ELABELA targets the MEK/ERK axis to enhance trophoblast invasion in early-onset preeclampsia

Abstract

The study aimed to investigate the role of ELABELA (ELA) in early-onset preeclampsia (EOPE), a condition characterized by dysregulated trophoblast invasivity leading to inadequate remodeling of uterine spiral arteries and shallow placental implantation. Using immunohistochemical staining, quantitative PCR (qPCR), and Western immunoblotting, placental ELA levels were evaluated in tissue samples. The invasive and migratory potential of trophoblastic cells in vitro was assessed using a Transwell system. Additionally, key kinases associated with the MEK (Mitogen-activated protein kinase kinase)/ERK (Extracellular-regulated protein kinases) signaling pathway in trophoblasts were examined through Western immunoblotting. The findings revealed that ELA was present in both cytotrophoblasts and syncytiotrophoblasts of the placenta. There were no significant differences in ELA mRNA or protein levels between normal pregnancies and preterm births; however, ELA levels were significantly lower in the EOPE group compared to the normal pregnancy group. Silencing of ELA in HTR-8/SVneo trophoblastic cells notably impaired their invasive and migratory abilities, accompanied by reduced levels of key kinases linked to the MEK/ERK pathway. Conversely, overexpression of ELA enhanced these cellular functions. The repression of MEK also inhibited HTR-8/SVneo cell migration and invasion while decreasing related kinase levels. The study concluded that ELA plays a critical role in promoting trophoblastic cell migration and invasion through the activation of the MEK/ERK signaling pathway. The observed decrease in ELA levels in EOPE suggests its potential involvement in the pathogenesis of this condition. These findings underscore the potential for novel therapeutic strategies targeting ELA and the MEK/ERK signaling pathway to address early-onset preeclampsia, a condition with significant maternal and fetal health implications. Further research into ELA's mechanisms and its regulatory pathways may offer insights for effective interventions.

Keywords: ERK; Invasion; MEK; Placenta; Preeclampsia.

Fig. 1

Correlation between EOPE and Reduced Placental ELA Levels. A The expression and localization of ELA in the placentas were detected using Immunohistochemical staining (IHC) at a × 400 magnification for the Normal, EOPE, and PD groups. (Line bar = 40 μm). B The expression of ELA mRNA was measured using qRT-PCR assay in placenta samples. C Western immunoblotting was used to assess the expression of the ELA protein. D Quantitative analysis of protein expression. Compare with indicated group: ** P < 0.01; ***P < 0.001; ns: no significance. Normal: n = 30, EOPE: n = 30, PD: n = 20.

Fig. 2

Impact of ELA Silencing on Trophoblast Migration and Invasivity. A Western blot analysis for ELA in si-ELA-transfected cells. The migration analysis B and quantitative analysis C. The invasion analysis D and quantitative analysis E Bar 100 μm. Compare with indicated group: * P < 0.05; ** P < 0.01; ***P < 0.001. n = 3.

Fig. 3

Effects of ELA Overexpression on Migration and Invasivity of HTR-8/SVneo Cells. A Western blot analysis for ELA in ELA-Overexpressed cells. The migration analysis B and quantitative analysis C The invasion analysis D and quantitative analysis E Bar 100 μm. Compare with indicated group: * P < 0.05; ** P < 0.01; ***P < 0.001. n = 3.

Fig. 4

The potential important role of MAPK pathway in EOPE. A The volcano plot showing the differential gene expression between EOPE and LOPE. B A heatmap displaying the differential gene expression between EOPE and LOPE. C Enrichment analysis of differentially expressed genes. Transcriptomic data (GSE74341) were used, with EOPE n = 7 and LOPE n = 5.

Fig. 5

Regulation of MEK/ERK Signaling by ELA in HTR-8/SVneo Cells. Key proteins in the MEK/ERK pathway were evaluated using western immunoblotting in HTR8/SVneo cells A The expression of pMEK1/2, MEK1/2, pERK1/2 and ERK1/2 were evaluated in ELA knocked down samples. B The expression of pMEK1/2, MEK1/2, pERK1/2 and ERK1/2 were evaluated in ELA overexpressed samples. C The expression of pMEK1/2, MEK1/2, pERK1/2 and ERK1/2 were evaluated in MEK1/2 knocked down samples. D The migration analysis and quantitative analysis. E Bar 100 μm. The invasion analysis and quantitative analysis. F Model diagram of ELA functioning by regulating MEK/ERK signaling. Compare with indicated group: * P < 0.05; ** P < 0.01. n = 3.