title: Targeting of Gamma-Glutamyl-Cysteine Ligase by miR-433 Reduces Glutathione Biosynthesis and Promotes TGF-β-Dependent Fibrogenesis.
abstract: OBJECTIVE
  Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ratio. Here, we investigated the regulation of the rate-limiting GSH biosynthetic heterodimeric enzyme γ-glutamyl-cysteine ligase (GCL) by microRNAs (miRNAs).
  
  
  RESULTS
  "In silico" analysis of the 3'- untranslated regions (UTRs) of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL enabled an identification of miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in human umbilical vein endothelial cells (HUVEC) showed a downregulation of both GCLc and GCLm in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-independent manner. Increases in pro-oxidant stimuli such as exposure to hydrogen peroxide or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation, and reduced endothelial nitric oxide synthase activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor β1 (TGF-β1)-related reduction of GCLc and GCLm levels that were miR-433 dependent.
  
  
  CONCLUSIONS
  We describe for the first time an miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels.
authors.affiliation: 1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .
authors.forename: Cristina
authors.initials: C
authors.lastname: Espinosa-Diez
authors.affiliation: 1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .
authors.forename: Marta
authors.initials: M
authors.lastname: Fierro-Fernández
authors.affiliation: 1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Gómez
authors.affiliation: 1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodríguez-Pascual
authors.affiliation: 2 Cellular Biology in Renal Diseases Laboratory, Universidad Autonoma de Madrid , Madrid, Spain .
authors.forename: Matilde
authors.initials: M
authors.lastname: Alique
authors.affiliation: 2 Cellular Biology in Renal Diseases Laboratory, Universidad Autonoma de Madrid , Madrid, Spain .
authors.forename: Marta
authors.initials: M
authors.lastname: Ruiz-Ortega
authors.affiliation: 3 Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd) , Bizkaia, Spain .
authors.forename: Naiara
authors.initials: N
authors.lastname: Beraza
authors.affiliation: 3 Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd) , Bizkaia, Spain .
authors.forename: Maria L
authors.initials: ML
authors.lastname: Martínez-Chantar
authors.affiliation: 4 Vascular Biology and Therapeutics Program, Department of Comparative Medicine, Yale University School of Medicine , New Haven, Connecticut.
authors.forename: Carlos
authors.initials: C
authors.lastname: Fernández-Hernando
authors.affiliation: 1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .
authors.forename: Santiago
authors.initials: S
authors.lastname: Lamas
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004794:Enzyme Repression; D005721:Glutamate-Cysteine Ligase / Q000235:genetics* / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D034622:RNA Interference
chemical_list: D020413:3' Untranslated Regions; C548883:MIRN433 microRNA, human; D035683:MicroRNAs; C000609392:GCLC protein, human; C546365:GCLM protein, human; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione