Dig Dis Sci DOI 10.1007/s10620-014-3208-1
ORIGINAL ARTICLE
R-Spondin2 Activates Hepatic Stellate Cells and Promotes Liver Fibrosis Xinguang Yin • Huixing Yi • Wanxin Wu Jing Shu • Xiaojun Wu • Linghua Yu
•
Received: 23 April 2013 / Accepted: 5 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Background The development of liver fibrosis is the fundamental stage toward a number of mortal complications of liver diseases, including cirrhosis and hepatocellular carcinoma. Canonical Wnt pathway is crucial in diverse biological processes and mediates the progression and regression of liver fibrosis. As a potent Wnt pathway agonist, the role of roof plate-specific spondin-2 (R-Spondin2) in the hepatic fibrosis has not been well elucidated. Aims The purpose of this study was to investigate whether R-Spondin2 contributes to hepatic stellate cells (HSCs) activation, the key event in liver fibrogenesis. Methods Human liver tissues, hepatic fibrosis mouse model, and freshly isolated mice HSCs were used. Protein expression and transcriptional level were analyzed by Western-blot assays and real-time PCR, respectively. Exogenous stimulation with recombinant R-Spondin2 and
knockdown of R-Spondin2 were performed to investigate functionality. Nuclear b-catenin level and T cell-specific transcription factors activity were analyzed, and HSC proliferation was tested by MTT assay. Results Overexpression of R-Spondin2 was observed in both human fibrotic liver tissues and hepatic fibrosis mouse model. Exogenous stimulation with R-Spondin2 in the freshly isolated mice HSCs induced a dose-dependent increase in Wnt pathway activities, HSC proliferation, and the expression of a-smooth muscle actin (a-SMA) and Collagen I. Additionally, Wnt pathway activities, HSC proliferation, and the expressions of a-SMA and Collagen I decreased in the R-Spondin2 knockdown HSCs. Conclusions These findings suggest that R-Spondin2 may promote HSC activation by enhancing the canonical Wnt pathway.
X. Yin W. Wu J. Shu X. Wu L. Yu (&) Department of Gastroenterology, The First Affiliated Hospital of Jiaxing College, 1882 Central-South Road, Jiaxing 314001, Zhejiang Province, People’s Republic of China e-mail: [email protected]
W. Wu Department of Pathology, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China
X. Yin e-mail: [email protected] W. Wu e-mail: [email protected] J. Shu e-mail: [email protected] X. Wu e-mail: [email protected] H. Yi Intensive Care Unit, The Second Affiliated Hospital of Zhejiang University, Hangzhou 310009, People’s Republic of China e-mail: [email protected]
J. Shu Intensive Care Unit, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China X. Wu Department of Hepatobiliary Surgery, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China L. Yu Department of Infectious Diseases and Hepatology, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China
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Keywords Catenin
Liver fibrosis R-Spondin2 Wnt HSC b-
Abbreviations R-Spondin2 Roof plate-specific Spondin-2 HSC Hepatic stellate cell ECM Extracellular matrix a-SMA a-Smooth muscle actin TCF T cell-specific transcription factors LEF Lymphoid enhancer-binding factor
Introduction Liver fibrosis is a reversible wound-healing response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drugs, or parasitic infection [1]. With chronic liver injury, this wound-healing process is presented as a progressive substitution of the functional parenchyma by scar tissue [2]. As the key fibrogenic cell population of the liver, the hepatic stellate cell (HSC) is the primary cell type responsible for scar formation [3]. In the normal liver, HSCs are in a resting state and serve as the main storage sites for vitamin A in the body. Following liver injury, HSCs undergo an activation process, which is a phenotypic transition from a quiescent, vitamin A-storing cell into a proliferative, a-smooth muscle actin (a-SMA)-positive, myofibroblast-like cell. The activated HSCs synthesize a large amount of extracellular matrix (ECM) proteins to encapsulate the injury, which subsequently results in liver fibrosis [4]. Many signaling pathways are implicated in HSC activation, perpetuation, and resolution, among which the Wnt pathway plays a pivotal role [5]. The Wnt pathway is crucial in diverse biological processes during embryogenesis, disease pathogenesis, and tissue homeostasis in adults [6–8]. Numerous studies have shown that the canonical Wnt pathway profoundly affects HSC activation and mediates HSC proliferation, resolution, and ECM accumulation [9–11]. Activation of the canonical Wnt pathway results in the stabilization of cytoplasmic b-catenin and its translocation into the nucleus and subsequent transactivation of Wnt target genes through interaction with the transcription factors T-cell factor/lymphoid enhancerbinding factor (TCF/LEF) [12, 13]. The roof plate-specific spondin (R-Spondin) family is implicated in the activation and regulation of the Wnt pathway [14–19]. The R-Spondin family is comprised of four members (R-Spondin1–4) that share overall 60 % sequence homology and a similar domain organization, of which R-Spondin2 plays a key role in the development of
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the gastrointestinal tract and myogenesis [20, 21]. Huch et al. [22] report a three-dimensional culture system for transplantable liver organoids with R-Spondin1 as the crucial component of the culture medium and the transcriptional level of R-Spondins increased three to *sixfold following liver injury. These studies illustrate the pivotal role of the R-Spondin family in regulating cell proliferation, differentiation, migration, and survival during embryonic development and tissue homeostasis. A mass of evidence has illustrated the vital influence of the Wnt pathway on liver fibrosis, whereas there is no related study about the effect of R-Spondin2 in the hepatic fibrosis to date. Therefore, the purpose of this study was to investigate the role of R-Spondin2 in the activation of HSC, the central event in liver fibrogenesis.
Materials and Methods Animals used Healthy Kunming mice (8–10 weeks old, body weight, 38–40 g) were purchased from Shanghai SLAC Laboratory Animal Co Ltd (Shanghai, China). The animals were housed under standard animal laboratory conditions and supplied with laboratory chow and water. All individuals involved in the animal research received instructions in experimental methods and in the care, maintenance, and handling of mice. All institutional and national guidelines for the care and use of laboratory animals were followed. The protocol of the experiments was approved by the Committee on the Ethics of Animal Experiments of Jiaxing College (No. AJX 2013-18). Patients and Liver Tissue Samples Human liver tissues were obtained from the First Affiliated Hospital of Jiaxing College with written consent and ethical approval from the local Ethics Committee of the college. A total of 48 fibrotic liver tissue samples were collected, with ten normal hepatic tissue specimens served as the control. Tissue samples were preserved at the temperature of -80 °C. Antibodies and Reagents Rabbits polyclonal to a-SMA, Collagen I, and b-Catenin were purchased from Abcam (Cambridge, MA, USA). Human R-Spondin2 antibody, mouse R-Spondin2 antibody, and recombinant mouse R-Spondin2 were purchased from R&D (R&D Systems, Minneapolis, MN, USA). NEPER Nuclear and Cytoplasmic Extraction Reagent Kit was obtained from Pierce (Pierce Biotechnology, Rockford, IL,
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USA). Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit IgG was obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lipofectamine 2000 transfection reagent and TRIzol were obtained from Invitrogen (Life Technologies, Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). TCF reporter plasmid TOPFLASH and the control inactive reporter FOPFLASH were purchased from Upstate (Upstate Biotechnology Inc., Lake Placid, NY, USA). CCl4, olive oil, collagenase IV, pronase E, Nycodenz, bovine serum albumin (BSA), MTT, and DMSO were obtained from Sigma-Aldrich (SigmaAldrich, St. Louis, MO, USA). HSC Isolation and Culture Primary stellate cells were isolated from the livers of Kunming mice as previously described [23]. Briefly, mouse liver was perfused in situ with DMEM (Gibco) to purge the liver of blood. After digestion of the liver with pronase E (Sigma) and collagenase IV (Sigma), dispersed cell suspensions were layered by Nycodenz (Sigma) density-gradient centrifugation according to the manufacturer’s protocol. The resulting upper layer contained the freshly isolated HSCs. The purity of isolated HSCs was examined by phase-contrast microscopy, and viability based on Trypan blue exclusion (purity [95 %, viability [95 %). The isolated HSCs were then cultured on uncoated plastic plates in DMEM (Gibco) supplemented with 10 % FBS (Gibco) at 37 °C at a density of 1 9 105 cells/cm2. The isolated HSCs were cultured for 1 or 7 days to examine quiescent or fully activated cells. Hepatic Fibrosis Model The hepatic fibrosis model of mice was induced by subcutaneous injection of 20 % solution of CCL4 in olive oil at a dose of 5 ml/kg twice per week for 10 weeks [24]. Twenty-four Kunming mice were randomly divided into two groups. The first group (n = 8) served as a normal control group, and the experimental group (n = 16) was hepatic fibrosis models. Four mice died during the induction of hepatic fibrosis, thus, the final experimental group contained 12 mice with CCL4-induced liver fibrosis. One week after the last infusion, the animals were killed by CO2 exposure and liver tissues were harvested.
tissue microarrays using a Beecher Instrument (Sun Prairie, WI, USA) as described previously [25]. Three tissue cylinders of 0.6 mm diameter were punched from each sample. After sectioning (4 mm for each section), tissue slides were baked at 60 °C for 2 h, and kept at 4 °C for further analysis. Immunohistochemistry Immunohistochemistry was performed on the TMA sections containing fibrotic liver tissues and normal liver tissues. After deparaffinization, rehydration, and antigen retrieval, TMA sections were immunostained with the primary antibodies R-Spondin2 (1:1,000 dilution, R&D Systems). The TMA sections were incubated with the primary antibody at 4 °C overnight, followed by HRPconjugated secondary anti-rabbit IgG (1:2,000 dilution, Santa Cruz) antibody for 1 h at room temperature. Immunohistochemical staining was carried out using the SABC kit (Boster, China). For quantification of the immunostain, cells were counted in six randomly chosen high-power fields at 400-fold magnification by two experienced researchers in a double-blinded manner. Results were scored by the percentage of R-Spondin2-stained cells as below: \10 % (score 0), 10–25 % (score 1), 25–50 % (score 2), 50–75 % (score 3), and 75–100 % (score 4). Results were considered positive with a score higher than 2. Quantification of Transcript Levels by Real-Time PCR The mRNA expressions were quantified by real-time PCR (StepOne-Plus, Applied Biosystems, Carlsbad, CA, USA) following the manufacturer’s instructions. Total RNA from the cells was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Thermocycling conditions consisted of an initial step of 2 min at 50 °C, denaturation of 10 min at 95 °C, followed by 35–40 cycles at 95 °C for 30 s, 60 °C for 45 s, and 72 °C for 1 min and a final elongation step at 72 °C for 10 min. Primers used for amplification are listed in Table 1. Each sample was analyzed in triplicate, with b-actin used for normalization. Comparative threshold (Ct) method was used for calculating the relative amount of mRNA of the sample compared with the control. Western-Blot Assays
Tissue Microarray Construction Tissue specimens (48 fibrotic liver tissue samples and ten normal hepatic tissue samples) were formalin-fixed and paraffin-embedded. The samples were used to construct
Standard Western-blot protocol was used, with b-actin serving as a loading control. Nuclear and cytoplasmic protein fractions were extracted using the NE-PER Extraction Reagent Kit (Pierce Biotechnology) according
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Dig Dis Sci Table 1 Primers sequences Gene
Forward sequence
R-Spondin2
50 -GCGGGTGTCGGCAAACTTTTTC-30 0
Reverse sequence
0
50 -CTTTTCCATGTCGTCCCAGT-30
a-SMA
5 -GCTGCTCCAGCTATGTGTGA-3
Collagen I
50 -TTCACCTACAGCACGCTTGTG-30
b-Catenin b-Actin
50 -GATGACTGTCTTGCCCCAAGTT-30
0
0
50 -TGTCAGGGGAGCTGTGGCTCC-30
0
0
50 -AACAGTCCGCCTAGAAGCAC-30
5 -CAGCAGTTTGTGGAGGGCGTG-3 5 -TCATCACTATTGGCAACGAGC-3
to the manufacturer’s instructions. The extracts were then cleared by centrifugation at 16,000 9 g for 15 min. After blocking with 5 % non-fat milk in PBS for 1 h at room temperature, membranes were incubated with primary antibodies against R-Spondin2 (1:1,000 dilution, R&D Systems), a-SMA (1:300 dilution, Abcam), Collagen I (1:200 dilution, Abcam), and b-catenin (1:200 dilution, Abcam) at 4 °C overnight. The membranes were then incubated with HRP-conjugated secondary anti-rabbit IgG (1:2,000 dilution, Santa Cruz) antibody for 1 h at room temperature. Results were visualized and quantified by enhanced chemiluminescence (ECL) using an electrochemiluminescence immunoblotting kit (Cell Signaling Technology) with a luminescent digital image analyzer Bio-Spectrum600 (UVP, Upland, CA, USA). Band intensity was assessed using a Gel-Pro analyzer.
Stimulation with R-Spondin2 For treatment with R-Spondin2, freshly isolated mice HSCs were maintained until 70 % confluence. Then, mediums were changed and HSCs were cultured for 24 h in DMEM (Gibco) with 5 % FBS (Gibco). HSCs were then exposed to different concentrations (0 ng/ml as control, 10, and 20 ng/ml) of recombinant mouse R-Spondin2 (R&D Systems) for 24 h. Finally, cells were harvested for further analysis.
siRNA-Mediated Gene Silencing Lipofectamine 2000 (Invitrogen) was used for transfection according to the manufacturer’s protocol. HSCs were seeded into six-well plates for 12 h before transfection. Next, siRNA plasmid was mixed with 5 ll Lipofectamine 2000 in 250 ll Opti-MEM I medium for 20 min. Then, the transfection mixture was added to each well with 1.5 ml FBS free DMEM (Gibco) at a concentration of 100 nmol/l. After 6 h of incubation, a liquid mixture containing siRNA was disposed. HSCs were then collected for further analyses after 48 h. The following siRNA sequences were used for gene silencing:
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50 -ATCTGGGGCTCG -GTGTCCATAATAC-30
Mouse R-Spondin2 siRNA: 50 - AAGCTGTTGTCACT AAGATAATG-30 Scrambled siRNA : 50 - GCTGCGTAATTTAAGAGTA ACAT -30 Luciferase Reporter Assay Cells were transiently transfected with TOPFLASH and FOPFLASH (Upstate Biotechnology Inc., Lake Placid, NY, USA) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were harvested and luciferase and Renilla luminescence were measured using the Dual-Luciferase Reporter Assay System (Promega, Wisconsin, WI, USA) on a luminometer (BioTek Instruments, Winooski, VT, USA). TCF reporter activity was presented as the ratio of firefly-to-Renilla luciferase activity. Two independent transfections were performed, with each sample tested in triplicate. HSCs Proliferation Assay The MTT assay was performed to determine the HSCs proliferation. Briefly, HSCs at the logarithmic growth phase were seeded at a density of 4 9 103 cells per pore into 96-well culture plates, with 100 ll cell suspension in each well. HSCs were simultaneously transfected by R-Spondin2 siRNA plasmid or co-cultured with different concentration of recombinant R-Spondin2 (0 ng/ml as control, 10, and 20 ng/ml). After treatment for 24, 48, and 72 h, 10 ll of MTT (Sigma) solution (5 mg/ml) was added into each well and incubated at 37 °C for 4 h, then centrifugation was performed at 3,000 rpm for 5 min. Finally, the precipitate was dissolved completely with 100 ll DMSO (Sigma), and the absorbance of the enzyme was measured at 570-nm wavelength in a Bio-Rad 550 microplate reader (Bio-Rad, Hercules, CA, USA). The experiments were performed in triplicate. Statistical Analysis The results of three independent experiments were recorded and expressed as mean ± SEM. The data was analyzed
Dig Dis Sci Fig. 1 R-Spondin2 was overexpressed in human fibrotic liver tissue. a Immunostaining showed R-Spondin2 was positive in human fibrotic liver tissue. b R-Spondin2 was negative in human normal liver tissue (bar 50 lm, magnification 9400). c Negative control. d The protein level of R-Spondin2 was significantly higher in human fibrotic liver tissue (n = 10) compared with normal liver tissue (n = 10). Quantification of levels of R-Spondin2 protein were normalized to b-actin, **p \ 0.01 compared to the normal liver tissue
using Student’s t test and considered significant at p \ 0.05.
Results R-Spondin2 Was Overexpressed in Liver Fibrosis Tissue Human liver tissue sections were immunostained for R-Spondin2 and the results demonstrated that R-Spondin2 protein was expressed in the cytoplasm and cell membrane. A total of 48 human fibrotic liver tissue samples were examined by immunohistochemistry experiment, with ten normal liver tissue samples serving as the control. The immunohistochemistry results showed that 83.3 % of the fibrotic liver tissue samples were positive for R-Spondin2 (Fig. 1a), whereas only 30 % of the normal liver tissue samples were positive for R-Spondin2 (Fig. 1b); the difference was significant at p \ 0.01. This finding indicated that R-Spondin2 was overexpressed in human fibrotic liver tissue. To verify this finding, the protein level of R-Spondin2 in the liver tissue was analyzed by Western-blot assay. Ten fibrotic liver tissue samples were examined, with ten normal liver tissue samples serving as the control. The result of Western-blot assay (Fig. 1d) was consistent with that of the immunohistochemistry experiment, and the protein level of R-Spondin2 was 7.71-fold that in the human fibrotic liver tissue compared with the normal liver tissue.
R-Spondin2 Was Upregulated in the Mouse Model of CCL4-Induced Hepatic Fibrosis Next, the expression of R-Spondin2 was examined in the CCL4-induced hepatic fibrosis mouse model (experimental group), with normal mice serving as the control. The protein expression and mRNA level of R-Spondin2, fibrosis biomarker a-SMA and Collagen I, and nuclear b-catenin were analyzed by Western-blot assay and real-time PCR, respectively. The results of Western blot showed that the protein expressions of a-SMA and Collagen I were 5.98fold and 4.17-fold in the experimental group, respectively, compared with the control group (Fig. 2a, b). The results of real-time PCR were aligned with those of the Western-blot assay, and the mRNA level of a-SMA and Collagen I were 7.16-fold and 5.33-fold in the experimental group compared with the control group (Fig. 2a, b). These findings indicated that the induction of liver fibrosis was successful. Both the protein expression and the mRNA level of R-Spondin2 were upregulated in the CCL4-induced hepatic fibrosis mouse model, which were 3.16-fold and 4.36-fold, respectively, compared with the control group (Fig. 2c). Prior studies have demonstrated that R-Spondins family of secreted proteins strongly potentiates the Wnt signaling pathway [14–17]. Thus, the expression of nuclear b-catenin was examined. The experimental results showed that protein expression of nuclear b-catenin increased 2.72-fold in the experimental group compared with the control group, and the mRNA level of total b-catenin increased 4.73-fold
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Fig. 2 R-Spondin2 was overexpressed in the hepatic fibrosis model of mouse. a The protein expression and mRNA level of a-SMA upregulated in the hepatic fibrosis mouse model. b The protein expression and mRNA level of Collagen I upregulated in the hepatic fibrosis mouse model. c The protein expression and mRNA level of
R-Spondin2 upregulated in the hepatic fibrosis mouse model. d The protein expression of nuclear b-catenin and mRNA level of a- total b-catenin upregulated in the hepatic fibrosis mouse model. Each experiment was performed in triplicate. **p \ 0.01 compared to the normal liver tissue
in the experimental group compared with the control group (Fig. 2d).
during the cultured-initiated activation of HSCs and R-Spondin2 was overexpressed in the fully activated HSCs.
The Expression of R-Spondin2 Was Elevated During the Activation of Mice HSCs Hepatic stellate cell (HSC) activation is the key event in liver fibrogenesis. Therefore, freshly isolated mice HSCs were cultured in vitro and the time-series expression change of R-Spondin2 during HSCs activation was observed. The primary HSC retains the quiescent phenotype when cultured on plastic plate for 1–2 days and become fully activated by day 7 in culture [3]. Thus, the protein level of a-SMA, Collagen I, and R-Spondin2 at day 1 and day 7 were examined by Western-blot assay (Fig. 3a). The results of Western blot showed that the protein level of a-SMA and Collagen I were 8.48-fold and 3.27-fold, respectively, in day 7 HSCs compared with day 1 HSCs (Fig. 3c), which indicated the freshly isolated HSCs were undergoing a phenotypic switch from quiescent to highly fibrogenic cells. The protein level of R-Spondin2 was also elevated with time (Fig. 3b), which increased 4.54-fold in the culture-activated HSCs (day 7). To confirm the findings of the Western-blot assay, the mRNA level of R-Spondin2 in day 1 HSCs (quiescent) and day 7 HSCs (fully activated) was examined by real-time PCR. The results of real-time PCR were in accordance with those of the Western-blot assay, and R-Spondin2 was detectable only in the fully activated HSCs (Fig. 3b). These findings suggested that the expression of R-Spondin2 was increased
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Increased Nuclear b-Catenin Level and TCF Activity in the Culture-Activated HSCs Previous studies have reported that R-Spondin2 protein is a potent Wnt pathway agonist [19, 20]. Thus, the protein level of nuclear b-catenin and the TCF activity in day 1 HSCs (quiescent) and day 7 HSCs (fully activated) were examined. The Western-blot result showed that the protein level of nuclear b-catenin was 4.47-fold in day 7 HSCs compared with day 1 HSCs (Fig. 3a, c), and Luciferase Reporter Assay showed that the TCF activity was 5.33-fold in day 7 HSCs compared with day 1 HSCs (Fig. 3d). These findings demonstrated stabilization and translocation of cytoplasmic b-catenin into the nucleus and the subsequent activation of TCF/LEF-dependent transcription in the fully activated HSCs. Exogenous Stimulation with Recombinant R-Spondin2 Enhanced the Wnt Activities in HSCs and Promoted HSCs Activation Having shown increased R-Spondin2 expression, elevated levels of nuclear b-catenin, and enhanced TCF activity in the culture-activated HSCs, we examined next the Wnt pathway activities stimulated exogenously by recombinant mouse R-Spondin2 in cultured HSCs. Freshly isolated
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Fig. 3 Time-series expression profiles of R-Spondin2, fibrosis biomarker a-SMA and Collagen I, and Wnt pathway activity during the culture-initiated activation of mice HSCs were analyzed. a Protein expression of R-Spondin2, a-SMA, Collagen I, and nuclear b-catenin were tested by Western blot assay. b The protein and mRNA level of R-Spondin2 elevated in the fully activated HSCs. c The protein level
of a-SMA, Collagen I, and nuclear b-catenin were significantly higher in the fully activated HSCs (day 7) compared with the quiescent HSCs (day 1). d TCF activity increased in culture-activated HSCs. Data represents the mean of three independent experiments, and error bars are standard deviation of means. *p \ 0.05 compared to the quiescent HSCs (day 1), **p \ 0.01 compared to the quiescent HSCs
Fig. 4 R-Spondin2 enhanced Wnt pathway activity and subsequently promoted HSCs fibrogenesis and proliferation in a dose-dependent manner. a–c The protein level of a-SMA, Collagen I, and nuclear b-catenin and increased in a dose-dependent manner (0 ng/ml as control, 10, and 20 ng/ml) when co-cultured with recombinant R-Spondin2. d Recombinant R-Spondin2 induced a dose-dependent
increase in TCF activity. e Recombinant R-Spondin2 up-regulated HSCs proliferation in a dose-dependent manner. Data represents the mean of three independent experiments, and error bars are standard deviation of means. *p \ 0.05 compared to the control (no treatment of recombinant R-Spondin2), **p \ 0.01 compared to the control
HSCs were exposed to recombinant R-Spondin2 at different concentration (0 ng/ml as control, 10, and 20 ng/ml), and the protein level of a-SMA, Collagen I, and nuclear
b-catenin were analyzed by Western-blot assay. The experiment results showed that the relative protein expression ratio of a-SMA (Fig. 4a) was 1:3.34:6.07
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(control: 10:20 ng/ml), the relative protein expression ratio of Collagen I (Fig. 4b) was 1:1.92:3.6 (control: 10:20 ng/ ml), and the relative ratio of nuclear b-catenin (Fig. 4c) was 1:2.2:4.77 (control: 10:20 ng/ml). TCF activities of HSCs stimulated by different concentration (control, 10, and 20 ng/ml) of recombinant R-Spondin2 were examined by Luciferase Reporter Assay. The results (Fig. 4d) showed that the relative ratio of TCF activity was 1:2.35:10.61 (control: 10:20 ng/ml). Because the activation of HSC induces a proliferative response [1–3], HSC proliferation was then tested by MTT assay. Results of HSCs proliferation assays (Fig. 4e) showed that the absorbance was increased in a dose-dependent manner. Taken together, these findings indicated that exogenous stimulation with R-Spondin2 resulted in dose-dependent nuclear b-catenin accumulation and TCF activity enhancement, which subsequently enhanced the synthesis of a-SMA and HSCs proliferation. The Knockdown of R-Spondin2 Down-Regulated the WNT Pathway Activities and Repressed HSCs Activation To further understand the role of R-Spondin2 in the activation of HSCs, the central event in the liver fibrogenesis, we knocked down the R-Spondin2 in the fully activated mice HSCs by siRNA and observed the expression change of a-SMA, Collagen I, and Wnt pathway activity. The effect of R-Spondin2 siRNA was validated by Western-blot assay and real-time PCR, respectively, and the results showed that both the protein expression and transcriptional level of R-Spondin2 were barely detectable in the R-Spondin2 knockdown HSCs (Fig. 5a, b). The protein level of a-SMA, Collagen I, and nuclear b-catenin in the R-Spondin2 knockdown HSCs were analyzed, with the HSCs transfected with scrambled siRNA (scrambled HSCs) serving as the control. The Western-blot results showed that the protein level of a-SMA, Collagen I, and nuclear b-catenin were 7.45-fold, 3.98-fold, and 4.61-fold, respectively, in the scrambled HSCs compared with the R-Spondin2 knockdown HSCs (Fig. 5a, d). Then, TCF activities of R-Spondin2 knockdown HSCs were examined by Luciferase Reporter Assay, and the results showed the TCF activity was 2.67-fold in the scrambled HSCs compared with the R-Spondin2 knockdown HSCs (Fig. 5c). Finally, HSCs proliferation was tested by MTT assay. Results of HSCs proliferation assays showed that the absorbance decreased in the R-Spondin2 knockdown HSCs compared with the scrambled HSCs (Fig. 5f). Taken together, these findings indicated that the siRNA-mediated gene silencing of R-Spondin2 suppressed the activity of Wnt pathway, and subsequently reduced the synthesis of a-SMA and HSCs proliferation.
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Discussion Most chronic hepatic diseases will progress to liver fibrosis, and some may even result in cirrhosis or hepatocellular carcinoma [26]. Thus, the development of liver fibrosis is the fundamental stage toward a number of mortal complications of liver disease. Continuing progress in the field of hepatic fibrosis research presents us a comprehensive and nuance picture of the liver fibrogenesis, especially the effects of signaling pathway in induction the HSC activation. Numerous studies have shown that the canonical Wnt pathway profoundly affects HSC activation, perpetuation, and resolution [9–11]. Prior studies also have documented that the R-Spondins family of secreted proteins strongly potentiates the Wnt pathway and regulates tissue patterning and differentiation [27–29]. Moreover, recent reports have revealed that R-Spondin2 plays a key role in the development of the gastrointestinal tract and myogenesis [16, 17, 20, 22]. Therefore, this study investigated whether R-Spondin2 contributed to HSC activation, the central event in fibrogenesis. We demonstrated that R-Spondin2 protein was overexpressed in human fibrotic liver tissues, whereas it was almost undetectable in the normal liver tissues. In addition, the expression of R-Spondin2 in the CCL4-induced liver fibrosis mice was significantly higher than that of normal mice. Based on this finding and prior reports, we hypothesized that R-Spondin2 might be implicated in HSC activation. Progressive activation occurs when freshly isolated HSCs are cultured in a plastic plate, and the cellular events of this spontaneous-activation are similar to those occurring in liver injury [3]. Observation on culture-initiated activation of isolated mice HSCs demonstrated that both the transcriptional level and protein expression of R-Spondin2 elevated with time, which was consistent with a prior report that the transcriptional level of R-Spondins increased three to *sixfold following liver injury [18]. This study also found that the protein level of nuclear b-catenin and TCF activity increased in the culture-activated HSCs. In brief, these findings suggested that R-Spondin2 might promote the HSC activation by enhancing the accumulation of nuclear b-catenin and subsequent transactivation of Wnt target genes. To prove our hypothesis, several functional analyses were performed on isolated mice HSCs, including exogenous stimulation with recombinant R-Spondin2 and siRNA-mediated gene silencing of R-Spond2. We found that stimulation with R-Spondin2 on freshly isolated HSCs upregulated HSCs fibrogenesis and Wnt pathway activity in a dose-dependent manner. Additionally, the knockdown of R-Spondin2 led to the down-regulation of Wnt pathway activity and repressed HSCs activation. Taken together, these findings supported our hypothesis that R-Spondin2
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Fig. 5 Knockdown of R-Spondin2 repressed Wnt pathway activity and HSCs activation. a Protein expression of R-Spondin2, a-SMA, Collagen I, and nuclear b-catenin in R-Spondin2 knockdown HSCs were examined, HSCs transfected with scrambled siRNA severed as the control. b The effect of R-Spondin2 knockdown was validated by Western-blot assay and real-time PCR. c TCF activity reduced significantly in R-Spondin2 knockdown HSCs. d Protein level of
a-SMA, Collagen I, and nuclear b-catenin decreased significantly in the R-Spondin2 knockdown HSCs compared with the control. e HSCs proliferation decreased significantly in R-Spondin2 knockdown HSCs. Data represents the mean of three independent experiments, and error bars are standard deviation of means. *p \ 0.05 compared to the control (HSCs transfected with scrambled siRNA), **p \ 0.01 compared to the control
promoted HSC activation by enhancing the canonical Wnt pathway. During the liver fibrosis recovery, the activated HSCs either revert to a quiescent phenotype or are cleared. TGFb1 has a well-established role as a fibrogenic mediator, and the synergistic activation of TGF-b1 and Wnt pathway profoundly affects the HSC activation, proliferation, and resolution [1–3]. However, development of anti-fibrotic therapy targeting the TGF-b1 is problematic owing to the pleiotropic nature of therapy TGF-b1. Therefore, our findings provided new insights into the function of Wnt pathway provoked by R-Spondin2 during hepatic fibrogenesis, and demonstrated R-Spondin2 as a potential antifibrotic therapeutic target for liver fibrosis. Our study focused on the role of R-Spondin2 in the activation of HSC. Due to the limitation of lab facility and the availability of normal human liver tissue, this study did not isolate human HSCs and examined the effect of R-Spondin2 on primary human HSCs. Although HSC activation is the dominant pathway leading to hepatic fibrosis, portal fibroblast may be especially prominent in cholestatic liver injury [1]. Further studies are required to investigate the effect of R-Spondin2 in the myofibroblast formation of portal fibroblast.
References 1. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology. 2008;134:1655–1669. 2. Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol. 2008;214:199–210. 3. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev. 2008;88:125–172. 4. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 2000;275:2247–2250. 5. Akhmetshina A, Palumbo K, et al. Activation of canonical Wnt signalling is required for TGF-b-mediated fibrosis. Nat Commun. 2012;3:735. 6. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009;17:9–26. 7. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810. 8. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127:469–480. 9. Cheng JH, She H, et al. Wnt antagonism inhibits hepatic stellate cell activation and liver fibrosis. Am J Physiol Gastrointest Liver Physiol. 2008;294:G39–G49. 10. Kordes C, Sawitza I, et al. Canonical Wnt signaling maintains the quiescent stage of hepatic stellate cells. Biochem Biophys Res Commun. 2008;367:116–123. 11. Jiang F, Parsons CJ, Stefanovic B. Gene expression profile of quiescent and activated rat hepatic stellate cells implicate Wnt signaling pathway in activation. J Hepatol. 2006;45:401–409.
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21. Kazanskaya O, et al. R-Spondin2 is a secreted activator of Wnt/bcatenin signaling and is required for Xenopus myogenesis. Dev Cell. 2004;7:525–534. 22. Huch M, Dorrell C, Boj SF, et al. In vitro expansion of single Lgr5? liver stem cells induced by Wnt-driven regeneration. Nature. 2013;494:247–250. 23. Ramm GA. Isolation and culture of rat hepatic stellate cells. J Gastroenterol Hepatol. 1998;13:846–851. 24. Wei YH, Jun L, Qiang CJ. Effect of losartan, an angiotensin II antagonist, on hepatic fibrosis induced by CCl4 in rats. Dig Dis Sci. 2004;49:1589–1594. 25. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4:844–847. 26. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet. 2008;371: 838–851. 27. Kim K-A, et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science. 2005;309:1256–1259. 28. Kazanskaya O, et al. The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development. 2008;135:3655–3664. 29. Blaydon DC, Ishii Y, et al. The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat Genet. 2006;38:1245–1247.
ORIGINAL ARTICLE
R-Spondin2 Activates Hepatic Stellate Cells and Promotes Liver Fibrosis Xinguang Yin • Huixing Yi • Wanxin Wu Jing Shu • Xiaojun Wu • Linghua Yu
•
Received: 23 April 2013 / Accepted: 5 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Background The development of liver fibrosis is the fundamental stage toward a number of mortal complications of liver diseases, including cirrhosis and hepatocellular carcinoma. Canonical Wnt pathway is crucial in diverse biological processes and mediates the progression and regression of liver fibrosis. As a potent Wnt pathway agonist, the role of roof plate-specific spondin-2 (R-Spondin2) in the hepatic fibrosis has not been well elucidated. Aims The purpose of this study was to investigate whether R-Spondin2 contributes to hepatic stellate cells (HSCs) activation, the key event in liver fibrogenesis. Methods Human liver tissues, hepatic fibrosis mouse model, and freshly isolated mice HSCs were used. Protein expression and transcriptional level were analyzed by Western-blot assays and real-time PCR, respectively. Exogenous stimulation with recombinant R-Spondin2 and
knockdown of R-Spondin2 were performed to investigate functionality. Nuclear b-catenin level and T cell-specific transcription factors activity were analyzed, and HSC proliferation was tested by MTT assay. Results Overexpression of R-Spondin2 was observed in both human fibrotic liver tissues and hepatic fibrosis mouse model. Exogenous stimulation with R-Spondin2 in the freshly isolated mice HSCs induced a dose-dependent increase in Wnt pathway activities, HSC proliferation, and the expression of a-smooth muscle actin (a-SMA) and Collagen I. Additionally, Wnt pathway activities, HSC proliferation, and the expressions of a-SMA and Collagen I decreased in the R-Spondin2 knockdown HSCs. Conclusions These findings suggest that R-Spondin2 may promote HSC activation by enhancing the canonical Wnt pathway.
X. Yin W. Wu J. Shu X. Wu L. Yu (&) Department of Gastroenterology, The First Affiliated Hospital of Jiaxing College, 1882 Central-South Road, Jiaxing 314001, Zhejiang Province, People’s Republic of China e-mail: [email protected]
W. Wu Department of Pathology, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China
X. Yin e-mail: [email protected] W. Wu e-mail: [email protected] J. Shu e-mail: [email protected] X. Wu e-mail: [email protected] H. Yi Intensive Care Unit, The Second Affiliated Hospital of Zhejiang University, Hangzhou 310009, People’s Republic of China e-mail: [email protected]
J. Shu Intensive Care Unit, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China X. Wu Department of Hepatobiliary Surgery, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China L. Yu Department of Infectious Diseases and Hepatology, The First Affiliated Hospital of Jiaxing College, Jiaxing 314001, People’s Republic of China
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Keywords Catenin
Liver fibrosis R-Spondin2 Wnt HSC b-
Abbreviations R-Spondin2 Roof plate-specific Spondin-2 HSC Hepatic stellate cell ECM Extracellular matrix a-SMA a-Smooth muscle actin TCF T cell-specific transcription factors LEF Lymphoid enhancer-binding factor
Introduction Liver fibrosis is a reversible wound-healing response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drugs, or parasitic infection [1]. With chronic liver injury, this wound-healing process is presented as a progressive substitution of the functional parenchyma by scar tissue [2]. As the key fibrogenic cell population of the liver, the hepatic stellate cell (HSC) is the primary cell type responsible for scar formation [3]. In the normal liver, HSCs are in a resting state and serve as the main storage sites for vitamin A in the body. Following liver injury, HSCs undergo an activation process, which is a phenotypic transition from a quiescent, vitamin A-storing cell into a proliferative, a-smooth muscle actin (a-SMA)-positive, myofibroblast-like cell. The activated HSCs synthesize a large amount of extracellular matrix (ECM) proteins to encapsulate the injury, which subsequently results in liver fibrosis [4]. Many signaling pathways are implicated in HSC activation, perpetuation, and resolution, among which the Wnt pathway plays a pivotal role [5]. The Wnt pathway is crucial in diverse biological processes during embryogenesis, disease pathogenesis, and tissue homeostasis in adults [6–8]. Numerous studies have shown that the canonical Wnt pathway profoundly affects HSC activation and mediates HSC proliferation, resolution, and ECM accumulation [9–11]. Activation of the canonical Wnt pathway results in the stabilization of cytoplasmic b-catenin and its translocation into the nucleus and subsequent transactivation of Wnt target genes through interaction with the transcription factors T-cell factor/lymphoid enhancerbinding factor (TCF/LEF) [12, 13]. The roof plate-specific spondin (R-Spondin) family is implicated in the activation and regulation of the Wnt pathway [14–19]. The R-Spondin family is comprised of four members (R-Spondin1–4) that share overall 60 % sequence homology and a similar domain organization, of which R-Spondin2 plays a key role in the development of
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the gastrointestinal tract and myogenesis [20, 21]. Huch et al. [22] report a three-dimensional culture system for transplantable liver organoids with R-Spondin1 as the crucial component of the culture medium and the transcriptional level of R-Spondins increased three to *sixfold following liver injury. These studies illustrate the pivotal role of the R-Spondin family in regulating cell proliferation, differentiation, migration, and survival during embryonic development and tissue homeostasis. A mass of evidence has illustrated the vital influence of the Wnt pathway on liver fibrosis, whereas there is no related study about the effect of R-Spondin2 in the hepatic fibrosis to date. Therefore, the purpose of this study was to investigate the role of R-Spondin2 in the activation of HSC, the central event in liver fibrogenesis.
Materials and Methods Animals used Healthy Kunming mice (8–10 weeks old, body weight, 38–40 g) were purchased from Shanghai SLAC Laboratory Animal Co Ltd (Shanghai, China). The animals were housed under standard animal laboratory conditions and supplied with laboratory chow and water. All individuals involved in the animal research received instructions in experimental methods and in the care, maintenance, and handling of mice. All institutional and national guidelines for the care and use of laboratory animals were followed. The protocol of the experiments was approved by the Committee on the Ethics of Animal Experiments of Jiaxing College (No. AJX 2013-18). Patients and Liver Tissue Samples Human liver tissues were obtained from the First Affiliated Hospital of Jiaxing College with written consent and ethical approval from the local Ethics Committee of the college. A total of 48 fibrotic liver tissue samples were collected, with ten normal hepatic tissue specimens served as the control. Tissue samples were preserved at the temperature of -80 °C. Antibodies and Reagents Rabbits polyclonal to a-SMA, Collagen I, and b-Catenin were purchased from Abcam (Cambridge, MA, USA). Human R-Spondin2 antibody, mouse R-Spondin2 antibody, and recombinant mouse R-Spondin2 were purchased from R&D (R&D Systems, Minneapolis, MN, USA). NEPER Nuclear and Cytoplasmic Extraction Reagent Kit was obtained from Pierce (Pierce Biotechnology, Rockford, IL,
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USA). Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit IgG was obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lipofectamine 2000 transfection reagent and TRIzol were obtained from Invitrogen (Life Technologies, Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). TCF reporter plasmid TOPFLASH and the control inactive reporter FOPFLASH were purchased from Upstate (Upstate Biotechnology Inc., Lake Placid, NY, USA). CCl4, olive oil, collagenase IV, pronase E, Nycodenz, bovine serum albumin (BSA), MTT, and DMSO were obtained from Sigma-Aldrich (SigmaAldrich, St. Louis, MO, USA). HSC Isolation and Culture Primary stellate cells were isolated from the livers of Kunming mice as previously described [23]. Briefly, mouse liver was perfused in situ with DMEM (Gibco) to purge the liver of blood. After digestion of the liver with pronase E (Sigma) and collagenase IV (Sigma), dispersed cell suspensions were layered by Nycodenz (Sigma) density-gradient centrifugation according to the manufacturer’s protocol. The resulting upper layer contained the freshly isolated HSCs. The purity of isolated HSCs was examined by phase-contrast microscopy, and viability based on Trypan blue exclusion (purity [95 %, viability [95 %). The isolated HSCs were then cultured on uncoated plastic plates in DMEM (Gibco) supplemented with 10 % FBS (Gibco) at 37 °C at a density of 1 9 105 cells/cm2. The isolated HSCs were cultured for 1 or 7 days to examine quiescent or fully activated cells. Hepatic Fibrosis Model The hepatic fibrosis model of mice was induced by subcutaneous injection of 20 % solution of CCL4 in olive oil at a dose of 5 ml/kg twice per week for 10 weeks [24]. Twenty-four Kunming mice were randomly divided into two groups. The first group (n = 8) served as a normal control group, and the experimental group (n = 16) was hepatic fibrosis models. Four mice died during the induction of hepatic fibrosis, thus, the final experimental group contained 12 mice with CCL4-induced liver fibrosis. One week after the last infusion, the animals were killed by CO2 exposure and liver tissues were harvested.
tissue microarrays using a Beecher Instrument (Sun Prairie, WI, USA) as described previously [25]. Three tissue cylinders of 0.6 mm diameter were punched from each sample. After sectioning (4 mm for each section), tissue slides were baked at 60 °C for 2 h, and kept at 4 °C for further analysis. Immunohistochemistry Immunohistochemistry was performed on the TMA sections containing fibrotic liver tissues and normal liver tissues. After deparaffinization, rehydration, and antigen retrieval, TMA sections were immunostained with the primary antibodies R-Spondin2 (1:1,000 dilution, R&D Systems). The TMA sections were incubated with the primary antibody at 4 °C overnight, followed by HRPconjugated secondary anti-rabbit IgG (1:2,000 dilution, Santa Cruz) antibody for 1 h at room temperature. Immunohistochemical staining was carried out using the SABC kit (Boster, China). For quantification of the immunostain, cells were counted in six randomly chosen high-power fields at 400-fold magnification by two experienced researchers in a double-blinded manner. Results were scored by the percentage of R-Spondin2-stained cells as below: \10 % (score 0), 10–25 % (score 1), 25–50 % (score 2), 50–75 % (score 3), and 75–100 % (score 4). Results were considered positive with a score higher than 2. Quantification of Transcript Levels by Real-Time PCR The mRNA expressions were quantified by real-time PCR (StepOne-Plus, Applied Biosystems, Carlsbad, CA, USA) following the manufacturer’s instructions. Total RNA from the cells was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Thermocycling conditions consisted of an initial step of 2 min at 50 °C, denaturation of 10 min at 95 °C, followed by 35–40 cycles at 95 °C for 30 s, 60 °C for 45 s, and 72 °C for 1 min and a final elongation step at 72 °C for 10 min. Primers used for amplification are listed in Table 1. Each sample was analyzed in triplicate, with b-actin used for normalization. Comparative threshold (Ct) method was used for calculating the relative amount of mRNA of the sample compared with the control. Western-Blot Assays
Tissue Microarray Construction Tissue specimens (48 fibrotic liver tissue samples and ten normal hepatic tissue samples) were formalin-fixed and paraffin-embedded. The samples were used to construct
Standard Western-blot protocol was used, with b-actin serving as a loading control. Nuclear and cytoplasmic protein fractions were extracted using the NE-PER Extraction Reagent Kit (Pierce Biotechnology) according
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Dig Dis Sci Table 1 Primers sequences Gene
Forward sequence
R-Spondin2
50 -GCGGGTGTCGGCAAACTTTTTC-30 0
Reverse sequence
0
50 -CTTTTCCATGTCGTCCCAGT-30
a-SMA
5 -GCTGCTCCAGCTATGTGTGA-3
Collagen I
50 -TTCACCTACAGCACGCTTGTG-30
b-Catenin b-Actin
50 -GATGACTGTCTTGCCCCAAGTT-30
0
0
50 -TGTCAGGGGAGCTGTGGCTCC-30
0
0
50 -AACAGTCCGCCTAGAAGCAC-30
5 -CAGCAGTTTGTGGAGGGCGTG-3 5 -TCATCACTATTGGCAACGAGC-3
to the manufacturer’s instructions. The extracts were then cleared by centrifugation at 16,000 9 g for 15 min. After blocking with 5 % non-fat milk in PBS for 1 h at room temperature, membranes were incubated with primary antibodies against R-Spondin2 (1:1,000 dilution, R&D Systems), a-SMA (1:300 dilution, Abcam), Collagen I (1:200 dilution, Abcam), and b-catenin (1:200 dilution, Abcam) at 4 °C overnight. The membranes were then incubated with HRP-conjugated secondary anti-rabbit IgG (1:2,000 dilution, Santa Cruz) antibody for 1 h at room temperature. Results were visualized and quantified by enhanced chemiluminescence (ECL) using an electrochemiluminescence immunoblotting kit (Cell Signaling Technology) with a luminescent digital image analyzer Bio-Spectrum600 (UVP, Upland, CA, USA). Band intensity was assessed using a Gel-Pro analyzer.
Stimulation with R-Spondin2 For treatment with R-Spondin2, freshly isolated mice HSCs were maintained until 70 % confluence. Then, mediums were changed and HSCs were cultured for 24 h in DMEM (Gibco) with 5 % FBS (Gibco). HSCs were then exposed to different concentrations (0 ng/ml as control, 10, and 20 ng/ml) of recombinant mouse R-Spondin2 (R&D Systems) for 24 h. Finally, cells were harvested for further analysis.
siRNA-Mediated Gene Silencing Lipofectamine 2000 (Invitrogen) was used for transfection according to the manufacturer’s protocol. HSCs were seeded into six-well plates for 12 h before transfection. Next, siRNA plasmid was mixed with 5 ll Lipofectamine 2000 in 250 ll Opti-MEM I medium for 20 min. Then, the transfection mixture was added to each well with 1.5 ml FBS free DMEM (Gibco) at a concentration of 100 nmol/l. After 6 h of incubation, a liquid mixture containing siRNA was disposed. HSCs were then collected for further analyses after 48 h. The following siRNA sequences were used for gene silencing:
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50 -ATCTGGGGCTCG -GTGTCCATAATAC-30
Mouse R-Spondin2 siRNA: 50 - AAGCTGTTGTCACT AAGATAATG-30 Scrambled siRNA : 50 - GCTGCGTAATTTAAGAGTA ACAT -30 Luciferase Reporter Assay Cells were transiently transfected with TOPFLASH and FOPFLASH (Upstate Biotechnology Inc., Lake Placid, NY, USA) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were harvested and luciferase and Renilla luminescence were measured using the Dual-Luciferase Reporter Assay System (Promega, Wisconsin, WI, USA) on a luminometer (BioTek Instruments, Winooski, VT, USA). TCF reporter activity was presented as the ratio of firefly-to-Renilla luciferase activity. Two independent transfections were performed, with each sample tested in triplicate. HSCs Proliferation Assay The MTT assay was performed to determine the HSCs proliferation. Briefly, HSCs at the logarithmic growth phase were seeded at a density of 4 9 103 cells per pore into 96-well culture plates, with 100 ll cell suspension in each well. HSCs were simultaneously transfected by R-Spondin2 siRNA plasmid or co-cultured with different concentration of recombinant R-Spondin2 (0 ng/ml as control, 10, and 20 ng/ml). After treatment for 24, 48, and 72 h, 10 ll of MTT (Sigma) solution (5 mg/ml) was added into each well and incubated at 37 °C for 4 h, then centrifugation was performed at 3,000 rpm for 5 min. Finally, the precipitate was dissolved completely with 100 ll DMSO (Sigma), and the absorbance of the enzyme was measured at 570-nm wavelength in a Bio-Rad 550 microplate reader (Bio-Rad, Hercules, CA, USA). The experiments were performed in triplicate. Statistical Analysis The results of three independent experiments were recorded and expressed as mean ± SEM. The data was analyzed
Dig Dis Sci Fig. 1 R-Spondin2 was overexpressed in human fibrotic liver tissue. a Immunostaining showed R-Spondin2 was positive in human fibrotic liver tissue. b R-Spondin2 was negative in human normal liver tissue (bar 50 lm, magnification 9400). c Negative control. d The protein level of R-Spondin2 was significantly higher in human fibrotic liver tissue (n = 10) compared with normal liver tissue (n = 10). Quantification of levels of R-Spondin2 protein were normalized to b-actin, **p \ 0.01 compared to the normal liver tissue
using Student’s t test and considered significant at p \ 0.05.
Results R-Spondin2 Was Overexpressed in Liver Fibrosis Tissue Human liver tissue sections were immunostained for R-Spondin2 and the results demonstrated that R-Spondin2 protein was expressed in the cytoplasm and cell membrane. A total of 48 human fibrotic liver tissue samples were examined by immunohistochemistry experiment, with ten normal liver tissue samples serving as the control. The immunohistochemistry results showed that 83.3 % of the fibrotic liver tissue samples were positive for R-Spondin2 (Fig. 1a), whereas only 30 % of the normal liver tissue samples were positive for R-Spondin2 (Fig. 1b); the difference was significant at p \ 0.01. This finding indicated that R-Spondin2 was overexpressed in human fibrotic liver tissue. To verify this finding, the protein level of R-Spondin2 in the liver tissue was analyzed by Western-blot assay. Ten fibrotic liver tissue samples were examined, with ten normal liver tissue samples serving as the control. The result of Western-blot assay (Fig. 1d) was consistent with that of the immunohistochemistry experiment, and the protein level of R-Spondin2 was 7.71-fold that in the human fibrotic liver tissue compared with the normal liver tissue.
R-Spondin2 Was Upregulated in the Mouse Model of CCL4-Induced Hepatic Fibrosis Next, the expression of R-Spondin2 was examined in the CCL4-induced hepatic fibrosis mouse model (experimental group), with normal mice serving as the control. The protein expression and mRNA level of R-Spondin2, fibrosis biomarker a-SMA and Collagen I, and nuclear b-catenin were analyzed by Western-blot assay and real-time PCR, respectively. The results of Western blot showed that the protein expressions of a-SMA and Collagen I were 5.98fold and 4.17-fold in the experimental group, respectively, compared with the control group (Fig. 2a, b). The results of real-time PCR were aligned with those of the Western-blot assay, and the mRNA level of a-SMA and Collagen I were 7.16-fold and 5.33-fold in the experimental group compared with the control group (Fig. 2a, b). These findings indicated that the induction of liver fibrosis was successful. Both the protein expression and the mRNA level of R-Spondin2 were upregulated in the CCL4-induced hepatic fibrosis mouse model, which were 3.16-fold and 4.36-fold, respectively, compared with the control group (Fig. 2c). Prior studies have demonstrated that R-Spondins family of secreted proteins strongly potentiates the Wnt signaling pathway [14–17]. Thus, the expression of nuclear b-catenin was examined. The experimental results showed that protein expression of nuclear b-catenin increased 2.72-fold in the experimental group compared with the control group, and the mRNA level of total b-catenin increased 4.73-fold
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Fig. 2 R-Spondin2 was overexpressed in the hepatic fibrosis model of mouse. a The protein expression and mRNA level of a-SMA upregulated in the hepatic fibrosis mouse model. b The protein expression and mRNA level of Collagen I upregulated in the hepatic fibrosis mouse model. c The protein expression and mRNA level of
R-Spondin2 upregulated in the hepatic fibrosis mouse model. d The protein expression of nuclear b-catenin and mRNA level of a- total b-catenin upregulated in the hepatic fibrosis mouse model. Each experiment was performed in triplicate. **p \ 0.01 compared to the normal liver tissue
in the experimental group compared with the control group (Fig. 2d).
during the cultured-initiated activation of HSCs and R-Spondin2 was overexpressed in the fully activated HSCs.
The Expression of R-Spondin2 Was Elevated During the Activation of Mice HSCs Hepatic stellate cell (HSC) activation is the key event in liver fibrogenesis. Therefore, freshly isolated mice HSCs were cultured in vitro and the time-series expression change of R-Spondin2 during HSCs activation was observed. The primary HSC retains the quiescent phenotype when cultured on plastic plate for 1–2 days and become fully activated by day 7 in culture [3]. Thus, the protein level of a-SMA, Collagen I, and R-Spondin2 at day 1 and day 7 were examined by Western-blot assay (Fig. 3a). The results of Western blot showed that the protein level of a-SMA and Collagen I were 8.48-fold and 3.27-fold, respectively, in day 7 HSCs compared with day 1 HSCs (Fig. 3c), which indicated the freshly isolated HSCs were undergoing a phenotypic switch from quiescent to highly fibrogenic cells. The protein level of R-Spondin2 was also elevated with time (Fig. 3b), which increased 4.54-fold in the culture-activated HSCs (day 7). To confirm the findings of the Western-blot assay, the mRNA level of R-Spondin2 in day 1 HSCs (quiescent) and day 7 HSCs (fully activated) was examined by real-time PCR. The results of real-time PCR were in accordance with those of the Western-blot assay, and R-Spondin2 was detectable only in the fully activated HSCs (Fig. 3b). These findings suggested that the expression of R-Spondin2 was increased
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Increased Nuclear b-Catenin Level and TCF Activity in the Culture-Activated HSCs Previous studies have reported that R-Spondin2 protein is a potent Wnt pathway agonist [19, 20]. Thus, the protein level of nuclear b-catenin and the TCF activity in day 1 HSCs (quiescent) and day 7 HSCs (fully activated) were examined. The Western-blot result showed that the protein level of nuclear b-catenin was 4.47-fold in day 7 HSCs compared with day 1 HSCs (Fig. 3a, c), and Luciferase Reporter Assay showed that the TCF activity was 5.33-fold in day 7 HSCs compared with day 1 HSCs (Fig. 3d). These findings demonstrated stabilization and translocation of cytoplasmic b-catenin into the nucleus and the subsequent activation of TCF/LEF-dependent transcription in the fully activated HSCs. Exogenous Stimulation with Recombinant R-Spondin2 Enhanced the Wnt Activities in HSCs and Promoted HSCs Activation Having shown increased R-Spondin2 expression, elevated levels of nuclear b-catenin, and enhanced TCF activity in the culture-activated HSCs, we examined next the Wnt pathway activities stimulated exogenously by recombinant mouse R-Spondin2 in cultured HSCs. Freshly isolated
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Fig. 3 Time-series expression profiles of R-Spondin2, fibrosis biomarker a-SMA and Collagen I, and Wnt pathway activity during the culture-initiated activation of mice HSCs were analyzed. a Protein expression of R-Spondin2, a-SMA, Collagen I, and nuclear b-catenin were tested by Western blot assay. b The protein and mRNA level of R-Spondin2 elevated in the fully activated HSCs. c The protein level
of a-SMA, Collagen I, and nuclear b-catenin were significantly higher in the fully activated HSCs (day 7) compared with the quiescent HSCs (day 1). d TCF activity increased in culture-activated HSCs. Data represents the mean of three independent experiments, and error bars are standard deviation of means. *p \ 0.05 compared to the quiescent HSCs (day 1), **p \ 0.01 compared to the quiescent HSCs
Fig. 4 R-Spondin2 enhanced Wnt pathway activity and subsequently promoted HSCs fibrogenesis and proliferation in a dose-dependent manner. a–c The protein level of a-SMA, Collagen I, and nuclear b-catenin and increased in a dose-dependent manner (0 ng/ml as control, 10, and 20 ng/ml) when co-cultured with recombinant R-Spondin2. d Recombinant R-Spondin2 induced a dose-dependent
increase in TCF activity. e Recombinant R-Spondin2 up-regulated HSCs proliferation in a dose-dependent manner. Data represents the mean of three independent experiments, and error bars are standard deviation of means. *p \ 0.05 compared to the control (no treatment of recombinant R-Spondin2), **p \ 0.01 compared to the control
HSCs were exposed to recombinant R-Spondin2 at different concentration (0 ng/ml as control, 10, and 20 ng/ml), and the protein level of a-SMA, Collagen I, and nuclear
b-catenin were analyzed by Western-blot assay. The experiment results showed that the relative protein expression ratio of a-SMA (Fig. 4a) was 1:3.34:6.07
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(control: 10:20 ng/ml), the relative protein expression ratio of Collagen I (Fig. 4b) was 1:1.92:3.6 (control: 10:20 ng/ ml), and the relative ratio of nuclear b-catenin (Fig. 4c) was 1:2.2:4.77 (control: 10:20 ng/ml). TCF activities of HSCs stimulated by different concentration (control, 10, and 20 ng/ml) of recombinant R-Spondin2 were examined by Luciferase Reporter Assay. The results (Fig. 4d) showed that the relative ratio of TCF activity was 1:2.35:10.61 (control: 10:20 ng/ml). Because the activation of HSC induces a proliferative response [1–3], HSC proliferation was then tested by MTT assay. Results of HSCs proliferation assays (Fig. 4e) showed that the absorbance was increased in a dose-dependent manner. Taken together, these findings indicated that exogenous stimulation with R-Spondin2 resulted in dose-dependent nuclear b-catenin accumulation and TCF activity enhancement, which subsequently enhanced the synthesis of a-SMA and HSCs proliferation. The Knockdown of R-Spondin2 Down-Regulated the WNT Pathway Activities and Repressed HSCs Activation To further understand the role of R-Spondin2 in the activation of HSCs, the central event in the liver fibrogenesis, we knocked down the R-Spondin2 in the fully activated mice HSCs by siRNA and observed the expression change of a-SMA, Collagen I, and Wnt pathway activity. The effect of R-Spondin2 siRNA was validated by Western-blot assay and real-time PCR, respectively, and the results showed that both the protein expression and transcriptional level of R-Spondin2 were barely detectable in the R-Spondin2 knockdown HSCs (Fig. 5a, b). The protein level of a-SMA, Collagen I, and nuclear b-catenin in the R-Spondin2 knockdown HSCs were analyzed, with the HSCs transfected with scrambled siRNA (scrambled HSCs) serving as the control. The Western-blot results showed that the protein level of a-SMA, Collagen I, and nuclear b-catenin were 7.45-fold, 3.98-fold, and 4.61-fold, respectively, in the scrambled HSCs compared with the R-Spondin2 knockdown HSCs (Fig. 5a, d). Then, TCF activities of R-Spondin2 knockdown HSCs were examined by Luciferase Reporter Assay, and the results showed the TCF activity was 2.67-fold in the scrambled HSCs compared with the R-Spondin2 knockdown HSCs (Fig. 5c). Finally, HSCs proliferation was tested by MTT assay. Results of HSCs proliferation assays showed that the absorbance decreased in the R-Spondin2 knockdown HSCs compared with the scrambled HSCs (Fig. 5f). Taken together, these findings indicated that the siRNA-mediated gene silencing of R-Spondin2 suppressed the activity of Wnt pathway, and subsequently reduced the synthesis of a-SMA and HSCs proliferation.
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Discussion Most chronic hepatic diseases will progress to liver fibrosis, and some may even result in cirrhosis or hepatocellular carcinoma [26]. Thus, the development of liver fibrosis is the fundamental stage toward a number of mortal complications of liver disease. Continuing progress in the field of hepatic fibrosis research presents us a comprehensive and nuance picture of the liver fibrogenesis, especially the effects of signaling pathway in induction the HSC activation. Numerous studies have shown that the canonical Wnt pathway profoundly affects HSC activation, perpetuation, and resolution [9–11]. Prior studies also have documented that the R-Spondins family of secreted proteins strongly potentiates the Wnt pathway and regulates tissue patterning and differentiation [27–29]. Moreover, recent reports have revealed that R-Spondin2 plays a key role in the development of the gastrointestinal tract and myogenesis [16, 17, 20, 22]. Therefore, this study investigated whether R-Spondin2 contributed to HSC activation, the central event in fibrogenesis. We demonstrated that R-Spondin2 protein was overexpressed in human fibrotic liver tissues, whereas it was almost undetectable in the normal liver tissues. In addition, the expression of R-Spondin2 in the CCL4-induced liver fibrosis mice was significantly higher than that of normal mice. Based on this finding and prior reports, we hypothesized that R-Spondin2 might be implicated in HSC activation. Progressive activation occurs when freshly isolated HSCs are cultured in a plastic plate, and the cellular events of this spontaneous-activation are similar to those occurring in liver injury [3]. Observation on culture-initiated activation of isolated mice HSCs demonstrated that both the transcriptional level and protein expression of R-Spondin2 elevated with time, which was consistent with a prior report that the transcriptional level of R-Spondins increased three to *sixfold following liver injury [18]. This study also found that the protein level of nuclear b-catenin and TCF activity increased in the culture-activated HSCs. In brief, these findings suggested that R-Spondin2 might promote the HSC activation by enhancing the accumulation of nuclear b-catenin and subsequent transactivation of Wnt target genes. To prove our hypothesis, several functional analyses were performed on isolated mice HSCs, including exogenous stimulation with recombinant R-Spondin2 and siRNA-mediated gene silencing of R-Spond2. We found that stimulation with R-Spondin2 on freshly isolated HSCs upregulated HSCs fibrogenesis and Wnt pathway activity in a dose-dependent manner. Additionally, the knockdown of R-Spondin2 led to the down-regulation of Wnt pathway activity and repressed HSCs activation. Taken together, these findings supported our hypothesis that R-Spondin2
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Fig. 5 Knockdown of R-Spondin2 repressed Wnt pathway activity and HSCs activation. a Protein expression of R-Spondin2, a-SMA, Collagen I, and nuclear b-catenin in R-Spondin2 knockdown HSCs were examined, HSCs transfected with scrambled siRNA severed as the control. b The effect of R-Spondin2 knockdown was validated by Western-blot assay and real-time PCR. c TCF activity reduced significantly in R-Spondin2 knockdown HSCs. d Protein level of
a-SMA, Collagen I, and nuclear b-catenin decreased significantly in the R-Spondin2 knockdown HSCs compared with the control. e HSCs proliferation decreased significantly in R-Spondin2 knockdown HSCs. Data represents the mean of three independent experiments, and error bars are standard deviation of means. *p \ 0.05 compared to the control (HSCs transfected with scrambled siRNA), **p \ 0.01 compared to the control
promoted HSC activation by enhancing the canonical Wnt pathway. During the liver fibrosis recovery, the activated HSCs either revert to a quiescent phenotype or are cleared. TGFb1 has a well-established role as a fibrogenic mediator, and the synergistic activation of TGF-b1 and Wnt pathway profoundly affects the HSC activation, proliferation, and resolution [1–3]. However, development of anti-fibrotic therapy targeting the TGF-b1 is problematic owing to the pleiotropic nature of therapy TGF-b1. Therefore, our findings provided new insights into the function of Wnt pathway provoked by R-Spondin2 during hepatic fibrogenesis, and demonstrated R-Spondin2 as a potential antifibrotic therapeutic target for liver fibrosis. Our study focused on the role of R-Spondin2 in the activation of HSC. Due to the limitation of lab facility and the availability of normal human liver tissue, this study did not isolate human HSCs and examined the effect of R-Spondin2 on primary human HSCs. Although HSC activation is the dominant pathway leading to hepatic fibrosis, portal fibroblast may be especially prominent in cholestatic liver injury [1]. Further studies are required to investigate the effect of R-Spondin2 in the myofibroblast formation of portal fibroblast.
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