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Localization of Thioredoxin Reductase and Thioredoxin in Normal Human Placenta and Their Protective Effect Against Oxidative Stress
Placenta (1999), 20, 95–101
Localization of Thioredoxin Reductase and Thioredoxin in Normal Human Placenta and Their Protective Effect Against Oxidative Stress K. Ejimaa,b,c, H. Nanria, N. Tokib, M. Kashimurab and M. Ikedaa Departments of a Health Development and Health, Kitakyushu 807, Japan Paper accepted 1 July 1998
b
Obstetrics and Gynecology, University of Occupational and Environmental
Recent studies have indicated that oxidative stress is involved in the pathogenesis of pre-eclampsia. Oxidative stress damages systemic tissues, and placental damage may result in intrauterine growth retardation and fetal distress. Thus, this study attempted to elucidate the placental localization of thioredoxin and thioredoxin reductase, substances that may reduce oxidative stress. Furthermore, it studied the defence mechanism of the thioredoxin–thioredoxin reductase system against oxidative stress in mitochondria of normal human placenta where reactive oxygen species are primarily produced. The examination of thioredoxin reductase activity in subcellular fractions of human placenta indicated that thioredoxin reductase was located not only in cytoplasm, but also in mitochondria. The existence of thioredoxin and thioredoxin reductase in human placenta was confirmed immunologically using antibodies raised against thioredoxin and thioredoxin reductase. Thioredoxin and thioredoxin reductase were localized histochemically in cytotrophoblasts, decidua, and stromal cells in the stem villi. The addition of exogenous thioredoxin and thioredoxin reductase to fumarase in mitochondria of human placenta displayed a protective effect against oxidative stress. In conclusion, this study confirmed the intracellular localization and the tissue distribution of thioredoxin and thioredoxin reductase in human placenta. Moreover, the complete thioredoxin–thioredoxin reductase system in human placenta may protect the placenta from damage caused by oxidative stress. 1999 W. B. Saunders Company Ltd Placenta (1999), 20, 95–101
INTRODUCTION The thioredoxin–thioredoxin reductase system is a general protein disulphide oxidoreductase ubiquitously present in all living cells (Luthman and Holmgren, 1982). Thioredoxin reduced to a dithiol by thioredoxin reductase and NADPH is involved in the reduction of ribonucleotides (Laurent, Moore and Reichard, 1964), the activation of several receptors (Grippo et al., 1983; Tagaya et al., 1989), the stimulation of cell proliferation (Wakasugi et al., 1990; Rubaretelli, Bonifaci and Suita, 1995), the regulation of transcriptional factors (Abate et al., 1990; Hayashi, Ueno and Okamoto, 1993), and the maintenance of normal pregnancy as a component of the early pregnancy factor described by Clarke et al. (1991) and Matsui et al. (1996). Furthermore, the thioredoxin– thioredoxin reductase system has recently been implicated in the defence against oxidative stress, for example, in the repair of oxidatively damaged proteins (Fernando et al., 1992; Yoshitake et al., 1994) and the elimination of peroxides (Björnstedt et al., 1994). However, the subcellular localization and function of thioredoxin reductase and thioredoxin in placenta were unknown. c To whom correspondence should be addressed at: Department of Health Development, University of Occupational and Environmental Health, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu 807, Japan.
0143–4004/99/010095+07 $12.00/0
All living tissues under aerobic conditions face the continuous threat of not only exogenous but also endogenous reactive oxygen species (ROS) (Gille, Vanberkel and Joenje, 1994; Richter et al., 1995). An imbalance between the production and elimination of ROS, which are unavoidable products of normal metabolic processes, can cause lethal dysfunction of systemic tissue cells as a result of damage to proteins (Ischiropoulos and al-Mehdi, 1995), nucleic acids (Janssen et al., 1993) and lipids (Ueda and Shah, 1992). It has recently been reported that lipid peroxidation may contribute to endothelial cell dysfunction in pre-eclampsia (Hubel et al., 1996) and placental damage due to oxidative stress might result in intrauterine growth retardation and fetal distress. The localization of thioredoxin and thioredoxin reductase in female genital organs is generally accepted (Rozell et al., 1985; Fujii et al., 1991). Furthermore, it has been reported that thioredoxin is highly expressed in cytotrophoblast cells and stromal cells in early placenta (Perkins et al., 1995). However, the localization of thioredoxin reductase in normal placenta remained unknown. Therefore, we examined the intracellular localization and tissue distribution of thioredoxin reductase in placenta, using antibodies raised against purified thioredoxin reductase, and investigated the defence mechanism of thioredoxin and thioredoxin reductase against oxidative stress in uteroplacental circulation. 1999 W. B. Saunders Company Ltd
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This study suggests that the thioredoxin–thioredoxin reductase system in placenta may provide a protective function against oxidative stress in mitochondria where reactive oxygen species are primarily produced as a byproduct of aerobic energy metabolism (Takeshige and Manakami, 1979).
MATERIALS AND METHODS Tissue preparation First trimester placentae (n=3) were collected from legal abortions at 10 weeks of gestation according to the Helsinki guidelines, and normal placentae (n=3) were obtained at term from normal healthy women with normal deliveries of caesarean sections under informed consent, confirmed in all patient cases. The basal membrane and blood vessels were teased away and adhering blood was removed by several washings with 0.9 per cent NaCl. The materials were chilled in ice, and processed immediately for fractionation and for the histochemical study.
Fractionation of human placenta The following procedures were performed at 4C. Human placentae were cut into pieces after removing blood and endothelium; the tissue was minced and suspended in 3 vol of cold 0.25 sucrose, 0.185 per cent K2HPO4. The pH of the mixture was adjusted to about 7.5 by quickly adding 6 KOH and the suspension was homogenized with a food mixer at maximal output for 2 min, following by centrifugation for 10 min at 2000 g. After discarding the precipitate, the supernatant (post-nuclear supernatant) was filtered through four layers of gauze and centrifuged for 40 min at 11 000 g, and then filtered again. The organelle pellet was homogenized with 0.25 sucrose using Potter–Elvehjem-type homogenizer, suspended in the same buffer, and centrifuged for 40 min at 11 000 g. This procedure was repeated twice. The washed pellet was suspended in 20 ml of 0.25 sucrose, 2 m EDTA (pH 7.5), and stored at 20C for the mitochondria preparation. The supernatant was centrifuged for 1 h at 77 000 g and washed with 0.25 sucrose three thimes. The organelle pellet was collected for the preparation of microsomes. The supernatant was collected for the preparation of cytosol. The activities of lactate dehydrogenase (Bergmeyer, Bernt and Hess, 1965), succinate cytochrome C reductase (Schnitman and Greenawalt, 1968) and fumarase (Gellera et al., 1990) were measured as described for validating the purity of cytosol, mitochondria and mitochondrial matrix.
Thioredoxin reductase assays Thioredoxin reductase was assayed routinely by measuring the rate of reduction of 5,5 -dithiobis-(2-nitrobenzoate) (DTNB) by NADPH spectrophotometrically at 412 nm (Holmgren, 1977). The assay mixture contained 100 m potassium phos-
phate (pH 7.0), 10 m EDTA, 0.2 m NADPH, 0.2 mg/ml of serum albumin, 2.5 m DTNB and enzyme in a final volume of 1 ml at 30C. Enzyme activity in this assay was expressed as ìmol of thionitrobenzoate produced per minute, which is equivalent to 2ìmol NADPH oxidized per minute. Purification of thioredoxin reductase and thioredoxin, and preparation of antiserum against thioredoxin reductase and thioredoxin Thioredoxin reductase and thioredoxin were purified from bovine heart and liver according to the method described by Luthman and Holmgren (1982). The preparations of purified thioredoxin reductase and thioredoxin were homogeneous as judged by SDS/PAGE. Each antiserum was prepared by immunization of an albino rabbit. For primary immunization, 100 ìg of thioredoxin reductase and the C-terminal polypeptide of thioredoxin were emulsified with Titer Max (an adjuvant), and injected subcutaneously. Four weeks later, each rabbit was boosted with 100 ìg of thioredoxin reductase and the C-terminal polypeptide of thioredoxin emulsified with Titer Max. The rabbits were boosted twice at intervals of 2 weeks. Antiserum was then collected 2 weeks after the last booster. Immunoblotting of thioredoxin reductase and thioredoxin in cytosolic and mitochondrial fractions from human placenta with antibodies raised against thioredoxin reductase and thioredoxin revealed almost a single immunoreactive band. Western blotting analysis of subcellular fractions Thioredoxin and thioredoxin reductase expressions were determined by immunoblotting. An equal protein concentration from each sample was then boiled in a mixture of 1 per cent 2-mercaptoethanol and 0.05 per cent bromophenol blue for 3 min. Proteins were separated by SDS/PAGE using a 10 per cent acrylamide resolving gel for thioredoxin reductase and a 15 per cent gel for thioredoxin. Separated proteins were then transferred to a nitrocellulose membrane for use in enhanced chemiluminescence (ECL) Western blotting and probed with the polyclonal rabbit anti-bovine heart thioredoxin reductase antibody and an antibody to the C-terminal polypeptide of thioredoxin. A horseradish peroxidase-conjugated goat antirabbit secondary antibody was used in conjunction with enhanced chemiluminescence to visualize the thioredoxin reductase and thioredoxin bands on autoradiography film. The intensity of the stained bands was quantified by densitometric analysis using the public-domain computer program NIH Image and Color Magician (Wayne Rasband, NIH, Research Service Branch, NIMH, USA). Immunohistochemical staining The specimens of normal placental tissues were frozen in isopentane, cooled with liquid nitrogen, and stored at 80C
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Table 1. Cytosolic and mitochondrial thioredoxin reductase activities of human early and term placenta Thioredoxin reductase
Cytosol E T Mitochondria E T
Total activity (per cent)
Specific activity (per cent)
LDH (per cent)
S-C (per cent)
100a 100b
28.61.9* 14.41.1
100c 100d
9.7 4.0
8.8 8.81.1
12.51.3* 6.50.9
ND 11.0
100e 100f
0.2 U/1 g of placenta; b0.4 U/1 g of placenta; c570 mU/mg protein; d192 mU/mg protein; e85.6 mU/mg protein; 35.3 mU/mg protein. LDH, lactate dehydrogenase; S-C, succinate-cytochrome C reductase; E, early placenta; T, term placenta; ND, not detectable. Total activities, defined as total amounts of thioredoxin reductase activities in subcellular fractions derived from 1 g of placenta, were expressed as per cent of the activity in cytosol. Specific activity was expressed as activity units per mg of protein in each fraction. The values are expressed as means of two independent experiments or meanss.d. Statistical comparison was performed by Student’s t-test. *P > 0.05 was considered statistically significant.
a f
as soon as possible. For immunohistochemical studies to demonstrate the localization of thioredoxin reductase and thioredoxin, the specimens were serially sectioned at 8 ìm. Primary antibodies used in this study were rabbit antisera against purified thioredoxin reductase and C-terminal thioredoxin. Bound antibodies were detected by the indirect avidinbiotin complex method, by using a Vectastain Elite kit (Vecter Laboratories Inc., Burlingame, CA, USA). The sections were rinsed with phosphate-buffered saline (PBS) (0.01 m NaPi, 0.15 NaCl, pH 7.4), fixed with acetone for 10 min, and then rinsed again with PBS. The sections were treated with 1 per cent hydrogen peroxide (H2O2) in methanol for 30 min at room temperature to eliminate endogenous peroxidase activity. After washing with PBS and incubating with normal goat serum (1 : 200) for 30 min, each section was incubated with serum from a rabbit immunized with purified thioredoxin reductase (1 : 100) for 1 h, followed by secondary biotinlabelled anti-rabbit Ig G (1 : 100) at room temperature. After washing with PBS, avidin-biotin complex (ABC) was applied for 30 min. Finally, brown staining was performed by a 5-min treatment with 0.05 per cent 3,3-diaminobenzidine tetrahydrochloride in 0.01 Tris-HCl buffer (pH 7.5) containing 0.01 per cent H2O2. Parallel sections of this study served as negative controls, in which the primary antiserum was omitted and replaced with either diluted normal rabbit serum or PBS. Counterstaining was performed with haematoxylin.
Exposure of mitochondria to hydrogen peroxide Mitochondria (3 mg/ml) were prepared from human placentae and treated with 880 m H2O2 for 0–60 min. Fumarase activities (Gellera et al., 1990) of the H2O2-treated mitochondria were measured in the presence and absence of the
complete thioredoxin–thioredoxin reductase system (composed of purified thioredoxin, 10 ì; thioredoxin reductase; 256 n; and NADPH, 2 m).
Other analytical methods Protein concentrations were determined by the Bio-Rad protein assay kit with bovine serum albumin as a standard. The values are presented as the mean or means.d. Statistical comparison was performed by Student’s t-test. P<0.05 was considered statistically significant.
RESULTS Thioredoxin reductase activity in human placenta Subcellular distribution of human placental thioredoxin reductase. Human placenta was separated into cytosolic and mitochondrial fractions characterized by the existence of specific marker enzymes; most lactate dehydrogenase was present in the cytosolic fraction, while almost all of the succinate-cytochrome C reductase was in the mitochondria (Table 1). The total activity of thioredoxin reductase in early placenta was primarily located in the cytosol, while about 10 per cent of the total thioredoxin reductase activity was localized in the mitochondrial fraction. The specific activity of thioredoxin reductase in cytosolic fractions from early placenta was higher than that in mitochondrial fractions. The total and specific activities of thioredoxin reductase in both cytosol and mitochondria of term placenta were similar to these activities in early placenta, whereas the specific activities of thioredoxin reductase in both cytosol and mitochondria from early placenta were significantly higher than in term placenta.
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Total content (per cent)
79 ± 4.1 E
100 T
Cytoplasm
35 ± 1.5 29 ± 1.9 E T Mitochondria
Total content 63 ± 3.7 (per cent) E
100 T
Cytoplasm
18 ± 1.1 12 ± 2.4 E
T
Mitochondria
66 kDa 55 kDa 45 kDa 36 kDa
29 kDa
12 kDa
24 kDa
Figure 1. Intracellular localization of thioredoxin reductase in placenta. Immunoblots using cytosolic and mitochondrial soluble fractions from human early (E) and term (T) placenta stained with the polyclonal antibody raised against thioredoxin reductase. The amount of protein applied per lane was the same in all cases (126 ìg). The value in each fraction is expressed as the means.d. and the percentage relative to the total content of cytosolic thioredoxin reductase from term placenta (given as 100 per cent) per 1 g. Molecular weight markers are shown on the left.
Western blots of thioredoxin and thioredoxin reductase in cytosolic and mitochondrial fractions of human placenta Immunological detection of cytosolic and mitochondrial thioredoxin reductase protein. To determine whether the activity of thioredoxin reductase measured in subcellular fractions actually represents the amount of thioredoxin reductase protein itself, we prepared an antibody against thioredoxin reductase and used it for immunological analysis. Each subcellular fraction was analysed by immunoblotting using anti-thioredoxin reductase (Figure 1). Nearly single bands corresponding to the 55-kDa polypeptide were detected in the cytosol and mitochondria. When non-immune IgG was used, no signal was detected (data not shown). The total content of thioredoxin reductase in term placenta was mainly located in the cytosol, while 29 per cent of the total thioredoxin reductase activity was localized in the mitochondrial fraction (Figure 1). This result almost paralleled the total activity pattern of thioredoxin reductase. The total content of thioredoxin reductase in the mitochondrial fraction from early placenta was higher than that from term placenta. Furthermore, the specific contents of thioredoxin reductase, which refer to the amount of thioredoxin reductase protein relative to total protein in subcellular fractions, in cytosolic and mitochondrial fractions in term placenta were almost the same level as those in the early placenta (Figure 1). Immunological detection of cytosolic and mitochondrial thioredoxin protein. We prepared an antibody against the C-terminal polypeptide of thioredoxin and used it for immunological analysis. Each subcellular fraction was analysed by immunoblotting with the antibody (Figure 2). A single band corre-
Figure 2. Intracellular localization of thioredoxin in placenta. Immunoblots using cytosolic and mitochondrial soluble fractions from human early (E) and term (T) placenta stained with the polyclonal antibody raised against C-terminal thioredoxin. The amount of protein applied per lane was the same (126 ìg). The value in each fraction is expressed as the means.d. and the percentage relative to the total content of cytosolic thioredoxin from term placenta (100 per cent) per 1 g.
sponding to the 12-kDa polypeptide was detected both in the cytosol and in the mitochondria. The total contents of thioredoxin in early and term placenta were mainly located in the cytosol, while about 10–20 per cent of the total thioredoxin reductase protein was localized in the mitochondrial fractions (Figure 2). Furthermore, the specific contents of thioredoxin in both cytosolic and mitochondrial fractions in term placenta tended to be higher than these contents in the early placenta. Immunohistochemical localization of thioredoxin reductase and thioredoxin in human placenta In chorionic villi of the first trimester, immunohistochemical localization of thioredoxin reductase was observed in most cytotrophoblastic cells (Figure 3). Syncytiotrophoblasts showed almost no immunoreactivity for anti-thioredoxin reductase antisera. Thioredoxin reductase in placental tissue was also found in decidual cells (Figure 4) and stromal cells in the stem villi (Figure 5). Thioredoxin was also immunohistochemically localized in cytotrophoblasts, decidua, and stromal cells in the stem villi (Figures 3, 4 and 5). In the terminal chorionic villi of mature term placenta, the reaction products of anti-thioredoxin reductase or thioredoxin antisera were inconspicuously detected. Protective effect of thioredoxin reductase and thioredoxin on mitochondria of human placenta Fumarase activities of mitochondria treated with hydrogen peroxide. Incubation of mitochondria treated with 880 m H2O2 for 0–60 min inhibited their fumarase activities up to 50 per cent.
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(a)
(b)
99
(c)
Figure 3. Immunolocalization of thioredoxin (Trx) and thioredoxin reductase (TR) in first trimester villi (124). (a) Negative control, (b) anti-Trx, (c) anti-Tr.
(a)
(b)
(c)
Figure 4. Immunolocalization of thioredoxin (Trx) and thioredoxin reductase (TR) in term decidua (124). (a) Negative control, (b) anti-Trx, (c) anti-TR.
However, co-incubation of mitochondria in the presence of the complete thioredoxin and thioredoxin reductase system (composed of thioredoxin reductase, thioredoxin, and NADPH) regenerated fumarase to almost the control level (Figure 6).
DISCUSSION In this study, thiorodoxin and thioredoxin reductase were demonstrated not only in cytosol, but also in mitochondria of both early and term placenta. Moreover, the mitochondrial soluble fraction in early placenta was more enriched with thioredoxin reductase and thioredoxin protein than term placenta. This result suggests that the thioredoxin–thioredoxin reductase system might play a protective role against oxidative stress in mitochondria of early placenta.
It has been reported that thioredoxin is a key component of an early pregnancy factor, which has been detected in maternal serum within hours after fertilization and remains during two-thirds of the pregnancy (Clarke et al., 1991). The expression of thioredoxin in normal term placenta, however, was as high as that in early placenta. One possible reason for this might be that the early pregnancy factor represents extracellular thioredoxin and the expression of intracellular thioredoxin in placenta differs from extracellular expression. Although the tissue distribution of thioredoxin in placenta has been reported by Perkins et al. (1995), and is almost the same as that described here, the distribution of thioredoxin reductase essential for keeping thioredoxin in the active reduced form has remained unknown. Thioredoxin reductase was localized in cytotrophoblasts, decidua, and stromal cells in
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(c)
(b)
(a)
Fumarase activity (per cent relative to control)
Figure 5. Immunolocalization of thioredoxin (Trx) and thioredoxin reductase (TR) in stromal cells in term stem villi (50). (a) Negative control, (b) anti-Trx, (c) anti-TR.
120
100
80
60
40
20
0
A
B
C
Figure 6. Protective effect of thioredoxin and thioredoxin reductase on fumarase activity. Mitochondria from normal term placenta pretreated with H2O2 for 60 min were incubated at 37C for 20 min with or without the complete thioredoxin–thioredoxin reductase system. Fumarase activities were spectrophotometrically measured at 340 nm. The values are means of two independent experiments. A, Untreated mitochondria (100 per cent); B, H2O2-exposed mitochondria; C, H2O2-exposed mitochondria with purified thioredoxin reductase, thioredoxin, and NADPH.
the stem villi. The thioredoxin–thioredoxin reductase system may play an important role in the maintenance of pregnancy, because knocking out the thioredoxin gene was reported to result in spontaneous abortion (Matsui et al., 1996). Interestingly, in placental tissue of the first trimester, cytotrophoblasts (but not syncytiotrophoblasts) in the chorionic villi showed positive reactivity for anti-thioredoxin reductase or thioredoxin antisera. Most villous cytotrophoblasts have differentiated to form the syncytium and are decreased in number in mature placental tissue (Sen, Kaufmann and Schweikhart, 1979). We also showed that the thioredoxin–thioredoxin reductase system regenerated mitochondrial fumarase that had been
exposed to hydrogen peroxide. Therefore, the thioredoxin– thioredoxin reductase system may act as an antioxidant defence mechanism in mitochondria of placental tissue. Recent studies have indicated that oxidative stress is involved in the pathogenesis of pre-eclampsia (Hubel et al., 1996). An increased number of villous cytotrophoblasts is a striking feature in several pathological conditions, such as maternal hypertensive disorders and pre-eclampsia (Wigglesworth, 1962; Fox, 1964). These facts have been interpreted as a response to hypoxia due to placental dysfunction, but another interpretation might be the acceleration of the antioxidant defence mechanism. It is known that natural defence mechanisms against oxidative stress consist of glutathione peroxidase, superoxide dismutase, catalase (Halliwell, 1987), and antioxodiants such as glutathione, ascorbate, urate, vitamin E, ubiquinone and betacarotene (Sies, 1985; Machlin and Bendich, 1987). The imbalance of these defence mechanisms against oxidative stress in placenta might result in placental dysfunction. Furthermore, pre-eclampsia is associated with endothelial cell dysfunction that may be caused by oxidative stress (Hubel, 1996). Unsaturated lipid- and thiol-containing proteins in cell membranes are susceptible to oxidative stress (Freeman and Crapo, 1982). Markers of lipid peroxidation have been reported to rise during the progression of normal pregnancy, with greater increases seen in association with pre-eclampsia (Ishihara, 1978; Wickens et al., 1981). Thiol groups are involved in the defence against oxidative stress (Sies, 1985). Oxidative stress will result in the conversion of thiol groups to disulphide forms, with a consequent decrease in the observed thiol levels. It has been reported that in normal pregnancy intracellular thiol increases, but in pre-eclampsia pregnancy, this intracellular thiol increase is not present (Wisdom et al., 1991). These reports indicate that in normal pregnancy antioxidants containing the thioredoxin–thioredoxin reductase system might function as a natural defence mechanism against oxidative stress
Ejima et al.: Thioredoxin Localization and its Protective Effect in Placenta
and that when there is an imbalance between production and degradation of ROS in the placenta, the abnormal conditions of pre-eclampsia, placental dysfunction and intrauterine retardation might result from abnormal uteroplacental circulation. In conclusion we confirmed the intracellular distribution and localization of thioredoxin and thioredoxin reductase in normal early and term placenta. Furthermore, the effects of the eoxogenously added thioredoxin–thioredoxin reductase system on damaged fumarase suggested that the system might serve as a protective mechanism against oxidative stress in human placenta.
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Localization of Thioredoxin Reductase and Thioredoxin in Normal Human Placenta and Their Protective Effect Against Oxidative Stress K. Ejimaa,b,c, H. Nanria, N. Tokib, M. Kashimurab and M. Ikedaa Departments of a Health Development and Health, Kitakyushu 807, Japan Paper accepted 1 July 1998
b
Obstetrics and Gynecology, University of Occupational and Environmental
Recent studies have indicated that oxidative stress is involved in the pathogenesis of pre-eclampsia. Oxidative stress damages systemic tissues, and placental damage may result in intrauterine growth retardation and fetal distress. Thus, this study attempted to elucidate the placental localization of thioredoxin and thioredoxin reductase, substances that may reduce oxidative stress. Furthermore, it studied the defence mechanism of the thioredoxin–thioredoxin reductase system against oxidative stress in mitochondria of normal human placenta where reactive oxygen species are primarily produced. The examination of thioredoxin reductase activity in subcellular fractions of human placenta indicated that thioredoxin reductase was located not only in cytoplasm, but also in mitochondria. The existence of thioredoxin and thioredoxin reductase in human placenta was confirmed immunologically using antibodies raised against thioredoxin and thioredoxin reductase. Thioredoxin and thioredoxin reductase were localized histochemically in cytotrophoblasts, decidua, and stromal cells in the stem villi. The addition of exogenous thioredoxin and thioredoxin reductase to fumarase in mitochondria of human placenta displayed a protective effect against oxidative stress. In conclusion, this study confirmed the intracellular localization and the tissue distribution of thioredoxin and thioredoxin reductase in human placenta. Moreover, the complete thioredoxin–thioredoxin reductase system in human placenta may protect the placenta from damage caused by oxidative stress. 1999 W. B. Saunders Company Ltd Placenta (1999), 20, 95–101
INTRODUCTION The thioredoxin–thioredoxin reductase system is a general protein disulphide oxidoreductase ubiquitously present in all living cells (Luthman and Holmgren, 1982). Thioredoxin reduced to a dithiol by thioredoxin reductase and NADPH is involved in the reduction of ribonucleotides (Laurent, Moore and Reichard, 1964), the activation of several receptors (Grippo et al., 1983; Tagaya et al., 1989), the stimulation of cell proliferation (Wakasugi et al., 1990; Rubaretelli, Bonifaci and Suita, 1995), the regulation of transcriptional factors (Abate et al., 1990; Hayashi, Ueno and Okamoto, 1993), and the maintenance of normal pregnancy as a component of the early pregnancy factor described by Clarke et al. (1991) and Matsui et al. (1996). Furthermore, the thioredoxin– thioredoxin reductase system has recently been implicated in the defence against oxidative stress, for example, in the repair of oxidatively damaged proteins (Fernando et al., 1992; Yoshitake et al., 1994) and the elimination of peroxides (Björnstedt et al., 1994). However, the subcellular localization and function of thioredoxin reductase and thioredoxin in placenta were unknown. c To whom correspondence should be addressed at: Department of Health Development, University of Occupational and Environmental Health, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu 807, Japan.
0143–4004/99/010095+07 $12.00/0
All living tissues under aerobic conditions face the continuous threat of not only exogenous but also endogenous reactive oxygen species (ROS) (Gille, Vanberkel and Joenje, 1994; Richter et al., 1995). An imbalance between the production and elimination of ROS, which are unavoidable products of normal metabolic processes, can cause lethal dysfunction of systemic tissue cells as a result of damage to proteins (Ischiropoulos and al-Mehdi, 1995), nucleic acids (Janssen et al., 1993) and lipids (Ueda and Shah, 1992). It has recently been reported that lipid peroxidation may contribute to endothelial cell dysfunction in pre-eclampsia (Hubel et al., 1996) and placental damage due to oxidative stress might result in intrauterine growth retardation and fetal distress. The localization of thioredoxin and thioredoxin reductase in female genital organs is generally accepted (Rozell et al., 1985; Fujii et al., 1991). Furthermore, it has been reported that thioredoxin is highly expressed in cytotrophoblast cells and stromal cells in early placenta (Perkins et al., 1995). However, the localization of thioredoxin reductase in normal placenta remained unknown. Therefore, we examined the intracellular localization and tissue distribution of thioredoxin reductase in placenta, using antibodies raised against purified thioredoxin reductase, and investigated the defence mechanism of thioredoxin and thioredoxin reductase against oxidative stress in uteroplacental circulation. 1999 W. B. Saunders Company Ltd
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This study suggests that the thioredoxin–thioredoxin reductase system in placenta may provide a protective function against oxidative stress in mitochondria where reactive oxygen species are primarily produced as a byproduct of aerobic energy metabolism (Takeshige and Manakami, 1979).
MATERIALS AND METHODS Tissue preparation First trimester placentae (n=3) were collected from legal abortions at 10 weeks of gestation according to the Helsinki guidelines, and normal placentae (n=3) were obtained at term from normal healthy women with normal deliveries of caesarean sections under informed consent, confirmed in all patient cases. The basal membrane and blood vessels were teased away and adhering blood was removed by several washings with 0.9 per cent NaCl. The materials were chilled in ice, and processed immediately for fractionation and for the histochemical study.
Fractionation of human placenta The following procedures were performed at 4C. Human placentae were cut into pieces after removing blood and endothelium; the tissue was minced and suspended in 3 vol of cold 0.25 sucrose, 0.185 per cent K2HPO4. The pH of the mixture was adjusted to about 7.5 by quickly adding 6 KOH and the suspension was homogenized with a food mixer at maximal output for 2 min, following by centrifugation for 10 min at 2000 g. After discarding the precipitate, the supernatant (post-nuclear supernatant) was filtered through four layers of gauze and centrifuged for 40 min at 11 000 g, and then filtered again. The organelle pellet was homogenized with 0.25 sucrose using Potter–Elvehjem-type homogenizer, suspended in the same buffer, and centrifuged for 40 min at 11 000 g. This procedure was repeated twice. The washed pellet was suspended in 20 ml of 0.25 sucrose, 2 m EDTA (pH 7.5), and stored at 20C for the mitochondria preparation. The supernatant was centrifuged for 1 h at 77 000 g and washed with 0.25 sucrose three thimes. The organelle pellet was collected for the preparation of microsomes. The supernatant was collected for the preparation of cytosol. The activities of lactate dehydrogenase (Bergmeyer, Bernt and Hess, 1965), succinate cytochrome C reductase (Schnitman and Greenawalt, 1968) and fumarase (Gellera et al., 1990) were measured as described for validating the purity of cytosol, mitochondria and mitochondrial matrix.
Thioredoxin reductase assays Thioredoxin reductase was assayed routinely by measuring the rate of reduction of 5,5 -dithiobis-(2-nitrobenzoate) (DTNB) by NADPH spectrophotometrically at 412 nm (Holmgren, 1977). The assay mixture contained 100 m potassium phos-
phate (pH 7.0), 10 m EDTA, 0.2 m NADPH, 0.2 mg/ml of serum albumin, 2.5 m DTNB and enzyme in a final volume of 1 ml at 30C. Enzyme activity in this assay was expressed as ìmol of thionitrobenzoate produced per minute, which is equivalent to 2ìmol NADPH oxidized per minute. Purification of thioredoxin reductase and thioredoxin, and preparation of antiserum against thioredoxin reductase and thioredoxin Thioredoxin reductase and thioredoxin were purified from bovine heart and liver according to the method described by Luthman and Holmgren (1982). The preparations of purified thioredoxin reductase and thioredoxin were homogeneous as judged by SDS/PAGE. Each antiserum was prepared by immunization of an albino rabbit. For primary immunization, 100 ìg of thioredoxin reductase and the C-terminal polypeptide of thioredoxin were emulsified with Titer Max (an adjuvant), and injected subcutaneously. Four weeks later, each rabbit was boosted with 100 ìg of thioredoxin reductase and the C-terminal polypeptide of thioredoxin emulsified with Titer Max. The rabbits were boosted twice at intervals of 2 weeks. Antiserum was then collected 2 weeks after the last booster. Immunoblotting of thioredoxin reductase and thioredoxin in cytosolic and mitochondrial fractions from human placenta with antibodies raised against thioredoxin reductase and thioredoxin revealed almost a single immunoreactive band. Western blotting analysis of subcellular fractions Thioredoxin and thioredoxin reductase expressions were determined by immunoblotting. An equal protein concentration from each sample was then boiled in a mixture of 1 per cent 2-mercaptoethanol and 0.05 per cent bromophenol blue for 3 min. Proteins were separated by SDS/PAGE using a 10 per cent acrylamide resolving gel for thioredoxin reductase and a 15 per cent gel for thioredoxin. Separated proteins were then transferred to a nitrocellulose membrane for use in enhanced chemiluminescence (ECL) Western blotting and probed with the polyclonal rabbit anti-bovine heart thioredoxin reductase antibody and an antibody to the C-terminal polypeptide of thioredoxin. A horseradish peroxidase-conjugated goat antirabbit secondary antibody was used in conjunction with enhanced chemiluminescence to visualize the thioredoxin reductase and thioredoxin bands on autoradiography film. The intensity of the stained bands was quantified by densitometric analysis using the public-domain computer program NIH Image and Color Magician (Wayne Rasband, NIH, Research Service Branch, NIMH, USA). Immunohistochemical staining The specimens of normal placental tissues were frozen in isopentane, cooled with liquid nitrogen, and stored at 80C
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Table 1. Cytosolic and mitochondrial thioredoxin reductase activities of human early and term placenta Thioredoxin reductase
Cytosol E T Mitochondria E T
Total activity (per cent)
Specific activity (per cent)
LDH (per cent)
S-C (per cent)
100a 100b
28.61.9* 14.41.1
100c 100d
9.7 4.0
8.8 8.81.1
12.51.3* 6.50.9
ND 11.0
100e 100f
0.2 U/1 g of placenta; b0.4 U/1 g of placenta; c570 mU/mg protein; d192 mU/mg protein; e85.6 mU/mg protein; 35.3 mU/mg protein. LDH, lactate dehydrogenase; S-C, succinate-cytochrome C reductase; E, early placenta; T, term placenta; ND, not detectable. Total activities, defined as total amounts of thioredoxin reductase activities in subcellular fractions derived from 1 g of placenta, were expressed as per cent of the activity in cytosol. Specific activity was expressed as activity units per mg of protein in each fraction. The values are expressed as means of two independent experiments or meanss.d. Statistical comparison was performed by Student’s t-test. *P > 0.05 was considered statistically significant.
a f
as soon as possible. For immunohistochemical studies to demonstrate the localization of thioredoxin reductase and thioredoxin, the specimens were serially sectioned at 8 ìm. Primary antibodies used in this study were rabbit antisera against purified thioredoxin reductase and C-terminal thioredoxin. Bound antibodies were detected by the indirect avidinbiotin complex method, by using a Vectastain Elite kit (Vecter Laboratories Inc., Burlingame, CA, USA). The sections were rinsed with phosphate-buffered saline (PBS) (0.01 m NaPi, 0.15 NaCl, pH 7.4), fixed with acetone for 10 min, and then rinsed again with PBS. The sections were treated with 1 per cent hydrogen peroxide (H2O2) in methanol for 30 min at room temperature to eliminate endogenous peroxidase activity. After washing with PBS and incubating with normal goat serum (1 : 200) for 30 min, each section was incubated with serum from a rabbit immunized with purified thioredoxin reductase (1 : 100) for 1 h, followed by secondary biotinlabelled anti-rabbit Ig G (1 : 100) at room temperature. After washing with PBS, avidin-biotin complex (ABC) was applied for 30 min. Finally, brown staining was performed by a 5-min treatment with 0.05 per cent 3,3-diaminobenzidine tetrahydrochloride in 0.01 Tris-HCl buffer (pH 7.5) containing 0.01 per cent H2O2. Parallel sections of this study served as negative controls, in which the primary antiserum was omitted and replaced with either diluted normal rabbit serum or PBS. Counterstaining was performed with haematoxylin.
Exposure of mitochondria to hydrogen peroxide Mitochondria (3 mg/ml) were prepared from human placentae and treated with 880 m H2O2 for 0–60 min. Fumarase activities (Gellera et al., 1990) of the H2O2-treated mitochondria were measured in the presence and absence of the
complete thioredoxin–thioredoxin reductase system (composed of purified thioredoxin, 10 ì; thioredoxin reductase; 256 n; and NADPH, 2 m).
Other analytical methods Protein concentrations were determined by the Bio-Rad protein assay kit with bovine serum albumin as a standard. The values are presented as the mean or means.d. Statistical comparison was performed by Student’s t-test. P<0.05 was considered statistically significant.
RESULTS Thioredoxin reductase activity in human placenta Subcellular distribution of human placental thioredoxin reductase. Human placenta was separated into cytosolic and mitochondrial fractions characterized by the existence of specific marker enzymes; most lactate dehydrogenase was present in the cytosolic fraction, while almost all of the succinate-cytochrome C reductase was in the mitochondria (Table 1). The total activity of thioredoxin reductase in early placenta was primarily located in the cytosol, while about 10 per cent of the total thioredoxin reductase activity was localized in the mitochondrial fraction. The specific activity of thioredoxin reductase in cytosolic fractions from early placenta was higher than that in mitochondrial fractions. The total and specific activities of thioredoxin reductase in both cytosol and mitochondria of term placenta were similar to these activities in early placenta, whereas the specific activities of thioredoxin reductase in both cytosol and mitochondria from early placenta were significantly higher than in term placenta.
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Total content (per cent)
79 ± 4.1 E
100 T
Cytoplasm
35 ± 1.5 29 ± 1.9 E T Mitochondria
Total content 63 ± 3.7 (per cent) E
100 T
Cytoplasm
18 ± 1.1 12 ± 2.4 E
T
Mitochondria
66 kDa 55 kDa 45 kDa 36 kDa
29 kDa
12 kDa
24 kDa
Figure 1. Intracellular localization of thioredoxin reductase in placenta. Immunoblots using cytosolic and mitochondrial soluble fractions from human early (E) and term (T) placenta stained with the polyclonal antibody raised against thioredoxin reductase. The amount of protein applied per lane was the same in all cases (126 ìg). The value in each fraction is expressed as the means.d. and the percentage relative to the total content of cytosolic thioredoxin reductase from term placenta (given as 100 per cent) per 1 g. Molecular weight markers are shown on the left.
Western blots of thioredoxin and thioredoxin reductase in cytosolic and mitochondrial fractions of human placenta Immunological detection of cytosolic and mitochondrial thioredoxin reductase protein. To determine whether the activity of thioredoxin reductase measured in subcellular fractions actually represents the amount of thioredoxin reductase protein itself, we prepared an antibody against thioredoxin reductase and used it for immunological analysis. Each subcellular fraction was analysed by immunoblotting using anti-thioredoxin reductase (Figure 1). Nearly single bands corresponding to the 55-kDa polypeptide were detected in the cytosol and mitochondria. When non-immune IgG was used, no signal was detected (data not shown). The total content of thioredoxin reductase in term placenta was mainly located in the cytosol, while 29 per cent of the total thioredoxin reductase activity was localized in the mitochondrial fraction (Figure 1). This result almost paralleled the total activity pattern of thioredoxin reductase. The total content of thioredoxin reductase in the mitochondrial fraction from early placenta was higher than that from term placenta. Furthermore, the specific contents of thioredoxin reductase, which refer to the amount of thioredoxin reductase protein relative to total protein in subcellular fractions, in cytosolic and mitochondrial fractions in term placenta were almost the same level as those in the early placenta (Figure 1). Immunological detection of cytosolic and mitochondrial thioredoxin protein. We prepared an antibody against the C-terminal polypeptide of thioredoxin and used it for immunological analysis. Each subcellular fraction was analysed by immunoblotting with the antibody (Figure 2). A single band corre-
Figure 2. Intracellular localization of thioredoxin in placenta. Immunoblots using cytosolic and mitochondrial soluble fractions from human early (E) and term (T) placenta stained with the polyclonal antibody raised against C-terminal thioredoxin. The amount of protein applied per lane was the same (126 ìg). The value in each fraction is expressed as the means.d. and the percentage relative to the total content of cytosolic thioredoxin from term placenta (100 per cent) per 1 g.
sponding to the 12-kDa polypeptide was detected both in the cytosol and in the mitochondria. The total contents of thioredoxin in early and term placenta were mainly located in the cytosol, while about 10–20 per cent of the total thioredoxin reductase protein was localized in the mitochondrial fractions (Figure 2). Furthermore, the specific contents of thioredoxin in both cytosolic and mitochondrial fractions in term placenta tended to be higher than these contents in the early placenta. Immunohistochemical localization of thioredoxin reductase and thioredoxin in human placenta In chorionic villi of the first trimester, immunohistochemical localization of thioredoxin reductase was observed in most cytotrophoblastic cells (Figure 3). Syncytiotrophoblasts showed almost no immunoreactivity for anti-thioredoxin reductase antisera. Thioredoxin reductase in placental tissue was also found in decidual cells (Figure 4) and stromal cells in the stem villi (Figure 5). Thioredoxin was also immunohistochemically localized in cytotrophoblasts, decidua, and stromal cells in the stem villi (Figures 3, 4 and 5). In the terminal chorionic villi of mature term placenta, the reaction products of anti-thioredoxin reductase or thioredoxin antisera were inconspicuously detected. Protective effect of thioredoxin reductase and thioredoxin on mitochondria of human placenta Fumarase activities of mitochondria treated with hydrogen peroxide. Incubation of mitochondria treated with 880 m H2O2 for 0–60 min inhibited their fumarase activities up to 50 per cent.
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(a)
(b)
99
(c)
Figure 3. Immunolocalization of thioredoxin (Trx) and thioredoxin reductase (TR) in first trimester villi (124). (a) Negative control, (b) anti-Trx, (c) anti-Tr.
(a)
(b)
(c)
Figure 4. Immunolocalization of thioredoxin (Trx) and thioredoxin reductase (TR) in term decidua (124). (a) Negative control, (b) anti-Trx, (c) anti-TR.
However, co-incubation of mitochondria in the presence of the complete thioredoxin and thioredoxin reductase system (composed of thioredoxin reductase, thioredoxin, and NADPH) regenerated fumarase to almost the control level (Figure 6).
DISCUSSION In this study, thiorodoxin and thioredoxin reductase were demonstrated not only in cytosol, but also in mitochondria of both early and term placenta. Moreover, the mitochondrial soluble fraction in early placenta was more enriched with thioredoxin reductase and thioredoxin protein than term placenta. This result suggests that the thioredoxin–thioredoxin reductase system might play a protective role against oxidative stress in mitochondria of early placenta.
It has been reported that thioredoxin is a key component of an early pregnancy factor, which has been detected in maternal serum within hours after fertilization and remains during two-thirds of the pregnancy (Clarke et al., 1991). The expression of thioredoxin in normal term placenta, however, was as high as that in early placenta. One possible reason for this might be that the early pregnancy factor represents extracellular thioredoxin and the expression of intracellular thioredoxin in placenta differs from extracellular expression. Although the tissue distribution of thioredoxin in placenta has been reported by Perkins et al. (1995), and is almost the same as that described here, the distribution of thioredoxin reductase essential for keeping thioredoxin in the active reduced form has remained unknown. Thioredoxin reductase was localized in cytotrophoblasts, decidua, and stromal cells in
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(c)
(b)
(a)
Fumarase activity (per cent relative to control)
Figure 5. Immunolocalization of thioredoxin (Trx) and thioredoxin reductase (TR) in stromal cells in term stem villi (50). (a) Negative control, (b) anti-Trx, (c) anti-TR.
120
100
80
60
40
20
0
A
B
C
Figure 6. Protective effect of thioredoxin and thioredoxin reductase on fumarase activity. Mitochondria from normal term placenta pretreated with H2O2 for 60 min were incubated at 37C for 20 min with or without the complete thioredoxin–thioredoxin reductase system. Fumarase activities were spectrophotometrically measured at 340 nm. The values are means of two independent experiments. A, Untreated mitochondria (100 per cent); B, H2O2-exposed mitochondria; C, H2O2-exposed mitochondria with purified thioredoxin reductase, thioredoxin, and NADPH.
the stem villi. The thioredoxin–thioredoxin reductase system may play an important role in the maintenance of pregnancy, because knocking out the thioredoxin gene was reported to result in spontaneous abortion (Matsui et al., 1996). Interestingly, in placental tissue of the first trimester, cytotrophoblasts (but not syncytiotrophoblasts) in the chorionic villi showed positive reactivity for anti-thioredoxin reductase or thioredoxin antisera. Most villous cytotrophoblasts have differentiated to form the syncytium and are decreased in number in mature placental tissue (Sen, Kaufmann and Schweikhart, 1979). We also showed that the thioredoxin–thioredoxin reductase system regenerated mitochondrial fumarase that had been
exposed to hydrogen peroxide. Therefore, the thioredoxin– thioredoxin reductase system may act as an antioxidant defence mechanism in mitochondria of placental tissue. Recent studies have indicated that oxidative stress is involved in the pathogenesis of pre-eclampsia (Hubel et al., 1996). An increased number of villous cytotrophoblasts is a striking feature in several pathological conditions, such as maternal hypertensive disorders and pre-eclampsia (Wigglesworth, 1962; Fox, 1964). These facts have been interpreted as a response to hypoxia due to placental dysfunction, but another interpretation might be the acceleration of the antioxidant defence mechanism. It is known that natural defence mechanisms against oxidative stress consist of glutathione peroxidase, superoxide dismutase, catalase (Halliwell, 1987), and antioxodiants such as glutathione, ascorbate, urate, vitamin E, ubiquinone and betacarotene (Sies, 1985; Machlin and Bendich, 1987). The imbalance of these defence mechanisms against oxidative stress in placenta might result in placental dysfunction. Furthermore, pre-eclampsia is associated with endothelial cell dysfunction that may be caused by oxidative stress (Hubel, 1996). Unsaturated lipid- and thiol-containing proteins in cell membranes are susceptible to oxidative stress (Freeman and Crapo, 1982). Markers of lipid peroxidation have been reported to rise during the progression of normal pregnancy, with greater increases seen in association with pre-eclampsia (Ishihara, 1978; Wickens et al., 1981). Thiol groups are involved in the defence against oxidative stress (Sies, 1985). Oxidative stress will result in the conversion of thiol groups to disulphide forms, with a consequent decrease in the observed thiol levels. It has been reported that in normal pregnancy intracellular thiol increases, but in pre-eclampsia pregnancy, this intracellular thiol increase is not present (Wisdom et al., 1991). These reports indicate that in normal pregnancy antioxidants containing the thioredoxin–thioredoxin reductase system might function as a natural defence mechanism against oxidative stress
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and that when there is an imbalance between production and degradation of ROS in the placenta, the abnormal conditions of pre-eclampsia, placental dysfunction and intrauterine retardation might result from abnormal uteroplacental circulation. In conclusion we confirmed the intracellular distribution and localization of thioredoxin and thioredoxin reductase in normal early and term placenta. Furthermore, the effects of the eoxogenously added thioredoxin–thioredoxin reductase system on damaged fumarase suggested that the system might serve as a protective mechanism against oxidative stress in human placenta.
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