Scholarly article on topic 'TGF-� Type I Receptor Kinase Inhibitor EW-7197 Suppresses Cholestatic Liver Fibrosis by Inhibiting HIF1a-Induced Epithelial Mesenchymal Transition'

TGF-� Type I Receptor Kinase Inhibitor EW-7197 Suppresses Cholestatic Liver Fibrosis by Inhibiting HIF1a-Induced Epithelial Mesenchymal Transition Academic research paper on "Biological sciences"

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Academic research paper on topic "TGF-� Type I Receptor Kinase Inhibitor EW-7197 Suppresses Cholestatic Liver Fibrosis by Inhibiting HIF1a-Induced Epithelial Mesenchymal Transition"

Cellular Physiology Ce" Physiol Biochem 2016;38:571-588

. DOI: 10.1.159/000038(551

and Biochemistry Publishedonline:Febbjary05, 2016

Accepted: December 21, 2015

© 2016 The Aubhor(s) Published by S. Karger AG, Basel 1321-9778/16/0382-0571$39.50/0 www.karger.com/cpb

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Original Paper

TGF-p Type I Receptor Kinase Inhibitor EW-7197 Suppresses Cholestatic Liver Fibrosis by Inhibiting HIFla-Induced Epithelial Mesenchymal Transition

Min-Jin Kima Sang-A Parka Chun Hwa Kimb So-Yeon Parka Jung-Shin Kima Dae-Kee Kima Jeong-Seok Namc Yhun Yhong Sheena

aCollege of Pharmacy, Ewha Womans University, Seoul, bNew Drug Discovery Lab, Hyundai Pharm, Yongin, cLaboratory of Tumor Suppressor, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea

Key Words

Cholestatic liver injury • Hepatic stellate cell • TGF-p • HIFla • Epithelial mesenchymal transition • EW-7197

Abstract

Background/Aims: Hypoxia is an environmental factor that aggravates liver fibrosis. HIFla activates hepatic stellate cells (HSCs) and increases transforming growth factor-p (TGF-P) signaling and the epithelial mesenchymal transition (EMT), accelerating the progression of fibrosis. We evaluated the anti-fibrotic therapeutic potential of a small-molecule inhibitor of TGF-p type I receptor kinase, EW-7197, on HIFla-derived TGF-p signaling in cholestatic liver fibrosis. Methods: We used a bile duct ligation (BDL)-operated rat model to characterize the role of HIFla-derived TGF-p signaling in liver fibrosis. Cellular assays were performed in LX-2 cells (human immortalized HSCs). The anti-fibrotic effects of EW-7197 in livertissues and HSCs were investigated via biochemical assays, immunohistochemistry (IHC), immunofluorescence (IF), chromatin immunoprecipitation (ChIP) assays, real-time PCR, and western blotting. Results: In our BDL rat model, orally administered EW-7197 inhibited fibrosis and attenuated HIFla-induced activation of HSCs and EMT in vivo. In addition, EW-7197 inhibited HIFla-derived HSC activation and expression of EMT markers in LX-2 cells in vitro. Conclusion: This study suggests that EW-7197 exhibits potential as a treatment for liver fibrosis because it inhibits HIFla-induced TGF-p signaling.

© 2016 The Author(s) Published by S. Karger AG, Basel

Introduction

Hepatic fibrosis is caused by various types of chronic liver injury. Fibrosis is characterized by enhanced accumulation of extracellular matrix (ECM] proteins, such as collagen (Colla] and fibronectin [1]. In response to injury, hepatic stellate cells (HSCs] are activated into myofibroblasts and become a major source of ECM proteins [2-4]. In addition, the epithelial

Yhun Y. Sheen, Ph.D. College of Pharmacy, Ewha Womans University, Seodaemun-gu, Seoul, 120-750, (Korea)

Tel. +82-2-3277-3028, Fax +82-2-3277-2851, E-Mail yysheen@ewha.ac.kr

KAHGEH i J

DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

mesenchymal transition (EMT] has been identified as an important mechanism in fibrotic progression [5]. Although recent studies have demonstrated that numerous mediators are essential for the initiation and progression of fibrosis, the association among these mediators remains unclear. Various studies have demonstrated that chemical and surgical liver injuries cause hypoxia in the liver [6-9]. Disruption of hepatic blood flow causes hypoxia, leading to deposition of ECM proteins [9, 10]. Hypoxia aggravates inflammation through generation of reactive oxygen species (ROS] [11], which might bring about the EMT [12]. Hypoxia may activate various transcription factors, such as hypoxia-inducible factor la (HIFla] and vascular endothelial growth factor (VEGF] in order to regulate gene expression and cellular signaling [13]. HIFla modulates the EMT in several cell lines; HIFla-induced EMT is involved in cancer progression, metastasis, and organ fibrosis [14-17]. In addition, transforming growth factor-p (TGF-P] signal transduction might play a pivotal role in HSC activation by HIFla [18,19]. TGF-p is a fibrogenic mediator involved in activation and transdifferentiation of HSCs [20,21]. TGF-p acts via canonical and non-canonical signaling pathways. Canonically, TGF-p binding to the TGF-p type II receptor (TpRII], recruits the TGF-p type I receptor (TpRI], and activates a TpRI kinase such as a activin-like kinase 5 (ALK5], ALK5 phosphorylates receptor-associated Smad2/3, after which p-Smad2/3 interacts with Smad4 to transactivate target genes [22]. Smad-dependent canonical TGF-p signaling plays a critical role in HSC activation and EMT progression during liver fibrosis [23-26]. Therefore, canonical TGF-p signal plays a central role in the molecular pathogenesis of liver fibrosis and might serve as a potential target for treatments for patients with hepatic fibrosis [27]. Recently, synthetic inhibitors of TGF-p have been reported to suppress hepatic fibrosis in experimental animal models [28,29].

We previously reported that a novel small-molecule inhibitor of ALK5, EW-7197, inhibited fibrosis in the liver, kidneys, and lungs by inhibiting TGF-p/Smad and ROS signaling [29]. In this study, we investigated the anti-fibrotic effect of EW-7197 (20 and 40 mg/kg] in bile duct-ligated (BDL] rats and showed that the higher dose of EW-7197 inhibited fibrosis by inhibiting HIFla-induced EMT.

Materials and Methods

Reagents

EW-7197, LY2157299, and IN-2001 were provided by Dr. D.K. Kim (Ewha Womans University, Seoul, Republic of Korea], Recombinant human TGF-pi was purchased from R&D Systems (Minneapolis, MN, USA], and CoCl2 was purchased from Sigma-Aldrich (St. Louis, MO, USA], Glucose oxidase (GOX] was obtained from Dr. H.A. Woo (EwhaWomans University],

Cell lines and culture

LX-2 immortalized human HSCs were maintained in Dulbecco's modified Eagle medium (DMEM] containing 10% fetal bovine serum. All of the media were purchased from Invitrogen (Carlsbad, CA, USA], The cells were maintained at 37°C in a humidified incubator in the presence of 5% C02.

Experimental designfor cholestatic Hverfibrosis models

Six-week-old male Sprague-Dawley (SD] rats were purchased from Orient Bio, Inc. (Seoul, Republic of Korea], SD rats weighing 180 to 200 g were randomly divided into five experimental groups: sham-operated rats (sham rats] as controls (n = 5]; sham rats treated with 40 mg/kg EW-7197 (n = 5]; BDL-operated rats (BDL rats] (n = 10]; BDL rats treated with 20 mg/kg EW-7197 (n = 10]; and BDL rats treated with 40 mg/kg EW-7197 (n = 10]. For BDL, the animals were anesthetized with Zoletil (20 mg/kg] andxylazine (10 mg/kg], after which the common bile duct was exposed and double-ligated using 4-0 silk sutures. The first ligature was placed below the junction of the hepatic ducts, whereas the second ligature was placed above the entrance of the pancreatic ducts. For the sham-operated rats, an incision was made in the abdomen and then closed without any further surgical procedure (Fig. 1A], The animals were maintained in a temperature-controlled room (21°C] and were supplied with autoclaved food and water. At 48 h after the last dosing, the

KARGER

DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

animals were sacrificed, after which serum, spleens, and the livers were removed. The livers were sliced into several parts, snap-frozen in liquid nitrogen, and maintained at-70°C. A portion of each liver was immersed in 10% neutral-buffered formalin for histopathological and immunohistochemical examinations. Body weight was measured weekly. In all animals used, body weight, wet liver and spleen weight, and the ratio of

Livft^i

Bile duct

Bile duct ligation

6-week-old S.D. rat

| T EW-7197 20 mgdkg or 40 mg?kg (P.O.) | Sacrificed

▼ ▼ ▼ ▼ ▼ t I

t I t I I > I t I < I I t I * I

4 (Weeks)

(B) Sham

Sham + EW(40)

(C) Sham

Sham ♦ EW(40)

0.0 BDC

+■ + + 40 - 20 40

Fig. 1. EW-7197 suppresses BDL-induced liver fibrosis. (A] Scheme of drug treatment. Six-week-old male SD rats were randomly divided into five experimental groups. Twenty-four hours after the BDL or sham operation, EW-7197 (EW: 20 or 40 mg/kg, qod) dissolved in vehicle (Veh] was administered to rats orally. EW was administered thrice weekly for 4 weeks. (B] Representative staining (top] and densitometric analysis (bottom] of Masson's trichrome staining of liver tissue from BDL rats. (C] IHC of Colla (top] and densitometric analysis (bottom] ofColla staining in liver tissue from BDL rats. (D] Hydroxyproline content in liver tissue from BDL rats was determined by hydroxyproline assay. (E] ALT and AST serum levels. AST and ALT serum levels of BDL rats were determined by enzyme-linked immunosorbent assay (ELISA], EW-7197 (EW: 20, 40 mg/kg, qod) dissolved in vehicle (Veh] was administered to rats orally thrice weekly for 4 weeks following BDL. (F] Effects of EW-7197 on body weight changes and relative liver and spleen weight. *p < 0.05 vs. sham, **p < 0.01 vs. sham,'"p < 0.001 vs. sham, #p < 0.05 vs. BDL,##p < 0.01 vs. BDL,###p < 0.001 vs. BDL.

KARGER

DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

liver and spleen weight to body weight were determined. Changes in body weight were calculated as follows.

„ J i . i ,„,-, Wsifffcr atAth woolT-H'srffPit at Isr u-selr

Body weight changes f%] =- X 100

Weight at *thv,-twk

(the weight ofanimals subjected to the sham operation was considered as 100%]

The experimental protocol was approved by The Institutional Animal Care and Use Committee (IACUC] of Ewha Womans University and complied with the NIH Guide for the Care and Use of Laboratory Animals (Institute ofLaboratoryAnimal Resources, National Research Council, WA, USA],

HSC-enriched cell preparation by collagen perfusion methods

Six-week-old male ICR mice were purchased from Orient Bio, Inc. (Seoul, Republic of Korea], HSC-enriched cells and primary hepatocytes were isolated from the livers of 6-to 7-week-old male mice weighing 20 - 25 g. Mouse primary hepatocytes and HSC-enriched cells were isolated by differential centrifugation at 1500 xg to collect HSC-enriched cells after gathering of hepatocytes via centrifugation at 500 x g for 5 min. Each liver was perfused with PBS using a non-circulating system for 10 min at 37°C (2.2 mL/min], followed by perfusion with a perfusion solution consisting of 0.03% (w/v] collagenase (Sigma-Aldrich] in PBS (pH 7.4] with 0.015 % (w/v] CaCl2^2H20 for 15 to 20 min. Perfusion solution was then circulated through each liver for 15 min to wash the tissue, after which each liver was cut into pieces in ice-cold MEM medium supplemented with 10% FBS and insulin (4 |ig/mL], The resulting suspension was filtered through nylon mesh and centrifuged at 400 x g for 5 min. The collected cells were plated onto collagen-coated plates at a density of 5 x 105 cells/mL of culture medium. Serum starvation was performed for 24 h, followed by stimulation with TGF-pi (2 ng/mL] and treatment with 0.5 |iM EW-7197 and 0.5 |iM pirfenidone for 24 h.

Proliferation assay

Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8] assay kit (Dojindo Laboratories, Kumamoto, Japan], Cells were seeded on 96-well plates at a density of 5 x 103 cells per well, after which they were treated with 10 nM to 10 |iM EW-7197 for 72 h. Next, CCK-8 solution (10 |iL] was added to each well, after which the wells were incubated for 1 h at 37°C. Absorbance was measured at 450 nm with an ELISA microplate reader (Versa Max; Molecular Devices, Sunnyvale, CA, USA],

Luciferase Reporter GeneAssay

HaCaT (3TP-Lux stable] cells were seeded on 96-well plates and treated with the indicated concentrations ofactivin receptor-like kinase 5 (ALK5] inhibitors in the presence or absence ofTGF-p (2 ng/ mL] for 24 h. Luminescence was measured in cell lysates with a luminometer (Micro Lumat Plus; Berthold Technologies Berthold Technologies, BadWildbad, Germany],

Determination of intracellular ROS generation

Intracellular ROS production was measured using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA] as described below. H2DCF-DA is a lipid-permeable, nonfluorescent compound that is oxidized by intracellular ROS to form the lipid-impermeable and fluorescent compound dichlorodihydrofluorescein (DCF], Cells were plated at density of 5 x 103 cells per well in 96-well plates (SPL, Pocheon, Korea], H2DCF-DA was added directly to the culture medium at a final concentration of 10 |iM, after which the cells were cultured under standard conditions. ROS arbitrary units were measured using fluorescence microplate reader (BIO-TEK, Winooski, USA] with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Upon the same cells, protein concentrations were determined using fluorescamine. The results of these experiments were expressed as arbitrary absorbance units/mg protein.

Histopathological examination

Tissues (approximately 9x9 mm sections from the left and caudate lobes ofthe liver] were fixed and stained using hematoxylinand eosin. Masson'strichrome staining for collagen was usedto quantify the fibrotic area. For immunohistochemical analysis, deparaffinized sections were incubated with primary/secondary antibodies. The sections were counterstained with diluted hematoxylin for immunohistochemistry or with 4', 6-diamidino-2-phenylindole (DAPI] for immunofluorescentstaining.

KARGER

DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

Table 1. Primer sequences

Immunofluorescence Cells and animal tissue samples fixed with 4% paraformaldehyde solution (pH 7.4] were blocked in 5% bovine serum albumin with normal serum in 0.1% Triton X-100 and subsequently incubated with primary/ secondary antibodies. The nuclei of the cells were stained with DAPI solution (blue]. Images were obtained using an LSM 510 META laser confocal microscopy system (Carl Zeiss, Oberkochen, Germany] at 400 x magnification.

Biochemical assays The hydroxyproline content of the tissue samples was quantified colorimetrically using the chloramine T method as described by Bergheim et al. [30]. Serum alanine aminotransferase (ALT] and aspartate aminotransferase (AST] activity levels were determined using a spectropho-tometric enzyme assay kit (Asan Pharm. Co., Hwaseong, Republic of Korea] according to the manufacturer's instructions.

RNA isolation and rever-se-transcriptase polymerase chain reaction (RT-PCR) analyses

Total RNA from tissue samples (sections of approximately 5x5 mm from the right lobe of the liver] and cells were isolated using TRIzol reagent (Invitrogen], cDNA was synthesized from 2 |ig of total RNA by M-MLV reverse transcriptase (Invitrogen] and random primers (Invitrogen], after which it was subjected to PCR amplification using Taq polymerase (Promega], For real-time quantitative RT-PCR, Power SYBR Green PCR Master Mix and a Step-One Real-time PCR system (Applied Biosystems] were used to amplify the isolated cDNA. The primers used for real-time quantitative RT-PCR are listed in Table 1.

Western blot analysis

Tissue samples (sections of approximately 5x5 mm from the right lobe of the liver] and cells were lysed and separated by 6 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE], after which their constituent proteins were transferred to polyvinylidene difluoride (PVDF] membranes. The antibodies used for western blotting are listed in Table 2. The primary antibodies were incubated with each PVDF membrane followed by incubation with appropriate peroxidase-conjugated second antibodies and detection using an enhanced chemiluminescence (ECL] kit. Band intensities were analyzed using a LAS-3000 densitometer (Fujifilm, Tokyo, Japan],

Speeles Sequence (S'-»3')

Rat Sense 5 GTG ATC ACC AT G G GG A ATG A - 3 '

asma Antlsense 5'-CAGCAATGCGTGGGTACATG-3'

Col-tal Rat Sense Antlsense 5*-GTG ATC ACC ATG G GG A ATG A - 3 ' 5'-CAG GAATGCCTGGGTACATG-3'

CTGF Rat Sense 5 A AG A C CTCTG G G ATG G CC- 3 '

Antlsense 5 '-TG GTG C AG C C AG A AA GGTC- 3 '

E-cadherln Rat Sense Antlsense 5' - A AG G A GG G C ATTCTG A A A AG A- 3 ' 5'-C A CTG TC ACG T G G A G A ATGT ACTG - 3'

Fn Rat Sense 5 G CTTC A AG CT G GG T GTAC G A - 3 '

Antlsense 5 ' - A AG TTG G TTGG GGG AG ACAG-3'

p-actin Rat Sense Antlsense 5'-AGCCATGTACGTAGCCATCC-3' 5*-TCT G A GCTGTG GTG GTG A AG -3 '

Pai-1 Rat Sense Antlsense 5'-CCTC ATGCTG G GCCTG GTTCTG G TCT-3 ' S'-G GTTTTCCCCCCTGTCGTCATCTGC-3'

Tgf-pi Rat Sense Antisense S'-AATATAGCAACAATTCCTGGCGTTA -3' S'-CTGCCGGACAACTCCAGTGA -3'

Snail Rat Sense Antlsense 5'-A C CA CT AT G GGG CG CT GTT -3' ü'-G GTGGTAGGGCTGCTGGAA -3'

a-SMA Human Sense Antlsense 5'- C ATC AT GCGT CTG G ATCTGG- 3 ' 5'- G G A C A ATCTC ACGCTC AGC A- 3 '

COL- 1A1 Human Sense Antlsense 5' -TCT GG AG AG G CTG GT ACTG C - 3 ' 5'-GAGCACCAAGAAGACCCTGA-3'

E-CADHERiN Humati Sense Antlsense S'-TCC ATTTCTTG CTCTACGCC-3' 5'- C A CCTTC A GCC ATC CTGTTT-3 '

N-CADHERIN Human Sense Antlsense 5' - GTG C C ATT AG C C A A GGG AATTC AG C - 3 ' 5 G C GTTC C T GTTGG AGTC AT A GG AG G - 3 '

VIMENTIN Human Sense Antisense S'-ACCCG CACCAACGAGAAGGT-31 S'-ATTCTGCTGCTCCAGGAAGCG-j'

PAI-1 Human Sense Antlsense 5'-CCGGAACGACCTG AAGAAGTG- 3' 5'-GTGTTTCAGCAGGTGGCGC-3'

PPIA Human Sense Antlsense 5'-TGCCATCGCCAAGGAGTAG-3" 5*-TGCACAGACGGTCACTGAAA-3'

TGF-pl Human Sense Antisense Sense S'-AAGGACCTCGGCTGGAAGTG-3' S'-CCGGGTTATGCTGGTTGTA-3' ü'-ATCGGAAGGCTAACTACAGC -3'

SNAIL Human Antisense S'-CAGAGTCGCAGATGAGGATT -3'

KARGER

S-Ï s

DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

Chromatin immunoprecip ita tio n (ChIP) assays

LX-2 cells were treated with EW-7197 or LY2157299 (or IN-2001] in the absence or presence TGF-p (or CoCl2] for 3 h. Cells were harvested and subjected to ChIP assays with anti-Smad2/3 (or anti-HIFla] at the promoter region upstream of the human PAI-1 gene. The promoter sequence ofPAI-1 is provided in Table 3.

Statistical analysis The results are expressed as the mean ± standard error ofthe mean (SEM] or as the mean ± standard deviation (SD]. Statistical comparisons were conducted using Student1 s t-test or one-way analysis of variance (ANOVA], followed by Dunnett's two-tailed post-hoc test (SPSS version 10.0; IBM Corporation, Armonk, NY, USA], Results with p-values less than 0.05 were considered statistically significant.

Table 2. Antibody list for Western blot, IHC, IF, and ChIP assay

Antibody Company Cat. #

Anti-a-SMA Sigma A2547

Anti-ß-actin Sigma A5316

Anti-CAIX Milipore ABC272

Anti-CTGF Santa Cruz Biotechnology sc-149 39

Anti-COL-1 Santa Cruz Biotechnology sc-59772

Antj-E-cadherin BD biosciences 610182

Anti-Fibronectin BD biosciences 610077

Anti-GAPDH Cell signaling 5174

Anti-GLUTl Abeam ab652

Anti-HIFla Abeam abl13642

Anti-Snail Cell Signaling C15D3

Anti-p-Smad2/3 Santa Cruz Biotechnology sc-11769

Anti-p-Smad3 Cell signaling 9520

Anti-Smad2/3 BD biosciences 610842

Anti-Smad3 Ab frontier AF9F7

Anti-N-Cadherin BD biosciences 610921

Antî-Vimentin Abeam 550513

Results

Table 3. PAI-1 promoter sequence

ChlP assay Species

Sequence

PAI-1 promoter Human

Sense Antisense

5 C C TCC A AC CTC AG C C A G AC A AG - 3 ' S'-CCCAUCCCAACAUCCACA-Ï

EW- 7197 suppresses induced liverfibrosis Previously we reported that ALK5 inhibitor EW-7197 strongly reduced fibrosis in various animal models and organs, including the liver, lung, and kidney, at a low dose (0.625 - 5.0 mg/kg] [29]. In this study, we assessed the anti-fibrotic effects of treatment with 20 and 40 mg/kg EW-7197 every other day in BDL rats, with the aim of examining the effects of higher doses of EW-7197 in vivo in order to ensure that such doses have efficacy similar to that of low doses of the drug. In other words, our goal was to confirm that EW-7197 showed a conventional dose-response effect rather than hormesis. Protein kinase and cellular reporter gene assays indicated that the high doses (20 - 40mg/ kg] of EW-7197 used in this study specifically inhibited ALK5 [31]. Moreover, a previous study found that a daily dose of 5 to 120 mg/kg EW-7197 for 4 weeks in normal SD rats produced no toxic effects [29]. We investigated the inhibitory effect of EW-7197 on BDL-induced ECM accumulation. Because liver fibrosis is characterized by excessive deposition of ECM proteins, we assessed the collagen content of the livers of BDL rats using various methods. First, we conducted histopathological analysis with Masson's trichrome staining, which stains collagen fibers blue. Staining of collagen fibers around the portal triad in the livers of BDL rats was increased 29-fold in comparison with that of sham rats. Treatment of BDL rats with 20 and 40 mg/kg EW-7197 significantly inhibited collagen accumulation in the liver by 20.43% and 8.25%, respectively, in comparison with that of untreated BDL rats (Fig. IB], Additionally, immunohistochemistry (IHC] was performed to examine Colla expression in the livers of BDL rats. Colla expression in BDL rat livers was increased 27-fold in comparison with that of sham rats. Treatment with 20 or 40 mg/kg EW-7197 decreased Colla expression in the livers of BDL rats (Fig. 1C], Furthermore, the total amountof collagen in the liver was determined by measuring the amount ofhydroxyproline, a major component of collagen protein. The hydroxyproline content of the livers of BDL rats was increased 2.74-

KARGER

DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

pSmad3 Smad3

+ + +■ 40 • 20 40

BDL EW E-Cadherin i N-Cadherin Vi men tin Fibroricctin Colla p-actin •

«tl.O

cp V ¿0.0 BDL EW

ft2"5 | I1'5

S §85

K to.o

E-cadherin 11 4

ft I Inn

5a 3 -O c ® '£ 2

3 V K £ 0 BDL EW

*** N-Cadherin

Br] [I #tm

Fibronectin

Si ll1

" o BDL EW

40 • 20 40 ■*» Colla

Qm i±u#

+ + + 40 ■ 20 40

. E-cadherin _6

Mi i I

KD.; E

BDL - - + + + EW - 40 - 20 40

z 2 QC E

BDL • • + + + EW - 40 - 20 40

Col-1a1

jijjjj ffffffffff

BDL .»+.* + EW . 40 - 20 40

is 0Ü

BDL • • + + + EW - 40 - 20 40

20. 40 Snail

titiii r. ■■ ■■

" " " IfffiF

+ + + 40 - 20 40

0J BDL EW

+ + 20 40

Fig. 2. EW-7197 attenuated the EMT in BDL rats. (A] Effects of EW-7197 on suppression of Smad3 phosphorylation in BDL rat livers. *p < 0.05 vs. sham, ##p < 0.01 vs. BDL. (B] Effects of EW-7197 on E-cadherin, N-cad-herin, vimentin, fibronectin, and Colla protein expression in BDL rat livers. p-Actin was used as a reference. *p < 0.05 vs. sham, "p < 0.01 vs. sham,**p < 0.001 vs. sham, #p < 0.05 vs. BDL,##p < 0.01 vs. BDL,###p < 0.001 vs. BDL. (C] Effects of EW-7197 on E-cadherin (E-cadherin], fibronectin {Fn), collagen type 1 {Col-1a1), and CTGF (Ctgf) mRNA levels in liver tissue from BDL rats. fi-Actin was used as a reference. *p <0.05 vs. sham, "p < 0.01 vs. sham,***p < 0.001 vs. sham, #p < 0.05 vs. BDL,##p < 0.01 vs. BDL,###p < 0.001 vs. BDL. (D] Effects of EW-7197 on Snail protein expression in BDL rat livers. **p < 0.01 vs. sham and ###p < 0.001 vs. BDL. (E] Snai1 mRNA levels in BDL rats were downregulated by EW-7197. fi-Actin was used as a reference. ***p < 0.001 vs. sham, ##p < 0.01 vs. BDL and###p < 0.001 vs. BDL.

fold in comparison with that of sham rats. Treatment of BDL rats with 20 and 40 mg/kg EW-7197 reduced hydroxyproline content by 55.94% and 25.41%, respectively, in comparison

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DOI: 10.1159/000438651

Cellular Physiology

and Biochemistry Published online: February 05, 2016

Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

with that of untreated BDL rats (Fig. ID], These results suggest that EW-7197 inhibited BDL-induced accumulation of collagen in vivo. Therefore, we examined the effects of EW-7197 on liver function by monitoring serum ALT and AST levels. The serum ALT and AST levels of BDL rats significantly increased in comparison with those of sham rats. Treatment of BDL rats with 20 mg/kg EW-7197 markedly decreased serum ALT and AST levels by 61.47% and 67.18%, respectively, in comparison with those of untreated BDL rats (Fig. IE], Treatment of BDL rats with 40 mg/kg EW-7197 markedly decreased serum ALT and AST levels by 44.14% and 35.75%, respectively, in comparison with those of untreated BDL rats (Fig. IF], The BDL rats exhibited a 10.95% loss of body weight in comparison with that of the sham rats. Additionally, the relative weights of the livers and spleens of the BDL rats were increased 2.26-fold and 3-fold, respectively, in comparison with those of the sham rats. Although EW-7197 did not affect the body weight or organ weight of healthy rats, EW-7197 attenuated BDL-induced changes in body weight and the relative weights of the liver and spleen (Fig. 1G], These data show that EW-7197 suppressed liver fibrosis and improved liver function in BDL rats without toxicity.

EW-7197 attenuated the EMT in BDL rats

To investigate the effects of EW-7197 on TGF-p signaling related to liver fibrosis, we assessed Smad3 phosphorylation in the livers of BDL rats. Smad3 phosphorylation was increased 2.15-fold in the livers of BDL rats in comparison with that of sham rats. EW-7197 inhibited Smad3 phosphorylation in the livers of BDL rats (Fig. 2A], The EMT plays a central role in the development of fibrosis [23, 25, 32]. To examine the effects of EW-7197 on the EMT in BDL rats, we measured the expression of EMT markers. As shown in Fig. 2B, EW-7197 restored expression of the epithelial marker E-cadherin, which was not evident in the livers of BDL rats. EW-7197 reduced the elevated protein levels of mesenchymal markers such as N-cadherin, vimentin, fibronectin, and Colla in the livers of BDL rats (Fig. 2B], Furthermore, EW-7197 attenuated the decrease in E-cadherin mRNA level and increase in fibronectin, Colla, and CTGF mRNA levels observed in the livers of BDL rats (Fig. 2C], These results show that EW-7197 inhibits the EMT in the livers of BDL rats. Snail is a master regulator of the EMT that plays an essential role in tumor metastasis [33, 34], but its role in liver fibrosis is unclear. Snail expression was elevated in the livers of BDL rats at the protein (Fig. 2D] and mRNA levels (Fig. 2E]; however, BDL rats treated with EW-7197 showed decreased protein and mRNA levels of Snail in comparison with those of untreated BDL rats. These results suggest that EW-7197 inhibits Snail-mediated EMT during BDL-induced fibrogenesis.

EW-7197 inhibits HSC activation

The pathogenesis of fibrosis involves various cell types in the liver, includinghepatocytes, Kupffer cells, sinusoidal endothelial cells, and HSCs. Although numerous cytokines and signaling pathways are involved in the progression of liver fibrosis, hypoxia and TGF-p drive injury-induced fibrosis by mediating injury-induced inflammation and contributing to hepatocyte apoptosis [35] and activation of quiescent HSCs [1, 32]. HSC activation and transformation into myofibroblasts play a key role in the progression ofliver fibrosis, which is accompanied by elevated expression of a-SMA [1, 36, 37]. As shown in Fig. 3A, IHC staining showed dramatic accumulation of a-SMA-positive cells around the portal triad of the livers of BDL rats; however, EW-7197 treatment decreased a-SMA-positive cell accumulation. In accordance with the IHC results, EW-7197 also decreased the elevated protein and mRNA levels of a-SMA in the livers of BDL rats (Fig. 3B and 3C], These results indicate that TGF-p is a strong driver of HSC activation in BDL-induced liver fibrosis and demonstrate that EW-7197 efficiently inhibits such activation in vivo. Because TGF-p induces the EMT in certain pathogenic situations, we tested whether HSCs and hepatocytes were involved in TGF-p-induced EMT. Therefore, we conducted in vitro experiments using mouse HSC-enriched cells and primary mouse hepatocytes. EW-7197 did not show cytotoxic effects in HSC-enriched cells, hepatocytes, or LX-2 cells (Fig. 3D]. Mouse HSC-enriched cells showed dramatic changes in expression levels of EMT markers E-cadherin, N-cadherin, and vimentin (Fig.

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

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Fig. 3. EW-7197 inhibits HSC activation and EMT. (A] IHC (left] and densitometry analysis (right] of a-SMA staining of liver tissue from BDL rats. (B] Representative western blot images of a-SMA protein in liver tissue from BDL rats (top]. Quantitative analysis of the relative density of a-SMA protein after normalization to p-actin (bottom], (C] Effects of EW-7197 on a-SMA (a-Sma) mRNA levels in liver tissue from BDL rats. fi-Actin was used as a reference. (D] Effect of EW-7197 on proliferation of isolated primary hepatocytes, HSC-enriched cells, and LX-2 cells. Cells were seeded at a density of 5 x 103 cells per well in 96-well plates and treated with EW-7197 at concentrations of 10 nM to 10 |iM for 72 h. Next, CCK-8 solution (10 |iL] was added to each well, after which the plates were incubated for 1 h at 37 °C. Absorbance was measured at 450 nm with an ELISA microplate reader. (E] Effects of EW-7197 and pirfenidone (Pir] on E-cadherin, N-cad-herin, and vimentin protein expression in isolated primary mouse HSC-enriched cells. Cells were treated with 0.5 |iM of the indicated drug with or without TGF-pi (2 ng/mL] and maintained for 24 h. Ponceau-S was used as a reference. (F] Effects of EW-7197 and Pir on E-cadherin, N-cadherin, PAI-I, and a-SMA protein expression in isolated primary mouse hepatocytes. Cells were treated with 0.5 |iM ofthe indicated drug with or without TGF-pi (2 ng/mL] and maintained for 24h. p-actin was used as a reference.

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

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Fig. 4. EW-7197 suppressed HIFla-mediated HSC activation and the EMT. (A] Effect of EW-7197 on BDL-in-duced TGF-p mRNA levels. Real-time PCR analysis was performed as described in the methods section. TGF-p mRNA levels in BDL rats were downregulated by EW-7197. fi-Actin was used as a reference, "p < 0.01 vs. sham and ###p < 0.001 vs. BDL (left]. Effects of EW-7197 and IN-2001 on HIFla-induced TGF-p mRNA levels in LX-2 cells. PPIA was used as a reference. *p < 0.05 vs. untreated control, "p < 0.01 vs. untreated control, ***p < 0.001 vs. untreated control, #p < 0.05 vs. CoCl2-treated control,##p < 0.01 vs. CoCl2-treated control, ###p < 0.001 vs. CoCl2-treated control (right], (B] Effects of EW-7197 on HIFla protein expression in BDL rat livers. *p < 0.05 vs. sham, ***p < 0.001 vs. sham, ##p < 0.01 vs. BDL. (C] Effects of EW-7197 and IN-2001 on HIFla and a-SMA expression in LX-2 cells. LX-2 cells were treated with 50 nM ofthe indicated drug with or without 100 |iM CoCl2 and maintained for 18 h. GAPDH was used as a reference. (D] Effects of EW-7197 and IN-2001 on a-SMA (a-SMA), collagen type 1 (_COl-1a1), and PAI-1 (PAI-1) mRNA levels in LX-2 cells. PPIA was used as a reference. (E] Effects of EW-7197 and IN-2001 on E-cadherin, N-cadherin, and vimentin protein expression in LX-2 cells. GAPDH was used as a reference. (F] Effects of EW-7197 on E-cadherin (CDH1),

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

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N-cadherin (CDH2), and vimentin (VIM) mRNA levels in LX-2 cells. PPIA was used as a reference. (G] Effects ofEW-7197 and IN-2001 on SNAIL mRNA expression in LX-2 cells. LX-2 cells were treated with 50 nM of the indicated drug with or without 100 |iM CoCl2 and maintained for 18 h. PPIA was used as a reference. (H] Effects ofEW-7197 and IN-2001 on promoter activation of PAI-1 as determined by ChIP assay. LX-2 cells were pretreated with 50 nM of the indicated drug for 30 min prior to the addition of 100 |iM CoCl2 and maintained for 3 h. ChIP assays were conducted at the PAI-1 promoter from LX-2 cells after immunoprecipitation with HIFla antibodies. Densitometric analysis of (bottom] revealed the band intensity.

3E] in response to TGF-p. In contrast, primary mouse hepatocytes did not undergo EMT in response to TGF-p, which did not affect expression levels of E-cadherin or N-cadherin in these cells (Fig. 3F], Taken together, these results show that HSCs are highly responsive to TGF-p, which induces transformation ofHSCs into myofibroblasts showing an EMT-like phenotype. Therefore, we focused on HSCs, rather than hepatocytes, in experiments intended to reveal the therapeutic mechanisms of ALK5 inhibitors in cholestatic liver fibrosis.

EW-7197suppressed HIFla-mediated HSC activation and the EMT

Hypoxia and elevated expression ofHIFla are typical phenomena observed in patients with obstructive cholestatic liver injury [38]. Because HIFla is a key regulator of gene expression in response to hypoxia [39], we measured mRNA levels ofTGF-p in BDL rats and LX-2 cells in order to gain insight into the role of cholestatic injury-mediated HIFla induction in fibrosis and the inhibitory effect of EW-7197 on such induction. TGF-p mRNA levels were increased in BDL rats and CoCl2-treated LX-2 cells (Fig. 4A]; however, EW-7197 treatment reduced TGF-p mRNA levels in vivo and in vitro. Because we expected EW-7197 to produce its effects via the same mechanism in LX-2 cells and BDL rats, we used a lower dose (50 nM] ofEW-7197 in comparison with that used in our previous study (0.5 ^M], Because EW-7197 has a relatively low IC50, the concentration used in this work should have resulted in 85% inhibition of ALK5 [40]. CoCl2 is used to stabilize HIFla, mimicking hypoxia [41]. Hypoxic conditions induce transcriptional activation of histone deacetylases (HDACs] in vitro, which downregulate expression of p53 and pVHL, leading to HIFla activation [42]. Therefore, we compared the inhibitory effect of potent HDAC inhibitor IN-2001 on HIFla expression with that of EW-7197. The effect of EW-7197 on HIFla expression was assessed in BDL rats. EW-7197 inhibited elevation of HIFla protein levels induced by cholestatic liver injury in BDL rats (Fig. 4B], To investigate the mechanism underlying hypoxia-dependent liver fibrosis, we measured HIFla-induced activation ofHSCs. In LX-2 cells, HIFla and a-SMA protein levels were increased by CoCl2 and reduced by treatment with EW-7197 or IN-2001 (Fig. 4C], Moreover, EW-7197 and IN-2001 decreased CoCl2-induced a-SMA, Colla, and PAI1 mRNA levels in LX-2 cells (Fig. 4D], To further examine the molecular mechanism of HIFla-induced liver fibrosis, we examined the EMT in LX-2 cells. Expression of E-cadherin was not evident in LX-2 cells, whereas N-cadherin and vimentin increased in response to CoCl2-induced hypoxic conditions. EW-7197 restored the protein and mRNAlevels of E-cadherin and reduced those of N-cadherin and vimentin in CoCl2-treated LX-2 cells. However, IN-2001 was not as potent as EW-7197 in CoCl2-treated LX-2 cells (Fig. 4E and 4F], mRNA levels of Snail in CoCl2-treated LX-2 cells are shown in Fig. 4G. Snail expression was increased by CoCl2 and inhibited by EW-7197. To verify the effects of EW-7197 on HIFla-induced transcription of plasminogen activator inhibitor-1 (PAI-1], a critical regulator of fibrosis, we performed ChIP assays in LX-2 cells. EW-7197 inhibited transactivation ofHIFla at the promoter region of PAI-1 (Fig. 4H], These data suggest that EW-7197 inhibits HIFla-induced EMT, HSC activation, and ECM accumulation.

EW-7197attenuates HIFla-mediated oxidative stress in BDL rats and LX-2 cells To determine whether oxidative stress was induced in the livers of BDL rats by hypoxia, we measured the expression of carbonic anhydrase IX (CAIX] and glucose transporter-1 protein (GLUT1] as intrinsic hypoxia markers [43-45]. The CAIX- and GLUTl-positive areas

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

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Fig. 5. EW-7197 attenuates HIFla-mediated oxidative stress. (A] IHC of CAIX (top] and (B] GLUT1 (bottom], Densitometry analysis of CAIX (upper right] and GLUT1 (lower right] staining in liver tissue from BDL rats. (C] IHC of 4-hydroxynonenal (4HNE] (top] and densitometry analysis (bottom] of 4HNE staining in liver tissue from BDL rats. ***p < 0.001 vs. sham and ##p < 0.01 vs. BDL. (D] Effect of EW-7197 on N0X4 protein expression in BDL rat livers.'p < 0.05 vs. sham, ##p < 0.01 vs. BDL, and ###p < 0.001 vs. BDL. (E] Effects of EW-7197 on peroxiredoxin I (Prdxl] protein expression in BDL rat livers. **p <0.01 vs. sham and ###p < 0.001 vs. BDL. (F] Effects ofEW-7197 and LY2157299 onintracellular ROS generationinducedbyGOX (5 mU] for 18 h in LX-2 cells. Cells were treated with the indicated concentration of the drug in the presence of GOX (5 mU]; ***p < 0.001 vs. control, ##p < 0.01 vs. GOX, and ###p < 0.001 vs. GOX. (G] Effects ofEW-7197 and LY2157299 on 3TP-Lux promoter activity induced by TGF-p in HaCaT (3TP-Lux stable] cells. Cells were treated with the indicated concentration ofthe drug in the presence ofTGF-p (2 ng/mL] for 24h.

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

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increased in size by 21.14-fold and 12.54-fold, respectively, in BDL rat livers in comparison with those of sham rats (Fig. 5A and 5B], These results indicate that hypoxia was induced in the livers ofBDL rats by the BDL procedure, because oxidative stress occurs downstream of hypoxia-mediated signal transduction [46, 47]. In Fig. 5C, we showed the expression of 4-hydroxynonenal (4HNE], a marker of lipid peroxidation and oxidative stress. IHC staining showed a dramatic increase in the size of the 4HNE-positive area around the portal triad of the livers of BDL rats; however, EW-7197 treatment decreased the size of the 4HNE-positive area. These results show that oxidative stress was elevated in accordance with the increase in the size of the hypoxic area in the livers of BDL rats. A previous report showed that NADPH oxidase (NOX] was expressed in activated HSCs, in which it generated ROS that mediated fibrogenesis [48]. Western blot analysis showed that EW-7197 treatment inhibited the increase in N0X4 expression produced in rats by BDL (Fig. 5D], We also found that expression of antioxidant peroxiredoxin I (Prdx I] was elevated in BDL rats, while EW-7197 reduced the protein level of Prdx I (Fig. 5E], To investigate the inhibitory effect ofEW-7197 on ROS generation, we performed an in vitro ROS assay in LX-2 cells. Administration of H202 or TGF-p to LX-2 cells increased the fluorescence of DCF which was inhibited by concomitant treatment with ALK5 inhibitors (Fig. 5F], Treatment of GOX that generates H202 to LX-2 cells increased fluorescence of DCF, which responds to ROS, by 8-fold however, treatment of GOX+EW-7197 and GOX+LY2157299 attenuated GOX-induced ROS generation in a dose-dependent manner. We used LY2157299 as a reference ALK5 inhibitor (Fig. 5G], The IC50 values of selected ALK5 inhibitors were determined by luciferase reporter gene assay in HaCaT (3TP-Lux stable] cells (Fig. 5H], The IC50 ofEW-7197 (18.14 nM] was lower than that of LY2157299 (128.6 nM], indicating that EW-7197 was a more potent inhibitor of ALK5. These results indicate that ALK5 inhibitor EW-7197 inhibits ROS generation induced by cholestatic liver injuries with high potency in vivo.

EW-7197inhibits liverfibrosis by blocking TGF-fi/Smad signaling

In order to understand the fibrotic mechanism of TGF-p and inhibitory effect of EW-7197, we conducted immunofluorescence (IF] staining to visualize nuclear translocation of p-Smad2/3 (Fig. 6A], EW-7197 inhibited TGF-p-induced nuclear translocation of pSmad2/3 in LX-2 cells. EW-7197 inhibited protein and mRNA expression of TGF-p-stimulated EMT markers E-cadherin, N-cadherin, and vimentin in LX-2 cells (Fig. 6B and 6C], Moreover, TGF-p increased the mRNA level of Snail, whereas treatment with EW-7197 decreased Snail mRNA expression (Fig. 6D], These results demonstrate that TGF-p-induced EMT was involved in regulation of Snail. In addition, these data show that EW-7197 inhibits EMT induced by TGF-p-mediated Snail activation. Western blot and qRT-PCR analyses showed that EW-7197 inhibited TGF-p-induced a-SMA, Colla, and PAI-1 protein and mRNA levels in LX-2 cells (Fig. 6E and 6F], Regulation of the expression ofPAI-1, a profibrotic and TGF-p-responsive target gene, was mediated by Smad activation through ALK5 [24]. ChIP assays showed that EW-7197 inhibited transactivation of Smad2/3 at the promoter region of PAI-1 (Fig. 6G], Taken together, these results suggest that EW-7197 attenuates fibrosis via suppression of TGF-p-mediated HIFla action, which ordinarily leads to HSC activation and the EMT (Fig. 6H],

Discussion

Liver fibrosis is a wound healing process that results in increased levels of ECM proteins [1, 32]. The pathogenesis of fibrosis involves TGF-p and hypoxia, which result in inflammation, activating quiescent HSCs and inducing their transformation into myofibroblasts [1, 32]. Following injury, vascular damage results in hypoxic conditions that lead to ROS generation [11], which might act as a mediator to bring about the EMT [12, 47, 49]. Cells respond to hypoxia by activating transcription factors, particularly HIFla [13, 47]. HIFla transcriptionally regulates the expression levels of ECM proteins (e.g., fibronectin and collagen], adhesion proteins (e.g., integrin], metabolic proteins (e.g., CAIX and GLUT1],

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

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Fig. 6. EW-7197 inhibits liver fibrosis by blocking TGF-p/Smad signaling. (A] Effects of EW-7197 on nuclear translocation of Smad2/3 and p-Smad2/3 in LX-2 cells. LX-2 cells were pretreated with 50 nM of the indicated drug for 30 min prior to the addition of TGF-pi (2 ng/mL] and maintained for 30 min. Smad2/3, p-Smad2/3, and nuclei were stained with Alexa 488 (green], Alexa 555 (red], and DAPI (blue], respectively, after which the confocal images were merged (n > 3]. The merged image reveals nuclear localization of p-Smad2/3 (pink]. The scalebarsare 50 |im. (B] Effects ofEW-7197 and LY2157299 onE-cadherin, N-cad-herin, and vimentin protein expression in LX-2 cells. LX-2 cells were treated with 50 nM of the indicated drug with or without TGF-pi (2 ng/mL] and maintained for 24 h. p-actin was used as a reference. (C] Effects ofEW-7197 and LY2157299 on E-cadherin (CDH1), N-cadherin (CDH2), and vimentin (VIM) mRNA levels in LX-2 cells. PPIA was used as a reference. (D] Effects ofEW-7197 or LY2157299 on SNAIL mRNA expression in LX-2 cells. LX-2 cells were treated with 50 nM of the indicated drug with or without 2 ng/mL TGF-p and

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maintained for 24 h. PPIA was used as a reference, "p < 0.01 vs. untreated control and ##p < 0.01 vs.TGF-P-treated control. (E] Effects of EW-7197 and LY2157299 on a-SMA, Colla, and PAI-1 protein expression in LX-2 cells. LX-2 cells were treated with 50 nM of the indicated drug with or without TGF-pi (2 ng/mL] and maintained for 24 h. GAPDH was used as a reference. (F] Effects of EW-7197 and LY2157299 on a-SMA [a-SMA], collagen type 1 (COl-1a1], and PAI-1(PAI-1] mRNA levels in LX-2 cells. PPIA was used as a reference. (G] Effects of EW-7197 and LY2157299 on promoter activation of PAI-1 as determined by ChIP assay. LX-2 cells were pretreated with 50 nM of the indicated drug for 30 min prior to the addition of TGF-pi (2 ng/mL] and maintained for 30 min. ChIP assays were conducted at the PAI-1 promoter in LX-2 cells after immunoprecipitation with the Smad2/3 antibody. Densitometry analysis of the ChIP assay (down] revealed the band intensity after electrophoresis. (H] Simplified scheme revealing the effects of EW-7197 on TGF-p and HIFla-induced EMT in cholestatic liver injury.

and growth factors (e.g., VEGF and TGF-P] [47]. TGF-p is modulated by HIFla and is an effective mediator of the EMT and the progression of fibrosis in various organs [15, 20, 50, 51]. Activation of HSCs by TGF-p is an important event in tissue fibrogenesis, but activation of hepatocytes during the development of fibrosis in the mouse liver has not been observed [2]. Quiescent HSCs express epithelial marker E-cadherin and enter the EMT because of the actions of HIFla and TGF-p [52-54]. These reports are in agreement with our results, in which only isolated mouse HSCs showed the EMT (Fig. 3 and 4]. TGF-p and HIFla are major cytokines that increase ROS generation and decrease concentrations of antioxidants during the progression of fibrosis [55-58]. In this paper we showed EW-7197 inhibited ROS generation by TGF-p or GOX (Fig. 5F and 5G] even though further study would be required to understand the mechanism of EW-7197. HIFla increases expression levels of NOXs, which results in ROS generation [59, 60], regulating the transcriptional activity of HIFla and drivingthe EMT [46, 61]. In addition, ROS generation induces Snail expression, subsequently leading to the EMT [62, 63] This study also showed that HIFla increased ROS generation via N0X4 and demonstrated that ROS induced the EMT by inducing Snail expression in the livers of BDL rats and LX-2 cells (Fig. 5 and 6]. Because TGF-p and HIFla also play important roles in modulating inflammation related to liver fibrosis [64, 65], we showed that EW-7197 decreased the elevated level of NFkB expression in the livers of BDL rats (data not shown]. However, further research is necessary to explain the manner in which TGF-p inhibition modulates the process of cholestatic fibrosis in terms of inflammation. Taken together, our results demonstrate that TGF-p inhibitor EW-7197 (20 and 40 mg/kg, po, in vivo] inhibited fibrosis by attenuating HIFla-induced EMT in BDL rats.

Acknowledgements

This work was supported by a Korea Science and Engineering Foundation (KOSEF] grant (MEST; No. 20090093972] and National Research Foundation (NRF-2014R1A1A2005644] of Korea funded by the Korean government.

Disclosure Statement

The authors state thatthey have no conflicts of interest. References

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DOI: 10.1159/000438651

Cellular Physiology

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Kim et al.: EW-7197 Suppresses Cholestatic Liver Fibrosis

© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

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DOI: 10.1159/000438651

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© 2016 The Author(s). Published by S. Karger AG, Basel www.karger.com/cpb

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