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Original article
The HLF/IL-6/STAT3 feedforward circuit drives hepatic stellate cell activation to promote liver fibrosis
  1. Dai-Min Xiang1,2,3,
  2. Wen Sun1,
  3. Bei-Fang Ning4,
  4. Teng-Fei Zhou1,
  5. Xiao-Feng Li1,
  6. Wei Zhong5,
  7. Zhuo Cheng1,
  8. Ming-Yang Xia1,
  9. Xue Wang1,
  10. Xing Deng4,
  11. Wei Wang4,6,
  12. Heng-Yu Li1,
  13. Xiu-Liang Cui1,
  14. Shi-Chao Li1,
  15. Bin Wu7,
  16. Wei-Fen Xie4,
  17. Hong-Yang Wang1,3,
  18. Jin Ding1,3
  1. 1 The International Cooperation Laboratory on Signal Transduction, Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China
  2. 2 Nelson Institute of Environmental Medicine, New York University School of Medicine, New York, USA
  3. 3 National Center for Liver Cancer, Shanghai, China
  4. 4 Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
  5. 5 Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University, Shanghai, China
  6. 6 Department of Gastroenterology, Lanzhou General Hospital of Lanzhou Military Command, Lanzhou, China
  7. 7 Department of Gastroenterology and Endoscopy, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China
  1. Correspondence to Hong-Yang Wang and Jin Ding, The International Cooperation Laboratory on Signal Transduction, Shanghai Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai Road, 200438 Shanghai, China; hywangk{at}vip.sina.com, dingjin1103{at}163.com

Abstract

Background and aims Liver fibrosis is a wound-healing response that disrupts the liver architecture and function by replacing functional parenchyma with scar tissue. Recent progress has advanced our knowledge of this scarring process, but the detailed mechanism of liver fibrosis is far from clear.

Methods The fibrotic specimens of patients and HLF (hepatic leukemia factor)PB/PB mice were used to assess the expression and role of HLF in liver fibrosis. Primary murine hepatic stellate cells (HSCs) and human HSC line Lx2 were used to investigate the impact of HLF on HSC activation and the underlying mechanism.

Results Expression of HLF was detected in fibrotic livers of patients, but it was absent in the livers of healthy individuals. Intriguingly, HLF expression was confined to activated HSCs rather than other cell types in the liver. The loss of HLF impaired primary HSC activation and attenuated liver fibrosis in HLFPB/PB mice. Consistently, ectopic HLF expression significantly facilitated the activation of human HSCs. Mechanistic studies revealed that upregulated HLF transcriptionally enhanced interleukin 6 (IL-6) expression and intensified signal transducer and activator of transcription 3 (STAT3) phosphorylation, thus promoting HSC activation. Coincidentally, IL-6/STAT3 signalling in turn activated HLF expression in HSCs, thus completing a feedforward regulatory circuit in HSC activation. Moreover, correlation between HLF expression and alpha-smooth muscle actin, IL-6 and p-STAT3 levels was observed in patient fibrotic livers, supporting the role of HLF/IL-6/STAT3 cascade in liver fibrosis.

Conclusions In aggregate, we delineate a paradigm of HLF/IL-6/STAT3 regulatory circuit in liver fibrosis and propose that HLF is a novel biomarker for activated HSCs and a potential target for antifibrotic therapy.

  • Liver fibrosis
  • Hepatic stellate cell
  • Hepatic leukemia factor
  • IL-6
  • STAT3
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Significance of this study

What is already known on this subject?

  • Liver fibrosis is a wound-healing response represented by excessive extracellular matrix deposition upon chronic liver injury. Liver-resident hepatic stellate cells (HSCs) are the major source of fibrogenic cells and play a central role in liver fibrosis.

What are the new findings?

  • Hepatic leukemia factor (HLF) was exclusively expressed in activated HSCs in patient fibrotic livers.

  • The loss of HLF dramatically attenuated the experimental liver fibrosis in mice.

  • The upregulation of HLF is an intrinsic response of HSC and is required for HSC activation.

  • The HLF/interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) feedforward regulatory circuit drives HSC activation.

  • The correlation between HLF expression and alpha-smooth muscle actin, IL-6 and p-STAT3 levels was observed in patient fibrotic livers.

How might it impact on clinical practice in the foreseeable future?

  • Our data suggest that HLF is a diagnostic biomarker for liver fibrosis and a potential target for antifibrotic therapy.

Introduction

Liver fibrosis is a wound-healing response represented by excessive extracellular matrix (ECM) deposition on chronic liver injury.1 As a scarring process, liver fibrosis usually results from viral hepatitis, cholestatic/biliary and metabolic diseases, including alcoholic steatohepatitis and non-alcoholic steatohepatitis.2–6 Liver fibrosis is primarily asymptomatic in the early stages; as it progresses, it disrupts the architecture and function of liver by replacing the functional parenchyma with scar tissues, ultimately leading to cirrhosis, liver failure and even hepatocellular carcinoma.7 8 Effective antiviral or other causal treatments that remove the underlying cause of liver injury may halt the process of liver fibrosis.9 Unfortunately, there is no sensitive and non-invasive approach to diagnose asymptomatic patients and to monitor the progression of fibrosis to date.9 Moreover, no antifibrotic therapy has been approved to date, largely due to the various side effects and limited patient response to the causal treatment.9 10 Recent experimental and preclinical research has advanced our knowledge on the pathogenesis of liver fibrosis. However, the translation of preclinical progress into clinical practice has been hampered due to the lack of specific targets, which thus emphasises the urgency of clarifying the detailed mechanism of liver fibrosis and identifying novel therapeutic targets.

Liver fibrosis is modulated by dynamic reciprocity between parenchymal and non-parenchymal cells, among which the ECM-producing myofibroblasts play prominent roles in this scarring process.11 Liver-resident hepatic stellate cells (HSCs) are the major source of fibrogenic cells and play a central role in liver fibrogenesis.12 Upon liver injury, quiescent HSCs receive stimuli from the microenvironment and then transdifferentiate from vitamin A and lipid storing cells into proliferating, contractile, pro-inflammatory and fibrogenic myofibroblasts.13 In recent years, we have witnessed significant progress in exploring the mechanism underlying liver fibrosis, and various signalling molecules and pathways involved in HSC activation have been identified.9 14 But as yet, our understanding on the regulation of HSC remains far from clear.

Hepatic leukemia factor (HLF) was initially discovered in a fusion protein encoding by the chimeric transcript E2A-HLF, which was created by the t(17;19)(q22;p13) translocation in pre-B-cell acute lymphoblastic leukemia.15 Moreover, HLF messenger RNA (mRNA) was also detected in liver; therefore, it was named as HLF.15 16 Subsequent studies revealed that HLF, together with two other basic leucine-zipper (bZIP) transcription factors, thyrotroph embryonic factor (TEF) and albumin promoter d-box binding protein (DBP), belongs to the proline and acidic amino acid-rich (PAR) protein family.16–18 Mice simultaneously devoid of HLF, TEF and DBP were prone to cardiovascular disorders,19 epilepsy and ageing with a decreased life span.20 As a bZIP PAR transcription factor, HLF was able to form homodimers or heterodimers with other family member to regulate the expression of target genes.16 18 21 Nevertheless, there have been few studies concerning the physiological or pathological role of HLF in the liver to date,22–24 and the precise localization and function of HLF remain vague.

Herein we found that, unlike previous reports, HLF was exclusively expressed in activated HSCs (aHSCs) and played an essential role in liver fibrosis, suggesting that HLF is a novel biomarker for aHSCs and a potential target for antifibrotic therapy.

Materials and methods

Mice and fibrosis models

The heterozygous HLFPB/+ (mouse strain, 090922043-HRA) mice on the FVB/Nj background were constructed at Fudan University.25 26 Briefly, the second intron of HLF on Chromosome11:90232906 was inserted with a piggyBac (PB) transposon encoding CAG-RFP (a red fluorescent protein sequence under CAG promoter), whose direction was opposite to that of HLF. The heterozygous HLFPB/+ mice were intercrossed to generate homozygous mutated HLFPB/PB mice. HLF+/+ wild-type (WT) siblings obtained from the offspring of these crosses were used as WT control in the experiments. The mice were genotyped using PCR with the primers P1/P2/P3, as indicated in online supplementary figure 2, and the primer sequences were listed as follows: P1, 5′-ttcgtggttcttcatagacagtgtg-3′; P2, 5′-ctgagatgtcctaaatgcacagcg-3′; P3, 5′-atatgtaccctccaccaggcag-3′. Male mice aged 6–8 weeks were intraperitoneally injected with carbon tetrachloride (CCl4) (0.25 mL/kg body weight) or vehicle (olive oil) three times per week for 4 weeks to induce fibrosis and were sacrificed 4 days after the last injection.27 The mice subjected to bile duct ligation (BDL) were anaesthetised with ketamine and xylazine followed by midline laparotomy. The common bile duct was ligated two times with 6–0 silk sutures and cut through between the ligations. Sham-operated mice were subjected to laparotomy without BDL. The mice that received BDL or the sham operation were sacrificed 15 days later. The mouse livers and serum were collected for subsequent experiments.28 All animal procedures were conducted with the approval of the Eastern Hepatobiliary Surgery Hospital (EHBH) and the Second Military Medical University.

Supplementary Material

Supplementary data

Human liver samples

The control liver tissues (n=8) used for the mRNA analysis were the distal para-haemangioma tissues without any abnormality from patients who underwent surgical resection for hepatic haemangioma at EHBH. The fibrotic liver tissues (n=66) were from patients with hepatic fibrosis. The liver tissues were snap-frozen and the RNA was extracted for further analysis.

The normal liver tissues (normal=28) used in this study for immunohistochemistry (IHC) or immunofluorescent staining were freshly obtained from patients who underwent surgical resection for hepatic haemangioma as described above at EHBH from 2012 to 2015. The fibrotic liver tissues (n=50) were from patients with hepatic fibrosis. The fresh tissues were immediately formalin fixed for IHC analysis or immunofluorescent staining. The patients’ informed consent was also obtained, and all procedures were approved by the ethical committee of EHBH.

Isolation and culture of primary cells

Primary murine HSCs were isolated from the livers of male HLFPB/PB or WT mice aged 6–10 weeks as previously described.29 The HSCs were then cultured on plastic culture plates in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Primary hepatocytes or Kupffer cells were isolated as previously described.12

IHC and immunofluorescent staining

The liver samples were fixed with neutral buffered formalin and embedded in paraffin. Briefly, the slides were incubated with the indicated antibody. A horseradish peroxidase-conjugated antibody was used as the secondary antibody. Finally, a diaminobenzidine colorimetric reagent solution was used, followed by haematoxylin counterstaining. The slides were then scanned and representative images were pictured. Immunofluorescent staining was performed using a tyramide signal amplification (TSA) fluorescence kit (TSA Plus Fluorescein, NEL741001KT; PerkinElmer, Waltham, Massachusetts, USA) according to the manufacturer’s instructions.

The rabbit antihuman HLF antibody was prepared by us as follows. Total RNA of the human HSC line Lx2 cells was extracted, and reverse transcriptase PCR was conducted as previously described.30 The complete coding sequence of human HLF was amplified by PCR from Lx2 complementary DNA and cloned into pET-21a(+) prokaryotic expression vector. The recombinant pET-21a(+)-HLF plasmid was sequenced to assure its accuracy and then used for transformation of BL21(DE3) competent Escherichia coli cells. The recombinant HLF protein was expressed in E. coli cells and was purified from the insoluble fractions of induced E. coli cells using the nickel-nitrilotriacetic acid (Ni-NTA) purification system (Life Technologies, Grand Island, New York, USA). The purified recombinant HLF protein was then used for immunisation of rabbits, and the antisera were purified by protein A and antigen affinity chromatography. The specificity of purified polyclonal antibody against human HLF was validated by ELISA, western blot and immunofluorescent assay, respectively. Moreover, western blot assay of HLF knockdown Lx2 cells showed the downregulated HLF expression compared with control cells, which further validated the specificity of our rabbit polyclonal antibody (see online supplementary figure 1A). Other antibodies used for IHC or immunofluorescent staining are listed in online supplementary table 1.

Cell migration and proliferation assays

For the cell proliferation analysis, the primary HSCs or Lx2 cells were seeded in 96-well plates (3×103 cells per well). ATP activity was measured using a Cell Counting Kit-8 at indicated time points. For the cell migration assay, 2×105 cells were seeded into the upper chamber of a polycarbonate Transwell in serum-free DMEM. DMEM containing 10% FBS as chemoattractant was added in the lower chamber. After 2 hours of incubation, the chamber was then fixed. The cell counts were expressed as the mean number of cells per field of view.

Western blot assay

Thirty micrograms of proteins was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated with the primary antibody overnight. The protein band, specifically bound to the primary antibody, was detected using an IRDye 800CW-conjugated secondary antibody and LI-COR imaging system (LI-COR Biosciences). The antibodies used for western blot are listed in online supplementary table 1.

Chromatin immunoprecipitation assays

The chromatin immunoprecipitation (ChIP) assay was performed using a Magna ChIP HiSens Kit (Millipore , Bedford, Massachusetts, USA) according to the manufacturer’s instructions. The chromatin was immunoprecipitated with IgG, anti-Flag or anti-STAT3 (signal transducer and activator of transcription 3) (Y705) antibodies. The DNA was then purified, and PCR was performed to assess the bound sequences. The primers used are listed in online supplementary table 2.

Picrosirius red staining and quantification

The formalin-fixed, paraffin-embedded sections were hydrated in distilled water and stained with 0.1% sirius red for 1 hour. The nuclei were counterstained with haematoxylin for 5 min. The slides were dehydrated in 100% ethanol and mounted. The slides were then scanned, and representative images were pictured. The staining was further quantified by a pathologist, and the proportion of the sirius red-positive area was calculated.

Adenoviruses

pFLAG-CMV2-HLF and pFLAG-CMV2 were cloned into the AdMax Shuttle vector according to the manufacturer’s instructions (Microbix, Ontario, Canada). The Shuttle vector was co-transfected with the genomic plasmid into HEK-293 cells to pack the adenoviral particles, and the particles were further amplified and purified. The anti-Flag antibody was used to recognise exogenous Flag-tagged HLF protein in Ad-HLF-infected cells.

Statistical analysis

The statistical analysis was performed using SPSS V.18.0 software. The differences between variables were assessed by a two-tailed Student’s t-test. Pearson’s correlation analysis was performed to determine the correlation between two variables. Data were presented as the means ± SD, unless otherwise indicated. A p value <0.05 was considered statistically significant.

Results

HLF is specifically expressed in aHSCs of fibrotic liver

To explore the role of HLF in liver fibrosis, we first examined the expression of HLF in human liver tissues. As shown in online supplementary figure 1B, the mRNA expression of HLF was significantly increased in fibrotic livers from patients compared with healthy individuals. Strikingly, HLF expression was absent in human healthy livers, but it was highly expressed in patient fibrotic livers (figure 1A). Notably, the expression pattern of HLF coincides with that of alpha-smooth muscle actin (α-SMA), a well-established marker of aHSCs in the fibrotic livers (figure 1A), implying the restricted expression of HLF in aHSCs. As expected, the upregulation of HLF was also observed in CCl4-induced or BDL-induced murine liver fibrosis (figure 1B,C). The HLF protein was only detected in mice fibrotic livers or primary aHSCs rather than parenchymal hepatocytes or primary Kupffer cells, further suggesting the restricted expression of HLF in aHSCs (see online supplementary figure 1C). Strikingly, dual immunofluorescence staining of liver tissues from patients with fibrosis displayed a close co-localization of HLF and aHSC marker α-SMA or desmin, whereas there was no notable overlap between HLF and other cell markers, including HNF4α (hepatocytes), CD31 (liver sinusoidal endothelial cells), CK19 (cholangiocytes) and CD68 (Kupffer cells), which further confirmed the HSC-specific expression pattern of HLF in fibrotic livers (figure 1D and see online supplementary figure 1D). Moreover, HLF was found to be mainly expressed in the nucleus of the cultured human HSCs (see online supplementary figure 1E). Collectively, these data suggest that HLF could serve as a novel biomarker for aHSCs and liver fibrosis.

Figure 1

HLF is highly expressed in HSCs of fibrotic livers. (A) Tissue sections of normal or fibrotic liver from patients were subjected to H&E staining, sirius red staining and immunohistochemistry, and representative images are shown. Scale bar=100 µm. (B and C) The FVB male mice were subjected to CCl4-induced or BDL-induced experimental fibrosis and sacrificed at the indicated times (n=4 for each group). The livers were collected as described in the Materials and methods section and subjected to real-time PCR and western blot analysis. *p<0.05. (D) Dual immunofluorescence staining of fibrotic liver of patient using anti-α-SMA, anti-desmin, anti-HNF4α, anti-CD31, anti-CD68, anti-CK19 and anti-HLF antibodies. Representative images are shown. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (scale bar, 25 µm). The white dashed boxes were further enlarged below to show the cellular distribution of HLF in HSCs. Corresponding images of HLF with HNF4α, CD31, CK19 or CD68 in small magnification are shown in the white dashed box in supplementary figure 1D. α-SMA, alpha-smooth muscle actin; BDL, bile duct ligation; CCl4, carbon tetrachloride; HLF, hepatic leukemia factor; HSC, hepatic stellate cell; WT, wild type.

HLF depletion ameliorates experimental liver fibrosis

DNA transposon PB-mediated insertional mutagenesis was recently reported as an efficient large-scale genetic manipulation approach in mice.25 26 In the present study, we used a PB insertion mutation strain, 090922043-HRA, in which CAG-RFP was inserted into the second intron of the HLF gene (see online supplementary figure 2A). The mice were genotyped using PCR (see online supplementary figure 2B), and HLF expression in healthy liver was examined by western blotting (see online supplementary figure 2C). Our data showed that HLF protein was undetectable in healthy livers from WT and HLFPB/PB mice. In addition, no overt phenotype change in liver of homozygous mice (HLFPB/PB) was observed (see online supplementary figure 2D).

To address the role of HLF in hepatic fibrosis, HLFPB/PB and WT mice were subjected to CCl4-induced or BDL-induced liver fibrosis. H&E and picrosirius red staining revealed the attenuated liver bridging fibrosis and collagen deposition in HLFPB/PB mice (figure 2A). Histological staining was also quantified, and consistent results were achieved (figure 2B,C). Moreover, the transcription of the predominant fibrotic genes, including α-SMA, α1(I)-collagen and TIMP-1, was significantly downregulated in the HLFPB/PB mice, whereas the transcription of the matrix degradation genes such as matrix metalloproteinase-3 (MMP3) and MMP13 was dramatically elevated (figure 2D,E). Collagen deposition during liver fibrosis is considered to be associated with the decreased collagen degradation in addition to the enhanced collagen synthesis. Our data revealed a notable increase in MMP3/MMP13 levels and a dramatic decrease in TIMP-1 levels in HLF-depleted mice compared with WT control, which might be responsible for the reduced collagen deposition. Collectively, these data suggested that HLF could play a promoting role in liver fibrosis.

Figure 2

HLF deletion ameliorated experimental liver fibrosis. (A) The HLFPB/PB and WT FVB male mice were subjected to CCl4-induced or BDL-induced experimental fibrosis (n=5 for each group), and the livers were collected as described in the Materials and methods section and subjected to H&E and sirius red staining. Scale bar=100 µm. (B and C). The proportion of the sirius red-positive area was quantified. **p<0.01. (D and E). Real-time PCR analysis of the fibrotic genes in the two experimental fibrosis models.

HLF is required for the activation of HSCs

As an aberrant wound-healing response, liver fibrosis is characterised by chronic injury-induced hepatocyte death, inflammation, regeneration and scar formation.11 31 As shown in online supplementary figure 3A, serum ALT and AST levels were equivalent between HLFPB/PB and WT mice, indicating that HLF depletion did not influence the liver injury upon pro-fibrotic stimuli. Histological Ki67 staining and quantification also demonstrated that HLF deficiency did not affect hepatocyte proliferation (see online supplementary figure 3B). Moreover, the difference of hepatic macrophage infiltration, which is known to be associated with fibrosis-related inflammation and HSC activation, was less noticeable between HLFPB/PB and WT mice (see online supplementary figure 3C). Nevertheless, impaired HSC activation as indicated by decreased α-SMA expression was observed in the HLFPB/PB mice compared with WT mice, suggesting that HLF could be involved in the activation of HSCs (figure 3A,B).

Figure 3

HLF is involved in the activation of HSCs. (A) The fibrotic livers of mice were subjected to H&E staining and immunohistochemistry of α-SMA. Scale bar=100 µm. (B) Quantification of the α-SMA-positive area (upper). The livers were subjected to western blotting, and the representative images are shown (lower). (C) The quiescent primary HSCs were isolated from the WT mice and underwent a culture-activated process in vitro. The cells were collected at indicated time points and subjected to western blot assay. Densitometry analysis of HLF levels relative to β-actin is shown. Representative images from three independent experiments are shown. (D) The proliferation of the primary HSCs isolated from the HLFPB/PB or WT mice was compared using the CCK8 assay. Representative results from three independent experiments are shown. (E) The migration capability of the primary HSCs isolated from the HLFPB/PB or WT mice was evaluated using the wound-healing assay. Representative results from three independent experiments are shown. (F) The primary HSCs isolated from the HLFPB/PB or WT mice were cultured for 96 hours and then subjected to real-time PCR analysis.

To elucidate the role of HLF in HSC activation, primary HSCs of mice were isolated as previously described.29 Importantly, HLF expression was undetectable in the isolated quiescent primary HSCs from WT mice, whereas evident HLF expression was noted in the culture-activated HSCs, which further supported the specific expression of HLF in aHSCs and the upregulation of HLF might be an early event in HSC activation (figure 3C). As shown in figure 3D,E, HLF depletion attenuated the proliferation and migration capability of primary HSCs. Moreover, expression of α-SMA and α1(I)-collagen, the classic fibrotic genes, remarkably decreased in HLF-depleted primary HSCs in comparison with WT HSCs (figure 3F). In addition, transforming growth factor (TGF)-β-triggered alteration of fibrotic gene expression was significantly compromised by HLF depletion in primary HSCs (see online supplementary figure 3D). Together, these data suggested that HLF was specifically expressed in aHSCs and was required for the activation of HSCs.

HLF overexpression promotes HSC activation

To further confirm the essential role of HLF in HSC activation, we delivered Flag-tagged HLF into the cultured Lx2 cells using adenovirus. Consistent with previous observation, exogenous HLF overexpression increased the proportion of S phase cells and promoted the cell proliferation (figure 4A,B). Moreover, forced HLF expression significantly enhanced the migration capability of Lx2 cells (figure 4C,D). Resistance to apoptosis is a distinct characteristic of aHSCs. As compared with control cells, Lx2 cells overexpressing HLF exhibited much less apoptosis during the prolonged culture in low-serum medium (figure 4E and see online supplementary figure 4A), which was a widely used approach to induce HSC apoptosis.32 33 Furthermore, interference of HLF led to a notable increase of spontaneous apoptosis in Lx2 cells (see online supplementary figure 4B,C), suggesting a pivotal role of HLF in the survival of aHSCs. Moreover, expression of classic fibrotic genes was enhanced by HLF overexpression in Lx2 cells (figure 4F). In addition, the role of HLF in the expression of lysyl oxidase (LOX), an important mediator for collagen stabilization, was investigated. As shown in online supplementary figure 4D,E, our results demonstrated that HLF did not regulate LOX expression in HSCs, which excluded the role of LOX in HLF-enhanced liver fibrosis. Together, these data further confirmed the promoting role of HLF in HSC activation.

Figure 4

Forced HLF expression promotes HSC activation. (A) The human HSC line Lx2 cells was infected with Ad-HLF or Ad-Con and then subjected to the CCK8 assay, and Flag-tagged exogenous HLF was recognized with anti-Flag antibody using western blot. (B) The cell cycle distribution of Lx2 cells infected with Ad-HLF or Ad-Con was analysed by flow cytometry, and the proportion of S phase cells are shown. (C) The migration capability of Lx2 cells infected with Ad-HLF or Ad-Con was measured using the wound-healing assay. Representative results from three independent experiments are shown. (D) The migration of the Lx2 cells infected with Ad-HLF or Ad-Con was compared using the Transwell assay, and the representative images are shown. The number of cells was counted from different fields. (E) The Lx2 cells infected with Ad-HLF or Ad-Con were cultured in low-serum (0.1%) medium for 24 hours, and the apoptotic cells was examined by flow cytometry. (F) Real-time PCR analysis of the fibrotic genes in the Lx2 cells infected with Ad-HLF or Ad-Con. *p<0.05.

HLF drives HSC activation through STAT3 signalling

Several signalling pathways including TGF-β/SMAD, PI3-K/Akt, MAPKs and JAK/STAT3 have been reported to feed into the activation of HSCs.9 Herein our data showed that TGF-β/SMAD, PI3-K/Akt or MAPKs pathway was not influenced by HLF overexpression (supplementary figure 5A). However, STAT3 phosphorylation (Y705) was enhanced by HLF in a dose-dependent and time-dependent manner (figure 5A and supplementary figure 5B). More importantly, the HLF-enhanced HSC proliferation and migration was entirely abrogated by the selective JAK2/STAT3 inhibitor AZD-1480 (figure 5B,C and supplementary figure 5C,D). Consistently, HLF-induced fibrotic gene expression was completely abrogated (figure 5D and see online Supplementary figure 5E), demonstrating that STAT3 activation was required for the pro-fibrogenic role of HLF. Moreover, IHC staining on serial liver sections from fibrotic tissues of patients revealed the correlation between HLF expression and STAT3 activation in the fibrotic areas (figure 5E). Noteworthy, the co-localization of HLF and p-STAT3 in human HSC line Lx2 cells and the fibrotic areas of patient liver was revealed by immunofluorescence staining (figure 5F,G), further supporting that HLF promoted HSC activation through intensifying STAT3 activation during liver fibrosis.

Figure 5

HLF promotes STAT3 signalling to activate HSCs. (A) The human HSC line Lx2 cells were infected with Ad-HLF or Ad-Con for 24 hours followed by western blot analysis. Flag-tagged exogenous HLF was recognized with anti-Flag antibody. Densitometry analysis of p-STAT3 levels relative to STAT3 is shown. Representative results from three independent experiments are shown. (B) The proliferation of the Lx2 cells infected with Ad-HLF or Ad-Con in the presence of AZD-1480 (2 µM) or DMSO, respectively, was measured using the CCK8 assay. Representative results from three independent experiments are shown. (C) The migration of Lx2 cells infected with Ad-HLF or Ad-Con were analyzed by the Transwell assay in the presence of AZD-1480 (2 µM) or DMSO. (D) The Lx2 cells infected with Ad-HLF or Ad-Con were treated with AZD-1480 (2 µM) or DMSO for 48 hours followed by real-time PCR analysis. Representative results from three independent experiments are shown. (E) H&E staining, sirius red staining and immunohistochemistry assay of serial tissue sections of patient fibrotic liver. Red arrowheads indicate p-STAT3 positive HSCs. Representative images are shown; scale bar=100 µm. (F) Dual immunofluorescence staining of HLF and p-STAT3 in human HSC line Lx2 cells. Representative images are shown. The nuclei were counterstained with DAPI. Scale bar=20 µm. (G) Dual immunofluorescence staining of fibrotic liver of patient using anti-p-STAT3 and anti-HLF antibodies. Representative images are shown. The nuclei were counterstained with DAPI. Scale bar=25 µm. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide.

HLF promotes interleukin 6 transcription to enhance STAT3 activation

It has been well accepted that the activation of STAT3 is predominantly regulated by the upstream interleukin 6 (IL-6) family and its receptor.34 In present study, transcription of IL-6 itself but not other family members or the receptor was remarkably increased upon HLF upregulation (figure 6A and supplementary figure 6A). As shown in supplementary figure 6B and figure 6B, forced HLF expression increased IL-6 mRNA and protein levels on a dose-dependent manner in concomitant with STAT3 activation. Furthermore, a putative homologous HLF binding site within the human IL-6 promoter (−220/–209) was uncovered by bioinformatics analysis and verified by ChIP assays (figure 6C). In addition, IL-6 expression was decreased in the primary HSCs and fibrotic livers from HLFPB/PB mice compared with that from WT mice (figure 6D,E). Moreover, the correlation between HLF levels and α-SMA or IL-6 expression was observed in patient fibrotic tissues (figure 6F).

Figure 6

HLF directly upregulates IL-6 transcription in HSCs. (A) Real-time PCR assay of the human HSC line Lx2 cells infected with Ad-HLF or Ad-Con for 24 hours. (B) The Lx2 cells were infected with Ad-HLF or Ad-Con for 48 hours followed by western blot analysis. Flag-tagged exogenous HLF was recognized with anti-Flag antibody. Densitometry analysis of IL-6 levels relative to β-actin is shown. Representative results from three independent experiments are shown. (C) Schematic diagram of the putative HLF binding site within the human IL-6 promoter. The Lx2 cells infected with Ad-HLF were subjected to ChIP assay with anti-Flag or IgG antibody. Representative results from three independent experiments are shown. (D) The primary HSCs isolated from the HLFPB/PB or WT mice were cultured for the indicated times followed by real-time PCR analysis. (E) The fibrotic livers from CCl4-induced murine experimental fibrosis in figure 2 were subjected to real-time PCR analysis. *p<0.05. (F) The correlation between HLF levels and IL-6 or α-SMA expression in patient fibrotic liver tissues was assessed using Pearson’s correlation analysis, n=66.ChIP, chromatin immunoprecipitation.

HLF/IL-6/STAT3 circuit drives HSC activation in liver fibrosis

Because HLF expression escalates with HSC activation and fibrosis progression, it is interesting to explore how HLF is upregulated during fibrosis. The Lx2 cells were first treated with TGF-β or PDGF, two crucial pro-fibrogenic factors; however, neither of them could trigger the upregulation of HLF (supplementary figure 7). Surprisingly, HLF expression was significantly enhanced upon IL-6 treatment (figure 7A). Moreover, IL-6-triggered HLF induction was attenuated by the selective IL-6R inhibitor tocilizumab or the JAK2/STAT3 inhibitor AZD1480 (figure 7B), suggesting that IL-6/STAT3 signalling could induce HLF expression in HSCs, thus completing an HLF/IL-6/STAT3 regulatory circuit. Bioinformatics analysis of the human HLF promoter also revealed a putative STAT3 binding site (−1055/–1045), which was further confirmed by ChIP assay (figure 7C). Consistently, exogenous IL-6 increased HLF expression and elicited the endogenous IL-6 expression in HSCs (figure 7D). As expected, exogenous IL-6-induced endogenous IL-6 expression could be blocked by IL-6R or JAK2/STAT3 inhibition (figure 7E), further supporting the existence of HLF/IL-6/STAT3 circuit in HSC activation (figure 7F).

Figure 7

The HLF/IL-6/STAT3 circuit promotes HSC activation. (A) The human HSC line Lx2 cells starved overnight were treated with IL-6 for 48 hours followed by real-time PCR and western blot analysis. Representative results from three independent experiments are shown. **p<0.01. (B) The Lx2 cells starved overnight were pretreated with AZD1480 (2 µM) or tocilizumab (20 µg/mL) for half an hour and were then stimulated with IL-6 (10 ng/mL) for additional 48 hours followed by western blot analysis. Representative results from three independent experiments are shown. (C) Schematic diagram of the putative STAT3 binding site within the human HLF promoter. The Lx2 cells were subjected to ChIP assay with anti-STAT3 (Y705) or IgG antibody. Representative results from three independent experiments are shown. (D) The primary HSCs isolated from WT mice were stimulated with IL-6 (10 ng/mL) for 48 hours followed by real-time PCR analysis. **p<0.01. (E) The Lx2 cells starved overnight were pretreated with AZD1480 (2 µM) or tocilizumab (20 µg/mL) for half an hour and were then stimulated with IL-6 (10 ng/mL) for additional 48 hours followed by real-time PCR analysis. **p<0.01. (F) Schematic diagram of the HLF/IL-6/STAT3 regulatory circuit in HSC activation of liver fibrosis. .

Discussion

Iterative hepatic injury triggers an aberrant wound-healing response that skews ECM turnover in favour of synthesis and deposition, eventually resulting in liver fibrosis and later cirrhosis. This scarring process serves to prevent the liver from disintegrating upon injury, which, in turn, may perturb its pliability and hepatic function.1 7 The resident liver HSCs represent the primary source of ECM-producing myofibroblasts in liver, and they undergo a well-established process to become activated.12 13 However, the in-depth mechanism of HSC activation remains largely unknown. In present study, we found that HLF was specifically expressed in aHSCs and enhanced HSC activation through the HLF/IL-6/STAT3 regulatory cascade to promote liver fibrosis.

Reports concerning the expression and function of HLF in liver physiology and pathology remain scarce so far. Inaba et al and Hunger et al proposed the expression of HLF in human liver based on their detection of HLF mRNA by northern blot.15 16 Falvey et al found that there were two HLF isoforms with distinct tissue-specific expression, promoter preferences and circadian rhythms in rat liver.22 Nonetheless, we did not detect the expression of HLF protein in normal liver of mouse or human in current study. The main cause of this divergence might be due to the different experimental models and the distinct technical approaches used among the studies. Currently, there is no commercially available HLF antibody suitable for pathological detection (data not shown). In the current study, we developed a new rabbit antihuman HLF antibody that is applicable for IHC and immunofluorescence detection in human liver tissues. Although we also detected the HLF transcript in hepatocytes by real-time PCR, HLF protein was undetectable in normal liver or in the cell types other than HSC in fibrotic liver by western blot or histological examination. Triple deletion of TEF, DBP and HLF in mice led to the impaired detoxification35 and lipid metabolism,36 whereas the specific role of HLF was missing. In addition, HLF was reported to be involved in viral replication23 and factor IX expression24 in HepG2 or Huh7 cancer cells. Nevertheless, the role of HLF in liver fibrosis remains unreported. Herein, we observed the upregulation of HLF in fibrotic liver and found that HLF expression was confined to aHSCs rather than other cell types in liver. More importantly, the loss of HLF attenuated HSC activation and liver fibrosis suggesting that the upregulation of HLF is an intrinsic response of HSC and is required for HSC activation. aHSCs are the predominant source of myofibroblasts in liver fibrosis. Except for producing ECM, they might also affect the liver injury, inflammation or regeneration via dynamic reciprocity with parenchymal and non-parenchymal cells.11 Nevertheless, accumulating studies revealed that HSC-conditional knockout of fibrosis-associated gene or the knockout of HSC-specific gene could attenuate the development of fibrosis without altering the levels of liver injury or inflammation.37–39 In our study, HLF was exclusively expressed in aHSCs rather than other cell types, and HLF depletion attenuated HSC activation and experimental liver fibrosis with little influence on liver injury, inflammation or regeneration.

Accumulating evidence have illustrated that IL-6/STAT3 pathway plays a pivotal role in HSC activation, but the detailed machinery has not been identified so far.40–44 In this study, our data showed that HLF directly bound to IL-6 promoter and enhanced the IL-6 transcription and subsequent STAT3 activation. Previous studies have demonstrated that IL-6 could reduce the expression of MMP3 and MMP13 in HSCs.45 46 Herein we observed a high increase in MMP3/MMP13 levels in the livers of HLF-depleted mice compared with WT mice that underwent the experimental fibrosis. Because HLF could directly upregulate the transcription of IL-6, we supposed that the high increase of MMP3/MMP13 could be due to, at least partially, the decreased IL-6 expression in HLF-depleted mice. Moreover, activated IL-6/STAT3 signalling upregulated the expression of HLF, thus completing an HLF/IL-6/STAT3 regulatory circuit. Numerous molecules and pathways have been reported to be involved in the transdifferentiation of HSCs during hepatic fibrosis to date.14 Nevertheless, the precise mechanism underlying the HSC activation in particular the perpetuation of HSC activation remains obscure. To our knowledge, this is the first study reporting a feedforward regulatory circuit connected by an HSC-specific protein. Moreover, it should be also important for the perpetuation of HSC activation, which is critical for liver fibrosis progression. These findings broaden the regulatory network of HSC activation and deepen our understanding on the molecular mechanism of liver fibrosis.

Hepatic fibrosis represents a mutual process of the majority of chronic liver diseases, and it disrupts the liver architecture and the hepatic function.7 8 Due to the lack of specific serum diagnostic biomarker and non-invasive detection technique, it is difficult to identify patients with symptom-less liver fibrosis and to monitor the progression or reversal during antifibrotic therapy.9 10 Meanwhile, the off-target side effects caused by antifibrotic therapies have severely hindered the translation of preclinical advances.47 In our study, HSC-specific HLF expression was detected in virtually all patients with fibrosis, and it correlated with fibrosis progression, indicating its diagnostic value for liver fibrosis. Although numerous molecules and pathways have been identified in HSC activation, most of them were expressed in the parenchymal or other non-parenchymal cells in liver and exerted certain functions. There would be inevitable off-target side effects in the fibrosis treatment targeting these molecules. Considering the specific expression pattern of HLF in aHSCs, pharmaceutical intervention targeting HLF in liver fibrosis treatment could yield less off-target effect in patients, which indeed merits further evaluation. Moreover, the potential role of HLF in fibrosis resolution is of interest and would be the future direction of this work.

Acknowledgments

The authors would like to thank Dong-Ping Hu, Huan-Lin Sun, Shan-Na Huang, Shan-Hua Tang, Dan-Dan Huang, Lin-Na Guo and Dan Cao in the International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital for their technical assistance.

Acknowledgments

The authors would like to thank Dong-Ping Hu, Huan-Lin Sun, Shan-Na Huang, Shan-Hua Tang, Dan-Dan Huang, Lin-Na Guo and Dan Cao in the International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, for their technical assistance.

References

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Footnotes

  • D-MX, WS and B-FN contributed equally.

  • Contributors Study concept and design: JD and H-YW. Acquisition of data: D-MX, WS, B-FN, X-FL, WZ, ZC, M-YX and XW. Analysis and interpretation of data: D-MX and WS. Statistical analysis: D-MX. Drafting of the manuscript: WS. Critical revision of the manuscript: JD. Obtained funding: JD and H-YW. Administrative, technical support: XD, WW, H-YL, T-FZ, X-LC, S-CL, BW and W-FX. Study supervision: JD and H-YW. D-MX, WS and B-FN contributed equally to this work.

  • Funding Supported by grants from the National Key Research and Development Program of China 2017YFA0504503, National Natural Science Foundation of China 81572412, 81372329, 81572897 and 81670546.

  • Competing interests None declared.

  • Patient consent Detail has been removed from this case description/these case descriptions to ensure anonymity. The editors and reviewers have seen the detailed information available and are satisfied that the information backs up the case the authors are making.

  • Ethics approval The Ethical Committee of Eastern Hepatobiliary Surgery Hospital.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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