FilGAP,a Rho/Rho-associated protein kinase–regulated GTPase-activating protein for Rac,controls tumor cell migration

Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and amoeboid migration. Mesenchymal mode is characterized by elongated morphology that requires high GTPase Rac activation, whereas amoeboid mode is dependent on actomyosin contractility induced by Rho/Rho-associated protein kinase (ROCK) signaling. While elongated morphology is driven by Rac-induced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement is not well understood. We identified FilGAP, a Rac GTPase-activating protein (GAP), as a mediator of Rho/ROCK-dependent amoeboid movement of carcinoma cells. We show that depletion of endogenous FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely, forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCK-dependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma cells, and FilGAP contributes to tumor invasion.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA

In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Galphaq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.     

RhoE Regulates Actin Cytoskeleton Organization and Cell Migration

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.     

Calpain mediates integrin-induced signaling at a point upstream of Rho family members.

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members which activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that mu-calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of mu-calpain increased the cleavage of these proteins, induced an overspread morphology and induced an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of mu-calpain resulted in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 could still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA could form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac- and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA.     

Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Rho; actin; swelling     

Pituicyte Stellation is Prevented by RhoA-or Cdc42-Dependent Actin Polymerization

Our aim was to shed light on different steps leading from metabotropic receptor activation to changes in cell shape, such as those that characterize the morphological plasticity of neurohypophysial astrocytes (pituicytes). Using explant cultures of adult rat pituicytes, we have previously established that adenosine A1 receptor activation induces stellation via inhibition of RhoA monomeric GTPase and subsequent disruption of actin stress fibers. Here, we rule out RhoA phosphorylation as a mechanism for that inhibition. Rather, our results are more consistent with involvement of a GTPase-activating protein (GAP). siRNA and pull-down experiments suggest that a step downstream of RhoA might involve Cdc42, another GTPase of the Rho family. However, RhoA activation, e.g., in the presence of serum, induces stress fibers, whereas direct Cdc42 activation appears to confine actin within a submembrane—i.e., cortical—network, which also prevents stellation. Therefore, we propose that RhoA may activate Cdc42 in parallel with an effector, such as p160Rho-kinase, that induces and maintains actin stress fibers in a dominant fashion. Rac1 is not involved in the stellation process per se but appears to induce a dendritogenic effect. Ultimately, it may be stated that pituicyte stellation is inducible upon mere actin depolymerization, and preventable upon actin organization, be it in the form of stress fibers or in a cortical configuration.     

Sdc1 negatively modulates carcinoma cell motility and invasion

During cancer progression, tumor cells eventually invade the surrounding collagen-rich extracellular matrix. Here we show that squamous cell carcinoma cells strongly adhere to Type I collagen substrates but display limited motility and invasion on collagen barriers. Further analysis revealed that in addition to the α2β1 integrin, a second collagen receptor was identified as Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan. We demonstrate that siRNA-mediated depletion of Sdc1 reduced adhesion efficiency to collagen I, whereas knockdown of Sdc4 was without effect. Importantly, silencing Sdc1 expression caused reduced focal adhesion plaque formation and enhanced cell spreading and motility on collagen I substrates, but did not alter cell motility on other ECM substrates. Sdc1 depletion ablated adhesion-induced RhoA activation. In contrast, Rac1 was strongly activated following Sdc1 knockdown, suggesting that Sdc1 may mediate the link between integrin-induced actin remodeling and motility. Taken together, these data substantiate the existence of a co-adhesion receptor system in tumor cells, whereby Sdc1 functions as a key regulator of cell motility and cell invasion by modulating RhoA and Rac activity. Downregulation of Sdc1 expression during carcinoma progression may represent a mechanism by which tumor cells become more invasive and metastatic.     

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.     

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.     

Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism

The Rho family GTPases Cdc42, Rac1 and RhoA control many of the changes in the actin cytoskeleton that are triggered when growth factor receptors and integrins bind their ligands [1] [2]. Rac1 and Cdc42 stimulate the formation of protrusive structures such as membrane ruffles, lamellipodia and filopodia. RhoA regulates contractility and assembly of actin stress fibers and focal adhesions. Although prolonged integrin engagement can stimulate RhoA [3] [4] [5], regulation of this GTPase by early integrin-mediated signals is poorly understood. Here we show that integrin engagement initially inactivates RhoA, in a c-Src-dependent manner, but has no effect on Cdc42 or Rac1 activity. Additionally, early integrin signaling induces activation and tyrosine phosphorylation of p190RhoGAP via a mechanism that requires c-Src. Dynamic modulation of RhoA activity appears to have a role in motility, as both inhibition and activation of RhoA hinder migration [6] [7] [8]. Transient suppression of RhoA by integrins may alleviate contractile forces that would otherwise impede protrusion at the leading edge of migrating cells.     

NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways

Glioblastoma is the most common and lethal primary intracranial tumor. As the key regulator of tumor cell volume, sodium-potassium-chloride cotransporter 1 (NKCC1) expression increases along with the malignancy of the glioma, and NKCC1 has been implicated in glioblastoma invasion. However, little is known about the role of NKCC1 in the epithelial-mesenchymal transition-like process in gliomas. We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape, polarity, and adhesion of cells in glioma. Here, we investigated whether NKCC1 promotes an epithelial–mesenchymal transition (EMT)-like process in gliomas via the RhoA and Rac1 signaling pathways. Pharmacological inhibition and knockdown of NKCC1 both decrease the expressions of mesenchymal markers, such as N-cadherin, vimentin, and snail, whereas these treatments increase the expression of the epithelial marker E-cadherin. These findings indicate that NKCC1 promotes an EMT-like process in gliomas. The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1, which results in a significant enhancement of the EMT-like process. Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA, and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas. Furthermore, we illustrated that NKCC1 knockdown abolished the dissemination and spread of glioma cells in a nude mouse intracranial model. These findings suggest that elevated NKCC1 activity acts in the regulation of an EMT-like process in gliomas, and thus provides a novel therapeutic strategy for targeting the invasiveness of gliomas, which might help to inhibit the spread of malignant intracranial tumors.     

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.     

DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells

BACKGROUND: Tumor cells can move in a three-dimensional (3D) environment in either mesenchymal-type or amoeboid modes. In mesenchymal-type movement, cells have an elongated morphology with Rac-induced protrusions at the leading edge. Amoeboid cells have high levels of actomyosin contractility, and movement is associated with deformation of the cell body through the matrix without proteolysis. Because signaling pathways that control the activation of GTPases for amoeboid movement are poorly understood, we sought to identify regulators of amoeboid movement by screening an siRNA library targeting guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. RESULTS: We identified DOCK10, a Cdc42 GEF, as a key player in amoeboid migration; accordingly, we find that expression of activated Cdc42 induces a mesenchymal-amoeboid transition and increases cell invasion. Silencing DOCK10 expression promotes conversion to mesenchymal migration and is associated with decreased MLC2 phosphorylation and increased Rac1 activation. Consequently, abrogating DOCK10 and Rac1 expression suppresses both amoeboid and mesenchymal migration and results in decreased invasion. We show that the Cdc42 effectors N-WASP and Pak2 are required for the maintenance of the rounded-amoeboid phenotype. Blocking Cdc42 results in loss of mesenchymal morphology, arguing that Cdc42 is also involved in mesenchymal morphology through different activation and effector pathways. CONCLUSIONS: Previous work has identified roles of Rho and Rac signaling in tumor cell movement, and we now elucidate novel roles of Cdc42 signaling in amoeboid and mesenchymal movement and tumor cell invasion.     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.     

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.     

Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.