SUMOylation Of KLF4 Promotes IL4 Induced Macrophage M2 Polarization

Objective and Background KLFs(Kruppel-like factors)are a subfamily of the zinc finger class of DNA-binding transcriptional regulators.So far,there are 17 members in KLFs family identified in mammals.KLFs can activate or repress target genes expression by binding to GC-and CACCC-boxes.KLFs are implicated in various biological processes,including cell proliferation,differentiation,development,migration,etc.KLF4,as an important member of the family plays an important role in many biological processes such as cell growth,differentiation and apoptosis.SUMO,a small ubiquitin-like protein,by conjugating to large numbers of proteins,regulates numerous cellular processes.SUMOylation of these target proteins is catalyzed by activating(E1),conjugating(E2)and ligating(E3)enzymes.Target proteins' SUMOylation can alter their biological activity as well as localization.Meanwhile,SUMO conjugation is a dynamic process that can be readily reversed by a family of SUMO-specific proteases(SENPs).Macrophage is the important component of the innate immunity,and plays a key role in the inflammatory response and immune resistance.Plasticity and flexibility are key features of mononuclear phagocytes and of their activation states.In response to various signals,macrophages may undergo classical M1 activation(stimulated by TLR ligands and IFN-?)or alternative M2 activation(stimulated by IL-4/IL-13).M1 macrophages stand for proinflammatory,and M2 macrophages stand for anti-inflammatory.SUMOylation of KLF4 has been reported and several previous studies haveinvestigated the role of KLF4 in macrophage polarization by using macrophage cell lines.Liao et al reported that KLF4 plays a key role in macrophage M2 polarization in vitro and in vivo.But the regulation of KLF4 in macrophage M2 polarization is not clear.For the reason of KLF4 critical roles in M2 macrophage polarization,we aimed to characterize the role of KLF4 SUMOylation in macrophage M2 polarization upon IL4 stimulus.Materials and Methods 1.Cell lines,plasmids and antibodies Celllines: HEK-293 T,HEK-293,RAW264.7,BMDM(Bone marrow derived macrophages),MEF(mouse embryonic fibroblast).Plasmids: p Shuttle-CMV,p Ad Easy-1,PXPAX2,PMD,PCDH-GFP vector,PCDNA3.1-vector,PCDNA3.1-HA-KLF4,PCDNA3.1-HA-KLF4-K278 R,PCDNA3.1-FLAG-SUMO1,FLAG-SENP1,FLAG-SENP2,PCDNA3.1-GFP,RGS-SENP1,RGS-SENP1 mutant.Antibodies: FLAG M2 and HA(Sigma),RGS(Qiagen),SUMO1(Abcam),KLF4(Santa Cruz),ACTIN(Cell Signaling Technology).2.Experimental methods Cell culture;Cell transfection technique;Establish KLF4 stable cell line by retrovirus;Establish KLF4 expression cell by adenovirus;Get and induce The bone marrow derived-macrophages(BMDMs);Western blotting technique;Co-Immunoprecipitation assay;RNA extraction technology;Reverse transcription experiment technology;Real time quantitative PCR;Chromatin immune coprecipitation experiment technology and Flow cytometry,etc.Results 1)Human KLF4 is SUMOylated at K278;2)Endogenous KLF4 is SUMOylated in RAW264.7 cells;3)SENP1,but not SENP2,can bind to KLF4;4)SENP1 de-SUMOylates KLF4;5)SUMOylated KLF4 is accumulated in Senp1-/-MEF cells;6)IL-4 treatment enhances KLF4 SUMOylation in RAW264.7 cells;7)KLF4 SUMOylation mediates the activated expression of M2 marker gene expression after IL-4 treatment in RAW264.7 cells;8)SUMOylation enhances the binding of KLF4 to the promoter of its target gene Arg-1 after IL-4 treatment in RAW264.7 cells;9)KLF4 SUMOylation mediates the activated expression of M2 marker gene expression after IL-4 treatment in BMDM;10)SUMOylation of KLF4 promotes IL-4 treated BMDM M2 polarization.Conclusion 1)KLF4 is a SUMOylated protein and specially de-SUMOylated by SENP1.2)IL-4 treatment enhances KLF4 SUMOylation in RAW264.7 cells.3)SUMOylation of KLF4 increases the expression of M2 marker genes in RAW264.7 cells after IL-4 treatment.4)SUMOylation of KLF4 promotes IL-4 treated BMDM M2 polarization.