The epidermal growth factor receptor mediates tumor necrosis factor-alpha-induced activation of the ERK/GEF-H1/RhoA pathway in tubular epithelium

Tumor necrosis factor (TNF)-α induces cytoskeleton and intercellular junction remodeling in tubular epithelial cells; the underlying mechanisms, however, are incompletely explored. We have previously shown that ERK-mediated stimulation of the RhoA GDP/GTP exchange factor GEF-H1/Lfc is critical for TNF-α-induced RhoA stimulation. Here we investigated the upstream mechanisms of ERK/GEF-H1 activation. Surprisingly, TNF-α-induced ERK and RhoA stimulation in tubular cells were prevented by epidermal growth factor receptor (EGFR) inhibition or silencing. TNF-α also enhanced phosphorylation of the EGFR. EGF treatment mimicked the effects of TNF-α, as it elicited potent, ERK-dependent GEF-H1 and RhoA activation. Moreover, EGF-induced RhoA activation was prevented by GEF-H1 silencing, indicating that GEF-H1 is a key downstream effector of the EGFR. The TNF-α-elicited EGFR, ERK, and RhoA stimulation were mediated by the TNF-α convertase enzyme (TACE) that can release EGFR ligands. Further, EGFR transactivation also required the tyrosine kinase Src, as Src inhibition prevented TNF-α-induced activation of the EGFR/ERK/GEF-H1/RhoA pathway. Importantly, a bromodeoxyuridine (BrdU) incorporation assay and electric cell substrate impedance-sensing (ECIS) measurements revealed that TNF-α stimulated cell growth in an EGFR-dependent manner. In contrast, TNF-α-induced NFκB activation was not prevented by EGFR or Src inhibition, suggesting that TNF-α exerts both EGFR-dependent and -independent effects. In summary, in the present study we show that the TNF-α-induced activation of the ERK/GEF-H1/RhoA pathway in tubular cells is mediated through Src- and TACE-dependent EGFR activation. Such a mechanism could couple inflammatory and proliferative stimuli and, thus, may play a key role in the regulation of wound healing and fibrogenesis.     

Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.     

A Highlights from MBoC Selection: Involvement of p114-RhoGEF and Lfc in Wnt-3a– and Dishevelled-Induced RhoA Activation and Neurite Retraction in N1E-115 Mouse Neuroblastoma Cells

The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a–induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF–silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a–induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a–induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a– and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.     

Cingulin regulates claudin-2 expression and cell proliferation through the small GTPase RhoA

In mouse embryoid bodies, mutation of the tight junction protein cingulin results in changes in gene expression. Here, we studied the function of cingulin using a gene silencing approach in Madin-Darby canine kidney (MDCK) cells. Cingulin-depleted cells show higher protein and mRNA levels of claudin-2 and ZO-3, increased RhoA activity, activation of G1/S phase transition, and increased cell density. The effects of cingulin depletion on claudin-2 expression, cell proliferation, and density are reversed by coexpression of either a dominant-negative form of RhoA (RhoAN19) or the Rho-inhibiting enzyme C3 transferase. However, the increase in ZO-3 protein and mRNA levels is not reversed by inhibition of either RhoA, p38, extracellular signal-regulated kinase (ERK), or c-Jun NH2-terminal kinase (JNK), suggesting that cingulin modulates ZO-3 expression by a different mechanism. JNK is implicated in the regulation of claudin-2 levels independently of cingulin depletion and RhoA activity, indicating distinct roles of RhoA- and JNK-dependent pathways in the control of claudin-2 expression. Finally, cingulin depletion does not significantly alter the barrier function of monolayers and the overall molecular organization of tight junctions. These results provide novel insights about the mechanisms of cingulin function and the signaling pathways controlling claudin-2 expression in MDCK cells.     

Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

Breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. In turn, matrix stiffness regulates RhoA activity. However, the upstream signaling mechanisms are poorly defined. Here we demonstrate that the Rho exchange factor GEF-H1 mediates RhoA activation in response to extracellular matrix stiffness. We demonstrate the novel finding that microtubule stability is diminished by a stiff three-dimensional (3D) extracellular matrix, which leads to the activation of GEF-H1. Surprisingly, activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway did not contribute to stiffness-induced GEF-H1 activation. Loss of GEF-H1 decreases cell contraction of and invasion through 3D matrices. These data support a model in which matrix stiffness regulates RhoA through microtubule destabilization and the subsequent release and activation of GEF-H1.     

GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA

The RhoA GTPase plays a vital role in assembly of contractile actin-myosin filaments (stress fibers) and of associated focal adhesion complexes of adherent monolayer cells in culture. GEF-H1 is a microtubule-associated guanine nucleotide exchange factor that activates RhoA upon release from microtubules. The overexpression of GEF-H1 deficient in microtubule binding or treatment of HeLa cells with nocodazole to induce microtubule depolymerization results in Rho-dependent actin stress fiber formation and contractile cell morphology. However, whether GEF-H1 is required and sufficient to mediate nocodazole-induced contractility remains unclear. We establish here that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole-induced cell contraction. Furthermore, the nocodazole-induced activation of RhoA and Rho-associated kinase (ROCK) that mediates phosphorylation of myosin regulatory light chain (MLC) is impaired in GEF-H1–depleted cells. Conversely, RhoA activation and contractility are rescued by reintroduction of siRNA-resistant GEF-H1. Our studies reveal a critical role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating nocodazole-induced cell contractility.     

Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression

Cingulin is a component of the cytoplasmic domain of vertebrate tight junctions (TJ). Mutation or down-regulation of cingulin in cultured cells results in changes in gene expression. Some of these changes are dependent on RhoA, whose activity is regulated by GEF-H1, which is inactivated by binding to cingulin at junctions. To gain further insights on the function of cingulin through dominant-negative effects, we cloned and sequenced canine cingulin, and developed stable MDCK cell lines where either full-length cingulin, or head or rod+tail domains were inducibly overexpressed. Surprisingly, analysis of these clones by immunoblotting, microarray, immunofluorescence, measurement of transepithelial resistance, and cell density showed that the overexpression of either full-length cingulin or its domains does not significantly affect TJ protein levels, gene expression, RhoA activity, cell density, doubling time, and the organization and function of TJ. These results suggest that compensatory mechanisms prevent dominant-negative effects in this model system, and that modulation of cellular functions by cingulin occurs within physiological protein levels.     

Contribution of guanine exchange factor H1 in phorbol ester-induced apoptosis

Phorbol-12-myristate-13-acetate (PMA) treatment induces erythroblastoma D2 cells kept in suspension to undergo RhoA-dependent contraction and to become proapoptotic, while attached cells are induced to differentiate accompanied by the reduction of RhoA activity. In this study, we found that guanine exchange factor H1 (GEF-H1) is highly expressed in D2 cells. Depletion of GEF-H1 expression in D2 cells decreased RhoA activity and prevented PMA-induced contraction and apoptosis. Upon PMA stimulation, GEF-H1 became associated with microtubules in cells that were induced to differentiate. As a contrast, in the proapoptotic population of cells GEF-H1 stayed in the cytoplasm without showing PMA-responsive microtubule translocation. Given that GEF-H1 is inactivated when associated with microtubules and its release into cytosol due to depolymerization of microtubules activates RhoA, our results demonstrated that nonmicrotubule-associated GEF-H1 in D2 cells contributes to the sustained activation of RhoA/ROCK signaling in suspension cells, making cells susceptible to PMA-induced apoptosis.     

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.     

Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis

Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.     

Guanine Nucleotide Exchange Factor-H1 Regulates Cell Migration via Localized Activation of RhoA at the Leading Edge

Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell–substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.     

GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.     

Extracellular Signal-Regulated Kinase Regulates RhoA Activation and Tumor Cell Plasticity by Inhibiting Guanine Exchange Factor H1 Activity

In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.     

Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac,ADAM17/TACE,and RhoA in tubular epithelial cells

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α–induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.     

Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.     

The Microtubule-Associated Rho Activating Factor GEF-H1 Interacts with Exocyst Complex to Regulate Vesicle Traffic

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.     

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.