Roles Of KLF4 In Regulating The Expression Of Inflammatory Mediator Genes Induced By LPS

Sepsis is the systemic inflammatory response syndrome(SIRS) induced by infection.It has been emphasized that lipopolysaechride(LPS) plays important role in the pathogenesis of sepsis by initiating the immune system.Many studies have demonstrated that LPS mediates activation of multiple signal-transduction pathways and some inflammation-related transcriptional factors,and then promotes the expression of multiple target inflammatory genes.However,the particular mechanisms of sepsis are still obscure.Kruppel-like factor 4(KLF4),a member of Sp1/KLF zinc-finger transcriptional factor family,binds to the binding sites including GC box, CACCC box and basic transcription elements on the promoters of target genes,to regulate the expression of target genes directly.By regulating the expression of the target genes,KLF4 plays important roles in cellular proliferation and differentiation,embryogenesis and the development of carcinoma.It is not delineated on the expression pattem of KLF4 under LPS treatment,and the roles and mechanisms of KLF4 in regulating the expression of inflammatory genes.Our previously research from cDNA microarray assays revealed that the expression level of KLF4 was up-regulated in lung tissues at 2 h,and further increased up to more than 10 times of the normal level at 20 h after LPS injection.These results indicate that KLF4 may be an important transcriptional factor participating in the expression regulation of inflammatory mediator genes induced by LPS.To verify the hypothesis mentioned above,we first investigated the expression pattern of KLF4 in LPS-mediated inflammatory cell models in human THP-1 monoeytes,human U937 monocytes/macrophages and mouse RAW264.7 macrophages by RT-PCR and Western blot.The results showed that the expression of KLF4 was up-regulated obviously in all the three kinds of monoeytes/maerophages in a time and dose-dependent manner after exposure to LPS.Meanwhile,the expression of KLF4 in RAW264.7 maerophages was increased significantly at the early phase (within 1-2 h)and further increased up to the late phase(after 24 h)after LPS stimulation.In order to understand the roles of KLF4 in regulating the expression of inflammatory mediator genes,we first overexpressed mouse KLF4 cDNA gene(pcDNA3.1-KLF4)into RAW264.7 macrophages by gene transfection and G418 screening,and inhibited the expression of KLF4 gene in RAW264.7 macrophages by transfection of KLF4 antisense oligonucleotides.Then,according to the screening results from bioinformatics and RT-PCR,we selected IL-1βand IL-10,two representative inflammatory mediator genes activated at early phase of inflammation,to investigate the effect of KLF4 on their expression and release under normal condition and LPS treatment.The results showed that,overexpression of KLF4 could inhibit the expression and release of IL-1βinduced by LPS,but promote the expression and release of IL-10 under the normal condition and after LPS stimulation;on the other hand, the inhibition of KLF4 by antisense oligonucleotides could promote the expression and release of IL-1β,and inhibit the expression and release of IL-10 under normal condition and after LPS stimulation.The results from EMSA demonstrated that KLF4 could bind to the KLF4 binding sites on the promoters of IL-1βand IL-10 genes,and the results from luciferase reporter gene assay demonstrated that KLF4 could inhibit the transcriptional activity of IL-1βgene and promote the transcriptional activity of IL- 10 gene.According to the up-regulation of KLF4 at the late phase after LPS stimulation,we predicted that KLF4 might also regulate the inflammatory mediator gene activated at the late phase.Therefore,we investigated the effect of KLF4 on the expression and release of HMGB1,an important inflammatory mediator gene released at the late phase of inflammation. The results showed that the levels of HMGB1 mRNA and protein were up-regulated under normal condition after KLF4 over-expression;after LPS stimulation,KLF4 overexpression promoted the expression, translocation and release of HMGB1.EMSA showed that KLF4 could bind to the sites at -736～-707 bp and -1317～-1304 bp on the HMGB1 promoter,and the binding activity was promoted by LPS stimulation.It is indicated that KLF4 plays a bidirectional role in the regulation of inflammation induced by LPS.It may play anti-inflammatory role at the early phase of SIRS induced by LPS by inhibiting the expression and release of early pro-inflammatory mediator IL-1βand promoting the expression and release of early anti-inflammatory mediator IL-10;and may play pro-inflammatory role at the late phase of SIRS induced by LPS by increasing the expression,translocation and release of late pro-inflammatory mediator HMGB1.Taken together,the present studies suggest a novel function of KLF4, that is,KLF4 can bind to the KLF4 binding sites on the promoters of inflammatory mediator genes including IL-1β,IL-10 and HMGB1 to regulate the expression and release of these genes.The bidirectional regulating effects of KLF4 on early or late inflammatory mediators provide novel insights into the pathogenesis of sepsis,and also new clues and information for the studies on the biological functions of KLF4,and the prevention and treatment of sepsis.