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      The Ser82 RAGE Variant Affects Lung Function and Serum RAGE in Smokers and sRAGE Production In Vitro

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          Abstract

          Introduction

          Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor ( AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model.

          Methods

          Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified.

          Results

          Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV 1, FEV 1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production.

          Conclusions

          This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.

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          Most cited references32

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          RAGE (Receptor for Advanced Glycation Endproducts), RAGE Ligands, and their role in Cancer and Inflammation

          The Receptor for Advanced Glycation Endproducts [RAGE] is an evolutionarily recent member of the immunoglobulin super-family, encoded in the Class III region of the major histocompatability complex. RAGE is highly expressed only in the lung at readily measurable levels but increases quickly at sites of inflammation, largely on inflammatory and epithelial cells. It is found either as a membrane-bound or soluble protein that is markedly upregulated by stress in epithelial cells, thereby regulating their metabolism and enhancing their central barrier functionality. Activation and upregulation of RAGE by its ligands leads to enhanced survival. Perpetual signaling through RAGE-induced survival pathways in the setting of limited nutrients or oxygenation results in enhanced autophagy, diminished apoptosis, and (with ATP depletion) necrosis. This results in chronic inflammation and in many instances is the setting in which epithelial malignancies arise. RAGE and its isoforms sit in a pivotal role, regulating metabolism, inflammation, and epithelial survival in the setting of stress. Understanding the molecular structure and function of it and its ligands in the setting of inflammation is critically important in understanding the role of this receptor in tumor biology.
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            The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system.

            The receptor for advanced glycation end products (RAGE), a newly-identified member of the immunoglobulin superfamily, mediates interactions of advanced glycation end product (AGE)-modified proteins with endothelium and other cell types. Survey of normal tissues demonstrated RAGE expression in situations in which accumulation of AGEs would be unexpected, leading to the hypothesis that under physiologic circumstances, RAGE might mediate interaction with ligands distinct from AGEs. Sequential chromatography of bovine lung extract identified polypeptides with M(r) values of approximately 12,000 (p12) and approximately 23,000 (p23) which bound RAGE. NH2-terminal and internal protein sequence data for p23 matched that reported previously for amphoterin. Amphoterin purified from rat brain or recombinant rat amphoterin bound to purified sRAGE in a saturable and dose-dependent manner, blocked by anti-RAGE IgG or a soluble form of RAGE (sRAGE). Cultured embryonic rat neurons, which express RAGE, displayed dose-dependent binding of 125I-amphoterin which was prevented by blockade of RAGE using antibody to the receptor or excess soluble receptor (sRAGE). A functional correlate of RAGE-amphoterin interaction was inhibition by anti-RAGE F(ab')2 and sRAGE of neurite formation by cortical neurons specifically on amphoterin-coated substrates. Consistent with a potential role for RAGE-amphoterin interaction in development, amphoterin and RAGE mRNA/antigen were co-localized in developing rat brain. These data indicate that RAGE has physiologically relevant ligands distinct from AGEs which are likely, via their interaction with the receptor, to participate in physiologic processes outside of the context of diabetes and accumulation of AGEs.
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              Identification, classification, and expression of RAGE gene splice variants.

              The receptor for advanced glycation end-products (RAGE) is a single-transmembrane, multiligand receptor of the immunoglobulin superfamily. RAGE up-regulation is implicated in numerous pathological states including vascular disease, diabetes, cancer, and neurodegeneration. The understanding of the regulation of RAGE is important in both disease pathogenesis and normal homeostasis. Here, we demonstrate the characterization and identification of human RAGE splice variants by analysis of RAGE cDNA from tissue and cells. We identified a vast range of splice forms that lead to changes in the protein coding region of RAGE, which we have classified according to the Human Gene Nomenclature Committee (HGNC). These resulted in protein changes in the ligand-binding domain of RAGE or the removal of the transmembrane domain and cytosolic tail. Analysis of splice variants for premature termination codons reveals approximately 50% of identified variants are targeted to the nonsense-mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the understanding of the gene in the normal and pathological state.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 October 2016
                2016
                : 11
                : 10
                : e0164041
                Affiliations
                [1 ]Division of Respiratory Medicine, University of Nottingham, Nottingham, United Kingdom
                [2 ]Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
                [3 ]Department of Biology, University of York, York, United Kingdom
                [4 ]Pfizer Worldwide Research & Development, Cambridge, Massachusetts, United States of America
                [5 ]Pfizer Worldwide Research & Development, Cambridge, United Kingdom
                [6 ]Institute for Ageing and Health, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne, United Kingdom
                [7 ]National Heart and Lung Institute, Imperial College London, London, United Kingdom
                [8 ]Institute for Lung Health, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom
                University of Pittsburgh, UNITED STATES
                Author notes

                Competing Interests: AM and IK are employees of Pfizer Ltd. PPP Healthcare Medical Trust funded sample collection for SGP for this publication. AJW is an advisory board member reporting personal fees from GlaxoSmithKline, Boehringer Ingelheim and Pulmocide outside of the submitted work. IK and AM's commercial affiliation with Pfizer does not alter our adherence to PLOS ONE policies on sharing data and materials.

                • Conceptualization: IPH IS

                • Data curation: SM APH EH AKK CKB SKB MO EM SKM CS CG CPN CES

                • Formal analysis: SM APH EH AKK CKB SKB MO EM SKM CS CG CPN CES

                • Funding acquisition: IK AM IPH IS

                • Investigation: SM APH EH AKK CKB TLR SKB MO EM SKM CS CG CPN CES

                • Methodology: SM APH EH AKK CKB SKB EM CS CES IPH IS

                • Project administration: SM IPH IS

                • Resources: EM CEB SGP MFM AJW IPH IS

                • Supervision: SM IPH IS

                • Validation: SM APH EH CKB TLR CS CG CPN CES

                • Visualization: SM EH AKK SKB MO EM SKM CS CES

                • Writing – original draft: SM IPH IS

                • Writing – review & editing: SM APH EH AKK CKB TLR SKB MO EM SKM CS CG CPN CES CEB IK AM SGP MFM AJW IPH IS.

                [¤a]

                Current address: James Hogg Research Centre, Institute for Heart and Lung Health, University of British Columbia, Vancouver, British Columbia, Canada

                [¤b]

                Current address: Sachs’ Children’s Hospital, Stockholm, Sweden

                Article
                PONE-D-16-16828
                10.1371/journal.pone.0164041
                5068780
                27755550
                9674aa52-247f-4624-8717-d41ca9520236
                © 2016 Miller et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 April 2016
                : 19 September 2016
                Page count
                Figures: 6, Tables: 2, Pages: 16
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: G1000861
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100009032, Pfizer UK;
                Award Recipient :
                Funded by: The Swedish Research Council
                Award Recipient :
                Funded by: Swedish Heart-Lung Foundation
                Award Recipient :
                Funded by: Stockholm County Council
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000272, National Institute for Health Research;
                Award Recipient :
                This study was funded by grants from the Medical Research Council, UK (G1000861) and Pfizer Inc. EM has received funding from The Swedish Research Council, The Swedish Heart-Lung Foundation and Stockholm County Council (ALF). AJW is supported by the National Institute for Health Research Leicester Respiratory Biomedical Research Unit. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The human embryonic and fetal material was provided by the Joint MRC/Wellcome Trust (grant # 099175/Z/12/Z) Human Developmental Biology Resource ( www.hdbr.org). The funder (Pfizer) provided support in the form of salaries for authors [IK and AM], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.
                Categories
                Research Article
                Biology and Life Sciences
                Developmental Biology
                Organism Development
                Organogenesis
                Lung Development
                Medicine and Health Sciences
                Pulmonology
                Chronic Obstructive Pulmonary Disease
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Epithelial Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Messenger RNA
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Custom metadata
                Data are available from Gene Expression Omnibus, GSE14334.

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