Effect of hydroxychloroquine on trophoblastic invasion in relation to autophagy mediated by ET-1 and ERK

Introduction: Hydroxychloroquine (HCQ), which has multiple effects, including autophagy inhibition, is used to prevent obstetric complications. However, the exact mechanism through which HCQ affects placentation remains unclear. We investigated the in vitro effects of HCQ on trophoblast invasion with respect to autophagy. Methods: HTR-8/SVneo cells were used to perform Matrigel invasion assays. They were treated with rapamycin to induce autophagy with or without HCQ treatment. We examined autophagy-related proteins (p62 and LC3-II) through western blotting and confocal microscopy, and measured proteins related to trophoblast invasion, including soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and endothelin-1(ET-1) by ELISA. We measured extracellular signal-regulated kinase (ERK) levels using western blotting to understand the signaling pathways related to trophoblastic invasion. Results: HCQ increased the expression of LC3-II and p62 proteins and decreased the LC3 puncta with less colocalization with LAMP2 compared to rapamycin-only treatment, indicating the inhibition of autophagy by HCQ in HTR-8/SVneo cells. HCQ restores rapamycin-induced decrease in trophoblast invasion. Of note, HCQ co-treatment decreased the level of ET-1 compared to rapamycin-only treatment, it did not affect the levels of MMP-2 and MMP-9. However, HCQ treatment increased sFlt-1/PlGF levels compared to those with rapamycin treatment alone. Additionally, it increased the level of phosphorylated ERK compared to that with rapamycin treatment alone. Discussion: Our data indicate that HCQ could improve trophoblast invasion by affecting ET-1 levels and the ERK signaling pathway.