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Tspan5 promotes the EMT process to regulate the syncytialization of trophoblast cells by activating Notch signalling

Published online by Cambridge University Press:  24 July 2023

Hai-Yu Tang*
Department of Reproductive Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou 514000, Guangdong Province, China.
Mei Lin
Department of Reproductive Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou 514000, Guangdong Province, China.
Yong-Qian Liang
Department of Reproductive Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou 514000, Guangdong Province, China.
Jin-Hua Wang
Department of Reproductive Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou 514000, Guangdong Province, China.
Hong-Gan Yi
Department of Reproductive Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou 514000, Guangdong Province, China.
Man Yang*
Department of Reproductive Medicine, Meizhou People’s Hospital (Huangtang Hospital), Meizhou 514000, Guangdong Province, China.
Corresponding authors: Hai-Yu Tang; Email: Man Yang; Email:
Corresponding authors: Hai-Yu Tang; Email: Man Yang; Email:


Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. β-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.

Research Article
© The Author(s), 2023. Published by Cambridge University Press

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Bahrampour, S. and Thor, S. (2020). The five faces of Notch signalling during Drosophila melanogaster embryonic CNS development. Advances in Experimental Medicine and Biology, 1218, 3958. doi: 10.1007/978-3-030-34436-8_3 CrossRefGoogle ScholarPubMed
Berditchevski, F. (2001). Complexes of tetraspanins with integrins: More than meets the eye. Journal of Cell Science, 114(23), 41434151. doi: 10.1242/jcs.114.23.4143 CrossRefGoogle ScholarPubMed
Berditchevski, F., Gilbert, E., Griffiths, M. R., Fitter, S., Ashman, L. and Jenner, S. J. (2001). Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis. Journal of Biological Chemistry, 276(44), 4116541174. doi: 10.1074/jbc.M104041200 CrossRefGoogle ScholarPubMed
Brosseau, C., Colas, L., Magnan, A. and Brouard, S. (2018). CD9 tetraspanin: A new pathway for the regulation of inflammation? Frontiers in Immunology, 9, 2316. doi: 10.3389/fimmu.2018.02316 CrossRefGoogle ScholarPubMed
Conley, S. M., Stuck, M. W. and Naash, M. I. (2012). Structural and functional relationships between photoreceptor tetraspanins and other superfamily members. Cellular and Molecular Life Sciences: CMLS, 69(7), 10351047. doi: 10.1007/s00018-011-0736-0 CrossRefGoogle ScholarPubMed
Crocker, I. P., Strachan, B. K., Lash, G. E., Cooper, S., Warren, A. Y. and Baker, P. N. (2001). Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro. Journal of the Society for Gynecologic Investigation, 8(6), 341346. doi: 10.1177/107155760100800606 CrossRefGoogle Scholar
Dornier, E., Coumailleau, F., Ottavi, J. F., Moretti, J., Boucheix, C., Mauduit, P., Schweisguth, F. and Rubinstein, E. (2012). TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals. Journal of Cell Biology, 199(3), 481496. doi: 10.1083/jcb.201201133 CrossRefGoogle ScholarPubMed
Gao, T., Liang, Y., Tang, H. and Quan, L. (2018). The increased level of Tspan5 in villi suggests more proliferation and invasiveness of trophoblasts in tubal pregnancy. European Journal of Obstetrics, Gynecology, and Reproductive Biology, 228, 3842. doi: 10.1016/j.ejogrb.2018.05.033 CrossRefGoogle ScholarPubMed
García-Frigola, C., Burgaya, F., Calbet, M., de Lecea, L. and Soriano, E. (2000). Mouse Tspan-5, a member of the tetraspanin superfamily, is highly expressed in brain cortical structures. NeuroReport, 11(14), 31813185. doi: 10.1097/00001756-200009280-00027 CrossRefGoogle ScholarPubMed
García-Frigola, C., Burgaya, F., de Lecea, L. and Soriano, E. (2001). Pattern of expression of the tetraspanin Tspan-5 during brain development in the mouse. Mechanisms of Development, 106(1–2), 207212. doi: 10.1016/s0925-4773(01)00436-1 CrossRefGoogle ScholarPubMed
Haining, E. J., Yang, J., Bailey, R. L., Khan, K., Collier, R., Tsai, S., Watson, S. P., Frampton, J., Garcia, P. and Tomlinson, M. G. (2012). The TspanC8 subgroup of tetraspanins interacts with A disintegrin and metalloprotease 10 (ADAM10) and regulates its maturation and cell surface expression. Journal of Biological Chemistry, 287(47), 3975339765. doi: 10.1074/jbc.M112.416503 CrossRefGoogle ScholarPubMed
Hemler, M. E. (2001). Specific tetraspanin functions. Journal of Cell Biology, 155(7), 11031107. doi: 10.1083/jcb.200108061 CrossRefGoogle ScholarPubMed
Huang, C., Hays, F. A., Tomasek, J. J., Benyajati, S. and Zhang, X. A. (2020). Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle-mediated release of ezrin to inhibit tumour cell movement. Journal of Extracellular Vesicles, 9(1), 1692417. doi: 10.1080/20013078.2019.1692417 CrossRefGoogle ScholarPubMed
Isaka, K., Usuda, S., Ito, H., Sagawa, Y., Nakamura, H., Nishi, H., Suzuki, Y., Li, Y. F. and Takayama, M. (2003). Expression and activity of matrix metalloproteinase 2 and 9 in human trophoblasts. Placenta, 24(1), 5364. doi: 10.1053/plac.2002.0867 CrossRefGoogle ScholarPubMed
Israels, S. J. and McMillan-Ward, E. M. (2010). Palmitoylation supports the association of tetraspanin CD63 with CD9 and integrin alphaIIbbeta3 in activated platelets. Thrombosis Research, 125(2), 152158. doi: 10.1016/j.thromres.2009.07.005 CrossRefGoogle ScholarPubMed
Jouannet, S., Saint-Pol, J., Fernandez, L., Nguyen, V., Charrin, S., Boucheix, C., Brou, C., Milhiet, P. E. and Rubinstein, E. (2016). TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization. Cellular and Molecular Life Sciences: CMLS, 73(9), 18951915. doi: 10.1007/s00018-015-2111-z CrossRefGoogle ScholarPubMed
Juenger, H., Holst, M. I., Duffe, K., Jankowski, J. and Baader, S. L. (2005). Tetraspanin-5 (Tm4sf9) mRNA expression parallels neuronal maturation in the cerebellum of normal and L7En-2 transgenic mice. Journal of Comparative Neurology, 483(3), 318328. doi: 10.1002/cne.20439 CrossRefGoogle ScholarPubMed
Kang, Y. and Massagué, J. (2004). Epithelial–mesenchymal transitions: Twist in development and metastasis. Cell, 118(3), 277279. doi: 10.1016/j.cell.2004.07.011 CrossRefGoogle ScholarPubMed
Kar, M., Ghosh, D. and SenGupta, J. (2007). Histochemical and morphological examination of proliferation and apoptosis in human first trimester villous trophoblast. Human Reproduction, 22(11), 28142823. doi: 10.1093/humrep/dem284 CrossRefGoogle ScholarPubMed
Knöfler, M. (2010). Critical growth factors and signalling pathways controlling human trophoblast invasion. International Journal of Developmental Biology, 54(2–3), 269280. doi: 10.1387/ijdb.082769mk CrossRefGoogle ScholarPubMed
Knöfler, M., Haider, S., Saleh, L., Pollheimer, J., Gamage, T. K. J. B. and James, J. (2019). Human placenta and trophoblast development: Key molecular mechanisms and model systems. Cellular and Molecular Life Sciences: CMLS, 76(18), 34793496. doi: 10.1007/s00018-019-03104-6 CrossRefGoogle ScholarPubMed
Kokkinos, M. I., Murthi, P., Wafai, R., Thompson, E. W. and Newgreen, D. F. (2010). Cadherins in the human placenta—Epithelial–mesenchymal transition (EMT) and placental development. Placenta, 31(9), 747755. doi: 10.1016/j.placenta.2010.06.017 CrossRefGoogle ScholarPubMed
Lamouille, S., Xu, J. and Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nature Reviews. Molecular Cell Biology, 15(3), 178196. doi: 10.1038/nrm3758 CrossRefGoogle ScholarPubMed
Langbein, M., Strick, R., Strissel, P. L., Vogt, N., Parsch, H., Beckmann, M. W. and Schild, R. L. (2008). Impaired cytotrophoblast cell–cell fusion is associated with reduced Syncytin and increased apoptosis in patients with placental dysfunction. Molecular Reproduction and Development, 75(1), 175183. doi: 10.1002/mrd.20729 CrossRefGoogle ScholarPubMed
Lash, G. E., Otun, H. A., Innes, B. A., Bulmer, J. N., Searle, R. F. and Robson, S. C. (2005). Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases. Biology of Reproduction, 73(2), 374381. doi: 10.1095/biolreprod.105.040337 CrossRefGoogle ScholarPubMed
Levy, S., Todd, S. C. and Maecker, H. T. (1998). CD81 (TAPA-1): A molecule involved in signal transduction and cell adhesion in the immune system. Annual Review of Immunology, 16, 89109. doi: 10.1146/annurev.immunol.16.1.89 CrossRefGoogle ScholarPubMed
Li, Y., Wang, L., Qiu, J., Da, L., Tiollais, P., Li, Z. and Zhao, M. (2012). Human tetraspanin transmembrane 4 superfamily member 4 or intestinal and liver tetraspan membrane protein is overexpressed in hepatocellular carcinoma and accelerates tumor cell growth. Acta Biochimica et Biophysica Sinica, 44(3), 224232. doi: 10.1093/abbs/gmr124 CrossRefGoogle ScholarPubMed
Lockwood, C. J., Oner, C., Uz, Y. H., Kayisli, U. A., Huang, S. J., Buchwalder, L. F., Murk, W., Funai, E. F. and Schatz, F. (2008). Matrix metalloproteinase 9 (MMP9) expression in preeclamptic decidua and MMP9 induction by tumor necrosis factor alpha and interleukin 1 beta in human first trimester decidual cells. Biology of Reproduction, 78(6), 10641072. doi: 10.1095/biolreprod.107.063743 CrossRefGoogle ScholarPubMed
Matthews, A. L., Szyroka, J., Collier, R., Noy, P. J. and Tomlinson, M. G. (2017). Scissor sisters: Regulation of ADAM10 by the TspanC8 tetraspanins. Biochemical Society Transactions, 45(3), 719730. doi: 10.1042/BST20160290 CrossRefGoogle ScholarPubMed
Moseley, G. W. (2005). Tetraspanin–Fc receptor interactions. Platelets, 16(1), 312. doi: 10.1080/09537100400004363 CrossRefGoogle ScholarPubMed
Ng, Y. H., Zhu, H. and Leung, P. C. (2011). Twist regulates cadherin-mediated differentiation and fusion of human trophoblastic cells. Journal of Clinical Endocrinology and Metabolism, 96(12), 38813890. doi: 10.1210/jc.2010-2714 CrossRefGoogle ScholarPubMed
Ng, Y. H., Zhu, H. and Leung, P. C. (2012). Twist modulates human trophoblastic cell invasion via regulation of N-cadherin. Endocrinology, 153(2), 925936. doi: 10.1210/en.2011-1488 CrossRefGoogle ScholarPubMed
Noy, P. J., Yang, J., Reyat, J. S., Matthews, A. L., Charlton, A. E., Furmston, J., Rogers, D. A., Rainger, G. E. and Tomlinson, M. G. (2016). TspanC8 tetraspanins and A disintegrin and metalloprotease 10 (ADAM10) interact via their extracellular regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS. Journal of Biological Chemistry, 291(7), 31453157. doi: 10.1074/jbc.M115.703058 CrossRefGoogle Scholar
Phillips, T. C., Dhaliwal, G. K., Verstegen-Onclin, K. M. and Verstegen, J. P. (2012). Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen. Theriogenology, 77(1), 3945. doi: 10.1016/j.theriogenology.2011.07.012 CrossRefGoogle ScholarPubMed
Prabha, B., Molykutty, J., Swapna, A., Rajalekshmi, T. N. and Gangadharan, V. P. (2001). Increased expression of interleukin-1 beta is associated with persistence of the disease and invasion in complete hydatidiform moles (CHM). European Journal of Gynaecological Oncology, 22(1), 5056.Google ScholarPubMed
Reichrath, J. and Reichrath, S. (2020a). Notch signaling and embryonic development: An ancient friend, revisited. Advances in Experimental Medicine and Biology, 1218, 937. doi: 10.1007/978-3-030-34436-8_2 CrossRefGoogle ScholarPubMed
Reichrath, J. and Reichrath, S. (2020b). Notch signaling and tissue patterning in embryology: An introduction. Advances in Experimental Medicine and Biology, 1218, 17. doi: 10.1007/978-3-030-34436-8_1 CrossRefGoogle ScholarPubMed
Reyes, R., Cardeñes, B., Machado-Pineda, Y. and Cabañas, C. (2018). Tetraspanin CD9: A key regulator of cell adhesion in the immune system. Frontiers in Immunology, 9, 863. doi: 10.3389/fimmu.2018.00863 CrossRefGoogle ScholarPubMed
Ruebner, M., Langbein, M., Strissel, P. L., Henke, C., Schmidt, D., Goecke, T. W., Faschingbauer, F., Schild, R. L., Beckmann, M. W. and Strick, R. (2012). Regulation of the human endogenous retroviral Syncytin-1 and cell–cell fusion by the nuclear hormone receptors PPARγ/RXRα in placentogenesis. Journal of Cellular Biochemistry, 113(7), 23832396. doi: 10.1002/jcb.24110 CrossRefGoogle ScholarPubMed
Schäfer-Somi, S. (2003). Cytokines during early pregnancy of mammals: A review. Animal Reproduction Science, 75(1–2), 7394. doi: 10.1016/s0378-4320(02)00222-1 CrossRefGoogle ScholarPubMed
Shin, J. C., Lee, J. H., Yang, D. E., Moon, H. B., Rha, J. G. and Kim, S. P. (2003). Expression of insulin-like growth factor-II and insulin-like growth factor binding protein-1 in the placental basal plate from pre-eclamptic pregnancies. International Journal of Gynaecology and Obstetrics: The Official Organ of the International Federation of Gynaecology and Obstetrics, 81(3), 273280. doi: 10.1016/s0020-7292(02)00444-7 CrossRefGoogle ScholarPubMed
Todd, S. C., Doctor, V. S. and Levy, S. (1998). Sequences and expression of six new members of the tetraspanin/TM4SF family. Biochimica et Biophysica Acta, 1399(1), 101104. doi: 10.1016/s0167-4781(98)00087-6 CrossRefGoogle ScholarPubMed
Tseng, A. M., Mahnke, A. H., Wells, A. B., Salem, N. A., Allan, A. M., Roberts, V. H., Newman, N., Walter, N. A., Kroenke, C. D., Grant, K. A., Akison, L. K., Moritz, K. M., Chambers, C. D. and Miranda, R. C. (2019) Maternal circulating miRNAs that predict infant FASD outcomes influence placental maturation. Life Science Alliance, 2(2). doi: 10.26508/lsa.201800252 CrossRefGoogle ScholarPubMed
Ullah, R., Dar, S., Ahmad, T., de Renty, C., Usman, M., DePamphilis, M. L., Faisal, A., Shahzad-Ul-Hussan, S. and Ullah, Z. (2018) CDK1 inhibition facilitates formation of syncytiotrophoblasts and expression of human chorionic gonadotropin. Placenta, 66, 5764. doi: 10.1016/j.placenta.2018.05.003 CrossRefGoogle ScholarPubMed
Wang, Y., Liang, Y., Yang, G., Lan, Y., Han, J., Wang, J., Yin, D., Song, R., Zheng, T., Zhang, S., Pan, S., Liu, X., Zhu, M., Liu, Y., Cui, Y., Meng, F., Zhang, B., Liang, S., Guo, H., Liu, Y., Hassan, M. K. and Liu, L. (2018). Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling. Journal of Experimental and Clinical Cancer Research: CR, 37(1), 300. doi: 10.1186/s13046-018-0969-y CrossRefGoogle ScholarPubMed
Yaseen, I. H., Monk, P. N. and Partridge, L. J. (2017). Tspan2: A tetraspanin protein involved in oligodendrogenesis and cancer metastasis. Biochemical Society Transactions, 45(2), 465475. doi: 10.1042/BST20160022 CrossRefGoogle ScholarPubMed
Zhou, J., Fujiwara, T., Ye, S., Li, X. and Zhao, H. (2014). Downregulation of Notch modulators, tetraspanin 5 and 10, inhibits osteoclastogenesis in vitro. Calcified Tissue International, 95(3), 209217. doi: 10.1007/s00223-014-9883-2 CrossRefGoogle ScholarPubMed
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