| [BACKGROUND]Nuclear factor kappa B(NF-?B),an inducible transcription factor,regulates the expression of a variety of genes,such as cytokines,chemokines,growth factors,oxidative stress-related enzymes and acute phase proteins and plays important roles in the inflammation,immune responses,cell survival and other life processes.So the activation of NF-?B signaling pathway in cells must be under strict control.In unstimulated cells,the NF-?B dimers bind to its inhibitor of kappa B(I?B)proteins and are retained in the cytoplasm in an inactive state by masking their nuclear localization sequence.As a result,they can't activate the transcription of downstream target genes.When exposed to lipopolysaccharide,tumor necrosis factor(TNFa),ultraviolet(UV)and other related stimuli,I?B kinase(IKK)kinase complexes in the cell are activated through their respective signaling pathways.The activated IKK? kinase then phosphorylates I?B proteins in specific serine residues and promotes their ubiquitination.Finally,ubiquitinated I?B proteins are degraded via the 26S proteasome pathway in an ATP dependent manner and release their binding NF-?B dimmers.The released NF-?B then translocates into the nucleus and induces the expression of its downstream genes.Ubiquitination is an important form of protein post-translational modification where ubiqutin is attached to a substrate protein.Ubiquitination is accomplished by the concerted action of three types of enzymes:ubiquitin activating enzymes(El),ubiquitin conjugating enzymes(E2),and ubiquitin ligases(E3).E3 ligase plays a central role in targeting protein for ubiqutination.It directly interacts with its substrate protein through variable protein-protein interacting domains and ensures the specificity of protein ubiquitination.Among the E3 ubiquitin ligases,one kind of E3 ligases based on Cul3 protein attract researcher's attention.These E3 ligases are composed of Cul3 protein,the RING protein Rbxl/Rocl/Hrtl and BTB(Bric-a-brac,Tramtrack and Broad Complex,referred to as BTB)proteins.BTB proteins directly bind to Cul3 through its N-terminal BTB domain and interact with substrate proteins through its protein-protein interaction domain in its C-terminus,such as Meprin and TRAF homology(MATH)and Kelch.Bioinformatics analysis reveals that human genome encodes more than 200 BTB proteins.But the functions of most of them are unclear at present.Among the proteins embedding BTB/POZ domain,a subset of proteins named as Kelch like(KLHL)attract much attention.Members of the family are highly conserved in evolution,suggesting that they may play important physiological roles in cells.Presently,42 KLHL genes have been classified by the Human Genome Organisation(HUGO).However,functions of most members in this subfamily are unclear except for a few.By searching for the NCBI GEO database,we found that the expression of KLHL21 mRNA decreased in macrophage and dendritic cells upon LPS treatment(GDS2856,GDS1043,GDS88),suggesting that it may be involved in the regulation of inflammation in macrophages.As a member of KLHL protein family,KLHL21 contains a BTB domain,a BACK domain and five Kelch repeats.Sarah Maerki's work revealed that KLHL21 inteacted with Cul3 and mediated the ubiquitination of protein kinase Aurora B.It is necessary for cytokinesis and regulates translocation of the chromosomal passenger complex from chromosomes to the spindle midzone in anaphase.Given that ubiquitination is indispensible in the activation of canonical NF-?B signaling pathway,we preliminarily analyzed the role of KLHL21 protein in inflammation,especially in the activation of NF-?B signaling pathway.[AIM AND SIGNIFICANCE]To analyze the role of KLHL21 in inflammation,especially in the activation of canonical NF-kB signaling pathway.Our study will shed new light on the function of KLHL21 in cells and may provide some clues for the functional analysis of other KLHL family members.It may also provide potential therapeutic targets and strategies for inflammation-related diseases in clinic medicine.[METHODS]1.Mononuclear macrophage RAW264.7 cells were treated with 100 ng/ml LPS and harvested at different time points(0 h,0.5 h,1 h,2 h,4 h,6 h,12 h,16 h,20 h,24 h)after stimulation.Total RNA were extracted and the dynamic change of KLHL21 mRNA in RAW264.7 cells upon LPS treatment was analyzed by using real-time PCR.2.Bone marrow derived macrophages(BMDM)were treated with 100 ng/ml LPS and harvested at different time points(0 h,1 h,2 h,4 h,8 h,12 h,24 h)after stimulation.Total RNA were extracted and the dynamic change of KLHL21 mRNA in RAW264.7 cells upon LPS treatment was analyzed by using real-time PCR.3.RAW264.7 cells were respectively treated with 100 ng/ml Pam3CSK4(TLR1/2 agonists)and Pam2CSK4(TLR2/6 agonists).The dynamic change of KLHL21 mRNA was analyzed by using real-time PCR.4.RAW264.7 cells were pretreated with 100 ng/ml LPS for 1 h.Then 5 ug/ml actinomycin D(ActD)was used to inhibit the synthesis of RNA and harvested at different time points(0 min,30 min,60 min,90 min,120 min).The level of KLHL21 mRNA was analyzed by using real-time PCR.The stability of KLHL21 mRNA in RAW264.7 cells upon LPS treatment was compared to that of untreated RAW264.7 cells.5.Tumor necrosis factor receptor-associated factor 2(TRAF2),TRAF6,constitutively activated IKK?(SS/EE)mutant and p65 were transiently co-expressed with KLHL21 in human embryonic kidney HEK293T cells respectively.The effects of co-expression of KLHL21 on the expression of luciferase reporter gene under the control of a promoter containing NF-?B binding sites induced by overexpressing TRAF2,TRAF6,constitutively activated IKK?(SS/EE)mutant and p65 were analyzed by using dual luciferase reporter gene system.6.KLHL21 were transiently co-expressed with constitutively activated IKK?(SS/EE)mutant in HEK293T cells.The effects of co-expressing KLHL21 on the expression of NFKBIA,CCL5,TNFAIP3(A20)and IL-8,which are downstream target genes of NF-?B and induced by expressing constitutively activated IKK?(SS/EE)mutant,were analyzed by using real-time PCR.7.HEK293T cells were transiently transfected with KLHL21.At 24 h after the transfection,the cells were treated with 20 ng/ml TNFa and harvested at different time points(0 min,10 min,30 min,60 min).The effects of overexpressiing KLHL21 on nuclear translocation of p65 upon TNFa treatment were analyzed by Western Blotting.8.HEK293T cells were transiently transfected with KLHL21.At 24 h after the transfection,the cells were treated with 20 ng/ml TNFa and harvested at different time points(0 min,15 min,30 min,45 min,60 min).The effect of overexpression of KLHL21 on I?B? degration induced by TNFa treatment was analyzed by Western blotting.9.HEK293T cells were transiently transfected with KLHL21 and the effect of overexpressing KLHL21 on the level of endogenous I?B? was analyzed by Western blotting.10.Chemically synthesized siRNAs targeted different region of human KLHL21 mRNA were transiently transfected into HEK293T cells.At 24 h after the transfection,the cells were treated with 20 ng/ml TNFa and harvested at different time points(0 min,15 min,30 min,45 min,60 min).The effect of knocking-down KLHL21 on I?B? degration induced by TNFa treatment was analyzed by Western blotting.11.HEK293T cells were transiently transfected with KLHL21.At 24h after transfection,the cells were treated with 20 ng/ml TNFa and harvested at different time points(0 min,5 min,10 min).The effect of overexpressing KLHL21 on the phosphorylated I?B? upon TNFa treatment was analyzed by Western blotting.12.HEK293T cells were transiently transfected with KLHL21 and the interaction between KLHL21 and endogenously expressed IKK?,IKK?,NEMO,I?B? and p65 which are involved in the activatin of NF-?B signaling pathway were analyzed by co-immunoprecipitation respectively.13.HEK293T cells were transiently transfected with KLHL21 and the possible competition between KLHL21 and NEMO for binding IKK? was analyzed by co-immunoprecipitation.14.HEK293T cells were transiently transfected with KLHL21 and its effect on the endogenous expression of IKK? were analyzed by Western blotting.[RESULTS]1.KLHL21 mRNA in both RAW264.7 cells and BMDM cells decreased quickly upon LPS treatment.It decreased to its lowest level after 4 h,about 10%of the normal level and then rose slowly,but not to the untreated level even after 24 h.2.The mRNA level of KLHL21 in RAW264.7 cells treated respectively with 100ng/ml Pam3CSK4 and Pam2CSK4 shows the similar dynamic change to LPS treatment.It also reached to its lowest level after treated for 4 h,about 20%of the normal level.3.Compared to the untreated cells,the stability of KLHL2 mRNA decreased in RAW264.7 cells pretreated with 100 ng/ml LPS for 1 h.4.Co-expressing KLHL21 suppressed the induced expression of luciferase report gene under the control of ?B sites containing promoter by overexpressing TRAF2,TRAF6 and constitutively activated IKK?(SS/EE)mutant respectively,but had no effects on the induced expression of luciferase report gene by overexpressing p65.5.Co-expressing KLHL21 inhibited the induced expression of NF-?B target genes such as CCL5,I?B? by overexpressing IKK?(SS/EE)mutant,but had no effect on the induced expression of TNFAIP3 and further upregulated the induced expression of IL-8.6.Overexpressing KLHL21 inhibited the nuclear translocation of p65 induced by TNF? treatment,but promoted the nuclear translocation of p65 in untreated cells.7.Overexpressing KLHL21 inhibited the degradation of I?B? in HEK293T cells upon TNF? treatment,but had no effects on the level of endogenous I?B?.8.Knocking-down KLHL21 by specific siRNAs promoted the degradation of I?B?in HEK293T cells upon TNF? treatment.9.Overexpression of KLHL21 inhibited the phosphorylation of I?B? in HEK293T cells upon TNF? treatment.10.KLHL21 directly interacted with IKK?,but not with IKKa,NEMO,p65 and I?B?.11.KLHL21 and NEMO didn't compete for biding to IKK? kinase.12.Overexpressiing KLHL21 didn't affect the expression of endogenous IKK?.[CONCLUSION]KLHL21 is involved in the regulation of inflammation.It inhibits the phosphorylation and ubiquitin-dependent degration of I?B?,and the nucleus translocation of p65(RelA)via directly binding to IKK? kinase.Therefore,it negatively regulates the activation of NF-?B signaling pathway in vivo. |
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