Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print January 30, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065433
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/6/976    most recent
biolreprod.107.065433v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nicola, C.
Right arrow Articles by Chakraborty, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nicola, C.
Right arrow Articles by Chakraborty, C.
Agricola
Right arrow Articles by Nicola, C.
Right arrow Articles by Chakraborty, C.
BIOLOGY OF REPRODUCTION 78, 976–982 (2008)
DOI: 10.1095/biolreprod.107.065433
© 2008 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action; 

Prostaglandin E2-Mediated Migration of Human Trophoblast Requires RAC1 and CDC421

Catalin Nicola 3, Peeyush K. Lala 3, and Chandan Chakraborty 2 4

Departments of Anatomy and Cell Biology3 and Pathology,4 Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1

ABSTRACT

The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E2 produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE2-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE2 or 17-phenyl trinor PGE2 (PGE receptor PTGER1 agonist) or PGE1 Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE2-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE2, PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE2-mediated migration of human EVTs.

mechanisms of hormone action, placenta, pregnancy, signal transduction

INTRODUCTION

In normal pregnancy, proliferation, migration and invasion of extravillous trophoblast (EVT) cells are stringently regulated in situ [1], but this regulation may fail in preeclampsia [2–4]. The availability of a pure human first-trimester EVT cell line propagated in vitro, HTR-8/SVneo [5], helped in understanding of receptor-mediated intracellular signaling events responsible for the regulation of migration of EVT cells by a variety of molecules produced at the maternal-fetal interface [1, 6–10]. Decidua-derived prostaglandin (PG) E2 is one such molecule that stimulates the migration of first-trimester human EVT cells, both in vitro and in situ [11].

PGE2, the most studied prostanoid, is a major product of prostaglandin-endoperoxide synthase (PTGS1, PTGS2) pathways. It exerts its autocrine or paracrine effects by interacting with four G protein-coupled receptors, called PTGER1–4 (also known as EP1–4) localized on the plasma membrane [12]. HTR-8/SVneo cells have been shown to express all these four receptors [11]. While PTGER1 signals through Gq proteins, activating phospholipase (PL) C and modulating the phosphatidylinositol pathway, leading to an increase in the intracellular concentration of Ca2+ [12], PTGER2 and PTGER4 activate Gs proteins, stimulating the cyclic AMP (cAMP) production via adenylate cyclase [12]. PGE2, signaling through PTGER4 but not through PTGER2, can also activate phosphatidyl inositol 3-kinase (PI3K) pathway, in a cAMP-independent manner [13]. PTGER3 has many splice variants that vary only in the carboxy-terminal tails, most of which activate Gi proteins, reducing the cAMP production, while others can stimulate Gs or Gq proteins [12]. PGE2, produced in vast amounts by maternal decidua has important functions in pregnancy, being required for implantation [14], parturition [15], and immune protection of the conceptus [16, 17]. The prevention of preeclampsia by treatment with linoleic acid, a precursor of the PTGS1 and PTGER 2 substrate arachidonic acid, suggests a possible derangement of PGE2 produced at the maternal-fetal interface in the pathophysiology of preeclampsia [18].

RHO GTPases are a ubiquitous protein family [19] that act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. They have essential biological functions in cell cycle progression, cell-cell interaction, cell polarity and cell migration, vesicle trafficking, controlling the expression of various genes, and regulating the activity of numerous enzymes. RHO GTPases play important roles in the functional organization of the actin cytoskeleton and the microtubule network and therefore may be critical for cell migration [20]. While GTP-bound RHOA induces the assembly of actin-myosin filaments, active RAC1 or CDC42 promotes the protrusion of lamellipodia or filopodia, respectively [21]. Lamellipodia are large cytoskeletal actin projections on the mobile edge of the cell, whereas filopodia are small spiky cytoplasmic projections that contain actin filaments cross-linked into bundles that extend from the leading edge of migrating cells and form focal adhesions with the substratum. Activation of the members of the RHO family GTPases, including RAC1 and CDC42, has been shown to mimic different extracellular factor-induced actin cytoskeletal remodeling required for various cellular functions including cell migration. However, whether either GTPase is required for PGE2-mediated cell migration remains unknown. The present study was designed to determine whether RAC1 and CDC42 are required for PTGER1- and PTGER4-mediated migration of human EVT cells and to examine the effects of PGE2 or PTGER1- or PTGER4-agonist on the activations of RAC1 and CDC42 GTPases in these cells.

MATERIALS AND METHODS

Reagents and Materials

RPMI 1640 and FBS were purchased from GIBCO (Burlington, ON). PGE2, 17-phenyl trinor Prostaglandin E2 (PTGER1 agonist) and PGE1 Alcohol (PTGER4 agonist) were obtained from Cayman Chemicals (Ann Arbor, MI). RAC1 inhibitor (NSC23766) was purchased from Calbiochem (Darmstadt, Germany). Bovine serum albumin (BSA) and monoclonal mouse anti β-tubulin (clone D66, catalog no. T0198) were obtained from Sigma (Oakville, ON). Anti-RAC1 antibodies, clone 23A8 (mouse monoclonal, catalog no. 05–389), were obtained from Upstate (Lake Placid, NY). Mouse anti-human CDC42 (catalog no. MAB3707) monoclonal antibodies were purchased from Chemicon International (Temecula, CA). Goat anti-mouse IgG-HRP (catalog no. 31430) secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated (FITC) goat anti-mouse (catalog no. CLCC30001) secondary antibodies were purchased from Cedarlane (Hornby, ON). Growth Factor Reduced Matrigel was obtained from Collaborative Biomedical Products (Bedford, MA).

EVT Cell Line and Culture

In the present study we used the well-characterized first-trimester EVT cell line HTR-8/SVneo derived by SV40 T-antigen immortalization of a short-lived EVT cell line HTR-8 [5]. HTR-8/SVneo cells express all the markers of EVT cells in situ—including cytokeratins 7, 8, and 18; ALPP (alkaline phosphatase, placental or Regan isoenzyme) high-affinity PLAUR (plasminogen activator, urokinase receptor; also known as uPAR); IGF2 mRNA and IGF2 protein; HLA framework antigen w6/32; integrins {alpha}1, {alpha}3, {alpha}5, β1, {alpha}vβ3/β5 [22]; and HLA-G when grown on laminin or Matrigel [23]—and mimic the phenotypic behavior of freshly isolated cytotrophoblast cells during Matrigel invasion [24]. The present study used HTR-8/SVneo cells with passages between 90 and 115 for all the described experiments cultured in RPMI 1640 supplemented with 10% FBS and 2% penicillin/streptomycin, unless specified otherwise.

Chorionic Villus Explant Culture

EVT migration and outgrowth was measured as reported earlier [11]. Placentae at 7–9 wk of gestation from elective pregnancy terminations (collected according to the approved institutional ethics committee guidelines) were dissected carefully, and small fragments of villus tips (10–15 mg wet weight) were placed on Millicell-CM culture dish inserts (Millipore Corp., Bedford, MA) precoated with 200 µl of 1:6 dilution of Growth Factor Reduced Matrigel (Collaborative Biomedical Products) in serum-free DMEM/F12 medium (Gibco, Grand Island, NY) supplemented with 2% penicillin/streptomycin and 0.25 mg/ml ascorbic acid (Sigma, Oakville, ON) at pH 7.4 and then placed in 24-well plates (Becton Dickinson, Franklin Lakes, NJ). For each experiment we used at least three placentae, and triplicate explants were set up for each treatment under control and experimental conditions. The explants were kept in culture for up to 72 h. Pictures were taken every 24 h using an inverted light microscope (40x objective). The area of EVT cell outgrowth was measured using Scion Image for Windows software (Scion Corporation, version beta 4.0.2).

Immunofluorescence Staining

Cells were allowed to grow in complete medium on 12-mm glass coverslips, serum starved overnight, and then treated as described in the Results section. The cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 (Alkylaryl Polyether Alcohol) for 10 min. Nonspecific binding sites were blocked using normal goat serum. Primary mouse anti-human RAC1 antibodies were added (10 µg/ml, 4 h at 4°C) and then secondary FITC goat anti-mouse (1/100 dilution, 1 h in the dark, at room temperature). The coverslips were mounted using fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). The pictures were taken with a Zeiss Axioplan 2 microscope.

Migration Assay

The random migration (chemokinesis) of HTR-8/SVneo cells was measured using a protocol as reported earlier [11]. In brief, 1 x 105 cells suspended in 200 µl serum-free RPMI 1640 medium supplemented with 0.1% BSA were plated on Falcon cell culture inserts with 8.0-µm pore size (Becton Dickinson) and then placed in 24-well Falcon notched plates. The lower chamber was filled with 800 µl serum-free medium with or without additional treatment. The cells were incubated for 48 h at 37°C and 5% CO2. Then the cells on the upper surface of the insert membranes were wiped, followed by staining with Harleco Hematocolor staining kit (EM Science, Gibbstown, NJ). The total number of cells attached to the lower surface of the membrane was counted using a Zeiss light microscope (400x magnification). The number of cells that migrated varied from day to day (from 80 to 200, when no treatment was made). However, the degree of effect of a test substance did not vary between days. For this reason, data have been presented as migration indices (relative changes with respect to controls).

Results of all the migration experiments were validated by examining the effect of various treatments on the proliferation/survival of HTR-8/SVneo cells as reported elsewhere [10] using Cell Proliferation Kit I (MTT) from Roche Diagnostics (Laval, QC). None of the test substances was found to have any effect on proliferation/survival of these cells when these assays were performed under same experimental conditions as those of the migration assays.

siRNA Preparation and Transfection

The siRNA oligonucleotides duplexes (Dharmacon, Lafayette, CO) specific for human RHO GTPases were designed according to the literature [25, 26] and were purchased from Dharmacon in deprotected and desalted form. The siRNA sequences with dTdT overhanging at their 3' terminus used were 5'-GAGGAAGAGAAAAUGCCUGTT-3' to inhibit RAC1 mRNA synthesis [25] and 5'-AAAGACUCCUUUCUUGCUUGUTT-3' to inhibit CDC42 mRNA synthesis [26]. Control siRNA, which is a pool of several control siRNAs, was purchased from Dharmacon. Transfection of siRNA was carried out using Silencer siRNA Transfection II Kit (Ambion, Austin, TX; catalog no. 1631). For transfection, 5 µl/ml of siPORT NeoFX (Ambion) and siRNA were diluted in OPTI-MEM I (Invitrogen Canada, Inc., Burlington, Ontario) medium. Both were preincubated for 10 min, after which the two mixtures were combined and incubated at room temperature for another 10 min for complex formation. The cells were lifted and resuspended in RPMI 1640 containing 10% FBS (1 x 105 cells/ml), and 2.3 ml of the suspension were added to the mixture to make final concentration of siRNA 100 nmol/L and total volume of 2.5 ml. Transfections were performed for 48 h, and the transfected cells were washed and subjected to either real-time RT-PCR to indirectly measure the "transfection efficiency" or migration assays. Western blot analysis for RAC1 and CDC42 proteins were also performed to document their successful downregulations.

RAC1 and CDC42 Activation Assays

RAC1 and CDC42 activities were determined using the pull-down assay kits from Pierce following their instructions [27, 28]. In brief, 50 µg of clarified cell lysate were incubated with GST-Pak1-PBD in resins at 4°C for 1 h in spin column, centrifuged, washed and eluted in SDS-PAGE buffer to pull down only the active form of RAC1 and CDC42. These eluted samples were run on 12% separating gel on PAGE and subjected to Western blot analyses using antibodies against RAC1 or CDC42. RAC1 and CDC42 activities were indicated by amounts of Pak1-PBD-bound RAC1 (GTP-RAC1) and CDC42 (GTP-CDC42). Total cell lysates were also directly immunoblotted for the levels of total RAC1 or CDC42 for normalization.

Western Blotting

Either the samples from RAC1 or CDC42 activation assays or whole-cell extracts from siRNA-transfected cells (20 µg/lane) were denatured and loaded onto SDS-PAGE gels. Then the proteins were resolved at 100 V for 1–2 h. The separated proteins were then transferred onto Immuno-Blot PVDF membrane (BioRad Laboratories, Mississauga, Ontario) at constant current (100 mA/gel) for 1 h. Nonspecific proteins on the PVDF membranes were blocked for 1 h using 20% nonfat dry milk in Tris-buffered saline (TBS). Primary antibodies to the RHO GTPases were diluted in 1x TBS, with 5% nonfat dry milk in the following ratios: 1:1000 for anti-RAC1 and 1:500 for anti-CDC42. The blots were incubated with the primary antibodies overnight at 4°C. After three washes in TBS with 0.1% Tween-20, the blots were incubated with horseradish peroxidase conjugated goat anti-mouse IgG (1:20 000 in 5% milk in TBS) secondary antibodies. Detection was performed using ECL plus Western blotting detection system from Amersham (Oakville, ON; catalog no. RPN2132).

Statistical Analysis

Absolute numbers of cells migrated were normalized as a percent of the control (migration indices), and migration scores were analyzed using two-way analysis of variance (ANOVA). Differences among treatment means were assessed by Student t-test. Statistical significance in the chorionic villus explant cultures was assessed by repeated measure two-way ANOVA with one "within-culture" factor (time) and one between treatments. Differences of P < 0.05 were considered significant.

RESULTS

RAC1 Inhibition Reduces PGE2-Induced Migration of EVT Cells

To examine the role of RAC1 in the migration of first trimester human extravillous trophoblast, we used NSC23766, a RAC1 inhibitor, on both our well-characterized HTR-8/SVneo cells [5, 22] and also on placental chorionic villi explants from first-trimester elective termination of pregnancy. Migration assays in serum-free conditions showed that NSC23766 (50 µmol/L) inhibited the basal migration of HTR-8/SVneo cells at 48 h as compared to controls (Fig. 1A). In the same experiment, the cells preincubated with RAC1 inhibitor for 2 h were challenged with 1 µmol/L PGE2, PTGER1 agonist (17-phenyl trinor PGE2), and PTGER4 agonist (PGE1 Alcohol) in the presence of the RAC1 inhibitor, but their migration could not be rescued (Fig. 1A), suggesting that RAC1 plays a very important role in basal and PGE2-mediated migration of HTR-8/SVneo cells. NSC23766 is a reversible RAC1 inhibitor that competitively inhibits interaction between RAC1 and RAC-specific guanine nucleotide exchange factors (GEFs) and has no effect on CDC42 or RHOA activation [29, 30]. Within the RAC subfamily, RAC1, RAC2, and RAC3 have a highly homologous primary sequence [31, 32], but their expression depends on the cell type [32]. NSC23766 effectively inhibited thrombin-induced activation of RAC1 and RAC2 in human platelets at 3 min, suggesting that it is not a RAC1 selective inhibitor [30]. The effects of NSC23766 on RAC3 activation have not been studied. Thus, our results with this inhibitor could not be attributed solely to RAC1 since our HTR-8/SVneo cells expressed the mRNA for all three RAC GTPases (data not shown). To evaluate the specific role of RAC1, we further characterized the effect of RAC1 knockdown using siRNA silencing. As seen in Figure 1B, HTR-8/SVneo cells transfected with 100 nmol/L specific RAC1 siRNA and used in migration assay showed a significantly reduced migration as compared to the cells transfected with a pool of scrambled siRNA. RAC1 siRNA transfected cells were also challenged with 1 µmol/L PGE2, PTGER1, or PTGER4 agonist (Fig. 1B), but the addition of these compounds did not restore their migratory abilities, suggesting again that RAC1 is an important mediator of PGE2-induced migration. In our previous report, we have documented successful knockdown of RAC1 protein following the same siRNA transfection of HTR-8/SVneo cells following 48 h posttransfection [33]. In the present study, we show that knockdown of RAC1 protein remains even 96 h posttransfection (Fig. 1C). The effectiveness of RAC1 siRNA transfection was also assessed by real-time PCR demonstrating a 70% inhibition in the mRNA levels at 48 h (data not shown). Neither NSC23766 nor RAC1 siRNA had any effect on proliferation/survival of HTR-8/SVneo cells (data not shown), suggesting that the migration assay results were not a consequence of changes in cell viability.


Figure 01
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1. The effect of RAC1 inhibition on HTR-8/SVneo cell migration induced by PGE2, 17-phenyl trinor PGE2 or PGE1 Alcohol. A) Migration assays were performed in serum-free conditions in presence or absence of RAC1 inhibitor NSC23766 (NSC, 50 µmol/L) plus and minus PGE2, 17-phenyl trinor PGE2 (17 Phenyl), or PGE1 Alcohol (PGE1 Alc) for 48 h (n = 3 ± SEM, P < 0.05). B) HTR-8/SVneo cells transfected for 48 h with 100 nmol/L RAC1 siRNA or scrambled siRNA, washed, and subjected to migration assays in presence or absence of PGE2, 17-phenyl trinor PGE2, or PGE1 Alcohol (n = 3 ± SEM, *P < 0.05). C) Western blotting using anti-RAC1 antibodies showing decreased protein levels at 96 h after the siRNA transfection.

CDC42 Inhibition Reduces PGE2-Induced Migration of EVT Cells

RAC1 and CDC42 can signal through either common pathways [25, 34] or divergent pathways [35–39], indicating that the effects of RAC1 and CDC42 downregulation on EVT cell migration might not be always similar. Therefore, we further examined the role of CDC42 in PGE2-mediated migration of HTR-8/SVneo cells by knocking down CDC42. Like RAC1-downregulated cells, CDC42-silenced HTR-8/SVneo cells also showed decreased migration in serum-free conditions at 48 h, as compared to controls (Fig. 2A). The addition of 1 µmol/L PGE2 or PTGER1 and PTGER4 agonists did not rescue EVT cell migration (Fig. 2A), suggesting that CDC42, similar to RAC1, is essential for PGE2-induced migration of first-trimester human extravillous trophoblast. The effect of siRNA silencing on the CDC42 protein levels is depicted in Figure 2B, which shows a significant decrease even at 96 h posttransfection, compared to both untransfected (control) and cells transfected with scrambled siRNA. The effectiveness of CDC42 siRNA transfection was also assessed by real-time PCR demonstrating a 70% inhibition in the mRNA levels at 48 h (data not shown). CDC42 siRNA transfection was found to have no effect on cell proliferation/survival (data not shown).


Figure 02
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2. The effect of CDC42 inhibition on HTR-8/SVneo cell migration induced by PGE2, 17-phenyl trinor PGE2, or PGE1 Alcohol. A) HTR-8/SVneo cells transfected for 48 h with 100 nmol/L CDC42 siRNA and scrambled siRNA, washed, and subjected to migration assays in presence or absence of PGE2, 17-phenyl trinor PGE2, or PGE1 Alcohol (n = 3 ± SEM, *P < 0.05). B) Western blotting using anti-CDC42 antibodies showing decreased protein levels at 96 h after the siRNA transfection.

PGE2 Activates RAC1 and CDC42 GTPases

To further elucidate whether these GTPases are simply permissive factors for PGE2-mediated EVT cell migration or can be activated by PGE2 acting through PTGER1 and/or PTGER4, we examined the effect of PGE2, PTGER1 or PTGER4 agonist on the activation status of these enzymes. We measured activities of these enzymes by using the pull-down assays for GTP-bound RAC1 or CDC42 using recombinant PAK1 (p21-activated kinase) fused with GST (glutathione S-transferase), followed by Western blotting with specific antibodies [28]. As shown in Figure 3, PGE2 or PTGER1 or PTGER4 agonist activated both RAC1 and CDC42 at 10 min, as compared to their respective controls. Together, these data thus suggest that PGE2 can activate RAC1 and CDC42 independent of other extracellular factors that can exert migration stimulatory effects on EVT cells.


Figure 03
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3. Effects of PGE2, 17-phenyl trinor PGE2, or PGE1 Alcohol on RAC1 and CDC42 activations in HTR-8/SVneo cells. A, C) 1 µmol/L of PGE2, 17-phenyl trinor PGE2, or PGE1 Alcohol was added in culture for 10 min to study the activations of RAC1 and CDC42, using pull-down assay kits (see Materials and Methods). B, D) Densitometric quantification of data from RAC1 (B) and CDC42 (D) activation assays (n = 3 ± SEM, *P < 0.01). Relative activity (GTP-RAC1/Total RAC1 or GTP-CDC42/Total CDC42) was first determined and expressed as activation index (relative activity with respect to control which is considered as 1.0).

Because it is generally believed that the majority of inactive RHO GTPases are coupled with RHO GDI in the cytosol, whereas the majority of active GTPases are membrane bound and these membrane-bound active enzymes are involved in cytoskeletal changes required for cell migration, we also determined RAC1 activation by studying the translocation of the enzyme to plasma membrane. We examined the translocation of RAC1 to the plasma membranes of HTR-8/SVneo cells following activation of PTGER1 or PTGER4, by immunofluorescence using RAC1-specific antibodies. HTR-8/SVneo cells grown on coverslips and serum starved overnight were challenged with 1 µmol/L PGE2 (Fig. 4B), 17-phenyl trinor PGE2 (Fig. 4C), or PGE1 Alcohol (Fig. 4D), showing increased staining on the plasma membrane, compared to controls (Fig. 4A). This suggests that PGE2, acting through PTGER1 or PTGER4, can translocate cytosolic RAC1 to the plasma membrane.


Figure 04
View larger version (80K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4. HTR-8/SVneo cells immunostained with RAC1 antibodies after 10 min of treatment with 1 µmol/L of PGE2 (B), 17-phenyl trinor PGE2 (C), or PGE1 Alcohol (D) as compared to controls (A). Arrows indicate RAC1 localization on the cell membrane. Increased RAC1 on the plasma membranes of treated cells (B–D) as compared to control (A) suggested translocation of RAC1 from cytosol to plasma membranes.

Treatment of Chorionic Villi Explant with NSC23766 Inhibits Spreading of First-Trimester Human Cytotrophoblast on Matrigel

The effect of RAC1 inhibition on first-trimester EVT cells was also assessed using chorionic villus explant cultures from first-trimester placentae. As shown in the third row of Figure 5A, the explants treated with 50 µmol/L NSC23766 showed no cell spreading at 24 and 48 h, as compared to controls (Fig. 5A, first row) or PGE2 treatments (Fig. 5A, second row). Also, explants incubated with 50 µmol/L NSC23766 for 2 h and then challenged with 1 µmol/L PGE2 did not show a restoration of cellular spreading (Fig. 5A, fourth row). Quantification of the areas covered by the migrating cells is represented in Figure 5B as outgrowth area index. These results confirm our earlier findings in EVT cell migration assays that RAC1 is essential for PGE2-mediated migration of first trimester EVT cells.


Figure 05
View larger version (96K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5. The effect of RAC1 inhibitor (NSC23766, 50 µmol/L) with or without PGE2 on first-trimester human cytotrophoblast spreading. A) Chorionic villus explant cultures of first-trimester human placentae were treated without (control, first row) or with NSC23766 (third row), with PGE2 (second row), or with NSC23766 plus PGE2 (fourth row). Arrows indicate spreading of cytotrophoblast cells. B) The graph quantifies the outgrowth area, plotting the outgrowth area of each treatment divided by the area of outgrowth in control experiments using Scion Image Software (see Materials and Methods) (n = 3 ± SEM, *P < 0.0001).

DISCUSSION

We have previously shown that PGE2 stimulates migration of first-trimester EVT cells, acting predominantly through PTGER1 and PTGER4 [11]. While EP1 is a Gq-coupled receptor that is involved in the rise of [Ca2+]i, activations of PLC and PI3K, PTGER4 is a Gs-coupled receptor involving activation of adenylate cyclase. PTGER4, however, is a unique Gs-coupled receptor that also activates PI3K in a cAMP-independent manner [13, 40]. These two receptors in many other cell types have also been shown to modulate mitogen-activated protein kinase1 (MAPK1, ERK1/2, MAPK p38) and PTK2 (protein tyrosine kinase2, also known as FAK or focal adhesion kinase). However, RHO GTPases, though regarded as major players in cellular migration, have never been shown before in any cell type to be modulated by PTGERs. The present study identifies for the first time that the activations of PTGER1 and PTGER4 can activate RAC1 and CDC42, which serve as mediators of PGE2-induced migration of first-trimester human extravillous trophoblast. These results may provide a possible mechanism underlying the hypoinvasive capabilities of the EVT cells in preeclampsia. Indeed, a recent report indicates decreased RAC1 activities in the placentae of women with preeclampsia [41].

Because we have examined activation of both GTPases at 10 min after agonist treatment, we were not able to determine the temporal sequence in the activation of RAC1 and CDC42. CDC42 and RAC1 activations have been shown to induce distinct actin cytoskeletal changes, signaling through either common or divergent pathways, depending on the cell type or specific cellular function [35–37]. In the context of cellular migration, available evidence suggests that activation of CDC42-mediating filopodial extensions may sequentially activate RAC1 that mediate lamellipodial extensions [42, 43]. While we can anticipate that PGE2-mediated migration of EVT cells would require CDC42 activation before RAC1 activation, different other types of cross talks between RAC1 and CDC42 cannot be excluded.

The fact that either PTGER1 or PTGER4 agonist activate both RAC1 and CDC42 may not necessarily mean that the mechanism of activation of these enzymes will be similar in each case. A number of studies indicate that the {alpha} subunit of Gq, which couples with PLC and induces an increase in the intracellular concentration of calcium, can activate p115RHOGEF (ARHGEF1) and, subsequently, RHOA [44–47]. Ligands like fMLP (FPR1, formal peptide receptor 1), bombesin, endothelin-1, and lysophosphatidic acid, signaling either through receptor tyrosine kinases or through GPCRs that activate the {alpha} subunit of Gi, have been shown to induce GEF-dependent activation of RAC1. Cyclic AMP-independent activation via PTGER4 of PI3K/ERK pathway has been documented by Fujino et al. [13, 40]. Active PI3K increases the formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which then can activate TIAM1, PREX1, and other GEFs that subsequently lead to RAC1 activation [48, 49]. Therefore, PTGER4, in either a cAMP-dependent or a cAMP-independent manner through PI3K/ERK pathway, could cause RAC1 activation. However, activation of PTGER2, another Gs-coupled receptor (that does not cause activation of PI3K), or increased intracellular cAMP was found to inhibit RAC1 activity and cellular migration [50, 51]. This may indicate that PTGER4-mediated RAC1 activation in EVT cells is due to the activation of PI3K. Our unpublished data have also shown that either PGE2 or PGE1 Alcohol can induce ERK1/2 phosphorylation in HTR-8/SVneo cells. PTGER4-induced ERK1/2 may in turn stimulate RAC1.

PTGER1-mediated activation of RAC1 and/or CDC42 could be through direct interaction of the G-protein subunits with GEFs, as demonstrated for G{alpha}12/G{alpha}13 and PDZ-Rho GEF (ARHGEF11) [52], or indirectly through [Ca2+]i, PLC, protein kinase C, PI3K, or a combination of some of these mechanisms. Elevated [Ca2+]i and protein kinase C have been shown before to cause activation of RAC1 and its translocation to the plasma membrane causing lamellipodial extensions necessary for cellular migration [53]. Although PTGER4 activation in EVT cells in the present study leads to the activation of CDC42, increased level of intracellular cAMP has been shown to stimulate activation of CDC42 in some other cell types [54, 55]. Therefore, CDC42 activation resulting from both Gq-coupled PTGER1 and Gs-coupled PTGER4 activation is likely to result from increased PI3K activity.

Besides its well-known function during parturition [15], decidua-derived PGE2 plays an important role in the implantation of the blastocyst [14, 56], and derangements in the homeostasis of this prostanoid metabolism have been reported in preeclampsia [18, 57, 58] where decreased levels of PGE2 may contribute to increased peripheral vascular resistance [59]. The present study suggests that PGE2 is an important regulator of EVT cell migration by activating RAC1 and CDC42 activity. Other signaling molecules have also been shown to play important roles in EVT cell migration during placentation [1, 60–62]. In vitro cultures of cytotrophoblast cells from pre-eclamptic pregnancies have shown a decreased activity of matrix metalloproteinase 9 (MMP9) as compared to normal pregnancies, and these cells have been shown to exhibit reduced invasive potential [63]. Other studies have indicated changes in MMP7 and MMP1 expression in placentas from pre-eclamptic pregnancies [64]. RAC1 has been shown to stimulate the activity of MMP2 [65] and MMP9, possibly through a MAPK8, also known as JNK-dependent pathway [41, 66], indicating a possible mechanism of reduced migratory capabilities of EVT cells in preeclampsia. Oxidative stress, an imbalance in the production of reactive oxygen species (ROS), and the ability of antioxidant defenses to scavenge them have important functions on placental development, including trophoblast proliferation and differentiation as well as vascular reactivity [67]. RAC activation has also been linked to ROS generation through activation of NADPH oxidase [68]. Our present data, along with the previous observation that an increased level of ROS is encountered during certain pathologic pregnancies, including preeclampsia [67], and the study that has indicated that RAC1 is downregulated in preeclampsia [41] suggest that RAC1 plays a major role in EVT migration and has a potential contribution to placental development. Results of these studies along with our present study suggest that RAC1 and CDC42 may control the migratory abilities of trophoblast cells, offering new possibilities for further investigation of the pathological mechanisms underlying various pregnancy-related disorders, including preeclampsia.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Mr. Larry W. Stitt for assistance in statistical analysis.

FOOTNOTES

1Supported by grant MOP 68997 (to C.C.) and grant MOP 69091 (to P.K.L.) of the Canadian Institutes of Health Research. C.N. is the recipient of a CIHR/STP scholarship in cancer research at UWO. Back

Correspondence: 2Chandan Chakraborty, Department of Pathology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada. FAX: 519 661 3370; e-mail: cchakrab{at}uwo.ca

Received: 5 September 2007.

First decision: 28 September 2007.

Accepted: 23 January 2008.

REFERENCES

  1. Chakraborty C, Gleeson LM, McKinnon T, Lala PK. Regulation of human trophoblast migration and invasiveness. Can J Physiol Pharmacol 2002; 80:116–124.[CrossRef][Medline]
  2. Meekins JW, Pijnenborg R, Hanssens M, McFadyen IR, van Asshe A. A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies. Br J Obstet Gynaecol 1994; 101:669–674.[Medline]
  3. Naicker T, Khedun SM, Moodley J, Pijnenborg R. Quantitative analysis of trophoblast invasion in preeclampsia. Acta Obstet Gynecol Scand 2003; 82:722–729.[CrossRef][Medline]
  4. de Groot CJ, O'Brien TJ, Taylor RN. Biochemical evidence of impaired trophoblastic invasion of decidual stroma in women destined to have preeclampsia. Am J Obstet Gynecol 1996; 175:24–29.[CrossRef][Medline]
  5. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, Lala PK. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 1993; 206:204–211.[CrossRef][Medline]
  6. McKinnon T, Chakraborty C, Gleeson LM, Chidiac P, Lala PK. Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK. J Clin Endocrinol Metab 2001; 86:3665–3674.[Abstract/Free Full Text]
  7. Gleeson LM, Chakraborty C, McKinnon T, Lala PK. Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through {alpha}5β1 integrin via mitogen-activated protein kinase pathway. J Clin Endocrinol Metab 2001; 86:2484–2493.[Abstract/Free Full Text]
  8. Chakraborty C, Barbin YP, Chakrabarti S, Chidiac P, Dixon SJ, Lala PK. Endothelin-1 promotes migration and induces elevation of [Ca2+]i and phosphorylation of MAP kinase of a human extravillous trophoblast cell line*1. Mol Cell Endocrinol 2003; 201:63–73.[CrossRef][Medline]
  9. Liu J, Chakraborty C, Graham CH, Barbin YP, Dixon SJ, Lala PK. Noncatalytic domain of uPA stimulates human extravillous trophoblast migration by using phospholipase C, phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Exp Cell Res 2003; 286:138–151.[CrossRef][Medline]
  10. Xu G, Guimond MJ, Chakraborty C, Lala PK. Control of proliferation, migration, and invasiveness of human extravillous trophoblast by decorin, a decidual product. Biol Reprod 2002; 67:681–689.[Abstract/Free Full Text]
  11. Nicola C, Timoshenko AV, Dixon SJ, Lala PK, Chakraborty C. EP1 receptor-mediated migration of the first trimester human extravillous trophoblast: the role of intracellular calcium and calpain. J Clin Endocrinol Metab 2005; 90:4736–4746.[Abstract/Free Full Text]
  12. Bos CL, Richel DJ, Ritsema T, Peppelenbosch MP, Versteeg HH. Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 2004; 36:1187–1205.[CrossRef][Medline]
  13. Fujino H, West KA, Regan JW. Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. J Biol Chem 2002; 277:2614–2619.[Abstract/Free Full Text]
  14. Psychoyos A, Nikas G, Gravanis A. The role of prostaglandins in blastocyst implantation. Hum Reprod 1995; 10(suppl 2):30–42.
  15. Keelan JA, Blumenstein M, Helliwell RJ, Sato TA, Marvin KW, Mitchell MD. Cytokines, prostaglandins and parturition—a review. Placenta 2003; 24(suppl A):S33–S46.[CrossRef][Medline]
  16. Lala PK, Kennedy TG, Parhar RS. Suppression of lymphocyte alloreactivity by early gestational human decidua. II. Characterization of the suppressor mechanisms. Cell Immunol 1988; 116:411–422.[CrossRef][Medline]
  17. Parhar RS, Kennedy TG, Lala PK. Suppression of lymphocyte alloreactivity by early gestational human decidua. I. Characterization of suppressor cells and suppressor molecules. Cell Immunol 1988; 116:392–410.[CrossRef][Medline]
  18. Herrera JA, Revalo-Herrera M, Herrera S. Prevention of preeclampsia by linoleic acid and calcium supplementation: a randomized controlled trial. Obstet Gynecol 1998; 91:585–590.[CrossRef][Medline]
  19. Aspenstrom P, Fransson A, Saras J. Rho GTPases have diverse effects on the organization of the actin filament system. Biochem J 2004; 377:327–337.[CrossRef][Medline]
  20. Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 2005; 21:247–269.[CrossRef][Medline]
  21. Etienne-Manneville S. Actin and microtubules in cell motility: which one is in control? Traffic 2004; 5:470–477.[CrossRef][Medline]
  22. Irving J, Lysiak J, Graham CH, Hearn S, Han V, Lala PK. Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture. Placenta 1995; 16:413–433.[CrossRef][Medline]
  23. Zdravkovic M, Boagye-Mathiesen G, Guimond M-J, Hager H, Ebbesen P, Lala PK. Susceptibility of MHC class i expressing extravillous trophoblast cell lines to killing by natural killer cells. Placenta 1999; 20:431–440.[CrossRef][Medline]
  24. Kilburn BA, Wang J, Duniec-Dmuchkowski ZM, Leach RE, Romero R, Armant DR. Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line. Biol Reprod 2000; 62:739–747.[Abstract/Free Full Text]
  25. Noritake J, Fukata M, Sato K, Nakagawa M, Watanabe T, Izumi N, Wang S, Fukata Y, Kaibuchi K. Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact. Mol Biol Cell 2004; 15:1065–1076.[Abstract/Free Full Text]
  26. Wilkinson S, Paterson HF, Marshall CJ. Cdc42-MRCK and Rho-ROCK signalling cooperate in myosin phosphorylation and cell invasion. Nat Cell Biol 2005; 7:255–261.[CrossRef][Medline]
  27. Zuo Y, Shields SK, Chakraborty C. Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase. Biochem Biophys Res Commun 2006; 351:361–367.[CrossRef][Medline]
  28. Benard V, Bokoch GM. Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol 2002; 345:349–359.[CrossRef][Medline]
  29. Desire L, Bourdin J, Loiseau N, Peillon H, Picard V, De OC, Bachelot F, Leblond B, Taverne T, Beausoleil E, Lacombe S, Drouin D, Schweighoffer F. RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo. J Biol Chem 2005; 280:37516–37525.[Abstract/Free Full Text]
  30. Akbar H, Cancelas J, Williams DA, Zheng J, Zheng Y. Rational design and applications of a Rac GTPase-specific small molecule inhibitor. Methods Enzymol 2006; 406:554–565.[Medline]
  31. Didsbury J, Weber RF, Bokoch GM, Evans T, Snyderman R. Rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem 1989; 264:16378–16382.[Abstract/Free Full Text]
  32. Haataja L, Groffen J, Heisterkamp N. Characterization of RAC3, a novel member of the Rho family. J Biol Chem 1997; 272:20384–20388.[Abstract/Free Full Text]
  33. Shields SK, Nicola C, Chakraborty C. Rho guanosine 5'-triphosphatases differentially regulate insulin-like growth factor I (IGF-I) receptor-dependent and -independent actions of IGF-II on human trophoblast migration. Endocrinology 2007; 148:4906–4917.[Abstract/Free Full Text]
  34. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 2005; 6:167–180.[CrossRef][Medline]
  35. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev 2001; 81:153–208.[Abstract/Free Full Text]
  36. Baird D, Feng Q, Cerione RA. The Cool-2/alpha-Pix protein mediates a Cdc42-Rac signaling cascade. Curr Biol 2005; 15:1–10.[CrossRef][Medline]
  37. ten Klooster JP, Jaffer ZM, Chernoff J, Hordijk PL. Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix. J Cell Biol 2006; 172:759–769.[Abstract/Free Full Text]
  38. Ridley AJ. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 2006; 16:522–529.[CrossRef][Medline]
  39. Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J 2000; 348(pt 2):241–255.[CrossRef][Medline]
  40. Fujino H, Xu W, Regan JW. Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. J Biol Chem 2003; 278:12151–12156.[Abstract/Free Full Text]
  41. Hannke-Lohmann A, Pildner von SS, Dehne K, Benard V, Kolben M, Schmitt M, Lengyel E. Downregulation of a mitogen-activated protein kinase signaling pathway in the placentas of women with preeclampsia. Obstet Gynecol 2000; 96:582–587.[CrossRef][Medline]
  42. Puls A, Eliopoulos AG, Nobes CD, Bridges T, Young LS, Hall A. Activation of the small GTPase Cdc42 by the inflammatory cytokines TNF(alpha) and IL-1, and by the Epstein-Barr virus transforming protein LMP1. J Cell Sci 1999; 112(pt 17):2983–2992.[Abstract]
  43. Nobes CD, Hall A. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 1999; 144:1235–1244.[Abstract/Free Full Text]
  44. Chikumi H, Vazquez-Prado J, Servitja JM, Miyazaki H, Gutkind JS. Potent activation of RhoA by Galpha q and Gq-coupled receptors. J Biol Chem 2002; 277:27130–27134.[Abstract/Free Full Text]
  45. Booden MA, Siderovski DP, Der CJ. Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA. Mol Cell Biol 2002; 22:4053–4061.[Abstract/Free Full Text]
  46. Vogt S, Grosse R, Schultz G, Offermanns S. Receptor-dependent RhoA activation in G12/G13-deficient cells: genetic evidence for an involvement of Gq/G11. J Biol Chem 2003; 278:28743–28749.[Abstract/Free Full Text]
  47. Lutz S, Freichel-Blomquist A, Yang Y, Rumenapp U, Jakobs KH, Schmidt M, Wieland T. The guanine nucleotide exchange factor p63RhoGEF, a specific link between Gq/11-coupled receptor signaling and RhoA. J Biol Chem 2005; 280:11134–11139.[Abstract/Free Full Text]
  48. Burridge K, Wennerberg K. Rho and Rac take center stage. Cell 2004; 116:167–179.[CrossRef][Medline]
  49. Welch HC, Coadwell WJ, Stephens LR, Hawkins PT. Phosphoinositide 3-kinase-dependent activation of Rac. FEBS Lett 2003; 546:93–97.[CrossRef][Medline]
  50. Nagasawa SY, Takuwa N, Sugimoto N, Mabuchi H, Takuwa Y. Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP. Biochem J 2005; 385:737–744.[CrossRef][Medline]
  51. Pelletier S, Julien C, Popoff MR, Lamarche-Vane N, Meloche S. Cyclic AMP induces morphological changes of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway. J Cell Physiol 2005; 204:412–422.[CrossRef][Medline]
  52. Fukuhara S, Murga C, Zohar M, Igishi T, Gutkind JS. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J Biol Chem 1999; 274:5868–5879.[Abstract/Free Full Text]
  53. Price LS, Langeslag M, ten Klooster JP, Hordijk PL, Jalink K, Collard JG. Calcium signaling regulates translocation and activation of Rac. J Biol Chem 2003; 278:39413–39421.[Abstract/Free Full Text]
  54. Feoktistov I, Goldstein AE, Biaggioni I. Cyclic AMP and protein kinase A stimulate Cdc42: role of A(2) adenosine receptors in human mast cells. Mol Pharmacol 2000; 58:903–910.[Abstract/Free Full Text]
  55. Chahdi A, Sorokin A. Endothelin 1 stimulates beta1Pix-dependent activation of Cdc42 through the G(salpha) pathway. Exp Biol Med (Maywood 2006; 231:761–765.[Abstract/Free Full Text]
  56. Yee GM, Squires PM, Cejic SS, Kennedy TG. Lipid mediators of implantation and decidualization. J Lipid Mediat 1993; 6:525–534.[Medline]
  57. Friedman SA. Preeclampsia: a review of the role of prostaglandins. Obstet Gynecol 1988; 71:122–137.[Medline]
  58. Chavarria ME, Lara-Gonzalez L, Gonzalez-Gleason A, Garcia-Paleta Y, Vital-Reyes VS, Reyes A. Prostacyclin/thromboxane early changes in pregnancies that are complicated by preeclampsia. Am J Obstet Gynecol 2003; 188:986–992.[CrossRef][Medline]
  59. Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol Rev 1978; 30:293–331.[Medline]
  60. Caniggia I, Grisaru-Gravnosky S, Kuliszewsky M, Post M, Lye SJ. Inhibition of TGF-β3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies. J Clin Invest 1999; 103:1641–1650.[Medline]
  61. Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, Kaufmann P. Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation. Placenta 2003; 24:181–190.[CrossRef][Medline]
  62. Lala PK, Chakraborty C. Factors regulating trophoblast migration and invasiveness: possible derangements contributing to pre-eclampsia and fetal injury. Placenta 2003; 24:575–587.[CrossRef][Medline]
  63. Lim KH, Zhou Y, Janatpour M, McMaster M, Bass K, Chun SH, Fisher SJ. Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia. Am J Pathol 1997; 151:1809–1818.[Abstract]
  64. Lyall F. Mechanisms regulating cytotrophoblast invasion in normal pregnancy and pre-eclampsia. Aust N Z J Obstet Gynaecol 2006; 46:266–273.[CrossRef][Medline]
  65. Laeeq S, Faust R. Modeling the cholesteatoma microenvironment: coculture of HaCaT keratinocytes with WS1 fibroblasts induces MMP-2 activation, invasive phenotype, and proteolysis of the extracellular matrix. Laryngoscope 2007; 117:313–318.[CrossRef][Medline]
  66. Gum R, Wang H, Lengyel E, Juarez J, Boyd D. Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene 1997; 14:1481–1493.[CrossRef][Medline]
  67. Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol 2004; 122:369–382.[CrossRef][Medline]
  68. Brown JH, Del Re DP, Sussman MA. The Rac and Rho hall of fame: a decade of hypertrophic signaling hits. Circ Res 2006; 98:730–742.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/6/976    most recent
biolreprod.107.065433v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nicola, C.
Right arrow Articles by Chakraborty, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nicola, C.
Right arrow Articles by Chakraborty, C.
Agricola
Right arrow Articles by Nicola, C.
Right arrow Articles by Chakraborty, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS