Alpha‐actinin‐4 is essential for maintaining normal trophoblast proliferation and differentiation during early pregnancy

Abstract

Background Proper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy. Method: To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6–10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown. Results ACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas. Conclusions Our findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.

Systematic proteomics analysis of lysine acetylation reveals critical features of placental proteins in pregnant women with preeclampsia

“…In addition, the differentiation of CTB is also related to changes in cytoskeletal organization and adhesion structures, indicating that cytoskeletal integrity and appropriate remodelling may affect trophoblast cell invasion 54 . ACTN4, an actin binding protein, is mainly expressed in placental CTB cells and participated in actin cytoskeleton dynamics and motility 54,55 . Previous evidence suggested that ACTN4 dysregulation may lead to compromised invasiveness in trophoblasts and abnormal cytoskeletal remodelling by inhibiting AKT phosphorylation, which may ultimately contribute to PE 54,55 .…”

“… 54 ACTN4, an actin binding protein, is mainly expressed in placental CTB cells and participated in actin cytoskeleton dynamics and motility. 54 , 55 Previous evidence suggested that ACTN4 dysregulation may lead to compromised invasiveness in trophoblasts and abnormal cytoskeletal remodelling by inhibiting AKT phosphorylation, which may ultimately contribute to PE. 54 , 55 Filamins (FLNs) as scaffolds for signalling proteins and adhesive receptors that regulate actin cytoskeleton remodelling.…”

“… 54 , 55 Previous evidence suggested that ACTN4 dysregulation may lead to compromised invasiveness in trophoblasts and abnormal cytoskeletal remodelling by inhibiting AKT phosphorylation, which may ultimately contribute to PE. 54 , 55 Filamins (FLNs) as scaffolds for signalling proteins and adhesive receptors that regulate actin cytoskeleton remodelling. 56 At present, three members of the filament protein family, namely FLNA, FLNB and FLNC, have been discovered.…”

Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells

“…O-GlcNAc regulation of translation 27 and the actin cytoskeleton 28 are of undoubted interest in relation to placental function. Reduced translation, as a consequence of increased eIF2α phosphorylation, reportedly contributes to placental dysfunction in pregnancies complicated by fetal growth restricted 29 , trophoblast turnover, fusion and motility are all influenced by cytoskeletal dynamics 30 – 33 , as is integrity of the endothelial barrier 34 . However, here we chose clathrin-mediated endocytosis (CME) for further study.…”

GWAS of preeclampsia and hypertensive disorders of pregnancy uncovers genes related to cardiometabolic, endothelial and placental function

“…The ACTN4 gene encodes for an actin-binding protein with multiple roles in different cell types, and is well known for causing focal segmental glomerulosclerosis [71][72][73] . The gene product is also involved in the maintenance of cytoskeletal structure, modulating cell motility and regulating endothelial cells 74,75 , and may play a role in placentation 76 . ACTN4 has been suspected to act as a regulator of trophoblast proliferation and differentiation during early pregnancy, and has been shown to have reduced expression in severe preeclampsia 76 .…”