Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

IKKγ/NEMO localization into multivesicular bodies.
S. Medunjanin1, R. Braun-Dullaeus1, A. Schmeißer1, S. Weinert1, J. Arendt1, P. Prazienka1
1Klinik für Kardiologie und Angiologie, Universität Magdeburg, Magdeburg;

Background: Atherosclerosis is initiated by vascular injury as a response to atherogenic risk factors such as dyslipidemia, diabetes, hypertension, and cigarette smoking. These risk factors induce endothelial cell activation through upregulation of adhesion molecules, particularly via NF-kB. The NF-kB pathway is a central signalling pathway for inflammatory and immune responses. IKKγ/NEMO is essential for NF-kB activation, and NEMO dysfunction in humans has been linked to immunodeficiency and inflammation. In a previous report, we identified glycogen synthase kinase-3b (GSK-3b) to participate in NF-kB activation by regulating IKKγ/NEMO. This study further examines the mechanism of this interaction. Results: We were able to demonstrate that NEMO phosphorylation by GSK-3b leads to NEMO localization into multivesicular bodies (MVBs). The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, this way, inhibited NF-kB signal transduction. Since the fusion of endosomes results in MVB formation, we performed colocalization studies of NEMO with intracellular organelles. Using the endosome marker Rab5, we observed the localization of NEMO into endosomes. In contrast, NEMO and Lamp1 did not colocalize, excluding that NEMO was directed to lysosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery, to facilitate its dissociation from endosomal membranes, as necessary for NEMO stability and NF-kB activation. The silencing of Vps4A led to a strong reduction of NEMO protein expression and consequently reduced NF-kB activation. Co-immunoprecipitation studies with NEMO and mutated NEMO demonstrated that Vps4A and NEMO interact directly and that this interaction requires the phosphorylation of NEMO. The transfection of HEK 293 cells with a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and a strong augmentation of NEMO expression in comparison to its expression in cells transfected with the control GFP-Vps4-WT. This upregulation of NEMO by the Vps4A-E233Q mutant was blocked by a specific GSK-3 inhibitor. In addition, overexpression of this mutated form of Vps4A led to increased NF-kB activation. These results reveal an unexpected role for GSK-3b and V-ATPase in the activation of NF-kB signalling.

Conclusion: Taken together, NF-κB/NEMO are regulators of inflammation as well as atherosclerosis. Our results demonstrate that NEMO requires its localization into multivesicular bodies and V-ATPase activity for its stabilization and subsequent NF-κB activation. Furthermore, our results suggest that MVBs are necessary for NEMO stability and that the ESCRT machinery is required for efficient NEMO degradation.

 


https://dgk.org/kongress_programme/jt2022/aP1956.html