Tissue factor enhances protease-activated receptor-2-mediated factor VIIa cell proliferative properties

In addition to its hemostatic functions, factor (F)VIIa exhibits cell proliferative properties as seen in angiogenesis and tumor growth. A role for tissue factor (TF) and protease-activated receptors (PAR)-1 and -2 in cell proliferation remain to be clarified. We tested the hypothesis that FVIIa induces cell proliferation by a mechanism involving TF and PAR-2. Human recombinant FVIIa induced cell proliferation of human BOSC23 cells transfected with plasmid containing human TF DNA sequence. Because DNA primase 1 (PRIM1) plays an essential role in cell proliferation, we used the cloned PRIM1 promoter upstream of the reporter gene chloramphenicol acetyl transferase (CAT) to elucidate the mode of action of FVIIa. FVIIa evoked a dose-dependent increase in cell proliferation and PRIM1 induction, which were markedly potentiated (4-5-fold) by the presence of TF and abrogated by TF antisense oligonucleotide. PRIM1 induction by FVIIa was also abolished by PAR-2 but not by PAR-1 antisense. In contrast, thrombin induced a small increase in CAT activity which was unaffected by TF, but was prevented only by PAR-1 antisense as well as the thrombin inhibitor hirudin. Proliferative properties of FVIIa were associated with a TF-dependent increase in intracellular calcium and were mediated by a concordant phosphorylation of p44/42 MAP kinase. In conclusion, data reveal that FVIIa induces PRIM1 and ensuing cellular proliferation via a TF- and of the PARs entirely PAR-2-dependent pathway, in distinction to that of thrombin which is PAR-1-dependent and TF-independent. We speculate that FVIIa-TF-PAR-2 inhibitors may be effective in suppressing cell proliferation.     

The pronociceptive effect of proteinase-activated receptor-4 stimulation in rat knee joints is dependent on mast cell activation

Proteinase-activated receptor-4 (PAR₄) is a G-protein-coupled receptor activated by serine proteinases released during tissue repair and inflammation. We have previously shown that PAR₄ activation sensitises articular primary afferents leading to joint pain. This study examined whether mast cells contribute to this PAR₄-induced sensitisation and consequent heightened pain behaviour. The expression of PAR₄ on synovial mast cells was confirmed with immunofluorescent staining of rat knee joint sections. Electrophysiological recordings were made from joint primary afferents in male Wistar rats during both nonnoxious and noxious rotations of the knee. Afferent firing rate was recorded for 15 minutes after close intra-arterial injection of 10⁻⁹ to 10⁻⁵ mol of the PAR₄ activating peptide, AYPGKF-NH₂, or the inactive peptide, YAPGKF-NH₂ (100-μl bolus). Rats were either naive or pretreated with the mast cell stabilise, cromolyn (20 mg/kg). Mechanical withdrawal thresholds were determined using a dynamic planter aesthesiometer and weight bearing determined using an incapacitance tester. These behavioural measurements were taken before and after intra-articular AYPGKF-NH₂, or the inactive peptide, YAPGKF-NH₂ (100 μg). Local administration of AYPGKF-NH₂ caused a significant increase in joint primary afferent firing rate and pain behaviour compared with the control peptide YAPGKF-NH₂. These effects were blocked by pretreatment with cromolyn. These data reveal that PAR₄ is expressed on synovial mast cells and the activation of PAR₄ has a pronociceptive effect that is dependent on mast cell activation.     

Signaling mechanisms involved in protease-activated receptor-1-mediated interleukin-6 production by human gingival fibroblasts

Human gingival fibroblasts (HGFs) express protease-activated receptor-1 (PAR-1) at high levels. In cultured HGFs, we studied the signaling pathway of thrombin-induced interleukin-6 (IL-6) production. The PAR-1 agonist peptide SFLLRN mimicked the thrombin-induced IL-6 production in the presence of amastatin, an aminopeptidase inhibitor. Thrombin or a combination of SFLLRN and amastatin also strikingly induced the expression of IL-6 mRNA. Although continuous exposure of HGFs to thrombin rapidly desensitized Ca(2+) signaling, the cells did not lose their ability to produce IL-6 in response to thrombin. Similarly, although treatment of HGFs with BAPTA-AM [1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester], an intracellular Ca(2+) chelator, markedly attenuated the thrombin-induced increase in intracellular Ca(2+) concentration, the same treatment did not suppress the thrombin-induced IL-6 production. However, thrombin-induced IL-6 production was strongly inhibited by the p38 mitogen-activated protein (MAP) kinase and tyrosine kinase inhibitors, and Western blotting analyses showed that thrombin stimulates p38 MAP kinase phosphorylation. Specific inhibitors that inhibit extracellular signal-regulated kinase 1/2 kinase, phosphatidylinositol 3-kinase, and RhoA kinase also partially suppressed the thrombin-induced IL-6 production, but the effects were smaller than those of the p38 MAP and tyrosine kinase inhibitors. Thus, thrombin induces HGFs to produce IL-6 by activating PAR-1, and the tyrosine kinase- and p38 MAP kinase-dependent pathways, rather than the Ca(2+) signaling pathway, may play a crucial role in the IL-6 production.     

Modified proteinase-activated receptor-1 and -2 derived peptides inhibit proteinase-activated receptor-2 activation by trypsin.

Trypsin activates proteinase-activated receptor-2 (PAR(2)) by a mechanism that involves the release of a tethered receptor-activating sequence. We have identified two peptides, FSLLRY-NH(2) (FSY-NH(2)) and LSIGRL-NH(2) (LS-NH(2)) that block the ability of trypsin to activate PAR(2) either in PAR(2)-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR(2) peptide, LRGILS-NH(2) (LRG-NH(2)) did not do so and FSY-NH(2) failed to block thrombin activation of PAR(1) in the aorta ring or in PAR(1)-expressing human embryonic kidney cells. Half-maximal inhibition (IC(50)) by FSY-NH(2) and LS-NH(2) of the activation of PAR(2) by trypsin in a PAR(2) KNRK calcium-signaling assay was observed at about 50 and 200 microM, respectively. In contrast, the activation of PAR(2) by the PAR(2)-activating peptide, SLIGRL-NH(2) (SL-NH(2)) was not inhibited by FSY-NH(2), LS-NH(2), or LRG-NH(2). In a casein proteolysis assay, neither FSY-NH(2) nor LS-NH(2) inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH(2) and LS-NH(2) were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR(2). Similarly, FSY-NH(2) and LS-NH(2) failed to block the ability of trypsin to release the PAR(2) N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH(2) and LS-NH(2) block the ability of trypsin to activate PAR(2) by a mechanism that does not involve a simple inhibition of trypsin proteolytic activity, but possibly by interacting with a tethered ligand receptor-docking site.     

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.     

Inhibitors of prostaglandin transport and metabolism augment protease-activated receptor-2-mediated increases in prostaglandin E2 levels and smooth muscle relaxation in mouse isolated trachea

Stimulants of protease-activated receptor-2 (PAR(2)), such as Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), cause airway smooth muscle relaxation via the release of the bronchodilatory prostanoid prostaglandin E(2) (PGE(2)). The principal aim of the current study was to determine whether compounds that inhibit PGE(2) reuptake by the prostaglandin transporter [bromocresol green and U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha) and PGE(2) metabolism by 15-hydroxyprostaglandin dehydrogenase (thiazolidenedione compounds rosiglitazone and ciglitazone) significantly enhanced the capacity of SLIGRL to elevate PGE(2) levels and produce relaxation in isolated segments of upper and lower mouse trachea. SLIGRL produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, although both effects were significantly greater in lower tracheal segments than in upper tracheal segments. SLIGRL-induced increases in PGE(2) levels were significantly enhanced in the presence of ciglitazone and rosiglitazone, and these effects were not inhibited by GW9662 (2-chloro-5-nitrobenzanilide), a peroxisome proliferator-activated receptor-gamma antagonist. SLI-GRL-induced relaxation responses were also significantly enhanced by ciglitazone and rosiglitazone, whereas responses to isoprenaline, a PGE(2)-independent smooth muscle relaxant, were unaltered. Ciglitazone and rosiglitazone alone produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, and these responses were inhibited by indomethacin, a cyclooxygenase inhibitor. Bromocresol green, an inhibitor of prostaglandin transport, significantly enhanced SLIGRL-induced increases in PGE(2) levels and relaxation. Immunohistochemical staining for 15-hydroxyprostaglandin dehydrogenase was relatively intense over airway smooth muscle, as was staining for the prostaglandin transporter over both airway smooth muscle and epithelium. In summary, inhibitors of PGE(2) reuptake and metabolism significantly potentiate PAR(2)-mediated increases in PGE(2) levels and smooth muscle relaxation in murine-isolated airways.     

Ribavirin inhibits mast cell mediator release

Ribavirin (1-beta-D-ribofuranosyl-1,2-4-triazole-3-carboxamide) is a promising antiviral agent as well as a structural analog of guanosine. Although at different concentrations it has been reported to induce either immunosuppression or immune stimulation, its effects upon immediate hypersensitivity reactions are largely unknown. Because purine metabolism appears to be important in mast cell secretion, the effects of ribavirin on mouse bone marrow-derived mast cell functions were investigated. When ribavirin was added to mast cells at the time of stimulation with A23187 or specific antigen, no effect on the release of beta-hexosaminidase, a preformed mediator, was evident. However, mast cells cultured in 1 to 20 microM ribavirin for 1 to 7 days exhibited dose- and time-dependent inhibitions of stimulated beta-hexosaminidase and leukotriene C4 releases without altering mast cell mediator content. This inhibition occurred even when ribavirin had no effect on cell growth. A concomitant decrease in antigen-challenged mast cell intracellular Ca concentration was also observed after ribavirin treatment. Chronic ribavirin exposure in vitro inhibits mast cell secretory processes stimulated by both immunoglobulin E- and nonimmunoglobulin E-related signals. Its precise mechanism of action and any potential efficacy as an antiallergic agent remain to be elucidated.     

Influence of dexamethasone on protease-activated receptor 2-mediated responses in the airways

Stimulants of protease-activated receptor (PAR)(2) promote the generation of the bronchoprotective prostanoid prostaglandin (PG) E(2) by airway epithelial cells. In contrast, glucocorticoids reduce the levels of PGE(2) in airway epithelial cell cultures by concomitantly inhibiting pathways required for PGE(2) synthesis and facilitating pathways involved in PGE(2) inactivation. The aim of this study was to determine whether glucocorticoids inhibited PAR(2)-mediated, PGE(2)-dependent responses in epithelial cell cultures, in intact airway preparations, and in whole animals. In cultures of A549 cells, a PAR(2)-activating peptide SLI-GRL-NH(2) produced concentration and time-dependent increases in PGE(2) levels, which were significantly enhanced after exposure to lipopolysaccharide (LPS). However, SLIGRL-NH(2)-induced increases in PGE(2) levels were abolished by pretreatment of cells with the glucocorticoid, dexamethasone. In mouse isolated tracheal preparations, SLIGRL-NH(2) and PGE(2) induced concentration-dependent relaxation responses that were unaffected by dexamethasone, irrespective of whether dexamethasone exposure occurred in vitro or in vivo. Intranasal administration of LPS produced a pronounced increase in the numbers of neutrophils recovered from the bronchoalveolar lavage fluid of BALB/c mice. Numbers of recovered neutrophils were 40 to 60% lower in mice that received f-LIGRL-NH(2) (PAR(2)-activating peptide, 30 microg intranasally), PGE(2) (10 mugintranasally), or dexamethasone (1 mg/kg i.p.). In the combined presence of dexamethasone and f-LIGRL-NH(2) or dexamethasone and PGE(2), the number of neutrophils was suppressed further (80-83% lower). Thus, although dexamethasone abolished PAR(2)-mediated generation of PGE(2) in A549 cells, neither the smooth muscle relaxant nor the anti-inflammatory effects of PAR(2)-activating peptides (and PGE(2)) were diminished by in vitro or in vivo exposure to dexamethasone.     

CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells

While interactions between CD28 and members of the B7 family costimulate and enhance T cell responses, recent evidence indicates that the CD28 homologue CTLA-4 plays a downregulatory role. The mechanism by which this occurs is not clear, but it has been suggested that CTLA-4 terminates ongoing responses of activated T cells, perhaps by induction of apoptosis. Here we demonstrate that CTLA-4 engagement by antibody cross-linking or binding to B7 inhibits proliferation and accumulation of the primary T cell growth factor, IL-2, by cells stimulated with anti-CD3 and anti-CD28. This inhibition is not a result of enhanced cell death. Rather it appears to result from restriction of transition from the G1 to the S phase of the cell cycle. Our observation that upregulation of both the IL-2R alpha chain and the CD69 activation antigen are inhibited by CTLA-4 engagement supplies further evidence that CTLA-4 restricts the progression of T cells to an activated state. Together this data demonstrates that CTLA-4 can regulate T cell activation in the absence of induction of apoptotic cell death.     

PAR-2、EGFR和Ki67在胃癌组织中的表达及其临床意义

目的 探讨PAR-2、EGFR和Ki67在胃癌组织中的表达及其临床病理意义.方 法应用免疫组织化学的方法检测80例胃癌组织中PAR-2、EGFR和Ki67的表达,并分析PAR-2表达与EGFR、Ki67及临床病理特征的关系.结果 80例胃癌组织标本中42例（52.5％） PAR-2表达阳性,44例（55％）Ki67表达阳性,39例（48.8％）EGFR表达阳性;PAR-2和EGFR蛋白主要表达在肿瘤细胞的胞膜和胞质中,而Ki67主要表达于细胞核上;PAR-2的表达与肿瘤浸润深度、远隔转移和病理分期呈正相关,但与组织类型、分化程度、淋巴结转移不相关;PAR-2的表达与EGFR和Ki67的表达呈正相关.结论 PAR-2、EGFR和Ki67在胃癌组织中的高表达与肿瘤增殖和进展相关.     

Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception

Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH₂ into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1β concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH₂ evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH₂, injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1β production are independent of this channel.     

Increased expression of toll-like receptor-2 and -4 on leukocytes from patients with sepsis

The reduced responsiveness of monocytes or granulocytes toward endotoxin (endotoxin tolerance) during sepsis may depend on Toll-like receptors (TLR). The expression of TLR-2 and TLR-4 was measured on neutrophils (PMN) and monocytes from patients with sepsis (n = 21) or healthy controls (n = 12). Leukocytes (1 x 10/mL) were incubated at 37 degrees C with or without a TLR-4 (LPS 1 microg/mL) or a TLR-2 ligand (MALP-2 2 nM). Surface expression of TLR-2 and TLR-4 at 0, 4, and 16 h was determined in FACS after staining with specific antibodies. The release of IL-8 and TNF-alpha was measured by ELISA. Freshly isolated PMN from patients with sepsis exhibited significantly (P < 0.05) higher mean fluorescence for TLR-2 (78.0 +/- 18.6) and TLR-4 (11.4 +/- 2.3) than controls (12.8 +/- 2.2 and 2.3 +/- 0.4). Similarly, monocytes from patients exhibited higher TLR-2 and TLR-4 expression (300.8 +/- 40.6 and 92.7 +/- 12.1) than cells from controls (149.5 +/- 27.1 and 52.2 +/- 7.6). In patients with sepsis, expression of TLR-2 and TLR-4 on PMN increased during 16 h of incubation (106.2 +/- 22.1 and 34.5 +/- 5.3), whereas it remained unchanged in controls (19.3 +/- 6.1 and 5.4 +/- 1.9). Incubation with LPS or MALP-2 had no effect on TLR-4 or TLR-2 expression in cells from either controls or patients. Despite increased TLR expression in cells from patients with sepsis, the endotoxin-induced release of TNF-alpha and IL-8 was indistinguishable from that in controls. Therefore, the endotoxin tolerance seen in patients with sepsis does not depend solely on TLR-2 or TLR-4 expression, and other mechanisms must be involved.     

Thrombin and protease-activated receptor-1 agonists promote lipopolysaccharide-induced hepatocellular injury in perfused livers

Bacterial lipopolysaccharide (LPS) is a potent inflammatory agent capable of producing liver injury, the pathogenesis of which depends on numerous mediators, including thrombin. Previous studies showed that thrombin promotes LPS-induced liver injury independent of its ability to form fibrin clots. In isolated, buffer-perfused livers from LPS-treated rats, thrombin added to the perfusion buffer caused dose-dependent liver injury with an EC(50) value of 0.4 nM, consistent with activation by thrombin of a protease-activated receptor (PAR). Actions of thrombin at PARs can be mimicked by thrombin receptor-activating peptides (TRAPs). TRAPs for PAR-1 reproduced the injury caused by thrombin in isolated livers, suggesting that one mechanism by which thrombin promotes LPS-induced liver injury is by activating PAR-1. Immunocytochemistry demonstrated the presence of PAR-1 on sinusoidal endothelial cells and Kupffer cells but not on parenchymal cells or neutrophils. Previous studies showed that thrombin interacts with neutrophils in the genesis of liver injury after LPS treatment. To explore this interaction further, the influence of thrombin on mediators that modulate neutrophil function were evaluated. Inhibition of thrombin in LPS-treated rats prevented liver injury but did not prevent up-regulation of cytokine-induced neutrophil chemoattractant-1, macrophage inflammatory protein-2, or intercellular adhesion molecule-1. Thrombin inhibition did, however, prevent neutrophil (PMN) degranulation in vivo as measured by plasma elastase levels. In addition, elastase concentration was increased in the perfusion medium of livers isolated from LPS-treated rats and perfused with TRAPs. These results suggest that activation of PAR-1 after LPS exposure promotes PMN activation and hepatic parenchymal cell injury.     

Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4-dependent mechanism

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.     

An antibody targeted to VEGFR-2 Ig domains 4-7 inhibits VEGFR-2 activation and VEGFR-2-dependent angiogenesis without affecting ligand binding

Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (K(D) < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC(50) of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo.