulmonary inflammation is important for the development of new therapies which focus on prevention of disease progression rather than symptom relief. Here we discuss the current understanding of miRNA involvement in innate immunity, specifically in LPS\\/TLR4 signalling and in the progression of the chronic inflammatory lung diseases cystic fibrosis, COPD and asthma. miRNA in lung cancer and IPF are also reviewed.
Mehrad, Borna; Burdick, Marie D; Strieter, Robert M
Fibrotic interstitial lung diseases are characterized by progressive decline in lung function and premature death from respiratory failure. Fibrocytes are circulating bone marrow-derived progenitor cells that traffic to the lungs and contribute to fibrosis and may represent novel therapeutic targets in these diseases. We have previously found the recruitment of fibrocytes to the lung to be dependent on the chemokine ligand CXCL12. Given that the expression of the CXCL12 receptor, CXCR4, can be modulated pharmacologically in other cell types, we tested the hypotheses that the regulation of CXCR4 expression on fibrocytes mediates their influx to the lung in the context of pulmonary fibrosis and that pharmacologic inhibition of this process results in attenuated disease severity. CXCR4 was the predominant chemokine receptor on human fibrocytes, and its expression on fibrocytes was enhanced by hypoxia and by growth factors including platelet-derived growth factor. Both hypoxia-induced and growth factor-induced CXCR4 expressions were attenuated by specific inhibition of PI3-kinase and mTOR. Finally, in the mouse model of bleomycin-induced pulmonary fibrosis, treatment with the mTOR inhibitor rapamycin resulted in reduced numbers of CXCR4-expressing fibrocytes in the peripheral blood and lung as well as reduced lung collagen deposition. Taken together, these experiments support the notion that pharmacologic inhibition of the CXCR4/CXCL12 biological axis is achievable in human fibrocytes and reduces the magnitude of pulmonary fibrosis in an animal model. This approach may hold promise in human fibrotic lung diseases.
Zhu, Haigang; Hou, Liyue; Liu, Jingjing; Li, Zhiming, E-mail: firstname.lastname@example.org
MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.
Full Text Available CUG-BP, Elav-like family member 1 (CELF1 is a highly conserved RNA binding protein that regulates pre-mRNA alternative splicing, polyadenylation, mRNA stability, and translation. In the heart, CELF1 is expressed in the myocardium, where its levels are tightly regulated during development. CELF1 levels peak in the heart during embryogenesis, and aberrant up-regulation of CELF1 in the adult heart has been implicated in cardiac pathogenesis in myotonic dystrophy type 1, as well as in diabetic cardiomyopathy. Either inhibition of CELF activity or over-expression of CELF1 in heart muscle causes cardiomyopathy in transgenic mice. Nonetheless, many of the cardiac targets of CELF1 regulation remain unknown. In this study, to identify cardiac targets of CELF1 we performed cross-linking immunoprecipitation (CLIP for CELF1 from embryonic day 8 chicken hearts. We identified a previously unannotated exon in MYH7B as a novel target of CELF1-mediated regulation. We demonstrated that knockdown of CELF1 in primary chicken embryonic cardiomyocytes leads to increased inclusion of this exon and decreased MYH7B levels. We also investigated global changes in the transcriptome of primary embryonic cardiomyocytes following CELF1 knockdown in a published RNA-seq dataset. Pathway and network analyses identified strong associations between CELF1 and regulation of cell cycle and translation. Important regulatory proteins, including both RNA binding proteins and a cardiac transcription factor, were affected by loss of CELF1. Together, these data suggest that CELF1 is a key regulator of cardiomyocyte gene expression.
Saito, Natsumi; Maeda, Michihisa; Tanaka, Kan; Ishihama, Akira
Leucine-responsive regulatory protein (Lrp) is a transcriptional regulator for the genes involved in transport, biosynthesis and catabolism of amino acids in Escherichia coli. In order to identify the whole set of genes under the direct control of Lrp, we performed Genomic SELEX screening and identified a total of 314 Lrp-binding sites on the E. coli genome. As a result, the regulation target of Lrp was predicted to expand from the hitherto identified genes for amino acid metabolism to a set of novel target genes for utilization of amino acids for protein synthesis, including tRNAs, aminoacyl-tRNA synthases and rRNAs. Northern blot analysis indicated alteration of mRNA levels for at least some novel targets, including the aminoacyl-tRNA synthetase genes. Phenotype MicroArray of the lrp mutant indicated significant alteration in utilization of amino acids and peptides, whilst metabolome analysis showed variations in the concentration of amino acids in the lrp mutant. From these two datasets we realized a reverse correlation between amino acid levels and cell growth rate: fast-growing cells contain low-level amino acids, whilst a high level of amino acids exists in slow-growing cells. Taken together, we propose that Lrp is a global regulator of transcription of a large number of the genes involved in not only amino acid transport and metabolism, but also amino acid utilization. PMID:28348809
Viegas, Sandra C; Silva, Inês J; Saramago, Margarida; Domingues, Susana; Arraiano, Cecília M
MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5' phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role.
Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B
Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.
Xiong, Bin; Lei, Xuefeng; Zhang, Lei; Fu, Jia
Our previous study showed olfactomedin 4 (OLFM4) suppressed triple-negative breast cancer cells migration, invasion and metastasis-associated protein MMP 9 expression. OLFM4 was identified as a potential target of miR-103 according to microRNA target databases and published studies. The aim of this study is to validate the relationship between miR-103 and OLFM4, and explore the function and clinical significance of miR-103 in triple-negative breast cancer patients. In our results, miR-103 negatively regulated OLFM4 expression by directly targeting its 3'-UTR. OLFM4 was a functional target of miR-103 to regulate triple-negative breast cancer cells migration, invasion and MMP 9 expression. Moreover, miR-103 overexpression was observed in triple-negative breast cancer tissues and cell lines, and associated with lymph node metastasis, distant metastasis and clinical stage. Univariate and multivariate analyses suggested that miR-103 overexpression was a poor independent prognostic factor for triple-negative breast cancer patients. In conclusion, miR-103 acts as an oncogene miRNA to promote triple-negative breast cancer cells migration and invasion through targeting OLFM4.
Full Text Available Abstract Background microRNAs (miRNAs represent a class of small (typically 22 nucleotides in length non-coding RNAs that can degrade their target mRNAs or block their translation. Recent research showed that copy number alterations of miRNAs and their target genes are highly prevalent in cancers; however, the evolutionary and biological functions of naturally existing copy number variable miRNAs (CNV-miRNAs among individuals have not been studied extensively throughout the genome. Results In this study, we comprehensively analyzed the properties of genes regulated by CNV-miRNAs, and found that CNV-miRNAs tend to target a higher average number of genes and prefer to synergistically regulate the same genes; further, the targets of CNV-miRNAs tend to have higher variability of expression within and between populations. Finally, we found the targets of CNV-miRNAs are more likely to be differentially expressed among tissues and developmental stages, and participate in a wide range of cellular responses. Conclusions Our analyses of CNV-miRNAs provide new insights into the impact of copy number variations on miRNA-mediated post-transcriptional networks. The deeper interpretation of patterns of gene expression variation and the functional characterization of CNV-miRNAs will help to broaden the current understanding of the molecular basis of human phenotypic diversity.
Markus, Steven M.; Plevock, Karen M.; St. Germain, Bryan J.; Punch, Jesse J.; Meaden, Christopher W.; Lee, Wei-Lih
LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik...
Zhang, Chi; Basta, Tamara; Jensen, Eric D; Klymkowsky, M W
In Xenopus laevis, beta-catenin-mediated dorsal axis formation can be suppressed by overexpression of the HMG-box transcription factor XSOX3. Mutational analysis indicates that this effect is due not to the binding of XSOX3 to beta-catenin nor to its competition with beta-catenin-regulated TCF-type transcription factors for specific DNA binding sites, but rather to SOX3 binding to sites within the promoter of the early VegT- and beta-catenin-regulated dorsal-mesoderm-inducing gene Xnr5. Although B1-type SOX proteins, such as XSOX3, are commonly thought to act as transcriptional activators, XSOX3 acts as a transcriptional repressor of Xnr5 in both the intact embryo and animal caps injected with VegT RNA. Expression of a chimeric polypeptide composed of XSOX3 and a VP16 transcriptional activation domain or morpholino-induced decrease in endogenous XSOX3 polypeptide levels lead to an increase in Xnr5 expression, as does injection of an anti-XSOX3 antibody that inhibits XSOX3 DNA binding. These observations indicate that maternal XSOX3 acts in a novel manner to restrict Xnr5 expression to the vegetal hemisphere.
Vilborg, Anna; Glahder, Jacob-Andreas Harald; Wilhelm, Margareta T
The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells...... exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link...
Tang, Dongdong; Huang, Yuanyuan; Liu, Weiqun; Zhang, Xiansheng
BACKGROUND MicroRNAs (miRNAs) play pivotal roles in spermatogenesis. MicroRNA-210 (miR-210) expression was up-regulated in the testes of sterile men with non-obstructive azoospermia (NOA). However, the underlying mechanisms of miR-210 involved in the spermatogenesis in patients with NOA are unknown. MATERIAL AND METHODS Expression of miR-210 and insulin-like growth factor II (IGF2) in the testes of NOA cases (only including maturation arrest and hypospermatogenesis) were detected in this study. We carried out in vitro experiments to determine if IGF2 was directly targeted by miR-210 in NT2 cells. RESULTS Compared with obstructive azoospermia (OA) as normal control, our results suggest that miR-210 was significantly up-regulated in testis of patients with NOA (Pspermatogenesis by targeting IGF2 in male infertility.
Fisher, Laura A.; Wang, Ling; Wu, Lan; Peng, Aimin
Mitotic progression is regulated largely through dynamic and reversible protein phosphorylation that is modulated by opposing actions of protein kinases and phosphatases. In this study, we show that phosphatase 1 nuclear targeting subunit (Pnuts) functions as a master regulator of mitosis by modulating protein phosphatase 1 (PP1). Overexpression of Pnuts in Xenopus egg extracts inhibited both mitotic and meiotic exit. Immunodepletion of Pnuts from egg extracts revealed its essential functions in mitotic entry and maintenance. The level of Pnuts oscillates during the cell cycle and peaks in mitosis. Pnuts destruction during M-phase exit is mediated by the anaphase-promoting complex/cyclosome (APC/C)-targeted ubiquitination and proteolysis, and conserved destruction motifs of Pnuts. Disruption of Pnuts degradation delayed M-phase exit, suggesting it as an important mechanism to permit M-phase exit. PMID:25002584
Villanueva, Eneida C.; Münzberg, Heike; Cota, Daniela; Leshan, Rebecca L.; Kopp, Keely; Ishida-Takahashi, Ryoko; Jones, Justin C.; Fingar, Diane C.; Seeley, Randy J.; Myers, Martin G.
The medial basal hypothalamus, including the arcuate nucleus (ARC) and the ventromedial hypothalamic nucleus (VMH), integrates signals of energy status to modulate metabolism and energy balance. Leptin and feeding regulate the mammalian target of rapamycin complex 1 (mTORC1) in the hypothalamus, and hypothalamic mTORC1 contributes to the control of feeding and energy balance. To determine the mechanisms by which leptin modulates mTORC1 in specific hypothalamic neurons, we immunohistochemicall...
Full Text Available The ZFP36/Tis11 family of zinc-finger proteins regulate cellular processes by binding to adenine uridine rich elements in the 3' untranslated regions of various mRNAs and promoting their degradation. We show here that ZFP36L1 expression is largely extinguished during the transition from B cells to plasma cells, in a reciprocal pattern to that of ZFP36 and the plasma cell transcription factor, BLIMP1. Enforced expression of ZFP36L1 in the mouse BCL1 cell line blocked cytokine-induced differentiation while shRNA-mediated knock-down enhanced differentiation. Reconstruction of regulatory networks from microarray gene expression data using the ARACNe algorithm identified candidate mRNA targets for ZFP36L1 including BLIMP1. Genes that displayed down-regulation in plasma cells were significantly over-represented (P = <0.0001 in a set of previously validated ZFP36 targets suggesting that ZFP36L1 and ZFP36 target distinct sets of mRNAs during plasmacytoid differentiation. ShRNA-mediated knock-down of ZFP36L1 in BCL1 cells led to an increase in levels of BLIMP1 mRNA and protein, but not for mRNAs of other transcription factors that regulate plasmacytoid differentiation (xbp1, irf4, bcl6. Finally, ZFP36L1 significantly reduced the activity of a BLIMP1 3' untranslated region-driven luciferase reporter. Taken together, these findings suggest that ZFP36L1 negatively regulates plasmacytoid differentiation, at least in part, by targeting the expression of BLIMP1.
David G Hendrickson
Full Text Available MicroRNAs (miRNAs regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes and ribosome density (the average number of ribosomes bound per unit length of coding sequence were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the
Tanwer, Pooja; Bauer, Susanne; Heinrichs, Elisabeth; Panda, Gurudutta; Saluja, Daman; Rudel, Thomas; Beier, Dagmar
Small non-coding RNAs (sRNAs) are well-established post-transcriptional regulators of gene expression in bacteria that respond to a variety of environmental stimuli. They usually act by base-pairing with their target mRNAs, which is commonly facilitated by the RNA chaperone Hfq. In this study we initiated the analysis of the sRNA FnrS of Neisseria gonorrhoeae, which is induced under anaerobic conditions. We identified four putative FnrS target genes using bioinformatics approaches and validated these target genes using translational reporter gene fusions in both Escherichia coli and N. gonorrhoeae, thereby demonstrating their downregulation by direct base-pairing between the respective mRNA and FnrS. We demonstrate deregulation of target mRNAs upon deletion of fnrS and provide evidence that the isc gene cluster required for iron-sulfur cluster biosynthesis, which harbours iscS, which is a direct target of FnrS, is coordinately downregulated by the sRNA. By mutational analysis we show that, surprisingly, three distinct regions of FnrS are employed for interaction with different target genes.
Gou, J.Y.; Liu, C.; Felippes, F. F.; Weigel, D.; Wang, J.-W.
Flavonoids are synthesized through an important metabolic pathway that leads to the production of diverse secondary metabolites, including anthocyanins, flavonols, flavones, and proanthocyanidins. Anthocyanins and flavonols are derived from Phe and share common precursors, dihydroflavonols, which are substrates for both flavonol synthase and dihydroflavonol 4-reductase. In the stems of Arabidopsis thaliana, anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. We show here that this accumulation pattern is under the regulation of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are deeply conserved and known to have important roles in regulating phase change and flowering. Increased miR156 activity promotes accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols. We further provide evidence that at least one of the miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Our results reveal a direct link between the transition to flowering and secondary metabolism and provide a potential target for manipulation of anthocyanin and flavonol content in plants.
Xu, Ya; Zhang, Yanfen; Wang, Lujing; Zhao, Ruiqi; Qiao, Yu; Han, Dong; Sun, Qian; Dong, Nazhen; Liu, Yicong; Wu, Dantong; Zhang, Xuemei; Huang, Ning; Ma, Ning; Zhao, Weiming; Liu, Yanhong; Gao, Xu
Microvesicle biogenesis is a highly regulated process. Aberrant release of microvesicles from cancer cells have been associated with their invasiveness and prognosis. However, the mechanism of aberrant release remains poorly understood. Herein, we found that hepatocellular carcinoma cells shed more microvesicles than normal hepatocytes and miR-200a were shown to inhibit the release of microvesicles in hepatocellular carcinoma cells. Then, we confirmed that miR-200a might target Gelsolin and change cytoskeleton to regulate microvesicles secretion. Further miR-200a may inhibit the proliferation of adjacent cells by inhibiting the release of microvesicles. Collectively, our findings indicate that miR-200a regulated the microvesicle biogenesis involved in the hepatocellular carcinoma progression.
Gopal, Udhayakumar; Gonzalez-Gronow, Mario; Pizzo, Salvatore Vincent
Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Gopal, Udhayakumar; Gonzalez-Gronow, Mario; Pizzo, Salvatore Vincent
Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu98–Leu115). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser62. Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser10. In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression. PMID:27002159
Kim, D.; Grun, D.; van Oudenaarden, A.
The complexity of multicellular organisms requires precise spatiotemporal regulation of gene expression during development. We find that in the nematode Caenorhabditis elegans approximately 2,000 transcripts undergo expression oscillations synchronized with larval transitions while thousands of gene
Huang, Hu; Kong, Dong; Byun, Kyung Hee; Ye, Chianping; Koda, Shuichi; Lee, Dae Ho; Oh, Byung-Chul; Lee, Sam W; Lee, Bonghee; Zabolotny, Janice M; Kim, Min Seon; Bjørbæk, Christian; Lowell, Bradford B; Kim, Young-Bum
Leptin regulates energy balance. However, knowledge of the critical intracellular transducers of leptin signaling remains incomplete. We found that Rho-kinase 1 (ROCK1) regulates leptin action on body weight homeostasis by activating JAK2, an initial trigger of leptin receptor signaling. Leptin promoted the physical interaction of JAK2 and ROCK1, thereby increasing phosphorylation of JAK2 and downstream activation of Stat3 and FOXO1. Mice lacking ROCK1 in either pro-opiomelanocortin (POMC) or agouti-related protein neurons, mediators of leptin action, displayed obesity and impaired leptin sensitivity. In addition, deletion of ROCK1 in the arcuate nucleus markedly enhanced food intake, resulting in severe obesity. Notably, ROCK1 was a specific mediator of leptin, but not insulin, regulation of POMC neuronal activity. Our data identify ROCK1 as a key regulator of leptin action on energy homeostasis.
CTCE-9908 inhibits breast cancer metastasis to lung and bone, Oncol. Rep. 21 (2009) 761–767.  N.T. Holm, F. Abreo, L.W. Johnson, B.D. Li, Q.D. Chu...Kawai, T. Inoue, H. Ito, M. Oshimura, T. Ochiya, MicroRNA-143 regulates human osteosarcoma metastasis by regulating matrix metalloprotease-13...cancers with increased potential for metastasis and recurrence (2). Basal-like breast carcinomas express genes associated with an EMT phenotype and
Fisher, Carolyn J.; Goswami, Prabhat C.
In recent years, cellular redox environment gained significant attention as a critical regulator of cellular responses to oxidative stress. Cellular redox environment is a balance between production of reactive oxygen species and their removal by antioxidant enzymes. We investigated the hypothesis that mitochondrial antioxidant enzyme activity regulates radioresistance in human pancreatic cancer cells. Vector-control and manganese superoxide dismutase (MnSOD) overexpressing human pancreatic c...
Zhang, Ling; Nemzow, Leah; Chen, Hua; Hu, Jennifer J; Gong, Feng
UV irradiation is known to cause cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), and plays a large role in the development of cancer. Tumor suppression, through DNA repair and proper cell cycle regulation, is an integral factor in maintaining healthy cells and preventing development of cancer. Transcriptional regulation of the genes involved in the various tumor suppression pathways is essential for them to be expressed when needed and to function properly. BRG1, an ATPase catalytic subunit of the SWI/SNF chromatin remodeling complex, has been identified as a tumor suppressor protein, as it has been shown to play a role in Nucleotide Excision Repair (NER) of CPDs, suppress apoptosis, and restore checkpoint deficiency, in response to UV exposure. Although BRG1 has been shown to regulate transcription of some genes that are instrumental in proper DNA damage repair and cell cycle maintenance in response to UV, its role in transcriptional regulation of the whole genome in response to UV has not yet been elucidated. With whole genome expression profiling in SW13 cells, we show that upon UV induction, BRG1 regulates transcriptional expression of many genes involved in cell stress response. Additionally, our results also highlight BRG1's general role as a master regulator of the genome, as it transcriptionally regulates approximately 4.8% of the human genome, including expression of genes involved in many pathways. RT-PCR and ChIP were used to validate our genome expression analysis. Importantly, our study identifies several novel transcriptional targets of BRG1, such as ATF3. Thus, BRG1 has a larger impact on human genome expression than previously thought, and our studies will provide inroads for future analysis of BRG1's role in gene regulation.
Full Text Available Long noncoding RNAs (lncRNAs are emerging as new players in gene regulation, but whether lncRNAs operate in the processing of miRNA primary transcript is unclear. Also, whether lncRNAs are involved in the regulation of the mitochondrial network remains to be elucidated. Here, we report that a long noncoding RNA, named mitochondrial dynamic related lncRNA (MDRL, affects the processing of miR-484 primary transcript in nucleus and regulates the mitochondrial network by targeting miR-361 and miR-484. The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484 and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels. In exploring the underlying molecular mechanism by which miR-361 is regulated, we identified MDRL and demonstrated that it could directly bind to miR-361 and downregulate its expression levels, which promotes the processing of pri-miR-484. MDRL inhibits mitochondrial fission and apoptosis by downregulating miR-361, which in turn relieves inhibition of miR-484 processing by miR-361. Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484. Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.
Full Text Available Real-time functional magnetic resonance imaging (rt-fMRI neurofeedback allows learning voluntary control over specific brain areas by means of operant conditioning and has been shown to decrease pain perception. To further increase the effect of rt-fMRI neurofeedback on pain, we directly compared two different target regions of the pain network i.e. the anterior insular cortex (AIC and the anterior cingulate cortex (ACC.Participants for this prospective study were randomly assigned to two age-matched groups of 14 participants each (7 females per group for AIC and ACC feedback. First, a functional localizer using block-design heat pain stimulation was performed to define the pain-sensitive target region within the AIC or ACC. Second, subjects were asked to down-regulate the feedback signal in four neurofeedback runs during identical pain stimulation. Data analysis included task-related and functional connectivity analysis.At the behavioral level, pain ratings significantly decreased during feedback versus localizer runs, but there was no difference between AIC and ACC groups. Concerning neuroimaging, ACC and AIC showed consistent involvement of the caudate nucleus for subjects that learned down-regulation (17/28 in both task-related and functional connectivity analysis. The functional connectivity towards the caudate nucleus is stronger for the ACC while the AIC is more heavily connected to the ventrolateral prefrontal cortex.Consequently, the ACC and AIC are suitable targets for real-time fMRI neurofeedback during pain perception as they both affect the caudate nucleus, although functional connectivity indicates that the direct connection seems to be stronger with the ACC. Additionally, the caudate, an important area involved in pain perception and suppression, could be a rt-fMRI target itself. Future studies are needed to identify parameters characterizing successful regulators and to assess the effect of repeated rt-fMRI neurofeedback on pain
Park, Spencer; Kang, Sungkwon; Veach, Alexander J; Vedvyas, Yogindra; Zarnegar, Rasa; Kim, Ju-Young; Jin, Moonsoo M
Site-specific delivery of drugs while minimizing unwanted distribution has been one of the pursued goals in cancer therapy. In this endeavor, we have developed targeted polymeric nanoparticles called amphiphilic urethane acrylate nonionomer (UAN) for encapsulation of diverse water-insoluble drugs and diagnostic agents, as well as for simple and reproducible surface conjugation of targeting ligands. Using monoclonal antibodies or lymphocyte function-associated antigen-1 (LFA-1) I domain engineered for varying affinities to intercellular adhesion molecule (ICAM)-1, we were able to deliver UAN nanoparticles to human cancer cells with the efficiency dependent on the strength of the molecular interactions and the degree of ICAM-1 expression on cell surface. Compared to non-specific uptake of free drugs, targeted delivery of UAN nanoparticles carrying equal amount of drugs produced more potent cytotoxicity. Notably, without the targeting ligands attached, UAN nanoparticles were largely precluded from non-specific uptake by the cells, resulting in much lower toxicity. The versatility of our UAN nanoparticles in both payload encapsulation and presentation of targeting ligands may facilitate developing a robust platform for evaluating various combinations of cancer drugs and molecular interactions toward developing effective cancer therapy formulations.
Dai, Xiaodan; Rao, Chunbao; Li, Huirong; Chen, Yu; Fan, Lilv; Geng, Huiqin; Li, Shuang; Qu, Jia; Hou, Ling
There is growing evidence that microRNAs are important regulators of gene expression in a variety of cell types. Using immortalized cell lines and primary neural crest cell explants, we show that microRNA-211, previously implicated in the regulation of melanoma proliferation and invasiveness, promotes pigmentation in melanoblasts and melanocytes. Expression of this microRNA is regulated by the key melanocyte transcription factor MITF and regulates pigmentation by targeting the TGF-β receptor 2. Transfection with pre-miR-211 precursor molecules in melb-a and melan-a cells leads to a decrease in the expression of TGF-β receptor 2 and reduces the TGF-β signaling-mediated downregulation of two melanogenic enzymes, tyrosinase and tyrosinase-related protein 1. Conversely, downregulation of microRNA-211 using specific microRNA inhibitors has the opposite effects. It appears, therefore, that microRNA-211 serves as a negative regulator of TGF-β signaling which is known to play a important roles in vivo in melanocyte stem cell maintenance and pigmentation.
Wang, Zhihao; Pang, Li; Zhao, Huiying; Song, Lei; Wang, Yuehui; Sun, Qi; Guo, Chunjie; Wang, Bin; Qin, Xiujiao; Pan, Aiqun
Human hair follicle mesenchymal stem cells (hHFMSCs) are an important source of cardiovascular tissue engineering for their differentiation potential into smooth muscle cells (SMCs), yet the molecular pathways underlying such fate determination is unclear. MicroRNAs (miRNAs) are non-coding RNAs that play critical roles in cell differentiation. In present study, we found that miR-128 was remarkably decreased during the differentiation of hHFMSCs into SMCs induced by transforming growth factor-β1 (TGF-β1). Moreover, overexpression of miR-128 led to decreased expression of SMC cellular marker proteins, such as smooth muscle actin (SMA) and calponin, in TGF-β1-induced SMC differentiation. Further, we identified that miR-128 targeted the 3'-UTR of SMAD2 transcript for translational inhibition of SMAD2 protein, and knockdown of SMAD2 abrogated the promotional effect of antagomir-128 (miR-128 neutralizer) on SMC differentiation. These results suggest that miR-128 regulates the differentiation of hHFMSCs into SMCs via targeting SMAD2, a main transcription regulator in TGF-β signaling pathway involving SMC differentiation. The miR-128/SMAD2 axis could therefore be considered as a candidate target in tissue engineering and regenerative medicine for SMCs.
Chen, Hongwei; Tian, Yun
Previous studies demonstrated that miR-15a-5p was probably associated with human hepatocellular carcinoma, while the function of miR-15a-5p in OA (Osteoarthritis) still remains unknown. Here, we uncovered the potential role of miR-15a-5p on OA pathogenesis and confirmed its predicted target VEGFA (Vascular Endothelial Growth Factor A). Measured by RT-PCR, miR-15a-5p expression increased remarkably while VEGFA expression was significantly decreased in OA chondrocytes compared with normal conditions. According to Luciferase activity assay, miR-15a-5p directly targeted the 3'-UTR of VEGFA to inhibit its expression. Functional analysis including CCK-8 assay and flow cytometry revealed that overexpression of VEGFA or inhibition of miR-15a-5p promoted cell proliferation, suppressed cell apoptosis and reduced matrix degradation in OA chondrocytes. Moreover, rescue assays carried out with both expression of VEGFA and miR-15a-5p demonstrated that miR-15a-5p contributes to cell apoptosis and matrix degradation via inhibiting VEGFA. We further provided evidence that multiple proteins related to matrix synthesis were regulated by miR-15a-5p and VEGFA using Western blot and ELISA assays. Taken together, our findings elucidated an underlying mechanism by which miR-15a-5p regulates viability and matrix degradation of OA and indicated a new target for OA diagnosis and therapy.
Oyallon, Justine; Apitz, Holger; Miguel-Aliaga, Irene; Timofeev, Katarina; Ferreira, Lauren; Salecker, Iris
During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail. PMID:22796650
Full Text Available Xiaoguang Zhang,1 Tong Zhang,2 Kuo Yang,3 Minghao Zhang,1 Keming Wang1 1Department of Urology, Tianjin Third Central Hospital, Tianjin, 2Department of Urology, Provincial Hospital Affiliated to Shandong University, Jinan, 3Tianjin Institute of Urology, Tianjin, People’s Republic of China Abstract: The most common cause of death from prostate cancer (PCa is metastases. There is an increasing body of evidence that microRNAs play an important role in the development of PCa by regulating target genes involved in tumor metastasis. Here, we identified that expression of miR-486-5p was decreased in metastatic C4-2 cells compared to non-metastatic LNCaP cells. Further validation in clinical samples showed that miR-486-5p expression was significantly decreased in metastatic PCa tissues compared to localized PCa tissues. Functional studies demonstrated that increased miR-486-5p expression can suppress cell migration and the invasive ability of C4-2 cells. Moreover, Snail, a key regulator of the epithelial–mesenchymal transition, was verified as a target gene of miR-486-5p. In conclusion, these findings suggest that miR-486-5p plays a suppressive role in mediating the migration and invasion of PCa by directly suppressing the protein expression of Snail and may provide a potential therapeutic target for the disease. Keywords: microRNA, prostate cancer, metastasis, EMT
Oyallon, Justine; Apitz, Holger; Miguel-Aliaga, Irene; Timofeev, Katarina; Ferreira, Lauren; Salecker, Iris
During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. We investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, we demonstrate that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, our findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail.
Gong, Xi [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Zhang, Kunshan [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Yanlu; Wang, Junbang; Cui, Yaru [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Li, Siguang, E-mail: email@example.com [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Luo, Yuping, E-mail: firstname.lastname@example.org [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China)
Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.
Rivero-Hinojosa, Samuel; Kang, Sungyun; Lobanenkov, Victor V.; Zentner, Gabriel E.
Despite sharing the same sequence specificity in vitro and in vivo, CCCTC-binding factor (CTCF) and its paralog brother of the regulator of imprinted sites (BORIS) are simultaneously expressed in germ cells. Recently, ChIP-seq analysis revealed two classes of CTCF/BORIS-bound regions: single CTCF target sites (1xCTSes) that are bound by CTCF alone (CTCF-only) or double CTCF target sites (2xCTSes) simultaneously bound by CTCF and BORIS (CTCF&BORIS) or BORIS alone (BORIS-only) in germ cells and in BORIS-positive somatic cancer cells. BORIS-bound regions (CTCF&BORIS and BORIS-only sites) are, on average, enriched for RNA polymerase II (RNAPII) binding and histone retention in mature spermatozoa relative to CTCF-only sites, but little else is known about them. We show that subsets of CTCF&BORIS and BORIS-only sites are occupied by several testis-specific transcriptional regulators (TSTRs) and associated with highly expressed germ cell-specific genes and histone retention in mature spermatozoa. We also demonstrate a physical interaction between BORIS and one of the analyzed TSTRs, TATA-binding protein (TBP)-associated factor 7-like (TAF7L). Our data suggest that CTCF and BORIS cooperate with additional TSTRs to regulate gene expression in developing male gametes and histone retention in mature spermatozoa, potentially priming certain regions of the genome for rapid activation following fertilization. PMID:28145452
Full Text Available ABSTRACT MicroRNAs (miRNAs are critical for both development and function of the central nervous system. Significant evidence suggests that abnormal expression of miRNAs is associated with neurodevelopmental disorders. MeCP2 protein is an epigenetic regulator repressing or activating gene transcription by binding to methylated DNA. Both loss-of-function and gain-of-function mutations in the MECP2 gene lead to neurodevelopmental disorders such as Rett syndrome, autism and MECP2 duplication syndrome. In this study, we demonstrate that miR-130a inhibits neurite outgrowth and reduces dendritic spine density as well as dendritic complexity. Bioinformatics analyses, cell cultures and biochemical experiments indicate that miR-130a targets MECP2 and down-regulates MeCP2 protein expression. Furthermore, expression of the wild-type MeCP2, but not a loss-of-function mutant, rescues the miR-130a-induced phenotype. Our study uncovers the MECP2 gene as a previous unknown target for miR-130a, supporting that miR-130a may play a role in neurodevelopment by regulating MeCP2. Together with data from other groups, our work suggests that a feedback regulatory mechanism involving both miR-130a and MeCP2 may serve to ensure their appropriate expression and function in neural development.
Bhajun, Ricky; Guyon, Laurent; Pitaval, Amandine; Sulpice, Eric; Combe, Stéphanie; Obeid, Patricia; Haguet, Vincent; Ghorbel, Itebeddine; Lajaunie, Christian; Gidrol, Xavier
MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues
Chen, Zhi; Shi, HuaiPing; Sun, Shuang; Xu, HuiFen; Cao, DuoYao; Luo, Jun
Milk fat metabolism is a complex procedure controlled by several factors. MiRNAs (microRNAs) regulate expression of genes and influence a series of biological procedures, such as fatty acid metabolism. Here we screened expression of goat mammary gland's miRNA during peak-lactation and late-lactation, and found that miR-181b expresses remarkably. Moreover, we illustrated that the over-expression of miR-181b impaired fat metabolism while the knockdown of miR-181b promoted fat metabolism in GMEC. These findings extend the discovery of miR-181b functioning in mediating adipocyte differentiation, by suggesting its role in impairing fat metabolism, which develops our cognition on the importance of miRNAs in milk fat metabolism and synthesis. In this study, we find that over expressed miR-181b impaired adipogenesis and inhibited miR-181b promoted adipogenesis in GMEC. Using Luciferase reporter assay and Western Blot, IRS2 was illustrated to be a miR-181b's potential target gene. What is interesting is that miR-181b regulates multiple key components in the Hippo pathway, such as LATS1 and YAP1 in GMECs. In conclusion, our findings indicated that miR-181b suppress fat metabolism by means of regulating multiple genes in the Hippo pathway and target IRS2, which promotes further study on the function of miRNAs in milk fat metabolism and synthesis.
Verdonk, J.C.; Vos, de C.H.; Verhoeven, H.A.; Haring, M.A.; Tunen, van A.J.; Schuurink, R.C.
Petunia hybrida line W115 (Mitchell) has large white flowers that produce a pleasant fragrance. By applying solid phase micro extraction (SPME) techniques coupled to GC-MS analysis, volatile emission was monitored in vivo using a targeted metabolomics approach. Mature flowers released predominantly
Bucha, Sudha; Mukhopadhyay, Debashis; Bhattacharyya, Nitai Pada
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the increase in CAG repeats beyond 36 at the exon1 of the gene Huntingtin (HTT). Among the various dysfunctions of biological processes in HD, transcription deregulation due to abnormalities in actions of transcription factors has been considered to be one of the important pathological conditions. In addition, deregulation of microRNA (miRNA) expression has been described in HD. Earlier, expression of microRNA-214 (miR-214) has been shown to increase in HD cell models and target HTT gene; the expression of the later being inversely correlated to that of miR-214. In the present communication, we observed that the expressions of several HTT co-expressed genes are modulated by exogenous expression of miR-214 or by its mutant. Among several HTT co-expressed genes, MFN2 was shown to be the direct target of miR-214. Exogenous expression of miR-214, repressed the expression of MFN2, increased the distribution of fragmented mitochondria and altered the distribution of cells in different phases of cell cycle. In summary, we have shown that increased expression of miR-214 observed in HD cell model could target MFN2, altered mitochondrial morphology and deregulated cell cycle. Inhibition of miR-214 could be a possible target of intervention in HD pathogenesis.
Gao, Feng; Kight, Alicia D.; Henderson, Rory; Jayanthi, Srinivas; Patel, Parth; Murchison, Marissa; Sharma, Priyanka; Goforth, Robyn L.; Kumar, Thallapuranam Krishnaswamy Suresh; Henry, Ralph L.; Heyes, Colin D.
Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP. PMID:25918165
Klironomos, Filippos D; Berg, Johannes
When small RNAs are loaded onto Argonaute proteins they can form the RNA-induced silencing complexes (RISCs), which mediate RNA interference (RNAi). RISC-formation is dependent on a shared pool of Argonaute proteins and RISC-loading factors, and is susceptible to competition among small RNAs. We present a mathematical model that aims to understand how small RNA competition for RISC-formation affects target gene repression. We discuss that small RNA activity is limited by RISC-formation, RISC-degradation, and the availability of Argonautes. We show that different competition conditions for RISC-loading result in different signatures of RNAi determined also by the amount of RISC-recycling taking place. In particular, we find that the small RNAs, although less efficient at RISC-formation, can perform in the low RISC-recycling range as well as their more effective counterparts. Additionally, we predict that under conditions of low RISC-loading efficiency and high RISC-recycling, the variation in target levels increases linearly with the target transcription rate. Furthermore, we show that RISC-recycling determines the effect that Argonaute scarcity conditions have on target expression variation. Our observations, taken together, offer a framework of predictions that can be used to infer from data the particular characteristics of underlying RNAi activity.
Pasini, Diego; Cloos, Paul A C; Walfridsson, Julian
The Polycomb group (PcG) proteins have an important role in controlling the expression of genes essential for development, differentiation and maintenance of cell fates. The Polycomb repressive complex 2 (PRC2) is believed to regulate transcriptional repression by catalysing the di- and tri-methy...
TOR is a major nutrition and energy sensor that regulates growth and lifespan in yeast and animals. In plants growth and lifespan are intertwined with not only nutrient acquisition but also nutrition generation and unique aspects of development and differentiation. How TOR functions in these process...
Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian;
Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600...
B. Schumacher (Björn); A. Gartner (Anton)
textabstractThe tumor suppressor p53 is a central player in apoptosis induction in response to oncogenic stimuli and DNA damage. As activation of p53 has been suggested as a prime strategy for future tumor therapy, inhibition of negative regulators of p53 activity would be a similarly desirable stra
Mukherjee, A; Koli, S; Reddy, K V R
Mechanistic target of rapamycin (mTOR) is a signal transduction pathway that modulates translation initiation in several animals including mammals. Rapamaycin, an allosteric inhibitor of mTOR pathway, is often used as an immunosuppressive drug following kidney transplantation and causes gonadal dysfunction and defects in spermatogenesis. The molecular mechanism behind rapamycin-mediated testicular dysfunction is not known. We have therefore explored the contribution of rapamycin in mTOR regulation and microRNA (miRNA) expression in mouse spermatocytes, the intermediate stage of spermatogenesis, where meiosis takes place. In the present study, we optimized the isolation of highly pure and viable spermatocytes by flow sorting, treated them with rapamycin, and investigated the expression of mTOR and downstream effector molecules. Western blot and immunocytochemical analysis confirm that rapamycin treatment suppresses mTOR and phopsphorylated P70S6 kinase activities in spermatocytes, but not that of phosphorylated 4E-binding protein 1. Also, rapamycin treatment modulates the expression of several spermatocyte-specific miRNAs. To complement these finding an in vivo study was also performed. In silico prediction of target genes of these miRNAs and their functional pathway analysis revealed that, several of them are involved in crucial biological process, cellular process and catalytic activities. miRNA-transcription factor (TF) network analysis enlisted different TFs propelling the transcription machineries of these miRNAs. In silico prediction followed by quatitative real-time PCR revealed two of these TFs namely, PU.1 and CCCTC binding factor (CTCF) are down and upregulated, respectively, which may be the reason of the altered expression of miRNAs following rapamycin treatment. In conclusion, for the first time, the present study provides insight into how rapamycin regulates mTOR pathway and spermatocyte-specific miRNA expression which in turn, regulate expression of
Sung Won Youn
Full Text Available Atherosclerosis arises when injury to the arterial wall induces an inflammatory cascade that is sustained by a complex network of cytokines, together with accumulation of lipids and fibrous material. Inflammatory cascades involve leukocyte adherence and chemotaxis, which are coordinated by the local secretion of adhesion molecules, chemotactic factors, and cytokines. Transcription factors are critical to the integration of the various steps of the cascade response to mediators of vascular injury, and are induced in a stimulus-dependent and cell-type-specific manner. Several small-nucleic-acid-based therapeutic strategies have recently been developed to target transcription factors: antisense oligodeoxynucleotides, RNA interference, microRNA, and decoy oligodeoxynucleotides. The aim of this review was to provide an overview of these particular targeted therapeutic strategies, toward regulation of the vascular inflammation, remodeling and fibrosis associated with atherosclerosis.
Schuhmacher, Marino; Eick, Dirk
The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc). c-Myc plays a crucial role in cell growth and proliferation. Here, we examined how expression of c-Myc target genes and cell proliferation depend on variation of c-Myc protein levels. We show that proliferation rates, the number of cells in S-phase, and cell size increased in a dose-dependent manner in response to increasing c-Myc levels. Likewise, the mRNA levels of c-Myc responsive genes steadily increased with rising c-Myc levels. Strikingly, steady-state mRNA levels of c-Myc target genes did not saturate even at highest c-Myc concentrations. These characteristics predestine c-Myc levels as a cellular rheostat for the control and fine-tuning of cell proliferation and growth rates.
Gensert, JoAnn M; Baranova, Oxana V; Weinstein, David E; Ratan, Rajiv R
Recent advances in cancer cell biology have focused on histone deacetylase inhibitors (HDACi's) because they target pathways critical to the development and progression of disease. In particular, HDACi's can induce expression of epigenetically silenced genes that promote growth arrest, differentiation and cell death. In glioma cells, one such repressed gene is the tetraspanin CD81, which regulates cytostasis in various cell lines and in astrocytes, the major cellular component of gliomas. Our studies show that HDACi's, trichostatin and sodium butyrate, promote growth arrest and differentiation with negligible cell death in glioma cells and induce expression of CD81 and cyclin-dependent kinase inhibitor 1A (p21(CIP/WAF-1)), another regulator of cytostasis in astrocytes. Interference RNA knock-down of CD81 abrogates cytostasis promoted by HDAC inhibition indicating that HDACi-induced CD81 is responsible for growth arrest. Induction of CD81 expression through HDAC inhibition is a novel strategy to promote growth arrest in glioma cells.
Karen G. Scheps
Full Text Available Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2. In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.
Brown, Zachary Z; Müller, Manuel M; Kong, Ha Eun; Lewis, Peter W; Muir, Tom W
Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native chromatin, each nucleosome can differ from its neighbors as a result of covalent modifications to both the DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation. A chemical approach to the production of heterotypic chromatin that can be used in such studies is introduced. This method involves the attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a peptide nucleic acid (PNA) targeting compound. This strategy was applied to dissect the effect of chromatin context on the activity of the histone methyltransferase PRC2. The results show that PRC2 can be stimulated to produce histone H3 methylation from a defined nucleation site.
Farzana, F; Zalm, R; Chen, N; Li, K W; Grant, Seth G N; Smit, A B; Toonen, R F; Verhage, M
Neurotransmission and synaptic strength depend on expression of post-synaptic receptors on the cell surface. Post-translational modification of receptors, trafficking to the synapse through the secretory pathway, and subsequent insertion into the synapse involves interaction of the receptor with A-kinase anchor proteins (AKAPs) and scaffolding proteins. Neurobeachin (Nbea), a brain specific AKAP, is required for synaptic surface expression of both glutamate and GABA receptors. Here, we investigated the role of Nbea-dependent targeting of postsynaptic receptors by studying Nbea interaction with synapse-associated protein 102 (SAP102/Dlg3) and protein kinase A subunit II (PKA II). A Nbea mutant lacking the PKA binding domain showed a similar distribution as wild-type Nbea in Nbea null neurons and partially restored GABA receptor surface expression. To understand the relevance of Nbea interaction with SAP102, we analysed SAP102 null mutant mice. Nbea levels were reduced by ~80% in SAP102 null mice, but glutamatergic receptor expression was normal. A single-point mutation in the pleckstrin homology domain of Nbea (E2218R) resulted in loss of binding with SAP102. When expressed in Nbea null neurons, this mutant fully restored GABA receptor surface expression, but not glutamate receptor expression. Our results suggest that the PKA-binding domain is not essential for Nbea's role in receptor targeting and that Nbea targets glutamate and GABA receptors to the synapse via distinct molecular pathways by interacting with specific effector proteins.
Heath, Jack M; Fernandez Esmerats, Joan; Khambouneheuang, Lucky; Kumar, Sandeep; Simmons, Rachel; Jo, Hanjoong
Calcific aortic valve disease (CAVD) is a major cause of morbidity in the aging population, but the underlying mechanisms of its progression remain poorly understood. Aortic valve calcification preferentially occurs on the fibrosa, which is subjected to disturbed flow. The side-specific progression of the disease is characterized by inflammation, calcific lesions, and extracellular matrix (ECM) degradation. Here, we explored the role of mechanosensitive microRNA-181b and its downstream targets in human aortic valve endothelial cells (HAVECs). Mechanistically, miR-181b is upregulated in OS and fibrosa, and it targets TIMP3, SIRT1, and GATA6, correlated with increased gelatinase/MMP activity. Overexpression of miR-181b led to decreased TIMP3 and exacerbated MMP activity as shown by gelatinase assay, and miR-181b inhibition decreased gelatinase activity through the repression of TIMP3 levels. Luciferase assay showed specific binding of miR-181b to the TIMP3 gene. Overexpression of miR-181b in HAVECs subjected to either LS or OS increased MMP activity, and miR-181b inhibition abrogated shear-sensitive MMP activity. These studies suggest that targeting this shear-dependent miRNA may provide a novel noninvasive treatment for CAVD.
Full Text Available Abstract Background The pyruvate dehydrogenase regulator protein (PdhR of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach. Results 22 new genes contained in two operons controlled by PdhR (previously only 20 regulatory targets in eight operons were known were identified by analysing a large-scale dataset of E. coli from the Many Microbes Microarray Database and novel expression data from a pdhR knockout strain, as well as a PdhR overproducing strain. We identified a regulation of the glycolate utilization operon glcDEFGBA using chromatin immunoprecipitation and gel shift assays. We show that this regulation could be part of a cross-induction between genes necessary for acetate and pyruvate utilisation controlled through PdhR. Moreover, a link of PdhR regulation to the replication machinery of the cell via control of the transcription of the dcw-cluster was verified in experiments. This augments our knowledge of the functions of the PdhR-regulon and demonstrates its central importance for further cellular processes in E. coli. Conclusions We extended the PdhR regulon by 22 new genes contained in two operons and validated the regulation of the glcDEFGBA operon for glycolate utilisation and the dcw-cluster for cell division proteins experimentally. Our results provide, for the first time, a plausible regulatory link between the nutritional status of the cell and cell replication mediated by PdhR.
Chen, Jiaying; Zhang, Can; Mi, Yang; Chen, Fuxue; Du, Dongshu
Glioma is stemmed from the glial cells in the brain, which is accounted for about 45% of all intracranial tumors. The characteristic of glioma is invasive growth, as well as there is no obvious boundary between normal brain tissue and glioma tissue, so it is difficult to resect completely with worst prognosis. The metabolism of glioma is following the Warburg effect. Previous researches have shown that GLUT1, as a glucose transporter carrier, affected the Warburg effect, but the molecular mechanism is not very clear. CREB1 (cAMP responsive element-binding protein1) is involved in various biological processes, and relevant studies confirmed that CREB1 protein regulated the expression of GLUT1, thus mediating glucose transport in cells. Our experiments mainly reveal that the CREB1 could affect glucose transport in glioma cells by regulating the expression of GLUT1, which controlled the metabolism of glioma and affected the progression of glioma.
Zhang, Zengli; Wang, Hongfeng; Ding, Qifeng; Xing, Yufei; Xu, Delai; Xu, Zhonghua; Zhou, Tong; Qian, Bin; Ji, Chenghong; Pan, Xue; Zhong, Anyuan; Ying, Zheng; Zhou, Caicun; Shi, Minhua
The cellular protein degradation system, such as proteasomal or autophagy-lysosomal system plays an important role in the pathogenesis of a variety of human diseases including cancer. Transcription factor EB (TFEB) is a master transcriptional factor in the regulation of autophagy-lysosome pathway (ALP), and it has multiple biological functions including protein degradation, cell homeostasis and cell survival. In the present study we show that the tumor suppressor p53 can regulate TFEB nuclear translocation and activity in lung cancer cells. We found p53 deletion or chemical inhibition of p53 using pifithrin-α could promote the translocation of TFEB from cytoplasm to the nucleus, thus increased the TFEB-mediated lysosomal and autophagosomal biogenesis in lung cancer cells. Moreover, re-expression of p53 could decrease the expression levels of TFEB-targeting genes involved in ALP, and knockdown of TFEB could abolish the effect of p53 on the regulation of ALP gene expression. Taken together, our data indicate that p53 affects ALP through regulating TFEB nuclear translocation in lung cancer cells. Importantly, our study reveals a critical link between two keys factors in tumourigenesis and autophagy, and suggests a potential important role of p53-TFEB signaling axis in lung cancer.
Eloi R. Verrier
Full Text Available Chronic hepatitis B, C, and D virus (HBV, HCV, and HDV infections are the leading causes of liver disease and cancer worldwide. Recently, the solute carrier and sodium taurocholate co-transporter NTCP has been identified as a receptor for HBV and HDV. Here, we uncover NTCP as a host factor regulating HCV infection. Using gain- and loss-of-function studies, we show that NTCP mediates HCV infection of hepatocytes and is relevant for cell-to-cell transmission. NTCP regulates HCV infection by augmenting the bile-acid-mediated repression of interferon-stimulated genes (ISGs, including IFITM3. In conclusion, our results uncover NTCP as a mediator of innate antiviral immune responses in the liver, and they establish a role for NTCP in the infection process of multiple viruses via distinct mechanisms. Collectively, our findings suggest a role for solute carriers in the regulation of innate antiviral responses, and they have potential implications for virus-host interactions and antiviral therapies.
Verrier, Eloi R; Colpitts, Che C; Bach, Charlotte; Heydmann, Laura; Zona, Laetitia; Xiao, Fei; Thumann, Christine; Crouchet, Emilie; Gaudin, Raphaël; Sureau, Camille; Cosset, François-Loïc; McKeating, Jane A; Pessaux, Patrick; Hoshida, Yujin; Schuster, Catherine; Zeisel, Mirjam B; Baumert, Thomas F
Chronic hepatitis B, C, and D virus (HBV, HCV, and HDV) infections are the leading causes of liver disease and cancer worldwide. Recently, the solute carrier and sodium taurocholate co-transporter NTCP has been identified as a receptor for HBV and HDV. Here, we uncover NTCP as a host factor regulating HCV infection. Using gain- and loss-of-function studies, we show that NTCP mediates HCV infection of hepatocytes and is relevant for cell-to-cell transmission. NTCP regulates HCV infection by augmenting the bile-acid-mediated repression of interferon-stimulated genes (ISGs), including IFITM3. In conclusion, our results uncover NTCP as a mediator of innate antiviral immune responses in the liver, and they establish a role for NTCP in the infection process of multiple viruses via distinct mechanisms. Collectively, our findings suggest a role for solute carriers in the regulation of innate antiviral responses, and they have potential implications for virus-host interactions and antiviral therapies. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
Full Text Available PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum, the mitochondria or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.
Kim, Jae-Eun; Jung, Hyun Jun; Lee, Yu-Jung; Kwon, Tae-Hwan
Mature microRNA (miRNA) acts as an important posttranscriptional regulator. We aimed to profile vasopressin-responsive miRNAs in kidney inner medullary collecting duct cells and to identify aquaporin-2 (AQP2)-targeting miRNAs. Microarray chip assay was carried out in inner medullary collecting duct tubule suspensions from rat kidneys in the absence or presence of desmopressin (dDAVP) stimulation (10(-9) M, 2 h). The results demonstrated 19 miRNAs, including both precursor and mature miRNAs, as potential candidates that showed significant changes in expression after dDAVP stimulation (P 1.3-fold changes in expression on the microarray (miR-127, miR-1, miR-873, miR-16, miR-206, miR-678, miR-496, miR-298, and miR-463) were further examined by quantitative real-time PCR, and target genes of the selected miRNAs were predicted. Next, to identify AQP2-targeting miRNAs, in silico analysis was performed. Four miRNAs (miR-32, miR-137, miR-216a, and miR-216b) target the 3'-untranslated region of rat AQP2 mRNA. Target seed regions of miR-32 and miR-137 were also conserved in the 3'-untranslated region of mouse AQP2 mRNA. Quantitative real-time PCR and immunoblot analysis demonstrated that dDAVP-induced AQP2 expression was significantly attenuated in mpkCCDc14 cells when cells were transfected with miRNA mimics of miR-32 or miR-137. Moreover, luciferase reporter assay demonstrated a significant decrease of AQP2 translation in mpkCCDc14 cells transfected with miRNA mimics of miR-32 or miR-137. The present study provides novel insights into the regulation of AQP2 by RNA interference; however, vasopressin-regulated miRNAs did not include miR-32 or miR-137, indicating that the interaction of miRNAs with the AQP2 regulatory pathway requires further analysis. Copyright © 2015 the American Physiological Society.
Full Text Available Abstract Background Artemin (ARTN is a neurotrophic factor belonging to the glial cell-derived neurotrophic factor family of ligands. To develop potential therapy targeting ARTN, we studied the roles of miR-223 in the migration and invasion of human esophageal carcinoma. Methods ARTN expression levels were detected in esophageal carcinoma cell lines KYSE-150, KYSE-510, EC-9706, TE13, esophageal cancer tissues and paired non-cancerous tissues by Western blot. Artemin siRNA expression vectors were constructed to knockdown of artemin expression mitigated migration and invasiveness in KYSE150 cells. Monolayer wound healing assay and Transwell invasion assay were applied to observe cancer cell migration and invasion. The relative levels of expression were quantified by real-time quantitative PCR. Results ARTN expression levels were higher in esophageal carcinoma tissue than in the adjacent tissue and was differentially expressed in various esophageal carcinoma cell lines. ARTN mRNA contains a binding site for miR-223 in the 3'UTR. Co-transfection of a mir-223 expression vector with pMIR-ARTN led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that ARTN is a target gene of miR-223. Overexpression of miR-223 decreased expression of ARTN in KYSE150 cells while silencing miR-223 increased expression of ARTN in EC9706 cells. Furthermore, overexpression of miR-223 in KYSE150 cells decreased cell migration and invasion. Silencing of miR-223 in EC9706 cells increased cell migration and invasiveness. Conclusions These results reveal that ARTN, a known tumor metastasis-related gene, is a direct target of miR-223 and that miR-223 may have a tumor suppressor function in esophageal carcinoma and could be used in anticancer therapies.
Weekes, D; Kashima, T G; Zandueta, C; Perurena, N; Thomas, D P; Sunters, A; Vuillier, C; Bozec, A; El-Emir, E; Miletich, I; Patiño-Garcia, A; Lecanda, F; Grigoriadis, A E
Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant owing to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signalling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 (fibroblast growth factor receptor 1) was identified as a novel c-Fos/activator protein-1(AP-1)-regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of mitogen-activated protein kinases (MAPKs), morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1-silenced osteosarcoma cells caused a marked twofold to fivefold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small-molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus deregulated FGFR signalling has an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy.
Cui, Tracy X.; Lin, Grace; LaPensee, Christopher R.; Calinescu, Anda-Alexandra; Rathore, Maanjot; Streeter, Cale; Piwien-Pilipuk, Graciela; Lanning, Nathan; Jin, Hui; Carter-Su, Christin; Qin, Zhaohui S.
Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein β (C/EBPβ). This study examines the role of C/EBPβ in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPβ depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPβ led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPβ mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPβ at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPβ and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPβ. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPβ, and recruitment of p300. Overall, these studies suggest that C/EBPβ, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH. PMID:21292824
Evans, Daniel S.; Cline, Thomas W.
The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). Here we show instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. We inferred the existence of this unknown Sxl target from the observation that a constitutively feminizing tra transgene that restores fertility to tra− females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra−. The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, we refer to this pathway as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, we find that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila. PMID:24191002
Li, Dengfeng; Jian, Wei; Wei, Chuankui; Song, Hongming; Gu, Yifan; Luo, Yi; Fang, Lin
MicroRNAs (miRNAs) are a small class of non-coding RNAs that are widely deregulated in various cancers. They act as either oncogenes or tumor suppressor genes in human cancer. The purpose of this study was to examine the potential role of miR-181b in human thyroid papillary cancer. The expression levels of different miRNAs were measured by micro array analysis in 10 thyroid papillary cancer specimens and adjacent normal thyroid cancer tissues. MTT assays, colony formation assays, apoptosis assays were used to explore the potential function of miR-181b inhibitor in TPC1 human thyroid papillary cancer cells. Luciferase reporter assays were performed to validate the regulation of a putative target of miR-181b, in corroboration with qPCR and western blot assays. We found that the expression of miR-181b was higher in thyroid papillary cancer specimens compared with adjacent normal tissues (P miR-181b inhibited cellular growth and promoted cellular apoptosis. Luciferase assays indicated that miR-181b can bind with its putative target site in the 3'-untranslated region (3'-UTR) of CYLD, suggesting that CYLD is a direct target of miR-181b. Western blot analysis indicated that downregulation of miR-181b results in the upregulation of CYLD at protein levels. Taken together, downregulation of miR-181b expression causes cellular growth inhibition, promoting cellular apoptosis by targeting CYLD. These findings suggest that downregulation of the expression of miR-181b may be a therapeutic target for the treatment of human thyroid papillary cancer.
Avkiran, Metin; Cook, Alexandra R; Cuello, Friederike
Extensive pre-clinical work indicates that inhibition of the Na(+)/H(+) exchanger (NHE) affords significant protection to myocardium subjected to ischaemia and reperfusion. By contrast, clinical studies with the NHE inhibitors cariporide, eniporide and zoniporide, in patients with evolving myocardial infarction and those at risk of myocardial infarction, have provided largely disappointing data. Nevertheless, some of these studies have confirmed that, in certain settings, NHE inhibition does indeed protect human myocardium. Furthermore, pre-clinical work suggests that NHE inhibition may provide therapeutic benefit in heart failure also. As an alternative to direct and global NHE inhibition, which may trigger non-cardiac adverse effects, the molecular mechanisms that stimulate cardiac NHE activity in disease may be targeted to attenuate such activity selectively in jeopardized tissue. Many factors associated with cardiac pathology activate RSK, an established NHE kinase, and several selective RSK inhibitors have been described recently. The role of RSK as a potential therapeutic target for indirectly suppressing cardiac NHE activity warrants further investigation.
Schickel, Robert; Park, Sun-Mi; Murmann, Andrea E; Peter, Marcus E
Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as an miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression. Copyright (c) 2010 Elsevier Inc. All rights reserved.
Full Text Available Eukaryotic transcription factors are grouped into families and, due to their similar DNA binding domains, often have the potential to bind to the same genomic regions. This can lead to redundancy at the level of DNA binding, and mechanisms are required to generate specific functional outcomes that enable distinct gene expression programmes to be controlled by a particular transcription factor. Here we used ChIP-seq to uncover two distinct binding modes for the ETS transcription factor ELK1. In one mode, other ETS transcription factors can bind regulatory regions in a redundant fashion; in the second, ELK1 binds in a unique fashion to another set of genomic targets. Each binding mode is associated with different binding site features and also distinct regulatory outcomes. Furthermore, the type of binding mode also determines the control of functionally distinct subclasses of genes and hence the phenotypic response elicited. This is demonstrated for the unique binding mode where a novel role for ELK1 in controlling cell migration is revealed. We have therefore uncovered an unexpected link between the type of binding mode employed by a transcription factor, the subsequent gene regulatory mechanisms used, and the functional categories of target genes controlled.
Yao, Qin; Cao, Siyu; Li, Chun; Mengesha, Asferd; Kong, Beihua; Wei, Mingqian
Transforming growth factor-β1 (TGF-β1) induces stromal fibroblast-to-myofibroblast transdifferentiation in the tumor-stroma interactive microenvironment via modulation of multiple phenotypic and functional genes, which plays a critical role in tumor progression. Up to now, the involvement of micro-RNAs (miRNAs) and their roles in TGF-β1-induced myofibroblast differentiation in tumor-stroma interaction are unclear. Using quantitative real-time RT-PCR, we demonstrated that the expression of micro-RNA-21 (miR-21) was upregulated in activated fibroblasts after treatment with TGF-β1 or conditioned medium from cancer cells. To determine the potential roles of miR-21 in TGF-β1-mediated gene regulation during myofibroblast conversion, we showed that miR-21 expression was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic. Interestingly, downregulation of miR-21 with the inhibitor effectively inhibited TGF-β1-induced myofibroblast differentiation while upregulation of miR-21 with a mimic significantly promoted myofibroblast differentiation. We further demonstrated that MiR-21 directly targeted and downregulated programmed cell death 4 (PDCD4) gene, which in turn acted as a negative regulator of several phenotypic and functional genes of myofibroblasts. Taken together, these results suggested that miR-21 participated in TGF-β1-induced myofibroblast transdifferentiation in cancer stroma by targeting PDCD4. Copyright © 2010 UICC.
Lucas, Keira J; Roy, Sourav; Ha, Jisu; Gervaise, Amanda L; Kokoza, Vladimir A; Raikhel, Alexander S
Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.
Lu, Cecilia S; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David
Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.
Karaca-Mandic, Pinar; Abraham, Jean M; Simon, Kosali
Effective January 1, 2011, individual market health insurers must meet a minimum medical loss ratio (MLR) of 80%. This law aims to encourage 'productive' forms of competition by increasing the proportion of premium dollars spent on clinical benefits. To date, very little is known about the performance of firms in the individual health insurance market, including how MLRs are related to insurer and market characteristics. The MLR comprises one component of the price-cost margin, a traditional gauge of market power; the other component is percent of premiums spent on administrative expenses. We use data from the National Association of Insurance Commissioners (2001-2009) to evaluate whether the MLR is a good target measure for regulation by comparing the two components of the price-cost margin between markets that are more competitive versus those that are not, accounting for firm and market characteristics. We find that insurers with monopoly power have lower MLRs. Moreover, we find no evidence suggesting that insurers' administrative expenses are lower in more concentrated insurance markets. Thus, our results are largely consistent with the interpretation that the MLR could serve as a target measure of market power in regulating the individual market for health insurance but with notable limited ability to capture product and firm heterogeneity. Copyright © 2013 John Wiley & Sons, Ltd.
Full Text Available The primary feature of the mammalian skin includes the hair follicle, inter-follicular epidermis and the sebaceous glands, all of which form pilo-sebaceous units. The epidermal protective layer undergoes an ordered/programmed process of proliferation and differentiation, ultimately culminating in the formation of a cornified envelope consisting of enucleated corneocytes. These terminally differentiated cells slough off in a cyclic manner and this process is regulated via induction or repression of epidermal differentiation complex (EDC genes. These genes, spanning 2 Mb region of human chromosome 1q21, play a crucial role in epidermal development, through various mechanisms. Each of these mechanisms employs a unique chromatin re-modelling factor or an epigenetic modifier. These factors act to regulate epidermal differentiation singly and/or in combination. Diseases like psoriasis and cancer exhibit aberrations in proliferation and differentiation through, in part, dysregulation in these epigenetic mechanisms. Knowledge of the existing mechanisms in the physiological and the aforesaid pathological contexts may not only facilitate drug development, it also can make refinements to the existing drug delivery systems.
Mitsiadis, Thimios A; Graf, Daniel; Luder, Hansueli; Gridley, Thomas; Bluteau, Gilles
The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism that is essential for cell fate specification and proper embryonic development. We have analysed the expression, regulation and function of the jagged 2 (Jag2) gene, which encodes a ligand for the Notch family of receptors, in developing mouse teeth. Jag2 is expressed in epithelial cells that give rise to the enamel-producing ameloblasts from the earliest stages of tooth development. Tissue recombination experiments showed that its expression in epithelium is regulated by mesenchyme-derived signals. In dental explants cultured in vitro, the local application of fibroblast growth factors upregulated Jag2 expression, whereas bone morphogenetic proteins provoked the opposite effect. Mice homozygous for a deletion in the Notch-interaction domain of Jag2 presented a variety of severe dental abnormalities. In molars, the crown morphology was misshapen, with additional cusps being formed. This was due to alterations in the enamel knot, an epithelial signalling structure involved in molar crown morphogenesis, in which Bmp4 expression and apoptosis were altered. In incisors, cytodifferentiation and enamel matrix deposition were inhibited. The expression of Tbx1 in ameloblast progenitors, which is a hallmark for ameloblast differentiation and enamel formation, was dramatically reduced in Jag2(-/-) teeth. Together, these results demonstrate that Notch signalling mediated by Jag2 is indispensable for normal tooth development.
Liang, Shuang; Chen, Rui-Ting; Zhang, Deng-Pan; Xin, Hu-Hu; Lu, Yan; Wang, Mei-Xian; Miao, Yun-Gen
Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the pathway in adipose development remains poorly understood. In this report, we found that Hh pathway components are expressed in the fat body of silkworm larvae. Functional analysis of these components in a BmN cell line model revealed that activation of the Hh gene stimulated transcription of Hh pathway components, but inhibited the expression of the adipose marker gene AP2. Conversely, specific RNA interference-mediated knockdown of Hh resulted in increased AP2 expression. This further showed the regulation of Hh signal on the adipose marker gene. In silkworm larval models, enhanced adipocyte differentiation and an increase in adipocyte cell size were observed in silkworms that had been treated with a specific Hh signaling pathway antagonist, cyclopamine. The fat-body-specific Hh blockade tests were consistent with Hh signaling inhibiting silkworm adipogenesis. Our results indicate that the role of Hh signaling in inhibiting fat formation is conserved in vertebrates and invertebrates.
Full Text Available Marburg virus (MARV has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1, a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2. Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses.
Mitsiadis, Thimios A.; Graf, Daniel; Luder, Hansueli; Gridley, Thomas; Bluteau, Gilles
The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism that is essential for cell fate specification and proper embryonic development. We have analysed the expression, regulation and function of the jagged 2 (Jag2) gene, which encodes a ligand for the Notch family of receptors, in developing mouse teeth. Jag2 is expressed in epithelial cells that give rise to the enamel-producing ameloblasts from the earliest stages of tooth development. Tissue recombination experiments showed that its expression in epithelium is regulated by mesenchyme-derived signals. In dental explants cultured in vitro, the local application of fibroblast growth factors upregulated Jag2 expression, whereas bone morphogenetic proteins provoked the opposite effect. Mice homozygous for a deletion in the Notch-interaction domain of Jag2 presented a variety of severe dental abnormalities. In molars, the crown morphology was misshapen, with additional cusps being formed. This was due to alterations in the enamel knot, an epithelial signalling structure involved in molar crown morphogenesis, in which Bmp4 expression and apoptosis were altered. In incisors, cytodifferentiation and enamel matrix deposition were inhibited. The expression of Tbx1 in ameloblast progenitors, which is a hallmark for ameloblast differentiation and enamel formation, was dramatically reduced in Jag2−/− teeth. Together, these results demonstrate that Notch signalling mediated by Jag2 is indispensable for normal tooth development. PMID:20685737
Fernandez, Ricardo; Nardocci, Gino; Navarro, Cristina; Reyes, Edison P; Acuña-Castillo, Claudio; Cortes, Paula P
Sepsis progresses to multiple organ dysfunction due to the uncontrolled release of inflammatory mediators, and a growing body of evidence shows that neural signals play a significant role in modulating the immune response. Thus, similar toall other physiological systems, the immune system is both connected to and regulated by the central nervous system. The efferent arc consists of the activation of the hypothalamic-pituitary-adrenal axis, sympathetic activation, the cholinergic anti-inflammatory reflex, and the local release of physiological neuromodulators. Immunosensory activity is centered on the production of pro-inflammatory cytokines, signals that are conveyed to the brain through different pathways. The activation of peripheral sensory nerves, i.e., vagal paraganglia by the vagus nerve, and carotid body (CB) chemoreceptors by the carotid/sinus nerve are broadly discussed here. Despite cytokine receptor expression in vagal afferent fibers, pro-inflammatory cytokines have no significant effect on vagus nerve activity. Thus, the CB may be the source of immunosensory inputs and incoming neural signals and, in fact, sense inflammatory mediators, playing a protective role during sepsis. Considering that CB stimulation increases sympathetic activity and adrenal glucocorticoids release, the electrical stimulation of arterial chemoreceptors may be suitable therapeutic approach for regulating systemic inflammation.
Sun, Hui; Wang, Huiyu; Zhang, Aihua; Yan, Guangli; Zhang, Yue; An, Na
Abstract Metabolomics has been increasingly applied to discovering biomarkers and identifying perturbed pathways. Berberine has been shown to exhibit anti-inflammatory, antioxidant, and anticancer properties, but its mechanisms for treating nonbacterial prostatitis (NBP) remain unclear completely. We developed the untargeted metabolomics approach based on UPLC-Q-TOF-HDMS to profile the metabolite changes in urine samples in order to discover novel potential biomarkers to clarify mechanisms of berberine in treating a rat model of capsaicin-induced nonbacterial prostatitis (NBP). The changes in metabolic profiling were restored to their base-line values after berberine treatment according to the principal component analysis (PCA) score plots. Fourteen different potential biomarkers and five acutely perturbed metabolic pathways contributing to the treatment of NBP were discovered and identified. Specifically, the berberine-treated rats are located closer to the normal group, indicating that the NBP-induced disturbances to the metabolic profile were partially reversed by berberine treatment. After treatment with berberine, the relative contents of 12 potential biomarkers were effectively regulated, which suggested that the therapeutic effects of berberine on NBP may involve regulating disturbances to the metabolism. Our results show that the protective effect of berberine occurs in part through a reversal of the NBP-caused disturbances. PMID:25588034
Abhishek, Sinha; Palamadai Krishnan, Suresh
The primary feature of the mammalian skin includes the hair follicle, inter-follicular epidermis and the sebaceous glands, all of which form pilo-sebaceous units. The epidermal protective layer undergoes an ordered/programmed process of proliferation and differentiation, ultimately culminating in the formation of a cornified envelope consisting of enucleated corneocytes. These terminally differentiated cells slough off in a cyclic manner and this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes. These genes, spanning 2 Mb region of human chromosome 1q21, play a crucial role in epidermal development, through various mechanisms. Each of these mechanisms employs a unique chromatin re-modelling factor or an epigenetic modifier. These factors act to regulate epidermal differentiation singly and/or in combination. Diseases like psoriasis and cancer exhibit aberrations in proliferation and differentiation through, in part, dysregulation in these epigenetic mechanisms. Knowledge of the existing mechanisms in the physiological and the aforesaid pathological contexts may not only facilitate drug development, it also can make refinements to the existing drug delivery systems.
Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi
Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27kip1 and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27kip1 mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. PMID:26926106
Song, Yichen, E-mail: email@example.com [Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004 (China); Wang, Ping, E-mail: firstname.lastname@example.org [Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001 (China); Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001 (China); Zhao, Wei, E-mail: email@example.com [Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001 (China); Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001 (China); Yao, Yilong, E-mail: firstname.lastname@example.org [Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004 (China); Liu, Xiaobai, E-mail: email@example.com [The 96th Class, 7-year Program, China Medical University, Shenyang, Liaoning Province 110001 (China); Ma, Jun, E-mail: firstname.lastname@example.org [Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001 (China); Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001 (China); Xue, Yixue, E-mail: email@example.com [Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001 (China); Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001 (China); Liu, Yunhui, E-mail: firstname.lastname@example.org [Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004 (China)
MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the association of miR-18a, an important member of this cluster, with glioblastoma remains unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human astrocytes, and the expression levels were increased along with the rising pathological grades of glioblastoma. Neogenin was identified as the target gene of miR-18a by dual-luciferase reporter assays. RT-PCR and western blot results showed that its expression levels were decreased along with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly up-regulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma. - Highlights: • MiR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines. • Neogenin was identified as the target gene of miR-18a. • Neogenin expressions were decreased along with the rising pathological grades of glioblastoma. • Inhibition of miR-18a suppressed biological behavior of glioma cells by up-regulating neogenin.
Background Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line. Materials and methods To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR). Results The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25) the second was characterized by miR-21 overexpression and RECK underexpression (N = 12), and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16). From men who presented the second profile (miR-21 overexpression and RECK underexpression) 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025). Conclusions Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion. PMID:22642976
Full Text Available Abstract Background Prognosis of prostate cancer (PCa is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line. Materials and methods To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR. Results The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25 the second was characterized by miR-21 overexpression and RECK underexpression (N = 12, and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16. From men who presented the second profile (miR-21 overexpression and RECK underexpression 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025. Conclusions Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion.
Ling Zhang; Jun Lei; Zi-Ling Fang; Jian-Ping Xiong
Gastric cancer (GC) is the fourth most prevalent type of cancer worldwide, which is usually caused by the interaction between environmental and genetic factors, or epigenetic aspects. Referring to the non-coding RNAs, miR-128b has been reported to be associated with many tumour cases, and exerts distinct functions in different types of cancers. However, the function of miR-128b in GC onset and progression largely remains unknown. In the present study, we found that miR-128b expression was down-regulated in tissues from 18 GC patients and 3 carcinoma cell lines. In turn, over-expression of miR-128b suppressed GC cell proliferation, invasion and promoted apoptosis. Moreover, miR-128b was predicted to bind the 3'UTR of PDK1 gene using bioinformatic target-screening tools. Accordingly, ectopic expression of miR-128b inhibited the PDK1 expression at both transcriptional and post-transcriptional levels, and furthermore, the expression of gene tailed by the 3′UTR of PDK1 gene was significantly decreased in a dualluciferase reporter assay, suggesting that PDK1 was a direct target of miR-128b in GC cells. In the conditon of miR-128b over-expression, we also observed spontaneous inactivation of the Akt/NF-κB signalling, implying PDK1 was a potential regulator of this pathway. In conclusion, our study shed some novel light on miR-128b-PDK1/Akt/NF-κB axis on GC progression.
Kaarniranta, Kai; Kauppinen, Anu; Blasiak, Janusz; Salminen, Antero
Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly in the developed countries. The number of AMD patients will double during the next decades due to increasing number of aged people. Chronic oxidative stress, inflammation and accumulation of protein-rich deposits both in the retinal pigment epithelium lysosomes and under the retinal pigment epithelium herald the onset of AMD. The disease can be divided into dry and wet AMD forms. The dry form of the disease is more prevalent accounting for up to 90% of all cases. Continued intraocular injections are the current treatment strategy to prevent progression of wet AMD. It is a major challenge to develop new drugs that could prevent or at least ease the symptoms of the increasing population of AMD patients. Since AMD pathology is clearly associated with accumulated protein deposits, the autophagy clearance system might represent a potential future therapeutic target for AMD as is thoroughly discussed here.
Fujisawa, Masaki; Nakano, Toshitsugu; Shima, Yoko; Ito, Yasuhiro
The fruit ripening developmental program is specific to plants bearing fleshy fruits and dramatically changes fruit characteristics, including color, aroma, and texture. The tomato (Solanum lycopersicum) MADS box transcription factor RIPENING INHIBITOR (RIN), one of the earliest acting ripening regulators, is required for both ethylene-dependent and -independent ripening regulatory pathways. Recent studies have identified two dozen direct RIN targets, but many more RIN targets remain to be identified. Here, we report the large-scale identification of direct RIN targets by chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) targeting the predicted promoters of tomato genes. Our combined ChIP-chip and transcriptome analysis identified 241 direct RIN target genes that contain a RIN binding site and exhibit RIN-dependent positive or negative regulation during fruit ripening, suggesting that RIN has both activator and repressor roles. Examination of the predicted functions of RIN targets revealed that RIN participates in the regulation of lycopene accumulation, ethylene production, chlorophyll degradation, and many other physiological processes. Analysis of the effect of ethylene using 1-methylcyclopropene revealed that the positively regulated subset of RIN targets includes ethylene-sensitive and -insensitive transcription factors. Intriguingly, ethylene is involved in the upregulation of RIN expression during ripening. These results suggest that tomato fruit ripening is regulated by the interaction between RIN and ethylene signaling.
Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs which can function as oncogenes or tumor suppressor genes in human cancers. Emerging evidence reveals that deregulation of miRNAs contributes to the human non-small cell lung cancer (NSCLC. In the present study, we demonstrated that the expression levels of miR-132 were dramatically decreased in examined NSCLC cell lines and clinical NSCLC tissue samples. Then, we found that introduction of miR-132 significantly suppressed the migration and invasion of lung cancer cells in vitro, suggesting that miR-132 may be a novel tumor suppressor. Further studies indicated that the EMT-related transcription factor ZEB2 was one direct target genes of miR-132, evidenced by the direct binding of miR-132 with the 3' untranslated region (3' UTR of ZEB2. Further, miR-132 could decrease the expression of ZEB2 at the levels of mRNA and protein. Notably, the EMT marker E-cadherin or vimentin, a downstream of ZEB2, was also down-regulated or up-regulated upon miR-132 treatment. Additionally, over-expressing or silencing ZEB2 was able to elevate or inhibit the migration and invasion of lung cancer cells, parallel to the effect of miR-132 on the lung cancer cells. Meanwhile, knockdown of ZEB2 reversed the enhanced migration and invasion mediated by anti-miR-132. These results indicate that miR-132 suppresses the migration and invasion of NSCLC cells through targeting ZEB2 involving the EMT process. Thus, our finding provides new insight into the mechanism of NSCLC progression. Therapeutically, miR-132 may serve as a potential target in the treatment of human lung cancer.
Robert J Lake
Full Text Available Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome.
Yu, Xiaoyun; An, Jingang; Hua, Yunhui; Li, Zihai; Yan, Ning; Fan, Weixin; Su, Chuan
MicroRNAs (miRNAs) are currently emerged as important regulators in psoriasis. Psoriasis is characterized by hyperproliferation and impaired differentiation of keratinocytes in skin lesions. miR-194 is a well-known regulator of cell proliferation and differentiation. However, the role of miR-194 in psoriasis pathogenesis remains unclear. In this study we aimed to investigate the role of miR-194 in keratinocyte hyperproliferation and differentiation. We found that miR-194 was significantly downregulated in psoriasis lesional skin. Overexpression of miR-194 inhibited the proliferation and promoted the differentiation of primary human keratinocytes, whereas miR-194 suppression promoted the proliferation and inhibited their differentiation. Bioinformatic analysis predicted that the Grainyhead-like 2 (GRHL2) was a target gene of miR-194, which we further validated with a dual-luciferase reporter assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot analysis. The effect of miR-194 on cell proliferation and differentiation was significantly reversed by overexpression of GRHL2. Moreover, the expression of miR-194 and GRHL2 was inversely correlated in psoriasis lesional skin. Taken together, our results suggest that miR-194 inhibits the proliferation and promotes the differentiation of keratinocytes through targeting GRHL2. The downregulation of miR-194 expression may contribute to the pathogenesis of psoriasis and targeting miR-194 may represent a novel and potential therapeutic strategy for psoriasis. Copyright © 2016 Elsevier GmbH. All rights reserved.
Zhou, Chusong; Tan, Wei; Lv, Hai; Gao, Fei; Sun, Jin
Osteosarcoma is a malignant bone cancer of which the survival rate is still low. One reason for this low survival rate is drug resistance. In the past, it has been shown that microRNAs may play critical roles in osteosarcoma development and drug resistance. The mechanisms by which osteosarcoma cells acquire this resistance have, however, remained largely unknown. Here, we aimed at assessing the role of microRNA-488 in the acquisition of drug resistance by osteosarcoma cells. Quantitative RT-PCR was used to measure the expression of microRNA-488 in primary osteosarcoma samples and in osteosarcoma-derived cells, whereas microRNA-488 mimics and inhibitors were used to modify its expression in these cells. Luciferase reporter, Western blotting, cell viability, apoptosis and ChIP assays were used to assess the various effects of modified microRNA-488 expression in osteosarcoma-derived cells. We found that microRNA-488 is over-expressed in primary osteosarcoma tissues and osteosarcoma-derived cells and that hypoxia can induce microRNA-488 expression via binding to the hypoxia response element (HRE) in its promoter. We also found that exogenous over-expression of microRNA-488 promotes the proliferation, reduces the apoptosis and decreases the sensitivity to chemotherapy (doxorubicin) of osteosarcoma cells via direct targeting of the tumor suppressor Bim, which is a mediator of apoptosis. In contrast, we found that transfection of a microRNA-488 inhibitor resulted in an increase in both apoptosis and drug sensitivity, and a decrease in proliferation. Our data suggest that miRNA-488 may serve as a predictor of response to chemotherapy and as a therapeutic target in human osteosarcomas.
Full Text Available Abstract Background Head and neck squamous cell carcinoma (HNSCC is a highly lethal cancer that contains cellular and functional heterogeneity. Previously, we enriched a subpopulation of highly tumorigenic head and neck cancer initiating cells (HN-CICs from HNSCC. However, the molecular mechanisms by which to govern the characteristics of HN-CICs remain unclear. GRP78, a stress-inducible endoplasmic reticulum chaperone, has been reported to play a crucial role in the maintenance of embryonic stem cells, but the role of GRP78 in CICs has not been elucidated. Results Initially, we recognized GRP78 as a putative candidate on mediating the stemness and tumorigenic properties of HN-CICs by differential systemic analyses. Subsequently, cells with GRP78 anchored at the plasma membrane (memGRP78+ exerted cancer stemness properties of self-renewal, differentiation and radioresistance. Of note, xenotransplantation assay indicated merely 100 memGRP78+ HNSCCs resulted in tumor growth. Moreover, knockdown of GRP78 significantly reduced the self-renewal ability, side population cells and expression of stemness genes, but inversely promoted cell differentiation and apoptosis in HN-CICs. Targeting GRP78 also lessened tumorigenicity of HN-CICs both in vitro and in vivo. Clinically, co-expression of GRP78 and Nanog predicted the worse survival prognosis of HNSCC patients by immunohistochemical analyses. Finally, depletion of GRP78 in HN-CICs induced the expression of Bax, Caspase 3, and PTEN. Conclusions In summary, memGRP78 should be a novel surface marker for isolation of HN-CICs, and targeting GRP78 signaling might be a potential therapeutic strategy for HNSCC through eliminating HN-CICs.
Sun, Shuyang; Wang, Yilong; Zhou, Rong; Deng, Zicheng; Han, Yong; Han, Xiao; Tao, Wenjie; Yang, Zi; Shi, Chaoji; Hong, Duo; Li, Jiang; Shi, Donglu; Zhang, Zhiyuan
In precision cancer nanomedicine, the key is to identify the oncogenes that are responsible for tumorigenesis, based on which these genetic drivers can be each specifically regulated by a nanovector-directed, oncogene-targeted microRNA (miRNA) for tumor suppression. Fibroblast Growth Factor Receptor 3 (FGFR3) is such an oncogene. The molecular tumor-subtype harboring FGFR3 genomic alteration has been identified via genomic sequencing and referred to as the FGFR3-driven tumors. This genomics-based tumor classification provides further rationale for the development of the FGFR3-targeted miRNA replacement therapy in treating patients with FGFR3 gene abnormity. However, successful miRNA therapy has been hampered by lacking of an efficient delivery vehicle. In this study, a nanovector is developed for microRNA-100 (miR-100) -mediated FGFR3 regulation. The nanovector is composed of the mesoporous magnetic clusters that are conjugated with ternary polymers for efficient miRNA in-vivo delivery. The miRNA-loading capacity of the nanovector is found to be high due to the polycation polymer functionalized mesoporous structure, showing excellent tumor cell transfection and pH-sensitive miRNA release. Delivery of miR-100 to cancer cells effectively down-regulates the expression of FGFR3, inhibits cell proliferation, and induces cell apoptosis in vitro. Patient-derived xenografts (PDXs) are used to evaluate the efficacy of miRNA delivery in the FGFR3-driven tumors. Notably, sharp contrasts are observed between the FGFR3-driven tumors and those without FGFR3 genomic alteration. Only the FGFR3-driven PDXs are significantly inhibited via miR-100 delivery while the non-FGFR3-driven PDXs are not affected, showing promise of precision cancer nanomedicine. PMID:28255359
Wada, Takeyoshi [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Asahi, Toru [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Sawamura, Naoya, E-mail: email@example.com [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan)
The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.
Song, Wencong; Mu, Hailong; Wu, Jiang; Liao, Mingzhi; Zhu, Haijing; Zheng, Liming; He, Xin; Niu, Bowen; Zhai, Yuanxin; Bai, Chunling; Lei, Anmin; Li, Guangpeng; Hua, Jinlian
The balance between the self-renewal and differentiation of male germline stem cells (mGSCs) is critical for the initiation and maintenance of mammalian spermatogenesis. The promyelocytic leukemia zinc finger (PLZF), a zinc finger protein, is a critical factor for maintaining the self-renewal of mGSCs, so, evaluation of the PLZF pathway in mGSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for the self-renewal and differentiation of mGSCs; however, there is currently no data indicating that which miRNA regulate the self-renewal and differentiation of mGSCs via PLZF. Here, we predicted the prospective miRNA targeting to PLZF using the online Bioinformatics database-Targetscan, and performed an analysis of the dual-luciferase recombinant vector, psiCHCEKTM-2-PLZF-3'UTR. miR-544 mimics (miR-544m), miR-544 inhibitors (miR-544i), Control (NC, scrambled oligonucleotides transfection), pPLZF-IRES2-EGFP or PLZF siRNA were transfected into mGSCs; the cells proliferation was evaluated by BRDU incorporation assay and flow cytometry, and the mGSC marker, GFRa1, PLZF, KIT, DAZL, and VASA expression were analyzed by RT-qPCR, immunofluorescence and Western blot. The results showed that miR-544 regulates dairy goat male germline stem cell self-renewal via targeting PLZF. Our study identifies a new regulatory pathway for PLZF and expands upon the PLZF regulatory network in mGSCs.
Alanazi, Ibrahim O; Ebrahimie, Esmaeil
Novel computational systems biology tools such as common targets analysis, common regulators analysis, pathway discovery, and transcriptomic-based hotspot discovery provide new opportunities in understanding of apoptosis molecular mechanisms. In this study, after measuring the global contribution of microRNAs in the course of apoptosis by Affymetrix platform, systems biology tools were utilized to obtain a comprehensive view on the role of microRNAs in apoptosis process. Network analysis and pathway discovery highlighted the crosstalk between transcription factors and microRNAs in apoptosis. Within the transcription factors, PRDM1 showed the highest upregulation during the course of apoptosis, with more than 9-fold expression increase compared to non-apoptotic condition. Within the microRNAs, MIR1208 showed the highest expression in non-apoptotic condition and downregulated by more than 6 fold during apoptosis. Common regulators algorithm showed that TNF receptor is the key upstream regulator with a high number of regulatory interactions with the differentially expressed microRNAs. BCL2 and AKT1 were the key downstream targets of differentially expressed microRNAs. Enrichment analysis of the genomic locations of differentially expressed microRNAs led us to the discovery of chromosome bands which were highly enriched (p < 0.01) with the apoptosis-related microRNAs, such as 13q31.3, 19p13.13, and Xq27.3 This study opens a new avenue in understanding regulatory mechanisms and downstream functions in the course of apoptosis as well as distinguishing genomic-enriched hotspots for apoptosis process.
Full Text Available Ruilin Lei,1,2,* Min Xue,2,* Lan Zhang,1 ZhongQiu Lin1 1Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Obstetrics and Gynecology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 2Department of Obstetrics and Gynecology, Third Xiangya Hospital, Central South University, Changsha, China *These authors contributed equally to this work Abstract: MALAT1, an important cancer-associated long noncoding RNA (lncRNA, contributes to the development and progression of several cancers. Disordered expression of MALAT1 has been observed in several cancers, including cervical cancer, breast cancer, and ovarian cancer. However, the exact effects and molecular mechanisms of MALAT1 in ovarian cancer progression are still unknown. Here, we investigated the role of MALAT1 in human ovarian cancer cell lines and clinical tumor samples, in order to determine the function of this molecule. In our research, lncRNA-MALAT1 was specifically upregulated in ovarian cancer cell lines and promoted ovarian cancer-cell growth through targeting microRNA (miR-506. Knockdown of MALAT1 inhibited the proliferation and DNA synthesis of human ovarian cancer cell in vitro. In addition, miR-506-dependent iASPP regulation was required in MALAT1-induced ovarian cancer-cell growth. These findings indicated that MALAT1 might suppress tumor growth via miR-506-dependent iASPP regulation. Taken together, our data indicated that MALAT1 might be an oncogenic lncRNA that promotes proliferation of ovarian cancer and could be regarded as a therapeutic target in human ovarian cancer. Keywords: lncRNA, MALAT1, miR-506, ovarian cancer, iASPP
Full Text Available Changes in the developmental or metabolic state of plastids can trigger profound changes in the transcript profiles of nuclear genes. Many nuclear transcription factors were shown to be controlled by signals generated in the organelles. In addition to the many different compounds for which an involvement in retrograde signaling is discussed, accumulating evidence suggests a role for proteins in plastid-to-nucleus communication. These proteins might be sequestered in the plastids before they act as transcriptional regulators in the nucleus. Indeed, several proteins exhibiting a dual localization in the plastids and the nucleus are promising candidates for such a direct signal transduction involving regulatory protein storage in the plastids. Among such proteins, the nuclear transcription factor WHIRLY1 stands out as being the only protein for which an export from plastids and translocation to the nucleus has been experimentally demonstrated. Other proteins, however, strongly support the notion that this pathway might be more common than currently believed.
Na, So Young; Park, Mi-Jin; Park, Sun; Lee, Eun-So
The goal of this study was to investigate whether microRNA-155 might be a potential therapeutic target for Behçet's disease (BD). Expression levels of miR-155 were quantified using TaqMan microRNA assays in peripheral blood mononuclear cells and in isolated CD4+ T cells from BD patients and healthy controls. To identify targets of miR-155, luciferase reporter assays and western blotting were performed. The effect of miR-155 on Th17 polarisation was analysed in patients with active BD by using flow cytometry and enzyme-linked immunosorbent assay. The expression of miR-155 and IL-17 was significantly increased in CD4+ T cells of patients with active BD. A luciferase reporter assay and western blot showed that Ets-1 expression was reduced by miR-155 mimics. Furthermore, the expression of Ets-1 was significantly decreased in patients with active BD compared to healthy controls. More importantly, repression of miR-155 in CD4+ T cells from active BD patients increased Ets-1 expression and reduced the number of IL- 17-expressing T cells and overall IL-17 production. MiR-155 regulates the Th17 immune response by targeting Ets-1. Suppression of miR-155 reduced the amount of pathogenic IL-17-expressing T cells and may be a potential therapeutic strategy for BD.
Ehret, Georg B; Ferreira, Teresa; Chasman, Daniel I; Jackson, Anne U; Schmidt, Ellen M; Johnson, Toby; Thorleifsson, Gudmar; Luan, Jian'an; Donnelly, Lousie A; Kanoni, Stavroula; Petersen, Ann-Kristin; Pihur, Vasyl; Strawbridge, Rona J; Shungin, Dmitry; Hughes, Maria F; Meirelles, Osorio; Kaakinen, Marika; Bouatia-Naji, Nabila; Kristiansson, Kati; Shah, Sonia; Kleber, Marcus E; Guo, Xiuqing; Lyytikäinen, Leo-Pekka; Fava, Cristiano; Eriksson, Niclas; Nolte, Ilja M; Magnusson, Patrik K; Salfati, Elias L; Rallidis, Loukianos S; Theusch, Elizabeth; Smith, Andrew J P; Folkersen, Lasse; Witkowska, Kate; Pers, Tune H; Joehanes, Roby; Kim, Stuart K; Lataniotis, Lazaros; Jansen, Rick; Johnson, Andrew D; Warren, Helen; Kim, Young Jin; Zhao, Wei; Wu, Ying; Tayo, Bamidele O; Bochud, Murielle; Absher, Devin; Adair, Linda S; Amin, Najaf; Arking, Dan E; Axelsson, Tomas; Baldassarre, Damiano; Balkau, Beverley; Bandinelli, Stefania; Barnes, Michael R; Barroso, Inês; Bevan, Stephen; Bis, Joshua C; Bjornsdottir, Gyda; Boehnke, Michael; Boerwinkle, Eric; Bonnycastle, Lori L; Boomsma, Dorret I; Bornstein, Stefan R; Brown, Morris J; Burnier, Michel; Cabrera, Claudia P; Chambers, John C; Chang, I-Shou; Cheng, Ching-Yu; Chines, Peter S; Chung, Ren-Hua; Collins, Francis S; Connell, John M; Döring, Angela; Dallongeville, Jean; Danesh, John; de Faire, Ulf; Delgado, Graciela; Dominiczak, Anna F; Doney, Alex S F; Drenos, Fotios; Edkins, Sarah; Eicher, John D; Elosua, Roberto; Enroth, Stefan; Erdmann, Jeanette; Eriksson, Per; Esko, Tonu; Evangelou, Evangelos; Evans, Alun; Fall, Tove; Farrall, Martin; Felix, Janine F; Ferrières, Jean; Ferrucci, Luigi; Fornage, Myriam; Forrester, Terrence; Franceschini, Nora; Duran, Oscar H Franco; Franco-Cereceda, Anders; Fraser, Ross M; Ganesh, Santhi K; Gao, He; Gertow, Karl; Gianfagna, Francesco; Gigante, Bruna; Giulianini, Franco; Goel, Anuj; Goodall, Alison H; Goodarzi, Mark O; Gorski, Mathias; Gräßler, Jürgen; Groves, Christopher; Gudnason, Vilmundur; Gyllensten, Ulf; Hallmans, Göran; Hartikainen, Anna-Liisa; Hassinen, Maija; Havulinna, Aki S; Hayward, Caroline; Hercberg, Serge; Herzig, Karl-Heinz; Hicks, Andrew A; Hingorani, Aroon D; Hirschhorn, Joel N; Hofman, Albert; Holmen, Jostein; Holmen, Oddgeir Lingaas; Hottenga, Jouke-Jan; Howard, Phil; Hsiung, Chao A; Hunt, Steven C; Ikram, M Arfan; Illig, Thomas; Iribarren, Carlos; Jensen, Richard A; Kähönen, Mika; Kang, Hyun; Kathiresan, Sekar; Keating, Brendan J; Khaw, Kay-Tee; Kim, Yun Kyoung; Kim, Eric; Kivimaki, Mika; Klopp, Norman; Kolovou, Genovefa; Komulainen, Pirjo; Kooner, Jaspal S; Kosova, Gulum; Krauss, Ronald M; Kuh, Diana; Kutalik, Zoltan; Kuusisto, Johanna; Kvaløy, Kirsti; Lakka, Timo A; Lee, Nanette R; Lee, I-Te; Lee, Wen-Jane; Levy, Daniel; Li, Xiaohui; Liang, Kae-Woei; Lin, Honghuang; Lin, Li; Lindström, Jaana; Lobbens, Stéphane; Männistö, Satu; Müller, Gabriele; Müller-Nurasyid, Martina; Mach, François; Markus, Hugh S; Marouli, Eirini; McCarthy, Mark I; McKenzie, Colin A; Meneton, Pierre; Menni, Cristina; Metspalu, Andres; Mijatovic, Vladan; Moilanen, Leena; Montasser, May E; Morris, Andrew D; Morrison, Alanna C; Mulas, Antonella; Nagaraja, Ramaiah; Narisu, Narisu; Nikus, Kjell; O'Donnell, Christopher J; O'Reilly, Paul F; Ong, Ken K; Paccaud, Fred; Palmer, Cameron D; Parsa, Afshin; Pedersen, Nancy L; Penninx, Brenda W; Perola, Markus; Peters, Annette; Poulter, Neil; Pramstaller, Peter P; Psaty, Bruce M; Quertermous, Thomas; Rao, Dabeeru C; Rasheed, Asif; Rayner, N William N W R; Renström, Frida; Rettig, Rainer; Rice, Kenneth M; Roberts, Robert; Rose, Lynda M; Rossouw, Jacques; Samani, Nilesh J; Sanna, Serena; Saramies, Jouko; Schunkert, Heribert; Sebert, Sylvain; Sheu, Wayne H-H; Shin, Young-Ah; Sim, Xueling; Smit, Johannes H; Smith, Albert V; Sosa, Maria X; Spector, Tim D; Stančáková, Alena; Stanton, Alice; Stirrups, Kathleen E; Stringham, Heather M; Sundstrom, Johan; Swift, Amy J; Syvänen, Ann-Christine; Tai, E-Shyong; Tanaka, Toshiko; Tarasov, Kirill V; Teumer, Alexander; Thorsteinsdottir, Unnur; Tobin, Martin D; Tremoli, Elena; Uitterlinden, Andre G; Uusitupa, Matti; Vaez, Ahmad; Vaidya, Dhananjay; van Duijn, Cornelia M; van Iperen, Erik P A; Vasan, Ramachandran S; Verwoert, Germaine C; Virtamo, Jarmo; Vitart, Veronique; Voight, Benjamin F; Vollenweider, Peter; Wagner, Aline; Wain, Louise V; Wareham, Nicholas J; Watkins, Hugh; Weder, Alan B; Westra, Harm-Jan; Wilks, Rainford; Wilsgaard, Tom; Wilson, James F; Wong, Tien Y; Yang, Tsun-Po; Yao, Jie; Yengo, Loic; Zhang, Weihua; Zhao, Jing Hua; Zhu, Xiaofeng; Bovet, Pascal; Cooper, Richard S; Mohlke, Karen L; Saleheen, Danish; Lee, Jong-Young; Elliott, Paul; Gierman, Hinco J; Willer, Cristen J; Franke, Lude; Hovingh, G Kees; Taylor, Kent D; Dedoussis, George; Sever, Peter; Wong, Andrew; Lind, Lars; Assimes, Themistocles L; Njølstad, Inger; Schwarz, Peter Eh; Langenberg, Claudia; Snieder, Harold; Caulfield, Mark J; Melander, Olle; Laakso, Markku; Saltevo, Juha; Rauramaa, Rainer; Tuomilehto, Jaakko; Ingelsson, Erik; Lehtimäki, Terho; Hveem, Kristian; Palmas, Walter; März, Winfried; Kumari, Meena; Salomaa, Veikko; Chen, Yii-Der I; Rotter, Jerome I; Froguel, Philippe; Jarvelin, Marjo-Riitta; Lakatta, Edward G; Kuulasmaa, Kari; Franks, Paul W; Hamsten, Anders; Wichmann, H-Erich; Palmer, Colin N A; Stefansson, Kari; Ridker, Paul M; Loos, Ruth J F; Chakravarti, Aravinda; Deloukas, Panos; Morris, Andrew P; Newton-Cheh, Christopher; Munroe, Patricia B
To dissect the genetic architecture of blood pressure and assess effects on target organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals were used for validation. We identified 66 blood pressure-associated loci, of which 17 were new; 15 harbored multiple distinct association signals. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target organ damage in multiple tissues but with minor effects in the kidney. Our findings expand current knowledge of blood pressure-related pathways and highlight tissues beyond the classical renal system in blood pressure regulation.
Chasman, Daniel I.; Jackson, Anne U.; Schmidt, Ellen M.; Johnson, Toby; Thorleifsson, Gudmar; Luan, Jian'an; Donnelly, Lousie A.; Kanoni, Stavroula; Petersen, Ann-Kristin; Pihur, Vasyl; Strawbridge, Rona J.; Shungin, Dmitry; Hughes, Maria F.; Meirelles, Osorio; Kaakinen, Marika; Bouatia-Naji, Nabila; Kristiansson, Kati; Shah, Sonia; Kleber, Marcus E.; Guo, Xiuqing; Lyytikäinen, Leo-Pekka; Fava, Cristiano; Eriksson, Niclas; Nolte, Ilja M.; Magnusson, Patrik K.; Salfati, Elias L.; Rallidis, Loukianos S.; Theusch, Elizabeth; Smith, Andrew J.P.; Folkersen, Lasse; Witkowska, Kate; Pers, Tune H.; Joehanes, Roby; Kim, Stuart K.; Lataniotis, Lazaros; Jansen, Rick; Johnson, Andrew D.; Warren, Helen; Kim, Young Jin; Zhao, Wei; Wu, Ying; Tayo, Bamidele O.; Bochud, Murielle; Absher, Devin; Adair, Linda S.; Amin, Najaf; Arking, Dan E.; Axelsson, Tomas; Baldassarre, Damiano; Balkau, Beverley; Bandinelli, Stefania; Barnes, Michael R.; Barroso, Inês; Bevan, Stephen; Bis, Joshua C.; Bjornsdottir, Gyda; Boehnke, Michael; Boerwinkle, Eric; Bonnycastle, Lori L.; Boomsma, Dorret I.; Bornstein, Stefan R.; Brown, Morris J.; Burnier, Michel; Cabrera, Claudia P.; Chambers, John C.; Chang, I-Shou; Cheng, Ching-Yu; Chines, Peter S.; Chung, Ren-Hua; Collins, Francis S.; Connell, John M.; Döring, Angela; Dallongeville, Jean; Danesh, John; de Faire, Ulf; Delgado, Graciela; Dominiczak, Anna F.; Doney, Alex S.F.; Drenos, Fotios; Edkins, Sarah; Eicher, John D.; Elosua, Roberto; Enroth, Stefan; Erdmann, Jeanette; Eriksson, Per; Esko, Tonu; Evangelou, Evangelos; Evans, Alun; Fall, Tove; Farrall, Martin; Felix, Janine F.; Ferrières, Jean; Ferrucci, Luigi; Fornage, Myriam; Forrester, Terrence; Franceschini, Nora; Duran, Oscar H. Franco; Franco-Cereceda, Anders; Fraser, Ross M.; Ganesh, Santhi K.; Gao, He; Gertow, Karl; Gianfagna, Francesco; Gigante, Bruna; Giulianini, Franco; Goel, Anuj; Goodall, Alison H.; Goodarzi, Mark O.; Gorski, Mathias; Gräßler, Jürgen; Groves, Christopher; Gudnason, Vilmundur; Gyllensten, Ulf; Hallmans, Göran; Hartikainen, Anna-Liisa; Hassinen, Maija; Havulinna, Aki S.; Hayward, Caroline; Hercberg, Serge; Herzig, Karl-Heinz; Hicks, Andrew A.; Hingorani, Aroon D.; Hirschhorn, Joel N.; Hofman, Albert; Holmen, Jostein; Holmen, Oddgeir Lingaas; Hottenga, Jouke-Jan; Howard, Phil; Hsiung, Chao A.; Hunt, Steven C.; Ikram, M. Arfan; Illig, Thomas; Iribarren, Carlos; Jensen, Richard A.; Kähönen, Mika; Kang, Hyun; Kathiresan, Sekar; Keating, Brendan J.; Khaw, Kay-Tee; Kim, Yun Kyoung; Kim, Eric; Kivimaki, Mika; Klopp, Norman; Kolovou, Genovefa; Komulainen, Pirjo; Kooner, Jaspal S.; Kosova, Gulum; Krauss, Ronald M.; Kuh, Diana; Kutalik, Zoltan; Kuusisto, Johanna; Kvaløy, Kirsti; Lakka, Timo A; Lee, Nanette R.; Lee, I-Te; Lee, Wen-Jane; Levy, Daniel; Li, Xiaohui; Liang, Kae-Woei; Lin, Honghuang; Lin, Li; Lindström, Jaana; Lobbens, Stéphane; Männistö, Satu; Müller, Gabriele; Müller-Nurasyid, Martina; Mach, François; Markus, Hugh S.; Marouli, Eirini; McCarthy, Mark I.; McKenzie, Colin A.; Meneton, Pierre; Menni, Cristina; Metspalu, Andres; Mijatovic, Vladan; Moilanen, Leena; Montasser, May E.; Morris, Andrew D.; Morrison, Alanna C.; Mulas, Antonella; Nagaraja, Ramaiah; Narisu, Narisu; Nikus, Kjell; O'Donnell, Christopher J.; O'Reilly, Paul F.; Ong, Ken K.; Paccaud, Fred; Palmer, Cameron D.; Parsa, Afshin; Pedersen, Nancy L.; Penninx, Brenda W.; Perola, Markus; Peters, Annette; Poulter, Neil; Pramstaller, Peter P.; Psaty, Bruce M.; Quertermous, Thomas; Rao, Dabeeru C.; Rasheed, Asif; Rayner, N William N.W.R.; Renström, Frida; Rettig, Rainer; Rice, Kenneth M.; Roberts, Robert; Rose, Lynda M.; Rossouw, Jacques; Samani, Nilesh J.; Sanna, Serena; Saramies, Jouko; Schunkert, Heribert; Sebert, Sylvain; Sheu, Wayne H.-H.; Shin, Young-Ah; Sim, Xueling; Smit, Johannes H.; Smith, Albert V.; Sosa, Maria X.; Spector, Tim D.; Stančáková, Alena; Stanton, Alice; Stirrups, Kathleen E.; Stringham, Heather M.; Sundstrom, Johan; Swift, Amy J.; Syvänen, Ann-Christine; Tai, E-Shyong; Tanaka, Toshiko; Tarasov, Kirill V.; Teumer, Alexander; Thorsteinsdottir, Unnur; Tobin, Martin D.; Tremoli, Elena; Uitterlinden, Andre G.; Uusitupa, Matti; Vaez, Ahmad; Vaidya, Dhananjay; van Duijn, Cornelia M.; van Iperen, Erik P.A.; Vasan, Ramachandran S.; Verwoert, Germaine C.; Virtamo, Jarmo; Vitart, Veronique; Voight, Benjamin F.; Vollenweider, Peter; Wagner, Aline; Wain, Louise V.; Wareham, Nicholas J.; Watkins, Hugh; Weder, Alan B.; Westra, Harm-Jan; Wilks, Rainford; Wilsgaard, Tom; Wilson, James F.; Wong, Tien Y.; Yang, Tsun-Po; Yao, Jie; Yengo, Loic; Zhang, Weihua; Zhao, Jing Hua; Zhu, Xiaofeng; Bovet, Pascal; Cooper, Richard S.; Mohlke, Karen L.; Saleheen, Danish; Lee, Jong-Young; Elliott, Paul; Gierman, Hinco J.; Willer, Cristen J.; Franke, Lude; Hovingh, G Kees; Taylor, Kent D.; Dedoussis, George; Sever, Peter; Wong, Andrew; Lind, Lars; Assimes, Themistocles L.; Njølstad, Inger; Schwarz, Peter EH.; Langenberg, Claudia; Snieder, Harold; Caulfield, Mark J.; Melander, Olle; Laakso, Markku; Saltevo, Juha; Rauramaa, Rainer; Tuomilehto, Jaakko; Ingelsson, Erik; Lehtimäki, Terho; Hveem, Kristian; Palmas, Walter; März, Winfried; Kumari, Meena; Salomaa, Veikko; Chen, Yii-Der I.; Rotter, Jerome I.; Froguel, Philippe; Jarvelin, Marjo-Riitta; Lakatta, Edward G.; Kuulasmaa, Kari; Franks, Paul W.; Hamsten, Anders; Wichmann, H.-Erich; Palmer, Colin N.A.; Stefansson, Kari; Ridker, Paul M; Loos, Ruth J.F.; Chakravarti, Aravinda; Deloukas, Panos; Morris, Andrew P.; Newton-Cheh, Christopher; Munroe, Patricia B.
To dissect the genetic architecture of blood pressure and assess effects on target-organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry and genotypes from an additional 140,886 individuals were used for validation. We identified 66 blood pressure loci, of which 17 were novel and 15 harbored multiple distinct association signals. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target-organ damage in multiple tissues, with minor effects in the kidney. Our findings expand current knowledge of blood pressure pathways and highlight tissues beyond the classic renal system in blood pressure regulation. PMID:27618452
Full Text Available Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome. TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5'-AGGTGTGAAGGTGTGA-3'. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq to further elucidate the
Shao-Ying Lu; Yan-Fang Sui; Zeng-Shan Li; Cheng-En Pan; Jing Ye; Wen-Yong Wang
AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene.METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-t. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations.RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells.The expression rate of EGFP in G418 screened cell line was 34.9±4.1%. 48 h after adding 1×10-7M retinoic acid, EGFP expression rate was 14.7±3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1×10-7M retinoic acid (P＜0.05, P=0.003, t=6.488).CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.
Castellanos, Raquel; Xie, Qing; Zheng, Deyou; Cvekl, Ales; Morrow, Bernice E
Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome). TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5'-AGGTGTGAAGGTGTGA-3'. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S) do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular
Lee, Junwoo; An, Sungkwan; Choi, Yeong Min; Jung, Jin Hyuk; Li, Li; Meng, Hong; Dong, Yinmao; Ahn, Kyu Joong; An, In-Sook; Bae, Seunghee
Nutlin-3a is a non-genotoxic, p53-activating, MDM2 inhibitor being investigated as an anticancer agent. Although Nutlin-3a selectively antagonizes the ubiquitin E3 ligase activity of MDM2, its efficacy is not entirely regulated by MDM2 levels in cancer cells. Here, we report that the cytotoxic effects of Nutlin-3a are regulated by TRIAD1 via a positive feedback loop with p53. We found that Nutlin-3a enhanced TRIAD1 transcription in a p53-dependent manner. Using in silico analysis and promoter luciferase assays, we demonstrated that p53-mediated transcription of TRIAD1 is mediated by a p53 consensus sequence in the TRIAD1 promoter region. Silencing TRIAD1 expression in wild-type p53 (p53(WT) ) cancer cells suppressed Nutlin-3a-mediated p53 activation and p53 target gene expression. These effects were enhanced in TRIAD1-overexpressing p53(WT) cancer cells, but not in p53-deficient cancer cells. Furthermore, TRIAD1 knockdown significantly reduced the growth inhibitory and cytotoxic effects of Nutlin-3a in p53(WT) cancer cells, as demonstrated by cell viability assays, cell cycle analysis, clonogenic growth, and soft-agar colony forming assays. Together, these data indicate that TRIAD1 regulates Nutlin-3a-mediated p53 activation and the cytotoxic activity of Nutlin-3a. J. Cell. Biochem. 9999: 1-8, 2016. © 2016 Wiley Periodicals, Inc.
Full Text Available ABSTRACT Gastric cancer is one of the most common malignancies worldwide; however, the molecular mechanism in tumorigenesis still needs exploration. BCL2L11 belongs to the BCL-2 family, and acts as a central regulator of the intrinsic apoptotic cascade and mediates cell apoptosis. Although miRNAs have been reported to be involved in each stage of cancer development, the role of miR-24 in GC has not been reported yet. In the present study, miR-24 was found to be up-regulated while the expression of BCL2L11 was inhibited in tumor tissues of GC. Studies from both in vitro and in vivo shown that miR-24 regulates BCL2L11 expression by directly binding with 3′UTR of mRNA, thus promoting cell growth, migration while inhibiting cell apoptosis. Therefore, miR-24 is a novel onco-miRNA that can be potential drug targets for future clinical use.
Feng, Yi; Wan, Mingxi
To discuss how the mitochondrion is involved in low intensity ultrasound induced apoptosis, HepG2 cells were irradiated by low intensity focused ultrasound (ISPTA = 3W/cm2, 1 min) and then cultured from 3-12 h post irradiation in the study. The morphological alteration was examined by light and fluorescent microscopy respectively. Cell viability and apoptosis were examined by trypan blue staining and flow cytometry with double staining of FITC-labelled Annexin-V/PI. Key proteins responded to irradiation were screened out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shotgun proteomic methods with Agilent 1100 HPLC-Chip-MS technology. Representative apoptotic morphological characteristics and increased percentage of apoptotic cells were achieved. Six important proteins (4 up-regulated and 2 down-regulated) were selected and analyzed. It revealed low intensity focused ultrasound could induce apoptosis in HepG2 cells and the US-induced apoptosis was mitochondria-dependent and caspases-dependent. Moreover, mitochondrial membrane permeability transition (MPT) is related to ultrasound induced apoptosis, but VDAC may be not the main MPT channel. Understanding it could help to assist the cancer therapy by regulating the MPT as the target.
Full Text Available Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR. The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3 or EAAT2 (SLC1A2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (IEAAT. EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable IEAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal IEAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.
Minh T N Le
Full Text Available MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain- and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.
Li, Zhi [Department of Human Anatomy and Histoembryology, College of Basic Medical Sciences, Jilin University (China); Li, Youjun, E-mail: firstname.lastname@example.org [Department of Human Anatomy and Histoembryology, College of Basic Medical Sciences, Jilin University (China); Wang, Nan; Yang, Lifeng; Zhao, Wei; Zeng, Xiandong [Central Hospital Affiliated to Shenyang Medical College (China)
miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.
Chen, Wen; Wang, Fangjuan; Zeng, Wen; Sun, Jun; Li, Li; Yang, Mingcan; Sun, Jiansen; Wu, Yangxiao; Zhao, Xiaohui; Zhu, Chuhong
Regulation of cellular response pattern to phosphorus ion (PI) is a new target for the design of tissue-engineered materials. Changing cellular response pattern to high PI can maintain monocyte/macrophage survival in TEBV and the signal of increasing PI can be converted by klotho to the adenosine signals through the regulation of energy metabolism in monocytes/macrophages.
Khanday, Imtiyaz; Das, Sanjukta; Chongloi, Grace L; Vijayraghavan, Usha
OsMADS1 controls rice (Oryza sativa) floral fate and organ development. Yet, its genome-wide targets and the mechanisms underlying its role as a transcription regulator controlling developmental gene expression are unknown. We identify 3112 gene-associated OsMADS1-bound sites in the floret genome. These occur in the vicinity of transcription start sites, within gene bodies, and in intergenic regions. Majority of the bound DNA contained CArG motif variants or, in several cases, only A-tracts. Sequences flanking the binding peak had a higher AT nucleotide content, implying that broader DNA structural features may define in planta binding. Sequences for binding by other transcription factor families like MYC, AP2/ERF, bZIP, etc. are enriched in OsMADS1-bound DNAs. Target genes implicated in transcription, chromatin remodeling, cellular processes, and hormone metabolism were enriched. Combining expression data from OsMADS1 knockdown florets with these DNA binding data, a snapshot of a gene regulatory network was deduced where targets, such as AP2/ERF and bHLH transcription factors and chromatin remodelers form nodes. We show that the expression status of these nodal factors can be altered by inducing the OsMADS1-GR fusion protein and present a model for a regulatory cascade where the direct targets of OsMADS1, OsbHLH108/SPT, OsERF034, and OsHSF24, in turn control genes such as OsMADS32 and OsYABBY5. This cascade, with other similar relationships, cumulatively contributes to floral organ development. Overall, OsMADS1 binds to several regulatory genes and, probably in combination with other factors, controls a gene regulatory network that ensures rice floret development. PMID:27457124
Goodliffe, Julie M; Cole, Michael D; Wieschaus, Eric
Background The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis. Results To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1. Conclusion Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications. PMID:17519021
Cole Michael D
Full Text Available Abstract Background The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis. Results To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1. Conclusion Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications.
Eskandani, Morteza; Vandghanooni, Somayeh; Barar, Jaleh; Nazemiyeh, Hossein; Omidi, Yadollah
Any dysfunctionality in maintaining the oxygen homeostasis by mammalian cells may elicit hypoxia/anoxia, which results in inescapable oxidative stress and possible subsequent detrimental impacts on certain cells/tissues with high demands to oxygen molecules. The ischemic damage in turn can trigger initiation of a number of diseases including organs ischemia, metabolic disorders, inflammatory diseases, different types of malignancies, and alteration in wound healing process. Thus, full comprehension of molecular mechanism(s) and cellular physiology of the oxygen homeostasis is the cornerstone of the mammalian cells metabolism, energetic pathways and health and disease conditions. An imbalance in oxygen content within the cellular microenvironment activates a cascade of molecular events that are often compensated, otherwise pathologic condition occurs through a complexed network of biomolecules. Hypoxia inducible factor-1 (HIF-1) plays a key transcriptional role in the adaptation of cell physiology in relation with the oxygen content within a cell. In this current study, we provide a comprehensive review on the molecular mechanisms of oxygen sensing and homeostasis and the impacts of HIF-1 in hypoxic/anoxic conditions. Moreover, different molecular and biochemical responses of the cells to the surrounding environment are discussed in details. Finally, modern technological approaches for targeting the hypoxia related proteins are articulated. Copyright © 2017. Published by Elsevier B.V.
Full Text Available Resistance to cytotoxic drugs is thought to be a major cause of treatment failure in childhood neuroblastoma, and members of the ATP-binding cassette (ABC transporter superfamily may contribute to this phenomenon by active efflux of chemotherapeutic agents from cancer cells. As a member of the C subfamily of ABC transporters, multidrug resistance-associated protein MRP4/ABCC4 has the ability to export a variety of endogenous and exogenous substances across the plasma membrane. In light of its capacity for chemotherapeutic drug efflux, MRP4 has been studied in the context of drug resistance in a number of cancer cell types. However, MRP4 also influences cancer cell biology independently of chemotherapeutic drug exposure, which highlights the potential importance of endogenous MRP4 substrates in cancer biology. Furthermore, MRP4 is a direct transcriptional target of Myc family oncoproteins and expression of this transporter is a powerful independent predictor of clinical outcome in neuroblastoma. Together these features suggest that inhibition of MRP4 may be an attractive therapeutic approach for neuroblastoma and other cancers that rely on MRP4. In this respect, existing options for MRP4 inhibition are relatively non-selective and thus development of more specific anti-MRP4 compounds should be a major focus of future work in this area.
Majidinia, Maryam; Yousefi, Bahman
The inability of cancer cells in taking care of DNA damages can lead to cancer development and/or progression. Due to the essential role of DNA repair in maintaining genomic stability, tightly controlled regulatory mechanism are required for these processes. Recent studies have shown a myriad of interactions among DNA damage response (DDR) components and miRNAs. While DDR modulates miRNA expression in transcriptional and post-transcriptional levels and affects miRNA degradation, miRNAs in turn, directly modulate the expression of multiple proteins in the DDR pathways, or indirectly fine-tune the expression of such proteins. A better understanding of DDR-miRNA interactions can facilitate the development of new anticancer agents targeting miRNAs involved in the DNA repair process. In this review, we provide a brief introduction about miRNA biogenesis and functions, DDR pathways, and recent findings about DDR-microRNA interactions. Finally, the therapeutic importance of miRNAs in modulation of DDR/DNA repair mechanisms will be discussed.
Full Text Available Obesity is associated with a low-grade systemic chronic inflammatory state, characterized by the abnormal production of pro- and anti-inflammatory adipocytokines. It has been found that immune cells such as macrophages can infiltrate adipose tissue and are responsible for the majority of inflammatory cytokine production. Obesity-induced inflammation is considered a potential mechanism linking obesity to its related pathologies, such as insulin resistance, cardiovascular diseases, type-2 diabetes, and some immune disorders. Therefore, targeting obesity-related inflammatory components may be a useful strategy to prevent or ameliorate the development of such obesity-related diseases. It has been shown that several food components can modulate inflammatory responses in adipose tissue via various mechanisms, some of which are dependent on peroxisome proliferator-activated receptor γ (PPARγ, whereas others are independent on PPARγ, by attenuating signals of nuclear factor-κB (NF-κB and/or c-Jun amino-terminal kinase (JNK. In this review, we introduce the beneficial effects of anti-inflammatory phytochemicals that can help prevent obesity-induced inflammatory responses and pathologies.
Leonard, Siobhán; Kinsella, Gemma K.; Benetti, Elisa; Findlay, John B. C.
Type 2 diabetes is a chronic metabolic disorder primarily caused by insulin resistance to which obesity is a major contributor. Expression levels of an orphan G protein-coupled receptor (GPCR), GPR21, demonstrated a trend towards a significant increase in the epididymal fat pads of wild type high fat high sugar (HFHS)-fed mice. To gain further insight into the potential role this novel target may play in the development of obesity-associated type 2 diabetes, the signalling capabilities of the receptor were investigated. Overexpression studies in HEK293T cells revealed GPR21 to be a constitutively active receptor, which couples to Gαq type G proteins leading to the activation of mitogen activated protein kinases (MAPKs). Overexpression of GPR21 in vitro also markedly attenuated insulin signalling. Interestingly, the effect of GPR21 on the MAPKs and insulin signalling was reduced in the presence of serum, inferring the possibility of a native inhibitory ligand. Homology modelling and ligand docking studies led to the identification of a novel compound that inhibited GPR21 activity. Its effects offer potential as an anti-diabetic pharmacological strategy as it was found to counteract the influence of GPR21 on the insulin signalling pathway.
Bazin, Jérémie; Langlade, Nicolas; Vincourt, Patrick; Arribat, Sandrine; Balzergue, Sandrine; El-Maarouf-Bouteau, Hayat; Bailly, Christophe
After-ripening is the mechanism by which dormant seeds become nondormant during their dry storage after harvest. The absence of free water in mature seeds does not allow detectable metabolism; thus, the processes associated with dormancy release under these conditions are largely unknown. We show here that sunflower (Helianthus annuus) seed alleviation of dormancy during after-ripening is associated with mRNA oxidation and that this oxidation is prevented when seeds are maintained dormant. In vitro approaches demonstrate that mRNA oxidation results in artifacts in cDNA-amplified fragment length polymorphim analysis and alters protein translation. The oxidation of transcripts is not random but selective, and, using microarrays, we identified 24 stored mRNAs that became highly oxidized during after-ripening. Oxidized transcripts mainly correspond to genes involved in responses to stress and in cell signaling. Among them, protein phosphatase 2C PPH1, mitogen-activated protein kinase phosphatase 1, and phenyl ammonia lyase 1 were identified. We propose that targeted mRNA oxidation during dry after-ripening of dormant seeds could be a process that governs cell signaling toward germination in the early steps of seed imbibition.
Yanfen Liu; Yihong Ye
To deal with the constant challenge of protein misfolding in the endoplasmic reticulum (ER), eukaryotic cells have evolved an ER protein quality control (ERQC) mechanism that is integrated with an adaptive stress response. The ERQC pathway is comprised of factors residing in the ER lumen that function in the identification and retention of aberrantly folded proteins, factors in the ER membrane for retrotranslocation of misfolded polypeptides, and enzymes in the cytosol that degrade retrotranslocated proteins. The integrated stress response (termed ER stress or unfolded protein response, UPR) contains several signaling branches elicited from the ER membrane, which fine-tune the rate of protein synthesis and entry into the ER to match the ER folding capacity. The fitness of the cell, particularly those bearing a high secretory burden, is critically dependent on functional integrity of the ER, which in turn relies on these stress-attenuating mechanisms to maintain protein homeostasis, or proteostasis. Aberrant proteostasis can trigger cellular apoptosis, making these adaptive stress response systems attractive targets for perturbation in treatment of cell malignancies. Here, we review our current understanding of how the cell preserves ER proteostasis and discuss how we may harness the mechanistic information on this process to develop new cancer therapeutics.
Pedraza-Alva, Gustavo; Lilia, B. Mérida; del Rio, Roxana; Nora, A. Fierro; Cruz-Muñoz, Mario E.; Olivares, Norma; Melchy, Erika; Igras, Vivian; Georg, A. Holländer; Steven, J. Burakoff; Rosenstein, Yvonne
SUMMARY T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and co-receptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Co-receptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of TCs by regulating TCR signals. In this report we show that TC pre-stimulation by the CD43 co-receptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ–dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced MAPK activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function. PMID:21905200
Gutteridge, Rosie Elizabeth Ann; Singh, Chandra K; Ndiaye, Mary Ann; Ahmad, Nihal
A limited number of studies have indicated an association of the mitotic kinase polo-like kinase 1 (PLK1) and cellular metabolism. Here, employing an inducible RNA interference approach in A375 melanoma cells coupled with a PCR array and multiple validation approaches, we demonstrated that PLK1 alters a number of genes associated with cellular metabolism. PLK1 knockdown resulted in a significant downregulation of IDH1, PDP2 and PCK1 and upregulation of FBP1. Ingenuity Pathway Analysis (IPA) identified that 1) glycolysis and the pentose phosphate pathway are major canonical pathways associated with PLK1, and 2) PLK1 inhibition-modulated genes were largely associated with cellular proliferation, with FBP1 being the key modulator. Further, BI 6727-mediated inhibition of PLK1 caused a decrease in PCK1 and increase in FBP1 in A375 melanoma cell implanted xenografts in vivo. Furthermore, an inverse correlation between PLK1 and FBP1 was found in melanoma cells, with FBP1 expression significantly downregulated in a panel of melanoma cells. In addition, BI 6727 treatment resulted in an upregulation in FBP1 in A375, Hs294T and G361 melanoma cells. Overall, our study suggests that PLK1 may be an important regulator of metabolism maintenance in melanoma cells. Published by Elsevier B.V.
Gabriely, Galina; Wurdinger, Thomas; Kesari, Santosh; Esau, Christine C; Burchard, Julja; Linsley, Peter S; Krichevsky, Anna M
Substantial data indicate that microRNA 21 (miR-21) is significantly elevated in glioblastoma (GBM) and in many other tumors of various origins. This microRNA has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis, and migration. We demonstrate that miR-21 regulates multiple genes associated with glioma cell apoptosis, migration, and invasiveness, including the RECK and TIMP3 genes, which are suppressors of malignancy and inhibitors of matrix metalloproteinases (MMPs). Specific inhibition of miR-21 with antisense oligonucleotides leads to elevated levels of RECK and TIMP3 and therefore reduces MMP activities in vitro and in a human model of gliomas in nude mice. Moreover, downregulation of miR-21 in glioma cells leads to decreases of their migratory and invasion abilities. Our data suggest that miR-21 contributes to glioma malignancy by downregulation of MMP inhibitors, which leads to activation of MMPs, thus promoting invasiveness of cancer cells. Our results also indicate that inhibition of a single oncomir, like miR-21, with specific antisense molecules can provide a novel therapeutic approach for "physiological" modulation of multiple proteins whose expression is deregulated in cancer.
Hontelez, Saartje; Karthaus, Nina; Looman, Maaike W; Ansems, Marleen; Adema, Gosse J
Dendritic cells (DCs) play a central role in the immune system; they can induce immunity or tolerance depending on diverse factors in the DC environment. Pathogens, but also tissue damage, hormones, and vitamins, affect DC activation and maturation. In particular, glucocorticoids (GCs) are known for their immunosuppressive effect on DCs, creating tolerogenic DCs. GCs activate the type I nuclear receptor (NR) glucocorticoid receptor (GR), followed by induced expression of the transcription factor glucocorticoid-inducible leucine zipper (GILZ). GILZ has been shown to be necessary and sufficient for GC-induced tolerogenic DC generation. Recently, we have identified the DC-specific transcript (DC-SCRIPT) as an NR coregulator, suppressing type I steroid NRs estrogen receptor and progesterone receptor. In this study, we analyzed the effect of DC-SCRIPT on GR activity. We demonstrate that DC-SCRIPT coexists with GR in protein complexes and functions as a corepressor of GR-mediated transcription. Coexpression of DC-SCRIPT and GR is shown in human monocyte-derived DCs, and DC-SCRIPT knockdown enhances GR-dependent upregulation of GILZ mRNA expression in DCs. This demonstrates that DC-SCRIPT serves an important role in regulating GR function in DCs, corepressing GR-dependent upregulation of the tolerance-inducing transcription factor GILZ. These data imply that by controlling GR function and GILZ expression DC-SCRIPT is potentially involved in the balance between tolerance and immunity.
Renna, Megan E.; Quintero, Jean M.; Fresco, David M.; Mennin, Douglas S.
“Distress disorders,” which include generalized anxiety disorder and major depression are often highly comorbid with each other and appear to be characterized by common temperamental features that reflect heightened sensitivity to underlying motivational systems related to threat/safety and reward/loss. Further, individuals with distress disorders tend to utilize self-referential processes (e.g., worry, rumination, self-criticism) in a maladaptive attempt to respond to motivationally relevant distress, often resulting in suboptimal contextual learning. Despite the success of cognitive behavioral therapies for emotional disorders, a sizable subgroup of patients with distress disorders fail to evidence adequate treatment response. Emotion Regulation Therapy (ERT) is a theoretically derived, evidence based, treatment that integrates principles (e.g., skills training, exposure) from traditional and contemporary therapies with findings from basic and translational affective science to offer a framework for improving intervention by focusing on the motivational responses and corresponding regulatory characteristics of individuals with high levels of chronic distress. Open and randomized controlled trials have demonstrated preliminary support for the utility of ERT as reflected by strong effect sizes comparable to and exceeding established intervention approaches. In addition, pilot findings support the role of underlying proposed mechanisms in this efficacious response. This article presents the functional model associated with ERT and describes the proposed mechanisms of the treatment. Additionally, a clinical case is presented, allowing the reader to gain a greater applied understanding of the different components of the ERT model and treatment. PMID:28220089
Wang, Shufen; Liu, Jiali; Li, Xinqiang; Ji, Xiaowen; Zhang, Jianfang; Wang, Yue; Cui, Sheng
The establishment of the primordial follicle pool is crucial for fertility in mammalian females, and the interruption of overall micro-RNA production byDicer1conditional knockout in the female reproductive system results in infertility. However, there are few reports about the functions of individual micro-RNA in regulating primordial follicle assembly. The present study aimed to investigate the function of miR-125b, which is conserved and preferentially expressed in mammalian ovary during primordial follicle assembly. Detection of miR-125b in the developing mouse ovaries by real-time PCR and in situ hybridization showed that it was highly expressed perinatally and specifically located in the ovarian somatic cells. MiR-125b overexpression blocked the process of primordial follicle assembly in cultured newborn mouse ovaries, while its knockdown promoted this process. Further studies showed that miR-125b regulated the activin/Smad2 signaling in neonatal mouse ovary by directly targeting the 3'-untranslated region of activin receptor type 2a (Acvr2a). Overexpression of miR-125b in neonatal mouse ovary suppressed theAcvr2aprotein level, attenuating activin/Smad2 signaling, while knockdown of miR-125b showed the opposite effects. In addition, recombinant human activin A (rh-ActA) down-regulated miR-125b in the neonatal mouse ovary. Overexpression of miR-125b attenuated the promoting effects of rh-ActA on primordial follicle assembly. Taken together, these data suggest that miR-125b blocks the process of primordial follicle assembly, and miR-125b may play this role by regulating the expression ofAcvr2ain the activin/Smad2 signaling pathway. © 2016 by the Society for the Study of Reproduction, Inc.
Sun, Zhenzhen; Zhang, Zhan; Ji, Minghui; Yang, Hongbao; Cromie, Meghan; Gu, Jun; Wang, Chao; Yang, Lu; Yu, Yongquan; Gao, Weimin; Wang, Shou-Lin
Cytochrome P450 3A (CYP3A) is the most abundant CYP450 enzyme in the liver and is involved in the metabolism of over 50% of xenobiotics. Our previous studies revealed that 2,2‧,4,4‧-tetrabromodiphenyl ether (BDE47) could induce rat CYP3A1 expression, but the molecular basis remains unclear. Using in silico analysis, we identified a potential miR-23b recognition element (MRE23b) in the 3‧-UTR region of CYP3A1 mRNA, which was verified by the luciferase assay. The miR-23b mimic and inhibitor significantly down- and up-regulated the expression of CYP3A1, respectively. Additionally, BDE47 significantly down-regulated the expression of miR-23b in rats and in hepatic H4IIE cells. Induction or blockage of CYP3A1 by a miR-23b inhibitor or mimic could correspondingly alter BDE47-induced expression of CYP3A1 and cytotoxicity in H4IIE cells. Furthermore, LV-anti-miR-23b significantly decreased endogenous levels of miR-23b and increased the expression and activity of CYP3A1 in rat liver. LV-anti-miR-23b also significantly increased the hydroxylated metabolites of BDE47 (3-OH-BDE47, 4-OH-BDE42, and 4‧-OH-BDE49) in rat serum. In conclusion, we first found that BDE47 induced rat CYP3A1 expression by targeting the transcriptional regulation of miR-23b. This study helps provide a better understanding of CYP3A regulation and offers novel clues for the role of miRNAs in the metabolism and distribution of environmental pollutants.
Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Song, Sang Heon [Department of Internal Medicine, Pusan National University Hospital, Busan (Korea, Republic of); Shin, Hwa Kyoung [Department of Anatomy, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Bae, Sun Sik, E-mail: email@example.com [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of)
Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.
Full Text Available Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10 is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl; Nestin-Cre(tg/+. This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.
Ciribilli, Yari; Singh, Prashant; Inga, Alberto; Borlak, Jürgen
c-Myc's role in pulmonary cancer metabolism is uncertain. We therefore investigated c-Myc activity in papillary lung adenocarcinomas (PLAC). Genomics revealed 90 significantly regulated genes (> 3-fold) coding for cell growth, DNA metabolism, RNA processing and ribosomal biogenesis and bioinformatics defined c-Myc binding sites (TFBS) at > 95% of up-regulated genes. EMSA assays at 33 novel TFBS evidenced DNA binding activity and ChIP-seq data retrieved from public repositories confirmed these to be c-Myc bound. Dual-luciferase gene reporter assays developed for RNA-Terminal-Phosphate-Cyclase-Like-1(RCL1), Ribosomal-Protein-SA(RPSA), Nucleophosmin/Nucleoplasmin-3(NPM3) and Hexokinase-1(HK1) confirmed c-Myc functional relevance and ChIP assays with HEK293T cells over-expressing ectopic c-Myc demonstrated enriched c-Myc occupancy at predicted TFBS for RCL1, NPM3, HK1 and RPSA. Note, c-Myc recruitment on chromatin was comparable to the positive controls CCND2 and CDK4. Computational analyses defined master regulators (MR), i.e. heterogeneous nuclear ribonucleoprotein A1, nucleolin, the apurinic/apyrimidinic endonuclease 1, triosephosphate-isomerase 1, folate transporter (SLC19A1) and nucleophosmin to influence activity of up to 90% of PLAC-regulated genes. Their expression was induced by 3-, 3-, 6-, 3-, 11- and 7-fold, respectively. STRING analysis confirmed protein-protein-interactions of regulated genes and Western immunoblotting of fatty acid synthase, serine hydroxyl-methyltransferase 1, arginine 1 and hexokinase 2 showed tumor specific induction. Published knock down studies confirmed these proteins to induce apoptosis by disrupting neoplastic lipogenesis, by endorsing uracil accumulation and by suppressing arginine metabolism and glucose-derived ribonucleotide biosynthesis. Finally, translational research demonstrated high expression of MR and of 47 PLAC up-regulated genes to be associated with poor survival in lung adenocarcinoma patients (HR 3.2 p < 0.001) thus
Benlhabib, Houda; Guo, Wei; Pierce, Brianne M; Mendelson, Carole R
Type II cell differentiation and expression of the major surfactant protein, SP-A, in mid-gestation human fetal lung (HFL) are induced by cAMP and inhibited by TGF-β. cAMP induction of SP-A promoter activity is mediated by increased phosphorylation and DNA binding of thyroid transcription factor-1 (TTF-1/Nkx2.1), a master regulator of lung development. To further define mechanisms for developmental induction of surfactant synthesis in HFL, herein, we investigated the potential roles of microRNAs (miRNAs, miRs). To identify and characterize differentially regulated miRNAs in mid-gestation HFL explants during type II pneumocyte differentiation in culture, we performed miRNA microarray of RNA from epithelial cells isolated from mid-gestation HFL explants before and after culture with or without Bt2cAMP. Interestingly, the miR-200 family was significantly up-regulated during type II cell differentiation; miR-200 induction was inversely correlated with expression of known targets, transcription factors ZEB1/2 and TGF-β2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-β2 and ZEB1 expression in type II cells. Overexpression of ZEB1 in type II cells decreased DNA binding of endogenous TTF-1, blocked cAMP stimulation of surfactant proteins, and inhibited miR-200 expression, whereas cAMP markedly inhibited ZEB1/2 and TGF-β. Importantly, overexpression of ZEB1 or miR-200 antagonists in HFL type II cells also inhibited LPCAT1 and ABCA3, enzymes involved in surfactant phospholipid synthesis and trafficking, and blocked lamellar body biogenesis. Our findings suggest that the miR-200 family and ZEB1, which exist in a double-negative feedback loop regulated by TGF-β, serve important roles in the developmental regulation of type II cell differentiation and function in HFL. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Sorek, Nadav; Henis, Yoav I; Yalovsky, Shaul
Rho of Plants (ROP) small G proteins function at discrete domains of the plasma and possibly endo membranes. ROPs are synthesized as soluble proteins and their attachment to membranes and partitioning in membrane microdomains are facilitated by the posttranslational lipid modifications prenylation and/or S-acylation. Based on their amino acid sequences, ROPs can be classified into two major subgroups: type-I ROPs terminate with a canonical CaaX box motif and are prenylated primarily by geranylgeranyltransferase-I (GGT-I) and to a lesser extent by farnesyltransferase (FT). Type-II ROPs terminate with a plant specific GC-CG box domain and are attached to the plasma membrane by stable S-acylation. In addition, type-I and possibly also type-II ROPs undergo activation dependent transient S-acylation in the G-domain and consequent partitioning into lipid rafts. Surprisingly, although geranylgeranylation is required for the membrane attachment of type-I ROPs and the γ subunits of heterotrimeric G proteins, Arabidopsis mutants lacking GGT-I function have a mild phenotype compared to wild type plants. The mild phenotype of the ggt-I mutants suggested that farnesylation by FT may compensate for the loss of GGT-I function and that possibly the prenylated type-I and S-acylated type-II ROPS have some overlapping functions. In a paper recently published in Plant Physiology we examined the role of the prenyl group type in type-I ROP function and membrane interaction dynamics and the functional redundancy between type-I and type-II ROPs. This study complements a second paper in which we examined the role of G-domain transient S-acylation in the membrane interaction dynamics and signaling by type-I ROPs. Together these two studies provide a framework for realizing the role of prenylation and S-acylation in subcellular targeting, membrane interaction dynamics and signaling by ROP GTPases.
Mir, Hina; Kapur, Neeraj; Singh, Rajesh; Sonpavde, Guru; Lillard, James W; Singh, Shailesh
Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB(-/-)) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.
Yan, Yuhua; Guo, Feifei; Li, Jian; Hu, Yali; Zhou, Huaijun; Xun, Qingying
MicroRNA-30c (miR-30c) has been reported to be a tumour suppressor in endometrial cancer (EC). We demonstrate that miR-30c is down-regulated in EC tissue and is highly expressed in estrogen receptor (ER)-negative HEC-1-B cells. MiR-30c directly inhibits MTA-1 expression and functions as a tumour suppressor via the miR-30c-MTA-1 signalling pathway. Furthermore, miR-30c is decreased upon E2 treatment in both ER-positive Ishikawa and ER-negative HEC-1-B cells. Taken together, our results suggest that miR-30c is an important deregulated miRNA in EC and might serve as a potential biomarker and novel therapeutic target for EC. PMID:24595016
Sarachana, Tewarit; Hu, Valerie W
We have recently identified the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha) as a novel candidate gene for autism spectrum disorder (ASD). Our independent cohort studies have consistently demonstrated the reduction of RORA transcript and/or protein levels in blood-derived lymphoblasts as well as in the postmortem prefrontal cortex and cerebellum of individuals with ASD. Moreover, we have also shown that RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. However, little is known about transcriptional targets of this nuclear receptor, particularly in humans. Here we identify transcriptional targets of RORA in human neuronal cells on a genome-wide level using chromatin immunoprecipitation (ChIP) with an anti-RORA antibody followed by whole-genome promoter array (chip) analysis. Selected potential targets of RORA were then validated by an independent ChIP followed by quantitative PCR analysis. To further demonstrate that reduced RORA expression results in reduced transcription of RORA targets, we determined the expression levels of the selected transcriptional targets in RORA-deficient human neuronal cells, as well as in postmortem brain tissues from individuals with ASD who exhibit reduced RORA expression. The ChIP-on-chip analysis reveals that RORA1, a major isoform of RORA protein in human brain, can be recruited to as many as 2,764 genomic locations corresponding to promoter regions of 2,544 genes across the human genome. Gene ontology analysis of this dataset of genes that are potentially directly regulated by RORA1 reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion and survival, synaptogenesis, synaptic transmission and plasticity, and axonogenesis, as well as higher level functions such as
Li, Jindong; Feng, Qingchuan; Wei, Xudong; Yu, Yongkui
Lung cancer remains a leading cause of cancer-related mortality, with metastatic progression remaining the single largest cause of lung cancer mortality. Hence, it is imperative to determine reliable biomarkers of lung cancer prognosis. MicroRNA-490-3p has been previously reported to be a positive prognostic biomarker for hepatocellular cancer. However, its role in human lung cancer has not yet been elucidated. Here, we report that hsa-miR-490-3p expression is significantly higher in human lung cancer tissue specimens and cell line. Gain- and loss-of-function studies of hsa-miR-490-3p showed that it regulates cell proliferation and is required for induction of in vitro migration and invasion-the latter being a hallmark of epithelial to mesenchymal transition. In situ analysis revealed that hsa-miR-490-3p targets poly r(C)-binding protein 1 (PCBP1), which has been previously shown to be a negative regulator of lung cancer metastasis. Reporter assays confirmed PCBP1 as a bona fide target of miR-490-3p, and metagenomic analysis revealed an inverse relation between expression of miR-490-3p and PCBP1 in metastatic lung cancer patients. In fact, PCBP1 expression, as detected by immunohistochemistry, was undetectable in advanced stages of lung cancer patients' brain and lymph node tissues. Xenograft tail vein colonization assays proved that high expression of miR-490-3p is a prerequisite for metastatic progression of lung cancer. Our results suggest that hsa-miR-490-3p might be a potential biomarker for lung cancer prognosis. In addition, we can also conclude that the lung cancer cells have evolved refractory mechanisms to downregulate the expression of the metastatic inhibitor, PCBP1.
Giacoppo, Sabrina; Pollastro, Federica; Grassi, Gianpaolo; Bramanti, Placido; Mazzon, Emanuela
This study was aimed to investigate whether treatment with purified cannabidiol (CBD) may counteract the development of experimental multiple sclerosis (MS), by targeting the PI3K/Akt/mTOR pathway. Although the PI3K/Akt/mTOR pathway was found to be activated by cannabinoids in several immune and non-immune cells, currently, there is no data about the effects of CBD in the PI3K/Akt/mTOR activity in MS. Experimental Autoimmune Encephalomyelitis (EAE), the most common model of MS, was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55. After EAE onset, which occurs approximately 14days after disease induction, mice were daily intraperitoneally treated with CBD (10mg/kg mouse) and observed for clinical signs of EAE. At 28days from EAE-induction, mice were euthanized and spinal cord tissues were sampled to perform immunohistochemical evaluations and western blot analysis. Our results showed a clear downregulation of the PI3K/Akt/mTOR pathway following EAE induction. CBD treatment was able to restore it, increasing significantly the phosphorylation of PI3K, Akt and mTOR. Also, an increased level of BNDF in CBD-treated mice seems to be involved in the activation of PI3K/Akt/mTOR pathway. In addition, our data demonstrated that therapeutic efficacy of CBD treatment is due to reduction of pro-inflammatory cytokines, like IFN-γ and IL-17 together with an up-regulation of PPARγ. Finally, CBD was found to promote neuronal survival by inhibiting JNK and p38 MAP kinases. These results provide an interesting discovery about the regulation of the PI3K/Akt/mTOR pathway by cannabidiol administration, that could be a new potential therapeutic target for MS management.
Feng, Cheng; Bai, Ming; Yu, Nan-Ze; Wang, Xiao-Jun; Liu, Zeng
Our study aims to explore the role of microRNA-181b (miR-181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty-eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non-lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real-time quantitative fluorescence polymerase chain reaction (RT-qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR-181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non-lesional skin respectively. The 3' untranslated region (3'UTR) of TLR4 combined with miR-181b was verified by a dual-luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR-181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non-lesional skin. In psoriatic lesional and non-lesional skin, TLR4-positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual-luciferase reporter assay verified that miR-181b targets TLR4. HEKs transfected with miR-181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR-181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR-181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR-181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4.
Xu, Yun-qiang; Zhang, Zhen-hui; Zheng, Yong-fa; Feng, Shi-qing
A study of lumbar ligamentum flavum (LF). The aim of this study was to identify LF hypertrophy related microRNAs (miRNAs) expression profile and to investigate the role of miRNAs in the development of LF hypertrophy in lumbar spinal stenosis (LSS). Although histologic and biologic literature on LF hypertrophy is available, the pathomechanism is still unknown. Accumulating evidence suggests that microRNAs (miRNAs) participate in many physiologic processes, including cell proliferation, differentiation, and fibrosis, but the role of specific miRNAs involved in LF hypertrophy remains elusive. An initial screening of LF tissues miRNA expression by miRNA microarray was performed using samples from 10 patients and 10 controls, respectively. Subsequently, differential expression was validated using qRT-PCR. Then, functional analysis of the miRNAs in regulating collagens I and III expression was carried out. Western blotting and luciferase reporter assay were also used to detect the target gene. In addition, the thickness of the LF at the level of the facet joint was measured on axial T1-weighted magnetic resonance images. We identified 18 miRNAs that were differentially expressed in patients compared with controls. Following qRT-PCR confirmation, miR-221 was significantly lower in LF tissues of patients than controls. The LF was significantly thicker in patients than that in controls. Bioinformatics target prediction identified tissue inhibitors of matrix metalloproteinase (TIMP)-2 as a putative target of miR-221. Furthermore, luciferase reporter assays demonstrated that miR-221 directly targets TIMP-2 and affects the protein expression of TIMP-2 in fibroblasts isolated from LF. Of note, miR-221 mimic reduced mRNA and protein expression of collagens I and collagen III in fibroblasts isolated from LF. The downregulation of miR-221 might contribute to LF hypertrophy by promoting collagens I and III expression via the induction of TIMP-2. Our study also underscores the
Li, Xiangquan; Wang, Hao; Yao, Biao; Xu, Weiting; Chen, Jianchang; Zhou, Xiang
We previously established a rat model of diabetic cardiomyopathy (DCM) and found that the expression of lncRNA H19 was significantly downregulated. The present study was designed to investigate the pathogenic role of H19 in the development of DCM. Overexpression of H19 in diabetic rats attenuated oxidative stress, inflammation and apoptosis, and consequently improved left ventricular function. High glucose was associated with reduced H19 expression and increased cardiomyocyte apoptosis. To explore the molecular mechanisms involved, we performed in vitro experiments using cultured neonatal rat cardiomyocytes. Our results showed that miR-675 expression was decreased in cardiomyocytes transfected with H19 siRNA. The 3′UTR of VDAC1 was cloned downstream of a luciferase reporter construct and cotransfected into HEK293 cells with miR-675 mimic. The results of luciferase assay indicated that VDAC1 might be a direct target of miR-675. The expression of VDAC1 was upregulated in cardiomyocytes transfected with miR-675 antagomir, which consequently promotes cellular apoptosis. Moreover, enforced expression of H19 was found to reduce VDAC1 expression and inhibit apoptosis in cardiomyocytes exposed to high glucose. In conclusion, our study demonstrates that H19/miR-675 axis is involved in the regulation of high glucose-induced apoptosis by targeting VDAC1, which may provide a novel therapeutic strategy for the treatment of DCM. PMID:27796346
Genc, Ozgür; Kochubey, Olexiy; Toonen, Ruud F; Verhage, Matthijs; Schneggenburger, Ralf
Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a 'conventional' PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001.
Li, Minmin; Li, Mei; Yin, Tao; Shi, Huashan; Wen, Yuan; Zhang, Binglan; Chen, Meihua; Xu, Guangchao; Ren, Kexin; Wei, Yuquan
Cancer‑associated fibroblasts (CAFs), key components of the tumor stroma, can regulate tumorigenesis by altering the tumor microenvironment in variety of ways to promote angiogenesis, recruit inflammatory immune cells and remodel the extracellular matrix. Using a murine xenograft model of colon carcinoma, the present study observed that oxaliplatin increased the accumulation of CAFs and stimulated the production of cytokines associated with CAFs. When oxaliplatin was combined with the small‑molecule dipeptidyl peptidase inhibitor PT‑100, which inhibits CAFs by targeting fibroblast activation protein (FAP), the accumulation of CAFs was markedly reduced, xenograft tumor growth was significantly suppressed and the survival of the mice increased, compared to those of mice treated with oxaliplatin or PT‑100 alone. Furthermore, the xenograft tumor tissues of mice treated with oxaliplatin and PT‑100 contained lower numbers of tumor‑associated macrophages and dendritic cells, expressed lower levels of cytokines associated with CAFs and had a lower density of CD31+ endothelial cells. The present study demonstrated that pharmacological inhibition of CAFs improved the response to chemotherapy, reduced the recruitment of immune tumor‑promoting cells and inhibited angiogenesis. Combining chemotherapy with agents which target CAFs may represent a novel strategy for improving the efficacy of chemotherapy and reducing chemoresistance.
Full Text Available Fibrodysplasia ossificans progressiva (FOP is a rare congenital disorder of skeletal malformations and progressive extraskeletal ossification. There is still no effective treatment for FOP. All FOP individuals harbor conserved point mutations in ACVR1 gene that are thought to cause ACVR1 constitutive activation and activate BMP signal pathway. The constitutively active ACVR1 is also found to be able to cause endothelial-to-mesenchymal transition (EndMT in endothelial cells, which may cause the formation of FOP lesions. MicroRNAs (miRNAs play an essential role in regulating cell differentiation. Here, we verified that miR-148a directly targeted the 3' UTR of ACVR1 mRNA by reporter gene assays and mutational analysis at the miRNA binding sites, and inhibited ACVR1 both at the protein level and mRNA level. Further, we veriﬁed that miR-148a could inhibit the mRNA expression of the Inhibitor of DNA binding (Id gene family thereby suppressing the BMP signaling pathway. This study suggests miR-148a is an important mediator of ACVR1, thus offering a new potential target for the development of therapeutic agents against FOP.
Kruse, Michael; Keyhani-Nejad, Farnaz; Isken, Frank; Nitz, Barbara; Kretschmer, Anja; Reischl, Eva; de las Heras Gala, Tonia; Osterhoff, Martin A; Grallert, Harald; Pfeiffer, Andreas F H
Maternal obesity is a worldwide problem associated with increased risk of metabolic diseases in the offspring. Genetic deletion of the gastric inhibitory polypeptide (GIP) receptor (GIPR) prevents high-fat diet (HFD)-induced obesity in mice due to specific changes in energy and fat cell metabolism. We investigated whether GIP-associated pathways may be targeted by fetal programming and mimicked the situation by exposing pregnant mice to control or HFD during pregnancy (intrauterine [IU]) and lactation (L). Male wild-type (WT) and Gipr(-/-) offspring received control chow until 25 weeks of age followed by 20 weeks of HFD. Gipr(-/-) offspring of mice exposed to HFD during IU/L became insulin resistant and obese and exhibited increased adipose tissue inflammation and decreased peripheral tissue substrate utilization after being reintroduced to HFD, similar to WT mice on regular chow during IU/L. They showed decreased hypothalamic insulin sensitivity compared with Gipr(-/-) mice on control diet during IU/L. DNA methylation analysis revealed increased methylation of CpG dinucleotides and differential transcription factor binding of promoter regions of genes involved in lipid oxidation in the muscle of Gipr(-/-) offspring on HFD during IU/L, which were inversely correlated with gene expression levels. Our data identify GIP-regulated metabolic pathways that are targeted by fetal programming.
Liu, Zhe; Long, Jin; Du, Ruixia; Ge, Chunlin; Guo, Kejian; Xu, Yuanhong
miR-204 was found to be downregulated in gastric cancer (GC) tissues, and the effect of miR-204 function on gastric cancer remains as a mystery. Therefore, this study was aimed at investigating the potential role of miR-204 involved in GC progression. Tissues collected from 60 gastric cancer patients were selected as the case group, while the matched normal paracancer tissues as controls. miR-204 expression levels in tissues and GC cells were detected using real-time fluorescent quantitative PCR. Luciferase assay was adopted to validate the interaction between potential gene targets and miR-204. Transwell assay was performed to evaluate the metastasis of GC cells. By building the epithelial-mesenchymal transition (EMT) model in vitro through the addition of transforming growth factor beta 1 (TGF-β1), expressions of miR-204 and snai1 in the EMT model together with their respective effects on EMT were evaluated. miR-204 was significantly downregulated in GC tissues and invasive GC cells (P EMT process in EMT in vitro models. Our study provided evidence that miR-204 may suppress the metastasis and invasion of GC cells through the regulation of the EMT process by targeting snai1.
Full Text Available Let-7 is crucial for both stem cell differentiation and tumor suppression. Here, we demonstrate a chromatin-dependent mechanism of let-7 in regulating target gene expression in cancer cells. Let-7 directly represses the expression of AT-rich interacting domain 3B (ARID3B, ARID3A, and importin-9. In the absence of let-7, importin-9 facilitates the nuclear import of ARID3A, which then forms a complex with ARID3B. The nuclear ARID3B complex recruits histone demethylase 4C to reduce histone 3 lysine 9 trimethylation and promotes the transcription of stemness factors. Functionally, expression of ARID3B is critical for the tumor initiation in let-7-depleted cancer cells. An inverse association between let-7 and ARID3A/ARID3B and prognostic significance is demonstrated in head and neck cancer patients. These results highlight a chromatin-dependent mechanism where let-7 regulates cancer stemness through ARID3B.
Full Text Available Protein arginine methyl transferase 5 (Prmt5 regulates various differentiation processes, including adipogenesis. Here, we investigated adipogenic conversion in cells and mice in which Copr5, a Prmt5- and histone-binding protein, was genetically invalidated. Compared to control littermates, the retroperitoneal white adipose tissue (WAT of Copr5 KO mice was slightly but significantly reduced between 8 and 16 week/old and contained fewer and larger adipocytes. Moreover, the adipogenic conversion of Copr5 KO embryoid bodies (EB and of primary embryo fibroblasts (Mefs was markedly delayed. Differential transcriptomic analysis identified Copr5 as a negative regulator of the Dlk-1 gene, a Wnt target gene involved in the control of adipocyte progenitors cell fate. Dlk-1 expression was upregulated in Copr5 KO Mefs and the Vascular Stromal Fraction (VSF of Copr5 KO WAT. Chromatin immunoprecipitation (ChIP show that the ablation of Copr5 has impaired both the recruitment of Prmt5 and β-catenin at the Dlk-1 promoter. Overall, our data suggest that Copr5 is involved in the transcriptional control exerted by the Wnt pathway on early steps of adipogenesis.
Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya
The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin.
Inaba, Kenji; Masui, Shoji; Iida, Hiroka; Vavassori, Stefano; Sitia, Roberto; Suzuki, Mamoru
In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine-tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b'-domain through its substrate-binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.
Kwan, Kelvin Y; Shen, Jun; Corey, David P
Sensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Loram, Ian D; Bate, Brian; Harding, Pete; Cunningham, Ryan; Loram, Alison
While individual muscle function is known, the sensory and motor value of muscles within the whole-body sensorimotor network is complicated. Specifically, the relationship between neck muscle action and distal muscle synergies is unknown. This work demonstrates a causal relationship between regulation of the neck muscles and global motor control. Studying violinists performing unskilled and skilled manual tasks, we provided ultrasound feedback of the neck muscles with instruction to minimize neck muscle change during task performance and observed the indirect effect on whole-body movement. Analysis of ultrasound, kinematic, electromyographic and electrodermal recordings showed that proactive inhibition targeted at neck muscles had an indirect global effect reducing the cost of movement, reducing complex involuntary, task-irrelevant movement patterns and improving balance. This effect was distinct from the effect of gaze alignment which increased physiological cost and reduced laboratory-referenced movement. Neck muscle inhibition imposes a proximal constraint on the global motor plan, forcing a change in highly automated sensorimotor control. The proximal location ensures global influence. The criterion, inhibition of unnecessary action, ensures reduced cost while facilitating task-relevant variation. This mechanism regulates global motor function and facilitates reinforcement learning to change engrained, maladapted sensorimotor control associated with chronic pain, injury and performance limitation.
Min He; Zhenqun Xu; Tong Ding; Dong-Ming Kuang; Limin Zheng
Macrophages (Mψ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mψ to trigger transient activation of monocytes, but the underlying regulatory mechanisms are incompletely understood. Here, we showed that the protein expression of transcription factor C/EBPβ was markedly elevated in tumor-associated Mψ both in vitro and human tumors in situ. The expression of C/EBPβ protein correlated with cytokine production in tumor-activated monocytes. Moreover, we found that C/EBPβ expression was regulated at the post-transcriptional level and correlated with sustained reduction of microRNA-155 (miR-155) in tumor-activated monocytes. Bioinformatic analysis revealed that C/EBPβ is a potential target of miR-155 and luciferase assay confirmed that C/EBPβ translation is suppressed by miR-155 through interaction with the 3'UTR of C/EBPβ mRNA. Further analysis showed that induction of miR-155 suppressed C/EBPβ protein expression as well as cytokine production in tumor-activated monocytes, an effect which could be mimicked by silencing of C/EBPβ. These results indicate that tumor environment causes a sustained reduction of miR-155 in monocytes/Mψ, which in turn regulates the functional activities of monocytes/Mψ by releasing the translational inhibition of transcription factor C/EBPβ. Cellular & Molecular Immunology. 2009;6(5):343-352.
Zhu, Yong-Chuan; Li, Dan; Wang, Lu; Lu, Bin; Zheng, Jing; Zhao, Shi-Lin; Zeng, Rong; Xiong, Zhi-Qi
The X-linked gene cyclin-dependent kinase-like 5 (CDKL5) is mutated in severe neurodevelopmental disorders, including some forms of atypical Rett syndrome, but the function and regulation of CDKL5 protein in neurons remain to be elucidated. Here, we show that CDKL5 binds to the scaffolding protein postsynaptic density (PSD)-95, and that this binding promotes the targeting of CDKL5 to excitatory synapses. Interestingly, this binding is not constitutive, but governed by palmitate cycling on PSD-95. Furthermore, pathogenic mutations that truncate the C-terminal tail of CDKL5 diminish its binding to PSD-95 and synaptic accumulation. Importantly, down-regulation of CDKL5 by RNA interference (RNAi) or interference with the CDKL5–PSD-95 interaction inhibits dendritic spine formation and growth. These results demonstrate a critical role of the palmitoylation-dependent CDKL5–PSD-95 interaction in localizing CDKL5 to synapses for normal spine development and suggest that disruption of this interaction by pathogenic mutations may be implicated in the pathogenesis of CDKL5-related disorders. PMID:23671101
Zhang, Wenjun; Shen, Xiaojun; Xie, Luyang; Chu, Maoping; Ma, Yanmei
Interleukin 6 (IL-6) is a major pro-inflammatory cytokine and dysregulation of IL-6 is relevant to many inflammatory diseases. Endotoxin induced tolerance of IL-6 is an important mechanism to avoid the excessive immune reaction. But to date, the molecular mechanisms of endotoxin tolerance of IL-6 remain unclear. Here we reported that IL-6 secretion and microRNA-181b (miR-181b) expression were inversely correlated following LPS stimulation. We also demonstrated that miR-181b targeting the 3'-UTR of IL-6 transcripts and up-regulation of miR-181b was associated with NF-kB. We further demonstrated that up-regulation of miR-181b in response to LPS was required for inducing IL-6 tolerance in macrophage. Our results suggested that the post-transcriptional control mediated by miR-181b could be involved in fine tuning the critical level of IL-6 expression in endotoxin tolerance.
Full Text Available Cyclooxygenase-2 (COX-2 is known to promote the carcinogenesis of esophageal squamous cell carcinoma (ESCC. There are no reports on whether microRNAs (miRNAs regulate COX-2 expression in ESCC. This study investigated the effect of miR-101 on ESCC through modulating COX-2 expression in ESCC.Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR was used to quantify miR-101 expression in ESCC clinical tissues and cell lines. The effects of miR-101 on ESCC progression were evaluated by cell counting kit-8 (CCK8, transwell migration and invasion assays, as well as by flow cytometry. The COX-2 and PEG2 levels were determined by western blot and enzyme-linked immunosorbent assays (ELISA. The luciferase reporter assay was used to verify COX-2 as a direct target of miR-101. The anti-tumor activity of miR-101 in vivo was investigated in a xenograft nude mouse model of ESCC.Downregulation of miR-101 was confirmed through comparison of 30 pairs of ESCC tumor and adjacent normal tissues (P < 0.001, as well as in 11 ESCC cell lines and a human immortalized esophageal cell line (P < 0.001. Transfection of miR-101 in ESCC cell lines significantly suppressed cell proliferation, migration, and invasion (all P < 0.001. The antitumor effect of miR-101 was verified in a xenograft model. Furthermore, COX-2 was shown to be a target of miR-101.Overexpression of miR-101 in ESCC inhibits proliferation and metastasis. Therefore, the miR-101/COX-2 pathway might be a therapeutic target in ESCC.
Full Text Available Background: MicroRNAs (miRNAs have been demonstrated to play a crucial role in tumorigenesis. Previous studies have shown that miR-520b/e acts as a tumor suppressor in several tumors. Other studies indicated that epidermal growth factor receptor (EGFR is highly expressed in many tumors, and involved in the development of tumors, such as cell proliferation, migration, angiogenesis and apoptosis. However, the correlation of miRNAs and EGFR in gastric cancer (GC has not been adequately investigated. Our aim was to explore the relationship. Methods: The expression levels of EGFR and miR-520b/e were examined by RT-PCR and Western blot. We also investigated the relationship between EGFR and miR-520b/e in GC cell lines by relevant experiments. Results: In this study, we found that miR-520b/e inhibits the protein expression of EGFR by directly binding with the 3'-untranslated region (3'-UTR. And it was shown that the down-regulation of miR-520b/e promotes cell proliferation and migration by negative regulation of the EGFR pathway, while over-expression of miR-520b/e inhibits these properties. In addition, the biological function of EGFR in GC cell lines was validated by silencing and over-expression assays respectively. Conclusions: Taken together, our results demonstrate that miR-520b/e acts as a tumor suppressor by regulating EGFR in GC, and provide a novel marker and insight for the potential therapeutic target of GC.
Shen, Yang; Meng, Linyi; Sun, Huajun; Zhu, Yizhun; Liu, Hongrui
Cochinchina Momordica Seed, which is the dried ripe seed of Momordica cochinchinensis (Lour.) Spreng, has been used as a mainly anticancer ingredient for many years in China. This study aims at investigating the roles of an ethanol-soluble extract of Cochinchina Momordica Seed (ECMS) in suppressing the proliferation and metastasis of human lung cancer cells, and further elucidating underlying molecular mechanisms. Our researches suggest that ECMS dose-dependently decreased the survival rates of A549 and H1299 cells, and inhibited the migration and invasion in A549 cells. ECMS-induced apoptosis was accompanied by up-regulation of p53, Bax and the down-regulation of Bcl-2, PI-3K/Akt signal pathway, and resulted in the dissipation of mitochondrial membrane potential (ΔΨm) and sequentially activated caspase-3 cascade. Pre-treated with specific inhibitors, LY294002 (PI-3K inhibitor) and BAY11-7082 (NF-κB inhibitor) could enhance the anti-proliferation effects of ECMS on A549 cells. Furthermore, ECMS could increase the level of E-cadherin and decrease of the level of STAT-3 and MMP-2, and scarcely affected the expression of VEGF, and resulted in the inhibition of migration and invasion. Pre-treated with specific inhibitors, WP1066 (STAT-3 inhibitor) and TIMP-2 (MMP-2 inhibitor) could enhance the inhibitory effects of ECMS on migration. In conclusion, the current data demonstrated ECMS inhibited the proliferation of A549 cells by inducing apoptosis, at least partly through the activation of p53 and inactivation of PI-3K/Akt signaling. STAT-3 and MMP-2 pathways may be partly involved in anti-metastasis activities of ECMS. Hence, ECMS might be a promising candidate for the therapy of the non-small cell lung cancer by regulating multiple molecular targets.
Villanueva, Eneida C; Münzberg, Heike; Cota, Daniela; Leshan, Rebecca L; Kopp, Keely; Ishida-Takahashi, Ryoko; Jones, Justin C; Fingar, Diane C; Seeley, Randy J; Myers, Martin G
The medial basal hypothalamus, including the arcuate nucleus (ARC) and the ventromedial hypothalamic nucleus (VMH), integrates signals of energy status to modulate metabolism and energy balance. Leptin and feeding regulate the mammalian target of rapamycin complex 1 (mTORC1) in the hypothalamus, and hypothalamic mTORC1 contributes to the control of feeding and energy balance. To determine the mechanisms by which leptin modulates mTORC1 in specific hypothalamic neurons, we immunohistochemically assessed the mTORC1-dependent phosphorylation of ribosomal protein S6 (pS6). In addition to confirming the modulation of ARC mTORC1 activity by acute leptin treatment, this analysis revealed the robust activation of mTORC1-dependent ARC pS6 in response to fasting and leptin deficiency in leptin receptor-expressing Agouti-related protein neurons. In contrast, fasting and leptin deficiency suppress VMH mTORC1 signaling. The appropriate regulation of ARC mTORC1 by mutant leptin receptor isoforms correlated with their ability to suppress the activity of Agouti-related protein neurons, suggesting the potential stimulation of mTORC1 by the neuronal activity. Indeed, fasting- and leptin deficiency-induced pS6-immunoreactivity (IR) extensively colocalized with c-Fos-IR in ARC and VMH neurons. Furthermore, ghrelin, which activates orexigenic ARC neurons, increased ARC mTORC1 activity and induced colocalized pS6- and c-Fos-IR. Thus, neuronal activity promotes mTORC1/pS6 in response to signals of energy deficit. In contrast, insulin, which activates mTORC1 via the phosphatidylinositol 3-kinase pathway, increased ARC and VMH pS6-IR in the absence of neuronal activation. The regulation of mTORC1 in the basomedial hypothalamus thus varies by cell and stimulus type, as opposed to responding in a uniform manner to nutritional and hormonal perturbations.
van de Werken, C; Avo Santos, M; Laven, J S E; Eleveld, C; Fauser, B C J M; Lens, S M A; Baart, E B
Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote. Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described. In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways. The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins. Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the
Full Text Available Abstract Background Myofibrillogenesis regulator 1 (MR-1 is overexpressed in human cancer cells and plays an essential role in cancer cell growth. However, the significance of MR-1 in human ovarian cancer has not yet been explored. The aim of this study was to examine whether MR-1 is a predictor of ovarian cancer and its value as a therapeutic target in ovarian cancer patients. Methods Reverse-transcription polymerase chain reaction (PCR and quantitative real-time PCR were used to detect MR-1 mRNA levels in tissue samples from 26 ovarian cancer patients and 25 controls with benign ovarian disease. Anti-MR-1 polyclonal antibodies were prepared, tested by ELISA and western blotting, and then used for immunohistochemical analysis of the tissue samples. Adhesion and invasion of 292T cells was also examined after transfection of a pMX-MR-1 plasmid. Knockdown of MR-1 expression was achieved after stable transfection of SKOV3 cells with a short hairpin DNA pGPU6/GFP/Neo plasmid against the MR-1 gene. In addition, SKOV3 cells were treated with paclitaxel and carboplatin, and a potential role for MR-1 as a therapeutic target was evaluated. Results MR-1 was overexpressed in ovarian cancer tissues and SKOV3 cells. 293T cells overexpressed MR-1, and cellular spread and invasion were enhanced after transfection of the pMX-MR-1 plasmid, suggesting that MR-1 is critical for ovarian cancer cell growth. Knockdown of MR-1 expression inhibited cell adhesion and invasion, and treatment with anti-cancer drugs decreased its expression in cancer cells. Taken together, these results provide the first evidence of the cellular and molecular mechanisms by which MR-1 might serve as a novel biological marker and potential therapeutic target for ovarian cancer. Conclusions MR-1 may be a biomarker for diagnosis of ovarian cancer. It may also be useful for monitoring of the effects of anti-cancer therapies. Further studies are needed to clarify whether MR-1 is an early
Full Text Available Transient receptor potential cation channel 6 (TRPC6 is a nonselective cation channel, and abnormal expression and gain of function of TRPC6 are involved in the pathogenesis of hereditary and nonhereditary forms of renal disease. Although the molecular mechanisms underlying these diseases remain poorly understood, recent investigations revealed that many signaling pathways are involved in regulating TRPC6. We aimed to examine the effect of the mammalian target of rapamycin (mTOR complex (mTOR complex 1 [mTORC1] or mTOR complex 2 [mTORC2] signaling pathways on TRPC6 in podocytes, which are highly terminally differentiated renal epithelial cells that are critically required for the maintenance of the glomerular filtration barrier. We applied both pharmacological inhibitors of mTOR and specific siRNAs against mTOR components to explore which mTOR signaling pathway is involved in the regulation of TRPC6 in podocytes. The podocytes were exposed to rapamycin, an inhibitor of mTORC1, and ku0063794, a dual inhibitor of mTORC1 and mTORC2. In addition, specific siRNA-mediated knockdown of the mTORC1 component raptor and the mTORC2 component rictor was employed. The TRPC6 mRNA and protein expression levels were examined via real-time quantitative PCR and Western blot, respectively. Additionally, fluorescence calcium imaging was performed to evaluate the function of TRPC6 in podocytes. Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes. However, ku0063794 down-regulated the TRPC6 mRNA and protein levels and suppressed TRPC6-dependent calcium influx in podocytes. Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes. These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear
Hansen, A L; Refsgaard, J C; Olesen, J E; Børgesen, C D
Denmark must further decrease the N-load to coastal waters from agricultural areas to comply with the Baltic Sea Action Plan and the EU Water Framework Directive. A new spatially targeted regulation is under development that focuses on locating N-mitigation measures in areas with low natural reduction of nitrate (N-reduction). A key tool in this respect is N-reduction maps showing how much N is removed by natural reduction processes, i.e. the ratio between the N-load out of the catchment and the N-leaching from the root zone for each spatial unit within the catchment. For the 85 km(2) groundwater dominated Norsminde catchment in Denmark we have analysed the potential benefits of a spatially targeted regulation and how its efficiency is affected by uncertainty in the N-reduction map. Our results suggest that there are potential benefits of implementing a spatially targeted regulation compared to a spatially uniform regulation. The total N-load at the catchment outlet can be decreased up to 8% by relocating the existing agricultural practice according to the N-reduction map and thus without decrease fertilization inputs. A further decrease in N-load can be obtained by identifying target areas with low N-reduction where N-mitigation measures must be applied. Uncertainty on the N-reduction map is found to lower the efficiency of spatially targeted regulation. This uncertainty can be lowered substantially by using the mean of an ensemble of N-reduction maps. The uncertainty decreases with coarser spatial resolution of the N-reduction map, but this will at the same time decrease the benefit from spatially targeted regulation. Copyright © 2017 Elsevier B.V. All rights reserved.
Full Text Available Dysregulation of the sonic hedgehog (Shh signaling pathway has been associated with cancer stem cells (CSC and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN, an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4 as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway
Duan, Shanhong; Wu, Ali; Chen, Zhengyu; Yang, Yarong; Liu, Liying; Shu, Qi
MicroRNAs (miRNAs) are small non-coding RNAs that involved in human carcinogenesis and progression. miR-204 has been reported to be a tumor suppressor in several cancer types. However, the function and underlying molecular mechanism of miR-204 in cervical cancer (CC) are still unclear. In the present study, the expression level of miR-204 was measured using qRT-PCR method in 30 paired CC clinical samples and 6 CC cell lines. We found that the expression of miR-204 was significantly down-regulated in CC tissues and cell lines compared to normal cervical tissues and cell line. miR-204 was overexpressed by transfection with miR-204 mimic in Hela and C33A cell lines in the following experiments. The results showed that overexpression of miR-204 dramatically suppressed cell proliferation, migration, and invasion, caused cell cycle arrest at G0/G1, promoted cell apoptosis in vitro and inhibited tumor growth in vivo. Western blot results indicated that overexpressing of miR-204 decreased the expressions of CDK2, cyclinE, MMP2, MMP9, Bcl2, while enhanced Bax expression and suppressed the activation of P13K/AKT signaling pathways in CC cells. Ephrin (Eph) B2 was identified as a direct target of miR-204 in CC cells according to bioinformatics analysis and luciferase reporter assay. Furthermore, knockdown of EphB2 mimicked the inhibitory effect of miR-204 on the proliferation, invasion and migration of CC cells. These findings suggested that miR-204 might serve as a tumor suppressor in the development of CC by directly targeting EphB2.
Kelly, Daniel M; Akhtar, Samia; Sellers, Donna J; Muraleedharan, Vakkat; Channer, Kevin S; Jones, T Hugh
Testosterone deficiency is commonly associated with obesity, metabolic syndrome, type 2 diabetes and their clinical consequences-hepatic steatosis and atherosclerosis. The testicular feminised mouse (non-functional androgen receptor and low testosterone) develops fatty liver and aortic lipid streaks on a high-fat diet, whereas androgen-replete XY littermate controls do not. Testosterone treatment ameliorates these effects, although the underlying mechanisms remain unknown. We compared the influence of testosterone on the expression of regulatory targets of glucose, cholesterol and lipid metabolism in muscle, liver, abdominal subcutaneous and visceral adipose tissue. Testicular feminised mice displayed significantly reduced GLUT4 in muscle and glycolytic enzymes in muscle, liver and abdominal subcutaneous but not visceral adipose tissue. Lipoprotein lipase required for fatty acid uptake was only reduced in subcutaneous adipose tissue; enzymes of fatty acid synthesis were increased in liver and subcutaneous tissue. Stearoyl-CoA desaturase-1 that catalyses oleic acid synthesis and is associated with insulin resistance was increased in visceral adipose tissue and cholesterol efflux components (ABCA1, apoE) were decreased in subcutaneous and liver tissue. Master regulator nuclear receptors involved in metabolism-Liver X receptor expression was suppressed in all tissues except visceral adipose tissue, whereas PPARγ was lower in abdominal subcutaneous and visceral adipose tissue and PPARα only in abdominal subcutaneous. Testosterone treatment improved the expression (androgen receptor independent) of some targets but not all. These exploratory data suggest that androgen deficiency may reduce the buffering capability for glucose uptake and utilisation in abdominal subcutaneous and muscle and fatty acids in abdominal subcutaneous. This would lead to an overspill and uptake of excess glucose and triglycerides into visceral adipose tissue, liver and arterial walls.
Full Text Available Abstract Background The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. Results We find that loss of Brahma associated protein 55 kD (Bap55 results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM and the Tat interactive protein 60 kD (TIP60 ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. Conclusions Our results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions.
Gao, Jun; Wagnon, Jacy L; Protacio, Reine M; Glazko, Galina V; Beggs, Marjorie; Raj, Vinay; Davidson, Mari K; Wahls, Wayne P
Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, posttranscriptional stability and decay of sets of target RNAs. Whole transcriptome RNA sequencing data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced loss. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This stress-activated RNA decay (SARD) provides a mechanism for reducing the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli.
Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L; Klein, David C
MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.
Wang, Youning; Wang, Lixiang; Zou, Yanmin; Chen, Liang; Cai, Zhaoming; Zhang, Senlei; Zhao, Fang; Tian, Yinping; Jiang, Qiong; Ferguson, Brett J.; Gresshoff, Peter M.; Li, Xia
MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean. PMID:25549672
Sumiya, Eri; Ogino, Yukiko; Toyota, Kenji; Miyakawa, Hitoshi; Miyagawa, Shinichi; Iguchi, Taisen
Embryo development in arthropods is accompanied by a series of moltings. A cladoceran crustacean Daphnia magna molts three times before reaching first instar neonate during embryogenesis. Previous studies argued ecdysteroids might regulate D. magna embryogenesis. However, no direct evidence between innate ecdysteroids fluctuation and functions has been forthcoming. Recently, we identified genes involved in ecdysteroid synthesis called, neverland (neverland1 and neverland 2) and shade and in the ecdysteroid degradation (Cyp18a1). To understand the physiological roles of ecdysteroids in D. magna embryos, we performed expression and functional analyzes of those genes. Examining innate ecdysteroids titer during embryogenesis showed two surges of ecdysteroids titer at 41 and 61 h after oviposition. The first and second embryonic moltings occurred at each ecdysteroid surge. Expression of neverland1 and shade began to increase before the first peak in ecdysteroid. Knockdown of neverland1 or shade by RNAi technique caused defects in embryonic moltings and subsequent development. The ecdysteroids titer seemingly decreased in nvd1-knowckdown embryos. Knockdown of Cyp18a1 resulted in early embryonic lethality before the first molting. Our in situ hybridization analysis revealed that nvd1 was prominently expressed in embryonic gut epithelium suggesting the site for an initial step of ecdysteroidgenesis, a conversion of cholesterol to 7-dehydrocholesterol and possibly for ecdysone production. Taken together, de novo ecdysteroid synthesis by nvd1 in the gut epithelial cells stimulates molting, which is indispensable for D. magna embryo development. These findings identify neverland as a possible target for chemicals, including various pesticides that are known to disrupt molting, development and reproduction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Zuiderveen Borgesius, F.J.
Information about millions of people is collected for behavioural targeting, a type of marketing that involves tracking people's online behaviour for targeted advertising. It is hotly debated whether data protection law applies to behavioural targeting. Many behavioural targeting companies say that,
Zuiderveen Borgesius, F.J.
Information about millions of people is collected for behavioural targeting, a type of marketing that involves tracking people's online behaviour for targeted advertising. It is hotly debated whether data protection law applies to behavioural targeting. Many behavioural targeting companies say that,
Casenghi, Martina; Barr, Francis A; Nigg, Erich A
When cells enter mitosis the microtubule (MT) network undergoes a profound rearrangement, in part due to alterations in the MT nucleating and anchoring properties of the centrosome. Ninein and the ninein-like protein (Nlp) are centrosomal proteins involved in MT organisation in interphase cells. We show that the overexpression of these two proteins induces the fragmentation of the Golgi, and causes lysosomes to disperse toward the cell periphery. The ability of Nlp and ninein to perturb the cytoplasmic distribution of these organelles depends on their ability to interact with the dynein-dynactin motor complex. Our data also indicate that dynactin is required for the targeting of Nlp and ninein to the centrosome. Furthermore, phosphorylation of Nlp by the polo-like kinase 1 (Plk1) negatively regulates its association with dynactin. These findings uncover a mechanism through which Plk1 helps to coordinate changes in MT organisation with cell cycle progression, by controlling the dynein-dynactin-dependent transport of centrosomal proteins.
Chen, Ting; Ozel, Duygu; Qiao, Yuan; Harbinski, Fred; Chen, Limo; Denoyelle, Séverine; He, Xiaoying; Zvereva, Nela; Supko, Jeffrey G.; Chorev, Michael; Halperin, Jose A.; Aktas, Bertal H.
Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2·GTP·Met-tRNAi translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. The chemical modifiers of the eIF2·GTP·Met-tRNAi ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining if this complex can be pharmacologically targeted for therapeutic purposes. Using a cell based assay, we identified N,N’-diarylureas as novel inhibitors of the ternary complex abundance. Direct functional-genetics and biochemical evidence demonstrated that the N,N’-diarylureas activate heme regulated inhibitor kinase, thereby phosphorylate eIF2α and reduce abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N’-diarylureas are potent and specific tools for studying the role eIF2·GTP·Met-tRNAi ternary complex in the pathobiology of human disorders. PMID:21765405
Ding, Yin-Xiu; Wei, Li-Chun; Wang, Ya-Zhou; Cao, Rong; Wang, Xi; Chen, Liang-Wei
Parkinson's disease (PD) is a severe deliberating neurological disease caused by progressive degenerative death of dopaminergic neurons in the substantia nigra of midbrain. While cell replacement strategy by transplantation of neural stem cells and inducement of dopaminergic neurons is recommended for the treatment of PD, understanding the differentiation mechanism and controlled proliferation of grafted stem cells remain major concerns in their clinical application. Here we review recent studies on molecular signaling pathways in regulation of dopaminergic differentiation and proliferation of stem cells, particularly Wnt/beta-catenin signaling in stimulating formation of the dopaminergic phenotype, Notch signaling in inhibiting stem cell differentiation, and Sonic hedgehog functioning in neural stem cell proliferation and neuronal cell production. Activation of oncogenes involved in uncontrolled proliferation or tumorigenicity of stem cells is also discussed. It is proposed that a selective molecular manipulation targeting strategy will greatly benefit cell replacement therapy for PD by effectively promoting dopaminergic neuronal cell generation and reducing risk of tumorigenicity of in vivo stem cell applications.
Qiu, H; Liu, N; Luo, L; Zhong, J; Tang, Z; Kang, K; Qu, J; Peng, W; Liu, L; Li, L; Gou, D
Myogenesis is an important biological process that occurs during both skeletal muscle regeneration and postnatal growth. Growing evidence points to the critical role of microRNAs (miRNAs) in myogenesis. Our analysis of miRNA expression patterns reveal that miRNAs of miR-17-92 cluster are dramatically downregulated in C2C12 cells after myogenesis stimulation, are strongly induced in mouse skeletal muscle after injury and decrease steadily thereafter and are downregulated with age in skeletal muscle during mouse and porcine postnatal growth. However, their roles in muscle developmental processes remain elusive. We show that the miR-17-92 cluster promotes mouse myoblast proliferation but inhibits myotube formation. miR-17, -20a and -92a target the actin-associated protein enigma homolog 1 (ENH1). The silencing of ENH1 increased the nuclear accumulation of the inhibitor of differentiation 1 (Id1) and represses myogenic differentiation. Furthermore, the injection of adenovirus expressing miR-20a into the tibialia anterior muscle downregulates ENH1 and delays regeneration. In addition, the downregulation of miR-17-92 during myogenesis is transcriptionally regulated by E2F1. Overall, our results reveal a E2F1/miR-17-92/ENH1/Id1 regulatory axis during myogenesis. PMID:27315298
Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong
Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.
Lin Zhang; Xing Cai; Yuan Yin; Cheng Wang; Tianfu Zhang; Dihan Zhu; Dianmu Zhang; Jie Xu; Qun Chen; Yi Ba; Jing Liu; Dongxia Hou; Qiang Wang; Jianqun Chen; Jin Wang; Meng Wang; Qipeng Zhang; Junfeng Zhang; Ke Zen; Chen-Yu Zhang; Xi Chen; Donghai Li; Lingyun Zhu; Yujing Zhang; Jing Li; Zhen Bian; Xiangying Liang
Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases,and act as signaling molecules in intercellular communication.Here,we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally,through food intake.MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects.Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA,inhibit LDLRAP1 expression in liver,and consequently decrease LDL removal from mouse plasma.These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.
Turrero García, Miguel; Chang, YoonJeung; Arai, Yoko; Huttner, Wieland B
The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal.
Thomas, S; Abdulhay, Enas; Baconnier, Pierre; Fontecave, Julie; Francoise, Jean-Pierre; Guillaud, Francois; Hannaert, Patrick; Hernandez, Alfredo; Le Rolle, Virginie; Maziere, Pierre; Tahi, Fariza; Zehraoui, Farida
We present progress on a comprehensive, modular, interactive modeling environment centered on overall regulation of blood pressure and body fluid homeostasis. We call the project SAPHIR, for "a Systems Approach for PHysiological Integration of Renal, cardiac, and respiratory functions". The project uses state-of-the-art multi-scale simulation methods. The basic core model will give succinct input-output (reduced-dimension) descriptions of all relevant organ systems and regulatory processes, and it will be modular, multi-resolution, and extensible, in the sense that detailed submodules of any process(es) can be "plugged-in" to the basic model in order to explore, eg. system-level implications of local perturbations. The goal is to keep the basic core model compact enough to insure fast execution time (in view of eventual use in the clinic) and yet to allow elaborate detailed modules of target tissues or organs in order to focus on the problem area while maintaining the system-level regulatory compensations.
Full Text Available MicroRNAs (miRNAs have been proven to play crucial roles in cancer, including tumor chemotherapy resistance and metastasis of non-small-cell lung cancer (NSCLC. TGFβ signal pathway abnormality is widely found in cancer and correlates with tumor proliferation, apoptosis and metastasis. Here, miR-17, 20a, 20b were detected down-regulated in A549/DDP cells (cisplatin resistance compared with A549 cells (cisplatin sensitive. Over-expression of miR-17, 20a, 20b can not only decrease cisplatin-resistant but also reduce migration by inhibiting epithelial-to-mesenchymal transition (EMT in A549/DDP cells. These functions of miR-17, 20a, 20b may be caused at least in part via inhibition of TGFβ signal pathway, as miR-17, 20a, 20b are shown to directly target and repress TGF-beta receptor 2 (TGFβR2 which is an important component of TGFβ signal pathway. Consequently, our study suggests that miRNA 17 family (including miR-17, 20a, 20b can act as TGFβR2 suppressor for reversing cisplatin-resistant and suppressing metastasis in NSCLC.
Katrin S. Reiners
Full Text Available Exosomes are endosomal-derived nanovesicles released by normal and tumor cells, which transfer functionally active proteins, lipids and nucleic acids between cells. They are important mediators of intercellular communication and act on the adjacent stroma as well as in the periphery. Recently, exosomes have been recognized to play a pathophysiological role in various diseases such as cancer or infectious diseases. Tumor cell-derived exosomes (Tex have been shown to act as tumor promotors by educating non-malignant cells to provide a tumor supporting microenvironment, which helps to circumvent immune detection by the host and supports metastasis. However, Tex with anti-tumor, immune-activating properties were also described reflecting the complexity of exosomes.Here, we assess the role of extracellular microvesicles/exosomes as messengers affecting NK cell function in health and disease and discuss the molecular basis for the differential impact of exosomes on NK cell activity. The molecular composition/load of exosomes and the mechanisms regulating their release remain unclear and need to be further analyzed to facilitate the development of new treatment options targeting the exosomal machinery.
Caro, Dario; Frederiksen, Pia; Thomsen, Marianne
In this paper we discuss how targets, policy instruments and accounting frameworks for greenhouse gas (GHG) reduction need to be complemented and aligned, to achieve a more effective road to reduce the GHG emission. We focus on gaps in the policy framework presently adopted by countries......, whether or not consumed inside or outside the country, policy regulations directed at consumption, such as a meat tax, may not effectively target the national GHG reduction obligations, and thereby they may not be sufficiently attractive to governments. In particular, emissions derived from...... internationally traded goods are insufficiently targeted by territorially oriented policy frameworks because a country might reduce the consumption of an imported good without contributing to the national GHG emission reduction target. If consumption-based reduction targets were implemented, a reduction of GHG...
Full Text Available In this study, in silico approaches, including multiple QSAR modeling, structural similarity analysis, and molecular docking, were applied to develop QSAR classification models as a fast screening tool for identifying highly-potent ABCA1 up-regulators targeting LXRβ based on a series of new flavonoids. Initially, four modeling approaches, including linear discriminant analysis, support vector machine, radial basis function neural network, and classification and regression trees, were applied to construct different QSAR classification models. The statistics results indicated that these four kinds of QSAR models were powerful tools for screening highly potent ABCA1 up-regulators. Then, a consensus QSAR model was developed by combining the predictions from these four models. To discover new ABCA1 up-regulators at maximum accuracy, the compounds in the ZINC database that fulfilled the requirement of structural similarity of 0.7 compared to known potent ABCA1 up-regulator were subjected to the consensus QSAR model, which led to the discovery of 50 compounds. Finally, they were docked into the LXRβ binding site to understand their role in up-regulating ABCA1 expression. The excellent binding modes and docking scores of 10 hit compounds suggested they were highly-potent ABCA1 up-regulators targeting LXRβ. Overall, this study provided an effective strategy to discover highly potent ABCA1 up-regulators.
Plosch, T.; Gellhaus, A.; van Straten, E. M. E.; Wolf, N.; Huijkman, N. C. A.; Schmidt, M.; Dunk, C. E.; Kuipers, F.; Winterhager, E.
Objectives: The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in th
Møller, Thea S. B.; Rau, Martin Holm; Bonde, Charlotte S;
The aim of the study was to determine how ESBL-producing Escherichia coli change the expression of metabolic and biosynthesis genes when adapting to inhibitory concentrations of cefotaxime. Secondly, it was investigated whether significantly regulated pathways constitute putative secondary target...
Wang, Pengchao; Zhao, Yuanyuan; Fan, Ruiwen; Chen, Tianzhi; Dong, Changsheng
MicroRNAs (miRNAs) play an important role in regulating almost all biological processes. miRNAs bind to the 3' untranslated region (UTR) of mRNAs by sequence matching. In a previous study, we demonstrated that miR-21 was differently expressed in alpaca skin with different hair color. However, the molecular and cellular mechanisms for miR-21 to regulate the coat color are not yet completely understood. In this study, we transfected miR-21a-5p into mouse melanocytes and demonstrated its function on melanogenesis of miR-21a-5p by targeting Sox5, which inhibits melanogenesis in mouse melanocytes. The results suggested that miR-21a-5p targeted Sox5 gene based on the binding site in 3' UTR of Sox5 and overexpression of miR-21a-5p significantly down-regulated Sox5 mRNA and protein expression. Meanwhile, mRNA and protein expression of microphthalmia transcription factor (MITF) and Tyrosinase (TYR) were up-regulated, which subsequently make the melanin production in melanocytes increased. The results suggest that miR-21a-5p regulates melanogenesis via MITF by targeting Sox5.
Dai, Shouping [Department of Diagnostic Imaging, Linyi People' s Hospital, Linyi, Shandong 276000 (China); Wang, Xianjun [Department of Neurology, Linyi People' s Hospital, Linyi, Shandong 276000 (China); Li, Xiao [Department of Pathology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Cao, Yuandong, E-mail: firstname.lastname@example.org [Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province (China)
MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays also revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. - Highlights: • miR-139-5p is downregulated in GBM. • miR-139-5p regulates cell proliferation through inducing apoptosis. • miR-139-5p regulates glioblastoma tumorigenesis by targeting 3′UTR of ELTD1. • miR-139-5p is involved in cell cycle regulation.
Full Text Available MicroRNAs (miRNAs play an important role in regulating almost all biological processes. miRNAs bind to the 3′ untranslated region (UTR of mRNAs by sequence matching. In a previous study, we demonstrated that miR-21 was differently expressed in alpaca skin with different hair color. However, the molecular and cellular mechanisms for miR-21 to regulate the coat color are not yet completely understood. In this study, we transfected miR-21a-5p into mouse melanocytes and demonstrated its function on melanogenesis of miR-21a-5p by targeting Sox5, which inhibits melanogenesis in mouse melanocytes. The results suggested that miR-21a-5p targeted Sox5 gene based on the binding site in 3′ UTR of Sox5 and overexpression of miR-21a-5p significantly down-regulated Sox5 mRNA and protein expression. Meanwhile, mRNA and protein expression of microphthalmia transcription factor (MITF and Tyrosinase (TYR were up-regulated, which subsequently make the melanin production in melanocytes increased. The results suggest that miR-21a-5p regulates melanogenesis via MITF by targeting Sox5.
The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 μmol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The ex- pression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regu- lated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation
Sun, Chuanzheng; Huang, Feizhou; Liu, Xunyang; Xiao, Xuefei; Yang, Mingshi; Hu, Gui; Liu, Huaizheng; Liao, Liangkan
Non-alcoholic fatty liver disease (NAFLD) has emerged as a public health issue with a prevalence of 15-30% in Western populations and 6-25% in Asian populations. Certain studies have revealed the alteration of microRNA (miRNA or miR) profiles in NAFLD and it has been suggested that miR-21 is associated with NAFLD. In the present study, we measured the serum levels of miR-21 in patients with NAFLD and also performed in vitro experiments using a cellular model of NAFLD to further investigate the effects of miR-21 on triglyceride and cholesterol metabolism. Furthermore, a novel target through which miR-21 exerts its effects on NAFLD was identified. The results revealed that the serum levels of miR-21 were lower in patients with NAFLD compared with the healthy controls. In addition, 3-hydroxy-3-methylglutaryl-co-enzyme A reductase (HMGCR) expression was increased in the serum of patients with NAFLD both at the mRNA and protein level. To mimic the NAFLD condition in vitro, HepG2 cells were treated with palmitic acid (PA) and oleic acid (OA). Consistent with the results obtained in the in vivo experiments, the expression levels of miR-21 were decreased and those of HMGCR were increased in the in vitro model of NAFLD. Luciferase reporter assay revealed that HMGCR was a direct target of miR-21 and that miR-21 exerted an effect on both HMGCR transcript degradation and protein translation. Furthermore, the results from the in vitro experiments revealed that miR-21 decreased the levels of triglycerides (TG), free cholesterol (FC) and total cholesterol (TC) in the PA/OA-treated HepG2 cells and that this effect was attenuated by HMGCR overexpression. Taken together, to the best of our knowledge, the present study is the first to report that miR-21 regulates triglyceride and cholesterol metabolism in an in vitro model of NAFLD, and that this effect is achieved by the inhibition of HMGCR expression. We speculate that miR-21 may be a useful biomarker for the diagnosis and
Zhao, Di; Chen, Youbin; Chen, Shunliang; Zheng, Chuangyi; Hu, Jun; Luo, Shaowei
MicroRNAs are small, endogenous, and non-coding RNAs that play important regulatory roles in multiple biological processes in cancers. Recent evidence has indicated that miR-19a participates in the cancer tumorigenic progression. However, the functional roles of miR-19a in cancer stem cells are still unclear. As the cancer stem cells are considered to be responsible for the tumor recurrence and treatment failure in osteosarcoma, the aim of this study is to investigate the molecular mechanism of miR-19a underlying osteosarcoma tumorigenesis. In this study, we observed significant upregulation of miR-19a in osteosarcoma patients' tumor tissues as well as the osteosarcoma cell lines in vitro. We showed that knockdown of miR-19a by its antisense oligonucleotide (anti-miR-19a) significantly decreased the population of cancer stem cells in osteosarcoma cell lines. Furthermore, we found the miR-19a regulated the cell proliferation, migration, and viability in the human osteosarcoma-cancer stem cells. The gene of phosphatase and tensin homolog deleted on chromosome 10, which is an important tumor suppressor, was found to be directly regulated by miR-19a in human osteosarcoma-cancer stem cells. We demonstrated that knockdown of miR-19a increased the expression of phosphatase and tensin homolog deleted on chromosome 10. As the anti-miR-19a inhibited the phosphatidylinositol 3-kinase/AKT pathway and induced apoptosis of human osteosarcoma-cancer stem cells, the phosphatase and tensin homolog deleted on chromosome 10 small interfering RNA inhibited the effect of it. Meanwhile, the phosphatase and tensin homolog deleted on chromosome 10 small interfering RNA also abolished the effect of anti-miR-19a on inhibiting the cell proliferation, migration, and viability in the human osteosarcoma-cancer stem cells. In conclusion, our findings demonstrated that dysregulation of miR-19a plays critical roles in the osteosarcoma stem cells, at least in part via targeting the phosphatase and
Han, Ye; Heuermann, Robert J.; Lyman, Kyle A.; Fisher, Daniel; Ismail, Quratul-Ain; Chetkovich, Dane M.
Major Depressive Disorder is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments and new pharmacologic targets are needed. HCN channels regulate excitability in neurons and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN channel auxiliary subunit TRIP8b also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN channel trafficking and its relationship to antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b-HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of Major Depressive Disorder. PMID:27400855
Nelson, Jessica Kristine; Koenis, Duco Steven; Scheij, Saskia; Cook, Emma Clare Laura; Moeton, Martina; Santos, Ana; Lobaccaro, Jean-Marc Adolphe; Baron, Silvere
Objective— The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3) and β (LXRβ, NR1H2), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study. Approach and Results— We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease–exonuclease–phosphatase family domain containing 1 (EEPD1/KIAA1706) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages. Conclusions— In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1. PMID:28082258
Nelson, Jessica Kristine; Koenis, Duco Steven; Scheij, Saskia; Cook, Emma Clare Laura; Moeton, Martina; Santos, Ana; Lobaccaro, Jean-Marc Adolphe; Baron, Silvere; Zelcer, Noam
The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3) and β (LXRβ, NR1H2), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study. We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease-exonuclease-phosphatase family domain containing 1 (EEPD1/KIAA1706) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages. In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1. © 2017 The Authors.
Han, Y; Heuermann, R J; Lyman, K A; Fisher, D; Ismail, Q-A; Chetkovich, D M
Major depressive disorder (MDD) is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments, and new pharmacologic targets are needed. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate excitability in neurons, and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN-channel auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN-channel trafficking and its relationship with antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN-channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN-channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b-HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small-molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of MDD.
Portal-Núñez, Sergio; Esbrit, Pedro; Alcaraz, María José; Largo, Raquel
Aging is a natural process characterized by the declining ability of the different organs and tissues to respond to stress, increasing homeostatic imbalance and risk of disease. Osteoarthritis (OA) is a multifactorial disease in which cartilage degradation is a central feature. Aging is the main risk factor for OA. In OA cartilage, a decrease in the number of chondrocytes and in their ability to regenerate the extracellular matrix and adequately respond to stress has been described. OA chondrocytes show a senescence secretory phenotype (SSP) consisting on the overproduction of cytokines (interleukins 1 and 6), growth factors (e.g., epidermal growth factor) and matrix metalloproteinases (MMP) (e.g., MMP-3, MMP-13). Reactive Oxygen Species (ROS) play a major role in the induction of the SSP. In chondrocytes, an increase in ROS production leads to hyper-peroxidation, protein carbonylation and DNA damage which alter chondrocyte function. ROS overproduction also induces changes in metabolic pathways such as PI3K-Akt and ERK. Autophagy is a key mechanism for maintaining cell homeostasis by adjusting cell metabolism to nutrient supply and removing damaged organelles. In cartilage, aging-related loss of autophagy leads to cell death and OA, while stimulation of autophagy exerts protective effects on cartilage deterioration. Aging also interferes with epigenetic mechanisms such as activity of histone acetylases that control the pattern of DNA methylation, and induces up- or down-regulation of microRNAs expression. A deeper knowledge of the mechanisms involved in chondrocyte aging could identify potential targets for the treatment of OA, a prevalent and therapeutic-orphan disease.
Hinsby, Klaus; Refsgaard, Jens Christian
even field scale based on estimated acceptable nutrient loadings to transitional and coastal waters (or any other protected aquatic ecosystem in the river basin) according to the requirements of the EU Water Framework and Groundwater directives, and use these to monitor and plan sustainable water and land use management, e.g. by differentiated practices in farming and land use within sub-catchments. Specifically, we suggest that monitoring of the nitrogen outputs from catchments, sub-catchment and even delineated specific fields with new cost-effective monitoring systems in combination with coupled soil-groundwater-surface water models has a large potential for the development of efficient differentiated measures controlling nutrient loadings to aquatic ecosystems. Here we present initial considerations and suggestions for the development of new governance concepts targeted at differentiated output based regulations using threshold values for N in groundwater and streams derived from acceptable loadings to a small Danish estuary.
Jian Zhou; Wei-Qiang Ju; Xiao-PengYuan; Xiao-Feng Zhu; Dong-PingWang; Xiao-Shun He
BACKGROUND: The deficiency of liver regeneration needs to be addressed in the fields of liver surgery, split liver transplan-tation and living donor liver transplantation. Researches of microRNAs would broaden our understandings on the mecha-nisms of various diseases. Our previous research confirmed that miR-26a regulated liver regeneration in mice; however, the relationship between miR-26a and its target, directly or in-directly, remains unclear. Therefore, the present study further investigated the mechanism of miR-26a in regulating mouse hepatocyte proliferation. METHODS: An established mouse liver cell line, Nctc-1469, was transfected with Ad5-miR-26a-EGFP, Ad5-anti-miR-26a-EGFP or Ad5-EGFP vector. Cell proliferation was assessed by MTS, cell apoptosis and cell cycle by flow cytometry, and gene expression by Western blotting and quantitative real-time PCR. Dual-luciferase reporter assays were used to test targets of miR-26a. RESULTS: Compared with the Ad5-EGFP group, Ad5-anti-miR-26a-EGFP down-regulated miR-26a and increased prolif-eration of hepatocytes, with more cells entering the G1 phase of cell cycle (82.70%±1.45% vs 75.80%±3.92%), and decreased apoptosis (5.50%±0.35% vs 6.73%±0.42%).CCND2 and CCNE2 were the direct targeted genes of miR-26a. miR-26a down-regulation up-regulated CCND2 and CCNE2 expressions and down-regulated p53 expression in Nctc-1469 cells. On the con-trary, miR-26a over-expression showed the opposite results. CONCLUSIONS: miR-26a regulated mouse hepatocyte pro-liferation by directly targeting the 3’ untranslated regions of cyclin D2/cyclin E2; miR-26a also regulated p53-mediated apoptosis. Our data suggested that miR-26a may be a promis-ing regulator in liver regeneration.
Full Text Available We have employed a novel approach for the identification of functionally important microRNA (miRNA-target interactions, integrating miRNA, transcriptome and proteome profiles and advanced in silico analysis using the FAME algorithm. Since miRNAs play a crucial role in the inner ear, demonstrated by the discovery of mutations in a miRNA leading to human and mouse deafness, we applied this approach to microdissected auditory and vestibular sensory epithelia. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. Functionally important miRNAs were determined by searching for enriched or depleted targets in the transcript and protein datasets with an expression consistent with the dogma of miRNA regulation. Importantly, quite a few of the targets were detected only in the protein datasets, attributable to regulation by translational suppression. We identified and experimentally validated the regulation of PSIP1-P75, a transcriptional co-activator previously unknown in the inner ear, by miR-135b, in vestibular hair cells. Our findings suggest that miR-135b serves as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells.
Targeted genetic modification, which enables scientists to genetically engineer plants more efficiently and precisely, challenges current process-based regulatory frameworks for genetically modified crops.
Devallière, Julie; Chatelais, Mathias; Fitau, Juliette; Gérard, Nathalie; Hulin, Philippe; Velazquez, Laura; Turner, Christopher E.; Charreau, Béatrice
Focal adhesion (FA) formation and disassembly play an essential role in adherence and migration of endothelial cells. These processes are highly regulated and involve various signaling molecules that are not yet completely identified. Lnk [Src homology 2-B3 (SH2B3)] belongs to a family of SH2-containing proteins with important adaptor functions. In this study, we showed that Lnk distribution follows that of vinculin, localizing Lnk in FAs. Inhibition of Lnk by RNA interference resulted in decreased spreading, whereas sustained expression dramatically increases the number of focal and cell-matrix adhesions. We demonstrated that Lnk expression impairs FA turnover and cell migration and regulates β1-integrin-mediated signaling via Akt and GSK3β phosphorylation. Moreover, the α-parvin protein was identified as one of the molecular targets of Lnk responsible for impaired FA dynamics and cell migration. Finally, we established the ILK protein as a new molecular partner for Lnk and proposed a model in which Lnk regulates α-parvin expression through its interaction with ILK. Collectively, our results underline the adaptor Lnk as a novel and effective key regulator of integrin-mediated signaling controlling endothelial cell adhesion and migration.—Devallière, J., Chatelais, M., Fitau, J., Gérard, N., Hulin, P., Velazquez, L., Turner, C. E. Charreau, B. LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets α-parvin to control cell adhesion and migration. PMID:22441983
Sandai, Doblin; Yin, Zhikang; Selway, Laura; Stead, David; Walker, Janet; Leach, Michelle D; Bohovych, Iryna; Ene, Iuliana V; Kastora, Stavroula; Budge, Susan; Munro, Carol A; Odds, Frank C; Gow, Neil A R; Brown, Alistair J P
Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients. IMPORTANCE Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the
Dou, Lin; Wang, Shuyue; Sun, Libo; Huang, Xiuqing; Zhang, Yang; Shen, Tao; Guo, Jun; Man, Yong; Tang, Weiqing; Li, Jian
Insulin resistance is a critical factor contributing to the pathogenesis of type 2 diabetes and other metabolic diseases. Recent studies have indicated that miR-338-3p plays an important role in cancer. Here, we investigated whether miR-338-3p mediates tumour necrosis factor-α (TNF-α)-induced hepatic insulin resistance. The activation of the insulin signalling pathway and the level of glycogenesis were examined in the livers of the db/db and high fat diet (HFD)-fed mice and in HEP1-6 cells transfected with miR-338-3p mimic or inhibitor. Computational prediction of microRNA target, luciferase assay and Western blot were used to assess the miR-338-3p target. Chromatin immunoprecipitation (ChIP) assay was used to determine the transcriptional regulator of miR-338-3p. miR-338-3p was down-regulated in the livers of the db/db, HFD-fed and TNF-α-treated C57BL/6J mice, as well as in mouse HEP1-6 hepatocytes treated with TNF-α. Importantly the down-regulation of miR-338-3p induced insulin resistance, as indicated by impaired glucose tolerance and insulin tolerance. Further research showed that the down-regulated miR-338-3p resulted in the impaired AKT/ glycogen synthase kinase 3 beta (GSl·Gβ) signalling pathway and glycogen synthesis. In contrast, hepatic over-expression of miR-338-3p rescued the TNF-α-induced insulin resistance. Moreover, protein phosphatase 4 regulator subunit 1 (PP4R1) was identified as a direct target of miR-338-3p that mediated hepatic insulin signalling by regulating protein phosphatase 4 (PP4). Finally we identified hepatic nuclear factor 4 alpha (HNF-4α) as the transcriptional regulator of miRNA-338-3p. Our studies provide novel insight into the critical role and molecular mechanism by which miR-338-3p is involved in TNF-α-induced hepatic insulin resistance. miR-338-3p might mediate TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression. © 2017 The Author(s). Published by S. Karger AG, Basel.
Neveu, Gregory; Ziv-Av, Amotz; Barouch-Bentov, Rina; Berkerman, Elena; Mulholland, Jon; Einav, Shirit
Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved ant...
Cifani, Carlo; Micioni Di Bonaventura, Maria V; Pucci, Mariangela; Giusepponi, Maria E; Romano, Adele; Di Francesco, Andrea; Maccarrone, Mauro; D'Addario, Claudio
.... To investigate the individual sensitivity to weight gain/resistance, we here studied gene transcription regulation of several hypothalamic neuropeptides involved in the control of energy balance...
G.B. Ehret (Georg); T. Ferreira (Teresa); D.I. Chasman (Daniel); A.U. Jackson (Anne); E.M. Schmidt (Ellen); T. Johnson (Toby); G. Thorleifsson (Gudmar); J. Luan (Jian'An); L.A. Donnelly (Louise); S. Kanoni (Stavroula); A.K. Petersen; V. Pihur (Vasyl); R.J. Strawbridge (Rona); D. Shungin (Dmitry); Hughes, M.F. (Maria F.); O. Meirelles; M. Kaakinen (Marika); N. Bouatia-Naji (Nabila); K. Kristiansson (Kati); S. Shah (Sonia); M.E. Kleber (Marcus); X. Guo (Xiuqing); L.-P. Lyytikäinen (Leo-Pekka); C. Fava (Cristiano); N. Eriksson (Niclas); I.M. Nolte (Ilja); P.K. Magnusson (Patrik); E. Salfati (Elias); L.S. Rallidis (Loukianos); Theusch, E. (Elizabeth); A.J.P. Smith; L. Folkersen (Lasse); H.E. Witkowska (Ewa); T.H. Pers (Tune); R. Joehanes (Roby); Kim, S.K. (Stuart K.); L. Lataniotis (Lazaros); R. Jansen; A.D. Johnson (Andrew); H. Warren (Helen); Y.J. Kim; Zhao, W. (Wei); Y. Wu (Ying); B. Tayo (Bamidele); M. Bochud (Murielle); D. Absher (Devin); L.S. Adair (Linda); N. Amin (Najaf); D.E. Arking (Dan); T. Axelsson (Tomas); D. Baldassarre (Damiano); B. Balkau (Beverley); S. Bandinelli (Stefania); M. Barnes (Michael); I. Barroso (Inês); Bevan, S. (Stephen); J.C. Bis (Joshua); Bjornsdottir, G. (Gyda); M. Boehnke (Michael); E.A. Boerwinkle (Eric); L.L. Bonnycastle (Lori); D.I. Boomsma (Dorret); S.R. Bornstein (Stefan); M.J. Brown (Morris); M. Burnier (Michel); Cabrera, C.P. (Claudia P.); J.C. Chambers (John); Chang, I.-S. (I-Shou); Cheng, C.-Y. (Ching-Yu); P.S. Chines (Peter); Chung, R.-H. (Ren-Hua); F.S. Collins (Francis); Connell, J.M. (John M.); A. Döring (Angela); J. Dallongeville; J. Danesh (John); U. de Faire (Ulf); G. Delgado; A. Dominiczak (Anna); A.S.F. Doney (Alex); F. Drenos (Fotios); T. Edkins (Ted); Eicher, J.D. (John D.); R. Elosua (Roberto); S. Enroth (Stefan); J. Erdmann (Jeanette); P. Eriksson (Per); T. Esko (Tõnu); E. Evangelou (Evangelos); A. Evans (Alun); M. Fall (Magnus); M. Farrall (Martin); J.F. Felix (Janine); J. Ferrieres (Jean); L. Ferrucci (Luigi); M. Fornage (Myriam); T. Forrester (Terrence); N. Franceschini (Nora); O.H. Franco (Oscar); A. Franco-Cereceda (Anders); R.M. Fraser (Ross); S.K. Ganesh (Santhi); Gao, H. (He); K. Gertow (Karl); F. Gianfagna (Francesco); B. Gigante (Bruna); F. Giulianini (Franco); A. Goel (Anuj); A.H. Goodall (Alison); M. Goodarzi (Mark); M. Gorski (Mathias); J. Gräßler (Jürgen); C.J. Groves (Christopher); V. Gudnason (Vilmundur); U. Gyllensten (Ulf); G. Hallmans (Göran); A.L. Hartikainen; Hassinen, M. (Maija); A.S. Havulinna (Aki); C. Hayward (Caroline); S. Hercberg (Serge); K.H. Herzig; A.A. Hicks (Andrew); A. Hingorani (Aroon); J.N. Hirschhorn (Joel); Hofman, A. (Albert); Holmen, J. (Jostein); O.L. Holmen (Oddgeir); J.J. Hottenga (Jouke Jan); P. Howard (Philip); Hsiung, C.A. (Chao A.); S.C. Hunt (Steven); M.K. Ikram (Kamran); T. Illig (Thomas); C. Iribarren (Carlos); Jensen, R.A. (Richard A.); M. Kähönen (Mika); H.M. Kang (Hyun Min); S. Kathiresan (Sekar); J. Keating (John); K.T. Khaw; Y.K. Kim (Yun Kyoung); E. Kim (Eric); M. Kivimaki (Mika); N. Klopp (Norman); Kolovou, G. (Genovefa); P. Komulainen (Pirjo); J.S. Kooner (Jaspal S.); Kosova, G. (Gulum); R.M. Krauss (Ronald); D. Kuh (Diana); Z. Kutalik (Zoltán); J. Kuusisto (Johanna); K. Kvaløy (Kirsti); T.A. Lakka (Timo); N.R. Lee (Nanette); I.T. Lee; W.-J. Lee (Wen-Jane); D. Levy (Daniel); X. Li (Xiaohui); Liang, K.-W. (Kae-Woei); Lin, H. (Honghuang); Lin, L. (Li); J. Lindström (Jaana); S. Lobbens (Stéphane); S. Männistö (Satu); G. Müller (Gabriele); M. Müller-Nurasyid (Martina); F. MacH (François); H.S. Markus (Hugh); E. Marouli (Eirini); M.I. McCarthy (Mark); C.A. McKenzie (Colin); P. Meneton (Pierre); C. Menni (Cristina); A. Metspalu (Andres); Mijatovic, V. (Vladan); L. Moilanen (Leena); M.E. Montasser (May E.); A.D. Morris (Andrew); A.C. Morrison (Alanna); Mulas, A. (Antonella); R. Nagaraja (Ramaiah); N. Narisu (Narisu); K. Nikus (Kjell); C.J. O'Donnell (Christopher); P.F. O'Reilly (Paul); K.K. Ong (Ken); Paccaud, F. (Fred); C. Palmer (Cameron); A. Parsa (Afshin); N.L. Pedersen (Nancy); B.W.J.H. Penninx (Brenda); M. Perola (Markus); A. Peters (Annette); N.R. Poulter (Neil); P.P. Pramstaller (Peter Paul); B.M. Psaty (Bruce); T. Quertermous (Thomas); D.C. Rao (Dabeeru C.); A. Rasheed (Asif); N.W. Rayner (Nigel William); F. Renström (Frida); R. Rettig (Rainer); K.M. Rice (Kenneth); R. Roberts (Robert); L.M. Rose (Lynda); Rossouw, J. (Jacques); N.J. Samani (Nilesh); S. Sanna (Serena); J. Saramies (Jouko); H. Schunkert (Heribert); S. Sebert (Sylvain); Sheu, W.H.-H. (Wayne H.-H.); Shin, Y.-A. (Young-Ah); X. Sim (Xueling); G.D. Smith; A.V. Smith (Albert Vernon); M.X. Sosa (Maria X.); T.D. Spector (Timothy); A. Stancáková (Alena); A. Stanton (Alice); K. Stirrups (Kathy); H.M. Stringham (Heather); Sundstrom, J. (Johan); A.J. Swift (Amy); A.C. Syvänen; Tai, E.-S. (E-Shyong); T. Tanaka (Toshiko); K.V. Tarasov (Kirill); A. Teumer (Alexander); U. Thorsteinsdottir (Unnur); M.D. Tobin (Martin); E. Tremoli (Elena); Uitterlinden, A.G. (Andre G.); M. Uusitupa (Matti); A. Vaez (Ahmad); D. Vaidya (Dhananjay); Van Duijn, C.M. (Cornelia M.); E.P.A. van Iperen (Erik); Vasan, R.S. (Ramachandran S.); G.C. Verwoert (Germaine); J. Virtamo (Jarmo); Vitart, V. (Veronique); B.F. Voight (Benjamin); P. Vollenweider (Peter); Wagner, A. (Aline); Wain, L.V. (Louise V.); N.J. Wareham (Nick); H. Watkins (Hugh); A.B. Weder (Alan); H.J. Westra (Harm-Jan); Wilks, R. (Rainford); T. Wilsgaard (Tom); J.F. Wilson (James F.); Wong, T.Y. (Tien Y.); T.-P. Yang (Tsun-Po); J. Yao (Jiefen); L. Yengo (Loic); W. Zhang (Weihua); J.H. Zhao; X. Zhu (Xiaofeng); P. Bovet (Pascal); Cooper, R.S. (Richard S.); K.L. Mohlke (Karen); Saleheen, D. (Danish); J.-Y. Lee (Jong-Young); P. Elliott (Paul); L.M. Gierman (Lobke); C.J. Willer (Cristen); L. Franke (Lude); G. Kees Hovingh; K.D. Taylor (Kent); G.V. Dedoussis (George); P. Sever (Peter); A. Wong (Andrew); W.H.L. Kao (Wen); T.L. Assimes (Themistocles); I. Njølstad (Inger); P.E.H. Schwarz (Peter); C. Langenberg (Claudia); H. Snieder (Harold); M. Caulfield (Mark); O. Melander (Olle); M. Laakso (Markku); J. Saltevo (Juha); R. Rauramaa (Rainer); J. Tuomilehto (Jaakko); Ingelsson, E. (Erik); T. Lehtimäki (Terho); K. Hveem (Kristian); W. Palmas (Walter); W. März (Winfried); M. Kumari (Meena); V. Salomaa (Veikko); Y.D. Chen (Y.); Rotter, J.I. (Jerome I.); P. Froguel (Philippe); M.-R. Jarvelin (Marjo-Riitta); E. Lakatta (Edward); K. Kuulasmaa (Kari); P.W. Franks (Paul); A. Hamsten (Anders); H.E. Wichmann (Heinz Erich); C.N.A. Palmer (Colin); Stefansson, K. (Kari); P.M. Ridker (Paul); R.J.F. Loos (Ruth); A. Chakravarti (Aravinda); P. Deloukas (Panagiotis); A.P. Morris (Andrew); C. Newton-Cheh (C.); P. Munroe (Patricia)
textabstractTo dissect the genetic architecture of blood pressure and assess effects on target organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals were used for validation. We
Ehret, Georg B.; Ferreira, Teresa; Chasman, Daniel I.; Jackson, Anne U.; Schmidt, Ellen M.; Johnson, Toby; Thorleifsson, Gudmar; Luan, Jian'an; Donnelly, Louise A.; Kanoni, Stavroula; Petersen, Ann -Kristin; Pihurl, Vasyl; Strawbridge, Rona J.; Shungin, Dmitry; Hughes, Maria F.; Meirelles, Osorio; Kaakinen, Marika; Bouatia-Naji, Nabila; Kristiansson, Kati; Shah, Sonia; Kleber, Marcus E.; Guo, Xiuqing; Lyytikainen, Leo-Pekka; Fava, Cristiano; Eriksson, Nidas; Nolte, Ilja M.; Magnusson, Patrik K.; Salfati, Elias L.; Rallidis, Loukianos S.; Theusch, Elizabeth; Smith, Andrew J. P.; Folkersen, Lasse; Witkowska, Kate; Pers, Tune H.; Joehanes, Roby; Kim, Stuart K.; Lataniotis, Lazaros; Jansen, Rick; Johnson, Andrew D.; Warren, Helen; Kim, Young Jin; Zhao, Wei; Wu, Ying; Tayo, Bamidele O.; Bochud, Murielle; Absher, Devin; Adair, Linda S.; Amin, Najaf; Arkingl, Dan E.; Axelsson, Tomas; Baldassarre, Damian; Balkau, Beverley; Bandinelli, Stefania; Barnes, Michael R.; Barroso, Ines; Bevan, Stephen; Bis, Joshua C.; Bjornsdottir, Gyda; Boehnke, Michael; Boerwinkle, Eric; Bonnycastle, Lori L.; Boomsma, Dorret I.; Bornstein, Stefan R.; Brown, Morris J.; Burnier, Michel; Cabrera, Claudia P.; Chambers, John C.; Chang, I-Shou; Cheng, Ching-Yu; Chines, Peter S.; Chung, Ren-Hua; Collins, Francis S.; Connell, John M.; Doring, Angela; Dallongeville, Jean; Danesh, John; de Faire, Ulf; Delgado, Graciela; Dominiczak, Anna F.; Doney, Alex S. F.; Drenos, Fotios; Edkins, Sarah; Eicher, John D.; Elosua, Roberto; Enroth, Stefan; Erdmann, Jeanette; Eriksson, Per; Esko, Tonu; Evangelou, Evangelos; Evans, Alun; Fai, Tove; Farra, Martin; Felixl, Janine F.; Ferrieres, Jean; Ferrucci, Luigi; Fornage, Myriam; Forrester, Terrence; Franceschinil, Nora; Franco, Oscar H.; Franco-Cereceda, Anders; Fraser, Ross M.; Ganesh, Santhi K.; Gao, He; Gertow, Karl; Gianfagna, Francesco; Gigante, Bruna; Giulianini, Franco; Goe, Anuj; Goodall, Alison H.; Goodarzi, Mark; Gorski, Mathias; Grassler, Jurgen; Groves, Christopher J.; Gudnason, Vilmundur; Gyllensten, Ulf; Hallmans, Goran; Hartikainen, Anna-Liisa; Hassinen, Maija; Havulinna, Aki S.; Hayward, Caroline; Hercberg, Serge; Herzig, Karl-Heinz; Hicks, Andrew A.; Hingorani, Aroon D.; Hirschhorn, Joel N.; Hofmanl, Albert; Holmen, Jostein; Holmen, Oddgeir Lingaas; Hottenga, Jouke-Jan; Howard, Phil; Hsiung, Chao A.; Hunt, Steven C.; Ikram, M. Arfan; Illig, Thomas; Iribarren, Carlos; Jensen, Richard A.; Kahonen, Mika; Kang, Hyun Min; Kathiresan, Sekar; Keating, Brendan J.; Khaw, Kay-Tee; Kim, Yun Kyoung; Kim, Eric; Kivimaki, Mika; Klopp, Norman; Kolovou, Genovefa; Komulainen, Pirjo; Kooner, Jaspal S.; Kosova, Gulum; Krauss, Ronald M.; Kuh, Diana; Kutalik, Zoltan; Kuusisto, Johanna; Kvaloy, Kirsti; Lakka, Timo A.; Lee, Nanette R.; Lee, I-Te; Lee, Wen-Jane; Levy, Daniel; Li, Xiaohui; Liang, Kae-Woei; Lin, Honghuang; Lin, Li; Lindstrom, Jaana; Lobbens, Stephane; Mannisto, Satu; Muller, Gabriele; Muller-Nurasyid, Martina; Mach, Francois; Markus, Hugh S.; Marouli, Eirini; McCarthy, Mark I.; McKenzie, Colin A.; Meneton, Pierre; Menni, Cristina; Metspalu, Andres; Mijatovic, Vladan; Moilanen, Leena; Montasser, May E.; Morris, Andrew D.; Morrison, Alanna C.; Mulas, Antonella; Nagaraja, Ramaiah; Narisu, Narisu; Nikus, Kjell; O'Donnell, Christopher J.; O'Reilly, Paul F.; Ong, Ken K.; Paccaud, Fred; Palmer, Cameron D.; Parsa, Afshin; Pedersen, Nancy L.; Penninx, Brenda W.; Perola, Markus; Peters, Annette; Poulter, Neil; Pramstaller, Peter P.; Psaty, Bruce M.; Quertermous, Thomas; Rao, Dabeeru C.; Rasheed, Asif; Rayner, N. William; Renstrom, Frida; Rettig, Rainer; Rice, Kenneth M.; Roberts, Robert; Rose, Lynda M.; Rossouw, Jacques; Samani, Nilesh J.; Sanna, Serena; Saramies, Jouko; Schunkert, Heribert; Sebert, Sylvain; Sheu, Wayne H-H; Shin, Young-Ah; Sim, Xueling; Smit, Johannes H.; Smith, Albert V.; Sosa, Maria X.; Spector, Tim D.; Stancakova, Alena; Stanton, Alice V.; Stirrups, Kathleen E.; Stringham, Heather M.; Sundstrom, Johan; Swift, Amy J.; Syvanen, Ann-Christine; Tai, E-Shyong; Tanaka, Toshiko; Tarasov, Kirill V.; Teumer, Alexander; Thorsteinsdottir, Unnur; Tobin, Martin D.; Tremoli, Elena; Uitterlinden, Andre G.; Uusitupa, Matti; Vaez, Ahmad; Vaidya, Dhananjay; van Duijn, Cornelia M.; van Iperen, Erik P. A.; Vasan, Ramachandran S.; Verwoert, Germaine C.; Virtamo, Jarmo; Vitart, Veronique; Voight, Benjamin F.; Vollenweider, Peter; Wagner, Aline; Wain, Louise V.; Wareham, Nicholas J.; Watldns, Hugh; Weder, Alan B.; Westra, Harm Jan; Wilks, Rainford; Wilsgaard, Tom; Wilson, James F.; Wong, Tien Y.; Yang, Tsun-Po; Yao, Jie; Yengo, Loic; Zhang, Weihua; Zhao, Jing Hua; Zhu, Xiaofeng; Bovet, Pascal; Cooper, Richard S.; Mohlke, Karen L.; Saleheen, Danish; Lee, Jong-Young; Elliott, Paul; Gierman, Hinco J.; Willer, Cristen J.; Franke, Lude; Hovingh, G. Kees; Taylor, Kent D.; Dedoussis, George; Sever, Peter; Wong, Andrew; Lind, Lars; Assimes, Themistocles L.; Njolstad, Inger; Schwarz, Peter E. H.; Langenberg, Claudia; Snieder, Harold; Caulfield, Mark J.; Melander, E.; Laakso, Markku; Saltevo, Juha; Rauramaa, Rainer; Tuomilehto, Jaakko; Ingelsson, Erik; Lehtimaki, Terho; Hveem, Kristian; Palmas, Walter; Marz, Winfried; Kumar, Meena; Salomaa, Veikko; Chen, Yii-Der I.; Rotter, Jerome I.; Froguel, Philippe; Jarvelin, Marjo-Riitta; Lakatta, Edward G.; Kuulasmaa, Kari; Franks, Paul W.; Hamsten, Anders; Wichmann, H-Erich; Palmer, Colin N. A.; Stefansson, Kari; Ridker, Paul M.; Loos, Ruth J. F.; Chalcravarti, Aravinda; Deloukas, Panos; Morris, Andrew P.; Newton-Cheh, Christopher; Munroe, Patricia B.
To dissect the genetic architecture of blood pressure and assess effects on target organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals were used for validation. We identified 66
Sangodkar, J, Terzo, EA, Leake, D, et al. (2009) KLF6-SV1 is a novel antiapoptotic protein that targets the BH3-only protein NOXA for degradation...et al. (2009) KLF6-SV1 is a novel antiapoptotic protein that targets the BH3-only protein NOXA for degradation and whose inhibition extends survival
Difeo, A, Huang, F, Sangodkar, J, Terzo, EA, Leake, D, et al. (2009) KLF6-SV1 is a novel antiapoptotic protein that targets the BH3-only protein NOXA ...2009) KLF6-SV1 is a novel antiapoptotic protein that targets the BH3-only protein NOXA for degradation and whose inhibition extends survival in an
Yao, Jingjing [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai, 200433 (China); Xu, Chen [Research Center of Developmental Biology, Second Military Medical University, 800th Xiangyin Road, Shanghai, 200433 (China); Department of Orthopedics, Changzheng Hospital Affiliated to Second Military Medical University, 415th Feng Yang Road, Shanghai, 200003 (China); Fang, Ziyu; Li, Yaoming [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai, 200433 (China); Liu, Houqi; Wang, Yue [Research Center of Developmental Biology, Second Military Medical University, 800th Xiangyin Road, Shanghai, 200433 (China); Translational Medicine Center, Second Military Medical University, 800th Xiangyin Road, Shanghai, 200433 (China); Xu, Chuanliang [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai, 200433 (China); Sun, Yinghao, E-mail: email@example.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai, 200433 (China)
Abstracts: MicroRNAs (miRNAs) are important endogenous gene regulators that play key roles in prostate cancer development and metastasis. However, specific miRNA expression patterns in prostate cancer tissues from Chinese patients remain largely unknown. In this study, we compared miRNA expression patterns in 65 pairs of prostate cancer and para-cancer tissues by RNA sequencing and found that miR-182-5p was the most up-regulated miRNA in prostate cancer tissues. The result was validated using realtime PCR in 18 pairs of prostate cancer and para-cancer tissues. In in vitro analysis, it was confirmed that miR-182-5p promotes prostate cancer cell proliferation, invasion and migration and inhibit apoptosis. In addition, the androgen receptor directly regulated the transcription of miR-182-5p, which could target to the 3′UTR of ARRDC3 mRNA and affect the expression of ARRDC3 and its downstream gene ITGB4. For the in vivo experiment, miR-182-5p overexpression also promoted the growth and progression of prostate cancer tumors. In this regard, we suggest that miR-182-5p may be a key androgen receptor-regulated factor that contributes to the development and metastasis of Chinese prostate cancers and may be a potential target for the early diagnosis and therapeutic studies of prostate cancer. -- Highlights: •miR-182-5p is the mostly up-regulated miRNA in Chinese prostate cancer. •miR-182-5p is regulated by androgen receptor. •miR-182-5p promotes prostate cancer progression. •miR-182-5p regulates ARRDC3/ITGB4 pathway.
Hu, Nan; Zhang, Jianli; Cui, Wenjing; Kong, Guangyao; Zhang, Shuai; Yue, Lin; Bai, Xiao; Zhang, Zhao; Zhang, Weiying; Zhang, Xiaodong; Ye, Lihong
MicroRNAs play important roles in tumor metastasis. Recently, we reported that the level of miR-520b is inversely related to the metastatic potential of breast cancer cells. In this study, we investigated the role of miR-520b in breast cancer cell migration. We found that miR-520b suppressed the migration of breast cancer cells with high metastatic potential, including MDA-MB-231 and LM-MCF-7 cells, although the inhibition of miR-520b enhanced the migration of low metastatic potential MCF-7 cells. We further discovered that miR-520b directly targets the 3'-untranslated region (3'UTR) of either hepatitis B X-interacting protein (HBXIP) or interleukin-8 (IL-8), which has been reported to contribute to cell migration. Surprisingly, tissue array assays showed that 75% (38:49) and 94% (36:38) of breast cancer tissues and metastatic lymph tissues, respectively, were positive for HBXIP expression. Moreover, overexpression of HBXIP was able to promote the migration of MCF-7 cells. Interestingly, HBXIP was able to regulate IL-8 transcription by NF-κB, suggesting that the two target genes of miR-520b are functionally connected. In addition, we found that miR-520b could indirectly regulate IL-8 transcription by targeting HBXIP. Thus, we conclude that miR-520b is involved in regulating breast cancer cell migration by targeting HBXIP and IL-8 via a network in which HBXIP promotes migration by stimulating NF-κB-mediated IL-8 expression. These studies point to HBXIP as a potential therapeutic target for breast cancer.
Pelkey, Kenneth A; McBain, Chris J
Individual axons of central neurons innervate a large number of distinct postsynaptic targets belonging to divergent functional categories such as glutamatergic principal cells and inhibitory interneurons. While each bouton along a common axon should experience the same activity pattern in response to action potential firing within the parent presynaptic neuron, accumulating evidence suggests that neighbouring boutons contacting functionally distinct postsynaptic targets regulate their release properties independently, despite being separated by only a few microns. This target-cell-specific autonomy of presynaptic function can greatly expand the computational prowess of central axons to allow for precise coordination of large neuronal ensembles within a given circuit. An excellent example of target-cell-specific presynaptic mechanisms occurs in the CA3 hippocampus where mossy fibre (MF) axons of dentate gyrus granule cells target both principal cells and local circuit inhibitory interneurons via both anatomically and functionally specialized terminals. Of particular interest, mechanisms of both short- and long-term plasticity remain autonomous at these divergent release sites due to an anatomical and biochemical segregation of discrete molecular signalling cascades. Here we review roughly a decades worth of research on the MF-CA3 pathway to showcase the target-cell dependence of presynaptically expressed NMDA receptor-independent synaptic plasticity.
Full Text Available Odontogenesis relies on the reciprocal signaling interactions between dental epithelium and neural crest-derived mesenchyme, which is regulated by several signaling pathways. Subtle changes in the activity of these major signaling pathways can have dramatic effects on tooth development. An important regulator of such subtle changes is the fine tuning function of microRNAs (miRNAs. However, the underlying mechanism by which miRNAs regulate tooth development remains elusive. This study determined the expression of miRNAs during cytodifferentiation in the human tooth germ and studied miR-34a as a regulator of dental papilla cell differentiation. Using microarrays, miRNA expression profiles were established at selected times during development (early bell stage or late bell stage of the human fetal tooth germ. We identified 29 differentially expressed miRNAs from early bell stage/late bell stage comparisons. Out of 6 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. miR-34a was selected for further investigation because it has been previously reported to regulate organogenesis. miR-34a mimics and inhibitors were transfected into human fetal dental papilla cells, mRNA levels of predicted target genes were detected by quantitative real-time PCR, and levels of putative target proteins were examined by western blotting. ALP and DSPP expression were also tested by qPCR, western blotting, and immunofluorescence. Findings from these studies suggested that miR-34a may play important roles in dental papilla cell differentiation during human tooth development by targeting NOTCH and TGF-beta signaling.
Sun, Feifei; Tang, Yin; Ye, Ling; Fan, Yi; Klein, Ophir D.; Zhou, Xuedong; Zheng, Liwei
Odontogenesis relies on the reciprocal signaling interactions between dental epithelium and neural crest-derived mesenchyme, which is regulated by several signaling pathways. Subtle changes in the activity of these major signaling pathways can have dramatic effects on tooth development. An important regulator of such subtle changes is the fine tuning function of microRNAs (miRNAs). However, the underlying mechanism by which miRNAs regulate tooth development remains elusive. This study determined the expression of miRNAs during cytodifferentiation in the human tooth germ and studied miR-34a as a regulator of dental papilla cell differentiation. Using microarrays, miRNA expression profiles were established at selected times during development (early bell stage or late bell stage) of the human fetal tooth germ. We identified 29 differentially expressed miRNAs from early bell stage/late bell stage comparisons. Out of 6 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. miR-34a was selected for further investigation because it has been previously reported to regulate organogenesis. miR-34a mimics and inhibitors were transfected into human fetal dental papilla cells, mRNA levels of predicted target genes were detected by quantitative real-time PCR, and levels of putative target proteins were examined by western blotting. ALP and DSPP expression were also tested by qPCR, western blotting, and immunofluorescence. Findings from these studies suggested that miR-34a may play important roles in dental papilla cell differentiation during human tooth development by targeting NOTCH and TGF-beta signaling. PMID:23226240
Wang, Qinhu; Li, Tingting; Xu, Ke; Zhang, Wei; Wang, Xiaolong; Quan, Junli; Jin, Weibo; Zhang, Meixiang; Fan, Guangjin; Wang, Ming-Bo; Shan, Weixing
Along with the well-studied microRNA (miRNA) and small interfering RNA (siRNA) is a new class of transfer RNA-derived small RNA (tsRNA), which has recently been detected in multiple organisms and is implicated in gene regulation. However, while miRNAs and siRNAs are known to repress gene expression through sequence-specific RNA cleavage or translational repression, how tsRNAs regulate gene expression remains unclear. Here we report the identification and functional characterization of tsRNAs in the oomycete pathogen Phytophthora sojae. We show that multiple tRNAs are processed into abundant tsRNAs, which accumulate in a similar developmental stage-specific manner and are negatively correlated with the expression of predicted target genes. Degradome sequencing and 5′ RLM RACE experiments indicate tsRNAs can trigger degradation of target transcripts. Transient expression assays using GUS sensor constructs confirmed the requirement of sequence complementarity in tsRNA-mediated RNA degradation in P. sojae. Our results show that the tsRNA are a class of functional endogenous sRNAs and suggest that tsRNA regulate gene expression through inducing sequence-specific degradation of target RNAs in oomycetes. PMID:28066490
Ehret, Georg B; Ferreira, Teresa; Chasman, Daniel I;
To dissect the genetic architecture of blood pressure and assess effects on target organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals were used for validation. We identified 66...... across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target organ damage in multiple tissues but with minor effects in the kidney. Our findings...
Kong, Ye; Zou, Shuiyan; Yang, Fenghua; Xu, Xia; Bu, Wenhong; Jia, Jihui; Liu, Zhifang
RUNX3 is a transcriptional factor that has been shown to regulate protein-coding gene expression at the transcriptional level. However, the regulation of RUNX3 on miRNAs is not fully understood. In this study, we used miRNA microarray to identify the miRNAs that are regulated by RUNX3 and found that miR-29b showed the most up-regulation in RUNX3 over-expressed cells compared with the control cells. We used qRT-PCR to confirm the miRNA microarray results in several gastric cancer cells and found that RUNX3 could bind to the miR-29b promoter directly and cooperate with Smad3 to increase the promoter activity of miR-29b. In the clinical setting, both RUNX3 and miR-29b are down-regulated significantly in human gastric cancer tissues. A positive correlation between miR-29b and RUNX3 was found in the gastric cancer tissues. Additionally, we found that miR-29b suppressed the proliferation and metastasis of gastric cancer cells by directly targeting KDM2A. The miR-29b/KDM2A axis was involved in the RUNX3-mediated inhibition of gastric cancer cell proliferation and metastasis. Taken together, our results suggested that RUNX3-mediated up-regulation of miR-29b inhibited the proliferation and migration of gastric cancer cells by targeting KDM2A, representing a novel molecular mechanism for the tumor suppression action of RUNX3.
Zhai, Lei; Wang, Chenchen; Chen, Yuanfan; Zhou, Shixin; Li, Lingsong
Embryonic stem cells (ESCs) are pluripotent cells and have the capability for differentiation into any of the three embryonic germ layers. The Wnt/β-Catenin pathway has been shown to play an essential role in ESC differentiation regulation. Activation of β-Catenin by post-translational modification has been extensively studied. However, mechanism(s) of post-transcriptional regulation of β-Catenin are not well defined. In this study, we report an RNA recognition motif-containing protein (RNA binding motif protein 46, RBM46) which regulates the degradation of β-Catenin mRNA. Our results show that Rbm46 is distributed primarily in the cytoplasm of mouse ESCs (mESCs) and is elevated during the process of ESC differentiation. In addition, overexpression of Rbm46 results in differentiation of mESCs into trophectoderm, while knock-down of Rbm46 leads to mESC differentiation into endoderm. β-Catenin, a key effector in the Wnt pathway which has been reported to play a significant role in the regulation of ESC differentiation, is post-transcriptionally regulated by Rbm46. Our study reveals Rbm46 plays a novel role in the regulation of ESC differentiation. PMID:28212427
Anthony J E Berndt
Full Text Available Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE that binds BMP-activated pMad, and a homeodomain-response element (HD-RE that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp, as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP
Cardoso, Angelo A; Jiang, Yanlin; Luo, Meihua; Reed, April M; Shahda, Safi; He, Ying; Maitra, Anirban; Kelley, Mark R; Fishel, Melissa L
Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3-APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3-APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.
Full Text Available Zinc oxide (ZnO nanoparticles (NPs have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 μg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 μg/ml and ZnSO4-10 μg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 μg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 μg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways.
Full Text Available The maintenance phase of memory-related long-term facilitation (LTF of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT-triggered presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22 that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC, another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.
Full Text Available The scandals that have tarnished the reputation of Wall Street and in Italy have damaged those who have invested their savings in Parmalat or Cirio corporate bonds or in Argentinean Government securities justify revisiting the objectives to be achieved by regulation and supervision of the financial sector. Stability is still regarded as paramount for banking, to be secured through an appropriate capital cushion, extensive supervisory arrangements, and market discipline, as agreed by Basel 2. Other financial intermediaries need to conduct their business with great transparency, openness and respect of the rules. However, an ever expanding and innovative financial sector, the blurring of traditional segmentations and globalisation make the task of regulating and supervising the financial sector more difficult and challenging, thus requiring new frameworks, for instance a single agency, in various jurisdictions and more international co-ordination and co-operation among regulating and supervising agencies. Up to now, the approach followed by the latter has been microeconomic, but the growing financial instability and the greater relevance of systemic risk may ask for a macroeconomic management of prudential regulation and supervision, thus complementing monetary policy in securing financial stability. JEL Codes: E52, G18, G21, G28Keywords: Banking; Regulation; Securities
Yang, Di; Okamura, Hirohiko; Teramachi, Jumpei; Haneji, Tatsuji
Posttranslational modifications including histone methylation regulate gene transcription through directly affecting the structure of chromatin. Trimethylation of histone H3K27 (H3K27me3) contributes to gene silencing and the histone demethylase Jumonji domain-containing 3 (Jmjd3) specifically removes the methylation of H3K27me3, followed by the activation of gene expression. In the present study, we explored the roles of Jmjd3 in regulating osteoblast apoptosis. Knockdown of Jmjd3 promoted osteoblast apoptosis induced by serum deprivation with decreased mitochondrial membrane potential and increased levels of caspase-3 activation, PARP cleavage, and DNA fragmentation. B cell lymphoma-2 (Bcl-2), an anti-apoptotic protein, was down-regulated by knockdown of Jmjd3 through retaining H3K27me3 on its promoter region. Knockdown of Jmjd3 increased the pro-apoptotic activity of Bim through inhibiting ERK-dependent phosphorylation of Bim. Protein kinase D1 (PKD1), which stimulates ERK phosphorylation, decreased in the Jmjd3-knockdown cells and introduction of PKD1 relieved osteoblast apoptosis in the Jmjd3-knockdown cells through increasing ERK-regulated Bim phosphorylation. These results suggest that Jmjd3 regulates osteoblast apoptosis through targeting Bcl-2 expression and Bim phosphorylation.
Full Text Available Abstract Background In breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer. Results First, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab, demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation. Conclusion In this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not
Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko
A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX acc...
Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko
A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ3-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ3-irradiation, including HP1β-, γ3H2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX ...
Bai, Lijing; Liang, Ruyi; Yang, Yalan; Hou, Xinhua; Wang, Zishuai; Zhu, Shiyun; Wang, Chuduan; Tang, Zhonglin; Li, Kui
MicroRNAs (miRNAs), which are short (22-24 base pairs), non-coding RNAs, play critical roles in myogenesis. Using Solexa deep sequencing, we detected the expression levels of 229 and 209 miRNAs in swine skeletal muscle at 90 days post-coitus (E90) and 100 days postnatal (D100), respectively. A total of 138 miRNAs were up-regulated on E90, and 31 were up-regulated on D100. Of these, 9 miRNAs were selected for the validation of the small RNA libraries by quantitative RT-PCR (RT-qPCR). We found that miRNA-21 was down-regulated by 17-fold on D100 (P<0.001). Bioinformatics analysis suggested that the transforming growth factor beta-induced (TGFβI) gene was a potential target of miRNA-21. Both dual luciferase reporter assays and western blotting demonstrated that the TGFβI gene was regulated by miRNA-21. Co-expression analysis revealed that the mRNA expression levels of miRNA-21 and TGFβI were negatively correlated (r = -0.421, P = 0.026) in skeletal muscle during the 28 developmental stages. Our results revealed that more miRNAs are expressed in prenatal than in postnatal skeletal muscle. The miRNA-21 is a novel myogenic miRNA that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene.
Full Text Available MicroRNAs (miRNAs, which are short (22-24 base pairs, non-coding RNAs, play critical roles in myogenesis. Using Solexa deep sequencing, we detected the expression levels of 229 and 209 miRNAs in swine skeletal muscle at 90 days post-coitus (E90 and 100 days postnatal (D100, respectively. A total of 138 miRNAs were up-regulated on E90, and 31 were up-regulated on D100. Of these, 9 miRNAs were selected for the validation of the small RNA libraries by quantitative RT-PCR (RT-qPCR. We found that miRNA-21 was down-regulated by 17-fold on D100 (P<0.001. Bioinformatics analysis suggested that the transforming growth factor beta-induced (TGFβI gene was a potential target of miRNA-21. Both dual luciferase reporter assays and western blotting demonstrated that the TGFβI gene was regulated by miRNA-21. Co-expression analysis revealed that the mRNA expression levels of miRNA-21 and TGFβI were negatively correlated (r = -0.421, P = 0.026 in skeletal muscle during the 28 developmental stages. Our results revealed that more miRNAs are expressed in prenatal than in postnatal skeletal muscle. The miRNA-21 is a novel myogenic miRNA that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene.
Full Text Available MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16 and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs, and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid and heat stress. The pri-miR160 was down-regulated in response to salicylic acid but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a* and -d* reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a* expression level. This suggests that they may function to abolish the binding between dlo-miR160a* and its targets. These two eTMs also participated in auxin and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The e
Sittka, A; Lucchini, S; Papenfort, K
explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant......-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria....
Tamura, M; Sasaki, Y; Koyama, R; Takeda, K; Idogawa, M; Tokino, T
p53 is an established tumor suppressor that can activate the transcription of multiple target genes. Recent evidence suggests that p53 may contribute to the regulation of cell invasion and migration. In this study, we show that the forkhead box transcription factor FOXF1 is a novel target of the p53 family because FOXF1 is upregulated by p53, TAp73 and TAp63. We show that FOXF1 is induced upon DNA damage in a p53-dependent manner. Furthermore, we identified a response element located within the FOXF1 gene that is responsive to wild-type p53, TAp73β and TAp63γ. The ectopic expression of FOXF1 inhibited cancer cell invasion and migration, whereas the inactivation of FOXF1 stimulated cell invasion and migration. We also show that FOXF1 regulates the transcriptional activity of E-cadherin (CDH1) by acting on its FOXF1 consensus binding site located upstream of the E-cadherin gene. Collectively, our results show that FOXF1 is a p53 family target gene, and our data suggest that FOXF1 and p53 form a portion of a regulatory transcriptional network that appears to have an important role in cancer cell invasion and migration.
He, Zhi-Mei; Zhang, Peng-Hui; Li, Xin; Zhang, Jian-Rong; Zhu, Jun-Jie
As catalytic nucleic acids, DNAzymes have been extensively used in the design of sensing platforms. However, their potentials as intelligent drug carriers for responsive drug release in gene therapy and chemotherapy were rarely explored. Herein, we report a dual-functional probe composed of gold nanoparticles (GNPs), catalytic Zn(2+)-dependent DNAzyme, anticancer drug doxorubicin (Dox), targeted AS1411 aptamer and acid-decomposable ZnO quantum dots (ZnO QDs) to achieve intracellular gene regulation and drug delivery in a controlled manner. By means of aptamer-guided targeting and receptor-mediated endocytosis, the probes were specifically internalized into the HeLa cells and trapped in the acidic endo-/lysosomes, where the ZnO QDs as the built-in Zn(2+) arsenal were promptly dissolved to offer Zn(2+), leading to the activation of DNAzyme to cleave the substrate strands, and subsequent drug release. Meanwhile, as designed, one part of the cleaved substrate, hybridized with the overexpressed miR-21 in the target cells, thereby declining its intracellular level. Taken together, the down-regulation of miR-21 has a synergistic effect with Dox to efficiently eradicate the cancer cells. Thus, the favorable biocompatibility, cancer cell specificity and combined treatment make the probe promising for therapy of multidrug-resistant cancer and in vivo application.
He, Zhi-Mei; Zhang, Peng-Hui; Li, Xin; Zhang, Jian-Rong; Zhu, Jun-Jie
As catalytic nucleic acids, DNAzymes have been extensively used in the design of sensing platforms. However, their potentials as intelligent drug carriers for responsive drug release in gene therapy and chemotherapy were rarely explored. Herein, we report a dual-functional probe composed of gold nanoparticles (GNPs), catalytic Zn2+-dependent DNAzyme, anticancer drug doxorubicin (Dox), targeted AS1411 aptamer and acid-decomposable ZnO quantum dots (ZnO QDs) to achieve intracellular gene regulation and drug delivery in a controlled manner. By means of aptamer-guided targeting and receptor-mediated endocytosis, the probes were specifically internalized into the HeLa cells and trapped in the acidic endo-/lysosomes, where the ZnO QDs as the built-in Zn2+ arsenal were promptly dissolved to offer Zn2+, leading to the activation of DNAzyme to cleave the substrate strands, and subsequent drug release. Meanwhile, as designed, one part of the cleaved substrate, hybridized with the overexpressed miR-21 in the target cells, thereby declining its intracellular level. Taken together, the down-regulation of miR-21 has a synergistic effect with Dox to efficiently eradicate the cancer cells. Thus, the favorable biocompatibility, cancer cell specificity and combined treatment make the probe promising for therapy of multidrug-resistant cancer and in vivo application. PMID:26956167
Li, Weina, E-mail: firstname.lastname@example.org [Department of Biomedical Engineering, Fourth Military Medical University, Xi’an 710032 (China); He, Fei, E-mail: email@example.com [Department of Hepatic Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032 (China)
Highlights: • Expression of MMD is increased in lung cancer tissues. • Knockdown of MMD inhibits growth of A549 and LLC cells in vitro and in vivo. • MMD is a direct functional target of miR-140-5p. • MiR-140-5p/MMD axis regulates Erk1/2 signaling. - Abstract: Monocyte to macrophage differentiation-associated (MMD) is identified in macrophages as a gene associated with the differentiation from monocytes to macrophages. Recent microarray analysis for non-small cell lung cancer (NSCLC) suggests that MMD is an important signature associated with relapse and survival among patients with NSCLC. Therefore, we speculate that MMD likely plays a role in lung cancer. In this study, we found that the protein level of MMD was increased in lung cancer compared to benign lung tissues, and knockdown of MMD inhibited the growth of A549 and Lewis lung cancer cells (LLC) in vitro and in vivo. Integrated analysis demonstrated that MMD was a direct functional target of miR-140-5p. Furthermore, we found that miR-140-5p/MMD axis could affect the cell proliferation of lung cancer cells by regulating Erk signaling. Together, our results highlight the significance of miR-140-5p/MMD axis in lung cancer, and miR-140-5p/MMD axis could serve as new molecular targets for the therapy against lung cancer.
Full Text Available Abstract Background Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal. Methods In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts. Results The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation. Conclusions These results indicate that P53 target genes involved in apoptosis and cell
Shao, Yi; Feng, Lihui; Rutherford, Steven T; Papenfort, Kai; Bassler, Bonnie L
Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1-5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, Qrr sRNAs-controlled genes are the most rapid to respond to quorum-sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.
Full Text Available BACKGROUND: MicroRNA-34a (miR-34a is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC and HDAC inhibitor Vorinostat (SAHA. METHODOLOGY/PRINCIPAL FINDINGS: Re-expression of miR-34a in human pancreatic cancer stem cells (CSCs and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT and invasion. In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. MiR-34a upregulation by these agents also induced acetylated p53, p21(WAF1, p27(KIP1 and PUMA in pancreatic CSCs. Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. In CSCs, SAHA inhibited Notch pathway, suggesting its suppression may contribute to inhibition of the self-renewal capacity and induction of apoptosis. Interestingly, treatment of pancreatic CSCs with SAHA resulted in the inhibition of EMT with the transcriptional up-regulation of E-Cadherin and down-regulation of N-Cadherin. Expression of EMT inducers (Zeb-1, Snail and Slug was inhibited in CSCs upon treatment with SAHA. 5-Aza-dC and SAHA also retard in vitro migration and invasion of CSCs. CONCLUSIONS: The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. The restoration of its expression by 5-Aza-dC and SAHA in CSCs will not only provide mechanistic
Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M
3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during...
Jansson, M D; Djodji Damas, Nkerorema; Lees, M
MicroRNAs (miRNAs) regulate many key cancer-relevant pathways and may themselves possess oncogenic or tumor-suppressor functions. Consequently, miRNA dysregulation has been shown to be a prominent feature in many human cancers. The p53 tumor suppressor acts as a negative regulator of cell...... proliferation in response to stress and represents the most commonly lost and mutated gene in human cancers. The function of p53 is inhibited by the MDM2 oncoprotein. Using a high-throughput screening approach, we identified miR-339-5p as a regulator of the p53 pathway. We demonstrate that this regulation...... inhibition of miR-339-5p function perturbs the p53 response in cancer cells, allowing an increased proliferation rate. In addition, miR-339-5p expression is downregulated in tumors harboring wild-type TP53, suggesting that reduction of miR-339-5p level helps to suppress the p53 response in p53-competent...
Lijun Liu; Matthew S. Zinkgraf; H. Earl Petzold; Eric P. Beers; Vladimir Filkov; Andrew Groover
The class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.
Pekcec, A.; Unkrüer, B.; Schlichtiger, J.; Soerensen, J.; Hartz, A.M.S.; Bauer, B.; van Vliet, E.A.; Gorter, J.A.; Potschka, H.
Up-regulation of the blood-brain barrier efflux transporter P-glycoprotein in central nervous system disorders results in restricted brain access and limited efficacy of therapeutic drugs. In epilepsies, seizure activity strongly triggers expression of P-glycoprotein. Here, we identified the prostag
Jasińska, Magdalena; Miłek, Jacek; Cymerman, Iwona A; Łęski, Szymon; Kaczmarek, Leszek; Dziembowska, Magdalena
Mir-132 is a neuronal activity-regulated microRNA that controls the morphology of dendritic spines and neuronal transmission. Similar activities have recently been attributed to matrix metalloproteinase-9 (MMP-9), an extrasynaptic protease. In the present study, we provide evidence that miR-132 directly regulates MMP-9 mRNA in neurons to modulate synaptic plasticity. With the use of luciferase reporter system, we show that miR-132 binds to the 3'UTR of MMP-9 mRNA to regulate its expression in neurons. The overexpression of miR-132 in neurons reduces the level of endogenous MMP-9 protein secretion. In synaptoneurosomes, metabotropic glutamate receptor (mGluR)-induced signaling stimulates the dissociation of miR-132 from polyribosomal fractions and shifts it towards the messenger ribonucleoprotein (mRNP)-containing fraction. Furthermore, we demonstrate that the overexpression of miR-132 in the cultured hippocampal neurons from Fmr1 KO mice that have increased synaptic MMP-9 level provokes enlargement of the dendritic spine heads, a process previously implicated in enhanced synaptic plasticity. We propose that activity-dependent miR-132 regulates structural plasticity of dendritic spines through matrix metalloproteinase 9.
Full Text Available The scandals that have tarnished the reputation of Wall Street and in Italy have damaged those who have invested their savings in Parmalat or Cirio corporate bonds or in Argentinean Government securities justify revisiting the objectives to be achieved by regulation and supervision of the financial sector. Stability is still regarded as paramount for banking, to be secured through an appropriate capital cushion, extensive supervisory arrangements, and market discipline, as agreed by Basel 2. Other financial intermediaries need to conduct their business with great transparency, openness and respect of the rules. However, an ever expanding and innovative financial sector, the blurring of traditional segmentations and globalisation make the task of regulating and supervising the financial sector more difficult and challenging, thus requiring new frameworks, for instance a single agency, in various jurisdictions and more international co-ordination and co-operation among regulating and supervising agencies. Up to now, the approach followed by the latter has been microeconomic, but the growing financial instability and the greater relevance of systemic risk may ask for a macroeconomic management of prudential regulation and supervision, thus complementing monetary policy in securing financial stability.
Kristina B. V. Døssing
Full Text Available Expression of miRNAs in Neuroendocrine Neoplasms (NEN is poorly characterized. We therefore wanted to examine the miRNA expression in Neuroendocrine Tumors (NETs, and identify their targets and importance in NET carcinogenesis. miRNA expression in six NEN primary tumors, six NEN metastases and four normal intestinal tissues was characterized using miRNA arrays, and validated by in-situ hybridization and qPCR. Among the down-regulated miRNAs miR-129-5p and the let-7f/let-7 family, were selected for further characterization. Transfection of miR-129-5p inhibited growth of a pulmonary and an intestinal carcinoid cell line. Analysis of mRNA expression changes identified EGR1 and G3BP1 as miR-129-5p targets. They were validated by luciferase assay and western blotting, and found robustly expressed in NETs by immunohistochemistry. Knockdown of EGR1 and G3BP1 mimicked the growth inhibition induced by miR-129-5p. let-7 overexpression inhibited growth of carcinoid cell lines, and let-7 inhibition increased protein content of the transcription factor BACH1 and its targets MMP1 and HMGA2, all known to promote bone metastases. Immunohistochemistry analysis revealed that let-7 targets are highly expressed in NETs and metastases. We found down-regulation of miR-129-5p and the let-7 family, and identified new neuroendocrine specific targets for these miRNAs, which contributes to the growth and metastatic potential of these tumors.
Yi, W; Ross, J M; Zarkower, D
Sex determination is controlled by global regulatory genes, such as tra-1 in Caenorhabditis elegans, Sex lethal in Drosophila, or Sry in mammals. How these genes coordinate sexual differentiation throughout the body is a key unanswered question. tra-1 encodes a zinc finger transcription factor, TRA-1A, that regulates, directly or indirectly, all genes required for sexual development. mab-3 (male abnormal 3), acts downstream of tra-1 and is known to be required for sexual differentiation of at least two tissues. mab-3 directly regulates yolk protein transcription in the intestine and specifies male sense organ differentiation in the nervous system. It encodes a transcription factor related to the products of the Drosophila sexual regulator doublesex (dsx), which also regulates yolk protein transcription and male sense-organ differentiation. The similarities between mab-3 and dsx led us to suggest that some aspects of sex determination may be evolutionarily conserved. Here we find that mab-3 is also required for expression of male-specific genes in sensory neurons of the head and tail and for male interaction with hermaphrodites. These roles in male development and behavior suggest further functional similarity to dsx. In male sensory ray differentiation we find that MAB-3 acts synergistically with LIN-32, a neurogenic bHLH transcription factor. Expression of LIN-32 is spatially restricted by the combined action of the Hox gene mab-5 and the hairy homolog lin-22, while MAB-3 is expressed throughout the lateral hypodermis. Finally, we find that mab-3 transcription is directly regulated in the intestine by TRA-1A, providing a molecular link between the global regulatory pathway and terminal sexual differentiation.
Rebecca L Skalsky
Full Text Available Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers.
Full Text Available So far little is known on the functional role of phosphorylation in the heat stress response of plants. Here we present evidence that heat stress activates the Arabidopsis mitogen-activated protein kinase MPK6. In vitro and in vivo evidence is provided that MPK6 specifically targets the major heat stress transcription factor HsfA2. Activation of MPK6 results in complex formation with HsfA2. MPK6 phosphorylates HsfA2 on T249 and changes its intracellular localisation. Protein kinase and phosphatase inhibitor studies indicate that HsfA2 protein stability is regulated in a phosphorylation-dependent manner, but this mechanism is independent of MPK6. Overall, our data show that heat stress-induced targeting of HsfA2 by MPK6 participates in the complex regulatory mechanism how plants respond to heat stress.
Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin
The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.
Ponente, Manfredi; Campanini, Letizia; Cuttano, Roberto; Piunti, Andrea; Delledonne, Giacomo A; Coltella, Nadia; Valsecchi, Roberta; Villa, Alessandra; Cavallaro, Ugo; Pattini, Linda; Doglioni, Claudio; Bernardi, Rosa
Elucidating the molecular basis of tumor metastasis is pivotal for eradicating cancer-related mortality. Triple-negative breast cancer (TNBC) encompasses a class of aggressive tumors characterized by high rates of recurrence and metastasis, as well as poor overall survival. Here, we find that the promyelocytic leukemia protein PML exerts a prometastatic function in TNBC that can be targeted by arsenic trioxide. We found that, in TNBC patients, constitutive HIF1A activity induces high expression of PML, along with a number of HIF1A target genes that promote metastasis at multiple levels. Intriguingly, PML controls the expression of these genes by binding to their regulatory regions along with HIF1A. This mechanism is specific to TNBC cells and does not occur in other subtypes of breast cancer where PML and prometastatic HIF1A target genes are underexpressed. As a consequence, PML promotes cell migration, invasion, and metastasis in TNBC cell and mouse models. Notably, pharmacological inhibition of PML with arsenic trioxide, a PML-degrading agent used to treat promyelocytic leukemia patients, delays tumor growth, impairs TNBC metastasis, and cooperates with chemotherapy by preventing metastatic dissemination. In conclusion, we report identification of a prometastatic pathway in TNBC and suggest clinical development toward the use of arsenic trioxide for TNBC patients.
Kim, Hyoji; Choi, Hoyun; Lee, Suk Kyeong
Although Epstein-Barr virus (EBV) BamHI A rightward transcript (BART) microRNAs (miRNAs) are ubiquitously expressed in EBV-associated tumors, the role of most BART miRNAs is unclear. In this study, we showed that Bcl-2-associated death promoter (BAD) expression was significantly lower in EBV-infected AGS-EBV cells than in EBV-negative AGS cells and investigated whether BART miRNAs target BAD. Using bioinformatics analysis, five BART miRNAs showing seed match with the 3' untranslated region (3'-UTR) of BAD were selected. Of these, only miR-BART20-5p reduced BAD expression when individually transfected into AGS cells. A luciferase assay revealed that miR-BART20-5p directly targets BAD. The expression of BAD mRNA and protein was decreased by miR-BART20-5p and increased by an inhibitor of miR-BART20-5p. PE-Annexin V staining and cell proliferation assays showed that miR-BART20-5p reduced apoptosis and enhanced cell growth. Furthermore, miR-BART20-5p increased chemoresistance to 5-fluorouracil and docetaxel. Our data suggest that miR-BART20-5p contributes to tumorigenesis of EBV-associated gastric carcinoma by directly targeting the 3'-UTR of BAD.
Ponente, Manfredi; Campanini, Letizia; Cuttano, Roberto; Piunti, Andrea; Delledonne, Giacomo A.; Coltella, Nadia; Valsecchi, Roberta; Villa, Alessandra
Elucidating the molecular basis of tumor metastasis is pivotal for eradicating cancer-related mortality. Triple-negative breast cancer (TNBC) encompasses a class of aggressive tumors characterized by high rates of recurrence and metastasis, as well as poor overall survival. Here, we find that the promyelocytic leukemia protein PML exerts a prometastatic function in TNBC that can be targeted by arsenic trioxide. We found that, in TNBC patients, constitutive HIF1A activity induces high expression of PML, along with a number of HIF1A target genes that promote metastasis at multiple levels. Intriguingly, PML controls the expression of these genes by binding to their regulatory regions along with HIF1A. This mechanism is specific to TNBC cells and does not occur in other subtypes of breast cancer where PML and prometastatic HIF1A target genes are underexpressed. As a consequence, PML promotes cell migration, invasion, and metastasis in TNBC cell and mouse models. Notably, pharmacological inhibition of PML with arsenic trioxide, a PML-degrading agent used to treat promyelocytic leukemia patients, delays tumor growth, impairs TNBC metastasis, and cooperates with chemotherapy by preventing metastatic dissemination. In conclusion, we report identification of a prometastatic pathway in TNBC and suggest clinical development toward the use of arsenic trioxide for TNBC patients. PMID:28239645
Giovannini, Catia; Gramantieri, Laura; Minguzzi, Manuela; Fornari, Francesca; Chieco, Pasquale; Grazi, Gian Luca; Bolondi, Luigi
CDKN1C/P57 is a cyclin-dependent kinase inhibitor implicated in different human cancers, including hepatocellular carcinoma (HCC); however, little is known regarding the role of CDKN1C/P57 and its regulation in HCC. In this study, we show that the down-regulation of Notch1 and Notch3 in two HCC cell lines resulted in Hes1 down-regulation, CDKN1C/P57 up-regulation, and reduced cell growth. In line with these data, we report that CDKN1C/P57 is a target of transcriptional repression by the Notch effector, Hes1. We found that the up-regulation of CDKN1C/P57 by cDNA transfection decreased tumor growth, as determined by growth curve, flow cytometry analysis, and cyclin D1 down-regulation, without affecting the apoptosis machinery. Indeed, the expression of Bax, Noxa, PUMA, BNIP(3), and cleaved caspase-3 was not affected by CDKN1C/P57 induction. Morphologically CDKN1C/p57-induced HCC cells became flat and lengthened in shape, accumulated the senescence-associated β-galactosidase marker, and increased P16 protein expression. Evaluation of senescence in cells depleted both for Hes1 and CDKN1C/P57 revealed that the senescent state really depends on the accumulation of CDKN1C/p57. Finally, we validated our in vitro results in primary HCCs, showing that Hes1 protein expression inversely correlates with CDKN1C/P57 mRNA levels. In addition, reduced Hes1 protein expression is accompanied by a shorter time to recurrence after curative resection, suggesting that Hes1 may represent a biomarker for prediction of patients with poor prognosis.
Full Text Available Several factors play a role in obesity (i.e. behavior, environment, and genetics and epigenetic regulation of gene expression has emerged as a potential contributor in the susceptibility and development of obesity. To investigate the individual sensitivity to weight gain/resistance, we here studied gene transcription regulation of several hypothalamic neuropeptides involved in the control of energy balance in rats developing obesity (diet-induced obesity, DIO or not (diet resistant, DR, when fed with a high fat diet. Rats have been followed up to 21 weeks of high fat diet exposure. After 5 weeks high fat diet exposure, the obese phenotype was developed and we observed a selective down-regulation of the orexygenic neuropeptide Y (NPY and peroxisome proliferator-activated receptor gamma (PPAR-γ genes. No changes were observed in the expression of the agouti-related protein (AgRP, as well as for all the anorexigenic genes under study. After long-term high fat diet exposure (21 weeks, NPY and PPAR-γ, as well as most of the genes under study, resulted not be different between DIO and DR, whereas a lower expression of the anorexigenic pro-opio-melanocortin (POMC gene was observed in DIO rats when compared to DR rats. Moreover we observed that changes in NPY and POMC mRNA were inversely correlated with gene promoters DNA methylation. Our findings suggest that selective alterations in hypothalamic peptide genes regulation could contribute to the development of overweight in rats and that environmental factor, as in this animal model, might be partially responsible of these changes via epigenetic mechanism.
Cifani, Carlo; Micioni Di Bonaventura, Maria V; Pucci, Mariangela; Giusepponi, Maria E; Romano, Adele; Di Francesco, Andrea; Maccarrone, Mauro; D'Addario, Claudio
Several factors play a role in obesity (i.e., behavior, environment, and genetics) and epigenetic regulation of gene expression has emerged as a potential contributor in the susceptibility and development of obesity. To investigate the individual sensitivity to weight gain/resistance, we here studied gene transcription regulation of several hypothalamic neuropeptides involved in the control of energy balance in rats developing obesity (diet-induced obesity, DIO) or not (diet resistant, DR), when fed with a high fat diet. Rats have been followed up to 21 weeks of high fat diet exposure. After 5 weeks high fat diet exposure, the obese phenotype was developed and we observed a selective down-regulation of the orexigenic neuropeptide Y (NPY) and peroxisome proliferator-activated receptor gamma (PPAR-γ) genes. No changes were observed in the expression of the agouti-related protein (AgRP), as well as for all the anorexigenic genes under study. After long-term high fat diet exposure (21 weeks), NPY and PPAR-γ, as well as most of the genes under study, resulted not be different between DIO and DR, whereas a lower expression of the anorexigenic pro-opio-melanocortin (POMC) gene was observed in DIO rats when compared to DR rats. Moreover we observed that changes in NPY and POMC mRNA were inversely correlated with gene promoters DNA methylation. Our findings suggest that selective alterations in hypothalamic peptide genes regulation could contribute to the development of overweight in rats and that environmental factor, as in this animal model, might be partially responsible of these changes via epigenetic mechanism.
Rosario, Fredrick J; Powell, Theresa L; Jansson, Thomas
Folate deficiency in fetal life is strongly associated with structural malformations and linked to intrauterine growth restriction. In addition, limited availability of methyl donors, such as folate, during pregnancy may result in abnormal gene methylation patterns and contribute to developmental programming. The fetus is dependent on placental transfer of folate, however the molecular mechanisms regulating placental folate transport are unknown. We used cultured primary human trophoblast cells to test the hypothesis that mechanistic target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate folate transport by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal folate uptake. Folate uptake stimulated by insulin + IGF-1 was mediated by mTORC2 but did not involve mTORC1. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of FR-α and RFC transporter isoforms without affecting global protein expression. Inhibition of the ubiquitin ligase Nedd4-2 had no effect on folate transport. In conclusion, we report for the first time that mTORC1/C2 are positive regulators of cellular folate uptake by modulating the cell surface abundance of specific transporter isoforms. We propose that regulation of placental folate transport by mTOR signaling provide a direct link between placental function, gene methylation and fetal programming.
Price, Tulin O; Sheibani, Nader; Shah, Gul N
Events responsible for cerebrovascular disease in diabetes are not fully understood. Pericyte loss is an early event that leads to endothelial cell death, microaneurysms, and cognitive impairment. A biochemical mechanism underlying pericyte loss is rapid respiration (oxidative metabolism of glucose). This escalation in respiration results from free influx of glucose into insulin-insensitive tissues in the face of high glucose levels in the blood. Rapid respiration generates superoxide, the precursor to all reactive oxygen species (ROS), and results in pericyte death. Respiration is regulated by carbonic anhydrases (CAs) VA and VB, the two isozymes expressed in mitochondria, and their pharmacologic inhibition with topiramate reduces respiration, ROS, and pericyte death. Topiramate inhibits both isozymes; therefore, in the earlier studies, their individual roles were not discerned. In a recent genetic study, we showed that mitochondrial CA VA plays a significant role in regulation of reactive oxygen species and pericyte death. The role of CA VB was not addressed. In this report, genetic knockdown and overexpression studies confirm that mitochondrial CA VA regulates respiration in pericytes, whereas mitochondrial CA VB does not contribute significantly. Identification of mitochondrial CA VA as a sole regulator of respiration provides a specific target to develop new drugs with fewer side effects that may be better tolerated and can protect the brain from diabetic injury. Since similar events occur in the capillary beds of other insulin-insensitive tissues such as the eye and kidney, these drugs may also slow the onset and progression of diabetic disease in these tissues.
Van de Poel, Bram; Bulens, Inge; Markoula, Aikaterina; Hertog, Maarten L A T M; Dreesen, Rozemarijn; Wirtz, Markus; Vandoninck, Sandy; Oppermann, Yasmin; Keulemans, Johan; Hell, Ruediger; Waelkens, Etienne; De Proft, Maurice P; Sauter, Margret; Nicolai, Bart M; Geeraerd, Annemie H
The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the postclimacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid oxidase. At the same time, 1-aminocyclopropane-1-carboxylic acid synthase activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during postclimacteric ripening. Postclimacteric red tomatoes on the plant showed only a moderate regulation of 1-aminocyclopropane-1-carboxylic acid synthase and Yang cycle genes compared with the regulation in detached fruit. Treatment of red fruit with 1-methylcyclopropane and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene biosynthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal.
Fernández, Lucía; Breidenstein, Elena B M; Taylor, Patrick K; Bains, Manjeet; de la Fuente-Núñez, César; Fang, Yuan; Foster, Leonard J; Hancock, Robert E W
Besides being a major opportunistic human pathogen, Pseudomonas aeruginosa can be found in a wide range of environments. This versatility is linked to complex regulation, which is achieved through the action of transcriptional regulators, and post-transcriptional regulation by intracellular proteases including Lon. Indeed, lon mutants in this species show defects in motility, biofilm formation, pathogenicity and fluoroquinolone resistance. Here, the proteomic approach stable isotope labeling by amino acids in cell culture (SILAC) was used to search for novel proteolytic targets. One of the proteins that accumulated in the lon mutant was the RNA-binding protein Hfq. Further experiments demonstrated the ability of Lon to degrade Hfq in vitro. Also, overexpression of the hfq gene in the wild-type strain led to partial inhibition of swarming, swimming and twitching motilities, indicating that Hfq accumulation could contribute to the phenotypes displayed by Lon mutants. Hfq overexpression also led to the upregulation of the small regulatory RNA PhrS. Analysis of the phenotypes of strains lacking or overexpressing this sRNA indicated that the Lon protease might be indirectly regulating the levels and activity of sRNAs via Hfq. Overall, this study revealed new links in the complex regulatory chain that controls multicellular behaviours in P. aeruginosa.
Bu, Pengcheng; Chen, Kai-Yuan; Chen, Joyce Huan; Wang, Lihua; Walters, Jewell; Shin, Yong Jun; Goerger, Julian P; Sun, Jian; Witherspoon, Mavee; Rakhilin, Nikolai; Li, Jiahe; Yang, Herman; Milsom, Jeff; Lee, Sang; Zipfel, Warren; Jin, Moonsoo M; Gümüş, Zeynep H; Lipkin, Steven M; Shen, Xiling
microRNAs regulate developmental cell-fate decisions, tissue homeostasis, and oncogenesis in distinct ways relative to proteins. Here, we show that the tumor suppressor microRNA miR-34a is a cell-fate determinant in early-stage dividing colon cancer stem cells (CCSCs). In pair-cell assays, miR-34a distributes at high levels in differentiating progeny, whereas low levels of miR-34a demarcate self-renewing CCSCs. Moreover, miR-34a loss of function and gain of function alter the balance between self-renewal versus differentiation both in vitro and in vivo. Mechanistically, miR-34a sequesters Notch1 mRNA to generate a sharp threshold response where a bimodal Notch signal specifies the choice between self-renewal and differentiation. In contrast, the canonical cell-fate determinant Numb regulates Notch levels in a continuously graded manner. Altogether, our findings highlight a unique microRNA-regulated mechanism that converts noisy input into a toggle switch for robust cell-fate decisions in CCSCs.
Kim, Yongsoon; Sun, Hong
In C. elegans, the highly conserved DAF-2/insulin/insulin-like growth factor 1 receptor signaling (IIS) pathway regulates longevity, metabolism, reproduction and development. In mammals, acid sphingomyelinase (ASM) is an enzyme that hydrolyzes sphingomyelin to produce ceramide. ASM has been implicated in CD95 death receptor signaling under certain stress conditions. However, the involvement of ASM in growth factor receptor signaling under physiological conditions is not known. Here, we report that in vivo ASM functions as a positive regulator of the DAF-2/IIS pathway in C. elegans. We have shown that inactivation of asm-3 extends animal lifespan and promotes dauer arrest, an alternative developmental process. A significant cooperative effect on lifespan is observed between asm-3 deficiency and loss-of-function alleles of the age-1/PI 3-kinase, with the asm-3; age-1 double mutant animals having a mean lifespan 259% greater than that of the wild-type animals. The lifespan extension phenotypes caused by the loss of asm-3 are dependent on the functions of daf-16/FOXO and daf-18/PTEN. We have demonstrated that inactivation of asm-3 causes nuclear translocation of DAF-16::GFP protein, up-regulates endogenous DAF-16 protein levels and activates the downstream targeting genes of DAF-16. Together, our findings reveal a novel role of asm-3 in regulation of lifespan and diapause by modulating IIS pathway. Importantly, we have found that two drugs known to inhibit mammalian ASM activities, desipramine and clomipramine, markedly extend the lifespan of wild-type animals, in a manner similar to that achieved by genetic inactivation of the asm genes. Our studies illustrate a novel strategy of anti-aging by targeting ASM, which may potentially be extended to mammals.
Orlando, Serena; Gallastegui, Edurne; Besson, Arnaud; Abril, Gabriel; Aligué, Rosa; Pujol, Maria Jesus; Bachs, Oriol
Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.
Chen, Cheng; Zhao, Yue; Yu, Yang; Li, Rong; Qiao, Jie
On the women undergoing IVF-ET with elevated progesterone on human chorionic gonadotrophin priming, the assisted reproductive technology outcome is poor. But, due to the unknown mechanism of this process, no effective method has been found to overcome this difficulty. Here, we investigated the roles of miR-125b and its target gene, MMP26, in endometrial receptivity (ER) in these women. The expression of miR-125b was significantly up-regulated in EECs in women with elevated progesterone during the window of implantation, and it showed a progesterone-dependent effect in vitro. Similarly, the expression of miR-125b was significantly up-regulated in the preimplantation period, and was down-regulated in the implantation period and the post-implantation period in mouse EECs. In addition, miR-125b showed a greater decrease at implantation sites than it did at interimplantation sites. The luciferase report assay demonstrated that MMP26 is a target gene of miR-125b. And the expression profile of MMP26 showed an inverse relationship with miR-125b in vivo and in vitro. Overexpression of miR-125b in human EECs inhibited cell migration and invasion. Gain-of-function of miR-125b induced a significant decrease in the number of implantation sites. In conclusion, these data shed new light on how miR-125b triggers ER decline through the regulation of MMP26 function. PMID:27143441
Wang, Lining; Patel, Pralit L.; Yu, Sha; Liu, Bo; Mcleod, Jeffrey D.; Clarke, Leon E.; Chen, Wenying
The rapid growth of energy consumption in China has led to increased emissions of air pollutants. As a response, in its 12th Five Year Plan the Chinese government proposed mitigation targets for SO2 and NOx emissions. Herein we have investigated mitigation measures taken in different sectors and their corresponding impacts on the energy system. Additionally, as non-fossil energy development has gained traction in addressing energy and environmental challenges in China, we further investigated the impact of non-fossil energy development on air pollutant emissions, and then explored interactions and co-benefits between these two types of policies. An extended Global Change Assessment Model (GCAM) was used in this study, which includes an additional air pollutant emissions control module coupling multiple end-of-pipe (EOP) control technologies with energy technologies, as well as more detailed end-use sectors in China. We find that implementing EOP control technologies would reduce air pollution in the near future, but with little room left to implement these EOP technologies, other cleaner and more efficient technologies are also effective. These technologies would reduce final energy consumption, increase electricity’s share in final energy, and increase the share of non-fossil fuels in primary energy and electricity consumption. Increasing non-fossil energy usage at China’s proposed adoption rate would in turn also reduce SO2 and NOx emissions, however, the reductions from this policy alone still lag behind the targeted requirements of air pollutant reduction. Fortunately, a combination of air pollutant controls and non-fossil energy development could synergistically help realize the respective individual targets, and would result in lower costs than would addressing these issues separately.
Bhatia, V; Saini, M. K.; Falzon, M.
Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines; expression correlates with colon carcinoma severity. PTHrP is synthesized as a prepro isoform and contains two targeting sequences – a signal sequence and a nuclear localization signal (NLS). The signal peptide (SP) directs PTHrP to the secretory pathway, where it exerts autocrine/paracrine effects. The NLS directs PTHrP to the nucleus/nucleolus, where it exerts intracrine effects. In this st...
Hirai, Takao; Kobayashi, Tatsuya; Nishimori, Shigeki; Karaplis, Andrew C; Goltzman, David; Kronenberg, Henry M
The blood calcium concentration during fetal life is tightly regulated within a narrow range by highly interactive homeostatic mechanisms that include transport of calcium across the placenta and fluxes in and out of bone; the mechanisms of this regulation are poorly understood. Our findings that endochondral bone-specific PTH/PTHrP receptor (PPR) knockout (KO) mice showed significant reduction of fetal blood calcium concentration compared with that of control littermates at embryonic day 18.5 led us to focus on bone as a possibly major determinant of fetal calcium homeostasis. We found that the fetal calcium concentration of Runx2 KO mice was significantly higher than that of control littermates, suggesting that calcium flux into bone had a considerable influence on the circulating calcium concentration. Moreover, Runx2:PTH double mutant fetuses showed calcium levels similar to those of Runx2 KO mice, suggesting that part of the fetal hypocalcemia in PTH KO mice was caused by the increment of the mineralized bone mass allowed by the formation of osteoblasts. Finally, Rank:PTH double mutant mice had a blood calcium concentration even lower than that of the either Rank KO or PTH KO mice alone at embryonic day 18.5. These observations in our genetic models suggest that PTH/PTHrP receptor signaling in bones has a significant role of the regulation of fetal blood calcium concentration and that both placental transport and osteoclast activation contribute to PTH's hypercalcemic action. They also show that PTH-independent deposition of calcium in bone is the major controller of fetal blood calcium level.
Natalia N Nalivaeva
Full Text Available Abnormal elevation of amyloid β-peptide (Aβ levels in the brain is the primary trigger for neuronal cell death specific to Alzheimer’s disease (AD. It is now evident that Aβ levels in the brain are manipulable due to a dynamic equilibrium between its production from the amyloid precursor protein (APP and removal by amyloid clearance proteins. Clearance can be either enzymic or non-enzymic (binding/transport proteins. Intriguingly several of the main amyloid-degrading enzymes (ADEs are members of the M13 peptidase family (neprilysin (NEP, NEP2 and the endothelin converting enzymes (ECE-1 and -2. A distinct metallopeptidase, insulin-degrading enzyme (IDE, also contributes to Aβ degradation in the brain. The ADE family currently embraces more than 20 members, both membrane-bound and soluble, and of differing cellular locations. NEP plays an important role in brain function terminating neuropeptide signals. Its decrease in specific brain areas with age or after hypoxia, ischaemia or stroke contribute significantly to the development of AD pathology. The recently discovered mechanism of epigenetic regulation of NEP (and other genes by the APP intracellular domain (AICD and its dependence on the cell type and APP isoform expression suggest possibilities for selective manipulation of NEP gene expression in neuronal cells. We have also observed that another amyloid-clearing protein, namely transthyretin (TTR, is also regulated in the neuronal cell by a mechanism similar to NEP. Dependence of amyloid clearance proteins on histone deacetylases and the ability of HDAC inhibitors to up-regulate their expression in the brain opens new avenues for developing preventive strategies in AD.
Ferreira, Rosana B R; Chodur, Daniel M; Antunes, Luis Caetano M; Trimble, Michael J; McCarter, Linda L
The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit.
Animesh Dhara; Anthony P Sinai; William J Sullivan
ABSTRACT The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicatin...
Full Text Available Initially identified in HL60 cells treated with Vitamin D3, thioredoxin-interacting protein (TXNIP is considered as a major redox regulator and a potential connector between cellular redox state and metabolism. TXNIP plays an important role in the control of glucose and lipid metabolism, and it has been defined as a tumor suppressor gene in various solid tumors and hematological malignancies. This review gives an overview of the mechanism of various medicines including Lycium barbarum polysaccharide, Quercetin, trans-resveratrol, metformin, purple sweet potato color, nobiletin, taurine, suberoylanilide hydroxamic acid, and Theophylline, and their potential applications in the clinical treatment of many diseases.
Parasramka, Mansi A.
Long non-coding RNAs (lncRNA) are critical regulators of gene expression, and can reprogram the transcriptome to modulate cellular processes involved in cellular growth and differentiation, and thereby contribute to tumorigenesis. In addition to effects on tumor cell growth, survival and cell signaling, lncRNA can modulate cancer stem cell (CSC) behavior, including the expression of pluripotency factors. The identification of lncRNA that are mechanistically linked to cancer stem cell self-renewal and differentiation, or aberrant signaling pathways associated with tumor growth or progression, offer new opportunities for therapeutic intervention. PMID:27358893
Oikawa, Hirotaka; Goh, Wilson W B; Lim, Vania K J; Wong, Limsoon; Sng, Judy C G
Valproic acid (VPA) is an anti-convulsant drug that is recently shown to have neuroregenerative therapeutic actions. In this study, we investigate the underlying molecular mechanism of VPA and its effects on Bdnf transcription through microRNAs (miRNAs) and their corresponding target proteins. Using in silico algorithms, we predicted from our miRNA microarray and iTRAQ data that miR-124 is likely to target at guanine nucleotide binding protein alpha inhibitor 1 (GNAI1), an adenylate cyclase inhibitor. With the reduction of GNAI1 mediated by VPA, the cAMP is enhanced to increase Bdnf expression. The levels of GNAI1 protein and Bdnf mRNA can be manipulated with either miR-124 mimic or inhibitor. In summary, we have identified a novel molecular mechanism of VPA that induces miR-124 to repress GNAI1. The implication of miR-124→GNAI1→BDNF pathway with valproic acid treatment suggests that we could repurpose an old drug, valproic acid, as a clinical application to elevate neurotrophin levels in treating neurodegenerative diseases.
Full Text Available Maternally Expressed Gene 3 (MEG3 encodes a lncRNA which is suggested to function as a tumor suppressor. Previous studies suggested that MEG3 functioned through activation of p53, however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation. Here, we try to illuminate the relationship of MEG3 and p53, and the consequence in hepatoma cells. We find that transfection of expression construct of MEG3 enhances stability and transcriptional activity of p53. Deletion analysis of MEG3 confirms that full length and intact structure of MEG3 are critical for it to activate p53-mediated transactivation. Interestingly, our results demonstrate for the first time that MEG3 can interact with p53 DNA binding domain and various p53 target genes are deregulated after overexpression of MEG3 in hepatoma cells. Furthermore, results of qRT-PCR have shown that MEG3 RNA is lost or reduced in the majority of HCC samples compared with adjacent non-tumorous samples. Ectopic expression of MEG3 in hepatoma cells significantly inhibits proliferation and induces apoptosis. In conclusion, our data demonstrates that MEG3 functions as a tumor suppressor in hepatoma cells through interacting with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes.
Varier, Radhika A; Carrillo de Santa Pau, Enrique; van der Groep, Petra; Lindeboom, Rik G H; Matarese, Filomena; Mensinga, Anneloes; Smits, Arne H; Edupuganti, Raghu Ram; Baltissen, Marijke P; Jansen, Pascal W T C; Ter Hoeve, Natalie; van Weely, Danny R; Poser, Ina; van Diest, Paul J; Stunnenberg, Hendrik G; Vermeulen, Michiel
Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.
Full Text Available The purpose of this study was to outline the timelines of mitochondrial function, oxidative stress and cytochrome c oxidase complex (COX biogenesis in cardiac muscle with age, and to evaluate whether and how these age-related changes were attenuated by exercise. ICR/CD-1 mice were treated with pifithrin-μ (PFTμ, sacrificed and studied at different ages; ICR/CD-1 mice at younger or older ages were randomized to endurance treadmill running and sedentary conditions. The results showed that mRNA expression of p53 and its protein levels in mitochondria increased with age in cardiac muscle, accompanied by increased mitochondrial oxidative stress, reduced expression of COX subunits and assembly proteins, and decreased expression of most markers in mitochondrial biogenesis. Most of these age-related changes including p53 activity targeting cytochrome oxidase deficient homolog 2 (SCO2, p53 translocation to mitochondria and COX biogenesis were attenuated by exercise in older mice. PFTμ, an inhibitor blocking p53 translocation to mitochondria, increased COX biogenesis in older mice, but not in young mice. Our data suggest that physical exercise attenuates age-related changes in mitochondrial COX biogenesis and p53 activity targeting SCO2 and mitochondria, and thereby induces antisenescent and protective effects in cardiac muscle.
Yin, Kaifeng; Lin, Wenting; Guo, Jing; Sugiyama, Toshihiro; Snead, Malcolm L; Hacia, Joseph G; Paine, Michael L
Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3'-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins. Finally, microinjection of miR-153 in the region of mouse first mandibular molar at postnatal day 8 (PN8) induced AI-like pathologies when the enamel development reached maturity (PN12). In conclusion, miR-153 regulates maturation-stage amelogenesis by targeting key genes involved in the endocytotic and endosomal/lysosomal pathways, and disruption of miR-153 expression is a potential candidate etiologic factor contributing to the occurrence of AI.