Background
Trophoblast cell invasion is essential for successful implantation and placentation. During the first trimester of gestation, trophoblast cells proliferate, invade the maternal decidua, and differentiate to form chorionic villi, composed of an inner layer of cytotrophoblasts (CTBs) and an outer layer of syncytiotrophoblasts [
1,
2]. A subpopulation of the proliferating CTBs subsequently streams out of the syncytiotrophoblasts to form mononuclear invasive extravillous cytotrophoblasts (EVTs). These EVTs invade the maternal tissues and penetrate the uterine arterioles, thereby ensuring a continuous blood supply to placenta and the developing fetus [
1,
2]. These processes require the action of metalloproteinases (MMPs), a family of zinc-dependent proteolytic enzymes that are the main mediators of extracellular matrix (ECM) degradation [
3].
MMP-26, also known as endometase or matrilysin-2, was recently identified as the smallest member of MMP family [
4‐
6]. It is widely expressed in villous CTBs, syncytiotrophoblasts and EVTs of human placenta [
7]. MMP-26 exhibits wide substrate specificity in cleaving ECM and basement membrane proteins including type IV collagen, fibronectin, fibrinogen, vitronectin and gelatin [
6,
8]. The fact that most of these ECM components are expressed in human villous trophoblasts [
9] suggests the involvement of MMP-26 in the degradation and remodelling of ECM at the feto-maternal interface. In addition, MMP-26 is able to process the lacent MMP-9, to generate its active form [
6,
10]. Our previous studies revealed that the spatiotemporal expression of MMP-26 was similar to that of MMP-9 in trophoblasts during the first trimester [
7,
11], implicating that MMP-26 may also indirectly contribute to ECM degradation through activation of proMMP-9 at the feto-maternal interface. Recently, we found that overexpression of MMP-26 increases invasive capacity of human trophoblast cells
in vitro[
12], suggesting the important role of MMP-26 in trophoblast invasion. However, little is known about the regulatory mechanism of MMP-26 expression in human trophoblasts.
An increasing body of evidence has shown the extrapituitary functions of gonadotropin-releasing hormone I (GnRH I) and the second mammalian form of this hormone (GnRH II). In human placenta, GnRH I is widely expressed among distinct subpopulations of trophoblasts throughout gestation [
13,
14], while GnRH II expression is restricted to mononuclear villous CTBs and EVTs during the first trimester [
13]. GnRH receptor (GnRHR) is highly expressed in both CTBs and syncytiotrophoblasts during early gestation [
15]. Recently, GnRH I and GnRH II have also been shown to regulate the expression of MMP-2, MMP-9/tissue inhibitor of metalloproteinases 1 (TIMP-1), and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) in human EVT cultures [
16,
17]. These observations suggest the paracrine and/or autocrine regulatory roles of these two hormones in modulating the activities of various protease systems at the feto-maternal interface.
Based on previous findings from our laboratory and others, we hypothesized that GnRH I and GnRH II induce MMP-26 expression in trophoblast cells. In the present study, we investigated the regulatory mechanism of MMP-26 expression by GnRH I and GnRH II using an
in vitro experimental model of an immortalized human cytotrophoblast-like cell line, B6Tert-1, which has been established in our laboratory [
18].
Methods
Materials
GnRH I and GnRH II native peptides were obtained from Peninsula Laboratories, Inc. (San Carlos, CA). ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). PD98059 and SP600125 were dissolved in DMSO. The antibodies specific against β-actin, phospho-ERK1/2, phospho-JNK, total-ERK1/2 and total-JNK were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The antibodies specific against GnRH receptor were obtained from Lab Vision Corp. (Fremont, CA). The polyclonal antibodies against MMP-26 [
5] are kind gifts from Dr. Qing-Xiang A. Sang of Florida State University, Florida, USA.
Cell culture and treatment
Immortalized human cytotrophoblast-like B6Tert-1 cells were cultured as described previously [
18]. In brief, the cells were cultured in collagen I (Cellmatrix Type I-A; Institute of Biochemistry, Osaka, Japan)-coated flasks with a defined serum-free medium [DMEM/F12 medium (Gibco-Invitrogen, San Diego, CA) supplemented with 10 ng/ml epidermal growth factor (EGF; Collaborative Research, Lexington, MA) and 10 mg/ml insulin (Sigma)]. Cells were incubated at 37°C in an atmosphere of 5% CO
2. To synchronize the cells, at least 18 hours before each hormone treatment, concentration of EGF in the culture medium was reduced from 10 ng/ml to 1 ng/ml and the concentration of insulin was decreased from 10 mg/ml to 1 mg/ml. When reagents were dissolved in DMSO, the same concentration of DMSO was added to medium for the control cells. After pretreatment with inhibitors or vehicle [0.1% (vol/vol) DMSO] for 30 minutes, the cells were treated with GnRH I or GnRH II at concentration of 100 nM for a further 24 hours. Cells were collected at various time points following treatment with GnRH I or GnRH II.
Cells from the human choriocarcinoma cell line, JEG-3, were purchased from American Type Culture Collection (ATCC, Rockville, MD). After thawing, the cells were maintained in DMEM medium (Gibco-Invitrogen) supplemented with antibiotics and 10% (vol/vol) heated-inactivated fetal bovine serum (FBS; Gibco-Invitrogen).
Isolation of human primary cytotrophoblast (CTB) cells
Human chorionic villi tissues were obtained from patients who underwent therapeutic termination of pregnancy at 6~7 weeks of gestation. Informed consent was provided by the patients, and the project received prior approval from the local ethics committee. The time of gestation was defined according to the first day of the last menstrual period, and further morphological examination by means of stereomicroscope. Primary CTB cells were isolated from chorionic villi tissues as previously described [
19]. Briefly, tissues were minced separately and digested with 0.25% (vol/vol) trypsin (Sigma) and DNase I (Sigma). Next, the dispersed cells were washed by DMEM/F12 medium (Gibco-Invitrogen) and then filtered through a nylon sieve to remove the gross villous core residues. The filtered cell suspension was then slowly added to the top of a BSA gradient [prepared by sequential addition of 3%, 2%, and 1% (wt/vol) BSA in DMEM/F12 medium]. The cells were sedimented for 1 h at unit gravity, and cytotrophoblast cells were collected from the bottom of the tube. The purified CTB cells were cultured in defined serum free medium and treated with GnRH I and GnRH II in the absence or presence of inhibitors for 24 h.
RNA preparation and synthesis of first-strand cDNA
Total RNA was extracted from the B6Tert-1 or CTB cells with TRIzol reagent (Invitrogen, San Diego, CA), according to the manufacturer's instructions. RNA was subjected to DNase I digestion to avoid possible genomic DNA contamination and then reverse transcribed with oligo-dT primers and Superscript II reverse transcriptase (Invitrogen).
Real-time PCR
The first-strand cDNA generated from the B6Tert-1 or CTB cells served as a template for Real-time PCR using the ABI PRISM 7000 sequence detection system (PerkinElmer Applied Biosystems, Foster City, CA) equipped with a 96-well optical reaction plate. The primers used for SYBR Green Real-time PCR were designed using the PRIMER3 software and were as follows: human MMP-26, 5'-TCC AGC AAG TGC AGA ATG GA-3'(forward) and 5'-GGG CCC ACT GCC AGA AA-3' (reverse); human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ATG GAA ATC CCA TCA CCA TCT T-3' (forward) and 5'-CGC CCC ACT TGA TTT TGG-3' (reverse). The primer specificity for MMP-26 and GAPDH was confirmed by running the PCR products on a 2.0% agarose gel. Each Real-time PCR reaction contained 12.5 μl SYBR Green ER qPCR SuperMix (Invirogen), 2.5 μl of primer mixture (400 nm), and 10 μl of cDNA template [10% (vol/vol) RT reaction product] under the following optimized conditions: 50°C for 2 min followed by 95°C for 10 min and 40 cycles of 95°C for 15 sec and 55°C for 1 min. All PCR reactions were performed in triplicate, with the mean being used to determine mRNA levels. Relative mRNA expression levels for MMP-26 were determined using the 2
-ΔΔCT method [
20] and normalized to the endogenous reference gene, GAPDH.
Western Blotting analysis
Total protein was isolated from B6Tert-1, JEG-3 or CTB cells using lysis buffer (Cell Signaling). The protein extracted from αT3-1 cells was kindly provided by Dr. Peter C.K. Leung in University of British Columbia, Canada. Total protein (30 μg) was run on 10% SDS-polyacrylamide gels. After electrotransferring the protein to the nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ), the membrane was immunoblotted using specific primary antibodies for MMP-26, GnRHR, phospho-ERK1/2 or phospho-JNK at 4°C overnight. The signals were detected with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour and visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). After stripping, the membranes were reprobed with β-actin, total-ERK1/2 and total-JNK antibodies, respectively. The relative density of MMP-26 was determined by normalization to the density value of β-actin. The relative densities of phosphorylated forms of ERK1/2 and JNK were normalized to total values of ERK1/2 and JNK. All densities were analyzed using the Gel-Pro Analyzer (software version 4.0; United Bio., Marlton, NJ).
Transwell insert invasion assay
In vitro cell invasion was assayed by determining the ability of cells to invade a synthetic basement membrane. Briefly, 24-well fitted transwell inserts with membranes (8- μm pore size, Millipore Corp, Bedford, MA) were coated with growth factor reduced Matrigel (BD Biosciences, Franklin, NJ) at a concentration of 200 μg/mL and placed in a 24-well plate. B6Tert-1 cells at a concentration of 2 × 104 were seeded in each insert containing defined medium supplemented with 1 ng/ml EGF and 1 mg/ml insulin and lower chambers were loaded with defined medium containing 10 ng/ml EGF and 10 mg/ml insulin. After incubating with or without GnRH I or GnRH II (100 nM) for 24 h, the cells were then fixed and stained with crystal violet. Non-invaded cells on the upper surface of the membrane were removed using a cotton swab. The membranes were cut from inserts and mounted onto glass slides. The number of stained cells was counted, in at least 15 randomly selected non-overlapping fields of the membranes, using a light microscope.
Statistical analysis
Data were shown as the mean ± SEM of three individual experiments performed in duplicate or triplicate. Statistical analysis was carried out using one-way ANOVA followed by Dunnett's test, and differences were considered significant for P < 0.05. Representative images of Western blot are shown.
Discussion
MMP-26 is a recently discovered and only partially characterized human proteinase [
4‐
6]. Unlike all other MMPs, the
Mmp-26 gene does not exist in the murine genome [
4‐
6]. Besides its extensive distribution in cancer cells of epithelial origin [
8], MMP-26 is also restricted in human placenta and uterus, but nor in other normal tissues [
4,
6]. Previous studies showed its intensive expression in human trophoblasts
in vivo and invasive-promoting effect on trophoblast cells
in vitro[
7,
12], suggesting the unique role of MMP-26 in trophoblast invasion at the feto-maternal interface. However, the regulation of MMP-26 expression in trophoblast cells remains to be determined. In this study, we utilized human trophoblast-like cell line, B6Tert-1, to show that 1) both GnRH I and GnRH II could up-regulate MMP-26 expression, 2) GnRH I and GnRH II had differential effects on MMP-26 expression, and 3) GnRH I and II-induced MMP-26 expression was mediated by JNK, but not ERK1/2 signaling pathway.
The B6Tert-1 cell line is an immortalized cytotrophoblast-like cell line developed from normal human placental villi during the first trimester. These cells are transfected with human telomerase catalytic subunit gene (
htert) and thus maintain reconstituted telomerase activity [
18]. B6Tert-1 cells exhibit the characteristics of normal EVTs by producing various biomarkers including CK8, HLA-G, integrin α1 and integrin β1 [
18]. They also exhibit the ability to invade matrix and express proteinase genes including MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2 and TIMP-3, which are important properties of EVTs [
18]. In this study, we further demonstrated that the expression of MMP-26 in B6Tert-1 cells was comparable to that in the primary CTB cells at early gestation. The invasiveness of B6Tert-1 cells was regulated by paracrine and/or autocrine factors such as EGF and transforming growth factor β [
18]. Here we showed that the invasive ability of B6Tert-1 cells was increased by GnRH I and GnRH II. This observation is also consistent with our previous report that GnRH I and GnRH II stimulated the invasiveness of primary EVT cells [
23]. Our data and other groups' studies strongly suggest that the B6Tert-1 cell line is a valuable
in vitro cellular model for the investigation of trophoblast-like/trophoblast cell behaviors [
25,
26]. In the present study, we utilized B6Tert-1 cells to determine the regulatory mechanism of GnRHs on MMP-26 expression in human trophoblast-like/trophoblast cells.
The classical mammalian GnRH (GnRH I) is a decapeptide that is well-known for its role in regulating the release of gonadotropins from the pituitary [
27]. Evidence shows that in addition to its classical endocrine functions, GnRH I has direct regulatory actions on the development and function of gonads and other reproductive tissues such as the ovary, endometrium and placenta [
16,
17,
27‐
29]. A distinct gene encoding a second form of GnRH, termed GnRH II, that is expressed in human extrapituitary tissues [
30], has been shown to imitate the paracrine/autocrine function of GnRH I in extrapituitary compartments [
16,
17,
27‐
29]. Recent studies demonstrated that GnRH I and GnRH II promoted the invasive capacity of human trophoblasts by regulating MMP-2, MMP-9/TIMP-1 and uPA/PAI protease systems [
16,
17,
23]. Consistent with these findings, our present work indicated that both GnRH I and GnRH II were capable of increasing the mRNA and protein levels of MMP-26 in trophoblast-like/trophoblast cells. In addition to its direct proteolytic action on ECM substrates, MMP-26 has also been reported to be an activator of proMMP-9 in prostate cancer cells and correlated to MMP-9 activity in esophageal carcinoma [
10,
31]. Considering the similar expression patterns between MMP-26 and MMP-9 in trophoblasts during early gestation [
7,
11], GnRH-induced MMP-26 production may, at least in part, participate in proteolysis of the ECM through activating the latent form of MMP-9. The coordinate induction of MMP-2, MMP-9 and MMP-26 by GnRHs suggests that these enzymes possibly elicit their functions as components of a proteolytic cascade in trophoblasts.
Although GnRH II is capable of mimicking the biological actions of GnRH I in extrapituitary tissues, evidence shows differential effects of these two hormones. For instance, GnRH II exhibited more potent anti-proliferative effects than an equimolar dose of GnRH I in human endometrial and ovarian cancer cells [
32]. In human placenta, GnRH I was more effective than GnRH II on hCG synthesis and secretion [
33], while GnRH II appeared to be more potent than GnRH I in stimulating leptin secretion [
34]. Recently, Chou et al. demonstrated that GnRH II was capable of eliciting its regulatory effects on MMP/TIMP systems at lower hormone concentrations than GnRH I in human EVTs [
17]. In this study, our data showed that the time course of the stimulatory effect of GnRH II on MMP-26 expression was earlier than that of GnRH I. In addition, GnRH I and GnRH II had different dosage effects on MMP-26 expression in B6tert-1 cells. These differential actions of GnRH I and GnRH II in regulating MMP-26 expression might be explained by different degradation pathways [
35], different signaling cascades [
23], different receptor affinities [
36] or even different types of GnRH receptor [
17,
23].
It has been well established in gonadotropic cells that the binding of GnRH with its receptor activates MAPK cascades, leading to phosphorylation of ERK1/2 and JNK [
37]. This process is an important link for the transmission of GnRH signals from the cell surface to the nucleus [
37]. MAPK also has been shown to mediate the action of GnRH in human trophoblast cells [
24]. In the present study, we demonstrated the rapid and transient activation of JNK by GnRH I and GnRH II in B6Tert-1 cells. GnRH I and II-induced MMP-26 expression was blocked by specific inhibition of JNK, but not ERK1/2 in B6Tert-1 and primary CTB cells. JNK signaling has been shown to be involved in the production of various MMPs that promote invasiveness and migration of cancer cells [
38‐
40]. A recent study showed that JNK, but neither ERK1/2 nor p38 MAPK, was critical for GnRH-mediated production of MMP-2 and MMP-9 in human ovarian cancer cells [
41]. In agreement with these observations, our data suggests that JNK, but not ERK1/2, may play a central role in mediating the effects of GnRHs on MMP-26 production in human trophoblast-like/trophoblast cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JL and YLW designed the study. JL and BC performed the research. YXL isolated human primary cytotrophoblast cells. XQW performed the Western blotting analyses of Figure
1C. JL and YLW interpreted the results and drafted the manuscript. All authors read and approved the final manuscript.