Protease-activated Receptor-2 Agonist Research Articles

Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

BackgroundMast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade.MethodsProliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV) was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs).ResultsTryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2), a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α.ConclusionsTaken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream activation of COX-2.

Acute PAR2 activation reduces GABAergic inhibition in the spinal dorsal horn

We investigated the mechanism underlying inhibition of spinal dorsal horn GABAergic neurotransmission to elucidate the role of protease-activated receptor-2 (PAR2). Initially, we confirmed that PAR2 agonist SL-NH2 applied intrathecally produced mechanical hyperalgesia. Then we performed patch-clamp experiments in substantia gelatinosa neurons of spinal cord slice, and found that spontaneous inhibitory post-synaptic currents (sIPSCs) were significantly decreased in both frequency and amplitude when neurons were incubated with PAR2 agonist SL-NH2 for a brief time period (2min). The GABA-mediated currents were significantly reduced, and there was no impact on glycine-mediated currents during this SL-NH2 treatment. These results suggest that PAR2 activation enhanced the pain response, potentially via inhibition of dorsal horn GABAergic neurotransmission.

Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice

BackgroundUnder conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of resistance arteries was attenuated by prolonged angiotensin II treatment in mice. We compared the vasodilatation of resistance-type arteries (mesenteric) from angiotensin II-treated PAR2 wild-type mice (WT) induced by PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) to the responses obtained in controls (saline treatment). We also investigated arterial vasodilatation in angiotensin II-treated PAR2 deficient (PAR2-/-) mice.Results2fly-induced relaxations of untreated arteries from angiotensin II-treated WT were not different than saline-treated WT. Treatment of arteries with nitric oxide synthase inhibitor and SK3.1 inhibitor (L-NAME + TRAM-34) blocked 2fly in angiotensin II-treated WT. Protein and mRNA expression of cyclooxygenase-1 and -2 were increased, and cyclooxygenase activity increased the sensitivity of arteries to 2fly in only angiotensin II-treated WT. These protective vasodilatation mechanisms were selective for 2fly compared with acetylcholine- and nitroprusside-induced relaxations which were attenuated by angiotensin II; PAR2-/- were protected against this attenuation of nitroprusside.ConclusionsPAR2-mediated vasodilatation of resistance type arteries is protected against the negative effects of angiotensin II-induced vascular dysfunction in mice. In conditions of endothelial dysfunction, angiotensin II induction of cyclooxygenases increases sensitivity to PAR2 agonist and the preserved vasodilatation mechanism involves activation of SK3.1.

Anatomical localization of protease‐activated receptor‐1 and protease‐mediated neuroglilal crosstalk on peri‐synaptic astrocytic endfeet

We studied the localization, activation and function of protease-activated receptor 1 (PAR-1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre-embedding diaminobenzidine (DAB) immunostaining found PAR-1 predominantly localized to the peri-synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti-PAR-1 antibody which co-stained mainly for glial-filament acidic protein compared with the neuronal markers synaptophysin and PSD-95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR-1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal-regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR-1 agonist (100 μM) and depolarization by high K(+) elevated extracellular thrombin-like activity in the synaptosomes preparation. The results indicate PAR-1 localized to the peri-synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron-glia pathway may be important in both physiological and pathological regulation of the synapse.

Glycyrrhetinic acid prevents cutaneous scratching behavior in mice elicited by substance P or PAR-2 agonist

Although glycyrrhetinic acid (GA) has been used for the prevention of itch in chronic dermatitis, the mechanism underlying the antipruritic effects of GA is still unclear. Recently, several mediators other than histamine, such as substance P and tryptase, were found to participate in chronic itch. Here, we investigated the effect of GA on pruritus induced by various pruritic agents including histamine in mice. We also determined the level of leukotriene (LT)B(4) in mouse skin injected with substance P in an effort to uncover part of the antipruritic mechanism of GA. Scratching events were counted for 10 min after intradermal injection of histamine, substance P (100 nmol per site each), protease-activated receptor-2 (PAR-2) agonistic peptide (50 nmol per site), or LTB(4) (0.03 nmol per site) with or without GA (4 nmol per site) into male ICR mice. Levels of LTB(4) in the skin after injection of substance P were determined by ELISA. GA did not suppress scratching behavior induced by histamine and LTB(4), but markedly and dose-dependently suppressed that induced by substance P and PAR-2 agonistic peptide. LTB(4) levels in skin elevated by substance P were lowered by GA. These data support the efficacy of GA in counteracting itch in chronic dermatitis because GA reduced scratching behavior induced by substance P and PAR-2 agonistic peptide. GA may exert antipruritic effects via inhibition of LTB(4) production in skin.

Trypsin Reduces Pancreatic Ductal Bicarbonate Secretion by Inhibiting CFTR Cl− Channels and Luminal Anion Exchangers

The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.

Preserved arterial vasodilatation via endothelial protease-activated receptor-2 in obese type 2 diabetic mice

BACKGROUND AND PURPOSEIn non-obese diabetic animals, protease-activated receptor-2 (PAR2) agonists are more effective vasodilators, which is attributed to increased COX-2 and endothelial NOS (eNOS) activities. Under conditions of diabetes and obesity, the effectiveness of PAR2 agonists is unknown. We compared the vasodilator responses of small calibre mesenteric arteries from obese diabetic B6.BKS(D)-Leprdb/J (db/db) induced by PAR2-activating agonists 2-furoyl-LIGRLO-amide (2fly) and trypsin to those obtained in controls [C57BL/6J (C57)], and assessed the contributions of COX, NOS and calcium-activated potassium channels (KCa) to these responses.EXPERIMENTAL APPROACHArteries mounted in wire myographs under isometric tension conditions were contracted submaximally by U46619 then exposed to vasodilators. mRNA and protein expression of PAR2, eNOS and soluble GC (sGC) were determined by real-time PCR and Western blots.KEY RESULTSACh- and nitroprusside-induced relaxations were attenuated in db/db compared with C57. In contrast, 2fly- and trypsin-induced relaxations were largely retained in db/db. A NOS inhibitor partly inhibited ACh- and 2fly-induced relaxations in C57, but not those in db/db. Inhibitors of the COX-cAMP pathway (FR122044, SC560, NS398, SC58125, SQ22536, CAY10441) did not affect these relaxation responses in either strain. Charybdotoxin (BKCa, SK3.1 blocker), but not iberiotoxin (BKCa blocker), inhibited responses to the PAR2 agonists in db/db. In db/db protein levels of eNOS were higher, whereas those of sGC were lower than in C57. PAR2 mRNA expression in db/db was higher than in C57.CONCLUSIONS AND IMPLICATIONSPAR2-mediated vasodilatation is protected against the negative effects of obesity and diabetes in mice. In diabetic vascular dysfunction, preserved PAR2 vasodilatation was linked to activation of SK3.1.

A matrix metalloprotease‐PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis

Sepsis is a deadly disease characterized by the inability to regulate the inflammatory–coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP-1 and active MMP-1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP-1 ortholog, MMP-1a. We identified mouse MMP-1a as an agonist of protease-activated receptor-1 (PAR1) on endothelial cells. MMP-1a was released from endothelial cells in septic mice. Blockade of MMP-1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1-deficient mice. Infusion of human MMP-1 increased lung vascular permeability in normal wild-type mice but not in PAR1-deficient mice. These findings implicate MMP-1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1-PAR1 may prove beneficial in the treatment of sepsis.

TGF- 1 induces proteinase-activated receptor 2 (PAR2) expression in endometriotic stromal cells and stimulates PAR2 activation-induced secretion of IL-6

Proteinase-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by several serine proteases. PAR2 activation in endometriotic stromal cells (ESCs) has been implicated in the development of endometriosis but the regulatory mechanism of PAR2 expression in ESC is unknown. Our objective was to study the mechanism by which PAR2 expression may be regulated in endometriotic lesions. Primary cultures of ESCs were treated with transforming growth factor-β (TGF-β) 1, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and the expression of PAR2 was examined by real-time quantitative PCR. ESCs pretreated with or without TGF-β1 were treated with PAR2 agonist peptide (PAR2AP) and the secretion of the pro-endometriotic cytokine, IL-6, was measured using a specific enzyme-linked immunosorbent assay. Effects of TGF-β type 1 inhibitor, SB431542, and PAR2 small interfering RNA (siRNA) on the TGF-β1 stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion were also evaluated. To study intracellular signaling, effects of inhibitors of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K) and of Smad4 siRNA on the TGF-β1-induced PAR2 gene expression were studied. Only TGF-β1, but neither TNF-α nor IL-1β, increased gene expression of PAR2. Activation of PAR2 with PAR2AP increased the secretion of IL-6 from ESCs. As expected, TGF-β1 pretreatment dose-dependently enhanced the PAR2AP-induced increase in IL-6 secretion from ESCs. Treatment of ESCs with the TGF-β type 1 inhibitor, SB431542, inhibited both TGF-β1-stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion. Transfection of ESCs with PAR2 siRNA produced a similar inhibition of IL-6 secretion. The TGF-β1-induced increase in PAR2 gene expression was repressed by inhibition of p38 MAPK, p42/44 MAPK or PI3K, but not by knockdown of Smad4 expression. In view of significant roles of PAR2 and IL-6 in endometriosis, the TGF-β1-induced increase in PAR2 expression may be an elaborate mechanism that augments the progression of the disease.

Vasodilator-stimulated phosphoprotein deficiency potentiates PAR-1-induced increase in endothelial permeability in mouse lungs

Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. The function of VASP in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP(-/-) mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP(-/-) mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may be correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function.

G12/13 Signaling Pathways Substitute for Integrin αIIbβ3-Signaling for Thromboxane Generation in Platelets

BackgroundWe have previously shown that ADP-induced TXA2 generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA2 generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G12/13 signaling pathways. Hence, we evaluated the role of these pathways in TXA2 generation.Principal FindingsInhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G12/13 pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G12/13 pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA2 generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA2 generation.ConclusionsTherefore, we conclude that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G12/13 pathways contributing to TXA2 generation in platelets remains elusive.

Involvement of Bradykinin in Trypsin-Induced Urinary Bladder Contraction in Cyclophosphamide-Treated Rats

Protease-activated receptor-2 (PAR-2) is activated by serine proteases, such as trypsin and mast cell tryptase. Previous studies have demonstrated that both trypsin and PAR-2 activating peptide contract isolated rat urinary bladder preparations, however, the mechanisms are not fully understood. In the present study, we examined the role of bradykinin in contractions induced by trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH(2) in urinary bladders isolated from control or cyclophosphamide (CYP)-induced cystitis rats. The contractile effects of trypsin were significantly greater in the preparations obtained from CYP-treated rats than in those from controls. The bradykinin B2 receptor antagonist Hoe 140 did not affect trypsin-induced contractions in control rat bladders, whereas it significantly reduced the contractile effects of trypsin on bladders from CYP-treated rats. On the other hand, Hoe 140 failed to affect contractions induced by the PAR-2 agonist 2-furoyl-LIGRL-NH(2). These results suggest that the actions of trypsin on urinary bladders in cystitis rats are partly exerted through stimulation of bradykinin B2 receptor in a PAR-2-independent manner. This mechanism seems to be involved in the enhancement of trypsin-induced bladder contractions observed after induction of cystitis with CYP in rats.

Genetic Enhancement of Behavioral Itch Responses in Mice Lacking Phosphoinositide 3-Kinase-γ (PI3Kγ)

Phosphoinositide 3-kinases (PI3Ks) are important for synaptic plasticity and various brain functions. The only class IB isoform of PI3K, PI3Kγ, has received the most attention due to its unique roles in synaptic plasticity and cognition. However, the potential role of PI3Kγ in sensory transmission, such as pain and itch has not been examined. In this study, we present the evidence for the first time, that genetic deletion of PI3Kγ enhanced scratching behaviours in histamine-dependent and protease-activated receptor 2 (PAR-2)-dependent itch. In contrast, PI3Kγ-deficient mice did not exhibit enhanced scratching in chloroquine-induced itch, suggesting that PI3Kγ selectively contributes to certain types of behavioal itch response. Furthermore, PI3Kγ-deficient mice exhibited normal acute nociceptive responses to thermal and mechanical noxious stimuli. Behavioral licking responses to intraplantar injections of formalin and mechanical allodynia in a chronic inflammatory pain model (CFA) were also not affected by PI3Kγ gene deletion. Our findings indicate that PI3Kγ selectively contributes to behavioral itching induced by histamine and PAR-2 agonist, but not chloroquine agonist.

Activation of PAR2 or/and TLR4 promotes SW620 cell proliferation and migration via phosphorylation of ERK1/2

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor and its activation has been associated with the pathogenetic progress in certain cancers. Toll-like receptor 4 (TLR4), one member of Toll-like receptors family, is mainly contributed to the innate immune response. However, recent studies have shown that TLR4 is aberrantly expressed in various types of carcinomas and may correlate with tumor progression. Previously, we reported that PAR2 could be expressed in human colon cancer cell line (SW620) and its activation by some stimulants was able to facilitate cell proliferation and migration. In our recent preliminary experiment, it was found that SW620 cells also had TLR4 expression. Thus, we considered that PAR2 and TLR4 could have some collaborative roles in SW620 cells. In the current study, the cross-inducible expression of PAR2 and TLR4 on SW620 cells was investigated, and the functional roles of their activation on the behavior of SW620 cells were evaluated. It was found that activation of PAR2 with PAR2-AP (PAR2 agonist, 100 μM) enhanced TLR4 releasement and vice versa. The activation of PAR2 or TLR4 (with LPS, 100 ng/ml) could promote SW620 cell proliferation and migration, and the phosphorylation of ERK1/2 but not p38MAPK, as well as the expression of interleukin 8 (IL-8) and tissue factor (TF). Whereas the caspase-7 expression was decreased under PAR2 or TLR4 activation. Furthermore, ERK1/2 inhibitor (U0126, at 10 μM) could intervene in all regulating effects of PAR2 or/and TLR4. Collectively, this study demonstrated that both PAR2 and TLR4 activation on SW620 cells can trigger the phosphorylation of ERK1/2, regulate the expression of IL-8, TF and caspase-7, thereby promote the proliferation and migration of cells.

Actin dynamics regulate immediate PAR-2-dependent responses to acute epidermal permeability barrier abrogation

Lamellar body (LB) secretion and terminal differentiation of stratum granulosum (SG) cells are signaled by both protease activated receptor-2 (PAR-2) and caveolin-1 (cav-1). To address the early dynamics of LB secretion, we examined cytoskeletal remodeling of keratinocytes in 3 mouse models following acute barrier abrogation: hairless mice, PAR-2 knockout (-/-) and cav-1 -/-. Under basal conditions, globular (G)-actin accumulates in SG cells cytosol, while filamentous (F)-actin is restricted to peri-membrane domains. Barrier abrogation induces the apical movement of F-actin and the retreat of the SG-G-actin front, paralleled by upstream cytoskeletal kinases activation. This phenomenon was both enhanced by PAR-2 agonist, and inhibited by cytochalasin-D and in PAR-2 knockout mice. We found that plasma membrane conformational changes causing LB secretion are controlled by PAR-2-dependent cytoskeletal rearrangements. We next addressed the interaction dynamics between cytoskeleton and plasma membrane following PAR-2-induced actin stress fiber formation in both cav-1 -/- and wildtype cells. Actin stress fiber formation is increased in cav-1 -/- cells prior to and following PAR-2 agonist peptide-treatment, while absence of cav-1 inhibits E-cadherin-mediated cell-to-cell adhesion. PAR-2 drives cytoskeletal/plasma membrane dynamics that regulate early LB secretion following barrier abrogation, stress fiber formation and keratinocyte adhesion.

Apical and basolateral pools of proteinase-activated receptor-2 direct distinct signaling events in the intestinal epithelium

Studies suggest that there are two distinct pools of proteinase-activated receptor-2 (PAR₂) present in intestinal epithelial cells: an apical pool accessible from the lumen, and a basolateral pool accessible from the interstitial space and blood. Although introduction of PAR₂ agonists such as 2-furoyl-LIGRL-O-NH₂ (2fAP) to the intestinal lumen can activate PAR₂, the presence of accessible apical PAR₂ has not been definitively shown. Furthermore, some studies have suggested that basolateral PAR₂ responses in the intestinal epithelium are mediated indirectly by neuropeptides released from enteric nerve fibers, rather than by intestinal PAR₂ itself. Here we identified accessible pools of both apical and basolateral PAR₂ in cultured Caco2-BBe monolayers and in mouse ileum. Activation of basolateral PAR₂ transiently increased short-circuit current by activating electrogenic Cl⁻ secretion, promoted dephosphorylation of the actin filament-severing protein, cofilin, and activated the transcription factor, AP-1, whereas apical PAR₂ did not. In contrast, both pools of PAR₂ activated extracellular signal-regulated kinase 1/2 (ERK1/2) via temporally and mechanistically distinct pathways. Apical PAR₂ promoted a rapid, biphasic PLCβ/Ca²(+)/PKC-dependent ERK1/2 activation, resulting in nuclear localization, whereas basolateral PAR₂ promoted delayed ERK1/2 activation which was predominantly restricted to the cytosol, involving both PLCβ/Ca²(+) and β-arrestin-dependent pathways. These results suggest that the outcome of PAR₂ activation is dependent on the specific receptor pool that is activated, allowing for fine-tuning of the physiological responses to different agonists.

Profiling Gene Expression Induced by Protease-Activated Receptor 2 (PAR2) Activation in Human Kidney Cells

Protease-Activated Receptor-2 (PAR2) has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD) and key events in tumor progression (angiogenesis, metastasis), but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293), a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2) and a PAR2 activating hexapeptide (2f-LIGRLO-NH2). Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes), the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2) and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15). Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4) known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

The proteinase/proteinase-activated receptor-2/transient receptor potential vanilloid-1 cascade impacts pancreatic pain in mice

Aims Proteinase-activated receptor-2 (PAR2) and transient receptor potential vanilloid-1 (TRPV1) are co-localized in the primary afferents, and the trans-activation of TRPV1 by PAR2 activation is involved in processing of somatic pain. Given evidence for contribution of PAR2 to pancreatic pain, the present study aimed at clarifying the involvement of TRPV1 in processing of pancreatic pain by the proteinase/PAR2 pathway in mice. Main methods Acute pancreatitis was created by repeated administration of cerulein in conscious mice, and the referred allodynia/hyperalgesia was assessed using von Frey filaments. Injection of PAR2 agonists into the pancreatic duct was achieved in anesthetized mice, and expression of Fos in the spinal cord was determined by immunohistochemistry. Key findings The established referred allodynia/hyperalgesia following cerulein treatment was abolished by post-treatment with nafamostat mesilate, a proteinase inhibitor, and with capsazepine, a TRPV1 antagonist, in mice. Injection of trypsin, an endogenous PAR2 agonist, or SLIGRL-NH 2, a PAR2-activating peptide, into the pancreatic duct caused expression of Fos protein in the spinal superficial layers at T8-T10 levels in the mice. The spinal Fos expression caused by trypsin and by SLIGRL-NH 2 was partially blocked by capsazepine, the former effect abolished by nafamostat mesilate. Significance Our data thus suggest that the proteinase/PAR2/TRPV1 cascade might impact pancreatic pain, in addition to somatic pain, and play a role in the maintenance of pancreatitis-related pain in mice.

Transient Receptor Potential Canonical 3 (TRPC3) Mediates Thrombin-Induced Astrocyte Activation and Upregulates Its Own Expression in Cortical Astrocytes

Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.

Novel Agonists and Antagonists for Human Protease Activated Receptor 2

Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH(2), SLIGRL-NH(2)), uncertain target selectivity, and poor bioavailability, limiting their usefulness for specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2 agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC(50) 0.28 μM) selectively induced PAR2-, but not PAR1-, mediated intracellular Ca(2+) release in HT29 human colorectal carcinoma cells. Antagonist GB83 (36, IC(50) 2 μM) is the first compound at micromolar concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g., trypsin, 2f-furoyl-LIGRLO-NH(2), 19). The new compounds are selective for PAR2 over PAR1, serum stable, and suitable for modulating PAR2 in disease models.