Roles for Rho/ROCK and vinculin in parietal endoderm migration

The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.     

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

The role of extracellular matrix in the migration and differentiation of parietal endoderm from teratocarcinoma embryoid bodies

Embryoid bodies formed from teratocarcinoma stem cells differentiate an outer layer consisting of parietal and visceral endoderm or of visceral endoderm exclusively. We have previously shown that when these embryoid bodies are plated on collagen-coated substrates a parietal endoderm-like cell migrates onto the substrate, whereas all of the visceral endoderm remains associated with the stem cell mass, suggesting a role for substrate contact in parietal endoderm differentiation. We now identify fibronectin as the migration-promoting component in these cultures, and note that laminin and collagen type IV are 10-fold less effective at promoting both attachment and endoderm outgrowth. The RGDS tetrapeptide (arg-gly-asp-ser) from the cell attachment domain of fibronectin can specifically block attachment and outgrowth on both fibronectin- and laminin-coated substrates. In addition, the involvement of the 140-kD fibronectin receptor is demonstrated using an antibody directed against this molecule.     

Migration of F9 parietal endoderm cells is regulated by the ERK pathway

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK).     

The outgrowth of parietal endoderm from mouse teratocarcinoma stem-cell embryoid bodies

Teratocarcinoma stem cells can be used to study certain events occurring during early mouse embryogenesis. We report that the outgrowth of parietal endoderm from teratocarcinoma stem-cell embryoid bodies in vitro is analogous to the same process in vivo in terms of the spatial distribution of endoderm types: only parietal endoderm migrates away from the aggregate, whereas visceral endoderm remains associated with the embryoid body. The outgrowths generated on a substrate of type-I collagen from PSA-1 and retinoic-acid-treated F 9 embryoid bodies were found to be comparable, even though these aggregates express different endoderm types. We demonstrated that retinoic-acid-treated F 9 embryoid bodies that contain essentially only visceral endoderm in suspension culture can nonetheless generate parietal-endoderm outgrowth when plated on type-I collagen, suggesting that substrate interaction plays an important role in inducing parietal-endoderm differentiation. These data indicate the usefulness and relevance of studying endoderm differentiation and outgrowth in vitro employing the teratocarcinoma model system.     

Distinct Actions and Cooperative Roles of ROCK and mDia in Rho Small G Protein-induced Reorganization of the Actin Cytoskeleton in Madin–Darby Canine Kidney Cells

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin-Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.     

Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.     

Roles of Necl-5/poliovirus receptor and Rho-associated kinase (ROCK) in the regulation of transformation of integrin alpha(V)beta(3)-based focal complexes into focal adhesions

Focal complexes are continuously formed and transformed into focal adhesions during cell movement. We previously demonstrated that Necl-5 co-localizes with integrin alpha(V)beta(3) at focal complexes, whereas Necl-5 does not localize at focal adhesions in moving NIH3T3 cells, suggesting that Necl-5 may be dissociated from integrin alpha(V)beta(3) during the transformation of focal complexes into focal adhesions, but the underlying mechanism remains unknown. Here, we explore the roles of Necl-5 and Rho-associated kinase (ROCK) in the regulation of the transformation of focal complexes into focal adhesions. We found that inhibition of Necl-5 expression and expression of a constitutively active mutant of ROCK1 enhanced, whereas treatment with a ROCK inhibitor Y-27632 inhibited the transformation of focal complexes into focal adhesions. In HEK293 cells ectopically expressing Necl-5 and integrin alpha(V)beta(3), treatment of cells with Y-27632 increased the binding of Necl-5 to clustered integrin alpha(V)beta(3). The experiments using inhibitors of myosin ATPase and actin polymerization revealed that actomyosin-driven contractility exerts a similar function as ROCK. The phosphorylation of integrin beta(3) at Tyr(747), which is known to be critical for the formation of focal adhesions, plays a pivotal role for the interaction between Necl-5 and integrin alpha(V)beta(3). These results indicate that the transformation of focal complexes into focal adhesions is negatively and positively regulated by Necl-5 and ROCK, respectively, and that ROCK-dependent actomyosin-driven contractility is a critical determinant for the regulation of the interaction between Necl-5 and integrin alpha(V)beta(3).     

Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS)

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

p160ROCK mediates RhoA activation of Na-H exchange.

The ubiquitously expressed Na-H exchanger, NHE1, acts downstream of RhoA in a pathway regulating focal adhesion and actin stress fiber formation. p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Here, stress fiber formation induced by p160ROCK was inhibited by the addition of a specific NHE1 inhibitor, ethylisopropylamiloride, in CCL39 fibroblasts, and was absent in PS120 mutant fibroblasts lacking NHE1. In CCL39 cells, NHE1 activity was stimulated by expression of mutationally active p160ROCK, but not by mutationally active protein kinase N, another RhoA target kinase. Expression of a dominant interfering p160ROCK inhibited RhoA-, but not Cdc42- or Rac-activation of NEH1. In addition, the p160ROCK-specific inhibitor Y-27632 inhibited increases in NHE1 activity in response to RhoA, and to lysophosphatidic acid (LPA), which stimulates RhoA, and it also inhibited LPA-increased phosphorylation of NHE1. A C-terminal truncation of NHE1 abolished both LPA-induced phosphorylation and activation of the exchanger. Furthermore, mutationally active p160ROCK phosphorylated an NHE1 C-terminal fusion protein in vitro, and this was inhibited in the presence of Y-27632. Phosphopeptide maps indicated that identical residues in NHE1 were phosphorylated by p160ROCK in vivo and in vitro. These findings identify p160ROCK as an upstream, possibly direct, activator of NHE1, and suggest that NHE1 activity and phosphorylation are necessary for actin stress fiber assembly induced by p160ROCK.     

Molecular characterization of the effects of Y-27632

Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extensions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.     

Role of lysophosphatidic acid and rho in glioma cell motility

We have studied the effects of the bioactive phospholipid lysophosphatidic acid (LPA) on cell lines derived from highly invasive human glioblastoma multiforme (GBM). Using transwell migration assays, we show that LPA stimulates both chemokinetic and chemotactic migration of glioma cells. Blood brain barrier breakdown and leakage of serum components that most likely include LPA are common features of GBM. Therefore, the effects of LPA on glioma cell motility are intriguing given the fact that, in vivo, GBM cells often migrate great distances from the main tumor, rendering successful therapy extremely difficult. We show here that LPA initiates a variety of signaling cascades in glioma cells. LPA-enhanced transwell migration was sensitive to pertussis toxin (PTX) treatment suggesting an important role for G(i) subtype of G proteins. LPA also stimulated Ca(2+) fluctuations and activation of extracellular signal-regulated kinases (ERKS) 1 and 2, although blocking either pathway had little effect on glioma cell migration. Exposure of glioma cells to LPA resulted in phosphorylation of the regulatory light chain (RLC) of myosin II and the formation of stress fibers and focal adhesions. These effects were blocked by Y-27632, an inhibitor of Rho-activated ROCK kinases. Time-lapse video microscopy revealed that Y-27632-treatment caused cells to assume long thin morphologies that suggested deficiencies in the contractile apparatus. Furthermore, many cells exhibited a conspicuous extension of processes when Rho/ROCK kinase cascades were inhibited. The above results suggest that LPA/Rho signaling cascades play important roles in glioma cell motility and that exposure of tumor cells to LPA in vivo may contribute to their invasive phenotype.     

Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer’s disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.     

Bidirectional signaling between the cytoskeleton and integrins

Clustering of integrins into focal adhesions and focal complexes is regulated by the actin cytoskeleton. In turn, actin dynamics are governed by Rho family GTPases. Integrin-mediated adhesion activates these GTPases, triggering assembly of filopodia, lamellipodia and stress fibers. In the past few years, signaling pathways have begun to be identified that promote focal adhesion disassembly and integrin dispersal. Many of these pathways result in decreased myosin-mediated cell contractility.     

Role of focal adhesion formation in migration and morphogenesis of endothelial cells

Cell motility and morphogenesis are regulated by a balance between formation and disassembly of stress fibers and focal adhesions. To understand the mechanisms underlying these cellular responses in angiogenesis, we studied the Rho family protein-driven pathways in FGF-2-induced chemotaxis and capillary morphogenesis of murine brain capillary endothelial cell line, IBE cells. Cells seeded onto fibronectin-coated surface migrated toward FGF-2. Expression of dominant negative Rho A (DNRho) or kinase-dead p21-activated kinase 1 (KDPAK1), or treatment with Y27632 inhibited chemotaxis in association with the lack of FGF-2-induced decrease in focal adhesions. On Matrigel, DNRho and Y27632 induced FGF-2-independent capillary morphogenesis despite loss of stress fiber formation. KDPAK1 cells formed stress fibers and showed capillary morphogenesis in response to FGF-2. Increase in focal adhesions was closely associated with capillary morphogenesis. Our results suggest that formation or disassembly of focal adhesions seems to determine the motility or morphogenesis of endothelial cells.