The Ubiquitination Modification Of PLA2G4A/cPLA2 By RNF8 Regulates Macrophage Polarization In Atopic Dermatitis

Objective:RNF8 is a ubiquitin ligase with multiple roles in DNA damage response as well as in other functions such as telomere protection,cell cycle regulation,and transcriptional regulation,but its mechanism of action in inflammation has been less well studied.Our previous study found that RNF8 also plays a role in skin inflammation.By multi-omics analysis of RNF8 knockout cells,we screened PLA2G4A/cPLA2,which is a rate-limiting enzyme of the production of arachidonic acid,prostaglandin and other inflammatory mediators,and the production of lipid mediators catalyzed by it plays an important role in the regulation of inflammation.At present,there are few studies on PLA2G4A posttranslational modification.Therefore,this study explored the effect of RNF8 on skin inflammation and its mechanism of action by discussing the regulatory mechanism of ubiquitination modification of RNF8 on PLA2G4A.Methods:The atopic dermatitis model was constructed by wild-type（WT）and RNF8 gene deletion(RNF8^-/-)mice,and the changes of back skin,skin lesion score,scratching behavior,skin thickness and skin barrier protein expression were observed to explore the effect of RNF8 loss on atopic dermatitis and skin barrier integrity in mice.The levels of M1 and M2 of macrophages in skin and abdominal cavity were detected by immunohistochemistry and western blotting to explore the effect of RNF8deficiency on the polarization of macrophages in mice.The downstream protein PLA2G4A associated with inflammation was identified by multi-omics analysis of RNF8 knockout cells.Protein levels of PLA2G4A in macrophages,arachidonic acid and prostaglandin E2 in skin tissue,and pro-inflammatory factors were measured.RNF8 overexpression and knockdown cell lines were constructed by lentivirus infection of A549 cell lines,and the relationship between RNF8 and PLA2G4A protein expression was verified by western blotting and immunofluorescence.The intracellular colocalization of PLA2G4A and RNF8 was detected by immunofluorescence,the interaction between endogenous and plasmid transfer PLA2G4A and RNF8 was detected by immunocoprecipitation,and the ubiquitination modification type of RNF8 on PLA2G4A was determined by western blotting.Results:By comparing the skin lesion score,scratching condition and skin thickening condition of WT and RNF8^-/-mice after atopic dermatitis,it was found that RNF8^-/-mice had more serious skin injury,more frequent scratching behavior and more obvious skin thickening than WT mice,and the difference between groups was significant.After dermatitis,the expression of skin barrier proteins CLDN1 and ZO1in RNF8^-/-mice was significantly reduced compared with that in WT mice.By comparing the number of M1 and M2 cells and the level of markers,it was found that M1 macrophages increased and M2 macrophages decreased in RNF8^-/-mice compared with WT mice with dermatitis,and the same results were also shown in untreated peritoneal macrophages.Compared with WT mice,it was found that the expression level of PLA2G4A in macrophages of RNF8^-/-mice was increased,the levels of arachidonic acid and prostaglandin E2 in skin tissue were increased,and the levels of pro-inflammatory factors IFN-γand IL-6 were increased.In A549 cell line,the expression level of RNF8 and PLA2G4A is negatively correlated,and the two interact.RNF8 regulates PLA2G4A by K48 polyubiquitination modification.Conclusion:In this study,it was found that the absence of RNF8 aggravated atopic dermatitis in mice,impaired the integrity of the skin barrier,and infiltrated macrophages increased M1 polarization and decreased M2 polarization in the skin tissue.The mechanism was that RNF8 regulated the degradation of PLA2G4A via proteasome dysmenorrhea through K48-type ubiquitination modification,thus regulating the polarization of macrophages.