The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.     

Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells

Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.     

An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival

Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.     

Pleiotropic role of Rac in mast cell activation revealed by a cell permeable Bordetella dermonecrotic fusion toxin

To activate the GTPase Rac in rat basophilic leukemia (RBL) cells and mouse bone marrow-derived mast cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell activation.     

A Rho-related GTPase is involved in Ca(2+)-dependent neurotransmitter exocytosis

Rho, Rac, and Cdc42 monomeric GTPases are well known regulators of the actin cytoskeleton and phosphoinositide metabolism and have been implicated in hormone secretion in endocrine cells. Here, we examine their possible implication in Ca(2+)-dependent exocytosis of neurotransmitters. Using subcellular fractionation procedures, we found that RhoA, RhoB, Rac1, and Cdc42 are present in rat brain synaptosomes; however, only Rac1 was associated with highly purified synaptic vesicles. To determine the synaptic function of these GTPases, toxins that impair Rho-related proteins were microinjected into Aplysia neurons. We used lethal toxin from Clostridium sordellii, which inactivates Rac; toxin B from Clostridium difficile, which inactivates Rho, Rac, and Cdc42; and C3 exoenzyme from Clostridium botulinum and cytotoxic necrotizing factor 1 from Escherichia coli, which mainly affect Rho. Analysis of the toxin effects on evoked acetylcholine release revealed that a member of the Rho family, most likely Rac1, was implicated in the control of neurotransmitter release. Strikingly, blockage of acetylcholine release by lethal toxin and toxin B could be completely removed in <1 s by high frequency stimulation of nerve terminals. Further characterization of the inhibitory action produced by lethal toxin suggests that Rac1 protein regulates a late step in Ca(2+)-dependent neuroexocytosis.     

A role for the small molecular weight GTPases, Rho and Cdc42, in muscarinic receptor signaling to focal adhesion kinase

An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elicited during neuronal growth cone remodeling and requires the maintenance of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate (PIP2). Rho family GTPases are putative regulators of both PIP2 synthesis and growth cone remodeling, including neurite outgrowth elicited by muscarinic cholinergic receptor (mAChR) stimulation. In this study, we investigated the interrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signaling to FAK in SH-SY5Y neuroblastoma cells. Preincubation with Clostridium difficile toxin B (Tox B), an inhibitor of Rho, Rac, and Cdc42, attenuated mAChR-stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidic acid (LPA)-induced FAK phosphorylation to a similar extent (75% decreases at 200 pg/ml Tox B) but did not affect mitogen-activated protein kinase activation elicited by either phorbol ester or an mAChR agonist. In contrast, preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rho kinase (HA-1 077) resulted in 80-90% reductions in LPA-induced FAK phosphorylation but only 40-50% decreases in mAChR-stimulated phosphorylation. Moreover, mAChR-mediated FAK phosphorylation was significantly attenuated in cells scrape-loaded with dominant-negative N17Cdc42 but not N17Rac1. Tox B had little or no effect on agonist-sensitive pools of PIP2 but inhibited mAChR-driven actin cytoskeletal remodeling. The results suggest that the Rho family GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK phosphorylation independently of effects on PIP2 synthesis.     

Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85Delta, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.     

PKB inhibition prevents the stimulatory effect of insulin on glucose transport and protein translocation but not the antilipolytic effect in rat adipocytes

We identified 1-(5 chloronaphthalenesulfonyl)-1H-hexahydro-1, 4-diazepine, also known as ML-9, as a powerful inhibitor of PKB activity in different cells as well as of recombinant PKB. It also inhibits other downstream serine/threonine kinases, such as PKA and p90 S6 kinase, but not upstream tyrosine phosphorylation or PI3-kinase activation in response to insulin. We compared the effects of ML-9 and wortmannin on several insulin-stimulated effects in isolated rat fat cells. Both ML-9 and wortmannin inhibited glucose transport and GLUT4/IGF II receptor translocation to the plasma membrane. In contrast, only wortmannin inhibited the antilipolytic effect and PDE3B activation by insulin. Thus, ML-9 inhibits PKB but not PI3-kinase activation in response to insulin and is useful to differentiate between these effects. Both PI3-kinase and PKB are important for glucose transport and intracellular protein translocation while PKB does not appear to play an important role for the antilipolytic effect or activation of PDE3B in response to insulin.     

Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C;G12V) double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V)-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42(G12V)-induced outgrowth did not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.     

Muscarinic receptors mediate phospholipase C-dependent activation of protein kinase B via Ca2+, ErbB3, and phosphoinositide 3-kinase in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, heterotrimeric G-protein-coupled receptors that activate phosphoinositide-specific phospholipase Cbeta (PLCbeta) isoforms via G(q), induced a prolonged activation of protein kinase B (PKB) after a short delay. For example, the effect of carbachol acting on M3 muscarinic receptors is blocked by wortmannin, suggesting it is mediated via a phosphoinositide 3-kinase (PI 3-kinase). In support of this, carbachol increased PI 3-kinase activity in PI 3-kinase (p85) immunoprecipitates. The pathway linking PLC-coupled receptors to PI 3-kinase was deduced to involve phosphoinositide hydrolysis and Ca2+-dependent ErbB3 transactivation but not protein kinase C on the basis of the following evidence: (i) inhibition of carbachol stimulated PLC by pretreatment with the phorbol ester phorbol 12-myristate 13-acetate concomitantly reduced PKB activity, whereas stimulation of other PLC-coupled receptors also activated PKB; (ii) Ca2+ ionophores and thapsigargin stimulated PKB activity in a wortmannin-sensitive manner, whereas bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked carbachol-stimulated PKB activity; (iii) phorbol 12-myristate 13-acetate alone did not activate PKB, whereas a protein kinase C inhibitor did not prevent the activation of PKB by carbachol; and (iv) carbachol stimulated ErbB3-tyrosine phosphorylation and association with p85, and both these and PKB activity were blocked by tyrphostin AG1478, an epidermal growth factor receptor-tyrosine kinase inhibitor. These experiments define a novel pathway linking G(q)-coupled G-protein-coupled receptors to the activation of PI 3-kinase and PKB.     

Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway

The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho family GTPase Cdc42. GTP-bound Cdc42 has been shown to facilitate neurite outgrowth elicited by activation of muscarinic cholinergic receptors (mAChRs). Because tyrosine kinase activity is a requirement for neuritogenesis in several cell systems, we investigated whether endogenous mAChRs (principally of the M3 subtype) expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1. Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold increase in the tyrosine phosphorylation of ACK-1 which was inhibited by atropine. ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, an inhibitor of Rho family GTPases. In contrast, disruption of the actin cytoskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreover, addition of Oxo-M to cells preincubated with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1. Although stimulation of M3 mAChRs induces both an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation. Instead, inhibition of PKC resulted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC with phorbol ester decreased ACK-1 phosphorylation. The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in an agonist-dependent manner. Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphorylation. The data are the first to show phosphorylation of ACK-1 after stimulation of a receptor coupled to neurite outgrowth and indicate that a Rho family GTPase (i.e. Cdc42) and Fyn are essential upstream elements of this signaling pathway.     

Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.     

Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway

Oxidized LDL (oxLDL) is known to induce endothelial adhesion molecule and monocyte chemoattractant protein 1 expression and this is thought to be involved in monocyte recruitment into atherosclerotic lesions. oxLDL has also been found to induce macrophage proliferation. The purpose of the present study was to determine whether oxLDL might also have the ability to increase macrophage populations by inhibiting apoptosis. We found that oxLDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages after macrophage colony-stimulating factor (M-CSF) withdrawal without inducing proliferation. Incubation of macrophages with either native LDL or acetylated LDL had no effect on apoptosis. The prosurvival effect of oxLDL was not inhibited by neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. oxLDL caused activation of the mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2) as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI 3-kinase). Furthermore, there was phosphorylation of two important prosurvival PKB targets, I-kappaBalpha(Ser-32) and Bad(Ser-136). The MEK inhibitors PD 98059 and U0126 blocked ERK1/2 activation but did not diminish survival. Conversely, the PI 3-kinase inhibitors LY 294002 and wortmannin blocked PKB activation, and the ability of oxidized LDL to promote macrophage survival.Taken together, these results indicate that oxLDL can directly activate a PI 3-kinase/PKB-dependent pathway that permits macrophage survival in the absence of growth factors.     

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.