A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.

Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice.

[Figure: see text].

Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases.

OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

Mechanopharmacology of Rho-kinase antagonism in airway smooth muscle and potential new therapy for asthma.

The principle of mechanopharmacology of airway smooth muscle (ASM) is based on the premise that physical agitation, such as pressure oscillation applied to an airway, is able to induce bronchodilation by reducing contractility and softening the cytoskeleton of ASM. Although the underlying mechanism is not entirely clear, there is evidence to suggest that large-amplitude stretches are able to disrupt the actomyosin interaction in the crossbridge cycle and weaken the cytoskeleton in ASM cells. Rho-kinase is known to enhance force generation and strengthen structural integrity of the cytoskeleton during smooth muscle activation and plays a key role in the maintenance of force during prolonged muscle contractions. Synergy in relaxation has been observed when the muscle is subject to oscillatory length change while Rho-kinase is pharmacologically inhibited. In this review, inhibition of Rho-kinase coupled to therapeutic pressure oscillation applied to the airways is explored as a combination treatment for asthma.

Resveratrol Attenuates Aortic Dissection by Increasing Endothelial Barrier Function Through the SIRT1 Pathway.

Aortic dissection (AD) is a serious condition and a health issue on a global scale. ß-Aminopropionitrile-induced AD in mice is similar to the pathogenesis of AD in humans. Resveratrol (RSV) is a natural polyphenolic substance that provides anti-inflammatory and cardiovascular effects, but the role of RSV in AD is unclear. In this study, we investigated the effects and mechanisms of RSV on ß-aminopropionitrile-induced AD in mice. Our results indicate that RSV can prevent the occurrence of AD. More meaningfully, we found that the protective effect comprises an increase in sirtuin 1 (SIRT1) expression in endothelial cells for the reconstruction of their structure, reducing the recruitment of inflammatory cells by endothelial cells and inhibiting the inflammation response, thereby suppressing the occurrence of AD.

Phospho-substrate profiling of Epac-dependent protein kinase C activity.

Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.

FGD5 Regulates VEGF Receptor-2 Coupling to PI3 Kinase and Receptor Recycling.

OBJECTIVE: VEGF (vascular endothelial growth factor-A) signaling to the endothelial cell (EC) through VEGFR2 (VEGF receptor-2) is the principal cue driving new blood vessel formation. FGD5 (faciogenital dysplasia-5)-a Rho-family guanine nucleotide exchange factor-is selectively expressed in EC. Deficiency of FGD5 is embryonically lethal in mice and perturbs angiogenesis and VEGF signal transduction. However, the mechanism of FGD5 regulation of VEGF signaling is poorly understood. APPROACH AND RESULTS: Angiogenic sprouting and EC cytoskeletal remodeling were evaluated in a 3-dimensional in vitro model. We examined the subcellular localization of FGD5 and VEGFR2 in EC by immunofluorescent staining and studied the association by immunoprecipitation. FGD5 deficiency reduced the number of angiogenic sprouts and tip cell filopodia by ≈80% and ≈70%, respectively. These defects were accompanied by downregulation of the expression of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting and VEGF-stimulated EC, FGD5 forms a complex with VEGFR2 and was enriched at the leading edge of the cell and among endosomes. FGD5 loss reduced mTORC2 (mammalian target of rapamycin complex-2)/Akt-dependent cortactin activation downstream of VEGFR2 but did not alter VEGFR2 plasma membrane expression, Y1175 phosphorylation, or endocytosis. However, FGD5 loss decreased endosomal VEGFR2 coupling to phosphoinositide-3 kinase and diverted VEGFR2 to lysosomal degradation. CONCLUSIONS: FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3 (phosphoinositide-3) kinase/mTORC2-dependent cytoskeletal remodeling.

Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and ß-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD.

Local VE-cadherin mechanotransduction triggers long-ranged remodeling of endothelial monolayers.

In this study, we present results demonstrating that mechanotransduction by vascular endothelial cadherin (VE-cadherin, also known as CDH5) complexes in endothelial cells triggers local cytoskeletal remodeling, and also activates global signals that alter peripheral intercellular junctions and disrupt cell-cell contacts far from the site of force application. Prior studies have documented the impact of actomyosin contractile forces on adherens junction remodeling, but the role of VE-cadherin in force sensation and its ability to influence endothelial cell and tissue mechanics globally have not been demonstrated. Using mechanical manipulation of VE-cadherin bonds and confocal imaging, we demonstrate VE-cadherin-based mechanotransduction. We then demonstrate that it requires homophilic VE-cadherin ligation, an intact actomyosin cytoskeleton, Rho-associated protein kinase 1 (ROCK1) and phosphoinositide 3-kinase. VE-cadherin-mediated mechanotransduction triggered local actin and vinculin recruitment, as well as global signals that altered focal adhesions and disrupted peripheral intercellular junctions. Confocal imaging revealed that VE-cadherin-specific changes appear to propagate across cell junctions to disrupt distant inter-endothelial junctions. These results demonstrate the central role of VE-cadherin adhesions and the actomyosin cytoskeleton within an integrated, mechanosensitive network that both induces local cytoskeletal remodeling at the site of force application and regulates the global integrity of endothelial tissues.

Structural pliability adjacent to the kinase domain highlights contribution of FAK1 IDRs to cytoskeletal remodeling.

Therapeutic protein kinase inhibitors are designed on the basis of kinase structures. Here, we define intrinsically disordered regions (IDRs) in structurally hybrid kinases. We reveal that 65% of kinases have an IDR adjacent to their kinase domain (KD). These IDRs are evolutionarily more conserved than IDRs distant to KDs. Strikingly, 36 kinases have adjacent IDRs extending into their KDs, defining a unique structural and functional subset of the kinome. Functional network analysis of this subset of the kinome uncovered FAK1 as topologically the most connected hub kinase. We identify that KD-flanking IDR of FAK1 is more conserved and undergoes more post-translational modifications than other IDRs. It preferentially interacts with proteins regulating scaffolding and kinase activity, which contribute to cytoskeletal remodeling. In summary, spatially and evolutionarily conserved IDRs in kinases may influence their functions, which can be exploited for targeted therapies in diseases including those that involve aberrant cytoskeletal remodeling.

Nonsteroidal anti-inflammatory drugs attenuate amyloid-ß protein-induced actin cytoskeletal reorganization through Rho signaling modulation.

Amyloid-ß protein (Aß) neurotoxicity occurs along with the reorganization of the actin-cytoskeleton through the activation of the Rho GTPase pathway. In addition to the classical mode of action of the non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin, and ibuprofen have Rho-inhibiting effects. In order to evaluate the role of the Rho GTPase pathway on Aß-induced neuronal death and on neuronal morphological modifications in the actin cytoskeleton, we explored the role of NSAIDS in human-differentiated neuroblastoma cells exposed to Aß. We found that Aß induced neurite retraction and promoted the formation of different actin-dependent structures such as stress fibers, filopodia, lamellipodia, and ruffles. In the presence of Aß, both NSAIDs prevented neurite collapse and formation of stress fibers without affecting the formation of filopodia and lamellipodia. Similar results were obtained when the downstream effector, Rho kinase inhibitor Y27632, was applied in the presence of Aß. These results demonstrate the potential benefits of the Rho-inhibiting NSAIDs in reducing Aß-induced effects on neuronal structural alterations.

A MORN1-associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding.

Apicomplexan parasites replicate by several budding mechanisms with two well-characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two-hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two-hybrid. HAD2a has demonstrated enzyme-activity in vitro, localizes to the nascent daughter buds, and co-localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex.

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant.

Exoenzyme Y (ExoY) was identified as a component of the Pseudomonas aeruginosa type 3 secretion system secretome in 1998. It is a common contributor to the arsenal of type 3 secretion system effectors, as it is present in approximately 90% of Pseudomonas isolates. ExoY has adenylyl cyclase activity that is dependent upon its association with a host cell cofactor. However, recent evidence indicates that ExoY is not just an adenylyl cyclase; rather, it is a promiscuous cyclase capable of generating purine and pyrimidine cyclic nucleotide monophosphates. ExoY's enzymatic activity causes a characteristic rounding of mammalian cells, due to microtubule breakdown. In endothelium, this cell rounding disrupts cell-to-cell junctions, leading to loss of barrier integrity and an increase in tissue edema. Microtubule breakdown seems to depend upon tau phosphorylation, where the elevation of cyclic nucleotide monophosphates activates protein kinases A and G and causes phosphorylation of endothelial microtubule associated protein tau. Phosphorylation is a stimulus for tau release from microtubules, leading to microtubule instability. Phosphorylated tau accumulates inside endothelium as a high molecular weight, oligomeric form, and is then released from the cell. Extracellular high molecular weight tau causes a transmissible cytotoxicity that significantly hinders cellular repair following infection. Thus, ExoY may contribute to bacterial virulence in at least two ways; first, by microtubule breakdown leading to loss of endothelial cell barrier integrity, and second, by promoting release of a high molecular weight tau cytotoxin that impairs cellular recovery following infection.

DAP12 couples c-Fms activation to the osteoclast cytoskeleton by recruitment of Syk.

We examined the mechanism by which M-CSF regulates the cytoskeleton and function of the osteoclast, the exclusive bone resorptive cell. We show that binding of M-CSF to its receptor c-Fms generates a signaling complex comprising phosphorylated DAP12, an adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) and the nonreceptor tyrosine kinase Syk. c-Fms tyrosine 559, the exclusive binding site of c-Src, is necessary for regulation of DAP12/Syk signaling. Deletion of either of these molecules yields osteoclasts that fail to reorganize their cytoskeleton. Retroviral transduction of null precursors with wild-type or mutant DAP12 or Syk reveals that the SH2 domain of Syk and the ITAM tyrosine residues and transmembrane domain of DAP12 mediate M-CSF signaling. Our data provide genetic and biochemical evidence that uncovers an epistatic signaling pathway linking the receptor tyrosine kinase c-Fms to the immune adaptor DAP12 and the cytoskeleton.

Cycling around cell-cell adhesion with Rho GTPase regulators.

The formation and stability of epithelial adhesive systems, such as adherens junctions, desmosomes and tight junctions, rely on a number of cellular processes that ensure a dynamic interaction with the cortical cytoskeleton, and appropriate delivery and turnover of receptors at the surface. Unique signalling pathways must be coordinated to allow the coexistence of distinct adhesive systems at discrete sub-domains along junctions and the specific properties they confer to epithelial cells. Rho, Rac and Cdc42 are members of the Rho small GTPase family, and are well-known regulators of cell-cell adhesion. The spatio-temporal control of small GTPase activation drives specific intracellular processes to enable the hierarchical assembly, morphology and maturation of cell-cell contacts. Here, we discuss the small GTPase regulators that control the precise amplitude and duration of the levels of active Rho at cell-cell contacts, and the mechanisms that tailor the output of Rho signalling to a particular cellular event. Interestingly, the functional interaction is reciprocal; Rho regulators drive the maturation of cell-cell contacts, whereas junctions can also modulate the localisation and activity of Rho regulators to operate in diverse processes in the epithelial differentiation programme.

Integrin-linked kinase regulates endothelial cell nitric oxide synthase expression in hepatic sinusoidal endothelial cells.

BACKGROUND & AIMS: Portal hypertension results from endothelial dysfunction after liver injury caused in part by abnormal production of endothelial cell derived nitric oxide synthase (eNOS). Here, we have postulated that endothelial mechanosensing pathways involving integrin-linked kinase (ILK) may play a critical role in portal hypertension, eNOS expression and function. In this study, we investigated the role of ILK and the small GTP-binding protein, Rho, in sinusoidal endothelial cell (SEC) eNOS regulation and function. METHODS: Primary liver SECs were isolated using standard techniques. Liver injury was induced by performing bile duct ligation (BDL). To examine the expression of Rho and ILK in vivo during wound healing, SECs were infected with constitutively active Rho (V14), a dominant negative Rho (N19) and constructs encoding ILK and a short hairpin-inhibiting ILK. RESULTS: Integrin-linked kinase expression was increased in SECs after liver injury; endothelin-1, vascular endothelial growth factor, and transforming growth factor beta-1 stimulated ILK expression in SECs. ILK expression in turn led to eNOS upregulation and to enhance eNOS phosphorylation and NO production. ILK knockdown or ILK (kinase) inhibition reduced eNOS mRNA expression, promoter activity, eNOS expression and ultimately NO production. In contrast, ILK overexpression had the opposite effect. Inhibition of ILK activity also disrupted the actin cytoskeleton in isolated SECs. Rho overexpression suppressed phosphorylation of the serine-threonine kinase, Akt and inhibited eNOS phosphorylation. Finally, inhibition of Rho function with the RGS domain of the p115-Rho-specific GEF (p115-RGS) significantly increased eNOS phosphorylation. CONCLUSIONS: Our data suggest a potential role for ILK, the cytoskeleton and ILK signalling partners including Rho in regulating intrahepatic SEC eNOS expression and function.

CTP synthase forms cytoophidia in the cytoplasm and nucleus.

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase.

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.

From skeleton to cytoskeleton: osteocalcin transforms vascular fibroblasts to myofibroblasts via angiotensin II and Toll-like receptor 4.

RATIONALE: The expression of osteocalcin is augmented in human atherosclerotic lesions. How osteocalcin triggers vascular pathogenesis and remodeling is unclear. OBJECTIVE: To investigate whether osteocalcin promotes transformation of adventitial fibroblast to myofibroblasts and the molecular mechanism involved. METHODS AND RESULTS: Immunohistochemistry indicated that osteocalcin was expressed in the neointima of renal arteries from diabetic patients. Western blotting and wound-healing assay showed that osteocalcin induced fibroblast transformation and migration, which were attenuated by blockers of the renin-angiotensin system and protein kinase Cδ (PKCδ), toll-like receptor 4 (TLR4) neutralizing antibody, and antagonist and inhibitors of free radical production and cyclooxygenase-2. Small interfering RNA silencing of TLR4 and PKCδ abolished fibroblast transformation. Angiotensin II level in the conditioned medium from the osteocalcin-treated fibroblasts was found elevated using enzyme immunoassay. Culturing of fibroblasts in conditioned medium collected from differentiated osteoblasts promoted fibroblast transformation. The expression of fibronectin, TLR4, and cyclooxygenase-2 is augmented in human mesenteric arteries after 5-day in vitro exposure to osteocalcin. CONCLUSIONS: Osteocalcin transforms adventitial fibroblasts to myofibroblasts through stimulating angiotensin II release and subsequent activation of PKCδ/TLR4/reactive oxygen species/cyclooxygenase-2 signaling cascade. This study reveals that the skeletal hormone osteocalcin cross-talks with vascular system and contributes to vascular remodeling.

Prostaglandin D2-DP signaling promotes endothelial barrier function via the cAMP/PKA/Tiam1/Rac1 pathway.

OBJECTIVE: Prostaglandin D(2) (PGD(2)) is one of the prostanoids produced during inflammation. Although PGD(2) is known to decrease endothelial permeability through D prostanoid (DP) receptor stimulation, the detailed mechanism is unknown. METHODS AND RESULTS: Treatment with PGD(2) (0.1-3 µmol/L) or the DP receptor agonist, BW245C (0.1-3 µmol/L), dose-dependently increased transendothelial electrical resistance and decreased the FITC-dextran permeability of human umbilical vein endothelial cells. Both indicated decreased endothelial permeability. These phenomena were accompanied by Tiam1/Rac1-dependent cytoskeletal rearrangement. BW245C (0.3 µmol/L) increased the intracellular cAMP level and subsequent protein kinase A (PKA) activity. Pretreatment with PKA inhibitory peptide, but not gene depletion of exchange protein directly activated by cAMP 1 (Epac1), attenuated BW245C-induced Rac1 activation and transendothelial electric resistance increase. In vivo, application of 2.5% croton oil or histamine (100 µg) caused vascular leakage indexed by dye extravasation. Pretreatment with BW245C (1 mg/kg) attenuated the dye extravasation. Gene deficiency of DP abolished, or inhibition of PKA significantly reduced, the DP-mediated barrier enhancement. CONCLUSIONS: PGD(2)-DP signaling reduces vascular permeability both in vivo and in vitro. This phenomenon is mediated by cAMP/PKA/Tiam1-dependent Epac1-independent Rac1 activation and subsequent enhancement of adherens junction in endothelial cell.