Modulation Of NRF2 Signaling Pathway By PML-RAR? And The Mechanism

BackgroundNRF2(NF-E2 p45-related factor 2)-KEAP1(Kelch-like ECH-associated protein 1)signaling pathway plays an important role in cyto-protection.KEAP1 is a potent repressor of NRF2,which mediates NRF2 degradation via ubiquitination,maintaining its homeostasis in normal cells.Nuclear receptors play a role in regulating NRF2 in a KEAP1-independent mechanism.However,in many tumors,somatic mutations in KEAP1 result in the loss of its function,which leads to increase in NRF2 stability and expression.Aberrantly activated NRF2 can promote malignant proliferation and drug resistance through regulating target proteins such as antioxidative enzymes,phase-II detoxification enzymes and metabolic enzymes.Therefore,NRF2 is regarded as an oncogene.Previously,RARa(Retinoic acid receptor alpha)and RXRa(Retinoid x receptor alpha)have been shown to inhibit NRF2 activity.In APL(acute promyelocytic leukemia),PML(promyelocytic leukemia protein)fused with RARa resulting in an oncogene known as PML-RARa,which disrupts the functions of PML and RARa,blocking transcription and differentiation of granulocytesAlthough previous study have shown that both RARa and PML inhibit NRF2 activity,however,how PML-RARa regulating NRF2 is not clear and require further investigation;furthermore,as thereare two major iso forms,PR-S(PML-RARa-Short iso form)and PR-L(PML-RARa-Long iso form),of PML-RARa that are present in APL cells,it is not clear whether they influence NRF2 activity in the same way.ObjectiveThis research focus on the modulation of NRF2 by PML-RAR?,and its mechanism.Methods1.Plasmids constructiona.Using the cDNA of NB4 cells,expressing PR-L in vivo as a template,genes encoding PR-S and PR-L were amplified by PCR and cloned into expression vector p3XFLAG and lentivirus vector pFuipw separately.b.Using the cDNA of NB4 cells as a template,genes encoding PR-S and PR-L mutants were amplified by PCR,and were cloned into p3XFLAG individually.c.Using the cDNA containing full length mRAR? as a template,the genes encoding mRARa mutants were amplified by PCR,and cloned into expression vector pET41a and pEGFP-C1 separately.2.Generation of stable cell linesEither pFuipw-PR-L,or pFuipw-PR-S,or pFuipw was transfected together with lentivirus packaging plasmids into HEK293T cells.Then the cell culture supernatant which contains lentiviral particles were collected,and the viral particles were concentrated by ultracentrifugation.Virus infection were monitored by immunofluorescence.To generate stable cell lines Hela cells infected with appropriate amount of supernatant,cells were selected by puromycin,subsequently single cell colonies were isolated and characterized.3.Investigating the effect of PR-S or PR-L on NRF2 signaling pathwaya.HO-1 expression by SDS-PAGE and WB.b.NRF2 transcriptional activity by luciferase assay.4.Analysis of the interaction of PR-S/PR-L and NRF2 signaling pathwaya.PR-L and NRF2 interaction by recombinant protein expression,immunoprecipitation(IP)and WB.b.Co-localization between PR-S/PR-L and NRF2 by recombinant protein expression and immunofluorescence staining assaysc.Mapping the interaction site between PR-S/PR-L and NRF2 using recombinant protein expression,IP,GST pull-down and WB.Results1.p3XFLAG and pFuipw plasmids encoding PR-S or PR-L were constructed successfully;p3XFLAG plasmids encoding PR-S or PR-L mutants were made successfully;pET41a or pEGFP-Cl plasmids encoding mRARa mutants were constructed successfully.For verification purposes,firstly,plasmids were sequenced and the NCBI BLAST tool were used to check the sequencing results;secondly,plasmids were overexpressed into prokaryote or eukaryote cells,and all the recombinant proteins were verified by WB.2.Hela cell lines,which overexpressed either PR-S(Hela-PR-S2,Hela-PR-S7),or PR-L(Hela-PR-L14,Hela-PR-L20)or empty vector(Hela-Fuipw),were generated successfully..To verify the cell lines,firstly,the intracellular localization of PR-S or PR-L in the cell lines was consistent with that of transient expression of PR-S or PR-L plasmid in Hela cells;secondly,although the expression level between PR-S and PR-L variable,similar results were obtained;thirdly,results of repeated experiments for the same cell line were comparable.In conclusion,we successfully generated Hela cell models which express recombinant proteins.3.Potentiation of NRF2 activity by PR-S/PR-La.Induction of the HO-1 expression by tBHQ in either polyclonal cell lines(Hela-PR-S,Hela-PR-L)or single cell lines(Hela-PR-S7,Hela-PR-L14)were much more pronounced than that in the control cell line.b.By treated with different doses(?M)of tBHQ for 24h,luciferase activity in PR-S or PR-L cell lines is higher than the control cell line,suggesting fusion proteins can potentiate NRF2 transcriptional activity.4.Interaction between PR-S/PR-L and NRF2a.Intracellular interaction Results from IP and WB showed that recombinant PR-L associated with NRF2 in Hela cells,which increased with NRF2 induction.b.Intracellular co-localization In HEK293T cells,recombinant PR-L and NRF2 were predominantly nuclear,and recombinant PR-S localized in both nucleus and cytoplasm.However,co-localization of PR-S/PR-L and NRF2 were primarily in the nucleus.Similar results were in Hela cells.In NB4 cells,PR-L and NRF2 co-localized in the nucleus,and the co-localization was enhanced with the increased expression of NRF2.c.Association in vitro Using full length RARa and its mutant recombinant proteins expressed in eukaryote or E.coli cells,and full length NRF2 and its mutant recombinant proteins expressed in E.coli cells,we performed systematic interaction analyses.The results revealed that recombinant PR-S or PR-L interacted with N-terminal region of NRF2 through interfinger region(IF)and C-terminal CII finger(CII)of RARa.ConclusionThis study revealed that PR-S/PR-L stimulated NRF2 transcriptional activity.Mechanistically,PR-S/PR-L interacted with NRF2 in the cells,and the interaction site of the two proteins were IF and C? regions of RARa.in the fusion proteins and N-terminal region in NRF2.Moreover,this study has laid the foundation for follow up study of the role of NRF2 in APL.