Unknown,Transcriptomics,Genomics,Proteomics

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ShRNA profiling of human hepatocyte HepG2 cells knocked down for HNF4 to investigate its role in inflammatory response

ABSTRACT: To obtain a genomic view of hepatocyte nuclear factor-4Î± (HNF-4Î±) in the regulation of the inflammatory response, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokines in a model of knock-down HNF-4Î± HepG2 cells. The results indicate an extensive role for HNF-4Î± plays in the regulation of a large number of the liver-specific genes. Majority of genes (71%) affected by cytokine treatment are also affected by HNF-4Î± knock-down. This significant overlap suggests that HNF-4Î± may play a role in regulating the cytokine-induced inflammatory response. Experiment Overall Design: The different treated HepG2 cells were grouped into 4 groups (four replicates in each group): Group1, Control; Group2, HNF-4Î± shRNA treated cells; Group3, cytokine treated group; Group4, HNF-4Î± shRNA and cytokine treatments. RNA extraction and hybridization on Affymetrix microarrays were processed. The expression profiles between different groups were analyzed and compared. In order to explore the function of HNF-4Î± in the inflammatory response, a set of 170 genes annotated as inflammatory response was obtained from GO (geneotology.org), the enrichments of these inflammatory genes were analyzed in different treated groups.

ORGANISM(S): Homo sapiens

SUBMITTER: Peter Burke

PROVIDER: E-GEOD-15991 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFÎ± signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1Î± and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis. We sought to determine the effect of co-exposure of certain drugs associated with idiosyncratic DILI with the cytokine TNFalpha to mimic drug exposure with inflammation signaling in HepG2 cells Human hepatoma HepG2 cells were obtained from American Type Culture Collection (ATCC, Wesel, Germany), cultured in DMEM supplemented with 10% (v/v) FBS, 25 U/mL penicillin and 25 Âµg/mL streptomycin and used for experiments between passage 5 and 20.

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ShRNA profiling of human hepatocyte HepG2 cells knocked down for HNF4 to investigate its role in inflammatory response

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