Peroxiredoxin 6 expression is inversely correlated with nuclear factor-κB activation during Clonorchis sinensis infestation

Free Radical Biology and Medicine 99 (2016) 273–285

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Original article

Peroxiredoxin 6 expression is inversely correlated with nuclear factorκB activation during Clonorchis sinensis infestation Jhang Ho Pak a,n, Woo Chan Son b, Sang-Beom Seo c, Sung-Jong Hong d, Woon-Mok Sohn e, Byoung-Kuk Na e, Tong-Soo Kim f a Department of Convergence Medicine University of Ulsan College of Medicine and Asan Institute for Life Sciences, Asan Medical Center, 388-1 Pungnap-2 dong, Songpa-gu, Seoul 138-736, Republic of Korea b Department of Pathology, University of Ulsan College of Medicine and Asan Institute for Life Sciences, Asan Medical Center, Seoul 138-736, Republic of Korea c Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Republic of Korea d Department of Medical Environmental Biology and Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul 156-756, Republic of Korea e Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea f Department of Parasitology, Inha University School of Medicine, Incheon 400-103, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 14 May 2016 Received in revised form 9 August 2016 Accepted 12 August 2016 Available online 20 August 2016

Clonorchis sinensis is a carcinogenic human liver fluke. Its infection promotes persistent oxidative stress and chronic inflammation environments in the bile duct and surrounding liver tissues owing to direct contact with worms and their excretory–secretory products (ESPs), provoking epithelial hyperplasia, periductal fibrosis, and cholangiocarcinogenesis. We examined the reciprocal regulation of two ESP-induced redox-active proteins, NF-κB and peroxiredoxin 6 (Prdx6), during C. sinensis infection. Prdx6 overexpression suppressed intracellular free-radical generation by inhibiting NADPH oxidase2 and inducible nitric oxide synthase activation in the ESP-treated cholangiocarcinoma cells, substantially attenuating NF-κB-mediated inflammation. NF-κB overexpression decreased Prdx6 transcription levels by binding to two κB sites within the promoter. This transcriptional repression was compensated for by other ESP-induced redox-active transcription factors, including erythroid 2-related factor 2 (Nrf2), hypoxia inducible factor 1α (HIF1α), and CCAAT/enhancer-binding protein β (C/EBPβ). Distribution of immunoreactive Prdx6 and NF-κB was distinct in the early stages of infection in mouse livers but shared concomitant localization in the later stages. The intensity and extent of their immunoreactive staining in infected mouse livers are proportional to lesion severity and infection duration. The constitutive elevations of Prdx6 and NF-κB during C. sinensis infection may be associated with more severe persistent hepatobiliary abnormalities mediated by clonorchiasis. & 2016 Elsevier Inc. All rights reserved.

Keywords: Clonorchis sinensis infection Cholangiocarcinoma Excretory-secretory products, Prdx6, NF- κB Free radicals Redox-active transcription factors

1. Introduction Persistent oxidative stress because of an imbalance between the generation and elimination of free radicals (reactive oxygen and nitrogen species; ROS/RNS) is implicated in the pathogenesis of numerous human diseases, including chronic inflammation. Acute inflammation functions as a host defense mechanism against infection or injury and persists for a short time; chronic inflammation lasts longer and is strongly associated with an increased risk of cancer. Cancer initiation and progression is linked to oxidative stress owing to genome instability, cell proliferation, and the increase in DNA mutations or induction of DNA lesions n

Corresponding author. E-mail address: [email protected] (J.H. Pak).

http://dx.doi.org/10.1016/j.freeradbiomed.2016.08.016 0891-5849/& 2016 Elsevier Inc. All rights reserved.

[1,2]. For example, infection with viruses, bacteria, and parasites can cause chronic inflammation-associated carcinogenesis, wherein oxidative and nitrative DNA lesion product accumulation occurs at the site of carcinogenesis [3]. In Southeast Asian countries, liver fluke (Clonorchis sinensis and Opisthorchis viverrini) infestation is a predominant risk factor for cholangiocarcinoma (CCA), which is a lethal adenocarcinoma arising from the biliary epithelia. Humans are infected by consuming raw/undercooked freshwater fish harboring their metacercariae [4]. Based on etiological and experimental evidence and several case-control studies, C. sinensis, with O. viverrini, is recently determined to be a Group 1 biological human carcinogen by the International Agency for Research on Cancer [5]. The proposed pathogenic mechanisms of chronic liver fluke infection-associated CCA include physical damage to the biliary epithelia caused by the feeding and migration of the worms, immunopathology owing to

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infection-related inflammation, and a direct effect of the excretory–secretory products (ESPs). These host–parasite interactions provoke inflammatory cell infiltration, fibrotic deposition, hyperplasia, and adenomatous and granulomatous changes in the biliary epithelia with subsequent malignant transformation of cholangiocytes into CCA [6]. O. viverrini infection increases the inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB) expressions in the inflammatory cells and epithelial bile ducts of hamster livers, with concomitant nitrative and oxidative DNA adduct accumulation. The increasing DNA lesion product and iNOS expression levels are proportional to the numbers of infections [7]. Exposure of HuCCT1 to C. sinensis ESPs triggered enzymatic free radical generation, leading to NF-κB-mediated inflammatory processes [8]. This suggests that persistent free radical generation during liver fluke infestation disturbs host redox homeostasis, creating a harmful environment that predisposes advanced hepatobiliary disease development, including that of chronic inflammation-associated CCA. Peroxiredoxins (Prdxs) belong to a superfamily of nonheme and nonselenium peroxidases, which reduce a wide range of peroxides. They occur in all phyla and are classified as 1/2-cys Prdxs according to the number of conserved cysteine residues involved in the catalytic mechanism [9]. Of the six mammalian Prdxs, Prdx6 is a sole 1-cys enzyme with both phospholipase A2 (PLA2) and peroxidase activities. Unique features of Prdx6 include using glutathione (GSH) instead of thioredoxin as a physiological reductant, heterodimerization with glutathione S-transferase π (GSTπ) to complete its catalytic cycle, and maintenance of phospholipid turnover [10]. Prdx6 expression was upregulated in HuCCT1 cells exposed to C. sinensis ESPs and in the livers of O. viverrini-infected hamsters, as demonstrated through proteomic analyses [11,12]. However, there are no studies on the regulatory mechanisms or pathophysiologic roles of Prdx6 in eliciting the host response to the infective milieu. Given that NF-κB-mediated inflammation is a crucial phenomenon for liver fluke-associated diseases [6] and NF-κB functions as a transcription factor for Prdx6 gene [13,14], we sought to examine the relative effects of these two proteins in HuCCT1 cells exposed to C. sinensis ESPs. We also aimed to determine the differential immunolocalization of NF-κB and Prdx6 proteins in the livers of infected mice according to the duration of infection.

2. Materials and methods 2.1. Materials Cell culture medium components were purchased from Life Technologies (Grand Island, NY) unless otherwise indicated. Polyclonal antibodies against the following proteins were obtained from the indicated sources: NF-κB p65, phospho-NF-κB p65, and IκB-α (Cell Signaling Technology, Beverly, MA); p47phox, p67phox, and iNOS (BD Biosciences, San Jose, CA); NF-κB p50, Keap1, Nrf2, C/EBPα, C/EBPβ, lamin B, and calnexin (Santa Cruz Biotechnology, Santa Cruz, CA); C/EBPγ and HIF1α (Abcam, Cambridge, MA); Prdx6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; AbFrontier Co., Seoul, Korea). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratory (West Grove, PA). The ESPs from C. sinensis adult worms were prepared as previously described [8]. The pcDNA3.1C/EBPβ was kindly provided by Dr. Sang Geon Kim (College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea).

2.2. Cell culture and ESP treatment HuCCT1, a human CCA cell line, was cultured in RPMI1640 and supplemented with 10% fetal bovine serum (FBS) and 0.05% penicillin/streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. For ESP treatment, cells were grown for 24 h under standard culture conditions. Cells were gradually deprived of serum by incubation in 1% FBS overnight, followed by incubation in serum-free medium for 3 h. These serum-starved cells were treated with 800 ng/ml ESPs and incubated for indicated times. 2.3. NF-κB p65-RFP fusion protein expression plasmid construction The coding region of p65 was amplified by PCR using the human p65 expression plasmid (pcDNA6-myc-p65 [13]) as a template. An XhoI restriction site was introduced into the forward primer (5′-CGCCTCGAGGCATGGACGAACTGTTCCCCCTC-3′) and a BamHI site was introduced into the reverse primer (5′CGCGGATCCTTAGGAGCTGATCTGACTCAGCAG-3′). The purified PCR product was double digested with XhoI and BamHI, and inserted in-frame into a pDsRed-C1 vector (Clontech Laboratories, Inc., Palo Alto, CA) to encode a fusion protein with RFP. 2.4. Transfection into HuCCT1 cells HuCCT1 cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with the appropriate expression plasmids. The same amount of empty vector was transfected as an internal control. Approximately 75% of the reproducible efficacy of transfection was confirmed by counting the number of GFP or RFP expressing cells from the total cells. Twenty-four hours after transfection, cells were treated with 800 ng/ml ESPs for 15 h. 2.5. Detection of intracellular free radicals Intracellular ROS and NO were detected using the fluorescent probes5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) and 4-amino-5-methylamino-2′7′-dichlorofluorescein diacetate (DAF-FM; Molecular Probes, Inc., Eugene, OR), respectively. Cells grown on 96-well plates were transfected with pcDNA3 as an internal control, pcDNA-Prdx6, or pcDNA-C47S, as described elsewhere [15,16]. Twenty-four hours after transfection, cells were treated with 800 ng/ml ESPs for 15 h. Cells were then loaded with 10 μM CM-H2DCFDA or 5 μM DAFFM. The DCF or DAF fluorescence levels were measured using a spectrofluorometer (Molecular Devices Corp., Sunnyvale, CA). The values were converted to percentages for comparison with the untreated control. 2.6. Luciferase assays HuCCT1 cells were seeded on 24-well culture plates and cotransfected with the pGL3-hPrdx6 reporter (100 ng each, [13]), and each transcription factor expression plasmid such as pcDNA6-mycp65 [13], pcDNA3-Nrf2 [17], pCDNA3.1-C/EBPβ [18], or HA- HIF1αpcDNA3 [19]. The simultaneously transfected pCMV-β-gal plasmid (200 ng) was used for the normalization of transfection efficiency. At 24 h after transfection, cells were treated with 800 ng/ml of ESPs for 15 h. Subsequently, total cell lysates were extracted with the Reporter Lysis Buffer (Promega, Madison, WI), and luciferase activities were measured with a microplate luminometer (MicroLumatPlus LB 96V, EG & G Berthold, Bad Wildbad, Germany). The luciferase activities of individual reporter plasmids were normalized to that of β-galactosidase.

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2.7. Total RNA isolation and RT-PCR Total RNA was extracted from HuCCT1 cells with an RNeasy mini kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Approximately 1 μg of total RNA was used to synthesize first-strand cDNA with the amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX), followed by amplification with TaKaRa Ex Taq™ (TaKaRa Bio, Inc., Shiga, Japan). The reaction was performed for 25 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, and extension at 72 °C for 45 s. The primer sequences were as follows: IL-1β 5′CCTGTGGCCTTGGGCCTCAA-3′ (forward) and 5′-GGTGCTGATGTACCAGTTGGG-3′ (reverse), IL-6 5′-TGAACTCCTTCTCCACAAGC-3′ (forward) and 5′-ATCCAGATTGGAAGCATCCA-3′ (reverse), and GADPH (internal control) 5′-AGAACATCATCCCTGCATCC-3′ (forward) and 5′-TTACTCCTTGGAGGCCATGT-3′ (reverse). The RT-PCR products were analyzed by electrophoresis on a 1.2% agarose gel and images were captured for quantification of DNA band densities. 2.8. Cytokine assay Immunoreactive IL-1β and IL-6 from untreated and ESP-treated culture supernatant were quantified using ELISA kits (Enzo Life Sciences Inc., Ann Arbor, MI), according to the manufacturer's instructions. A standard curve for each cytokine was generated using a known concentration of human recombinant IL-1β or IL-6 protein. Absorbance at 450 nm was used to calculate levels from the standard curve and adjusted by dilution factor.

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(256 bp; position
782 to
526). The mean threshold cycle (Ct) and standard error were calculated from individual Ct values obtained from triplicate determinations per stage. The normalized mean Ct was estimated as ΔCt by subtracting the mean Ct of input from that of each target. 2.12. Immunoblot analysis Thirty micrograms of total soluble proteins, cytosolic and membranous proteins, or 10 μg of nuclear proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (ATTO, Tokyo, Japan). Membranes were probed with primary antibodies specific for target proteins. After incubating with host-specific HRP-conjugated secondary antibodies, the immunoreaction was detected with a West-Q Chemiluminescent Substrate Kit (GenDEPOT) and quantified by densitometric scanning of the X-ray film with a Fluor-S Multimager (Bio-Rad, Hercules, CA). Blots were normalized for protein loading by washing in BlotFresh Western Blot Stripping Reagent (SignaGen Laboratories, Gaithersburg, MD) and reprobing with antibodies against GAPDH (for cytosolic proteins), calnexin (for membranous proteins), or lamin B (for nuclear proteins). 2.13. Collection of C. sinensis metacercariae and infection

XO activity was measured using the Amplex Red Xanthine/ Xanthine Oxidase Assay Kit (Molecular Probes), according to the manufacturer's instructions. XO activity in each sample was calculated by reference to a standard curve. NO production was accessed by measuring nitrite/nitrate concentration in culture medium using a Total Nitric Oxide Assay Kit (Assay Designs Inc., Ann Arbor, MI), according to the manufacturer's instructions. Nitrite/nitrate concentrations were determined using a standard curve.

The metacercariae of C. sinensis were collected from naturally infected freshwater fish (Pseudorasbora parva) obtained in Korea. The metacercariae were collected under a stereoscopic microscope as described elsewhere [8] and kept in physiological saline at 4 °C until infection. Animal care and experimental procedures were conducted according to institutional guidelines, and protocols were approved by the Animal Care and Use Committee of the Korea National Institutes of Health. Male FVB/N mice at 5–6 weeks of age were infected with 25 C. sinensis metacercariae by intragastric intubation, whereas the control mice received an equal volume of saline solution via the same route. Without any further treatment, these mice were fed with a sterilized commercial diet and water ad libitum. After 1, 2, 4, and 8 weeks, livers were excised, fixed in 10% buffered formaldehyde, and embedded in paraffin.

2.10. Subcellular fractionation of cell lysates

2.14. Immunohistochemical analysis

2.9. Enzymatic activity assays s

Membranous and nuclear proteins were isolated using the Plasma Membrane Protein Extraction Kit (Abcam) and the NEs PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, IL) according to the manufacturers’ instructions. The protein concentration of extracts was measured and extracts were stored in aliquots at
80 °C. 2.11. Chromatin immunoprecipitation assay Transfection of pcDNA6-myc-p65 into HuCCT1 cells and ESP treatment were performed as described above. At 24 h after ESP exposure, cells were fixed by adding formaldehyde directly into the medium. The sonicated chromatin was immunoprecipitated with anti-NF-κB p65 (Abcam) antibodies and control rabbit IgG. After reversing the cross-links, the eluted DNA fragments served as templates for real-time quantitative PCR using a LightCycler 480 system (Roche Diagnostics Inc., Basel, Switzerland). Primers utilized for the Prdx6 promoter were 5′-AGTGTCCAGCACACAGCAGG3′ (forward) and 5′-GAAGGGAAACCGGTGACAGAC-3′ (reverse) as the κB binding site1 (269 bp; position
1235 to
966), and 5′GGTTCATAACAAACAGAAAGGGGAG-3′ (forward) and 5′-AGAGGAGTGGAGTGACTGCTC-3′(reverse) as the κB binding site2

Paraffin sections (5-μm thick) were deparaffinized and incubated with primary antibodies against Prdx6 or phospho-NF-κB p65 overnight at 4 °C. The sections were incubated with a REAL Envision þ System-HRP labeled polymer anti-rabbit kit (DaKo North America, Inc., Carpinteria, CA) for 1 h and developed using a VECTASTAIN ABC Elite Kit (Vector Laboratories, Burlingame, CA), followed by counterstaining with hematoxylin. The sections were air-dried and coverslips were sealed with mounting medium before being photographed with an upright microscope (Nikon Eclipse Ci, Tokyo, Japan). 2.15. Statistical analysis Data are expressed as means 7standard error of three independent experiments and analyzed by one-way analysis of variance or a Student's t-test. Statistical significance was accepted at P o0.05. Statistical analyses were performed using SPSS 12.0 (SPSS Science, Chicago, IL). 3. Results 3.1. Inhibition of ESP-triggered free radical accumulation by Prdx6

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Fig. 1. Inhibitory effect of Prdx6 overexpression on ESP-triggered free radical generation. (A) Representative immunoblot showing Prdx6 expression in each transfected cell. Cells were harvested 24 h after transient transfection with pcDNA3 (mock), pcDNA-Prdx 6 (WT), or pcDNA-C47S (C47S), and were then analyzed for Prdx6 expression using a polyclonal antibody to Prdx6. Prdx6 protein bands were densitometrically quantified and expressed relative to GAPDH band density. The ratio of Prdx6 protein to GAPDH is presented as percentage of pcDNA3 transfectant. *Po 0.05 compared topcDNA3. Each transfectant was treated with 800 ng/ml for 15 h, and the levels of ROS and NO were determined by measuring CMH2DCFDA (B) and DAF-FM (C), respectively. Values are presented as means 7SE for three independent experiments, expressed as a percentage of that in the untreated control. *Po 0.05 compared with untreated control; #P o 0.05, wild-type versus mock and C47S mutant transfected cells.

overexpression We previously reported that the treatment of HuCCT1 cells with C. sinensis ESPs enzymatically generated free radicals and increased Prdx expressions. Furthermore, this free radical accumulation led to NF-κB-mediated inflammation [8,11]. To examine whether a change in free radical generation could affect the ESPinduced host cell immune response, Prdx6 was overexpressed to attenuate ESP-induced free radical generation. HuCCT1 cells were transiently transfected with wild-type Prdx6 (pcDNA-Prdx6), a mutant defective in catalytic Cys residue of Prdx6 (pcDNA-C47S), or an empty vector (pcDNA3, mock). Twenty-four hours posttransfection, cells were exposed to ESPs for 15 h. Immunoblot analysis revealed 200% increase in Prdx6 protein expression of both the wild-type and mutant transfectants compared with the expression of empty vector-transfected cells (Fig. 1A). ROS and RNS levels in ESP-treated wild-type transfectants were elevated significantly less than those in the mutant and mock transfectants (  63% and  74%, respectively), indicating that an increase in peroxidase activity of Prdx6 exerted the diminution of intracellular ESP-induced free radicals in its overexpressing cells (Fig. 1B, C). Because ESP-induced NADPH oxidase (NOX), xanthine oxidase (XO), and inducible nitric oxide synthase (iNOS) are responsible for free radical generation [8], we next examined whether Prdx6

overexpression affected the activation of these enzymes. Increased accumulation of two NOX2 regulatory components (p47phox and p67phox) in the membrane fraction was evident in the mock and mutant transfectants exposed to ESPs, whereas there was a  50% reduction of these subunits in the wild-type transfectants (Fig. 2). ESP-induced iNOS expression level in Prdx6-overexpressing cells was similar to that in untreated control cells, whereas there was a significant elevation in the levels of the mutant and mock transfectants. An ESP-mediated change in iNOS expression of each transfectant was further confirmed by measuring nitrite/nitrate concentrations in the culture media. Nitrite/nitrate formation in the mock and mutant transfectants was  1.5-fold greater than that in the wild-type transfectants. Increased XO activity was essentially unchanged despite different vector transfection. These results indicate that the change in Prdx6 expression influences NOX2 and iNOS, but not XO activities. 3.2. Inhibitory effect of Prdx6 overexpression on ESP-induced NF-κB activation Because NF-κB activation is controlled by free radicals produced in response to various stimuli [20,21], we examined ESPinduced NF-κB activation in Prdx6-overexpressing cells. The cytosolic and nuclear fractions of each transfectant treated with ESPs

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Fig. 2. Effect of Prdx6 overexpression on ESP-induced NOX, iNOS, and XO activation. Cells were transfected with mock, wild-type, or C47S mutant plasmids, followed by treatment with 800 ng/ml ESPs for 15 h. Cells were then harvested for immunoblot analysis and enzyme activity assays. (A) Accumulation of NOX subunits (p47phox and p67phox) in the membrane was assessed by immunoblotting. Calnexin was used as a control for membrane protein loading. (B) Total protein was immunoblotted using a polyclonal anti-iNOS antibody. GAPDH was used as a control for protein loading. (C) NO generation in culture media was measured using the Griess reaction after enzymatic conversion of nitrate to nitrite. (D) XO activity was measured by comparing the absorbance of each sample with that of XO standard samples and calculated as mU/mg after normalization to total protein content. Data in graphs C and D are presented as means 7 SE of three independent experiments.*Po 0.05 compared with untreated control. # P o 0.05, wild-type versus mock and C47S mutant transfected cells.

were used to determine the degradation of IκB-α and nuclear translocation of NF-κB subunits, p50 and p65, by immunoblot analysis. ESP-induced IκB-α degradation was significant in mutant and mock transfectants compared with that in the untreated control cells, whereas this degradation occurred substantially less in the wild-type transfectants (Fig. 3A, B). Nuclear p50 and p65 accumulation in mutant and mock transfectants was much greater than that in the untreated control cells, whereas it was significantly reduced in the wild-type transfectants. The inhibitory effect of Prdx6 overexpression on ESP-induced NF-κB activation was further confirmed by a cotransfection experiment examining the intracellular localization of Prdx6 and p65. HuCCT1 cells were cotransfected with pEGFP-C1-Prdx6 [15] and pDsRed2-C1-NF-κB p65 fusion constructs and treated with ESPs for 15 h. As negative controls, an empty vector (pEGFP-C1/pDsRed2-C1) was cotransfected with p65 or Prdx6 fusion construct. Green fluorescent protein (GFP)- and red fluorescent protein (RFP)-derived fluorescence was detected in the cytosol without ESP exposure (Fig. 3C).

In the presence of ESPs, the NF-κBp65-RFP fusion protein in cells cotransfected with pDsRed2-C1-NF-κB/p65 and pEGFP-C1 was translocated into the nucleus, but it stayed in the cytosol of the cells cotransfected with pDsRed2-C1-NF-κB/p65 and pEGFP-C1Prdx6. These results demonstrate that the suppression of ESP-induced NF-κB activation was associated with wild-type Prdx6 overexpression. 3.3. Effect of ESP-induced Prdx6 overexpression on IL-1β and IL6 expression We examined the effect of Prdx6 overexpression on proinflammatory cytokine expression, including that of interleukin (IL)1β and IL-6, which was upregulated by ESP-induced NF-κB activation [8]. The mRNA levels of IL-1β and IL-6 in each ESP-treated transfectant were analyzed using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). In comparison with that in the untreated control cells, a significant elevation in the

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Fig. 3. Inhibitory effect of Prdx6 overexpression on ESP-induced NF-κB activation Mock, wild-type, or C47S transfected cells were cultured in the presence of 800 ng/ml ESPs for 15 h. Cytosolic and nuclear proteins from each transfectant were subjected to immunoblot analysis for the expression levels of IκB-α (A) and NF-κB subunits p50 and p65 (B). Protein bands were densitometrically quantified, and the densities of IκB-α and p50/p65 bands were normalized to those of GAPDH and lamin B bands, respectively. The normalized IκB-α, p50, and p65 band densities from each group are presented as a percentage of those in ESP-untreated controls. Values are means 7SE of three independent experiments (*Po 0.05, compared with ESPs-untreated controls; #Po 0.05, wild-type versus mock and C47S mutant transfected cells). (C) Cotransfection of a RFPNF-κB/p65 fusion protein expression plasmid with a GFP-Prdx6 fusion protein expression plasmid in HuCCT1 cells. As controls, an empty vector, pEGFP-C1 or pDsRed2-C1, was used for a counterpart to pDsRed2-C1RFP-NF-κB/p65 and pEGFP-C1-Prdx6, respectively. At 24 h after cotransfection, cells were treated with 800 ng/ml of ESPs for a further 15 h. Nuclei were counter-stained with DAPI. The presence of red and green fluorescence was observed with the appropriate filter of a fluorescence microscope. The overlapping GFP and RFP signals (yellow) in the merged image indicates localization of RFP by itself or the NF-κB p65 subunit (arrow). The scale bar is 100 mm. Original magnification  100.

cytokine mRNA expression was obvious in the mock and mutant transfectants (Fig. 4A). This ESP-induced elevation was significantly attenuated in the wild-type transfectants. To validate the correlation of mRNA expression with protein level, we measured

the level of each cytokine in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). IL-1β and IL-6 secretion from ESP-treated mock and mutant transfected cells was significantly increased compared with that from the untreated

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Fig. 4. IL-1β and IL-6 expression in mock, wild-type, and mutant Prdx6 overexpressing cells after ESP treatment. (A) Each transfected cell was treated with 800 ng/ml of ESPs for 15 h and subjected to semi-quantitative RT-PCR analysis. Each band was densitometrically quantified and normalized to GAPDH band density. The ratios of IL-1β and IL-6 mRNA to GAPDH from each transfected cell are presented as a fold of that in the untreated control. (B and C) Cells were incubated with 800 ng/ml of ESPs for 15 h, and the production of IL-1β or IL-6 in culture supernatants was determined by ELISA. The concentration of each cytokine was calculated based on the absorbance of an IL-1β or IL-6 standard curve (inset). Data are presented as means7 SE of three independent experiments. *Po 0.05, compared with untreated controls; #P o0.05, wild-type versus mock and C47S mutant transfected cells.

control cells, but the increase was not as marked in the wild-type transfectants (Fig. 4B,C). This result revealed that Prdx6 overexpression transcriptionally and translationally inhibited NF-κBinduced IL-1β and IL-6 expressions, indicating that Prdx6 peroxidase activity contributed to ESP-mediated inflammation attenuation. 3.4. Transcriptional repression of Prdx6 by ESP-mediated NF-κB activation Previous reports indicated that NF-κB suppressed Prdx6 expression [22] and that there are κB binding sites within the human Prdx6 promoter at positions
1166 to
1157 and
750 to
741, respectively (Fig. 5A [13]). This prompted us to examine the NF-κB regulatory mechanism in Prdx6 expression. Transient cotransfection experiments were conducted using the pGL3-hPrdx6 luciferase reporter and pcDNA6-myc-p65 constructs in HuCCT1 cells. Prdx6 promoter-driven luciferase activities were inversely proportional to the amount of NF-κB p65 subunit expression plasmid (Fig. 5B), indicating that NF-κB functions as a negative transcription factor for Prdx6 gene. To demonstrate that ESP treatment recruited p65 to the Prdx6 promoter region, a chromatin immunoprecipitation (ChIP) assay was performed using an anti-NFκB p65 antibody, followed by real-time quantitative PCR for the levels of each κB binding site. Using a constant level of input DNA from pcDNA6-myc-p65-transfected cells, ESP treatment resulted in 2.5-fold increase in κB1 and κB2 levels at sites occupied by p65, compared with untreated controls (Fig. 5C, D). The binding frequency of the κB1 region was similar to that of κB2, suggesting that two putative κB binding sites were substantially recruited by p65. These results demonstrate that ESP treatment led to NF-κB

activation, increased binding activity to the Prdx6 promoter, and subsequent transcriptional repression of Prdx6 gene. 3.5. Transactivation of the Prdx6 promoter by ESP-induced redoxactive transcription factors Although ESP-induced NF-κB activation transcriptionally suppressed Prdx6 expression, ESP treatment resulted in the upregulation of the expression [11], indicating the existence of positive transcription factor(s) for the expression in response to ESPs. In addition to validated nuclear factor erythroid 2-related factor 2 (Nrf2) binding to antioxidant response element (ARE, [23]), a webbased computer analysis (MatInspector; Genomatrix) of a 1506 bp region (Prdx6 promoter) disclosed the presence of putative redoxactive transcription factor binding sites, including two CCAAT/enhancer binding proteins (C/EBPs) and a hypoxia inducible factor 1α (HIF1α) at positions,
1255 to
1245,
697 to
687, and
346 to
342, respectively (Fig. 6A). Therefore, we examined whether expression of these transcription factors was regulated by ESP exposure. Immunoblot analysis revealed that cytosolic Keap1 (a repressor of Nrf2) was gradually degraded, concomitant with time-dependent nuclear accumulation of Nrf2 (Fig. 6B). We also examined the changes in expression of C/EBP isoforms, including α, β, and γ, in the nucleus. Increased nuclear C/EBPα, β, and γ accumulations were detected in ESP-treated cells and elevated expressions were maintained until 24 h (Fig. 6C). Nuclear accumulation of HIF1α was evident 1 h into ESP treatment, reached a maximum level at 15 h, and although it declined at 24 h, it was still higher than that in the untreated control cells (Fig. 6D). To examine whether these ESP-induced transcription factors regulated Prdx6 gene expression, Nrf2, HIF1α, or C/EBPβ

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luciferase reporter assay. Nrf2, HIF1α, and C/EBPβ-driven luciferase activities were markedly elevated even in the absence of ESPs compared with that in the mock transfectants, indicating that these transcription factors function as positive transcriptional regulators of Prdx6 expression (Fig. 7). In particular, the activity induced by C/EBPβ overexpression was  15-fold higher than that induced byNrf2 and HIF1α, suggesting that C/EBPβ is the most active transcription factor. In the presence of ESPs, the luciferase activities enhanced by these transcription factors were further increased by  2-fold. A significant increase in ESP-induced luciferase activity was also noted in a mock vector transfectant, probably because of endogenous Nrf2, C/EBP isoforms, and HIF1α activation. These results indicate that ESP-induced Nrf2, HIF1α, and C/EBPβ transactivate Prdx6 gene expression, thus compensating for its NF-κB-mediated repression. 3.6. Differential distribution of Prdx6 and phospho-NF-κB p65 expression in mouse livers infected with C. sinensis Histopathological changes in the livers of mice infected with C. sinensis are characterized by epithelial hyperplasia, stromal edema, periductal fibrosis, and inflammatory infiltration. Lesion severities are proportional to infective time [24]. To investigate the association of Prdx6 and NF-κB expression with liver lesions, we examined the immunohistochemical Prdx6 and phospho-NF-κB p65 expression in C. sinensis-infected mouse livers. Expression of both proteins was very low in the livers of uninfected mice (Fig. 8). Two weeks post-infection, Prdx 6 was mainly observed in the biliary epithelium surrounding the worm, but phospho-p65 was barely detected in this region. Prdx6 strongly extended into the hyperplasia and inflammatory region at 4 weeks, whereas reactivity of phospho-p65 was intense in the inflammatory region and weak in epithelial and hyperplasia region. By week 8, Prdx6 was still observed in the bile duct epithelium, expanding hyperplasia, and inflammatory region, whereas phospho-p65 reactivity was mainly observed in the inflammatory and neighboring hyperplasia regions and relatively weak in the bile duct epithelium. These results indicated that Prdx6 and NF-κB were distinctively expressed in the early phase after infection, but were later localized together with different expression levels.

4. Discussion Fig. 5. Transcriptional repression of Prdx6-luciferase activity by NF-κB p65 subunit recruitment to κB binding sites within the Prdx6 promoter in ESP-treated HuCCT1 cells. (A) Schematic diagram of the Prdx6 promoter containing two κB binding sites (black boxes). Arrows indicate the positions of primer pairs used for RT-PCR. (B) Cells were cotransfected with the pGL3-hPrdx6 luciferase reporter (100 ng) and the indicated amounts of pcDNA6-myc-p65. The pCDNA6-myc empty vector was added to maintain equal amounts of total transfected DNA. Twenty-four hours post-transfection, cells were harvested and luciferase activity was measured. The average of the relative luciferase activity was divided into the relative reporter luciferase activity without pcDNA6-myc-p65, and the calculated average is shown as a fold change with an SE. *P o 0.05, compared with pGL3-hPrdx6 luciferase activity. (C) ESP-induced occupancy of the NF-κB p65 subunit in the Prdx6 promoter region. Cell lysates transfected with pcDNA6-myc-p65 and treated with 800 ng/ml ESPs for 15 h were analyzed by ChIP using NF-κB p65 antibody or rabbit IgG as a negative control. The immunoprecipitated Prdx6 promoter DNA fragment was PCRamplified using the primer sets specific for κB1 and κB2 regions, respectively. The recruitment of NF-κB p65 to κB1 and κB2 regions were normalized by the input. Data in the graph are presented as means 7 SE for three independent experiments, expressed as a fold enrichment normalized by input (refers to amplification of total cell lysates). *Po 0.05 compared with untreated control. (D) A representative gel image for ChIP assay. The PCR products were separated using 1.5% agarose gel electrophoresis.

expression plasmids and luciferase reporter plasmids were cotransfected into HuCCT1 cells. At 24 h post-cotransfection, cells were incubated for 15 h with or without ESPs and subjected to the

Liver fluke infection is closely associated with inflammation, free radical production, and cholangiocarcinogenesis. This free radical generation constitutes the NF-κB-mediated inflammatory cascade and induction of antioxidant proteins such as Prdxs in both animal and cell infection models [8,11,25]. We investigated the pathophysiologic relationship of two redox-active proteins, Prdx6 and NF-κB, induced by liver fluke infection. Prdx6 overexpression protects various cell types against oxidative stress-induced apoptosis [15,16,26,27], whereas the antisense suppression of this enzyme [28] or ablation of the gene in mice leads to increased sensitivity to oxidative injury [29]. Here wild-type Prdx6 overexpression in HuCCT1 cells, but not the C47S catalytic mutant, markedly decreased intracellular free radical accumulation by attenuating ESP-induced NOX2 and iNOS activation (Figs. 1 and 2), indicating that peroxidase activity is required to reduce ROS and RNS. Peroxynitrite reductase activity of Prdx6 was reported in primary murine macrophages stimulated with IFN-γ and LPS [30]. Decreased NOX2 and iNOS activation is probably associated with Prdx6 overexpression because ROS-mediated NF-κB activity is reduced. This is supported by studies showing that oxidative stress and free radical producing agents are strong inducers of NF-κB activation [31], and that NF-κB functions as a

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Fig. 6. Effects of ESP treatment on the expressions of redox active transcription factors. (A) Schematic diagram of the locations of ARE (vertical striped box), κB-1 and-2 sites (black boxes), two C/EBP responsive elements (white boxes), and a hypoxia-responsive element (HRE, HIF1α binding site, horizontal striped box) within a 1506 bp Prdx6 promoter. (B) After treatment with 800 ng/ml of ESPs for the indicated times, degradation of cytosolic Keap1 and nuclear accumulation of Nrf2 were determined by immunoblot analysis. The membranes were then stripped and re-probed with GAPDH and lamin B antibodies (for controls for protein loading). (C, D) Nuclear proteins from ESP-treated cells for 0–24 h were immunoblotted using polyclonal antibodies against C/EBPα, β, γ, and HIF1α. Lamin B was used as a control for protein loading. Individual data were quantified as densitometric units and normalized with expected loading control proteins. Each dataset represents the relative percentage of the zero time point. Data in graphs are presented as means 7 SE for three independent experiments (*Po 0.05, compared with the zero time point).

positive transcription factor by binding to κB sites present at the promoter region of genes encoding NOX subunits and iNOS [32– 34]. Moreover, it is plausible to speculate that elevated amounts of Prdx6 protein may reduce the translocation of available p67phox protein to the plasma membrane through their cytosolic interactions with consequent attenuation of ESP-induced NOX2

activation, because the interaction of Prdx6 with p67phox has been reported to terminate NOX2 activation signal [35]. We noticed that Prdx6 overexpression had no direct effect on ESP-induced XO activation (Fig. 2D), although it may reduce ROS generated by XO. Free radicals promote NF-κB nuclear translocation and also induce several post-translational modifications that are necessary

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Fig. 7. Role of Nrf2, HIF1α, and C/EBPβ in ESP-induced Prdx6 gene expression. HuCCT1 cells were transiently cotransfected with the luciferase reporter vector (100 ng) and each transcription factor expression (200 ng) plus the β-galactosidase plasmid. After 24 h, cells were treated with 800 ng/ml ESPs for 15 h, and luciferase activities were determined. Luciferase activities were normalized to that of β-galactosidase. (A) Transactivation of Prdx6-luciferase by Nrf2 and HIF1α. (B) Transactivation of Prdx6-luciferase by C/EBPβ. Values shown are means 7 SE for three independent experiments, expressed as a fold change. *P o0.05, compared with mock transfected cells; #P o0.05, ESPtreated versus untreated cells.

for full NF-κB-mediated transcription [36]. In the context of liver fluke infection, treatment with C. sinensis or O. viverrini ESPs results in NF-κB activation with subsequent proinflammatory cytokine generation in CCA cells or human cholangiocytes (H69 cells), respectively [8,37]. Here increased antioxidant ability in Prdx6overexpressing cells attenuated ESP-induced nuclear translocation of p50 and p65 as well as degradation of cytosolic IκB-α, leading to decreased production of proinflammatory cytokines (Figs. 3 and 4). This indicates that Prdx6 is an essential negative regulator of ESP-induced inflammatory signaling through modulation of NF-κB activation via controlling free radical generation. Consistent with this result, a previous study showed that Prdx6 overexpression protected retinal ganglion cells from hypoxia-evoked oxidative stress by eliminating ROS and inhibiting the NF-κB-mediated death pathway [38]. Moreover, targeted Prdx6 gene disruption exacerbated lipopolysaccharide-induced acute lung injury and

inflammation by modulating proinflammatory cytokine release through endogenous ROS-dependent activation of NF-κB, suggesting that Prdx6 functions as an anti-inflammatory enzyme [39]. Analyses of the proximal promoter of the Prdx6 gene have identified binding sites for multiple transcription factors [14,23,40–42], suggesting that complex transcriptional machinery regulates Prdx6 expression. Our study found that NF-κB bound to two κB binding sites in the 1506 bp region of the Prdx6 promoter, leading promoter activity repression, as shown by luciferase reporter and ChIP assays (Fig. 5). The suppressive effect of NF-κB on Prdx6 transcription was also reported in previous studies [13,22]. In addition to NF-κB, expression of other redox-active transcription factors, including Nrf2 and HIF1α, and C/EBPα, β, and γ was increased in response to ESP exposure (Fig. 6). Cotransfection of the Prdx6 promoter construct with each expression plasmid revealed that vector-driven luciferase activities were further

Fig. 8. Time profiles of Prdx6 and NF-κB expression in the liver of C. sinensis infected mice. Expression of Prdx6 and phospho-NF-κBp65 were analyzed by immunohistochemistry, as described in Section 2. Prdx6 and phospho-NF-κB p65 expression is marked with brown-colored deposits and nuclei are counterstained with hematoxylin. The W and CON represent week postinfection and uninfected control, respectively. BD, bile duct; PV, portal vein; Cs, C. sinensis worm; E, epithelial region; H, hyperplasia region; I, inflammatory region. Original magnification  400, scale bar ¼20 mm.

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increased in response to ESPs, reflecting the combined effect of the exogenous addition and endogenous ESP-induced upregulation of each transcription factor on Prdx6 expression (Fig. 7). These results suggest that the coordinated actions of Nrf2, HIF1α, and C/EBPβ as transactivators overcome NF-κB-mediated repression, leading to an increase in ESP-induced Prdx6 expression. Keap1/Nrf2/ARE pathway-mediated upregulation of Prdx6 transcription has been reported [23]. We found two consensus DNA binding sequences of C/EBP (RTTGC.GYAAY; R ¼ A or G, Y ¼C or T) in the Prdx6 promoter, which were located at
1255/
1245 and
698/
687 (Fig. 6A). The former distal sequence appears to be highly conserved and present in almost the same position of porcine, bovine, and human Prdx6 promoters, where C/EBPβ binds to this sequence and upregulates the porcine Prdx6 expression [43]. The latter proximal sequence was located within highly conserved regions and was highly similar to those in rhesus monkey, rat, mouse, canine, bovine, and horse (Pak et al., unpublished data). Moreover, ChIP analysis using a primer set containing this region and a C/EBPβ antibody indicated that the binding ability of C/EBPβ was substantially increased by ESP treatment (data not shown), implying that this sequence may be functionally active as a cis element binding site for C/EBPβ. Because ESP exposure also induced C/ EBPα and γ expression (Fig. 6C), we cannot eliminate the possible involvement of these isoforms in the transcriptional regulation of the Prdx6 gene. A perfectly matched hypoxia response element (HRE) consensus sequence (RCGTG) was observed at position
346 to
341 in the Prdx6 promoter (Fig. 6A). Cells transfected with a promoter/reporter gene construct with an HRE deletion showed a slight but significant decrease in basal and ESP-mediated luciferase activity (data not shown), providing evidence that HIF1α recognizes the HRE within the Prdx6 promoter and contributes to its ESP-induced activity. Multiple redox-active transcription factors are involved in the transcriptional regulation of antioxidant and phase II detoxifying genes such as heme oxygenase-1 (HO-1) and glutathione S-transferase alpha 2 (GSTA2). Transactivation of HO-1 gene is mediated by the binding of Nrf2, HIF1α, and the c-Jun/AP-1 protein complex to their respective motifs within the HO-1 promoter, and transcriptional upregulation of GSTA2 gene is by both Nrf2 and C/EBPβ [44,45]. AREs and C/EBP response elements are notably present in both the Prdx6 and GST promoters. Because the heterodimerization of Prdx6 and GST appears to be required for the peroxidase activity of Prdx6 [46], their common regulatory mechanism would allow efficient dimer formation to reduce various oxidants. The functional interaction between C/EBPβ and NFκB can either synergistically promote or repress transcriptional gene expression involved in immune or acute phase responses, depending on their interactions and their proximal binding sites within the target promoters. [47–49]. The similar interaction of C/ EBPβ with NF-κB may occur during transcriptional regulation of Prdx6 based on the close proximity between the NF-κB- and C/ EBP-binding sites within the Prdx6 promoter (Fig. 6A) and high basal luciferase reporter activity in C/EBPβ-overexpressing cells (Fig. 7B) compared with that in the Nrf2 or HIF1α transfectants. By forming heterodimers, excess C/EBPβ may stop p65 from binding to the κB sites within the Prdx6 promoter, thus abrogating the suppressive effect of NF-κB on Prdx6 transcription. Here the immunoreactivity of Prdx6 and active NF-κB p65 was observed in the bile duct epithelia and the inflammatory region, respectively, of mouse livers infected with C. sinensis in the early phase. By the late phase of infection, Prdx6 expression extended into the hyperplasia and inflammatory regions, and NF-κB p65 was observed in the hyperplasia regions and bile duct epithelia (Fig. 8), consistent with previous reports on O. viverrini-infected hamsters [12,25]. This indicates that the intensity and extension of the expression of both proteins are proportional to lesion severity

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and infection duration. Increased NF-κB activation during the early infection phase may reflect a protective immune response as part of the host's defense. Bile duct epithelial enrichment of Prdx6 during early infection suggests that it is involved in repairing wounds caused by the worm migratory and feeding activities. This is supported by previous reports showing strong induction of Prdx6 in the hyperproliferative epidermis of mouse skin wounds [50] and actively proliferating epithelia and stroma of photorefractive keratectomized rat corneas [51]. Moreover, elevated Prdx6 expression may be a consequence of maintaining host redox homeostasis because C. sinensis infection-mediated mouse liver lesions correlate with the extent of oxidative insults [24]. Therefore, NF-κB and Prdx6 activation during early infection is essential for normal host defense activities such as generating innate immune responses and protecting the host from oxidative stressmediated injuries, respectively. Increased constitutive activation of these proteins during prolonged infection, however, may lead to more severe chronic hepatobiliary abnormalities. Aberrant upregulation of NF-κB and its signaling pathway have been implicated in the pathogenesis of several liver diseases [52]. In liver flukeassociated CCA, NF-κB subunits are strongly expressed in the cytoplasm of hyperplastic/dysplastic and CCA bile duct epithelia, and inhibition of its activation suppresses the growth of CCA cell lines and CCA in inoculated mice via apoptotic signal induction [53]. Given its major role in antioxidant detoxification, it is likely that constitutive upregulation of Prdx6 during chronic infection leads to surviving injured cells adapting to unbalanced redox environments. This then promotes malignant progression through developments such as resistance to oxidative stress-induced apoptosis, enhanced proliferation, and accumulation of mutagenic DNA adducts. Supporting this, Prdx6 overexpression has been reported to attenuate cisplatin-induced apoptosis in ovarian cancer cells [15] and amyloid β-induced neuronal apoptotic cell death [16]. Prdx6transfected breast cancer cells grew much faster and metastasized more readily to lungs than control cells [54]. Prdx6 transgenic mice displayed a greater increase in the growth of lung tumors, compared with normal mice [42]. We have recently shown that the histopathological phenotypes of infected mouse livers have a precancerous appearance [24]. In this regard, perhaps dysregulated Prdx 6 and NF-κB expression facilitates an increased frequency of malignant transformation in chronically infected livers, which favors the development of advanced hepatobiliary diseases such as clonorchiasis-associated CCA. In conclusion, we have shown the inverse correlation between NF-κB activation and Prdx6 expression during C. sinensis infection. Enforced Prdx6 expression suppressed NF-κB-mediated inflammation by attenuating the accumulation of enzymatically generated free radicals. Transcriptional repression of Prdx6 via NFκB activation was compensated for by the activation of other redox-active transcription factors such as Nrf2, HIF1α, and C/EBPβ in response to ESP exposure. The intensity and extent of immunoreactive NF-κB and Prdx6 staining in infected livers were proportional to lesion severity and infection duration, suggesting that their constitutive elevations may be associated with more severe persistent hepatobiliary abnormalities. A more complete understanding of the pathophysiologic roles of NF-κB and Prdx6 in malignant progression in the biliary system during chronic infection provides opportunities to prevent advanced clonorchiasisassociated disease pathogenesis and cholangiocarcinogenesis.

Author disclosure statement No competing financial interests exist.

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Acknowledgments The authors thank Ju Hyun Moon for technical assistance and Sejung Maeng for figure rearrangement and reference formatting. This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (No. 2012R1A2A2A01014237) and the Korea National Institute of Health, Ministry of Health and Welfare, Korea (2011E5401100).

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