Mahmoud Saifeddine

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR1 and PAR4, the endogenous enzymes responsible for activating PAR2 in settings other than the gastrointestinal system, where trypsin can activate PAR2, are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR1, PAR2, and PAR4; 2) PAR-dependent calcium signaling responses in cells expressing PAR1, PAR2, and PAR4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR4-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR1, PAR2, and PAR4 and to disarm/inhibit PAR1. Although hK5 and -6 were also able to activate PAR2, they failed to cause PAR4-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR2-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR1, PAR2, and PAR4.

Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue.

1 The biological activities of the proteinase‐activated receptor number 2 (PAR‐2)‐derived peptides, SLIGRL (PP6) SLIGRL‐NH2 (PP6‐NH2) and SLIGR‐NH2 (PP5‐NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR‐2‐derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR‐NH2 (TP5‐NH2). 2 From a neonatal rat intestinal cDNA library, and from intestinal and kidney‐derived cDNA, the coding region of the rat PAR‐2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR‐2 receptor; and this information, along with a reverse‐transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR‐2 and thrombin receptor mRNA in both tissues. 3 In the mouse and rat gastric preparations, the PAR‐2‐derived polypeptides, PP6, PP6‐HN2 and PP5‐NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml−1; 10 nM) and that were equivalent to contractions caused by TP5‐NH2. 4 The cumulative exposure of the rat LM tissue to PP6‐NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5‐NH2 and vice versa, so as to indicate a lack of cross‐desensitization between the receptors responsive to the PAR‐2 and thrombin receptor‐derived peptides. 5 In the rat gastric preparation, the potencies of the PAR‐2‐activating peptides were lower than the potency of TP5‐NH2 (potency order: TP5‐NH2 > > PP6‐NH2 ≥ PP6 > PP5‐NH2); PP6 was a partial agonist in this preparation. 6 The contractile actions of PP6 and PP6‐NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo‐oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7 In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR‐2‐derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, Nω‐nitro‐L‐arginine‐methyl ester (L‐NAME) but not by D‐NAME; in an endothelium‐free preparation, which possessed mRNA for both the PAR‐2 and thrombin receptors, the PAR‐2‐activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5‐NH2. The relaxation response to PP6‐NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8 In the rat aortic preparation, the potencies of PP6, PP6‐NH2 and PP5‐NH2 were greater than those of the thrombin receptor activating peptide, TP5‐NH2 (potency order: PP6‐NH2 ≥ PP6 > PP5‐NH2 > TP5‐NH2). 9 In the rat aortic preparation, the relaxant actions of the PAR‐2‐derived peptides were mimicked by trypsin, at concentrations (0.5‐1 u ml−1; 1–2 nM) lower than those that can activate the thrombin receptor. 10 The bioassay data obtained with the PAR‐2 peptides and with trypsin, along with the molecular cloning/RT‐PCR analysis, point to the presence of functional PAR‐2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR‐2‐activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.

Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs).

Abstract We tested the hypothesis that human tissue kallikreins (hKs) may regulate signal transduction by cleaving and activating proteinase-activated receptors (PARs). We found that hK5, 6 and 14 cleaved PAR N-terminal peptide sequences representing the cleavage/activation motifs of human PAR1 and PAR2 to yield receptor-activating peptides. hK5, 6 and 14 activated calcium signalling in rat PAR2-expressing (but not background) KNRK cells. Calcium signalling in HEK cells co-expressing human PAR1 and PAR2 was also triggered by hK14 (via PAR1 and PAR2) and hK6 (via PAR2). In isolated rat platelets that do not express PAR1, but signal via PAR4, hK14 also activated PAR-dependent calcium signalling responses and triggered aggregation. The aggregation response elicited by hK14 was in contrast to the lack of aggregation triggered by hK5 and 6. hK14 also caused vasorelaxation in a phenylephrine-preconstricted rat aorta ring assay and triggered oedema in an in vivo model of murine paw inflammation. We propose that, like thrombin and trypsin, the kallikreins must now be considered as important ‘hormonal’ regulators of tissue function, very likely acting in part via PARs.

Proteinase-mediated cell signalling: targeting proteinase-activated receptors (PARs) by kallikreins and more.

Abstract Serine proteinases, like trypsin, can play a hormone-like role by triggering signal transduction pathways in target cells. In many respects these hormone-like actions of proteinases can now be understood in terms of the pharmacodynamics of the G protein-coupled ‘receptor’ responsible for the cellular actions of thrombin (proteinase-activated receptor-1, or PAR1). PAR1, like the other three members of this receptor family (PAR2, PAR3 and PAR4), has a unique mechanism of activation involving the proteolytic unmasking of an N-terminally tethered sequence that can activate the receptor. The selective activation of each PAR by short synthetic peptides representing these sequences has demonstrated that PAR1, PAR2 and PAR4 play important roles in regulating physiological responses ranging from vasoregulation and cell growth to inflammation and nociception. We hypothesise that the tissue kallikreins may regulate signal transduction via the PARs. Although PARs can account for many of their biological actions, kallikreins may also cause effects by mechanisms not involving the PARs. For instance, trypsin activates the insulin receptor and thrombin can act via a mechanism involving its non-catalytic domains. Based on the data we summarise, we propose that the kallikreins, like thrombin and trypsin, must now be considered as important ‘hormonal’ regulators of tissue function.

Neutrophil elastase and proteinase-3 trigger G-protein biased signaling through proteinase activated receptor-1 (PAR1)

Background: Proteinase-activated receptor-1 (PAR1) is a proteolytically activated G protein-coupled receptor. Neutrophil-derived enzymes might regulate PAR1 signaling. Results: Neutrophil elastase and proteinase-3 cleave and activate PAR1 signaling that is distinct from thrombin-triggered responses. Neutrophil elastase and proteinase-3 signaling through PAR1 modulates endothelial cell signaling. Conclusion: Neutrophil enzymes are Gαi-biased agonists for PAR1. Significance: Biased PAR1-activating compounds may prove of value as therapeutic agents to treat cardiovascular and inflammatory diseases. Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gαi-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity.

Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells.

Proteinase‐activated receptors (PARs) can activate a number of signalling events, including T‐cell signal‐transduction pathways. Recent data suggest that the activation of PARs 1, 2 and 3 in Jurkat T‐leukaemic cells induces tyrosine phosphorylation of the haematopoietic signal transducer protein, VAV1. To activate the PARs, this study used the agonist peptides SFLLRNPNDK, SLIGKVDGTS and TFRGAPPNSF, which are based on the sequences of the tethered ligand sequences of human PARs 1, 2 and 3, respectively. Here, we show that peptides based on either the human or murine PAR3‐derived tethered ligand sequences (TFRGAP‐NH2 or SFNGGP‐NH2) do not activate PAR3, but rather activate PARs 1 and 2, either in Jurkat or in other PAR‐expressing cells. Furthermore, whilst thrombin activates only Jurkat PAR1, trypsin activates both PARs 1 and 2 and also disarms Jurkat PAR1 for thrombin activation. We conclude therefore that in Jurkat or related T cells, signalling via PARs that can affect VAV1 phosphorylation is mediated via PAR 1 or 2, or both, and that distinct serine proteinases may potentially differentially affect T‐cell function in the settings of inflammation.

Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides : evidence for receptor heterogeneity

The vascular actions of the proteinase‐activated receptor‐2‐activating peptides (PAR2APs), SLIGRL‐NH2 (SL‐NH2) and SLIGKV‐NH2 (KV‐NH2) as well as the reverse‐sequence peptide, LSIGRL‐NH2 (LS‐NH2) and an N‐acylated PAR2AP derivative, trans‐cinnamoyl‐LIGRLO‐NH2 (tcLI‐NH2), were studied in rat intact and endothelium‐denuded artery ring preparations, primarily from the pulmonary artery (RPA). In RPA rings with but not without a functional endothelium, SL‐NH2 (but not LS‐NH2) caused either an endothelium‐dependent relaxation (at concentrations: <10 μm) or (at higher concentrations: >10 μm), an endothelium‐dependent contraction. No contractile response was observed in endothelium‐denuded preparations, that otherwise contracted in response to the PAR1AP, TFLLR‐NH2. The endothelium‐dependent contractile response to SL‐NH2 was not blocked by the α‐adrenoceptor antagonist prazosin, the endothelin antagonist BQ123, the angiotensin II antagonist DuP753, by tetrodotoxin; nor by the enzyme inhibitors, Nω‐nitro‐l‐arginine‐methylester (NO‐synthase), indomethacin (cyclo‐oxygenase), SKF‐525A (epoxygenase) and MK886 (leukotriene synthesis inhibitor). In the relaxation assay, KV‐NH2 was 5 fold less potent than SL‐NH2, whereas in the contractile assay KV‐NH2 was about equipotent with SL‐NH2. However, the maximal contractile response to KV‐NH2 was lower than that of SL‐NH2. The PAR2AP analogue, tcLI‐NH2, was as active as SL‐NH2 in the relaxation assay but was inactive as a contractile agonist in the endothelium‐intact RPA. The relaxant responses caused by SL‐NH2 and trypsin, as well as the contractile response caused by SL‐NH2, did not desensitize in the course of repeated exposures of the tissue to agonist; whereas the contractile response to trypsin, only observed at concentrations greater than 30 u ml−1, was desensitized by previous exposure of the tissue to either thrombin or trypsin. In a contractile assay, where the tissue was desensitized to a concentration of trypsin that would otherwise cause a relaxant response, the preparation still contracted in response to SL‐NH2. However, the trypsin‐desensitized preparations were no longer contracted by thrombin. From the cross‐desensitization by thrombin of the contractile response to trypsin (and vice versa), we concluded that the contractile effect of trypsin was due to activation of the thrombin receptor and not PAR2. We concluded that the endothelium‐dependent contraction caused by high concentrations of SL‐NH2 is due to an as yet unidentified contracting factor; whereas the endothelium‐dependent relaxation response observed at low concentrations of SL‐NH2 (10 μm) is mediated by nitric oxide. The distinct structure activity profiles for the contractile response (potency of KV‐NH2SL‐NH2) compared with the relaxant response (potency of KV‐NH2<5SL‐NH2); the contractile responsiveness to SL‐NH2 of an endothelium‐intact RPA preparation, that did not contract in response to trypsin; and the lack of contractile activity of the PAR2AP analogue tcLI‐NH2, that was as active as SL‐NH2 in the relaxation assay all argue in favour of receptor heterogeneity in the vasculature for the PAR2APs. It remains to be determined if the distinct endothelial receptor responsible for the contractile action of SL‐NH2 can be proteolytically activated, like PAR1 and PAR2.

Kallikreins and proteinase-mediated signaling: proteinase-activated receptors (PARs) and the pathophysiology of inflammatory diseases and cancer.

Abstract Proteinases such as thrombin and trypsin can affect tissues by activating a novel family of G protein-coupled proteinase-activated receptors (PARs 1–4) by exposing a ‘tethered’ receptor-triggering ligand (TL). Work with synthetic TL-derived PAR peptide sequences (PAR-APs) that stimulate PARs 1, 2 and 4 has shown that PAR activation can play a role in many tissues, including the gastrointestinal tract, kidney, muscle, nerve, lung and the central and peripheral nervous systems, and can promote tumor growth and invasion. PARs may play roles in many settings, including cancer, arthritis, asthma, inflammatory bowel disease, neurodegeneration and cardiovascular disease, as well as in pathogen-induced inflammation. In addition to activating or disarming PARs, proteinases can also cause hormone-like effects via PAR-independent mechanisms, such as activation of the insulin receptor. In addition to proteinases of the coagulation cascade, recent data suggest that members of the family of kallikrein-related peptidases (KLKs) represent endogenous PAR regulators. In summary: (1) proteinases are like hormones, signaling in a paracrine and endocrine manner via PARs or other mechanisms; (2) KLKs must now be seen as potential hormone-like PAR regulators in vivo; and (3) PAR-regulating proteinases, their target PARs, and their associated signaling pathways appear to be novel therapeutic targets.

Regulation of vascular and gastric smooth muscle contractility by pervanadate

1 The contractile actions of vanadate (VO4) and pervanadate (PV, peroxide(s) of vanadate) were studied in rat gastric longitudinal muscle strips and in aortic rings. The roles of extracellular sodium and calcium were evaluated and the potential effects of nerve‐released agonists were considered. The possibility that these responses were due to the potentiation of tyrosine kinase activity, as a result of PV‐mediated tyrosine phosphatase inhibition was explored with the use of tyrosine kinase inhibitors (genistein, tyrphostin) and by Western blot analysis of phosphotyrosyl proteins in PV‐treated tissues. The ability of PV to mimic the action of the tyrosine kinase receptor‐associated agonist, epidermal growth factor‐urogastrone (EGF‐Uro), in the gastric preparation was also studied. 2 PV caused concentration‐dependent contractions in both gastric and aorta‐derived tissues, with a potency that was 1 to 2 orders of magnitude greater than that of VO4. 3 Although repeated exposure of gastric and aortic tissues to a fixed concentration of VO4 caused reproducible contractions in both tissues, repeated exposure of gastric tissue to PV caused an increased contractile response plateauing after 3 exposures. In contrast, a single exposure of aortic tissue to PV (20 μm) caused a prolonged desensitization of the tissue to the subsequent contractile actions of PV or other agonists. 4 The contractile responses to PV were unaffected in both preparations by tetrodotoxin, atropine, yohimbine and phenoxybenzamine; and in the aortic preparation, the responses to VO4 and PV were the same in the presence or absence of a functional endothelium. 5 PV‐induced contractions in both tissues were observed in the absence of extracellular sodium but required extracellular calcium and were attenuated by 1 μm nifedipine. 6 In the gastric preparation, the characteristics of the contractile actions of PV paralleled those of EGF‐Uro in terms of (1) inhibition by genistein, (2) inhibition by indomethacin and (3) a requirement for extracellular calcium. These response characteristics differed from those of other contractile agonists such as carbachol. 7 In both the gastric and aortic preparations genistein was able to inhibit PV‐induced contractions selectively without causing comparable inhibition of KC1‐induced contractions. Tyrphostin (AG 18) also selectively blocked PV‐induced contractions in the gastric, but not in the aortic preparation. 8 In both the gastric and aortic tissue, in step with an increased contractile response, PV caused increases in tissue phosphotyrosyl protein content, as detected by Western blot analysis using a monoclonal antiphosphotyrosine antibody; the increases in phosphotyrosyl protein content were reduced when tissues were treated with PV at the same time as a tyrosine kinase inhibitor. 9 PV, at sub‐contractile concentrations, potentiated the contractile action of angiotensin II in both the gastric and aorta tissue. 10 We conclude that the growth factor‐mimetic agent, PV, is a much more potent contractile agonist than VO4 in both vascular and gastric smooth muscle tissue. PV can cause enhanced tissue phos‐photyrosyl protein content most likely via the inhibition of tissue protein tyrosine phosphatases. The contractile actions of PV, which require extracelullar calcium and are independent of extracellular sodium, would appear not to be due either to Na+/Ca2+ exchange, promoted by Na+/K+‐ATPase inhibition or to the inhibition of Ca2+‐ATPase and might be best explained by the ability of PV, via tyrosine phosphatase inhibition, to potentiate a tyrosine kinase pathway linked to calcium entry and to the contractile process.

Endothelium‐dependent contractile actions of proteinase‐activated receptor‐2‐activating peptides in human umbilical vein: release of a contracting factor via a novel receptor

1 The contractile actions of the proteinase‐activated receptor‐2‐activating peptides (PAR2APs), SLIGRL‐NH2 (SL‐NH2), SLIGKV‐NH2 (KV‐NH2), trans‐cinnamoyl‐LIGRLO‐NH2 (tc‐NH2), and the PAR1‐AP, TFLLR‐NH2 (TF‐NH2) as well as trypsin and thrombin were studied in endothelium‐denuded and intact human umbilical vein (HUV) ring preparations. 2 In HUV rings with, but not without an intact endothelium, PAR2APs caused a concentration‐dependent contractile response, whereas LSIGRL‐NH2 trypsin and PAR1APs were inactive. The contractile response was not affected by the endothelin ETA receptor antagonist, BQ123, the cyclooxygenase inhibitor, indomethacin, the leukotriene synthesis inhibitor, MK886, or the epoxygenase/P450 inhibitor, SKF‐525A. Other pharmacological antagonists (prazosin, Losartan®) were similarly inactive. 3 The order of potencies of the PAR2APs to cause a contraction in the endothelium‐intact preparation was: SL‐NH2>>KV‐NH2tc‐NH2. 4 Using an endothelium‐free rat aorta ring as a reporter tissue, surrounded with endothelium‐intact HUV as a donor tissue in a ‘sandwich assay,’ we also monitored the ability of SL‐NH2, TF‐NH2, trypsin and thrombin to release either contractile (EDCF) or relaxant (EDRF) factors. 5 In the ‘sandwich assay’ done in the presence of L‐NAME (0.1 mm), the endothelium‐intact HUV tissue (but not endothelium‐denuded HUV) released a contractile factor (EDCF) in response to SL‐NH2 (50 μm) but not to trypsin or LSIGRL‐NH2. The SL‐NH2‐mediated release/action of the EDCF was not affected by BQ123, indomethacin, MK886 or SKF‐525A. 6 In the ‘sandwich assay’, trypsin (4–10 nm), SL‐NH2, KV‐NH2 and tc‐NH2 caused the release of a relaxant activity (EDRF) from the endothelium‐intact (but not the denuded) HUV preparation. The release of EDRF was blocked by 0.1 mm ωnitro‐L‐arginine‐methylester (L‐NAME). Neither thrombin (10 u ml−1, 100 nm) nor TF‐NH2 (50 μm) were active in this EDRF‐release assay. 7 The relative potencies of the PAR2 agonists for causing the release of EDRF in the HUV sandwich assay were: trypsin>>SL‐NH2>>tc‐NH2>KV‐NH2. This order of potencies differed from the one observed for the same agonists in the HUV contraction assay (above) and in an intracellular calcium signalling assay, conducted with cloned human PAR2 that was expressed in cultured rat kidney KNRK cells: trypsin>>SL‐NH2=tc‐NH2>KV‐NH2. 8 We conclude that PAR2APs (but not PAR1APs) via a receptor distinct from PAR2, can cause a contractile response in endothelium‐intact HUV tissue via the release of a diffusable EDCF, that is different from previously recognized smooth muscle agonists (e.g. prostanoid metabolites, endothelin, noradrenaline, angiotensin‐II, acetylcholine).