Abstract
The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs) has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC) functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO) generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS). This suppression of NO generation was independent of changes in NO synthase (NOS) enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS) transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65), and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT) phosphorylation and activator protein-1 (AP-1) transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun) were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of excessive iNOS/NO-linked immune responses.
Highlights
Peptidylarginine deiminase (PAD) enzymes mediate the citrullination process—which involves the conversion of arginine to citrulline on a protein or peptide but not to free arginine—through the generation of an active site cleft via conformational changes in the presence of high calcium concentrations both in vivo and in vitro [1]
nitric oxide (NO) productiNonG-.meWtheyl-aL-lasroginfionue n(Ld-NtMhMatAt)hweasduescerdeasseadpoNsiOtivepcroondtruolcatinodncoimndpluetceleydinbhyibitCedl-aNmO idine occurs at an early timpreodpuoctiionnt. (W6eha)lsoanfodunidnthaattitmheed-edcreepaseend dNeOnptrmoduacntinonerin(dFuicgedubrey C1lB-a)m. iTdihneesoeccruerssuatlatsn suggest that early time point (6 h) and in a time-dependent manner (Figure 1B)
We found that the decreased NO production induced by Cl-amidine occurs at an early time point (6 h) and in a time-dependent manner (Figure 1B)
Summary
Peptidylarginine deiminase (PAD) enzymes mediate the citrullination process—which involves the conversion of arginine to citrulline on a protein or peptide but not to free arginine—through the generation of an active site cleft via conformational changes in the presence of high calcium concentrations both in vivo and in vitro [1]. Citrullination, a class of post-translational modifications, plays key roles in chromatin remodeling, gene transcription, conformational stability, the generation of autoantibodies to self-proteins, and neutrophil extracellular trap (NET) formation. Rheumatoid arthritis (RA) is characterized by a chronic inflammation of synovial joints with high amounts of citrullinated proteins and infiltration of inflammatory cells, including PAD2/PAD4-expressing macrophages, neutrophils, and dendritic cells [2,6]. Immune complexes containing ACPA and citrullinated proteins stimulate a cytokine release, and glycosylation of ACPA leads to the differentiation of osteoclasts and bone loss [6,7]
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