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Current Molecular Medicine 2014, 14, 772-782

The Mitochondrial Thioredoxin is Required for Liver Development in Zebrafish J. Zhang1,2,§, X. Cui2,§, L. Wang1, F. Liu1, T. Jiang1, C. Li1, D. Li1, M. Huang1, S. Liao1, J. Wang1, J. Chen1, H. Jia1, J. He3, Z. Tang1, Z. Yin*,3 and M. Liu*,1 1

Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China

2

Key Laboratory of Cellular and Molecular Immunology, Institute of Immunology, Medical College of Henan University, Kaifeng, Henan, P.R. China 3

Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, P.R. China Abstract: Thioredoxins (Trxs) are a class of small molecular redox proteins that play an important role in scavenging abnormally accumulated reactive oxygen species (ROS). Thioredoxin 2 (Trx2) is one member of this family located in mitochondria. Trx2 protects cells from increased oxidative stress and has anti-apoptosis function. Knockout of Trx2 in mice led to early embryonic lethality. However, the essential role of Trx2 during embryogenesis remains unclear. To further investigate the role of Trx2 during embryonic development, we performed Trx2 knockdown in zebrafish and investigated the regulation role of Trx2 during embryonic development. Our results indicate that Trx2 had a high expression in early zebrafish embryos and its knockdown in zebrafish led to defective liver development mainly due to increased hepatic cell death. The increased ROS and the imbalance of members of the Bcl-2 family were involved in cell death induced by Trx2 suppression in zebrafish. The dysregulation of Bax, puma and Bcl-xl promoted the reduction of mitochondrial trans-membrane potential and the mitochondria membrane permeabilization (MMP), which initiated the mitochondrial apoptosis pathway. Additionally, we found that the increase of relocated GAPDH in mitochondria may be another factor responsible for the mitochondrial catastrophe.

Keywords: Apoptosis, Bcl-2 family, liver morphogenesis, MPP, Trx2, zebrafish.

INTRODUCTION Thioredoxins (Trxs) maintain the balance of cellular redox status to prevent cells from oxidative stress. They also have a crucial role in apoptosis, neuroprotection and inflammatory reaction [1-3]. To execute these functions, Trxs reduce oxidized substrate proteins by breaking the disulfide bonds with the Trp-Cys-Gly-Pro-Cys catalytic center [4]. Trx2 is a member of the Trxs family and is specifically localized in the mitochondria [5]. The mitochondria are the main source of reactive oxygen species (ROS) production due to the existence of electron transport chain and oxidative phosphorylation [6]. With the cooperation of TrxR2 and peroxiredoxin, the Trx2 anti-oxidative system plays an important role in scavenging aberrant excessive ROS in the mitochondria [5, 7, 8]. To date, several reports mentioned the anti-apoptosis biological function of Trx2. Overexpression of Trx2 in *Address correspondence to these authors at the (M. Liu) College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, 1037 Luoyu Rd, Wuhan, Hubei, 430074, P.R. China; Tel.: 86-027-87792649; Fax: 8627-87794549; E-mail: [email protected] and (Z. Yin) Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, P.R. China; E-mail: [email protected] §

Equal contribution authors.

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osteosarcoma cells protected against tBH (tert butyl hydroperoxide) induced cell cytotoxicity [9]. Trx2 protected cells from etoposide-induced cell death and increased MMP mitochondrial membrane potential (MMP) in HEK-293 cell [10]. Conditional knockout of Trx2 in chicken DT40 cells caused elevated ROS levels and cell apoptosis [11]. In homozygous null mice, the absence of mitochondrial thioredoxin 2 led to massive apoptosis and early embryonic lethality [12]. The -/TrxR2 mice embryos could not survive at embryonic day 13 and showed increased apoptosis in the liver +/mice indicated that Trx2 [13]. Studies in Trx2 haploinsufficiency increased oxidative stress and impaired mitochondria function, especially in liver [14]. It is important that the mentioned results above mainly relate to the function of Trx2 against oxidative stress. Also utilizing the DT40 cell model, Wang et al. proposed that Trx2 controlled the Bcl-xl levels and this concerned the cell viability. Conditional Trx2-deficient DT40 cells had a lower expression level of Bcl-xl and showed more cell apoptosis. Meanwhile, the regulation of Bcl-xl levels by Trx2 seemed a little relationship with the conserved active center [15]. Bcl-xl belongs to the BCL-2 family, which could be divided into three groups: pro-apoptosis proteins with multiple BH regions such as Bax/Bak, anti-apoptosis proteins with three or four BH3 domains such as Bcl-2, Bcl-xl and Mcl-2, and those with only one BH3 domain such as puma, Bid © 2014 Bentham Science Publishers

Liver Development in Zebrafish

and Bim [16]. The members of the BCL-2 family play a crucial role in inducing MMP and the intrinsic mitochondrial apoptotic pathway [16-18]. Bax, a famous pro-apoptosis protein, could relocate to the mitochondrial outer membrane with the help of BH3only puma, Bid and /or Bim and then trigger MMP [17, 19]. Bcl-xl could directly interact with Bax on the mitochondria outer membrane (MOM) or release Bax into cytoplasm to prevent Bax oligomerization [16, 20]. Bcl-xl also could prevent Bid from interacting with Bax [21]. Puma, a BH3-only protein, transduces death signals to mitochondria through bax/bak, directly or indirectly. It could activate bax by relieving its interaction with anti-apoptotic members of the Bcl-2 family [22]. Puma could also trigger apoptosis by directly activating bax or cytoplasmic p53 in some cell lines and even induce mitochondrial autophagy depending on bax [23-25]. To thoroughly understand the biological function of Trx2, we conducted Trx2 knockdown in zebrafish using the morpholino technology. Our results showed that Trx2 had a wide expression pattern in zebrafish and an obvious expression signal in early liver. The liver morphogenesis in zebrafish could be divided into two phases: the budding stage and the growth stage. The budding stage begins from 24 hpf (hours post fertilization) and completes at about 50 hpf. At the budding stage, firstly the intestinal rod at the first somite thickens and the liver bud emerges, then the furrow between the liver bud and the adjacent esophagus forms, and lastly hepatic duct is shaped. In the growth stage, the hepatic cells have a rapid proliferation and the liver changed dramatically in size [26]. Knocking down the expression of Trx2 in zebrafish by morpholino led to their livers’ reduction in shape and size mainly due to liver cell death. We demonstrated that the increased ROS was one of the reasons for the hepatic cell death. Furthermore, there were abnormal levels of Bcl-xl, Bax and puma, which accounted for the elevated MMP and the activation of mitochondrial apoptotic pathway. Our results were the first study on the relationship between Trx2 and the BCL-2 family in vivo. This suggested that in addition to ROS, we may need to pay more attention to the genes related to MMP in further Trx2 biological functions research. Surprisingly, we found that GAPDH relocated more to the mitochondria when Trx2 was cut off. This may worsen the mitochondrial catastrophe.

METHODS AND MATERIALS

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were synthesized by GeneTools. 1-2 nl volume (0.5 mM) MOs were injected into one-two cell stage zebrafish embryos. Whole-Mount In Situ Hybridization The whole-mount in situ hybridization was performed as described in previous studies [27]. Briefly, zebrafish embryos were collected and fixed overnight at 4°C. The first day, embryos were gradiently rehydrated with methanol and PBST. Then embryos were digested with proteinase K, and prehybridized for 1-3 hours and then incubated with proper probes. The second day, probes were recycled and embryos were incubated with hybridization buffer (without tRNA and heparin) at 65°C, 30 min for 4 times. Embryos were washed with 2×SSCT for 15 min and 0.2×SSCT for 30 min. After that, embryos were washed with PBST, blocked for at least 2 hours and incubated in anti-DIG antibodies (Roche) overnight at 4°C. The third day embryos were washed with PBST and stained with NBT/NCIP staining buffer (Roche). All antisense RNA probes were synthesized using T7 or SP6 RNA polymerases and labeled with digoxigenin-UTP (Roche, Mannheim, Germany). Probes used in the study include: lfabp (liver), ifabp (intestine), trypsin (exocrine pancreas), insulin (endocrine pancreas), foxA3, gata6, hhex and prox1. The primers for Trx2 probe are as followings: 5’-GCAGCTCCAGCAGGT CCTCGT-3’ and 5’-GG CGGGCGGAGACTGACAG-3’. Immunostaining and TUNEL Assay Zebrafish embryos at 33 hpf were fixed in 4% PFA at 4°C overnight, infiltrated by gradient sucrose and embedded in OCT. Then 6 µm cryosections were prepared for immunostaining using LEICA CM1900. The sections were blocked with PBDT (PBS containing 1% BSA, 1% DMSO and 0.1% TritonX-100) for 1 hour at room temperature, followed by incubation with PH3 antibodies at 4 °C overnight to detect cell proliferation [28, 29]. After washing with PBDT, the appropriate secondary antibodies were applied. The nuclei were stained with DAPI for 5 minutes. The TUNEL assay was carried out using the DeadEnd™ Fluorometric TUNEL System (Promega) [28, 29]. Briefly, the cryosections were fixed in 4% PFA for 15 minutes, then washed with PBS and immersed in 20 µg/ml proteinase K solution (8-10 minutes) for permeabilization. Then cryosections were equilibrated and labeled with reagents according to the product manual followed by DAPI staining.

Zebrafish Lines, Antisense MO Oligonucleotides Zebrafish were maintained under standard laboratory conditions. The embryos were collected from naturally mating zebrafish and the developmental stages were measured according to hpf. A translation blocking (ATG) morpholino (MO1, 5’-GCGAGCAGCC GGAACGCCATCGTGA-3’) and a splicing morpholino (MO2, 5’-GCCATCTGAGAAACACACAGAGAGA-3’) were applied to suppress Trx2. A stand control morpholino (CMO, 5’-CCTCTTACCTCAGTTACAATTTATA-3’) was also used in our experiment. All morpholinos (MOs)

Mitochondria Purification and JC-1 Staining Mitochondria Isolation Kit was used to isolate mitochondria in zebrafish embryos. First, zebrafish yolk were deprived by the deyolked buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3) and washed twice with the wash buffer (110 mM NaCl 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris-HCl PH 8.5). About 0.5 mg (about 250-300 injected embryos) embryos at 60 hpf were homogenized in 800 ul extraction buffer A followed by 600 g and 11000 g centrifugation. The mitochondrial

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pellet was resuspended in storage buffer. Part of the mitochondrial suspension was used for a BCA-based protein concentration assay. Suitable mitochondrial suspension (about 100 ug protein) was used for JC-1 staining (Mitochondrial membrane potential assay kit, Beyotime). Red and green fluorescence were detected using Multi-Function Measuring Instrument (SpectraMax M5, Molecular Devices) setting as followings: Excitation wavelength = 490 nm, Emission wavelength = 590 nm for red fluorescence and Excitation wavelength = 490 nm, Emission wavelength = 538 nm for green fluorescence. H2O2 Concentration Detection and Caspase Activity Assay H2O2 production levels were measured by Hydrogen Peroxide Assay Kit (Beyotime). Fifty embryos at desired stages were homogenized with 200 ul lysis buffer in the kit. After 12000 g centrifugation for 4 min at 4°C, supernatant was incubated with Hydrogen Peroxide assay buffer for 50 min. A560 was detected and then H2O2 concentration was calculated by standard curve. Fifty injected embryos were used to generate protein lysates as described [30]. Briefly, 48 hpf embryos were homogenized in 200 ul lysis buffer (20 mM Hepes-KOH, pH 7.5, 250 mM saccharose, 50 mM KCl, 2.5 mM MgCl2, and 1 mM dithiothreitol) and after centrifuged at 10000 rpm for 15 min at 4°C, supernatant was diluted 20 fold. Caspase-3 and caspase-9 activities in the lysates were measured with the Caspase-Glo kit (Promega), according to the manufacturer's protocol. Luminescence was measured on a Microplate Luminometer (Orion–L2, BERTHORD). Immunoblot Analysis and RT-PCR We performed immunoblot analysis and RT-PCR as we described previously [31]. Mitochondrial lysis buffer in the kit was used to prepare mitochondrial protein for immunoblot. The VDAC1 antibody as the control mitochondria protein was purchased from Proteintech (#10866-1-AP), and Bcl-xl (#10783-1-AP), Bcl-2 (#12789-1-AP), Bax (#50599-2-Ig), puma (#55120-1AP) and GAPDH (#10494-1-AP) antibodies were also from Proteintech. The polyclonal Trx2 antibody for HepG2 cell line was from Abnova (#PAB20026), and PH3 for Immunostaining was purchased from Santa Cruz. The HepG2 cells were cultured in DMEM medium with 10% FBS (Invitrogen). The small interfering RNA against human Trx2 was synthesized by RIBOBIO, and the sequence is: 5’-GGACGUGGUGGA CAAGUUU-3’.

RESULTS Trx2 Expressed in the Liver of Zebrafish Zebrafish Trx2 (NM_205641) has 166 amino acids and the conserved redox active site Trp-Cys-Gly-ProCys, the same as human Trx2. The mature protein shares significant homology with human Trx2 (75%

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identical, blasted in NCBI). Reverse transcription PCR (RT-PCR) analysis revealed that Trx2 is expressed in all tissues of adult zebrafish, including the liver and gut (Fig. 1A). Results from Real-time PCR also indicated that Trx2 maintained high levels throughout the embryogenesis (Fig. 1B). Then whole mount in situ hybridization (WISH) was performed and the results suggested that Trx2 had a maternal expression pattern at 4-cell stage and expressed ubiquitously at 50%epiboly stage (Fig. 1C1, C2). At 24 hours post fertilization (hpf), Trx2 expressed widely, and especially in somites and brain in embryos (Fig. 1C3). The Trx2 signals in somites weakened while strong signals could be observed in the liver bud of zebrafish embryos at 36 hpf (Fig. 1C4, C5). Signals could be found in the liver and also the gut of embryos at 72h (Fig. 1C6). High expression of Trx2 in liver was seen from the sections of Trx2 WISH embryos at 36 hpf and 72 hpf (Fig. 1D). Because of Trx2’s highest expression level in infant +/human liver and the vulnerable liver in Trx2 mice, we mainly analyzed zebrafish liver in this study to figure out Trx2’s function during embryogenesis [10, 14]. Trx2 is Required for Liver Morphogenesis in Zebrafish To determine the function of Trx2 during the liver development, an antisense morpholino oligonucleotide (Trx2 MO1) targeted to the translation start site of Trx2 was synthesized. The efficiency of Trx2 MO1 (ATG MO) was detected by co-injection with Trx2: EGFP reporters (Fig. S1A). As shown in Fig. (S1), EGFP was abolished by the co-injection of Trx2 MO1, showing that Trx2 MO1 specifically binds to the available binding site. There was no obvious development delay during gastrula stage and early segmentation period: Trx2 morphants all developed to 5 somites at the same stages as control embryos did (Fig. S1B). Then WISH was performed with the marker lfabp (liver fatty acid binding protein) to visualize the liver. As shown in Fig. (2A), the expression of lfabp sharply diminished in Trx2 morphants (more than 90% embryos) at 72 hpf and the liver in morphants was much smaller in shape and size. We also carried out WISH using the marker ifabp (intestinal fatty acid binding protein) for intestines, trypsin and insulin for pancreas. The results suggested that except for endocrine pancreas, intestines and exocrine pancreas were also reduced in size in Trx2 morphants (Fig. 2B-2D). Moreover, the liver reduction was clearly shown in the Trx2 MO1 injected EGFP transgenic embryos of the MP760 line (a kind gift from Shuo Lin, Fig. 2E). To further determine the specificity of liver defect, a splicing MO targeted to the intron 1/exon 2 boundary of Trx2 (Trx2 MO2) was synthesized and injected into zebrafish embryos. WISH analysis with lfabp probe indicated that a significant reduction in liver (about 70%) could also be observed in the splicing MO injected embryos at 72 hpf (Fig. 2F). Meanwhile, the liver defect could be partially rescued by injection of Trx2 mRNA (Fig. 2F). The intestine and exocrine



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Fig. (1). Trx2 expression in adult and embryonic zebrafish. (A) RT-PCR analysis was carried to determine the Trx2 expression levels in major tissues of adult zebrafish (top panel), with the mRNA levels of β-actin as the control. (B) Real-time PCR were performed to detect the expression levels of Trx2 during the zebrafish embryos development. (C) Trx2 expression patterns were detected in embryos at 4-cells (C1), 6 hpf (C2), 24 hpf (C3), 36 hpf (C4 and C5) and 72 hpf (C6) by in situ hybridization, respectively. Embryos in C2, C3, C4 and C6 were lateral view with the anterior to the left, and embryo in C1 and C5 was dorsal view with anterior to the top. Black arrows indicate the liver bud. (D) In situ hybridization of frozen sectioned zebrafish embryos at 36 and 72 hpf. L, liver; nt, neural tube; bs, body somites; nc, notochord; ai, anterior intestine.

pancreas were also affected in embryos injected with Trx2 MO2 (Fig. S2). However, as Trx2 MO2 had a lower efficiency than MO1,Trx2 MO1 was preferred in the following studies. Knockdown of Trx2 Blocks the Liver Growth, Not the Budding During zebrafish embryonic development, the liver morphogenesis could be divided into two general phases: budding (composed of specification and initiation) and growth [32]. Failure in liver budding and liver growth could both lead to liver reduction in Trx2 morphants. To test these possibilities, WISH was first performed on Trx2 morphants using markers foxA3 and gata6, which are responsible for hepatic cells specification. At 30 hpf, there was minimal difference in signal intensity of foxA3 and gata6 between WT and

Trx2 morphants. However, they stayed in the similar expression patterns. At 48 hpf, hepatic primodium of the Trx2 morphants stay almost the same size and shape as at 30 hpf, while a normal expansion growth of liver was observed in the control morphants (Fig. 3A, B). Meanwhile, the signals of hhex and prox1, which are required for the liver budding, suggested a complete liver budding but an abnormal liver growth in embryos injected with Trx2 MO1 (Fig. 3C, D). These results suggested that the Trx2 MO1 knockdown had little effect on liver budding, but affected liver outgrowth and expansion instead. Suppression of Zebrafish Trx2 Induced Apoptosis of Liver Cells The liver size reduction in Trx2 morphants could be resulted from either the reduced proliferation or the increased apoptosis or both. To unveil the detailed

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Fig. (2). Trx2 is required for liver morphogenesis. (A to D) In situ hybridization assay with specific markers for liver (lfabp, A), intestine (ifabp, B), exocrine pancreas (tryspin, C) and endocrine pancreas (insulin, D) in CMO (control morpholino) and Trx2 MO1 morphants at 72 hpf. (E) Trx2 knockdown in EGFP transgenic embryos of MP760 line also showed a much smaller liver than those in control group at 72 hpf. (F) Trx2 splicing morpholino (Trx2 MO2) showed identical liver defect compared with Trx2 MO1. lfabp expression liver tissue became restricted in Trx2 MO2 morphants at 72 hpf, and the liver reduction could be partially rescued by Trx2 mRNA. The embryos with small liver were counted and the error bars mean +/- 1.00 SD (F, right). Embryos in A-D and F were dorsal view with anterior to the top; embryos in E, lateral view with the anterior to the left. White arrows indicate the liver.

mechanism of the phenotype, immunofluorescence analysis was carried out with anti-phosphorylated Histone 3 antibody (PH3), a marker of proliferating cells. As shown in Fig. (4), the liver buds were discriminated by the process of liver primodium formation as described before [26, 29]. Our data showed that the number of PH3 positive cells in frozen sections of Trx2 morphants is in

close proximity to that in control embryos (Fig. 4A). Then we performed TUNEL analysis to assay the apoptotic liver cells in Trx2 morphants at 33 hpf. Our observation showed that there were enhanced apoptosis signals in the liver bud of Trx2 morphant sections, whereas apoptotic cells could barely be identified in controls (Fig. 4B).



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Fig. (3). Trx2 knockdown blocked the liver growth not the budding in zebrafish embryos. (A to D) In situ hybridization on the control and Trx2 MO1 injected embryos at 30 hpf and 48 hpf using hepatic specification markers foxA3 (A) and gata6 (B), and liver budding markers hhex (C) and prox1 (D). Upper-left arrow: liver; Right arrow: pancreas; Lower-left arrow: intestine. Dorsal view of embryos was with anterior to the top.

The major biological function of Trx2 is to ensure the redox balance in mitochondria, and hydrogen peroxide is a reactive oxygen metabolic product. So, we investigated the H2O2 release levels to measure the production of ROS. As shown in Fig. (4C), suppression of Trx2 induced more H2O2 release, which indicated more ROS production. These results suggest that the block of liver growth in Trx2 morphants is mainly due to apoptosis of liver cells rather than the defect of cell proliferation. Meanwhile, ROS accumulation was also one of the reasons for the small liver. Trx2 Control Bcl-xl, Bax and Puma Levels in Mitochondria The reported anti-apoptosis biological function of Trx2 was mainly scavenging the abnormally increased ROS. Wang et al. reported that Trx2 could control the Bcl-xl levels, which correlated with the DT40 cells viability [15]. The question whether BCL-2 family members were regulated by Trx2 still remains debatable, and especially requires studies in vivo. It was necessary to know the exactly change of BCL-2 family members in mitochondria, not in the whole cells. Zebrafish mitochondria were purified and Bcl-xl levels were first detected. We found that in Trx2 morphants,

Bcl-xl protein levels significantly decreased while Bcl-2, another anti-apoptosis member, changed slightly (Fig. 5A). Next we wondered how the expression of proapoptosis members of Bcl-2 family in mitochondria changed. As shown in Fig. (5B), the levels of Bax and puma increased significantly in mitochondria of Trx2 morphants. Bax and puma are well known proapoptosis members located in mitochondria whose significant increase induces the mitochondrial apoptotic pathway. To confirm the increase of Bax and puma induced by TRX2 deletion, small interfering RNA (siRNA) were explored to block human TRX2 in HepG2 cells. We found that human Bax and puma also increased when TRX2 was knockdown in HepG2 cells (Fig. S3). These results indicated that Trx2 was involved in maintaining the rational expression levels of BCL-2 family and the imbalance may account for the impaired mitochondria function. Trx2 Knockdown Induced Reduced ΔΨm and Activation of Mitochondrial Apoptotic Pathway MMP (mitochondrial membrane permeabilization) is a decisive event during the activation of the cell death pathway and a complex process closely related to BCL-2 family. It is reported that Bax could permeabilize

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Fig. (4). Trx2 knockdown induced cell apoptosis in liver cells and high ROS levels. (A) An anti-PH3 antibody was used to detect proliferated cells (green) in CMO and Trx2 morphants. (B) TUNEL assay was performed to detect the cell apoptosis in CMO and Trx2 morphants. The liver primordium and the adjacent endoderm region at 33 hpf were labeled with white circles. The nuclei were stained with DAPI (blue) in A and B. The PH3 and TUNEL positive liver cells were counted (5 embryos) and the error bars mean +/- 1.00 SD, *P