To investigate the mechanism of wild-type p53-induced phosphatase (Wip1) in regulating p53-dependent apoptosis of trophoblasts for further understanding the etiology of preeclampsia (PE).

Placenta tissues were collected from normal (n=15) and PE (n=13) gravidas who underwent caesarean section in the First Affiliated Hospital of Chongqing Medical University from June 2017 to December 2018. Chorionic villus and decidua tissues were collected from another 10 women who aborted in early pregnancy. Two in vitro trophoblastic hypoxia cultures were established by subjecting human chorionic trophoblast cells (HTR8/SVneo) to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 expressions at the transcriptional and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The localization of Wip1 in placental tissues and HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Cell apoptosis was assessed by flow cytometry after viral infection and hypoxia. And the changes of pathway-related molecules including p53, phospho-p53 (p-p53), mouse double minute 2 homolog (Mdm2) and cleaved caspase3 (cl-cas3) were measured by Western blotting. The impact of Wip1 on Mdm2-p53 interaction was examined by co-immunoprecipitation. NVP-CGM097, an Mdm2-p53 specific inhibitor, was administered in PE cell models to verify the regulation of Wip1 on trophoblastic apoptosis through Mdm2-p53 pathway. Independent student's t-test, Welch's t-test and one-way analysis of variance were used as statistical methods.

(1) Wip1 expression, which was mainly in trophoblast cells, was significantly elevated in human PE placentas (mRNA: 1.711±0.141 vs 0.860±0.126, t=4.496; protein: 0.449±0.027 vs 0.192±0.019, t=7.902) and in both in vitro trophoblastic PE models (protein in HII: 1.376±0.086 vs 0.977±0.114, t=2.792; SIB: 1.243±0.057 vs 0.381±0.045, t=11.910) compared with the corresponding control groups (all P<0.05). (2) Compared with corresponding control groups, overexpression of Wip1 suppressed the hypoxia-induced upregulation of p53 (HII: 0.185±0.024 vs 0.572±0.072; SIB: 0.400±0.067 vs 0.803±0.064), cl-cas3 (HII: 0.243±0.034 vs 0.529±0.072; SIB: 0.179±0.011 vs 0.368±0.025) and p-p53/p53 protein expression (HII: 1.326±0.129 vs 2.100±0.187; SIB: 0.473±0.028 vs 0.925±0.036) and also reduced the apoptosis rate [HII: (8.925±1.092)% vs (17.610±1.980)%; SIB: (13.910±1.886)% vs (24.650±1.622)%], which in turn promoted Mdm2-p53 binding (all P<0.05). However, knockdown of Wip1 gene expression in HTR8/SVneo cells brought about opposite effects (all P<0.05). (3) Neither overexpression nor knockdown of Wip1 influenced p53 or cl-cas3 expression when Mdm2-p53 interaction was blocked by NVP-CGM097.

Mdm2-p53 interaction promoted by Wip1 upregulation could compensate for the trophoblastic p53 accumulation in response to hypoxia, while exogenous upregulation of Wip1 in trophoblasts may reverse hypoxia-induced apoptosis. Therefore, this might provide a new therapeutic target for PE.

Pre-eclampsia; Cell hypoxia; Trophoblasts; Protein phosphatase 2C; Apoptosis