Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in16, HSC Activation and Liver Fibrosis Accepted: December 2014 1421-9778/15/0353-0885$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Original Paper
PTPRO-Associated Hepatic Stellate Cell Activation Plays a Critical Role in Liver Fibrosis Xudong Zhanga Zhongming Tana Youjing Wanga Junwei Tanga Runjiu Jianga Jiajie Houa Han Zhuoa Xiaochen Wanga Jie Jia Xihu Qinb Beicheng Suna Liver Transplantation Center of the First Affiliated Hospital, Nanjing Medical University, Nanjing, The Affiliated Changzhou NO.2 People’s Hospital of Nanjing Medical University, Changzhou, Jiangsu Province, P.R. China a
b
Key Words PTPRO • Liver fibrosis • Bile duct ligation • CCl4 • Platelet-derived growth factor Abstract Background/Aims: PTPRO (protein tyrosine phosphatase, receptor type O) is implicated in diverse physiological and pathological processes in cancer and hepatic ischemia/ reperfusion injury, although little is known about its role in hepatic fibrosis. Methods: Here, by using genetically deficient mice, we reported that PTPRO knockout (PTPRO–/–) significantly attenuated liver injury, release of inflammatory factors, tissue remodeling, and liver fibrosis in two experimental mouse models of fibrogenesis induced by bile-duct ligation or carbon tetrachloride administration. Results: However, we proved that PTPRO expression was strongly downregulated in clinical and experimental liver fibrosis specimens. Further investigations revealed that stimulation of primary hepatic stellate cells (HSCs) and hepatocytes with specific activator platelet-derived growth factor (PDGF)-BB increased PTPRO transcription in HSCs but had the opposite effect in primary hepatocytes. More importantly, synthetic short hairpin RNA targeting PTPRO significantly neutralized PDGF-BB-induced HSC proliferation and myofibroblast marker expression through downregulated phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. Conclusion: These observations confirm that PTPRO plays a critical role in liver fibrogenesis by affecting PDGF signaling in HSC activation and might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases. Copyright © 2015 S. Karger AG, Basel
X. Zhang and Z. Tan contributed equally to this work. Beicheng Sun, M.D., Ph.D., or Xihu Qin, M.D., Ph.D.
Liver Transplantation Center of the First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, 210029 Nanjing, Jiangsu Province (P.R. China) or The Affiliated Changzhou NO.2 People’s Hospital of Nanjing Medical University, Changzhou, Jiangsu Province, (P.R. China) E-Mail [email protected] or E-Mail [email protected]
885
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Introduction
Liver fibrosis is a pathological process arising from continuous wound-healing responses to liver injury, which encompasses infection-related hepatic fibrosis, metabolic-type fibrosis, and biliary-type fibrosis. As the injury proceeds, the extracellular matrix (ECM) such as collagen and fibronectin accumulates and forms a fibrous scar to nodules of regenerating hepatocytes, which predisposes to liver failure and hepatocellular carcinoma [1]. Hepatic stellate cells (HSCs) resident in the space between hepatocytes and liver sinusoidal endothelial cells (LSECs) are viewed as a predominant cellular source of ECM when activated [2, 3]. They normally store vitamin (retinoid) droplets in their cytoplasm, and are differentiated by injury-associated immunological processes into fibrogenic myofibroblasts that produce ECM and affect the function of hepatocytes and LSECs [4, 5]. Fibrosis progression and regression need specific and complicated signaling pathways, including chemokines and cytokines such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β and leptin [68]. Many recent studies have focused on the early events in liver fibrosis, such as immune interactions with interleukin (IL)-17 and IL-33 [9-11], but how they support fibrosis and activate HSCs requires verification. Protein-tyrosine phosphatases (PTPs) can act in an opposite but equally important manner to protein-tyrosine kinases (PTKs), as a key part of post-translational modification strategies to regulate protein activities [12]. Protein tyrosine phosphatase receptor type O (PTPRO), also named glomerular epithelial protein 1 (GLEPP), is an important member of the receptor-type PTPs family that is expressed in humans, rats and rabbits. There are two types of PTPRO, full-length PTPRO (PTPRO-FL) expressed in kidney, brain, lung, liver and breast, and truncated PTPRO (PTPROt) which is predominantly expressed in B lymphocytes and macrophages [13]. PTPRO has been implicated in several biological processes, including neuronal growth and branching [14, 15], mammary epithelial cell morphogenesis [16], and as a biomarker in various types of carcinoma [17-20]. Previous research from our center has indicated that PTPRO expression promoted by estrogen receptor α contributes to the sex difference in incidence of hepatocellular carcinoma [21]. Furthermore, we also reported that PTPRO played a bidirectional role in hepatic ischemia/reperfusion (I/R) injury by affecting signal transducer and activator of transcription (STAT)3 activity [22]. However, the role of PTPRO in liver fibrosis is still unclear. Here, we identified PTPRO as a key mediator of hepatic fibrosis, and further explored the idea of therapeutic modulation of PTPRO in hepatic inflammation and fibrosis. Materials and Methods
Patient samples All human liver samples were collected at First Affiliated Hospital of Nanjing Medical University, China (Table 1). Informed consent was obtained from the patients before the specimens were obtained. Cirrhosis specimens (n=18) were collected from patients undergoing liver transplantation for end-stage liver cirrhosis. Normal liver tissue (n=8) was collected from patients receiving hepatoectomy for hepatic cavernous hemangioma. Liver samples were analyzed by quantitative real-time PCR, or fixed in 4% paraformaldehyde followed by paraffin embedding for immunoblotting. Mouse models Specific pathogen-free, male C57BL/6 mice, aged 8–10 weeks were obtained from the Experimental Animal Center of Nanjing Medical University. PTPRO knockout (KO) C57BL/6 mice were kindly donated by Dr. Bixby, University of Miami, USA and bred in the Model Animal Research Center of Nanjing University. Carbon tetrachloride (CCl4; 0.6 g/kg) was administered intraperitoneally twice weekly for 4 weeks, mixed with corn oil (or oil for control), and the mice were sacrificed 3 days after the final dose, when transaminase levels should return to near normal, as described previously [23]. For bile duct ligation (BDL), mice were anesthetized with 1% pentobarbital. After midline laparotomy, the common bile duct was dissociated and ligated twice with 6-0 silk sutures, and the abdomen was closed. Sham operation was performed similarly,
886
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Table 1. Clinical characteristics. Data are presented as mean (minimum-maximum). ALT, alanine aminotransferase; HBV, hepatitis B virus (Others are alcoholic cirrhosis)
except that the bile duct was not ligated [24]. To eliminate the influence of other complications, animals were sacrificed 3 or 14 days after BDL. Serum and liver were prepared for other research. Indicated groups of BDL challenged mice were observed until 21 days for overall survival rate analysis, Microscopic investigation of liver after BDL or CCl4 treatment Fragments of liver tissues were fixed in 4% buffered formaldehyde. Sections (3 µm) of liver were cut and stained with hematoxylin and eosin (H&E). Liver H&E sections were analyzed for I/R injury under a Leica DM4000 B Upright Research Microscope (100×).
Measurement of plasma alanine aminotransferase (ALT) activity Blood was obtained by cardiac puncture at the time of sacrifice for analysis of serum ALT as an index of hepatocellular injury. Measurements of serum ALT were made using a diagnostic kit (Sigma, St. Louis, MO, USA).
HSCs and hepatocytes isolation and restimulation assay Primary mouse HSCs were isolated from livers of C57BL/6 mice, as described previously, with modification [10]. Livers were perfused in situ with 45 ml Gibco Liver Perfusion Media (Invitrogen, Carlsbad, CA, USA) followed by 45 ml Gibco Liver Digestion Media (Invitrogen). The liver digests were filtered through a cell strainer and washed with Gey’s Balanced Salt Solution (GBSS) (Sigma) containing DNase I (2 mg/ml, Roche Diagnostics). The homogenate was centrifuged at 25 g for 5 min at room temperature to remove the hepatocytes. The supernatant was transferred to a new tube and centrifuged at 400 g for 10 min at 4°C. The cell pellet was resuspended in 5 ml 15% OptiPrep (Sigma), and loaded carefully with 5 ml of 11.5% OptiPrep and 2 ml GBSS. After centrifugation at 1400 g for 17 min at 4°C, the cell fraction in the GBSS and 11.5% OptiPrep interphase was gently aspirated, mixed with GBSS, and centrifuged at 1400 g for 10 min at 4°C. After another wash, the final cell pellet was resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing penicillin, streptomycin and 10% fetal bovine serum (FBS) (complete medium), and then plated on uncoated plastic at a density of 5×106 per 10-cm diameter plate. After the first 24 h, nonadherent cells and debris were removed by washing. Cell viability was >90% as assessed by Trypan blue exclusion. Purity was 90–95% as assessed by light microscopy. Primary murine hepatocytes were isolated from male C57BL/6 mice aged 6–8 weeks, as previously reported [22]. Isolated HSCs and hepatocytes were placed into the 96 well plate (8×104 cells/well) in DMEM and exposed to recombinant (rm) PDGF-BB (Peprotech, UK) (5ng/ ml) [25] . After 12 h incubation at 37°C, cells were harvested for other tests. RNA interference analysis PTPRO silencing by mouse PTPRO small hairpin RNA (shRNA) (5′-GCG CTC ATA CCG AAT GTC AAT CTC GAG ATT GAC ATT CGG TAT GAGCGC-3′) or scrambled sequences (5′-TGACAGTCGATTGATGTGATT-3′) designed and synthesized by Wuhan Genesial Corporation (Wuhan, China) was tested in HSCs. PTPRO levels following shRNA silencing were analyzed by RT-PCR and immunoblotting using anti-PTPRO antibodies. Such analysis revealed that the PTPRO-shRNA silenced PTPRO expression by ~90% at a relatively low concentration (50 nmol/l) using Lipofectamine 2000 (Invitrogen) for three independent transfection experiments.
887
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Table 2. Primer used
Real-time quantitative PCR RNA was isolated from mouse or human liver tissue by homogenization and purification using TRIzol reagent (Invitrogen). RNA was isolated from HSCs using RNeasy. Following DNase treatment and reverse transcription, real-time quantitative PCR of mouse and human samples was performed for 40 cycles of 15 s at 95°C and 1 min at 60°C using a one-step RT-PCR kit (TaKaRa, Dalian, China). The specific primers used are summarized in Table 2. Quantification was performed by comparing the Ct values of each sample with a standard curve and normalization to GAPDH. Values were expressed as fold induction in comparison with untreated or sham controls. Immunohistochemistry Immunohistochemistry of α-smooth muscle actin (SMA) was performed using 5-µm paraffin sections. The sections were deparaffined in xylene and rehydrated in alcohol and distilled water. Antigen retrieval was performed by heating sections in solution (10 mM citrate buffer, pH 6.0) in a pressure cooker. H2O2 (3%) was used to eliminate endogenous peroxidase. Slides were washed with Tris-buffered saline (TBS)– Triton three times and once with TBS for 5 min. Nonspecific binding was blocked using 5% goat serum for 30 min. The blocking buffer was removed and sections were incubated with a polyclonal α-SMA antibody (AB5694; Abcam, Cambridge, UK). Control experiments were incubated with rabbit serum replacing the first antibody. After incubation overnight at 4°C and washing, the sections were incubated with biotinylated goat anti-rabbit IgG antibody. After washing, peroxidase-coupled antibody was applied for 30 min at room temperature. Bound antibody was detected with 3, 3′-diaminobenzidine tetrachloride (DAB) (Sigma– Aldrich, St. Louis, MO, USA). All sections were counterstained with hematoxylin. Image-ProPlus version 6.0 was used to evaluate the areas of positive α-SMA as computer-assisted semi-quantitative. Collagen detection assay Supernatants of liver homogenate samples were collected from mice; cell medium were obtained from 5 ng/ml rmPDGF-BB restimulation for 12 h (1×105 cells/ml in 6-well plates). The collagen concentration was measured using the Sircol assay according to the manufacturer’s recommended procedure (S1000, Biocolor, Carrickfergus, UK). Masson’s trichrome stain and hydroxyproline assay were used to focus on the change in hepatic collagen content in vitro and in vivo. The detailed methods were according to the manufacturer’s recommended procedure (HT15; Sigma–Aldrich and KGT030-2; KeyGEN Biotech, Nanjing, China).
Cytotoxicity and viability assays Cell viability was assessed using the MTT reduction assay. Formation of blue formazan was measured spectrophotometrically from the metabolism of MTT by mitochondrial dehydrogenases that are active only in live cells. Isolated HSCs were seeded in 96-well plates (1×104 cells per well) and then incubated in DMEM containing penicillin, streptomycin and 10% FBS for 24 h. The cells were pretreated with various concentrations of rmPDGF-BB for 12 h, and MTT (5 mg/ml) was added to each of the wells, and the plate was incubated for an additional 1 h at 37°C. The medium was removed, and the intracellular formazan product was dissolved in 250 µl DMSO. The absorbance of each well was measured at 540 nm using a microplate reader. OD values from untreated control cells were designated as 100% viability. Triplicate specimens were
888
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Fig. 1. PTPRO is required for hepatic fibrogenesis in BDL. Mice underwent BDL and were sacrificed 3 or 14 days later. (A) Serum ALT values and total bilirubin (TBil) were measured 3 and 14 days after BDL from WT and PTPRO–/– mice (n=8 per group) compared to each sham-operated control (n=4). TBil in BDL mice was almost identical, indicating a similar cholestatic effect of BDL between the groups of animals. (B) H&E showed that the WT group had more widespread bile infarctions and cholestatic hepatitis than the PTPRO–/– group had. Fibrillar collagen was determined by Masson staining (Collagen fibers stained as blue and cytoplasm stained as red, original magnification, 100×). (C) Quantification of positive area by Masson staining (left). Hydroxyproline quantification indicated that collengen deposition was increased in BDL-treated liver (right). (D) Expression of α-SMA was measured by immunohistochemistry. Western blotting of α-SMA between the two groups. Average integrated optical density (IOD) was obtained by analyzing five fields for each slide evaluated by Image-Pro Plus software (version 5.0) for immunohistochemical staining (*P
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in16, HSC Activation and Liver Fibrosis Accepted: December 2014 1421-9778/15/0353-0885$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Original Paper
PTPRO-Associated Hepatic Stellate Cell Activation Plays a Critical Role in Liver Fibrosis Xudong Zhanga Zhongming Tana Youjing Wanga Junwei Tanga Runjiu Jianga Jiajie Houa Han Zhuoa Xiaochen Wanga Jie Jia Xihu Qinb Beicheng Suna Liver Transplantation Center of the First Affiliated Hospital, Nanjing Medical University, Nanjing, The Affiliated Changzhou NO.2 People’s Hospital of Nanjing Medical University, Changzhou, Jiangsu Province, P.R. China a
b
Key Words PTPRO • Liver fibrosis • Bile duct ligation • CCl4 • Platelet-derived growth factor Abstract Background/Aims: PTPRO (protein tyrosine phosphatase, receptor type O) is implicated in diverse physiological and pathological processes in cancer and hepatic ischemia/ reperfusion injury, although little is known about its role in hepatic fibrosis. Methods: Here, by using genetically deficient mice, we reported that PTPRO knockout (PTPRO–/–) significantly attenuated liver injury, release of inflammatory factors, tissue remodeling, and liver fibrosis in two experimental mouse models of fibrogenesis induced by bile-duct ligation or carbon tetrachloride administration. Results: However, we proved that PTPRO expression was strongly downregulated in clinical and experimental liver fibrosis specimens. Further investigations revealed that stimulation of primary hepatic stellate cells (HSCs) and hepatocytes with specific activator platelet-derived growth factor (PDGF)-BB increased PTPRO transcription in HSCs but had the opposite effect in primary hepatocytes. More importantly, synthetic short hairpin RNA targeting PTPRO significantly neutralized PDGF-BB-induced HSC proliferation and myofibroblast marker expression through downregulated phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. Conclusion: These observations confirm that PTPRO plays a critical role in liver fibrogenesis by affecting PDGF signaling in HSC activation and might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases. Copyright © 2015 S. Karger AG, Basel
X. Zhang and Z. Tan contributed equally to this work. Beicheng Sun, M.D., Ph.D., or Xihu Qin, M.D., Ph.D.
Liver Transplantation Center of the First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, 210029 Nanjing, Jiangsu Province (P.R. China) or The Affiliated Changzhou NO.2 People’s Hospital of Nanjing Medical University, Changzhou, Jiangsu Province, (P.R. China) E-Mail [email protected] or E-Mail [email protected]
885
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Introduction
Liver fibrosis is a pathological process arising from continuous wound-healing responses to liver injury, which encompasses infection-related hepatic fibrosis, metabolic-type fibrosis, and biliary-type fibrosis. As the injury proceeds, the extracellular matrix (ECM) such as collagen and fibronectin accumulates and forms a fibrous scar to nodules of regenerating hepatocytes, which predisposes to liver failure and hepatocellular carcinoma [1]. Hepatic stellate cells (HSCs) resident in the space between hepatocytes and liver sinusoidal endothelial cells (LSECs) are viewed as a predominant cellular source of ECM when activated [2, 3]. They normally store vitamin (retinoid) droplets in their cytoplasm, and are differentiated by injury-associated immunological processes into fibrogenic myofibroblasts that produce ECM and affect the function of hepatocytes and LSECs [4, 5]. Fibrosis progression and regression need specific and complicated signaling pathways, including chemokines and cytokines such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β and leptin [68]. Many recent studies have focused on the early events in liver fibrosis, such as immune interactions with interleukin (IL)-17 and IL-33 [9-11], but how they support fibrosis and activate HSCs requires verification. Protein-tyrosine phosphatases (PTPs) can act in an opposite but equally important manner to protein-tyrosine kinases (PTKs), as a key part of post-translational modification strategies to regulate protein activities [12]. Protein tyrosine phosphatase receptor type O (PTPRO), also named glomerular epithelial protein 1 (GLEPP), is an important member of the receptor-type PTPs family that is expressed in humans, rats and rabbits. There are two types of PTPRO, full-length PTPRO (PTPRO-FL) expressed in kidney, brain, lung, liver and breast, and truncated PTPRO (PTPROt) which is predominantly expressed in B lymphocytes and macrophages [13]. PTPRO has been implicated in several biological processes, including neuronal growth and branching [14, 15], mammary epithelial cell morphogenesis [16], and as a biomarker in various types of carcinoma [17-20]. Previous research from our center has indicated that PTPRO expression promoted by estrogen receptor α contributes to the sex difference in incidence of hepatocellular carcinoma [21]. Furthermore, we also reported that PTPRO played a bidirectional role in hepatic ischemia/reperfusion (I/R) injury by affecting signal transducer and activator of transcription (STAT)3 activity [22]. However, the role of PTPRO in liver fibrosis is still unclear. Here, we identified PTPRO as a key mediator of hepatic fibrosis, and further explored the idea of therapeutic modulation of PTPRO in hepatic inflammation and fibrosis. Materials and Methods
Patient samples All human liver samples were collected at First Affiliated Hospital of Nanjing Medical University, China (Table 1). Informed consent was obtained from the patients before the specimens were obtained. Cirrhosis specimens (n=18) were collected from patients undergoing liver transplantation for end-stage liver cirrhosis. Normal liver tissue (n=8) was collected from patients receiving hepatoectomy for hepatic cavernous hemangioma. Liver samples were analyzed by quantitative real-time PCR, or fixed in 4% paraformaldehyde followed by paraffin embedding for immunoblotting. Mouse models Specific pathogen-free, male C57BL/6 mice, aged 8–10 weeks were obtained from the Experimental Animal Center of Nanjing Medical University. PTPRO knockout (KO) C57BL/6 mice were kindly donated by Dr. Bixby, University of Miami, USA and bred in the Model Animal Research Center of Nanjing University. Carbon tetrachloride (CCl4; 0.6 g/kg) was administered intraperitoneally twice weekly for 4 weeks, mixed with corn oil (or oil for control), and the mice were sacrificed 3 days after the final dose, when transaminase levels should return to near normal, as described previously [23]. For bile duct ligation (BDL), mice were anesthetized with 1% pentobarbital. After midline laparotomy, the common bile duct was dissociated and ligated twice with 6-0 silk sutures, and the abdomen was closed. Sham operation was performed similarly,
886
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Table 1. Clinical characteristics. Data are presented as mean (minimum-maximum). ALT, alanine aminotransferase; HBV, hepatitis B virus (Others are alcoholic cirrhosis)
except that the bile duct was not ligated [24]. To eliminate the influence of other complications, animals were sacrificed 3 or 14 days after BDL. Serum and liver were prepared for other research. Indicated groups of BDL challenged mice were observed until 21 days for overall survival rate analysis, Microscopic investigation of liver after BDL or CCl4 treatment Fragments of liver tissues were fixed in 4% buffered formaldehyde. Sections (3 µm) of liver were cut and stained with hematoxylin and eosin (H&E). Liver H&E sections were analyzed for I/R injury under a Leica DM4000 B Upright Research Microscope (100×).
Measurement of plasma alanine aminotransferase (ALT) activity Blood was obtained by cardiac puncture at the time of sacrifice for analysis of serum ALT as an index of hepatocellular injury. Measurements of serum ALT were made using a diagnostic kit (Sigma, St. Louis, MO, USA).
HSCs and hepatocytes isolation and restimulation assay Primary mouse HSCs were isolated from livers of C57BL/6 mice, as described previously, with modification [10]. Livers were perfused in situ with 45 ml Gibco Liver Perfusion Media (Invitrogen, Carlsbad, CA, USA) followed by 45 ml Gibco Liver Digestion Media (Invitrogen). The liver digests were filtered through a cell strainer and washed with Gey’s Balanced Salt Solution (GBSS) (Sigma) containing DNase I (2 mg/ml, Roche Diagnostics). The homogenate was centrifuged at 25 g for 5 min at room temperature to remove the hepatocytes. The supernatant was transferred to a new tube and centrifuged at 400 g for 10 min at 4°C. The cell pellet was resuspended in 5 ml 15% OptiPrep (Sigma), and loaded carefully with 5 ml of 11.5% OptiPrep and 2 ml GBSS. After centrifugation at 1400 g for 17 min at 4°C, the cell fraction in the GBSS and 11.5% OptiPrep interphase was gently aspirated, mixed with GBSS, and centrifuged at 1400 g for 10 min at 4°C. After another wash, the final cell pellet was resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing penicillin, streptomycin and 10% fetal bovine serum (FBS) (complete medium), and then plated on uncoated plastic at a density of 5×106 per 10-cm diameter plate. After the first 24 h, nonadherent cells and debris were removed by washing. Cell viability was >90% as assessed by Trypan blue exclusion. Purity was 90–95% as assessed by light microscopy. Primary murine hepatocytes were isolated from male C57BL/6 mice aged 6–8 weeks, as previously reported [22]. Isolated HSCs and hepatocytes were placed into the 96 well plate (8×104 cells/well) in DMEM and exposed to recombinant (rm) PDGF-BB (Peprotech, UK) (5ng/ ml) [25] . After 12 h incubation at 37°C, cells were harvested for other tests. RNA interference analysis PTPRO silencing by mouse PTPRO small hairpin RNA (shRNA) (5′-GCG CTC ATA CCG AAT GTC AAT CTC GAG ATT GAC ATT CGG TAT GAGCGC-3′) or scrambled sequences (5′-TGACAGTCGATTGATGTGATT-3′) designed and synthesized by Wuhan Genesial Corporation (Wuhan, China) was tested in HSCs. PTPRO levels following shRNA silencing were analyzed by RT-PCR and immunoblotting using anti-PTPRO antibodies. Such analysis revealed that the PTPRO-shRNA silenced PTPRO expression by ~90% at a relatively low concentration (50 nmol/l) using Lipofectamine 2000 (Invitrogen) for three independent transfection experiments.
887
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Table 2. Primer used
Real-time quantitative PCR RNA was isolated from mouse or human liver tissue by homogenization and purification using TRIzol reagent (Invitrogen). RNA was isolated from HSCs using RNeasy. Following DNase treatment and reverse transcription, real-time quantitative PCR of mouse and human samples was performed for 40 cycles of 15 s at 95°C and 1 min at 60°C using a one-step RT-PCR kit (TaKaRa, Dalian, China). The specific primers used are summarized in Table 2. Quantification was performed by comparing the Ct values of each sample with a standard curve and normalization to GAPDH. Values were expressed as fold induction in comparison with untreated or sham controls. Immunohistochemistry Immunohistochemistry of α-smooth muscle actin (SMA) was performed using 5-µm paraffin sections. The sections were deparaffined in xylene and rehydrated in alcohol and distilled water. Antigen retrieval was performed by heating sections in solution (10 mM citrate buffer, pH 6.0) in a pressure cooker. H2O2 (3%) was used to eliminate endogenous peroxidase. Slides were washed with Tris-buffered saline (TBS)– Triton three times and once with TBS for 5 min. Nonspecific binding was blocked using 5% goat serum for 30 min. The blocking buffer was removed and sections were incubated with a polyclonal α-SMA antibody (AB5694; Abcam, Cambridge, UK). Control experiments were incubated with rabbit serum replacing the first antibody. After incubation overnight at 4°C and washing, the sections were incubated with biotinylated goat anti-rabbit IgG antibody. After washing, peroxidase-coupled antibody was applied for 30 min at room temperature. Bound antibody was detected with 3, 3′-diaminobenzidine tetrachloride (DAB) (Sigma– Aldrich, St. Louis, MO, USA). All sections were counterstained with hematoxylin. Image-ProPlus version 6.0 was used to evaluate the areas of positive α-SMA as computer-assisted semi-quantitative. Collagen detection assay Supernatants of liver homogenate samples were collected from mice; cell medium were obtained from 5 ng/ml rmPDGF-BB restimulation for 12 h (1×105 cells/ml in 6-well plates). The collagen concentration was measured using the Sircol assay according to the manufacturer’s recommended procedure (S1000, Biocolor, Carrickfergus, UK). Masson’s trichrome stain and hydroxyproline assay were used to focus on the change in hepatic collagen content in vitro and in vivo. The detailed methods were according to the manufacturer’s recommended procedure (HT15; Sigma–Aldrich and KGT030-2; KeyGEN Biotech, Nanjing, China).
Cytotoxicity and viability assays Cell viability was assessed using the MTT reduction assay. Formation of blue formazan was measured spectrophotometrically from the metabolism of MTT by mitochondrial dehydrogenases that are active only in live cells. Isolated HSCs were seeded in 96-well plates (1×104 cells per well) and then incubated in DMEM containing penicillin, streptomycin and 10% FBS for 24 h. The cells were pretreated with various concentrations of rmPDGF-BB for 12 h, and MTT (5 mg/ml) was added to each of the wells, and the plate was incubated for an additional 1 h at 37°C. The medium was removed, and the intracellular formazan product was dissolved in 250 µl DMSO. The absorbance of each well was measured at 540 nm using a microplate reader. OD values from untreated control cells were designated as 100% viability. Triplicate specimens were
888
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2015;35:885-898 DOI: 10.1159/000369746 Published online: January 30, 2015
© 2015 S. Karger AG, Basel www.karger.com/cpb
Zhang et al.: PTPRO in HSC Activation and Liver Fibrosis
Fig. 1. PTPRO is required for hepatic fibrogenesis in BDL. Mice underwent BDL and were sacrificed 3 or 14 days later. (A) Serum ALT values and total bilirubin (TBil) were measured 3 and 14 days after BDL from WT and PTPRO–/– mice (n=8 per group) compared to each sham-operated control (n=4). TBil in BDL mice was almost identical, indicating a similar cholestatic effect of BDL between the groups of animals. (B) H&E showed that the WT group had more widespread bile infarctions and cholestatic hepatitis than the PTPRO–/– group had. Fibrillar collagen was determined by Masson staining (Collagen fibers stained as blue and cytoplasm stained as red, original magnification, 100×). (C) Quantification of positive area by Masson staining (left). Hydroxyproline quantification indicated that collengen deposition was increased in BDL-treated liver (right). (D) Expression of α-SMA was measured by immunohistochemistry. Western blotting of α-SMA between the two groups. Average integrated optical density (IOD) was obtained by analyzing five fields for each slide evaluated by Image-Pro Plus software (version 5.0) for immunohistochemical staining (*P
