Potential role for nectin-4 in the pathogenesis of pre-eclampsia: a molecular genetic study

Background Nectins are cell adhesion molecules that play a pivotal role in adherens junctions and tight junctions. Our previous study using whole-genome oligonucleotide microarrays revealed that nectin-4 was upregulated in pre-eclamptic placentas. We investigated the role of nectin-4 in the etiology of pre-eclampsia. Methods We investigated the expression of nectin-4 using real-time RT–PCR, western blot and immunostaining. Additionally, we performed matrigel invasion assay and cytotoxicity assay using cells overexpressing the nectin-4. Results NECTIN4 transcripts were elevated in pre-eclamptic placentas relative to uncomplicated pregnancies. Nectin-4 protein levels in pre-eclamptic placentas were higher on a semi-quantitative western blot. Nectin-4 was localized at the apical cell membrane in syncytiotrophoblast cells and not at the adherens junctions. Nectin-4 was also detected in cytotrophoblasts and a subset of cells in the decidua. Nectin-4 overexpressing trophoblast cells migrated normally in the matrix. However, Natural killer (NK) cells showed a strong cytotoxic effect against nectin-4 overexpressing trophoblast cells. No causative genetic variation was evident in the NECTIN4 gene from a pre-eclamptic placenta. Conclusions There are as yet unknown factors that induce nectin-4 overexpression in trophoblast cells that may contribute to abnormal placentation via an aberrant immune response and the onset of a pre-eclamptic pregnancy. Electronic supplementary material The online version of this article (10.1186/s12881-018-0681-y) contains supplementary material, which is available to authorized users.


Background
Pre-eclampsia is one of the most common obstetrical problems and accounts for almost 15% of pregnancyassociated disorders. Pre-eclampsia is simply defined by a pregnancy-induced hypertension with proteinuria [1], but is actually not a simple disorder as it involves multiple organs including the liver, kidneys, lungs, coagulation and neural systems. There is now an emerging consensus that pre-eclampsia is a complex polygenetic trait in which maternal and fetal genes, as well as environmental factors, contribute to its onset [2][3][4].
Accumulating evidence has also now indicated that the aberrant expression of placental microRNAs is involved in the pathogenesis of pre-eclampsia [5,6]. Further to this, the pathogenetic processes underlying this disorder involve numerous factors such as oxidative stress, endothelial dysfunction, vasoconstriction, metabolic changes, thrombotic disorders and inflammatory responses, although the precise mechanisms have remained elusive [7,8].
Nectins are Ca2 + −independent, immunoglobulin-like cell adhesion molecules that play a pivotal role in both adherens junctions and tight junctions [9]. Nectins were originally described as homologs of poliovirus receptorlike receptors (PVRL) [10]. Some of these molecules interact with the domain of the filamentous-actin (F-actin)-associated molecule (known as afadin) and are involved in signal transduction [11]. Although nectin-1, 2 and 3 have been biologically well characterized as adhesion molecules, the functional properties of nectin-4 are still unknown. Recently, nectin-4 has been demonstrated to be an epithelial receptor for the measles virus [12]. In addition, since certain cancers, (breast, lung and ovarian cancer) overexpress nectin-4, it has been described as a new biomarker for these cancer types [13][14][15].
We previously performed gene expression profiling of placental tissue from women with and without preeclampsia using whole-genome oligonucleotide microarrays [16,17]. Our results from these analyses subsequently identified~150 genes with significantly altered expression in pre-eclampsia. NECTIN4 was identified as one of the most significantly upregulated of these genes in pre-eclamptic placentas. In our current study, we have examined the localization of the nectin-4 protein in placental tissues. We then compared the nectin-4 protein levels between women with uncomplicated pregnancies and women with severe pre-eclampsia. Further, we performed some functional assays using nectin-4 overexpressing trophoblast cells. Our present findings provide some additional insights into the role of nectin-4 in the etiology of pre-eclampsia.

Samples
All of the clinical samples analyzed in this study were collected at the Department of Obstetrics and Gynecology, Fujita Health University Hospital, Japan. Placental biopsy samples were obtained during Caesarean sections from both normotensive pregnancy (n = 20) and those with severe pre-eclampsia (n = 20). Severe pre-eclampsia was defined as a systolic blood pressure of 160 mmHg or higher, or diastolic blood pressure of 110 mmHg or higher on two occasions at least 4 h apart while the patient is on bed rest (unless antihypertensive therapy is initiated before this time) [18]. Normotensive subjects were matched for maternal age, gestational age, and maternal body mass index at pre-pregnancy. In addition to the normotensive term subjects, we collected preterm normotensive control samples from pregnancies with a premature rupture of the membrane due to a breech presentation or a previous caesarean section without evidence of intrauterine infection. The early gestation placental tissues were obtained from elective surgical terminations of pregnancies. Clinical details of these subjects are presented in Table 1.
To avoid any possible effects of labor on NECTIN4 expression, only placental samples that were obtained through Caesarean section from women who had not undergone labor were analyzed. A central area of chorionic tissue was dissected and the maternal deciduas and amnionic membranes were removed from these samples. After vigorous washing of the maternal blood with saline, tissues were immediately frozen in liquid nitrogen and stored until use. Informed consent was obtained from each patient and this study was approved by the Ethical Review Board for Clinical Studies at Fujita Health University.

Antibodies
Polyclonal goat antibodies raised against the C-terminal cytoplasmic domain of human nectin-4 was used for immunostaining and western blotting (AF2659; R&D, Minneapolis, MN). In addition, rabbit polyclonal anti-human nectin-4 antibodies were raised against amino acid residues 399-415 (CRRLHSHHTDPRSQPEES) and residues 463-479 (CPGSGRAEEEEDQDEGIK) within the cytoplasmic domain of human nectin-4. The resulting antisera were affinity purified on columns coupled to the peptide. To detect cells of the trophoblast lineage, anti-pancytokeratin monoclonal antibodies (Nichirei, Tokyo, Japan) were used.

Western blot analysis
Total cell lysates from placental tissue were prepared using T-PER (Pierce, Rockford, IL) and the proteins were separated by SDS-PAGE in 14% Tris-glycine gels (TEFCO, Tokyo, Japan). After electrophoresis, the proteins were blotted onto a nitrocellulose membrane and blocked with 5% skimmed milk powder diluted in Tris-buffered saline (TBS) with 0.05% Tween20. Membranes were incubated with diluted antibody preparations overnight at 4°C. After washing the next day, membranes were incubated with horseradish peroxidase (HRP)-conjugated affinity-purified donkey anti-rabbit or anti-goat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. The blots were then developed using SuperSignal (Pierce) and images were captured using Light-Capture with a cooled CCD camera (ATTO, Tokyo, Japan). MagicMark XP Western Protein Standard (Invitrogen) and Precision Plus Protein Standards (BIO-RAD) were used as size markers. Recombinant human nectin-4 (H00081607-Q01, Abnova) and nectin-4293 T cell transient overexpression lysate (H00081607-T01, Abnova) were also used. For semi-quantitative analysis, the membranes were reprobed with an anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO) as an internal control for the relative loading of the samples.

Immunostaining
Placental samples were fixed with 10% formaldehyde overnight, and then embedded with paraffin. Tissue sections (2 μm) were placed on silane-coated slides and dried in a conventional oven at 60°C for 24 h. After deparaffinization and rehydration, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol. Polyclonal goat or rabbit antibodies against human nectin-4 were used for detection. Bound antibodies were detected with a peroxidase-based method using Simple Stain MAX-PO (Nichirei, Tokyo, Japan) with 3,3-diaminobenzidine as a substrate. Non-specific goat or rabbit IgG (Zymed, San Francisco, CA) was used as the negative control. Counterstaining was performed with Mayer's hematoxylin solution. We performed immunostaining for 8 placental samples from preeclampsia and 8 controls. We stained three sections for each sample, and we examined 5 fields per each section. The immunostained specimens were examined by an observer who was not aware of the expression data and clinical outcomes. The signal intensities were evaluated by a method proposed previously [19].

Overexpression of NECTIN4
The cDNA encoding human NECTIN4 was chemically synthesized and cloned into the pCMV-3Tag-8 vector.

Statistical analysis
Intergroup comparisons were made using the Student's t test or one way analysis of variance method and P values of less than 0.05 were considered statistically significant.

NECTIN4 mRNA and protein are upregulated in a preeclamptic placenta
We performed quantitative RT-PCR for NECTIN4 using 20 placental samples from severe pre-eclampsia patients and 20 from subjects with an uncomplicated pregnancy. The NECTIN4 transcript level was found to be significantly increased in placentas from the pre-eclampsia cases (Fig. 1, Additional file 1: Figure S1). We performed western blotting to then examine the expression of nectin-4 protein. Although the molecular weight of nectin-4 is 55.5 kDa based upon its amino acid sequence, total cell lysates from placental samples showed two expressed nectin-4 isoforms of 55.5 kDa and7 5 kDa (Fig. 2a, left panel). To confirm that these two species were derived from nectin-4, we used a different nectin-4 antibody and obtained similar immunoblotting results (Additional file 2: Figure S2). To evaluate the nectin-4 protein level, the western blots were subjected to semi-quantitative analyses using β-actin as an internal control. Both isoforms of the nectin-4 protein were found to be more abundant in pre-eclamptic placentas (Fig. 2b).

Nection-4 protein is produced by syncytiotrophoblasts in the placenta
To localize the nectin-4 protein within the placental tissue, we examined placental histological specimens stained with a rabbit polyclonal anti-nectin-4 antibody of confirmed specificity on a western blot. This analysis revealed signals in the cytoplasm and stronger signals at the cell membrane on the apical side in the syncytiotrophoblast cells within the chorionic villi from uncomplicated term pregnancies ( Fig. 3a and b, Additional file 3: Figure S3). Similar observations were made using alternate goat anti-nectin-4 antibodies (Fig. 3c). In preeclamptic placentas however, the nectin-4 protein signals in the syncytiotrophoblasts were stronger than those detected in uncomplicated pregnancies. Notably, the signal intensities at the apical cell membranes of the syncytiotrophoblast cells in the pre-eclamptic subjects were much stronger than those in the normal pregnancy controls (Fig. 3d, e and f ). Cytotrophoblast cells were also positive for the nectin-4, which was more prominent in samples from early gestation ( Fig. 4a and b). Further, we observed the presence of nectin-4-positive cells in maternal decidua ( Fig. 4c and d).
These cells might be extravillous trophoblasts since they were also positive for cytokeratin ( Fig. 4e and f). The signal intensity was stronger in samples from pre-eclampsia than from controls ( Fig. 4c and d).

Nectin-4 protein overexpression does not affect cell migration
Since HTR-8/SVneo cells do not strongly express NECTIN4 (data not shown), we produced a nectin-4 overexpressing HTR-8/SVneo cell line to use as a model system for invasive trophoblasts in pre-eclampsia (Additional file 4: Figure S4). In the early gestational period when placentation occurs, fetal trophoblasts that have invaded the basal membrane of the maternal uterus and then migrate to the uterine interstitium. These cells then migrate to the uteroplacental spiral artery and finally replace the maternal endovascular endothelial cells [20]. It has been proposed that the failure of this vascular remodeling system may lead to the onset of pre-eclampsia [20]. We compared the cell migration ability of trophoblasts overexpressing nectin-4 relative to control trophoblasts. However, the up-regulation of NECTIN4 did not affect the invasiveness of the HTR-8 cells in the basal membrane-type extracellular matrix (Fig. 5a).
We also speculate that invading fetal trophoblasts may be a target for the maternal immune system, such as NK cells, and that the migration of these cells might be strictly regulated by natural immunity. We thus compared NK cell cytotoxicity towards trophoblasts overexpressing nectin-4 relative to control trophoblasts. Trophoblasts overexpressing nectin-4 showed more sensitivity to the cytotoxic effects of NK cells (Fig. 5b). The NECTIN4 gene is not mutated in pre-eclamptic placental tissue Given the possibility that the up-regulation of NECTIN4 might be the primary etiology of pre-eclampsia, we hypothesized that mutations in this gene may underlie its up-regulation. We sequenced an 18 kb genomic region that contains the promoter region and 9 exons of the NECTIN4 gene but found no rare variants in placental samples from pre-eclampsia patients (data not shown).

Discussion
We have analyzed the overexpression of the NECTIN4 gene, both at the RNA and protein levels, in pre-eclamptic and normal placentas to test whether alterations in the  Although other members of the nectin family are expressed ubiquitously, NECTIN4 is specifically expressed in the skin, esophagus, urinary bladder and placenta. The nectins are known to be adhesion molecules that may have key functions at adherens junctions and tight junctions [9]. However, our current data indicate that the nectin-4 protein is localized at the apical cell membrane of syncytiotrophoblasts, suggesting different functional properties from other nectins. Further, we observed the presence of nectin-4-positive cells in maternal decidua, which might be extravillous trophoblasts. It is thus feasible to suspect a functional linkage between nectin-4 and trophoblast invasion in the placental bed and in maternal uteroplacental arteries [20]. We do not know however if invasive trophoblasts actually express nectin-4 in the plasma membrane during early gestation. We hypothesize from our current analysis that trophoblast invasion is regulated by the surface expression of nectin-4 on these cells.
It has long been accepted that placentation is abnormal in pre-eclampsia [21]. Trophoblast invasion of the interstitial uterine compartment is frequently shallow, and invasion into the spiral artery is often incomplete, during a pre-eclamptic pregnancy. Likewise, the impaired invasion of trophoblasts is believed to underlie the etiology of this disorder. Our current results indicate that nectin-4 expression is upregulated at the apical cell membrane of the syncytiotrophoblasts in pre-eclampsia. The overexpression of nectin-4 in extravillous trophoblast in maternal decidua might reflect invasive trophoblast in early gestation. We hypothesized that trophoblast invasion might be impaired in pre-eclampsia by the overexpression of NECTIN4.
However, an invasion assay using HTR-8 cell lines did not support this possibility.
In further experiments however, HTR-8 cells stably overexpressing exogenous NECTIN4 were significantly more sensitive to NK cell cytotoxicity than control HTR-8 cells. These data suggest the possibility that the nectins might regulate NK-target interactions and that the impairment of these interactions might induce the onset of pre-eclampsia. Consistently, there is some evidence that nectins play an important role in the recognition of target cells by NK cells. It has been reported previously that NK cells recognize and kill human glioblastoma cells that express high levels of nectin-2 [22]. Another study has demonstrated that the interaction of nectin-2 and its ligand inhibits NK cell cytotoxicity [23]. Similarly, it has been reported that the interaction of nectin-4 and its ligand might activate NK cell cytotoxity towards trophoblasts, leading to their impaired invasion [24,25].
It was demonstrated recently that mutations in NEC-TIN4 resulted in ectodermal dysplasia-syndactyly syndrome in humans [26,27]. This is a rare autosomal recessive disorder that might be caused by a loss-of-function of NECTIN4. We thus hypothesized that this disorder and pre-eclampsia might represent allelic heterogeneity and the mutation in NECTIN4 may cause its overexpression of NECTIN4 and lead to the onset of pre-eclampsia. However, our current data did not reveal any NECTIN4 variants in pre-eclamptic placentas. Taken together, our present data suggest the possibility that some unknown factors induce nectin-4 protein overexpression in trophoblasts that might cause abnormal placentation via aberrant immunity, and ultimately a pre-eclamptic pregnancy. On the other hand, it is also possible that the elevated expression of the nectin-4 is secondary to the dysfunction of syncytiotrophoblast cells.

Conclusion
Our current data highlight an old proposition i.e. the direct involvement of NK cells in the etiology of preeclampsia. Further detailed analysis of the functional roles of nectin-4 in placentation will be needed to properly assess the association of its overexpression with the etiology of pre-eclampsia.