title: NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation.
abstract: NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-κB activation. However, the exact mechanism of its function has not been fully understood. Here, we report that knockdown of NEMO by RNA interference in Jurkat E6.1 cells enhanced TCR-induced NF-κB report gene activity and IL-2 production by promotion of IκBα degradation and p65 nuclear translocation, whereas inhibited TNF-α and LPS-induced IκBα degradation without influencing the phosphorylation of MAPKs. In human primary T and Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-κB activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-κB report gene activity was increased along with ascending doses of transfected siNEMO and reached the highest activity when knockdown about 70% of NEMO, then turned to decline and gradually be blocked once almost thoroughly knockdown of NEMO. Meanwhile, TNF-α induced NF-κB was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKKβ may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKKβ continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKKβ. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-κB activation and differentially regulates TNF-α and TCR-induced NF-κB pathways.
authors.affiliation: State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.forename: Liang-Hui
authors.initials: LH
authors.lastname: Diao
authors.forename: Yu
authors.initials: Y
authors.lastname: Gong
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Yingqiu
authors.initials: Y
authors.lastname: Li
mesh_terms: D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: C491731:IKBKG protein, human; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D051550:I-kappa B Kinase