Journal of Perinatology (2015) 35, 16–22 © 2015 Nature America, Inc. All rights reserved 0743-8346/15 www.nature.com/jp ORIGINAL ARTICLE Increased expression of prostasin contributes to early-onset severe preeclampsia through inhibiting trophoblast invasion Y Yang1,3, J Zhang1,3, Y Gong1, X Liu1, Y Bai1,WXu1,2 and R Zhou1 OBJECTIVE: To investigate the potential role of prostasin, as an invasion suppressor, in the process of trophoblast invasion in preeclampsia. STUDY DESIGN: This case-control study included 19 early-onset severe preeclampsia (⩽34 weeks), 20 late-onset severe preeclampsia (434 weeks) and 20 normal term pregnant women. Immunohistochemistry was conducted to identify the cellular localization of prostasin, as well as the matrix metalloproteinase 2 (MMP2) and MMP9 in the placenta tissues. Enzyme-linked immunosorbent assay was performed to analyze the expression of these three proteins in placental homogenates. The effect of prostasin on the invasive and migratory ability of trophoblast cells was detected by transwell assays. We also examined the regulation of the prostasin antibody in the MMP2 and MMP9 secretion by HTR-8/SVneo cells via blocking the prostasin activity. RESULT: This study demonstrated that the prostasin, MMP2 and MMP9 were all expressed in the placental syncytiotrophoblasts. Increased expression of prostasin was detected in cases with early-onset severe preeclampsia compared with the late-onset and control groups (Po0.05), whereas the expression patterns of MMP2 and MMP9 in placental homogenates were opposite to that of prostasin (Po0.05). Recombinant prostasin inhibited the invasion and migration of trophoblast cells, whereas prostasin antibody enhanced the MMP2 and MMP9 secretion in a dose- and time-dependent manner. CONCLUSION: These findings suggest that prostasin may suppress the invasion process in preeclampsia by attenuating MMP2 and MMP9 secretion. Journal of Perinatology (2015) 35, 16–22; doi:10.1038/jp.2014.136; published online 31 July 2014 INTRODUCTION ‘gelatinase A and B’, are rate-limiting enzymes in invasion pro- Preeclampsia, especially early-onset severe preeclampsia, con- cesses. It has been demonstrated that patients with preeclampsia 13,14 tinues to be a leading cause of maternal and perinatal morbidity exhibit high levels of placental MMP2 and MMP9, whereas and mortality.1,2 Although the exact etiology remains elusive, it is conflicting results were also observed, highlighting the signifi- 15,16 commonly believed that inadequate trophoblast invasion of the cance in invasion-regulatory function. When treated with maternal spiral arteries represents one characteristic pathologic functional antibody against prostasin, human choriocarcinoma factor.3 In the development of placenta, reduced invasion ability JEG-3 cells exhibit an increase in the production of MMP2 and 17 results in the trophoblasts failing to invade the myometrium MMP26, as well as enhanced invasion ability. This finding raises deeply and to remodel the uterine spiral arteries appropriately.4 the possibility that prostasin may participate in the regulation of Consequently, the transformation of spiral arteries is suppressed trophoblast invasion in preeclampsia via the changes of MMP2 from high-resistance, low-flow vessels into dilated vessels.5 The and MMP9 expression. However, the role of prostasin as mediators decreased blood flow and fetoplacental perfusion induce placen- of trophoblast invasion in preeclampsia is still unclear. tal ischemia, subsequent system endothelial dysfunction and Given the presence of impaired trophoblast invasion in preeclampsia.6,7 Furthermore, studies have indicated that the preeclampsia, we hypothesized that abnormal high expression interaction of trophoblast with the extracellular matrix is one of of prostasin in preeclamptic placental tissues may impede tro- the most important factors in trophoblastic invasion, through phoblast invasion through altering MMP2 and MMP9 secretion. To providing a substrate for attachment, growth and migration.4 test this hypothesis, we investigated the localization and expres- However, the exact mechanism that underlies incomplete tro- sion of prostasin, MMP2 and MMP9 in preeclamptic placental phoblastic invasion in preeclampsia remains to be elucidated. tissues, and determined the effects of recombinant human Prostasin is a glycosylphosphatidyl inositol-anchored active prostasin on the invasion and migration of HTR-8/SVneo cells, a serine protease, displaying trypsin-like enzymatic activities.8 It is human trophoblast-derived cell line. widely distributed in the prostate, ovaries, mammary glands, lungs, placenta and other tissues.9 Prostasin has been identified as an invasion suppressor for human prostate and breast cancers.10,11 METHODS In rhesus monkeys, prostasin was also shown to be involved in Study subjects placental trophoblastic invasion and endometrial tissue remodel- The study included 19 early-onset severe preeclampsia (⩽34 weeks, early- ing.12 Matrix metalloproteinase 2 (MMP2) and MMP9, also called onset group), 20 late-onset severe preeclampsia (434 weeks, late-onset 1Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, China and 2Sichuan University-The Chinese University of Hong Kong Joint Laboratory for Reproductive Medicine, West China Institute of Women and Children’s Health, West China Second University Hospital, Sichuan University, Chengdu, China. Correspondence: Dr R Zhou, Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu 610041, China. E-mail: [email protected] 3These authors contributed equally to this work. Received 16 March 2014; revised 13 June 2014; accepted 23 June 2014; published online 31 July 2014 Prostasin inhibits trophoblast invasion in preeclampsia Y Yang et al 17 group) and 20 normal term pregnant women (as the control group). All the The concentrations of MMP2 and MMP9 in supernatants were determined participants were enrolled at the time of diagnosis from the Department of using ELISA kits purchased form R&D Systems (Minneapolis, MN, USA) Obstetrics & Gynecology, West China Second University Hospital, Sichuan University during the period from August 2011 to October 2012, and they were all Han population living in Sichuan Province, southwest China. In the IHC early-onset group, there were all preterm deliveries; in the late-onset IHC was conducted with a biotin-streptavidin-peroxidase kit (Gene Tech, group, there were 10 term deliveries and 10 preterm deliveries; and in the Shanghai, China) according to the manufacturer’s instructions. The sections normal group, there were all term deliveries. Exclusion criteria include of placental samples were deparaffinized completely, rehydrated in vaginal delivery (avoiding potential influence on protein levels), multiple decreasing series of alcohol and subjected to antigen retrieval in citrate pregnancy, diabetes, heart diseases, chronic hypertension, fetal malforma- buffer (10 mM citrate sodium, 10 mM citric acid, pH 6.0) in a microwave tion, chronic nephritis and HELLP syndrome (hemolysis, elevated liver oven at 92 to 98 °C for 15 min followed by washing with 1 × phosphate- enzymes and low platelet count). The study was approved by the ethics buffered saline three times at room temperature for 5 min each. Sections committee of the West China Second University Hospital, Sichuan were then incubated with rabbit anti-human prostasin polyclonal antibody University. All participants gave written informed consent form at entry (1:50), mouse anti-human MMP2 monoclonal antibody (Cat. Number: of the study. Preeclampsia was defined as blood pressure4140/90 mm Hg ab86607, 1: 150, Abcam) and mouse anti-human MMP9 monoclonal on two separate occasions 6 h apart or a single recording of a diastolic antibody (Cat. Number: ab119906, 1:400, Abcam) at 4 °C overnight. The pressure of ⩾ 110 mm Hg, in association with proteinuria ⩾ 1+ on dipstick tissue sections were further incubated with biotinylated antibodies and testing or proteinuria ⩾ 300 mg per 24 h after 20 weeks’ gestation.18 horseradish peroxidase-labeled streptavidin. The substrate was stained Patients with severe preeclampsia were further classified as either having with diaminobenzidine, and counterstained with hematoxylin. early-onset (⩽34 weeks) or late-onset (4 34 weeks) disease according to 19 the gestational age at which onset preeclampsia was diagnosed. ELISA Gestational age was based on the last menstrual period and/or was confirmed by ultrasound examination conducted at the early stage of The placental homogenates of early-onset, late-onset and control groups, pregnancy. Blood pressure was measured on the right arm with a mercury and the cell culture supernatants collected at different end points (24, 48 sphygmomanometer with the subjects seated and after a 5-min rest. Three and 72 h) were analyzed by ELISA with commercially available ELISA kits. times of measurement were conducted at 1-min intervals, and the values (Uscn Life Science (Wuhan, China) for placental homogenates; R&D were averaged. Systolic blood pressure (SBP) and diastolic blood pressure Systems for cell supernatants). The concentrations of prostasin, MMP2 were recorded as phase I and V Korotkoff sounds. All the patients were and MMP9 in the culture supernatants were determined by comparison delivered by undergoing elective cesarean section, with the indications of with the standard curve using a spectrophotometer, which was set at a previous cesarean