DOCSLIB.ORG

Transmembrane Protein with Unknown Function 16A Overexpression Promotes Glioma Formation Through the Nuclear Factor‑Κb Signaling Pathway

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Transmembrane Protein with Unknown Function 16A Overexpression Promotes Glioma Formation Through the Nuclear Factor‑Κb Signaling Pathway 1068 MOLECULAR MEDICINE REPORTS 9: 1068-1074, 2014 Transmembrane protein with unknown function 16A overexpression promotes glioma formation through the nuclear factor‑κB signaling pathway JUN LIU1, YU LIU2, YINGANG REN1, LI KANG1 and LIHUA ZHANG1 Departments of 1Geriatrics and 2Neurology, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710038, P.R. China Received July 18, 2013; Accepted January 2, 2014 DOI: 10.3892/mmr.2014.1888 Abstract. Ion channels have been suggested to be important in Introduction the development and progression of tumors, however, chloride channels have rarely been analyzed in tumorigenesis. More In previous years, the association between ion channels and recently, transmembrane protein with unknown function 16A tumors has drawn particular attention. Increasing evidence has (TMEM16A), hypothesized to be a candidate calcium-acti- demonstrated that ion channels are involved in the regulation vated Cl- channel, has been found to be overexpressed in a of tumor progression, including potassium (1-3), calcium (4) number of tumor types. Although several studies have impli- and sodium channels (5,6). Therefore, understanding the cated the overexpression of TMEM16A in certain tumor types, underlying molecular mechanisms of ion channels in tumori- the exact role of TMEM16A in gliomas and the underlying genesis, and tumor progression and migration provides novel mechanisms in tumorigenesis, remain poorly understood. In insights into tumor pathogenesis, and also identifies potential the present study, the role of TMEM16A in gliomas and the targets for tumor prevention and treatment. potential underlying mechanisms were analyzed. TMEM16A Chloride channels are expressed ubiquitously and are was highly abundant in various grades of gliomas and important in various cellular processes, including the cell cycle cultured glioma cells. Knockdown of TMEM16A suppressed and proliferation, cell volume modulation, epithelial secretion cell proliferation, migration and invasion. Furthermore, and membrane excitability (7). To date, chloride channel nuclear factor-κB (NF-κB) was activated by overexpression of family members, including CLC proteins, ionotropic receptors TMEM16A. In addition, TMEM16A regulated the expression for γ‑aminobutyric acid and glycine, and the cystic fibrosis of NF-κB-mediated genes, including cyclin D1, cyclin E and transmembrane conductance regulator (CFTR), have been c-myc, involved in cell proliferation, and matrix metallopro- described (8). Calcium-activated chloride channels (CaCCs) teinases (MMPs)-2 and MMP-9, which are associated with the are reported to be major modulators of cell volume and epithe- migration and invasion of glioma cells. Collectively, results lial secretion (9). However, the composition of CaCCs remains of the present study provide evidence for the involvement of unknown. More recently, transmembrane protein with unknown TMEM16A in gliomas and the potential mechanism through function 16 (TMEM16) A, also known as anoctamin 1, has which TMEM16A promotes glioma formation. been hypothesized to be a candidate CaCC (10-12). TMEM16A belongs to the TMEM16 family, which consists of nine other members, including TMEM16B-K, characterized by eight transmembrane segments and a highly conserved domain of unknown function. TMEM16A has been demonstrated to be Correspondence to: Dr Lihua Zhang, Department of Geriatrics, involved in the regulation of gastrointestinal tract motility, Tangdu Hospital, Fourth Military Medical University, 1 Xinsi Road, epithelial fluid transport and saliva production (13-16). CaCCs Baqiao, Xi'an, Shanxi 710038, P.R. China have been found to be proapoptotic, acting as tumor suppressor E-mail: [email protected] genes in mammary epithelium (17). However, bestrophin, Abbreviations: CaCCs, calcium-activated chloride channels; a putative CaCC that promotes cell proliferation, has been TMEM16A, transmembrane protein with unknown function 16A; shown to be overexpressed in colon cancer (18). Notably, MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; CFTR, TMEM16A is localized on chromosome 11q13, a region that cystic fibrosis transmembrane conductance regulator; BrdU, is frequently amplified in human cancers, including those 5-bromo-2-deoxyuridine of the head and neck, esophagus, bladder and breast (19-21). Therefore, TMEM16A dysregulation may be associated with Key words: calcium-activated chloride channels, nuclear factor-κB, tumor progression. As hypothesized, several studies have glioma, transmembrane protein with unknown function 16A revealed that TMEM16A is overexpressed in various tumor types, including gastrointestinal stromal tumors, esophageal cancers, and head and neck cancers (22-24). However, the LIU et al: ROLE OF TMEM16A IN GLIOMAS 1069 molecular mechanisms of TMEM16A in the regulation of reverse, 5'-GTCCATGTTCTGCTGGGCCTG-3'; cyclin E tumors remains poorly understood. forward, 5'-GTCCTGGCTGAATGATACATC-3' and Several studies have hypothesized that chloride channels reverse, 5'-CCCTATTTTGTTCAGACAACATGGC-3'; are associated with the nuclear factor-κB (NF-κB) signaling c-myc forward 5'-ACACATCAGCACAACTACGC-3' and pathway (25-29). NF-κB, which is tightly controlled, regulates reverse, 5'-CCTCTTGACATTCTCCTCGGT-3'; GAPDH a wide range of cellular processes and is extensively involved forward, 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and in cancer progression (30). Normally, the binding of NF-κB reverse 5'-AGCCTTCTCCATGGTGGTGAA-3'. The qPCR to inhibitory inhibitor of κB (IκB) proteins inactivates NF-κB mixture system contained 5 µl SsoFast™ EvaGreen Supermix in the cytoplasm (31). Degradation of IκB proteins releases (Bio-Rad, Hercules, CA, USA), 1 µl cDNA (diluted at 1:50) NF-κB and enables it to translocate to the nucleus and activate and 2 µl each of the forward and reverse primers (1 µM), to a target genes. Dysregulation of this process is involved in a final volume of 10 µl. The PCR procedure was as follows: 94˚C number of diseases (32,33). NF-κB has also been demonstrated for 4 min; 94˚C for 20 sec, 55˚C for 30 sec and 72˚C for 20 sec; to be a critical regulator of tumorigenesis involved in cell 2 sec for plate reading for 35 cycles; melting curve, 65‑95˚C. survival, metastasis and angiogenesis (31). However, although GAPDH was used as a control for normalizing the gene TMEM16A has been hypothesized to be a transmembrane expression. Three independent experiments were performed. protein regulating cellular ion exchanges, its role in the regula- The data obtained were analyzed by the 2-ΔΔCt method and tion of the NF-κB signal pathway remains unclear. statistically analyzed as described previously (35), followed by To date, no studies have determined the exact role of Student's unpaired sample t-test. TMEM16A in gliomas. The aim of the present study was to determine whether TMEM16A is involved in gliomas and the Nuclear protein extraction. Nuclear proteins were extracted potential underlying mechanisms. using a Nuclear Extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Briefly, Materials and methods cells were lysed in cytoplasmic buffer containing protease inhibitors, mixed and incubated for 15 min at 4˚C. Next, cells Cell lines and cell culture. One normal human astrocyte line were centrifuged at 12,000 rpm for 20 min at 4˚C. Supernatants (SVGp12) and four glioma cell lines (U87MG, U118, U251 containing cytoplasmic extraction products were collected. Cell and SHG44) were obtained from the American Type Culture sediments were collected and resuspended in nucleus buffer for Collection (Manassas, VA, USA). All cells were maintained 10 min at 4˚C. Next, the sample was centrifuged at 12,000 rpm according to standard protocols. Briefly, cells were cultured in for 10 min at 4˚C and the supernatant was collected for analysis. Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum to which 100 U/ml penicillin, Western blotting. A total of 20-30 µg protein was fractionated by 100 µg/ml streptomycin and 2 mM L-glutamate were added. 12% SDS-PAGE electrophoresis and transferred to nitrocellulose All cells were cultured at 37˚C with 5% CO2 in an incubator membranes (Amersham, Little Chalfont, UK). The membrane (Invitrogen Life Technologies, Carlsbad, CA, USA). was treated under agitation and blocking at room temperature, with 2% non-fat dry milk in Tris-buffered saline (TBS) for Tumor tissue preparation. All tumor tissues were obtained 1 h, followed by incubation with primary rabbit anti-human from Tangdu Hospital (Xi'an, China) according to institutional TMEM16A, IκB, p65, MMP-2, MMP-9, GAPDH and Histone guidelines for consent with the approval of patients and the polyclonal antibodies. (Santa Cruz Biotechnology, CA, USA). hospital. Brain tissue was obtained from 20 patients with Antibodies were diluted in blocking buffer (1:2,000) and incu- gliomas (eight females and 12 males; median age, 43.6 years; bated with the membrane at 4˚C overnight. Next, membranes range, 16‑78 years; five each of grade I, II, III and IV). Normal were washed three times with TBS and Tween 20 [TBST; brain tissue samples were obtained from five patients with 10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% Tween-20] brain trauma (median age, 42 years; range, 21-68 years). for 10 min at room temperature. Subsequently, the membrane All samples were obtained from the initial surgery, prior to was incubated in horseradish peroxidase (HRP)-conjugated chemotherapy and radiotherapy. The malignant grades of the secondary
Load more

Recommended publications
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Transcriptomic Analysis of Native Versus Cultured Human and Mouse Dorsal Root Ganglia Focused on Pharmacological Targets Short
    bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Transcriptomic analysis of native versus cultured human and mouse dorsal root ganglia focused on pharmacological targets Short title: Comparative transcriptomics of acutely dissected versus cultured DRGs Andi Wangzhou1, Lisa A. McIlvried2, Candler Paige1, Paulino Barragan-Iglesias1, Carolyn A. Guzman1, Gregory Dussor1, Pradipta R. Ray1,#, Robert W. Gereau IV2, # and Theodore J. Price1, # 1The University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, 800 W Campbell Rd. Richardson, TX, 75080, USA 2Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine # corresponding authors [email protected], [email protected] and [email protected] Funding: NIH grants T32DA007261 (LM); NS065926 and NS102161 (TJP); NS106953 and NS042595 (RWG). The authors declare no conflicts of interest Author Contributions Conceived of the Project: PRR, RWG IV and TJP Performed Experiments: AW, LAM, CP, PB-I Supervised Experiments: GD, RWG IV, TJP Analyzed Data: AW, LAM, CP, CAG, PRR Supervised Bioinformatics Analysis: PRR Drew Figures: AW, PRR Wrote and Edited Manuscript: AW, LAM, CP, GD, PRR, RWG IV, TJP All authors approved the final version of the manuscript. 1 bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Expression Profiling of Ion Channel Genes Predicts Clinical Outcome in Breast Cancer
    UCSF UC San Francisco Previously Published Works Title Expression profiling of ion channel genes predicts clinical outcome in breast cancer Permalink https://escholarship.org/uc/item/1zq9j4nw Journal Molecular Cancer, 12(1) ISSN 1476-4598 Authors Ko, Jae-Hong Ko, Eun A Gu, Wanjun et al. Publication Date 2013-09-22 DOI http://dx.doi.org/10.1186/1476-4598-12-106 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Ko et al. Molecular Cancer 2013, 12:106 http://www.molecular-cancer.com/content/12/1/106 RESEARCH Open Access Expression profiling of ion channel genes predicts clinical outcome in breast cancer Jae-Hong Ko1, Eun A Ko2, Wanjun Gu3, Inja Lim1, Hyoweon Bang1* and Tong Zhou4,5* Abstract Background: Ion channels play a critical role in a wide variety of biological processes, including the development of human cancer. However, the overall impact of ion channels on tumorigenicity in breast cancer remains controversial. Methods: We conduct microarray meta-analysis on 280 ion channel genes. We identify candidate ion channels that are implicated in breast cancer based on gene expression profiling. We test the relationship between the expression of ion channel genes and p53 mutation status, ER status, and histological tumor grade in the discovery cohort. A molecular signature consisting of ion channel genes (IC30) is identified by Spearman’s rank correlation test conducted between tumor grade and gene expression. A risk scoring system is developed based on IC30. We test the prognostic power of IC30 in the discovery and seven validation cohorts by both Cox proportional hazard regression and log-rank test.
    [Show full text]
  • What Biologists Want from Their Chloride Reporters
    © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs240390. doi:10.1242/jcs.240390 REVIEW SUBJECT COLLECTION: TOOLS IN CELL BIOLOGY What biologists want from their chloride reporters – a conversation between chemists and biologists Matthew Zajac1,2, Kasturi Chakraborty1,2,3, Sonali Saha4,*, Vivek Mahadevan5,*, Daniel T. Infield6, Alessio Accardi7,8,9, Zhaozhu Qiu10,11 and Yamuna Krishnan1,2,‡ ABSTRACT inhibitory synaptic action potential (Kaila et al., 2014; Medina − + − Impaired chloride transport affects diverse processes ranging from et al., 2014). Under normal conditions, [Cl ]i is kept low by a K -Cl SLC12A5 neuron excitability to water secretion, which underlie epilepsy and cotransporter (KCC2, encoded by the gene ), allowing γ cystic fibrosis, respectively. The ability to image chloride fluxes with activation of the -aminobutyric acid (GABA) receptor (GABAAR) fluorescent probes has been essential for the investigation of the roles to drive chloride down the electrochemical gradient into the neuron of chloride channels and transporters in health and disease. Therefore, (Doyon et al., 2016). Improper chloride homeostasis is therefore developing effective fluorescent chloride reporters is critical to associated with several severe neurological disorders and epilepsies characterizing chloride transporters and discovering new ones. (Ben-Ari et al., 2012; Huberfeld et al., 2007; Payne et al., 2003). In However, each chloride channel or transporter has a unique epithelial cells, the chloride channel
    [Show full text]
  • Chloride Intracellular Channel 4 Is Critical for the Epithelial Morphogenesis of RPE Cells and Retinal Attachment Jen-Zen Chuang,* Szu-Yi Chou,* and Ching-Hwa Sung*†
    Molecular Biology of the Cell Vol. 21, 3017–3028, September 1, 2010 Chloride Intracellular Channel 4 Is Critical for the Epithelial Morphogenesis of RPE Cells and Retinal Attachment Jen-Zen Chuang,* Szu-Yi Chou,* and Ching-Hwa Sung*† *Department of Ophthalmology, and †Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10021 Submitted October 29, 2009; Revised June 1, 2010; Accepted June 29, 2010 Monitoring Editor: Keith E. Mostov Retinal detachment is a sight-threatening condition. The molecular mechanism underlying the adhesion between the RPE and photoreceptors is poorly understood because the intimate interactions between these two cell types are impossible to model and study in vitro. In this article, we show that chloride intracellular channel 4 (CLIC4) is enriched at apical RPE microvilli, which are interdigitated with the photoreceptor outer segment. We used a novel plasmid-based transfection method to cell-autonomously suppress CLIC4 in RPE in situ. CLIC4 silenced RPE cells exhibited a significant loss of apical microvilli and basal infoldings, reduced retinal adhesion, and epithelial-mesenchymal transition. Ectopically expressing ezrin failed to rescue the morphological changes exerted by CLIC4 silencing. Neural retinas adjacent to the CLIC4-suppressed RPE cells display severe dysplasia. Finally, a high level of aquaporin 1 unexpectedly appeared at the apical surfaces of CLIC4-suppressed RPE cells, together with a concomitant loss of basal surface expression of monocar- boxylate transporter MCT3. Our results suggested that CLIC4 plays an important role in RPE-photoreceptor adhesion, perhaps by modulating the activity of cell surface channels/transporters. We propose that these changes may be attributable to subretinal fluid accumulation in our novel retinal detachment animal model.
    [Show full text]
  • Proteomic Identification of Mediators Implicated in the Metastatic Progression of Ovarian Cancer
    Proteomic Identification of Mediators Implicated in the Metastatic Progression of Ovarian Cancer by Natasha Musrap A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Natasha Musrap 2015 Proteomic Identification of Mediators Implicated in the Metastatic Progression of Ovarian Cancer Natasha Musrap Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2015 Abstract Ovarian cancer (OvCa) is the leading cause of death among gynecological malignancies, and is characterized by peritoneal metastasis and increased resistance to chemotherapy. Acquired drug resistance is often attributed to the formation of multicellular aggregates (MCAs) in the peritoneal cavity, which seed abdominal surfaces, particularly, the mesothelial lining of the peritoneum. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of the underlying biology surrounding OvCa metastasis may lead to the identification of key molecules that are integral to the progression of the disease, which therefore, may serve as practicable therapeutic targets. To that end, in vitro cell line models of cancer-peritoneal interaction and aggregate formation were used to identify proteins that are differentially expressed during cancer progression, using mass spectrometry-based approaches. First, we performed a proteomics analysis of a co-culture model of ovarian cancer and mesothelial cells, in which we identified numerous proteins that were differentially regulated during cancer-peritoneal interaction. We further validated one protein, MUC5AC, and confirmed its expression at the cancer-peritoneal interface. Next, we conducted a quantitative proteomics analysis of a cell line grown as a monolayer and as MCAs.
    [Show full text]
  • Viewed and Edited the Knowledge of the PT and Urinary Proteome (31–33)
    Original Investigation Functionally Essential Tubular Proteins Are Lost to Urine-Excreted, Large Extracellular Vesicles during Chronic Renal Insufficiency Ryan J. Adam,1 Mark R. Paterson,1 Lukus Wardecke,1 Brian R. Hoffmann,1,2,3,4 and Alison J. Kriegel1,3,5 Abstract Background The 5/6 nephrectomy (5/6Nx) rat model recapitulates many elements of human CKD. Within weeks of surgery, 5/6Nx rats spontaneously exhibit proximal tubular damage, including the production of very large extracellular vesicles and brush border shedding. We hypothesized that production and elimination of these structures, termed large renal tubular extracellular vesicles (LRT-EVs), into the urine represents a pathologic mechanism by which essential tubule proteins are lost. Methods LRT-EVs were isolated from 5/6Nx rat urine 10 weeks after surgery. LRT-EV diameters were measured. LRT-EV proteomic analysis was performed by tandem mass spectrometry. Data are available via the Proteo- meXchange Consortium with identifier PXD019207. Kidney tissue pathology was evaluated by trichrome staining, TUNEL staining, and immunohistochemistry. Results LRT-EV size and a lack of TUNEL staining in 5/6Nx rats suggest LRT-EVs to be distinct from exosomes, microvesicles, and apoptotic bodies. LRT-EVs contained many proximal tubule proteins that, upon disruption, are known to contribute to CKD pathologic hallmarks. Select proteins included aquaporin 1, 16 members of the solute carrier family, basolateral Na1/K1-ATPase subunit ATP1A1, megalin, cubilin, and sodium-glucose cotransporters (SLC5A1 and SLC5A2). Histologic analysis confirmed the presence of apical membrane proteins in LRT-EVs and brush border loss in 5/6Nx rats. Conclusions This study provides comprehensive proteomic analysis of a previously unreported category of extracellular vesicles associated with chronic renal stress.
    [Show full text]
  • NIH Public Access Author Manuscript FEBS Lett
    NIH Public Access Author Manuscript FEBS Lett. Author manuscript; available in PMC 2011 May 17. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: FEBS Lett. 2010 May 17; 584(10): 2102±2111. doi:10.1016/j.febslet.2010.01.037. Chloride Channels of Intracellular Membranes John C. Edwards* and Christina R. Kahl UNC Kidney Center and the Division of Nephrology and Hypertension, Department of Medicine, University of North Carolina at Chapel Hill Abstract Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. Keywords chloride channel; ClC; CLIC; bestrophin; GPHR The study of chloride channels of intracellular membranes has seen enormous advances over the past two decades and exciting recent developments have sparked renewed interest in this field. The discovery of important roles for intracellular chloride channels in human disease processes as diverse as retinal macular dystrophy, osteopetrosis, renal proximal tubule dysfunction, and angiogenesis have highlighted the importance of these molecules in critical cellular activities. Startling discoveries regarding the intracellular ClC family of proteins have forced a re-examination
    [Show full text]
  • A Single-Cell Transcriptome Atlas of the Mouse Glomerulus
    RAPID COMMUNICATION www.jasn.org A Single-Cell Transcriptome Atlas of the Mouse Glomerulus Nikos Karaiskos,1 Mahdieh Rahmatollahi,2 Anastasiya Boltengagen,1 Haiyue Liu,1 Martin Hoehne ,2 Markus Rinschen,2,3 Bernhard Schermer,2,4,5 Thomas Benzing,2,4,5 Nikolaus Rajewsky,1 Christine Kocks ,1 Martin Kann,2 and Roman-Ulrich Müller 2,4,5 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Three different cell types constitute the glomerular filter: mesangial depending on cell location relative to the cells, endothelial cells, and podocytes. However, to what extent cellular heteroge- glomerular vascular pole.3 Because BP ad- neity exists within healthy glomerular cell populations remains unknown. aptation and mechanoadaptation of glo- merular cells are key determinants of kidney Methods We used nanodroplet-based highly parallel transcriptional profiling to function and dysregulated in kidney disease, characterize the cellular content of purified wild-type mouse glomeruli. we tested whether glomerular cell type sub- Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identi- sets can be identified by single-cell RNA fied the three known glomerular cell types. We provide a comprehensive online sequencing in wild-type glomeruli. This atlas of gene expression in glomerular cells that can be queried and visualized using technique allows for high-throughput tran- an interactive and freely available database. Novel marker genes for all glomerular scriptome profiling of individual cells and is cell types were identified and supported by immunohistochemistry images particularly suitable for identifying novel obtained from the Human Protein Atlas.
    [Show full text]
  • Antisense Suppression of the Chloride Intracellular Channel Family Induces Apoptosis, Enhances Tumor Necrosis Factor A-Induced Apoptosis, and Inhibits Tumor Growth
    Research Article Antisense Suppression of the Chloride Intracellular Channel Family Induces Apoptosis, Enhances Tumor Necrosis Factor a-Induced Apoptosis, and Inhibits Tumor Growth Kwang S. Suh, Michihiro Mutoh, Michael Gerdes, John M. Crutchley, Tomoko Mutoh, Lindsay E. Edwards, Rebecca A. Dumont, Pooja Sodha, Christina Cheng, Adam Glick, and Stuart H. Yuspa Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland Abstract CLICs are also found in a soluble form in the cytoplasm (2–5). mtCLIC/CLIC4 is a p53 and tumor necrosis factor a (TNFa) Crystallographic analysis of the structure of soluble CLIC1 regulated intracellular chloride channel protein that local- indicates homology to the glutathione transferase family of izes to cytoplasm and organelles and induces apoptosis proteins. It is hypothesized that soluble CLICs may become when overexpressed in several cell types of mouse and activated as anion channels or channel regulators when ‘‘auto- humanorigin.CLIC4iselevatedduringTNFa-induced inserted’’ into intracellular membranes (6). apoptosis in human osteosarcoma cell lines. In contrast, Among the CLIC family proteins, the biological functions of inhibition of NFKB results in an increase in TNFa-mediated CLIC4 have been most thoroughly studied. CLIC4 is expressed in apoptosis with a decrease in CLIC4 protein levels. Cell lines many cell types. In skin keratinocytes, CLIC4 was first localized to expressing an inducible CLIC4-antisense construct that also mitochondria and cytoplasm and later was localized specifically reduces the expression of several other chloride intracellular to the inner mitochondrial membrane by immunogold electron channel (CLIC) family proteins were established in the microscopy (7, 8). Other reports have localized CLIC4 in the trans- human osteosarcoma lines SaOS and U2OS cells and Golgi network in pancreatic cells, endoplasmic reticulum in rat a malignant derivative of the mouse squamous papilloma hippocampal HT-4 cells, and large dense core vesicles in line SP1.
    [Show full text]
  • The Integrated RNA Landscape of Renal Preconditioning Against Ischemia-Reperfusion Injury
    BASIC RESEARCH www.jasn.org The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury Marc Johnsen,1 Torsten Kubacki,1 Assa Yeroslaviz ,2 Martin Richard Späth,1 Jannis Mörsdorf,1 Heike Göbel,3 Katrin Bohl,1,4 Michael Ignarski,1,4 Caroline Meharg,5 Bianca Habermann,6 Janine Altmüller,7 Andreas Beyer,3,8 Thomas Benzing,1,3,8 Bernhard Schermer,1,3,8 Volker Burst,1 and Roman-Ulrich Müller 1,3,8 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Although AKI lacks effective therapeutic approaches, preventive strategies using precondi- tioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. Methods To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. Results The gene expression signatures induced by both preconditioning strategies involve distinct com- mon genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI.
    [Show full text]
  • CREB-Dependent Transcription in Astrocytes: Signalling Pathways, Gene Profiles and Neuroprotective Role in Brain Injury
    CREB-dependent transcription in astrocytes: signalling pathways, gene profiles and neuroprotective role in brain injury. Tesis doctoral Luis Pardo Fernández Bellaterra, Septiembre 2015 Instituto de Neurociencias Departamento de Bioquímica i Biologia Molecular Unidad de Bioquímica y Biologia Molecular Facultad de Medicina CREB-dependent transcription in astrocytes: signalling pathways, gene profiles and neuroprotective role in brain injury. Memoria del trabajo experimental para optar al grado de doctor, correspondiente al Programa de Doctorado en Neurociencias del Instituto de Neurociencias de la Universidad Autónoma de Barcelona, llevado a cabo por Luis Pardo Fernández bajo la dirección de la Dra. Elena Galea Rodríguez de Velasco y la Dra. Roser Masgrau Juanola, en el Instituto de Neurociencias de la Universidad Autónoma de Barcelona. Doctorando Directoras de tesis Luis Pardo Fernández Dra. Elena Galea Dra. Roser Masgrau In memoriam María Dolores Álvarez Durán Abuela, eres la culpable de que haya decidido recorrer el camino de la ciencia. Que estas líneas ayuden a conservar tu recuerdo. A mis padres y hermanos, A Meri INDEX I Summary 1 II Introduction 3 1 Astrocytes: physiology and pathology 5 1.1 Anatomical organization 6 1.2 Origins and heterogeneity 6 1.3 Astrocyte functions 8 1.3.1 Developmental functions 8 1.3.2 Neurovascular functions 9 1.3.3 Metabolic support 11 1.3.4 Homeostatic functions 13 1.3.5 Antioxidant functions 15 1.3.6 Signalling functions 15 1.4 Astrocytes in brain pathology 20 1.5 Reactive astrogliosis 22 2 The transcription
    [Show full text]