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Tumor Biology |
Division of Hematology/Oncology [A. M., F. F., A. K.], Molecular Cardiology Research Institute, New England Medical Center, and Departments of Medicine and Biochemistry, Tufts University School of Medicine [L. K., A. K.], Boston, Massachusetts 02111
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSCell Surface Expression of...PARs Generate Intracellular Ca2+...Activation of PAR1 by...Activation of PAR1 Inhibits...Inhibition of Chemotaxis in...DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSCell Surface Expression of...PARs Generate Intracellular Ca2+...Activation of PAR1 by...Activation of PAR1 Inhibits...Inhibition of Chemotaxis in...DISCUSSIONREFERENCES |
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PAR1 has also been proposed to play a role in the pathological invasion processes of breast cancer (13
, 14)
. Like the majority of cancers, invasive breast cancers originate from epithelial tissues, which are enveloped by the ECM. Normal breast epithelial cells do not have the capacity to migrate efficiently in response to chemotactic signals. Examination of preinvasive breast carcinoma biopsy specimens reveals high expression levels of the RANTES chemokine both in the carcinoma cells and the infiltrating leukocytes (15)
. Application of RANTES, MIP-1
, MIP-1ß, or MCP-1 chemokines that are secreted by leukocytes (16)
stimulates chemotaxis of MCF-7 cells. Thus, breast carcinoma cell lines including MCF7, T47D, and ZR-75–1 have somehow acquired or reacquired the ability to migrate but still lack invasive capabilities (16)
. To be invasive, the cancer cell must produce matrix metalloproteases and have signaling pathways that can coordinate the cyclic cytoskeletal motions required for attachment and traction through integrin-ECM contacts (17
, 18)
. Because proteases are essential for cancer cells to invade through the ECM, there has been a concerted effort (13
, 14
, 19
, 20)
to determine whether PARs are also involved in the invasion and metastasis processes. For instance, studies (21)
have shown that thrombin activation of PAR1 promotes cell adhesion to vitronectin, whereas trypsin activation of PAR2 stimulates 5ß1-dependent adhesion to fibronectin in human gastric carcinoma cells.
Recently, Even-Ram et al. (13) demonstrated that PAR1 expression levels are directly correlated with degree of invasiveness in both primary breast tissue specimens and established cancer cell lines. High levels of PAR1 mRNA were found in infiltrating ductal carcinoma and very low amounts in normal and premalignant atypical intraductal hyperplasia. Transfection of the invasive breast cancer cells with antisense PAR1 DNA abolished invasion implying that PAR1 expression was somehow involved in the invasion process. However, the effect of activation of PAR1 by thrombin or the contribution of the other PARs, if any, in the breast cancer cell invasion process was not evaluated.
Here, we determined that invasive breast cancer cells express high levels of PAR1, PAR2, and PAR4. We present the first evidence that PAR2 and PAR4 can act as chemokine receptors. Moreover, we make the unanticipated observation that thrombin and PAR1 peptide ligands strongly inhibit migration and invasion of MDAMB231 breast cancer cells. This PAR1-mediated inhibition requires that the agonist is placed in the direction of cell migration. Analysis of G-protein dependent signaling pathways lead us to conclude that the inhibitory effects of PAR1 on invasion are mediated by a Gi/PI(3)kinase-dependent pathway. Furthermore, selective stimulation of Gi-signaling by other pathways can also prevent cell migration and invasion. These findings suggest a novel strategy to interdict migration and invasion of breast cancer cells.
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MATERIALS AND METHODS |
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-thrombin was obtained from Hematological Technologies (Essex Junction, VT). The PAR agonists T-(pf-F)LLRN (TFLLRN), SFLLRN, SLIGKV, GYPGKF, and AYPGKF were synthesized as COOH-terminal amides at the Tufts University peptide core facility. Recombinant hirudin, PMA, GFX, wortmannin, LY, genistein, mastoparan M5280, and trypsin were obtained from Calbiochem (La Jolla, CA). MTT, PTx, and epinephrine were from Sigma Chemical Co. (St. Louis, MO). Fura2/AM was from Molecular Probes (Eugene, OR), PPACK and TLCK were from Boehringer Mannheim (Indianapolis, IN).
Cell Culture.
MDAMB231 and MCF7 cells were obtained from the National Cancer Institute (NIH, Bethesda, MD). MDAMB231 cells were maintained in RPMI 1640 supplemented with 10% FBS, 0.15% sodium bicarbonate, and 1% penicillin and streptomycin under conditions of 5% CO2 at 37°C. MCF7 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin and streptomycin. NIH3T3 fibroblast cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin.
Flow Cytometry.
Rabbit polyclonal PAR1-Ab (raised against residues S42FLLRNPNDKYEPF55C), PAR3-Ab (raised against residues T42FRGAPPNSFEEFP55C), and PAR4-Ab (raised against residues G42YPGQVSANDSDTLELP58C) were generated from keyhole limpet hemocyanin conjugates and purified on the corresponding antigen peptide-conjugated columns by affinity chromatography as described previously (12)
. Goat anti-PAR2 antibody and FITC-conjugated donkey antigoat antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Flow cytometry was performed on MDAMB231 and MCF7 cells as before (12)
. The reactivity of each of the PAR-antibodies was confirmed using tissue culture cells that expressed the individual PAR receptors. The PAR antibodies were not cross-reactive.
Invasion Assays.
Invasion of cells through Matrigel was determined using a Transwell system (6.5 mm diameter, 8-µm pore size with polycarbonate membrane; Corning Costar) as described previously (22)
with the following modifications. Cells were starved overnight in serum-free medium containing 0.1% BSA. Matrigel (40 µg) was polymerized in the upper well at 37°C for 2 h. Cells (25,000) were added to the upper well of each chamber in 200 µl of serum-free medium containing 0.1% BSA. CM (600 µl) from NIH3T3 fibroblasts was added to the lower well with or without various concentrations of agonists or inhibitors. The cells were incubated for 40 h at 37°C. Cells in the top well were completely removed by swiping with cotton swabs and the cells on the underside of the membrane were stained using the Diff-Quik (Dade Boehringer) stain kit. Cells on 15–30% of the membrane area were counted at x20–40 and extrapolated to 100% of the membrane surface. Percentage of basal invasion was defined as number of cells migrating in the presence of Matrigel/number of cells migrating in the absence of Matrigel x 100, and invasion in the presence of agonist was defined as (number of cells migrating in the presence of Matrigel + agonist) number of cells migrating in the absence of Matrigel x 100. Statistical analyses were performed using a two-tailed Student’s t test. Statistical significance was assumed to occur when P 
0.05.
Migration Assays.
Transwell migration assays were performed essentially as the invasion assays above except that Matrigel was omitted. Cells (50,000/200 µl in serum-free medium) were added to the upper wells and agonists (thrombin, SFLLRN, TFLLRN, SLIGKV, GYPGKF, or AYPGKF) were added to the upper and/or lower wells of the transwell chambers for the "checkerboard" (23)
migration studies. Cells were incubated for 5 h at 37°C before being stained and counted and statistical analyses conducted as in the invasion studies.
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RESULTS |
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Cell Surface Expression of PAR1, PAR2, PAR3, and PAR4 in Breast Cancer Cells. |
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, MDAMB231 cells express relatively high levels of PAR1 and PAR4 and lower levels of PAR2. The MCF7 breast cell line expresses low levels of PAR2 and PAR4 (Fig. 1B)
and trace levels of PAR1 (Fig. 1B)
. Little or no PAR3 was present in the MCF7 or MDAMB231 cells. A second noninvasive breast carcinoma cell line, T47D, gave essentially identical PAR expression profiles as MCF7 (data not shown) and was not studied further.
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PARs Generate Intracellular Ca2+ Fluxes in Breast Cancer Cells. |
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(Gi), which causes production of inositol triphosphate and mobilization of Ca2+ from intracellular stores. The SFLLRN peptide agonist activates both PAR1 and PAR2; TFLLRN activates PAR1; SLIGKV activates PAR2; GYPGKF and AYPGKF activate PAR4; and thrombin activates PAR1, PAR3, and PAR4. Synthetic agonists for PAR3 have not yet been described. As shown in Fig. 2A
, the MDAMB231 cells exhibit robust intracellular calcium fluxes when stimulated with PAR1, PAR2, or PAR4 agonists. Thrombin and SFLLRN cause sharp increases in intracellular Ca2+ that quickly fall back to baseline levels. The PAR1-selective peptide TFLLRN gives a Ca2+ response similar in profile to that elicited by thrombin. The PAR2-selective agonist SLIGKV also gives a sharp Ca2+ response, and the PAR4-selective ligand GYPGKF gives a robust Ca2+ response. The Ca2+ responses were markedly different in the MCF7 cells (Fig. 2B)
. Addition of TFLLRN gave a nearly undetectable Ca2+ response consistent with the flow cytometry data, which showed trace levels of PAR1 expression. A larger Ca2+ response was elicited with SLIGKV and SFLLRN indicating that PAR2 is functional in the MCF7 cells. The PAR4 agonists GYPGKF and thrombin both gave very slow but appreciable rises in intracellular Ca2+ demonstrating that PAR4 is functionally active in the MCF7 cells.
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Activation of PAR1 by Thrombin or Peptide Ligands Inhibits Invasion of MDAMB231 Cells. |
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. By comparison, CM elicits extremely low levels of invasion (1.5%) in the MCF7 cells, which is consistent with previous studies (13
, 14)
. Neither MDAMB231 nor MCF7 cells invade the Matrigel (<0.01%) when CM is omitted from the bottom wells in the presence or absence of any of the PAR agonists (data not shown). Thrombin and SFLLRN had no effect on cell migration when added to the top well (Fig. 3
, 
).
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, low concentrations of thrombin added to the bottom well blocks 80% of cell invasion with an IC50 value of 
5 nM. Addition of hirudin, a direct thrombin inactivator, reversed the inhibition of invasion by exogenously added thrombin (Fig. 4)
. The PAR1 peptide agonist SFLLRN inhibits 93% of invasion with an IC50 of 200 nM (Fig. 3B)
. However, high amounts of the serine protease-alkylating agents PPACK or TLCK, hirudin alone, or a PAR1-blocking antibody (3)
did not inhibit basal invasion of the MDAMB231 cells (Fig. 4)
. This effectively rules out the possibility (13
, 14)
that activation of PAR1 by endogenously produced serine proteases, including thrombin, is necessary for the invasion of MDAMB231 cells through the ECM.
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, or trypsin (data not shown) did not inhibit basal invasion of the MDAMB231 cells but slightly stimulated invasion (20–30%) at higher concentrations of peptide ligand. Identical results (data not shown) were obtained for all of the PAR agonists from independently maintained MDAMB231 cells (gift of Vimla Band, NEMC, Boston, MA). Next, we determined whether the inhibitory effects of thrombin or SFLLRN on the invasion of the MDAMB231 cells were attributable to impaired growth or apoptosis (2
, 26)
. As shown in Table 1
, 0.1–100 nM of thrombin or 0.1–100 µM of SFLLRN had little or no effect on the viability of the MDAMB231 breast cancer cells during the time period of the invasion experiments (40–48 h).
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Activation of PAR1 Inhibits Chemotactic Migration of MDAMB231 Cells. |
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, thrombin inhibits migration of the MDAMB231 cells when present at 
1 nM concentration in the bottom well. Addition of thrombin to the upper well (Fig. 5, C and D)
did not inhibit cell migration. Similar results were observed with the PAR1 ligand, SFLLRN. Placement of SFLLRN (1 µM) in the lower well inhibited 80% of migration of the MDAMB231 cells (Fig. 5, E and F)
. Placement of SFLLRN in the upper well had little or no effect on migration (Fig. 5, G and H)
. Addition of the PAR2 or PAR4 ligands SLIGKV and GYPGKF to the upper or lower wells did not inhibit migration of the MDAMB231 cells (data not shown).
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, the CM is strongly chemotactic to the MDAMB231 cells, does not stimulate chemokinesis (Fig. 6C)
, and, thus, must be added to the bottom well for migration to occur. Therefore, activation of PAR1 with a concentration gradient of thrombin or SFLLRN in the same direction as the chemotactic agent(s) inhibits chemotaxis in these highly invasive cells.
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. The PAR2- and PAR4-selective ligands SLIGKV and GYPGKF (and AYPGKF, data not shown) stimulate 1.5–2-fold increases in migration of the MCF7 cells indicating that these PARs can act as chemotactic receptors. PAR1 did not mediate the chemotactic effects of thrombin, because the PAR1-specific agonist TFLLRN had no effect on MCF7 migration (Fig. 7B)
. It is important to stress that the basal levels of cell migration are quite different in MCF7 cells (2.5–5%) versus MDAMB231 (50–70%). Therefore, each of the PARs, depending on the cellular context, i.e., normal versus invasive, could conceivably operate as either chemoinhibitory or chemotactic receptors.
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Inhibition of Chemotaxis in the MDAMB231 Cells Is Mediated Through Gi and PI(3)K. |
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subunits from Gi-coupled chemokine receptors (23
, 27)
. The free ß subunits then stimulate PI(3)K and generate polarized lipid signals to direct cell movement (28
, 29)
. We found that pretreatment of MDAMB231 cells with PTx, which inactivates Gi, results in complete inhibition of invasion in the presence or absence of thrombin (Fig. 8)
. Likewise, the specific PI(3)K inhibitor LY blocks 70% of the basal invasion of the MDAMB231 cells. Addition of thrombin to the LY-treated cells showed no additional inhibition of invasion, which is evidence that the inhibitory effects of thrombin require the activity of PI(3)K.
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. Therefore, PAR1-mediated inhibition of MDAMB231 cell invasion is independent of PKC nor does the basal invasion process require PKC. Tyrosine kinases such as focal adhesion kinase and the src kinase family members fyn, yes, and lck have been shown to be essential for regulating the assembly and disassembly of focal adhesion complexes in migrating cells (18
, 32)
. Accordingly, addition of the tyrosine kinase inhibitor genistein blocked 80–85% of cell invasion in the absence or presence of thrombin.
Next, we tested whether other activators of Gi-signaling besides PAR1 could inhibit cell migration and invasion. As shown in Fig. 9
, addition of epinephrine, which activates the Gi-coupled 2-ARs (33)
blocks 60% of MDAMB231 cell invasion. Expression of the Gi-coupled 2C-AR in MDAMB231 cells was confirmed by a search of the NCI60 cDNA microarray database (34)
. The wasp venom mastoparan which is a direct activator of Gi-dependent nucleotide exchange, also inhibits 60% of cell invasion and migration of the MDAMB231 cells (Fig. 9A
and data not shown). Moreover, mastoparan potently inhibits (IC50 6 nM) essentially all migration of the poorly invasive breast cell line MCF7 (Fig. 9B)
. Thus, it is evident that inhibition of breast cancer cell invasion and migration is not restricted to PAR1 and may be elicited to a greater or lesser extent by other Gi activators. Cell viability experiments were performed, and the inhibitors or activators used above (PTx, GFX, PMA, genistein, mastoparan, or LY) caused little or no growth-inhibitory or stimulatory effects (0.73–1.32-fold relative to control) over the 40–48 h time period encompassed by the invasion experiments (Table 1)
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DISCUSSION |
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We identified the G-protein signaling pathways that are responsible for both chemotaxis and the PAR1-dependent chemoinhibition of breast cancer cells. Many studies using hematopoietic cells have shown that cell movement is directed by polyphosphoinositide lipid signals such as PIP3, which are internally polarized to the side of the cell nearest to the chemotactic agent (28)
. This spatially restricted signal emanates from free ß subunits released from Gi-coupled chemokine receptors, which, in turn, activate PI(3)K to generate PIP3. The transiently produced PIP3 binds pleckstrin homology domain-containing proteins such as phosphoinositide-dependent kinase and serine/threonine protein kinase B, which then control Rac/Rho/Cdc42-dependent pathways. In accordance with the studies in inflammatory cells, we showed that both chemotaxis and inhibition of chemotaxis in breast cancer cells require Gi and PI(3)K. Activation of Gi-signaling with the 2-AR ligand epinephrine or by the wasp venom mastoparan blocked basal invasion of the MDAMB231 cells. Mastoparan was also a very potent inhibitor of migration of the poorly invasive MCF7 cells (Fig. 9)
. Thus, the observed chemoinhibition seen on activation of PAR1 is not restricted to PAR1 alone, but may be achieved by other Gi-coupled receptors.
We propose three possible pathways, which are not mutually exclusive, that would explain the inhibition of cell motility and invasion of the breast cancer cells by activated PAR1 (Fig. 10)
. The first explanation is that by activating PAR1 we have simply desensitized the chemokine receptors by receptor phosphorylation (31)
. We ruled out participation of PKC in the PAR1-mediated chemoinhibition of the MDAMB231 cells, because addition of the PKC inhibitor GFX did not block the inhibitory effects of thrombin on invasion (Fig. 8)
. If cross-desensitization of chemokine receptors is an important contributor to the PAR1-dependent chemoinhibition, it was not elicited on equally robust stimulation of the homologous PAR2 and PAR4 receptors (Fig. 2)
. A second possibility is that activated PAR1 steals downstream components that are necessary for proper chemokine signaling. These sequestered components could include certain Gi(ß) isoforms or other immediate effectors in the PAR1 microenvironment that are not accessible to PAR2 or PAR4. A third possibility is that activation of PAR1 causes inappropriate or hyperstimulation of signaling pathways, which interfere with the proper cyclic pattern of signaling invoked by ligand-internalized and recycled chemokine receptors (31)
. Hyperstimulation could occur at essentially any level, from the immediate downstream effectors of PAR1 such as ß
PI(3)K to the more temporally remote components that regulate focal adhesion turnover and the contraction/relaxation apparatus. Hyperstimulation by PAR1 could be exacerbated by the fact that PARs are irreversibly cleaved by their cognate proteases. Because stimulation of PAR1, the 2-AR, or the addition of the direct Gi activator mastoparan all cause inhibition of MDAMB231 cell migration and invasion, we should consider the likelihood that a common mechanism through the GißPI(3)KRho family GTPases disrupts coordinated cell motion.
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and in MCF7 cells (Fig. 7)
. This is relevant because tumors would be expected to generate trypsin-like proteases, which can activate these two receptors. Like PAR1 (35
, 36)
, PAR2 is a known Gi-coupler (21)
, and addition of PTx to the MDAMB231 cells causes a 50% reduction in Ca2+ levels in response to the PAR2 agonist SLIGKV.5
Thus, to the extent that chemotaxis requires a receptor that can activate Gi to release the appropriate ß subunits (23)
, both PAR2 and PAR4 likely can also interact with Gi in both invasive and noninvasive breast cancer cells. Hence the ability to couple to Gi may be a necessary but not sufficient condition to cause chemoinhibition of a highly invasive breast cancer cell such as MDAMB231. Subtle differences in the intracellular loop structures of the PAR1 versus PAR4 and/or perhaps the magnitude and duration of signals emanating from Gi/ß subunits or from other immediate effectors may dictate whether a particular PAR can act as either a chemotactic or chemoinhibitory receptor.
From an analysis of the gene expression patterns of the NCI-60 cancer cell panel (34)
, we note that unlike their epithelial/carcinoma progenitors, highly invasive and motile breast cancer cells such as MDAMB231 gain high levels of expression of Gi-couplers such as PAR1 and the 2-ARs. These receptors are expressed in platelets and other vascular cells and are essential for adhesion of the platelets to the vascular wall during blood coagulation. A compelling biological rationale for our results is that the breast cancer cells use these receptors to adhere to the endothelium and underlying matrix on entering the blood stream during the later vascular invasive and metastatic stage. Corroborating evidence for this mechanism has been shown in melanoma cells, which express PAR1 and tissue factor (20)
. The trypsin-activated PAR2 and PAR4 receptors are also expressed in the invasive cancer cells. Therefore, it is possible that proteases with trypsin-like specificity could be used to facilitate invasion of cancer cells by recruiting the chemotactic abilities of PAR2 and PAR4. Once the cell has penetrated into the vasculature and encounters the highly specific serum protease thrombin, then PAR1 would activate adhesion proteins for attachment at distant sites and metastasis. This scenario presents an exciting opportunity to impact the initial mobilization of breast cancer cells prior to their invasion into the vascular system. If Gi-couplers are critical for the evolution of breast cancer cells as they acquire the ability to mobilize and invade the vasculature, then therapeutics that target selected Gi-couplers, such as PAR1, represent a novel strategy to alter the biological behavior of tumor cells in the context of hormonal or other ongoing treatments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The costs of publication of this article were defrayed in part by 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. 
2 Supported by NIH Grant R01HL57905 and by a Scholar Award from the PEW Scholars Program in the Biomedical Sciences.
3 To whom requests for reprints should be addressed, at New England Medical Center, Division of Hematology/Oncology, Box 8486, 750 Washington Street, Boston, MA 02111. Fax: (617) 636-4833; E-mail: athan.kuliopulos{at}tufts.edu
4 The abbreviations used are: ECM, extracellular matrix; PAR, protease-activated receptor; PI(3)K, phosphoinositide-3-OH kinase; PKC, protein kinase C; PMA, phorbol-12,13-dibutyrate; MTT, 3-(4,5-dimethylthiazole-2-yl-2,5-diphenyl tetrazolium; PTx, pertussis toxin; PPACK, D-phenylalanyl-L-propyl arginine chloromethylketone; TLCK, L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride; FBS, fetal bovine serum; CM, conditioned medium; GFX, GF109203X; LY, LY294002; PIP3, phosphatidyl inositol-3,4,5-triphosphate; 2-AR, 2-adrenergic receptor.
5 L. Kamath, and A. Kuliopulos, unpublished results.
Received 12/28/00. Accepted 5/31/01.
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REFERENCES |
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5ß1-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2. J. Biol. Chem., 275: 4592-4598, 2000. subunit of Gi, not by activating Gq or Gs. Proc. Natl. Acad. Sci. USA, 94: 14489-14494, 1997. dimers released by activation of Gi-coupled receptors. Proc. Natl. Acad. Sci. USA, 94: 14495-14499, 1997. in chemoattractant-mediated signal transduction. Science (Wash. DC), 287: 1046-1049, 2000.(2A)-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels. J. Biol. Chem., 275: 1763-1772, 2000. subunit. J. Biol. Chem., 275: 2627-2635, 2000.This article has been cited by other articles:
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X. Zhang, J. L. Hunt, D. P. Landsittel, S. Muller, K. Adler-Storthz, R. L. Ferris, D. M. Shin, and Z.(G. Chen Correlation of Protease-Activated Receptor-1 With Differentiation Markers in Squamous Cell Carcinoma of the Head and Neck and Its Implication in Lymph Node Metastasis Clin. Cancer Res., December 15, 2004; 10(24): 8451 - 8459. [Abstract] [Full Text] [PDF]
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 L. Hu, M. Lee, W. Campbell, R. Perez-Soler, and S. Karpatkin Role of endogenous thrombin in tumor implantation, seeding, and spontaneous metastasis Blood, November 1, 2004; 104(9): 2746 - 2751. [Abstract] [Full Text] [PDF]
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 D. Darmoul, V. Gratio, H. Devaud, F. Peiretti, and M. Laburthe Activation of Proteinase-Activated Receptor 1 Promotes Human Colon Cancer Cell Proliferation Through Epidermal Growth Factor Receptor Transactivation Mol. Cancer Res., September 1, 2004; 2(9): 514 - 522. [Abstract] [Full Text] [PDF]
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X. Shi, B. Gangadharan, L. F. Brass, W. Ruf, and B. M. Mueller Protease-Activated Receptors (PAR1 and PAR2) Contribute to Tumor Cell Motility and Metastasis Mol. Cancer Res., July 1, 2004; 2(7): 395 - 402. [Abstract] [Full Text] [PDF]
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B.-C. Lee, T.-H. Lee, S. Avraham, and H. K. Avraham Involvement of the Chemokine Receptor CXCR4 and Its Ligand Stromal Cell-Derived Factor 1{alpha} in Breast Cancer Cell Migration Through Human Brain Microvascular Endothelial Cells Mol. Cancer Res., June 1, 2004; 2(6): 327 - 338. [Abstract] [Full Text] [PDF]
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G. M. Hjortoe, L. C. Petersen, T. Albrektsen, B. B. Sorensen, P. L. Norby, S. K. Mandal, U. R. Pendurthi, and L. V. M. Rao Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration Blood, April 15, 2004; 103(8): 3029 - 3037. [Abstract] [Full Text] [PDF]
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 V. S. OSSOVSKAYA and N. W. BUNNETT Protease-Activated Receptors: Contribution to Physiology and Disease Physiol Rev, April 1, 2004; 84(2): 579 - 621. [Abstract] [Full Text] [PDF]
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B.-H. Koo and D.-S. Kim Factor Xa Induces Mitogenesis of Vascular Smooth Muscle Cells via Autocrine Production of Epiregulin, J. Biol. Chem., December 26, 2003; 278(52): 52578 - 52586. [Abstract] [Full Text] [PDF]
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 P. J. O'Brien, H. Koi, S. Parry, L. F. Brass, J. F. Strauss III, L.-P. Wang, J. E. Tomaszewski, and L. K. Christenson Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation: Receptor Activation Mediates Extravillous Trophoblast Invasion in Vitro Am. J. Pathol., October 1, 2003; 163(4): 1245 - 1254. [Abstract] [Full Text] [PDF]
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L. Ge, Y. Ly, M. Hollenberg, and K. DeFea A {beta}-Arrestin-dependent Scaffold Is Associated with Prolonged MAPK Activation in Pseudopodia during Protease-activated Receptor-2-induced Chemotaxis J. Biol. Chem., September 5, 2003; 278(36): 34418 - 34426. [Abstract] [Full Text] [PDF]
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Y.-J. Yin, Z. Salah, S. Grisaru-Granovsky, I. Cohen, S. C. Even-Ram, M. Maoz, B. Uziely, T. Peretz, and R. Bar-Shavit Human Protease-Activated Receptor 1 Expression in Malignant Epithelia: A Role in Invasiveness Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 940 - 944. [Abstract] [Full Text] [PDF]
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M. Mareel and A. Leroy Clinical, Cellular, and Molecular Aspects of Cancer Invasion Physiol Rev, April 1, 2003; 83(2): 337 - 376. [Abstract] [Full Text] [PDF]
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 N. Sugimoto, N. Takuwa, H. Okamoto, S. Sakurada, and Y. Takuwa Inhibitory and Stimulatory Regulation of Rac and Cell Motility by the G12/13-Rho and Gi Pathways Integrated Downstream of a Single G Protein-Coupled Sphingosine-1-Phosphate Receptor Isoform Mol. Cell. Biol., March 1, 2003; 23(5): 1534 - 1545. [Abstract] [Full Text] [PDF]
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 M. D. Hollenberg and S. J. Compton International Union of Pharmacology. XXVIII. Proteinase-Activated Receptors Pharmacol. Rev., June 1, 2002; 54(2): 203 - 217. [Abstract] [Full Text] [PDF]
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 Q.-D. NGUYEN, S. FAIVRE, E. BRUYNEEL, C. RIVAT, M. SETO, T. ENDO, M. MAREEL, S. EMAMI, and C. GESPACH RhoA- and RhoD-dependent regulatory switch of G{alpha} subunit signaling by PAR-1 receptors in cellular invasion FASEB J, April 1, 2002; 16(6): 565 - 576. [Abstract] [Full Text] [PDF]
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