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Stimulation of hepatic stellate cells (HSCs) by platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) is an essential pathway of proliferation and fibrogenesis, respectively, in liver fibrosis. We provide evidence that PTK787/ZK222584 (PTK/ZK), a potent tyrosine kinase inhibitor that blocks vascular endothelial growth factor receptor (VEGFR), significantly inhibits PDGF receptor expression, as well as PDGF-simulated HSC proliferation, migration and phosphorylation of ERK1/2, Akt and p70S6 kinase. Interestingly, PTK/ZK also antagonizes the TGF-β1-induced expression of VEGF and VEGFR1. Furthermore, PTK/ZK downregulates TGF-β receptor expression, which is associated with reduced Akt, ERK and p38MAPK phosphorylation. Furthermore, PDGF-induced TGF-β1 expression is inhibited by PTK/ZK. These findings provide evidence that PTK/ZK targets multiple essential pathways of stellate cell activation that provoke proliferation and fibrogenesis. Our study underscores the potential use of PTK/ZK as an antifibrotic drug in chronic liver disease.
Liver fibrosis is a pathological response of the liver to a variety of chronic stimuli. Hepatic stellate cells (HSCs) play an essential role in the development of liver fibrosis. After chronic liver injury, HSCs undergo a process of activation, developing a myofibroblast-like phenotype that proliferates and becomes fibrogenic1,2 and produces increased extracellular matrix proteins.3
Several cytokine mediates are central to the fibrotic process, including platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1). PDGF is the most potent proliferative cytokine toward HSCs, whereas TGF-β1 primarily functions in the stimulation of extracellular matrix production.3 In the liver, expression of PDGF and its receptors is increased both in experimental fibrosis in rats and in human fibrotic liver.4–6 Both PDGF-B and PDGFR-β are rapidly induced in vivo and in culture.4,7 Moreover, the genetic overexpression of PDGF leads to liver fibrosis in mice.8 Remarkably, very few studies have assessed the impact of PDGF antagonism on hepatic fibrosis.9 Recent reports using imatinib mesylate (Gleevec) show promise,10–12 but it is uncertain whether PDGFR is its main target of action in these studies.
TGF-β1 is the most potent stimulus to hepatic fibrogenesis.13 Increased levels of TGF-β have been described in chronic liver diseases, and activated HSCs represent a major cellular source of TGF-β in injured liver. In HSCs, TGF-β promotes HSC transformation into myofibroblasts, simulates the synthesis of extracellular matrix proteins and inhibits their degradation. Antagonism of TGF-β1 signaling pathways markedly decreases fibrosis in experimental models.14,15
Because of their combined roles in fibrosis, inhibiting PDGF and TGF-β1 signal transduction is an attractive target for antifibrotic therapy. An emerging strategy is to generate small-molecule inhibitors of receptor tyrosine kinase domains. PTK787/ZK22258 (PTK/ZK) is a potent tyrosine kinase inhibitor of both vascular endothelial growth factor receptor 1 (VEGFR1, also named Flt-1) and VEGFR2 (Flk-1), and also inhibits the tyrosine kinase activity of PDGFR-β, Flt-4, c-kit and c-fms, although with less potency.16 PTK/ZK inhibits endothelial cell migration and proliferation without cytotoxic or antiproliferative effects on cells that do not express VEGF receptors.16 Oral administration of PTK/ZK at a dose of 25–100 mg/kg/day was previously shown to inhibit tumor growth in human cancer xenografts, including hepatocellular carcinoma.16–18 Very recently, we reported that PTK/ZK inhibited liver fibrosis in mice and downregulated stellate cell activation.19 In this study, we uncover the molecular mechanisms of PTK/ZK in attenuating HSC activation.
PTK/ZK (succinate salt) was provided by Novartis Pharma AG. (Basel, Switzerland). A stock solution of 50 mM PTK/ZK was prepared in DMSO, and the concentration of DMSO for all assays did not exceed 0.1%.18,19 PTK/ZK (dihydrochloride salt) was synthesized as previously described.20 Dihydrochloride salt was dissolved in distilled water.
HSCs were purified from normal rats purchased from the Laboratory Animal Unit. Nonparenchymal cell suspension was obtained by a single-step density gradient centrifugation with Nycodenz, characterized and cultured as described in detail previously.21 Experimental manipulations were performed with cells at passage 4–7. Research protocol was approved by the Institutional Ethics Committee.
HSCs were pretreated overnight with PTK/ZK at various concentrations before labeling for PDGFR-β antibody. HSCs were incubated with PDGFR-β antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at 4°C, washed with ice-cold PBS and then incubated with anti-mouse PE (BD PharMingen, San Diego, CA, USA) for 30 min. Cells were washed and then subjected to flow cytometry analysis by FACS calibur (Becton Dickinson, San Jose, CA, USA). Mouse IgG1 isotype (R&D Systems) was included as a negative control.
Proliferation of HSCs was measured by bromodeoxyuridine (BrdU) incorporation using a BrdU labeling and detection kit (Roche Diagnostics Corporation, Indianapolis, IN, USA). Cells were plated at a density of 2 × 103 cells/well into 96-well plates and were cultured overnight, followed by washing of cells with PBS twice and replacing the growth medium with a medium containing 0.1% FBS. PTK/ZK in serial dilutions was added 3 h before PDGF (10 ng/ml) (R&D Systems) and incubated with cells for 48 h. BrdU labeling solution was added to cells, followed by incubation for another 16 h before fixation, and addition of nucleases, anti-BrdU-POD and peroxidase substrate. The absorbance at 405 nm (with a reference wavelength at 490 nm) was measured using an ELISA plate reader (Molecular Devices Corp., Sunnyvale, CA, USA).
The migratory capacity of HSCs was investigated using a BIOCOAT MATRIGEL Invasion Chamber (Becton Dickinson). Confluent HSCs at the top chamber were incubated in serum-free medium for 24 h. The lower chamber was filled with PDGF (10 ng/ml) in the presence or absence of PTK/ZK at incremental concentrations. After incubation for 24 h, cells from the upper surface of membranes were completely removed with gentle swabbing. The remaining migrated cells on the lower surface of membranes were fixed and stained with hematoxylin and eosin. Cellular migration was determined by counting the number of stained cells on membranes in five randomly selected fields at high power. HSCs that migrated through the membrane were enumerated by flow cytometry as described before.22
For measurement of cell migration during wound healing, rat HSCs were seeded in 6-well plates and grown to confluence in a growth medium containing 10% FBS. Confluent HSCs were deprived of serum for 24 h, and then disrupted to generate a linear wound, followed by incubation in a medium containing PDGF (10 ng/ml) (R&D Systems) in the presence or absence of increasing doses of PTK/ZK for 20 h. HSCs were subsequently fixed and observed under phase contrast microscopy. For evaluation of wound closure under different experimental conditions, five randomly selected points along each wound were marked, and the vertical distance of migrating cells from the initial wound was measured. Experiments were carried out in duplicate, and five fields of each well were recorded.
For analysis of the expression of PDGFR-β, TGF-β type I receptor (TGFβ RI) and TGF-β type II receptor (TGFβ RII), HSCs were treated with PTK/ZK at incremental concentrations in the medium with 1% FBS for 24 h and washed twice with PBS before lysis. To evaluate the effect of TGF-β1 on the phosphorylation of Smad2, Akt, ERK and p38MAPK in HSCs, HSCs were serum deprived overnight, and then stimulated with TGF-β1 at various concentrations for 20 min. To examine the effect of PTK/ZK on PDGF-induced expression of ERK, Akt and p70S6 kinase, or TGF-β1-induced expression of Akt, ERK and p38MAPK, HSCs were serum deprived overnight, and were then treated with PTK/ZK for 3 h, followed by stimulation with PDGF 10 ng/ml or TGF-β1 1 ng/ml for 10 or 20 min, respectively, before lysis. To detect the Flt-1 expression induced by TGF-β, HSCs grown to subconfluence were rendered quiescent in serum-free medium for 24 h, followed by treatment with TGF-β1 at various concentrations for 20 h. In the experiments using PTK/ZK, cells were preincubated for 4 h in the absence or presence of 5 μM, or 10 μM PTK/ZK, before treatment with TGF-β1. After blocking, membranes were immunoblotted with the following antibodies: PDGFRβ, TGF-β1, TGFβRI, TGFβRII, Smad2 (Santa Cruz Biotechnology), ERK, phospho-ERK, Akt, phospho-Akt, p70S6 kinase, phospho-p70S6 kinase, p38 MAPK, phospho-p38 MAPK, Raf, phospho-Raf (Cell Signaling) and phospho-Smad2 (Chemicon) for 18 h at 4°C, followed by detection using an HRP-conjugated secondary antibody (Santa Cruz Biotechnology) (1 h at room temperature). Immunoreactive protein bands were visualized by the ECL system (Amersham Biosciences).
HSCs were cultured for 24 h in a serum-free medium and were then stimulated with TGF-β1 for 12 h at indicated concentrations. For the effect of PTK/ZK, cells were incubated with PTK/ZK at 5 μM, or 10 μM PTK/ZK for 4 h, before treatment with TGF-β1. Total RNA was isolated from HSCs using an RNAeasy Mini kit (QIAGEN Inc. Hilden, Germany), according to the manufacturer’s instructions. For cDNA synthesis, Taqman reverse transcription reagents were used as described in the manufacturer’s protocol (PE Applied Biosystems, Foster City, California, USA). The mRNA expression of VEGF and Flt-1 was evaluated using quantitive real-time PCR using TaqMan Gene Expression Assays and Taqman Universal PCR Master Mix kit (PE Applied Biosystems) on a PE Applied Biosystems 7700 Sequence Detector. The amplification conditions on the PE Applied Biosystems 7700 sequence detector were the following: 2 min at 50°C, 10 min at 95°C, 40 cycles of 95°C for 15 s and 60°C for 1 min for all reactions. Relative gene expression was calculated using 18S as internal control.
Continuous data were expressed as mean±standard error (s.e.) of mean. One-way ANOVA was used where appropriate. A P-value <0.05 was considered as statistically significant.
Flow cytometry analysis on gated active cells (Figure 1a) confirmed the expression of PDGFR-β by HSCs (Figure 1b), as determined by both the percentage of PDGFR-β-positive cells and PDGFR-β expression levels (mean channel fluorescence) (Figure 1c); PDGFR-β expression was confirmed using western blot (Figure 1d). PTK/ZK treatment significantly suppressed the PDGFR-β expression as measured by both flow cytometry (Figure 1b and c) and western blot (Figure 1d).
As shown in Figure 2a, PDGF induced a marked increase in HSC proliferation, which was significantly inhibited by PTK/ZK in a dose-dependent manner. Incubation of HSCs with PDGF also dramatically increased HSC migration in BIOCOAT MATRIGEL chamber systems, whereas PTK/ZK significantly blocked the migration of HSC induced by PDGF (Figure 2b, P<0.01). Figure 2c shows wounded HSC monolayers incubated in a medium containing PDGF without or with PTK/ZK. PDGF induced cell migration leading to wound closure 20 h after wounding. In the presence of PTK/ZK, PDGF-induced HSC migration in the wounded area was inhibited.
To assess the effect of PTK/ZK on Raf, ERK1/2, Akt and p70S6 kinase phosphorylation in HSCs, cultured activated HSCs were serum starved for 24 h and were then treated with 5 or 10 μM PTK/ZK for 3 h. Cells were subsequently stimulated with 10 ng/ml PDGF for 10 min. Western blot analysis showed that 10 ng/ml PDGF led to phosphorylation of Raf, ERK1/2, Akt and p70S6 kinase. PTK/ZK significantly blocked Raf, ERK1/2, Akt and p70S6 kinase phosphorylation (Figure 3).
Based on its importance as a fibrogenic cytokine, we examined the effect of TGF-β1 on the expression of VEGF mRNA in HSCs. HSCs treated with TGF-β1 expressed higher VEGF mRNA (Figure 4a). PTK/ZK antagonized the augmented VEGF mRNA expression induced by TGF-β1 (Figure 4b).
Analyzed by real-time PCR, Flt-1 mRNA expression increased significantly after TGF-β1 treatment compared with that in untreated cells (Figure 5a). This induction was significantly suppressed by PTK/ZK (Figure 5b). The results were further confirmed at the protein level; TGF-β1-induced upregulation of Flt-1 expression was suppressed by PTK/ZK (Figure 5c and d).
We observed that TGF-β1 signaled through Akt, ERK and p38MAPK pathways (Figure 7a), consistent with earlier evidence indicating that TGF-β1 activates the PI3K-Akt, ERK and p38MAPK pathways in stellate cells.23–25 In HSCs exposed to TGF-β1, PTK/ZK inhibited Akt, ERK and p38MAPK phosphorylation (Figure 7b). Although Smads are the preferred substrates and signal transducers of TGF-β receptors, PTK/ZK did not show a significant inhibition of Smad2 phosphorylation induced by TGF-β (Figure 8a and b).
An increased TGF-β1 expression in HSCs was observed after PDGF-BB (10 ng/ml) induction for 16 h. The elevated TGF-β1 expression could be inhibited by PTK/ZK added 3 h before TGF-β1 (Figure 9).
Interest in treating hepatic fibrosis has continued to accelerate. PTK/ZK, a potent receptor tyrosine kinase inhibitor, was initially developed as a potent antiangiogenic agent binding directly to the ATP-binding sites of VEGF receptors, but it also inhibits PDGFR-β with less potency.16 Our study demonstrated that PTK/ZK not only inhibits liver cancer18 but also liver fibrosis both in vivo and in vitro.19 In a separate report, PTK/ZK inhibits HSC activation by attenuating HSC proliferation, migration and collagen synthesis through the VEGF pathway.19 In this study, we further explore molecular targets of PTK/ZK in HSCs. This study addresses the novel mechanisms and molecular signaling pathways of PTK/ZK as an antifibrotic agent, which were not included in our earlier publication.19
This study has revealed that PTK/ZK inhibits PDGFR-β expression in activated HSCs, and proliferation and motility of activated HSCs induced by PDGF, as well as activation of Raf, ERK, Akt and p70S6 kinase stimulated by PDGF. In HSCs, it was very likely that the activation of Ras-Raf-ERK, induced by PDGF binding to PDGFR-β, was the signal involved in the mitogenic response to PDGF.26 ERK activation induced by PDGF was also associated with HSC proliferation and migration,27 whereas Akt activation not only stimulates HSC proliferation and migration but also increases collagen production by HSC.28 In addition, Akt signaling also mediated HSC survival and resistance to apoptosis.29 The p70S6 kinase is a downstream target of Akt, and is activated by mitogens and growth factors in a PI3K-dependent manner. In HSCs, p70S6 kinase is critical to cell proliferation, collagen expression and cell cycle control after PDGF stimulation.30 Our findings uncover a molecular link between PTK/ZK as a PDGF receptor tyrosine kinase inhibitor and ERK-, Akt- and p70S6 kinase-mediated HSC proliferation, migration, collagen expression, apoptosis and cell cycle distribution.
Our data have extended previous studies examining the role of TGF-β1 in HSC activation in three important areas. First, we report that TGF-β1 stimulates VEGF gene expression in HSCs in a dose-dependent manner, whereas previous studies indicated that TGF-β treatment induces VEGF mRNA in fibroblasts and epithelial cells, but not in endothelial cells.31 Second, we demonstrate that TGF-β1 also induces both VEGFR1 (Flt-1) gene and protein expression in HSCs. TGF-β1 induction of Flt-1 was reported previously in bovine retinal endothelial cells.32 Third, PTK/ZK inhibited both VEGF and VEGFR1 expression induced by TGF-β1. Interestingly, PTK/ZK also suppressed the expression of both TGFβRI and TGFβRII. The signaling of the TGF-β family is mediated through TGFβRI and TGFβRII to phosphorylate receptor-activated Smad, which is the best characterized downstream target of the TGF-β pathway.33 In addition to the Smad-mediated canonical TGF-β signaling pathway, evidence over the past few years suggested that TGF-β may signal through non-Smad pathways to mediate cellular effects. For example, TGF-β can activate ERK,34 PI3K/Akt and p38MAPK.23–25,35,36 It is reported that p38MAPK could be activated by TGFβR to mediate Smad-independent TGF-β responses.35 Furthermore, PI3K/Akt activity could be potently induced by the activation of TGFβRI.36 Indeed, we observed that TGF-β1 stimulated the phosphorylation of Akt, ERK and p38MAPK, which could be inhibited by PTK/ZK. It is reported that the phosphorylation of p38MAPK is augmented in activated HSC, which is associated with increased collagen production.25 In addition, p38MAPK is also involved in TGF-β-stimulated synthesis of VEGF in aortic smooth muscle cells.37 Furthermore, both Akt and p38MAPK are involved in TGF-β1-downregulated matrix metalloproteinase-13 (MMP-13) expression, as well as in upregulated type I collagen expression.23 MMP-13 plays an important role in the resolution of liver fibrogenesis augmented by macrophages through increased matrix degradation.6 Therefore, PTK/ZK inhibits HSC activation by complex mechanisms involving Akt, ERK, p70S6kinase, as well as p38MAPK. Unlike PDGF, we could not detect Raf activation upon TGF-β1 stimulation. Therefore, PTK/ZK might exert an inhibitory effect on ERK induced by TGF-β1, rather than through the Ras–Raf cascade. Although Smad signaling is important in fibrogenesis,24,38 PTK/ZK failed to significantly inhibit Smad2 signaling induced by TGF-β. As the antibody we used interacts not only with Smad2 but also with Smad3 upon addition of TGF-β, it is unlikely that PTK/ZK inhibited both Smad2 and Smad3 activation by TGF-β. Consistent with our findings, Wang et al39 also showed that renal fibrosis was ameliorated through a non-Smad TGF-β pathway by imatinib mesylate. Several reasons may account for the failure of PTK/ZK to inhibit Smad2/3 phosphorylation. First, Smad2/3 is a receptor substrate not only for TGF-β/TGFβR but also for Activin/Activin receptor (ActR).40,41 Activin and its receptors are expressed in both hepatic and pancreatic stellate cells, and activin serves as an autocrine activator for stellate cell activation.42,43 Although PTK/ZK inhibited TGFβR, Smad2/3 could still be activated through Activin/ActR. Second, Smad phosphorylation is directly mediated through TGF-βRI; however, other kinase pathways also regulate Smad signaling. For example, phosphorylation of Smad2 can also result from a stimulation of EGF or hepatocyte growth factor (HGF), which acts through its own cognate receptor tyrosine kinase receptors.44,45 Indeed, HGF is expressed in cultured HSCs,46 and therefore Smad2 could still be activated through HGF, even if PTK/ZK blocked TGFβ/TGFβR signaling. Third, phosphorylation of Smad2/3 could also be activated directly by IGF-binding protein-3 (IGFBP-3),47 although IGFBP-3 reportedly acts through TGFβRI in smooth muscle cells.47 IGFBP-3 is expressed by HSCs, and an increased expression of IGFBP-3 is observed during transformation to myofibroblast-like cells in culture.48 PTK/ZK at 5 or 10 μM only partially inhibited the expression of TGFβRI; therefore, phospho-Smad2 could still be induced by an incomplete inhibition of the pathway by PTK/ZK and by the possible presence of alternative Smad2 phosphorylation events that are not sensitive to PTK/ZK.
In support of our findings, Bisping et al49 also reported that BIBF 1000, a novel receptor tyrosine kinase inhibitor targeting VEGFR1 through VEGFR3, FGFR1, FGFR3, as well as PDGFRα, also interferes with TGF-β-mediated effects in bone marrow stromal cells. However, the mechanism by which the receptor tyrosine kinase inhibitor interfered with signaling pathways triggered by TGF-β was not elucidated. In an earlier study, PDGF reportedly upregulates TGF-β1 expression in human mesangial cells and modulates mesangial cell proliferation and mesangial matrix production.50 As the mesangial cell is a cell type in kidney related to HSC, we investigated whether PDGF stimulates TGF-β1 expression in activated HSCs. Indeed, we found that PDGF stimulated TGF-β1 expression, and the induced TGF-β1 expression was suppressed by PTK/ZK. The mechanism by which PTK/ZK inhibited TGF-β1-mediated effects could also be through a blockade of the PDGF/PDGFR system by PTK/ZK. Therefore, PTK/ZK suppresses HSC proliferation and survival not only through a direct inhibition of VEGFR,19 PDGFR and TGFβR-mediated Akt activation but also through an indirect inhibition of the TGF-β1-mediated Akt pathway. In HSCs, inhibition of proliferation and collagen production by PTK/ZK was related to the inhibition of VEGF, PDGF and TGF-β1 signaling and their downstream target, Akt.
In conclusion, this study provides a critical molecular analysis of a comprehensive analysis that uncovers the attenuating development of liver fibrosis through the inhibition of PDGF and TGF-β1, two most potent stimuli of liver fibrosis, by a receptor tyrosine kinase inhibitor PTK/ZK, which is completely distinct from our published work.19 Furthermore, it is unlikely that PTK/ZK has a toxic effect on hepatocytes as immortalized human hepatocytes were not affected by PTK/ZK in our previous report.18 Importantly, PTK/ZK is capable of inhibiting liver fibrosis and blocking VEGF,19 as well as PDGF and TGF-β1-regulated HSC activation. Thus, PTK/ZK may involve the blockage of multiple essential signal pathways in connection with HSC activation that provoke proliferation and fibrogenesis. Our study underscores the therapeutic potential of PTK/ZK as an antifibrotic drug for patients with chronic liver disease. We believe that this study provides even more compelling molecular mechanisms that are distinct from our earlier work.19 It establishes a novel approach and reveals novel insights into understanding and treating hepatic fibrosis.
The authors thank Professor Mien-Chie Hung (Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.), Dr Jeremy Hughes (MRC Center for Inflammation Research, University of Edinburgh, UK) and Dr Nai-Sum Wong (Department of Biochemistry, The University of Hong Kong) for their valuable advice and comments, as well as Xueming Qian for technical assistance. We acknowledge financial support from the Hong Kong Research Grants Council (Projects: PolyU 5407/06M; PolyU 5638/07M); Shenzhen Bureau of Science, Technology and Information (Shenzhen Key Laboratory Advancement Scheme); Small Project Funding Programme of the University of Hong Kong; and NIH Grant DK56621.
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conflict of interest. The authors do not have any financial interest related to the development of PTK/ZK.