Angiotensin II Inhibits Human Trophoblast Invasion through AT1 Receptor Activation*

Trophoblast implantation depends, in part, on the controlled production of plasmin from plasminogen, a process regulated by plasminogen activators and plasminogen activator inhibitors. We have determined that angiotensin II (Ang II) stimulates plasminogen activator inhibitor-1 (PAI-1) synthesis and secretion in human trophoblasts in a time- and concentration-dependent manner. Our results indicate that Ang II activates PAI-1 gene expression through the AT1 receptor and involves the calcium-dependent activation of calcineurin and the nuclear translocation of NFAT. Increased PAI-1 synthesis and secretion is associated with reduced trophoblast invasion as judged by an in vitro invasion assay. These studies are the first to link the renin-angiotensin system with the fibrinolytic system to regulate trophoblast invasion.

The fibrinolytic system is best known for its role in the regulated digestion of fibrin clots. A key component of this system is plasminogen, an inactive zymogen, which is converted into the active protease, plasmin, by the action of a plasminogen activator termed tissue-type plasminogen activator (tPA). 1 The ability of tPA to activate plasminogen is substantially enhanced by its association with the substrate, fibrin (1). Plasminogen can also be activated (i.e. converted to plasmin) by the action of another plasminogen activator, urokinase-type plasminogen activator (uPA) (2)(3)(4)(5)(6). In contrast to tPA, uPA is not associated with fibrin clots but instead is often associated with a specific cell surface receptor (uPAR) (7), which is thought to play a key role in cell migration and invasion (8). In this role, uPA is synthesized as an inactive single-chain proenzyme that can be stored or secreted. Secreted uPA is cleaved into the active two-chain molecule upon binding to uPAR on the surface of cells. Following activation, receptorbound uPA is capable of converting plasminogen into plasmin, the latter of which is able to degrade several key components of the extracellular matrix (ECM) (9). It is widely accepted that uPA initiates a cascade of proteolysis at the cell surface, which in turn leads to the degradation of the ECM, thereby promoting cellular migration. It is now clear that the uPA-regulated aspect of the fibrinolytic system plays a key role in mediating ECM degradation and cell invasion. Plasminogen activator activity is controlled by plasminogen activator inhibitors (PAIs) of which PAI-1 is the predominant physiological inhibitor (1,10,11). PAI-1 is an ECM glycoprotein that is capable of inhibiting both free and receptor-bound uPA through the formation of an irreversible covalent complex (12). In this way PAI-1 is believed to control cell migration and invasion in tumor growth and angiogenesis (13)(14)(15)(16).
Numerous studies suggest that PAI-1 production by human trophoblasts plays an important role in trophoblast invasion and placental development (17,18). The human hemochorial placenta is a highly invasive structure in which a subpopulation of placental trophoblast cells invades the uterus, its underlying stroma, and local blood vessels to achieve a physiological union between maternal and fetal circulatory systems (19). Plasminogen activators and plasminogen activator inhibitors are believed to regulate trophoblast invasion of the uterus. Key players in this process include uPA, PAI-1, and PAI-2 that together govern the production of plasmin from circulating plasminogen (20 -24). Recent studies suggest that the reninangiotensin system (RAS) exerts an important role in the regulation of PAI-1 gene expression in human mesangial cells (25)(26)(27)(28), vascular smooth muscle cells (29,30), and rat aortic and cardiac cells (31). Renin, angiotensinogen, angiotensinconverting enzyme, and angiotensin receptors are all present in the human placenta, suggesting that angiotensin II (Ang II) synthesized in the placenta may serve as a local modulator of placental function (32,33). Although it is clear that PAI-1 is elevated during normal pregnancy and even higher in preeclampsia (34 -36), the direct correlation of the RAS with PAI-1 production during human pregnancy is not well studied. We show here that Ang II stimulates PAI-1 synthesis and secretion in human trophoblasts in a time-and concentration-dependent manner. Our results indicate that Ang II activates PAI-1 gene expression through the AT1 receptor and involves the calciumdependent activation of the phosphatase, calcineurin, and the nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT). We show that increased PAI-1 synthesis and secretion result in reduced trophoblast invasion as judged by an in vitro Matrigel invasion assay. These studies are the first to link the renin-angiotensin system with the fibrinolytic system to regulate trophoblast invasion.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture medium, fetal bovine serum, antibioticantimycotic (100ϫ), Superscript II RNase H reverse transcriptase, Platinum Taq DNA polymerase, and a total RNA isolation kit were purchased from Invitrogen (Grand Island, NY). The PAI-1 enzyme-linked immunosorbent assay (ELISA) kit was obtained from American Diagnostic, Inc. (Greenwich, CT). The nylon membranes used in RNA transfer were from Bio-Rad Laboratories (Hercules, CA). Radiochemicals were obtained from ICN Radiochemicals (Irvine, CA). BD Biocoat Matrigel invasion chambers and control inserts were obtained from Becton Dickinson Labware (Bedford, MA). Ang II and bovine calf serum were obtained from Sigma Chemical Co. (St. Louis, MO). Polyclonal antibodies specific for human PAI-1, AT1, and ␤-actin were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Losartan and prazosin were generous gifts from Merck Sharp and Dohme (Rahway, NJ).
Cells-The HTR-8/SVneo trophoblast cell line (a kind gift from Dr. Charles H. Graham, Queen's University, Ontario, Canada) was established from explant cultures of human first trimester placentas and immortalized by transfection with the gene encoding simian virus 40 large T antigen, as previously described (37). These cells exhibit a high proliferation index and share phenotypic similarities with the nontransfected parent HTR-8 cells, including in vitro invasive ability and lack of tumorigenicity in nude mice. Furthermore, the parent cell population has also been shown to express a variety of other markers characteristic of extravillous invasive trophoblasts in situ (37).
Ang II Treatment of Cells-HTR-8/SVneo trophoblasts were plated in 6-well dishes in RPMI 1640 medium supplemented with 5% fetal bovine serum at a plating density of 0.5 ϫ 10 5 cells per well. After 24 h, the serum-containing medium was removed, and the cells were washed and subsequently maintained in serum-free RPMI 1640, containing 0.5% bovine serum albumin (fraction V) for 2 h and then stimulated with 0, 10, 50, and 100 nM Ang II for 24 h. Other cells were treated with 100 nM Ang II for 0, 24, and 48 h, respectively, for time-dependent studies. In some experiments, losartan, prazosin, and cyclosporin A (CSA), at 1 M, were added to the Ang II (100 nM)-stimulated cells at the initiation of Ang II treatment.
RNA Isolation and Northern Blot Analysis-Total RNA was isolated using an RNA Isolation Kit (Trizon) at the indicated times from control or Ang II-stimulated cells. For Northern analysis, 10 g of total RNA was fractionated by electrophoresis on 1% agarose gels containing 0.6% formaldehyde at 50 V for 3 h. Fractionated RNA was transferred onto a Duralon-UV nitrocellulose membrane by capillary action overnight. After blotting, the RNA was cross-linked to the filter by UV irradiation. Following prehybridization (50% formamide, 25 mM potassium phosphate, 5ϫ SSC, 5ϫ Denhardt's solution, and 100 g/ml salmon sperm DNA) overnight at 42°C, the blot was hybridized in the prehybridization solution plus 1% dextran sulfate and the labeled DNA fragment. After washing, the blots were visualized by autoradiography following storage at Ϫ70°C for various times.
Preparation of DNA Probes for Northern Blot Analysis-The nucleotide sequence of the oligonucleotides used to generate the cDNA probe for human PAI-1 mRNA was as follows: P1 5Ј-AGGATGCAGAT-GTCTCCAGC-3Ј and P2 5Ј-GGTTCCATCACTTGGCCCAT-3Ј. The probe was obtained using total RNA from HTR-8/Svneo trophoblast cells by Superscript II RNase H reverse transcriptase and Platinum Taq DNA polymerase. The cDNA probe was sequenced by the dideoxymediated chain termination method, and sequence analysis was performed using the Genetics computer group sequence analysis package, version 7.2 (University of Wisconsin, Madison, WI).
Cellular Protein Extraction and Western Blot Analysis-Cellular protein was suspended in 0.1 ml of lysis buffer containing 0.9% NaCl, 0.2% SDS, 4 mM Tris-HCl with proteinase inhibitors phenylmethylsulfonyl fluoride (40 g) and leupeptin (5 g) on ice and then centrifuged at 14,000 rpm for 5 min. The supernatant (50 -100 g of protein) was denatured in sample buffer and electrophoresed at 100 V on 10% polyacrylamide SDS-denaturing gels for 1 h. The separated proteins were transferred from gels onto nitrocellulose membranes at 100 V for 1 h. The blot was blocked with 5% milk in TBST (0.05% Tween-20 in Tris-buffered saline) solution for 1 h at room temperature and then incubated with polyclonal rabbit antibodies to human renin, PAI-1, or ␤-actin at 1:1500 dilution in 1% milk in TBST for 1 h at room temperature followed by three 15-min washes in TBST. Finally, the blot was incubated with goat anti-rabbit IgG at 1:2000 dilution in 1% milk-TBST for 1 h at room temperature. Rabbit antibody was detected by an enhanced chemiluminescence detection system (Amersham Biosciences, ECL blotting system).
Detection of Secreted PAI-1-The secreted PAI-1 concentration was detected by enzyme-linked immunosorbent assay (ELISA) as previously described (30). Preparation of Plasmids for Transfection-Plasmids used to express NFAT-GFP and ⌬NFAT-GFP were constructed in pEGFP-1 (CLON-TECH, Palo Alto, CA) and were generous gifts from Dr. R. Sanders Williams (Duke University, Durham, NC). The expression constructs pCMV-NFAT and pCMV-⌬NFAT were digested by AgeI and NotI to release the GFP fragment. The correct size fragments of CMV-⌬NFAT and CMV-NFAT were isolated and purified from the gel, blunt-ended, and self-ligated as described before (38).
PCMV-NFAT and pCMV-⌬NFAT Transient Transfection-HTR-8/ SVneo trophoblast cells were cultured and transfected with pCMV-NFAT and pCMV-⌬NFAT plasmid (2 g) in the absence of Ang II. The pCMVlacZ plasmid (2 g) was used as an internal control to monitor transfection efficiency. After 48 h, the cellular extract was isolated, and Western blot analysis was performed to detect PAI-1 protein production using rabbit polyclonal anti human PAI-1 antibody as describe before (39).
NFAT-GFP Transient Transfection and Microscopic Techniques-For cells expressing the NFAT-GFP fusion protein, HTR-8/SVneo human trophoblasts were cultured on laminin-coated (10 g) glass coverslips inserted into the multiwell dishes. After 24 h cells were transfected with 2 g of NFAT-GFP construct and 6 h later washed twice with PBS. Half of the cells were treated by 100 nM Ang II for 48 h, and half were maintained under the same conditions in the absence of Ang II. After 48 h all cells were washed with 1ϫ PBS, and the cells on the coverslips were viewed using an Olympus BX 60 fluorescence microscope equipped with darkfield optics with a green filter and photographed using a SPOT digital camera (Diagnostics Instruments Inc., Sterling Height, MI).

Matrigel Transwell Invasion Assay-Invasiveness of HTR-8/SVneo was measured with a Matrigel Invasion Chamber (Becton Dickinson
Labware, Bedford, MA) as described before (40). Briefly, confluent cells were harvested with 0.25% trypsin and 0.2% EDTA then centrifuged at 800 ϫ g for 10 min. Cells were washed with 1ϫ phosphate-buffered saline (PBS) once and resuspended in RPMI 1640 containing 0.5% bovine serum albumin. The lower compartment of the invasion chamber was filled with RPMI 1640 containing 5% fetal bovine serum as chemoattractant. The lower compartment was overlaid with an 8-m pore size polyethylene phthalate membrane precoated with Matrigel basement membrane matrix at 125 g/cm 2 . The cells (2.5 ϫ 10 5 per 6-well plate) were seeded in the upper compartment of the prehydrated Matrigel-coated invasion chambers in the absence or presence of Ang II. The control insert chambers contained 8-m pore size polyethylene phthalate membrane without being precoated with Matrigel basement membrane were seeded at the same concentration of cells and treated the same as the invasion chamber. Percent invasion is expressed as the percent invasion through the Matrigel matrix-coated membrane relative to cell migration through the control membrane. The upper surface of the filter was scraped with moist cotton swabs to remove Matrigel and non-migrated cells. Then both the control insert membranes, which were not precoated with Matrigel, and the Matrigel-coated insert membrane were stained with Diff-Quick stain (Biochemical Sciences Inc., Swedesboro, NJ) for 3 min. The membranes were washed three times with water, and the cells in the membranes were viewed by using an Olympus BX 60 fluorescence microscope equipped with brightfield optics and photographed using a SPOT digital camera (Diagnostics Instruments Inc., Sterling Height, MI). In some experiments, losartan (1 M), CSA (1 M), and human anti-PAI-1 antibody (1:100) were added to the cells at the initiation of Ang II treatment.
Statistics-All values are expressed as mean Ϯ S.E. Data were analyzed for statistical significance using GraphPad Prism software. Statistical significance was determined by analysis of variance test. A value of p Ͻ 0.05 was interpreted to mean that observed experimental differences were statistically significant.

PAI-1 mRNA Abundance Is Increased by Ang II in Human
Trophoblast Cells-Because of the potentially important role of PAI-1 in regulating trophoblast invasion, it is important to identify signal transduction pathways regulating PAI-1 gene expression. Realizing that the maternal fetal interface has a locally active renin-angiotensin system (32,41) and that PAI-1 gene expression is regulated by Ang II in other systems (25,28,29) we conducted experiments to determine if the abundance of PAI-1 mRNA is induced by Ang II in human trophoblasts. For these studies we used HTR-8/Svneo cells, an immortalized line of human trophoblasts. These cells were incubated with different concentrations of Ang II for 24 h, total RNA was prepared, and the abundance of PAI-1 mRNA was determined by Northern blot analysis. A typical result is shown in Fig. 1A and indicates that PAI-1 mRNA abundance increased in response to increasing concentrations of Ang II. Desitometric analysis of PAI-1 mRNA hybridization signals from repeated experiments, normalized to those for 28 S rRNA, demonstrated that PAI-1 mRNA levels were 1.1-, 2.2-, and 3.2-fold that of control untreated cells at 10, 50, and 100 nM, respectively (Fig. 1A). To determine the kinetics of Ang II-induced PAI-1 mRNA abundance, we treated human trophoblast cells with 100 nM Ang II for 24 and 48 h. Northern analysis was used to determine PAI-1 mRNA abundance in the presence and absence of Ang II for each time point. The results showed that PAI-1 mRNA is induced in response to Ang II treatment and is higher at 48 h than at 24 h of treatment (Fig. 1B). Densitometric analysis of PAI-1 hybridization signals from repeated experiments, normalized to those of 28 S rRNA, demonstrated that PAI-1 mRNA levels in Ang II-treated cells were 2.9-and 3.6-fold greater than those of the control cells without Ang II treatment at 24 and 48 h, respectively (Fig. 1D). These studies are the first to link the renin-angiotensin system with the fibrinolytic system in trophoblasts.
The Abundance of PAI-1 Protein and Its Secretion Are Induced by Ang II-We next wished to determine the impact of Ang II on the abundance and secretion of PAI-1 protein by human trophoblasts. For these experiments cells were incubated with increasing concentrations of Ang II for 24 h, and the abundance of PAI-1 in cell extracts was determined by Western blotting. PAI-1 protein from cell extracts was detected as a single band of ϳ52 kDa ( Fig. 2A). Densitometric analyses of the specific bands from repeated experiments, normalized to that of ␤-actin, demonstrated that PAI-1 protein levels were 1.1-, 1.9-, and 3.1-fold that of control untreated cells at 10, 50, and 100 nM, respectively ( Fig. 2A). Incubation of cells with 100 nM Ang II for 24 and 48 h resulted in a 2.7-and 3.6-fold increase in the abundance of PAI-1 protein over that of untreated cells, respectively (Fig. 2B). The increased abundance of PAI-1 protein in cells following incubation with Ang II treatment was consistent with the increase in PAI-1 mRNA abundance following Ang II treatment.
PAI-1 protein presumably exerts its physiological effects following secretion from cells where it is associated with plasminogen activators such as uPA (42). To determine the effect of Ang II on the secretion of PAI-1, we determined the concentration of PAI-1 in cell culture medium following 24 h of incubation with 100 nM Ang II. The concentration of PAI-1 in cell culture medium was determined using an enzyme-linked immunosorbent assay (ELISA) specific for human PAI-1. The results (Fig. 2C) show that the concentration of PAI-1 in cell culture fluid increased ϳ2.5-fold in response to 24-h incubation with 100 nM Ang II. These findings show that trophoblasts secrete increased amounts of PAI-1 in response to Ang II treatment.
Ang II Acts through AT1 Receptors on Human Trophoblasts-To determine if induction of PAI-1 production is through the AT1 receptor on HTR-8/SVneo cells, losartan, an AT1 receptor antagonist, was used to treat the cells. Treatment with losartan (1 M) completely prevented PAI-1 mRNA abundance (Fig. 3A), protein synthesis (Fig. 3B), and protein secretion (Fig. 3C) in response to treatment with 100 nM Ang II. These results indicate that HTR-8/SVneo cells possess the AT1 receptor and that PAI-1 mRNA abundance and protein synthesis and secretion are enhanced as a result of AT1 receptor activation.
Ang II-mediated Induction of PAI-1 Gene Expression and Protein Secretion Is Inhibited by CSA-Intracellular signaling downstream of AT1 receptor activation in cardiomyocytes involves calcium mobilization and the activation of calcineurin, a calcium/calmodulin-dependent cytoplasmic phosphatase (43).
To determine if calcineurin is involved in AT1 receptor signaling in human trophoblasts we used the calcineurin inhibitor, CSA. The effect of CSA on Ang II-induced stimulation of PAI-1 gene expression and protein secretion was determined. PAI-1 gene expression was assessed by Northern analysis to determine the abundance of PAI-1 mRNA and by Western analysis to determine the abundance of cellular PAI-1 protein. PAI-1 secretion was quantified by the use of an ELISA to determine the concentration of PAI-1 in cellular fluid following incubation with Ang II. The results show that Ang II induction of PAI-1 mRNA abundance (Fig. 4A), PAI-1 protein abundance (Fig.  4B), and PAI-1 protein secretion (Fig. 4C) were consistently inhibited by about 45-55% in the presence of 1 M CSA. These results indicate that the induction of PAI-1 gene expression by Ang II is mediated in part through a calcineurin-dependent signaling pathway in human trophoblast cells.
Evidence for the Involvement of NFAT in the Activation of PAI-1 Gene Expression-NFAT transcription factors translocate from the cytoplasm to the nucleus following dephosphorylation by calcineurin, a Ca 2ϩ /calmodulin-activated phosphatase (44). Thus, the fact that CSA inhibits the induction of PAI-1 gene expression following Ang II treatment of human trophoblasts suggests that this induction may be mediated, in part, through the action of NFAT. To evaluate this possibility, we transfected a mammalian expression construct encoding a constitutively active NFAT (NFATc), which is known to localize preferentially to the nucleus and in this way mimic the action of dephosphorylated NFAT (38). Transfection of wild type NFAT did not activate PAI-1 gene expression and did not result in an increase in PAI-1 protein concentration. However, transfection of the constitutively active NFATc did result in increased PAI-1 protein levels (Fig. 5A). These results provide strong evidence that NFAT transcriptional activators are involved in activation of PAI-1 gene expression.
Based on the results presented above, we propose that Ang II treatment results in dephosphorylation of NFAT followed by its translocation to the nucleus. To test this hypothesis, we transfected an expression construct encoding an NFAT-GFP fusion protein into control and Ang II-treated human trophoblast cells. After 48 h, cells were washed with phosphate-buffered saline and viewed by fluorescence microscopy to determine the cellular localization of green fluorescence. In control cultures, green fluorescence was cytoplasmic and was excluded from nuclei (Fig. 5B). However, in Ang II-treated cells, the green fluorescence became concentrated in nuclei in 65% of transfected cells. In control cells, the GFP fluorescence in the transfected cells was only found in the cytosol. These results indicate that human trophoblasts are capable of appropriate compart- mentalization of NFAT proteins in the nucleus following Ang II stimulation.
Ang II-induced PAI-1 Production Results in Reduced Trophoblast Invasiveness-Evidence provided above indicates that Ang II induces PAI-1 gene expression and protein secretion. Based on the fact that PAI-1 functions as an inhibitor of plasminogen activators resulting in a decreased degradation of the ECM, we propose that induction of PAI-1 protein synthesis and secretion by Ang II reduces invasiveness of human trophoblast cells. To test this hypothesis, trophoblast invasiveness was measured by an in vitro assay based on the ability of cells to penetrate a Matrigel-coated filter in an invasion chamber over a 24-h period. The ability of the trophoblasts to invade Matrigel-coated membranes is presented relative to the migration of cells through a control membrane that was not coated with Matrigel matrix. Ang II treatment decreased the invasion of the cells by 52% compared with control cells without Ang II treatment (Fig. 6, A, B, and F). To test whether the decreased invasion of trophoblasts following Ang II treatment is caused by a calcineurin-dependent PAI-1 induction following AT1 receptor activation, we treated the cells with losartan, CSA, and specific PAI-1 antibody, respectively, plus Ang II. The results (Fig. 6, A, C, and F) show that losartan completely abolished the inhibition of invasion by Ang II. Human PAI-1 antibody and CSA treatment also reduced the inhibition of invasion by Ang II relative to that of control cells without Ang II treatment (Fig. 6, A, D, E, and F). These results suggest that decreased trophoblast invasiveness following Ang II treatment is through AT1-receptor activation via calcineurin-dependent PAI-1 gene activation. DISCUSSION Trophoblast invasion depends, in part, on the regulated production of proteolytic enzymes such as plasmin, which function to degrade the extracellular matrix and to activate other proteases such as matrix metalloproteinases (24,45). Trophoblasts are equipped to activate plasminogen, leading to the production of plasmin, though the action of urokinase-type plasminogen activator (uPA) associated with specific cell surface receptors, uPARs (24). This system appears to be well controlled during normal intrauterine pregnancies in which the activity of uPA is regulated by plasminogen activator inhibitors, particularly PAI-1. In a number of cell types PAI-1 production is controlled through activation of the AT1 receptor (26 -31). Realizing that a complete RAS is present at the maternal fetal interface of humans (32) and other mammals (39), we tested the effect of Ang II on various aspects of PAI-1 gene expression in human trophoblasts. We have shown here that Ang II stimulates PAI-1 production by human trophoblasts in a time-and dosage-dependent manner, acting through the AT1 receptor. We have also shown that activation of AT1 receptors decreases trophoblast invasion. Our studies are the first to link the RAS with the fibrinolytic system to regulate placental development and function. These findings suggest that the local RAS plays an important role in trophoblast invasion.
The renin-angiotensin system (RAS) is a signaling cascade that plays a key role in regulating blood pressure and electrolyte balance. Traditionally, the RAS has been considered primarily as a circulating system involved in the regulation of blood pressure and salt and fluid homeostasis. In addition to this classic view of the RAS, accumulating evidence indicates that the components of the RAS are synthesized in many tissues, such as brain, heart, ovary, and placenta, and that Ang II levels can be controlled locally, independent of circulating Ang II (32,33). Renin, angiotensinogen, angiotensin-converting enzyme, and angiotensin receptors are all present in the human placenta. These findings suggest that Ang II synthesized in the placenta may serve as an autocrine/paracrine modulator of placental function (32,33). Activation of the RAS has previously been shown to induce multiple growth factors that have been implicated in fibrosis, such as transforming growth fac-tor-␤ (TGF-␤) and platelet-derived growth factor-␤ (46 -48). Induction of these growth factors appears to be mediated by Ang II acting at the AT1 receptor (49). TGF-␤ itself is known to regulate PAI-1 gene expression through the Smad-dependent signaling pathway (50). However, the induction of PAI-1 by Ang II in vitro in mesangial cells is TGF-␤-independent (49). Hypoxia, TGF-␤, and endothelin-1 are known to regulate PAI-1 gene expression in the trophoblast (51,52). However, the link between the RAS and the PAI-1 proteolytic pathway has not been recognized and fully understood in physiological and pathological placental development. Our current studies provide strong evidence that a local RAS links with the fibrinolytic system to control key aspects of placenta development.
We have also shown that the intracellular signaling pathway linking AT1 receptor activation with PAI-1 gene expression involves calcium-mediated activation of calcineurin, a cytoplasmic phosphatase. Numerous recent studies have shown that a variety of physiological and pathological stimuli elicit cellular responses through calcium-mediated signaling pathways involving the activation of calcineurin (43,44,53). This cytoplasmic phosphatase acts to dephosphorylate the phosphorylated form of NFAT, thereby allowing the dephosphorylated NFAT to enter the nucleus and activate genes. A common strategy for probing this pathway involves the use of CSA, a compound that binds with cyclophilin, forming a complex that binds to the catalytic subunit of calcineurin, inhibiting its activity (54). The role of the Ca 2ϩ -calcineurin-NFAT signaling pathway in the induction of PAI-1 gene expression in trophoblasts has not been addressed prior to our studies. We have presented three lines of evidence for the involvement of the calcineurin/NFAT pathway in the activation of PAI-1 gene expression in human trophoblasts. First, we have shown that CSA partially blocked the induction of PAI-1 gene expression by Ang II in human trophoblasts. Second, we have shown that a constitutively active mutant of NFAT activates PAI-1 gene expression. Third, we have shown that AT1 receptor activation leads to nuclear translocation of an NFAT-GFP fusion protein. Together, these results provide strong evidence for the role of the calcineurin/ NFAT-signaling pathway in PAI-1 gene expression following AT1 receptor activation in human trophoblasts.
CSA only partially blocked the induction of PAI-1 gene expression following AT1 receptor activation. This finding is consistent with previous studies in other systems showing important roles for protein kinase C (PKC) signaling pathways in the up-regulation of PAI-1 gene expression following AT1 receptor activation (28). The involvement of calcineurin and PKC signaling downstream of AT1 receptor activation is consistent with the activation of phospholipase C (PLC). The action of PLC generates two intracellular messenger molecules, inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. IP 3 contributes to intracellular calcium mobilization, and diacylglycerol activates PKC. The fact that CSA only partially blocks the AT1 receptormediated activation of PAI-1 gene expression indicates that signaling downstream of PKC may also contribute to PAI-1 gene activation in human trophoblasts.
Our results are consistent with a molecular pathway for a local RAS regulating trophoblast invasion via PAI-1 production as shown in Fig. 7. According to this model, Ang II from the local RAS leads to mobilization of intracellular Ca 2ϩ , resulting in activation of calcineurin. NFAT within the cytoplasm is dephosphorylated by calcineurin, enabling it to translocate to the nucleus where it participates in the activation of PAI-1 gene expression to control trophoblast invasion in normal placental development.
In both human and mouse the RAS system undergoes major changes in response to pregnancy (32,39). In normal human pregnancy, Ang II is increased in the maternal circulation (41,55). As we have shown here, one of the local functions of the increased Ang II in the placenta may be to induce local PAI-1 production. Because PAI-1 is the key regulator of uPA activity and uPA is important for cell invasion, our studies suggest that the local RAS regulates PAI-1 production to regulate trophoblast invasion. In this regard, we have recently reported that high levels of renin gene expression occur at the maternal fetal interface throughout pregnancy in mice, initially in the deciduum and subsequently in the placenta (39). Increased renin gene expression in the deciduum could lead to increased Ang II production and in this way could limit trophoblast invasion. Likewise, increased renin gene expression in trophoblasts in near term placentas could lead to increased local Ang II concentration to reduce trophoblast invasion near the end of gestation. Thus, local renin gene expression at the maternal-fetal interface may regulate trophoblast invasion through the activation of the AT1 receptor.
Pre-eclampsia is one of the leading causes of maternal and fetal morbidity and mortality during pregnancy (56). The condition is characterized by severe maternal hypertension, proteinuria, and is often associated with shallow trophoblast invasion and improper spiral arterial remodeling. Elevated PAI-1 occurs in the maternal circulation in pre-eclampsia and has been implicated as a contributing risk factor for hypercoagulation and fibrinolytic imbalance (6,34). In our studies, we have shown that Ang II can induce PAI-1 gene expression in human trophoblasts, leading to increased PAI-1 secretion and decreased trophoblast invasiveness. Thus, our results implicate increased PAI-1 production by trophoblasts as the basis of two features of pre-eclampsia: shallow trophoblast invasion and increased maternal circulating PAI-1. In this regard, a recent report presented evidence that pre-eclampsia is associated with the production of an autoantibody capable of binding to, and activating, the AT1 receptor (57,58). We recently determined that IgG from women with pre-eclampsia, but not IgG from normotensive pregnant women, can activate AT1 receptors on human trophoblasts resulting in increased production and secretion of PAI-1 protein and reduced trophoblast invasiveness. The ability of IgG from pre-eclamptic women to stimulate PAI-1 production and to reduce trophoblast invasiveness was blocked by losartan, an AT1 receptor-specific antagonist, and by the presence of a 7-amino acid peptide corresponding to an epitope associated with the second extracellular loop of the AT1 receptor. These findings indicate that the AT1 receptor agonistic autoantibody associated with pre-eclampsia is efficient at activating AT1 receptors on human trophoblasts. 2 Overall, our studies suggest that the local RAS may play an important role in normal placental development and that abnormalities of the RAS in pre-eclampsia, i.e. the presence of an autoantibody masquerading as Ang II, may be an essential cause of shallow trophoblast invasion, increased maternal PAI-1, and increased thrombosis associated with pre-eclampsia. Our current efforts focus on testing the pathophysiological impact of pre-eclampsia-associated AT1 receptor agonistic autoantibodies on trophoblast physiology and development.