PAR-1 Activation Research Articles

Acyl-Protein Thioesterase 1 Functions in Palmitoylation/Depalmitoylation Cycles of G Proteins and Regulates Platelet Activation

Abstract 1062Protein palmitoylation is a reversible post-translational modification that regulates both lipid-protein and protein-protein interactions. During the palmitoylation cycle, palmitoylation occurs through a thioester linkage to a cysteine residue. Depalmitoylation occurs primarily through cleavage of this bond by acyl-protein thioesterase 1 (APT1). We have previously demonstrated the presence of APT1 in platelets and showed that APT1 translocates to membranes in an activation-dependent manner. However, the function of APT1 in platelet activation is not known. To determine whether APT1 functions in platelet signal transduction we evaluated the effect of palmostatins, novel small molecule inhibitors of APT1, on platelet function. Palmostatins B and M both inhibited platelet aggregation and α -granule secretion induced through protease-activated receptor (PAR) 1 with an IC50 of 15 μM. To assess which signaling pathways were affected by APT1 inhibition, we screened palmostatins for their ability to inhibit activation induced by several agonists. Palmostatins blocked platelet aggregation induced by a PAR1 agonist, a PAR4 agonist, TxA2, or epinephrine. In contrast, palmostatins failed to inhibit aggregation induced by collagen, PMA, or ionophore. Palmostatins also inhibited α -granule exocytosis induced by a PAR1 agonist or TxA2, but not exocytosis induced by PMA or ionophore. These results suggested that palmostatins blocked proximal signaling events mediated through G protein coupled receptors (GPCRs). To evaluate this supposition, we tested the effect of palmostatin B on PAR1-mediated [Ca2+]i flux. Palmostatin B inhibited PAR1-induced Ca2+ signaling with and IC50 of 15 μM, the same concentration required for inhibition of platelet aggregation and α -granule secretion. We have recently described the platelet palmitoylome (Dowal et al., Blood, 118:e62-73) and found several components of the proximal G protein signaling pathway that are palmitoylated, including many Gα subunits. To directly assess the effect of APT1 inhibition on palmitoylation/depalmitoylation cycles on a target Gα subunit, we evaluated Gα q palmitoylation using acyl biotin exchange chemistry. Total Gα q palmitoylation decreased substantially with activation of platelets through PAR1. In the presence of palmostatin B, however, Gα q palmitoylation increased following PAR1 activation. These results demonstrate that Gα q is a substrate for APT1. Our studies demonstrate a role for palmitoylation/depalmitoylation cycles in proximal signaling pathways downstream of GPCRs and implicate APT1 as an essential regulator of G protein signaling in platelets. Disclosures:No relevant conflicts of interest to declare.

A Large Cluster of Micrornas At 14q32 Defines an RNA Expression Module That Accounts for Racial Differences in Protease Activated Receptor 4-Mediated Platelet Reactivity

AbstractAbstract 380Myocardial infarction and stroke typically result from an occlusive platelet thrombus formed at the site of atherosclerotic plaques. There is reproducible variation in platelet reactivity among different individuals – a variation that likely contributes to thrombosis risk. The inter-individual variation in platelet reactivity is highly heritable, and this heritability is greater in African Americans (AAs) than European Americans (EAs), but there is limited understanding of the genetic mechanisms responsible for this variability. The Platelet RNA And eXpression-1 (PRAX1) study was designed to identify novel genes and miRNAs responsible for inter-individual variation in platelet reactivity. We assessed platelet aggregation in 70 AA and 84 EA healthy subjects and profiled miRNA and mRNA from highly purified blood platelets. The richness of these data provided unique opportunities to establish miRNA-mRNA-physiology relationships that led to a number of novel and unexpected results.Although 607 miRNAs were detected above background, some were virtually absent in some samples. Using highly stringent criteria based on a biphasic distribution of expression in the 154 samples, we defined 178 platelet miRNAs present in at least 65% of subjects. Statistical analysis identified miRNAs that were differentially expressed (DE) by age, gender and race. A miRNA-miRNA correlation matrix uncovered a cluster of 52 miRNAs located at 14q32 that were highly co-regulated and that were expressed at higher levels in platelets from EAs than AAs. Unexpectedly, platelet aggregation in response to PAR4-AP was significantly higher in AAs than in EAs, whereas no racial difference was observed for the platelet aggregation response to arachidonic acid, ADP or PAR1-AP. Having established that 14q32 miRNAs were DE by race and that PAR4-mediated platelet reactivity was different by race, we sought to determine whether there were mRNAs that linked these two discoveries. Using a simple linear regression with an FDR q<.25, we found 119 mRNAs that were positively correlated with PAR4 reactivity. Notably, the gene encoding PAR4 (F2RL3) was not DE by race. A substantially higher proportion of 14q32 miRNAs were represented among the miRNAs enriched for targets among PAR4-positively-correlated mRNAs than would be expected by chance (p = 0.0092, OR=3.71). In other words, the racially-differentially expressed miRNAs located on 14q32 target mRNAs that are positively associated with PAR4 reactivity. A conditional correlation analysis showed that the significant association between PAR4 reactivity and race vanished after accounting for RNA expression, indicating that these two traits are highly collinear. We were especially intrigued with the gene, PCTP, which encodes phosphatidylcholine (PC) transfer protein (TP), because of the well-established importance of fatty acids, phospholipids and PC in platelet reactivity and because PC-TP catalyzes the transfer of PC between membranes. PCTP was DE by race (q value=10−19) and by PAR4 reactivity (p=10−6), and PC-TP protein was higher in AAs than EAs. PCTP is predicted to be targeted by 14q32 miRNA, hsa-miR-376c, and we validated that PC-TP mRNA and protein were knocked down by hsa-miR-376c.In summary, we have discovered racial differences in PAR4-mediated platelet activation and uncovered a genetic regulatory module in which racial differences in expression of miRNAs located at 14q32 and their target mRNAs account for this racial difference in platelet function. High levels of 14q32 miRNAs in EAs specifically target mRNAs that positively regulate PAR4 activity, resulting in lower sensitivity to PAR4-mediated activation. The inverse is true of AAs. Compared to EAs, AAs have a 2-fold increase in the incidence of CHD and a lower long-term survival. In addition, it is unknown whether the racial difference in platelet reactivity impacts the benefits and risks of anti-platelet therapies. Because novel inhibitors of PAR1 and 4 are currently in clinical development, it is critical to know whether dosing of such agents should be adjusted by race to maximize benefit and avoid toxicity. Lastly, since PAR4 is expressed in tissues other than platelets (e.g., heart, brain, liver), if 14q32 miRNA targets are DE by race in non-platelet tissues, it is possible that the consequent altered gene expression could contribute to non-thrombotic diseases known to show racial differences. Disclosures:No relevant conflicts of interest to declare.

PAK and Akt Interactions Regulate PAR-Mediated Akt Translocation to Membrane in Platelets: A Novel PIP3-Independent Mechanism.

Abstract 2166Akt has been shown to be an important signaling molecule regulating platelet aggregation. It was proposed that Akt translocates to the membrane for its activation where it is phosphorylated on Thr308 by PDK1 and Ser473 by mTORC2. However, the specific signaling events that regulate the Akt phosphorylation and its mechanism of translocation to the membrane are not clearly defined. It has been shown that Akt phosphorylation by many agonists requires signaling events downstream of Gi activation, involving PI3-kinaseb. In addition, it was proposed that Akt translocates to the plasma membrane by binding to PIP3 (Phosphatidylinositol-3,4,5-trisphosphate) generated by PI3-kinase. Our previous studies have revealed that Gq and G12/13 pathways enhance Akt phosphorylation in platelets, suggesting a role for these pathways independently of PIP3. Hence we investigated the mechanisms of Akt translocation in platelets. Stimulation of platelets with the PAR4 activation peptide (AYPGKF) caused translocation of Akt to the membrane rapidly (as early as 20 seconds) whereas, the phosphorylation of Akt occurred at the later time points (Figure 1). Pan-phoshatidylinositide 3-kinase (PI3-Kinase) inhibitors, LY-294002 (Figure 2) and Wortmannin inhibited Ser473 phosphorylation but failed to affect translocation of Akt to the membrane indicating that Akt translocation can occur independent of PI3-kinase activity. However, translocation of Akt was dramatically inhibited in the presence of a Gq-selective inhibitor, YM254890, indicating that Akt translocation is regulated downstream of Gqsignaling pathways (Figure 3).The p21-activated kinases (PAKs) are a family of serine/threonine kinases and are key regulators of actin polymerization and cell migration. PAK functions as a scaffolding protein and is reported to interact with numerous proteins including Akt. Here, we evaluated whether PAK activated by the Gqpathways plays a significant role in the translocation of Akt to the membrane. The co-immunoprecipitation studies revealed that Akt associates with PAK upon stimulation with AYPGKF (Figure 4). This association was abolished when platelets were pre-treated with YM254890 (Figure 5).These results suggest that PAK1/2 activation downstream of Gq pathway mediates Akt translocation to the membrane when stimulated by PAR agonist in platelets and that this translocation is PIP3- independent. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Tissue Factor Signals Airway Epithelial Basal Cell Survival via Coagulation and Protease-Activated Receptor Isoforms 1 and 2

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

Residual platelet activation through protease-activated receptors (PAR)-1 and ‐4 in patients on P2Y12 inhibitors

BackgroundDual antiplatelet therapy with aspirin and thienopyridines has improved outcomes of patients after coronary stent implantation. However, current knowledge suggests that thrombin generation is not affected by inhibition of the P2Y12 receptor, and therefore, platelet activation may still occur. MethodsThe response to agonists specific for protease-activated receptors (PAR)-1 and ‐4 was tested by multiple electrode impedance aggregometry in 82 patients on stable doses of clopidogrel or prasugrel, and in 55 healthy controls. ResultsBased on the consensus cut-off value for adenosine diphosphate (ADP) responsiveness, only one out of 19 patients on prasugrel, but 22 out of 63 patients on clopidogrel had high on-treatment residual platelet reactivity in response to exogenous ADP (p=0.01). Among the patients with adequate ADP P2Y12 receptor inhibition (n=59), we still observed 32 patients (54.2%) with normal response to the PAR-1 activator SFLLRN (26 patients on clopidogrel, 81.2%; 6 patients on prasugrel, 18.8%), and 37 patients (63.8%) with a normal response to the PAR-4 activator AYPGKF (29 patients on clopidogrel, 78.4%; 8 patients on prasugrel, 21.6%). The degree of PAR-agonists inducible platelet activation was directly correlated with the activation induced by ADP (r>0.5 and p<0.001 for both agonists). Moreover, SFLLRN and AYPGKF inducible platelet reactivities were strongly correlated (r=0.75, p<0.001). ConclusionPAR responsiveness is preserved in the majority of patients with adequate clopidogrel-mediated inhibition of the platelet P2Y12 receptor, and still in about 20% of those with adequate inhibition by prasugrel.

Displacement of basolateral Bazooka/PAR-3 by regulated transport and dispersion during epithelial polarization inDrosophila

Polarity landmarks guide epithelial development. In the early Drosophila ectoderm, the scaffold protein Bazooka (Drosophila PAR-3) forms apicolateral landmarks to direct adherens junction assembly. However, it is unclear how Bazooka becomes polarized. We report two mechanisms acting in concert to displace Bazooka from the basolateral membrane. As cells form during cellularization, basally localized Bazooka undergoes basal-to-apical transport. Bazooka requires its three postsynaptic density 95, discs large, zonula occludens-1 (PDZ) domains to engage the transport mechanism, but with the PDZ domains deleted, basolateral displacement still occurs by gastrulation. Basolateral PAR-1 activity appears to act redundantly with the transport mechanism. Knockdown of PAR-1 sporadically destabilizes cellularization furrows, but basolateral displacement of Bazooka still occurs by gastrulation. In contrast, basolateral Bazooka displacement is blocked with disruption of both the transport mechanism and phosphorylation by PAR-1. Thus Bazooka is polarized through a combination of transport and PAR-1-induced dispersion from basolateral membranes. Our work complements recent findings in Caenorhabditis elegans and thus suggests the coupling of transport and dispersion is a common protein polarization strategy.

β-Arrestin-2 mediates the proinflammatory effects of proteinase-activated receptor-2 in the airway.

Proteinase-Activated receptor-2 (PAR(2)), a G-protein-coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR(2) have been proposed for treating asthma. PAR(2) can signal through two independent pathways: a β-arrestin-dependent one that promotes leukocyte migration, and a G-protein/Ca(2+) one that is required for prostaglandin E(2) (PGE(2)) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR(2) activation are mediated by β-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR(2)-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in β-arrestin-2(-/-) mice. In contrast, PAR(2)-mediated PGE(2) production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR(2) "protective" effects) were independent of β-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not β-arrestin-2(-/-) mice. Using the forced oscillation technique to measure airway resistance reveals that PAR(2) activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR(2)-enhanced inflammatory process is β-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR(2) may be β-arrestin independent.

Trypsin impaired epithelial barrier function and induced IL-8 secretion through basolateral PAR-2: a lesson from a stratified squamous epithelial model

Immune-mediated injury by the protease-activated receptor-2-interleukin-8 (PAR-2-IL8) pathway may underlie the development of gastroesophageal reflux disease (GERD). However, the localization of PAR-2 and the mechanism of PAR-2 activation remain unclear. This study aimed to address these questions on an esophageal stratified squamous epithelial model and in the human esophageal mucosa of GERD patients. Normal human esophageal epithelial cells were cultured with the air-liquid interface system to establish the model. SLIGKV-NH2 (PAR-2 synthetic agonist), trypsin (PAR-2 natural activator), and weak acid (pH 4, 5, and 6) were added to either the apical or basolateral compartment to evaluate their effects on transepithelial electrical resistance (TEER) and IL-8 production. PAR-2 localization was examined both in the cell model and biopsies from GERD patients by immunohistochemistry. Apical trypsin stimulation induced IL-8 accompanied by decreased TEER in vitro, whereas the effective concentration from the basolateral side was 10 times lower. SLIGKV-NH2 from basolateral but not apical stimulation induced IL-8 production. Apical weak acid stimulation did not influence TEER or IL-8 production. Immunohistochemistry showed intense reactivity of PAR-2 in the basal and suprabasal layers after stimulation with trypsin. A similar PAR-2 reactivity that was mainly located at the basal and suprabasal layers was detected in GERD patients. In conclusion, the activation of the PAR-2-IL-8 pathway probably occurred at the basal and suprabasal layers, while the esophageal epithelial barrier may influence the activation of PAR-2. Under proton pump inhibitor therapy, refluxed trypsin may remain active and be a potential agent in the pathogenesis of refractory GERD.

Protease-activated receptor 2 signaling upregulates angiogenic growth factors in renal cell carcinoma

Renal cell carcinoma (RCC) is a highly vascular tumor associated with expression of various angiogenic growth factors. The precise process of how these growth factors are regulated in RCC is not fully understood. Recent evidence suggests that protease activated receptors (PARs), a new family of G-protein coupled receptors, play a crucial role in vascular development and tumor progression through a variety of mechanisms. However, the nature of PAR expression in human RCC tissues and its function in regulating angiogenesis in RCC are largely unknown. In this study, we investigated the expression and function of PAR-2 in RCC. RT-PCR and immunohistochemistry assays show that PAR-2 expression is significantly increased in human RCC tissue compared with the adjacent non-neoplastic kidney tissue. In RCC derived cells, PAR-2 is functional as evidenced by robust signaling through MAP kinases including ERK1/2 and JNK. Furthermore, activation of PAR-2 significantly upregulates several angiogenic cytokines, including interleukin-6 (IL-6), IL-8, monocytes chemotactic protein-1 (MCP-1) and growth-related oncogene (GRO). To our knowledge, this is the first report that characterized PAR-2 expression in RCC tissue and further demonstrated that PAR-2 has a critical role in regulating angiogenesis in RCC.

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.

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-NH2 and SLIGKV-NH2, 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.

Granzyme B-Induced Neurotoxicity Is Mediated via Activation of PAR-1 Receptor and Kv1.3 Channel

Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target.

Susceptibility to intracerebral hemorrhage-induced brain injury segregates with low aerobic capacity in rats

Although low exercise capacity is a risk factor for stroke, the exact mechanisms that underlie this connection are not known. As a model system for exploring the association between aerobic capacity and disease risks we applied two-way artificial selection over numerous generations in rats to produce low capacity runners (LCR) and high capacity runners (HCR). Here we compared intracerebral hemorrhage (ICH)-induced brain injury in both genders of these rat lines. HCR and LCR rats had 100μl blood injected into the right caudate and were killed at days 1, 3, 7 and 28 for brain water content determination, immunohistochemistry, histology, Western blot, and behavioral tests. Compared to male HCRs, male LCRs had more severe ICH-induced brain injury including worse brain edema, necroptosis, brain atrophy, and neurological deficits, but not increased numbers of Fluoro-Jade C positive cells or elevated cleaved caspase-3 levels. This was associated with greater microglial activation, and heme oxygenase-1 and protease activated receptor (PAR)-1 upregulation. In females, edema was also greater in LCRs than in HCRs, although it was less severe in females than in males for both LCRs and HCRs. Thus, ICH-induced brain injury was more severe in LCRs, a model of low exercise capacity, than in HCRs. Increased activation of microglia and PAR-1 may participate mechanistically in increased ICH-susceptibility. Females were protected against ICH-induced brain edema formation in both HCRs and LCRs.

P152 Synergistic activation of proteinase-activated receptor 2 and toll-like receptor 4 induces production of thymic stromal lymphopoietin production by human keratinocytes

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic inflammation by instructing myeloid dendritic cells (mDCs) to induce inflammatory T helper 2 (Th2) cells. TSLP is highly expressed by keratinocytes in skin lesions from patients with atopic dermatitis (AD), and act as a master switch of AD pathogenesis. However factors inducing TSLP production from human keratinocytes are uncertain. Protease-activated receptors (PARs) and Toll-like receptors (TLRs) play a role in cytokine secretion from kertinocytes. Recent studies described synergistic PAR2- and TLR4-mediated cytokine production; but little information is available on TSLP production. In this study, we investigated the secretion of TSLP from human keratinocytes after combined stimulation with PAR2- and TLR4-agonists. Normal human epidermal keratinocytes (NHEK) were stimulated with PAR2-activating peptide (AP) and/or Kdo2-Lipid A in vitro . TSLP secretion from NHEK cells was measured by enzyme-linked immunosorbent assay (ELISA). The expression of TSLP mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Combined stimulation of NHEK cells with PAR2-AP and Kdo2-Lipid A resulted in a marked secretion of TSLP. The stimulation with Kdo2-Lipid A alone showed a moderate and lower increase in TSLP production compared to the synergistic stimulation. The stimulation with PAR2-AP alone failed to induce TSLP production by human keratinocytes. RT-PCR analysis confirmed these results in TSLP gene transcriptional levels. It appears that PAR2 and TLR4 synergistically up-regulate TSLP production by human keratinocytes. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. This implies that PAR2 activation and TLR4 triggering may amplify Th2 inflammation via the induction of TSLP in AD. The knowledge about the molecular basis of AD will allow the development of novel, rational-based therapeutic strategies.

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial-specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE.

PAR‐1‐dependent and PAR‐independent pro‐inflammatory signaling in human lung fibroblasts exposed to thrombin

Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (∼0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation.

PKCα Activation of p120-Catenin Serine 879 Phospho-Switch Disassembles VE-Cadherin Junctions and Disrupts Vascular Integrity

Adherens junctions (AJs) are the primary intercellular junctions in microvessels responsible for endothelial barrier function. Homophilic adhesion of vascular endothelial (VE) cadherin forms AJs, which are stabilized by binding of p120-catenin (p120). p120 dissociation from VE-cadherin results in loss of VE-cadherin homotypic interaction and AJ disassembly; however, the signaling mechanisms regulating p120 dissociation from VE-cadherin are not understood. To address the mechanism of protein kinase C (PKC)-α function in increasing endothelial permeability, we determined the role of PKCα phosphorylation of p120 in mediating disruption of AJ integrity. We showed that PKCα phosphorylation of p120 at serine (S)879 in response to thrombin or lipopolysaccharide challenge reduced p120 binding affinity for VE-cadherin and mediated AJ disassembly secondary to VE-cadherin internalization. In studies in mouse lung vessels, expression of the phosphodeficient S879A-p120 mutant prevented the increase in vascular permeability induced by activation of the thrombin receptor PAR-1. PKCα phosphorylation of p120 at S879 is a critical phospho-switch mediating disassociation of p120 from VE-cadherin that results in AJ disassembly. Therefore, blocking PKCα-mediated p120 phosphorylation represents a novel targeted anti-inflammatory strategy to prevent disruption of vascular endothelial barrier function.

Crosstalk between Inflammation and Coagulation: The Lessons of Sepsis

Sepsis results in the concurrent activation of inflammatory and procoagulant pathways. Bacterial products and proinflammatory cytokines trigger the coagulation system primarily via induction of tissue factor. During sepsis, activation of coagulation is accompanied by impaired function of major anticoagulant mechanisms, including antithrombin, the protein C system and fibrinolysis. Protease activated receptors (PARs) form the molecular connection between coagulation and inflammation, and especially PAR1 seems to play an eminent role in sepsis pathogenesis. Activated protein C (APC) can cleave PAR1 when associated with either the endothelial protein C receptor (EPCR) or CD11b/CD18, resulting in broad cytoprotective effects mediated by sphingosine 1 phosphate (S1P) receptor 1 (S1P1). In contrast, activation of PAR1 by high dose thrombin results in barrier disruptive effects in endothelial cells via an S1P3 dependent mechanism. Recombinant APC protects against mortality in experimental endotoxemia and sepsis by effects that can be mediated by either EPCR - PAR1 dependent (endothelial cells, dendritic cells) or CD11b/CD18 - PAR1 dependent (macrophages) mechanisms. These protective APC effects do not rely on the anticoagulant properties of this protein. APC mutants that lack anticoagulant properties but retain the capacity to activate PAR1 are promising new drugs for sepsis treatment.

Immunomodulatory role of proteinase-activated receptor-2

ObjectiveProteinase-activated receptor-2 (PAR2) has been implicated in inflammatory articular pathology. Using the collagen-induced arthritis model (CIA) the authors have explored the capacity of PAR2 to regulate adaptive immune pathways that...

Abstract 367: PAR3 Negatively Regulates PAR4-Mediated Gq Signaling Without Affecting the G12/13 Pathway in Mouse Platelets

Protease activated receptors (PAR) are G-protein-coupled receptors (GPCR) that are activated by proteolytic cleavage of the N-terminus. Mouse platelets use PAR3 and PAR4 to respond to thrombin. PAR3 itself does not signal, but it is a cofactor that enhances cleavage and activation of PAR4 by thrombin ∼ 10-fold. PAR4 is the signaling thrombin receptor and is sufficient to induce platelet activation at &gt;10 nM thrombin. The aim of this study is to determine how PAR3 regulates PAR4 signaling in mouse platelets. In this study we show that platelets from PAR3 knockout mice (PAR3-/-) had a 1.6-fold increase in the maximum calcium mobilization at thrombin concentrations above 10 nM, compared to wild type platelets. The PAR3+/- had an intermediate increase (∼ 1.2-fold) in the maximum calcium mobilization. PAR3 -/- platelets also had an increase in PKC activation, as measured by serine phosphorylation of PKC substrates in response to thrombin (10-100nM). The PAR4 agonist peptide (AYPGKF) also induced an increase in maximum calcium mobilization and PKC activation in PAR3 -/- platelets, indicating that PAR3 may be influencing PAR4 at the level of the receptor. PAR4 is coupled to Gq and G12/13. PAR3 influences PAR4-mediated calcium mobilization and PKC activation which are both downstream of Gq. We next, determined the influence of PAR3 on PAR4 mediated G12/13 signaling by measuring the activation of small GTPase RhoA protein in response to thrombin (1-100nM). The level of RhoA activation measured was not different in PAR3 -/- compared to wild-type platelets in response to thrombin. In conclusion, PAR3 negatively regulates PAR4 signaling through the Gq pathway without affecting the G12/13 pathway. Since the increase of Gq signaling is independent of PAR4 agonist, PAR3 is influencing PAR4 signaling at the level of the receptors, perhaps by forming heterodimers. The crosstalk between platelet GPCRs may be a general mechanism for regulating platelet signaling and uncovering the details of these interactions may lead to novel insights for developing therapeutics.