Enhancement of adult liver regeneration in mice through the hepsin-mediated epidermal growth factor receptor signaling pathway

Tsai, Yu-Fei; Fang, Mo-Chu; Chen, Chien-Hung; Yu, I-Shing; Shun, Chia-Tung; Tao, Mi-Hua; Sun, Cheng-Pu; Lu, Jean; Sheu, Jin-Chuan; Hsu, Yu-Chen; Lin, Shu-Wha

doi:10.1038/s42003-024-07357-1

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Published: 19 December 2024

Enhancement of adult liver regeneration in mice through the hepsin-mediated epidermal growth factor receptor signaling pathway

Communications Biology volume 7, Article number: 1672 (2024) Cite this article

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Abstract

Given the widespread use of partial hepatectomy for treating various liver pathologies, understanding the mechanisms of liver regeneration is vital for enhancing liver resection and transplantation therapies. Here, we demonstrate the critical role of the serine protease Hepsin in promoting hepatocyte hypertrophy and proliferation. Under steady-state conditions, liver-specific overexpression of Hepsin in adult wild-type mice triggers hepatocyte hypertrophy and proliferation, significantly increasing liver size. This effect is predominantly driven by the catalytic activity of Hepsin, engaging the EGFR-Raf-MEK-ERK signaling pathway. Significantly, administering Hepsin substantially enhances hepatocyte proliferation and facilitates liver regeneration following a 70% partial hepatectomy. Crucially, the proliferation induced by Hepsin is a transient event, without leading to long-term adverse effects such as liver fibrosis or hepatocellular carcinoma, as evidenced by extensive observation. These results offer substantial potential for future clinical applications and translational research endeavors in the field of liver regeneration post-hepatectomy.

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Introduction

The liver has a remarkable regenerative response that allows this vital organ to rapidly recover from tissue loss due to factors such as partial hepatectomy, toxin exposure, infection, or other forms of injury. The liver regeneration process is mainly facilitated by the dynamic behavior of hepatocytes and the coordinated activation of various signaling pathways and cellular mechanisms. However, the mechanisms that govern the intricate processes underlying liver regeneration remain incompletely understood¹.

Liver regeneration can be classified into three phases, namely priming, proliferation, and termination^2,3,4. In the priming stage, hepatocytes are stimulated to enter the G1 phase of the cell cycle under the influence of various cytokines⁵. Subsequently, hepatocyte proliferation is primarily driven by mitogens such as hepatocyte growth factor (HGF) and epidermal growth factor (EGF), initiating the proliferation process^6,7. Previous studies have demonstrated impaired regenerative capacity in mice lacking hepatic epidermal growth factor receptor (EGFR) following partial hepatectomy, underscoring the significance of EGFR signaling⁸. A similar regenerative role for the endogenous HGF-c-Met axis (c-Met is a receptor tyrosine kinase) was also evident in mice with liver-specific c-Met deficiency⁹. Furthermore, investigations have shown that systemic elimination of both MET and EGFR signaling abolishes liver regeneration and results in liver decompensation, emphasizing their crucial contributions and compensatory functions in promoting hepatocyte proliferation during liver regeneration¹⁰.

We previously demonstrated that hepsin serves as an activator of pro-HGF (HGF precursor) and induces MET signal transduction in hepatocytes¹¹. Hepsin, a type II transmembrane serine protease, is primarily expressed in the liver, where it plays a pivotal role in cell growth. Moreover, various tissues in the developing mouse embryo have significantly elevated hepsin levels, particularly in regions undergoing cell proliferation¹². Additionally, hepsin cleaves the EGFR extracellular domain, independent of tyrosine (Tyr) phosphorylation, leading to subsequent phosphorylation at Tyr1068¹³. Although many reports have indicated that hepsin has several important substrates that serve as hepatocyte mitogens, such as HGF and EGFR, and extracellular matrix turnover is associated with hepatocyte proliferation^14,15,16,17, the physiological role of hepsin in hepatocyte proliferation and liver regeneration remains incompletely characterized in mice.

To investigate how the serine protease hepsin induces hepatocyte proliferation, we overexpressed hepsin in the mouse liver. Our data show that hepsin overexpression initially causes hepatocyte hypertrophy and proliferation, eventually resulting in an increased liver size. In wild-type adult mice, hepsin overexpression resulted in EGFR phosphorylation at Tyr1068, activating downstream signaling. Overall, the results demonstrate that hepsin-induced hepatocyte proliferation is primarily achieved through the activation of the EGFR-Raf-MEK-ERK signaling pathway, facilitating liver regeneration after partial hepatectomy.

Results

Overexpression of human hepsin results in increased liver size in adult wild-type mice

To investigate the physiological functions of hepsin in adult wild-type mice, we utilized the AAV2/8 delivery system, combining AAV2 inverted terminal repeat DNA with the AAV serotype 8 capsid, to overexpress the wild-type human hepsin (hHPN^WT) or a loss-of-function mutant form (hHPN^RS) in the livers of adult mice¹¹. After intravascular delivery of AAV-hHPN^WT, AAV-hHPN^RS, or AAV-EGFP, human hepsin levels in both the liver and serum were analyzed at designated time points using ELISA and immunoblotting (Supplementary Fig. 1a-d). Human hepsin was detectable 1 day after AAV-hHPN^WT administration, peaking on Day 4 and gradually decreasing thereafter. Notably, only hHPN^RS accumulated over time, whereas hHPN^WT did not, even with the same dose of AAV-hHPN. The administration of AAV-hHPN^RS resulted in a 3 to 4-fold increase in the expression of the mutant form compared to the AAV-hHPN^WT group, with this elevated expression persisting until Day 14. The differing expression levels of hHPN^WT and hHPN^RS were primarily due to the inability of the loss-of-function mutant form of hHPN^RS to undergo auto-activation, thereby preventing its subsequent degradation, as previously described^14,18. Immunoblotting analysis of both liver and serum samples consistently identified the full-length Hepsin protein at 45 kDa. Remarkably, in the serum samples, a distinct band was exclusively observed, indicative of the cleaved/activated form of Hepsin, with molecular weights approximately around 17 and 10 kDa following hHPN^WT expression. The significant decrease in serum hHPN^WT from Day 4 to Day 7 was hypothesized to be due to hepsin auto-activation and subsequent degradation.

Analysis of liver gross anatomy revealed a notable increase in size on Day 4 and Day 7 following administration of AAV-hHPN^WT, whereas no such increase was observed for the AAV-hHPN^RS or AAV-EGFP (control AAV) group (Fig. 1a). Body weight did not differ significantly among the three groups (Supplementary Fig. 1e), although the ratio of liver weight to body weight was substantially greater for the AAV-hHPN^WT group (range, 6–9%) compared with the AAV-hHPN^RS or AAV-EGFP group (range, 4–5%). This difference remained evident over the entire 8- to 9-week observation period following AAV administration. Over time, however, the difference gradually decreased, indicating that the increase in liver size due to hepsin overexpression in mice was not sustained indefinitely (Fig. 1b).

**Fig. 1: Induction of hepatocyte hypertrophy and proliferation in adult mice upon overexpression of human hepsin.**

The size of individual hepatocytes was quantified by measuring the dimensions of hepatocytes with phalloidin to label F-actin^19,20. Administration of AAV-hHPN^WT led to a noticeable increase in hepatocyte size on each of Days 1, 2, and 4, whereas no such increase was observed for the other two control groups (Fig. 1c). Given that hepatocyte hypertrophy is a well-established initial process during normal liver regeneration^19,21, we analyzed hepatocyte proliferation at each time point after AAV-hHPN^WT administration. Immunoblotting revealed significant upregulation of the cellular level of cyclin D1, indicating the G1 to S phase transition, starting after Day 1 following AAV-hHPN^WT administration in comparison with the two control groups (Fig. 1d). Similarly, the assessment of cell proliferation through immunoblotting for proliferating cell nuclear antigen (PCNA) indicated a significant increase in levels from Day 4 post-AAV-hHPN^WT administration (Fig. 1e). Notably, cyclin D1 and PCNA levels decreased significantly from Day 7 to Day 14 after AAV-hHPN^WT administration, aligning with the observed decline in human hepsin expression in both the liver and serum (Supplementary Fig. 1b).

Next, we conducted immunohistochemical staining for phosphorylated histone H3, a marker for chromatin condensation during mitosis, to assess the proportion of mitotic hepatocytes²². AAV-hHPN^WT administration resulted in a significant increase in phosphorylated histone H3-positive cells on Days 4, 7, and 14, while no such increase occurred in the other two AAV groups (Fig. 1f). Immunohistochemical staining for another mitosis marker, Ki67, supported these findings (Supplementary Fig. 2). Histological examination conducted by the pathologist showed no apparent pathological abnormalities in any of the groups. Furthermore, there was no significant accumulation of intrahepatic leukocytes, as assessed by CD45 staining. Additionally, minimal hepatocyte death was observed, whether through apoptosis or necrosis, as indicated by TUNEL staining or HMGB1 staining, respectively, despite a transient increase in both alanine aminotransferase and total bilirubin on Day 4. (Supplementary Fig. 3).

In summary, the data clearly indicate that the overexpression of human hepsin in the adult mouse liver led to hepatocyte hypertrophy and increased proliferation. These effects were evident only in the wild-type hepsin group, not the loss-of-function mutant group, suggesting that hepsin’s serine protease activity primarily drives the observed phenomena.

Hepsin-induced hepatocyte proliferation with preserved liver function and absence of fibrosis and hepatocellular carcinoma in long-term observations

To elucidate the association between hepatocyte proliferation and hepsin overexpression levels, we conducted experiments using a series of titrated low doses of AAV-hHPN (ranging from 10¹¹ to 5 × 10⁹ vg per mouse). As expected, the level of human hepsin in serum decreased proportionally with the AAV dose on both Day 4 and Day 14 after AAV administration (Fig. 2a). The liver-to-body weight ratio increased significantly in the AAV-hHPN^WT group at doses as low as 2.5 × 10¹⁰ vg per mouse but not at 5 × 10⁹ vg (Fig. 2b). This suggests that hepatocyte proliferation was still stimulated despite the lower hepsin levels produced by AAV-hHPN^WT administration at 2.5 × 10¹⁰ vg per mouse, with concentrations of approximately 38.78 ± 7.8 ng/mL on Day 4 and 11.68 ± 4.88 ng/mL on Day 14 (Fig. 2b). Furthermore, the liver-to-body weight ratio showed a significant positive correlation with serum human hepsin levels on Day 14 following AAV administration (Fig. 2c).

**Fig. 2: Low levels of hepsin result in increased liver size in adult mice by inducing hepatocyte proliferation while maintaining normal liver function.**

In addition to the possibility of hepatocyte hypertrophy induced on Days 1, 2, and 4 after the administration of AAV-hHPN^WT (Fig. 1c), the immunoblotting data demonstrated a significant increase in cyclin D1 levels on Day 14 following low-dose AAV-hHPN^WT administration, indicating that hepatocyte proliferation also played a major role in the increase in liver size (Supplementary Fig. 4a and Fig. 2d). The use of a two-step perfusion technique—a well-established primary hepatocyte isolation protocol widely applied in studies involving mouse hepatocytes and hepatocyte transplantation^23,24,25—resulted in approximately 1.8-fold greater total hepatocyte yield from AAV-hHPN^WT mice compared with the AAV-hHPN^RS and AAV-EGFP groups on Day 14 after low-dose AAV administration (Supplementary Fig. 4b). Notably, the levels of alanine aminotransferase and total bilirubin did not differ significantly among the three groups of mice expressing low levels of human hepsin (Fig. 2e, f), indicating no obvious liver damage in these mice during hepatocyte proliferation.

For the long-term observation, we compared the histology results of mouse livers at 8, 27, and 82 weeks post-administration of 10¹² vg per mouse of AAV-hHPN^WT. Notably, neither liver fibrosis nor increased incidence of hepatocellular carcinoma was observed in the AAV-hHPN^WT group. Additionally, there were no pathological changes in major organs, including heart, kidney, lung, spleen and prostate, among the three groups at 82 weeks after AAV administration (Supplementary Fig. 5). These results further supported the safety and lack of adverse effects associated with hepsin-induced hepatocyte proliferation over an extended observation period.

Comparative transcriptome analysis of the entire liver following human hepsin overexpression via AAV delivery

To investigate the mechanism of hepsin-induced hepatocyte proliferation, we extracted total RNA from the livers of mice on Days 1, 2, and 4 after administration of AAV-hHPN^WT or AAV-EGFP. Gene chip data were employed for a comprehensive analysis, and principal component analysis was performed on probe-level gene expression data from raw CEL files. The results showed significant differences between the AAV-hHPN^WT and AAV-EGFP groups over time. A scatter plot of the first two principal components (PC1: 33.3%, PC2: 33.3% of explained variance) revealed distinct gene cluster separation as early as Day 1 of AAV-hHPN^WT administration (Fig. 3a), indicating an early and robust response of genes involved in hepatocyte proliferation.

**Fig. 3: Expression pattern of the liver transcriptome after overexpression of human hepsin.**

A total of 777 differentially expressed genes (DEGs) were identified on Day 1, 647 on Day 2, and 517 on Day 4 following AAV-hHPN^WT administration compared to AAV-EGFP, with 212 DEGs common across all three days (Fig. 3b, and Supplementary Data 1). Among the 212 commonly shared DEGs, 144 of them have assigned Gene Ontology (GO) terms, with those lacking any GO terms excluded. Through clustering analysis of the 144 common DEGs, illustrated in the heatmap (Fig. 3c and Supplementary Data 2), we identified two distinct groups of transcripts in livers treated with AAV-hHPN^WT compared to control groups. This analysis clearly distinguished between upregulated and downregulated transcripts. Subsequent Gene Ontology analysis of these DEGs revealed a selective identification of transcripts associated with metabolic processes and cell proliferation. The analysis revealed an upregulation in lipid biosynthesis processes, supported by genes like Serpina12, Cers6, Dhcr24, and Acaca (ACC1). In addition, transcripts associated with lipid metabolism, such as Acsl5, Angptl8, Pctp, Nucb2, Apom, and Ddit3 (CHOP), were upregulated (Fig. 3d, and Supplementary Data 2). Furthermore, the upregulation of proliferation-related transcripts, including Foxa3 (FoxP3), Avpi1, Pold4, and Dhcr24, suggested an increased proliferation status in AAV-hHPN^WT-treated livers (Fig. 3e, and Supplementary Data 2).

These findings provide valuable insights into the dynamic alterations in gene expression associated with the effect of hepsin overexpression on cellular metabolism, highlighting the critical role of hepsin during the early stages of both liver hypertrophy and hepatocyte proliferation²⁶. These effects of hepsin were observed at various time points following AAV-hHPN^WT administration.

Functional Enrichment Analyses of the total DEGs from Day 1, 2, and 4 were conducted to identify over-represented biological functions utilizing WikiPathways (Supplementary Fig. 6 and Supplementary Data 2). The selected subsets of over-represented biological functions and pathways from Day 1 were found to be enriched in energy metabolism-related functions, including Cholesterol Biosynthesis, Fatty Acid Biosynthesis, Lipid metabolic process, and the PPAR signaling pathway. Additionally, signaling pathways related to cell proliferation were substantially upregulated on Days 2 and 4 after AAV-hHPN^WT administration, such as DNA replication, Cell cycle, and G1 to S cell cycle control. Significantly, at each of the analyzed time points, there was no observed elevation in pathways related to inflammation, apoptosis, or necrosis.

Transcriptome data underwent Gene Set Enrichment Analysis (GSEA) using WikiPathways, assessing gene contributions to pathways based on fold change and P-value rankings. This analysis validated potential functional pathway regulation and offered a complementary perspective on gene sets post-AAV-hHPN^WT administration (Fig. 3f, g, and Supplementary Data 2). Consistent with the results of the Functional Enrichment Analysis, several metabolism-related pathways, including cholesterol biosynthesis, fatty acid biosynthesis, insulin signaling, glycogen metabolism, and adipogenesis, were identified as significantly altered on Day 1 or Day 4 after AAV-hHPN^WT delivery. Additionally, pathways related to liver regeneration and hepatocyte proliferation, such as G1 to S cell cycle control, Notch signaling, p53 signaling, and the Hypertrophy model, aligned with observed hepatocyte proliferation in AAV-hHPN^WT-administered mice. Furthermore, the upregulation of EGFR and MAPK pathways, well-recognized for their pivotal roles in liver regeneration and hepatocyte proliferation post liver injury^27,28,29, was observed as early as Day 1 or Day 4 after AAV-hHPN^WT delivery. These findings provide compelling support for the involvement of these pathways in the process of hepatocyte proliferation following hepsin overexpression.

Hepsin-induced hepatocyte proliferation is mediated by the EGFR-Raf-MEK-ERK signaling pathway

To validate the microarray analysis and functional pathway/gene set findings, immunoblotting was performed to assess the cellular level and activation status of EGFR and downstream components of the MAPK pathway at various time points after AAV-hHPN^WT, AAV-hHPN^RS, or AAV-EGFP administration. Notably, phosphorylation at EGFR’s C-terminal sites, specifically Tyr1068 and Tyr1148, crucial for ligand-induced activation³⁰, showed a significant increase as early as Day 0.75 and Day 1 after AAV-hHPN^WT administration (Fig. 4a). Additionally, a notable decrease in total EGFR levels starting from Day 2, following the pattern of EGFR phosphorylation, suggests the possibility of ubiquitination and subsequent degradation of activated EGFRs, as reported previously^31,32.

**Fig. 4: Hepsin-induced hepatocyte proliferation is activated by the EGFR-Raf-MEK-ERK signaling pathway.**

While no significant difference was observed in the N-region phosphorylation at serine 338 (Ser338) of c-Raf, the AAV-hHPN^WT group exhibited a significant increase in the phosphorylation of both MEK1/2 and ERK1/2 compared to control groups on Day 0.75 and Day 1 post AAV delivery (Fig. 4b–d). Previous studies indicate that c-Raf activity is modulated through feedback regulation of MEK-ERK signaling at specific phosphorylated sites, including Ser289, Ser296, and Ser301^33,34. In line with this, an increase in phosphorylation at the Ser289/Ser296/Ser301 site was observed from Day 0.75 to Day 2 after AAV-hHPN^WT administration (Fig. 4b). This heightened phosphorylation is associated with negative-feedback suppression of c-Raf-MEK1/2-ERK1/2 signaling, providing evidence for EGFR activation and downstream c-Raf-MEK1/2-ERK1/2 signaling following human hepsin overexpression in the adult mouse liver. To confirm the critical role of hepsin in hepatocyte proliferation, we performed immunoblotting of lysates of wild-type mouse primary hepatocytes following transduction with AAV-hHPN, focusing on EGFR phosphorylation, the downstream Raf-MEK-ERK signaling response, and cyclin D1 expression (Supplementary Fig. 7). Consistent with the immunoblotting results for mouse liver lysates, these primary hepatocytes exhibited a similar trend in the activation of the EGFR-Raf-MEK-ERK signaling pathway (Supplementary Fig. 7b). Additionally, cyclin D1 expression was elevated in the hepsin-overexpressing primary hepatocytes after AAV-hHPN^WT administration.

We previously reported the involvement of hepsin in the processing of pro-HGF and its influence on the activation of the downstream receptor, c-Met, in the liver¹¹. Given the critical role of the HGF/Met axis in liver regeneration and hepatocyte proliferation^35,36, we explored whether the overexpression of human hepsin in mouse liver would affect HGF/c-Met activation. However, there were no significant differences in either the serum HGF level or the phosphorylation of c-Met between the AAV-hHPN^WT and control groups (Supplementary Fig. 8). These results suggest that the overexpression of human hepsin in the adult wild-type mouse liver did not significantly impact the HGF-cMet signaling pathway.

Inhibition of EGFR activity blocks hepsin-induced hepatocyte proliferation

To elucidate the mechanism by which hepsin induces hepatocyte proliferation via EGFR activation and subsequent downstream signal transduction, we used the potent and irreversible EGFR inhibitor canertinib dihydrochloride (CI-1033). Canertinib was administered intraperitoneally on a daily basis, commencing 2 days prior to AAV-hHPN^WT administration and continuing for 2 days post-AAV delivery, for a total of 5 days of treatment (Fig. 5a). Immunoblotting was conducted to assess the extent of EGFR phosphorylation at Tyr1068 and Tyr1148 in the absence or presence of canertinib (CI-1033). At 2 days post-AAV-hHPN^WT administration, canertinib (CI-1033) treatment significantly reduced phosphorylation at Tyr1148 but not at Tyr1068 (Fig. 5b, c). This may be due to timing, as samples taken 2 days after AAV-hHPN^WT administration likely reflect a period beyond the peak phosphorylation at Tyr1068, when levels may already be declining (Fig. 4a). To confirm the inhibitory effect of canertinib (CI-1033) on EGFR phosphorylation at both Tyr1068 and Tyr1148, we conducted the analysis on day 1 post-AAV-hHPN^WT administration. The data showed that canertinib significantly inhibited EGFR phosphorylation at both sites on day 1 compared to the PBS-treated group, confirming that canertinib induces a rapid and robust attenuation of EGFR signaling (Supplementary Fig. 9).

**Fig. 5: Effect of an EGFR inhibitor on hepatocyte proliferation induced by hepsin overexpression in adult mice.**

Consistent with our hypothesis, our observations reveal that canertinib-mediated inhibition of EGFR substantially reduced the phosphorylation of c-Raf at the Ser289/Ser296/Ser301 site after AAV-hHPN^WT administration (Fig. 5d). Phosphorylation of c-Raf at any of these three sites is associated with downstream signaling pathways that engage in a negative-feedback loop that modulates c-Raf-MEK1/2-ERK1/2 signaling. Furthermore, administration of mice with AAV-hHPN^WT and treatment with canertinib resulted in a significant decrease in the cellular level of the early proliferation marker cyclin D1 as well as a decrease in the ratio of liver weight to body weight (Fig. 5e, f). This effect was not apparent in the PBS control group, in which mice administered AAV-hHPN^WT had a larger liver-to-body weight ratio. These outcomes underscored the hypothesis that inhibition of EGFR signaling could effectively counteract the hepsin-induced increase in liver size in adult wild-type mice.

Hepsin administration promotes liver regeneration in adult wild-type mice after 70% partial hepatectomy

After partial hepatectomy, the regenerative capacity of the liver allows the remaining liver to expand, resulting in the complete restoration of both liver mass and functionality³⁷. Consequently, we assessed the promotional effect of hepsin-induced hepatocyte proliferation in adult wild-type mice following partial hepatectomy. The surgical procedure involved the resection of the left lobe and middle lobe of the liver in adult wild-type mice. After surgery, mice were immediately intravenous administration of AAV-hHPN^WT, AAV-hHPN^RS, or AAV-EGFP (Fig. 6a). The extent of liver tissue removal was similar across these three groups (Fig. 6b). Importantly, at 3 days post-surgery, the liver-to-body weight ratio of AAV-hHPN^WT mice (3.37 ± 0.32%) was significantly greater than the value calculated for the other two groups (2.39 ± 0.45% and 2.53 ± 0.49%), indicating more complete liver recovery in the AAV-hHPN^WT administered group (Fig. 6c). Similarly, immunoblotting of liver lysates revealed that the level of the proliferation marker PCNA in the AAV-hHPN^WT mice was substantially higher than that measured for the AAV-hHPN^RS and AAV-EGFP control groups at 3 days after the 70% partial hepatectomy (Fig. 6d). Immunofluorescence and Immunohistochemical staining for proliferation markers revealed a significant increase in the proportion of Ki67-positive (Fig. 6e, and Supplementary Fig. 10a) and phosphorylated histone H3-positive hepatocytes in the AAV-hHPN^WT group compared to the other two groups (Fig. 6f).

**Fig. 6: Enhanced liver regeneration in adult mice following partial hepatectomy induced by hepsin-mediated hepatocellular proliferation.**

A pathology assessment, conducted on the third day following a 70% partial hepatectomy, examined hepatocytes and infiltrated immune cells in the remaining liver tissue. Consistent with immunostaining assay results for proliferation markers, the AAV-hHPN^WT group displayed significantly higher hepatocyte mitosis (Supplementary Fig. 10b) compared to the control groups. However, no notable differences were observed in hepatocyte cytoplasmic vacuolation or in the infiltration of neutrophils and mononuclear cells between these groups (Supplementary Fig. 10c). While alanine aminotransferase levels were comparable, total bilirubin significantly decreased in the AAV-hHPN^WT group, indicating a gradual improvement in liver function (Supplementary Fig. 10d). In conclusion, hepsin-induced stimulation of hepatocyte proliferation enhances liver regeneration after 70% partial hepatectomy in adult wild-type mice, suggesting a potential innovative approach for supporting liver regeneration in patients undergoing liver resection.

Discussion

This study is the first to demonstrate the crucial role of hepsin overexpression in promoting liver cell proliferation under normal physiological conditions and following liver surgery in mice within a specific timeframe. Figure 7a illustrates the signaling pathways and timing of events after AAV-hHPN^WT treatment, as summarized in the corresponding table in Fig. 7b. Our results indicate that hepsin introduction can trigger hepatocyte hypertrophy and proliferation, forming a foundational basis for liver regeneration within a defined period. The work provides several novel insights with significant biological and therapeutic implications. First, using an optimized transduction system, hepsin overexpression in the adult mouse liver initiated hepatocyte proliferation, leading to an increase in liver size. Second, hepsin overexpression induced the phosphorylation of EGFR, effectively activating the downstream Raf-MEK-ERK signaling pathway and promoting hepatocyte proliferation in vivo. Finally, hepsin overexpression was found to have potential applications for promoting liver regeneration following 70% partial hepatectomy. Importantly, this approach did not lead to long-term adverse effects such as liver fibrosis or hepatocellular carcinoma.

**Fig. 7: Characteristics and experimentally verified signaling pathway of hepsin-induced hepatocyte proliferation.**

Despite the multitude of potential proteolytic substrates predicted through in vitro studies³⁸, the physiological role of hepsin remains elusive. Previous studies suggested that using anti-sense oligonucleotides or hepsin-blocking antibodies could increase hepatoma cell size and reduce cell growth rate^11,12. Similarly, administering anti-hepsin antibodies to mice, mimicking a hepsin-deficient phenotype, resulted in enlarged hepatocytes and narrowing of liver sinusoids¹¹. These findings suggest that endogenous hepsin may regulate liver hemostasis, hepatocyte morphology, and cell growth. However, hepsin-deficient mice exhibited comparable survival and liver mass gain rates to wild-type mice after partial hepatectomy, indicating that endogenous hepsin might not be crucial or compensatory for liver regeneration in mice³⁹.

A previous study reported that overexpression of hepsin and EGFR in FT-293 cells (human embryonal kidney line) resulted in hepsin-mediated cleavage of the extracellular domain of EGFR¹³. The cleaved EGFR was then phosphorylated at Tyr1068 and thus no longer initiated downstream signaling, such as Erk1/2 phosphorylation, which typically responds to EGF stimulation in this in vitro system. Consequently, the precise mechanism by which activated hepsin promotes cell proliferation remains unclear. Our current results show that phosphorylation of EGFR at both Tyr1068 and Tyr1148 increased significantly within 1 day of hepsin overexpression in mouse liver, leading to the initiation of downstream Raf-MEK-ERK signaling. Importantly, inhibiting EGFR signaling mitigated the hepsin-induced increase in liver size in adult wild-type mice. These results suggest that EGFR and downstream c-Raf-MEK-ERK signaling constitute the primary mechanism driving hepatocyte proliferation in response to hepsin.

The function of c-Raf is attenuated through negative-feedback mechanisms within the MEK-ERK pathway at phosphorylated sites Ser289/Ser296/Ser301³³. In our study, we observed simultaneous negative-feedback phosphorylation at c-Raf Ser289/Ser296/Ser301, aiming to mitigate the activity of the Raf-MEK-ERK pathway. This suggests that the hepsin-triggered hepatocyte proliferation we observed was temporary and had a limited duration. Supporting this, the liver-to-body weight ratio peaked on Day 7 after hepsin overexpression and gradually decreased thereafter. Moreover, over an extended observation period, there was no liver fibrosis or hepatocellular carcinoma observed. The overall histology of major organs remained preserved even after 82 weeks of AAV-hHPN^WT administration. These findings collectively demonstrate the safety profile and potential application of hepsin for liver regeneration, highlighting its promise as a viable candidate for future clinical investigation.

Our study has several limitations. First, our results are based on mouse models, which may not fully replicate human liver physiology. Further studies using human liver cells and clinical trials are necessary to confirm the translational potential of hepsin for liver regeneration. Additionally, whereas the current study utilized an AAV-mediated gene delivery system that proved effective in mice, this approach may encounter challenges in human applications, such as efficiency, targeting specificity, and immune response. Future studies should explore alternate delivery methods, such as mRNA nanoparticles or small molecules that can upregulate hepsin, to overcome these potential challenges. We previously showed that hepsin-deficient mice did not exhibit defects in liver size or regeneration capacity after partial hepatectomy³⁹, suggesting that endogenous hepsin may not be essential or could be compensated for in liver regeneration in mice. In the present study, we modulated the hepsin level varying the dose of AAV administration. Our findings indicated that even low levels of exogenous hepsin in serum (as low as 38.78 ± 7.8 ng/ml) could induce hepatocyte proliferation and increase liver size. Although this concentration appears optimal and suitable for potential clinical application, we cannot rule out the possibility that other hepsin-related mechanisms or associated factors might be involved beyond the direct cleavage of EGFR by hepsin in hepatocytes. Hepsin plays critical roles in various processes, including maintaining liver architecture, regulating hepatocyte growth, and interacting with other serine proteases and modulating lipid metabolism^11,12,38. Despite its potential, further research is needed to fully understand hepsin’s mechanisms of action and safety profile. Therefore, ongoing research is essential to elucidate these underlying mechanisms and validate the therapeutic potential of hepsin for liver regeneration.

Given its promising potential for clinical applications, our study elucidates the pivotal role of hepsin-induced hepatocyte proliferation in efficiently driving liver regeneration and preserving optimal liver function in adult wild-type mice after partial hepatectomy. Immediate administration of AAV-hHPN^WT after 70% partial hepatectomy further accentuated these effects, evidenced by the restoration of the liver-to-body weight ratio and concurrent upregulation of key proliferation markers such as PCNA and Ki67. Histological assessments corroborated these findings, revealing remarkable augmentation of liver recovery in the AAV-hHPN^WT administrated mice compared with control groups after 70% partial hepatectomy. Our comprehensive safety evaluations provide additional support for the validity of these outcomes, demonstrating the potential of hepsin as a versatile player in liver regeneration. Beyond its role in liver regeneration, our study underscores the translational potential of hepsin as a candidate worthy of clinical investigation. This dual role of hepsin, as both a mechanistic driver of liver regeneration and a potential clinical asset, underscores its significance for shaping future therapeutic interventions for liver-related ailments.

Methods

Preparation of single-stranded adeno-associated virus (AAV) vectors 2/8

The Vector pAAV-MCS-hAAT/EGFP was designed for maximum transgene expression and contains a hepatic locus control region from the ApoE gene locus (ApoE-HCR), the human α1-antitrypsin (hAAT) promoter, a portion of the first intron of coagulation factor IX (FIX intron A) and the 3’ mRNA transcription termination/polyadenylation signal (bghpA, bovine growth hormone polyadenylation signal), and the reporter gene EGFP (encoding enhanced green fluorescent protein)⁴⁰. The hAAT promoter is well-established and recognized for its hepatocyte-specific expression in previous gene therapy studies^41,42, thus ensuring the targeted expression of human hepsin in hepatocytes with minimal off-target effects and enhancing the relevance of our study. The cDNA sequences for wild-type (hHPN^WT) human hepsin and a mutant form (hHPN^RS; R162A and S353Y double mutant) were previously described and were used to replace EGFP to produce AAV-hHPN^WT and AAV-hHPN^RS, respectively. The hHPN^RS mutant was engineered to produce a non-functional hepsin by introducing two specific mutations in critical regions essential for its proteolytic activity. We previously validated the loss of protease activity of hHPN^RS, where this mutant was used as a control in our experiments¹¹. An AAV-EGFP served as the vector control.

Administration of hepsin to adult wild-type mice

Male C57BL/6 mice (7-9 weeks old) were obtained from the Laboratory Animal Center of National Taiwan University College of Medicine and housed under specific-pathogen-free conditions with a 12-hour light/dark cycle in the same facility. Mice underwent retro-orbital injections with AAV-hHPN^WT, AAV-hHPN^RS, or AAV-EGFP at 5 × 10⁹, 2.5 × 10¹⁰, 10¹¹ or 10¹² vg per mouse. Each was dissolved in 50 μl of sterile phosphate-buffered saline. After the injections, the mice were sacrificed at pre-determined time points for further analysis. All animal experiments conducted were approved by the Board of Animal Welfare of the National Taiwan University College of Medicine and were performed in accordance with its guidelines (IACUC No. 20200110, IACUC No. 20220389). We have complied with all relevant ethical regulations for animal use.

Preparation of liver lysate samples

For the human hepsin ELISA, approximately 100 mg mouse liver tissue was lysed in Triton X-100 lysis buffer (100 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% w/v Triton X-100, 0.5% sodium deoxycholate, 2 mM EDTA, with freshly added 1% protease inhibitor (Protease Inhibitor Cocktail, Sigma, P8340) and 1 mM phenylmethanesulfonyl fluoride (Sigma, P7626). For immunoblotting, approximately 100 mg mouse liver tissue was lysed in RIPA lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% w/v NP-40 (CA630), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA), with freshly added 1% protease inhibitor, 1 mM phenylmethanesulfonyl fluoride, 1 mM dl-dithiothreitol (Sigma, D9163), and 1–2 × PhosSTOP working solution (Sigma, 4906837001). The liver tissue was shaken at high speed using a TissueLyser II (QIAGEN), centrifuged (16,000 × g) at 4°C for 20 min, and each resultant supernatant (liver lysate) was stored at –80°C for later analysis with the BCA assay (Pierce BCA Protein Assay kit).

ELISA for human hepsin

Antibodies that we produced and are specific for human hepsin were described before¹¹. Antibodies were diluted in coating buffer (0.05 M NaHCO₃/Na₂CO₃, pH 8.6) and incubated in a plate overnight at 4 °C. The next day, the coating buffer was removed and blocking buffer (5% fat-free milk in TBS containing 0.5% w/v Tween-20 (TBST) with 0.02% sodium azide) was added to the plate with subsequent incubation for at least 2 h before use. The blocking buffer was removed and the plate washed four times with wash buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.005% Triton X-100) before use.

For diluted liver lysate samples, the liver lysate was adjusted to a concentration of 3 mg/ml. Loading samples from Day 0.5 to Day 4 were diluted at ratios of 1:5 or 1:10 in TBS, while samples from Day 7 and 14 were diluted at ratios of 1:20 or 1:40. For diluted serum samples, those from Day 4 were diluted 1:20 or 1:40, and others were diluted 1:5 to 1:20. After adding the samples to the plate, they were incubated overnight at 4°C. The next day, each sample was removed, and each plate was washed four times with wash buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.005% Triton X-100). Horseradish peroxidase–conjugated secondary antibodies were then added and incubated for 2 h at room temperature. After washing four times with wash buffer, Enhanced K-Blue Substrate was added (TMB; NEOGEN, 308177) with incubation for 10–15 min at room temperature. Each reaction was terminated by addition of 1 N H₂SO4, and then the optical density at 450 nm was measured using a Spectramax 340PC Microplate Reader.

Immunoblotting

Diluted serum (1:10 in PBS) or mouse liver lysate containing 50 µg total protein were mixed with 3 × SDS-PAGE loading dye and subjected to SDS-PAGE (10% polyacrylamide gel). The separated proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane (100 V, 90 min, 4 °C), and the membrane was then blocked in TBST containing 5% fat-free milk for 0.5–1 h. The membrane was incubated with appropriate primary antibodies (diluted 1:500–1:1000 in TBST) overnight at 4 °C. The following primary antibodies were used: rabbit anti-hepsin from Abcam (#189246) for liver lysate and Cayman (#100022) for serum; all other antibodies were from Cell Signaling: rabbit anti-cyclin D1 (#2978), rabbit anti-PCNA (#2586), rabbit anti-p-Met (Tyr1234/1235; p, phosphorylated) (#3077), mouse anti-Met (#3127), rabbit anti-p-EGFR (Tyr1068) (#3777), rabbit anti-p-EGFR (Tyr1148) (#4404), rabbit anti-EGFR (#4267), rabbit anti-p-c-Raf (Ser338) (#9427), rabbit anti-p-c-Raf (Ser289/296/301) (#9431), rabbit anti--c-Raf (#9422), rabbit anti-p-MEK1/2 (Ser217/221) (#9121), rabbit anti-MEK1/2 (#9122), rabbit anti-p-ERK1/2 (Thr202/Tyr204) (#9101), rabbit anti-ERK1/2 (#9102), and rabbit anti-α-tubulin (#2144). Anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (1:5000) was used as the secondary antibody, which was incubated with the polyvinylidene difluoride membrane at room temperature for 1 h. Images of immunoblots were captured with a LAS-4000 IR (Fujifilm).

Immunofluorescence staining

For preparation of cryo blocks, the right posterior lobe of each liver was removed and fixed with 4% paraformaldehyde for 30 min followed by dehydration with a serial-dilution series of sucrose (10% for 1 h, 20% for 1 h, 30% overnight); samples were embedded in Tissue-Tek OCT Compound (Sakura Finetek USA) and then frozen at –80 °C. Each cryo block was cut into 8-μm-thick sections using a cryostat for immunodetection of F-actin, HMGB1, CD45, and Ki67. Reagents and antibodies used included phalloidin-tetramethylrhodamine B isothiocyanate (stock: 0.5 mg/ml, 1:500–1:1000, Sigma, P1951), anti-HMGB1 (1:100; Abcam, ab18256), anti-CD45 (1:100, Abcam, ab10558), anti-Ki67 (1:400; Abcam, ab15580). The secondary antibody was Cy3-conjugated anti-rabbit IgG (1:100; Jackson ImmunoResearch). Immunofluorescence staining was observed and quantified with an Olympus fluorescence microscope. Five fields in each section were subjected to quantification using a computer-based system equipped with ImageJ software (NIH). This process was repeated for two sections per mouse.

Immunohistochemical staining

Wax blocks were cut into 5-μm-thick paraffin-embedded liver sections, which were then dewaxed, rehydrated, and subjected to antigen retrieval for 15 min in 0.01 M sodium citrate buffer (pH 6.0) using the TintoRetriever Pressure Cooker (Bio SB). After washing three times with PBS, the 3% H₂O₂ for 10–15 min was used to inhibit the activity of endogenous peroxidase. After blocking for 1 h with 1% Fish Gelatin Blocking Agent (Biotum) in PBS containing 5% bovine serum albumin, anti-p-histone H3 (1:100; Cell Signaling, 9701) or anti-Ki67 (1:400; Abcam, ab16667) was applied with incubation at 4°C overnight. The secondary antibody was biotin-conjugated goat anti-rabbit IgG (1:200; Vector, 111065003), and the ABC system (ABC kit, Vector, PK-6100) was used to amplify the Nova Red (Vector, SK-4800) positive signal. Images were captured with an Olympus fluorescence microscope. Five fields in each section were subjected to quantification using a computer-based system equipped with ImageJ software (NIH). This process was repeated for two sections per mouse. The average number of p-histone H3-positive or Ki67-positive cells per mm² was calculated.

TUNEL assay

Paraffin-embedded liver sections were analyzed for hepatocyte apoptosis using the in-situ DNA terminal deoxynucleotidyl transferase dUTP nick end-labeling method (TUNEL Assay kit, HRP-DAB, Abcam, ab206386). The absolute number of TUNEL-positive cells was scored under a microscope.

Biochemistry tests and measurement of mouse serum HGF

Serum samples were collected via puncture from the inferior vena cava and analyzed for alanine aminotransferase and total bilirubin using a cobas c 111 analyzer (Roche) or a Fuji Dri-Chem 4000i. Hepatocyte growth factor (HGF) levels in the serum were measured using an ELISA kit (RayBiotech, ELM-HGF).

Isolation of primary hepatocytes from mouse liver

Hepatocytes were obtained through a retrograde two-step collagenase perfusion method⁴³. In short, mice were anesthetized via i.p. injection of avertin (25 mg 2,2,2-tribromoethanol/ml diethyldithiocarbamate) at a dosage of 0.25–0.3 ml per 20 g body weight. Following perfusion with Ca²⁺- and Mg²⁺-free HBSS (Cytiva, SH30588.01) for 2 minutes, additional perfusion was conducted at 37°C for 10 min with HBSS containing 0.5 mM EGTA and then 10 minutes of HBSS containing 5 mM CaCl₂ and 0.05% Type IV collagenase (Sigma, C5138). This perfusion method was performed through the vena cava and utilized the vasculature to allow the collagenase to come into close contact with the majority of hepatocytes while maintaining their structural integrity⁴⁴. After digestion with collagenase, each liver was excised and triturated using a syringe piston in Dulbecco’s minimal essential medium (DMEM). The filtrate underwent centrifugation at 50 × g for 2 minutes to gather the pellet, distinguishing hepatocytes from non-parenchymal cells due to their different cell densities^23,45. The pellet was then washed with 20 ml of DMEM and subsequently resuspended in DMEM for cell counting.

Total RNA extraction from mouse liver for microarrays

Mouse liver tissue samples were lysed and homogenized via the liquid nitrogen grinding method⁴⁶ with 1 ml RLT buffer from RNeasy Mini kit (QIAGEN, 74104). Liver lysates were collected in 1.5 ml tubes for RNA extraction using the RNeasy Mini kit (QIAGEN, 74104). Prepared RNA samples were quantified with a NanoDrop spectrophotometer (Thermo Fisher Scientific), and RNA quality was checked by 1% agarose electrophoresis to assure distinct 28S and 18S bands.

Microarrays

Microarray analysis was conducted by using the Affymetrix GeneChip Mouse Transcriptome Assay 1.0 (Thermo Fisher Scientific), and raw data were analyzed by Transcriptome Analysis Console Software (Thermo Fisher Scientific). The principal component analysis was performed on whole probe-level gene expression data from raw CEL files. The criteria employed for the selection of differentially expressed genes (DEGs) following AAV-hHPN^WT administration in comparison to AAV-EGFP on Day 1, 2, and 4 post-AAV transduction included a log2 fold change threshold of ±2, a significance level of p < 0.05, and the classification of gene groups based on “Coding” and “Multiple_Complex” criteria. A total of 777 DEGs were detected on Day 1, 647 on Day 2, and 517 on Day 4. The entire set of DEGs underwent Venn diagram analysis and Functional Enrichment Analysis based on WikiPathways conducted on DAVID online database. Subsequently, among the 212 commonly shared DEGs, 144 of them have assigned Gene Ontology (GO) terms, with those lacking any GO terms excluded. These 144 common DEGs were subjected to clustering for heatmap generation and further Gene Ontology analysis. The transcriptome data underwent Gene Set Enrichment Analysis (GSEA) using version 4.3.2 of the GSEA software. This analysis, based on WikiPathways, assessed the contributions of genes to specific pathways by considering their fold change and P-value rankings. All results from the aforementioned analyses were plotted using http://www.bioinformatics.com.cn/srplot, an online platform for data analysis and visualization.

Raw data processed with the TAC Software (Thermo Fisher Scientific) has been further analyzed, yielding results such as the list of DEGs, clustering, FEA, and GSEA. These outcomes have been documented in Supplementary Data 1 and 2 as Excel files. All aforementioned data have been deposited in NCBI GEO under accession number GSE245233 and are publicly accessible.

Canertinib treatment

Canertinib dihydrochloride (LC Laboratories, Woburn, MA; also known as CI-1033) was administered daily starting on Day -2 and continued through Day 0 of the AAV transduction (AAV-hHPN^WT, AAV-hHPN^RS, or AAV-EGFP) at a dose of 10¹² vector genomes (vg) per mouse, with the final dose of Canertinib given on either Day 1 or Day 2, for a total treatment period of 4 to 5 days. Canertinib was prepared by dissolving it in normal saline, heated to approximately 55°C, and administered via a daily intraperitoneal injection at a dose of 100 mg/kg body weight in a volume of 250 µl. Control mice received a daily intraperitoneal injection of 250 µl of normal saline.

Administration of hepsin to adult wild-type mice after 70% partial hepatectomy

The mice underwent isoflurane anesthesia, followed by a 70% partial hepatectomy. After detaching the liver-diaphragm ligament, the left lobe was manipulated and excised, and blood flow was blocked using a 4-0 silk suture. The left and median lobes were then removed, and the surgical site was sutured and secured. A retro-orbital injection of 10¹² vg per mouse of AAV and subcutaneous administration of 1 ml electrolyte solution (Otsuka, TAITA No. 5) were performed. Body temperature was maintained during recovery with a warm light.

Pathological score

The severity of histopathological changes was scored based on hematoxylin and eosin (H&E) staining. The frequency represents the proportion of individual mice in each group displaying different stages of pathological change. For hepatocyte mitosis, the pathological score was determined by examining 10 high-power fields (HPFs; 400 × magnification) of H&E-stained sections per mouse. Mitotic hepatocytes in 10 HPFs were categorized as follows: 0: no positive cells; 1: 1–5 mitotic cells; 2: 6–10 mitotic cells; 3: 11–15 mitotic cells; 4: 16–20 mitotic cells. The pathological score for hepatocyte hypertrophy, cytoplasmic vacuolation, and immune cell infiltration was determined by the percentage of cells exhibiting these morphological changes per section, screened at 100x magnification on bright-field H&E-stained sections per mouse. The scoring categories are as follows: 0: no positive cells; 1: <1% of cells; 2: 1-25% of cells; 3: 26–50% of cells; 4: 51–75% of cells.

Statistics and reproducibility

The number of mice used is indicated in the figure legends. Detailed and exact sample sizes for each group are provided in the Supplementary Data file. Prism 10 (GraphPad Software Inc.) was used for all statistical analyses. Data are expressed as the mean ± SD. The Kruskal–Wallis test with Dunn’s post-test, two-way ANOVA with Tukey’s post-test, Mann–Whitney test, and unpaired t test with Welch’s correction were used for the statistical analysis. P-values of <0.05 were considered to reflect statistical significance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings are included in the main text and Supplementary Information. Source data for the plots in the main figures are provided as an Excel file in the Supplementary Data, and uncropped, unedited blot images are included in the Supplementary Information. The raw microarray data have been deposited in NCBI GEO under accession number GSE245233 and are publicly accessible. Additional details and reagents are available from the corresponding author upon reasonable request.

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Acknowledgements

This study is supported by grants from the National Science and Technology Council (NSTC 112-2740-B-002-002, MOST 111-2740-B-002-004, NSTC 112-2314-B-002-140, NSTC 113–2314-B-002–240). We acknowledge the AAV Core Facility of Academia Sinica for generating recombinant AAV (Grant AS-CFII112-204). We are thankful for the technical services provided by the Transgenic Mouse Models Core Facility of the National Core Facility for Biopharmaceuticals, National Science and Technology Council, Taiwan as well as the Gene Knockout Mouse Core Laboratory of the National Taiwan University Center of Genomic and Precision Medicine. We are grateful to the Laboratory Animal Center of the National Taiwan University College of Medicine and Taiwan Mouse Clinic, Academia Sinica was well as the Taiwan Animal Consortium for technical support with pathology determination. We appreciate the Laboratory Animal Center of the National Taiwan University College of Medicine and the National Laboratory Animal Center (NLAC), NARLabs, Taiwan for animal supply. We sincerely thank the Biomedical Institute, Trineo Biotechnology Co., LTD, for their technical support. Additionally, we are grateful to Ying-Te Lee and Ching-I Lin from the Liver Disease Prevention and Treatment Research Foundation for their valuable technical assistance. Figures 5, 6, and Supplementary Fig. 9 include icons created using BioRender. A publication license has been obtained for each figure. Created in BioRender. Hsu, J. (2024). Available at: https://BioRender.com/o65j055.

Author information

These authors contributed equally: Yu-Chen Hsu, Shu-Wha Lin.

Authors and Affiliations

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan
Yu-Fei Tsai, Mo-Chu Fang, Mi-Hua Tao, Yu-Chen Hsu & Shu-Wha Lin
Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan
Chien-Hung Chen & Jin-Chuan Sheu
Department of Medicine, National Taiwan University Cancer Center, Taipei, Taiwan
Chien-Hung Chen
Laboratory Animal Center, College of Medicine, National Taiwan University, Taipei, Taiwan
I-Shing Yu
Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
Chia-Tung Shun
Department of Pathology, Good Liver Clinic, Taipei, Taiwan
Chia-Tung Shun
Department of Pathology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan
Chia-Tung Shun
Biomedical Translation Research Center (BioTReC), Academia Sinica, Taipei, Taiwan
Mi-Hua Tao & Cheng-Pu Sun
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Mi-Hua Tao & Cheng-Pu Sun
Genomics Research Center, Academia Sinica, Taipei, Taiwan
Jean Lu
Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan
Jin-Chuan Sheu & Yu-Chen Hsu
Department of Laboratory Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan
Shu-Wha Lin

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Contributions

Y.C.H. and S.W.L. conceptualized the study, orchestrated experimental procedures, and authored the manuscript. Y.F.T. and M.C.F. conducted the experiments and subsequent analyses. I.S.Y. and J.L. offered valuable insights during the experimental phase. C.T.S. conducted the pathological analyses. C.H.C. and J.C.S. provided essential guidance for translating the study’s findings into clinical applications. M.H.T. and C.P.S. provided professional consultation and support for the production of recombinant AAVs.

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Correspondence to Yu-Chen Hsu or Shu-Wha Lin.

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Tsai, YF., Fang, MC., Chen, CH. et al. Enhancement of adult liver regeneration in mice through the hepsin-mediated epidermal growth factor receptor signaling pathway. Commun Biol 7, 1672 (2024). https://doi.org/10.1038/s42003-024-07357-1

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Received: 04 April 2024
Accepted: 03 December 2024
Published: 19 December 2024
DOI: https://doi.org/10.1038/s42003-024-07357-1