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Cancer Research 68, 1851, March 15, 2008. doi: 10.1158/0008-5472.CAN-07-5793
© 2008 American Association for Cancer Research
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Tumor Microenvironment

Proteinase-Activated Receptor-1–Triggered Activation of Tumor Progression Locus-2 Promotes Actin Cytoskeleton Reorganization and Cell Migration

Maria Hatziapostolou, Christos Polytarchou, Dimitrios Panutsopulos, Lidija Covic and Philip N. Tsichlis

Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts

Requests for reprints: Philip N. Tsichlis, Molecular Oncology Research Institute, Tufts-New England Medical Center, 750 Washington Street, #5609, Boston, MA 02111. Phone: 617-636-6111; Fax: 617-636-6127; E-mail: ptsichlis{at}tufts-nemc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor progression locus 2 (Tpl2), a mitogen-activated protein kinase kinase kinase (MAP3K) that is activated by provirus insertion in retrovirus-induced rodent lymphomas and mammary adenocarcinomas, is known to transduce Toll-like receptor, interleukin 1, tumor necrosis factor {alpha}, and CD40 signals and to play an important role in inflammation. Here we show that Tpl2 is also required for the transduction of cell migration and gene expression signals originating in the G-protein–coupled receptor proteinase-activated receptor 1 (PAR1). PAR1 signals transduced by Tpl2 activate Rac1 and focal adhesion kinase, and they are required for reorganization of the actin cytoskeleton and cell migration. PAR1 expressed in fibroblasts can be triggered by proteinases produced by tumor cells, and PAR1 expressed in tumor cells can be triggered by proteinases produced by fibroblasts. These data suggest that signals that regulate cell migration and gene expression flow between stromal and tumor cells in both directions and that Tpl2 plays a pivotal role in this process. [Cancer Res 2008;68(6):1851–61]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor progression locus 2 (Tpl2), also known as Cot, is a mitogen-activated protein kinase kinase kinase (MAP3K) that is activated by virus integration in Moloney murine leukemia virus–induced T-cell lymphomas and mouse mammary tumor virus–induced mammary adenocarcinomas in rodents (1, 2). Overexpression of Tpl2 activates all the mitogen-activated protein kinase (MAPK) pathways, nuclear factor of activated T cells, and nuclear factor {kappa}B, and promotes cell transformation and proliferation (3, 4). A COOH-terminally truncated, constitutively active mutant of Tpl2, which is overexpressed in retrovirus-induced rodent neoplasms, induces T-cell lymphomas in mice when overexpressed in developing thymocytes (5). Knocking out Tpl2 in mice allowed us to show that Tpl2 is required for the transduction of Toll-like receptor, interleukin 1 (IL-1), tumor necrosis factor-{alpha} (TNF-{alpha}), and CD40 signals, and that it plays a very important role in both cancer and inflammation (6–8).

The four proteinase-activated receptors, PAR1 to PAR4, belong to a subfamily of tethered-ligand G-protein–coupled receptors (GPCR) that are activated by a select group of proteinases. Earlier studies had shown that PAR1, the prototypic member of the PAR family, is expressed in platelets and is activated via NH2-terminal cleavage by thrombin, which targets the bond between residues R41 and S42. Cleavage at this site creates a new NH2 terminus, S42FLLRN47, which acts as an intramolecular ligand (9).

To date, we know that PAR1, as well as other members of the PAR receptor family, are expressed in a variety of cell types. Moreover, PAR1 promotes transformation and tumor cell invasion and metastasis (10). Thrombin, the strongest activator of PAR1, functions as a potent mesenchymal cell mitogen and chemoattractant, promoting the recruitment and proliferation of mesenchymal cells at sites of injury and in tumors (11, 12). However, thrombin is not the only proteinase that activates PAR1. Recent studies revealed that PAR1 is activated by several additional proteinases, including matrix metalloproteinase-1 (MMP-1). MMP-1 is commonly present in the tumor microenvironment, induces the expression of proinflammatory and proangiogenic genes, and promotes tumor cell migration and invasiveness (13).

The downstream effects of PAR1 can be blocked by SCH-79797, which inhibits ligand binding, and by inhibitory pepducins, palmitoylated peptides that target the intracellular surface of the receptor. Studies based on the use of pepducin inhibitors in animals were instrumental in showing that PAR1 plays important roles in inflammation and in tumor cell migration and invasiveness (14, 15).

In this article, we present evidence that Tpl2, which is known to transduce Toll-like receptor, IL-1, TNF-{alpha}, and CD40 signals, is also required for the transduction of PAR1 signals. Specifically, we show that PAR1 signals activate Tpl2 and that Tpl2 is required for the activation of extracellular signal–regulated kinase (ERK)-1/2 downstream of PAR1. Given the importance of PAR1 signals in the regulation of cell migration, we addressed the role of Tpl2 in the PAR1-induced activation of this process. Data presented here show that Tpl2 regulates the organization of the actin cytoskeleton and is required for the stimulation of cell migration by PAR1 signals via Rac and focal adhesion kinase (FAK). Moreover, they show that Tpl2-transduced PAR1 signals regulate the expression of MMPs and other secreted molecules both in fibroblasts and tumor cells. These data suggest that Tpl2-transduced signals generate information that flows between tumor cells and the surrounding stroma in both directions and regulates the migratory properties of both the stromal and tumor cells in a Tpl2-dependent manner. They therefore confirm that Tpl2 plays an important role in regulating intracellular communication, and they expand our understanding of the role the Tpl2 kinase plays in both cancer and inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Constructs, reagents, and antibodies. Wild-type and mutated kinase-inactive (K167M) Tpl2, tagged with the hemagglutinin (HA) epitope, were cloned in the retroviral vector pBabe puro, following standard procedures. HA-tagged FAK-related nonkinase (FRNK), cloned in pCMV5, was kindly provided by T.O. Chan (Thomas Jefferson University, Philadelphia, PA). The plasmid encoding the glutathione S-transferase (GST)-Pak3 Rac-binding domain was a generous gift by Larry Feig (Tufts University School of Medicine, Boston, MA). The T7-tagged N17Rac1 (dominant negative Rac1) and the GST-tagged V12Rac1 (constitutively active Rac1) were kindly provided by Rachel Buchsbaum (Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA). To knock down Tpl2, we used MAP3K8 Stealth RNAi DuoPak and Stealth RNAi Negative Universal Control (Invitrogen).

The PAR1 inhibitor SCH-79797 and thrombin were purchased from Tocris and Haematologic Technologies, Inc., respectively. Tpl2 kinase inhibitor 4-(3-chloro-4-fluorophenylamino)-6-(pyridine-3-yl-methylamino)-3-cyano-[1,7]-napthyridine was purchased from Calbiochem. The PAR1 agonist TFLLRN-NH2 and the DNA primers were synthesized at the DNA/Protein core facility (Tufts University School of Medicine).

Polyclonal antibodies to Tpl2 and Rac1 and monoclonal antibodies to total FAK were purchased from Santa Cruz. Polyclonal antibodies to phospho-ERK1/2, total ERK1/2, phospho-p38MAPK, total p38MAPK, phospho–c-jun NH2-terminal kinase (JNK), total JNK, phospho-Y925 FAK, and phospho–MAPK/ERK kinase (MEK)-1/2 and monoclonal antibodies to HA-tag were purchased from Cell Signaling. The polyclonal antibody to phospho-Y576 FAK and the monoclonal antibody to phospho-Y397 FAK were purchased from Invitrogen and Upstate, respectively. Monoclonal antibodies to tubulin and horseradish peroxidase–conjugated secondary antibodies were purchased from Sigma.

Cell culture, retroviral infections, and transient transfection. Mouse embryonic fibroblasts (MEF) from 13.5-day-old wild-type and Tpl2–/– C57BL6 embryos were used to establish immortalized cell lines according to the 3T3 protocol (16). Immortalized MEFs, MDA-MB 231 breast cancer cells, SKMEL-2 melanoma cells, and NIH-3T3 fibroblasts were cultured under standard conditions.

Retrovirus constructs were packaged via transient transfection of 293T cells. Immortalized Tpl2–/– MEFs were infected in the presence of polybrene (Sigma) and selected for 10 days in puromycin (Sigma). Cells generated from three independent infections were analyzed for HA expression. Tpl2–/– cells reconstituted with wild-type Tpl2 will be referred to as Rec-WT. Similarly, the cells infected with the construct of the kinase-dead mutant of Tpl2 or with the empty vector will be referred to as Rec-KD and Rec-EV, respectively.

For the transient transfection, Lipofectamine 2000 (Invitrogen) was used.

Phalloidin staining. Following thrombin or TFLLRN-NH2 stimulation, cells were stained with fluorescent phalloidin-FITC (Sigma) and images were obtained using a Nikon Eclipse 80i microscope with a 20x objective and a Spot charge-coupled device camera (Diagnostic Instruments).

Immunoblotting. Immunoblotting was done following standard procedures and the protein levels were quantified using the ImagePC image analysis software (Scion Corporation).

Transwell filter and wound healing motility assays. These assays were carried out following standard procedures (17, 18). For details, see Supplementary data.

In vitro kinase assay, zymography, and Rho GTPase GTP loading assays. These assays were carried out following standard procedures (19–22). For details, see Supplementary data.

Quantitative real-time reverse transcriptase PCR. Purification of total RNA from the cells and first-strand cDNA synthesis were carried out with the RNeasy mini kit (Qiagen) and RETROscript (Ambion), respectively. PCRs were done as triplicates in a 25-µL final volume containing template cDNA, iQ SYBRGreen Supermix (Bio-Rad), and specific primers. Primer sequences are shown in Supplementary Table S1. Cycling conditions were as follows: 95°C for 10 minutes followed by 40 amplification cycles (95°C for 15 seconds, 55°C for 35 seconds, and 72°C for 30 seconds). Data were analyzed using an Opticon2 continuous fluorescence detector (MJ Research). Results were expressed as the amount of cDNA for the gene under investigation, divided by the mean of glyceraldehyde-3-phosphate dehydrogenase and β-actin cDNA, in the same sample.

Statistical analysis. The significance of variability between a given group and its corresponding control was determined with the unpaired t test. All results are expressed as the mean ± SE from at least three independent experiments, unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tpl2 is required for the transduction of PAR1-induced cell migration signals in fibroblasts exposed to cancer cell conditioned media. Recent studies produced evidence supporting the concept of the functional activation of the tumor stroma by tumor-derived factors (23). To mechanistically dissect the process and the consequences of tumor stroma activation, we have initiated studies addressing the genetic control of stroma activation by tumor cell–derived factors and the genetic control of the response of the tumor cells to the activated stroma. Here we report the results of our ongoing studies addressing the role of Tpl2 in these processes.

To determine whether Tpl2 is required for the stimulation of fibroblast migration by cancer cell–derived factors, we examined the migration of fibroblasts in response to tumor cell conditioned media, using the transwell filter assay. The fibroblasts were derived from spontaneously immortalized Tpl2–/– MEFs, infected with pBabe puro-Tpl2.HA (Rec-WT), pBabe puro-K167MTpl2.HA (Rec-KD), or pBabe puro (Rec-EV). All cell lines expressed Tpl2.HA at levels only slightly higher than Tpl2+/+ MEFs (Fig. 1A ). The results showed that cancer cell–induced fibroblast migration is significantly impaired in Rec-EV fibroblasts. Conditioned media from MDA-MB 231, SKMEL-2, and NIH 3T3 cells induced the migration of Rec-WT cells by 288 ± 12%, 220 ± 16%, and 276 ± 27%, respectively, in comparison with the Rec-EV fibroblasts (Fig. 1A).


Figure 1
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  		Figure 1. Fibroblast migration induced by tumor cell–derived conditioned media depends on PAR1 signals transduced via Tpl2. A, top left, expression of wild-type (wt) and kinase-dead (KD) Tpl2 in spontaneously immortalized Tpl2–/– fibroblasts. Western blots of cell lysates from two Rec-WT fibroblast cultures and two Rec-KD cultures were probed with an anti-HA monoclonal antibody. Bottom left, Western blot of cell lysates derived from Rec-WT and Tpl2+/+ immortalized MEFs, probed with the anti-Tpl2 antibody. Right, transwell filter assay measuring the migration of Rec-WT and Rec-EV fibroblasts. Cells were exposed to conditioned media from the indicated cancer cell lines or from NIH 3T3 cells. Columns, mean number of migrating cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9). B, migration of Rec-WT fibroblasts in response to conditioned media from the indicated cancer cell lines. Migration was measured with the transwell filter assay. Cells were preincubated in the presence or absence of the PAR1-specific inhibitor SCH-79797 (1 µmol/L) for 30 min. Columns, mean number of migrating cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9).

 
Tumor cells produce a variety of factors that may promote the migration of stromal fibroblasts. Prominent among them are serine and nonserine proteinases, which hydrolyze structural proteins of the extracellular matrix. The same proteinases stimulate PARs, which function as sensors of extracellular proteinase gradients, promoting a cellular response to the proteolytically altered microenvironment. Activation of PAR1 stimulates cell migration in a variety of cell types in culture and in animals (13, 24). We therefore proceeded to determine whether cell migration induced by tumor cell–derived conditioned media depends on the activation of PAR1. Before addressing this question, we used real-time reverse transcriptase PCR (RT-PCR) to determine the expression of all PAR isoforms in immortalized Rec-WT and Rec-EV fibroblasts. The results showed that PAR1 was expressed at higher levels than the other isoforms. However, the expression pattern of all four PAR isoforms was similar in both cell types (Supplementary Fig. S1). To determine whether the induction of cell migration in fibroblasts exposed to tumor cell conditioned media depends on PAR1, we repeated the experiment in Fig. 1A in the presence or absence of the peptide mimetic antagonist of PAR1 SCH-79797 (25). The results (Fig. 1B) showed that inhibition of PAR1 interfered significantly with cell migration induced by both the MDA-MB 231– and SKMEL-2–derived conditioned media (174 ± 25% and 129 ± 41%, respectively). We conclude that PAR1 activation by tumor cell–derived conditioned media plays a major role in promoting fibroblast migration.

Tpl2 is activated by PAR1 and transduces obligatory PAR1-induced ERK activation signals. The preceding data suggested that Tpl2 may transduce PAR1 migratory signals. To address this hypothesis, we examined the activation of Tpl2 by thrombin and addressed the role of Tpl2 in the activation of ERK, JNK, and p38MAPK by thrombin or by the PAR1 peptide agonist TFLLRN-NH2.

Previous studies had shown that Tpl2 is stoichiometrically bound to nuclear factor {kappa}B1/p105 and that p105-associated Tpl2 is stable but inactive. Stimulation of macrophages with lipopolysaccharide or TNF-{alpha} promotes the dissociation of the p58 isoform of Tpl2 from p105. Tpl2 released from p105 is catalytically active but is also unstable and rapidly degraded (26–28). The rapid degradation of the p58 isoform of Tpl2, therefore, can be used as a surrogate marker of activation. Probing Tpl2+/+ MEF lysates with the anti-Tpl2 antibody revealed that the p58 isoform of Tpl2 undergoes rapid degradation in response to thrombin (Fig. 2A ). To confirm the Tpl2 activation by thrombin, we carried out in vitro kinase assays on Tpl2.HA immunoprecipitated with a monoclonal anti-HA antibody from lysates of Rec-WT and Rec-KD fibroblasts using purified GST-MEK1 as the substrate. The results (Fig. 2A) confirmed that Tpl2 is indeed activated rapidly by thrombin. Western blots of lysates from Rec-WT and the respective control cell lines (Rec-EV), harvested before and after thrombin stimulation, were probed with antibodies against phosphorylated and total ERK, JNK, and p38MAPK. The results showed that whereas the phosphorylation of ERK was impaired in the absence of Tpl2, the phosphorylation of p38 was not affected. Finally, whereas the phosphorylation of JNK1 was also impaired in thrombin-treated Rec-EV (at 1 and 2 minutes), the phosphorylation of JNK 2 was not affected (Fig. 2B). To confirm the physiologic involvement of Tpl2 in PAR1 signaling, the same experiment was repeated in Tpl2+/+ and Tpl2–/– MEFs. The results confirmed the preceding data (Supplementary Fig. S2).


Figure 2
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  		Figure 2. Tpl2 is activated on PAR1 stimulation and transduces obligatory ERK and JNK1 activation signals. A, top, thrombin stimulation of immortalized Tpl2+/+ MEFs promotes the rapid degradation of the p58 isoform of Tpl2. Western blot of cell lysates harvested at the indicated time points following thrombin stimulation (1 unit/mL), probed with a polyclonal anti-Tpl2 antibody. Representative picture of three independent experiments. Bottom, thrombin stimulation of fibroblasts rapidly activates Tpl2. Tpl2 in vitro kinase assay, using fibroblast cell lysates harvested at the indicated time points following stimulation with thrombin. Phosphorylation of the GST-MEK1 substrate was detected by probing a Western blot of the product of the kinase reaction with a phospho-MEK1/2 antibody. Representative picture of four independent experiments. B, Tpl2 transduces PAR1 signals that activate ERK1/2 and JNK 1 but not p38MAPK. Rec-WT and Rec-EV cells were stimulated with thrombin (1 unit/mL). Western blots of cell lysates harvested at the indicated time points were probed with antibodies specific for ERK, JNK, and p38MAPK, or their phosphorylated forms. Representative picture of three independent experiments. C, the PAR1-specific agonist TFLLRN-NH2 activates ERK1/2 in a Tpl2-dependent manner. Rec-WT and Rec-EV cells were stimulated with TFLLRN-NH2 (1 µmol/L). Western blot of cell lysates, harvested at the indicated time points, was probed with anti-ERK1/2 and anti–phospho-ERK1/2 antibodies. Representative picture of three independent experiments.

 
PAR1 has been identified as the main thrombin receptor in a variety of cell types (29, 30). However, non-PAR thrombin receptors, responsible for the activation of PAR1-independent signaling pathways by thrombin, have also been identified (31). To determine whether the activation of Tpl2 and its targets by thrombin is due to the activation of PAR1, we treated the cells with the PAR1 agonist peptide TFLLRN-NH2, and we examined the effects of Tpl2 ablation on the phosphorylation of ERK. The results showed that the phosphorylation of ERK in response to PAR1 activation is indeed Tpl2 dependent (Fig. 2C).

Tpl2 is required for the transduction of PAR1 signals that promote the reorganization of the actin cytoskeleton and cell migration. To determine whether the thrombin/PAR1 signaling defect observed in Tpl2-negative cells translates into a defect in cell migration, we repeated the transwell filter experiment using thrombin to deliver the migration signal. The results showed that cell migration induced by thrombin is indeed Tpl2 dependent. Thus, whereas the migration of Rec-WT cells was stimulated significantly (167 ± 19%) when the cells were exposed to thrombin, the migration of Rec-KD or Rec-EV cells was barely affected by the same treatment. Interestingly, the basal cell migration was also enhanced in Rec-WT cells (Fig. 3A ).


Figure 3
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  		Figure 3. Tpl2 is required for the transduction of PAR1 signals that promote cell migration and the reorganization of the actin cytoskeleton. A, Tpl2 is required for the directional movement of fibroblasts toward thrombin. Thrombin-induced directional migration of Rec-WT, Rec-KD, and Rec-EV fibroblasts. Migration was measured with the transwell filter assay. Columns, mean number of migrating cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9). B, the Tpl2-dependent cell migration signals induced by thrombin are triggered by PAR1. The Rec-WT fibroblasts were pretreated for 30 min with the PAR1-specific antagonist SCH-79797 (1 µmol/L). The migration of pretreated and not pretreated fibroblasts in response to thrombin (1 unit/mL) or in response to the PAR1-specific agonist TFLLRN-NH2 (1 µmol/L) was measured using the transwell filter assay. Columns, mean number of migrating cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9). C, Tpl2 ablation diminishes thrombin-induced random cell motility. Wound-healing assays were carried out on a monolayer of confluent cultures of Tpl2–/– or Rec-WT fibroblasts. Cells were either treated with thrombin (Th; 1 unit/mL) or they were left untreated. The images show the wounded areas at 6 and 24 h after wounding. Cell were fixed and stained with phalloidin-FITC. Representative pictures of two independent experiments. D, Tpl2 is required for the reorganization of the actin cytoskeleton by PAR1 signals. Rec-WT and Tpl2–/– cells were seeded on glass coverslips and incubated overnight in media containing 2% fetal bovine serum. The cells were stimulated for 15 min with thrombin (1 unit/mL) or with TFFLRN-NH2 (1 µmol/L). Cells were fixed in 4% formaldehyde, permeabilized with 0.2% Triton X-100, and actin was visualized with phalloidin-FITC. Representative pictures of two independent experiments. Arrows, lamellipodia and fillopodia.

 
To determine whether thrombin triggered cell migration through PAR1 activation, we used the PAR1 agonist TFLLRN-NH2 as the stimulus. To further validate the results of this experiment, we also examined the ability of SCH-79797 to block the effects of TFLLRN-NH2. The results (Fig. 3B) confirmed that PAR1 activation indeed promotes cell migration.

The transwell filter experiments addressed the role of Tpl2 in directional migration induced by thrombin or TFLLRN-NH2. To address the role of Tpl2 in the regulation of random cell motility, we examined cell migration following wounding of confluent cell monolayers. The results showed that thrombin significantly enhanced random cell motility in a Tpl2-dependent manner. Interestingly, the migration of unstimulated cells was also impaired by Tpl2 ablation (Fig. 3C).

Cell migration depends on the reorganization of the actin cytoskeleton and on the assembly/disassembly of focal adhesion complexes (32). In an attempt to identify morphologic features that could help clarify the role of Tpl2 in cell migration, we surveyed the actin cytoskeleton on stimulation with thrombin or TFLLRN-NH2. Stimulation of Rec-WT cells evoked rearrangements of the actin cytoskeleton, characterized by the loss of stress fibers and the formation of lamellipodia and fillopodia. The same treatment of Tpl2-negative cells failed to induce the actin cytoskeleton reorganization observed in Rec-WT cells. These cells instead continued to display a prominent network of stress fibers (Fig. 3D).

Tpl2 is required for the induction and activation of Rac1 by thrombin. Rearrangement of the actin cytoskeleton and the dynamics of focal adhesions are regulated by Rho GTPases (33). We therefore proceeded to determine the expression and the GTP loading of the Rho GTPases Rac1, Cdc42, and RhoA in the Rec-WT and Rec-EV fibroblasts in response to thrombin. The expression of Rho GTPases was measured by quantitative real-time RT-PCR. The results revealed that Rac1 expression is induced at least 2-fold by thrombin, and this induction depends on Tpl2. The effects of thrombin on the expression of Cdc42 and RhoA were small and the differences between Rec-WT and Rec-EV cells were not statistically significant (Fig. 4A ).


Figure 4
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  		Figure 4. Tpl2 is required for the activation of Rac1 and FAK by thrombin. A, Rac1 expression is induced by thrombin in Rec-WT but not in Rec-EV fibroblasts. Cells were stimulated with thrombin (1 unit/mL) for 3 h. The expression of Rac1 was measured in thrombin-treated and untreated cells by quantitative real-time RT-PCR. Results are expressed as mean fold change of mRNA levels in stimulated versus unstimulated cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9). The expression of all tested genes was given the arbitrary value of 1 in untreated Rec-EV cells, which were used as the basis of all comparisons. B, Tpl2 is required for the activation of Rac1, but not RhoA, by thrombin. Rec-WT and Rec-EV cells were stimulated with thrombin (1 unit/mL). The GTP loading of Rac1, Cdc42, and RhoA was then measured in cell lysates harvested at the indicated time points following stimulation, using pull-down assays as described in Materials and Methods. Western blots of the total cell lysates and of the GTPases pulled down from the same lysates were probed with anti-Rac1, anti-Cdc42, and anti-RhoA antibodies. Representative picture of two independent experiments. C, top, schematic diagram of FAK showing the map position of the kinase and FRNK domains as well as the map position of the phosphorylation sites Y397, Y576/577, and Y925. Bottom, FAK phosphorylation at Y397 and Y576 in response to thrombin stimulation depends on Tpl2. Western blots of lysates of Rec-WT and Rec-EV fibroblasts, harvested before and at the indicated time points after thrombin stimulation (1 unit/mL), were probed with antibodies against total FAK or against FAK phosphorylated at Y397, Y576, or Y925. Representative picture of three independent experiments. D, FAK is required both for basal and thrombin-induced cell migration in Tpl2-positive fibroblasts. The Rec-WT fibroblasts were transiently transfected with HA-FRNK, a naturally occurring dominant inhibitor of FAK. The expression of both Tpl2.HA and HA-FRNK was confirmed by probing a Western blot of cell lysates, harvested 24 h after the transfection, with an anti-HA antibody. Cell migration was measured in HA-FRNK– and vector-transfected cells in the presence (1 unit/mL) and absence of thrombin. Columns, number of migrating cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9).

 
The GTP-loaded forms of Rho GTPases were measured by pull-down assays, based on the ability of the GTPases to interact with their downstream targets only when they are bound to GTP. For Rac1 and Cdc42, we measured their binding to the p21 binding domain of p21-activated kinase 1, whereas for RhoA, we measured its binding to the Rho binding domain of Rhotekin. The results (Fig. 4B) showed that thrombin stimulates the rapid loading of Rac1 with GTP (584 ± 86% increase at 5 minutes; Supplementary Fig. S3) via a process that is Tpl2 dependent. Thrombin also stimulated the GTP loading of RhoA, but in a Tpl2-independent manner. Finally, due to the weakness of Cdc42 activation, we could not determine with certainty its dependence on Tpl2.

FAK is activated in a Tpl2-dependent manner and is required for cell migration. Rearrangements of the actin cytoskeleton, the assembly and disassembly of focal adhesions, and cell migration also depend on the activation of FAK, a 125-kDa tyrosine kinase characterized by an NH2-terminal domain that mediates its interactions with membrane proteins, a centrally located kinase domain, and the COOH-terminal focal adhesion targeting domain. A larger region of the COOH terminus is known as the FRNK domain, which is expressed through alternative splicing and acts as a negative regulator of FAK (Fig. 4C; refs. 34, 35). FAK is activated by tyrosine phosphorylation at multiple sites following stimulation, among them, Y397, an autophosphorylation site, and Y925, which provides docking for SH2 domain-containing proteins (34, 35). FAK phosphorylation at Y397 creates a high-affinity binding site for members of the Src family of tyrosine kinases, which phosphorylate FAK at Y576 and activate the kinase (34, 35). To determine whether Tpl2 is required for the transduction of thrombin-generated signals that activate FAK, cells were treated with thrombin and lysates were probed with antibodies recognizing total FAK or FAK phosphorylated at Y397, Y576, and Y925. The relative phosphorylation levels were calculated from the ratio of phosphorylated FAK to total FAK. The results (Fig. 4C and Supplementary Fig. S4) showed that phosphorylation at Y397 occurs within 1 minute from the start of the stimulation and is Tpl2 dependent. Phosphorylation at Y576 takes place within 2 minutes from the start of the stimulation, reaches maximum levels at 5 minutes, and is Tpl2 dependent. Phosphorylation of Y925 was increased in both Rec-WT and Rec-EV cells, suggesting that phosphorylation at this site is Tpl2 independent.

Activated FAK recruits the p130Cas-Crk complex, whose assembly seems to be regulated by Ajuba. Elevated expression of these proteins may limit the maturation of focal adhesion complexes or may promote their rapid turnover, enhancing cell motility (35). To determine the expression of the genes encoding these proteins, we used quantitative real-time RT-PCR. The results (Supplementary Fig. S5) showed that thrombin-induced FAK expression is more robust in Rec-WT cells, although not statistically significant. The expression of all the other genes was induced by thrombin, but in a Tpl2-independent manner.

The preceding data prompted us to investigate whether FAK activation is required for thrombin-induced cell migration. To address this question, we applied the transwell filter assay to measure the migration of Rec-WT cells, before and after transient transfection with FRNK. The results showed that thrombin induces cell migration and that FAK activation is indeed required for the transduction of both basal and thrombin-induced cell migration signals (Fig. 4D).

Epistatic relationship between Rac1 and FAK in the transduction of thrombin-induced cell migration signals. The preceding data raised the question of the epistatic relationship between Rac1 and FAK in the transduction of thrombin/Tpl2 signals that promote cell migration. Previous studies using various stimuli in a variety of cell types placed Rac1 upstream (36–39) or downstream (40–43) of FAK. To address this question, we first examined the ability of thrombin to stimulate the GTP loading of Rac1 in Rec-WT fibroblasts, transiently transfected with FRNK or the empty vector. The results showed that FRNK does not affect the GTP binding of Rac1 in either unstimulated or thrombin-stimulated cells (Fig. 5A ), indicating that Rac1 functions upstream of FAK. To confirm these data, we examined the effects of the constitutively active GST-V12Rac1 and the dominant negative T7-N17Rac1 on the phosphorylation of FAK at Y397 and Y576, on stimulation with thrombin. This experiment was done in Rec-WT fibroblasts transiently transfected with the Rac1 constructs or the empty vector. The results (Fig. 5B, left) revealed that the T7-N17 Rac1 construct attenuates thrombin-induced phosphorylation of FAK at both Y397 and Y576, indicating that Rac1 is required for the activation of FAK. To further explore the epistatic relationship between Rac1 and FAK, we transiently transfected Rec-WT and Rec-EV MEFs with the constitutively active GST-V12Rac1. The results revealed that V12Rac1 induces the phosphorylation of FAK at Y397 and Y576 in a Tpl2-independent manner (Fig. 5B, right). Moreover, V12Rac1 promoted cell migration in both cell types (Supplementary Fig. S6). We conclude that Rac1 functions upstream of FAK and downstream of Tpl2 in thrombin signaling (Fig. 5C).


Figure 5
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  		Figure 5. Rac1 is epistatically upstream of FAK in the PAR1/Tpl2 pathway. A, HA-FRNK expression does not affect thrombin-induced Rac1 activation. Left, Western blots of whole-cell lysates of HA-FRNK– or vector-transfected Rec-WT fibroblasts, harvested before and 5 min after stimulation with thrombin (1 unit/mL), were probed with anti-HA (recognizing Tpl2.HA and HA-FRNK) antibody. Right, the same lysates were used in a GTP-Rac1 pull-down assay as described in Supplementary data. A Western blot of the pull-down sample was also probed with the anti-Rac1 antibody. This experiment was repeated twice with similar results. B, left, the N17 dominant negative mutant of Rac1 inhibits the phosphorylation of FAK at Y397 and Y576. The Rec-WT fibroblasts were transiently transfected with GST-V12Rac1 or T7 N17Rac1 and stimulated with thrombin (1 unit/mL) as indicated. Western blots of whole lysates of these cells harvested before and after stimulation with thrombin were probed with antibodies against total FAK and FAK phosphorylated at Y397. Western blots of whole lysates of the same cells, harvested after stimulation with thrombin, were probed with antibodies against FAK phosphorylated at Y576 or Y925. Middle, Western blots of the same cell lysates were probed with an anti-Rac1 antibody. Right, constitutively active Rac1 induces the phosphorylation of FAK at Y397 and Y576 in both Rec-WT and Rec-EV fibroblasts. The cells were transiently transfected with GST-V12Rac1 or the empty vector and harvested 24 h later. Western blots of whole-cell lysates were probed with antibodies against total FAK and FAK phosphorylated at Y397 and Y576. C, schematic diagram showing the epistatic relationship between PAR1, Tpl2, Rac1, and FAK.

 
Tpl2 is required for the induction of MMP-1, MMP-2, MMP-3, and vascular endothelial growth factor by PAR1 signals. MMPs are nonserine proteinases that are abundantly expressed by both cancer cells and the stroma of invasive and metastatic tumors. MMPs promote tumor cell invasiveness by hydrolyzing structural proteins of the extracellular matrix and by direct stimulation of cell migration. For example, MMP-1 promotes cell migration by activating PAR1 (13). Another abundant proteinase that contributes to tumor cell migration and invasiveness is the serine proteinase urokinase-type plasminogen activator (uPA). Previous studies had shown that MMPs and uPA can be induced by thrombin (10, 44, 45). To determine the role of Tpl2 in the induction of MMPs and uPA, we used real-time RT-PCR to measure the expression of the genes encoding these proteins, before and after thrombin stimulation. The results showed that the expression of MMP-1, MMP-2, and MMP-3 was higher in the Rec-WT cells, both before and after thrombin treatment (Fig. 6A ). Similar results were observed in Tpl2+/+ and Tpl2–/– fibroblasts (Supplementary Fig. S7). We conclude that the activity of Tpl2 is necessary for the induction of these proteinases in the context of thrombin signaling, and that it is sufficient in the absence of additional signals.


Figure 6
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  		Figure 6. Tpl2 transduces PAR1 signals that promote the up-regulation of MMP-2, MMP-3, MMP-1, and VEGF in fibroblasts and mediates tumor cell migration. A, left, Tpl2 ablation down-regulates the basal expression and abolishes the induction of MMP-2, MMP-3, and MMP-1 by thrombin. mRNA levels of MMP-2, MMP-9, MMP-3, MMP-7, MMP-1, and uPA were measured by quantitative real-time RT-PCR in Rec-WT and Rec-EV fibroblasts before and 3 h after the start of thrombin (1 unit/mL) stimulation. Columns, mean fold induction of mRNA levels in thrombin-treated cells, calculated from the combined results of three independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 9). The expression of all tested genes was given the arbitrary value of 1 in untreated Rec-EV cells, which were used as the basis of all comparisons. Right, MMP-2 and MMP-3 basal activity and thrombin-mediated induction are impaired in the absence of Tpl2. Cells were serum starved and treated with thrombin (1 unit/mL) for 24 h. The levels of secreted MMP-2 and MMP-3 were measured by gelatin and casein zymography, respectively, in conditioned media harvested before and after thrombin stimulation. Top, arrows, inactive pro-MMP-2 (72 kDa) and active MMP-2 (62 kDa). The amounts of the enzymes were quantitated by densitometric analyses of the corresponding bands. Results are expressed as mean fold induction of enzyme levels, calculated from the combined results of two independent experiments (n = 2); bars, SE. The secretion of all tested proteinases was given the arbitrary value of 1 in untreated Rec-EV cells, which were used as the basis of all comparisons. B, Tpl2 ablation abolishes the induction of VEGF by thrombin and down-regulates the basal expression of Cyr61 and SDF1a. Expression of the indicated genes was measured by real-time RT-PCR before and 3 h after thrombin stimulation (1 unit/mL) and the results are presented as in A. C, tumor cell migration induced by fibroblast cell–derived conditioned media depends on PAR1 signals transduced via Tpl2. Rec-WT and Rec-EV fibroblasts were stimulated with thrombin (1 unit/mL) either before or 30 min after pretreatment with the PAR1-specific inhibitor SCH-79797 (1 µmol/L). Conditioned media were harvested 24 h following thrombin treatment and they were transferred to the lower compartment of the transwell filter unit. Four hours later, cells migrating to the lower surface of the filter were stained and counted. Columns, mean number of migrating cells, calculated from the combined results of two independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 6). The migrating cells are indicated in the graph. D, MDA-MB 231 cells were transfected with Tpl2 siRNA (siTpl2; 40 nmol/L) or control siRNA (siControl; 40 nmol/L), and 36 h later, they were harvested and transferred to the upper compartment of the transwell filter unit. Conditioned media from Rec-WT and Rec-EV fibroblasts were added to the lower compartment of the chamber, and 4 h later, cells migrating to the lower surface of the filter were stained and counted. Columns, mean number of migrating cells, calculated from the combined results of two independent experiments; bars, SE. Each experiment included triplicate measurements for each condition tested (n = 6). Knockdown efficiency was evaluated by Western blot (left) and quantitative real-time RT-PCR (70%).

 
To further explore the role of Tpl2 in MMPs expression, we applied zymography to measure their activity in media harvested from Rec-WT and Rec-EV fibroblasts. The results showed that thrombin promotes the activation of MMP-2, MMP-3, and MMP-9. However, whereas the activation of MMP-2 and MMP-3 was Tpl2 dependent, the activation of MMP-9 was not. The basal activity of these enzymes was also slightly higher in Rec-WT cells (Fig. 6A).

Cell migration in tumors may be promoted by additional factors, including the vascular endothelial growth factor (VEGF), which is known to be induced by thrombin (25); Cyr61, a member of the CCN family of proteins that is known to enhance PAR1-mediated cell migration (46); and stromal cell–derived factor 1a (SDF1a), a chemokine for hematopoietic progenitor cells, which promotes the growth and metastasis of solid tumors (47, 48). To determine the role of Tpl2 in the expression of genes encoding these molecules, we applied real-time RT-PCR in Rec-WT and Rec-EV cells. The results showed that VEGF is induced by thrombin in a Tpl2-dependent manner (Fig. 6B). The same data were obtained from Tpl2+/+ and Tpl2–/– fibroblasts (Supplementary Fig. S7). Cyr61 and SDF1a were up-regulated in Rec-WT cells equally well in the presence and absence of thrombin, suggesting that their up-regulation maybe caused by Tpl2 signals that are independent of PAR1 (Fig. 6B).

MMPs induced in fibroblasts by Tpl2-transduced PAR1 signals promote cancer cell migration. The Tpl2-dependent induction of MMPs and VEGF by PAR1 signals in fibroblasts raised the question on whether these factors promote tumor cell migration, and if so, whether the promotion of tumor cell migration is also mediated by Tpl2-dependent PAR1 signals. To address this question, we first harvested conditioned media from Rec-WT and Rec-EV fibroblast cultures, before and 24 hours after stimulation with thrombin. Half of the thrombin-stimulated Rec-WT cell cultures were pretreated with SCH-79797. Culture supernatants were used in transwell filter experiments to induce the migration of MDA-MB 231, SKMEL-2, and NIH 3T3 cells (Fig. 6C). To determine whether the effects of the supernatants on tumor cell migration were PAR1 or Tpl2 dependent, MDA-MB 231 cells were pretreated with SCH-79797 or the Tpl2 inhibitor (Supplementary Fig. S8). The effect of supernatants on cancer cell migration was also evaluated by using Tpl2 siRNA (Fig. 6D). The results showed that the fibroblast-derived culture supernatants induced the migration of both the tumor and NIH 3T3 cells, and that both the induction of the cell migration–promoting factors in the fibroblasts and the response of the tumor cells to these factors were PAR1 and Tpl2 dependent. We conclude that fibroblasts secrete MMP-1, MMP-2, and MMP-3 in response to PAR1 signals transduced by Tpl2, and that they, in turn, promote the migration of cancer cells, also via Tpl2-transduced PAR1 signals. The Tpl2-PAR1 axis, therefore, plays an important role in defining the mutual interactions between tumor cells and the surrounding stroma. The data presented here may have important implications on our understanding of the role of these interactions in tumor cell invasiveness and metastasis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our earlier studies had shown that Tpl2 is required for the transduction of Toll-like receptor, IL-1, TNF-{alpha}, and CD40 signals in a variety of cell types, and that it plays a very important role in inflammation. The data presented in this article show that Tpl2 is required for the transduction of signals that originate in the GPCR PAR1. Tpl2 is activated rapidly following PAR1 stimulation with thrombin or with the specific PAR1 agonist TFLLRN-NH2. Tpl2 activation, in turn, is required for the transduction of PAR1 signals that activate ERK and JNK, but not p38MAPK.

Moreover, PAR1 is activated not only by thrombin but also by other proteinases such as MMP-1 (13), which is expressed by neoplastic and inflammatory cells and may therefore activate PAR1 at sites of neoplasia and inflammation. Thus, it has been shown that stromal cells in tumors produce MMP-1, which activates PAR1 in the neighboring tumor cells, and activation of PAR1 enhances its own expression and promotes tumor cell migration and invasiveness. Activation of PAR1 also promotes the expression of a variety of proinflammatory and proangiogenic molecules such as VEGF and Cyr61 (25, 46), both of which enhance the expression of MMP-1 by the tumor stroma.

The observation that Tpl2 contributes to the transduction of PAR1 signals, viewed in the context of the studies addressing the biological output of these signals, led us to explore the role of Tpl2 in PAR1-induced cell migration and in PAR1-induced expression of proinflammatory and proangiogenic molecules. Our data confirmed that Tpl2 is required for the transduction of PAR1 signals that regulate both cell migration and gene expression, and suggested that the role of Tpl2 in oncogenesis and inflammation may be mediated, at least in part, by PAR1 signals that are transduced via Tpl2.

The stimulation of tumor cell migration by Tpl2-transduced PAR1 signals is a process that is clearly important for tumor cell invasiveness in vivo. Earlier studies suggested that the information for tumor cell invasiveness flows from the stroma, which produces the PAR1 activator MMP-1, to the tumor cells, which express PAR1 (13, 46). The data presented in this report provide evidence supporting the results of the earlier reports. In addition, however, they showed that the information may flow in both directions because both fibroblasts and tumor cells produce MMP-1 and PAR1 and both migrate in response to MMP-1 signals transduced via Tpl2.

Cell migration is a complex process that is regulated by an extensive array of signaling molecules. In this report, we examined the role of Tpl2 in the activation of Rho GTPases and FAK, both of which are critically involved in the regulation of cell migration. These experiments showed that Tpl2-transduced PAR1 signals activate Rac1 but not RhoA, a finding that is consistent with the observation that these signals are also associated with the loss of stress fibers and the formation of lamellipodia and fillopodia. The same experiments showed that Tpl2 is required for the transduction of signals that phosphorylate FAK at Y397 and Y576, but not Y925. Because phosphorylation at Y576 is required for the activation of FAK, we conclude that the activation of FAK by PAR1 depends on Tpl2. Interestingly, overexpression of FRNK, a naturally occurring dominant inhibitor of this kinase, blocked PAR1-induced cell migration and confirmed that activation of FAK via Tpl2 is required for the transduction of PAR1-induced cell migration signals.

To determine the epistatic relationship between Rac1 and FAK in the transduction of PAR1/Tpl2 signals, we first examined the effects of FRNK on thrombin-induced GTP binding of Rac1 in Tpl2-positive fibroblasts. The results showed that FRNK did not affect the activation of Rac1 by thrombin and suggested that Rac1 may function epistatically upstream of FAK. This was confirmed by additional experiments addressing the effects of the dominant negative N17 or the constitutively active V12 mutant of Rac1 on the phosphorylation of FAK at Y397 and Y576. However, mapping the epistatic relationship between Tpl2 and other molecules in this pathway needs to be investigated further. Initial preliminary studies along these lines suggested that Tpl2 interacts with several signaling molecules involved in the regulation of the actin cytoskeleton and cell migration.1

PAR1 is one of the ~1,000 GPCRs in the mammalian genome. These receptors respond to a great variety of signals and are responsible for a large array of biological phenomena (49, 50). Mutations of GPCRs have also been linked to various types of cancer (50). Interestingly, there are significant similarities between pathways activated by signals induced by the triggering of different GPCRs. This suggests that although we have not yet addressed the role of Tpl2 in signaling from GPCRs other than PAR1, Tpl2 may have a role in the regulation of many biological phenomena that are under GPCR control.

In summary, this report presents evidence that Tpl2 is required for the transduction of signals originating in the GPCR PAR1. These signals target the actin cytoskeleton and the gene expression machineries and play important roles in the communication between different cell types in both inflammatory and neoplastic sites.


   Acknowledgments
 
Grant support: NIH grant RO1 CA 095431. Some of the core services were provided by the Tufts-NEMC GRASP Center, supported by grant P30DK34928.

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.

We thank T.O. Chan, L. Feig, and R. Buchsbaum for providing expression constructs used in the course of this work; and S. Rajagopal, W. Ren, O. Serebrenikova, S.C. Kampranis, and A. Tzatsos (Molecular Oncology Research Institute, Tufts-New England Medical Center).


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Current address for D. Panutsopulos: Department of Nursing, Faculty of Human Movement and Quality of Life Sciences, University of Peloponnese, Sparti, Laconia, Greece.

1 Unpublished data. Back

Received 10/ 7/07. Revised 1/14/08. Accepted 1/15/08.


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 Results
 Discussion
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