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.

Keywords: ACTN4; Invasion and migration; Preeclampsia; Proliferation; Trophoblast.

Fig. 1

ACTN4 expression in placental CTBs. a IHC staining of ACTN4 in the human first trimester placenta villi. CTBs were stained for CK7; Scale bars: 100 μm. b EdU labeling in explant cultures. Villous explants isolated from the first trimester placentas were treated with the control siRNA (si-NC) or ACTN4 siRNA (si-ACTN4). Representative pictures showed EdU (pink) labeling in the villous cytotrophoblasts of floating villi following 72-h treatment. Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. All data are presented as the means ± SEM of three independent experiments. ^*p < 0.05

Fig. 2

ACTN4 expression in the process of CTB differentiation into STBs. a Immunofluorescence staining for ACTN4 (red) and CK7 (green) in the freshly isolated primary human CTB cells. Staining was performed after 3 or 48 h of culture. Nuclei were counterstained with DAPI (blue). White dashed circles and white arrows indicate syncytialized cells and unsyncytialized cells, respectively. Scale bars: 100 μm. b Western blotting for ACTN4 and GCM-1 (a syncytiotrophoblast marker) in the primary human CTB cells after 3 or 48 h of culture. All data are presented as the means ± SEM of three independent experiments. ^**P < 0.01; ^***P < 0.001

Fig. 3

Role of ACNT4 in villous explants outgrowth. a IHC staining of ACTN4 in the human first trimester placenta villi. iEVTs were stained for HLA-G. Scale bars: 200 μm. b EdU labeling in explant cultures. Representative pictures showed EdU (pink) labeling in the CCTs of the anchoring villi after 72-h treatment of ACNT4 with siRNA. Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. c Outgrowth of induced EVTs in a villous explant culture model. Left panel, bright and fluorescent field views showing that small interfering RNA labeled with FAM successfully penetrated the villous explants. Right panel, the outgrowth of induced EVTs from villous explants treated with control siRNA (si-NC) or ACTN4 siRNA (si-ACTN4) for 24 and 48 h. Scale bar: 1000 μm. All data are presented as the means ± SEM of three independent experiments, ^*P < 0.05; ^**P < 0.01

Fig. 4

Effects of ACTN4 on HTR8/SVneo cell invasion and migration. a TRITC-Phalloidin staining on si-NC and si-ACTN4 of HTR8/SVneo cells. Scale bar, 200 μm. b Representative images and quantification of cells from the HTR8/SVneo cells with invasion in the presence or absence of a Matrigel-coated membrane for 24 h. Scale bar: 200 μm. c HTR8/SVneo cells attached to the culture plates after wounding and treatment with ACTN4 siRNA. The percentage of wound closure was calculated at 24 h. Scale bar: 400 μm. All data are presented as the means ± SEM of three independent experiments. ^**P < 0.01

Fig. 5

ACTN4 regulation of the AKT/GSK3β/Snail pathway in trophoblasts. Western blotting for ACTN4, p-AKT^Ser473, AKT, p-GSK3β, Snail, N-cadherin, and Vimentin in the first trimester villi (left panel) and HTR-8/SVneo cells (right panel) transfected with si-NC or si-ACTN4 for 72 h. All data are presented as the means ± SEM of three independent experiments. ^*P < 0.05; ^**P < 0.01

Fig. 6

ACTN4 expression in the iEVTs from sPE placentas. a IHC staining of ACTN4 on the maternal side of normal and sPE placentas. Staining was performed on serial sections; iEVTs were stained for HLA-G. DS: decidual side. Scale bars: 200 μm. b Western blotting for ACTN4 on the maternal side of normal and sPE placentas. All data are presented as the means ± SEM of three independent experiments. ^***P < 0.001