Thesis
Reference
Role of GRP78 in trophoblastic cell fusion and differentiation
BASTIDA RUIZ, Daniel
Abstract
Le placenta est un organe généré pendant la grossesse qui a pour objectif d'assurer le bon développement fœtal. L'un des principaux types cellulaires placentaires est le syncytiotrophoblaste formé par un processus appelé syncytialisation, consistant en la fusion et différentiation de cytotrophoblastes. Nous avons établi que la «glucose-regulated protein 78» (GRP78), une protéine connue pour réguler la «unfolded protein response» (UPR) et pour agir comme récepteur à la surface cellulaire, est impliquée dans la syncytialisation. Nous avons démontré que l'UPR est activée pendant la syncytialisation et qu'elle permet ainsi de réguler l'autophagie et la survie cellulaire nécessaires à la syncytialisation. Par ailleurs, nous avons démontré que la GRP78 à la surface cellulaire interagit avec l'α2-macroglobuline et permet ainsi l'activation de la voie de signalisation ERK1/2-CREB-UPR conduisant à la modulation de la fusion trophoblastique. En conclusion, nous avons démontré le rôle essentiel de la GRP78 dans la régulation de la syncytialisation trophoblastique.
BASTIDA RUIZ, Daniel. Role of GRP78 in trophoblastic cell fusion and differentiation. Thèse de doctorat : Univ. Genève, 2020, no. Sc. Vie 51
DOI : 10.13097/archive-ouverte/unige:144709 URN : urn:nbn:ch:unige-1447093
Available at:
http://archive-ouverte.unige.ch/unige:144709
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE FACULTÉ DE MÉDECINE Section de médecine clinique
Département de pédiatrie, gynécologie et obstétrique Professeure Marie Cohen
ROLE OF GRP78 IN TROPHOBLASTIC CELL FUSION AND DIFFERENTIATION
THÈSE
présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en sciences de la vie,
mention Sciences biomédicales
par
Daniel BASTIDA RUIZ
de
Logroño (Espagne)
Thèse No 51
GENÈVE
2020
UFE SCIENCES PhD SCHOOL
GENEVA
DocroRAT Ès sctENcES EN ScTENCES DE LAVrE DES
FAcUITÉs DE MÉoTcINE ET DES SCIENCES MENTIoN SCIENCES BIOMÉoICnIrS
Thèse de Mr Daniel BASTIDA RUIZ
intitulée :
<<
Role of GRP78 in trophoblastic cell fusion and differentiation >
Les Facultés de médecine
et
des sciences,sur
le préavisde
Madame Marie COHEN, professeure assistante et directrice de thèse (Département de Pédiatrie, Gynécologie et Obstétrique), MonsieurSerge
NEF, professeur ordinaire (Départementde
Médecine Génétiqueet
Développement), Madame Patrycja NOWAK-SLIWINSKA, professeure assistante (Départementdes
Sciences Pharmaceutiques), Madame Nadia ALFAIDY professeure (U1036:
Biologiedu
Canceret de
I'infection, INSERM, Grenoble, France) autorisent I'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées.Genève, le 24 février 2020
Thèse -
51Le
Faculté de médecine
N.B.
-
La thèse doit porter la déclaration précédente et remplir les conditions énumérées dans les "lnformations relatives aux thèses de doctorat à I'Université de Genève".Le Décanat
Faculté des sciences
FACULTE DE MEDECINE FACULTÉ DES SCIENCES
a
UN IVERSITE
DE GENEVÊ
T ABLE OF CONTENTS
ACKNOWLEDGEMENTS ... 1
ABSTRACT ... 9
RÉSUMÉ ... 11
ABBREVIATION LIST ... 14
FIGURE LIST ... 22
1 INTRODUCTION ... 24
1.1 Placenta ... 25
1.1.1 vCTB cell fusion ... 35
1.1.2 Syncytiotrophoblast ... 41
1.1.3 Pregnancy pathologies ... 44
1.1.3.1 Preeclampsia ... 45
1.2 GRP78 ... 48
1.2.1 GRP78 structure ... 49
1.2.2 GRP78 localisation and functions ... 50
1.2.2.1 Secreted ... 51
1.2.2.2 Cytosolic GRP78 ... 51
1.2.2.3 Mitochondrial GRP78 ... 52
1.2.2.4 Nuclear GRP78 ... 52
1.2.2.5 Cell membrane-localised GRP78 ... 52
1.2.2.5.1 α2M* interaction... 54
1.2.2.5.2 CRIPTO interaction ... 56
1.2.2.5.3 Integrin β-1 interaction ... 57
1.2.2.5.4 Par-4 interaction ... 58
1.2.2.5.5 Kringle 5 interaction ... 58
1.2.2.6 ER lumen-localised GRP78 ... 58
1.2.2.6.1 Chaperone function ... 59
1.2.2.6.2 ER-translocator helper ... 60
1.2.2.6.3 UPR regulator ... 60
1.3 The UPR ... 61
1.3.1 The UPR branches ... 63
1.3.1.1 ATF6 ... 63
1.3.1.2 PERK ... 65
1.3.1.3 IRE1α ... 65
1.3.2 UPR-triggered mechanisms ... 67
1.3.2.1 Autophagy ... 67
1.3.2.2 Apoptosis ... 70
1.3.2.2.1 Intrinsic pathway ... 71
1.3.2.2.2 Extrinsic pathway ... 73
2 AIMS OF THE STUDY ... 76
3 RESULTS ... 78
3.1 CHAPTER I: The fine-tuning of endoplasmic reticulum stress response and autophagy activation during trophoblast syncytialization ... 79
3.2 CHAPTER II: Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion ... 101
4 DISCUSSION ... 140
4.1 Cell surface-localised GRP78 ... 145
4.2 Preeclampsia ... 149
4.3 GRP78 as a therapeutic approach and the UPR ... 153
5 CONCLUSIONS AND PERSPECTIVES ... 155
6 REFERENCES ... 156
7 ANNEXES ... 187
1
A CKNOWLEDGEMENTS
First, I would like to thank Marie Cohen for giving me the opportunity to work in her lab during the last for years and a half. Thank you for trusting me since the beginning and for teaching me so many things, not only at the working level, but also at the personal one. We have had our difficulties during this time, but I will always remember the good moments and, overall, the experience have been nothing but positive. I also have to acknowledge your patience with me and the flexibility you have demonstrated during this time. It has been a pleasure working with you.
I would also like to thank Nadia Alfaidy, Serge Nef and Patricja Nowak for accepting to be part of my PhD thesis committee. Thank you for spending part of your time reading my manuscript and evaluating my presentation.
Of course, I am also very grateful to all the people I have work with in the lab during my PhD. Christine, it has been a pleasure working with you. Thank you for all the experiments you have performed and analysed in order to make my investigation advance. You were in the lab since the beginning and you were always there to teach new techniques and useful things for working in the lab. But most importantly, thank you for giving me personal support and help during these years, facilitating my life and making my days easier. Lucile, you have been the perfect lab colleague. You were always willing to help with an experiment, to provide new ideas, to teach me how to use some software, to correct my writing in french, to attend presentation rehearsals and propose interesting changes… to sum up, thank you for losing some of your time to help me, I have really appreciate it. Also, I would like to thank you for all the discussions we have had, for all the times you have listened to me complaining,
2 for all the advices you have given me, and for tearing me up during my low moments, it has been very nice working back-to-back with you. Pascale, thank you for being the calm in the middle of the storm. We have not work that much together in the term of experiments, but your presence have given me relaxing vibes, and that has been essential in many moments.
By observing you I have learned how to be meticulous and organized, I just need to apply that into my own work, but you have given me some guidelines that may be very helpful for my future. I also would like to thank you for being so kind to me and for always be willing to help by proofreading manuscripts, giving new ideas, suggesting interesting changes… I would also like to thank Sonia, you lighten the lab days with your amazing personality. As a PhD student you understood me the best and always had good advices, on top of that, every time we have seen each other after you left; you have had the right thing to say to give me the strength that I needed. Thank you for being such an amazing person and making working next to you so easy. Finally, I would like to thank all the people that have been in the lab during these years and have had some positive impact: Kelly, Robin, Emily, Giorgia, Camelia, Aline, Nolwenn, Marylise, Belinda, Maxime, Mélanie, Melia…
I would also like to thank Robin for being such a nice next-door lab colleague. You have always been there when I needed to laugh a bit to cut the tension. I will miss a lot to gossip with you and Wednesdays will never be the same without our conversation in repeat just before you leave. I also want to thank you for being such a strong support in the last months, for believing in me when some other people have doubts and for helping me to stay focused.
Erika, I would like to thank you for all the conversations we have had while sharing the office and for the company we have made to each other during the long evenings/nights. It is great to have someone with such a positive personality around, but also so hard worker and
3 intelligent. Francesca and Soline, you have the ability to understand when I was feeling low, and most importantly, you have the ability to cheer me up, thank you.
Some other important people, who have helped me a lot, giving me support in the distance to keep on going every day, are my Erasmus friends. Your positive vibes and your encouraging words have pushed me through this whole process several times. You are the example of true friendship, thank you Aldin, Anni, Caoimhe, Emmi, Francesca, Jemima, Meltem and Perry; for much more moments together!
Luca, your moving to Zurich was one of the best things that happened last year. You were the perfect scape from reality, the weekends with you gave me the fuel to continue. You are a nice friend that knows how to listen and gives the best advice, but you are also the craziest person I know, making me laugh as no one else does.
Monday dinners crew, thank you very much for everything. You have been a big support during these years, scientifically and personally. Thank you for always listening to me, for making my problems your own problems, for being there whenever I needed someone to talk to. You are good friends and I am very lucky to have found you. Amy, thank you for your support and for making me feel worth it when I did not trust myself anymore. Damian, thank you for bringing all us together and for being such a supporting friend. Morgan, thank you for understanding me so much, for the laughs and for having the right words to say in the right moment. Lou, thank you very much for all the conversations, for giving me so many advice and for all the love and support.
Stefan, thank you for helping me with my English whenever I asked for help and for always being welcoming me in Berlin for a disconnecting weekend. Albert, graciés pels sopars dels
4 dijous, m’has ajudat moltíssim a desconnectar i carregar les piles. Fabien, merci pour m’encourager, surtout pendant les derniers mois quand je n’étais pas dans mes meilleurs moments. Nico, merci pour être toujours prêt pour parler autour d’un verre ou une pizza, des fois c’est tout qu’il faut pour changer une mauvaise journée en une bonne journée. Dailo, gracias por tu energía y por estar siempre dispuesto a hacer algo para despejarme la cabeza.
Sara, muchísimas gracias por tu amistad durante estos años, has sido una persona muy importante a lo largo de este doctorado y has sabido escucharme y entenderme siempre.
A mis amigos de la carrera en Tarragona, sin vosotros llegar hasta aquí hubiera sido mucho más complicado, gracias Cris, Roger, Maceira, Kathe, Maria, Mace, Carol, Gemma, Manu, Susanna, Anna, Nuria… En especial, gracias a Ana Maria y Ferran, habéis sido apoyos muy importantes estos años. Y a ti Juncal, gracias por sacar siempre un momento para verme, y un ratito para hablar y preguntarme como estoy, eres única.
A mi familia en Tarragona, mi equipo Ficus, mis niños, mis residencieros, infinitas gracias por todo. Carmen, mi andaluza, tu buen humor y tu risa son contagiosos, gracias por haberlos compartido conmigo. Cris, mi Chipsa, gracias por tantísimos momentos únicos llenos de felicidad, dejamos el listón de los riojanos muy alto en Tarragona. Piki, gracias por ser tan especial, por ser ese oso amoroso que tantas veces he necesitado y que siempre ha estado allí.
Lore, menos mal que estabas tú para aportar un poco de dulzura a mi vida, gracias por darme tanto cariño. Marta, gracias por ser la payasa que toda persona necesita, eres capaz de cambiarme el ánimo con solo una mirada. Naroa, gracias por todas las risas. Rober, simplemente gracias por existir. Eres una de las personas más especiales que conozco y que más me han llegado al corazón. Gracias por esos 3 años de convivencia y por todos los demás de amistad. Tu humor me ha levantado miles de veces y sé que siempre estarás ahí cuando te
5 necesite. Sandra, mi galleguiña, gracias por preocuparte por mí, por ser tan detallista y estar presente en los momentos que más falta hace. Tener una amiga como tú, es como que te toque la lotería. Xabel, gracias por todo, por entenderme tan bien sin decirte nada y saber que decir en todo momento. En muchas ocasiones has sido el bastón en el que apoyarme para no caerme y nunca te lo podré agradecer suficiente con palabras. Gracias a todos por los momentos vividos y por los que nos quedan por vivir, os quiero amigos.
Mis niñas, muchas gracias a vosotras por ese gran año que pasamos juntos y todos los momentos vividos después. Gracias a María por ser ese apoyo tan sólido siempre, gracias a Mónica por ser la cordura que tantas veces me ha hecho falta y gracias a Sara por ser la locura que en tantos momentos he necesitado. Gracias Silvia por estar tan atenta siempre a mí, por saber escuchar y darme tan buenos consejos. Pero, sobre todo, gracias por darme todas las risas y los momentos de desconexión que tanto he necesitado.
Muchas gracias Arepada, por haberme hecho muchísimo más fáciles y llevaderos los días en Ginebra. Alberto, Ana, Anaid, Helena, Luzma, Manu, Maria, Mely, Olga, Raquel y Samu. Gracias Manu por haber sido ese amigo tan necesario, siempre me sorprende como eres capaz de entenderme y decir lo que hace falta cuando hace falta. Gracias Raquel por ser ese apoyo en la distancia que aparece de sorpresa un día y lo mejora de repente. Ana, gracias por esas escapadas, esas conversaciones que tanto me sirven para desconectar y por todo el apoyo. Gracias Mely por tantísimo, no puedo ni enumerar todo lo que has hecho por mí y todo lo que te quiero. Eres esa amiga fiel a la que cuesta entender al principio, pero una vez la puedes llamar amiga, sabes que es para siempre y que no importa lo que pase, ella está ahí.
Anaid, chochete mío, mil veces gracias por existir y por haberte cruzado en mi camino. Nadie me entiende como tú me entiendes ni nadie es tan parecido a mí. Gracias por haberme sacado
6 del hoyo tantas veces y tener siempre algo bueno que decir en los peores momentos para que todo se minimice. Gracias por dejarme llamarte amiga, te quiero.
Roberto e Irene, gracias por todos los tececitos, los martecitos, las cenas, los juegos, las quedadas espontaneas… habéis sido un apoyo muy importante estos años y me habéis animado y ayudado infinitas veces. Cris, gracias por tu espontaneidad, tu humor, tus locuras y tu empatía. Tanto en Ginebra como en la distancia me has dado muchísimo apoyo y fuerzas.
Fernandita, mil gracias por ser ese pequeño rayo de luz que llega a tu vida y la mejora por completo. Me has dado muchísimo en estos años de amistad y me has ayudado siempre a tirar para adelante. Juno con Astrid y Moni, me habéis alegrado muchísimos días y me habéis demostrado que la distancia no es impedimento para nada.
Gracias Ainoa por las conversaciones tanto científicas como no. Por tu apoyo y tus ánimos, por entender tanto por lo que he pasado y decir las palabras que hacen falta cuando hacen falta. Anais, gracias por haber pasado esos meses con nosotros y haberme hecho la vida en el laboratorio mucho más fácil, y sobre todo, muchas gracias por las risas en los momentos de desesperación que tan bien entendiste. Cris, gracias en mayúsculas por estos años, y sobre todo por estos últimos meses. Sin ti me hubiera vuelto loco. Has sido un pilar básico sin el que toda esta tesis no hubiera podido levantarse. Gracias por haberme dejado desahogarme tantísimas veces, gracias por los consejos, gracias por los ánimos, gracias por todo.
Marta, no hay palabras suficientes para agradecerte todo lo que has hecho por mí en estos 4 años. Nadie ha estado tan al pie del cañón como lo has estado tú, y sobretodo, nadie ha estado tan presente cada día. Gracias por ser ese hombro en el que llorar, por saber sacarme una sonrisa cuando todo parecía negro, por entenderme tanto, por apoyarme, por creer en mí y por estar siempre a mi lado.
7 Gracias Freskas por todo lo que me habéis dado a lo largo de la vida. Algunas habéis estado ahí desde hace más de 26 años y sé que estaréis ahí por siempre. Sois únicas, solo vosotras conseguís sacarme una carcajada en los momentos de bajón con tanta facilidad. Gracias por estar a mi lado apoyándome durante tanto tiempo y por no dejarme caer nunca. Sois un pilar básico en todo lo que hago, y por supuesto en esta tesis también lo habéis sido. Es difícil encontrar unas amigas tan buenas como vosotras y nunca podré agradeceros suficiente todo lo que hacéis por mí. Gracias por todo; Ana, Angélica, Bea, Iris, Macarena, Paola, Piti, Sandra y Teresa. Os quiero mucho.
Ángela Martoc, Ángela Orío, Borja, Elena Cruz, Elena Moreno, Eva, Héctor, Lucía, María Martínez y María Presedo, infinitas gracias. Es increíble la suerte que he tenido de encontraros y que hayamos creado algo tan bonito entre todos. Sois el apoyo con el que sé que siempre puedo contar, no importa el momento ni el lugar. Gracias por todo lo que me habéis dado, ya no solo en estos 4 años, sino a lo largo de toda mi vida. Sois capaces de trasformar un mal día en uno bueno con un par de palabas. Espero poder devolveros todo lo que me habéis dado porque no tenéis ni idea de cómo de importantes habéis sido en que esto llegue a buen puerto. Os quiero muchísimo.
Por supuesto, también quiero darle las gracias a toda mi familia por haber creído en mí durante todo este tiempo. Sobretodo quiero darle las gracias a mi primo Tomi por comprenderme, entenderme y apoyarme tanto. Y las gracias más grandes son para la Titi, como tú misma dices, mucho más que una tía. Gracias porque con tus pequeños mensajes me has demostrado todo lo que me apoyabas y lo que confiabas en mí. Te quiero mucho titi.
Un agradecimiento muy especial es para Hernán, la persona que más presente ha estado en estos 4 años y que más me ha escuchado, aconsejado y entendido. Hay tantas cosas por las
8 que darte las gracias que no sabría ni por dónde empezar. Solo decirte que sin ti esto no hubiera sido posible. Muchas gracias por tu paciencia, no te lo he puesto fácil muchas veces, pero has sabido entender la situación y siempre me has apoyado en mis decisiones. Gracias por darme tanto cariño, aunque yo no te lo devuelva en la misma medida. Nunca podré agradecerte todo lo que has hecho y lo que haces por mí. Te quiero.
Por último, quiero darle las gracias y dedicarle esta tesis a mi red de apoyo incondicional, las personas que siempre están allí, las que más creen en mí y en todo lo que hago. Gracias Raúl por todos estos años de tira y afloja, pero sobretodo, de cariño y amor. Somos muy diferentes, pero somos capaces de entendernos a la perfección. Me alegro muchísimo de tenerte como hermano y de que nos queramos y protejamos tanto. Gracias papá por absolutamente todo lo que me has dado. Sin ti esto hubiera sido imposible, así que, en gran parte, esta tesis es tuya. Muchas gracias por todo el amor que me das cada día. Tus consejos y recomendaciones me han ayudado muchísimo a sobrellevar esta experiencia tan difícil. Gracias por haberla hecho más sencilla. Gracias mamá por ser la otra luz que junto con papá y Raúl sé que siempre está ahí. Tus mensajes diarios me dan la fuerza para afrontar los días más duros y me alegran en general cada día. Gracias por darme tantísimo en esta vida, en todas las facetas, pero sobre todo por darme tantísimo amor y cariño. Gracias por preocuparte tanto de mí, por hacer tuyos mis problemas y por ayudarme siempre ha salir de ellos.
Gracias por haber entendido que por desgracia no he tenido tanto tiempo para dedicaros estos años como hubiera querido, pero, aun así, cada instante que he necesitado, habéis estado ahí.
Gracias a los 3 por creer en mi incluso cuando yo no lo hacía. Os quiero.
9
A BSTRACT
Placenta formation is a complex process involving different cell types, resulting in the formation of a functional organ that can ensure correct foetal development. The function of the placenta is to protect, oxygenise, and nourish the foetus. In addition, the placenta is a temporary organ that produces and secretes hormones necessary during pregnancy. One of the main structures involved in performing some of these functions is the syncytiotrophoblast (STB), a multinucleated cell layer formed by the fusion of several villous cytotrophoblasts (vCTBs). The STB is the external cell layer of the chorionic villi, which are tree-like structures completely surrounded by maternal blood. An alteration in STB formation has been reported in some placental disorders such as preeclampsia. The underlying mechanisms implicated in vCTB cell fusion and differentiation, also called as syncytialisation, are still not completely understood. Nevertheless, some proteins such as glucose-regulated protein 78 (GRP78) have been reported to be involved in these processes. The aim of this study is to determine the role of GRP78 in vCTB cell fusion and differentiation.
GRP78 is a chaperone protein found in multiple locations in a cell. It is mainly located in the lumen of the endoplasmic reticulum (ER), where it regulates the unfolded protein response (UPR). The UPR is an adaptive mechanism that cells have developed in order to overcome ER stress. Firstly, we investigated UPR activation during syncytialisation. We observed enhanced UPR activation during cell fusion and differentiation in BeWo cells and in primary vCTB cells purified from a term placenta. UPR activation during syncytialisation was accompanied by the activation of autophagy. Autophagy was found to be an essential mechanism during vCTB cell fusion and differentiation since changes in the homeostatic
10 regulation of the cells negatively impacted STB formation. Finally, we observed that the blockage of the UPR in primary vCTB cells reduced autophagy and cell survival, suggesting that syncytialisation requires UPR activation leading to the activation of autophagy and cell survival.
We then decided to investigate the role of cell surface GRP78 in syncytialisation, since the blockage of membrane GRP78 was shown to reduce trophoblastic syncytialisation. GRP78, located on the cell surface, is known to interact with α2-macroglobulin (α2M) in prostate cancer cells. The GRP78–α2M interaction activates several intracellular signalling pathways implicated in the expression of proteins involved in syncytialisation, such as human chorionic gonadotropin and syncytins. In this study, we evaluated the role of α2M in syncytialisation.
We first observed that the expression of α2M in primary vCTB cells purified from placenta increases with gestational age, while BeWo cells do not express this protein. We then used BeWo cells to determine the role of exogenous α2M in syncytialisation. We demonstrated that α2M treatment induced trophoblastic fusion but not differentiation of BeWo cells. The effect of α2M on cell fusion was dependent on cell surface GRP78. BeWo cell treatment with α2M led to the phosphorylation of ERK1/2 and CREB proteins and UPR activation, which had no impact on the expression of syncytins by BeWo cells. These observations reinforce the importance of the UPR in trophoblastic fusion.
In conclusion, we demonstrated the essential role of GRP78 in trophoblastic syncytialisation, which may point towards this protein being a therapeutic target for placental disorders such as preeclampsia.
11
R ÉSUMÉ
La formation du placenta est un processus complexe impliquant différents types cellulaires afin d’obtenir un organe fonctionnel assurant un bon développement fœtal. Les fonctions du placenta sont de protéger, oxygéner et nourrir le fœtus. Par ailleurs, il s’agit d’un organe endocrine qui produit et sécrète les hormones nécessaires au maintien de la grossesse et au développement fœtal. L’un des principaux types cellulaires en charge d’exécuter une partie de ces fonctions est le syncytiotrophoblaste (STB), des cellules multi-nucléées formées par la fusion de plusieurs cytotrophoblastes villeux (vCTB). Le STB compose la couche externe des villosités choriales et est en contact direct avec le sang maternel. Une altération de la formation du STB est observée dans des pathologies de la grossesse telles que la pré- éclampsie. Les mécanismes de fusion et de différentiation des vCTB en STB, aussi appelée syncytialisation, ne sont pas encore complètement compris. Néanmoins, il a été établi que certaines protéines comme la « glucose-regulated protein 78 » (GRP78) sont impliquées dans ce processus. Le but de cette étude est d’étudier le rôle de la GRP78 dans la fusion et la différentiation des vCTB.
La GRP78 est une protéine chaperonne ayant plusieurs localisations cellulaires. Sa localisation principale est dans la lumière du réticulum endoplasmique (RE), où elle est connue pour agir en tant que régulateur de la « unfolded protein response » (réponse UPR).
La réponse UPR est un mécanisme d’adaptation que les cellules ont développé afin de surmonter un stress du RE. Dans un premier temps, nous avons étudié l’activation de la réponse UPR pendant la syncytialisation. Nous avons ainsi observé une activation de la réponse UPR pendant le processus de fusion et différentiation des cellules BeWo, une lignée
12 cellulaire de choriocarcinome, et dans les vCTB primaires purifiées à partir de placentas à terme. L’activation de la réponse UPR pendant la syncytialisation est accompagnée d’une activation de l’autophagie. Nous avons identifié l’autophagie comme un mécanisme essentiel pour la fusion des vCTB et leur différentiation, puisque des changements dans la régulation homéostatique de la cellule impacte négativement la formation des STB. Finalement, nous avons observé que le blocage de la réponse UPR dans les vCTB primaires a réduit l’activation de l’autophagie et la survie cellulaire, suggérant que la syncytialisation requiert l’activation de la réponse UPR menant à l’activation de l’autophagie afin d’assurer la survie cellulaire.
Nous avons ensuite décidé d’étudier le rôle de la GRP78 à la surface cellulaire dans la syncytialisation, puisqu’il avait été établi que le blocage de la GRP78 membranaire réduisait significativement la fusion des cellules trophoblastiques. A la surface cellulaire, la GRP78 est connue pour interagir avec l’α2-macroglobuline (α2M) dans les cellules cancéreuses de la prostate. Dans ces cellules, l’interaction GRP78-α2M active plusieurs voies de signalisation intracellulaires dont certaines sont connues pour être impliquées dans l’expression de protéines impliquées dans la syncytialisation telles que les syncytines et l’hormone chorionique gonadotrope. Dans cette étude, nous avons donc évalué le rôle de l’α2M dans la syncytialisation. Nous avons d’abord observé que l’expression de l’α2M dans les vCTB primaires purifiés à partir de placentas augmente avec l’âge gestationnel, tandis que les cellules BeWo n’expriment pas ce gène. Nous avons donc choisi d’utiliser les cellules BeWo pour déterminer le rôle de l’α2M exogène dans la syncytialisation. Nous avons démontré qu’un traitement avec l’α2M induit la fusion trophoblastique, mais pas la différentiation des cellules BeWo. Nous avons également confirmé que l’’effet de l’α2M sur la fusion cellulaire est dépendant de la GRP78 à la surface cellulaire. Le traitement des cellules avec l’α2M induit
13 la phosphorylation des protéines ERK1/2 et CREB conduisant à l’activation de la réponse UPR. Néanmoins, l’activation de ces voies n’impacte pas l’expression des syncytines dans les cellules BeWo. Cette observation renforce le rôle de la réponse UPR dans le mécanisme de fusion cellulaire.
En conclusion, nous avons démontré le rôle essentiel de la GRP78 dans la syncytialisation trophoblastique, suggérant que cette protéine pourrait être une cible thérapeutique pour les anomalies placentaires telle que la pré-éclampsie.
14
A BBREVIATION LIST
11-βHSD2: 11-β-hydroxysteroid dehydrogenase 2 4TM: 4-pass transmembrane
α2M: α2-macroglobulin
α2M*: activated α2-macroglobulin
ADAM12: a disintegrin and metalloproteinase domain-containing protein 12 ADP: adenosine diphosphate
AEBSF: 4-2-aminoethyl benzenesulfonyl fluoride hydrochloride AMP: adenosine monophosphate
AMPK: AMP-activated protein kinase Apaf-1: apoptotic protease activating factor-1 ARE: antioxidant responsive element
ASCT1/2: alanine, serine and cysteine selective transporter 1 or 2 ASK1: apoptosis signal-regulating kinase 1
ATF3: activating transcription factor 3 ATF4: activating transcription factor 4 ATF6: activating transcription factor 6 ATG: autophagy-related gene
15 ATG/ULK1: autophagy related protein/Unc-51 like autophagy activating kinase 1
ATP: adenosine triphosphate BAF: bafilomycin A1
Bak: Bcl-2 homologous antagonist killer Bax: Bcl-2 associated X
Bcl-2: B-cell lymphoma 2
Bcl-xL: B-cell lymphoma-extra large
Bim: Bcl-2 interacting mediator of cell death BiP: immunoglobulin heavy-chain-binding protein c-FLIP: cellular FLICE inhibitory protein
CHOP: CCAAT-enhancer-binding homologous protein CQ: chloroquine
CRE: cyclic adenosine monophosphate response element CREB: cyclic AMP-response element binding protein Cyt: cytosolic tail
DISC: death-inducing signaling complex DMSO: dimethyl sulfoxide
DNA: deoxyribonucleic acid
16 eIF2α: eukaryotic translation initiation factor 2α
ER: endoplasmic reticulum
ERAD: ER-associated degradation
ERK1/2: extracellular signal-regulated kinase 1/2 ERS: ER stress
ERSE-1: ERS response element-1 evCTB: extravillous cytotrophoblasts
FADD: Fas-associated protein with death domain FAK: focal adhesion kinase
FasL: Fas ligand FI: fusion index
FLICE: FADD-like interleukin-1β-converting enzyme FP: fusion peptide
Fsk: forskolin GA: golgi apparatus
GADD34: growth arrest and DNA damage-inducible 34 GDP: guanosine diphosphate
GPI: glycosylphosphatidylinositol
17 GRP78: glucose-regulated protein 78
GRP94: glucose-regulated protein 94 GSK: GSK2656157
GTP: guanosine triphosphate
hCG: human chorionic gonadotropin HERV: human endogenous retroviruses HR: heptad repeats
HRP: horseradish peroxidase
HSPA5: heat shock 70-Ka protein A member 5 IAPs: inhibit apoptosis
IRE1α: inositol-requiring enzyme 1α IUGR: intrauterine growth restriction JNK: c-Jun N-terminal kinase
K5: Kringle 5
LC3b: microtubule-associated proteins 1A/1B light chain 3B LIM: Lin11, Isl-1 & Mec-3
MFSD2: major facilitator superfamily domain containing 2 MHC-I: major histocompatibility complex class I
18 MPT: mitochondrial permeability transition
mTOR: mammalian target of rapamycin NBD: nucleotide-binding domain
NFkB: nuclear factor kappa-light-chain-enhancer of activated B cells NRF2: nuclear factor erythroid 2-related factor 2
NuMa: nuclear mitotic apparatus protein
p38 MAPK: p38 mitogen-activated protein kinase PAK2: p21-activated protein kinase-2
Par-4: prostate apoptosis response protein-4 PARP: Poly ADP-ribose polymerase PBS: phosphate-buffered saline PCR: polymerase chain reaction PE: preeclampsia
PEA: phosphatidylethanolamine
PERK: protein kinase RNA-like endoplasmic reticulum kinase PGCCs: polyploid giant cancer cells
PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase PI3KC3: phosphatidylinositol 3-kinase class III
19 PI3P: phosphatidylinositol 3-phosphate
PKA: protein kinase A PlGF: placental growth factor PZP: pregnancy zone protein
qPCR : quantitative polymerase chain reaction Raf-1: rapidly accelerated fibrosarcoma-1 RBD: receptor binding domain
RIP: receptor-interacting protein ROS: reactive oxygen species SBD: substrate-binding domain sGRP78: cell surface located GRP78 siRNA : small interfering RNA
SMACs: second mitochondria-derived activator of caspases
SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor SP1: serine protease 1
SP2: serine protease 2 SQSTM1: sequestosome 1 STB: syncytiotrophoblast
20 STF: STF-083010
STOX1: storkhead box 1 SU: surface unit
TGF-β: transforming growth factor-β Tm: transmembrane anchorage domain TM: transmembrane unit
TNF: tumor necrosis factor TNRF1: TNF receptor 1
TRADD: TNFR1-associated death domain protein TRAF2: TNF receptor-associated factor 2
TRI: trichostatin A
t-SNARE: target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor TUDCA: tauroursodeoxycholic acid
UDCA: ursodeoxycholic acid UPR: unfolded protein response VA: valproic Acid
vCTB: villous cytotrophoblast Vps34: vacuolar protein sorting 34
21 v-SNARE: vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptor XBP-1: x-box binding protein-1
22
F IGURE LIST
Figure 1. First steps of embryo development: from fertilised egg to blastocyst ... 25 Figure 2. Representative image of a human placenta after delivery ... 26 Figure 3. Development of the fertilised egg and transit through the oviduct to the uterus. . 26 Figure 4. Human decidualisation during the menstrual cycle ... 27 Figure 5. Representation of some key molecules implicated in the apposition and stable adhesion of the human blastocyst in the maternal endometrium... 28 Figure 6. Representation of some key molecules implicated in the invasion of the human blastocyst into the maternal endometrium ... 29 Figure 7. Syncytiotrophoblast expansion and trophoblastic lacunae formation ... 29 Figure 8. Chorionic villi formation... 30 Figure 9. Cytotrophoblast differentiation diagram ... 32 Figure 10. Maternal spiral artery remodelling ... 34 Figure 11. Structure of the placenta ... 35 Figure 12. Villous cytotrophoblast fusion and syncytiotrophoblast formation ... 36 Figure 13. Schematic representation of the structure of syncytin-1 ... 38 Figure 14. Schematic representation of syncytin-1-dependent cell fusion ... 39 Figure 15. Syncytiotrophoblast differentiation gives rise to protein profile modification ... 42 Figure 16. Maternal spiral artery remodelling during early-onset preeclampsia ... 46 Figure 17. Schematic representation of the GRP78 structure ... 49 Figure 18. GRP78 localisation and function ... 50 Figure 19. Cell surface GRP78 conformation ... 53 Figure 20. Effects of the α2-macroglobulin (α2M) interaction with cell surface GRP78 ... 56
23 Figure 21. Effects of cell surface-localised GRP78 interacting with CRIPTO, integrin β-1, prostate apoptosis response protein-4 (Par-4), Kringle 5 (K5) and viruses... 57 Figure 22. Functions of endoplasmic reticulum (ER)-localised GRP78. ... 59 Figure 23. Endoplasmic reticulum stress (ERS) response induction and overactivation ... 63 Figure 24. Autophagy ... 68 Figure 25. Apoptosis intrinsic pathway ... 72 Figure 26. Apoptosis extrinsic pathway ... 74
24
1 I NTRODUCTION
Pregnancy is defined as a state in which a female carries a developing embryo or foetus within her body. Human pregnancy normally lasts around 40 weeks. During this time, prenatal development takes place to form a functional multicellular individual [1]. Prenatal development is divided in three stages: fertilisation, embryonic development (until the 10th week after the last menstrual period) and foetal development. Fertilisation is the process by which the ovum and sperm fuse, forming a zygote. The genome of the zygote is a combination of the deoxyribonucleic acid (DNA) from the ovum and sperm and contains all the genetic material necessary to form a new organism [2]. Embryonic development begins when the zygote undergoes the first cell division, which is followed by many rounds of cell division and differentiation. When the zygote divides, it forms a blastocyst, which is a multicellular structure possessing an outer layer, known as the trophoblast, and an inner cell mass [3]. The trophoblast goes on to form the placenta, and the inner cell mass forms the amniotic sac and the developing embryo (Figure 1). After the 9th week of conception, all of the embryo’s major structures have been formed and the embryo becomes a foetus. Foetal development is characterised by growth and development of all the major foetal structures [4].
The stages of embryonic development have been simplified in the previous paragraph to give an overall vision of prenatal development and the origin of the different structures. As mentioned, the outer layer of the blastocyst gives rise to the trophoblast, which goes on to form the placenta [3]. We will now focus on placental formation.
25
Figure 1. First steps of embryo development: from fertilised egg to blastocyst. The fertilised egg undergoes several cell divisions until it reaches the blastocyst state. The blastocyst consists of an inner cell mass (embryoblast) that will form the embryo and the amniotic sac, a trophoblast layer that will form the placenta and a blastocyst cavity. Adapted from [5].
1.1 P
LACENTAThe placenta is the transient organ that nourishes, oxygenates and protects the foetus [6]. It is a discoid organ weighing approximately 500 g and measuring approximately 22 cm in diameter, with a thickness at the centre of around 2.5 cm (Figure 2) [7]. The placenta forms the maternal–foetal interface. It presents a basal plate that is tightly linked to the maternal endometrium, and a chorionic plate that is attached to the umbilical cord and connects to the foetus [6]. As mentioned, the placenta originates from the trophoblast cell layer of the blastocyst. Until day 20–23after the last menstrual period, the blastocyst is a free structure that is able to move from the fallopian tube towards the uterus [3]. However, the embryo requires nutrients and oxygen to develop correctly, emphasising the importance of the maternal–foetal connection [8]. The placenta facilitates this connection, making blastocyst implantation into the endometrium and subsequent placental formation essential steps in prenatal development.
26
Figure 2. Representative image of a human placenta after delivery. On the right, the foetal side of the placenta with the umbilical cord can be observed. On the left, the maternal side of the placenta can be observed.
Scale bar represents 5 cm. Adapted from [9].
Figure 3. Development of the fertilised egg and transit through the oviduct to the uterus. The fertilised egg travels through the oviduct while undergoing multiple division rounds. The blastocyst arrives to the uterus at day 5 and starts the implantation process in the maternal endometrium at day 7. Taken from [10].
To receive the blastocyst, the endometrium undergoes adaptations during a process called decidualisation, which plays an important role in successful pregnancy [11,12]. This process allows preparation of the endometrium for placentation by enlarging and specialising the maternal endometrial epithelium [11]. The endometrial stromal cells undergo morphological and functional changes, while the maternal arteries undergo vascular changes, leading to
27 complete tissue remodelling [13]. In humans, enlargement of the endometrium occurs during the menstrual cycle, and in case of blastocyst implantation, the decidua further develops (Figure 4) [14]. Additionally, the blastocyst needs to undergo certain changes prior to implantation. First, the blastocyst needs to enzymatically hatch from the zona pellucidaa layer of glycoproteins that surrounds and protects the blastocyst [15]. After hatching, the blastocyst is naked, and the trophoblast is exposed [16]. At this point, the trophoblast cells at the embryonic pole of the blastocyst can directly interact with the enlarged endometrium, allowing apposition and adhesion of the blastocyst to the decidua [17]. This adhesion is achieved by formation of a strong protein attachment, which involves adhesion molecules such as L-selectin [18], integrins [19,20], trophinin [21] or osteopontin [22] (Figure 5). For successful adhesion of the blastocyst, the decidua contains special structures called endometrial pinopodes that facilitate adhesion and are preferential for the blastocyst to adhere [23].
Figure 4. Human decidualisation during the menstrual cycle. During the first phases of the menstrual cycle, the endometrium becomes enlarged, and cell proliferation and spiral artery vascularisation occur. Endometrial stromal cells differentiate morphologically and functionally, leading to the formation of decidual cells. These changes lead to the formation of the decidua, which can host the blastocyst for implantation and underpin further development. Adapted from [24].
28
Figure 5. Representation of some key molecules implicated in the apposition and stable adhesion of the human blastocyst in the maternal endometrium. L-selectin, integrins, osteopontin and trophinin are some of the adhesion molecules implicated in these processes. Adapted from [14].
After attachment of the blastocyst to the receptive decidua, the differentiated primitive syncytiotrophoblast (STB) starts to invade the decidua [25,26]. The primitive syncytium produces and secretes several enzymes that promote degradation of the extracellular matrix between the cells of the decidua, allowing blastocyst infiltration [27]. Serine proteases [28], metalloproteases [29-31] and collagenase type IV [32] are some of the degenerative enzymes that are involved in this process. Additionally, to facilitate the invasion and generate space for the growing embryo, the STB induces decidual apoptosis by Fas/Fas ligand (FasL)- mediated mechanisms (Figure 6) [33]. By day 7after fertilisation, the human blastocyst is completely pulled into the decidua and placental formation begins (Figure 7) [34].
29
Figure 6. Representation of some key molecules implicated in the invasion of the human blastocyst into the maternal endometrium. The primitive syncytium secretes enzymes that promote extracellular matrix degradation such as serine proteases, metalloproteases or collagenase type IV. It also induces decidual apoptosis through Fas/FasL-mediated mechanisms. Adapted from [14].
Figure 7. Syncytiotrophoblast expansion and trophoblastic lacunae formation. After implantation, the syncytiotrophoblast expands and surrounds the embryo. Empty spaces known as trophoblastic lacunae form within the syncytiotrophoblast. Adapted from [34].
30 Once the blastocyst is implanted, the STB expands and surrounds the embryo. Vacuoles, also known as trophoblastic lacunae, are formed within the STB (Figure 8) [34]. Connections between maternal capillaries and trophoblastic lacunae are formed, filling the vacuoles with maternal blood. At this point, the first branched structures that will increase the STB contact surface with maternal blood start to be formed in the lacunae [14,34].
Figure 8. Chorionic villi formation. a. Primary villi formation (11–13 days after conception). Villous cytotrophoblasts (vCTBs) proliferate and generate columns, pushing the syncytiotrophoblast (STB) into the trophoblastic lacunae. b. Secondary villi formation (16 days after conception). Extraembryonic mesoderm starts to proliferate, penetrating the centre of the primary villi. c. Tertiary villi formation (21 days after conception).
The extraembryonic mesoderm gives rise to blood vessels inside the secondary villi, connecting with the embryonic vessels and establishing the maternal–foetal circulation. Taken from [34].
31 The first step in the formation of the branched structure is orchestrated by the villous cytotrophoblasts (vCTBs) that start to proliferate, generating columns of cells that push the STB layer into the lacunae [34]. These protrusions are known as primary chorionic villi and are formed between day 25 and 28 after the last menstrual period (11–13 days after conception) or 5 days after the implantation process begins (Figure 8A) [35]. At day 30 after the last menstrual period (16 days after conception), the extraembryonic mesoderm that is associated with the vCTBs starts to proliferate and penetrate the centre of each primary chorionic villi, forming the secondary chorionic villi (Figure 8B) [34,35]. The extraembryonic mesoderm is the tissue from which fibroblasts, resident macrophages and the vascular network originate [6]. At day 35 after the last menstrual period (21 days after conception), the extraembryonic mesoderm gives rise to blood vessels inside the secondary villi. These are known as the umbilical arteries and veins, which connect with the vessels forming in the embryo, establishing the placental–foetal circulation (Figure 8C) [34,36]. At the same time, the chorionic villi inside the trophoblastic lacunae that are irrigated with maternal blood, underpin the maternal–foetal circulation. At this point, the chorionic villi are in a tertiary state and are capable of undergoing exchange functions. Nevertheless, these structures continue to grow and develop throughout pregnancy [37,38]. Additional branches grow from the tertiary chorionic villi, increasing the surface area in contact with the maternal blood. Primary branching takes place between the 9th week and the 16th week after conception, and the branches are known as immature intermediate villi. Mature intermediate villi form near the end of the secondary trimester, by re-branching of the immature intermediate villi. Finally, small branches form from the mature intermediate villi by week 32 after conception, which are referred to as terminal villi [39]. The terminal villi exhibit capillary dilatation, which reduces the thickness of the scaffolding tissue and decreases the
32 distance between the capillary and the STB. The distance between the maternal and foetal circulations can be reduced to 2–3 µm, facilitating the exchange of oxygen and nutrients. The mature placenta contains around 30–40 terminal villi, displaying a total surface area of 12–
14 m2. Each of the chorionic villi forms a globular lobule of 1–3 cm in diameter and represents an independent maternal–foetal exchange unit [6,37].
The trophoblast layer proliferates and differentiates along two different pathways: the extravillous pathway or the villous pathway [14,40,41]. The extravillous pathway generates extravillous cytotrophoblasts (evCTBs) and the villous pathway gives rise to the previously mentioned vCTBs that fuse and differentiate to form the STB that is in charge of embryonic protection, hormone secretion and exchanges between the mother and the foetus (Figure 9) [14,40,41].
Figure 9. Cytotrophoblast differentiation diagram. Cytotrophoblasts differentiate into extravillous cytotrophoblasts through the extravillous pathway, gaining invasive and proliferative properties. On the other side, cytotrophoblasts differentiate into villous cytotrophoblasts (trophoblastic cells with the capacity to fuse and differentiate), giving rise to the syncytiotrophoblast.
33 We previously mentioned that the primary chorionic villi grow in the trophoblastic lacunae [14,34]. The trophoblastic lacunae increase in size and in blood capacity, and at this point, they are termed intervillous spaces. The maternal blood enters these chambers around week 10–12 of gestation, bathing the chorionic villi with blood through about 100 remodelled spiral arteries, which leaves via the endometrial veins [14,38]. Remodelling of the maternal arteries is a critical step in placentation since it has been reported that correct remodelling is involved in the development of future placental disorders [42,43]. This process is orchestrated by the evCTBs, which invade the decidua and migrate towards the maternal spiral arteries that supply blood to the endometrium [44]. Due to the large blood supply required by the foetus during pregnancy, enlargement of the maternal spiral arteries is needed to increase blood flow into the placenta [45]. First, evCTBs colonise the arteries, replacing and phenocopying the endothelial cells [46], while accumulating and creating a plug in the vessel lumen (Figure 10) [44]. When the trophoblastic plug occludes the maternal spiral arteries, the blood vessels enlarge, allowing a better pouring of the blood into the intervillous space [43,47]. The generation of trophoblastic plugs and vessel dilatation is an essential part of placental formation [42,48-50]. Trophoblastic plugs allow only blood plasma to permeate and create a hypoxic environment in the placenta, which is essential for correct placental formation [44,51]. By week 10–12 after gestation, the trophoblastic plug disappears and full blood flow into the intervillous space occurs [52,53].
34
Figure 10. Maternal spiral artery remodelling. The extravillous cytotrophoblast (evCTB) invades the decidua and colonises the spiral arteries, replacing and phenocopying the endothelial cells. At the same time, a plug of evCTB forms in the lumen of the vessels, generating a low oxygen environment and allowing dilatation of spiral arteries. Adapted from [54].
The total capacity for maternal blood in the mature placenta is approximately 150 ml, which is completely replaced three or four times per minute [55]. The final chorionic villi that reside in the intervillous spaces display an enormous surface area of STB, which is in direct contact with maternal blood and allows exchange of nutrients, oxygen and waste products between the mother and the foetus (Figure 11) [56]. Generation of chorionic villi has been described
35 for a long time, however, STB formation from vCTB fusion and differentiation is not completely understood and is essential to better understand placental formation.
Figure 11. Structure of the placenta. The maternal arteries pour the blood into the intervillous space where the chorionic villi reside. The most external cell layer of the chorionic villus, the syncytiotrophoblast, performs nutrient, gas and waste exchange. The exchanged products are transported to the foetus through the foetal arteries that reside inside the chorionic villus.
1.1.1 vCTB cell fusion
Cell fusion is a rare event in human cells that has been reported in physiological events such as fecundation [57], placental formation [58], muscle differentiation [59] and bone maintenance, remodelling and repair [60]. Additionally, in pathophysiological circumstances such as viral entry [61] and cancer [62], cell–cell fusion also takes place. Although certain common features have been described in cell fusion events, each type of cell fusion has its own mechanism.
36 Previous studies have identified proteins that are implicated in vCTB cell fusion and a trophoblastic fusion mechanism has been proposed and accepted by the research community.
The vCTBs fuse upon implantation of the blastocyst into the endometrium to create a multinucleated cell layerthe STB (Figure 12). However, underlying vCTBs continue to fuse with the STB during pregnancy, allowing renewal of the syncytia [63]. In this way, the expression of fusogenic proteins must be stable over time and cell fusion should be tightly controlled. The expression of cell–cell adhesion proteins, such as E-cadherin, is increased in vCTBs prior to cell fusion [64]. Moreover, gap junction proteins such as connexin 43 [65], and tight junction proteins such as zona occludens 1 (ZO-1) [66], are expressed in vCTBs allowing functional inter-trophoblastic communication and favouring cell fusion.
Nevertheless, the most well-studied proteins that are essential for correct cell fusion are the syncytins and their associated receptors [67].
Figure 12. Villous cytotrophoblast fusion and syncytiotrophoblast formation. On the left, a schematic view of a chorionic villous showing the localisation of the different cell types: syncytiotrophoblast (STB), villous cytotrophoblast (vCTB) and extravillous cytotrophoblast (evCTB). On the right, an in-vitro fusion model of vCTB, which leads to formation of the STB. The trophoblast was stained after 24 h and 72 h of culture for desmoplakin (magenta) and nuclei (blue). Scale bar represents 15 µm. Adapted from [68].
37 The syncytins are class I envelope human endogenous retroviruses (HERVs) that were acquired by humans from retroviruses [69-71]. Syncytin-1 (HERV-W) was inserted into the human genome 19–28 million years ago [69,70,72], while syncytin-2 (HERV-FDR) was inserted 40 million years ago [71]. The fusion mechanisms of these proteins are very similar to vesicle–lysosome-facilitated fusion, which is achieved by target soluble N- ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) and vesicular SNARE (v-SNARE) [73]. In fact, they form bundles of alpha helices, resulting in intracellular membrane apposition and fusion, and their engineered expression in the surface cell membrane promotes cell–cell fusion [73-75]. The syncytin receptors are the alanine-, serine- and cysteine-selective transporter 1 or 2 (ASCT1/2) receptor for syncytin-1 [76] and the major facilitator superfamily domain-containing 2 (MFSD2) receptor for syncytin-2 [77].
The viral origin of the syncytin proteins facilitated the understanding of their mechanism in vCTB cell fusion since it was largely studied in the context of virus–cell fusion. The syncytins in vCTBs bind to their receptors and achieve membrane fusion in a pH-independent manner, just as their homologues do in viruses [78,79]. Fortunately, the structure of syncytin- 1 and the syncytin-1-dependent fusion mechanism have been very well characterised.
As expected from its retroviral envelope protein origin, syncytin-1 is a glycoprotein composed of two units: a surface unit (SU) and a transmembrane (TM) unit. At the same time, the SU is subdivided into a receptor-binding domain (RBD), a CΦΦC (186−CX2C−189) motif, a furin cleavage site (314−RNKR−317) and six N-glycosylation sites. The TM unit is also subdivided into different domains, including a fusion peptide (FP), two heptad repeats (HR1 and HR2), a 397−Cx6C−407 domain, a TM anchorage domain (tm), a cytosolic tail (cyt) and one N-glycosylation site. To become a fully functional protein, post-transcriptional
38 modifications need to take place in the endoplasmic reticulum (ER). In fact, the SU and the TM unit are translated together as one unit; however, the SU contains a furin cleavage site that can be cleaved by the furin-convertase enzyme in the ER, separating both subunits.
Subsequently, a disulphide bond between the 397−Cx6C−407 domain of the TM unit and a 186−CX2C−189 motif of the SU is formed (Figure 13) [78-81]. Finally, syncytin-1 forms a functional protein when it exists as a trimer. Trimerisation takes place in the ER, while prior maturation takes place in the Golgi apparatus (GA), and the protein is transported to the cell membrane where it can trigger cell–cell fusion [79].
Figure 13. Schematic representation of the structure of syncytin-1. The signal peptide domain is represented in light blue. The surface unit (SU) is represented in yellow and contains a receptor binding domain (RBD;
SDGGGX2DX2R) and a CXXC motif. The transmembrane unit (TM) contains a fusion peptide and is represented in red, heptad repeats 1 (HR1) are represented in purple, heptad repeats 2 (HR2) are represented in pink, a transmembrane domain is represented in black, an intracytoplasmic domain is represented in green and a CX6CC domain. Between the SU and the TM unit is a furin cleavage site (RNKR) represented in light red.
The Y indicates N-glycosylation sites and numbers indicate the amino acid position. A disulphide bond is formed between the CXXC motif of the SU and the CX6CC domain of the TM unit. Taken from [82].
The mechanism in charge of approaching the cell membranes and accomplish cell–cell fusion begins when syncytin-1 recognises ASCT-1 or ASCT-2 in the target membrane. The RBD of syncytin-1 recognises the receptor located in the target membrane and the first conformational change in the trimer takes place. More concretely, the SU and the TM unit
39 dissociate from each other by breakage of the disulphide bond. The structural modification causes a projection of syncytin-1 fusion peptide towards the top of the protein, allowing it to interact with the target membrane and insert itself into the target membrane [78,79]. The connection of both cell membranes through syncytin-1 generates folding of the HR2 domain, which interacts with the HR1 domain. This interaction reverses the direction of the cell membrane and brings both membranes into close proximity, reducing the free energy needed to overcome the merging barrier (Figure 14) [78,79,82].
Figure 14. Schematic representation of syncytin-1-dependent cell fusion. a. Resting stage. b. RBD (yellow) binding to the hASCT2 receptor (light green). c. Disulphide bond breaking and removing of SU domains, producing a conformational change in syncytin-1 protein leading to insertion of the fusion peptide (red) into the cell membrane. d. Assembly of HR2 (pink) and HR1 (purple). e. Final stage with the membranes in close proximity and initiation of membrane bending. Taken from [82].
Membrane merging is a multistep process where different forces and energies take part. First, the close proximity of cell membranes and contact of opposing outer cell membranes cause
40 dehydration of the contact site. This dehydration reduces hydration repulsion between the outer leaflets of the membranes, leading to fusion of the outer leaflets or hemifusion [78,79,83]. Nevertheless, the proximity of the cell membranes and the reduction in hydration are not sufficient for hemifusion to take place. It has been reported that negatively charged phospholipids, such as phosphatidylserine, need to be located in the outer membrane.
Phosphatidylserine is actively held at the cytosolic side of the cell membrane and internal signals need to be unleashed to achieve the phosphatidylserine flip. [78,79,83-86] The localisation of phosphatidylserine at the outer leaflet of the membrane reduces the energy needed for the cell membranes to fuse, facilitating the process. For successful cell fusion, partial hemifusion or the fusion stalk need to radially expand. The expansion allows the inner leaflets to fuse as well, completing the merge of the vCTB cell membranes and the opening of a pore by which the cell content can mix [79].
As previously mentioned, other factors distinct from the syncytins and their receptors have also been implicated in cell fusion. Some of these factors are the phosphatidylserine flip [87], cadherin-11 [88], caspase-8 [89], CD98 [90], a disintegrin and metalloproteinase domain- containing protein 12 (ADAM12) [91], connexin-43 [65], ZO-1 [66] and glucose-regulated protein 78 (GRP78) [92,93]. Concretely, reduced expression of GRP78 by siRNA leads to a decrease in cell fusion, suggesting that GRP78 plays a role in trophoblastic cell fusion [93].
However, the mechanism of cell fusion linked to GRP78 remains to be investigated.
Importantly, the cell fusion process not only involves merging of the cell membranes and cell content mixing, but several modifications need to take place for the fused cells to act as a new entity. Cessation of the cell cycle takes place during vCTB fusion and STB formation since the newly form cell layer is a terminally differentiated tissue [94]. The STBs do not
41 perform cell cycle divisions. Instead, the material that needs to be eliminated from the cell, including apoptotic nuclei, is released in the form of syncytial knots into the maternal blood flow [95]. In exchange, new mononuclear vCTBs fuse and incorporate into the STB to expand during pregnancy and maintain a constant STB density at the mature state [63].
Moreover, the recently fused cells need to perform cytoskeletal rearrangements to organise the cytoskeleton of the STB according to the new cell necessities. It was previously reported that caspase-8 activation is involved in cytoskeletal rearrangement of vCTBs during syncytialisation. Additionally, caspase-8 is implicated in the phosphatidylserine flip that occurs during STB formation [89]. The occurrence of the recently mentioned events leads to fusion of vCTBs; however, several changes need to take place in the newly formed cell for it to become a mature and functional STB.
1.1.2 Syncytiotrophoblast
The unique functions that the STB performs cannot be explained by cell fusion origins alone.
Rather, a whole differentiation process accompanies the morphological changes [96]. First, the transport of oxygen, nutrients and waste materials is performed through diffusion, transporter-mediated mechanisms and endocytosis/exocytosis [97]. The protein expression profile of STB cells completely changes to permit molecular transport. The expression of transporter proteins is increased in STB cells, especially glucose [98-101] and amino acid transporters [102-104]. Additionally, changes in the internal vesicle transport machinery are needed in STB cells to confront the high endocytosis rates that have been reported during lipid transport (Figure 15) [97].
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Figure 15. Syncytiotrophoblast differentiation gives rise to protein profile modification. Exchange function. An increase in the expression of transporter proteins such as glucose or amino acid transporters is observed, together with increased internal vesicle transport machinery. Endocrine function. Several hormones are secreted by the syncytiotrophoblast after differentiation including progesterone, oestrogen, human chorionic gonadotropin, human placental lactogen, placental growth hormone, human chorionic thyrotropin, prostaglandins, insulin-like growth factor, prolactin and human chorionic corticotropin. Immunological barrier. The syncytiotrophoblast acts as a physical barrier. The expression of several proteins is modified to achieve foetal protection. The expression of multidrug resistance transporters and transporters that allow maternal antibody transport is increased.
Nevertheless, STB cells are not only in charge of transporting molecules, they also carry out the endocrine functions of the placenta [97,105,106]. To maintain the pregnant state, prevent miscarriage or preterm labour and prepare the mammary glands for lactation, STB cells produce progesterone and oestrogen [105,107,108]. Another intrinsic hormone that is secreted by STB cells is human chorionic gonadotropin (hCG) [105,109,110]. This glycosylated molecule is a multifunctional hormone for which positive expression is used as a diagnostic marker for pregnancy [111] and it is known to be essential for correct gestational development [106,112]. It is secreted very early during pregnancy; it has been detected at the
43 eight-cell embryo stage, suggesting that the STB precursors also produce it [113,114].
Nevertheless, it is generally accepted that the release of hCG by STB cells starts at the 7th day of gestation and its secretion increases during the first trimester of pregnancy, showing a peak of 10,352 ng/ml in maternal serum at 10 weeks [110]. After 10 weeks, hCG secretion is gradually reduced until week 27 when the detected hCG concentration in maternal serum is 1,911 ng/ml, which is maintained until the end of pregnancy [110]. Human chorionic gonadotropin is involved in maintenance of the corpus luteum during the beginning of pregnancy, allowing secretion of progesterone and oestradiol during the first trimester [115,116]. Progesterone and hCG have angiogenic functions. Progesterone is involved in enriching the uterus with blood vessels and capillaries, while hCG is involved in stimulating new vessel formation in the placenta [117-120]. Additionally, hCG promotes steroidogenesis in the placenta by stimulating the conversion of cholesterol into pregnenolonea precursor/metabolic intermediate in the biosynthesis of most steroid hormones [121]. Other functions of hCG include blockage of the maternal immune response against the invading placental cells [122,123] and promotion of vCTB fusion and subsequent differentiation to generate the STB [124]. The STB also secretes other important hormones during pregnancy, such as the human placental lactogen [38], placental growth hormone [125], human chorionic thyrotropin [38], prostaglandins [126], insulin-like growth factor [38], prolactin [127] and human chorionic corticotropin [128]. The differentiation process needed to transform a non- endocrine cell into a hormone-secreting cell requires modification of the protein expression profile of STB cells (Figure 15) [129,130].
Finally, STB cells form the selective barrier between the foetus and the mother and are considered the main foetal immunological barrier [131]. First, the STB is a physical barrier
