S1P induces FA remodeling in human pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin.

Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.     

Influence of oxidatively modified LDL on monocyte-macrophage differentiation

Cellular cytoskeletal remodeling reflects alterations in local biochemical and mechanical changes in terms of stress that manifests relocation of signaling molecules within and across the cell. Although stretching due to load and chemical changes by high homocysteine (HHcy) causes cytoskeletal re-arrangement, the synergism between stretch and HHcy is unclear. We investigated the contribution of HHcy in cyclic stretch-induced focal adhesion (FA) protein redistribution leading to cytoskeletal re-arrangement in mouse aortic endothelial cells (MAEC). MAEC were subjected to cyclic stretch (CS) and HHcy alone or in combination. The redistribution of FA protein, and small GTPases were determined by Confocal microscopy and Western blot techniques in membrane and cytosolic compartments. We found that each treatment induces focal adhesion kinase (FAK) phosphorylation and cytoskeletal actin polymerization. In addition, CS activates and membrane translocates small GTPases RhoA with minimal effect on Rac1, whereas HHcy alone is ineffective in both GTPases translocation. However, the combined effect of CS and HHcy activates and membrane translocates both GTPases. Free radical scavenger NAC (N-Acetyl-Cysteine) inhibits CS and HHcy-mediated FAK phosphorylation and actin stress fiber formation. Interestingly, CS also activates and membrane translocates another FA protein, paxillin in HHcy condition. Cytochalasin D, an actin polymerization blocker and PI3-kinase inhibitor Wortmannin inhibited FAK phosphorylation and membrane translocation of paxillin suggesting the involvement of PI3K pathway. Together our results suggest that CS- and HHcy-induced oxidative stress synergistically contribute to small GTPase membrane translocation and focal adhesion protein redistribution leading to endothelial remodeling.     

Rap-afadin axis in control of Rho signaling and endothelial barrier recovery

Activation of the Rho GTPase pathway determines endothelial cell (EC) hyperpermeability after injurious stimuli. To date, feedback mechanisms of Rho down-regulation critical for barrier restoration remain poorly understood. We tested a hypothesis that Rho down-regulation and barrier recovery of agonist-stimulated ECs is mediated by the Ras family GTPase Rap1. Thrombin-induced EC permeability driven by rapid activation of the Rho GTPase pathway was followed by Src kinase–dependent phosphorylation of the Rap1-specific guanine nucleotide exchange factor (GEF) C3G, activation of Rap1, and initiation of EC barrier recovery. Knockdown experiments showed that Rap1 activation was essential for down-regulation of Rho signaling and actin stress fiber dissolution. Rap1 activation also enhanced interaction between adherens junction (AJ) proteins VE-cadherin and p120-catenin and stimulated AJ reannealing mediated by the Rap1 effector afadin. This mechanism also included Rap1-dependent membrane translocation of the Rac1-specific GEF Tiam1 and activation of Rac1-dependent peripheral cytoskeletal dynamics, leading to resealing of intercellular gaps. These data demonstrate that activation of the Rap1-afadin axis is a physiological mechanism driving restoration of barrier integrity in agonist-stimulated EC monolayers via negative-feedback regulation of Rho signaling, stimulation of actin peripheral dynamics, and reestablishment of cell–cell adhesive complexes.     

Edaravone mimics sphingosine-1-phosphate-induced endothelial barrier enhancement in human microvascular endothelial cells

Edaravone is a potent scavenger of hydroxyl radicals and is quite successful in patients with acute cerebral ischemia, and several organ-protective effects have been reported. Treatment of human microvascular endothelial cells with edaravone (1.5 microM) resulted in the enhancement of transmonolayer electrical resistance coincident with cortical actin enhancement and redistribution of focal adhesion proteins and adherens junction proteins to the cell periphery. Edaravone also induced small GTPase Rac activation and focal adhesion kinase (FAK; Tyr(576)) phosphorylation associated with sphingosine-1-phosphate receptor type 1 (S1P(1)) transactivation. S1P(1) protein depletion by the short interfering RNA technique completely abolished edaravone-induced FAK (Tyr(576)) phosphorylation and Rac activation. This is the first report of edaravone-induced endothelial barrier enhancement coincident with focal adhesion remodeling and cytoskeletal rearrangement associated with Rac activation via S1P(1) transactivation. Considering the well-established endothelial barrier-protective effect of S1P, endothelial barrier enhancement as a consequence of S1P(1) transactivation may at least partly be the potent mechanisms for the organ-protective effect of edaravone and is suggestive of edaravone as a therapeutic agent against systemic vascular barrier disorder.     

The proto-oncogene Fgr regulates cell migration and this requires its plasma membrane localization

Fgr participates in integrin signaling in myeloid leukocytes. To examine the role of its specific domains in regulating cell migration, we expressed various Fgr molecules in COS-7 cells. Full-length, membrane-bound Fgr, but not an N-terminal truncation mutant that distributed to an intracellular compartment, increased cell migration on fibronectin and enhanced phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K), cortactin and focal adhesion kinase (FAK) at Y397 and Y576. Fgr increased Rac GTP loading, and phosphorylation of the Rac GEF Vav2, and bound to a protein complex formed by the Rho inhibitor p190RhoGAP and FAK, increasing p190RhoGAP phosphorylation, in a manner absolutely dependent on membrane localization. A kinase-defective truncation mutant of Fgr increased cell migration, albeit to a much lower extent than full-length Fgr, and was found to associate with the plasma membrane, to activate Rac and to form complexes with p190RhoGAP/FAK. Formation of complexes between p190RhoGAP, Fgr, and the FAK-related protein Pyk2 were also detected in murine macrophages. These findings suggest that the proto-oncogene Fgr regulates cell migration impinging on a signaling pathway implicating FAK/Pyk2 and leading to activation of Rac and the Rho inhibitor p190RhoGAP.     

Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.     

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

 Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK^−/− mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130^CAS/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.     

Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

Prostaglandin E(2) (PGE(2)) and prostacyclin are lipid mediators produced by cyclooxygenase and implicated in the regulation of vascular function, wound repair, inflammatory processes, and acute lung injury. Although protective effects of these prostaglandins (PGs) are associated with stimulation of intracellular cAMP production, the crosstalk between cAMP-activated signal pathways in the regulation of endothelial cell (EC) permeability is not well understood. We studied involvement of cAMP-dependent kinase (PKA), cAMP-Epac-Rap1 pathway, and small GTPase Rac in the PGs-induced EC barrier protective effects and cytoskeletal remodeling. PGE(2) and PGI(2) synthetic analog beraprost increased transendothelial electrical resistance and decreased dextran permeability, enhanced peripheral F-actin rim and increased intercellular adherens junction areas reflecting EC barrier-protective response. Furthermore, beraprost dramatically attenuated thrombin-induced Rho activation, MLC phosphorylation and EC barrier dysfunction. In vivo, beraprost attenuated lung barrier dysfunction induced by high tidal volume mechanical ventilation. Both PGs caused cAMP-mediated activation of PKA-, Epac/Rap1- and Tiam1/Vav2-dependent pathways of Rac1 activation and EC barrier regulation. Knockdown of Epac, Rap1, Rac-specific exchange factors Tiam1 and Vav2 using siRNA approach, or inhibition of PKA activity decreased Rac1 activation and PG-induced EC barrier enhancement. Thus, our results show that barrier-protective effects of PGE(2) and prostacyclin on pulmonary EC are mediated by PKA and Epac/Rap pathways, which converge on Rac activation and lead to enhancement of peripheral actin cytoskeleton and adherens junctions. These mechanisms may mediate protective effects of PGs against agonist-induced lung vascular barrier dysfunction in vitro and against mechanical stress-induced lung injury in vivo.     

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.     

Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.     

Cross talk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids

We previously reported that the barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cells (ECs) delineate the role of Rac- and Cdc42-dependent mechanisms and described the involvement of the focal adhesion (FA) protein paxillin in enhancement of the EC barrier upon OxPAPC challenge. This study examined a potential role of paxillin in the feedback mechanism of Rac regulation by FAs in OxPAPC-stimulated ECs. Our results demonstrate that OxPAPC induced Rac-dependent, Rho-independent peripheral accumulation of paxillin-containing FAs and time-dependent paxillin phosphorylation. Molecular inhibition of Rac decreased association of paxillin with the Rac-specific guanine nucleotide exchange factor beta-PIX. Molecular inhibition of paxillin also attenuated OxPAPC-induced enhancement of adherens junctions critical for the EC barrier-protective response, accumulation of vascular endothelial cadherin in the membrane fractions, and decreased activation of Rac and its effector p21-activated kinase (PAK1). Expression of paxillin with a mutated PAK1-dependent phosphorylation site (S273A) attenuated OxPAPC-induced PAK1 activation and the EC barrier-protective response. These results suggest that PAK1-specific paxillin phosphorylation at Ser(273) is critically involved in the positive-feedback regulation of the Rac-PAK1 pathway and may contribute to sustained enhancement of the EC barrier caused by oxidized phospholipids.     

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.     

Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr(418), Rac1 at Ser(71), FAK at Tyr(397) and Tyr(576), and ERK1/2 at Thr(202)/Tyr(204). The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr(576) and ERK phosphorylation but not FAK phosphorylation at Tyr(397). Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.     

VE-cadherin trans-interactions modulate Rac activation and enhancement of lung endothelial barrier by iloprost

Small GTPase Rac is important regulator of endothelial cell (EC) barrier enhancement by prostacyclin characterized by increased peripheral actin cytoskeleton and increased interactions between VE-cadherin and other adherens junction (AJ) proteins. This study utilized complementary approaches including siRNA knockdown, culturing in Ca(2+) -free medium, and VE-cadherin blocking antibody to alter VE-cadherin extracellular interactions to investigate the role of VE-cadherin outside-in signaling in modulation of Rac activation and EC barrier regulation by prostacyclin analog iloprost. Spatial analysis of Rac activation in pulmonary EC by FRET revealed additional spike in iloprost-induced Rac activity at the sites of newly formed cell-cell junctions. In contrast, disruption of VE-cadherin extracellular trans-interactions suppressed iloprost-activated Rac signaling and attenuated EC barrier enhancement and cytoskeletal remodeling. These inhibitory effects were associated with decreased membrane accumulation and activation of Rac-specific guanine nucleotide exchange factors (GEFs) Tiam1 and Vav2. Conversely, plating of pulmonary EC on surfaces coated with extracellular VE-cadherin domain further promoted iloprost-induced Rac signaling. In the model of thrombin-induced EC barrier recovery, blocking of VE-cadherin trans-interactions attenuated activation of Rac pathway during recovery phase and delayed suppression of Rho signaling and restoration of EC barrier properties. These results suggest that VE-cadherin outside-in signaling controls locally Rac activity stimulated by barrier protective agonists. This control is essential for maximal EC barrier enhancement and accelerated barrier recovery.     

Focal adhesion kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.     

Downregulation of FAK-related non-kinase mediates the migratory phenotype of human fibrotic lung fibroblasts

Fibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseased condition. To examine the role of focal-adhesion-kinase (FAK)-related non-kinase (FRNK) in regulation of fibrotic lung fibroblast migration, we examined cell migration, FRNK expression, and activation of focal adhesion kinase (FAK) and Rho GTPase (Rho and Rac) in primary lung fibroblasts derived from both idiopathic pulmonary fibrosis (IPF) patients and normal human controls. Fibrotic (IPF) lung fibroblasts have increased cell migration when compared to control human lung fibroblasts. FRNK expression is significantly reduced in IPF lung fibroblasts, while activation of FAK, Rho and Rac is increased in IPF lung fibroblasts. Endogenous FRNK expression is inversely correlated with FAK activation and cell migration rate in IPF lung fibroblasts. Forced exogenous FRNK expression abrogates the increased cell migration, and blocked the activation of FAK and Rho GTPase (Rho and Rac), in IPF lung fibroblasts. These data for the first time provide evidence that downregulation of endogenous FRNK plays a role in promoting cell migration through FAK and Rho GTPase in fibrotic IPF lung fibroblasts.     

Long-term cyclic stretch controls pulmonary endothelial permeability at translational and post-translational levels

We have previously described differential effects of physiologic (5%) and pathologic (18%) cyclic stretch (CS) on agonist-induced pulmonary endothelial permeability. This study examined acute and chronic effects of CS on agonist-induced intracellular signaling and cell morphology in the human lung macro- and microvascular endothelial cell (EC) monolayers. Endothelial permeability was assessed by analysis of morphological changes, parameters of cell contraction and measurements of transendothelial electrical resistance. Exposure of both microvascular and macrovascular EC to 18% CS for 2-96 h increased thrombin-induced permeability and monolayer disruption. Interestingly, the ability to promote thrombin responses was present in EC cultures exposed to 48-96 h of CS even after replating onto non-elastic substrates. In turn, physiologic CS preconditioning (72 h) attenuated thrombin-induced paracellular gap formation and MLC phosphorylation in replated EC cultures. Long-term preconditioning at 18% CS (72 h) increased the content of signaling and contractile proteins including Rho GTPase, MLC, MLC kinase, ZIP kinase, PAR1, caldesmon and HSP27 in the pulmonary microvascular and macrovascular cells. We conclude that short term CS regulates EC permeability via modulation of agonist-induced signaling, whereas long-term CS controls endothelial barrier at both post-translational level and via magnitude-dependent regulation of pulmonary EC phenotype, signaling and contractile protein expression.     

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori- mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori -induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.     

Tiam1 and betaPIX mediate Rac-dependent endothelial barrier protective response to oxidized phospholipids

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibits potent barrier protective effects on pulmonary endothelium, which are mediated by small GTPases Rac and Cdc42. However, upstream mechanisms of OxPAPC-induced small GTPase activation are not known. We studied involvement of Rac/Cdc42-specific guanine nucleotide exchange factors (GEFs) Tiam1 and betaPIX in OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective responses in the human pulmonary endothelial cells (EC). OxPAPC induced membrane translocation of Tiam1, betaPIX, Cdc42, and Rac, but did not affect intracellular distribution of Rho and Rho-specific GEF p115-RhoGEF. Protein depletion of Tiam1 and betaPIX using siRNA approach abolished OxPAPC-induced activation of Rac and its effector PAK1. EC transfection with Tiam1-, betaPIX-, or PAK1-specific siRNA dramatically attenuated OxPAPC-induced barrier enhancement, peripheral actin cytoskeletal enhancement, and translocation of actin-binding proteins cortactin and Arp3. These results show for the first time that Tiam1 and betaPIX mediate OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective response in pulmonary endothelium.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.