title: Tumor necrosis factor (TNF)-α induction of CXCL10 in endothelial cells requires protein arginine methyltransferase 5 (PRMT5)-mediated nuclear factor (NF)-κB p65 methylation.
abstract: The chemokine CXCL10/IP-10 facilitates recruitment of Th1-type leukocytes to inflammatory sites. In this study, we show that the arginine methyltransferase PRMT5 is critical for CXCL10 transcription in TNF-α-activated human endothelial cells (EC). We found that depletion of PRMT5 results in significantly reduced levels of CXCL10 mRNA, demonstrating a positive role for PRMT5 in CXCL10 induction. Chromatin immunoprecipitation experiments revealed the presence of the symmetrical dimethylarginine modification catalyzed by PRMT5 associated with the CXCL10 promoter in response to TNF-α. However, symmetrical dimethylarginine-modified proteins were not detected at the promoter in the absence of PRMT5, indicating that PRMT5 is essential for methylation to occur. Furthermore, NF-κB p65, a critical driver of TNF-α-mediated CXCL10 induction, was determined to be methylated at arginine residues. Crucially, RNAi-mediated PRMT5 depletion abrogated p65 methylation and CXCL10 promoter binding. Mass spectrometric analysis in EC identified five dimethylated arginine residues in p65, four of which are uncharacterized in the literature. Expression of Arg-to-Lys point mutants of p65 demonstrated that both Arg-30 and Arg-35 must be dimethylated to achieve full CXCL10 expression. In conclusion, we have identified previously uncharacterized p65 post-translational modifications critical for CXCL10 induction.
authors.affiliation: From the Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195 and Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Harris
authors.affiliation: From the Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195 and.
authors.forename: Smarajit
authors.initials: S
authors.lastname: Bandyopadhyay
authors.affiliation: From the Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195 and.
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Maxwell
authors.affiliation: From the Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195 and.
authors.forename: Belinda
authors.initials: B
authors.lastname: Willard
authors.affiliation: From the Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195 and Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 dicorlp@ccf.org.
authors.forename: Paul E
authors.initials: PE
authors.lastname: DiCorleto
mesh_terms: D001120:Arginine / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000235:genetics* / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: C114753:CXCL10 protein, human; D054357:Chemokine CXCL10; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D001120:Arginine; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases
keywords: Endothelial Cell; Gene Expression; NF-Kappa B (NF-KB); Post-Translational Modification (PTM); Protein Methylation; Tumor Necrosis Factor (TNF)
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