A NOVEL MECHANISM FOR NF-κB-ACTIVATION VIA IκB-AGGREGATION: IMPLICATIONS FOR HEPATIC MALLORY-DENK-BODY INDUCED INFLAMMATION

Background & AimsMallory-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-{kappa}B activation. However, the precise mechanism that links protein aggregation to NF{kappa}B-activation and inflammatory response remains unclear.\n\nMethodsHerein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an I{kappa}B-loss with consequent NF-{kappa}B 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 I{kappa}B.\n\nResultsFour known mechanisms of I{kappa}B-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this I{kappa}B-loss was due to its sequestration along with I{kappa}B{beta} into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with I{kappa}B 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 I{kappa}B-nuclear import.\n\nConclusionWe discovered a novel mechanism of inflammatory NF-{kappa}B activation through I{kappa}B-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 a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.\n\nLay SummaryMallory-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 NF{kappa}B 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 NF{kappa}B activation via cytosolic I{kappa}B-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with I{kappa}B. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.