Abstract
Objective: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. Methods: The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, 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 plate. Results: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 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 PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 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 PADI2 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 PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.
Highlights
Peptidylarginine deiminases (PADIs) are a group of enzyme that converts peptidyl-arginine to peptidyl-citrulline, called protein citrullination, in the presence of Ca2+ [1]
The protein expression of the cit-p65 in the immunoprecipitates was analyzed by Western blotting using anti-p65 antibodies as a probe. (D) Representative images showing the relative protein expression levels of peptidylarginine deiminase 2 (PADI2) and PADI4; citrullinated histone H3 of the U937 cells, the differentiated macrophages, and the macrophages stimulated with LPS and cit-p65 in the immunoprecipitates of the LPS-stimulated macrophages in the PADI2 knockout group and the control group
Effects of PADI2 Knockout on Proinflammatory Cytokines Expression and Secretion In Figure 2A, we found that the gene expression levels of IL-1β, IL-6, and TNF-α were increased in the differentiated macrophages compared with in the U937 cells in both the PADI2 knockout group and the control group
Summary
Peptidylarginine deiminases (PADIs) are a group of enzyme that converts peptidyl-arginine to peptidyl-citrulline, called protein citrullination, in the presence of Ca2+ [1]. There are five members in the human PADI family, and each member has its own tissue distribution and substrate specificity [2]. PADIs and protein citrullination is known to contribute to the pathogenesis of. 2 2oof f1132 seovfesreavlearuatloaiumtmoimunmeudniseedasiseesa, sseusc,hsuacshrhaesurmheautomidataoritdhrairttihsr(iRtiAs )(RaAnd) amnudltmipuleltispclleersocsliesro[1s,i3s,4[1],,3b,4u]t, tbhuety wPaAntPaleihndpADredeoyDPIp2aAPIwo2laAsDlenyaoDrIdnse4arId4ePacwcAcPlwsehaAoDnasaDrtrIslr4ieIydecr4ameeeframsnoeartue(arlhLyknrhikPgadfiaogSbhbuh)tllyoyllnyiyndeifnaceixntrxccpoecirprlarefeiresaatesaaecssssdtieseleeediddttdcah[datd1[eenu1]u.]ccrlr.eiaeOiOnvnnrugeucgilrenrsmmrpvpoiararanfecescvvrivrcooaioiiostnoppriuuhuohassnalanlssggiadtnteeuunamdddddteyyiiemdfffftsseeaehhprrstooereatwowannsttstteeaeiiidaasdsinittsti[itsoho5h.[n,an5a6Wt,t]6wwt.te]hh.hheIeIfenenuererxreehhxtpaahpuusrsermmeetrtshsahaspsenineioroalonalendevduoudiodkfdikfiotPetoiPcdiAocAoynyDnttDethoIesoI2efs,2f, liepvoipdoelnycseatchcahtaPriAdDesI2(LmPiSg)htinpclraeyaasecdrittihcael lreovleelisnothf eciitnrfulallminmataetdorpyrroetsepinosn.seWuesinfugrpthlaesrmpirdo-venidceoddinthge evshidoernt cheaitrhpaitnPRANDAI2-tmariggehtitnpglaPyAaDcIr2it[i7c]a.lTrhoilse rinestuhlet iisncfloanmsimstaetnotrwy irtehspthoantsreepuosirntegdpblaysBmaiwd-aedneckoadrinetg shaol.r,twhhaiircphindeRmNoAn-sttarragteedtinthgaPtAPDAID2 I[27-]d. ETfhicisiernetsumlticies csohnoswisetdenat rweidtuhctehdatjoreinptoirntefldambymBaatwioandienkmaruertinael., wthuimchodr enmecornosstisraftaecdtotrh-aatlpPhAaD(TI2N-dFe-fiαc)-ieinndt umciecde asrhtohwrietids [a8r].educed joint inflammation in murine tumor necrosRisecfaecnttolyr-,acllpuhsate(rTedNFre-αgu)-lianrdlyucinetderasrpthacreitdiss[h8o].rt palindromic repeats (CRISPR) and the CRISPR-. Ckout on various functions of macrophages, including inflammation, cell survival, and adhesion capacity ThPeArDefIo2rek,nwocekuosuetdotnhevCarRioISuPs Rfu–nCcatsio9nssysotfemmatocrkonpohcakgoeus,t iPnAclDudI2ining minaflcarmopmhaatgioens ,acnedlltosuervvaivluaal,teanthde effadechtessoiof nPAcaDpIa2ckitnyo. ckout on various functions of macrophages, including inflammation, cell survival, and adhesion capacity
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