The unfolded protein response regulates hepatocellular injury during the pathogenesis of nonalcoholic steatohepatitis

Abstract

Non-alcoholic steatohepatitis (NASH), which is characterized by the induction of hepatocellular death and inflammation, is associated with the activation of cellular stress pathways such as the Unfolded Protein Response (UPR), an adaptive response to disruptions in endoplasmic reticulum (ER) homeostasis. Because the role of the UPR in the progression of liver disease is not well understood, we established an in vitro model to evaluate the role of the UPR in NASH and translated results to clarify disease progression in human liver biopsy samples. Treating HepG2 cells and primary human hepatocytes with saturated, but not unsaturated free fatty acids (FFAs), at physiologic concentrations induced hepatotoxicity by inhibiting autophagic flux. Saturated FFA treatment activated the UPR, including the transcription factors CHOP (GADD153/DDIT3) and NF-κB, leading to increased expression and secretion of cytokines such as TNFα and IL-8 that contributed to hepatic cell death and inflammation. Depletion of either CHOP or the RELA subunit of NF-κB in hepatocytes alleviated autophagy and cytokine secretion, resulting in enhanced cell viability and lowered inflammatory responses during exposure to saturated FFAs. We carried out next generation sequencing on cells deleted for either CHOP or RELA and identified IBTKα as a novel UPR member directly regulated by CHOP and NF-κB. In response to saturated FFAs, loss of IBTKα increased cell survival through lowered phagophore formation and reduced cytokine secretion. We also identified binding partners of IBTKα by immunoprecipitation and LC/MS, indicating that that IBTKα is part of a protein complex which functions at ER exit sites to facilitate initiation of autophagy and protein secretion. Furthermore, we discovered that CHOP and RELA coordinately regulate proteasome activity through NRF2 as an adaptive response to an inhibition of autophagic flux following palmitate exposure. To validate our model, we utilized human liver biopsy samples and demonstrated up-regulation of the UPR coincident with accumulation of autophagy markers, as well as secretion of cytokines IL 8 and TNFα in serum of NASH patients. Our study provides a mechanistic understanding of the roles of the UPR and autophagy in regulating saturated FFA induced hepatotoxicity at the cellular level.