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.     

Complement activates the c-Jun N-terminal kinase/stress-activated protein kinase in glomerular epithelial cells

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A(2) (cPLA(2)), and products of cPLA(2)-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA(2) in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and alpha-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H(2)O(2) activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA(2) and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.     

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.     

Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of cytosolic phospholipase A(2) (cPLA(2)). This study addresses the role of endoplasmic reticulum (ER) stress proteins (bip, grp94) in GEC injury. GEC that overexpress cPLA(2) (produced by transfection) and "neo" GEC (which expresses cPLA(2) at a lower level) were incubated with complement (40 min), and leakage of constitutively expressed bip and grp94 from ER into cytosol was measured to monitor ER injury. Greater leakage of bip and grp94 occurred in complement-treated GEC that overexpress cPLA(2), as compared with neo, implying that cPLA(2) activation perturbed ER membrane integrity. After chronic incubation (4-24 h), C5b-9 increased bip and grp94 mRNAs and proteins, and the increases were dependent on cPLA(2). Expression of bip-antisense mRNA reduced stimulated bip protein expression and enhanced complement-dependent GEC injury. Glomerular bip and grp94 proteins were up-regulated in proteinuric rats with PHN, as compared with normal control. Pretreatment of rats with tunicamycin or adriamycin, which increase ER stress protein expression, reduced proteinuria in PHN. Thus, C5b-9 injures the ER and enhances ER stress protein expression, in part, via activation of cPLA(2). ER stress protein induction is a novel mechanism of protection from complement attack.     

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.     

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.     

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.     

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 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.     

Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition

The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. Here we show that GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with cingulin, a junctional adaptor. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling.     

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.     

Polarity-regulating kinase partitioning-defective 1b (PAR1b) phosphorylates guanine nucleotide exchange factor H1 (GEF-H1) to regulate RhoA-dependent actin cytoskeletal reorganization

Partitioning-defective 1b (PAR1b), also known as microtubule affinity-regulating kinase 2 (MARK2), is a member of evolutionally conserved PAR1/MARK serine/threonine kinase family, which plays a key role in the establishment and maintenance of cell polarity at least partly by phosphorylating microtubule-associated proteins (MAPs) that regulate microtubule stability. PAR1b has also been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system. Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). This observation suggests a functional link between PAR1b and GEF-H1. Here we show that PAR1b induces phosphorylation of GEF-H1 on serine 885 and serine 959. We also show that PAR1b-induced serine 885/serine 959 phosphorylation inhibits RhoA-specific GEF activity of GEF-H1. As a consequence, GEF-H1 phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b not only regulates microtubule stability through phosphorylation of MAPs but also influences actin stress fiber formation by inducing GEF-H1 phosphorylation. The dual function of PAR1b in the microtubule-based cytoskeletal system and the actin-based cytoskeletal system in the coordinated regulation of cell polarity, cell morphology, and cell movement.     

Cytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.     

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.     

Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton

Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.     

Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.     

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.     

Cellular functions of GEF-H1, a microtubule-regulated Rho-GEF: is altered GEF-H1 activity a crucial determinant of disease pathogenesis?

The Rho guanine nucleotide exchange factor GEF-H1 is uniquely regulated by microtubule binding and is crucial in coupling microtubule dynamics to Rho-GTPase activation in a variety of normal biological situations. Here, we review the roles of GEF-H1 in epithelial barrier permeability, cell motility and polarization, dendritic spine morphology, antigen presentation, leukemic cell differentiation, cell cycle regulation, and cancer. GEF-H1 might also contribute to pathophysiological signaling involved in leukemias, and in cancers associated with mutated p53 tumor suppressor gene, epithelial and endothelial cell dysfunction, infectious disease, and cardiac hypertrophy. We suggest that GEF-H1 could be a novel therapeutic target in multiple human diseases.     

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.     

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.