The expression of thioredoxin-1 in preterm delivery placenta

Abstract

Preterm delivery (PTD) is the leading cause of infant mortality and morbidity. However, the mechanism at the molecular level is still unknown. Placental inflammatory response and oxidative stress are associated with PTD. Thioredoxin-1 (TRX-1) regulates oxidative stress, inflammation, and the activities of transcription factors.

Objectives

The objective was to detect in placental tissues the expressions of TRX-1 and the TRX-1-related molecules: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), thioredoxin-1-binding protein-2 (TBP-2), hypoxia inducible transcription factor 1α (HIF-1α), and forkhead box protein O3A (FoxO3A).

Methods

PTD was defined as gestation of <37 weeks and term delivery (TD) as ≥37 weeks. The expressions of TRX-1 and TRX-1-related molecules were examined in placental tissues by real-time polymerase chain rection and western blot.

Results

The expressions of TRX-1, TNF-α, COX-2, HIF-1α, and FoxO3A in the placenta of PTD were significantly higher as compared with TD, but no difference was observed in TBP-2 expression.

Discussion

These results indicate that TRX-1 may be adaptively induced by the effects of inflammation and oxidative stress, suggesting protective roles for TRX-1 against these effects in the placenta of PTD.

Keywords:

• Thioredoxin-1
• Inflammation
• Oxidative stress
• Preterm delivery
• Term delivery

Introduction

Preterm delivery (PTD) is the delivery prior to 37 weeks of gestation. The frequency of PTD varies from 5 to 10% in the developed regions of the world, but it is much higher (40%) in certain poor areas.^1,2 Although the survival rate for PTD has recently improved due to technological advances, PTD remains the leading cause of infant mortality and morbidity. It accounts for 75% of perinatal mortality.³ The pathogenesis of PTD is multifactorial, associated with cervical insufficiency, hormonal imbalance, genetic predisposition, and altered immune surveillance.^4,5

Approximately 45–50% of PTD is idiopathic or spontaneous, which is closely associated with intrauterine infection and/or inflammation.^6,7 Inflammation induces oxidative stress by up-regulating proinflammatory cytokines and impairing the antioxidant defense systems. Although oxidative stress is involved in normal pregnancies and spontaneous parturition at term, there is growing evidence that oxidative stress has been implicated in the pathogenesis of many complications of human pregnancy, including miscarriage, pre-eclampsia, and preterm labor.^4,8,9 Moreover, oxidative stress occurs with the overproduction of reactive oxygen species (ROS), and placental antioxidant enzymes increase to compensate for the increase in oxidative stress.^10,11 Antioxidant proteins, such as glutathione peroxidase, superoxide dismutase (SOD), thioredoxin, and thioredoxin reductase, participate in placental oxidative stress.^12–14

Thioredoxin-1 (TRX-1) is a small molecular weight protein (12 kDa) with a conserved dithiol/disulphide active site, Cys-Gly-Pro-Cys.¹⁵ TRX-1, also known as the ‘early pregnancy factor’ (EPF), modifies lymphocyte activity in pregnancy sera.¹⁶ Genetic knockout of TRX-1 in mice is embryonically lethal.¹⁷ In the placenta, TRX-1 may protect against lipopolysaccharide (LPS)-induced oxidative stress.¹⁸ TRX-1 expression is reduced in pre-eclamptic placenta¹⁹, but it has not been previously reported in human placenta of PTD. Therefore, the aim of this study is to examine the hypothesis that TRX-1 may be implicated in PTD.

Thioredoxin-1-binding protein-2 (TBP-2), also known as vitamin-D3 up-regulated protein-1 (VDUP1) or thioredoxin-interacting protein (TXNIP), has a molecular weight of 50 kDa. TBP-2 binds to reduced TRX-1 but not to oxidized TRX-1 or mutant TRX-1 in which two redox-active cysteine residues are substituted with serine.^20,21 TBP-2 is the endogenous inhibitor of TRX-1 which binds to the reduced TRX-1 and inhibits TRX-1 activity.

The aim of this study was to examine the expression of TRX-1 in late gestation with other molecular signals expressed co-temporally.

Materials and methods

Placental biopsies

Fifty-eight pregnant women were recruited for this study and divided into two groups: (i) 29 cases with PTD (29–37 weeks, average age 28.7 ± 5.2 years, average gestational age 34.2 ± 2.0 weeks) and (ii) 29 cases with term delivery (TD) (≥37 weeks, average age 28.7 ± 2.9 years, average gestational age 38.7 ± 1.4 weeks). There was no significant difference in the average age of pregnant women between PTD and TD (P > 0.05). All patients were hospitalized in the First People's Hospital of Yunnan Province, Kunming, China. Pregnant women with chronic hypertension, renal disease, diabetes mellitus, pre-eclampsia, major fetal anomalies, infection, fever, or intrauterine fetal death were excluded from the study. All patients underwent normal vaginal delivery without surgery. All of the placental biopsies were collected from patients who agreed to donate their placenta to the hospital. The ethics committee of the First People's Hospital of Yunnan Province approved this study and all procedures were performed in accordance with the Code of Ethics of the World Medical Association.

Placental biopsies were collected from the same location (the maternal surface side of the placenta to a depth of approximately 1 cm and about 500 mg) immediately after delivery. Abnormal areas and infarcts were avoided. Biopsies were immediately frozen in liquid nitrogen and stored at –80°C for later biochemical analysis.

Quantitative reverse transcriptase-polymerase chain reaction

Total RNA was extracted from 0.1 g placental tissues using a Trizol reagent kit (CWBIO Corporation, Beijing, China) and converted to cDNA using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Walldorf Baden, Germany). The product was analyzed using a Prism 7300 Sequence Detection System (Applied Biosystems, Foster, CA, USA). The following primer pairs were selected for quantitative real-time polymerase chain reaction (PCR): human TRX-1 forward: 5′-AAG CCT TGG ACG CTG CAG-3′, reverse: 5′-CAT CCT GAC AGT CAT CCA CAT CTA CT-3′, probe: TGA TCA AGC CTT TCT TTC ATT CCC TCT C; human TBP-2 forward: 5′- GGA TCC CAG CAG TGC AAA C-3′, reverse: 5′-AAG CCG AAC TTG TAC TCA TAT TTG T-3′, probe: AGT ACC TGC GCT ATG AAG ACA CGC TT; human GAPDH forward: 5′-CAA GGC TGA GAA CGG GAA G-3′, reverse 5′-GGT GAA GAC GCC AGT GGA CT-3′, probe: ATC CCA TCA CCA TCT TCC AGG AGC G; human hypoxia inducible transcription factor 1α (HIF-1α) forward: 5′-ACA GCA GCC AGA CGA TCA TGC AG-3′, reverse: 5′-AAC TGG TCA GCT GTG GTA ATC CAC T-3′; human cyclooxygenase-2 (COX-2) forward: 5′-TGC ATT CTT TGC CCA GCA CT-3′, reverse: 5′-AAA GGC GCA GTT TAC GCT GT-3′; human tumor necrosis factor- α (TNF-α) forward: 5′-GCC CAG GCA GTC AGA TCA TC-3′, reverse: 5′-CGG TTC AGC CAC TGG AGC T-3′. Reaction mixtures containing Premix Ex Taq™ (TaKaRa code: DRR039) and SYBR Green (TaKaRa code: DRR041; TaKaRa Biotechnology, Dalian, China) were prepared according to the manufacturer's protocol. GAPDH was used as an internal standard for all samples.

Western blotting

Placental tissues from 58 pregnant women were homogenized and lysed in a solubilizing solution (150 mM NaCl, 0.5% NP-40, 10 mM Tris-HCl, pH 7.2, 0.1 mM PMSF, 2 µg/ml aprotinin, and 200 µg/ml NaN₃) on ice for 1 hour and centrifuged at 20 600 g (Allegra® X-22 Series Benchtop Centrifuge, Beckman Coulter, Fullerton, CA, USA) for 15 minutes. The protein concentration in the supernatant was determined using the Bio-Rad Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein from each sample (10 µg for TRX-1, 20 µg for COX-2, and forkhead box protein O3A (FoxO3A)) were subjected to electrophoresis in 15% (TRX-1) and 10% (COX-2 and FoxO3A) sodium dodecyl sulfate polyacrylamide gels under reducing conditions and transferred with polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA) using a humid transfer machine. Membranes were incubated in a blocking solution (PBS-T [Tween-20-phosphate buffered saline] containing 10% powdered non-fat milk and 0.1% Tween-20) at 4°C overnight followed by incubation in the same solution containing the primary antibody (anti-human TRX-1, dilution ratio 1:5000 (Redox Bioscience, Inc., Kyoto, Japan); anti-human COX-2, dilution ratio 1:1000 (Santa Cruz Biotechnology, CA, USA); anti-human FoxO3A, dilution ratio 1:5000, (Epitomics, CA, USA)) at room temperature for 70 minutes. Membranes were washed in TBS-T and incubated with a horseradish peroxidase-conjugated secondary antibody (KPL, Gaithersburg, MD, USA) at room temperature for 1 hour. The immunoreactive bands were visualized using an enhanced chemiluminescence system (Millipore Corporation) according to the manufacturer's instructions. Each sample was normalized to β-actin. Image analysis software (ImageJ) assessed band intensity and provided the quantitative analysis.

Statistical analysis

Data are expressed as the means ± SD. Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA) software. A one-way analysis of variance followed by a post hoc comparisons test was performed. Comparisons between PTD and TD were analysed by multiple-samples comparison. Differences were considered statistically significant at either *P < 0.05, **P < 0.01, or ***P < 0.001.

Results

Placental inflammation in PTD placenta

Although the mechanism of PTD remains unknown, it has been reported that the placental inflammation is associated with the premature onset of labor.²² Therefore, we first examined whether inflammation existed in PTD. TNF-α was detected by using real-time PCR. The mRNA level of TNF-α was higher in PTD than TD (P < 0.05; Fig. 1A), which suggested that inflammation was involved in the pathogenesis of PTD. This result is consistent with previous studies.^23,24

Figure 1. The expressions of TNF-α mRNA levels and COX-2 mRNA levels and protein in placenta of PTD (n = 29) and TD (n = 29). (A) The mRNA levels of TNF-α in placenta of PTD and TD by real-time PCR. (B) The mRNA levels of COX-2 in placenta of PTD and TD by real-time PCR. (C) The expression of COX-2 protein in placenta of PTD and TD by western blot. The relative protein density was normalized to β-actin. Bars indicate the mean value ± SD. Asterisks indicate statistical significance (*P < 0.05, ***P < 0.001). PTD, preterm delivery; TD, term delivery.

COX-2 is an inducible enzyme that is up-regulated by the inflammatory responses. It is involved in LPS- and glucocorticoid-induced PTD.^25–27 Further examination of the expression of COX-2 in the placenta of PTD showed that the levels of COX-2 mRNA and protein in PTD were higher compared with that in TD (Figs. 1B and 1C). This result further demonstrated that inflammation occurred in the placenta of PTD in the present study.

TRX-1 expression in PTD placenta

TRX-1, also known as the ‘EPF’, modifies lymphocyte activity in pregnancy sera.¹⁶ TRX-1 expressions in PTD and TD placenta were examined, and the results showed that TRX-1 mRNA and protein levels were significantly increased (P < 0.05) in PTD compared to TD (Figs. 2A and 2B).

Figure 2. The expressions of TRX-1 in placenta of PTD (n = 29) and TD (n = 29). (A) The mRNA levels of TRX-1 in placenta of PTD and TD by real-time PCR. (B) The expression of TRX-1 protein in placenta of PTD and TD by western blot. The relative protein density was normalized to β-actin. Bars indicate the mean value ± SD. Asterisks indicate statistical significance (*P < 0.05, ***P < 0.001). PTD, preterm delivery; TD, term delivery.

The expressions of TRX-1-associated molecules (TBP-2, HIF-1α, and FoxO3A)

Although TRX-1 can be induced by inflammation and oxidative stress, it was needed to detect the mechanisms or roles of TRX-1 induction in PTD. TBP-2, as an endogenous inhibitor of TRX-1, was detected using real-time PCR and western blot. The results showed that there were no significant differences (P > 0.05) in placental TBP-2 mRNA and protein levels between PTD and TD (Figs. 3A and 3B).

Figure 3. The expression of TBP-2 in placenta of PTD and TD (n = 29) and TD (n = 29). (A) The mRNA levels of TBP-2 in placenta of PTD and TD by real-time PCR. (B) The expression of TBP-2 protein in placenta of PTD and TD by western blot. The relative protein density was normalized to β-actin. Bars indicate the mean value ± SD. PTD, preterm delivery; TD, term delivery.

Oxidative stress usually occurs in hypoxic conditions. As a result, the expression of HIF-1α in the placenta was detected, and the HIF-1α mRNA levels were found to be significantly higher in PTD as compared with TD (P < 0.05; Fig. 4). Therefore, the increase in HIF-1α expression may be associated with placental oxidative stress in PTD.

Figure 4. The mRNA levels of HIF-1α in placenta of PTD (n = 29) and TD (n = 29) by real-time PCR. Bars indicate the mean value ± SD. Asterisks indicate statistical significance (*P < 0.05). PTD, preterm delivery; TD, term delivery.

The overexpression of FoxO3A reduces the abnormal accumulation of ROS, enhances cellular resistance to oxidative stress, and increases antioxidant gene expression.²⁸ Thus, we further examined FoxO3A expression in the placenta of PTD and found that FoxO3A expression was significantly higher in the placenta of PTD than TD (P < 0.01; Fig. 5). The increased expression of FoxO3A may play a role in resisting placental oxidative stress of PTD by inducing TRX-1 expression.

Figure 5. The protein expression of FoxO3A in placenta of PTD (n = 29) and TD (n = 29) by western blot. The relative protein density was normalized to β-actin. Bars indicate the mean value ± SD. Asterisks indicate statistical significance (0.001 < **P < 0.01). PTD, preterm delivery; TD, term delivery.

Discussion

As reported previously, the pathogenesis of PTD is closely associated with inflammation.²⁹ TNF-α is an inflammatory stressor. As the proinflammatory cytokine, TNF-α could induce oxidative stress in various cells and provoke a variety of biological effects on the placenta.^23,24,30 COX-2 is also a factor in the inflammatory response and is involved in LPS and glucocorticoid-induced PTD, and it is responsible for prostaglandin (PG) production during TD and PTD.^26,27,31,32 In this study, we demonstrated the increasing expressions of TNF-α and COX-2, as shown in Figs. 1A–1C. These results indicate that inflammation is involved in PTD. The increase of TNF-α and COX-2 could stimulate the synthesis and release of PG, which ultimately leads to uterine contraction and irreversible cervical changes.³³ COX-2 is also known to be involved in the initiation and augmentation of parturition.³⁴ Therefore, the increase of TNF-α and COX-2 is associated with PTD of the fetus.

Although infection and/or inflammation are the main pathological processes, placental oxidative stress has been implicated in the pathogenesis of many complications of human pregnancy, including miscarriage, pre-eclampsia, and PTD. TRX-1 is one of the major components of the thiol-reducing system and is important for the regulation of the redox balance.³⁵ TRX-1 has an essential role in maintaining their surface thiol density as the principal mechanism for antioxidative defense, and is sensitive to proinflammatory stimuli, mainly TNF-α. Increased TRX-1 production in the naturally occurring human regulatory T cells confers enhanced tolerance to oxidative stress.³⁶

TRX-1 is localized in the cytosol of normal early and term placenta.^37–39 The expression of TRX-1 protein and mRNA levels were increased in PTD placenta in the present study (Fig. 2). The increase of TRX-1 may play roles in protecting the placenta from oxidative stress and inflammation.

TBP-2 expression is regulated by environmental conditions, various stresses and inflammation, and its expression affects cellular growth and apoptosis.^20,21,40 We examined TBP-2 expression in the placenta of PTD and found that TBP-2 mRNA and protein levels in placenta were not significantly different between the groups (Figs. 3A and 3B), suggesting that TRX-1 may not be regulated by TBP-2 in PTD placenta.

The expression of TRX-1 can be induced by various stimulations through AP-1, antioxidant responsive element, and cAMP-responsive element sites in the TRX promoter.⁴¹ The forkhead-box (Fox) gene family of transcription factors, which includes more than 100 members, includes FoxO3A that binds to the TRX-1 promoter to induce TRX-1 expression through the AMP-activated protein kinase pathway during oxidative stress responses.⁴² Thus, it was important to further examine the expression of FoxO3A. This study is the first peer-reviewed report of an increase in FoxO3A in the placenta of PTD (Fig. 5). Mammalian FoxO proteins play numerous roles in cellular proliferation/arrest, survival/death, metabolism, and autophagy. FoxO3A-null mice exhibit age-dependent infertility due to abnormal ovarian follicular development, leading to degeneration.^43,44 FoxO3A functions as a major regulator of oxidative stress, and this transcription factor increases oxidative stress resistance by the up-regulation of mitochondrial SOD and peroxisomal catalase.^45,46 FoxO3A is expressed in human placenta⁴⁷ and our data demonstrate that the increased expression of FoxO3A might be associated with oxidative stress in PTD placenta.

Besides TRX-1, many proteins are also induced by oxidative stress, such as angiogenic molecules.^4,8,9 HIF-1α, an angiogenic molecule, is a redox sensitive transcription factor and was detected by reverse transcriptase (RT)-PCR. Our results showed a significant increase in the expression of HIF-1α in the placenta of PTD compared with those of TD (Fig. 4). In normal pregnancy, a low oxygen environment in the placenta is physiological and necessary in the first trimester, but it is pathological and associated with common complications of pregnancy in later gestation.⁴⁸ HIF-1α could be expressed during early pregnancy, but its amount decreases with gestational age.⁴⁹ Therefore, hypoxia is implicated as a key regulator of placental morphogenesis and function.^49,50 HIF-1α was induced by the oxidative stress occurred in the PTD placenta. TRX-1 regulates the activation of transcription factors, including HIF-1α. In addition, TRX-1 enhances HIF-1α activity and protein expression under both normoxic and hypoxic conditions. In contrast, a redox-inactive TRX (C32S/C35S) markedly decreases HIF-1α protein levels.⁵¹ In current clinical development, an inhibitor of TRX-1 has been found to decrease tumor levels of the HIF-1α transcription factor.⁵² Thus, HIF-1α may be induced by the redox response in the PTD placenta and regulated by TRX-1. In addition, TNF-α and interleukin-1β have been shown to stabilize HIF-1α protein, and thus HIF-1α can be recruited by tissue inflammation.^53–55

In conclusion, this study showed that TNF-α, COX-2, FoxO3A, TRX-1, and HIF-1α were significantly increased in the human placenta of PTD. These results suggest that oxidative stress and inflammation are involved in the pathological process of PTD.

Acknowledgements

This study was supported by The Candidates of Young and Middle Aged Academic Leaders of Yunnan Province 2006PY01–07, 2008PY045. Key Laboratory of Medical Neurobiology of Kunming University of Science and Technology.