Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion

Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.     

Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal–regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

Inhibition of integrin-mediated cell adhesion but not directional cell migration requires catalytic activity of EphB3 receptor tyrosine kinase. Role of Rho family small GTPases

Genetic studies have shown that Eph receptor tyrosine kinases have both kinase-dependent and kinase-independent functions through incompletely understood mechanisms. We report here that ephrin-B1 stimulation of endogenous EphB kinases in LS174T colorectal epithelial cells inhibited integrin-mediated adhesion and HGF/SF-induced directional cell migration. Using 293 cells stably transfected with wild type (WT)- or kinase-deficient (KD-EphB3), we found that inhibition of integrin-mediated cell adhesion and induction of cell rounding was kinase-dependent. Unexpectedly, in two independent assays, both KD- and WT-EphB3 significantly inhibited directional cell migration. Upon ephrin-B1 stimulation, the activities of Rac1 and Cdc42 were reduced in both WT- and KD-EphB3-expressing cells that were induced to migrate. Pharmacological evidence demonstrates that a relative increase in RhoA signaling as a result of decreased Rac1/Cdc42 activities contributes to the inhibitory effects. Furthermore, EphB3-mediated inhibitory effect on cell adhesion but not migration was abolished by the integrin activating antibodies, suggesting that the inhibition of cell migration is not because of down-regulation of integrin function. These results uncover a differential requirement for EphB3 catalytic activity in the regulation of cell adhesion and migration, and suggest that while catalytic activity of EphB3 is required for inhibition of integrin-mediated cell adhesion, a distinct signaling pathway to Rho GTPases shared by WT- and KD-EphB3 receptor mediates inhibition of directional cell migration.     

Transforming growth factor beta activates Rac1 and Cdc42Hs GTPases and the JNK pathway in skeletal muscle cells

The transforming growth factor beta (TGFbeta) plays an important role in cell growth and differentiation. However, the intracellular signaling pathways through which TGFbeta inhibits skeletal myogenesis remain largely undefined. By measuring GTP-loading of Rho GTPases and the organization of the F-actin cytoskeleton and the plasma membrane, we analyzed the effect of TGFbeta addition on the activity of three GTPases, Rac1, Cdc42Hs and RhoA. We report that TGFbeta activates Rac1 and Cdc42Hs in skeletal muscle cells, two GTPases previously described to inhibit skeletal muscle cell differentiation whereas it inactivates RhoA, a positive regulator of myogenesis. We further show that TGFbeta activates the C-jun N-terminal kinases (JNK) pathway in myoblastic cells through Rac1 and Cdc42Hs GTPases. We propose that the activation of Rho family proteins Rac1 and Cdc42Hs which subsequently regulate JNK activity participates in the inhibition of myogenesis by TGFbeta.     

Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGD-dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis.     

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.     

Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8

Blood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (alpha4beta1gamma1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through alpha6beta1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectin showed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for alpha6beta1 integrin-mediated cell spreading and migration on laminin-8.     

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.     

Cdc42: an important regulator of neuronal morphology

Regulation of neuronal morphology and activity-dependent synaptic modifications involves reorganization of the actin cytoskeleton. Dynamic changes of the actin cytoskeleton in many cell types are controlled by small GTPases of the Rho family, such as RhoA, Rac1 and Cdc42. As key regulators of both actin and microtubule cytoskeleton, Rho GTPases have also emerged as important regulators of dendrite and spine structural plasticity. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and growth cone protrusion, while the activation of RhoA induces stress fiber formation, leading to growth cone collapse and neurite retraction. This review focuses on recent advances in our understanding of the molecular mechanisms underlying physiological and pathological functions of Cdc42 in the nervous system. We also discuss application of different FRET-based biosensors as a powerful approach to examine the dynamics of Cdc42 activity in living cells.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

PTP-PEST couples membrane protrusion and tail retraction via VAV2 and p190RhoGAP

Cell motility is regulated by a balance between forward protrusion and tail retraction. These phenomena are controlled by a spatial asymmetry in signals at the front and the back of the cell. We show here that the protein-tyrosine phosphatase, PTP-PEST, is required for the coupling of protrusion and retraction during cell migration. PTP-PEST null fibroblasts, which are blocked in migration, exhibit exaggerated protrusions at the leading edge and long, unretracted tails in the rear. This altered morphology is accompanied by changes in the activity of Rho GTPases, Rac1 and RhoA, which mediate protrusion and retraction, respectively. PTP-PEST null cells exhibit enhanced Rac1 activity and decreased RhoA activity. We further show that PTP-PEST directly targets the upstream regulators of Rac1 and RhoA, VAV2 and p190RhoGAP. Moreover, we demonstrate that the activities of VAV2 and p190RhoGAP are regulated by PTP-PEST. Finally, we present evidence indicating the VAV2 can be regulated by integrin-mediated adhesion. These data suggest that PTP-PEST couples protrusion and retraction by acting on VAV2 and p190RhoGAP to reciprocally modulate the activity of Rac1 and RhoA.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.     

Small rho GTPases regulate antigen presentation in dendritic cells

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms maintaining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was significantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo.