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1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan
2 Department of Hematology, Graduate School of Medicine, Nagoya University, Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan
3 Department of Cardiology, Graduate School of Medicine, Nagoya University, Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan
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Abstract |
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Introduction |
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Upon stimulation of cells by agonists, the GDP-bound form of Rho family small GTPases are converted to the GTP-bound form, and they interact with their effector molecules (Hall 1998). Rho regulates myosin phosphorylation through its effector molecules, such as Rho-kinase/ROCK/ROK (Leung et al. 1995; Ishizaki et al. 1996; Matsui et al. 1996) and the myosin binding subunit (MBS) of myosin phosphatase (Kimura et al. 1996). Activated Rho interacts with Rho-kinase and MBS and activates Rho-kinase (Matsui et al. 1996). Subsequently, the activated Rho-kinase phosphorylates the myosin light chain (MLC) (Amano et al. 1996) and MBS. Phosphorylation of MBS inactivates myosin phosphatase (Kimura et al. 1996). Both processes may contribute to increase MLC phosphorylation. The phosphorylation of MLC is essential for contraction of smooth muscle cells (SMCs) and the actin–myosin interaction leading to stress fiber formation in non-muscle cells (Kaibuchi et al. 1999).
Rho is also thought to play an important role in cell migration. However, our present understanding of the relationship between the Rho/Rho-kinase pathway and cell migration is unclear because it is derived from studies of various types of cells in different conditions. The Rho-kinase inhibitors suppress cell migration of SMCs (Ai et al. 2001), neutrophils (Niggli 1999), and monocytes (Worthylake & Burridge 2001) but not NIH3T3 cells (Magdalena et al. 2003).
Atherosclerotic lesions begin as fatty streaks underlying the arterial endothelium (Glass & Witztum 2001). Monocytes are recruited into the arterial intima and then differentiate into macrophages. Subsequently, macrophages uptake low-density lipoprotein-derived cholesterol. This uptake of cholesterol ultimately leads to foam cell formation. This is the major cellular event contributing to fatty streak formation. Interactions among macrophage foam cells and T cells establish a chronic inflammatory state (Hansson 1997; Lusis 2000). Secretion of cytokines from these cells induces dedifferentiation and migration of SMCs from the medial portion of the arterial wall to the intima. SMCs proliferate and secrete extracellular matrix proteins and form the fibrous plaque (Libby 2002). Thus, migration of macrophages and SMCs is thought to play a pivotal role in the development of atherosclerotic lesions.
Using a porcine coronary artery model, Shimokawa et al. (1996) showed that long-term treatment with an inflammatory cytokine from the adventitia causes the development of coronary vascular lesion with the accumulation of macrophages. In this model, vascular lesion formation is mostly enhanced at the coronary segment by simultaneous treatment with monocyte chemoattractant protein-1 (MCP-1) and oxidized low-density lipoprotein-derived cholesterol. Under these conditions, fasudil, a Rho-kinase inhibitor, inhibits the macrophage invasion into the adventitia and media (Miyata et al. 2000). In addition, long-term treatment with fasudil inhibits the neointimal formation and development of vascular remodeling in vivo (Shimokawa et al. 2001). Consistently, Mallat et al. (2003) showed Rho-kinase inhibitor reduced early atherosclerotic plaque development in mice model. These results strongly suggest that Rho-kinase plays critical roles in migration of macrophages and SMCs in atherosclerosis and is a therapeutic target. If Rho-kinase inhibitors prevent migration of all types of cells, however, the Rho-kinase is not an appropriate therapeutic target for certain diseases.
These observations led us to examine whether Rho-kinase inhibitors are cell type specific. We show here that Rho-kinase inhibitors suppressed cell migration in a cell type-dependent and migration type-dependent manner, and myosin II inhibitor suppressed cell migration in a similar fashion. Our results indicate that Rho-kinase inhibitors specifically suppress the migration of SMCs and macrophages through the regulation of myosin II activity.
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Results |
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The Boyden chamber assay was performed for ECV304 (endothelial-like cells), HeLa (epithelial-like cells), and Vero (fibroblast) cells co-expressing EGFP and the dominant negative form of Rac1 (Rac DN), Cdc42 (Cdc42 DN), or RhoA (Rho DN). The number of EGFP-positive cells on the bottom side of the filter was counted. Transfection efficiency of each cell was almost the same. The expression of Rac DN and Cdc42 DN inhibited the migration of ECV304, HeLa, and Vero cells (Fig. 1). In contrast, the expression of Rho DN inhibited the migration of ECV304 cells, but not that of HeLa, or Vero cells (Fig. 1).
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To elucidate the role of the Rho/Rho-kinase pathway more precisely, we investigated the effects of Y-27632, a specific inhibitor of Rho-kinase (Uehata et al. 1997), on the migration of various cell types. We examined the basal migratory activity of SMCs (primary cultured smooth muscle cells), HUVECs (primary cultured endothelial cells), HeLa cells, and Vero cells without chemoattractant. Because macrophages, which were isolated from mouse peritoneum, did not migrate in the absence of the chemoattractants, we added 25 ng/mL of MCP-1 to the lower chamber as a chemoattractant. In this assay, Y-27632 inhibited migration of macrophages, SMCs and HUVECs in a dose-dependent manner, but not that of HeLa and Vero cells (Fig. 2A). Furthermore, to examine the effects of Rho-kinase inhibitor on cell migration, various kinds of cells (ECV304 cells, HL-60 cells (promyelocytic leukaemia cells), Fibro cells (primary fibroblasts), KB cells (epithelial-like cells) and NIH3T3 (fibroblasts)) were subjected to the 3D migration assay on various coating material (Matrigel, collagen type I, and fibronectin). Migration of SMCs, HUVECs, ECV304 cells, macrophages, and HL-60 cells was inhibited by treatment with Y-27632, but that of Vero cells, NIH3T3 cells, Fibro cells, KB cells, and HeLa cells was not inhibited (Fig. 2B). In addition, migration of SMC was inhibited by treatment with fasudil, other Rho-kinase inhibitor, comparing to that of control, whereas Vero cells’ migration was not affected by fasudil (Fig. 2C). Fasudil also showed the inhibitory effect on migration of HUVECs and macrophages but not on that of KB cells (data not shown). These results suggest that Rho-kinase inhibitors suppress three-dimensional migration of SMCs, endothelial cells, and macrophages, whereas they do not inhibit epithelial cells or fibroblasts.
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We next examined whether the phosphorylation of Rho-kinase substrates was inhibited by treatment with Y-27632 in each cell line. Rho-kinase is thought to phosphorylate MBS at Thr697, Ser854, and Thr855 (Kawano et al. 1999). To examine the effects of Y-27632 on various cells, we monitored the activity of Rho-kinase by measuring the phosphorylation of MBS at Thr855. In every cell type, Y-27632 reduced the phosphorylation levels of MBS at Thr855 in a dose-dependent manner (Fig. 3A). Similar IC50 values of Y-27632 were obtained for these cell lines: 2.0 µM (Vero), 2.1 µM (SMC), and 3.9 µM (HUVEC) (Fig. 3B).
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The effects of myosin II inhibitor on cell migration
To elucidate the role of myosin II, whose activity is regulated by MLC phosphorylation in cell migration, we used the myosin II-specific inhibitor blebbistatin (Straight et al. 2003) in this assay. Blebbistatin inhibited migration of SMCs, HUVECs, and macrophages in a dose-dependent manner, whereas it did not inhibit HeLa or Vero cells (Fig. 4A). Furthermore, we examined the effects of blebbistatin on migration of various cells on various coating materials (Fig. 4B). Blebbistatin inhibited cell migration with specificity similar to that of Rho-kinase inhibitors. These results suggest that the cell type-specific inhibitory effects of Rho-kinase inhibitors on three-dimensional migration are mediated by myosin II activity.
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When a scratch was made in the sheet of the cells in the wound-healing assay, cells at the edges started migrating to sites close to the wound, thus allowing us to estimate the rate of two-dimensional cell migration. We examined the effects of Y-27632 on cell migration in the wound-healing assay by monitoring velocity of cell movement (Fig. 5). Y-27632 did not affect the migration rates of SMCs, HUVECs, and Vero cells, and enhanced that of HeLa cells. Treatment with blebbistatin did not affect the two-dimensional cell migration rates (data not shown). These results are quite different from those of the Boyden chamber assay, and indicate that the activity of Rho-kinase is not essential for cell migration of SMCs, endothelial cells, fibroblasts, and epithelial cells during wound healing.
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To address the issue of which step of cell migration was inhibited by treatment with Rho-kinase inhibitors in the Boyden chamber assay, we examined whether the cells and their nuclei passed through the pores of the membranes (Fig. 6). SMCs were subjected to the Boyden chamber assay and stained with Hoechst 33342 and DiI. Some cells treated with Y-27632 were stuck in the pores (Fig. 6A), and we counted the number of cells that failed to migrate. The majority of the migrating SMCs passed through the pore completely, whereas one-third of the cells treated with Y-27632 failed to pass through (Fig. 6B). Similar results were observed in HUVECs and macrophages (data not shown). These results suggest that Rho-kinase activity is necessary to pass the nuclei of SMCs, endothelial cells, and macrophages through pores.
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Discussion |
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In this study, we characterized the relationship between cell migration and Rho family small GTPases, especially the Rho/Rho-kinase pathway. In the Boyden chamber assay, the activities of Rac and Cdc42 were necessary for migration of endothelial cells, fibroblasts, and epithelial cells, and the activity of Rho was also necessary for migration of endothelial cells. These results indicate that the activity of Rho, unlike Rac and Cdc42, is required for migration of specific cell types.
Similar results were observed in the case of Rho-kinase inhibitors. The three-dimensional migration of endothelial cells, SMCs, and macrophages was inhibited by treatment with Rho-kinase inhibitors, whereas migration of fibroblasts and epithelial cells was not inhibited. Blebbistatin, the specific inhibitor of myosin II, inhibited the cell migration in a similar fashion. Treatment with Y-27632 in the wound-healing assay did not inhibit two-dimensional migration of any cell type examined. Thus, Rho/Rho-kinase and myosin II appear to be involved in the migration of specific cell types in a particular environment.
What is the difference between three-dimensional and two-dimensional cell migration? More dramatic, continuous morphological changes are thought to occur during three-dimensional cell migration as compared to two-dimensional cell migration. In the Boyden chamber assay, the cells must initially migrate to the pores of the membrane and then remodel their shapes dramatically to intercalate into the pores. After the nuclei pass through the pores, the cells must detach their tails from the matrix. During these processes, cytoskeletons are continuously remodeled. Tail retraction and detachment are also important steps for cell migration. Rho-kinase is necessary for Rho-mediated tail retraction during migration of monocytes through endothelial monolayer, and Rho-kinase signalling negatively regulates integrin adhesions (Worthylake et al. 2001). In addition, the cells have to squeeze themselves through the pores, which are smaller than the nuclei of the cells. In this study, we showed that the number of cells whose nuclei could not pass through the pores was increased by treatment with Rho-kinase inhibitor and Rho-kinase activity affected the morphology of nucleus. The morphology of nuclei of epithelial cells and fibroblasts was also affected by Rho-kinase activity; however, the nuclei of epithelial cells and fibroblast were not stuck in the pore by treatment of Rho-kinase inhibitors. Thus, it appears that SMCs, macrophages, and HUVECs stuck in the membrane of the chamber because Rho-kinase inhibitors and myosin II inhibitor inhibited proper morphological change of the cells to pass through. Alternatively, it is possible that Rho-kinase inhibitor inhibits the cell migration by suppressing the detachment of the tail from the matrix.
The migration of macrophages and endothelial cells is pivotal in immunoresponse and physiological angiogenesis, respectively. The motility of these cells is regulated by the actomyosin system (Rousseau et al. 2000; Worthylake & Burridge 2001), which also plays an important role in the contractility of SMCs (Kaibuchi et al. 1999; Riento & Ridley 2003). These cells might have a more highly developed actomyosin system than fibroblasts and epithelial cells, which may explain why Rho-kinase inhibitors suppress migration in a cell type-specific manner. Further analysis is necessary to understand how Rho-kinase and myosin II regulate cell shape in the specific types of cells in a particular environment during cell migration.
Suppressing the development of arteriosclerosis by Rho-kinase inhibitors
Arteriosclerosis is characterized by abnormal vascular SMC proliferation and migration (Liu et al. 1989). It has been shown that constrictive remodeling is important for luminal narrowing after coronary angioplasty (Currier & Faxon 1995; Andersen et al. 1996). Recent studies also reveal that long-term inhibition of Rho-kinase causes a marked regression of constrictive remodeling of the coronary artery in vivo (Shimokawa et al. 2001). Collagen accumulation in the vessel wall and endothelial dysfunction are both associated with constrictive remodeling (Lafont et al. 1999), and collagen accumulation alters the properties of SMCs, playing a pivotal role in pathogenesis of constrictive remodeling. Miyata et al. (2000) showed that Rho-kinase inhibitor fasudil suppresses the Rho-kinase activity and inhibits the macrophage accumulation in the adventitia, migration into media, and vascular lesion formation. In addition, it was previously reported that fasudil inhibits many vascular diseases including hypertension (Mukai et al. 2001) and cardiac allograft vasculopathy in mice (Hattori et al. 2003), and Y-27632 prevents nephritis (Nagatoya et al. 2002). These findings suggest the benefits of developing Rho-kinase inhibitors to treat inflammatory diseases. Our findings suggest that migration of macrophages and/or SMCs is a novel therapeutic target for arteriosclerosis (Wettschureck & Offermanns 2002). Our results indicate that Rho-kinase inhibitors specifically suppress the migration of macrophages and SMCs through the regulation of myosin II activity, which is expected to result in arteriosclerosis regression.
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Experimental procedures |
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Fasudil (HA1077) was kindly provided by Asahi Chemical Industry (Shizuoka, Japan). Y-27632 was kindly provided by Mitsubishi Pharma. Co. (Osaka, Japan). Blebbistatin was purchased from Toronto Research Chemicals Inc. (Toronto, Canada).
Plasmid construction
Various constructs of pEF-BOS-small GTPases were produced as previously described (Kuroda et al. 1996). pEGFP-C1 was purchased from Clontech Laboratories, Inc. (Palo Alto, CA, USA). The cDNA encoding the catalytic fragment of Rho-kinase (6–553 amino acids, Rho-kinase cat) was inserted into the BamHI site of pEGFP-C1 (pEGFP-Rho-kinase cat) (Kawano et al. 1999).
Cell culture
Macrophages were isolated from the mouse peritoneum as previously described (Xie et al. 1994). Collected macrophages were cultured in serum-free RPMI 1640 (Invitrogen Corp., Carlsbad, CA, USA). Human umbilical vein endothelial cell (HUVEC) pooled from several donors, Fibro cell, and smooth muscle cell (SMC) were purchased from Cascade Biologics, Inc. (PO) and grown in Humedia-EG, Medium 106S with LSGS, and Humedia-SG2 (Cascade Biologics, Inc. PO) according to the manufacturer's instructions. In all experiments, HUVECs, Fibro cells, and SMCs were used between passages 5 and 6. Vero, ECV304, KB, and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) at 37 °C in an air/5% CO2 atmosphere at constant humidity (Shishido et al. 1995). NIH3T3 cells were grown in DMEM containing 10% calf serum (CS) at 37 °C in an air/5% CO2 atmosphere at constant humidity. HL-60 promyelocytic leukaemia cells were cultured in RPMI 1640 containing 10% FBS. For differentiation, HL-60 cells were plated at a density of 1 x 105 cells/mL and grown for 4 days until they reached 1.5 x 105 cells/mL. Cells (2 mL) were then diluted with 16 mL of fresh medium and 2 mL of a 13% stock solution of DMSO was added to the cell suspension. Cells were propagated for 6 or 7 days.
Transfection into cells
ECV304, Vero and HeLa cells were seeded on 60-mm dishes at a density of 5 x 105 cells/dish and cultured overnight. The medium was renewed 2 h prior to transfection. Transfection of plasmid into each cell was carried out using the Lipofectamine‘ reagent (Invitrogen) according to the manufacturer's protocol. SMCs were seeded on cover slips coated with Matrigel at a density of 5 x 105 cells/30-mm dish and cultured overnight. The medium was renewed 2 h prior to transfection. Transfection of pEGFP-Rho-kinase cat was carried out using the Effectene Transfection Reagent (QIAGEN Inc, Valencia, CA, USA) according to the manufacturer's protocol. After transfection, cells were incubated for 15 h.
Three-dimensional cell migration assay
A three-dimensional cell migration (Boyden chamber) assay was performed using Transwell (Costar, Cambridge, MA, USA) 24-well tissue culture plates composed of a polycarbonate membrane containing 8-µM, 5-µM, or 3-µM pores. The membrane was coated with 10 µg/mL of Fibronectin (Sigma-Aldrich Co, St. Louis, MO, USA) for 2 h at 37 °C, 40 µg/mL of Collagen Type I (Sigma-Aldrich Co) for 2 h at 37 °C, or 500 µg/mL of Matrigelô Basement Membrane Matrix (Becton, Dickinson and Co., San Jose, CA, USA) for 1 h at room temperature as indicated. The lower chamber was filled with 500 µL of serum-free medium containing 0.1% bovine serum albumin (BSA), various doses of drugs. The cells were pretreated with various doses of Rho-kinase inhibitors for 30 min and seeded on the upper chamber of the Transwell at 1 x 105 cells in 100 µL of serum-free medium containing 0.1% BSA and incubated for 4 h.
The cells in upper chamber were removed with a cotton swab. The cells that migrated to the lower side of the membrane were fixed with 3.0% formaldehyde in phosphate buffer saline (PBS) for 10 min and then treated with PBS containing 0.2% Triton X-100 and 2 mg/mL BSA for 10 min. The fixed cells were stained with 100 µg/mL of Hoechst 33342 (Sigma-Aldrich Co) and 20 µg/mL of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Inc., Eugene, OR, USA). After being washed with PBS three times, the cells were examined under a Zeiss axiophoto microscope (Carl Zeiss, Oberkochen, Germany). The number of the Hoechst 33342-positive structures was counted in three randomly chosen fields of three independent experiments at 200x magnification.
Observation of MBS phosphorylation
The cells were seeded on 60-mm dishes at a density of 8 x 105 cells/dish and cultured overnight. The cells were then treated with various doses of Rho-kinase inhibitor for 30 min and harvested with 10% (w/v) trichloroacetic acid. The resulting precipitates were subjected to immunoblotting with anti-phospho-MYPT-1 (Thr850) (Upstate, Waltham, MA, USA). The region containing MBS was visualized using an ECL Western blotting system (Amersham Biosciences, Piscateway, NJ, USA). Quantitative evaluation of phosphorylated MBS was performed by densitometric analysis using ATTO Densitograph (ATTO, Tokyo, Japan).
Observation of MLC phosphorylation
MLC phosphorylation was determined using the urea/glycerol-polyacrylamide gel electrophoresis (PAGE), as previously described (Persechini et al. 1986). The cells were seeded on 60-mm dishes at a density of 8 x 105 cells/dish and cultured overnight. Then cells were treated with various doses of Rho-kinase inhibitor for 30 min and harvested with 10% (w/v) trichloroacetic acid. The resulting precipitates were suspended in urea sample buffer, processed for urea/glycerol-PAGE, and immunoblot analysis with anti-MLC monoclonal antibody (Sigma-Aldrich Co). The region containing MLC was visualized using an ECL Western blotting system. Quantitative evaluation of the non-phosphorylated, monophosphorylated, and diphosphorylated MLC was performed by denstometric analysis using ATTO Densitograph.
Two-dimensional cell migration assay
A two-dimensional cell migration assay using a wound-healing model was performed as previously described (Santos et al. 1997). A confluent monolayer of each cell type was treated with various doses of Rho-kinase inhibitor for 30 min and was scraped with a chip. After wounding, time-lapse images were captured using a confocal laser microscopy system (LSM 510, Carl Zeiss) for 10 h.
Immunostaining
After transfection, SMCs were fixed with 3.0% formaldehyde in phosphate buffer saline (PBS) for 10 min and then treated with PBS containing 0.2% Triton X-100 and 2 mg/mL BSA for 10 min. The fixed cells were stained with anti-lamin antibody, followed by second antibodies. Fluorescent images were taken with a confocal laser microscopy system (Carl Zeiss LSM 510; Carl Zeiss) built around a Zeiss Axiovert 100M.
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Acknowledgements |
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Footnotes |
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*Correspondence: E-mail: kaibuchi{at}med.nagoya-u.ac.jp
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References |
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Received: 30 September 2004
Accepted: 14 November 2004
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), HUVECs (•), Vero cells (
), HeLa cells (
) and macrophages (
) were incubated with Y-27632 (0.1–100 µM) for 30 min, then subjected to the Boyden chamber assay. The membrane of the chamber was coated with Matrigel for SMCs, HUVECs, and macrophages, or with fibronectin for Vero cells and HeLa cells. Each cell type was incubated for 4 h, then stained with Hoechst 33342. The number of migrating cells was counted. The ratio of migrating cells was compared with a control. Each value represents the mean ± SD of three independent experiments. The mean value of the cells not treated with Y-27632 was defined as 100%. (B) SMCs, HUVECs, ECV304 cells, macrophages, HL-60 cells, Fibro cells, NIH3T3 cells, Vero cells, KB cells, and HeLa cells were incubated with 30 µM of Y-27632 for 30 min, then subjected to the Boyden chamber assay. The membrane of the chamber was coated with Matrigel, Collagen Type I, or fibronectin. The ratio of migrating cells was compared with a control. Each value represents the mean ± SD of three independent experiments. The mean value of the cells not treated with Y-27632 was defined as 100%. (C) SMCs and Vero cells, which were preincubated with 10 µM of fasudil, were subjected to the Boyden chamber assay. Migrating cells were stained with Hoechst 33342. The number of migrating cells was counted, and the ratio of migrating cells was compared with a control. The membrane of the chamber was coated with Matrigel for SMCs or with fibronectin for Vero cells. Each value represents the mean ± SD of three independent experiments. The mean value of the cells not treated with fasudil was defined as 100%. (*t-test, P < 0.01)
