title: PRMT5-Mediated Methylation of NF-κB p65 at Arg174 Is Required for Endothelial CXCL11 Gene Induction in Response to TNF-α and IFN-γ Costimulation.
abstract: Inflammatory agonists differentially activate gene expression of the chemokine family of proteins in endothelial cells (EC). TNF is a weak inducer of the chemokine CXCL11, while TNF and IFN-γ costimulation results in potent CXCL11 induction. The molecular mechanisms underlying TNF plus IFN-γ-mediated CXCL11 induction are not fully understood. We have previously reported that the protein arginine methyltransferase PRMT5 catalyzes symmetrical dimethylation of the NF-κB subunit p65 in EC at multiple arginine residues. Methylation of Arg30 and Arg35 on p65 is critical for TNF induction of CXCL10 in EC. Here we show that PRMT5-mediated methylation of p65 at Arg174 is required for induction of CXCL11 when EC are costimulated with TNF and IFN-γ. Knockdown of PRMT5 by RNAi reduced CXCL11 mRNA and protein levels in costimulated cells. Reconstitution of p65 Arg174Ala or Arg174Lys mutants into EC that were depleted of endogenous p65 blunted TNF plus IFN-γ-mediated CXCL11 induction. Mass spectrometric analyses showed that p65 Arg174 arginine methylation is enhanced by TNF plus IFN-γ costimulation, and is catalyzed by PRMT5. Chromatin immunoprecipitation assays (ChIP) demonstrated that PRMT5 is necessary for p65 association with the CXCL11 promoter in response to TNF plus IFN-γ. Further, reconstitution of p65 Arg174Lys mutant in EC abrogated this p65 association with the CXCL11 promoter. Finally, ChIP and Re-ChIP assays revealed that symmetrical dimethylarginine-containing proteins complexed with the CXCL11 promoter were diminished in p65 Arg174Lys-reconstituted EC stimulated with TNF and IFN-γ. In total, these results indicate that PRMT5-mediated p65 methylation at Arg174 is essential for TNF plus IFN-γ-mediated CXCL11 gene induction. We therefore suggest that the use of recently developed small molecule inhibitors of PRMT5 may present a therapeutic approach to moderating chronic inflammatory pathologies.
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute and Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America.|Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Harris
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute and Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Unnikrishnan M
authors.initials: UM
authors.lastname: Chandrasekharan
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute and Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Smarajit
authors.initials: S
authors.lastname: Bandyopadhyay
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute and Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Belinda
authors.initials: B
authors.lastname: Willard
authors.affiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute and Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America.|Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Paul E
authors.initials: PE
authors.lastname: DiCorleto
mesh_terms: D002478:Cells, Cultured; D054371:Chemokine CXCL11 / Q000235:genetics*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008745:Methylation; D009154:Mutation; D011401:Promoter Regions, Genetic; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D054371:Chemokine CXCL11; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases