MNSFβ regulates placental development by conjugating IGF2BP2 to enhance trophoblast cell invasiveness

Abstract Objectives Success in pregnancy in mammals predominantly depends on a well‐developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFβ) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFβ in regulating placentation and pregnancy maintenance remains to be elucidated. Methods Trophoblast‐specific knockout of MNSFβ was generated using Cyp19‐Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFβ and insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) at the foeto‐maternal interface. The interaction and expression of MNSFβ, IGF2BP2 and invasion‐related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real‐time polymerase chain reaction (qRT‐PCR). The cell invasion ability was measured by the Transwell insert assay. Results We found that deficiency of MNSFβ in trophoblasts led to embryonic growth retardation by mid‐gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFβ with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion‐promoting effect of MNSFβ. In the placentas from MNSFβ‐deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression. Conclusions The findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.


| INTRODUC TI ON
As a transient organ that is particularly formed during pregnancy in mammals, the placenta acts as an indispensable safeguard for both foetal growth and maternal health. 1,2 The recent integrative investigation in genetically manipulated mice has strongly revealed that defects in placentation are highly prevalent in abnormal embryo development. 3 Placental trophoblast cell differentiation along the invasive pathway is one of the fundamental processes for placental development. The invasive extravillous trophoblasts (EVTs) infiltrate into decidua to anchor the embryo to the uterine wall, reconstruct uterine blood vessels to ensure blood supply and establish an immune-tolerant environment through interaction with maternal immune cells. Defects in trophoblast cell invasiveness and subsequent insufficient remodelling of maternal spiral arteries have been well recognized as the dominant pathological factors of pregnancyassociated disorders including PE. 2,4,5 We previously identified a remarkable differential gene, MNSFβ, in mouse implantation site, 6,7 which encodes a non-antigen-specific immunosuppressive factor with 97.8% homology between human and mouse. Structurally, MNSFβ consists of 133 amino acids which form a ubiquitin-like (Ubi-like) domain and a ribosomal S30 domain. [8][9][10] Conventional knockout of MNSFβ gene in mice led to embryonic lethality of homozygotes (MNSFβ −/− ) and significantly retarded in utero growth of heterozygotes (MNSFβ +/− ) after embryonic day 10.5 (E10.5). 11 In addition, we found the invasion-promoting effect of MNSFβ on human trophoblasts, and its markedly decreased expression in the placentas of recurrent miscarriage patients. 11,12 These evidences highly indicate the dominant roles of MNSFβ in regulating trophoblast differentiation and therefore governing pregnancy outcomes.
To further address the regulatory mechanisms of MNSFβ in the process of placental development, here we generated a mouse model with specific deletion of MNSFβ gene in placental trophoblasts (PHTs) using the Cyp19-Cre tool. Phenotypical analysis of embryonic and placental growth as well as trophoblast differentiation was conducted. Glutathione S-transferase (GST) pull-down, mass spectrometry and invasion assay in human trophoblast cells were carried out to determine the interaction between MNSFβ and IGF2BP2 that can regulate trophoblast cell invasiveness. What's more, the aberrant expressions of MNSFβ and IGF2BP2 in severe PE placentas were examined. The findings reveal MNSFβ as a novel factor governing trophoblast cell invasiveness and therefore foetal development.

| Collection of human placental and decidual tissues
The study was approved by the Local Ethical Committees in IOZ, the Second Hospital Affiliated to Tianjin Medical University (Tianjin, China) and Peking University Third Hospital (Beijing, China). Written IGF2BP2 essentially mediated the invasion-promoting effect of MNSFβ. In the placentas from MNSFβ-deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression.

Conclusions:
The findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.
informed consent was obtained from the enrolled pregnant women.
The decidual and villous tissues from healthy pregnant women at early gestation were collected at the Second Hospital Affiliated to Tianjin Medical University (Tianjin, China), and the clinical features of these pregnant women are summarized briefly in Table S1.
Human placental tissues from patients with severe PE (PE, n = 7) or unexplained preterm labour (PTL, n = 7) were collected at Peking University Third Hospital (Beijing, China). The placentas from PTL were used as the gestation-matched controls for severe PE, as described previously. 14 The clinical features of the pregnant women enrolled in this study are summarized in Table S2. Severe PE was defined as a pregnancy having no history of preexisting or chronic hypertension but showing systolic blood pressure of ≥160 mmHg or diastolic blood pressure of ≥110 mmHg on at least two occasions, accompanied by significant proteinuria (≥2 g/24 h or 3+ by dipstick in two random samples collected at >4 h intervals) or problems in multiple organs (such as pulmonary oedema, seizures, oliguria, abnormal liver enzymes associated with persistent epigastric or right upper quadrant pain, or persistent and severe central nervous system (CNS) symptoms) after the 20th week of gestation. 15 PTL was defined as labour earlier than the 34th week, but without clinical or pathological features of other maternal or placental complications.
Specimens of the placenta were obtained immediately after caesarean section and subjected to snap freezing. The results were recorded on a Zeiss LSM780 confocal microscope system (Zeiss, Germany) and processed with ZEN 2012 software (Zeiss).

| In situ hybridization (ISH)
MNSFβ-specific riboprobe was designed to recognize nucleo- The ISH was carried out, as described previously. 17,18 Briefly, serial frozen sections at 10 μm were fixed in 4% PFA, treated with protease K and hybridized with sense or antisense probes for MNSFβ at 55°C.

| Statistical analysis
The statistical analysis was performed with GraphPad Prism version 5.01 (GraphPad Software). Data were shown as mean ± SEM according to at least three independently repeated experiments.
The differences between groups were analysed by independent Student's t-test or unpaired one-way analysis of variance (ANOVA) with Sidak's correction. The P values less than 0.05 were considered statistically significant.

| Expression pattern of MNSFβ at the foetomaternal interface of human and mouse
Immunofluorescent staining and/or ISH for MNSFβ was performed to clearly demonstrate its distribution at the foeto-maternal interface of human and mouse. In human placental villous and decidual tissues at early gestation, immunofluorescent signals were intensively observed in villous cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs) and EVTs, as well as many other cell types in placenta and decidua ( Figure 1A). At the mouse foeto-maternal interface at E10.5 and E13.5, the MNSFβ expression could be extensively ob-

| Placental-specific deficiency in MNSFβ leads to retarded embryonic and placental development at early-to-mid gestation in mice
To specifically elucidate the role of MNSFβ in placental trophoblasts, ing that cKO embryos could survive till mid-gestation ( Figure 2C and Table S3). However, the embryonic weight and placental weight of cKO embryos were remarkably lowered at E13.5 compared to those of the Het or Control group, although the decreased tendency was not significant at E10.5 ( Figure 2D,E).
The phenotype observations reveal that trophoblast-specific deficiency in MNSFβ results in retarded embryonic and placental development and subsequent foetal lethality.

| Deletion of MNSFβ in the placenta impairs trophoblast cell invasiveness
We further examined the placental structures at E10.5 and E13.5 in the mice. Histological analysis revealed little difference between the placentas of the Het and Control groups in the area of the three functional layers -Dec, Sp and Lab at E10.5 or E13.5 ( Figure 3A,B).
Whereas in cKO placentas, the areas of Dec, Sp and Lab at E10.5 were separately reduced to approximately 90%, 85% and 80% of the corresponding Control ( Figure 3C), which were more severe at E13.5, being approximately 80%, 65% and 60% of the corresponding Control ( Figure 3D).
Immunohistochemistry (IHC) for CK7 was performed to mark trophoblast cells and thus the cell invasiveness was analysed ( Figure 3A,B). As shown, both the number of trophoblast cells that invaded into the decidual layer and their invasion distance were markedly declined in cKO placentas, which were approximately half of the Control at E10.5 ( Figure 3E) and less than one third of the Control at E13.5 ( Figure 3F). In parallel, the results of quantitative real-time PCR showed a remarkable downregulation of the invasionassociated marker genes (prl3a1, prl3b1, prl2c2 and prl3d1) in cKO placentas, relative to the Het or Control group ( Figure 3G).
These data demonstrate that the impaired trophoblast cell invasion due to MNSFβ deficiency is a primary cellular defect in association with retarded embryonic and placental development.
Among the 24 candidates as shown in Table S4, IGF2BP2 has been reported as an important intracellular factor for female fertility and trophoblast invasion. 22,23 We carried out IP analysis in both JEG3 cells and human trophoblast stem cells (hTSCs) and proved the direct binding of MNSFβ to IGF2BP2 ( Figure 4A). Immunofluorescent staining for IGF2BP2 was then performed to clarify its distribution at the foeto-maternal interface of human and mouse. In human decidual tissues at early gestation, immunofluorescent signals were primarily observed in EVT cells ( Figure 4B). At mouse foeto-maternal interface at E10.5, IGF2BP2 staining was found in multiple subtypes of trophoblasts, including the trophoblasts that invaded into the decidual tissue ( Figure 4C). These observations suggest the probable involvement of IGF2BP2 in MNSFβ-regulated trophoblastic invasiveness.

| MNSFβ binds with IGF2BP2 to protect its degradation by ubiquitin-proteasome in trophoblast cell
Interestingly, knockdown of MNSFβ in JEG3 cells using specific siRNA did not alter the mRNA level of IGF2BP2, but led to a markedly reduced IGF2BP2 protein level, being less than half of that in the NC group in which scramble siRNA was transfected ( Figure 5A-C). On the contrary, knockdown or overexpression of IGF2BP2 had little influence on MNSFβ expression ( Figure 5A-C). These data indicate that the direct binding of MNSFβ to IGF2BP2 may increase the stability of IGF2BP2 protein in trophoblasts.
In JEG3 cells, we found a gradually elevated IGF2BP2 protein level upon the treatment of the proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132), whereas the autophagy inhibitor 3-methyladenine (3-MA) had little influence ( Figure 5D,E and Figure S1). In addition, knocking-down of MNSFβ significantly increased the ubiquitination level of IGF2BP2 in JEG3 cells ( Figure 5F).
These data demonstrate that MNSFβ binding with IGF2BP2 protects the degradation of IGF2BP2 by the ubiquitin-proteasome pathway.

| MNSFβ promotes trophoblast cell invasion via interaction with IGF2BP2
To interpret whether the interaction of MNSFβ with IGF2BP2 regulates trophoblast cell invasion, we carried out the Transwell insert invasion assay in JEG3 and HTR8/SVneo cells that were subjected to knockdown or overexpression of MNSFβ or IGF2BP2. As shown in Figure 6, Figure S2

| Downregulation of IGF2BP2 is associated with repressed MNSFβ in the placentas derived from cKO mice and severe preeclamptic (PE) patients
Based on the above evidence, we measured the protein levels of IGF2BP2 and MNSFβ in the placentas from the MNSFβ-cKO mice Control or Het groups ( Figure 7A,B). The level of IGF2BP2 protein in these cKO placentas was reduced by nearly 60% relative to Control or Het groups ( Figure 7A,B). Statistical analysis demonstrated a tight association between the protein level of MNSFβ and IGF2BP2 in cKO placentas ( Figure 7C). In parallel, the ubiquitination level of IGF2BP2 in cKO placentas at E13.5 obviously elevated compared to the Control group ( Figure S4A).
It has been well accepted that compromised trophoblast cell invasion and subsequent insufficient spiral artery remodelling are predominant pathological changes in PE placenta, especially in those cases with severe symptoms. We thus examined the protein levels of MNSFβ and IGF2BP2 in the placentas from severe PE patients.
Considering most of the severe PE placentas were delivered at earlier than the 37th gestational week, we collected placentas from unexplained PTL as the gestational-week-matched control. 14 As expected, the levels of MNSFβ and IGF2BP2 were significantly lower in PE placentas, being approximately 40% and 35% of the corresponding PTL control, respectively ( Figure 7D,E). Statistical analysis showed a tight association between the protein level of MNSFβ and IGF2BP2 in PE placentas ( Figure 7F). As expected, the ubiquitination level of IGF2BP2 obviously increased in PE placentas compared to PTL control ( Figure S4B).

| DISCUSS ION
A recent systematic study in mice with embryonic-lethal mutations demonstrates that a considerable number of embryonic defects may result from defective placentation, robustly revealing the vital role of a healthy placenta for embryonic development. 3 In this study, the mice with trophoblastic deficiency of MNSFβ exhibited a similar phenotype of embryonic lethality with that of cKO mice. 11 As stated, the deficiency of MNSFβ in trophoblasts from E6.5 causes an apparently limited trophoblast cell invasiveness at E10.5, leading to great retardation in embryonic and placental development and eventually no surviving foetus. MNSFβ is widely expressed in multiple cell types at the foeto-maternal interface, the early placental dysplasia in trophoblast-specific deletion of MNSFβ strongly indicating the critical roles of this molecule for early differentiation of PHTs and thus for pregnancy maintenance.
The appropriate trophoblast differentiation towards invasive pathway is important to anchor the embryo into uterine wall and to build up proper blood perfusion into the foeto-maternal interface.
Limited invasiveness has been recognized as the major pathological factor for various adverse pregnancy outcomes, including PE, foetal growth restriction or early foetal loss. 24-27 IGF2BP2 has been found to stimulate invasion and migration of trophoblast cell, and its protein level is significantly lower in severe PE placentas. 22 Our F I G U R E 3 Defect in trophoblastic invasiveness in trophoblast-specific deletion of monoclonal nonspecific suppressor factor beta (MNSFβ) at embryonic day 10.5 (E10.5) and embryonic day 13.5 (E13.5). (A, B) Typical results of haematoxylin and eosin (HE) and immunohistochemistry (IHC) for cytokeratin 7 (CK7) in placentas from Control, Het and conditional knockout (cKO) groups at E10.5 (A) and E13.5 (B), respectively. Based on the HE and CK7 staining, decidua, sponge zone and labyrinth layers are separately marked by blue, red and yellow solid lines. Scale bars indicate 500 μm (upper panels) and 100 μm (lower panels). (C, D) Statistical analysis of the areas of decidua, sponge zone and labyrinth layers in Control, Het and cKO placentas at E10.5 (C) and E13.5 (D) (n = 5 in each group). (E, F) Statistical analysis of the number of trophoblasts that infiltrated into decidua and the invasion distance at E10.5 (E) and E13.5 (F) (n = 5 in each group). (G) Statistical results of quantitative real-time PCR for invasion-related marker genes (prl3a1, prl3b1, prl2c2 and prl3d1) in the placentas from Control, Het and cKO at E13.5 (n = 5 in each group). The experiments are independently repeated for three times, and data are presented as mean ± SEM. Comparison between groups is carried out with one-way analysis of variance (ANOVA) with Sidak's correction. *p < 0.05, **p < 0.01 MNSFβ has long been known as a non-antigen-specific immunosuppressive factor. It was first found to be a lymphokine produced by murine T cell hybridoma which can inhibit the secretion of immunoglobulin (Ig) from LPS-induced mononuclear cells. 28 (Table S4), which were not similar to those found in macrophages. Such a difference also indicated that the effect of MNSFβ may largely depend on the intracellular microenvironment.
Here in our study, in vitro and in vivo experiments proved a direct effect of MNSFβ on trophoblast cell invasion, partly through an intracellular interaction with IGF2BP2. However, it is notable that among the candidates that potentially bind to MNSFβ, ribosomal protein S3A (RPS3A) was reported to promote the biological processes related to tumourigenesis, metastasis and immunosuppression in hepatocellular carcinoma patients, 31  in endophilin II, K481 in heat shock protein 60 (HSP60) and K72 in formate dehydrogenase (FDH). 30,[33][34][35] K139 in IGF2BP2 has been the predominant site for its ubiquitination. 36 We thus assume that the covalent binding between G74 in MNSFβ with K139 in IGF2BP2 may be the recognition site to protect the degradation of IGF2BP2.
Interestingly, our results showed that the areas of all three functional layers, decidua, sponge zone and labyrinth, significantly decreased in MNSFβ-cKO mice, indicating that the embryonic lethality, placental and foetal maldevelopment in MNSFβ-deficient mice may not be solely caused by an impaired EVT invasion, but may also involve compromised trophoblast syncytialization and thus placental haemodynamics or spongiotrophoblast differentiation. On the other hand, considering the extensive expression of MNSFβ in diverse cell types at the foeto-maternal interface and its lymphokine property, there is a possibility that MNSFβ from other cells may, to a certain extent, compensate its deletion in trophoblasts, leading to a relatively late appearance of the placental phenotypes in cKO mice. Further investigation using models such as trophoblast subtype-specific deletion or tetraploid compensation is needed to address these issues.
In general, our findings reveal the crucial role of MNSFβ in modulating trophoblast differentiation at the early stage of gestation.
Particularly, it affects trophoblastic invasiveness by binding and stabilizing IGF2BP2. The study also indicates the participation of functionally deficient MNSFβ in the pathogenesis of pregnancy disease such as PE.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T S
The authors declare no competing or financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data of this study are available from the corresponding author upon reasonable request.