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VASCULAR BIOLOGY
1Department of Clinical Pharmacology and 2Department of Biochemistry, Royal College of Surgeons, Dublin; and 3Cork Cancer Research Centre, Mercy University Hospital, University College Cork, Cork, Ireland
Submitted 14 December 2005 ; accepted in final form 22 February 2006
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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v
3-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior. cytoskeletal remodeling; dynamic blebs
The driving force for cell movement is provided by the dynamic reorganization of the actin and tubulin cytoskeleton. The Rho GTPases, in particular Rho, Rac, and Cdc42, play important roles in modulating actin and microtubule activity (32). Like all members of the small GTPase superfamily, these proteins cycle between an inactive GDP-bound and an active GTP-bound state. They act as molecular switches that can be activated by a variety of extracellular stimuli to promote the formation of integrin-containing adhesion complexes, which mediate attachment to the extracellular matrix. Because Rho GTPases have been implicated in vascular endothelial responses to fluid shear (26), we hypothesized that they may also play a critical role in the behavior of metastatic cells in blood flow. Within the blood, tumor cells are exposed to shear stress resulting from the frictional force of blood against the vessel wall and shear rates resulting from the velocity of flowing fluid layers. In adherent vascular endothelial cells, shear stress induces translocation of Cdc42 and Rho from the cytosol to the membrane, while the Rho-ROCK pathway regulates cytoskeletal remodeling in response to shear (26). Tzima et al. (29) recently showed that shear stress rapidly stimulates conformational activation of integrin v3 in adherent vascular endothelial cells, followed by increased cell adhesion to extracellular matrix components and transient downregulation of Rho activity. Although shear responses through Rho-GTPases have only recently been demonstrated in adherent vascular endothelial cells, the direct effect of shear alone on metastatic cells is not well characterized.
Rho GTPases interact with different oncogenic signaling cascades, including those downstream of Ras. The primary products of RAS genes are sequestered in the cytosol until activation by growth factors, which trigger recruitment to the plasma membrane or Golgi (12). The Raf-mitogen-activated protein kinases (MAPKs) pathway has been identified as a key effector in Ras signaling (5). However, the recruitment of many other Ras targets is critical to elicit a full Ras response. It has been shown that shear stress induces a rapid and transient activation of Ras in adherent vascular endothelial cells, which in turn regulates the MAPKs, including extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs) (17, 27). Although Rho GTPases are required for Ras signaling (22), the contributions they make to metastatic motility remain unclear.
In the present study, we show that ROCK plays a critical role in the morphological and cytoskeletal changes observed in OC-1 cells under shear. We show that shear induces specific changes in the distribution of actin and tubulin in OC-1 cells. Under static conditions, actin and tubulin colocalize to the cortical ring of the cell. By contrast, a mechanosensing mechanism signals tubulin polymerization to the cell edge under shear. Furthermore, we show that dynamic bleb formation is induced by shear alone, independent of integrin ligation. Y-27632, a specific ROCK inhibitor, inhibits both shear-induced dynamic blebs and integrin v3-Ras colocalization at the leading cell edge. Further analysis shows that Ras activity is increased in the presence of shear, an effect inhibited by Y-27632. Finally, we demonstrate that shear alone enhances invasion of metastatic cells, a process inhibited by the presence of Y-27632. In summary, we demonstrate a critical role for ROCK and Ras in metastatic behavior under physiological shear conditions.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Lipopolysaccharide (LPS), bovine serum albumin (BSA), phosphate-buffered saline (PBS), and human plasma fibronectin (Fn) were purchased from Sigma-Aldrich; Dulbecco's modified Eagle's medium (DMEM) was from GIBCO; and ROCK inhibitor (Y-27632) was purchased from CN Biosciences.
Antibodies and Cells
Monoclonal antibody directed against tubulin was purchased from Sigma-Aldrich. Monoclonal antibody against Ras was from BD Biosciences. Monoclonal antibody against the integrin receptor V3 (LM609) was obtained from Chemicon International. Phalloidins (Alexa Fluor 488 and 647) were from Molecular Probes. The metastatic esophageal cancer cell line OC-1 was established from the ascites of a male patient with metastatic squamous cell cancer of the esophagus (23). Human umbilical vascular endothelial cells (HUVECs) were isolated and cultured as previously described (24).
Parallel-Plate Flow Chamber Assays
The interaction between adherent OC-1 cells and shear was monitored in a parallel-plate flow chamber (Glycotech, Rockville, MD) mounted on the stage of an Axiovert-200 epifluorescence microscope (Zeiss, Gottingen, Germany). A syringe pump (Harvard Apparatus, Holliston, MA) was used to generate shear rates between 50 and 800 s–1. All experiments were maintained at 37°C.
Analysis of effects of shear on actin and tubulin remodeling in adherent OC-1 cells with or without Y-27632.
To examine the effects of shear on actin and tubulin in metastatic cells, adherent OC-1 cells were exposed to static (no shear) or sheared conditions (continuous shear rate of 200 s–1, corresponding to a low venous shear rate). Fn was used as a ligand-binding substrate, because OC-1 cells bind to Fn through the integrin V3 (16). Glass slides were coated with Fn (100 µg/ml) for 2 h at room temperature, blocked with 1% BSA in PBS for 1 h, and then washed three times with PBS before use. OC-1 cells were resuspended in serum-free DMEM at a concentration of 1 x 106 cells/ml. Tumor cell suspension (400 µl) was added to each Fn-coated slide and incubated for 1 h at room temperature. To evaluate the effect of shear on adherent cells, OC-1 cells were allowed to adhere to Fn for 30 min and then exposed to a continuous shear rate of 200 s–1 for 30 min at 37°C. Adherent OC-1 cells were then washed three times with PBS, fixed in 3.7% formaldehyde for 15 min at room temperature, and permeabilized with ice-cold acetone for 2 min. After permeabilization, the OC-1 cells were incubated with 5 µg/ml tubulin antibody for 1 h at room temperature, washed, and incubated with a secondary rhodamine goat anti-mouse IgG antibody for 10 min. Phalloidin Alexa Fluor 488 was added at a concentration of 5 µg/ml and incubated at room temperature for 20 min to stain actin. The cells were then washed with PBS and mounted with DAKO fluorescent mounting medium. Both resting adherent cells and adherent cells subject to shear were analyzed with a LSM510 Axioplan 2 upright confocal microscope (Zeiss). Fluorescence was detected at 546 and 488 nm.
In additional experiments, we examined the combined effects of shear and ROCK inhibition on shear-induced tubulin and actin remodeling in adherent OC-1 cells. OC-1 cells were pretreated with Y-27632 (10 µg/ml) for 2 h and resuspended in DMEM at a concentration of 1 x 106 cells/ml. The OC-1 cells were harvested either resting or after shear and evaluated for the effect of ROCK inhibition on actin and tubulin remodeling.
Analysis of effects of ROCK inhibition on dynamic bleb formation. The behavior of OC-1 cells under physiologically relevant shear conditions was assayed as previously described (16). In brief, OC-1 cells in the presence and absence of Y-27632 were resuspended in DMEM (1 x 106 cells/ml) and allowed to settle for 5 min on LPS-stimulated HUVECs in the parallel-plate flow chamber before being exposed to shear rates of 50–800 s–1. As soon as flow was initiated, images were captured along the center of the chamber every 200 ms up to 1 min by a liquid-chilled Quantix-57 charge-coupled device camera (Photometrics, Tucson, AZ). Flow was then stopped for 5 min to analyze the tumor adhesion. After the 5-min interval, the shear rate was increased incrementally to 100, 200, 400, 600, and 800 s–1, with 5-min intervals of no flow. The interaction of the OC-1 cells and HUVECs was analyzed offline with the commercial software package MetaMorph (version 4.6.8, Universal Imaging, Downingtown, PA). Flow rates were calculated with the equation for laminar flow to give a venous shear rate between 50 and 800 s–1.
Analysis of effects of ROCK inhibition on localization of integrin V3 and Ras in adherent OC-1 cells in presence of shear.
OC-1 cells with or without Y-27632 (10 µM) treatment were allowed to adhere to Fn for 30 min and then exposed to a continuous shear rate of 200 s–1 for 30 min at 37°C. Adherent OC-1 cells were then washed three times with PBS, fixed in 3.7% formaldehyde for 15 min at room temperature, and permeabilized with ice-cold acetone for 2 min. Monoclonal antibodies against v3 (LM609, 10 µg/ml) and Ras (anti-Ras, 5 µg/ml) were conjugated to fluorescent IgGs (Molecular Probes), FITC, and rhodamine, respectively, and incubated with OC-1 cells for 1 h at room temperature. The cells were then washed with PBS and mounted with DAKO fluorescent mounting medium.
Cone-Plate Shearing
To evaluate the direct effect of shear alone on tumor behavior, OC-1 cells were sheared in a cone-plate viscometer (Haake RheoStress RS600). The cone-plate viscometer has been used to study both cell surface receptor function and shear-induced cellular activation phenomena (23). The device consists of a stationary plate placed beneath a rotating cone maintained at a constant temperature of 37°C. A cone angle of 4° was used in the current study, with a gap between the cone and plate of 0.146 mm. The geometry of the viscometer enables application of a uniform, linear shear rate to the entire cell suspension. OC-1 cells suspended in serum-free DMEM (1 x 106 cells/ml) were placed in the viscometer, and a shear rate of 200 s–1 was applied immediately for 15 min. The OC-1 cells were then washed and pelleted at 100 g for 3 min and fixed in 3.7% formaldehyde for 15 min. In additional experiments, OC-1 cells pretreated with or without Y-27632 (10 µM) were sheared for 15 min and assayed for their ability to invade or lysed for Ras activity as described below.
Cell Viability Assay
Cell viability was measured by Trypan blue exclusion. Cells in 200 µl of DMEM were mixed with 50 µl of Trypan blue stain. The number of live and dead cells was counted with a hemocytometer. Results were expressed as the means of four independent experiments.
Measuring Ras-Bound GTP and GDP
Ras activation in the presence and absence of Y-27632 was measured in cells sheared with a cone-plate viscometer. The activation status of Ras was assayed as previously described (6). Ras-bound GTP and GDP were measured using an EZ-Detect Ras activation kit (Pierce). OC-1 cell suspensions were exposed to a shear rate of 200 s–1 for 15 min via cone-plate viscometry or to static conditions for 15 min. The cells were pelleted at 100 g for 5 min, resuspended in ice-cold Tris-buffered saline, and pelleted at 100 g for 5 min. The pellet was then lysed in lysis buffer (25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT, and 5% glycerol) on ice for 5 min. OC-1 lysates were centrifuged at 16,000 g at 4°C for 15 min. Activated Ras was pulled down with the glutathione S-transferase (GST)-Raf1-Ras-binding domain (RBD) complex followed by Western blotting for active Ras.
Invasion and Migration Assay
The effects of shear and ROCK inhibition on OC-1 cell invasion were determined with a modified Boyden chamber assay (BD BioCoat Matrigel Invasion Chamber, BD Biosciences). Briefly, OC-1 tumor cell suspensions containing 5 x 104 cells/ml were sheared at 200 s–1 for 15 min via cone-plate viscometry and added immediately to inserts within the invasion chamber. A standard Matrigel invasion protocol was followed that recommended a standard time point of 22 h to assess tumor invasion. Hence, the OC-1 cells were placed for 22 h in a humidified tissue culture chamber at 37°C in 5% CO2 atmosphere. Nonsheared OC-1 cell suspensions were used as a control. After the 22-h incubation period, OC-1 cells that had invaded the Matrigel layer were stained with crystal violet and quantified by light microscopy. Cells from 10 randomly chosen fields (x10, x40, and x63 magnification) on the lower side of the filter were counted, and all experiments were run in triplicate.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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In the present investigation, we examined the role of shear in modulating actin and tubulin in OC-1 cells adherent to the matrix protein Fn. In previous studies, we analyzed the adhesion and activation of OC-1 cells on a range of individual matrix components under flow and found that maximal adhesion occurred on the matrix protein Fn (data not shown). We allowed OC-1 cells to adhere to Fn and compared the cellular morphology and localization patterns of actin and tubulin under static and sheared conditions. For adherent OC-1 cells, shear was generated in a parallel plate-flow chamber.
Under static conditions, adherent OC-1 cells exhibited minimal lamellipodial activity and maintained a rounded and smooth morphology after 1-h adhesion to Fn (Fig. 1A). When a constant shear stress of 200 s–1 was applied for 30 min, a significant increase in lamellipodial protrusion and cell surface area was observed (Fig. 1B). A marked difference in the distribution of actin and tubulin staining was observed in OC-1 cells exposed to static and venous shear conditions. Colocalization of both actin and tubulin was observed in static and sheared OC-1 cells. Whereas actin stained more prominently at the cortical cell edge in the absence of shear, actin staining was far more diffuse and not localized to the cell edge in the presence of shear. By contrast, tubulin rapidly translocated to the leading cell edge and lamellipodia, forming a meshwork under shear (Fig. 1B).
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Shear Alone Induces Dynamic Bleb Formation in OC-1 Cells
Because shear stress is a critical determinant in metastasis progression and induces dynamic bleb formation in adherent metastatic esophageal cells (16), we wanted to determine the effect of shear alone on cellular morphology. Therefore, we investigated the effects of shear on OC-1 cells with a cone-plate viscometer. This system is commonly used for measuring the dynamic response of cells under a controlled shear field. OC-1 cells were subjected to a shear rate of 200 s–1 for 5, 10, 15, and 30 min via cone-plate viscometry and examined for dynamic bleb formation. When OC-1 cells were subjected to a constant shear rate of 200 s–1 for 10, 15, and 30 min, there was a marked increase in the percentage of cells exhibiting both large and small dynamic blebs (P < 0.008 or P < 0.001) compared with nonsheared OC-1 cells (Fig. 2A; n = 4; any degree of blebbing was characterized as a blebbing cell). A representative cell is shown in Fig. 2B. These morphological changes were not due to cell death, because there was no difference in the uptake of Trypan blue between sheared (15 min, 200 s–1) and nonsheared (Fig. 2C) cells.
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Shear alone caused dynamic bleb formation in OC-1 cells. This dynamic blebbing phenomenon has been associated with movement through a three-dimensional matrix and utilizes the Rho-ROCK signaling pathway (9). We therefore investigated the effect of Y-27632, a specific ROCK inhibitor, on shear-induced dynamic bleb formation in adherent OC-1 cells under shear. Y-27632 caused no toxicity at a concentration of 10 µM as assessed by Trypan blue staining (data not shown). To simulate physiological shear conditions, we investigated the morphology of OC-1 cells adherent to LPS-stimulated HUVECs under shear conditions in the parallel-plate flow chamber as previously described (16). The tumor cells were allowed to settle on the inflamed endothelium for 5 min, and then perfusion was initiated. We and others (20a) have documented that a brief period of minutes facilitates the interactions between receptors and their ligands under various shear conditions. Exposing adherent OC-1 cells to a shear rate of 200 s–1 for 5, 10, 15, 20, 25, and 30 min resulted in a significant increase in dynamic blebbing at 15, 20, 25, and 30 min (P < 0.001 or P < 0.0001), in contrast to static adherent OC-1 cells (Fig. 3A).
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Y-27632 Interrupted Intercellular Meshworks Between OC-1 Cells Adherent to Fn Under Flow
Because the addition of Y-27632 inhibited dynamic bleb formation and OC-1 cell spreading on the HUVEC matrix under flow, we investigated the effect of Y-27632 on the cytoskeletal rearrangement of actin and tubulin in blebbing OC-1 cells under static and sheared conditions. OC-1 cells pretreated with or without Y-27632 were allowed to adhere to Fn in the presence and absence of shear and stained for both actin and tubulin.
In untreated cells, a striking arrangement of a tubulin meshwork was observed around the cell periphery, specifically under shear. However, in the presence of Y-27632 and shear, this tubulin meshwork was significantly reduced (Fig. 3C). Redistribution of actin from the cortical ring to the cell body was inhibited in the presence of Y-27632, in contrast to untreated cells, which show distinct actin rich blebs.
Y-27632 Inhibits Integrin v3-Ras Colocalization in the Cell Bleb
The mechanochemical mechanisms of fluid shear-induced signal transduction are thought to be mediated through the integrins (29) and the Ras-MAPK pathway (5). Although Rho GTPases are required for Ras signaling, the contributions they make to metastatic motility remain unclear. To investigate the effect of ROCK inhibition on Ras activity, we first examined the localization of Ras with integrin v3 in OC-1 cells with or without Y-27632 treatment. Our results show that clustered integrin v3 colocalized with Ras in OC-1 cell blebs and at the cell periphery under shear conditions in the absence of Y-27632 (Fig. 4). By contrast, in the presence of Y-27632 and shear, this colocalization was inhibited and the clustering activity of Ras was less distinct. Furthermore, Ras translocated from the cell periphery to the cytosol, indicative of an inactive Ras state.
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Our results demonstrate that shear activates metastatic cells through a ROCK-mediated signaling mechanism. Therefore, to investigate the effect of shear on the ability of OC-1 cells to invade and the involvement of ROCK in this process, we used a BioCoat Matrigel Invasion Chamber assay. This assay utilizes a mixture of extracellular matrix components coated on a filter as a model of the basement membrane. Matrix and growth factor components were kept constant throughout the invasion experiments, to assess the direct effect of shear alone and Y-27632 drug treatment. A time point of 22 h was recommended by the Matrigel invasion protocol to assess tumor invasion.
OC-1 cells pretreated with or without Y-27632 were sheared in the cone-plate viscometer for 15 min and then seeded onto the matrix coating. Invasion into the extracellular matrix barrier was then determined. Single OC-1 cell invasion was mainly observed; however, sheets of invading cells were also detected, which may have resulted from possible cell proliferation. Analysis revealed that sheared OC-1 cells are significantly more invasive (P < 0.007) in contrast to nonsheared samples (Fig. 6A). The enhanced invasiveness of sheared OC-1 cells was abrogated in the presence of Y-27632. By contrast, there were no significant differences in tumor invasion between static and Y-27632-treated static cells.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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v3 and Ras at the leading edge of the cell. Moreover, we show that shear activates Ras and enhances OC-1 cell invasion, processes inhibited by the presence of Y-27632. In summary, we demonstrate a novel role for shear in metastatic motility and invasion through ROCK and Ras signaling. Esophageal cancer is one of the least studied and deadliest cancers worldwide. Its pathogenesis is unclear; however, more than 90% of patients exhibit metastatic disease at the time of diagnosis (7). Adhesion of tumor cells to the vasculature of host organs is a prerequisite for further metastatic spread (11) and occurs primarily in venous circulation (14). Tumor responses to shear forces include morphological alterations of cell shape and deformation, induction of signaling cascades, and cell activation. Recently, von Sengbusch et al. (30) showed that shear forces caused by hydrodynamic shear flow can induce tyrosine hyperphosphorylation of focal adhesion kinase in colon carcinoma cells. Key processes in the mobility of metastatic cells are dynamic bleb formation regulated through ROCK activity (22) and lamellipodia extension regulated by the small GTPase Rac (24). These processes have been well characterized under static conditions; however, the role of shear in regulating this dynamic behavior in metastatic cells is less well understood. We used flow chamber adhesion assays to assess the effect of shear on adherent OC-1 cells. By contrast, a cone-plate viscometer was used to assess the effect of shear alone on OC-1 cell suspensions, independent of adhesion.
Microtubule-actin interactions are basic phenomena that underlie many fundamental cellular processes, including cell motility, cell division, and cortical flow. Recently, Korb et al. (15) demonstrated that pharmacological disruption of actin and microtubules can result in modification of metastatic tumor cell adhesion within the hepatic microcirculation. To investigate the role of shear in OC-1 cell cytoskeletal remodeling, we compared the localization of actin and tubulin in static and sheared OC-1 cells. We demonstrated a marked difference in the localization patterns of actin and tubulin in OC-1 cells exposed to static and sheared conditions. Under static conditions, actin localized to the cortical ring of the cell. By contrast, a polymerized meshwork of tubulin translocated to the leading cell in the presence of shear. The role of microtubules in controlling the polarity of a migrating cell has only recently been shown (33). They may serve as tracks for membrane and organelle transport toward the leading edge of a cell (18), while growing microtubules may directly promote lamellipodial protrusion (20) and locally regulate adhesion and contraction (1). Moreover, it was demonstrated recently that microtubules specifically invade regions where stress is locally increased (13). In the present study, we have shown that tubulin rapidly polymerizes under the shear stress of fluid flow, suggesting a dynamic mode for tubulin in the metastatic process.
The Rho family of GTPases are critical in the mobility of tumor cells, regulating the cytoskeleton and cell migration. RhoA, Rac1, and Cdc42, and their downstream effectors, including ROCK, are the best-characterized members of the Rho family. We recently demonstrated (16) that metastatic esophageal cells activate in response to defined venous shear rates, characterized by the formation of dynamic blebs. This dynamic bleb formation is associated with a rounded mode of motility and is promoted through Rho and ROCK activity (21). In previous work (data not shown), we demonstrated that once shear activates blebbing, this morphology becomes independent of shear and can interchange to form a spreading lamellipodium or can be retained for up to 22 h in an invading cell as shown in Fig. 6. Furthermore, inhibition of the integrin v3 in OC-1 cells with blocking antibody does not inhibit dynamic blebbing (data not shown). Recently, Charras et al. (2) described blebbing as ballooning out of the plasma membrane when it detaches from the actin cortex and the cell membrane. In the present investigation, we demonstrate that shear alone (200 s–1) can induce dynamic bleb formation in OC-1 cells. However, shearing OC-1 cells for periods of >30 min at 200 s–1 via cone-plate viscometry resulted in significant apoptosis (data not shown), compared with adherent OC-1 cells, which are fully viable after exposure to 1-h shear at 200 s–1 (data not shown). This suggests that the interaction between metastatic cells and matrix is critical in the survival of metastatic cells under shear.
Pretreatment of OC-1 cells with Y-27632 resulted in loss of dynamic blebs and promoted cell rounding, suggesting that these morphological responses are dependent on ROCK function. In addition, we showed that shear stress potentiates the invasiveness of OC-1 cells across a Matrigel barrier, an effect inhibited by the presence of Y-27632. ROCK promotes contractility by increasing phosphorylation of the regulatory light chain of myosin-2 and the ezrin/radixin/moesin family of proteins that link the actin cytoskeleton to the plasma membrane (19). ROCK inhibition with Y-27632 has been reported to inhibit transcellular invasion and reduce the extent of local peritoneal metastasis by MM1 rat hepatoma and also in vivo dissemination of prostate cancer cells (25). Recently, Wang et al. (31) demonstrated how migration and invasion of hepatoma cells are inhibited with Y-27632. These findings suggest that Y-27632 may be important in the prevention of tumor motility and invasion.
By cycling between an inactive GDP- and a active GTP-bound state, Ras can transduce signals originating from membrane receptors, such as integrins (3). The primary products of RAS genes are cytosolic, which become secondarily associated with membranes through a series of posttranslational modifications (4). It has been shown that shear stress induces rapid activation of Ras and integrin, which in turn activates MAPKs, ERKs, and JNKs (17, 27, 29). Therefore, we examined the localization of Ras with the integrin v3 in sheared OC-1 cells. In the presence of shear, clustered integrin v3 colocalized with Ras in OC-1 cell blebs and at the cell periphery, indicative of an activated Ras and integrin state. This plaquelike distribution of integrin was also detected by Korb et al. (15) in metastatic colon carcinoma cells under shear. In the presence of Y-27632 and shear, the colocalization of integrin v3 and Ras was inhibited and Ras translocated from the cell periphery to the cytosol, representative of an inactive Ras state. To further elucidate the role of Rho/ROCK in Ras activation, we examined the total Ras-GTP with or without Y-27632 treatment in the presence or absence of shear. A basal level of Ras activity was detected in OC-1 cells, which increased with shear. This elevation in Ras activity coincided with bleb formation and increased invasion. Our results showed that shear stress induced an increase in Ras activity that was inhibited by the presence of Y-27632. Rho GTPases interact with different oncogenic signaling cascades, including those downstream of Ras. One mechanism by which ROCK inhibition may inhibit Ras activation is via guanine nucleotide exchange factors (GEFs). GEFs must colocalize with Ras at the cell membrane to activate Ras (8). ROCK inhibition may function in preventing GEF translocation to the plasma membrane, thereby preventing Ras activation.
In summary, we highlight a critical role for shear in metastatic motility and invasion through ROCK and Ras signaling. We show that shear induces specific changes in actin and tubulin remodeling in OC-1 cells. Both actin and tubulin colocalize to the cortical ring of the cell under static conditions. By contrast, under shear, actin acquires a more diffuse distribution, while tubulin translocates to the leading edge of the cell. We demonstrate that dynamic bleb formation is induced by shear alone, independent of integrin ligation. Y-27632, a specific ROCK inhibitor, inhibits both shear-induced dynamic blebs and integrin v3-Ras colocalization at the leading cell edge. We show that Ras activity is increased in the presence of shear, an effect inhibited by Y-27632. Finally, we demonstrate that shear alone enhances invasion of metastatic cells, a process inhibited by the presence of Y-27632. Given the significant contributions that Ras and Rho make to tumor growth and progression, ROCK inhibition may be an effective therapeutic agent in metastatic disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* A. Long and D. Kenny contributed equally to this work. 
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5-fold increase in % of OC-1 cells exhibiting dynamic bleb formation at 15 and 30 min, in contrast to nonsheared OC-1 cells (*P < 0.008, **P < 0.0001). Values represent means ± SD for 5 independent experiments. B: representative quiescent and dynamic blebbing OC-1 cells. C: Trypan blue exclusion assays confirmed that the direct application of shear (15 min, 200 s–1) does induce apoptosis in OC-1 cells. Scale bar, 7 µm.
