TGF-β serves as a critical signaling determinant of liver progenitor cell activation and function
Chenhao Tong, Tao Lin, Han Wang, Luyao Jiang, Xiaodong Yuan, Wenwu Luo, Minghan Zhou, Carolina De La Torre, Hui Liu, Chen Shao, Seddik Hammad, Hui Gao, Jiarong Xie, Lei Xu, Roman Liebe, Zuguang Gu, Matthias P. Ebert, Huiguo Ding, Steven Dooley, Hong-Lei Weng
Chenhao Tong, Tao Lin, Han Wang, Luyao Jiang, Xiaodong Yuan, Wenwu Luo, Minghan Zhou, Carolina De La Torre, Hui Liu, Chen Shao, Seddik Hammad, Hui Gao, Jiarong Xie, Lei Xu, Roman Liebe, Zuguang Gu, Matthias P. Ebert, Huiguo Ding, Steven Dooley, Hong-Lei Weng
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Research Article Cell biology Hepatology

TGF-β serves as a critical signaling determinant of liver progenitor cell activation and function

Abstract

When massive hepatic necrosis–associated (MHN-associated) acute liver failure (ALF) occurs following severe damage, liver progenitor cells (LPCs) exit quiescence and enter differentiation programs during which they acquire hepatocyte-like functions. How LPCs maintain quiescence under physiological conditions and orchestrate activation following MHN remains largely unknown. We elucidate an essential role of TGF-β in regulating LPC quiescence and activation. Spatial transcriptomics and single-cell sequencing revealed that LPCs receive multiple signals, particularly TGF-β, HGF, and EGF, from surrounding hepatic stellate cells and macrophages in patients and zebrafish with MHN-induced ALF. Physiologically, TGF-β inhibits LPC proliferation by blocking G1-to-S phase transition, an effect reversed by Smad7 overexpression in a murine injury model. We observed extensive LPC proliferation in patients with ALF despite strong TGF-β/phosphorylated SMAD signaling. Immunostaining revealed concurrent activation of HGF/MET, EGF/EGFR, and downstream STAT3/ERK pathways in LPCs. In vitro, HGF or EGF overcame TGF-β–mediated growth arrest and promoted LPC proliferation. Beyond acting as a mitogen, HGF additionally induced hepatocyte gene programs (e.g., Hnf4a, Hnf1a) in LPCs. Strikingly, TGF-β signaling was required for HGF-dependent hepatocyte gene induction, indicating a dual role in restraining LPC proliferation and promoting functional maturation. These findings position TGF-β as a context-dependent determinant of LPC activation and lineage specification during ALF.

Authors

Chenhao Tong, Tao Lin, Han Wang, Luyao Jiang, Xiaodong Yuan, Wenwu Luo, Minghan Zhou, Carolina De La Torre, Hui Liu, Chen Shao, Seddik Hammad, Hui Gao, Jiarong Xie, Lei Xu, Roman Liebe, Zuguang Gu, Matthias P. Ebert, Huiguo Ding, Steven Dooley, Hong-Lei Weng

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Figure 6

HGF overrides TGF-β–mediated cell cycle arrest in LPCs.

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HGF overrides TGF-β–mediated cell cycle arrest in LPCs. (A) Cell viabili...
(A) Cell viability was assessed in HepaRG cells treated with different concentrations of HGF for 48 hours using the CCK-8 assay. (B) A heatmap displays transcriptomic changes in HepaRG cells treated with 20 ng/mL HGF for 0, 1, 8, and 24 hours. Genes were grouped into 6 clusters (C1–C6) based on distinct expression patterns. Altered cell cycle– and transcription factor–related genes are annotated within each cluster. Corresponding enrichment curves and gene counts illustrate dynamic expression trends over time. (C) GSVA scores were calculated to assess E2F and MYC target gene sets at indicated time points. (D) qRT-PCR was performed to examine mRNA expression of MYC, E2F1, CCND1, and MKI67 in HepaRG cells treated with HGF for the indicated durations. (E) Western blot analysis was conducted to assess phosphorylated and total MET, AKT, ERK, and FOXO1 in HepaRG cells treated with 20 ng/mL HGF for the indicated time points. (F) Western blotting was used to analyze protein expression of c-MYC, c-JUN, c-FOS, cyclin D1, and phosphorylated and total RB in HepaRG cells stimulated with 20 ng/mL HGF for different periods. (G) IF staining for Ki-67 and Draq5 was performed in HepaRG cells treated with TGF-β1, HGF, or a combination of both. Scale bar: 50 μm. (H) Flow cytometry was used to analyze cell cycle distribution in HepaRG cells under TGF-β1, HGF, and TGF-β1+HGF treatment. The right panel summarizes the percentage of cells in the G0/G1, S, and G2/M phase. Data are presented as mean ± SD. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****). 1-way ANOVA with Dunnett’s test (A and C); 2-tailed Welch’s t test vs. Con (D and H).