Browsing by Subject "CHOP"

Now showing 1 - 4 of 4
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    CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of nonalcoholic steatohepatitis
    (2015) Willy, Jeffrey A.; Young, Sara K.; Stevens, James L.; Masuoka, Howard C.; Wek, Ronald C.; Department of Biochemistry and Molecular Biology, IU School of Medicine
    Free fatty acid induction of inflammation and cell death is an important feature of nonalcoholic steatohepatitis (NASH) and has been associated with disruption of the endoplasmic reticulum and activation of the Unfolded Protein Response (UPR). Following chronic UPR activation, the transcription factor CHOP (GADD153/DDIT3) triggers cell death; however, the mechanisms linking the UPR or CHOP to hepatoceullular injury and inflammation in the pathogenesis of NASH are not well understood. Using HepG2 and primary human hepatocytes, we found that CHOP induces cell death and inflammatory responses following saturated free fatty acid exposure by activating NF-κB through a pathway involving IRAK2 expression, resulting in secretion of cytokines IL-8 and TNFα directly from hepatocytes. TNFα facilitates hepatocyte death upon exposure to saturated free fatty acids and secretion of both IL-8 and TNFα contribute to inflammation. Interestingly, CHOP/NF-κB signaling is not conserved in primary rodent hepatocytes. Our studies suggest that CHOP plays a vital role in the pathophysiology of NASH through induction of secreted factors that trigger inflammation and hepatocellular death via a signaling pathway specific to human hepatocytes.
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    The effect of alternative splicing on key regulators of the integrated stress response
    (2016-08) Alzahrani, Mohammed; Wek, Ronald C.; Goebl, Mark G.; Mosley, Amber L.
    The protein kinase General control non-derepressible-2 (GCN2) is a key regulator of the Integrated stress response that responds to various stress signals, including nutritional deprivation. As a result of high levels of uncharged tRNAs during amino acid depletion, GCN2 phosphorylates serine-51 of the α subunit of eukaryotic initiation factor-2 (eIF2), a translation factor that delivers initiator tRNA to ribosomes. Phosphorylation of eIF2α inhibits general translation, which conserves energy and nutrients and facilitates reprogramming of gene expression for remediation of stress damage. Phosphorylation of eIF2α also directs preferential translation of specific transcription factors, such as ATF4. ATF4 reprograms gene expression to alleviate stress damage; however, under chronic stress, ATF4 directs the transcriptional expression of CHOP, which can trigger apoptosis. Because multiple stresses can induce eIF2α phosphorylation and translational control in mammals, this pathway is referred to as the Integrated stress response. GCN2 and CHOP are subject to alternative splicing that results in multiple transcripts that differ in the 5'-end of the gene transcripts. However, the effect of the different GCN2 and CHOP isoforms on their function and regulation have not been investigated. Our data suggests that GCN2 is alternatively spliced into five different transcripts and the beta isoform of GCN2 is most abundant. Also alternative splicing of CHOP creates two CHOP transcripts with different 5'-leaders encoding inhibitory upstream open reading frames that are critical for translational control of CHOP during stress. This study suggests that alternative splicing can play an integral role in the implementation and regulation of key factors in the Integrated stress response.
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    Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response
    (American Society for Biochemistry and Molecular Biology, 2016-03-18) Young, Sara K.; Palam, Lakshmi Reddy; Wu, Cheng; Sachs, Matthew S.; Wek, Ronald C.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Upon exposure to environmental stress, phosphorylation of the α subunit of eIF2 (eIF2α-P) represses global protein synthesis, coincident with preferential translation of gene transcripts that mitigate stress damage or alternatively trigger apoptosis. Because there are multiple mammalian eIF2 kinases, each responding to different stress arrangements, this translational control scheme is referred to as the integrated stress response (ISR). Included among the preferentially translated mRNAs induced by eIF2α-P is that encoding the transcription factor CHOP (DDIT3/GADD153). Enhanced levels of CHOP promote cell death when ISR signaling is insufficient to restore cell homeostasis. Preferential translation of CHOP mRNA occurs by a mechanism involving ribosome bypass of an inhibitory upstream ORF (uORF) situated in the 5'-leader of the CHOP mRNA. In this study, we used biochemical and genetic approaches to define the inhibitory features of the CHOP uORF and the biological consequences of loss of the CHOP uORF on CHOP expression during stress. We discovered that specific sequences within the CHOP uORF serve to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP coding sequence. As a consequence, deletion of the CHOP uORF substantially increases the levels and modifies the pattern of induction of CHOP expression in the ISR. Enhanced CHOP expression leads to increased expression of key CHOP target genes, culminating in increased cell death in response to stress.
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    The unfolded protein response regulates hepatocellular injury during the pathogenesis of nonalcoholic steatohepatitis
    (2016-08) Willy, Jeffrey Allen; Wek, Ronald C.
    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.