Prothymosin α activates type I collagen to develop a fibrotic placenta in gestational diabetes

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Introduction
Gestational diabetes mellitus (GDM) refers to the phenomenon of hyperglycemia first detected during pregnancy. GDM is becoming more common due to a greater number of women conceiving later in life and the obesity epidemic [1]. The prevalence of GDM varies among populations, ranging from 1.7% to 11.6% [2].
Numerous studies have established that GDM is associated with significantly higher risks of maternal and fetal complications, such as type 2 diabetes and cardiovascular disease [3,4]. The current screening strategy for GDM of offering oral glucose tolerance test (OGTT) to high-risk patients is inadequate due to the inconvenience of OGTT. Therefore, biomarkers for predicting GDM are attractive approaches that require further investigation.
The pathogenic mechanisms of GDM remain elusive. The placenta acts as a natural selective barrier between the maternal and fetal circulatory systems. It is believed to be the main source of hormone and cell regulatory factors including placental growth factor, estrogen, glucocorticoids, and progesterone [5]. These hormones and regulatory factors antagonize the effects of insulin, leading to glucose metabolism dysfunction, low insulin sensitivity, and high blood glucose levels [6]. In addition, hyperglycemia creates hypoxia which causes placental damage and induces Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 the production of proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) [7,8]. Moreover, histological changes, including marked hyperplasia of cytotrophoblasts, villous immaturity, villous fibrinoid necrosis, chorangiosis, and increased angiogenesis were observed in placentae from patients with poorly controlled GDM [9]. GDM placentae are enlarged, thick, and plethoric with an increased placental to fetal weight ratio [10,11]. Although histological changes in GDM placenta are well documented and are related to the development of GDM, the underlying pathophysiology of GDM remains obscure [9].
Hyperglycemia induces mitochondria dysfunction and increases reactive oxygen species (ROS) production, and increased ROS level is associated with GDM [12,13].
Prior research has shown that placentae from women with GDM exhibit oxidative stress [14]. Furthermore, hyperglycemia-induced GDM placental changes, including increased MMP-9 expression, have been shown to be mediated by ROS [15]. Another study also demonstrated that placental MMP-9 activity is modulated by ROS which may result in the placental anomalies observed in diabetic pregnancies [16]. Although increased ROS level induces fibrosis, and fibrosis is observed in placentae from diabetic pregnancies [17], the factors that link these observations in GDM have yet to be elucidated. Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 Prothymosin α (ProT) is an acidic protein that plays roles in regulating cell fates, oxidative stress, and immunomodulation [18,19]. ProT increases acetylation of histones and NFκB, and contributes to emphysema, a chronic disease characterized by inflammation and oxidative stress [20]. In addition, increased ROS might activate the apoptotic pathway, which further leads to the release of ProT into the circulation, and further induces insulin resistance through the toll-like receptor 4 (TLR4) pathway [21]. Although ProT expression can be detected in the placenta [22], the role of ProT in GDM is still unknown.
In this study, we compared the expression of ProT in both the placenta and blood in women with and without GDM. Furthermore, we investigated the role of ProT in the development of GDM.

Human study
This study was reviewed and approved by the Institutional Review Board of National Cheng Kung University Hospital (B-ER-106-416). The research has been carried out in accordance with the World Medical Association Declaration of Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 Helsinki, and that all subjects provided written informed consent for their samples and data to be used. In total, 141 subjects (n=39 subjects with GDM and n=102 without GDM) were recruited for this study. All patients who attended the outpatient department of National Cheng Kung University Hospital for prenatal examination were screened. After an 8-h fast, study participants who were 24~28 weeks pregnant received OGTTs. Fasting, one hour-, and two hours-blood samples after loading of 75-g glucose were collected to measure blood glucose concentrations. GDM was diagnosed if one or more of the following criteria were met: fasting plasma glucose (FPG) of 5.1~6.9 mmol/l (92~125 mg/dl); one hour-plasma glucose of ≥10.0 mmol/l (180 mg/dl) following a 75-g oral glucose load; or two hour-plasma glucose of 8.5~11.0 mmol/l (153~199 mg/dl) following a 75-g oral glucose load. The body-mass index (BMI) (in kg/m 2 ) was calculated as the weight (in kilograms) divided by the height (in meters) squared. For blood pressure measurements, subjects were asked to rest in a supine position in a quiet location, and measurements were obtained in a fasting state between 08:00 and 10:00 AM. Serum ProT concentrations were measured as previously described [21]. We excluded women with preeclampsia, eclampsia, pregnancy-induced hypertension, preexisting hypertension or alcohol Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 misuse prior to pregnancy. After delivery, the placentae were collected, weighed, and then fixed in 10% formalin overnight, dehydrated, and embedded in paraffin.

Animals
All animal experiments were carried out at National Cheng Kung University in Taiwan, and approved by the Institutional Animal Care and Use Committee (IACUC no: 104101) of National Cheng Kung University. Ten to 12-week-old pregnant mice were purchased from the animal center of National Cheng Kung University. ProT transgenic mice with a friend virus B-type (FVB) background were backcrossed with mice with a C57BL/6J genetic background for ten generations [21]. After euthanasia by injection of pentobarbital (Sigma-Aldrich), the placental tissues of mice were collected and weighed on days 9, 11, 13, 15, 17, and 19 after the mice had become pregnant. Placental tissues were then fixed in 10% formalin overnight, dehydrated, and embedded in paraffin.
On day 14, blood samples were collected, and glucose levels were determined using a commercial assay kit (Biosystems, Barcelona, Spain).

Cell culture
The human 3A-Sub-E placental trophoblast cell line was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA) containing heat-inactivated 10% fetal bovine serum (Gibco) at 5% CO 2 and 37°C.

Reactive Oxygen Species (ROS) Detection
Intracellular ROS were determined using 2'7'-dichlorofluorescin diacetate (DCFDA) (Thermo Fisher, Vantaa, Finland). 3A-sub-E (4×10 5 /well) were grown in a 6-well plate and cultured at 37 ℃ and 5% CO 2 until confluency. Cells were maintained in DMEM with high glucose (25 mM glucose) or low glucose (5.5 mM glucose). After 48 hours incubation, 25 M DCFDA were added into medium and incubated for 30 minutes. Cells were rinsed with serum free medium two times and analyzed using a fluorescence microscope (Olympus, Tokyo, Japan) with a 10X objective lens.

Ribonucleic acid (RNA) extraction
The RNA of samples was extracted with 1.0 ml TRIzol ® Reagent (Invitrogen, Carlsbad, CA, USA) at room temperature for 5 min until the cells had completely dissolved and then well mixed with 0.2 ml chloroform (Merck, Kenilworth, NJ, USA). After centrifugation (12,000 rpm for 10 min at 4 °C), the supernatant was removed, and then 0.2 ml 100% isopropanol (Merck) was added at room temperature for sedimentation of RNA. Absolute ethanol (Merck) was used to wash the RNA. The supernatant was removed, and RNA was air-dried and then reconstituted with DEPC-

Quantitative real-time polymerase chain reaction (qPCR)
Five micrograms of complementary (c)DNA was added to 10 mM of a forward primer and 10 mM of a reverse primer, and then water was added to reach 12.5 µl. A quantitative (q)PCR was performed using a SYBR Green master mix (SYBR Green Premix Ex Taq, Takara, Japan). The reaction was performed for 25 cycles: initial denaturation at 95°C for 150 seconds, denaturation at 95°C for 15 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 30 seconds, with a final extension Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 at 72°C for 7 min. Cepheid Smart Cycler vers. 2.0 software (Thermo Scientific, Rockford, IL, USA) was used to analyze the results.

Western blot analysis
Proteins were extracted using a RIPA lysis buffer (VWR Chemicals, Sobn, OH, USA). After being centrifuged (12,000 rpm for 10 min at 4 °C), the supernatant was collected. The protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Scientific); protein lysates (30 µg) were separated using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes. The membranes were blocked at room temperature for one hour in PBST (phosphate-buffered saline (PBS) + Tween 20) containing 10% skimmed milk, and probed with 1:1000 primary antibodies at 4°C overnight. Blots were rinsed with PBST and incubated with a 1:5000 dilution of secondary antibodies at room temperature for two hour, and then washed with PBST again. Protein bands were visualized using a chemiluminescence horseradish peroxidase substrate. The relative signal intensity was quantified using the BioSpectrum Imaging System (UVP, Upland, CA, USA).

Immunohistochemistry
Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 At the end of the experiments, each group of the mice was euthanized, and the placentae were removed. Tissue samples were fixed in 10% formaldehyde at 4°C, and fixed specimens were then dehydrated and embedded in paraffin. Specimens were cut into 5 µm-thick sections at 50 μm intervals and stained with hematoxylin and eosin (Muto Pure Chemicals, Tokyo, Japan) or picro-Sirius red stain (Abcam, Cambridge, UK), and sections were observed under a microscope (100×). In addition, the target proteins in the sections were quantified using TissueFAXS Plus (TissueGnostics, Vienna, Austria), and analyzed by HistoQuest (TissueGnostics).

Statistical analysis
Data were analyzed with two-way repeat-measures analysis of variance (ANOVA) and unpaired Student's t-test. All results are presented as the mean±standard error of the mean (SEM), and p<0.05 was considered as significant.

Expressions of fibrosis-related genes and proteins are increased in the placentae of GDM patients
Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 As shown in Table 1, age-and BMI-matched subjects with (n=39) or without (n=102) GDM were enrolled in this study. Blood glucose levels, including FPG, and plasma glucose levels at one or two-hour post-loading with oral glucose showed significant differences between GDM and Normal groups, while there were no differences in their blood pressure. Consistent with previous studies and observations, we found that the weight of the placenta after delivery was significantly heavier in GDM patients (n=39) compared to normal pregnant women (n=102) ( Figure 1A). To identify the possible sources that contributed to the increased placental weight, we tested the placental expression of Col-1. Figure 1B shows that the increased protein expression of Col-1 in the placenta of GDM patients by immunohistochemical staining ( Figure 1B). In addition, the gene expressions of Col-1 ( Figure 1C), transforming growth factor  (TGFβ) ( Figure 1D), and  smooth muscle actin (SMA) ( Figure 1E) expressions were significantly increased in GDM patients.

Levels of ProT are increased in GDM patients and positively correlated with Col-1 expression in the placenta
Because ProT plays an important role in fibrogenesis, we investigated the relationship between ProT and placental fibrosis. As shown in Figure 2A, Col-1 Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 expression was significantly increased in the placentae of ProT transgenic mice compared to wild-type mice. From another perspective, both the protein ( Figure 2B) and RNA ( Figure 2C) expression levels of ProT, as well as the ProT concentration in the plasma ( Figure 2D), were also significantly elevated in GDM patients. We further investigated the relationship between placental fibrosis and ProT expression. Figure 2E shows that ProT expression in the placenta was positively correlated with Col-1 levels implying that ProT may play a role in enriching the placental extracellular matrix.

Expression of ProT is increased in the placenta of GDM mice
To elucidate the role of ProT in the increased GDM placenta weight, we first investigated changes in the expression of placental ProT during pregnancy in mice.
As shown in Figure 3A and 3B, ProT expression was gradually increased during pregnancy, and reached a peak on day 15 of pregnancy, but then dramatically decreased before delivery. In view of the increment in ProT expression during pregnancy and the fact that ProT plays an important role in insulin resistance, we speculated that the increased placental ProT expression might be related to the development of GDM. We established a GDM mouse model to test this hypothesis Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 further. After the injection of STZ into pregnant mice, decreased plasma insulin levels ( Figure 3C) led to hyperglycemia ( Figure 3D). In addition, the weight of the placenta significantly increased in pregnant mice with GDM ( Figure 3E). Moreover, placental ProT expression was significantly higher compared to the control group at the same stage during pregnancy, indicating that the increased ProT expression may play a crucial role in the enlargement of the placenta in GDM and the development of GDM ( Figure 3F). In addition, the expressions of fibrosis-related proteins, such as Col-1, TGFβ andSMA, were significantly increased in placentae at gestational day 19 of pregnant mice ( Figure 3G).

ProT induces fibrosis through an NFκB-dependent pathway
Following the investigation of the role of ProT in the development of GDM, we then investigated the mechanism of ProT-induced Col-1 expression in trophoblasts.
Overexpression of ProT using lentivirus-mediated gene delivery in 3A-sub-E trophoblasts significantly increased the expressions of fibrosis-related proteins, such as Col-1, TGFβ andSMA ( Figure 4A), whereas knockdown of ProT expression by different lentiviral vectors containing short hairpin (sh)RNA targeted to ProT significantly decreased the expressions of fibrosis-related proteins ( Figure 4B). We Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 further investigated the mechanism of increased ProT expression in GDM in detail.
As shown in Figure 5A, we found increased macrophage infiltration in placentae from GDM patients, as determined by CD68 staining. Furthermore, expressions of proinflammatory cytokines in the placenta, such as IL-1β, IL-6, TNFα, and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in GDM patients compared to the normal group ( Figure 5B). Given the observed inflammation in the placentae of GDM patients and the fact that ProT activates NFκB to induce insulin resistance, we evaluated the role of NFκB in ProT-induced fibrosis-related protein expressions. Overexpression of ProT in 3A-sub-E trophoblasts increased the activity of NFκB p65 ( Figure 5C), and inhibition of NFκB activity by the NFκB inhibitor Bay117082 significantly reversed the effect on fibrosis-related protein expressions by ProT, indicating that ProT induces fibrosis through an NFκB pathway ( Figure 5D).

Hyperglycemia-induced ROS regulates ProT expression
In this study, we found that high glucose at a concentration of 25 mM significantly induced ROS production in trophoblasts ( Figure 6A). We then investigated the effects of hyperglycemia on ProT expression. As shown in Figure   6B, high glucose significantly increased the expression of both ProT and Col-1 in Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 3A-sub-E trophoblasts. On the other hand, pretreatment with N-acetyl-L-cysteine (NAC) to inhibit ROS reversed methylglyoxal, an advanced glycation end-product (AGE) that induces ProT expression, indicating that ROS plays roles in regulating ProT expression in GDM ( Figure 6C).

Discussion
To the best of our knowledge, this is the first report to investigate the physiological effects of ProT on the placenta and the pathological role of ProT in the development of GDM. In this study, we found that ProT expression in the placenta and circulating ProT concentrations were both significantly elevated in GDM patients. Furthermore, hyperglycemia and AGEs might contribute to placental inflammation and the increased expression of ProT. Increased ProT expression further induces Col-1 expression and contributes to placental fibrosis through an NFκB-dependent pathway. This effect may be associated with the increased placental mass observed in GDM.
An abnormal secretion of placental hormones may be attributed, in part, to the pathogenesis of gestational trophoblastic diseases, gestational diabetes, and preterm delivery. However, few biochemical and biophysical markers from the placenta have Consistent with a previous study on GDM rats, fetal weights were significantly lower and placental weights higher along with increased placental fibrosis and ischemia [24]. Moreover, both GDM and preexisting diabetes in pregnancy change the structure of the placenta, including increased calcium and fibrin deposits, and is associated with higher incidences of placental infarction, hematoma, and fibrosis [17].
Although certain factors, such as the cystic fibrosis transmembrane regulator, regulate the functions of trophoblasts, the crosstalk between placenta fibrosis and GDM is unknown. In the present study, we provide mechanistic evidence for the role of ProT in the link between placenta fibrosis and GDM. ProT is ubiquitously expressed in various cell types and tissues [25]. In the lung, ProT contributes to the pathogenesis of emphysema. ProT inhibits the association of histone deacetylases with histones and NFκB, and ProT overexpression increases the expression of NFκB-dependent matrix metalloproteinase-2 (MMP2) and MMP9, which are found in the lungs of Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20200147/892840/cs-2020-0147.pdf by guest on 15 September 2020 patients with the chronic obstructive pulmonary disease [20]. In the liver, the silencing of hepatic ProT expression ameliorates high-fat diet-induced insulin resistance in C57BL/6 mice through a TLR4/NFκB-dependent pathway. Serum ProT levels of patients with type 2 diabetes are significantly higher than those of normal individuals [21]. In the present study, we investigated a novel role of ProT in the placenta that contributes to fibrosis and its link with GDM.
Hyperglycemia is a key symptom of GDM, while high glucose level is related to inflammation and increased ROS. However, the role of ProT in ROS production remains unclear. ProT regulates the expression of oxidative stress-protective genes