title: The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes.
abstract: OBJECTIVE
  Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes.
  
  
  METHODS
  Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile.
  
  
  RESULTS
  Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM).
  
  
  CONCLUSIONS
  The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.
authors.affiliation: Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA) and The National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina.
authors.forename: Ángel G
authors.initials: ÁG
authors.lastname: Valdivieso
authors.forename: Mariángeles
authors.initials: M
authors.lastname: Clauzure
authors.forename: Macarena
authors.initials: M
authors.lastname: Massip-Copiz
authors.forename: Tomás A
authors.initials: TA
authors.lastname: Santa-Coloma
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects*; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007476:Ionophores / Q000032:analysis / Q000737:chemistry; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects*; D016415:Sequence Alignment
chemical_list: D000838:Anions; D002712:Chlorides; C516011:GLRX5 protein, human; D054477:Glutaredoxins; D007476:Ionophores; D008667:Metalloproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C083686:RPS27 protein, human; D012269:Ribosomal Proteins