Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.     

Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction

Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-alpha or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-alpha-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83(+) DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.     

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.     

beta-Amyloid peptide induces formation of actin stress fibers through p38 mitogen-activated protein kinase

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.     

Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin remodeling and barrier function in endothelial cells

In vivo and in vitro studies indicate that 4-hydroxy-2-nonenal (4-HNE), generated by cellular lipid peroxidation or after oxidative stress, affects endothelial permeability and vascular tone. However, the mechanism(s) of 4-HNE-induced endothelial barrier function is not well defined. Here we provide evidence for the first time on the involvement of mitogen-activated protein kinases (MAPKs) in 4-HNE-mediated actin stress fiber formation and barrier function in lung endothelial cells. Treatment of bovine lung microvascular endothelial cells with hydrogen peroxide (H(2)O(2)), as a model oxidant, resulted in accumulation of 4-HNE as evidenced by the formation of 4-HNE-Michael protein adducts. Exposure of cells to 4-HNE, in a dose- and time-dependent manner, decreased endothelial cell permeability measured as transendothelial electrical resistance. The 4-HNE-induced permeability changes were not because of cytotoxicity or endothelial cell apoptosis, which occurred after prolonged treatment and at higher concentrations of 4-HNE. 4-HNE-induced changes in transendothelial electrical resistance were calcium independent, as 4-HNE did not alter intracellular free calcium levels as compared with H(2)O(2) or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 microm) resulted in phosphorylation of ERK1/2, JNK, and p38 MAPKs, and actin cytoskeleton remodeling. Furthermore, pretreatment of bovine lung microvascular endothelial cells with PD 98059 (25 microm), an inhibitor of MEK1/2, or SP 600125 (25 microm), an inhibitor of JNK, or SB 202190 (25 microm), an inhibitor of p38 MAPK, partially attenuated 4-HNE-mediated barrier function and cytoskeletal remodeling. These results suggest that the activation of ERK, JNK, and p38 MAP kinases is involved in 4-HNE-mediated actin remodeling and endothelial barrier function.     

Protease-activated receptor-2 regulates vascular endothelial growth factor expression in MDA-MB-231 cells via MAPK pathways

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is cleaved and activated by serine proteases including the coagulation protease factor VIIa (FVIIa). There is evidence that PAR2 function contributes to angiogenesis, but the mechanisms involved are poorly defined. Here we show that PAR2 activation in human breast cancer cells leads to the upregulation of vascular endothelial growth factor (VEGF). Activation of PAR2 with agonist peptide (AP), trypsin or FVIIa results in a robust increase of VEGF message and protein. Incubation of cells with PAR1-AP, PAR3-AP, PAR4-AP, or thrombin has only a modest effect on VEGF production. Cleavage blocking antibodies show that FVIIa-mediated VEGF production is PAR2 mediated. Mitogen-activated protein kinase (MAPK) pathway inhibitors U0126 and SB203580 inhibit PAR2-mediated VEGF production. Incubation of cells with PAR2-AP leads to significant extracellular regulated kinase1/2 (ERK1/2) and p38 MAPK phosphorylation and activation. Collectively, these data suggest that PAR2 signaling through MAPK pathways leads to the production of proangiogenic VEGF in breast cancer cells.     

Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.     

Proteinase-activated receptors (PARs)--the PAR3 Neo-N-terminal peptide TFRGAP interacts with PAR1

Thrombin activates proteinase-activated receptor (PAR)1, PAR3 and PAR4 by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR1 or PAR4 independently of receptor cleavage. However, corresponding peptides for PAR3 have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR3 induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR1 and PAR3. This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR1 antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A-498 cells we speculate that TFRGAP does signal MAPK via interaction with PAR1. This was underlined by experiments on PAR1-/- mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR1 and PAR3, respectively. While TFRGAP was without effect on ERK activation in PAR3+ KOLF cells, it induced MAPK activation in KOLF cells transfected with PAR1. These studies provide evidence that analogues of the PAR3 tethered ligand can mediate cell signaling by interaction with PAR1-type thrombin receptors.     

Protease-activated receptor 2 induces migration and promotes Slug-mediated epithelial-mesenchymal transition in lung adenocarcinoma cells

Protease-activated receptor 2 (PAR2), a G protein-coupled receptor for trypsin, contributes to growth, anti-apoptosis, and migration in lung cancer. Given that PAR2 activation in airway epithelial cells compromises the airway epithelium barrier by disruption of E-cadherin adhesion, PAR2 may be involved in epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. Although PAR2 is known to promote the migration of lung cancer cells, the detailed mechanism of this event is still not clear. Here, we found that PAR2 is highly expressed in several lung adenocarcinoma cell lines. In two lung adenocarcinoma cell lines, CL1-5 and H1299 cells, activation of PAR2 induces migration and Slug-mediated EMT. The underlying mechanisms involved in PAR2-induced migration and EMT in CL1-5 cells were further investigated. We showed that PAR2-induced migration of CL1-5 cells is mediated by the Src/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. β-arrestin 1, not G protein, is involved in this PAR2-mediated Src/p38 MAPK signaling pathway. PAR2-induced EMT in CL1-5 cells is dependent on the activation of extracellular-signal-regulated kinase 2 (ERK2). The activation of ERK2 further mediates Slug stabilization through suppressing the activity of glycogen synthase kinase 3β. In addition, a poor prognosis was observed in lung adenocarcinoma patients with a high expression of PAR2. Thus, PAR2 regulates migration through β-arrestin 1-dependent activation of p38 MAPK and EMT through ERK2-mediated stabilization of Slug in lung adenocarcinoma cells. Our finding also suggests that PAR2 might serve as a therapeutic target for metastatic lung adenocarcinoma and a potential biomarker for predicting the prognosis of lung adenocarcinoma.     

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.     

Cortactin activation by FVIIa/tissue factor and PAR2 promotes endothelial cell migration

Cellular migration is a complex process that requires the polymerization of actin filaments to drive cellular extension. Smooth muscle and cancer cell migration has been shown to be affected by coagulation factors, notably the factor VII (FVIIa) and tissue factor (TF) complex. The present studies delineated mediators involved with the process of FVIIa/TF-induced cell migration and utilized a simple, precise, and reproducible, migration assay. Both FVIIa and protease-activated receptor-2 (PAR2)-activating peptide, SLIGRL, increased the migration rate of porcine cerebral microvascular endothelial cells (pCMVECs) overexpressing human TF. Ras homolog gene family member A (RhoA) and cortactin were upregulated during the process; expression of HIF, actin polymerization nuclear diaphanous-related formin-1 and -2 (Dia1, and Dia2) were unaffected. Gene silencing by shRNA to PAR2, RhoA, and cortactin attenuated this gene upregulation and migration induced by FVIIa/TF. Utilizing immunocellular localization, we demonstrate that during FVIIa/TF and PAR2 activation, cortactin molecules translocate from the cytoplasm to the cell periphery and assist in lamellipodia formation of pCMVECs. Overall, we demonstrate a novel regulation and role for cortactin in FVIIa/TF-mediated endothelial cell migration that occurs through a PAR2 and RhoA dependent mechanism.     

Protease-activated receptor (PAR)-induced interleukin-8 production in airway epithelial cells requires activation of MAP kinases p44/42 and JNK

The mechanism underlying protease-activated receptor (PAR)-activation and subsequent interleukin (IL)-8 production in airway epithelial cells is not yet understood. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 airway epithelial cells. We studied the consequence of activation of PARs with simultaneous exposure to LPS. Thrombin, PAR-2-activating peptide and LPS, were tested alone and in combination. They induced significant synthesis of IL-8. However, only activation of PAR triggered phosphorylation of ERK1/2 and JNK. The application of the inhibitors of these two MAPKs resulted in reduction of IL-8 production. Thus, activation of PARs but not stimulation with LPS leads to ERK1/2 and JNK-mediated production of IL-8.     

Direct ANP inhibition of hypoxia-induced inflammatory pathways in pulmonary microvascular and macrovascular endothelial monolayers

Atrial natriuretic peptide (ANP) has been shown to reduce hypoxia-induced pulmonary vascular leak in vivo, but no explanation of a mechanism has been offered other than its vasodilatory and natriuretic actions. Recently, data have shown that ANP can protect endothelial barrier functions in TNF-alpha-stimulated human umbilical vein endothelial cells. Therefore, we hypothesized that ANP actions would inhibit pulmonary vascular leak by inhibition of TNF-alpha secretion and F-actin formation. Bovine pulmonary microvascular (MVEC) and macrovascular endothelial cell (LEC) monolayers were stimulated with hypoxia, TNF-alpha, or bacterial endotoxin (LPS) in the presence or absence of ANP, and albumin flux, NF-kappa B activation, TNF-alpha secretion, p38 mitogen-activated protein kinase (MAPK), and F-actin (stress fiber) formation were assessed. In Transwell cultures, ANP reduced hypoxia-induced permeability in MVEC and TNF-alpha-induced permeability in MVEC and LEC. ANP inhibited hypoxia and LPS increased NF-kappa B activation and TNF-alpha synthesis in MVEC and LEC. Hypoxia decreased activation of p38 MAPK in MVEC but increased activation of p38 MAPK and stress fiber formation in LEC; TNF-alpha had the opposite effect. ANP inhibited an activation of p38 MAPK in MVEC or LEC. These data indicate that in endothelial cell monolayers, hypoxia activates a signal cascade analogous to that initiated by inflammatory agents, and ANP has a direct cytoprotective effect on the pulmonary endothelium other than its vasodilatory and natriuretic properties. Furthermore, our data show that MVEC and LEC respond differently to hypoxia, TNF-alpha-stimulation, and ANP treatment.     

Trypsin induces activation and inflammatory mediator release from human eosinophils through protease-activated receptor-2.

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.     

Role of CaMKII in hydrogen peroxide activation of ERK1/2, p38 MAPK, HSP27 and actin reorganization in endothelial cells

Growing evidence suggests that reactive oxygen species such as hydrogen peroxide (H(2)O(2)) can function as important signaling molecules in vascular cells. H(2)O(2)-activated redox-sensitive pathways mediate both physiological and pathological responses given the location and concentration of H(2)O(2). We showed previously for the first time that calcium/calmodulin-dependent protein kinase II (CaMKII) is redox-sensitive in endothelial cells, mediating H(2)O(2) upregulation of endothelial nitric oxide synthase. This response is always accompanied by an elongation phenotype of endothelial cells, implying modulation of actin cytoskeleton. In the present study, we investigated the role of CaMKII in H(2)O(2) activation of p38 MAPK/heat shock protein 27 (HSP27) pathway and ERK1/2, both of which have been known to regulate actin reorganization in endothelial cells. Addition of H(2)O(2) to bovine aortic endothelial cells increased ERK1/2 phosphorylation and activity, which was attenuated by a specific inhibitor of CaMKII, KN93. KN93 also prevented H(2)O(2) activation of p38 MAPK. Transfection of endothelial cells with a CaMKII-specific inhibitory peptide (AA 281-309) reduced H(2)O(2) phosphorylation of p38 MAPK and ERK1/2. Furthermore, blockade of CaMKII or janus kinase 2 (JAK2, downstream of CaMKII) prevented H(2)O(2) activation of HSP27. KN93 attenuated, whereas AG490 (JAK2 inhibitor) abolished, H(2)O(2)-induced formation of actin stress fibers. Blockade of ERK1/2 inhibited H(2)O(2) phosphorylation of HSP27 transiently. It also partially prevented H(2)O(2) induction of actin stress fibers. In summary, redox-sensitive activation of p38 MAPK/HSP27 pathway or ERK1/2 in endothelial cells requires CaMKII. These pathways are at least partially responsible for H(2)O(2) induced reorganization of actin cytoskeleton.     

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH(2) and SLIGKV-NH(2,) respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.     

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH(2) terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 mug/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.     

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).     

Protease-activated receptor-2 activation induces acute lung inflammation by neuropeptide-dependent mechanisms

Protease-activated receptors (PARs) and tachykinin-immunoreactive fibers are located in the lung as sentries to respond to a variety of pathological stimuli. The effects of PAR activation on the lung have not been adequately studied. We report on the effects of instilling PAR-activating peptides (PAR-APs, including PAR1-, PAR2-, and PAR4-AP) into the lungs of ventilated or spontaneously breathing mice. PAR2-AP, but not PAR1-AP or PAR4-AP, caused a sharp increase in lung endothelial and epithelial permeability to protein, extravascular lung water, and airway tone. No responses to PAR2-AP were detected in PAR2 knockout mice. In bronchoalveolar lavage, PAR2 activation caused 8- and 5-fold increase in MIP-2 and substance P levels, respectively, and a 12-fold increase in the number of neutrophils. Ablation of sensory neurons (by capsaicin) markedly decreased the PAR2-mediated airway constriction, and virtually abolished PAR2-mediated pulmonary inflammation and edema, as did blockade of NK1 or NK2 receptors. Thus, PAR2 activation in the lung induces airway constriction, lung inflammation, and protein-rich pulmonary edema. These effects were either partly or completely neuropeptide dependent, suggesting that PAR2 can cause lung inflammation by a neurogenic mechanism.