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Decidual ACVR2A regulates extravillous trophoblast functions of adhesion, proliferation, migration and invasion in vitro
Pregnancy Hypertension xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Pregnancy Hypertension journal homepage: www.elsevier.com/locate/preghy
Short communication
Decidual ACVR2A regulates extravillous trophoblast functions of adhesion, proliferation, migration and invasion in vitro ⁎
Hannah E.J. Yong , Padma Murthi1, Bill Kalionis, Rosemary J. Keogh2, Shaun P. Brennecke2 The University of Melbourne, Department of Obstetrics and Gynaecology and Department of Maternal-Fetal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital, Locked Bag 300, Corner Grattan Street and Flemington Road, Parkville 3052, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords: ACVR2A Decidual stromal cells Extravillous trophoblast
Decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface. Our aim was to investigate the role of the pre-eclampsia associated decidual activin receptor, ACVR2A, in regulating trophoblast functions at this interface. St-T1b and HTR-8/SVneo cell lines were used to model decidual stromal and trophoblast cells respectively. St-T1b conditioned medium inhibited HTR-8/SVneo adhesion, proliferation, migration and invasion; all effects that were attenuated by decidual ACVR2A siRNA transfection. These findings suggest that altered decidual ACVR2A expression perturbs the maternal-fetal crosstalk involved in regulating trophoblast function at the interface, which may affect placentation and lead to pre-eclampsia.
1. Introduction In pregnancy, decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface, where they regulate multiple functions of fetal extravillous trophoblast cells. The decidualised stromal cells produce and release secretory factors [1,2], which can inhibit trophoblast gelatinolytic activity [3] by modifying expression of metalloproteinases [4], integrins and tissue inhibitors of metalloproteinases [5] that are necessary for invading the decidual matrix. Past studies show that activin A, a TGFβ family member, is actively involved in inducing and amplifying the decidualisation process, which prepares the uterine endometrium for pregnancy [6,7]. Expression of the activin A βA subunit is significantly upregulated in uterine stromal cells during the secretory phase of the menstrual cycle, when decidualisation begins in vivo and in early pregnancy [8,9]. A similar temporal expression pattern is also observed for the activin A binding receptor, ACVR2A [10]. Nevertheless, while the role of activin A in preparing and supporting a healthy pregnancy at the maternal-fetal interface is well-established, the role of its receptor ACVR2A in mediating its effects is currently unknown. Additionally, genetic variants of ACVR2A show associations with the hypertensive disorder – preeclampsia [11–15], where ACVR2A expression is significantly reduced at the maternal-fetal interface [13,16]. Therefore, our study aim was to
explore the functional effects of altered decidual ACVR2A at this interface using an in vitro cell culture model. 2. Materials & methods 2.1. Cell culture St-T1b cells were maintained in DMEM medium and Ham’s F12 medium in a 1:1 ratio, with 10% charcoal-stripped fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin under humidified conditions (5% CO2/95% air) at 37 °C; and decidualised in vitro with 0.5 mM 8-Br-cAMP (Sigma-Aldrich Corp.) to model the decidual stromal cells [17]. HTR-8/SVneo cells were maintained in RPMI-1640 medium with 10% FBS, 2 mM L-glutamine, 100 U/ ml penicillin and 100 µg/ml streptomycin under humidified conditions (5% CO2/95% air) at 37 °C; and used to model the extravillous trophoblast cells [18]. 2.2. Decidualisation of St-T1b cells St-T1b cells were seeded 4 × 105 cells per wells in a 6-well plate in normal growth medium. When confluent, new medium containing 0.5 mM 8-Br-cAMP in DMEM medium and Ham’s F12 medium in a 1:1
⁎ Corresponding author at: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. E-mail address: [email protected] (H.E.J. Yong). 1 Present address: Department of Medicine and Department of Obstetrics and Gynaecology, School of Clinical Sciences, The Ritchie Centre, Monash University, Monash Medical Centre, Clayton 3168, Victoria, Australia. 2 Joint senior authors.
https://doi.org/10.1016/j.preghy.2017.11.002 Received 30 May 2017; Received in revised form 11 August 2017; Accepted 13 November 2017 2210-7789/ © 2017 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Yong, H.E.J., Pregnancy Hypertension (2017), https://doi.org/10.1016/j.preghy.2017.11.002
Pregnancy Hypertension xxx (xxxx) xxx–xxx
H.E.J. Yong et al.
3. Results
ratio with 2% (v/v) charcoal-stripped FCS, 2 mM L-glutamine, 100 U/ ml penicillin and 100 μg/ml streptomycin was used to decidualise the cells for 72 h. Concurrently, a separate batch of St-T1b cells from the same passage received the same treatment with the exception of no 8Br-cAMP addition to determine the effects of decidualisation.
The ACVR2A receptor was expressed by the St-T1b cell line and was localised primarily to the cytoplasm in both non-decidualised and decidualised cells (Fig. 1A). Decidualisation of St-T1b cells with 8-BrcAMP was verified by significant induction of prolactin PRL mRNA by over 1,000 fold (Fig. 1B). Decidualisation with 8-Br-cAMP significantly increased ACVR2A mRNA expression by 2.2 fold (Fig. 1C). The representative immunoblot demonstrates an increase of immunoreactive ACVR2A protein after decidualisation of St-T1b with 8-Br-cAMP (Fig. 1D). After correcting for protein loading with the GAPDH control, a significant increase of 1.2 fold was observed at the protein level (Fig. 1E). Trophoblast functions of adhesion, proliferation, migration and invasion were determined by xCELLigence assays. Treatment with conditioned medium from decidualised St-T1b cells significantly inhibited HTR-8/SVneo adhesion at 1 hour, and proliferation, migration and invasion at 24 h by 45%, 62%, 58% and 72% respectively (Fig. 1F–I). To model decreased decidual ACVR2A expression in PE, siRNA transfection was used to silence ACVR2A expression of decidualised StT1b cells. ACVR2A mRNA expression was significantly reduced by 75% in decidualised St-T1b cells transfected with ACVR2A siRNA compared with the negative control after 72 h (Fig. 2A). The representative immunoblot demonstrates an evident knockdown of immunoreactive ACVR2A protein (Fig. 2B). A knockdown of approximately 31% was achieved at the protein level (Fig. 2C). Additionally, prolactin expression was compared to examine the effect of ACVR2A receptor knockdown on decidualisation. Transfection of ACVR2A siRNA into decidualised St-T1b cells significantly lowered PRL mRNA induction by 83% (Fig. 2D). Conditioned medium from decidualised ACVR2A siRNAtransfected St-T1b cells significantly increased HTR-8/SVneo trophoblast adhesion at 1 hour and proliferation, migration and invasion at 24 h by 14%, 17%, 41% and 28% respectively (Fig. 2E–H).
2.3. Immunocytochemistry Immunocytochemistry was performed as described previously [19]. Rabbit polyclonal activin receptor type IIA ab71521 antibody (5 μg/ml, Abcam) was used to detect ACVR2A. 2.4. Western immunoblotting Western immunoblotting was performed as described previously with either ACVR2A antibody (1.7 μg/ml) or rabbit anti-GAPDH Polyclonal IgG (0.5 μg/ml, Imgenex) [20]. 2.5. RNA extraction, cDNA synthesis and real-time polymerase chain reaction RNA extraction, cDNA synthesis and real-time PCR were performed as described previously [19,21]. PRL and ACVR2A mRNA transcripts were probed using inventoried FAM-labelled TaqMan® Gene Expression Assays (Applied Biosystems) Hs00168730_m1 and Hs00155658_m1 respectively. Relative mRNA expression levels were then calculated using the 2−ΔΔCT method [22]. 2.6. siRNA transfection ACVR2A expression was silenced over 72 h using a pool of ACVR2Aspecific siRNA (Santa Cruz Biotechnology Inc.). Non-targeting siRNA (Santa Cruz Biotechnology Inc.) was used as the negative control (NC). St-T1b cells were seeded at 4 × 105 cells per well in 6-well plates and allowed to reach confluence prior to siRNA transfection. HiPerFect transfection reagent (Qiagen) was then used to transfect 5 nM siRNA into St-T1b cells according to the manufacturer’s protocol. Wells also contained 0.5 mM 8-Br-cAMP to decidualise the cells during the transfection period.
4. Discussion To test the suitability of the St-T1b model for in vitro functional analyses, expression of the ACVR2A receptor in St-T1b cells was first verified by immunocytochemistry (Fig. 1A). Decidualisation was then confirmed by significantly increased mRNA expression of prolactin (Fig. 1B), which is a marker of decidualisation levels in pregnancy [23]. The significant increase in ACVR2A expression with in vitro decidualisation (Fig. 1C–E) was consistent with a previous report of increased receptor expression during the secretory phase of the menstrual cycle, when decidualisation occurs in vivo [10]. Conditioned medium collected from decidualised St-T1b cells significantly inhibited HTR-8/ SVneo trophoblast adhesion, proliferation, migration and invasion (Fig. 1F–I), which is also consistent with past studies [2,3]. Having established the suitability of the model, further functional analyses were performed to examine the effects of altered decidual ACVR2A expression observed in pre-eclampsia on decidualisation and its ability to regulate trophoblast functions. Silencing of decidual ACVR2A by siRNA transfection significantly reduced production of the decidualisation marker prolactin (Fig. 2D), suggesting that altered ACVR2A expression can impair decidualisation of stromal cells. A recent microarray study showed that insufficient decidualisation or endometrial maturation in the first trimester is implicated in the development of pre-eclampsia [24]. Another study demonstrated that pre-eclamptic decidua produced significantly less prolactin [25]. This study also found that serum from pre-eclamptic women did not affect normotensive decidua explant prolactin production, suggesting an inherent genetic defect in pre-eclamptic decidua rather than the influence of a circulatory factor [25]. Hence, these observations suggest that adequate ACVR2A expression plays a role in the optimal conditioning of the decidua for pregnancy. Conversely, the genetic dysregulation of ACVR2A expression may lead to suboptimal
2.7. Conditioned medium collection and treatment Conditioned medium was aspirated from St-T1b culture plates after 72 h of either 8-Br-cAMP, siRNA or control treatment and centrifuged at 600×g for 5 min at room temperature. The supernatant was then aliquoted and stored at −80 °C. For in vitro functional assays, HTR-8/ SVneo cells were treated with 25% conditioned medium pooled from a minimum of n = 3 batches collected under sterile conditions. 2.8. Functional assays Adhesion, proliferation, migration and invasion assays were performed over 24 h using the xCELLigence RTCA DP Analyzer (ACEA Biosciences Inc.) with E-16 plates and CIM-16 plates for adhesion/ proliferation and migration/invasion respectively. Invasion assays were performed with wells pre-coated with Matrigel™ Basement Membrane Matrix (Becton Dickinson). xCELLigence results were then analysed as described previously [19]. 2.9. Statistical analyses Paired Student’s t test and 2 X 2 contingency tables with Fisher’s Exact Test were used as appropriate. All data are expressed as mean ± SEM unless stated otherwise. GraphPad Prism 5 (GraphPad Software Inc.) was used for statistical analyses. A value of p < .05 was considered statistically significant. 2
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Fig. 1. Effect of decidualisation on St-T1b ACVR2A and PRL expression, and HTR-8/SVneo trophoblast functions. A: Representative ACVR2A immunostaining in both undecidualised and decidualised St-T1b cells with negative IgG control inset. B: Induction of PRL mRNA expression with 8-Br-cAMP treatment after 72 h. C: Increased ACVR2A mRNA expression with 8-BrcAMP treatment after 72 h. D: Representative immunoblot of St-T1b ACVR2A protein with GAPDH loading control. E: Protein quantitation of ACVR2A in St-T1b cells. F: HTR-8/SVneo trophoblast adhesion at 1 hour. G: HTR-8/SVneo trophoblast proliferation at 24 h. H: HTR-8/SVneo trophoblast migration at 24 h. I: HTR-8/SVneo trophoblast invasion at 24 h. The graphs depict results from n ≥ 3 independent experiments. Data presented as mean ± SEM. *p < .05, **p < .01, ***p < .001, paired Student’s t test. Scale bar represents 100 µm.
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Fig. 2. Effect of ACVR2A siRNA knockdown on decidualised St-T1b cells and HTR-8/SVneo trophoblast functions. A: Reduction of ACVR2A mRNA expression with siRNA transfection. B: Representative immunoblot of ACVR2A protein and GAPDH loading control. C: Protein quantitation of ACVR2A in St-T1b cells. D: Reduction of PRL mRNA expression with ACVR2A siRNA transfection after 72 h. E: HTR-8/SVneo trophoblast adhesion at 1 hour. F: HTR-8/SVneo trophoblast proliferation at 24 h. G: HTR-8/SVneo trophoblast migration at 24 h. H: HTR8/SVneo trophoblast invasion at 24 h. NC – negative control non-targeting siRNA; siRNA – ACVR2A siRNA. The graphs depict results from n ≥ 3 independent experiments. Data presented as mean ± SEM. *p < .05, ***p < .001, paired Student’s t test.
beneficial for trophoblast invasion and subsequent spiral arterial remodelling at first glance, any perturbation in the maternal-fetal cross talk is likely to have detrimental effects on placentation. For example, undecidualised cells produce factors that can elicit a pro-inflammatory response from extravillous trophoblast cells [1]. As such, it is probable that an excessive inflammatory response may lead to an improper maternal immune interaction with the invading fetal trophoblast cells, which could ultimately restrict trophoblast invasion and impair spiral arteriole remodelling. Thus, altered decidual ACVR2A expression may
decidualisation and contribute to the development of pre-eclampsia. Additionally, conditioned medium collected from the ACVR2A siRNA transfected decidualised St-T1b cells significantly promoted HTR-8/SVneo trophoblast adhesion, proliferation, migration and invasion above that of the negative control decidualised St-T1b cells (Fig. 2E–H), thereby resembling the stimulatory effects of the undecidualised St-T1b cells (Fig. 1F–I). Normal trophoblast invasion into the maternal decidua is a tightly regulated process. Therefore, although decreased decidual ACVR2A expression appears paradoxically 4
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also contribute to aberrant regulation of trophoblast functions, with potentially negative consequences for placentation that may lead to pre-eclampsia. In conclusion, these findings collectively suggest that altered decidual ACVR2A expression impairs the ability of stromal cells to properly decidualise and regulate trophoblast function at the maternalfetal interface, which may result in abnormal placentation that can lead to poor pregnancy outcomes such as pre-eclampsia.
[9]
[10]
[11]
Declaration of interest [12]
The authors report no conflict of interest. [13]
Acknowledgements We would like to acknowledge Dr Birgit Gellersen, Endokrinologikum Hamburg, Hamburg, Germany for her generous gift of the St-T1b cell line and Prof Charles Graham, Queen’s University, Kingston, Canada for his generous gift of the HTR-8/SVneo cell line.
[14]
[15]
Funding sources [16]
This study was funded by the Royal Women’s Hospital, Melbourne, Australia. H.E.J. Yong was supported by the Melbourne International Fee Remission Scholarship and the Felix Meyer Scholarship in Obstetrics and Gynaecology from the University of Melbourne, Melbourne, Australia.
[17]
[18]
References [19] [1] E.M. Menkhorst, N. Lane, A.L. Winship, P. Li, J. Yap, K. Meehan, A. Rainczuk, A. Stephens, E. Dimitriadis, Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation, PLoS One. 7 (2) (2012) e31418. [2] H. Singh, Y. Endo, G. Nie, Decidual HtrA3 negatively regulates trophoblast invasion during human placentation, Human Reprod. 26 (4) (2011) 748–757. [3] P. Bischof, A. Meisser, A. Campana, L. Tseng, Effects of decidua-conditioned medium and insulin-like growth factor binding protein-1 on trophoblastic matrix metalloproteinases and their inhibitors, Placenta 19 (7) (1998) 457–464. [4] X.M. Zhu, T. Han, I.L. Sargent, Y.L. Wang, Y.Q. Yao, Conditioned medium from human decidual stromal cells has a concentration-dependent effect on trophoblast cell invasion, Placenta 30 (1) (2009) 74–78. [5] G. Godbole, P. Suman, S.K. Gupta, D. Modi, Decidualized endometrial stromal cell derived factors promote trophoblast invasion, Fertil Steril. 95 (4) (2011) 1278–1283. [6] R.L. Jones, L.A. Salamonsen, J.K. Findlay, Activin A promotes human endometrial stromal cell decidualization in vitro, The Journal of clinical endocrinology and metabolism. 87 (8) (2002) 4001–4004. [7] E. Menkhorst, L.A. Salamonsen, J. Zhang, C.A. Harrison, J. Gu, E. Dimitriadis, Interleukin 11 and activin A synergise to regulate progesterone-induced but not cAMP-induced decidualization, J. Reprod. Immunol. 84 (2) (2010) 124–132. [8] R.L. Jones, L.A. Salamonsen, H.O. Critchley, P.A. Rogers, B. Affandi, J.K. Findlay, Inhibin and activin subunits are differentially expressed in endometrial cells and
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leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception, Mol. Human Reprod. 6 (12) (2000) 1107–1117. T. Otani, S. Minami, K. Kokawa, T. Shikone, M. Yamoto, R. Nakano, Immunohistochemical localization of activin A in human endometrial tissues during the menstrual cycle and in early pregnancy, Obstet Gynecol. 91 (5 Pt 1) (1998) 685–692. R.L. Jones, L.A. Salamonsen, Y.C. Zhao, J.F. Ethier, A.E. Drummond, J.K. Findlay, Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization, Mol. Human Reprod. 8 (4) (2002) 363–374. L.C. Ferreira, C.E. Gomes, A.C. Araujo, P.F. Bezerra, P. Duggal, S.M. Jeronimo, Association between ACVR2A and early-onset preeclampsia: replication study in a Northeastern Brazilian population, Placenta 36 (2) (2015) 186–190. E. Fitzpatrick, M.P. Johnson, T.D. Dyer, S. Forrest, K. Elliott, J. Blangero, S.P. Brennecke, E.K. Moses, Genetic association of the activin A receptor gene (ACVR2A) and pre-eclampsia, Mol. Human Reprod. 15 (3) (2009) 195–204. E.K. Moses, E. Fitzpatrick, K.A. Freed, T.D. Dyer, S. Forrest, K. Elliott, M.P. Johnson, J. Blangero, S.P. Brennecke, Objective prioritization of positional candidate genes at a quantitative trait locus for pre-eclampsia on 2q22, Mol Human Repro. 12 (8) (2006) 505–512. L.T. Roten, M.P. Johnson, S. Forsmo, E. Fitzpatrick, T.D. Dyer, S.P. Brennecke, J. Blangero, E.K. Moses, R. Austgulen, Association between the candidate susceptibility gene ACVR2A on chromosome 2q22 and pre-eclampsia in a large Norwegian population-based study (the HUNT study), Eur. J. Human Genetics: EJHG. 17 (2) (2009) 250–257. B. Zeybek, H.A. Celik, H.H. Aydin, N. Askar, Polymorphisms in the activin A receptor type 2A gene affect the onset time and severity of preeclampsia in the Turkish population, J. Perinatal Med. 41 (4) (2013) 389–399. U. Manuelpillai, M. Schneider-Kolsky, A. Dole, E.M. Wallace, Activin A and activin receptors in gestational tissue from preeclamptic pregnancies, J. Endocrinol. 171 (1) (2001) 57–64. A. Samalecos, K. Reimann, S. Wittmann, H.M. Schulte, J.J. Brosens, A.M. Bamberger, B. Gellersen, Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b, Reprod. Biol. Endocrinol: RB&E. 7 (2009) 76. C.H. Graham, T.S. Hawley, R.G. Hawley, J.R. MacDougall, R.S. Kerbel, N. Khoo, P.K. Lala, Establishment and characterization of first trimester human trophoblast cells with extended lifespan, Exp. Cell Res. 206 (2) (1993) 204–211. H.E. Yong, P. Murthi, M.H. Wong, B. Kalionis, J.E. Cartwright, S.P. Brennecke, R.J. Keogh, Effects of normal and high circulating concentrations of activin A on vascular endothelial cell functions and vasoactive factor production, Pregnancy Hypertens. 5 (4) (2015) 346–353. H.E. Yong, P. Murthi, M.H. Wong, B. Kalionis, S.P. Brennecke, R.J. Keogh, Antiangiogenic collagen fragment arresten is increased from 16 weeks' gestation in preeclamptic plasma, Placenta 36 (11) (2015) 1300–1309. H.E. Yong, P. Murthi, A. Borg, B. Kalionis, E.K. Moses, S.P. Brennecke, R.J. Keogh, Increased decidual mRNA expression levels of candidate maternal pre-eclampsia susceptibility genes are associated with clinical severity, Placenta 35 (2) (2014) 117–124. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (4) (2001) 402–408. W.X. Wu, J. Brooks, A.F. Glasier, A.S. McNeilly, The relationship between decidualization and prolactin mRNA and production at different stages of human pregnancy, J Mol Endocrinol. 14 (2) (1995) 255–261. M.B. Rabaglino, E.D. Post Uiterweer, A. Jeyabalan, W.A. Hogge, K.P. Conrad, Bioinformatics approach reveals evidence for impaired endometrial maturation before and during early pregnancy in women who developed preeclampsia, Hypertension 65 (2) (2015) 421–429. A. Golander, R. Kopel, N. Lazebnik, Y. Frenkel, Z. Spirer, Decreased prolactin secretion by decidual tissue of pre-eclampsia in vitro, Acta Endocrinol. (Copenh). 108 (1) (1985) 111–113.
Contents lists available at ScienceDirect
Pregnancy Hypertension journal homepage: www.elsevier.com/locate/preghy
Short communication
Decidual ACVR2A regulates extravillous trophoblast functions of adhesion, proliferation, migration and invasion in vitro ⁎
Hannah E.J. Yong , Padma Murthi1, Bill Kalionis, Rosemary J. Keogh2, Shaun P. Brennecke2 The University of Melbourne, Department of Obstetrics and Gynaecology and Department of Maternal-Fetal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital, Locked Bag 300, Corner Grattan Street and Flemington Road, Parkville 3052, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords: ACVR2A Decidual stromal cells Extravillous trophoblast
Decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface. Our aim was to investigate the role of the pre-eclampsia associated decidual activin receptor, ACVR2A, in regulating trophoblast functions at this interface. St-T1b and HTR-8/SVneo cell lines were used to model decidual stromal and trophoblast cells respectively. St-T1b conditioned medium inhibited HTR-8/SVneo adhesion, proliferation, migration and invasion; all effects that were attenuated by decidual ACVR2A siRNA transfection. These findings suggest that altered decidual ACVR2A expression perturbs the maternal-fetal crosstalk involved in regulating trophoblast function at the interface, which may affect placentation and lead to pre-eclampsia.
1. Introduction In pregnancy, decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface, where they regulate multiple functions of fetal extravillous trophoblast cells. The decidualised stromal cells produce and release secretory factors [1,2], which can inhibit trophoblast gelatinolytic activity [3] by modifying expression of metalloproteinases [4], integrins and tissue inhibitors of metalloproteinases [5] that are necessary for invading the decidual matrix. Past studies show that activin A, a TGFβ family member, is actively involved in inducing and amplifying the decidualisation process, which prepares the uterine endometrium for pregnancy [6,7]. Expression of the activin A βA subunit is significantly upregulated in uterine stromal cells during the secretory phase of the menstrual cycle, when decidualisation begins in vivo and in early pregnancy [8,9]. A similar temporal expression pattern is also observed for the activin A binding receptor, ACVR2A [10]. Nevertheless, while the role of activin A in preparing and supporting a healthy pregnancy at the maternal-fetal interface is well-established, the role of its receptor ACVR2A in mediating its effects is currently unknown. Additionally, genetic variants of ACVR2A show associations with the hypertensive disorder – preeclampsia [11–15], where ACVR2A expression is significantly reduced at the maternal-fetal interface [13,16]. Therefore, our study aim was to
explore the functional effects of altered decidual ACVR2A at this interface using an in vitro cell culture model. 2. Materials & methods 2.1. Cell culture St-T1b cells were maintained in DMEM medium and Ham’s F12 medium in a 1:1 ratio, with 10% charcoal-stripped fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin under humidified conditions (5% CO2/95% air) at 37 °C; and decidualised in vitro with 0.5 mM 8-Br-cAMP (Sigma-Aldrich Corp.) to model the decidual stromal cells [17]. HTR-8/SVneo cells were maintained in RPMI-1640 medium with 10% FBS, 2 mM L-glutamine, 100 U/ ml penicillin and 100 µg/ml streptomycin under humidified conditions (5% CO2/95% air) at 37 °C; and used to model the extravillous trophoblast cells [18]. 2.2. Decidualisation of St-T1b cells St-T1b cells were seeded 4 × 105 cells per wells in a 6-well plate in normal growth medium. When confluent, new medium containing 0.5 mM 8-Br-cAMP in DMEM medium and Ham’s F12 medium in a 1:1
⁎ Corresponding author at: Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. E-mail address: [email protected] (H.E.J. Yong). 1 Present address: Department of Medicine and Department of Obstetrics and Gynaecology, School of Clinical Sciences, The Ritchie Centre, Monash University, Monash Medical Centre, Clayton 3168, Victoria, Australia. 2 Joint senior authors.
https://doi.org/10.1016/j.preghy.2017.11.002 Received 30 May 2017; Received in revised form 11 August 2017; Accepted 13 November 2017 2210-7789/ © 2017 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Yong, H.E.J., Pregnancy Hypertension (2017), https://doi.org/10.1016/j.preghy.2017.11.002
Pregnancy Hypertension xxx (xxxx) xxx–xxx
H.E.J. Yong et al.
3. Results
ratio with 2% (v/v) charcoal-stripped FCS, 2 mM L-glutamine, 100 U/ ml penicillin and 100 μg/ml streptomycin was used to decidualise the cells for 72 h. Concurrently, a separate batch of St-T1b cells from the same passage received the same treatment with the exception of no 8Br-cAMP addition to determine the effects of decidualisation.
The ACVR2A receptor was expressed by the St-T1b cell line and was localised primarily to the cytoplasm in both non-decidualised and decidualised cells (Fig. 1A). Decidualisation of St-T1b cells with 8-BrcAMP was verified by significant induction of prolactin PRL mRNA by over 1,000 fold (Fig. 1B). Decidualisation with 8-Br-cAMP significantly increased ACVR2A mRNA expression by 2.2 fold (Fig. 1C). The representative immunoblot demonstrates an increase of immunoreactive ACVR2A protein after decidualisation of St-T1b with 8-Br-cAMP (Fig. 1D). After correcting for protein loading with the GAPDH control, a significant increase of 1.2 fold was observed at the protein level (Fig. 1E). Trophoblast functions of adhesion, proliferation, migration and invasion were determined by xCELLigence assays. Treatment with conditioned medium from decidualised St-T1b cells significantly inhibited HTR-8/SVneo adhesion at 1 hour, and proliferation, migration and invasion at 24 h by 45%, 62%, 58% and 72% respectively (Fig. 1F–I). To model decreased decidual ACVR2A expression in PE, siRNA transfection was used to silence ACVR2A expression of decidualised StT1b cells. ACVR2A mRNA expression was significantly reduced by 75% in decidualised St-T1b cells transfected with ACVR2A siRNA compared with the negative control after 72 h (Fig. 2A). The representative immunoblot demonstrates an evident knockdown of immunoreactive ACVR2A protein (Fig. 2B). A knockdown of approximately 31% was achieved at the protein level (Fig. 2C). Additionally, prolactin expression was compared to examine the effect of ACVR2A receptor knockdown on decidualisation. Transfection of ACVR2A siRNA into decidualised St-T1b cells significantly lowered PRL mRNA induction by 83% (Fig. 2D). Conditioned medium from decidualised ACVR2A siRNAtransfected St-T1b cells significantly increased HTR-8/SVneo trophoblast adhesion at 1 hour and proliferation, migration and invasion at 24 h by 14%, 17%, 41% and 28% respectively (Fig. 2E–H).
2.3. Immunocytochemistry Immunocytochemistry was performed as described previously [19]. Rabbit polyclonal activin receptor type IIA ab71521 antibody (5 μg/ml, Abcam) was used to detect ACVR2A. 2.4. Western immunoblotting Western immunoblotting was performed as described previously with either ACVR2A antibody (1.7 μg/ml) or rabbit anti-GAPDH Polyclonal IgG (0.5 μg/ml, Imgenex) [20]. 2.5. RNA extraction, cDNA synthesis and real-time polymerase chain reaction RNA extraction, cDNA synthesis and real-time PCR were performed as described previously [19,21]. PRL and ACVR2A mRNA transcripts were probed using inventoried FAM-labelled TaqMan® Gene Expression Assays (Applied Biosystems) Hs00168730_m1 and Hs00155658_m1 respectively. Relative mRNA expression levels were then calculated using the 2−ΔΔCT method [22]. 2.6. siRNA transfection ACVR2A expression was silenced over 72 h using a pool of ACVR2Aspecific siRNA (Santa Cruz Biotechnology Inc.). Non-targeting siRNA (Santa Cruz Biotechnology Inc.) was used as the negative control (NC). St-T1b cells were seeded at 4 × 105 cells per well in 6-well plates and allowed to reach confluence prior to siRNA transfection. HiPerFect transfection reagent (Qiagen) was then used to transfect 5 nM siRNA into St-T1b cells according to the manufacturer’s protocol. Wells also contained 0.5 mM 8-Br-cAMP to decidualise the cells during the transfection period.
4. Discussion To test the suitability of the St-T1b model for in vitro functional analyses, expression of the ACVR2A receptor in St-T1b cells was first verified by immunocytochemistry (Fig. 1A). Decidualisation was then confirmed by significantly increased mRNA expression of prolactin (Fig. 1B), which is a marker of decidualisation levels in pregnancy [23]. The significant increase in ACVR2A expression with in vitro decidualisation (Fig. 1C–E) was consistent with a previous report of increased receptor expression during the secretory phase of the menstrual cycle, when decidualisation occurs in vivo [10]. Conditioned medium collected from decidualised St-T1b cells significantly inhibited HTR-8/ SVneo trophoblast adhesion, proliferation, migration and invasion (Fig. 1F–I), which is also consistent with past studies [2,3]. Having established the suitability of the model, further functional analyses were performed to examine the effects of altered decidual ACVR2A expression observed in pre-eclampsia on decidualisation and its ability to regulate trophoblast functions. Silencing of decidual ACVR2A by siRNA transfection significantly reduced production of the decidualisation marker prolactin (Fig. 2D), suggesting that altered ACVR2A expression can impair decidualisation of stromal cells. A recent microarray study showed that insufficient decidualisation or endometrial maturation in the first trimester is implicated in the development of pre-eclampsia [24]. Another study demonstrated that pre-eclamptic decidua produced significantly less prolactin [25]. This study also found that serum from pre-eclamptic women did not affect normotensive decidua explant prolactin production, suggesting an inherent genetic defect in pre-eclamptic decidua rather than the influence of a circulatory factor [25]. Hence, these observations suggest that adequate ACVR2A expression plays a role in the optimal conditioning of the decidua for pregnancy. Conversely, the genetic dysregulation of ACVR2A expression may lead to suboptimal
2.7. Conditioned medium collection and treatment Conditioned medium was aspirated from St-T1b culture plates after 72 h of either 8-Br-cAMP, siRNA or control treatment and centrifuged at 600×g for 5 min at room temperature. The supernatant was then aliquoted and stored at −80 °C. For in vitro functional assays, HTR-8/ SVneo cells were treated with 25% conditioned medium pooled from a minimum of n = 3 batches collected under sterile conditions. 2.8. Functional assays Adhesion, proliferation, migration and invasion assays were performed over 24 h using the xCELLigence RTCA DP Analyzer (ACEA Biosciences Inc.) with E-16 plates and CIM-16 plates for adhesion/ proliferation and migration/invasion respectively. Invasion assays were performed with wells pre-coated with Matrigel™ Basement Membrane Matrix (Becton Dickinson). xCELLigence results were then analysed as described previously [19]. 2.9. Statistical analyses Paired Student’s t test and 2 X 2 contingency tables with Fisher’s Exact Test were used as appropriate. All data are expressed as mean ± SEM unless stated otherwise. GraphPad Prism 5 (GraphPad Software Inc.) was used for statistical analyses. A value of p < .05 was considered statistically significant. 2
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Fig. 1. Effect of decidualisation on St-T1b ACVR2A and PRL expression, and HTR-8/SVneo trophoblast functions. A: Representative ACVR2A immunostaining in both undecidualised and decidualised St-T1b cells with negative IgG control inset. B: Induction of PRL mRNA expression with 8-Br-cAMP treatment after 72 h. C: Increased ACVR2A mRNA expression with 8-BrcAMP treatment after 72 h. D: Representative immunoblot of St-T1b ACVR2A protein with GAPDH loading control. E: Protein quantitation of ACVR2A in St-T1b cells. F: HTR-8/SVneo trophoblast adhesion at 1 hour. G: HTR-8/SVneo trophoblast proliferation at 24 h. H: HTR-8/SVneo trophoblast migration at 24 h. I: HTR-8/SVneo trophoblast invasion at 24 h. The graphs depict results from n ≥ 3 independent experiments. Data presented as mean ± SEM. *p < .05, **p < .01, ***p < .001, paired Student’s t test. Scale bar represents 100 µm.
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Fig. 2. Effect of ACVR2A siRNA knockdown on decidualised St-T1b cells and HTR-8/SVneo trophoblast functions. A: Reduction of ACVR2A mRNA expression with siRNA transfection. B: Representative immunoblot of ACVR2A protein and GAPDH loading control. C: Protein quantitation of ACVR2A in St-T1b cells. D: Reduction of PRL mRNA expression with ACVR2A siRNA transfection after 72 h. E: HTR-8/SVneo trophoblast adhesion at 1 hour. F: HTR-8/SVneo trophoblast proliferation at 24 h. G: HTR-8/SVneo trophoblast migration at 24 h. H: HTR8/SVneo trophoblast invasion at 24 h. NC – negative control non-targeting siRNA; siRNA – ACVR2A siRNA. The graphs depict results from n ≥ 3 independent experiments. Data presented as mean ± SEM. *p < .05, ***p < .001, paired Student’s t test.
beneficial for trophoblast invasion and subsequent spiral arterial remodelling at first glance, any perturbation in the maternal-fetal cross talk is likely to have detrimental effects on placentation. For example, undecidualised cells produce factors that can elicit a pro-inflammatory response from extravillous trophoblast cells [1]. As such, it is probable that an excessive inflammatory response may lead to an improper maternal immune interaction with the invading fetal trophoblast cells, which could ultimately restrict trophoblast invasion and impair spiral arteriole remodelling. Thus, altered decidual ACVR2A expression may
decidualisation and contribute to the development of pre-eclampsia. Additionally, conditioned medium collected from the ACVR2A siRNA transfected decidualised St-T1b cells significantly promoted HTR-8/SVneo trophoblast adhesion, proliferation, migration and invasion above that of the negative control decidualised St-T1b cells (Fig. 2E–H), thereby resembling the stimulatory effects of the undecidualised St-T1b cells (Fig. 1F–I). Normal trophoblast invasion into the maternal decidua is a tightly regulated process. Therefore, although decreased decidual ACVR2A expression appears paradoxically 4
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also contribute to aberrant regulation of trophoblast functions, with potentially negative consequences for placentation that may lead to pre-eclampsia. In conclusion, these findings collectively suggest that altered decidual ACVR2A expression impairs the ability of stromal cells to properly decidualise and regulate trophoblast function at the maternalfetal interface, which may result in abnormal placentation that can lead to poor pregnancy outcomes such as pre-eclampsia.
[9]
[10]
[11]
Declaration of interest [12]
The authors report no conflict of interest. [13]
Acknowledgements We would like to acknowledge Dr Birgit Gellersen, Endokrinologikum Hamburg, Hamburg, Germany for her generous gift of the St-T1b cell line and Prof Charles Graham, Queen’s University, Kingston, Canada for his generous gift of the HTR-8/SVneo cell line.
[14]
[15]
Funding sources [16]
This study was funded by the Royal Women’s Hospital, Melbourne, Australia. H.E.J. Yong was supported by the Melbourne International Fee Remission Scholarship and the Felix Meyer Scholarship in Obstetrics and Gynaecology from the University of Melbourne, Melbourne, Australia.
[17]
[18]
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