Ezrin and Its Phosphorylated Thr567 form Are Key Regulators of Human Extravillous Trophoblast Motility and Invasion

Abstract

The protein ezrin has been shown to enhance cancer cell motility and invasion leading to malignant behaviours in solid tumours, but a similar regulatory function in the early physiological reproduction state is, however, much less clear. We speculated that ezrin may play a key role in promoting first-trimester extravillous trophoblast (EVT) migration/invasion. Ezrin, as well as its Thr567 phosphorylation, were found in all trophoblasts studied, whether primary cells or lines. Interestingly, the proteins were seen in a distinct cellular localisation in long, extended protrusions in specific regions of cells. Loss-of-function experiments were carried out in EVT HTR8/SVneo and Swan71, as well as primary cells, using either ezrin siRNAs or the phosphorylation Thr567 inhibitor NSC668394, resulting in significant reductions in both cell motility and cellular invasion, albeit with differences between the cells used. Our analysis further demonstrated that an increase in focal adhesion was, in part, able to explain some of the molecular mechanisms involved. Data collected using human placental sections and protein lysates further showed that ezrin expression was significantly higher during the early stage of placentation and, importantly, clearly seen in the EVT anchoring columns, further supporting the potential role of ezrin in regulating migration and invasion in vivo.

Ezrin Thr567 phosphorylation participates in mouse oocyte maturation, fertilization, and early embryonic development

“…NSC668394 speci cally inhibits the phosphorylation of ezrin T567 [19]. Here, we found that the treatment with NSC668394 only during the oocyte maturation not only signi cantly reduced the in vitro maturation rate of oocytes but also affected the in vitro fertilization and early embryo development potential.…”

“…Ezrin has multiple phosphorylation sites, such as Ser66 [9][10][11] Thr213 [12] Thr332 [13,14] Thr567 [15][16][17] Tyr145 Tyr353 and Tyr477 [18]. Phosphorylation of Thr567 is currently considered a sign of ezrin activation and is also the most studied phosphorylation site [19].…”

An Extracellular/Membrane-Bound S100P Pool Regulates Motility and Invasion of Human Extravillous Trophoblast Lines and Primary Cells

“…The protocol for isolating EVTs has been previously described [ 8 ] and adapted from [ 31 ]. After washing first trimester placental samples in Ham’s F12 (Sigma, Hertfordshire, UK), chorionic villi were physically separated and digested in 0.25% ( w / v ) trypsin solution (Sigma, Hertfordshire, UK).…”

“…The pellet containing plasma membranes was resuspended in 250 mM sucrose/50 mM Tris pH 7.4. Samples were stored at −20 °C prior to analysis via Western blot as previously described [ 8 , 9 ]. Original, uncropped, and unadjusted images are available as Supporting Information Supplementary Figure S1 .…”

“…Interestingly, migration and invasion characteristics of trophoblast cells have been likened to those seen in cancer cells. Consequently numerous proteins, whose functions have been highlighted as markers for carcinogenesis, have been reported to be important regulators of trophoblast migration/invasion (recently reviewed [ 7 ]), of which ezrin [ 8 ] and S100P [ 9 ] are two of the newest additions.…”