GnRH Regulates Trophoblast Invasion Via Twist Induced N-cadherin Expression

Embryo implantation is a crucial step during early pregnancy. Gonadotropin-releasing hormone (GnRH), normally considered a key neuroendocrine regulator of the reproductive system, is expressed by trophoblastic cells during implantation and exerts pro-invasive effects on extravillous trophoblast (EVT) cells. While the mechanisms involved in these effects are unclear, expression of the mesenchymal adhesion molecule N-cadherin, normally regulated in partby the transcription factor Twist, is associated with invasive phenotypes in many other cells. Thus, the aim of our study was to investigate the involvement of Twist-mediated N-cadherin expression in the pro-invasive effects of GnRH on trophoblastic cells.

Methods: Immunohistochemistry was performed to examine the cellular localization of GnRH receptor, Twist and N-cadherin in first trimester placenta. In vitro studies were performed with primary EVT cells, prepared from the tips of firsttrimester placental villi, or the human immortalized EVT cell line HTR-8/SVneo. Cells were treated with or without GnRH in the presence or absence of the GnRH receptor antagonist Antide or the phosphoinositide3-kinase inhibitor LY294002.Alternatively, cells transfected with small interfering RNA (siRNA) targeting Twist or N-cadherin were treated with or without GnRH. Reverse transcription-PCR and Western blot were performed to examine Twist and N-cadherin expression. Phosphoinositide3-kinase/Akt signaling was monitored by immunoblotting with a phospho-specific Akt antibody. Transwell invasion assays were used to investigatethe effects of treatment with GnRH and/or siRNAs on the invasiveness of trophoblastic cells.

Results: GnRH receptor, Twist and N-cadherin were detected at invasion sites of human first-trimester placenta. Treatment of HTR-8/SVneo cells with GnRH significantly increased the mRNA and protein levels of Twistand N-cadherin in a concentration- and time-dependent manner. Pre-treatment with the GnRH receptor antagonist Antide attenuated the effects of GnRH on Twist and N-cadherin expression. The effects of GnRH on Twist and N-cadherin are likely mediated by Akt signaling because treatment with GnRH increases Akt phosphorylation and the phosphoinositide3-kinase inhibitor LY294002 attenuates the effects of GnRH on Twist and N-cadherin expression. HTR-8/SVneo cell invasion was reduced following siRNA-mediated knockdown of Twist or N-cadherin. Importantly, down-regulation of Twist reduced N-cadherin expression as well as GnRH-induced HTR-8/SVneo cell invasion.

Conclusion: Our results suggest that GnRH acts via its receptor to induce Akt phosphorylation which contributes to elevated Twist expression. Increased levels of Twist subsequently induce N-cadherin expression which promotes trophoblastic cell invasion during human placentation.