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Cancer Research 68, 115, January 1, 2008. doi: 10.1158/0008-5472.CAN-07-1018
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Thromboxane A2 Receptors in Prostate Carcinoma: Expression and Its Role in Regulating Cell Motility via Small GTPase Rho

Daotai Nie1, Yande Guo2, Dianer Yang1, Yong Tang1, Yakun Chen1, Man-Tzu Wang1, Alex Zacharek2, Yan Qiao2, Mingxin Che2 and Kenneth V. Honn2

1 Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine and SimmonsCooper Cancer Institute, Springfield, Illinois and 2 Department of Pathology, Wayne State University School of Medicine and Karmanos Cancer Institute, Detroit, Michigan

Requests for reprints: Daotai Nie, Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine and the SimmonsCooper Cancer Institute, P. O. Box 19626, Springfield, IL 62794. Phone: 217-545-9702; Fax: 217-545-3227; E-mail: dnie{at}siumed.edu or Kenneth V. Honn, Department of Pathology, Wayne State University, 431 Chemistry Building, Detroit, MI 48202. Fax: 313-577-0798; E-mail: k.v.honn{at}wayne.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Thromboxane A2 (TxA2) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product prostaglandin H2 as the substrate. Previously, increased expression of thromboxane synthase was found in prostate tumors, and tumor cell motility was attenuated by inhibitors of thromboxane synthase. This study was undertaken to elucidate how tumor motility is regulated by TxA2. Here, we report that human prostate cancer cells express functional receptors for TxA2 (TP). Ligand binding assay found that PC-3 cells binded to SQ29548, a high-affinity TP antagonist, in a saturable manner with Kd of 3.64 nmol/L and Bmax of 120.4 fmol per million cells. Treatment of PC-3 cells by U46619, a TP agonist, induced PC-3 cell contraction, which was blocked by pretreatment with the TP antagonist SQ29548 or pinane TxA2. The migration of prostate cancer cells was significantly inhibited either by sustained activation of TP or by blockade of TP activation, suggesting that TP activation must be tightly controlled during cell migration. Further studies found that small GTPase RhoA was activated by TP activation, and pretreatment of PC-3 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. A dominant-negative mutant of RhoA also blocked U46619-induced cell contraction. Taken together, the data suggest that TPs are expressed in prostate cancer and activation of TPs regulates prostate cancer cell motility and cytoskeleton reorganization through activation of Rho. [Cancer Res 2008;68(1):115–21]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Cyclooxygenases (COX) convert arachidonic acid to prostaglandin H2, which can give rise to various bioactive prostanoids via different downstream isomerases. The second isoform of COX (COX-2) is frequently up-regulated in various cancers, including prostate cancer (1). Oncogenes such as ras or Her-2 stimulate COX-2 expression (2), whereas tumor suppressors such as p53 down-regulate COX-2 expression (3). COX-2 has been a promising target for prevention and treatment of cancer (4). However, there are some caveats targeting COX-2 directly for cancer chemoprevention or treatment by using nonsteroidal anti-inflammatory drugs (NSAID), such as aspirin or COX-2–specific inhibitors. Long-term use of NSAIDs, especially aspirin, can have serious side effects, such as gastrointestinal bleeding. In addition, the use of COX-2–specific inhibitors for arthritic pains has been linked to an increased risk of cardiovascular events (5, 6). Therefore, alternative approaches are highly desirable to inhibit COX-mediated cancer initiation or progression.

Among five primary prostanoids from the COX pathway of arachidonic acid metabolism, prostaglandin E2 is reported to promote tumor angiogenesis (7) and represents a novel angiogenic switch in mammary cancer progression (8). Emerging evidences suggest a possible involvement of another prostanoid, TxA2, in tumor progression. TxA2 is a potent vasoconstrictor, mitogen, and platelet activator (9–11). Increased thromboxane synthase expression and/or elevated levels of TxB2, the stable product of TxA2, were found in papillary thyroid carcinoma (12), larynx squamous cell carcinoma (13), and renal carcinoma (14). Previously, we reported an increase in thromboxane synthase at mRNA level in renal carcinoma, breast carcinoma, prostate cancer, and uterine cancer when compared with their matched normal tissues in a cancer profiling array (15). In prostate cancer, the expression of thromboxane synthase was increased in tumor specimens of advanced stage and grade and particularly in the areas of perineural invasion (15, 16). Thromboxane synthase expressed in prostate cancer cells was enzymatically active and may play a contributory role in tumor progression, especially tumor cell motility (15, 17, 18).

It is unknown, however, how thromboxane synthase affects tumor migratory phenotype. Herein, we report that functional TP(s), the G protein–coupled receptor (GPCR) for TxA2, is expressed in prostate tumor cells as well as in tumor specimens. Further, activation of this receptor by TxA2 mimetics has profound effects on tumor cell cytoskeleton organization and causes contraction through the small GTPase RhoA. Our studies suggest that TxA2 and its cognate receptor(s) play a signaling role in tumor progression associated with thromboxane synthase expression in prostate cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Materials and reagents. SQ29548, pinane thromboxane A2 (TxA2), and U46619 were from Cayman Chemical Co. or Biomol. [3H]SQ29548 was purchased from NEN Life Science Products, Inc. Y27632 was from Calbiochem. Rho activation assay kit was obtained from Pierce. Rabbit polyclonal antibodies against TP were kindly provided by Dr. Tai (University of Kentucky, Lexington, KY) or purchased from Cayman Chemical or Santa Cruz Biotechnology.

Cell culture. PC-3 and DU145 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 100 µg/mL each of penicillin/streptomycin at 37°C under an atmosphere of 95% air, 5% CO2, as previously described (15).

Immunohistochemistry. For tissue immunostaining for TP expression, paraffin-embedded tissue sections or tissue arrays were deparaffinized, rehydrated, and antigen retrieved by placing in Declere working solution (Cell Marque) in an electric pressure cooker for 15 min. After a hot rinse with boiling Declere, slides were cooled for 5 min. After washing in deionized water, slides were processed for immunohistochemical staining using a Zymed Histostain-SP kit according to the manufacturer's instructions.

Immunocytochemistry. Cells were grown on glass coverslips in RPMI 1640-10% FBS overnight and changed into serum-free medium. Cells were fixed for 10 min in 3.7% paraformaldehyde solution at room temperature, washed, and then blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. For immunostaining for TPs or other proteins, antibodies were diluted in 1% BSA in PBS, added onto cell monolayers, and incubated for 45 min at room temperature. After washing in PBS-1% BSA thrice, cells were incubated with Alexa Fluor 488–conjugated or Alexa Fluor 568–conjugated secondary antibodies (Invitrogen) for 30 min at room temperature. After staining, the cells on coverslips were washed and mounted with Gold antifade mounting medium (Invitrogen).

For live cell staining, cells cultured on cover glass were blocked with 3% BSA for 20 min and then incubated with primary antibody raised against the first 120 amino acids at the NH2 terminus of TP (H-120, 1:25; Santa Cruz Biotechnology) for 45 min at 4°C. After washing with ice-cold PBS, the cells were incubated with Alexa Fluor 488–conjugated secondary antibody. The staining was immediately examined under an epifluorescence microscope. Cells not incubated with the primary antibody but with the secondary antibody were used as negative controls.

Western blot. PC-3 and DU145 cells were grown to 90% confluence on 10-cm dishes and collected with ice-cold complete radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH 8.0)] containing protease inhibitor cocktail (Sigma). Cell debris was removed by centrifugation. Protein concentration was measured using bicinchoninic acid protein assay kit (Pierce). Protein samples (30 µg) were mixed with 6x SDS sample buffer and subjected to electrophoresis in 8% SDS-PAGE gels and transferred to 0.45 µm of polyvinylidene difluoride membranes. After transfer, the membrane was blocked with TBS containing 5% low fat milk for 60 min and then incubated with the primary antibody against TP receptor (Cayman Chemical) at the dilution of 1:1,000 in TBS-Tween 20 with 5% low fat milk at 4°C overnight. The membrane was washed (thrice) with TBS-Tween 20 (0.1%) and probed with goat anti-rabbit fluorescently labeled secondary antibody (1:5,000) for 1 h at room temperature and washed (thrice) with TBS-Tween 20 for a total of 15 min. The immunoblots were visualized and the target bands were quantified by IR imaging system (Odyssey).

Determination of the TxA2 receptor binding sites. PC-3 cells were seeded in 24-multiwell plates at a density of 1.5 x 105 per well. When the cells reached confluence, the binding test was performed. The binding buffer was prepared by combining 9 volumes of modified HEPES-Tyrode's buffer [10 mmol/L HEPES, 129 mmol/L NaCl, 2.8 mmol/L KCl, 8.9 mmol/L NaHCO3, 1.8 mmol/L KH2PO4, 0.8 mmol/L MgCl2, 5.6 mmol/L dextrose, 1 mmol/L CaCl2 (with pH adjusted to 7.4)] and 1 volume of 3.8% sodium citrate. The confluent PC-3 cells in the 24-well plate were washed twice with the binding buffer. Then, the cells were treated with 0.625, 1.25, 2.5, 5, 10, and 20 nmol/L of the TxA2 receptor antagonist [3H]SQ29548 at room temperature for total binding samples. [3H]SQ29548 plus unlabeled SQ29548 (10 µmol/L) were coincubated in the nonspecific binding samples. After 30 min, the labeled buffer was removed, and the cells were quickly rinsed thrice with 2 mL of washing buffer [20 mmol/L HEPES (pH 7.4), 0.38% sodium citrate]. The cell layer was then solubilized with 0.5 mL of 2% SDS and transferred to scintillation vial with 2.5 mL of scintillation liquid (EcoLite, ICN) to measure the bound radioactivities. Specific bindings were obtained by subtracting the nonspecific bindings from the total bindings. The binding data were analyzed using Prism 4 software to obtain Kd and Bmax (GraphPad Software).

Cell contraction assay. PC-3 cells were plated at 2 x 105 per well in six-well culture dishes in serum-free medium. Twenty-four hours after plating, the cells were treated with U46619 or IBOP (300 nmol/L) or vehicle solvent (ethanol) for durations indicated, with or without 15-min pretreatment with TP antagonists, such as SQ29548 (10 µmol/L) or pinane TxA2 (PTA2; 10 µmol/L), or other inhibitors of downstream effectors as described in the text. Cells were fixed in 3.7% paraformaldehyde for 20 min at room temperature and photographed using a Nikon Eclipse TE200 inverted fluorescence microscope connected to a Sony DKC5000 digital camera. Occasionally, cells were stained with tetramethylrhodamine isothiocyanate-phalloidin (TRITC)-phalloidin. Cells displaying rounded morphology with length less than twice the width were counted. A minimum of 300 cells was scored in a double-blind approach.

Rhotekin pull-down assay for RhoA activation. Ten million PC-3 cells were plated out in serum-free medium in a 10-cm culture dish and, after overnight culture, treated with graded levels of U46619, with or without pretreatment of SQ29548 to block the activation of TP by U46619. At different time intervals, cells were rapidly lysed on ice and processed for Rhotekin pull-down assay for the levels of GTP-bound RhoA according to the instruction from the manufacturer (Pierce).

Transient transfection of RhoA expression constructs. PC-3 cells (2 x 105) were plated on cover glass in RPMI 1640 with 10% FBS and then cotransfected with equal amounts of pEGFP-C2 and one of RhoA allelic constructs (Millipore). Twenty-four hours after transfection, cells were cultured in serum-free medium overnight and treated with 300 nmol/L U46619 or vehicle control for 15 min. The cells were then fixed in 3% paraformaldehyde for 10 min and stained with TRITC-phalloidin (Sigma). The cover glasses were then mounted on a slide and observed under epifluorescence microscopy. The contraction of transfected cells, as indicated by green fluorescent protein, was analyzed.

Cell migration assay. Modified Boyden chamber assay was used to assess the effects of TP activation or inhibition on prostate cancer cell migration essentially as previously described (15). Briefly, both sides of membrane were coated with 10 µg/mL fibronectin for 2 h. After rinsing, 0.5 x 106 prostate cancer cells in 0.5 mL serum-free RPMI 1640 were placed on the top chamber. After the cells settled (1 h), 1 mL RPMI 1640 with compounds for treatment was added to the bottom chamber. The migration was terminated after 6 h, and the cells at the top chamber were removed by cotton swabs. The membranes were fixed, stained, and mounted for enumeration of cells migrated to the underside of membrane. At least six fields were counted in a double-blind approach.

Statistical analysis. Student's t test (two tailed) was used to analyze the difference between two groups. For comparisons among three or more groups, ANOVA test was used. The statistical tests were performed using GraphPad Prism 4.00 for Windows (GraphPad Software). A P value, if <0.05, is considered statistically significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
TP expression in human prostate carcinoma. Previously, we found that thromboxane synthase is expressed in prostate cancer cells and its expression is positively related to tumor progression (15). The arachidonate product of thromboxane synthase, TxA2, can elicit cellular signaling through its cognate GPCRs (TP). To study whether TP is involved in prostate cancer progression, we first evaluated the expression of TP at protein level in normal and cancerous human prostate tissues by immunohistochemistry. Tissue slides were stained with rabbit anti-TP antibody. As shown in Fig. 1A , the benign gland (left) showed weak cytoplasmic staining for TP (the stain on luminal surface is likely artifact), whereas the neoplastic glands from the same patient showed strong cytoplasmic staining for TP (right). The clinical relevance of TP expression is also supported by the observations that increased TP expression was associated with extracapsular extension and invasion of the seminal vesicles by prostate tumors (16).


Figure 1
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  		Figure 1. Expression of TPs in prostate carcinoma cells. A, immunohistochemical evaluation of TP expression in benign and malignant human prostate tissues. Micrographs of a malignant tissue with Gleason grade 2 (right) and its matched benign gland from the same patient (left). Brown, TP staining; blue, nucleus staining with hematoxylin. Original magnification, x200. B, immunocytochemistry analysis of TP. Left, negative control; right, cells stained with a polyclonal antibody recognizing both isoforms of TP. Note the predominant intracellular localization of TP in PC-3 cells. C, Western blot analysis of TP expression at protein level. Top, blot probed with a polyclonal antibody against both isoforms of TP. The annotation of TP isoforms was determined by the molecular weight. Bottom, loading control as revealed by actin. D, densitometry analysis of differential expression of TP isoforms in prostate cancer cells. The density of TP bands was quantified and normalized with actin. Note that whereas PC-3 cells express both TP{alpha} and TPβ, DU145 cells primarily express TPβ.

 
Next, we determined the expression of TPs in human prostate carcinoma cells by immunocytochemistry. As shown in Fig. 1B, TPs were mainly localized in the perinuclear region of the cytosol of PC-3 cells. Western blot analysis confirmed the expression of both TP{alpha} and TPβ isoforms in PC-3 cells (Fig. 1C). The level of total TP proteins in DU145 cells (6.3) was approximately a half of those in PC-3 cells (12.07). Further, in PC-3 cells, TP{alpha} constituted 28% of total TP proteins. In contrast, TP{alpha} accounted for only 7% of total TP proteins in DU145 cells (Fig. 1D).

Surface expression and ligand binding activities of TPs expressed in prostate cancer. The predominant localization of TP in the cytosol of prostate cancer cells led us to examine whether a subset of TPs is expressed at cell surface. A polyclonal antibody raised against the NH2-terminal 120-amino acid residues of TP was used to stain live, nonpermeabilized cells. As shown in Fig. 2A , PC-3 and, to a less extent, DU145 cells had positivity of staining at cell surface, suggesting that a subset of TPs could be localized at plasma membrane.


Figure 2
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  		Figure 2. Ligand binding of TPs expressed in prostate cancer cells. A, live cell staining. DU145 and PC-3 cells were grown on cover glass and stained with an antibody against the NH2 terminus of TP followed by Alexa Fluor 488–conjugated secondary antibody. Note the surface expression of TP in PC-3 and DU145 cells. B, specific binding of PC-3 cells toward SQ29548. Nonlinear regression of the binding data revealed a Kd of 3.641 nmol/L and a Bmax of 120.4 fmol per million cells. Inset, Scatchard plot.

 
To examine the functionality of TPs expressed, ligand binding assays were performed using SQ29548, a high-affinity antagonist of TP (19). Confluent PC-3 or DU145 cells in the 24-well plates were treated with [3H]SQ29548 alone (for total binding) or with [3H]SQ29548 plus excess unlabeled SQ29548 (for nonspecific binding). The specific binding was obtained by subtracting nonspecific binding from total binding. As shown in Fig. 2B, live PC-3 cells can bind SQ29548 in a saturable manner, suggesting the presence of receptors on cell surface. Nonlinear regression analysis of the specific binding data revealed that PC-3 cells bind to SQ29548 with Bmax of 120.4 fmol per million cells and Kd of 3.641 nmol/L. The Bmax and Kd of DU145 cells toward SQ29548 were 51.47 fmol per million cells and 8.06 nmol/L, respectively. The lower Bmax of DU145 cells, when compared with that of PC-3 cells, is consistent with the pattern of TP expression at protein levels (Fig. 1C and D) and TP localization at cell surface (Fig. 2A).

Taken together, the data suggest that at least a subset of TPs is expressed at the cell surface and further prostate cancer cells bind to TP ligand in a specific and saturable manner. Interestingly, the Kd values of PC-3 and DU145 cells are slightly lower than the Kd values of SQ29548 for TP{alpha} (12.4 nmol/L) and for TPβ (10 nmol/L; ref. 20). Both isoforms are expressed in PC-3 cells, whereas in DU145 cells TPβ is predominantly expressed. Further studies are needed to determine whether the increased affinity of TP(s) expressed in prostate cancer cells is due to mutations/variations in primary sequences and/or to potential alterations in their binding partners that may lead to alterations in its affinity toward agonists or antagonists, such as SQ29548.

Induction of cell contraction by TP activation. When plated in serum-free medium, PC-3 cells presented a spindle-shaped morphology. On treatment with U46619 (300 nmol/L), an agonist of TP (21–23), cells contracted and presented a "round" shape within 5 to 15 min (Fig. 3A ). Similar results were also obtained with another TP agonist, IBOP (data not shown; refs. 24, 25). To study whether the induction of cell contraction by U46619 was mediated by TP receptors, we pretreated PC-3 cells with SQ29548 (10 µmol/L) or PTA2 (10 µmol/L), two TxA2 receptor antagonists that have higher affinity to TPs than U46619 does (19, 26). As shown in Fig. 3B, SQ29548 and PTA2 blocked the induction of PC-3 cell contraction by U46619. The blockade of U46619-induced cell contraction by TP antagonists suggests that TxA2 mimetic U46619 induces cell contraction through TP receptors and that the induction of cell contraction by U46619 can be blocked by high-affinity antagonists of TPs, such as SQ29548. The data also suggest that TP(s) expressed in prostate cancer cells is able to transduce extracellular signals to the inside of the cells.


Figure 3
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  		Figure 3. Induction of PC-3 cell contraction by U46619 through TP. A, induction of cell contraction by TP agonist U46619. Left, PC-3 cells treated with ethanol as solvent control; right, PC-3 cells treated with 300 nmol/L U46619 for 15 min. Note the cell contraction as revealed by filamentous actin by TRITC-phalloidin staining. B, blockade of U46619-induced cell contraction by SQ29548 and PTA2, antagonists of TPs. Cells were pretreated with compounds denoted for 15 min before the treatment with 300 nmol/L U46619. **, P < 0.01, when compared with its respective vehicle control [ethanol (EtOH)].

 
Regulation of tumor cell motility by TxA2-TP signaling. Cell contraction is an integral part of cell migration. During the migratory process, cells form lamellipodia or filopodia at the leading edge to charter cells forward. At the trailing edge, however, cells must detach and retract to enable cells to move forward (27). To study whether cell contraction, mediated by TxA2-TP, has effects on prostate cancer cell migration, the effects of U46619 and SQ29548 on PC-3 cell migration were examined using the modified Boyden chamber assay. As shown in Fig. 4A , U46619 stimulated the migration of PC-3 cells at very low concentration (30 nmol/L). However, at higher concentration (300 nmol/L or 3 µmol/L), the migration of PC-3 cells was significantly compromised. In DU145 cells, the effects of U46619 were modest; only at high concentration (3 µmol/L), U46619 had significant inhibition on cell migration (Fig. 4A). Interestingly, blockade of TP activation with SQ29548 or PTA2 also significantly reduced PC-3 migration (Fig. 4B), suggesting that TP activation is required for PC-3 cell motility.


Figure 4
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  		Figure 4. TP regulation of tumor cell migration. Modified Boyden chamber assay was used to assess the effects of U46619, SQ29548 (10 µmol/L), or PTA2 (10 µmol/L) on PC-3 and DU145 cell migration on fibronectin as described in Materials and Methods. A, modulation of PC-3 and DU145 cell migration by U46619. Points, average number of cells migrated expressed as percentage of the vehicle controls; bars, SD. B, inhibition of PC-3 cell migration by TP antagonists. Columns, average number of cells migrated; bars, SD. *, P < 0.05; **, P < 0.01, when compared with the vehicle solvent control.

 
Taken together, the data suggest that TP activation has to be controlled in a spatial and temporal manner in its regulation of cell migration. On one hand, blockade of TP activation with SQ29548 or PTA2 was found inhibitory for cell migration, suggesting that TP activation is required for tumor cells (PC-3) to migrate. On the other hand, sustained activation of TP with high levels of U46619 significantly compromised tumor cell motility. Further studies are required to determine when and how TP is activated in a temporal and spatial manner facilitative for cell migration and whether TP activation requires endogenous synthesis of TxA2 or other TP ligands, such as prostaglandin H2.

Activation of RhoA by TxA2-TP signaling. As a GPCR, TP activation can elicit a multitude of signaling cascades, including the activation of phospholipase C, mobilization of Ca2+, phosphorylation of p38 and p42/44 mitogen-activated protein kinases (MAPK), formation of cyclic AMP, and activation of protein kinase G, C, and A (28). We examined the involvement of different effectors of TP activation in cell contraction induced by U46619 using various pharmacologic inhibitors. It was found that the activities of p42/44 MAPK, p38 MAPK, protein kinase G (Fig. 5A ), phosphatidylinositol 3-kinase, or pertussis toxin-sensitive Gi subunit were not required for U46619 to induce cell contraction (data not shown). The screening revealed that Y27632 (10 µmol/L), a Rho kinase inhibitor (29), was able to block U46619-induced contraction in PC-3 cells (Fig. 5A), suggesting a possible involvement of small GTPase RhoA in cell contraction induced by TP activation.


Figure 5
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  		Figure 5. RhoA in TP-mediated cell contraction. A, blockade of U46619-induced cell contraction by Y27632, an inhibitor of Rho kinase (ROCK), but not by KT5823, an inhibitor of protein kinase G. **, P < 0.05, when compared with its respective vehicle controls (ethanol). B, activation of RhoA by U46619, a TxA2 receptor agonist, in PC-3 cells. Cells were pretreated with ethanol (left four lanes) or 3 µmol/L SQ29548 (right three lanes) for 15 min before the addition of graded levels of U46619. After 15 min, cells were lysed and active RhoA (GTP bound) was evaluated using Rhotekin pull-down assay as described in Materials and Methods. Top, levels of GTP-bound RhoA; bottom, RhoA proteins in the supernatants after Rhotekin pull-down assay. Note the activation of RhoA by U46619 and in cells pretreated with SQ29548, RhoA activation by U46619 was inhibited. C, active RhoA is sufficient to cause cell contraction, whereas dominant-negative RhoA blocks U46619-induced cell contraction. Green cells denote the transfected cells with the constructs indicated at the left. D, effects of various RhoA allelic forms on U46619-induced cell contraction. Cells were transfected with the constructs as indicated and then treated with 300 nmol/L U46619. *, P < 0.05, when compared with its respective vehicle controls (ethanol). Note the abolishing of U46619-induced cell contraction by either constitutively active RhoA (Rho-Act) or dominant-negative RhoA (Rho-DN) but not the wild-type RhoA (Rho-WT).

 
To elucidate whether RhoA participates in TxA2 cellular signaling, we studied whether activation of TP can lead to the activation of RhoA. As shown in Fig. 5B, U46619 treatment stimulated the activation of RhoA as shown by Rhotekin pull-down assay. This activation was attenuated by pretreatment of cells with SQ29548, a high-affinity antagonist for TPs. The data suggest that U46619 stimulated the activation of RhoA through TxA2 receptor activation.

To further confirm the involvement of RhoA in U46619-induced cell contraction, we evaluated the effects of ectopic expression of RhoA or its mutants on U46619-induced cell contraction. As shown in Fig. 5C and D, the constitutively active mutant of RhoA was sufficient to cause cell contraction, whereas the dominant-negative mutant of RhoA blocked cell contraction induced by U46619. The data affirm that activation of RhoA is required for TxA2-TP to induce cell contraction and that RhoA may serve as intermediary for TxA2-TP signaling axis to regulate tumor cell motility.

Rho GTPases, which include RhoA, Cdc42, and Rac, are critical for the dynamic changes in cell shape and adhesions that drive migration (30–33). During the highly coordinated process of migration, cells form lamellipodia or filopodia at the leading edge to charter cells forward (27). At the trailing edge, however, cells must detach and retract to enable cells to move forward (27). Cdc42 is required for cell polarity and filopodial protrusions, whereas Rac1 promotes lamellipodial protrusions. The contraction at the trailing edge is mediated by the small GTPase RhoA (27). Recently, it has been shown that RhoA was activated by GPCR signaling, including those elicited by U46619, through G12 heterotrimeric G proteins during invasion of cancer cells, including PC-3 (34, 35). Our study suggests that TP can regulate cell migration and cytoskeleton reorganization through inducing cell contraction via activating RhoA.

In summary, the present study reports that functional receptors for TxA2 were expressed in prostate cancer cells. Activation of TP by receptor agonists, such as U46619, caused profound reorganization in cytoskeleton and induced cell contraction. Cell motility was significantly inhibited either by sustained activation of TP with high concentrations of U46619 or by blockade of TP activation with SQ29548. Further studies found that RhoA was activated by U46619 through TP activation, which was required for U46619 to induce cell contraction. The present study suggests that TxA2-TP signaling axis may regulate cell migration through elaborating Rho activation and cytoskeleton reorganization. Further studies are required, however, to determine how TxA2-TP signaling is activated and regulated in a temporal and spatial manner optimal for cell migration and whether the TxA2-TP signaling axis can be intervened to impede the invasion and metastasis of prostate carcinoma cells.


   Acknowledgments
 
Grant support: NIH grant R01CA114051 (K.V. Honn), U.S. Department of Defense Prostate Cancer Research Program New Investigator Award No. W81XWH-04-1-0143 (D. Nie), and Illinois Department of Public Health Prostate Cancer Research Program Grant (D. Nie).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/19/07. Revised 10/ 2/07. Accepted 10/30/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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