The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage.

OBJECTIVE:The study aims to investigate the functional roles of peptidylarginine deiminase 2 in macrophages. METHODS:The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated knockout could markedly suppress the duanyu1563I2 protein expression, but not the protein expression. duanyu1563I2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1β and TNF-α in the U937 cells and IL-1β and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1β, IL-6, and TNF-α were all dramatically decreased in the duanyu1563I2 knockout group compared with in the controls. duanyu1563I2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. duanyu1563I2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. CONCLUSION:This study showed that duanyu1563I2 could promote IL-1β, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting duanyu1563I2 could be used as a novel strategy for controlling inflammation caused by macrophages.

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