Trophoblast invasion is crucial for the development of normal placentas.
During placental development, fetal cytotrophoblasts proliferate to form aggregates known as cell columns that anchor peripheral villi to the maternal decidua. These cytotrophoblasts, like stem cells of trophoblasts in cell column, just beneath the anchoring basal lamina, start to proliferate and differentiate into the extravillous cytotrophoblast and syncytiotrophoblast.
One of the primary placental defect in preeclampsia and partly intrauterine fetal growth restriction is shallow invasion of the extravillous trophoblast into the decidua, which leads to incomplete spiral artery remodeling and inadequate uteroplacental perfusion. The syncytiotrophoblast covers the chorionic villi and is bathed by maternal blood in the intervillous space. Its primary functions are to remove wastes, exchange oxygen and nutrients, and produce hormones. Its well controlled
differentiation and maintenance are also essential for successful pregnancy.
Mucins are highly glycosylated proteins expressed by epithelial cells of various organs. They are classified into two classes: secreted mucins and membrane-bound mucins. The secreted gel-forming mucins, which lack a transmembrane domain, include MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, and MUC19. The
membrane-bound mucins, which possess a single transmembrane domain and a highly conserved cytoplasmic tail, include MUC1, MUC3A, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, and MUC20. These membrane-bound mucins are postulated to serve as sensors of the external environment and can transmit signals into the cell.
Among the known mucins, MUC1 is best studied and plays crucial roles in regulating many cellular properties, including cell proliferation, apoptosis, adhesion, and invasion. Although MUC1 has been found to be expressed by trophoblasts of various species, its expression in the human placenta throughout pregnancy and its potential role in trophoblast invasion remain unclear.
MUC15 is a membrane-bound mucin which was originally isolated from bovine milk fat globule membranes. The mRNA of MUC15 has been detected by RT–PCR in various human tissues. The predicted protein product of human MUC15 (334 amino acids) contains an extracelluar domain, a small transmembrane domain, and a highly conserved cytoplasmic tail. In addition, a splice variant of MUC15 encoding a secreted mucin has been detected. So far, physiological functions of MUC15 have never been investigated.
In the present study, we therefore investigated MUC1 and MUC15 expression in the human placenta and the effect on the invasion of trophoblast cells.
Materials and Methods
Clinical tissue collection
The first trimester, second trimester, and third trimester placentas were obtained from the Department of Obstetrics and Gynecology, National Taiwan University Hospital.
The use of human placentas for this study was approved by the local hospital ethic committees and written consent was obtained from patients before the collection of samples. The placental tissue sections were obtained by dissecting a 1.5-cm square segment, 0.5-cm thick, from the maternal part of placentas.
Cell lines and cell culture
Human choriocarcinoma cell lines JEG-3 and JAR were purchased from BCRC
(Hsinchu,Taiwan).Cellsweremaintained with Dulbecco’smodified Eagle’smedium
containingm 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 mg/ml streptomycin in a humidified tissue culture incubator at 370C, in 5% CO2 atmosphere.
Northern blot
Multiple Tissue Northern (MTNTM) Blot was purchased from Clontech (BD Biosciences, CA, USA). The blot was probed with32P-labeled random-primed full-length MUC15 comlementary DNA (cDNA). Hybridization was performed at 650C overnight. The blot was washed at 550C for 1 h in a buffer containing
2xstandard saline citrate and 0.5% sodium dodecyl sulphate, and signals were
visualized by autoradiography using Kodak BioMax film.
RNA extraction and RT–PCR
Total RNA was extracted with Trizol reagent (InVitrogen, California, USA)
according to themanufacturer’sprotocol.In both conventionaland quantitativePCR,
we used a 2-ml aliquot of the cDNA and the following primer sets. PCR primers are indicated in Table 1. PCR products were run on a 2% agarose gel and visualized with ethidium bromide.
Matrigel invasion assays
Cell invasion assays were analyzed in a BD BioCoatTM MatrigelTM Invasion Chamber (BD Bioscienses, Massachusetts, USA) according to manufacturer’s protocol. The non-invading cells on the upper surface of the membrane were removed by scrubbing with a cottontipped swab, and the invaded cells on the lower surface of the membrane were fixed with 100% methanol and then stained with 0.5% crystal violet. After two washes with distilled water, the chamber was allowed to air dry, and the numbers of invaded cells per field were counted under a phase contrast
microscope. The means ±SD were calculated from six random fields per invasion chamber.
Overexpression of MUC1 or MUC15
A total of 5x 105JAR and JEG-3 cells were transiently transfected with 4μg of
pcDNA3.1/myc-His control plasmids (Mock) or MUC15/pcDNA3.1/myc-His (MUC15) or 2μg of control expression vector pHb-Apr1-neo (Mock) or
pHb-Apr1-neo containing full-length human MUC1 (MUC1) with Lipofectamine 2000. After 48 h of transfection, cells were harvested for experiments.
Quantitative Real-Time PCR
Quantitative PCR system Mx3000P (Stratagene) was used to analyze gene expression in human placentasaccording to themanufacturer’sprotocol.PCR reactionswere
incubated at 950C for 15 min, followed by 40 amplification cycles with 30-sec
denaturation at 95 C, 50-sec annealing at 54 C, and 30-sec extension at 72 C. Samples were analyzed in triplicate, and product purity was checked through dissociation curves at the end of real-time PCR cycles. PCR products were confirmed to be correct by DNA sequencing. Relative quantity of specific gene expression normalized to ACTB was analyzed with MxPro Software (Stratagene).
Western Blot
Human placental tissues or cells were homogenized in lysis buffer containing 1% (v/v) Triton X-100, 20 mM Tris-HCl (pH 8.0), 160 mM NaCl, 1 mM CaCl2, and 1 mM PMSF, followed by ultrasonication (three 5-sec bursts). Insoluble materials were removed by centrifugation. Forty micrograms of total proteins were separated on a 6% SDSPAGE and transferred to a Hybond enhanced chemiluminescence
nitrocellulose membrane. MUC1 protein was detected with mouse anti-MUC1 mAbs.
MUC15 proteins were detected with rabbit anti-MUC15 polyclonal antibodies. Bands were visualized by incubation with anti-mouse HRPconjugated secondary antibodies and enhanced chemiluminescence reagents. The signals were quantified and
normalized to ACTB signals by the use of ImageQuant 5.1 software.
Immunohistochemistry
Paraffin-embedded human placental sections were deparaffinized in xylene and rehydrated in a series of graded alcohol. After quenching the activity of endogenous peroxidase with 3% (v/v) H2O2 in PBS for 30 min, the sections were rinsed twice with PBS for 10 min and then blocked with 5% (w/v) nonfat milk/PBS for 1 h to reduce nonspecific bindings. Sections were incubated with primary antibodies (1:400 for anti-MUC1 mAb VU4H5; 1:100 for anti-MUC1 mAb M2C5; 1:100 for
anti-KRT7 mAb, anti-MUC15 polyclonal antibody (1:400)) diluted in 5% (w/v) nonfat milk/PBS for 16 h at 48C. Negative controls were performed by replacing primary antibodies with an isotype-matched control IgG at the same concentration.
After rinsing twice with PBS, Super Sensitive Link-Label IHC Detection System (BioGenex, California) was used, and the specific immunostaining was visualized with 3,3-diaminobenzidine liquid substrate system (Sigma, Missouri). All sections were counterstained with hematoxylin for 30 sec and mounted with UltraKitt .
SiRNA knockdown of MUC15 expression
Small interfering RNA (siRNA) oligonucleotides against MUC15 and control siRNA were synthesized by Dharmacon Research, Inc. (Illinois, USA) using their custom SMARTpool and non-targeting siRNA pool, respectively. For knockdown of MUC15, JAR and JEG-3 cells were transfected with siRNA using Lipofectamine 2000
(InVitrogen) with a final concentration of 100 nmol siRNA. The cells were incubated for 4 h and then serum-free DMEM medium was replaced with complete DMEM medium (10% FBS). After 48 h incubation, cells were harvested for analysis.
Gelatin Zymography
Conditioned media of mock- or MUC1 or MUC15-transfected cells cultured in serum free DMEM for 48 h were electrophoresed on an 8% SDS-PAGE copolymerized with 1% (w/v) gelatin. After electrophoresis, the gels were washed twice for 30 min in 2.5% (v/v) Triton X-100 and incubated for 16 h in developing buffer (50 mM Tris, 200 mM NaCl, 10 mM CaCl2, and 0.05% Brij35; pH 7.5). After incubation, gels were stained with Coomassie Brilliant Blue R-250 for 2 h and destained to reveal zones of gelatinase activity.
Statistical Analysis
Student t-test or Mann-Whitney U-test was used for statistical analyses. Data were presented as mean 6 SD. P , 0.05 was considered statistically significant
Results
MUC1 and MUC15 mRNA Expression in the Human Placenta
MUC1 mRNA expression in the third trimester placenta was significantly higher than that in the first (P< 0.01) and second(P < 0.05) trimester placenta. These results indicate that the MUC1 mRNA expression in the human placenta is increased during placental development. Similar results were seen for MUC15。
MUC1 and MUC15 Protein Expression in the Human Placenta
Two major bands (170 kDa and >250 kDa) of MUC1 protein were detected by clone VU4H5 of anti- MUC1 mAb in human placentas; these bands represent two isoforms of MUC1. In addition, our results showed that the third trimester placenta expressed significantly higher levels of MUC1 protein than did the first or second trimester placenta. Placentas at term expressed much higher levels of MUC15 than those in early pregnancy, in agreement with the results from real-time RT–PCR.
Immunohistochemistry of MUC1 and MUC15 in Human Placental Villi
MUC1 in human placental villi is mainly expressed by syncytiotrophoblasts
throughout pregnancy and is increased during placental development. The expression of MUC15 protein in syncytiotrophoblasts is increased during placental development.
MUC1-Positive and MUC1-Negative Extravillous Trophoblasts Are Present in
Human Decidua
Extravillous trophoblasts were present in decidua throughout pregnancy. Interestingly, numbers of MUC1-positive stained extravillous trophoblasts in decidua were
increased during placental development.
MUC1 and MUC15 overexpression inhibits invasion of JAR and JEG-3 cells
MUC1-high expressers indeed exhibit less invasive ability than do MUC1-low expressers in JAR cells. Overexpression of MUC15 substantially decreased invasion of JAR and JEG-3 cells by 87.5±1.1 and 83.8±5.7%, respectively.
MMP9 activity was suppressed by MUC1 overexpression. However, MMP2 activity did not change.
MUC15 overexpression increases TIMP-1 and TIMP-2 expression,
MUC15 overexpression increased TIMP-1 and TIMP-2 mRNA expression in JAR cells by an average of 4.1- and 3.1-fold, respectively. In addition, TIMP-1 and TIMP-2 mRNA expression in JEG cells were increased by an average of 4.7- and 3.7-fold, respectively.
Discussion
The data show that MUC1 mRNA and MUC1 protein increase with gestational stage of human pregnancy. MUC1 protein is mainly expressed by
syncytiotrophoblasts in placental villi throughout pregnancy. In addition, we demonstrate that two populations of extravillous trophoblasts, MUC1-postive and MUC1-negative cells, are present in human decidua, and the numbers of
MUC1-positive extravillous trophoblasts are increased during human placental development. This study also demonstrates that MUC15 is differentially expressed in human placentas throughout gestation. Both MUC15 mRNA and protein are
maximally expressed in term placentas. Results of immunohistochemistry further reveal that trophoblasts of placentas in early pregnancy express lower levels of MUC15 than those at term.
Interestingly, MUC1 overexpression suppressesinvasion of trophoblast-like JAR cells in vitro, which is associated with inhibition of MMP9 activity. Our results suggest that MUC1 is differentially expressed in the placenta and decidua during human placental development and may regulate extravillous trophoblast invasion in vitro. Furthermore, MUC15 overexpression significantly suppresses invasion of trophoblast-like JAR and JEG-3 cells, suggesting that MUC15 could also act as a negative regulator of trophoblast invasion in vivo.
To date, mucins have been reported to participate in various physiological functions, such as immune reactions, and cell adhesion, migration, and invasion. Here, we demonstrate that overexpression of MUC1 and MUC15 significantly suppresses invasion of both trophoblast-like JAR and/or JEG-3 cells. Among members of MMP family, MMP-2 and MMP-9 were suggested to regulate trophoblast invasion. MUC1 overexpression suppresses invasion of trophoblast-like JAR cells in vitro, which is associated with inhibition of MMP9 activity. We found that MUC15 overexpression can suppress invasion of both JAR and JEG-3 cells. However, MMP-2 and MMP-9 activity were not significantly affected by MUC15 overexpression. We found that MUC15 overexpression significantly increased TIMP-1 and TIMP-2 expression in both JAR and JEG-3 cells.
In the present study, we observed that two populations of extravillous
trophoblasts, MUC1-positive and MUC1-negative cells, in decidua and the numbers of MUC1-positive extravillous trophoblasts are increased during human placental development. In addition, we demonstrate that MUC1 overexpression suppresses invasion of trophoblast-like JAR cells. These results suggest that MUC1 could be a negative regulator of trophoblast invasion and that MUC1-positive extravillous trophoblasts in decidua may exhibit less invasive ability than MUC1-negative extravillous trophoblasts in vivo. It is therefore reasonable to speculate that MUC1
could act as a restraint or brake in trophoblast invasion and could control the invasion of trophoblasts to an appropriate depth in the endometrium, which is essential for normal placental development.
MUC1 has been found to be overexpressed by various typesof tumors. Many studies support that MUC1 overexpression enhances tumor cell invasion and
promotes tumor malignancy. In sharp contrast, we found that MUC1 overexpression suppresses invasion of trophoblast-like JAR cells in vitro. Interestingly, MUC15 can also suppress invasion of the trophoblast-like cells JAR and JEG-3. These results imply that the role of mucins in trophoblast-lineage cells in terms of invasion is different from that in other types of epithelia. The difference in MUC1 function in regulating cell invasiveness between trophoblast-like and other types of tumor cells may be a result of differential expression of certain cellular factors in trophoblast-like cells.
Our data show that MUC1 is highly expressed by syncytiotrophoblasts in the term placenta. Muc1-null mice (Muc1tm1Gend) exhibit chronic infection and
inflammation of the uterus as a result of increased infection by normal bacteria of the reproductive tract. In addition, MUC1 has been shown to protect cancer cells from immune cell attack. Therefore, we speculate that MUC1 expression on the surface of syncytiotrophoblasts could protect the human placenta from microbial and immune
cell attack. Furthermore, it has been found that MUC1 can interact directly with estrogen receptor alpha (ESR1) and epidermal growth factor receptor (EGFR) and regulate their signalings. It has been shown that estrogen, via interaction with the estrogen receptor, regulates functional differentiation of the syncytiotrophoblast in the primate placenta. EGF, through interaction with EGFR, is able to regulate trophoblast apoptosis and Nat/Ht exchanger activity in the syncytiotrophoblast. Moreover, ESR1 and EGFR have been reported to be expressed by the human syncytiotrophoblast.
Therefore, it is interesting to investigate whether MUC1 can regulate the differentiation or biological functions of the syncytiotrophoblast through the regulation of ESR1 and EGFR signalings.
MUC15 protein is highly conserved in its cytoplasmic domain (74 amino acids), which shares 81.1% identity and 94.6% similarity between human and mouse amino acid sequences. The cytoplasmic tail contains four conserved tyrosine and seven serine/threonine phosphorylation sites. We therefore speculate that the potential phosphorylation sites in MUC15 could regulate the interactions of its cytoplasmic tail with proteins involved in signal transduction and thereby modulate trophoblast invasion.
In conclusion, the present studies report the spatial and temporal expression of MUC1 and MUC15 in the human placenta throughout pregnancy. MUC1 expression
in syncytiotrophoblasts and the numbers of MUC1-positive extravillous trophoblasts increase with gestational age during human placental development. In addition, MUC1 overexpression suppresses MMP9 activity and decreases invasion of
trophoblast-like JAR cells. Based on these findings, we suggest that MUC1 could play a critical role in trophoblast invasion and may contribute to human placental
development. MUC15 overexpression suppresses trophoblast-like cell invasion, which is associated with increased TIMP-1 and TIMP-2 expression.
For further investigation, it will be of great interest to study the roles of MUC1, MUC15 and other mucins in pregnancy disorders, such as pre-eclampsia and
intrauterine growth restriction, which are related to dysregulation of trophoblast invasion.
第七章 參考文獻