Gonadotropin-Releasing Hormone Induces Actin Cytoskeleton Remodeling and Affects Cell Migration in a Cell-Type-Specific Manner in TSU-Pr1 and DU145 Ce
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《内分泌学杂志》
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan
Abstract
GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In TSU-Pr1, GnRH-I and -II induced the filopodia formation of the cells and promoted cell migration, whereas in DU145, GnRH-I and -II induced the formation of the cells with stress fiber and inhibited cell migration. In our previous studies, we reported the stimulatory and inhibitory effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells. This study provides the first evidence for the effects of GnRH on actin cytoskeleton remodeling and cell migration of cells in which cell proliferation was affected by GnRH at the same time. Moreover, we also demonstrated that the same human GnRH receptor subtype, human type I GnRH receptor, is essential for the effects of GnRH-I and -II on actin cytoskeleton remodeling and cell migration in both TSU-Pr1 and DU145 cells using the technique of gene knock-down by RNA interference.
Introduction
GnRH IS THE central regulator of the reproductive functions in vertebrates through its activity of inducing gonadotropin release from the pituitary gonadotropes. In addition to its well-known function, diverse physiological functions have been suggested in extrapituitary tissues and organs, such as cerebrum, ovary, testis, placenta, thymus, and adrenal cortex (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Through studies using heterologous expression systems and the cells that natively express GnRH receptors, the diverse signaling pathways of the GnRH receptors have been demonstrated (15, 16), and this diversity of the signaling pathways is thought to contribute to the various physiological functions of GnRH. Regarding the autocrine and/or paracrine extrapituitary effects of GnRH, important direct effects of GnRH on the cells have been reported. GnRH promotes or inhibits cell proliferation depending on the cell type. There have been numerous studies demonstrating that GnRH directly inhibits cell proliferation in hormone-related tumors (17, 18). On the other hand, several studies have demonstrated that GnRH directly promotes cell proliferation of lymphocytes, mononuclear cells, and a human prostatic carcinoma cell line (19, 20, 21, 22). The differing effects of GnRH on cell proliferation depending on the cell type are interesting, and we have studied and are continuing to study the mechanisms of these effects, especially what determines the direction of the effects (22, 23, 24, 25). During our studies, we found that GnRH affects the cell migration as well as the cell proliferation of TSU-Pr1 cells (derived from human prostatic carcinoma). Recently, various effects of GnRH on cell migration and cellular morphology have been reported (26, 27); however, it has not hitherto been reported that GnRH affects cell migration simultaneously with cell proliferation. This fact is very important for considering the common features and specificity of the diverse signaling pathways mediating the effects of GnRH and for understanding the overall aspects of the GnRH signaling systems.
In the present study, we investigated the mechanisms of the effects of GnRH on cell migration and actin cytoskeleton remodeling, focusing on the Rho family monomeric G proteins. Moreover, we examined the contribution of human type I GnRH receptor, which is the only GnRH receptor demonstrated to be a functional seven-transmembrane receptor in humans, to the two opposite effects of GnRH on cell migration and actin cytoskeleton remodeling.
Materials and Methods
Cell culture
DU145, a human prostatic carcinoma cell line, was a gift from the Cell Resource Centre for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). TSU-Pr1, a human prostatic carcinoma cell line, was established by Dr. T. Iizumi et al. (28) (Department of Urology, Teikyo University School of Medicine, Tokyo, Japan). Each cell line was maintained at 37 C in appropriate medium (DMEM-low glucose for TSU-Pr1 and RPMI 1640 for DU145), containing 10% fetal bovine serum in a humidified atmosphere of 5% CO2 in air at 37 C. In all assays performed in this study, RPMI 1640 containing 10% Nu-serum I, a semisynthetic serum supplement (Collaborative Biomedical Products, Bedford, MA), was used. The usefulness of Nu-serum I for investigating the effects of GnRH was described in our previous study (29).
Visualization of two-dimensional distribution of cells
Glass coverslips (25 mm) coated with 0.01% solution of poly-L-lysine (Sigma, St. Louis, MO) were placed in six-well plates and 2 ml cell suspension (200,000 cells/ml) and were inoculated on each coverslip. After overnight incubation, the glass coverslips with cells were transferred into new six-well plates to avoid the effects of the activities of cells attached to the bottom of the initial six-well plates. After cells were left untreated or incubated with GnRH-I for 8 h, they were fixed in methanol and stained with hematoxylin and eosin. To transform the localizations of cells into digital photos, the stained glass coverslips were scanned using an image scanner (SEICO EPSON Corp., Suwa, Nagano, Japan). After scanning, coverslips were mounted on the slide glasses and observed using an optical microscope.
Modified Boyden chamber assay
For investigating the effects of GnRH on cell migration, we used a modified Boyden chamber (Transwell 6.5-mm diameter, Corning, Acton, MA) (see Fig. 2A). This assay was carried out according to the method used by Furanca et al. (30). The chambers contain two compartments divided by a polycarbonate membrane filter (8-μm pore size). Cells were cultured in serum-free RPMI 1640 medium for 18 h and then harvested from the dishes using 1 mM EDTA in PBS. After centrifugation, the cell pellet was suspended in fresh RPMI 1640 medium with 10% Nu-serum I, and 50,000 cells were seeded into each Transwell upper chamber. Lower chambers were supplied with fresh RPMI 1640 medium with 10% Nu-serum I or conditioned medium of TSU-Pr1 or DU145 cells. Cells were incubated for 8 h in a humidified atmosphere of 5% CO2 in air at 37 C. Then, they were fixed in methanol and stained with hematoxylin and eosin. The filters were gently removed from the inserts with scalpel blades and placed on glass slides with the lower side down. To keep only migrated cells in the filter, the upper monolayer was completely wiped off with a cotton swab. The filters were then mounted with glass coverslips using Eukitt (O Kindler GmbH and Co., Schwabach, Germany). Each condition was assayed in triplicate. The number of cells that migrated into the lower chamber was counted using a hemocytometer. The stained filters were viewed with an optical microscope, and three random fields per filter were captured with a digital CCD camera connected to an IBM-compatible PC, and the number of cells in the field was counted. Statistical analyses were performed using one- or two-way ANOVA (see Figs. 2, 5, 6, and 8).
Conditioned medium
Conditioned medium of TSU-Pr1 or DU145 cells was collected as follows. Cells were inoculated at the density of 10,000 cells/ml. After overnight incubation, the medium was replaced with fresh RPMI 1640 medium with 10% Nu-serum I. Then, conditioned medium was collected after 24 h of incubation and stored at –80 C.
Phalloidin staining and confocal laser microscopy
Glass coverslips (12 mm) coated with 0.01% solution of poly-L-lysine (Sigma) were placed in 24-well plates, and 1 ml cell suspension (100,000 cells/ml) was inoculated per well. The cells were cultured in serum-free RPMI 1640 medium for 18 h and then left untreated or incubated with GnRH-I or -II for 4 h. The cells were fixed with 4.0% paraformaldehyde in PBS with 1 mM Ca2+ and 0.5 mM Mg2+ for 30 min and then permeabilized for 10 min with PBS (Ca2+/Mg2+-free) containing 0.1% Triton X-100. F-actin was stained with Alexa Fluor 488 phalloidin (1:50) (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were viewed with a laser scanning confocal microscope (FluoView 500, Olympus, Tokyo, Japan).
Quantification of cellular morphologies
Filopodial cells, lamellar cells, cells with few protrusions, and cells with stress fiber were scored with reference to the following reports by Aarts et al.(31), Gauthier-Campbell et al. (32), Niu et al. (33), and Jaffe et al. (34). Cells with a minimum of five filopodia measuring at least 10 mm or cells with 20 filopodia measuring at least 5 mm in length were scored as being filopodial cells. At least 100 cells were counted in each experiment, and experiments were performed in triplicate. Statistical analyses were performed using a two-tailed Student’s t test.
Dominant-negative clones of Rac1, Cdc42, and RhoA and transient transfection experiments
Dominant-negative mutant clones of Rac1 and Cdc42 and RhoA (Rac1 T17N, Cdc42 T17N, and RhoA T19N), which were cloned in pcDNA3.1(+) (Invitrogen, Carlsbad, CA), were obtained from the University of Missouri-Rolla cDNA Resource Center (http://www.cdna.org). The cells in a 100-mm dish were washed with Opti-MEM I Reduced-Serum Medium (Invitrogen) before transfection. Twelve micrograms of plasmid DNA and 36 μl TransFast Transfection Reagent (Promega) were mixed with 2 ml Opti-MEM I Reduced-Serum Medium. The mixture was incubated for 15 min at room temperature for complex formation. The entire mixture was added to the cells. The cells were incubated for 1 h in a humidified atmosphere of 5% CO2 in air at 37 C and supplied with 8 ml fresh culture medium. Transfection efficiency was measured using -gal staining kit (Invitrogen) according to the manufacturer’s manual.
Transfection of the mammalian expression vector for siRNA-induced gene knock-down
To establish cell lines in which human type I GnRH receptor was stably knocked down by RNA interference (RNAi), a mammalian expression vector for siRNA-induced gene knock-down, pSilencer 2.1-U6 hygro (Ambion Inc., Austin, TX), was used. The siRNA target region was selected referring to The siRNA User Guide (35), and the sequence of the hairpin siRNA insert for pSilencer vector was designed according to the manufacturer’s instructions. The sequences of the siRNA inserts were as follows: sense strand, 5'-GATCCCGATCCGAGTGACGGTTACTTTCAAGAGAAGTAACCGTCACTCGGATCTTTTTTGGAAA-3'; and antisense strand, 5'-AGCTTTTCCAAAAAAGATCCGAGTGACGGTTACTTCTCTTGAAAGTAACCGTCACTCGGATCGG-3'. In our previous study, the efficacy and specificity of the designed siRNA insert were confirmed by transient expression using pSilencer 2.0-U6 (Ambion Inc.) (24). The cells in a 60-mm dish were washed with Opti-MEM I Reduced-Serum Medium before transfection. Eight micrograms pSilencer vector containing the siRNA insert and 20 μl Lipofectamine 2000 (Invitrogen) were each mixed with 500 μl Opti-MEM I Reduced-Serum Medium. The two mixtures were combined and incubated for 20 min at room temperature for complex formation. The entire mixture was added to the cells. The cells were incubated for 6–8 h in a humidified atmosphere of 5% CO2 in air at 37 C, washed once, and supplied with 8 ml fresh culture medium.
Antibiotic selection and cloning of stably human type I GnRH receptor (hGnRHR-1) knocked-down cells
The appropriate concentrations of hygromycin were examined in a preliminary experiment using nontransfected TSU-Pr1 and DU145 cells. The selection of transfected TSU-Pr1 and DU145 cells was performed using the following procedures. First, the transfected cells (500,000 cells/well) were inoculated into 24-well plates 24 h after transfection. Next, culture media containing various concentrations of hygromycin were supplied: 0, 100, 300, and 500 μg/ml for TSU-Pr1 cells and 0, 100, and 500 μg/ml for DU145 cells. The duration of the exposure to each concentration of hygromycin was determined according to the death rate of nontransfected cells cultured with the same exposure schedule. Then, the selected cells were diluted to 5 or 10 cells/ml, and 100 μl cell suspension was inoculated in each well of 96-well plates. After 1 wk, plates were observed, and the wells containing only one colony were selected. The selected wells were incubated for 2–3 wk in a humidified atmosphere of 5% CO2 in air at 37 C; finally, hygromycin (500 μg/ml) was added again. After 1 wk of culture, the cells in the wells with viable cells were subcultured and allowed to proliferate.
Monoclonal antibody against GnRH
In our previous study, we prepared a monoclonal antibody (LRH13) directed against the common amino acid sequence of mammalian, avian, and fish GnRH. The properties of LRH13 were described in detail in the report by Park and Wakabayashi (36). In this study, we used LRH13 (1:500) to absorb GnRH released from TSU-Pr1 cells.
RT-PCR analysis
Total RNA was extracted from cells using ISOGEN (NIPPON GENE, Tokyo, Japan). Two micrograms of total RNA was used for the first-strand synthesis of cDNA using Moloney murine leukemia virus reverse transcriptase (Promega). The primer sets used for RT-PCR analysis of hGnRHR-1 and -2 were as follows: hGnRHR-1, sense primer, 5'-AGTCCAATGGTATGCTGGAG-3' and antisense primer, 5'-ACCCGTGTCAGGGTGAAGAT-3'; hGnRHR-2, sense primer, 5'-TTCATCCTCCTCAGTTTCTCTCC-3' and antisense primer, 5'-ATGGCAGTCAGTGGCAGCAGA-3'. These primer sets were designed to span one exon-intron boundary, so any products resulting from genomic contamination could be eliminated. The reaction conditions for PCR were as follows: 94 C for 5 min, 45 cycles of 94 C for 60 sec, 60 C for 60 sec, and 72 C for 60 sec, and 72 C for 5 min.
Statistical analyses
A two-tailed Student’s t test and one- or two-way ANOVA were performed using the statistical software GraphPad Prism version 4.0 (GraphPad Software Inc., San Diego, CA) with default settings.
Results
Effects of GnRH on two-dimensional distribution of cells
We previously investigated the mechanisms of the stimulatory and inhibitory effects of GnRH on cell proliferation using human tumor cell lines, including TSU-Pr1 and DU145 cells (22, 23, 24, 25). During such studies, we found that GnRH affects the two-dimensional distribution of TSU-Pr1 cells in a culture dish as well as cell proliferation. To visualize this phenomenon, we cultured TSU-Pr1 cells on glass coverslips with or without GnRH-I treatment. The same experiment was performed in DU145 cells because in terms of cell proliferation, TSU-Pr1 and DU145 cells show opposite responses to GnRH treatment, and it is of interest to determine whether such a difference of effect is also observed on the distribution of cells.
Figure 1 shows the effects of GnRH-I treatment on the two-dimensional distribution of TSU-Pr1 and DU145 cells. TSU-Pr1 cells were unevenly distributed on the coverslip after 8 h of treatment with GnRH-I compared with the control (Fig. 1, A1 and B1). The photos (Fig. 1, A2, B2, and B3) are magnified images (x100) of the fields designated by the identically labeled boxes. It is clear that there are larger number of TSU-Pr1 cells in the field of B3 than in the fields of A2 and B2. In contrast, in DU145 cells, the clear difference between nontreatment and treatment group was not observed (Fig. 1, C1, C2, D1, and D2). This experiment was repeated in triplicate, and the similar results were obtained (data not shown). These results suggest that GnRH affects the cell migration of TSU-Pr1 cells. To investigate the role of GnRH in this phenomenon, next we carried out a modified Boyden chamber assay.
Effects of GnRH on cell migration
Firstly, to check the hypothesis that TSU-Pr1 cells showed chemotaxis to GnRH and were consequently unevenly distributed, cell migration was quantified under the condition that fresh RPMI 1640 medium containing GnRH-I or -II (final concentration, 1 nM) was added to the lower chamber. This examination was also performed in DU145 cells, which were not affected by GnRH-I treatment in Fig. 1. In our previous study, it was demonstrated that the potencies of GnRH-I and -II are different with regard to mediating the effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells; thus, both of these GnRH species were tested. Consequently, the results showed no significant difference between the effects on the migration of TSU-Pr1 and DU145 cells being independent of added GnRH species (Fig. 2, B and C).
Next, we presumed that GnRH affected the cellular morphology and consequently affected the cellular motility, and cell migration was investigated under the condition that GnRH-I or -II (final concentration, 1 nM) was added to the upper chamber with cells. In the lower chamber, fresh RPMI 1640 medium with 10% Nu-serum I or conditioned medium collected as described in Materials and Methods was added. Several reports demonstrated the chemotaxis of cells to an autocrine factor and that cell migration was enhanced in a modified Boyden chamber assay under the condition that conditioned medium was supplied in the lower chamber (37, 38). We found that when conditioned medium was supplied in the lower chamber, GnRH-I and -II significantly increased the number of migrated TSU-Pr1 cells (Fig. 2, D and E). In contrast, in DU145 cells, GnRH-I and -II significantly decreased the number of migrated cells (Fig 2, F and G). In both cell lines, the number of migrated cells on the lower side of the filter when conditioned medium was supplied in the lower chamber was significantly greater than that when fresh medium was supplied in the lower chamber (P < 0.001). Regarding the number of cells that migrated to the lower chamber, only the experimental groups in which conditioned medium was supplied in the lower chamber were statistically analyzed because too few cells were observed in the lower chamber when fresh medium was supplied in the lower chamber. Figure 2, D and E, shows that GnRH-I was more effective than GnRH-II in promoting the migration of TSU-Pr1 cells, whereas GnRH-II was more effective than GnRH-I in inhibiting the migration of DU145 cells (Fig. 2, F and G).
Effects of GnRH on actin cytoskeleton remodeling
The results shown in Fig. 2 suggest that GnRH affects the structure of the cytoskeleton, especially the actin cytoskeleton, and consequently alters the cell motility. Thus, we investigated the effects of GnRH on actin cytoskeleton remodeling by fluorostaining of polymerized actin filaments using Alexa Fluor 488 phalloidin. In both TSU-Pr1 and DU145 cells, the observed structures of the actin cytoskeleton were classified into four types with reference to the report by Aarts et al. (31): filopodial cells, lamellar cells, cells with few protrusions, and cells with stress fiber (Figs. 3, A–D, and 4, A–D). The typical cells of each classified group are indicated by white arrows. Then, we performed the quantitative analyses of the percentages of the four types of cells in both TSU-Pr1 and DU145 cultured with or without GnRH-I and -II. As shown in Fig. 3, E and F, in TSU-Pr1 cells, GnRH-I increased the percentage of the filopodial cells in a dose-dependent manner and 10 pM to 10 nM of GnRH-I had the significant effects. GnRH-II showed the similar effect, and the dose-responsibility (data not shown) was observed (Fig. 3, E and F). GnRH-I was more effective (10–100-fold) than GnRH-II. The decrease of the percentages of cells with few protrusions, and cells with stress fiber accompanied the increase of the percentage of filopodial cells by the treatment of GnRH-I or -II. The percentage of lamellar cells showed no significant change. In DU145 cells, GnRH-II increased the percentage of the cells with stress fiber in a dose-dependent manner, and 10 pM to 10 nM GnRH-II had the significant effects (Fig. 4, E and F). GnRH-I showed the similar effect, and the dose-responsibility (data not shown) was observed (Fig. 4, E and F). GnRH-II is more effective (approximately 10-fold) than GnRH-I. The decrease of the percentages of filopodial cells and the increase of the percentage of cells with few protrusions accompanied the increase of the percentage of cells with stress fiber by the treatment of GnRH-I or -II. The percentage of lamellar cells showed no significant change.
Effects of the expression of dominant-negative mutants of Rho family G proteins on the effects of GnRH on cell migration
A number of reports have demonstrated that the activation of Rho family small monomeric G proteins induces actin cytoskeleton remodeling (39, 40, 41). Thus, our results in Figs. 5 and 6 strongly suggest that Rho family small monomeric G proteins are activated by the GnRH treatment. Then, to clarify the relationship between the activation of Rho family G proteins and the enhancement or attenuation of the cell migration by GnRH treatment, a modified Boyden chamber assay was performed using dominant-negative mutants of Rho family small monomeric G proteins (Rac1 T17N, Cdc42 T17N, and RhoAT19N) transiently transfected into TSU-Pr1 and DU145 cells. Consequently, in TSU-Pr1 cells, the expression of Rac1 T17N and Cdc42 T17N diminished the promotive effects of GnRH-I and -II on cell migration (Fig. 5, A–F). On the other hand, the expression of RhoA T19N diminished the inhibitory effects of GnRH-I and -II in DU145 cells (Fig. 6, A–F). The expression of the control vector pcDNA3.1(+) had no effect on either cell line (Figs. 5, G and H, and 6, G and H). We also measured the transfection efficiency in TSU-Pr1 and DU145 cells using -gal staining kit (Invitrogen), and more than 90% of transfection efficiencies (TSU-Pr1, 94.8%; DU145, 92.0%) were obtained by the protocol of the transient transfection described in Materials and Methods. These high transfection efficiencies in these cell lines can explain the approximately 100% attenuation of the effects of GnRH-I and -II on cell migration by the transfection of the dominant-negative mutants of Rac1, Cdc42, or RhoA.
Establishment of human type I GnRH receptor knocked-down clones
Humans possess two types of GnRH receptor genes. The type I receptor has been demonstrated to be a functional receptor. However, the type II receptor gene is disrupted by a frame shift and a premature stop codon, suggesting that a conventional receptor is not translated from this gene (42, 43, 44, 45, 46). In our previous report, we demonstrated that hGnRHR-1 is indispensable for both the stimulatory and inhibitory effects of GnRH on cell proliferation using cells with transient knock-down of hGnRHR-1 by the technique of RNAi (24). It was demonstrated that RNAi is an effective technique to investigate the contribution of the two types of the human GnRH receptors to the effects of GnRH; thus, we have established multiple clones in which human GnRH receptor is stably knocked down following the methods described in Materials and Methods. To date, we have confirmed the previous results that hGnRHR-1 is indispensable for both the stimulatory and inhibitory effects of GnRH on cell proliferation (Utsumi, M., M. Enomoto, and M. K. Park, unpublished data). In this study, the contributions of hGnRHR-1 to the effects of GnRH on cell migration and actin cytoskeleton remodeling were examined using hGnRHR-1 stably knocked-down TSU-Pr1 and DU145 clones. In this study, the clones examined are designated as TSU-Pr1_R1(–)a,b and DU145_R1(–)a,b. Figure 7 shows the expression patterns of the two types of human GnRH receptors mRNAs in TSUPr1, TSU-Pr1_R1(–)a, DU145, and DU145_R1(–)a. Neither of the knocked-down clones expressed the hGnRHR-1 mRNA (the upper panel of Fig. 7), whereas for hGnRHR-2 mRNA, both knocked-down clones showed the same expression pattern as the original cells (the lower panel of Fig. 7). The shorter 426-bp band is a splice variant of hGnRHR-2 that has been reported in a number of studies (24, 43, 45). Another clone [TSU-Pr1_R1(–)b and DU145_R1(–)b] showed the same expression patterns (data not shown). These results indicate that hGnRHR-1 was specifically knocked-down in these clones.
Effects of GnRH on cell migration and actin cytoskeleton remodeling in human type I GnRH receptor knocked-down clones
Figure 8 shows the effects of GnRH-I and -II on cell migration in TSU-Pr1_R1(–)a and DU145_R1(–)a. In both clones, GnRH-I and -II had no significant effects in both TSU-Pr1_R1(–)a and DU145_R1(–)a, indicating that hGnRHR-1 is indispensable for both the stimulatory and inhibitory effects of GnRH-I and -II on cell migration. These results were also confirmed in TSU-Pr1_R1(–)b and DU145_R1(–)b (data not shown).
To examine whether GnRH did not induce the actin cytoskeleton remodeling in hGnRHR-1 knocked-down clones and consequently had no effect on cell migration, we stained the polymerized actin filaments with Alexa Fluor 488 phalloidin and performed the quantitative analyses of the percentages of the four types of cells in both TSU-Pr1 and DU145 cultured with or without GnRH-I and -II. In TSU-Pr1_R1(–)a, the shapes of more than 90% cells were like the shapes of the cells in Fig. 9A, being independent of the GnRH treatment. Short protrusions (less than 5 μm, indicated by arrowheads) were observed but were not identified as filopodia according to the criteria described in Materials and Methods. The cellular morphologies are clearly different from those in the original TSU-Pr1 cells (Fig. 9C). We presumed that the knock-down of hGnRHR-1 caused the attenuation of the autocrine activity of GnRH and consequently affected the structure of the actin cytoskeleton. Then, the original TSU-Pr1 cells were cultured with LRH13 (a monoclonal antibody against GnRH) and stained. Results similar to those in TSU-Pr1_R1(–)a were observed. Cells like that in Fig. 9B constituted more than 90% of the cells. The cells with short protrusions like Fig. 9, A and B, were expediently classified into cells with few protrusions in the quantitative analyses (Fig. 9, D and E). Figure 9, D and E, show the results of quantification of the percentages of the four types of cells, and no significant differences were detected among any pairs of the experimental groups. Similar results were confirmed in TSU-Pr1_R1(–)b cells (data not shown), whereas in DU145_R1(–)a, the morphologies of the cells were classified into four types (Fig. 10, A–D), and no significant differences were detected among any pairs of the experimental groups (Fig. 10, E and F). Similar results were confirmed in DU145_R1(–)b cells (data not shown).
Discussion
GnRH was originally identified as a hypothalamic decapeptide that promotes gonadotropin secretion from pituitary gonadotropes (47, 48). However, there is increasing evidence of extrapituitary effects of GnRH on a number of peripheral tissues, such as the cerebrum, ovary, testis, placenta, thymus, and adrenal cortex (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). This multifunctional nature of GnRH is considered to be due to the divergent signaling pathways of the GnRH receptors (15, 16). In fact, it is reported that GnRH receptor is coupled to small monomeric G protein signaling as well as multiple heterotrimeric G proteins (27, 49). In this study, we provided the first evidence that GnRH induces different orientations of the actin cytoskeleton remodeling dependent on the cell type through a common GnRH receptor subtype, human type I GnRH receptor, and consequently differentially modulates cell migration.
In our previous studies, we studied the two opposite (stimulatory and inhibitory) effects of GnRH on cell proliferation, focusing on the roles of GnRH receptors, using human tumor cell lines (24). In the present study, we found that in TSU-Pr1 cells, GnRH affects the two-dimensional distribution of cells in a culture dish as well as the cell proliferation (Fig. 1). A few reports showed that GnRH affects cell attachment or cell migration (26, 27), but there have been no reports investigating the effects of GnRH on the actin cytoskeleton remodeling and cell migration of the cells in which cell proliferation is simulated or inhibited by the GnRH treatment. From this point of view, the results in Fig. 1 were very interesting and suggestive, but it was difficult to perform the quantitative analyses by the method used in Fig. 1. Thus, we adopted the other quantitative assay, a modified Boyden chamber assay, and performed more detailed analyses.
One major finding in this study is that GnRH oppositely modulates the cell’s migratory behavior between TSU-Pr1 and DU145 cells. Cell migration of TSU-Pr1 was enhanced by GnRH treatment, whereas in contrast, that of DU145 cells was attenuated (Fig. 2, D–G). The results of phalloidin staining (Figs. 3 and 4) showed that GnRH induces the formation of filopodia in TSU-Pr1 cells and stress fiber in DU145 cells, strongly suggesting that this difference of the induction of actin cytoskeleton remodeling by GnRH causes the opposite effects on cell migration. This possibility was investigated in experiments using cells transfected with dominant-negative mutants of Rac1, Cdc42, and RhoA. The results of Figs. 5 and 6 demonstrated that in TSU-Pr1 cells, the activation of Rac1 and Cdc42 was necessary for the enhancement of cell migration by GnRH and that the activation of RhoA was necessary for the attenuation of cell migration by GnRH in DU145 cells. It is known that the localized activation of Rac and/or Cdc42 in concert with other regulators such as WASP/WAVE family proteins, and the Arp2/3 complex stimulates the formation of a branching actin filament network at the leading edge, which in turn induces a protrusion in the direction of migration (50). Thus, it is presumed that the activation of both Rac1 and Cdc42 by GnRH induced the formation of protrusions; as a consequence, cell migration was enhanced in TSU-Pr1 cells.
Another important finding of this study is that the opposite effects of GnRH-I and -II on cell migration occur through the same type human GnRH receptor, human type I GnRH receptor. In the human genome, two types of GnRH receptor genes exist (42, 43, 44, 45, 46). One is the type I GnRH receptor gene, which has been demonstrated to encode a functional seven-transmembrane-spanning G protein-coupled receptor (GPCR). The other is the type II GnRH receptor gene, which contains a frame shift in coding exon 1 and a premature stop codon in exon 2. To date, all studies have failed to produce direct evidence of the transcription or translation of a functional full-length seven-transmembrane-spanning G protein-coupled type II GnRH receptor (16). However, there is indirect evidence that the human type II GnRH receptor functions by itself in the inhibitory effects of GnRH on cell proliferation; this evidence was obtained using cell lines in which the expression of human type I GnRH receptor was inhibited by the antisense fragment transfection method (51). As shown in Figs. 8–10, GnRH had no significant effects on cell migration or actin cytoskeleton remodeling in hGnRHR-1 knocked-down TSU-Pr1 and DU145 clones. We also confirmed that in hGnRHR-1 transiently and stably knocked-down Jurkat (derived from human mature leukemia), TSU-Pr1, HHUA (derived from human endometrial carcinoma), and DU145 cell lines, GnRH had no significant effects on cell proliferation (Ref.24 ; and Utsumi, M., M. Enomoto, and M. K. Park, unpublished data). These results strongly suggest that human type II GnRH receptor does not function, at least as the functional GnRH receptor. The discrepancy between our results and the results of Grundker et al. (52, 53), may be due to the methods used for the inhibition of the expression of hGnRHR-1. It was reported that double-stranded RNA longer than about 30 bp in mammalian cells has major side effects (i.e. interferon responses and global transcription shutdown). In our experiments, TSU-Pr1 and DU145 clones in which hGnRHR-1 was knocked-down by RNAi grew normally; thus, we think that our results are credible. Such a discrepancy due to the methods used for the inhibition of gene expression was also reported for the inositol 1,4,5-triphosphate receptors (54). Moreover, the knock-down of hGnRHR-1 altered the morphology of the TSU-Pr1 cells (Fig. 9A), suggesting that the autocrine activity of GnRH via hGnRHR-1 is important for maintaining the cell morphology. This hypothesis was confirmed by the result that the original TSU-Pr1 cells cultured with LRH13 (a monoclonal antibody against GnRH) showed a similar staining result. In contrast, in DU145 cells, there are no remarkable morphological differences between the original DU145 cells and hGnRHR-1 knocked-down DU145 cells. It is of interest whether this difference has a relationship with the response to the exogenous GnRH treatment or not.
In the present study, the two human prostatic carcinoma cell lines, TSU-Pr1 and DU145, exhibited completely opposite results as response to GnRH stimulation. It is very important how the same cell-type responses in such an opposite manner to the same stimulation. Both TSU-Pr1 and DU145 cells are androgen insensitive and independent and do not produce prostate-specific antigen, and it is thought that the characteristics of TSU-Pr1 are similar to those of DU145 (28, 55). However, in our previous study, we have demonstrated that TSU-Pr1 and DU145 cells exhibited the distinct patterns of the ligand selectivities in the effects of GnRH on colony formation (24, 25). In TSU-Pr1 cells, GnRH-I was more effective (about 10-fold); in contrast, GnRH-II is much more effective (1000–10,000-fold) than GnRH-I in DU145 cells. This difference was observed in the effects of GnRH on cell migration and actin cytoskeleton remodeling (Figs. 3–6). These distinct ligand selectivities between TSU-Pr1 and DU145 cells suggest that the ligand stimulation is mediated via the different types of human GnRH receptors between TSU-Pr1 and DU145 cells. However, our previous study and the present study demonstrated that human type I GnRH receptor is indispensable for both the stimulatory and inhibitory effects of GnRH-I and -II on cell proliferation and cell migration (24). This fact is difficult to explain by the classical ligand receptor theory; however, several studies on human type II GnRH receptor give us an important suggestion. It is known that the human type II GnRH receptor gene is transcriptionally active and has several alternative splice variants (45). Recently, it was reported that the expression of a human type II GnRH receptor fragment inhibited the human type I GnRH receptor function (56). These reports have led us to hypothesize that the several transcripts that are produced may encode functionally modulatory proteins and thereby contribute to the diverse signaling pathways of human type I GnRH receptor. To understand the overall aspects of the signaling pathways of human GnRH receptors, comprehensive investigation of the human type II GnRH receptor alternative splice variants will be necessary in the near future. It is also very important how human type I GnRH receptor is essential for both the stimulatory and inhibitory effects of GnRH on cell proliferation and cell migration. Furthermore, we presented the results strongly suggesting that GnRH affects cell migration via the activation of Rho family G proteins (Figs. 5 and 6). In this study, we did not try to elucidate the detailed signaling pathway from human type I GnRH receptor to Rho family G proteins. It is well known that human type I GnRH receptor is one of the GPCRs that are coupled with heterotrimeric G proteins. Recently, the activation and inactivation of monomeric G proteins, such as Rho family G proteins, via GPCRs have been focused on (57), and GnRH receptor is not an exception. To elucidate this point, the comprehensive studies using inhibitors, toxins, and constitutively active mutants of signaling molecules that is thought to be relevant to the human GnRH receptor signaling will be necessary.
So far, the diverse signaling pathways of the GnRH receptors have been studied (15, 16). Particularly, GnRH receptor expression has been demonstrated in several tumors and tumor-derived cell lines in which the addition of GnRH and its analogs resulted in the inhibition of cell growth, and the molecular mechanisms underlying this effect have been intensively studied. In our previous study, we also demonstrated that GnRH inhibits cell proliferation of DU145 cells (24). Recently, it was reported that GnRH agonist induces apoptosis of DU145 cells and that this requires the activation and/or inhibition of multiple pathway, such as c-Src, protein kinase B, and c-Jun N-terminal kinase (58). These signaling molecules are known to be in the downstream of RhoA (59). Thus, it is possible that our result that RhoA is involved in the GnRH signaling of DU145 cells can be applied to the case of the effects of GnRH on cell proliferation, including apoptosis of DU145 cells, whereas it was reported that early G1 phase expression of cyclin D1 is mediated by Rac1 and Cdc42 (60). Our result that Rac1 and Cdc42 are involved in the effects of GnRH on cell migration of TSU-Pr1 cells can be relevant to the stimulatory effects of GnRH on TSU-Pr1 cell proliferation. Based on the results of this study and our previous studies, the common features and specificity of the signaling pathways of the effects of GnRH on cell proliferation and cell migration will be clarified, and these approaches will provide important clues for understanding the overall aspects of the signaling pathways of the GnRH receptors.
In most vertebrates, GnRH-immunoreactive neurons are detected in the epithelium of the medial olfactory pit soon after its formation. The GnRH-immunoreactive neurons migrate out of the placodal epithelium and into the brain along a migration route that consists of the central processes of the terminal, olfactory, and vomeronasal nerves (61). Recently, it was also reported that in human olfactory GnRH-secreting neurons, GnRH triggered axon growth, actin cytoskeleton remodeling, and an increase in migration, suggesting that GnRH acts in an autocrine manner to promote differentiation and migration of those cells (26).
In the present study, we showed the effects of GnRH in only prostatic carcinoma cell lines; however, the similar effects of GnRH on cell migration were obtained using human T-cell leukemia cell line and human endometrial carcinoma cell line (Enomoto, M., and M. K. Park, unpublished data). In these tissues and organs, expressions of GnRH receptor were demonstrated in normal cells (21, 61, 62). These facts lead us to hypothesize that the ontogenic GnRH activity on cell migration may be observed in physiological and pathological settings of extrapituitary tissues and organs in adults. Furthermore, cell migration orchestrates morphogenesis throughout embryonic development (50). To date, the presence of GnRH and its receptor in preimplantation embryos and effects of GnRH on early embryonic development have been demonstrated in cattle, mice, pigs, and humans, suggesting that GnRH may play a substantial autocrine or paracrine role in human fertilization, early embryonic development, and implantation (62, 63, 64, 65, 66). From this point of view, elucidating the molecular mechanisms of the GnRH effects on actin cytoskeleton remodeling and cell migration will lead to the understanding of the physiological roles of GnRH in embryogenesis and morphogenesis.
Acknowledgments
We express our gratitude to Prof. H. Takeda and Dr. K. Horikawa (Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan) for lending a laser scanning confocal microscope and advice on phalloidin staining and observation using it. We are also grateful to Dr. T. Iizumi (Department of Urology, Teikyo University School of Medicine, Tokyo, Japan) and Dr. J.Y. Seong and Ms. D. Y. Oh (Laboratory of G protein-coupled receptors, Graduate School of Medicine, Korea University, Seoul, Korea) for advice on culturing TSU-Pr1 cells. Finally, we are thankful to Prof. Y. Oka, Dr. Y. Akazome, Dr. H. Abe, and Ms. M. Kyokuwa (Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan) for valuable discussions throughout this study.
Footnotes
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
First Published Online September 29, 2005
Abbreviations: GPCR, G protein-coupled receptor; hGnRHR-1, human type I GnRH receptor; RNAi, RNA interference.
Accepted for publication September 15, 2005.
References
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Abstract
GnRH was first identified as the hypothalamic decapeptide that promotes gonadotropin release from pituitary gonadotropes. Thereafter, direct stimulatory and inhibitory effects of GnRH on cell proliferation were demonstrated in a number of types of primary cultured cells and established cell lines. Recently, the effects of GnRH on cell attachment, cytoskeleton remodeling, and cell migration have also been reported. Thus, the effects of GnRH on various cell activities are of great interest among researchers who study the actions of GnRH. In this study, we demonstrated that GnRH induces actin cytoskeleton remodeling and affects cell migration using two human prostatic carcinoma cell lines, TSU-Pr1 and DU145. In TSU-Pr1, GnRH-I and -II induced the filopodia formation of the cells and promoted cell migration, whereas in DU145, GnRH-I and -II induced the formation of the cells with stress fiber and inhibited cell migration. In our previous studies, we reported the stimulatory and inhibitory effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells. This study provides the first evidence for the effects of GnRH on actin cytoskeleton remodeling and cell migration of cells in which cell proliferation was affected by GnRH at the same time. Moreover, we also demonstrated that the same human GnRH receptor subtype, human type I GnRH receptor, is essential for the effects of GnRH-I and -II on actin cytoskeleton remodeling and cell migration in both TSU-Pr1 and DU145 cells using the technique of gene knock-down by RNA interference.
Introduction
GnRH IS THE central regulator of the reproductive functions in vertebrates through its activity of inducing gonadotropin release from the pituitary gonadotropes. In addition to its well-known function, diverse physiological functions have been suggested in extrapituitary tissues and organs, such as cerebrum, ovary, testis, placenta, thymus, and adrenal cortex (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Through studies using heterologous expression systems and the cells that natively express GnRH receptors, the diverse signaling pathways of the GnRH receptors have been demonstrated (15, 16), and this diversity of the signaling pathways is thought to contribute to the various physiological functions of GnRH. Regarding the autocrine and/or paracrine extrapituitary effects of GnRH, important direct effects of GnRH on the cells have been reported. GnRH promotes or inhibits cell proliferation depending on the cell type. There have been numerous studies demonstrating that GnRH directly inhibits cell proliferation in hormone-related tumors (17, 18). On the other hand, several studies have demonstrated that GnRH directly promotes cell proliferation of lymphocytes, mononuclear cells, and a human prostatic carcinoma cell line (19, 20, 21, 22). The differing effects of GnRH on cell proliferation depending on the cell type are interesting, and we have studied and are continuing to study the mechanisms of these effects, especially what determines the direction of the effects (22, 23, 24, 25). During our studies, we found that GnRH affects the cell migration as well as the cell proliferation of TSU-Pr1 cells (derived from human prostatic carcinoma). Recently, various effects of GnRH on cell migration and cellular morphology have been reported (26, 27); however, it has not hitherto been reported that GnRH affects cell migration simultaneously with cell proliferation. This fact is very important for considering the common features and specificity of the diverse signaling pathways mediating the effects of GnRH and for understanding the overall aspects of the GnRH signaling systems.
In the present study, we investigated the mechanisms of the effects of GnRH on cell migration and actin cytoskeleton remodeling, focusing on the Rho family monomeric G proteins. Moreover, we examined the contribution of human type I GnRH receptor, which is the only GnRH receptor demonstrated to be a functional seven-transmembrane receptor in humans, to the two opposite effects of GnRH on cell migration and actin cytoskeleton remodeling.
Materials and Methods
Cell culture
DU145, a human prostatic carcinoma cell line, was a gift from the Cell Resource Centre for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). TSU-Pr1, a human prostatic carcinoma cell line, was established by Dr. T. Iizumi et al. (28) (Department of Urology, Teikyo University School of Medicine, Tokyo, Japan). Each cell line was maintained at 37 C in appropriate medium (DMEM-low glucose for TSU-Pr1 and RPMI 1640 for DU145), containing 10% fetal bovine serum in a humidified atmosphere of 5% CO2 in air at 37 C. In all assays performed in this study, RPMI 1640 containing 10% Nu-serum I, a semisynthetic serum supplement (Collaborative Biomedical Products, Bedford, MA), was used. The usefulness of Nu-serum I for investigating the effects of GnRH was described in our previous study (29).
Visualization of two-dimensional distribution of cells
Glass coverslips (25 mm) coated with 0.01% solution of poly-L-lysine (Sigma, St. Louis, MO) were placed in six-well plates and 2 ml cell suspension (200,000 cells/ml) and were inoculated on each coverslip. After overnight incubation, the glass coverslips with cells were transferred into new six-well plates to avoid the effects of the activities of cells attached to the bottom of the initial six-well plates. After cells were left untreated or incubated with GnRH-I for 8 h, they were fixed in methanol and stained with hematoxylin and eosin. To transform the localizations of cells into digital photos, the stained glass coverslips were scanned using an image scanner (SEICO EPSON Corp., Suwa, Nagano, Japan). After scanning, coverslips were mounted on the slide glasses and observed using an optical microscope.
Modified Boyden chamber assay
For investigating the effects of GnRH on cell migration, we used a modified Boyden chamber (Transwell 6.5-mm diameter, Corning, Acton, MA) (see Fig. 2A). This assay was carried out according to the method used by Furanca et al. (30). The chambers contain two compartments divided by a polycarbonate membrane filter (8-μm pore size). Cells were cultured in serum-free RPMI 1640 medium for 18 h and then harvested from the dishes using 1 mM EDTA in PBS. After centrifugation, the cell pellet was suspended in fresh RPMI 1640 medium with 10% Nu-serum I, and 50,000 cells were seeded into each Transwell upper chamber. Lower chambers were supplied with fresh RPMI 1640 medium with 10% Nu-serum I or conditioned medium of TSU-Pr1 or DU145 cells. Cells were incubated for 8 h in a humidified atmosphere of 5% CO2 in air at 37 C. Then, they were fixed in methanol and stained with hematoxylin and eosin. The filters were gently removed from the inserts with scalpel blades and placed on glass slides with the lower side down. To keep only migrated cells in the filter, the upper monolayer was completely wiped off with a cotton swab. The filters were then mounted with glass coverslips using Eukitt (O Kindler GmbH and Co., Schwabach, Germany). Each condition was assayed in triplicate. The number of cells that migrated into the lower chamber was counted using a hemocytometer. The stained filters were viewed with an optical microscope, and three random fields per filter were captured with a digital CCD camera connected to an IBM-compatible PC, and the number of cells in the field was counted. Statistical analyses were performed using one- or two-way ANOVA (see Figs. 2, 5, 6, and 8).
Conditioned medium
Conditioned medium of TSU-Pr1 or DU145 cells was collected as follows. Cells were inoculated at the density of 10,000 cells/ml. After overnight incubation, the medium was replaced with fresh RPMI 1640 medium with 10% Nu-serum I. Then, conditioned medium was collected after 24 h of incubation and stored at –80 C.
Phalloidin staining and confocal laser microscopy
Glass coverslips (12 mm) coated with 0.01% solution of poly-L-lysine (Sigma) were placed in 24-well plates, and 1 ml cell suspension (100,000 cells/ml) was inoculated per well. The cells were cultured in serum-free RPMI 1640 medium for 18 h and then left untreated or incubated with GnRH-I or -II for 4 h. The cells were fixed with 4.0% paraformaldehyde in PBS with 1 mM Ca2+ and 0.5 mM Mg2+ for 30 min and then permeabilized for 10 min with PBS (Ca2+/Mg2+-free) containing 0.1% Triton X-100. F-actin was stained with Alexa Fluor 488 phalloidin (1:50) (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were viewed with a laser scanning confocal microscope (FluoView 500, Olympus, Tokyo, Japan).
Quantification of cellular morphologies
Filopodial cells, lamellar cells, cells with few protrusions, and cells with stress fiber were scored with reference to the following reports by Aarts et al.(31), Gauthier-Campbell et al. (32), Niu et al. (33), and Jaffe et al. (34). Cells with a minimum of five filopodia measuring at least 10 mm or cells with 20 filopodia measuring at least 5 mm in length were scored as being filopodial cells. At least 100 cells were counted in each experiment, and experiments were performed in triplicate. Statistical analyses were performed using a two-tailed Student’s t test.
Dominant-negative clones of Rac1, Cdc42, and RhoA and transient transfection experiments
Dominant-negative mutant clones of Rac1 and Cdc42 and RhoA (Rac1 T17N, Cdc42 T17N, and RhoA T19N), which were cloned in pcDNA3.1(+) (Invitrogen, Carlsbad, CA), were obtained from the University of Missouri-Rolla cDNA Resource Center (http://www.cdna.org). The cells in a 100-mm dish were washed with Opti-MEM I Reduced-Serum Medium (Invitrogen) before transfection. Twelve micrograms of plasmid DNA and 36 μl TransFast Transfection Reagent (Promega) were mixed with 2 ml Opti-MEM I Reduced-Serum Medium. The mixture was incubated for 15 min at room temperature for complex formation. The entire mixture was added to the cells. The cells were incubated for 1 h in a humidified atmosphere of 5% CO2 in air at 37 C and supplied with 8 ml fresh culture medium. Transfection efficiency was measured using -gal staining kit (Invitrogen) according to the manufacturer’s manual.
Transfection of the mammalian expression vector for siRNA-induced gene knock-down
To establish cell lines in which human type I GnRH receptor was stably knocked down by RNA interference (RNAi), a mammalian expression vector for siRNA-induced gene knock-down, pSilencer 2.1-U6 hygro (Ambion Inc., Austin, TX), was used. The siRNA target region was selected referring to The siRNA User Guide (35), and the sequence of the hairpin siRNA insert for pSilencer vector was designed according to the manufacturer’s instructions. The sequences of the siRNA inserts were as follows: sense strand, 5'-GATCCCGATCCGAGTGACGGTTACTTTCAAGAGAAGTAACCGTCACTCGGATCTTTTTTGGAAA-3'; and antisense strand, 5'-AGCTTTTCCAAAAAAGATCCGAGTGACGGTTACTTCTCTTGAAAGTAACCGTCACTCGGATCGG-3'. In our previous study, the efficacy and specificity of the designed siRNA insert were confirmed by transient expression using pSilencer 2.0-U6 (Ambion Inc.) (24). The cells in a 60-mm dish were washed with Opti-MEM I Reduced-Serum Medium before transfection. Eight micrograms pSilencer vector containing the siRNA insert and 20 μl Lipofectamine 2000 (Invitrogen) were each mixed with 500 μl Opti-MEM I Reduced-Serum Medium. The two mixtures were combined and incubated for 20 min at room temperature for complex formation. The entire mixture was added to the cells. The cells were incubated for 6–8 h in a humidified atmosphere of 5% CO2 in air at 37 C, washed once, and supplied with 8 ml fresh culture medium.
Antibiotic selection and cloning of stably human type I GnRH receptor (hGnRHR-1) knocked-down cells
The appropriate concentrations of hygromycin were examined in a preliminary experiment using nontransfected TSU-Pr1 and DU145 cells. The selection of transfected TSU-Pr1 and DU145 cells was performed using the following procedures. First, the transfected cells (500,000 cells/well) were inoculated into 24-well plates 24 h after transfection. Next, culture media containing various concentrations of hygromycin were supplied: 0, 100, 300, and 500 μg/ml for TSU-Pr1 cells and 0, 100, and 500 μg/ml for DU145 cells. The duration of the exposure to each concentration of hygromycin was determined according to the death rate of nontransfected cells cultured with the same exposure schedule. Then, the selected cells were diluted to 5 or 10 cells/ml, and 100 μl cell suspension was inoculated in each well of 96-well plates. After 1 wk, plates were observed, and the wells containing only one colony were selected. The selected wells were incubated for 2–3 wk in a humidified atmosphere of 5% CO2 in air at 37 C; finally, hygromycin (500 μg/ml) was added again. After 1 wk of culture, the cells in the wells with viable cells were subcultured and allowed to proliferate.
Monoclonal antibody against GnRH
In our previous study, we prepared a monoclonal antibody (LRH13) directed against the common amino acid sequence of mammalian, avian, and fish GnRH. The properties of LRH13 were described in detail in the report by Park and Wakabayashi (36). In this study, we used LRH13 (1:500) to absorb GnRH released from TSU-Pr1 cells.
RT-PCR analysis
Total RNA was extracted from cells using ISOGEN (NIPPON GENE, Tokyo, Japan). Two micrograms of total RNA was used for the first-strand synthesis of cDNA using Moloney murine leukemia virus reverse transcriptase (Promega). The primer sets used for RT-PCR analysis of hGnRHR-1 and -2 were as follows: hGnRHR-1, sense primer, 5'-AGTCCAATGGTATGCTGGAG-3' and antisense primer, 5'-ACCCGTGTCAGGGTGAAGAT-3'; hGnRHR-2, sense primer, 5'-TTCATCCTCCTCAGTTTCTCTCC-3' and antisense primer, 5'-ATGGCAGTCAGTGGCAGCAGA-3'. These primer sets were designed to span one exon-intron boundary, so any products resulting from genomic contamination could be eliminated. The reaction conditions for PCR were as follows: 94 C for 5 min, 45 cycles of 94 C for 60 sec, 60 C for 60 sec, and 72 C for 60 sec, and 72 C for 5 min.
Statistical analyses
A two-tailed Student’s t test and one- or two-way ANOVA were performed using the statistical software GraphPad Prism version 4.0 (GraphPad Software Inc., San Diego, CA) with default settings.
Results
Effects of GnRH on two-dimensional distribution of cells
We previously investigated the mechanisms of the stimulatory and inhibitory effects of GnRH on cell proliferation using human tumor cell lines, including TSU-Pr1 and DU145 cells (22, 23, 24, 25). During such studies, we found that GnRH affects the two-dimensional distribution of TSU-Pr1 cells in a culture dish as well as cell proliferation. To visualize this phenomenon, we cultured TSU-Pr1 cells on glass coverslips with or without GnRH-I treatment. The same experiment was performed in DU145 cells because in terms of cell proliferation, TSU-Pr1 and DU145 cells show opposite responses to GnRH treatment, and it is of interest to determine whether such a difference of effect is also observed on the distribution of cells.
Figure 1 shows the effects of GnRH-I treatment on the two-dimensional distribution of TSU-Pr1 and DU145 cells. TSU-Pr1 cells were unevenly distributed on the coverslip after 8 h of treatment with GnRH-I compared with the control (Fig. 1, A1 and B1). The photos (Fig. 1, A2, B2, and B3) are magnified images (x100) of the fields designated by the identically labeled boxes. It is clear that there are larger number of TSU-Pr1 cells in the field of B3 than in the fields of A2 and B2. In contrast, in DU145 cells, the clear difference between nontreatment and treatment group was not observed (Fig. 1, C1, C2, D1, and D2). This experiment was repeated in triplicate, and the similar results were obtained (data not shown). These results suggest that GnRH affects the cell migration of TSU-Pr1 cells. To investigate the role of GnRH in this phenomenon, next we carried out a modified Boyden chamber assay.
Effects of GnRH on cell migration
Firstly, to check the hypothesis that TSU-Pr1 cells showed chemotaxis to GnRH and were consequently unevenly distributed, cell migration was quantified under the condition that fresh RPMI 1640 medium containing GnRH-I or -II (final concentration, 1 nM) was added to the lower chamber. This examination was also performed in DU145 cells, which were not affected by GnRH-I treatment in Fig. 1. In our previous study, it was demonstrated that the potencies of GnRH-I and -II are different with regard to mediating the effects of GnRH on the cell proliferation of TSU-Pr1 and DU145 cells; thus, both of these GnRH species were tested. Consequently, the results showed no significant difference between the effects on the migration of TSU-Pr1 and DU145 cells being independent of added GnRH species (Fig. 2, B and C).
Next, we presumed that GnRH affected the cellular morphology and consequently affected the cellular motility, and cell migration was investigated under the condition that GnRH-I or -II (final concentration, 1 nM) was added to the upper chamber with cells. In the lower chamber, fresh RPMI 1640 medium with 10% Nu-serum I or conditioned medium collected as described in Materials and Methods was added. Several reports demonstrated the chemotaxis of cells to an autocrine factor and that cell migration was enhanced in a modified Boyden chamber assay under the condition that conditioned medium was supplied in the lower chamber (37, 38). We found that when conditioned medium was supplied in the lower chamber, GnRH-I and -II significantly increased the number of migrated TSU-Pr1 cells (Fig. 2, D and E). In contrast, in DU145 cells, GnRH-I and -II significantly decreased the number of migrated cells (Fig 2, F and G). In both cell lines, the number of migrated cells on the lower side of the filter when conditioned medium was supplied in the lower chamber was significantly greater than that when fresh medium was supplied in the lower chamber (P < 0.001). Regarding the number of cells that migrated to the lower chamber, only the experimental groups in which conditioned medium was supplied in the lower chamber were statistically analyzed because too few cells were observed in the lower chamber when fresh medium was supplied in the lower chamber. Figure 2, D and E, shows that GnRH-I was more effective than GnRH-II in promoting the migration of TSU-Pr1 cells, whereas GnRH-II was more effective than GnRH-I in inhibiting the migration of DU145 cells (Fig. 2, F and G).
Effects of GnRH on actin cytoskeleton remodeling
The results shown in Fig. 2 suggest that GnRH affects the structure of the cytoskeleton, especially the actin cytoskeleton, and consequently alters the cell motility. Thus, we investigated the effects of GnRH on actin cytoskeleton remodeling by fluorostaining of polymerized actin filaments using Alexa Fluor 488 phalloidin. In both TSU-Pr1 and DU145 cells, the observed structures of the actin cytoskeleton were classified into four types with reference to the report by Aarts et al. (31): filopodial cells, lamellar cells, cells with few protrusions, and cells with stress fiber (Figs. 3, A–D, and 4, A–D). The typical cells of each classified group are indicated by white arrows. Then, we performed the quantitative analyses of the percentages of the four types of cells in both TSU-Pr1 and DU145 cultured with or without GnRH-I and -II. As shown in Fig. 3, E and F, in TSU-Pr1 cells, GnRH-I increased the percentage of the filopodial cells in a dose-dependent manner and 10 pM to 10 nM of GnRH-I had the significant effects. GnRH-II showed the similar effect, and the dose-responsibility (data not shown) was observed (Fig. 3, E and F). GnRH-I was more effective (10–100-fold) than GnRH-II. The decrease of the percentages of cells with few protrusions, and cells with stress fiber accompanied the increase of the percentage of filopodial cells by the treatment of GnRH-I or -II. The percentage of lamellar cells showed no significant change. In DU145 cells, GnRH-II increased the percentage of the cells with stress fiber in a dose-dependent manner, and 10 pM to 10 nM GnRH-II had the significant effects (Fig. 4, E and F). GnRH-I showed the similar effect, and the dose-responsibility (data not shown) was observed (Fig. 4, E and F). GnRH-II is more effective (approximately 10-fold) than GnRH-I. The decrease of the percentages of filopodial cells and the increase of the percentage of cells with few protrusions accompanied the increase of the percentage of cells with stress fiber by the treatment of GnRH-I or -II. The percentage of lamellar cells showed no significant change.
Effects of the expression of dominant-negative mutants of Rho family G proteins on the effects of GnRH on cell migration
A number of reports have demonstrated that the activation of Rho family small monomeric G proteins induces actin cytoskeleton remodeling (39, 40, 41). Thus, our results in Figs. 5 and 6 strongly suggest that Rho family small monomeric G proteins are activated by the GnRH treatment. Then, to clarify the relationship between the activation of Rho family G proteins and the enhancement or attenuation of the cell migration by GnRH treatment, a modified Boyden chamber assay was performed using dominant-negative mutants of Rho family small monomeric G proteins (Rac1 T17N, Cdc42 T17N, and RhoAT19N) transiently transfected into TSU-Pr1 and DU145 cells. Consequently, in TSU-Pr1 cells, the expression of Rac1 T17N and Cdc42 T17N diminished the promotive effects of GnRH-I and -II on cell migration (Fig. 5, A–F). On the other hand, the expression of RhoA T19N diminished the inhibitory effects of GnRH-I and -II in DU145 cells (Fig. 6, A–F). The expression of the control vector pcDNA3.1(+) had no effect on either cell line (Figs. 5, G and H, and 6, G and H). We also measured the transfection efficiency in TSU-Pr1 and DU145 cells using -gal staining kit (Invitrogen), and more than 90% of transfection efficiencies (TSU-Pr1, 94.8%; DU145, 92.0%) were obtained by the protocol of the transient transfection described in Materials and Methods. These high transfection efficiencies in these cell lines can explain the approximately 100% attenuation of the effects of GnRH-I and -II on cell migration by the transfection of the dominant-negative mutants of Rac1, Cdc42, or RhoA.
Establishment of human type I GnRH receptor knocked-down clones
Humans possess two types of GnRH receptor genes. The type I receptor has been demonstrated to be a functional receptor. However, the type II receptor gene is disrupted by a frame shift and a premature stop codon, suggesting that a conventional receptor is not translated from this gene (42, 43, 44, 45, 46). In our previous report, we demonstrated that hGnRHR-1 is indispensable for both the stimulatory and inhibitory effects of GnRH on cell proliferation using cells with transient knock-down of hGnRHR-1 by the technique of RNAi (24). It was demonstrated that RNAi is an effective technique to investigate the contribution of the two types of the human GnRH receptors to the effects of GnRH; thus, we have established multiple clones in which human GnRH receptor is stably knocked down following the methods described in Materials and Methods. To date, we have confirmed the previous results that hGnRHR-1 is indispensable for both the stimulatory and inhibitory effects of GnRH on cell proliferation (Utsumi, M., M. Enomoto, and M. K. Park, unpublished data). In this study, the contributions of hGnRHR-1 to the effects of GnRH on cell migration and actin cytoskeleton remodeling were examined using hGnRHR-1 stably knocked-down TSU-Pr1 and DU145 clones. In this study, the clones examined are designated as TSU-Pr1_R1(–)a,b and DU145_R1(–)a,b. Figure 7 shows the expression patterns of the two types of human GnRH receptors mRNAs in TSUPr1, TSU-Pr1_R1(–)a, DU145, and DU145_R1(–)a. Neither of the knocked-down clones expressed the hGnRHR-1 mRNA (the upper panel of Fig. 7), whereas for hGnRHR-2 mRNA, both knocked-down clones showed the same expression pattern as the original cells (the lower panel of Fig. 7). The shorter 426-bp band is a splice variant of hGnRHR-2 that has been reported in a number of studies (24, 43, 45). Another clone [TSU-Pr1_R1(–)b and DU145_R1(–)b] showed the same expression patterns (data not shown). These results indicate that hGnRHR-1 was specifically knocked-down in these clones.
Effects of GnRH on cell migration and actin cytoskeleton remodeling in human type I GnRH receptor knocked-down clones
Figure 8 shows the effects of GnRH-I and -II on cell migration in TSU-Pr1_R1(–)a and DU145_R1(–)a. In both clones, GnRH-I and -II had no significant effects in both TSU-Pr1_R1(–)a and DU145_R1(–)a, indicating that hGnRHR-1 is indispensable for both the stimulatory and inhibitory effects of GnRH-I and -II on cell migration. These results were also confirmed in TSU-Pr1_R1(–)b and DU145_R1(–)b (data not shown).
To examine whether GnRH did not induce the actin cytoskeleton remodeling in hGnRHR-1 knocked-down clones and consequently had no effect on cell migration, we stained the polymerized actin filaments with Alexa Fluor 488 phalloidin and performed the quantitative analyses of the percentages of the four types of cells in both TSU-Pr1 and DU145 cultured with or without GnRH-I and -II. In TSU-Pr1_R1(–)a, the shapes of more than 90% cells were like the shapes of the cells in Fig. 9A, being independent of the GnRH treatment. Short protrusions (less than 5 μm, indicated by arrowheads) were observed but were not identified as filopodia according to the criteria described in Materials and Methods. The cellular morphologies are clearly different from those in the original TSU-Pr1 cells (Fig. 9C). We presumed that the knock-down of hGnRHR-1 caused the attenuation of the autocrine activity of GnRH and consequently affected the structure of the actin cytoskeleton. Then, the original TSU-Pr1 cells were cultured with LRH13 (a monoclonal antibody against GnRH) and stained. Results similar to those in TSU-Pr1_R1(–)a were observed. Cells like that in Fig. 9B constituted more than 90% of the cells. The cells with short protrusions like Fig. 9, A and B, were expediently classified into cells with few protrusions in the quantitative analyses (Fig. 9, D and E). Figure 9, D and E, show the results of quantification of the percentages of the four types of cells, and no significant differences were detected among any pairs of the experimental groups. Similar results were confirmed in TSU-Pr1_R1(–)b cells (data not shown), whereas in DU145_R1(–)a, the morphologies of the cells were classified into four types (Fig. 10, A–D), and no significant differences were detected among any pairs of the experimental groups (Fig. 10, E and F). Similar results were confirmed in DU145_R1(–)b cells (data not shown).
Discussion
GnRH was originally identified as a hypothalamic decapeptide that promotes gonadotropin secretion from pituitary gonadotropes (47, 48). However, there is increasing evidence of extrapituitary effects of GnRH on a number of peripheral tissues, such as the cerebrum, ovary, testis, placenta, thymus, and adrenal cortex (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). This multifunctional nature of GnRH is considered to be due to the divergent signaling pathways of the GnRH receptors (15, 16). In fact, it is reported that GnRH receptor is coupled to small monomeric G protein signaling as well as multiple heterotrimeric G proteins (27, 49). In this study, we provided the first evidence that GnRH induces different orientations of the actin cytoskeleton remodeling dependent on the cell type through a common GnRH receptor subtype, human type I GnRH receptor, and consequently differentially modulates cell migration.
In our previous studies, we studied the two opposite (stimulatory and inhibitory) effects of GnRH on cell proliferation, focusing on the roles of GnRH receptors, using human tumor cell lines (24). In the present study, we found that in TSU-Pr1 cells, GnRH affects the two-dimensional distribution of cells in a culture dish as well as the cell proliferation (Fig. 1). A few reports showed that GnRH affects cell attachment or cell migration (26, 27), but there have been no reports investigating the effects of GnRH on the actin cytoskeleton remodeling and cell migration of the cells in which cell proliferation is simulated or inhibited by the GnRH treatment. From this point of view, the results in Fig. 1 were very interesting and suggestive, but it was difficult to perform the quantitative analyses by the method used in Fig. 1. Thus, we adopted the other quantitative assay, a modified Boyden chamber assay, and performed more detailed analyses.
One major finding in this study is that GnRH oppositely modulates the cell’s migratory behavior between TSU-Pr1 and DU145 cells. Cell migration of TSU-Pr1 was enhanced by GnRH treatment, whereas in contrast, that of DU145 cells was attenuated (Fig. 2, D–G). The results of phalloidin staining (Figs. 3 and 4) showed that GnRH induces the formation of filopodia in TSU-Pr1 cells and stress fiber in DU145 cells, strongly suggesting that this difference of the induction of actin cytoskeleton remodeling by GnRH causes the opposite effects on cell migration. This possibility was investigated in experiments using cells transfected with dominant-negative mutants of Rac1, Cdc42, and RhoA. The results of Figs. 5 and 6 demonstrated that in TSU-Pr1 cells, the activation of Rac1 and Cdc42 was necessary for the enhancement of cell migration by GnRH and that the activation of RhoA was necessary for the attenuation of cell migration by GnRH in DU145 cells. It is known that the localized activation of Rac and/or Cdc42 in concert with other regulators such as WASP/WAVE family proteins, and the Arp2/3 complex stimulates the formation of a branching actin filament network at the leading edge, which in turn induces a protrusion in the direction of migration (50). Thus, it is presumed that the activation of both Rac1 and Cdc42 by GnRH induced the formation of protrusions; as a consequence, cell migration was enhanced in TSU-Pr1 cells.
Another important finding of this study is that the opposite effects of GnRH-I and -II on cell migration occur through the same type human GnRH receptor, human type I GnRH receptor. In the human genome, two types of GnRH receptor genes exist (42, 43, 44, 45, 46). One is the type I GnRH receptor gene, which has been demonstrated to encode a functional seven-transmembrane-spanning G protein-coupled receptor (GPCR). The other is the type II GnRH receptor gene, which contains a frame shift in coding exon 1 and a premature stop codon in exon 2. To date, all studies have failed to produce direct evidence of the transcription or translation of a functional full-length seven-transmembrane-spanning G protein-coupled type II GnRH receptor (16). However, there is indirect evidence that the human type II GnRH receptor functions by itself in the inhibitory effects of GnRH on cell proliferation; this evidence was obtained using cell lines in which the expression of human type I GnRH receptor was inhibited by the antisense fragment transfection method (51). As shown in Figs. 8–10, GnRH had no significant effects on cell migration or actin cytoskeleton remodeling in hGnRHR-1 knocked-down TSU-Pr1 and DU145 clones. We also confirmed that in hGnRHR-1 transiently and stably knocked-down Jurkat (derived from human mature leukemia), TSU-Pr1, HHUA (derived from human endometrial carcinoma), and DU145 cell lines, GnRH had no significant effects on cell proliferation (Ref.24 ; and Utsumi, M., M. Enomoto, and M. K. Park, unpublished data). These results strongly suggest that human type II GnRH receptor does not function, at least as the functional GnRH receptor. The discrepancy between our results and the results of Grundker et al. (52, 53), may be due to the methods used for the inhibition of the expression of hGnRHR-1. It was reported that double-stranded RNA longer than about 30 bp in mammalian cells has major side effects (i.e. interferon responses and global transcription shutdown). In our experiments, TSU-Pr1 and DU145 clones in which hGnRHR-1 was knocked-down by RNAi grew normally; thus, we think that our results are credible. Such a discrepancy due to the methods used for the inhibition of gene expression was also reported for the inositol 1,4,5-triphosphate receptors (54). Moreover, the knock-down of hGnRHR-1 altered the morphology of the TSU-Pr1 cells (Fig. 9A), suggesting that the autocrine activity of GnRH via hGnRHR-1 is important for maintaining the cell morphology. This hypothesis was confirmed by the result that the original TSU-Pr1 cells cultured with LRH13 (a monoclonal antibody against GnRH) showed a similar staining result. In contrast, in DU145 cells, there are no remarkable morphological differences between the original DU145 cells and hGnRHR-1 knocked-down DU145 cells. It is of interest whether this difference has a relationship with the response to the exogenous GnRH treatment or not.
In the present study, the two human prostatic carcinoma cell lines, TSU-Pr1 and DU145, exhibited completely opposite results as response to GnRH stimulation. It is very important how the same cell-type responses in such an opposite manner to the same stimulation. Both TSU-Pr1 and DU145 cells are androgen insensitive and independent and do not produce prostate-specific antigen, and it is thought that the characteristics of TSU-Pr1 are similar to those of DU145 (28, 55). However, in our previous study, we have demonstrated that TSU-Pr1 and DU145 cells exhibited the distinct patterns of the ligand selectivities in the effects of GnRH on colony formation (24, 25). In TSU-Pr1 cells, GnRH-I was more effective (about 10-fold); in contrast, GnRH-II is much more effective (1000–10,000-fold) than GnRH-I in DU145 cells. This difference was observed in the effects of GnRH on cell migration and actin cytoskeleton remodeling (Figs. 3–6). These distinct ligand selectivities between TSU-Pr1 and DU145 cells suggest that the ligand stimulation is mediated via the different types of human GnRH receptors between TSU-Pr1 and DU145 cells. However, our previous study and the present study demonstrated that human type I GnRH receptor is indispensable for both the stimulatory and inhibitory effects of GnRH-I and -II on cell proliferation and cell migration (24). This fact is difficult to explain by the classical ligand receptor theory; however, several studies on human type II GnRH receptor give us an important suggestion. It is known that the human type II GnRH receptor gene is transcriptionally active and has several alternative splice variants (45). Recently, it was reported that the expression of a human type II GnRH receptor fragment inhibited the human type I GnRH receptor function (56). These reports have led us to hypothesize that the several transcripts that are produced may encode functionally modulatory proteins and thereby contribute to the diverse signaling pathways of human type I GnRH receptor. To understand the overall aspects of the signaling pathways of human GnRH receptors, comprehensive investigation of the human type II GnRH receptor alternative splice variants will be necessary in the near future. It is also very important how human type I GnRH receptor is essential for both the stimulatory and inhibitory effects of GnRH on cell proliferation and cell migration. Furthermore, we presented the results strongly suggesting that GnRH affects cell migration via the activation of Rho family G proteins (Figs. 5 and 6). In this study, we did not try to elucidate the detailed signaling pathway from human type I GnRH receptor to Rho family G proteins. It is well known that human type I GnRH receptor is one of the GPCRs that are coupled with heterotrimeric G proteins. Recently, the activation and inactivation of monomeric G proteins, such as Rho family G proteins, via GPCRs have been focused on (57), and GnRH receptor is not an exception. To elucidate this point, the comprehensive studies using inhibitors, toxins, and constitutively active mutants of signaling molecules that is thought to be relevant to the human GnRH receptor signaling will be necessary.
So far, the diverse signaling pathways of the GnRH receptors have been studied (15, 16). Particularly, GnRH receptor expression has been demonstrated in several tumors and tumor-derived cell lines in which the addition of GnRH and its analogs resulted in the inhibition of cell growth, and the molecular mechanisms underlying this effect have been intensively studied. In our previous study, we also demonstrated that GnRH inhibits cell proliferation of DU145 cells (24). Recently, it was reported that GnRH agonist induces apoptosis of DU145 cells and that this requires the activation and/or inhibition of multiple pathway, such as c-Src, protein kinase B, and c-Jun N-terminal kinase (58). These signaling molecules are known to be in the downstream of RhoA (59). Thus, it is possible that our result that RhoA is involved in the GnRH signaling of DU145 cells can be applied to the case of the effects of GnRH on cell proliferation, including apoptosis of DU145 cells, whereas it was reported that early G1 phase expression of cyclin D1 is mediated by Rac1 and Cdc42 (60). Our result that Rac1 and Cdc42 are involved in the effects of GnRH on cell migration of TSU-Pr1 cells can be relevant to the stimulatory effects of GnRH on TSU-Pr1 cell proliferation. Based on the results of this study and our previous studies, the common features and specificity of the signaling pathways of the effects of GnRH on cell proliferation and cell migration will be clarified, and these approaches will provide important clues for understanding the overall aspects of the signaling pathways of the GnRH receptors.
In most vertebrates, GnRH-immunoreactive neurons are detected in the epithelium of the medial olfactory pit soon after its formation. The GnRH-immunoreactive neurons migrate out of the placodal epithelium and into the brain along a migration route that consists of the central processes of the terminal, olfactory, and vomeronasal nerves (61). Recently, it was also reported that in human olfactory GnRH-secreting neurons, GnRH triggered axon growth, actin cytoskeleton remodeling, and an increase in migration, suggesting that GnRH acts in an autocrine manner to promote differentiation and migration of those cells (26).
In the present study, we showed the effects of GnRH in only prostatic carcinoma cell lines; however, the similar effects of GnRH on cell migration were obtained using human T-cell leukemia cell line and human endometrial carcinoma cell line (Enomoto, M., and M. K. Park, unpublished data). In these tissues and organs, expressions of GnRH receptor were demonstrated in normal cells (21, 61, 62). These facts lead us to hypothesize that the ontogenic GnRH activity on cell migration may be observed in physiological and pathological settings of extrapituitary tissues and organs in adults. Furthermore, cell migration orchestrates morphogenesis throughout embryonic development (50). To date, the presence of GnRH and its receptor in preimplantation embryos and effects of GnRH on early embryonic development have been demonstrated in cattle, mice, pigs, and humans, suggesting that GnRH may play a substantial autocrine or paracrine role in human fertilization, early embryonic development, and implantation (62, 63, 64, 65, 66). From this point of view, elucidating the molecular mechanisms of the GnRH effects on actin cytoskeleton remodeling and cell migration will lead to the understanding of the physiological roles of GnRH in embryogenesis and morphogenesis.
Acknowledgments
We express our gratitude to Prof. H. Takeda and Dr. K. Horikawa (Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan) for lending a laser scanning confocal microscope and advice on phalloidin staining and observation using it. We are also grateful to Dr. T. Iizumi (Department of Urology, Teikyo University School of Medicine, Tokyo, Japan) and Dr. J.Y. Seong and Ms. D. Y. Oh (Laboratory of G protein-coupled receptors, Graduate School of Medicine, Korea University, Seoul, Korea) for advice on culturing TSU-Pr1 cells. Finally, we are thankful to Prof. Y. Oka, Dr. Y. Akazome, Dr. H. Abe, and Ms. M. Kyokuwa (Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan) for valuable discussions throughout this study.
Footnotes
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
First Published Online September 29, 2005
Abbreviations: GPCR, G protein-coupled receptor; hGnRHR-1, human type I GnRH receptor; RNAi, RNA interference.
Accepted for publication September 15, 2005.
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