Overexpression of the STOX1B isoform of STOX1 triggers preeclampsia-like symptoms through HNF4α-dependent alterations of coagulation cascades in mice

Abstract

STOX1 has been involved in genetic forms of preeclampsia. The gene encodes two major isoforms coined STOX1A and STOX1B (989 and 227 amino-acids, respectively), sharing the same DNA binding domain. The two isoforms have opposite function on major genes involved in trophoblast syncytialization and oxidative stress management. Placenta-fetal overexpression of STOX1A induces preeclampsia in mice. Here we explore the effects of STOX1B placenta-fetal overexpression. A STOX1B transgenic mouse line (expression restricted to the foeto-placental unit through ad hoc crosses) was analyzed in terms of blood pressure, proteinuria, soluble antiangiogenic factors, placental and fetal weights, maternal heart weight, and placental histology. Placental gene expression was explored by RNA-sequencing, followed by a bioinformatics analysis. Female mice carrying STOX1B placentas displayed preeclampsia features with a striking increase of genes involved in coagulation, presumably under the control of the HNF4α transcription factor. Genes specific of the spongiotrophoblast were strongly down-regulated consistently with a junctional zone reduction. This new model of preeclampsia seems connected with an enhancement of coagulation processes, similar to preeclamptic patients living at high altitude. Our model could be useful to study the features of preeclampsia in this context, and be a convenient complement to other animal models of preeclampsia.

Fig. 1

Phenotype of the pregnant mice carrying STOX1B embryo/placental units. (A) upper left, blood pressure curves normalized for each mouse at pre-gestation blood pressures during the whole gestation (18.5 days in FVB/N mice), upper right: blood pressures averages upon three consecutive periods (early, mid and late-gestation). Down left and down middle Albumin/Creatinin ratio measured in the urine of the mice; Down right, concentration of soluble Endoglin in the mouse plasma at the end of gestation. (B) weights of the fetuses (upper part) and placentas (down part) at 15.5 dpc, 16.5 dpc and 17.5 dpc. Statistical tests were carried out using Student T-tests (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001).

Fig. 2

Bioinformatics analysis of the STOX1B transcriptome in the placentas compared to controls. (A) Principal Component Analysis; this is semi-supervised, since the 500 more deregulated genes were used to build the graph. Each SB refers to a mouse number and P to a placenta. Each dot corresponds to a bulk RNA-seq from three pooled placentas assembled, taking the sex into consideration (circles are for female pools, while triangles are for male pools). Red is for STOX1B pools and blue green for controls. As indicated, the first axis captures over 78% of the variance and separates unambiguously the two types of pools. (B) Volcano plot showing the degree of variation of the affected genes (in red, more expressed in the STOX1B placentas and the contrary in blue); thresholds on the abscissa represents genes modified more than twice, while the ordinate represents a threshold of p < 0.05). Some highly deregulated genes are shown. (C) EnrichR analysis of the upregulated genes (380), allowed to reveal that in different databases, there was a very strong enrichment in ‘Coagulation and Complement cascades’ and in ‘Cholesterol metabolism’ (directly or not). The length of the bars is proportional with the p-value of the enrichment. For instance, the adjusted p value is 2.82 10^− 13 and 1.26 10^− 8 for ‘Complement and Coagulation cascades’ and ‘Statin pathway’ in the case of the BioPlanet 2019 database.

Fig. 3

Promoter enrichment analyses reveals the role of HNF4α in modulating up-regulated genes. (A) Without a priori, EnrichR revealed quasi systematically an enrichment in genes up-regulated through the HNF4α transcription factor (left). On the right the genes targeted by HNF4α that permitted its identification are represented. It is the only significant enrichment in Encode, based upon ChiP-seq experiments (adjusted p-value = 0.0031). In the case of the KO databases, the p value for connection with the HNF4α mouse KO was 2.87 10^− 38 adjusted p-value. (B) correlation between the present dataset and the most correlated dataset identified by Rummagen, which was from Ducat et al. (2019). The latter is composed of 80 genes that are modified in preeclamptic placentas (STOX1A mouse model) compared to the same when the mice were treated with aspirin. There was a quasi-perfect negative correlation between the two datasets. The induction by aspirin is presented in the abscissae as Logarithm of fold change (base 2), while the vertical axis (ordinate) corresponds to the present dataset (comparison between STOX1B placentas and WT placentas). HNF4α is present amongst these gene and presented in a red circle. (C) RT-qPCR validation of HNF4α on individual placentas. The deltaCt were normalized to the average of the WT placentas, and tested by a Mann-Whitney non-parametric test (since exponentiation does not allow to maintain the use of a T-test). On average, the increase of expression was estimated at 2.46-fold in this experiment.

Fig. 4

(A) Decreased mRNAs are strongly enriched specifically in the invasive spongiotrophoblast cells. Dark orange and Dark blue correspond to significant enrichments. While the induction of genes is not specific of a given cell type, in the decreased genes a unique cluster is recognized as represented in the GSEA enrichment plot (B,C) part of the spongiotrophoblast-specific genes is located on mouse chromosome 7 (syntenic to human chromosome 19), and are present as a specific cluster between 16,800 and 18,600 kilobases on the chromosome. The graph shows that all these genes have a reduced mRNA level by a factor of ~ 2, suggesting a common regulation.

Fig. 5

Morphometric analyses of the placentas following Trichrome Masson staining. (A) measures of the ratio between the Junctional Zone/ and the total placenta thickness. At the three time points when the placentas were collected, there was a systematic reduction of the junctional zone. (B) examples of WT (in blue) and STOX1B (in red) placentas. The blue arrow marks the junctional zone thickness, while the red arrow marks the labyrinth thickness. Each placenta originates from a different mouse (identified by the numbers at the left).