Protease-activated Receptor-1 Signaling Research Articles

Activated protein C prevents glutamate- and thrombin-induced activation of nuclear factor-κB in cultured hippocampal neurons

Brain injury is associated with neuroinflammation, neurodegeneration, and also blood coagulation with thrombin formation and generation of activated protein C (APC). We have previously shown that APC, a serine protease of hemostasis, at very low concentrations has protective effects in rat hippocampal and cortical neurons at glutamate-induced excitotoxicity through protease-activated receptor-1 (PAR-1) or endothelial receptor of protein C (EPCR)/PAR-1. The transcription factor nuclear factor κB (NF-κB) takes part in regulating neuronal survival in several pathological conditions. To elucidate the impact of NF-κB in APC-mediated cell survival, we investigated nuclear translocation of NF-κB p65 at glutamate- or thrombin-induced toxicity in hippocampal neurons. We used immunoassay and immunostaining with confocal microscopy with anti-NF-κBp65 antibody. We show that APC at concentrations as low as 1–2 nM inhibits translocation of NF-κB p65 into the nucleus of cultured rat hippocampal neurons, induced by 100 μM glutamate or 50 nM thrombin (but not 10 nM). The blocking effect of APC on NF-κB p65 translocation was observed at 1 and 4 h after treatment of neurons with glutamate, when the NF-κBp 65 level in the nucleus was significantly above the basal level. Then we investigated whether the binding of APC to EPCR/PAR-1 is required to control NF-κB activation. Antibodies blocking PAR-1 (ATAP2) or EPCR (P-20) abolished the APC-induced decrease of nuclear level of NF-κB p65 at glutamate-induced toxicity, whereas control antibodies to PAR-1 (S-19) and EPCR (IgG) exerted no effect. Thus, we suggest that the activation of NF-κB in rat hippocampal neurons mediates the glutamate- and thrombin-activated cell death program, which is reduced by exposure of cells to APC. APC induces the reduction of the nuclear level of NF-κB p65 in hippocampal neurons at glutamate-induced excitotoxicity via binding to EPCR and subsequent PAR-1 activation and signaling.

Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells

We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Coagulation factor Xa cleaves protease-activated receptor-1 and mediates signaling dependent on binding to the endothelial protein C receptor.

Coagulation is intrinsically tied to inflammation, and both proinflammatory and anti-inflammatory responses are modulated by coagulation protease signaling through protease-activated receptor-1 (PAR1). Activated factor X (FXa) can elicit cellular signaling through PAR1, but little is known about the role of cofactors in this pathway. Endothelial protein C receptor (EPCR) supports PAR1 signaling by the protein C pathway, and in the present study we tested whether EPCR mediates surface recruitment and signaling of FXa. Here, we show that FXa binds to overexpressed as well as native endothelial EPCR. PAR1 cleavage by FXa as analyzed with conformation-sensitive antibodies and a tagged PAR1 reporter construct was strongly enhanced if EPCR was available. Anti-EPCR failed to affect the tissue factor-dependent activation of FX, but high concentrations of FXa decreased EPCR-dependent protein C activation. Most importantly, the FXa-mediated induction of Erk1/2 activation, expression of the transcript for connective tissue growth factor and barrier protection in endothelial cells required binding to EPCR. Our results demonstrate that EPCR plays an unexpected role in supporting cell surface recruitment, PAR1 activation, and signaling by FXa.

A matrix metalloproteinase-1/protease activated receptor-1 signaling axis promotes melanoma invasion and metastasis

Hallmarks of malignant melanoma are its propensity to metastasize and its resistance to treatment, giving patients with advanced disease a poor prognosis. The transition of melanoma from non-invasive radial growth phase (RGP) to invasive and metastatically competent vertical growth phase (VGP) is a major step in tumor progression, yet the mechanisms governing this transformation are unknown. Matrix Metalloproteinase-1 (MMP-1) is highly expressed by VGP melanomas, and is thought to contribute to melanoma progression by degrading type I collagen within the skin to facilitate melanoma invasion. Protease activated receptor-1 (PAR-1) is activated by MMP-1, and is also expressed by VGP melanomas. However, the effects MMP-1 signaling through PAR-1 have not been examined in melanoma. Here, we demonstrate that an MMP-1/PAR-1 signaling axis exists in VGP melanoma, and is necessary for melanoma invasion. Introduction of MMP-1 into RGP melanoma cells induced gene expression associated with tumor progression and promoted invasion in vitro, and enhanced tumor growth and conferred metastatic capability in vivo. This study demonstrates that both the type I collagenase and PAR-1 activating functions of MMP-1 are required for melanoma progression, and suggests that MMP-1 may be a major contributor to the transformation of melanoma from non-invasive to malignant disease.

Zyxin is involved in thrombin signalingviainteraction with PAR‐1 receptor

Protease-activated receptor 1 (PAR-1) mediates thrombin signaling in human endothelial cells. As a G-protein-coupled receptor, PAR-1 transmits thrombin signal through activation of the heterotrimeric G proteins, Gi, Gq, and G12/13. In this study, we demonstrated that zyxin, a LIM-domain-containing protein, is involved in thrombin-mediated actin cytoskeleton remodeling and serum response element (SRE)-dependent gene transcription. We determined that zyxin binds to the C-terminal domain of PAR-1, providing a possible mechanism of involvement of zyxin as a signal transducer in PAR-1 signaling. Data showing that disruption of PAR-1-zyxin interaction inhibited thrombin-induced stress fiber formation and SRE activation supports this hypothesis. Similarly, depletion of zyxin using siRNA inhibited thrombin-induced actin stress fiber formation and SRE-dependent gene transcription. In addition, depletion of zyxin resulted in delay of endothelial barrier restoration after thrombin treatment. Notably, down-regulation of zyxin did not affect thrombin-induced activation of RhoA or Gi, Gq, and G12/13 heterotrimeric G proteins, implicating a novel signaling pathway regulated by PAR-1 that is not mediated by G-proteins. The observation that zyxin targets VASP, a partner of zyxin in regulation of actin assembly and dynamics, to focal adhesions and along stress fibers on thrombin stimulation suggests that zyxin may participate in thrombin-induced cytoskeletal remodeling through recruitment of VASP. In summary, this study establishes a crucial role of zyxin in thrombin signaling in endothelial cells and provides evidence for a novel PAR-1 signaling pathway mediated by zyxin.

Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

BackgroundUsing human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes.Methodology/Principal FindingsUsing RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%–49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified.Conclusions/SignificanceTogether, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

Cathepsin G Recruits Osteoclast Precursors via Proteolytic Activation of Protease-Activated Receptor-1

Metastatic breast cancer shows extreme tropism for the bone microenvironment, leading to the establishment of osteolytic metastases. Perpetuation of tumor-induced osteolysis requires a continuous supply of osteoclast precursors migrating into the bone microenvironment that can subsequently differentiate into mature osteoclasts and resorb bone. Thus, identification and subsequent targeting of chemoattractants of osteoclast precursors that are up-regulated at the tumor-bone interface represents a potential avenue to interrupt osteolysis. We report that cathepsin G, a serine protease, plays a vital role in the bone microenvironment by modulating tumor-stromal interaction in a manner that favors tumor establishment and regulates chemotaxis of monocytes, a subset of which has the potential to differentiate into osteoclasts. Our data show that cathepsin G-induced chemotaxis of monocytes is mediated by proteolytic activation of protease-activated receptor-1 (PAR-1). Attenuation of PAR-1 activation abrogates cathepsin G-mediated induction of monocyte chemotaxis. We also show that in vivo inhibition of cathepsin G reduces the number of CD11b(+) osteoclast precursors and mature osteoclasts at the tumor-bone interface. Together, these data suggest that therapeutic targeting of both PAR-1 signaling in osteoclast precursors as well as cathepsin G at the tumor-bone interface has the potential to reduce osteolysis by inhibiting the recruitment, differentiation, and activation of osteoclast precursors.

The complex role of fibrin in acute lung injury

in the last decade there have been many advances in our understanding of the pathogenesis of acute lung injury (ALI), but there have been frustratingly few successful therapeutic interventions. In fact, the only treatment proven to reduce mortality is low tidal volume lung-protective mechanical

Differential Effects of Serine Proteases on the Migration of Normal and Tumor Cells: Implications for Tumor Microenvironment

The supporting role of proteases in tumor progression and invasion is well known; however, the use of proteases as therapeutic agents has also been demonstrated. In this article, the authors report on the differential effects of exogenous serine proteases on the motility of tumor and normal cells. The treatment of normal and tumor cells with a single dose of pancreatic serine proteases, trypsin (TR) and chymotrypsin (CH), leads to a concentration-dependent response by cells, first accelerating and then slowing mobility. Tumor cells are 10 to 20 times more sensitive to exogenous TR/CH, suggesting that a single dose of proteases may cause discordant movements of normal and tumor cells within the tumor environment. The inhibitory effects of TR on cell motility are contradicted by thrombin (TH), particularly in the regulation of normal cells' migration. The purpose of this investigation was to ascertain the role of protease-activated receptors (PARs) in terms of normal and tumor cell motility. Duplicate treatments with proteases resulted in diminished mobility of both normal and tumor cells. Repeated application of TR and TH in 1-hour treatment intervals initially desensitizes cell surface PARs. However, cell surface PARs reappear regardless of subsequent protease treatments in both normal and tumor cells. The resensitization process is retarded in tumor cells when compared with normal cells. This is evidenced by lower expression of PARs as well as by their relocalization at the tumor cell surfaces. Under these conditions, normal cells remain responsive to exogenous proteases in terms of cell motility. Exogenous proteases do not modulate motility of repeatedly stimulated tumor cells, and consequently, the migration of tumor cells appears disconnected from the PAR signaling pathways. The use of activating peptides in lieu of the cognate proteases for a given PAR system indicated that proteases may act through additional targets not regulated by PAR signaling. We hypothesize that the divergent migration patterns of normal and tumor cells due to exposure to proteases is in part mediated by PARs. Thus, treatment with exogenous proteases may cause rearrangement of the tumor and stromal cells within the tumor microenvironment. Such topographical effects may lead to the inhibition of tumor progression and metastasis development.

Inhibition of the ADP/P2Y12 Pathway Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice

Clopidogrel, a potent anti-thrombotic drug widely used for the secondary prevention of myocardial infarction and ischemic stroke, inhibits the ADP/P2Y12 pathway by inducing irreversible alteration of the platelet receptor P2Y12, ultimately uncoupling ADP from adenylyl cyclase inhibition. Clinical trials have demonstrated additional benefits of dual antiplatelet therapy, such as combined treatment with clopidogrel and aspirin, in the prevention of ischemic cardiovascular disease. We previously established that protease-activated receptor (PAR) signaling is necessary for platelet activation by thrombin and plays an important role in mouse models of hemostasis and thrombosis. Indeed a PAR antagonist is currently being used as an anti-thrombotic agent in clinical trials.In order to evaluate the possible interaction between the thrombin/PAR and ADP/P2Y12 pathways, we orally administered clopidogrel (30mg/kg) or vehicle to PAR-4 null, heterozygous and wild type littermates, and evaluated arterial thrombus formation and bleeding during surgical challenge. In a FeCl3-induced carotid thrombosis model, PAR-4 deficient mice treated with clopidogrel were completely protected from thrombosis compared to their wild type treated littermates. Interestingly, clopidogrel treated PAR-4 heterozygotes showed an intermediate level of protection compared to treated wild type and null mice that was similar to vehicle-treated PAR-4 deficient mice. Additionally, we scored bleeding during the surgical procedure while blinded to genotype and treatment. The bleeding score was increased in clopidogrel treated mice compared to vehicle treated mice and was exacerbated with increasing haplotype deficiency of Par-4.To better define the relationship between these two activation pathways, we generated mice lacking both PAR-4 and P2Y12. All genetic combinations were born at the expected Mendelian distribution. Females lacking both receptors carried offspring to term, but signs of bleeding were observed during parturition. Preliminary data from PAR-4/P2Y12 double null mice, in both the tail bleeding assay and in a FeCl3-induced carotid thrombosis model, is in agreement with the results obtained in clopidogrel treated PAR-4 deficient mice. This study provides useful information to evaluate the efficacy and safety of dual antiplatelet therapy inhibiting the thrombin/PAR and ADP/P2Y12 pathways and explores the relationship between these two critical pro-thrombotic pathways in an in vivo setting.

Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

The ATP-gated P2X1 Receptor Plays a Pivotal Role in Activation of Aspirin-treated Platelets by Thrombin and Epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2.

Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)-bound protein C is activated by thrombomodulin-bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease-activated receptor-1 (PAR-1). Binding of APC to EPCR both modifies and induces PAR-1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. To determine whether platelets possess receptors for protein C that can generate intracellular signals. Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca(2+). Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2' (ApoER2') and by receptor-associated protein (RAP), an inhibitor of the low-density lipoprotein receptor family. Under shear, surface-bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibalpha-dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2' or RAP. APC bound to purified recombinant ApoER2' or GPIbalpha. Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbalpha and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbalpha. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbalpha.

LIGR, a protease‐activated receptor‐2‐derived peptide, enhances skin pigmentation without inducing inflammatory processes

The protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR-2 with Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), a known PAR-2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu-Ile-Gly-Arg-NH(2) (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno-deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho-GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR-2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR-2 signaling pathways, including [Ca(2+)] mobilization, cAMP induction, the induction of cyclooxgenase-2 (COX-2) expression and the secretion of prostaglandin E2, interleukin-6 and -8. PAR-2 siRNA inhibited LIGR-induced phagocytosis, indicating that LIGR signals via PAR-2. Our data suggest that LIGR is a more specific regulator of PAR-2-induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned-like skin color, without enhancing inflammatory processes, and without the need of UV exposure.

Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Galpha(i/o) and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.

Tissue Factor and Protease-Activated Receptor Signaling in Cancer

The activation of the coagulation cascade in the tumor microenvironment is a key feature of advanced malignancies. On tumor cells, tissue factor (TF) plays a central role to initiate cross-talk through the release of procoagulant microparticles or through direct, protease-activated receptor (PAR)-mediated cell signaling that leads to the production of soluble cytokines and angiogenic growth factors. In addition, the hemostatic system in the host compartment sustains crucial circuits that promote metastasis and support tumor growth and angiogenesis. Experimental tumor and genetic models have defined specific pathways that are supported by tumor cell and host TF and have identified potential therapeutic modalities to specifically interrupt TF signaling in tumor biology without impairment of hemostatic functions.

The contribution of PARs to inflammation and immunity to fungi

During inflammation, host- and microbial-derived proteases trigger the activation of protease-activated receptors (PARs), a family of G-protein-coupled receptors. We report here that activation of Toll-like receptors (TLRs) by fungi unmasks an essential and divergent role for PAR1 and PAR2 in downstream signaling and inflammation. TLRs activated PARs and triggered distinct signal transduction pathways involved in inflammation and immunity to Candida albicans and Aspergillus fumigatus. Inflammation was promoted by PAR1 and PAR2 activation in response to Candida and by PAR2 inhibition in response to Aspergillus. This occurred by TLR regulation of PAR signaling, with TLR2 promoting PAR1 activity, and TLR4 suppressing PAR2 activity. Thus, tissue injury and pathogens induce signals that are integrated at the level of distinct TLR/PAR-dependent pathways, the exploitation or subversion of which contributes to divergence in microbial promotion of inflammatory response.

Dendritic cell PAR1–S1P3 signalling couples coagulation and inflammation

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Proteinase-activated Receptor-2 Induces Cyclooxygenase-2 Expression through β-Catenin and Cyclic AMP-response Element-binding Protein

Chronic inflammation of mucosae is associated with an increased cancer risk. Tumorigenesis in these tissues is associated with the activity of some proteinases, cyclooxygenase-2 (COX-2), and beta-catenin. Serine proteinases participate in both inflammation and tumorigenesis through the activation of proteinase-activated receptor-2 (PAR(2)), which up-regulates COX-2 by an unknown mechanism. We sought to determine whether beta-catenin participated in PAR(2)-induced COX-2 expression and through what cellular mechanism. In A549 epithelial cells, we showed that PAR(2) activation increased COX-2 expression through the beta-catenin/T cell factor transcription pathway. This effect was dependent upon ERK1/2 MAPK, which inhibited the beta-catenin-regulating protein, glycogen synthase kinase-3beta, and induced the activity of the cAMP-response element-binding protein (CREB). Knockdown of CREB by small interfering RNA revealed that PAR(2)-induced beta-catenin transcriptional activity and COX-2 expression were CREB-dependent. A co-immunoprecipitation assay revealed a physical interaction between CREB and beta-catenin. Thus, PAR(2) up-regulated COX-2 expression via an ERK1/2-mediated activation of the beta-catenin/Tcf-4 and CREB pathways. These findings reveal new cellular mechanisms by which serine proteinases may participate in tumor development and are particularly relevant to cancers associated with chronic mucosal inflammation, where serine proteinases are abundant and COX-2 overexpression is a common feature.

Thrombin and protease-activated receptors (PARs) in atherothrombosis

Thrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including endothelial cells, vascular smooth muscle cells (VSMC), monocytes, T lymphocytes and fibroblasts. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage. Thrombin signalling in platelets contributes to haemostasis and thrombosis. In normal arteries PARs are mainly expressed in endothelial cells, while their expression in VSMC is limited. Endothelial PARs participate in the regulation of vascular tone, vascular permeability and endothelial secretory activity while in VSMC they mediate contraction, migration, proliferation, hypertrophy and production of extracellular matrix. PARs contribute to the pro-inflammatory phenotype observed in endothelial dysfunction and their up-regulation in VSMC seems to be a key element in the pathogenesis of atherosclerosis and restenosis. In the last years a myriad of studies have emphasized the critical role of PAR signalling in thrombin mediated effects in haemostasis, inflammation, cancer and embryonic development. Lately, PARs have become a therapeutic target to inhibit platelet aggregation and thrombosis. Early data from a clinical trial (TRA-PCI) to evaluate safety and efficacy of a potent new oral thrombin receptor antagonist (TRA) have promisingly indicated that overall TRA treatment reduces adverse event rates without an increase in bleeding risk. In this paper we review cellular responses triggered by thrombin and their implication in vascular pathophysiology.