FAP和Rho 家族蛋白在卵巢癌侵袭迁移中的作用

目的：明确FAP 是否通过RhoA/ROCK、Rac1-GTP 通路发挥促增殖、侵袭和迁移作用。方法：用MTT 实验,Transwell 实验 和迁移实验检测FAP、RhoA/ROCK、Rac1-GTP 对卵巢癌细胞系HO-8910PM 的增殖,侵袭和迁移的影响。结果：1、MTT 法,迁移和 侵袭实验证实用Y-27632 抑制RhoA/ROCK 途径能够促进卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用时促进作用增强。 2、MTT 法, 迁移和侵袭实验证实NSC23766 抑制Rac1 途径能够抑制卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用使FAP 的 促进作用减弱。结论：1、RhoA/ROCK 通路抑制HO-8910PM 细胞增殖、迁移和侵袭;Rac1-GTP 促进HO-8910PM 细胞增殖、迁移 和侵袭。2、FAP不是通过RhoA/ROCK而是通过Rac1-GTP 信号通路在HO-8910PM细胞发挥促增殖、迁移和侵袭作用的。     

Negative regulation of endothelial morphogenesis and angiogenesis by S1P2 receptor

We speculated that the sphingosine-1-phosphate (S1P) receptor S1P(2), which uniquely inhibits cell migration, might mediate inhibitory effects on endothelial cell migration and angiogenesis, different from S1P(1) and S1P(3). Mouse vascular endothelial cells, which endogenously express S1P(2) and S1P(3), but not S1P(1), responded to S1P and epidermal growth factor (EGF) with stimulation of Rac, migration, and the formation of tube-like structures on the Matrigel. The S1P(3)-antagonist VPC-23019 abolished S1P-induced, G(i)-dependent Rac stimulation, cell migration, and tube formation, whereas the S1P(2)-antagonist JTE-013 enhanced these S1P-induced responses, suggesting that S1P(2) exerts inhibitory effects on endothelial Rac, migration, and angiogenesis. S1P(2) overexpression markedly augmented S1P-induced, G(i)-independent inhibition of EGF-induced migration and tube formation. Finally, the blockade of S1P(2) by JTE-013 potentiated S1P-induced stimulation of angiogenesis in vivo in the Matrigel implant assay. These observations indicate that in contrast to S1P(1) and S1P(3), S1P(2) negatively regulates endothelial morphogenesis and angiogenesis most likely through down-regulating Rac.     

S1P(2) receptors mediate inhibition of glioma cell migration through Rho signaling pathways independent of PTEN

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P₂ receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P₂ receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P₂ receptors/G_12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P₂ receptor-mediated action in glioma cells.     

Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho-like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (10(-7)-10(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S1P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73122, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.     

Activin B Antagonizes RhoA Signaling to Stimulate Mesenchymal Morphology and Invasiveness of Clear Cell Renal Cell Carcinomas

Activin B belongs to the TGFβ family of growth factors and is upregulated in clear cell renal cell carcinoma cells by hypoxia inducible factors. Expression of Activin B is required for tumor growth in vivo and tumor cell invasion in vitro. Here we show that activation of RhoA signaling counteracts Activin B mediated disassembly of actin stress fibers, mesenchymal cell morphology and invasiveness, whereas inhibition of RhoA rescues these effects in Activin B knockdown cells. Conversely, Activin B inhibits RhoA signaling suggesting that there is an antagonistic connection between both pathways. In addition we found that Rac1 plays an opposite role to RhoA, i.e. activation of Rac1 initiates loss of actin stress fibers, promotes a mesenchymal cell morphology and induces invasion in Activin B knockown cells, whereas inhibition of Rac1 abolishes these Activin B effects. Collectively, our data provide evidence that reduction of RhoA signaling by Activin B together with persistent Rac1 activity is a prerequisite for inducing an invasive phenotype in clear cell renal cell carcinoma.     

Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase

Rac GTPases are known to play a crucial role in regulating cytoskeletal changes necessary for cell migration. Migration has been shown to be positively regulated by Rac in most cell types. However, there is also a large body of conflicting evidence in some other cell types with respect to the role of Rac in migration, suggesting that Rac GTPases regulate cell migration in a cell type-dependent manner. In the present study, we have characterized the effects of Rac1 GTPase inhibition on the migratory abilities of a number of breast cancer cell lines with differential degrees of tumorigenic and metastatic potentials. We show that Rac1 inhibition in non-metastatic (MCF-7, T-47D) or moderately metastatic (Hs578T) cell lines results in inhibition of migration, whereas in highly metastatic cell lines (MDA-MB-435, MDA-MB-231, and C3L5) Rac1 inhibition results in stimulation of migration. This stimulation of migration following Rac1 inhibition is also accompanied by the enhanced RhoA activity, suggesting a possible existence of a dominating role of RhoA over Rac1 in regulating intrinsic migration of the highly metastatic breast cancer cells.     

Sphingosine 1-phosphate inhibits migration and RANTES production in human bronchial smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been shown to be increased in bronchoalveolar lavage fluid after allergen challenge in asthmatic patients. Here, we examined S1P actions and their intracellular signalings in cultured human bronchial smooth muscle cells (BSMCs). Expression of mRNAs of three subtypes of S1P receptors, including S1P(1), S1P(2), and S1P(3), was detected in BSMCs, and exposure of the cells to S1P inhibited platelet-derived growth factor (PDGF)-induced migration and tumor necrosis factor-alpha-induced RANTES production. S1P also inhibited PDGF-induced Rac1 activation, and dominant negative Rac1 inhibited PDGF-induced migration. On the other hand, dominant negative Galpha(q) attenuated the S1P-induced inhibition of RANTES production. Finally, an S1P(2)-selective antagonist, JTE-013, suppressed the S1P-induced inhibition of migration response and RANTES production. These results suggest that S1P attenuates cell migration by inhibiting a Rac1-dependent signaling pathway and decreases RANTES production by stimulating a Galpha(q)-dependent mechanism both possibly through the S1P(2) receptors.     

Sphingosine-1-phosphate and interleukin-1 independently regulate plasminogen activator inhibitor-1 and urokinase-type plasminogen activator receptor expression in glioblastoma cells: implications for invasiveness

Glioblastoma multiforme is an invasive primary brain tumor, which evades the current standard treatments. The invasion of glioblastoma cells into healthy brain tissue partly depends on the proteolytic and nonproteolytic activities of the plasminogen activator system proteins, including the urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and a receptor for uPA (uPAR). Here we show that sphingosine-1-phosphate (S1P) and the inflammatory mediator interleukin-1 (IL-1) increase the mRNA and protein expression of PAI-1 and uPAR and enhance the invasion of U373 glioblastoma cells. Although IL-1 enhanced the expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, down-regulation of SphK1 had no effect on the IL-1-induced uPAR or PAI-1 mRNA expression, suggesting that these actions of IL-1 are independent of S1P production. Indeed, the S1P-induced mRNA expression of uPAR and PAI-1 was blocked by the S1P(2) receptor antagonist JTE013 and by the down-regulation of S1P(2) using siRNA. Accordingly, the inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and Rho-kinase, two downstream signaling cascades activated by S1P(2), blocked the activation of PAI-1 and uPAR mRNA expression by S1P. More importantly, the attachment of glioblastoma cells was inhibited by the addition of exogenous PAI-1 or siRNA to uPAR, whereas the invasion of glioblastoma cells induced by S1P or IL-1 correlated with their ability to enhance the expression of PAI-1 and uPAR. Collectively, these results indicate that S1P and IL-1 activate distinct pathways leading to the mRNA and protein expression of PAI-1 and uPAR, which are important for glioblastoma invasiveness.     

Follicular fluid high-density lipoprotein-associated sphingosine 1-phosphate (S1P) promotes human granulosa lutein cell migration via S1P receptor type 3 and small G-protein RAC1

Coordinated migration and progesterone production by granulosa cells is critical to the development of the corpus luteum, but the underlying mechanisms remain obscure. Sphingosine 1-phosphate (S1P), which is associated with follicular fluid high-density lipoprotein (FF-HDL), was previously shown to regulate ovarian angiogenesis. We herein examined the effects of S1P and FF-HDL on the function of granulosa lutein cells. Both FF-HDL and S1P induced migration of primary human granulosa lutein cells (hGCs) and the granulosa lutein cell line HGL5. In addition, FF-HDL but not S1P promoted progesterone synthesis, and neither of the two compounds stimulated proliferation of granulosa lutein cells. Polymerase chain reaction and Western blot experiments demonstrated the expression of S1P receptor type 1 (S1PR1), S1PR2, S1PR3, and S1PR5 but not S1PR4 in hGCs and HGL5 cells. The FF-HDL- and S1P-induced granulosa lutein cell migration was emulated by FTY720, an agonist of S1PR1, S1PR3, S1PR4, and S1PR5, and by VPC24191, an agonist of S1PR1 and S1PR3, but not by SEW2871 and phytosphingosine 1-phosphate, agonists of S1PR1 and S1PR4, respectively. In addition, blockade of S1PR3 with CAY1044, suramine, or pertussis toxin inhibited hGC and HGL5 cell migration toward FF-HDL or S1P, while blockade of S1PR1 and S1PR2 with W146 and JTE013, respectively, had no effect. Both FF-HDL and S1P triggered activation of small G-protein RAC1 and actin polymerization in granulosa cells, and RAC1 inhibition with Clostridium difficile toxin B or NSC23766 abolished FF-HDL- and S1P-induced migration. The FF-HDL-associated S1P promotes granulosa lutein cell migration via S1PR3 and RAC1 activation. This may represent a novel mechanism contributing to the development of the corpus luteum.     

Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform

The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha12 and Galpha13, but not of Galphaq, by expression of respective C-terminal peptides abolished S1P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P2 and either Galpha12 or Galpha13, but not Galphaq, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Galphai, by contrast, specifically antagonized S1P2-mediated inhibition of Rac and migration. The S1P2 actions were mimicked by expression of V14Rho and were abolished by C3 toxin and N19Rho, but not Rho kinase inhibitors. In contrast to S1P2, S1P3 mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G12/13. Upon inactivation of Gi by pertussis toxin, S1P3 mediated inhibition of Rac and migration just like S1P2. These results indicate that integration of counteracting signals from the Gi- and the G12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.     

Protein kinase C-epsilon regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-zeta, and Rac1

The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P(1) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P(1) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P(1) and G(i) to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Fluid shear stress induces cell migration and invasion via activating autophagy in HepG2 cells

Fluid shear stress (FSS) regulates the metastasis of hepatocellular carcinoma (HCC). In the present study, we aimed to study the role of autophagy in HCC cells under FSS. The results showed that FSS upregulated the protein markers of autophagy, induced LC3B aggregation and formation of autophagosomes. Inhibition of integrin by Cliengitide (Cli) or inhibition of the microfilaments formation both inhibited the activation of autophagy in HepG2 under FSS. In addition, Cli inhibited the microfilaments formation and expressions of Rac1 and RhoA in HepG2 cells under FSS. Finally, inhibition of autophagy suppressed the cell migration and invasion in HepG2 under FSS. In conclusion, FSS induced autophagy to promote migration and invasion of HepG2 cells via integrin/cytoskeleton pathways.     

FAP和Rho家族蛋白在卵巢癌侵袭迁移中的作用

目的：明确FAP是否通过RhoA／ROCK、Racl-GTP通路发挥促增殖、侵袭和迁移作用。方法：用MTT实验,Transwell实验和迁移实验检测FAP、RhoA／ROCK、Racl-GTP对卵巢癌细胞系HO-8910PM的增殖,侵袭和迁移的影响。结果：1、MTT法,迁移和侵袭实验证实用Y-27632抑制RhoA／ROCK途径能够促进卵巢癌细胞的增殖、迁移和侵袭,与FAP联合作用时促进作用增强。2、MTT法,迁移和侵袭实验证实NSC23766抑制Racl途径能够抑制卵巢癌细胞的增殖、迁移和侵袭,与FAP联合作用使FAP的促进作用减弱。结论：l、RhoA／ROCK通路抑制HO一8910PM细胞增殖、迁移和侵袭;Racl-GTP促进H0—8910PM细胞增殖、迁移和侵袭。2、FAP不是通过RhoA／ROCK而是通过Racl—GTP信号通路在HO．8910PM细胞发挥促增殖、迁移和侵袭作用的。     

The requirement for polyamines for intestinal epithelial cell migration is mediated through Rac1

The rapid migration of intestinal epithelial cells is important to the healing of mucosal ulcers and wounds. This cell migration requires the presence of polyamines and the activation of RhoA. RhoA activity, however, is not sufficient for migration because polyamine depletion inhibited the migration of IEC-6 cells expressing constitutively active RhoA. The current study examines the role of Rac1 and Cdc42 in cell migration and whether their activities are polyamine-dependent. Polyamine depletion with alpha-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of alpha-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vector-transfected cells, whereas cells expressing dominant negative Rac1 and Cdc42 migrated more slowly. Polyamine depletion had no effect on the migration of cells expressing Rac1 and only partially inhibited the migration of those expressing Cdc42. Although polyamine depletion caused the disappearance of actin stress fibers in cells transfected with empty vector, it had no effect on cells expressing Rac1. Constitutively active Rac1 increased RhoA and Cdc42 activity in both normal and polyamine-depleted cells. These results demonstrate that Rac1, RhoA, and Cdc42 are required for optimal epithelial cell migration and that Rac1 activity is sufficient for cell migration in the absence of polyamines due to its ability to activate RhoA and Cdc42 as well as its own effects on the process of cell migration. These data imply that the involvement of polyamines in cell migration occurs either at Rac1 itself or upstream from Rac1.     

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

Contact inhibition of locomotion (CIL) occurs when a cell ceases moving in the same direction following contact with another cell. Homotypic and heterotypic CIL occur between cells of the same and different types, respectively. Using Abercrombie's confronted explants assay we studied the effect of changing Rac1 or RhoA activities on heterotypic CIL between NIH3T3 and chicken heart fibroblasts. Both dominant active (L61) and dominant negative (N17) Rac1 expressed in NIH3T3 cells resulted in loss of heterotypic CIL. N17Rac1 expression caused RhoA activation. Increasing RhoA activity directly (V14RhoA) or indirectly (downregulation of N-cadherin or p120-catenin) also resulted in loss of CIL. High RhoA activity has been associated with tumour invasion and our results are consistent with loss of heterotypic CIL playing a role.     

RhoA and RhoC have distinct roles in migration and invasion by acting through different targets

Several studies suggest that RhoA and RhoC, despite their sequence similarity, have different roles in cell migration and invasion, but the molecular basis for this is not known. Using RNAi, we show that RhoA-depleted cells became elongated and extended multiple Rac1-driven narrow protrusions in 2D and 3D environments, leading to increased invasion. These phenotypes were caused by combined but distinct effects of the Rho-regulated kinases ROCK1 and ROCK2. Depletion of ROCK2 induced multiple delocalized protrusions and reduced migratory polarity, whereas ROCK1 depletion selectively led to cell elongation and defective tail retraction. In contrast, RhoC depletion increased cell spreading and induced Rac1 activation around the periphery in broad lamellipodia, thereby inhibiting directed migration and invasion. These effects of RhoC depletion are mediated by the formin FMNL3, which we identify as a new target of RhoC but not RhoA. We propose that RhoA contributes to migratory cell polarity through ROCK2-mediated suppression of Rac1 activity in lamellipodia, whereas RhoC promotes polarized migration through FMNL3 by restricting lamellipodial broadening.     

鞘氨醇-1-磷酸对人脐带间充质干细胞增殖及表面标记物表达的影响

鞘氨醇－1－磷酸（sphingosine－1 phosphate, S1P）是一种脂质信号分子,与细胞增殖、凋亡和迁移等有密切关系．本研究发现,S1P促进人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC－MSCs)增殖,但目前关于其作用信号通路及S1P对hUC－MSCs表面标记表达的影响尚不十分清楚．Real－time PCR检测hUC－MSCs中S1P受体mRNA表达情况,发现在hUC－MSCs中优势表达S1PR1 3,而S1PR4、S1PR5的表达很少．MTT法检测S1PR1/3拮抗剂VPC23019、S1PR2拮抗剂JTE013、S1PR3拮抗剂CAY10444、Gi蛋白抑制剂PTX和ERK抑制剂PD98059对S1P诱导hUC－MSCs增殖的影响．结果显示,VPC23019完全抑制S1P诱导的hUC MSCs增殖,JTE013对此没有明显影响,CAY10444部分抑制S1P诱导的hUC MSCs增殖,PTX、PD98059完全抑制S1P诱导hUC－MSCs增殖．进一步用Western印迹检测ERK1/2磷酸化水平揭示,S1P通过促进ERK1/2磷酸化进而促进hUC MSCs增殖．流式细胞术检测发现,S1P对hUC MSCs表面标记物(CD45、CD34、CD90、CD29、CD105、CD44、CD73、CD71)表达没有明显影响．本研究证明,S1P通过S1PR1/3、Gi偶联蛋白及ERK1/2信号通路促进hUC MSCs增殖,而对hUC MSCs表面标记物表达无明显影响．     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.