Missed abortion is considered to be the most common complications of pregnancy. From the immunological point, pregnancy is somewhat analogous to organ transplantation. The fetus possesses the antigens of both maternal and paternal, it is semi-allogeneic. A normal immune system of pregnant woman can not only protect mother and fetus from foreign pathogens, but also tolerate semi-alogenous[17]. A appropriate immune status, embryo quality and the physiological status of decidua are indispensable for a successful pregnancy. According to the previous study, normal pregnancy has a certain degree of trophoblast cell apoptosis, which is conducive to the formation and development of villi and chorionic villi branches[18]. However, excessive apoptosis of trophoblasts may lead to abnormal villi development or cytotrophoblast degeneration, and even lead to pregnancy failure[19, 20, 21]. In addition, a study by Chen reported that adequate angiogenesis in the villi plays an important role in maintaining early pregnancy[22]. Therefore, apoptosis and angiogenesis may be closely related to the occurrence of missed abortion.
Arrestins were originally discovered to be the main protein component of the retinal photoreceptor region, and also called “retinal S antigen” because of their involvement in uveitis. There are four members of the arrestin family:β-arrestin1, β-arrestin2, x-arrestin, and s-arrestin[23]. The classical functions of β-arrestin1 and β-arrestin2 were initially identified to mediate desensitization, sequestration, and recycling of G protein-coupled receptors (GPCRs), whereas s-arrestin and x-arrestin can only be found in the vision system[24]. Further investigations established that β-arrestin system is a major hub that controls almost the entire GPCR signal network. There is increasing evidence suggests that β-arrestin1 acts as modulator in a number of intracellular signaling pathways, such as extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK3), apoptosis-signal-regulating kinase 1(ASK1), insulin-like growth factor I (IGF-1), phosphoinositide 3-kinase (PI3K-Akt), and wingless-type MMTV integration site family members 5A and 3A (Wnt5a and Wnt3A)[25–29], which play an important role in the regulation of various cellular functions in both normal and malignant cells.
In addition, recent studies have shown that the downstream of IGF-1R has an attractive β-arrestin1 antagonistic effect on p53 activation. β-arrestin1 moves to the nucleus and acts as an adaptor protein to promote the binding and degradation of p53 through E3-ubiquitin ligase Mdm2, thereby accumulating DNA damage[30]. However, when Mdm2 recognizes the target p53 and binds to the HIF-lα subunit, it mediates the degradation of HIF-lα, reduces the expression of VEGF, inhibits angiogenesis, stops the cell cycle and increases apoptosis and eventually leads to missed abortion[31]. Our previous studies have confirmed that p53 and Mdm2 genes play a vital role in the development of villus tissue during early pregnancy, and ultimately affect the outcome of pregnancy[32]. However, there was no study have been made regarding the role of β-arrestin1 in early pregnancy. Based on the former studies, we speculated that β-arrestin1 may be closely related with missed abortion.
In the present study, we collected the villous samples of missed abortion and healthy controls. At first, we detected the expression of β-arrestin1 by qRT-PCR. We found that the expression of β-arrestin1 was significantly downregulated in missed abortion group. To further investigate the expression sites and protein levels of β-arrestin1, we measured the expression of β-arrestin1 of missed abortion and induced abortion groups by immunohistochemistry. We found that β-arrestin1 positive staining mainly expressed in the nucleuses of villous cytotrophoblast、syneytiotrophoblast and extravillous trophoblastic cell column༎Consistent with the qRT-PCR results, the expression of β-arrestin1 in missed abortion group was significantly lower than normal controls. The results evidently proved the correlation between the low expression of β-arrestin1 and undesirable pregnancy outcome.
We also detected the expressions of p53, Mdm2 immunoreactivity in both the missed abortion groups and normal pregnancy, and we found that there was significant differences between the two groups. P53 is a vital apoptosis-regulating gene which is precisely controlled to keep the balance between cell death and proliferation. Mdm2 is the main negative regulator of p53, it ubiquitinates p53 and promotes its proteasome degradation. In turn, p53 stimulates Mdm2 transcription[33]. Therefore, Mdm2 and p53 form a regulatory feedback loop which strongly affected by cellular stress. Our former studies have confirmed that Mdm2 and p53 may be a genetic risk factor for missed abortion[11]. This fact strongly supports the critical role of p53 and Mdm2 in the regulation of embryogenesis. In the present study, the expressions of p53 and Mdm2 protein in villous samples of missed-abortion patients are significantly higher than in those from normal pregnancies. The result suggests that the lack of β-arrestin1 in villous samples may lead to increased apoptosis, and then subsequently activates P53-mediated apoptosis.
To further understand the relationship between β-arrestin1 and p53, Mdm2 in missed abortion, we silenced and up-regulated the expression of β-arrestin1 by lentiviral transfection. Through cell experiments, we found that knockdown of β-arrestin1 by siRNA transfection significantly suppressed p53 expression, stimulated Mdm2 activiation. Furthermore, overexpression of β-arrestin1 exhibited the adverse effect. These data suggested that β-arrestin1 was associated with cell apoptosis may be dependent on p53/Mdm2 interaction.
In the early stage of embryonic development, angiogenesis is the most basic condition for embryo implantation and endometrial development. Angiogenesis requires the interaction of a variety of factors, including various hormones, growth factors, and soluble extracellular matrix molecules. VEGF is the most important regulator of angiogenesis[34]. The formation of placental trophoblast vessels is strictly regulated by oxygen concentration [35]. HIF-1αcan regulate local oxygen pressure. HIF-lα-mediated transcriptional activation contributes to the increase of VEGF gene expression during hypoxia, but high levels of HIF-lα may inhibit the secretion of VEGF[36]. Excessive and prolonged hypoxia can induce apoptosis, the mechanism of HIF-1α inducing apoptosis is that p53 protein can bind to HIF-lα through oxygen degradation domain (ODD) during hypoxia, enhance the stability of p53. To study the correlation between VEGF and HIF-1α expression and missed abortion, we further detected the expression of VEGF and HIF-1α by immunohistochemistry. We found that HIF-1α expresssion in the missed abortion villous samples to be significantly higher than in control samples, but there was significantly lower VEGF in the samples from missed abortions. In this way, these findings corroborate those of previous reports showing that β-arrestin1 deficiency may form a negative effect in neovascularization.
Previous studies have revealed that β-arrestin1 bounds to mGluR1 to activate ERK and mediate the effect of TNFR1 on the NF-ΚB activity, and β-arrestin1 interacts with GSK3β to regulate the activity of p-Akt [37]. We therefore assessed ERK, Akt and NF-Κ expression and phosphorylation status in cells at differential expression level ofβ-arrestin1 in the HTR-8 cells. We found that down-regulation of β-arrestin1 cause high levels of p-NF-ΚB expression and a remarkable decrease in the level of p-ERK、p-AKT. Overexpression of β-arrestin1 showed the adverse effect. The results demonstrated that β-arrestin1 overexpression stimulats ERK, AKT signaling, but has a suppression effect on NF-ΚB signaling pathway.
In the first trimester of pregnancy, the placenta-derived villous cytotrophoblast cells proliferate or differentiate through two different ways to form the external layer of the multinucleated syncytiotrophoblast or the extremely invasive extravillous trophoblasts (EVTs)[38, 39]. Acquiring invasiveness is essential for EVT to constitute a connection between the uterine wall and placenta, which ensure enough blood flow to the growing fetus. Insufficient EVTs invasion of the spiral artery wall was identified to be closley related with many early pregnancy complications, such as missed abortion, intrauterine growth restriction or pre-eclampsia [40]. However, the molecular mechanisms of EVT differentiation and invasion are still poorly understood. Recently, β-arrestin1 has been reported to act as regulators of EMT and be involved in cell invasion, migration and tumor metastasis. Zeng etal found thatβ-arrestin1 facilitate the cell migration and invasion of prostate cancer cells by interacting withβ- catenin [37]. In this study, we explored the role of β-arrestin1 in HTR-8 cell invasion, the present data indicated that overexpression ofβ-arrestin1 promote cell invasion, silencing of β-arrestin1 significantly inhibited the invasive abiltiy. The results suggested that β-arrestin1 may serve a promotive role in cell invasion.
In conclusion, for the first time, we confirmed that there may be a significantly association between missed abortion and reduced expression of β-arrestin1. The β-arrestin1 depletion leads to increased apoptosis and decreased invaion ability. Furthermore, β-arrestin1 was found to be involved in the regulation of the Akt, NF-ΚB, ERK pathway. Exploring the exact mechanism will hopefully identify more new and effective strategies to diagnose and treat the patients with missed abortion.