NEMO differentially regulates TCR and TNF-α induced NF-κB pathways and has an inhibitory role in TCR-induced NF-κB activation

• Published in: Cellular signalling 2012-08, Vol.24 (8), p.1556-1564
• Main Authors: Wang, Kai, Diao, Liang-Hui, Gong, Yu, Liu, Xin, Li, Yingqiu
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Differentially regulation; HEK293 Cells; Humans; I-kappa B Kinase - metabolism; Jurkat Cells; Molecular Sequence Data; NEMO; NF-kappa B - metabolism; NF-κB; Receptors, Antigen, T-Cell - antagonists & inhibitors; Receptors, Antigen, T-Cell - metabolism; T cell receptor; TNF-α; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2012.03.022

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. ► RNA silencing NEMO in Jurkat E6.1 cells enhanced TCR-induced NF-κB reporter activity and IL-2 production ► In human primary T cells, CD3/CD28-induced NF-κB activation showed a para-curve correlation with the dosage of siRNA targeting NEMO ► TNF-α induced NF-κB activation showed a negative correlation with the knockdown extent of NEMO ► CD3/CD28 costimulation promoted cytoskeleton translocation of NEMO and IKKβ and the stoichiometric ratio of NEMO to IKKβ continuously shifted from high to low in cytoskeleton along with stimulation till 60min.