A Novel Mechanism for NF-κB-Activation via IκB-Aggregation: Implications for Hepatic Mallory-denk-body Induced Inflammation
Authors: Liu Yi , Trnka Michael J. , Guan Shenheng , Kwon Doyoung , Kim Do-Hyung , Chen J-J. , Greer Peter A. , Burlingame A. L. , Correia Maria Almira 
Published: March 2019

Background & Aims

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NFκB-activation and inflammatory response remains unclear.

Methods

Herein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an IκBα-loss with consequent NF-κB activation. We characterized the underlying mechanism in detail using hepatocytes from various knockout mice and MEF cell lines and multiple approaches including immunoblotting, EMSA, RT-PCR, confocal immunofluorescence microscopy, affinity immunoprecipitation, and protein solubility assays. Additionally, we performed rigorous proteomic analyses to identify the proteins aggregating upon PPIX treatment and/or co-aggregating with IκBα.

Results

Four known mechanisms of IκBα-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with IκBα through direct interaction or proximity. Of these ZnPPIX-aggregation targets, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as likely mediators of IκBα-nuclear import.

Conclusion

We discovered a novel mechanism of inflammatory NF-κB activation through IκB-sequestration into insoluble aggregates along with interacting aggregation-prone proteins. This mechanism may account for the protein aggregate-induced inflammation observed in MDB-associated liver diseases, thereby identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is abona fideprotoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

Lay Summary

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates commonly featured in many liver diseases. MDB-presence is associated with the induction of inflammatory responses both clinically and in all MDB-inducing models. Similar protein aggregation in neurodegenerative diseases is also known to trigger inflammation and NFκB pathway activation via an as yet to be characterized non-canonical mechanism. Herein using a MDB-inducing cell model, we uncovered a novel mechanism for NFκB activation via cytosolic IκB-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with IκBα. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.