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BOR - Papers in Press, published online ahead of print May 25, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040436
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BIOLOGY OF REPRODUCTION 73, 619–626 (2005)
DOI: 10.1095/biolreprod.105.040436
© 2005 by the Society for the Study of Reproduction, Inc.

Adrenomedullin Enhances Invasion by Trophoblast Cell Lines1

Xiaoquan Zhang , Kortney E. Green , Chandra Yallampalli , and Yuan Lin Dong 2 

Department of Obstetrics and Gynecology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have tested the hypothesis that adrenomedullin (ADM), a multifunctional peptide hormone, works as a trophoblast proinvasion factor. Our results showed that ADM receptor components—the mRNA and proteins of calcitonin receptor-like receptor (CALCRL) and receptor activity modifying proteins (RAMPs)—were expressed by human choriocarcinoma JAr cells and first-trimester cytotrophoblast HTR-8/SV neo cells. ADM stimulates both JAr and HTR-8/SV neo cell proliferation. The invasion capabilities of JAr cells and HTR-8/SV neo cells were also enhanced by ADM, and this was associated with increased gelatinolytic activity and reduced plasminogen activator inhibitor-1 mRNA expression (SERPINE1). Our data support the notion that ADM may be involved in the human implantation process via regulating trophoblast proliferation and differentiation.

adrenomedullin, cell proliferation, growth factors, implantation, placenta, pregnancy, trophoblast, trophoblast invasion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The placenta is a unique, temporary organ developed solely for the fetus. A fetus survives with a wide diameter, low-resistance vascular system for adequate blood flow. The cytotrophoblast cells play a major role in establishing maternal-fetal circulation for the development of the fetus. They do this by invading and reconstructing blood vessels during placental formation. The cytotrophoblast cells are derived from the trophectoderm, the outermost epithelial cell layer of the blastocyst, and behave like tumor cells as they invade endometrial tissue. Cytotrophoblast stem cells in the human placenta differentiate into two pathways. On one pathway, the cytotrophoblasts migrate away from the endometrium to form the amorphous, multinucleate syncytiotrophoblast [1], which covers the floating chorionic villi. The syncytiotrophoblast is in direct contact with maternal blood and functions as a metabolic organ. On another pathway, the cytotrophoblasts penetrate the basement membrane and form fingerlike, multilayered columns that adhere to the uterus, invade the decidua, the inner third myometrium, and finally the uterine spiral arteries [2]. Cytotrophoblast cell invasion peaks at an early stage of pregnancy and then declines as gestation progresses [3].

Cytotrophoblast cell migration is impeded by the extracellular matrix. The cells must degrade these matrix components before intruding into other tissues. This invasive behavior is mediated primarily by matrix metalloproteinases (MMPs). These are Zn+2-dependent proteolytic enzymes that specifically degrade protein components of the extracellular matrix [4]. The MMP superfamily is categorized by their substrates and structures. They are collagenases, gelatinases, elastinases, stromelysins, and membrane-type metalloproteinases [5]. The gelatinase group has two enzymes, gelatinase A (MMP2, 72 kDa) and gelatinase B (MMP9, 92 kDa); both of which have specificities for collagen IV (a component of the vascular base membrane) and affect trophoblast invasion [6, 7]. Inactive proenzyme MMPs are secreted into the extracellular matrix by the cytotrophoblasts. The proenzyme then acquires its full proteolytic activity by the removal of a fragment from the N-terminus [8]. Plasmin cleavage is required for activation of MMPs. There are two types of plasminogen activators, urokinase-type plasminogen activators (PLAUs) [9] and tissue-type plasminogen activators (PLATs) [10]. PLAU is secreted as an inactive form (proenzyme), whereas PLAT is secreted ready for binding to fibrin. Plasminogen is the preferred substrate for both PLAT and PLAU, and is present in plasma and extracellular fluid associated with fibrin or other proteins via the lysine-binding site [11]. The plasminogen activator cleavage of an Arg-Val bond yields the active form of plasmin that consists of two polypeptide chains linked by a disulfide bond. Plasmin belongs to the serine protease family but has a broad spectrum of substrates, including fibrin, matrix protein, and metalloprotease precursors to achieve extracellular matrix degradation [12]. Physiological inhibitors of cytotrophoblast invasion have been identified, such as the plasminogen activator inhibitors 1 and 2 (SERPINE1 and SERPINE2) [12, 13]. This system is at work not only during homeostasis, but it is also involved in tumor cell metastasis, migration, and tissue remodeling [14–16]. It was recently reported that during mouse implantation, the decidua protects the uterus from inappropriate invasion of the embryo through a physical barrier, as well as by the production of inhibitors of proteolytic enzymes such as the tissue inhibitors of metalloproteinases (TIMPs) and SERPINE1 [17]. Further, treatment of plasminogen-deficient mice with the broad spectrum MMP inhibitor galardin leads to a high rate of embryonic lethality [18], indicating that the combination of MMPs and plasminogen is essential for the proper development of the fetus.

The upstream cytokine actions on cytotrophoblast cells and the patterns of actions in early gestation are ambiguous. Cytotrophoblast cells respond to many growth factors [19– 23]. Among them, epidermal growth factor significantly increases cytotrophoblast invasion [17, 19], whereas tumor necrosis factor and TGFB1 have negative effects [24, 25]. In addition, calcitonin generated by the pregnant uterus binds to a G-protein-coupled receptor on mouse blastocysts, and stimulates primary trophoblast differentiation through intracellular cAMP and Ca2+ signaling [26]. Adrenomedullin (ADM) is a polypeptide that was first identified in pheochromocytoma [27]. It belongs to the calcitonin gene-related peptide (CGRP) superfamily [28]. ADM is expressed in several vital organs [29, 30] and shows multiple functions [31–39]. ADM is also expressed by human placental trophoblast tissues throughout pregnancy. The expression of ADM in cytotrophoblasts is most abundant in first-trimester placenta and becomes least abundant during the course of pregnancy [40]. Elevated ADM levels in maternal plasma appear to be derived partly via placental tissue production [41]. The existence of both ADM mRNA and ADM binding sites in the trophoblast suggest that ADM may play a role as an autocrine and paracrine factor in the regulation of trophoblast differentiation [40]. The present study was designed to determine the expression of ADM receptor components, calcitonin receptor-like receptor (CALCRL), and receptor activity-modifying protein 2 (RAMP2) in a choriocarcinoma cell line (JAr cells) and first-trimester cytotrophoblast HTR-8/SV neo cells, and to examine the effects of ADM on trophoblast proliferation and invasion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

Human choriocarcinoma JAr cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM Hepes, 1.0 mM sodium pyruvate (RPMI 1640 plus) and 10% fetal bovine serum (FBS). The first-trimester cytotrophoblast HTR-8/SV neo cells (a gift from Dr. C.H. Graham, Queen University, Kingston, ON, Canada) were cultured in RPMI 1640 containing 5% FBS. Cells were starved in serum-free media for 2 h before being treated with varying doses of ADM (10–9, 10–8, or 10–7 M) in the presence of 1.5% FBS for HTR-8/SV neo cells or 4% FBS for JAr cells. Cells were harvested at 24, 48, and 72 h after treatment. In some experiments, ADM antagonist (ADM22–52,10–7 M) was added 30 min before ADM (10–8 M) treatment to determine the involvement of ADM receptors. ADM and ADM22–52 were purchased from Sigma-Aldrich Co. (St. Louis, MO).

Reverse Transcription-Polymerase Chain Reaction

Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA). The cDNA was synthesized by reverse transcription (RT). All procedures followed those the protocols described by Promega Corporation (Madison, WI). Polymerase chain reaction (PCR) was initiated by the specific primer sets shown in Table 1. PCR reactions followed the conditions described in Table 2, and the cycles were determined by the optimal amount of PCR products. All other reagents were obtained from Fisher Scientific (Pittsburgh, PA). PCR products were subjected to gel electrophoresis. The bands were densitometrically scanned and analyzed using a Fluorchem Analysis System (Sigma Gel Software; Sigma). The relative densities were measured and normalized to that of 18S. The identity of the amplified sequences was verified by sequencing the gel-extracted PCR products.


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  		TABLE 1. Sequence of oligonucleotides synthesized for PCR detection of mRNA in trophoblast


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  		TABLE 2. PCR condition and cycle numbers

Immunofluorescence

Cells were cultured in Lab-Tek chambers (Nalge Nunc International, Naperville, IL) for 24 h. Slides were fixed with 70% acetone for 10 min. PBS containing 3% goat serum and 0.1% Triton X-100 was then applied for 30 min at room temperature before incubation with avidin-biotin blocking buffer for 10 min. The primary polyclonal CALCRL and RAMP2 antibodies were produced and well-characterized by this laboratory [42, 43] in 1% goat serum and then added to the slides and incubated overnight at 4°C. The specificity of the affinity-purified antisera was checked by blocking the antibody with the corresponding antigen and using the blocked antibody for Western blotting, in which total blockage of the respective band was observed, and linearity of the antibodies was examined by loading increasing concentrations of the antigen. Western blot analysis showed a linear increase in the intensity of the bands [42, 43]. Fluorescein-conjugated secondary antibody (Alexa Fluor 594; Molecular Probes, Eugene, OR) was then added and incubated for 4 h at room temperature. Cell morphology was then observed and photographed (Olympus Optical Co. Ltd., Tokyo, Japan).

Cell Proliferation Assay

JAr cells (6 x 103 cells per well) in RPMI 1640 with 10% serum and HTR-8/SV neo cells (1.5 x 103 cells per well) in RPMI 1640 with 5% serum were placed in 96-well plates for 24 h. Cells were treated with varying doses of ADM (10–9, 10–8, and 10–7 M) in 4% (JAr) or 1.5% (HTR-8/SV neo) serum after 2 h of serum starvation. Ten microliters of methylthiazoltetrazolium (MTT, 5 mg/ml) per well was added at 24, 48, and 72 h after ADM treatment. Experiments were stopped 4 h later by application of dimethylsulfoxide (DMSO), which dissolved the formazan crystal. The absorbance at 550 nm was then measured by using a microplate reader (SOFTmaxPRO; Molecular Devices Corp., Sunnyvale, CA).

Matrigel-Invasion Assay

Invasion was measured using 24-well Matrigel-invasion chamber plates (35-4480; Becton Dickinson Labware, Bedford, MA) according to published methods [44]. Cells (2.5 x 104 cells/well) in 500 µl of 0.5% BSA were seeded in the upper compartment of the Matrigel-coated insert in the presence of ADM (10–8 M) with or without ADM22–52 (10–7 M). RPMI 1640 (750 µl) with 5% serum was added to the lower chambers to induce chemotaxis. After 48 h of incubation, noninvasive cells on the upper surface of the insert were wiped away with a cotton applicator. Invasive cells that penetrated to the lower surface of the insert were cultured with MTT for 4 h. MTT-formazan crystals were resolved by adding 200 µl of DMSO. The lysates were then transferred to 96-well plates. The optical absorbance at 550 nm was measured in a microplate reader. For morphological study, Matrigel-coated insert membranes were stained with Diff-Quick stain (Biochemical Science, Inc., Swedesboro, NJ) and viewed under a microscope. The percentage of invasion and invasive index were calculated as follows [44]:

Gelatin Zymography

The culture media were harvested at 48 h and standardized against the amount of protein in cell lysates [45]. Ten to twenty microliters of medium equivalent to 6 µg of cell lysate protein was mixed with sample buffer and loaded onto a 15% SDS-PAGE gel (polymerized with 1 mg/ml gel atin) without boiling. The electrophoretic gels were washed with 2.5% Triton X-100 and 50 mM Tris-HCl (pH 7.5) to remove SDS and then incubated at 37°C in reaction buffer (150 mM NaCl, 5 mM CaCl2, and 50 mM Tris-HCl at pH 7.5) for 2–3 days. Thereafter, gels were stained with 0.1% Coomassie Brilliant blue R-250 for 30–60 min and destained in 10% methanol with 5% glacial acetic acid. The gelatinolytic activity could be seen as a clear band on a uniform blue background.

Data Analysis

Data are presented as mean ± SEM and are followed by the Bonferroni t-test. Unless specified, we used six replicates per group for the invasion assay and three replicates for RT-PCR as determined by the power analysis for each parameter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Messenger RNA Expression of CALCRL and RAMP2 by Jar and HTR-8/SV neo Cell Lines

To find the molecular target of ADM on JAr cells and HTR-8/SV neo cells, the existence of the ADM receptor complex was verified by mRNA analysis using RT-PCR. Figure 1 shows the abundant expression of ADM receptor components CALCRL and RAMP2 mRNA in JAr and HTR-8/SV neo cells, and human placental villous tissues. We also detected ADM mRNA in JAr and HTR-8/SV neo cell lines (data not shown). These results indicated the existence of both ADM and its receptors on trophoblast cells.



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  		FIG. 1. RT-PCR analysis of mRNA expression for ADM receptor components CALCRL (496 base pairs) and RAMP2 (282 base pairs) in JAr cell, HTR-8/SV neo cells, and human placental villous tissue

Immunofluorescent Identification of CALCRL and RAMP2 in JAr and HTR-8/SV neo Cell Lines

CALCRL and RAMP2 proteins were located in the trophoblast by immunofluorescence. Figures 2 and 3 show the presence of the ADM receptor components CALCRL and RAMP2 on both JAr and HTR-8/SV neo cells. No immunofluorescent signal was detected on negative controls, which ensures the specificity of the immunofluorescence staining.



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  		FIG. 2. Immunofluorescent localization of CALCRL (A1, A2) and RAMP2 (C1, C2) in JAr cells. Omission of the primary polyclonal antibodies served as the negative control (A3, C3). Original magnification x400



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  		FIG. 3. Immunofluorescent localization of CALCRL (A1, A2) and RAMP2 (C1, C2) in HTR-8/SV neo cell. Omission of the primary polyclonal antibodies served as the negative control (A3, C3). Original magnification x400

ADM Stimulation of JAr and HTR-8/SV neo Cell Proliferation

The presence of ADM receptor complex mRNAs and proteins on JAr and HTR-8/SV neo cells led us to examine their proliferative responses to ADM. Addition of ADM (10–9 to 10–7 M) to culture media stimulated JAr cell proliferation in a dose-dependent manner (Fig. 4). However, the dose-related response of HTR-8/SV neo cells to ADM was somewhat different than that of the JAr cells. Proliferation of these cells reached maximal at the 10–8 M dose and declined at 10–7 M dose (Fig. 5). Furthermore, ADM-induced HTR-8/SV neo cell proliferation could be blocked by the addition of the competitive ADM antagonist (ADM22–52), thus confirming the hypothesis that ADM-induced cytotrophoblast proliferation is mediated by ADM receptors (Fig. 5).



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  		FIG. 4. ADM-induced JAr cell proliferation. JAr cells (6 x 103/well) were plated in RPM1 1640 plus 4% serum, and with varying doses of ADM (10–9, 10–8, and 10–7 M) for 48 h. Cell growth was evaluated by MTT assay as described in Materials and Methods. Error bars indicate ± SEM. Asterisks indicate P < 0.01 vs. control (n = 3) (ANOVA)



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  		FIG. 5. ADM-induced HTR-8/SV neo cells proliferation. HRT-8/SV neo cells (1.5 x 103/well) were plated in RPMI 1640 with 1.5% serum and ADM (10–9, 10–8, and 10–7 M) for 48 h. Cell growth was measured by MTT assay as described in Materials and Methods. Error bars indicate ± SEM. Bar with different letter at the top differ significantly (P < 0.005; n = 6)

ADM Enhances JAr and HTR-8/SV neo Cell Invasion

A Matrigel-based assay was carried out to test the effects of ADM on JAr and HTR-8/SV neo cell invasion. As shown in Figure 6, very few JAr cells penetrated the Matrigel-coated membrane in the control, but addition of ADM substantially increased JAr cell migration and invasion. Figure 7 shows that the JAr cell invasion index was significantly (P < 0.05) higher (1.8-fold) in ADM-treated cells than in untreated controls, and this increase was blocked by ADM22–52. Similar to the JAr cells, ADM also increased the invasion index (1.5-fold) of HTR-8/SV neo cells, suggesting that ADM can stimulate first-trimester trophoblast cell migration and invasion.



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  		FIG. 6. Effects of ADM on trophoblast invasion. Matrigel-invasion assay was performed to evaluate JAr cell invasion of controls, and cultured with ADM (10–8 M) and ADM22–52 (10–7 M) plus ADM (10–8 M) for 48 h. The cell invasions were quantified by counting the cells penetrating the filter pores and appearing on the underside of the filters as described in Materials and Methods. Original magnification x100



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  		FIG. 7. Invasion index of ADM-treated JAr cells. JAr cells were cultured in medium with ADM or ADM22–52 plus ADM on Matrigel-coated transwell filters for 48 h. Invasive cells penetrated to the downward surface of the insert were cultured with MTT, and the optical absorbance at 550 nm was measured with a microplate reader. The invasion index was calculated as the ratio of the percentage of invasion in ADM-treated cells and the invasion of control cells. Data are expressed as mean ± SEM (n = 6). Bars with different letters at the top of the bar differ significantly (P < 0.05, ANOVA)

ADM Induces JAr Cell Gelatinolytic Activity

To examine the involvement of MMPs in cytotrophoblast invasion, we initially measured the MMP-2 and MMP-9 mRNA and protein expressions. Results showed that no significant changes were found in either cell line with regard to MMP-2 and MMP-9 mRNA or protein expression by using RT-PCR and Western blot analysis (data not shown). This led us to measure their activity by gelatin zymography. Figure 8 shows that cytotrophoblast cells displayed a substantial increase in MMP-2 activity (72 kDa) when cultured with ADM (10–8 M), without changes in MMP-9 (92 kDa) activity. These results suggested that ADM-stimulated trophoblast invasion is primarily dependent on MMP-2 activation.



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  		FIG. 8. ADM-induced JAr cell gelatinolytic activity. JAr cells were cultured with ADM (10–8 M) for 48 h. The culture medium was loaded onto SDS-PAGE gel containing 1 mg/ml gelatin and electrophoresed. After incubation with Tris (50 mM) and CaCl2 (5 mM), the gels were stained, destained, and analyzed. A digested clear band can be seen in a blue background, as shown by the arrow

ADM Decreases Trophoblast SERPINE1 Expression

In this study, we also tested the effects of ADM on the mRNA expression of plasminogen activator/plasmin system components such as PLAU, PLAU receptor (PLAU-R), PLAT mRNAs, and TIMPs -1, -2, -3, and -4 in the trophoblast cells. No significant alterations were noted with ADM treatment in these cells (data not shown). However, we demonstrate that SERPINE1 levels decreased in JAr cells when they were treated with ADM (Fig. 9). Further exploration using the HTR-8/SV neo cells showed a time-dependent decrease in SERPINE1 mRNA expression in the presence of ADM (Fig. 10). These results suggested that ADM inhibited SERPINE1 production, thus activated plasmin can be produced during cytotrophoblast invasion. The relative expression of SERPINE1 mRNA to 18S mRNA in HTR-8/ SV neo cells decreased when treated with ADM, and ADM22–52 reversed these ADM actions (Fig. 11), confirming that ADM-induced inhibition in SERPINE1 expression is mediated via ADM receptors.



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  		FIG. 9. Time-related changes in mRNA expression for SERPINE1 in JAr cells. JAr cells were cultured in RPMI 1640 with ADM (10–8 M) for 0, 4, 8, 12, 24, 48, and 72 h. Total RNA were isolated from the cells and RT-PCR were performed using specific primers. Representative bands of RT-PCR for SERPINE1 (465 base pairs) and 18S (209 base pairs) show clearly in the ethidium bromide agarose gel



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  		FIG. 10. Time-related changes in mRNA expression for SERPINE1 in HTR-8/SV neo cells. Cells were cultured in RPMI 1640 with ADM (10–8 M) for different periods of time. a) Representative bands of RT-PCR for SERPINE1 (405 base pairs) and 18S (209 base pairs). b) Comparison of density ratio measured by densitometric analysis of bands. Data are expressed as mean ± SEM (n = 6). The error bars indicate ± SEM. Asterisk indicates P < 0.05 vs. other time points (ANOVA)



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  		FIG. 11. a) ADM22–52 reversed ADM-induced SERPINE1 inhibition in JAr (48 h, n = 4). RT-PCR results for SERPINE1 and 18S. ADM22–52 was added 30 min before ADM treatment as described in Materials and Methods. Control, no treatment; ADM, ADM (10–8 M); ADM22–52, ADM22–52 (10–7 M) + ADM (10–8 M). b) Density of bands for SERPINE1 are expressed relative to that of 18S, and the error bars indicate ± SEM. The bars with different letters at the top differ significantly (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Local hypoxia and poor angiogenesis before 10–12 wk of gestation makes it difficult for cytotrophoblast cells to be affected by hormones and maternal mechanisms. For years, cytokines were believed to be involved in cytotrophoblast invasion in an autocrine, paracrine, or juxtacrine pattern. Epidermal growth factor shows a positive effect on cytotrophoblast invasion, whereas the roles of other cytokines in pathophysiology have long been questioned [16, 20, 25]. Originally, it was suggested that the cytotrophoblast is the builder of the bridge between mother and fetus; developing along either the syncytiotrophoblast, or extravillous trophoblasts, which possess a more aggressive invasive behavior to provide maternal-fetus interface. ADM is known as a potent vasodilator [28] and an angiogenic factor [46]. It is expressed in many tissues and has the potential to possess more functions such as cell natriuretic, diuretic, and antiapoptosis functions besides vasodilation [31–39]. To examine the effect of ADM on trophoblast cells, we first measured ADM receptor components CALCRL and RAMP2 mRNA in JAr cells by RT-PCR, and show their presence in HTR-8/SV neo cells and villous tissue as well. The presence of CALCRL and RAMP2 proteins on JAr and HTR-8/SV neo cells was further supported by immunofluorescence studies. This is consistent with a report by Moriyama et al. that a specific ADM binding site is found on these cells [40]. These are the proteins that constitute the ADM receptor complex and mediate ADM activity.

Because nearly nothing is known about the effect of ADM on cytotrophoblast, three approaches were employed in our experiment: cell proliferation, invasive behavior, and gelatinolytic activity. In cell proliferation, JAr cells responded in a dose-dependent pattern when treated with ADM (10–9 to 10–7 M), and the effects of ADM on HTR-8/SV neo cell (a transformed first-trimester human trophoblast) are analogous to those of the JAr cell. However, dose responses to ADM were not similar in HTR-8/SV cells compared to those of JAr cells, as the maximal effects of ADM occurred at lower concentrations (10–8 M).

Preliminary results led us to determine the influence of ADM on trophoblast invasion by using a Matrigel-based invasion chamber. ADM indeed increases in JAr and HTR-8/SV cell invasion. As a quantitative measurement, invasive index was calculated based on optical absorbance at 550 nm. The JAr cell invasive index was 1.8 times higher in ADM-treated cells than in controls, and the HTR-8/SV cells were 1.5 times higher in ADM-treated cells than controls. The proinvasion effect can be antagonized by ADM22–52, a competitive ADM antagonist. Thus, we suggest that ADM induces trophoblast cell proliferation and invasion.

Successful implantation and trophoblast invasion are closely linked to the expression of MMPs, which are able to degrade basement membranes. MMP-2 and MMP-9 have been identified on trophoblast cells, and are therefore regarded as key enzymes in the invasion process. Plasma MMP-2 levels were significantly higher in women with preeclampsia compared with those in women with uncomplicated pregnancies. MMP-9 levels were below the levels of detection [47], suggesting that the disturbances in plasma MMP-2 and MMP-9 may be involved in impaired placental implantation. The present study showed that trophoblast cells displayed a substantial increase in MMP-2 activity (72 kDa) when cultured with ADM (10–8 M), without changes in MMP-9 (92 kDa) activity, suggesting that ADM-stimulated trophoblast cell invasion in vitro is primarily dependent on MMP-2 activation.

Plasmin cleavage is necessary for MMP-2 and MMP-9 to function. Evidence shows that increased SERPINE1 mRNA expression in placenta and elevated plasma SERPINE1 concentration may play a role in the pathophysiology of preeclampsia [48, 49]. It therefore became the natural candidate for resolving the puzzle of upstream control. In the time-course experiment, SERPINE1 was found to be decreased in JAr cells, with a steep nadir at 48 and 72 h. Results from HTR-8 /SV neo cells mirrored this result. The inhibition was time-dependent and initiated at 4–8 h after ADM treatment, and with maximal effect at 72 h. Further, ADM22–52, an N-terminally truncated ADM fragment lacking the disulfide bridge-formed six-member ring in the 16– 21 position [50], induced a decrease in SERPINE1, indicating the involvement of specific ADM receptors in trophoblast cell functions.

Kobayashi et al. found that maternal circulatory ADM levels are higher in all three trimesters and early puerperium, then peaks in the third trimester [41]. After examining placental tissues via immunohistochemistry and Northern blot analysis, they suggested that ADM in maternal plasma is partly generated by placental tissues [41]. Moriyama and coworkers reported that ADM is expressed by cytotrophoblasts and the syncytiotrophoblast in all three trimesters, but that ADM is more abundant in first-trimester placenta [40]. Placenta-borne ADM accounts for only a small portion of ADM elevation in maternal plasma during pregnancy. This limited effect is hardly reflected by the maternal plasma level of ADM [41]. Placental ADM is more likely to act locally on the placental tissue itself. In addition, SERPINE1 is secreted as an active antiprotease and is present in plasma [48]. It is also the primary constituent of the components of extracellular milieu and is associated with extracellular vitronectin, which stabilizes the SERPINE1 in active conformation. The transcriptional mechanism also plays a role in determining SERPINE1 expression and regulation [12]. As we know, exocytotic inactive pro-PLAU must attach to PLAU receptors on the cell surfaces if it is to be transformed to active PLAU, which in turn, activates plasminogen. SERPINE1 inhibits PLAU at this stage and blocks extracellular matrix degradation. In our study, and as in many cases of tumor metastasis, ADM treatment inhibits SERPINE1 mRNA expression. We hypothesized that ADM might remove the SERPINE1 obstacle to plasminogen activation and accelerate the accumulation of plasmin, and result in increased MMP activity and subsequent increases in downstream biochemical reactions such as the digestion of collagen, degradation of matrix, and the release of cells from the matrix. Our results indicate an extension of the starting site for the biochemical cascades to cytotrophoblast from syncytiotrophoblast as indicated by Estelles et al. [48]. We propose that SERPINE1 can be transcriptionally regulated in the cytotrophoblast, probably by an autocrine mode, and in syncytiotrophoblast by paracrine or juxtacrine modes of action. In pregnancy, elevated ADM concentration in maternal circulation may be responsible for the inhibition of SERPINE1 expression at the maternal-fetus interface, especially in syncytiotrophoblasts.

Accumulative evidence has implied that MMPs are blocked primarily by TIMPs, and that serine proteinases are inhibited by SERPINE1 [17, 18]. Because there is cross-talk in MMP and serine proteinase catalytic and autocatalytic cascades, blocking serine proteinases with SERPINE1 may also block some activation of the MMPs. Apparently, the interaction between MMPs and SERPINE1 in the process of trophoblast invasion in normal and complicated pregnancies warrant further investigation.


   ACKNOWLEDGMENTS
 
We thank Dr. C.H. Graham, Queen University, Kingston, ON, Canada, for providing us with HTR-8/SV neo cell lines. We also thank Alice Fussner-Coker for her editorial assistance and Cheryl Welch for administrative support.


   FOOTNOTES
 
1 Supported by grant HL70883 from the National Institutes of Health to Y.-L.D. Back

2 Correspondence: Yuan-Lin Dong, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., MRB, 11.138, Route 1062, Galveston, TX 77555-1062. FAX: 409 747 0475;ydong{at}utmb.edu Back

Received: 28 January 2005.

First decision: 2 March 2005.

Accepted: 24 May 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bentin-Ley U, Horn T, Sjogren A, Sorensen S, Falck LJ, Hamberger L. Ultrastructure of human blastocyst-endometrial interactions in vitro. J Reprod Fertil 2000 120:337-350
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