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

NF-kappa B essential modulator (NEMO), the regulatory subunit of the I kappa B kinase (IKK) complex, is an essential adaptor both for inflammation stimuli and TCR-induced NF-kappa B 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-kappa B report gene activity and IL-2 production by promotion of I kappa B alpha degradation and p65 nuclear translocation, whereas inhibited TNF-alpha and LPS-induced I kappa B alpha degradation without influencing the phosphorylation of MAPKs. In human primary land Jurkat E6.1 cells, both CD3/CD28 and PMA/Ionomycin induced NF-kappa B activation showed a para-curve correlation with the dosage of small interfering RNA targeting NEMO (siNEMO): the NF-kappa B 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-alpha induced NF-kappa B was always inhibited no matter how much NEMO was knockdown. Subcellular fractionation results suggested that upon CD3/CD28 costimulation, NEMO and IKK beta may not cotranslocate to cytoskeleton fraction as a conventional NEMO/IKK complex with a static stoichiometric ratio, instead the ratio of NEMO: IKK beta continuously shift from high to low. Depletion of NEMO accelerated TCR-induced cytoskeleton translocation of IKK beta. Altogether, this study suggests that NEMO may function as a rheostat exerting a negative action on TCR-induced NF-kappa B activation and differentially regulates TNF-alpha and TCR-induced NF-kappa B pathways. (C) 2012 Elsevier Inc. All rights reserved.