Knockdown of activated Cdc42-associated kinase inhibits human extravillous trophoblast migration and invasion and decreases protein expression of pho-Akt and matrix metalloproteinase

Abstract

Introduction: The sufficient invasion and migration of human extravillous trophoblast (EVTs) cells are crucial for placentation. Inadequate invasion of trophoblasts may correlate with the development of preeclampsia. Many studies have suggested that activated Cdc42-associated kinase (ACK1) is associated with tumor metastasis and invasion. This study investigated the ACK1 expression and its function in trophoblasts during placental development.Methods: ACK1 expression in human placentas was determined through immunofluorescence. We investigated the migration/invasion of the immortalized human first-trimester EVT cell line HTR8/SVneo. Hypoxia–reoxygenation (H/R) conditions were applied to mimic preeclampsia model in vitro. Lentiviral vector-based short-hairpin RNA directed against the sequence of ACK1 (ACK1 shRNA) was used to knock down ACK1 expression in HTR8/SVneo cells. Cell apoptosis and proliferation were determined through flow cytometry and cell counting Kit-8 (CCK-8) assays, respectively. The expression of matrix metalloproteinase (MMP) 2/9 and tissue inhibitors of metalloproteinase (TIMP) 1/2 was measured by western blotting.Results: ACK1 localized within trophoblasts of human placental villi, decidual cells in the maternal decidua. ACK1 levels in preeclampsia (PE) placentas were significantly lower than those in controls. ACK1 shRNA significantly inhibited HTR8/SVneo cells migration and invasion but did not affect their apoptosis and proliferation. ACK1 knockdown decreased MMP2/9 and increased TIMP1/2 expression, as well as downregulated the phosphorylation of AKt (p-Akt). In addition, ACK1 and MMP2/9 were downregulated following treatment with LY294002, whereas ACK1 shRNA had no effect on phosphorylation of PI3K(p-PI3K). After exposed in H/R condition, ACK1 expression, MMP2/9 protein, and p-Akt were also significantly decreased.Discussion and conclusions: ACK1 expression is lowered in preeclamptic placentas and promotes trophoblast cell invasion, migration. H/R conditions decrease ACK1 expression and appear to decouple the positive relationship between ACK1 expression and Akt activation.