Peptidylarginine Deiminase 2 Suppresses Inhibitory κB Kinase Activity in Lipopolysaccharide-stimulated RAW 264.7 Macrophages

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 mu g/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 mu M). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-kappa B activity, determined by NF-kappa B-driven luciferase activity. The effect of PAD2 on NF-kappa B activity was further examined by using HEK 293 cells transfected with NF-kappa B luciferase, I kappa B beta/gamma kinase (IKK beta/gamma) subunits, and PAD2. IKK beta increased NF-kappa B activity, but this increase was markedly suppressed when PAD2 was present in cells. IKK kappa-mediated NF-kappa B activation was further enhanced by IKK beta in the presence of calcium ionophore A23187. However, this stimulatory effect of IKK beta/gamma was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKK gamma and PAD2 can coimmunoprecipitate in the presence of the Ca2+ ionophore. IKK gamma coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca2+ binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKK gamma(1-196) and IKK gamma(197419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKK gamma. In vitro citrullination assay showed that incubation of purified PAD2 and IKK gamma proteins in the presence of Ca2+ citrullinated IKK gamma. These results demonstrate that PAD2 interacts with IKK gamma and suppresses NF-kappa B activity.