ESA-SRB-AOTA 2019

A placenta-specific protease that is critical for trophoblast syncytialization (#137)

Mary Mansilla 1 2 , Yao Wang 1 2 , Guiying Nie 1 2 3
  1. Implantation and Placental Development, Hudson Institute of Medical Research, Melbourne, Victoria, Australia
  2. Department of Molecular and Translational Science, Monash University, Melbourne, Victoria, Australia
  3. Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia

INTRODUCTION: The placenta is a pregnancy-specific organ that functions to nourish the developing fetus. The outer layer of the placenta contains the syncytiotrophoblast (STB), which forms through fusion of cytotrophoblasts and the process is called syncytialization. Impaired syncytialization may lead to abnormal release of STB-derived proteins into the maternal circulation, causing pregnancy complications such as preeclampsia (PE). HtrA4 is a placenta-specific protease that is highly expressed in STB and significantly up-regulated in PE. However, the functional importance of HtrA4 during normal syncytialization is unknown. The aim of this study was to determine whether HtrA4 is essential for syncytialization.

METHOD: Primary human cytotrophoblasts were isolated from healthy term placentas, and cultured under a condition that promotes spontaneous syncytialization. Trophoblast BeWo cell line was treated with forskolin to induce syncytialization. Real-time RT-PCR, western blot (WB) and immunocytochemistry (ICC) were performed to monitor changes in HtrA4 as well as syncytial markers in both cell models. HtrA4 was also analysed by ELISA. To determine if HtrA4 is critical for syncytialization, a stable HtrA4 knockout (KO) BeWo line was established using the CRISPR/Cas9 technology, and used to determine the consequences of HtrA4 KO on syncytialization.

RESULTS: Primary cytotrophoblasts spontaneously syncytialized in culture as expected. BeWo cells also syncytialized following forskolin treatment as reported. HtrA4 expression was significantly up-regulated during syncytialization in both cell models. When treated with forskolin, control BeWo cells displayed an expression profile of syncytial markers similar to that of un-transfected BeWo cells. However, HtrA4 KO BeWo cells failed to syncytialize following forskolin treatment, as real-time RT-PCR, ELISA, WB and ICC all revealed minimal changes in syncytial markers in these cells.

CONCLUSION: HtrA4 increased during syncytialization in both primary trophoblasts and BeWo cells. Knockout of HtrA4 prevented BeWo cells from syncytialization. These data strongly suggest that HtrA4 plays an essential role in syncytialization.