Hepatic Protoporphyrin IX activates NF‐kB inflammatory pathway through a non‐canonical mechanism
Authors: Liu Yi , Correia Maria Almira 
Published: April 2020

Mallory‐Denk‐bodies (MDBs) are hepatic protein aggregates featured in various liver diseases. MDB‐presence is known to induce inflammatory responses 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 unknown mechanism. To mechanistically examine this association we used a cell‐based MDB‐inducing model of cultured mouse hepatocytes treated with the heme‐synthetic inhibitor N‐methylprotoporphyrin (NMPP). NMPP‐treatment, as expected from its ferrochelatase inhibition, resulted in cellular PPIX accumulation and p62 aggregation (markers of MDBs). A time‐dependent IκBα loss was also observed in hepatic lysates upon NMPP‐treatment with consequent NFkB activation. This IκBα‐loss was not inhibited upon inclusion of the proteasomal inhibitor MG‐132 or the dual autophagic‐lysosomal degradation (ALD) inhibitors (ALIs) 3‐methyladenine + NH4Cl. These findings thus excluded both the canonical ubiquitin‐dependent proteasomal degradation (UPD) and ALD involvement in this IκBα‐loss. HepG2 cells transfected with HA‐tagged wild type (WT) IkBa or a S32A/S36A‐IκBα mutant that is incapable of being phosphorylated for its canonical UPD, showed a loss of both IkBa WT and its mutant upon NMPP treatment, further excluding UPD. Then we considered whether NMPP‐elicited heme depletion led to this IκBα loss since heme depletion could activate the hepatic HRI eIF2α kinase, resulting in translational suppression of IκBα. Thus we examined HRI eIF2α kinase activity in mouse hepatocytes treated with NMPP and found increased ratio of phosphorylated eIF2α (eIF2αP) over basal (eIF2α) levels in parallel with the profound IkBα‐loss. However, in cultured hepatocytes from HRI WT (HRI+/+) and HRI knockout (KO; HRI−/−) mice showed that NMPP elicited a similar IκBα‐loss, revealing the independence of IκBα loss from HRI activation. This led us to check if the accumulation of PPIX from NMPP‐elicited ferrochelatase inhibition was actually responsible for the IκBα loss and found that PPIX and ZnPP are more potent than NMPP as inducers of IκBα‐loss and NFκB‐activation and p62 oligomerization/aggregation. Given the reported IκBα‐degradation via ALD, we next employed ATG5 WT and KO (ATG5−/−) MEF cells to conclusively exclude ALD in this process. As with ALD inhibitors, ZnPP‐elicited IκBα loss was not at all mitigated by knockout of the essential autophagic gene ATG5. Because calpain‐mediated degradation of IκBα also occurs, we employed CAPN4 WT, capn4 KO and CAPN4‐rescued capn4 KO MEF cells and found that knockout of the calpain‐degradation pathway (capn4 KO) failed to abrogate this IκBα loss induced by ZnPP. We additionally used ALD and calpain inhibitors and further excluded ALD and calpain degradation in this process. In summary, four known mechanisms of IκBα loss i.e. the canonical ubiquitin‐dependent proteasomal degradation, autophagic‐lysosomal degradation, calpain degradation and translational inhibition, were all examined and excluded, thus inferring a possibly novel mechanism of IκBα loss.Support or Funding InformationThese studies were supported by NIH Grants DK26506 and GM44037.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.