title: Negative regulation of the peptidylarginine deiminase type IV promoter by NF-κB in human myeloid cells.
abstract: High titers of anti-citrullinated protein/peptide antibodies (ACPAs) have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of citrullinating enzymes and has been linked to RA. Therefore, our aim was to determine how transcription of PAD4 is regulated in the human myeloid lineage. We located the PAD4 transcription start site and promoter and phylogenetic comparisons of the area identified a 200 bp conserved region. Bioinformatics analysis predicted the presence of a NF-κB binding site and we tested this via luciferase assays. Intriguingly, mutation of the predicted NF-κB site significantly increased biological activity. We used RT-qPCR to quantify PAD4 expression in HL-60 cells treated with TNF-α to activate the canonical NF-κB pathway and found that PAD4 mRNA was reduced in response to TNF-α treatment. Finally, we used chromatin immunoprecipitation (ChIP) to determine NF-κB enrichment at the PAD4 promoter and the p50 subunit of NF-κB was more highly enriched than p65 at the PAD4 promoter. These results suggest that the p50 subunit of NF-κB may play a role in the repression of PAD4 transcription during inflammation.
authors.affiliation: Department of Microbiology and Immunology, Schulich School of Medicine & Dentistry, London, Ontario, Canada; Centre for Human Immunology, Schulich School of Medicine & Dentistry, London, Ontario, Canada.
authors.forename: Ali K
authors.initials: AK
authors.lastname: Abbas
authors.forename: Kevin
authors.initials: K
authors.lastname: Le
authors.forename: Virginia L
authors.initials: VL
authors.lastname: Pimmett
authors.forename: David A
authors.initials: DA
authors.lastname: Bell
authors.forename: Ewa
authors.initials: E
authors.lastname: Cairns
authors.forename: Rodney P
authors.initials: RP
authors.lastname: Dekoter
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018922:HL-60 Cells; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D022423:Myeloid Cells / Q000201:enzymology / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D012689:Sequence Homology, Nucleic Acid
chemical_list: D016328:NF-kappa B; D004247:DNA; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; C552151:peptidylarginine deiminase 4, mouse
keywords: 5′ RACE; 5′ rapid amplification of cDNA ends; ACPA; Arthritis; Autoimmune diseases; ChIP; DNA transcription; EDTA; GAPDH; IL8; IgG; LPS; NET; NF-κB; NF-κB transcription factor; Neutrophil; PAD4; Protein citrullination; RA; RT-qPCR; SDS; TNF-α; anti-citrullinated protein/peptide antibody; chromatin immunoprecipitation; ethylenediaminetetraacetic acid; glyceraldehyde 3-phosphate dehydrogenase; immunoglobulin G; interleukin 8; lipopolysaccharide; neutrophil extracellular trap; nuclear factor-kappa B; peptidylarginine deiminase type IV; reverse transcriptase quantitative polymerase chain reaction; rheumatoid arthritis; sodium dodecyl sulfate; tumor necrosis factor alpha