TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury
Amber W. Wang, … , Noam Erez, Klaus H. Kaestner
Amber W. Wang, … , Noam Erez, Klaus H. Kaestner
Published June 1, 2018
Citation Information: J Clin Invest. 2018;128(6):2297-2309. https://doi.org/10.1172/JCI95120.
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Categories: Research Article Gastroenterology Hepatology

TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury

Abstract

Understanding the molecular basis of the regenerative response following hepatic injury holds promise for improved treatment of liver diseases. Here, we report an innovative method to profile gene expression specifically in the hepatocytes that regenerate the liver following toxic injury. We used the Fah–/– mouse, a model of hereditary tyrosinemia, which conditionally undergoes severe liver injury unless fumarylacetoacetate hydrolase (FAH) expression is reconstituted ectopically. We used translating ribosome affinity purification followed by high-throughput RNA sequencing (TRAP-seq) to isolate mRNAs specific to repopulating hepatocytes. We uncovered upstream regulators and important signaling pathways that are highly enriched in genes changed in regenerating hepatocytes. Specifically, we found that glutathione metabolism, particularly the gene Slc7a11 encoding the cystine/glutamate antiporter (xCT), is massively upregulated during liver regeneration. Furthermore, we show that Slc7a11 overexpression in hepatocytes enhances, and its suppression inhibits, repopulation following toxic injury. TRAP-seq allows cell type–specific expression profiling in repopulating hepatocytes and identified xCT, a factor that supports antioxidant responses during liver regeneration. xCT has potential as a therapeutic target for enhancing liver regeneration in response to liver injury.

Authors

Amber W. Wang, Kirk J. Wangensteen, Yue J. Wang, Adam M. Zahm, Nicholas G. Moss, Noam Erez, Klaus H. Kaestner

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Figure 1

TRAP enables cell type–specific isolation of RNA from quiescent and repopulating hepatocytes.

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TRAP enables cell type–specific isolation of RNA from quiescent and repo...
(A) The approach for isolating repopulating hepatocyte RNA with the Fah–/– model involves use of the FAH expression construct to mediate liver repopulation and the GFP-tagged ribosomal protein L10a (GFP-L10a) to specifically isolate translating mRNAs with TRAP. Injection of the RosaLSL-GFP-L10a mouse with the AAV8-TBG-Cre virus, which has a tropism for hepatocytes and has a hepatocyte-specific promoter driving Cre expression in nearly all hepatocytes, allows for immunoprecipitation of translating mRNA from quiescent hepatocytes. (B) Bioanalyzer tracings of affinity-purified RNA from mice treated with or without the TRAP vector. FU, fluorescence units. (C) Representative (n = 3) IHC images of GFP show progressive repopulation over time in Fah–/– mice as well as complete labeling of quiescent hepatocytes in RosaLSL-GFP-L10a mice 1 week after injection of AAV8-TBG-Cre. No GFP expression was observed in livers from the uninjected mice. IF of Ki67 and GFP confirmed successful liver repopulation in Fah–/– mice injected with the TRAP vector, as all Ki67-positive hepatocytes express GFP. IF costaining also showed global GFP-expressing and rare Ki67-positive hepatocytes, indicating that the control tissue was truly quiescent. Note that a subset of mice showed only partial repopulation at 4 weeks (4-week regeneration after severe injury). Scale bars: 1 mm (top) and 100 μm (bottom).