TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury
Amber W. Wang, Kirk J. Wangensteen, Yue J. Wang, Adam M. Zahm, Nicholas G. Moss, Noam Erez, Klaus H. Kaestner
Amber W. Wang, Kirk J. Wangensteen, Yue J. Wang, Adam M. Zahm, Nicholas G. Moss, Noam Erez, Klaus H. Kaestner
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Concise Communication 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 4

Slc7a11 enhances hepatocyte repopulation.

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Slc7a11 enhances hepatocyte repopulation. (A) The Slc7a11 gene product ...
(A) The Slc7a11 gene product (xCT) imports cystine, which is used for GSH synthesis to alleviate oxidative stress. Several GSH metabolic enzymes were significantly (FDR ≤ 5%) upregulated (red) in repopulating hepatocytes from Fah–/– mice. GSSG, glutathione disulfide; GCL, glutamate-cysteine ligase; GSS, glutathione synthetase; GST, glutathione S-transferase; GSR, glutathione reductase; GPX, glutathione peroxidase. (B) Schematic of the competition assay to determine the effects of Slc7a11 overexpression on repopulation. (C) The Fah-Slc7a11 plasmid was significantly enriched after 4 weeks of repopulation. A 1-sample, 2-tailed Student’s t test was used to compare the ratio of 2 plasmids before and after repopulation (n = 8). (D) Representative IF staining and quantification showing a significant increase in xCT-positive hepatocytes. A paired, 2-tailed Student’s t test was used to compare HA- and GFP-expressing hepatocytes (n = 5). Scale bar: 100 μm. (E) Schematic of the CRISPR/Cas9 system used to inactivate Slc7a11 in Fah–/– mice. sgCtl, sgRNAs targeting firefly luciferase. (F) Representative IHC and IF images and quantification showing a significant reduction in repopulation nodules and replicating hepatocytes in mice treated with sgRNAs targeting Slc7a11 (sgSlc7a11) compared with control mice treated with sgCtl. A 2-sample, 2-tailed Student’s t test was used to compare groups (n = 4 each). Scale bars: 300 μm (top) and 100 μm (bottom).