help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0476
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shields, S.-K.
Right arrow Articles by Chakraborty, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shields, S.-K.
Right arrow Articles by Chakraborty, C.
Endocrinology Vol. 148, No. 10 4906-4917
Copyright © 2007 by The Endocrine Society

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

Sarah-Kim Shields, Catalin Nicola and Chandan Chakraborty

Departments of Pathology (S.-K.S., C.C.) and Anatomy and Cell Biology (C.N.), Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1

Address all correspondence and requests for reprints to: Dr. Chandan Chakraborty, Department of Pathology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: cchakrab{at}uwo.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both IGF-I and IGF-II stimulate migration of human extravillous trophoblast (EVT) cells. Although IGF-I is known to signal through IGF type 1 receptor (IGF1R), IGF-II signals through IGF1R as well as in an IGF1R-independent manner. The purpose of this study was to investigate the roles of Rho GTPases in IGF1R-independent and -dependent actions of IGF-II on EVT cell migration. To distinguish IGF1R-dependent and -independent actions, we used picropodophyllin, a selective inhibitor of IGF1R tyrosine kinase, and IGF analogs with differential affinities for IGF1R, IGF-II/cation-independent mannose 6-phosphate receptor, and IGF-binding proteins. IGF1R-dependent actions of IGF-II were confirmed by showing the effects of IGF1R-selective agonist Des1–3 IGF-I. We used pharmacological inhibitors or selective small interfering RNAs to investigate the roles of RhoA, RhoC, Rac1, Cdc42, and Rho effector kinases called ROCK-I and -II in IGF-induced EVT cell migration. Although basal migration of EVT cells required each member of the Rho GTPase family studied, IGF1R-dependent and -independent EVT cell migration exhibited differential requirements for these enzymes. IGF1R-mediated EVT cell migration was found to depend on RhoA and RhoC but not on Rac1 or Cdc42. However, IGF1R-independent effect of IGF-II on EVT cell migration required ROCKs but not RhoA, RhoC, Rac1, or Cdc42. Most importantly, IGF1R-independent action of IGF-II was found to be exaggerated when RhoA or RhoC was down-regulated. Thus, different members of the Rho GTPase family regulate IGF-II-mediated EVT cell migration differentially, depending upon whether it signals through IGF1R or in an IGF1R-independent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN PLACENTA contains a migratory/invasive subpopulation of trophoblast cells, known as extravillous trophoblast (EVT). Migration of these cells, like other migratory cells, is thought to be initiated by factors produced by the same cell type or neighboring cell types. IGF-II, produced by trophoblast cells, has been regarded to be important for placental development in the human as well as other placental mammals and, therefore, is important for fetal growth. Whereas igf2-null mutations in the mouse cause placental and fetal growth retardation (1, 2, 3, 4), its overexpression causes placental and fetal overgrowth (5). Furthermore, placentas from preeclamptic pregnancies in women were found to exhibit increased IGF-II expression in EVT cells surrounding placental infarcts, suggesting a role in placental remodeling (6). In healthy pregnancies, IGF-II was found to be abundantly expressed by the EVT cells at the invading front of the anchoring chorionic villi of the placenta (7), indicating its potential role in invasion. Indeed, IGF-II was shown to stimulate migration of EVT cells in vitro (8, 9). These effects of IGF-II were similar in cells migrating on two- and three-dimensional matrices (8). Furthermore, extracellular matrix-degrading enzymes were unaffected by IGF-II. These results suggest that IGF-II stimulates EVT cell invasion by increasing migration.

Both IGF-I and IGF-II propagate intracellular signals after binding with tyrosine kinase IGF type 1 receptor (IGF1R) on the cell surface, although IGF-II binds with much lower affinity than IGF-I. IGF-II, on the other hand, can bind with high affinity to a nonclassical receptor called IGF-II/cation-independent mannose 6-phosphate receptor (IGF-II/CI-M6PR). IGF-II/CI-M6PR is neither a seven-transmembrane domain G protein-coupled receptor nor a kinase-containing receptor (10). Signaling function of this receptor is controversial. However, several investigators have provided evidence for signaling events through IGF-II/CI-M6PR (9, 10, 11, 12, 13, 14). Interaction of IGF-II with IGF-II/CI-M6PR has been postulated to activate Gi protein(s) and trigger the MAPK (ERK1/2) signaling cascade during migration of EVT and endothelial cells (9, 11, 12). IGF-II has also been shown to bind to the insulin receptor (IR)-A isoform with strong affinity, activate its tyrosine kinase, and induce proliferation and migration of several cell types (15, 16). The present study was designed to further investigate the intracellular signaling events underlying IGF1R-dependent and -independent actions of IGF-II on EVT cell migration.

Rho GTPases are members of the Ras superfamily of small (20–30 kDa) monomeric guanine nucleotide-binding proteins. The Rho subfamily includes RhoA, -B, and -C isoforms; the Rac subfamily includes Rac1, -2, and -3 isoforms; and the Cdc42 subfamily includes Cdc42Hs and -G25K isoforms. Many other members of the Rho GTPase family have been identified (17); however, the most well characterized members are RhoA, Rac1, and Cdc42. Rho GTPases cycle between an inactive GDP-bound state and an active GTP-bound state. Most of these members play crucial roles in cell migration. For example, active RhoA or RhoC induces formation of focal adhesion complexes and stress fibers. Active Rac promotes the formation of broad cell extensions or lamellipodia, whereas active Cdc42 promotes the extension of long and thin microspikes (filopodia), both at the leading edge. Active RhoA, RhoB, and RhoC interact with a number of downstream effectors, the most important being Rho-associated coiled-coil protein kinase, or Rho kinase (ROCK). Activation of ROCK leads to phosphorylation of myosin light chain (MLC) and MLC phosphatase. These phosphorylated molecules in turn lead to myosin filament assembly and F-actin bundling, resulting in stress fiber formation (18, 19). Rac and Cdc42 interact with a common target, p21-activated kinase (Pak1), whose activation can lead to phosphorylation of MAPK kinase 1, thus activating the MAPK pathway. A great deal is known about the role of Rho GTPases in actin cytoskeletal rearrangements during cellular migration, and their regulation by many receptor tyrosine kinases and G protein-coupled receptors (18, 19). However, nothing is known about the role of Rho GTPases in receptor-mediated cellular functions triggered by IGF-II. We hypothesized that Rho GTPases play important roles in IGF-II-mediated EVT cell migration and that these effects of IGF-II are mediated through both IGF1R and other receptors. The present study investigated the roles of Rho GTPases and Rho kinases in basal and IGF-II-induced EVT cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
RPMI 1640 and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. (Burlington, Canada) and BSA from Sigma (Oakville, Canada). Recombinant human IGF-II, [Leu27] IGF-II (L27 IGF-II), [Arg6] IGF-II (R6 IGF-II), Des1–6 IGF-II, and Des1–3 IGF-I were purchased from GroPep (Adelaide, Australia). Human insulin was obtained from Eli Lilly (Indianapolis, IN). IGF1R inhibitor picropodophyllin (PPP) and duel inhibitor of IGF1R and IR tyrphostin (AG1024) were purchased from Calbiochem (La Jolla, CA). Rho kinase inhibitor Y27632 was purchased from Biomol International, LP (Plymouth Meeting, PA). NSC23766, which inhibits Rac1, was purchased from Calbiochem. Multiporous transwells were purchased from Costar (Corning, NY). WST-1 cell survival kit was obtained from Roche Diagnostics (Laval, Canada). RhoA, -B, and -C inhibitor Clostridium botulinum exoenzyme C3 transferase (cell permeable) and a luminescence based G-LISA RhoA activation assay kit were obtained from Cytoskeleton Inc. (Denver, CO). EZ-Detect Rac1 and Cdc42 activation kits, which detect active small GTPases, were purchased from Pierce (Rockford, IL). SYBR Jump Start Taq Ready Mix for real-time PCR was obtained from Sigma (St. Louis, MO). Anti-RhoA (mouse monoclonal) and anti-RhoC (goat polyclonal) IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Cdc42 (mouse monoclonal) and anti-ROCK-I and anti-ROCK-II (rabbit polyclonal) IgGs were from Chemicon International (Temecula, CA), anti-Rac1 (mouse monoclonal) IgG was from UpState Biotechnology, Inc. (Lake Placid, NY), and anti-GAPDH IgG was from HyTest Ltd. (Turku, Finland). Peroxidase-labeled goat antirabbit, rabbit antimouse, and rabbit antigoat IgG were purchased from Vector Laboratories Inc. (Burlingame, CA). Small interfering RNA (siRNA) oligonucleotide duplexes specific for human RhoA, RhoC, Rac1, Cdc42, ROCK-I, and ROCK-II and DharmaFECT transfecting reagent were obtained from Dharmacon Inc. (Lafayette, CO). Alexa Fluor 488 phalloidin, which binds F-actin, and Toto-3 iodide 642/660, which binds nucleic acids, were from Invitrogen Molecular Probes (Eugene, OR), and Vectashield mounting medium was obtained from Vector.

Human trophoblast cell line
The present study used the human EVT cell line HTR-8/SVneo produced by Graham et al. (20) and obtained from Dr. P. K. Lala. A short-lived, first-trimester EVT cell line (HTR-8) made from explants of chorionic villi from human placentas has been immortalized by simian virus 40 large tumor antigen transfection to produce HTR-8/SVneo cells. HTR-8/SVneo cells show the same phenotypic and functional characteristics as the parental cell line. They were shown to express on several different occasions their signature markers such as cytokeratin 7, 8, and 18; several integrins ({alpha}1, {alpha}3, {alpha}5, ß1, {alpha}vß3, and {alpha}vß5 but not {alpha}6ß4), IGF-II mRNA and protein, urokinase type plasminogen activator, HLA framework antigen, and placental-type alkaline phosphatase. Also, these cells were negative for macrophage marker 63/D3 and endothelial cell marker factor VIII. Finally, when grown on laminin or Matrigel, these cells expressed HLA-G mRNA. They were also shown to be nontumorigenic in nude mice and to require anchorage for growth. The present work used HTR-8/SVneo cells, at passages 85–130, grown in RPMI 1640 complete medium (with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin supplements). Cells within these passages were verified by immunostaining for expressions of two important markers of EVT cell, i.e. cytokeratin 7 and {alpha}5ß1 integrins. For experiments under serum-free conditions, cells were cultured in RPMI 1640 with 0.1% BSA [serum-free medium (SFM)], a concentration that does not have any nonspecific effect on survival of these EVT cells.

Migration assay
Chemokinetic migrations of HTR-8/SVneo cells were assessed following the protocol used before (21). EVT cells (100 µl cell suspension containing 1 x 105 cells/ml) were plated on the upper wells of the Transwell chambers in the presence or absence of the test agent(s) in SFM to a final volume of 200 µl in the upper well. The respective lower wells of the Transwell chambers, in which the upper wells were immersed, contained the same agent(s), in equal concentration(s), as the upper well, with a total volume of 800 µl. Various IGFs were added, and cells were incubated at 37 C in 5% CO2 and allowed to migrate for 24 h. At 24 h, all the media were discarded, nonmigrant cells were gently swabbed off the insert membranes, and then the remaining migratory cells were fixed and stained using the Harleco Hematocolor staining kit (EM Science, Gibbstown, NJ). Migration scores were obtained by counting total migratory cells on each insert membrane using a light microscope (x400 magnification). Each assay was performed in triplicate and reproduced on two to three different occasions. The actual number of cells that migrated varied from day to day; for example, the number of cells migrated in control experiments varied from 84 ± 3 to 258 ± 17. However, the extent of stimulation or inhibition due to different treatments did not vary between days. For this reason, migration assay results were expressed as migration index (i.e. relative change with respect to control).

Human recombinant IGF-II was previously shown to promote EVT cell migration, with optimal effects at concentrations of 7–10 nmol/liter (9). This study, therefore, used a concentration of 10 nmol/liter to investigate the possible involvement of Rho GTPases downstream of IGF-II. The same concentration of the other IGF-II analogs was also used for comparison. We found that ROCK inhibitor Y27632 at 20 µmol/liter was more effective than 10 µmol/liter concentration to inhibit EVT cell migration. Therefore, for all the experiments that used Y27632, 20 µmol/liter was chosen as the optimal concentration. Use of this concentration of Y27632 for the purpose of ROCK inhibition in EVT cells is in favor of its reported IC50 value of 5 µmol/liter for inhibition of Rho kinase and migration of fibroblasts (22). C3 exoenzyme, at the concentration of 800 nmol/liter, has been recommended by the supplier for the purpose of inhibition of RhoA, RhoB, and RhoC (23). After performing a number of time-course studies, we determined that the cells, after a pretreatment with Y27632 for 30 min and with C3 exoenzyme for 3 h at the concentrations indicated above, exhibited distinct actin cytoskeletal perturbations, which were comparable with those of 24 h treatment. The cells were found to be viable at all time points. Therefore, we chose these time points to examine the effects of Y27632 and C3 exoenzyme in IGF-mediated migration of EVT cells. To investigate the effect of Rac1 inhibition on IGF-induced EVT cell migration, we used 100 µmol/liter NSC23766 because we have previously shown this concentration to be optimal for inhibition of Rac1 GTPase and cellular migration (24). Migration experiments in EVT cells with all these inhibitors have further been confirmed by repeating the same experiments in cells where individual enzymes in the Rho GTPase family were down-regulated by the respective siRNAs.

Results of each migration assay experiment were validated by examining the effect of each of the treatments that were used for migration assays on the proliferation/survival of HTR-8/SVneo cells using WST-1 assay (Roche Diagnostics). Like the migration assays, these assays were reproduced on two to three different occasions with three replicates on every occasion. None of the test substances was found to have any significant effect on proliferation/survival of these cells when these assays were performed under the same experimental conditions as those of the migration assays.

siRNA preparation and transfection
siRNA oligonucleotide duplexes specific for human RhoA and RhoC were designed according to Pille et al. (25); those specific for human Rac1 were designed according to Noritake et al. (26); those specific for Cdc42 according to Wilkinson et al. (27); and ROCK-I and -II 21-nucleotide siRNAs were purchased from Dharmacon (SiGenome reagents D-003536-05 and D-004610-05, respectively) in deprotected and desalted form. The siRNA sequences with dTdT overhanging at their 3' terminus are presented in Table 1Go. Control siRNA, which is a pool of several control siRNAs from Dharmacon, served as controls of all the siRNA experiments in the present study. Transfection of test siRNA or control siRNA was carried out using DharmaFECT (Dharmacon). Cells were seeded and grown in six-well plates at 1 x 105 cells per well in 2 ml RPMI 1640 supplemented with 10% FBS. For transfection, 4 µl/ml DharmaFECT was diluted in SFM without antibiotics. siRNA was also diluted in SFM without antibiotics. Both were preincubated for 5 min, after which the two mixtures were combined and incubated at room temperature for 20 min for complex formation. Finally, RPMI 1640 (10% FBS) was added to the mixture for 100 nmol/liter final concentration of siRNA (as recommended by Dharmacon), and a total volume of 2 ml was added to each well. Transfections were performed for 48 h.


View this table:
[in this window]
[in a new window]

  		TABLE 1. The siRNA sequences with their respective target genes

 
RNA isolation and real-time-PCR analyses of cDNAs
Total RNA from siRNA-transfected HTR-8/SVneo cells was isolated using Trizol reagent following the manufacturer’s protocol. cDNA was synthesized using 3 µg total RNA primed with oligo(dT) as described in SuperScript (Invitrogen). Quantitative PCR analyses for ROCK-I, ROCK-II, RhoA, RhoC, Rac1, Cdc42, and GAPDH were then performed using the SYBR Green JumpStart Taq ReadyMix (Sigma, St. Louis, MO) in single microcapillary tubes using the LightCycler (Roche Diagnostics). The specific primer pairs for the respective genes used for cDNA amplifications are presented in Table 2Go. Cycling parameters were optimized as follows: denaturation at 95 C (0 sec), annealing at 55 C (5 sec), extension at 72 C (24 sec), and detection at 80 C (1 sec). Each microcapillary contained 10 µl SYBR reagent, 2 µl template cDNA, 1.6 µl 25 nmol/liter MgCl2, and 0.8 µl 25 pmol/µl primer mixture. The value of each mRNA level was normalized to the GAPDH mRNA level using the Cycler software to account for the differences in RT efficiencies and the amount of template in the reaction mixture.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Sequences of primers used for amplifications of the cDNAs by real-time PCR

 
Rho GTPases activation assays
To measure RhoA activity, cell lysates were immediately frozen in liquid N2 and stored at –70 C until the assay was performed. RhoA activity was measured using a luminescence-based G-LISA kit from Cytoskeleton, Inc., following the manufacturer’s protocol as described before (24).

Rac1 and Cdc42 activities were determined using the pull-down assay kits from Pierce following their instructions (26). In brief, 50 µg clarified cell lysate was incubated with GST-Pak1-PBD in resins at 4 C for 1 h in a spin column, centrifuged, washed, and eluted in SDS-PAGE buffer. These eluted samples were run on a 4–16% separating gel on PAGE and subjected to Western blot analyses using antibodies against Rac1 or Cdc42.

Western blotting
Either the samples from Rac1 or Cdc42 activation assays or cell extracts from siRNA-transfected cells (20 µg/lane) were denatured and loaded onto SDS-PAGE gels. Then, the proteins were resolved at 120 V for 1–2 h using the MiniProtean-3 kit (Bio-Rad, Hercules, CA). The separated proteins were then transferred onto Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad) at constant current (200 mA) for 1 h, on ice.

Nonspecific proteins on the polyvinylidene difluoride membranes were blocked for 1 h using 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.1% Tween 20. Primary antibodies to the Rho GTPases were diluted in 1x TBS, 0.1% Tween 20 with 5% BSA in the following ratios: anti-RhoA and anti-RhoC (1:200), anti-Rac1 (1:1000), anti-Cdc42 (1:250), anti-ROCK-I (1:2000), anti-ROCK-II (1:500), and anti-GAPDH antibodies (1:10,000, clone Mab 6C5; HyTest) and then incubated overnight at 4 C. After three washes in TBS with 0.1% Tween 20, the blots were incubated with horseradish peroxidase-conjugated goat antimouse IgG (for RhoA, Rac1, and Cdc42) or rabbit antigoat IgG (for RhoC) or goat antirabbit IgG (for ROCKs) (1:20,000 in 5% milk in TBS-Tween 20) secondary antibodies (Cedarlane Laboratories, Hornby, Ontario, Canada). Detection was performed using enhanced chemiluminescence (ECL plus Western blotting detection system; Amersham, Oakville, Ontario, Canada).

Immunofluorescent staining for F-actin
Cells were seeded on 12-mm glass coverslips, allowed to attach overnight in complete medium (RPMI 1640 supplemented with 10% FBS), and then pretreated in SFM with 800 nmol/liter C3 exoenzyme (3 h) or 20 µmol/liter Y27632 (30 min) or only vehicle. They were then treated with PPP or only vehicle for 30 min followed by R6 IGF-II for 2 h. The cells were then washed, fixed in 4% formaldehyde in PBS (10 min), washed with PBS, permeabilized with 0.1% Triton-X in PBS (15 min), and washed again. These cells were incubated with Alexa Fluor 488 phalloidin (40 min) to stain F-actin and with Toto-3 iodide 642/660 (5 min) to stain nucleic acids (Molecular Probes). Cover slips were mounted in Vectashield mounting medium (Vector). Images were captured using confocal microscopy at x40 magnification.

Statistical analysis
Statistical analyses were performed using SPSS software (version 12.0; SPSS, Chicago, IL). Migration and proliferation assay results were converted to indices by normalizing each value as a percentage of the control. All the results were expressed as means ± SEM. Fold increases of migration indices in IGF-II- or its analog-treated cells compared with controls without inhibitor treatments were compared with those with inhibitor treatments, and the significant differences between these two groups were identified using two-way ANOVA. Dunnett’s test was performed for comparing each experimental mean with the control mean. Student’s t tests were used to compare the mRNA levels after gene-specific siRNA transfections with those of their respective controls, as obtained by real-time RT-PCR. Differences at P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential effects of Y27632 or C3 exoenzyme on migration of EVT cells induced by IGF-II and its analogs
Migration assays were performed in the presence or absence of either Y27632 (20 µmol/liter), an inhibitor of Rho kinases ROCK-I and -II, or C. botulinum C3 exoenzyme (800 nmol/liter), which inhibits Rho activity by ADP ribosylation of asparagine 41, which is conserved in RhoA, RhoB, and RhoC but not Rac1 or Cdc42 (23, 28). These inhibitors significantly reduced basal EVT cell migration, Y27632 by 50% and C3 exoenzyme by over 25% (Fig. 1Go, A and E), suggesting that Rho (-A, -B, and -C) as well their effector ROCK are required for basal EVT cell migration. We next investigated whether these enzymes were required by IGF-II to stimulate EVT cell migration. Pretreatment of the cells with Y27632 increased the extent (i.e. fold stimulation) of IGF-II-induced migration (P < 0.01) (Fig. 1AGo). This suggests that IGF-II does not require active Rho kinase for its stimulatory effect on EVT cell migration. Similarly, the extent of stimulation of migration by IGF-II in C3 exoenzyme-treated cells was also more pronounced than in the cells that were not treated with C3 exoenzyme (Fig. 1EGo), suggesting that IGF-II does not require Rho to stimulate migration of EVT cells. However, because IGF-II is known to have strong affinity for IGF1R, IGF-II/CI-M6PR, IR-A, and IGF-binding proteins (IGFBPs), it was difficult to make any conclusion from this set of experiments regarding the type(s) of receptor(s) mediating these actions of IGF-II.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 1. Effects of IGF-II or its analogs on migration of EVT cells after ROCK inhibition by Y27632 treatment and after Rho inhibition by C3 exoenzyme treatment. Fold increase after treatment of IGF-II or its analogs in Y27632-treated cells is either significantly more than (A) or equal to (B) or less than (C and D) that in untreated cells. Fold increase after treatment of IGF-II or its analogs in C3 exoenzyme-treated cells is always significantly more (E–H) than in untreated cells. Inhibition of ROCK significantly impedes L27IGF-II-mediated effects on EVT migration, but inhibition of Rho does not prevent IGF-II or any of its analogs from promoting migration. Values are the mean ± SEM of triplicate determinations. The experiments were reproduced three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control.

 
To control for potential interactions of IGF-II with IGF1R or IGFBPs, and to examine possible involvement of IGF-II/CI-M6PR in IGF-II-mediated cell migration, we used IGF-II analogs of differential affinities for IGF-II/CI-M6PR, IGF1R, and IGFBPs. We previously provided evidence for the predominance of IGF-II/CI-M6PR in mediating IGF-II-induced EVT cell migration (9). However, these migration assays were performed in the presence of 1% FBS. In the present study, migration assays were performed under serum-free conditions to avoid interference due to any confounding factor present in the serum. Under serum-free conditions, IGF-I/IGF1R interactions have also been demonstrated to be involved in EVT cell migration (29, 30). We used three IGF-II analogs in the present study: R6 IGF-II, Des1–6 IGF-II, and L27 IGF-II. As tested in a variety of cell lines, R6 IGF-II has 2- to 3-fold more and Des1–6 IGF-II has 2- to 4-fold less biological potency to bind with CI-M6P/IGIIR when compared with IGF-II (31, 32, 33, 34). Again, R6 IGF-II has 60- to 120-fold less and Des1–6 IGF-II has 150–300-fold less potency to bind with IGFBPs when compared with IGF-II (31, 33). But both these analogs can also bind strongly with IGF1R (31, 32, 33, 34). L27 IGF-II, on the other hand, although has strong affinity to IGFBPs, has 25-fold reduced affinity to IGF1R but only 3-fold reduced affinity to CI-M6P/IGF-IIR as compared with IGF-II (32, 33, 34). We found that the extent of migration stimulation by R6 IGF-II and Des1–6 IGF-II in control cells was more than that by L27 IGF-II (Fig. 1Go, B–D and F–H), indicating that IGF1R accounted for a significant component of the observed effect on EVT cell migration by IGF-II. Some of the L27 IGF-II effects could also be tempered by IGFBPs made by the EVT cells. The effects of Des1–6 IGF-II (Fig. 1Go, C and G), on the other hand, were IGFBP independent, although its M6P/IGF-IIR-mediated effects were most likely to be confounded more by IGF1R than the other two analogs. The extent of EVT cell migration stimulation by R6 IGF-II and Des1–6 IGF-II under ROCK inhibition by Y27632 were either equal to (Fig. 1BGo) or less than (Fig. 1CGo) those in control cells (Fig. 1Go, B and C). However, L27 IGF-II, the most selective IGF-II analog toward IGF-II/CI-M6PR, failed to stimulate migration in Y27632-treated cells (Fig. 1DGo). Under Rho inhibition by C3 exoenzyme, however, each of the IGF-II analogs was found to promote migration (Fig. 1Go, F–H), and the extent of migration stimulation by each of the analogs was significantly higher in C3 exoenzyme-treated cells than in control cells. Interestingly, the most selective M6P/IGFRII-agonist, L27 IGF-II, which is least potent among all the IGF-II analogs to stimulate migration of the EVT cells that were not pretreated with any inhibitor (Fig. 1Go, A–H), became highly efficient to stimulate migration of EVT cells in which Rho was inhibited by C3 exoenzyme (Fig. 1HGo). Although it is difficult to postulate the requirements of ROCK and Rho in IGF1R-dependent action of IGF-II from these experiments, the results suggest that the IGF1R-independent effect of IGF-II (likely the M6P/IGFRII-mediated effect) requires ROCK but not Rho for EVT cell migration.

IGF1R-dependent and -independent effects of IGFs on migration of EVT cells pretreated with Y27632 or C3 exoenzyme
To distinguish the IGF1R-independent action of IGF-II from its IGF1R-dependent action, we first inhibited IGF1R-mediated action and then investigated the effect of an IGF-II analog. A recently discovered plant cyclolignan, PPP, at 0.5–2.5 µmol/liter concentrations, inhibits tyrosine kinase activity of IGF1R but not the tyrosine kinases of other growth factor receptors (35, 36). This compound was therefore used in this study to inhibit IGF1R-mediated actions of IGFs in EVT cells. Indeed, PPP at 0.5 µmol/liter concentration was consistently found to inhibit migration that was stimulated by Des1–3 IGF-I (Fig. 2AGo) without affecting basal migration. R6 IGF-II was selected as the IGF-II/CI-M6PR ligand because this analog has strong affinity for this receptor and extremely poor affinity for IGFBPs. The presence of PPP did not affect R6 IGF-II-mediated migration (Fig. 2AGo). IGF1R-independent action of IGF-II on EVT migration was almost completely abolished under inhibition of Rho kinase or ROCK with Y27632 (Fig. 2BGo), which is consistent with the results obtained from L27 IGF-II treatment (Fig. 1DGo). However, under inhibition of Rho by C3 exoenzyme, IGF1R-independent action of IGF-II on migration was not affected (Fig. 2CGo). These assays therefore confirmed our previous observations in Fig. 1Go of IGF1R-independent action of IGF-II requiring Rho kinase but not RhoA, RhoB, or RhoC GTPases. We also showed that insulin at the same concentration (10 nmol/liter) did not stimulate migration of these cells (data not shown). Furthermore, tyrphostin AG1024 at a concentration (100 µmol/liter) that was shown to inhibit basal and insulin-induced phosphorylation of IR and IR-mediated cellular functions (37) did not block IGF-II-mediated migration of these cells (data not shown). In summary, our results suggest that although IGF1R-dependent effects are dependent on Rho and ROCK, IGF1R-independent and possibly IR-independent effect of IGF-II on EVT cell migration require ROCK, but not Rho.


Figure 2
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 2. IGF1R-dependent and -independent effects IGFs on EVT cell migration with or without pretreatment of Y27632 or C3 exoenzyme. A, PPP treatment does not significantly affect basal EVT migration; R6 IGF-II-mediated effects on EVT migration are only slightly decreased, whereas Des IGF-I-mediated EVT migration is reduced to control levels. B–E, Inhibition of ROCK significantly impedes R6 IGF-II stimulatory effects on EVT migration, but inhibition of Rho does not prevent R6 IGF-II from promoting migration. On the other hand, pretreatment with neither Y27632 (D) nor C3 exoenzyme (E) prevents Des1–3 IGF-I-mediated migration of EVT cells. F, Effects of PPP and R6 IGF-II on actin polymerization in EVT cells before and after Y27632 or C3 exoenzyme treatment. R6 IGF-II treatment induces changes in actin distribution only after Rho inhibition. The dotted arrows point to actin stress fibers, and the solid arrows point to cortical actin. Values are the mean ± SEM of triplicate determinations. The experiments were reproduced three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control.

 
Actin polymerization and reorganization of F-actin-based cytoskeleton are absolutely required for cell migration. Rho GTPases play a key role in actin cytoskeletal remodeling. Therefore, we also studied the roles of ROCK and Rho in IGF-mediated actin cytoskeletal changes. F-actin organization and overall cellular morphology were visualized using confocal microscopy. Treatments of the EVT cells with the IGFs or the inhibitors of Rho kinase or Rho exhibited distinct changes in morphology and actin reorganizations. These changes were consistent with their effects on EVT cell migration. Under control conditions, EVT cells have polyhedral morphology with a cortical shell of F-actin and clear actin stress fibers (Fig. 2FGo), in keeping with the migratory phenotype of the EVT cells. Treatment with R6 IGF-II in PPP-pretreated cells slightly increased the intensity of these stress fibers. In response to Y27632, these cells lost stress fibers and F-actin was reorganized into multiple, longer spiky processes. F-actin stress fibers were completely lost in response to C3 exoenzyme, and the spreading of the cells was inhibited. Y27632 prevented R6 IGF-II from promoting stress fiber formation. However, addition of PPP plus R6 IGF-II to the C3 exoenzyme-treated cells resulted in spreading and reappearance of stress fibers and cortical F-actin (Fig. 2FGo). These results provide morphological evidence for the requirement of ROCK but not Rho in IGF1R-independent EVT migration induced by IGF-II.

Effect of Des1–3 IGF-I or PPP and R6 IGF-II on EVT cell migration after ROCK-I or ROCK-II gene silencing
The two Rho kinases, ROCK-I and -II, although homologous, were shown to have distinct cellular functions (38). Furthermore, Y27632 is a nonselective ROCK inhibitor and also may have unknown nonspecific effects. Therefore, we examined whether both ROCK-I and -II regulated IGF1R-dependent and -independent effects of IGF-II on EVT cell migration. A gene-silencing technique was used to specifically knock down the expression of ROCK-I or ROCK-II. Successful knockdown by their respective siRNA was documented by substantial depletion (more than 75%) of mRNA assessed by real-time-PCR (Fig. 3Go, A and E) and of protein assessed by Western blot analyses (Fig. 3Go, B and F). These RNA-silenced cells were then subjected to migration assays in the presence or absence of Des1–3 IGF-I or R6 IGF-II plus IGF1R tyrosine kinase inhibitor PPP. Silencing of either ROCK-I or ROCK-II inhibited basal migration of these cells by 80% (Fig. 3Go, C, D, G, and H). Silencing of ROCK-I or -II resulted in significant attenuation of IGF1R-dependent and -independent actions of IGF (Fig. 3Go, C, D, G, and H). Thus, IGF-II signaling through IGF1R or receptor(s) independent of IGF1R requires each of the Rho kinases to induce EVT cell migration.


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 3. Effect of Des1–3 IGF-I or PPP and R6 IGF-II on EVT cell migration after ROCK-I or ROCK-II silencing. Real-time PCR and Western blot analyses show significant reductions of EVT cell mRNA and protein levels for each of the ROCKs (A, B, E, and F). Inhibition of either of the ROCKs significantly impedes stimulatory effects of both PPP plus R6 IGF-II (C and G) and Des1–3 IGF-I (D and H) on EVT migration. Values are the mean ± SEM of triplicate determinations. The experiments were reproduced twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control.

 
Effects of RhoA or RhoC gene silencing on EVT cell migration
Although RhoA, RhoB, and RhoC are highly homologous, they have distinct cellular functions (28). Although RhoB has been implicated in intracellular trafficking and cell survival, both RhoA and RhoC have been implicated in cell migration. C3 exoenzyme inhibits all these three Rho GTPases. Therefore, to determine the role of RhoA or RhoC independently, we silenced each in HTR8/SVneo cells by respective siRNA. We observed 70% down-regulations of their mRNA (Fig. 4Go, A and E) and strong down-regulation of protein levels (Fig. 4Go, B and F) after transfection of these siRNAs. siRNA-mediated effects were specific because real-time PCR data showed no change in RhoA mRNA levels after RhoC siRNA transfection and vice versa (data not shown). Down-regulation of RhoA inhibited basal migration of these cells by 60% (Fig. 4Go, C and D) and that of RhoC by 80% (Fig. 4Go, G and H). However, the R6 IGF-II effect on RhoA- or RhoC-silenced PPP-treated cells was at least 2-fold more when compared with its effect on the control PPP-treated cells (Fig. 4Go, C and G). This indicates that RhoA and RhoC are not required for IGF1R-independent actions of IGF-II on EVT cell migration. On the contrary, IGF1R-dependent actions of IGF were found to depend on both RhoA and RhoC (Fig. 4Go, D and H). RhoA activation assays performed with the EVT cells after treatment with Des1–3 IGF-I or PPP plus R6 IGF-II showed only a modest (but not statistically significant) increase in active RhoA as compared with the controls (data not shown). These results confirm that the IGF1R-independent actions of IGF-II do not require RhoA and indicate a permissive role of RhoA in the IGF1R-dependent actions of IGFs on EVT cell migration.


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 4. Effect of Des1–3 IGF-I or PPP and R6 IGF-II on migration of EVT cells after RhoA or RhoC gene silencing. Real-time PCR and Western blot analyses show significant reductions of EVT cell mRNA and protein levels for each Rho GTPase silencing (A, B, E, and F). Inhibition of either RhoA or RhoC significantly impedes Des1–3 IGF-I-induced migration but not PPP plus R6 IGF-II-mediated EVT migration. Values are the mean ± SEM of triplicate determinations. The experiments were reproduced twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control.

 
Effects of Rac1 or Cdc42 inhibition on EVT cell migration
To down-regulate Rac1, we either used Rac1-specific siRNA or the cell-permeable, small-molecule Rac1 inhibitor NSC23766. Rac1 siRNA caused significant knockdown of Rac1 mRNA and protein (Fig. 5Go, A and B). Treatment with either Rac1 siRNA or NSC23766 inhibited basal migration of HTR-8/SVneo cells by about 70% (Fig. 5Go, C–E). However, Des1–3 IGF-I or PPP plus R6 IGF-II significantly stimulated migration of these Rac1-down-regulated cells (Fig. 5Go, C–E). Cdc42 siRNA also caused significant down-regulation of its mRNA (Fig. 5FGo) and protein (Fig. 5GGo) and inhibition of basal migration of these EVT cells (Fig. 5Go, H and I). Des1–3 IGF-I or PPP and R6 IGF-II caused significant stimulation of migration in Cdc42-silenced cells (Fig. 5Go, H and I). These results suggest that although Rac1 and Cdc42 are important for basal EVT cell migration, neither of these may be required for IGF1R-dependent or -independent action of IGFs on EVT cell migration. However, neither Des1–3 IGF-I nor R6 IGF-II was able to restore migration even close to basal levels (Fig. 5Go, C, D, H, and I) when either of these Rho GTPases was down-regulated. This means that although IGF-II may not require these enzymes for its IGF1R-dependent or -independent actions, some other intracellular factors that depend on Rac1 and Cdc42 are required for EVT cell migration. Neither Des1–3 IGF-I nor PPP plus R6 IGF-II was shown to activate Rac1 or Cdc42 in the EVT cells (data not shown), further substantiating the Rac1- and Cdc42-independence of IGF1R-dependent and -independent effects of IGF-II on EVT cell migration.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 5. Effect of Des1–3 IGF-I or PPP and R6 IGF-II on migration of EVT cells after Rac1 or Cdc42 inhibition. Real-time PCR and Western blot analyses show significant reductions of EVT cell mRNA and protein levels for Rac1 and Cdc42 silencing (A, B, F, and G). Fold increase after treatment of Des1–3 IGF-I or PPP plus R6 IGF-II in Rac1- or Cdc42-inhibited cells is either not different from (C, E, and H) or more than (D and I) the controls. Therefore, inhibition of either Rac1 or Cdc42 does not significantly impede Des1–3 IGF-I-induced or PPP plus R6 IGF-II-induced effects on EVT cell migration. Values are the mean ± SEM of triplicate determinations. The experiments were reproduced twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study examined the roles of the Rho family GTPases and Rho kinases in regulating IGF1R-dependent and -independent actions of IGF-II on cellular migration. The key findings of the present study are the following. 1) Rho, Rac, and Cdc42 are required for basal migration of human EVT cells. 2) Whereas the IGF1R-independent effect of IGF-II on migration of these cells does not require RhoA or RhoC, IGF1R-mediated migration depends on RhoA and RhoC. 3) When RhoA or RhoC is inhibited, the IGF1R-independent effect of IGF-II on cellular migration is exaggerated. 4) Rho kinase, which is considered one of the main effectors of RhoA and RhoC, is required for both the IGF1R-dependent and -independent effects of IGF-II on cellular migration. 5) Both IGF1R-dependent and -independent effects of IGF-II on migration do not require Rac1 or Cdc42.

Unlike the present study, where the migration assays were performed under serum-free conditions, Hamilton et al. (8) and McKinnon et al. (9) performed the assays under serum-reduced conditions. IGF-I was found to be ineffective in stimulating EVT cell migration under serum-reduced conditions. It is possible that the presence of serum either saturated the IGF1R sites on the cell membranes or inhibited IGF1R expression in EVT cells. In fact, Yang et al. (39) have reported that the presence of serum down-regulates, whereas serum withdrawal up-regulates, IGF1R mRNA (39). Our present assay system used serum-free conditions to avoid the possible confounding factors present in serum. Indeed, both IGF1R-dependent and -independent effects of IGFs on EVT cell migration were observed in our present study.

Either IGF-II/CI-M6PR or IR-A or both might mediate IGF1R-independent cellular functions of IGF-II in EVT cells. IGF-II has been shown to have strong affinity for both IR-A (15, 16) and IGF-II/CI-M6PR (9, 10, 11, 12, 13, 14). Both these receptors have also been shown to propagate intracellular signals leading to cellular migration. IR-A results from alternative splicing of 36-nucleotide exon 11 from pre-mRNA encoding the IR (40, 41). Because exon 11 is devoid of any kinase activity, tyrosine kinase inhibitor that would inhibit the kinase activity of IR-B should also inhibit IR-A. For this reason, we preincubated the EVT cells with the concentration of tyrphostin AG 1024 that was previously shown to effectively inhibit IR-B phosphorylation (37) before performing IGF-II-induced cell migration assay. Failure of AG 1024 to attenuate IGF-II-induced migration and inability of insulin to stimulate EVT cell migration indicate participation of some receptor other than IR-A in the IGF1R-independent actions of IGF-II. Although we cannot totally exclude the possible involvement of residual kinase activity of IR-A after treatment with AG 1024 in IGF-II-induced EVT cell migration, involvement of the IGF-II/CI-M6PR remains a strong possibility.

By using blocking antibodies against IGF-II/CI-M6PR and IGF-II/CI-M6PR-selective IGF-II analogs in our previous study, we were able to provide evidence for the signaling function of IGF-II/CI-M6PR in IGF-II-induced EVT cell migration (9). To provide evidence for IGF1R- and IGFBP-independent but IGF-II/CI-M6PR-dependent action of IGF-II in the present study, we chose three IGF-II analogs having differential selectivity toward IGF1R, IGF-II/CI-M6PR, and IGFBPs and compared their effects on EVT cell migration. This approach suggested the role of IGF-II/CI-M6PR in propagating cellular motility signals in an IGFBP-independent manner. We also took another approach to confirm the IGF1R-independent action of IGF-II throughout our study. The use of a newly identified selective inhibitor of IGF1R tyrosine kinase (PPP) along with R6 IGF-II helped us identify IGF1R-independent actions of IGF-II.

Rho GTPases have been regarded as the major downstream targets of a number of membrane-bound receptors. We therefore explored Rho GTPases as the downstream targets of the receptors for IGF-II action in relation to EVT cell migration. C3 exoenzyme-induced inhibition of Rho or siRNA-induced inhibition of RhoA or RhoC impairs basal migration of EVT cells. However, IGF1R-independent action of IGF-II on migration of these Rho-inhibited cells have totally overcome the impairment of migration. This indicates that RhoA and RhoC are not required for IGF1R-independent cellular migration induced by IGF-II. Furthermore, the extent of stimulation of migration due to the interaction of IGF-II with the receptor(s) other than IGF1R in control cells was much less than that in RhoA- or RhoC-inhibited cells. This compensatory response of IGF-II in Rho-inhibited cells suggests the existence of a tonic inhibitory influence of RhoA/RhoC on another signaling pathway, which is required for IGF-II-mediated cellular migration. Activation of MAPK (ERK1/2) was shown to cause extensive stress fiber formation and lamellipodial extensions (42) and stimulation of MLC kinase (43), enhancing cellular migration. Rho-independent stress fiber formation has also been documented by Ory et al. (44). Two totally separate pathways of Rho and ERK1/2 regulation of cell migration have been documented (45). However, we cannot exclude the possibility that these two pathways regulate cell migration in a coordinated manner. On the other hand, interaction of IGF-II with receptor(s) other than IGF1R might directly activate signaling molecules downstream of RhoA/C. Furthermore, agents that would cause direct activation of protein kinase C{delta} can activate MLC kinase (46) and subsequent reorganization of the actin cytoskeleton required for cellular migration. The results of the present study also show formation of stress fibers, cortical distribution of actin, and change in cell shape to an elongated morphology after addition of IGF-II (but not IGF-I) in C3 exoenzyme-treated cells. IGF1R-dependent actions of IGF-II on EVT cell migration, however, depend largely on RhoA or RhoC, a phenomenon that was confirmed by investigating the action of the IGF1R-selective agonist Des1–3 IGF-I. However, Des1–3 IGF-I failed to stimulate RhoA activity in EVT cells. Factors like urokinase type plasminogen activator and lysophosphatidic acid that are known to be produced in the maternal-fetal interface are likely to keep RhoA activated (47) in EVT cells so that IGF-I can use this activated enzyme to mediate its migratory signals.

Activated forms of RhoA, -B, and -C perform their functions by interacting with their effectors. Numerous Rho effectors have been identified (28). One of these effectors called Rho-associated kinase (Rho kinase/ROK/ROCK) has been implicated in a variety of Rho-dependent cellular functions including migration. Both isoforms of Rho kinase (Rho kinase ß, or ROCK-I; and Rho kinase {alpha}, or ROCK-II) are expressed ubiquitously and perform similar and distinct functions. In most cases, impairment of a cellular function after inhibition of Rho kinase(s) mimics the inhibitory effects of RhoA, -B, and -C. Likewise, the IGF1R-mediated action of IGF-I in the present study was shown to depend on Rho kinases. However, IGF1R-independent actions of IGF-II in the present study differ in ROCK-inhibited and Rho-inhibited cells. Although IGF1R-independent actions of IGF-II on EVT cell migration can occur in a Rho-independent manner, they are dependent on ROCK. Indeed, evidence for Rho-independent activation of ROCK has been documented (48).

Rac and Cdc42 are known to play important roles in cellular migration by promoting extensions of lamellipodia and filopodia, respectively. The presence of IGF-I or IGF-II significantly abrogated migration-inhibitory effects of Rac1 or Cdc42 inhibition in EVT cells, although these effects were far away from reaching the full migratory potential. EVT cells may produce some factors that largely depend on Rac1 and Cdc42 for their effects on cell migration. Furthermore, any other signaling pathway in the EVT cells that is activated by IGFs may not be able to compensate for the inhibition of migration caused by the down-regulation of Rac1 or Cdc42.

In summary, the results of our present study show that when IGF-II interacts with a receptor other than IGF1R in human EVT cells, it does not require RhoA or RhoC but requires Rho kinases to migrate, whereas IGF1R requires all these enzymes to mediate its migratory signals. Furthermore, none of the IGF receptors is likely to require Rac1 or Cdc42 to mediate its migratory signals in the EVT cells. We can speculate from our studies that very high levels of IGF-II expression in the fetal-maternal interface during pregnancy may be able to compensate for any loss or reduction of RhoA or RhoC expression in the cytotrophoblast. This indeed might explain the reason for normal embryogenesis in RhoC-null mice (49).


   Acknowledgments
 
We thank Dr. S. J. Dixon for editing the manuscript and Dr. Biju George for assisting us in statistical analyses.


   Footnotes
 
This work was supported by Grant MOP 68997 (to C.C.) from Canadian Institutes of Health Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: CI-M6PR, Cation-independent mannose 6 phosphate receptor; EVT, extravillous trophoblast; FBS, fetal bovine serum; IGFBP, IGF-binding protein; IGF1R, IGF type 1 receptor; IR, insulin receptor; L27, Leu27; MLC, myosin light chain; PPP, picropodophyllin; R6, Arg6; ROCK, Rho-associated coiled-coil protein kinase; SFM, serum-free medium; siRNA, small interfering RNA; TBS, Tris-buffered saline.

Received April 13, 2007.

Accepted for publication July 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. DeChiara TM, Efstratiadis A, Robertson EJ 1990 A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345:78–80[CrossRef][Medline]
  2. Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  3. Constancia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, Fundele R, Stewart F, Kelsey G, Fowden A, Sibley C, Reik W 2002 Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 417:945–948[CrossRef][Medline]
  4. Sibley CP, Coan PM, Ferguson-Smith AC, Dean W, Hughes J, Smith P, Reik W, Burton GJ, Fowden AL, Constancia M 2004 Placental-specific insulin-like growth factor 2 (Igf2) regulates the diffusional exchange characteristics of the mouse placenta. Proc Natl Acad Sci USA 101:8204–8208[Abstract/Free Full Text]
  5. Ferguson-Smith AC, Cattanach BM, Barton SC, Beechey CV, Surani MA 1991 Embryological and molecular investigations of parental imprinting on mouse chromosome 7. Nature 351:667–670[CrossRef][Medline]
  6. Gratton RJ, Asano H, Han VK 2002 The regional expression of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) in the placentae of women with pre-eclampsia. Placenta 23:303–310[CrossRef][Medline]
  7. Han VH, Bassett N, Walton J, Challis JR 1996 The expression of insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) genes in the human placenta and membranes: evidence for IGF-IGFBP interactions at the feto-maternal interface. J Clin Endocrinol Metab 81:2680–2693[Abstract]
  8. Hamilton GS, Lysiak JJ, Han VKM, Lala PK 1998 Autocrine-paracrine regulation of human trophoblast invasiveness by insulin-like growth factor (IGF)-II and IGF-binding protein (IGFBP)-I. Exp Cell Res 244:147–156[CrossRef][Medline]
  9. McKinnon T, Chakraborty C, Gleeson LM, Chidiac P, Lala PK 2001 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 86:3665–3674[Abstract/Free Full Text]
  10. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[Abstract/Free Full Text]
  11. Volpert O, Jackson D, Bouck N, Linzer DI 1996 The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis. Endocrinology 1996 137:3871–3876[Abstract]
  12. Groskopf JC, Syu LJ, Saltiel AR, Linzer DI 1997 Proliferin induces endothelial cell chemotaxis through a G protein-coupled, mitogen-activated protein kinase-dependent pathway. Endocrinology 138:2835–2840[Abstract/Free Full Text]
  13. Gallicchio MA, Kaun C, Wojta J, Binder B, Bach LA 2003 Urokinase type plasminogen activator receptor is involved in insulin-like growth factor-induced migration of rhabdomyosarcoma cells in vitro. J Cell Physiol 197:131–138[CrossRef][Medline]
  14. Hawkes C, Jhamandas JH, Harris KH, Fu W, MacDonald RG, Kar S 2006 Single transmembrane domain insulin-like growth factor-II/mannose-6-phosphate receptor regulates central cholinergic function by activating a G-protein-sensitive, protein kinase C-dependent pathway. J Neurosci 26:585–596[Abstract/Free Full Text]
  15. Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, Goldfine ID, Belfiore A, Vigneri R 1999 Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells. Mol Cell Biol 19:3278–3288[Abstract/Free Full Text]
  16. Sciacca L, Mineo R, Pandini G, Murabito A, Vigneri R, Belfiore A 2002 In IGF-I receptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor isoform A. Oncogene 21:8240–8250[CrossRef][Medline]
  17. Ridley AJ 2000 Rho. In: Hall A, ed. GTPases: frontiers in molecular biology. Oxford, UK: Oxford University Press; 89–136
  18. Kjoller L, Hall A 1999 Signalling to Rho GTPase. Exp Cell Res 253:166–179[CrossRef][Medline]
  19. Sah VP, Seasholtz TM, Sagi SA, Brown JH 2000 The role of Rho in G protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol 40:459–489[CrossRef][Medline]
  20. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, Lala PK 1993 Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 206:204–211[CrossRef][Medline]
  21. Nicola C, Timoshenko AV, Dixon SJ, Lala PK, Chakraborty C 2005 EP1 receptor-mediated migration of the first trimester human extravillous trophoblast: the role of intracellular calcium and calpain. J Clin Endocrinol Metab 90:4736–4746[Abstract/Free Full Text]
  22. Yarrow JC, Totsukawa, Charras GT, Mitchison TJ 2005 Screening for cell migration via automated microscopy reveals a Rho-kinase inhibitor. Chem Biol 12:385–395[CrossRef][Medline]
  23. Genth H, Gerhard R, Maeda A, Amano M, Kaibuchi K, Aktories K, Just I 2003 Entrapment of Rho GDP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-guanine nucleotide dissociation inhibitor-1 complex. J Biol Chem 278:28523–28527[Abstract/Free Full Text]
  24. Zuo Y, Shields SK, Chakraborty C 2006 Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase. Biochem Biophys Res Commun 351:361–367[CrossRef][Medline]
  25. Pille JY, Denoyelle C, Varet J, Bertrand JR, Soria J, Opolon P, Lu H, Pritchard LL, Vannier JP, Malvy C, Soria C, Li H 2005 Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo. Mol Ther 11:267–274[Medline]
  26. Noritake J, Fukata M, Sato K, Nakagawa M, Watanabe T, Izumi N, Wang S, Fukata Y, Kaibuchi K 2004 Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact. Mol Biol Cell 15:1065–1076[Abstract/Free Full Text]
  27. Wilkinson S, Paterson HF, Marshall CJ 2005 Cdc42-MRCK and Rho-ROCK signaling cooperate in myosin phosphorylation and cell invasion. Nat Cell Biol 7:255–261[CrossRef][Medline]
  28. Wheeler AP, Ridley AJ 2004 Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res 301:43–49[CrossRef][Medline]
  29. Kabir-Salmani M, Shiokawa S, Akimoto Y, Sakai K, Iwashita M 2004 The role of {alpha}5ß1-integrin in the IGF-I-induced migration of extravillous trophoblast cells during the process of implantation. Mol Hum Reprod 10:91–97[Abstract/Free Full Text]
  30. Kabir-Salmani M, Shiokawa S, Akimoto Y, Sakai K, Nagamatsu S, Sakai K, Nakamura Y, Lotfi A, Kawakami H, Iwashita M 2003 {alpha}vß3 Integrin signaling pathway is involved in insulin-like growth factor I-stimulated human extravillous trophoblast cell migration. Endocrinology 144:1620–1630[Abstract/Free Full Text]
  31. Francis GL, Aplin SE, Milner SJ, McNeil KA, Ballard FJ, Wallace JC 1993 Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II. Biochem J 293:713–719[Medline]
  32. Burgisser DM, Roth BV, Giger R, Luthi C, Weigl S, Zarn J, Humbel RE 1991 Mutants of human insulin-like growth factor II with altered affinities for the type 1 and type 2 insulin-like growth factor receptor. J Biol Chem 266:1029–1033[Abstract/Free Full Text]
  33. Forbes BE, Hartfield PJ, McNeil KA, Surinya KH, Milner SJ, Cosgrove LJ, Wallace JC 2002 Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis. Eur J Biochem 269:961–968[Medline]
  34. Roth BV, Burgisser DM, Luthi C, Humbel RE 1991 Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62–67. Biochem Biophys Res Commun 181:907–914[CrossRef][Medline]
  35. Vasilcanu D, Girnita A, Girnita L, Vasilcanu R, Axelson M, Larsson O 2004 The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway. Oncogene 23:7854–7862[CrossRef][Medline]
  36. Ulfarsson E, Karstrom A, Yin S, Girnita A, Vasilcanu D, Thoren M, Kratz G, Hillman J, Axelson M, Larsson O, Girnita L 2005 Expression and growth dependency of the insulin-like growth factor I receptor in craniopharyngioma cells: a novel therapeutic approach. Clin Cancer Res 11:4674–4680[Abstract/Free Full Text]
  37. Gunton JE, Delhanty PJ, Takahashi S, Baxter RC 2003 Metformin rapidly increases insulin receptor activation in human liver and signals preferentially through insulin-receptor substrate-2. J Clin Endocrinol Metab 88:1323–1332[Abstract/Free Full Text]
  38. Yoneda A, Multhaupt HA, Couchman JR 2005 The Rho kinases I and II regulate different aspects of myosin II activity. J Cell Biol 170:443–453[Abstract/Free Full Text]
  39. Yang Y, Hoeflich A, Butenandt O, Kiess W 1996 Opposite regulation of IGF-I and IGF-I receptor mRNA and concomitant changes of GH receptor and IGF-II/M6P receptor mRNA in human IM-9 lymphoblasts. Biochem Biophys Acta 1310:317–324[Medline]
  40. Seino S, Seino M, Nishi S, Bell GI 1989 Structure of the human insulin receptor gene and characterization of its promoter. Proc Natl Acad Sci USA 86:114–118[Abstract/Free Full Text]
  41. Panz VR, Ruff P, Joffe BI, Kedda MA, Seftel HC 1996 SSCP analysis of the tyrosine kinase domain of the insulin receptor gene: polymorphisms detected in South African black and white subjects. Hum Genet 97:438–440[Medline]
  42. Vaidya RJ, Ray RM, Johnson LR 2005 MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm. Am J Physiol Cell Physiol 288:350–359[CrossRef]
  43. Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA 1997 Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 137:481–492[Abstract/Free Full Text]
  44. Ory S, Destaing O, Jurdic P 2002 Microtubule dynamics differentially regulates Rho and Rac activity and triggers Rho-independent stress fiber formation in macrophage polykaryons. Eur J Cell Biol 81:351–362[CrossRef][Medline]
  45. Brahmbhatt AA, Klemke RL 2003 ERK and RhoA differentially regulate pseudopodia growth and retraction during chemotaxis. J Biol Chem 278:13016–13025[Abstract/Free Full Text]
  46. Iwabu A, Smith K, Allen FD, Lauffenburger DA, Wells A 2004 Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C{delta}-dependent pathway. J Biol Chem 279:14551–14560[Abstract/Free Full Text]
  47. Jo M, Thomas KS, Somlyo AV, Somlyo AP, Gonias SL 2002 Cooperativity between the Ras-ERK and Rho-Rho kinase pathways in urokinase-type plasminogen activator-stimulated cell migration. J Biol Chem 277:12479–12485[Abstract/Free Full Text]
  48. Ueda H, Morishita R, Itoh H, Narumiya S, Mikoshiba K, Kato K, Asano T 2001 G{alpha}11 induces caspase-mediated proteolytic activation of Rho-associated kinase, ROCK-I, in HeLa cells. J Biol Chem 276:42527–42533[Abstract/Free Full Text]
  49. Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G, Wakeham A, Khokha R, Mak TW 2005 RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev 19:1974–1979[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
P. Fafet, C. Rebouissou, T. Maudelonde, and M.-L. Vignais
Opposite Effects of Transforming Growth Factor-{beta} Activation and Rho-Associated Kinase Inhibition on Human Trophoblast Migration in a Reconstituted Placental-Endometrial Coculture System
Endocrinology, September 1, 2008; 149(9): 4475 - 4485.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. Nicola, P. K. Lala, and C. Chakraborty
Prostaglandin E2-Mediated Migration of Human Trophoblast Requires RAC1 and CDC42
Biol Reprod, June 1, 2008; 78(6): 976 - 982.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Nicola, A. Chirpac, P. K. Lala, and C. Chakraborty
Roles of Rho Guanosine 5'-Triphosphatase A, Rho Kinases, and Extracellular Signal Regulated Kinase (1/2) in Prostaglandin E2-Mediated Migration of First-Trimester Human Extravillous Trophoblast
Endocrinology, March 1, 2008; 149(3): 1243 - 1251.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
R. Vasilcanu, D. Vasilcanu, B. Sehat, S. Yin, A. Girnita, M. Axelson, and L. Girnita
Insulin-Like Growth Factor Type-I Receptor-Dependent Phosphorylation of Extracellular Signal-Regulated Kinase 1/2 but not Akt (Protein Kinase B) Can Be Induced by Picropodophyllin
Mol. Pharmacol., March 1, 2008; 73(3): 930 - 939.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shields, S.-K.
Right arrow Articles by Chakraborty, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shields, S.-K.
Right arrow Articles by Chakraborty, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals