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. 2021 Jul 6:9:615973.
doi: 10.3389/fcell.2021.615973. eCollection 2021.

STX2 Promotes Trophoblast Growth, Migration, and Invasion Through Activation of the PI3K-AKT Pathway in Preeclampsia

Yan Li  1 , Xian-Li Sun  2 , Chun-Ling Ma  1 , Chao Li  1 , Ying Zhan  1 , Wen-Ting Li  1 , Can Li  1 , Yi-Hao Wang  3
Affiliations

STX2 Promotes Trophoblast Growth, Migration, and Invasion Through Activation of the PI3K-AKT Pathway in Preeclampsia

Yan Li et al. Front Cell Dev Biol. .

Abstract

Objectives: Abnormal trophoblast behaviors during pregnancy contribute to the development of preeclampsia (PE). Syntaxin2 (STX2) has been shown to be a crucial epithelial mediator in numerous diseases. However, the functions of STX2 and the mechanisms underlying its role in PE remain largely unknown. The aim of this study was to explore the role of STX2 on trophoblast biology and unravel the molecular mechanisms that contribute to the development and progression of PE.

Materials and methods: We first compared the expression of STX2 in placental tissues from women with PE and women with normal pregnancies. Then, we investigated the role of STX2 on trophoblast proliferation, migration and invasion in HTR-8/SVneo and primary human trophoblast cells by loss or gain of function experiments. In addition, co-immunoprecipitation, pulldown and immunofluorescence assays were performed to investigate the co-localization of STX2 with other proteins, and to help clarify the mechanisms underlying STX2-mediated functions on trophoblasts.

Results: We demonstrated that STX2 expression was downregulated in placental tissues of women with PE compared with those from normal pregnancies. Loss and gain of function experiments further confirmed a role for STX2 in cell proliferation, migration and invasion in trophoblasts. By co-immunoprecipitation, pulldown and immunofluorescence co-localization assays, we revealed that STX2 selectively interacted with p85, a subunit of PI3K, and directly recruited p85 to the cytomembrane, thereby activating the AKT signaling pathway. We further demonstrated that the AKT activation was abolished by the use of a PI3K inhibitor (LY294002), which negatively affected STX2-mediated functions on trophoblasts.

Conclusion: All together, our findings point to a crucial role for STX2 in PE progression. Our new insights also suggest that STX2 may be a potential diagnostic tool and a novel therapeutic target for treating PE.

Keywords: AKT; PI3K; preeclampsia; syntaxin2; trophoblast.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
STX2 is downregulated in the placenta of PE pregnancies. (A) Representative images of IHC staining of STX2 in normal control (NC; n = 35) tissues and PE (n = 40) tissues. Scale bar: 100 μm (100×); 50 μm (400×). Student’s t-test: ***P < 0.001. (B) Relative STX2 mRNA levels of placenta tissues from pregnant women with PE or normal control pregnancy (NC) as detected by qRT-PCR (n = 4/group). (C) Relative STX2 protein levels of placenta tissues from pregnant women with PE or normal control pregnancy as detected by western blot (n = 4/group). β-Tubulin was used for normalization. All the experiments were repeated three times independently.
FIGURE 2
FIGURE 2
STX2 promotes proliferation, migration and invasion of trophoblast cells. (A) qRT-PCR analysis and (B) western blot analysis of STX2 expressions in HTR-8/SVneo and primary human trophoblast cells transfected with Sh-STX2 and STX2-expressing lentivirus. (C) EdU assay and (D) colony forming assay of control and experimental cells in which STX2 was knocked down or overexpressed. Down-regulated STX2 reduced cell proliferation, whereas up-regulated STX2 increased cell proliferation. (E) Transwell assays of control and experimental cells in which STX2 was knocked down or overexpressed. Down-regulated STX2 inhibited cell migration and invasion, whereas up-regulated STX2 promoted cell migration and invasion. All the experiments were repeated three times independently. Data are represented as the mean ± SEM. Student’s t-test: **P < 0.01.
FIGURE 3
FIGURE 3
STX2 activates the AKT signaling pathway via membrane recruitment of PI3K p85. (A) Western blot analysis of p-AKT (ser473), AKT, p-GSK3β(ser9), and β-catenin in stably transfected HTR-8/SVneo and primary human trophoblast cells. β-Tubulin was used for normalization. (B) Co-IP results showing cell lysates were immunoprecipitated (IP) with antibodies for STX2, followed by western blot using PI3K p85, PI3K p110α, PI3K p110β, or STX2 antibodies. (C) GST pull-down assay was performed to clarify the binding between STX2 and PI3K p85 in vitro. (D) Confocal microscopy detection of subcellular distribution of STX2 (red) and PI3K p85 (green) in HTR-8/SVneo and primary human trophoblast cells transfected with Sh-STX2. Scale bar: 25μm. (E) Western blot analysis of the expression of p85 in membrane protein of control and experimental cells in which STX2 was knocked down. (F) Representative images of IHC staining for STX2 and p-AKT (ser473) in patients with PE (n = 4/group). Data are represented as the mean ± SEM. Student’s t-test: **P < 0.01. All the experiments were repeated three times independently.
FIGURE 4
FIGURE 4
STX2 promotes trophoblasts proliferation, migration and invasion through activating PI3K-AKT pathway. (A,B) Western blot analysis of p-AKT (ser473), AKT in indicated stably transfected HTR-8/SVneo and primary human trophoblast cells treated with LY294002 (PI3K inhibitor, 10 μM) or DMSO (negative control) for 48 h. (C) EdU assay and (D) colony forming assay were used to evaluated the proliferation of control and experimental cells in which STX2 was stably transfected with or without LY294002 treatment. (E) Transwell assays were used to evaluated the migration and invasion of control and experimental cells in which STX2 was stably transfected with or without LY294002 treatment. All the experiments were repeated three times independently. Data are represented as the mean ± SEM. Student’s t-test: ***P < 0.01.
FIGURE 5
FIGURE 5
The diagrammatic presentation of the underlying mechanism of STX2 promoting PE development. STX2 activated the PI3K-AKT pathway by interacting with PI3K p85 to prompt the trophoblast proliferation, migration and invasion. Therefore, down-regulation of STX2 in placenta contributed to the development of PE.
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