Phosphorylated E2F1 Is Stabilized By Nuclear USP11 To Drive Peg10 Gene Expression And Activate Lung Epithelial Cells

Background: The re-epithelialization of lung epithelial cells is very important for the recovery of ARDS.Phosphorylation affects ubiquitination,stability,and activity of transcriptional factors,thus regulating various cellular functions.E2 F transcriptional factor 1(E2F1)regulates paternally expressed imprinted gene 10(Peg10)expression,thereby promoting cell proliferation.However,the effect of E2F1 stability on Peg10 expression and the molecular regulation of E2F1 stability by its phosphorylation have not been well demonstrated.USP11 is a kind of DUBs belongs to cysteine protease can reverse poly-ubiquitin of substrates and has a significance for regulating protein function and signal transduction.DUBs UCH37 and POH1 mediated E2F1 deubiquitinating,and increased the stability of E2F1.Here,we mainly describe USP11 deubiquitinated E2F1 and increased the stability of E2F1,and study the mechanism of USP11 on the role of repair after lung injury.This study provides new therapeutic targets to lessen lung injury by improving lung epithelial cell repair and remodeling after injury.Methods: [1] The protein and m RNA level of PEG10 were determined by immunoblotting and q RT-PCR after transfected with Usp11 Sh RNA.[2] A549 cells were transfected with E2f1 si RNA or HA-E2f1 after knockdown USP11,the protein and m RNA level of PEG10 were examined by immunoblotting and q RT-PCR.The protein and m RNA level of E2F1,E2F2,E2F4 were determined by immunoblotting and q RT-PCR.The protein and m RNA level of PEG10 and E2F1 after MX treatment were examined by immunoblotting and q RT-PCR.[3] Ubiquitin immunoprecipitation(IP)was used to detect the polyubiquitination of E2F1 after downregulation or overexpression of USP11.Co-immunoprecipitation(Co-IP)was performed to detect the binding effect between USP11 and E2F1.The protein level of E2F1 was examined by immunoblotting followed by CHX treatment.[4] The immunofluorescence staining was performed to detect cellular location of USP11 or the co-localization of between USP11 and E2F1 after transfected with USP11-V5 and USP11?NLS-V5 or overexpressed USP11-V5 and HA-E2F1.Ubiquitin(IP)was used to detect the polyubiquitination of E2F1 after overexpressed USP11-V5 or USP11?NLS-V5.[5] The phosphorylation and ubiquitination level of E2F1 were detected by IP and immunoblotting after transfected with GSK3?S9A.The protein and ubiquitination level of E2F1 were detected by IP and immunoblotting after TWS119 treatment.[6] Co-IP was performed to detect the binding effect between USP11 and E2F1 after TWS119 treatment or overexpressed GSK3?.The phosphorylation and ubiquitination level of HA or the interaction of USP11 and HA were detected by IP and immunoblotting after overexpressed GSK3? or HA-E2F1S403A/ HA-E2F1S403 D.[7] The proliferation and migration of cells were detected by ECIS after knockdown USP11 or inhibited USP11 by overexpressing Usp11C318 S.The wound-healing of cells were examined by ECIS after MX or TWS119 treatment or overexpressed USP11 followed by knockdown PEG10.The proliferation of cells were determined by scratch assay after overexpressed E2F1 followed by downregulated USP11.Results: [1] USP11 regulates Peg10 expression.[2] USP11 up-regulates Peg10 expression by increasing E2F1 levels.[3] USP11 deubiquitinates and stabilizes E2F1.[4] Nuclear USP11 is essential for regulating E2F1.[5] GSK3?-mediated phosphorylation of E2F1 enhancing E2F1 deubiquitination and stability.[6] GSK3? promotes association between E2F1 and USP11.[7] USP11 promotes cells proliferation and migration through stabilization of E2F1.Conclusions: 1)The transcription factor E2F1 regulates PEG10 to promote lung epithelial cells proliferation and migration.2)Downregulating USP11 reduces the deubiquitination of E2F1,and decreases the stability of E2F1,and finally reduces the m RNA level of PEG10.3)The phosphorylation of E2F1 by GSK3? promotes the interaction between E2F1 and USP11,preventing the degradation of E2F1 in the nucleus.4)Physiologically,the absence of USP11 inhibits the cells proliferation and lung injury repair,but the effect can be reversed by overexpression of E2F1 and PEG10.