Indiana University-Purdue University Indianapolis (IUPUI) / 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.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/10999 |
| Date | 17 June 2016 |
| Creators | Willy, Jeffrey Allen |
| Contributors | Wek, Ronald C. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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