Elsevier

Gene

Volume 533, Issue 1, 1 January 2014, Pages 123-131
Gene

Negative regulation of the peptidylarginine deiminase type IV promoter by NF-κB in human myeloid cells

https://doi.org/10.1016/j.gene.2013.09.108Get rights and content

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.

Introduction

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by inflammation and progressive destruction of synovial joints (Bax et al., 2011). Although RA etiology remains unclear, the presence of anti-citrullinated protein/peptide antibodies (ACPAs) has been demonstrated in the synovia of RA patients both to precede RA development and to predict RA severity (Brink et al., 2013, Lundberg et al., 2005, Rantapaa-Dahlqvist et al., 2003, Suzuki et al., 2007, van Boekel et al., 2002, van Gaalen et al., 2004). Indeed, diagnostic tests for ACPAs show > 97% specificity and sensitivity of at least 82% (van Boekel et al., 2002, Wegner et al., 2010). The presence of ACPAs and the high titers of their targets in the synovia of RA patients bring into question the role of the family of enzymes responsible for catalyzing the citrullination reaction: peptidylarginine deiminases (PAD). In particular, PAD4 is an enzyme present in granulocytes and has been investigated for its role in the immune response and links to autoimmune diseases (Asaga et al., 2001, Rohrbach et al., 2012).

The PAD4 gene is located on chromosome 1p36, and encodes the PAD4 enzyme — a 74 kDa enzyme that exists as a dimer and is the only PAD family member demonstrated to localize to the nucleus (Arita et al., 2004, Liu et al., 2011). The citrullination reaction catalyzed by PAD enzymes is an important physiologic method of post-translational modification of proteins and occurs in a variety of cells and tissues (Wegner et al., 2010). Studies on neutrophils have demonstrated that PAD4 is essential to the histone citrullination that precedes formation of neutrophil extracellular traps (NETs) — an important component of the inflammatory response to bacteria as well as fungi (Brinkmann et al., 2004, Li et al., 2010, Urban et al., 2006, Wang et al., 2004). Indeed, the neutrophils of PAD4-deficient mice are unable to generate NETs, resulting in greater susceptibility to bacterial infections (Li et al., 2010). Thus, PAD4 is an important mediator of innate immunity and may play a role in a variety of autoimmune diseases including RA (Rohrbach et al., 2012).

Studies in recent years have established that PAD4 is present in high levels in RA synovia and is itself a target of autoantibodies (Chang et al., 2005, Kolfenbach et al., 2010). Furthermore, a meta-analysis of RA patients in Japan, North America, and Europe has established a positive correlation between polymorphisms in the PAD4 gene and RA incidence (Iwamoto et al., 2006). Intriguingly, a recently published pan-PAD inhibitor, Cl-amidine, was used to treat mice with collagen-induced arthritis, inducing an ~ 50% reduction in disease activity (Willis et al., 2011).

While studies of polymorphisms in the PAD4 have linked the gene with rheumatoid arthritis, little is known about how PAD4 is transcriptionally regulated. The major aim of this study was therefore to determine how transcription of the PAD4 gene is regulated in human myeloid cells. To that end, transcription start sites of the murine PAD4 gene were identified and phylogenetic comparisons revealed a conserved region that was analyzed for possible transcription factor binding sites. This analysis predicted a potential binding site for NF-κB transcription factors. The predicted NF-κB binding site in the PAD4 promoter was tested for biological activity and, interestingly, mutation of the site resulted in lower promoter activity in murine cells but higher promoter activity in human cells. Moreover, in vitro treatment of different cell cultures with inflammatory stimuli resulted in significant changes in PAD4 expression patterns. Finally, the p50 subunit of NF-κB was more highly enriched at the PAD4 promoter than p65 in cells with activated NF-κB. Taken together, our results suggest that NF-κB may play an important role in the repression of PAD4 gene transcription.

Section snippets

Cell culture

The human HL-60 cell line (American Type Culture Collection [ATCC], Manassas, VA) was cultured in media consisting of Iscove's Modified Dulbecco's Medium (IMDM) with 4.5 g/L glucose, HEPES buffer, and l-glutamine (Lonza, Shawinigan, QC) supplemented with 10% charcoal stripped fetal bovine serum (Wisent, St. Bruno, QC), penicillin (100 U/mL)/streptomycin (100 μg/mL)/l-glutamine (.292 mg/mL) stock combination (Mediatech, Manassas, VA), and 5 × 10 5 M β-2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO).

Identification of the PAD4 promoter

It has previously been reported that PAD4 is expressed in granulocytes as well as monocytes (Asaga et al., 2001, Vossenaar, 2004). We tested this by isolating RNA from WEHI-3B cells, RAW 264.7 macrophages, C57BL/6 macrophages and dendritic cells and performed two separate RT-PCR amplifications, targeting exons 5–8 and 9–11 of the PAD4 gene. PAD4 transcripts were detected in WEHI-3B cells and dendritic cells but were not detected in RAW 264.7 or C57BL/6 macrophages (Fig. 1A), suggesting that PAD4

Discussion

Antibodies towards citrullinated proteins/peptides have become the gold standard in diagnosing RA and their presence is predictive of disease severity, implicating the PAD family of citrullinating enzymes in disease pathogenesis (Rantapaa-Dahlqvist et al., 2003, van Gaalen et al., 2004). One family member, PAD4, is of particular interest in the context of RA because it is found in the extracellular synovial fluid of RA patients, and has also been shown to be necessary for neutrophil

Conflict of interest

The authors disclose no conflict of interest.

Acknowledgments

We thank Sung O. Kim for contribution of cell lines.

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    This work was supported by grants from the Canadian Institutes of Health Research (MOP-106581) to R.P.D. and from the Arthritis Society of Canada to E.C. and D.A.B. (RG-10-010).

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