Protease-activated receptor-1 (PAR1) and PAR2 but not PAR4 mediate relaxations in lower esophageal sphincter

Protease-activated receptor-1 (PAR1), PAR2 and PAR4 activation can alter the gastrointestinal motility. To investigate effects mediated by PARs in the lower esophageal sphincter, we measured contraction or relaxation of transverse strips from the guinea-pig lower esophageal sphincter caused by PAR1 (TFLLR-NH2 and SFLLRN-NH2), PAR2 (SLIGKV-NH2 and SLIGRL-NH2) and PAR4 peptide agonists (GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2) as well as PAR protease activators (thrombin and trypsin). In resting lower esophageal sphincter strips, TFLLR-NH2 and SFLLRN-NH2 caused moderate concentration-dependent relaxation whereas thrombin did not cause any relaxation or contraction. Furthermore, in carbachol-contracted strips, TFLLR-NH2 and SFLLRN-NH2 caused marked whereas thrombin caused mild concentration-dependent relaxation. These indicate the existence of PAR1 mediating relaxation. Similarly, in resting lower esophageal sphincter strips, trypsin caused moderate concentration-dependent relaxation whereas SLIGRL-NH2 and SLIGKV-NH2 did not cause any relaxation or contraction. In addition, in carbachol-contracted strips, trypsin caused marked whereas SLIGRL-NH2 and SLIGKV-NH2 caused mild concentration-dependent relaxation. These indicate the existence of PAR2 mediating relaxation. The relaxant response of thrombin, TFLLR-NH2, trypsin and SLIGKV-NH2 was insensitive to atropine or tetrodotoxin, suggesting a direct effect. The relaxant response of trypsin was not affected by apamin, charybdotoxin, indomethacin and capsaicin but was attenuated by NG-nitro-L-arginine methyl ester, indicating involvement of NO. FSLLR-NH2, a PAR1 control peptide, and VKGILS-NH2, a PAR2 control peptide, as well as all three PAR4 peptide agonists, GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2, did not cause any relaxation or contraction. Taken together, these results demonstrate that PAR1 and PAR2 but not PAR4 mediate relaxations in the guinea-pig lower esophageal sphincter.     

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2? mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y?? receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.     

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR₂) at Arg³⁶↓Ser³⁷ reveal a tethered ligand that binds to the cleaved receptor. PAR₂ activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR₂ at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR₂-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR₂, causes inflammation and pain by activating PAR₂ and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR₂ at Ala⁶⁶↓Ser⁶⁷ and Ser⁶⁷↓Val⁶⁸_. Elastase stimulated PAR₂-dependent cAMP accumulation and ERK1/2 activation, but not Ca²⁺ mobilization, in KNRK cells. Elastase induced PAR₂ coupling to Gα_s but not Gα_q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR₂, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR₂-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR₂-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR₂- and TRPV4-mediated influx of extracellular Ca²⁺ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR₂- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR₂ causes Gα_s-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR₂ as a mediator of protease-driven inflammation and pain.     

Proteinase-activated receptors (PARs)--the PAR3 Neo-N-terminal peptide TFRGAP interacts with PAR1

Thrombin activates proteinase-activated receptor (PAR)1, PAR3 and PAR4 by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR1 or PAR4 independently of receptor cleavage. However, corresponding peptides for PAR3 have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR3 induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR1 and PAR3. This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR1 antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A-498 cells we speculate that TFRGAP does signal MAPK via interaction with PAR1. This was underlined by experiments on PAR1-/- mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR1 and PAR3, respectively. While TFRGAP was without effect on ERK activation in PAR3+ KOLF cells, it induced MAPK activation in KOLF cells transfected with PAR1. These studies provide evidence that analogues of the PAR3 tethered ligand can mediate cell signaling by interaction with PAR1-type thrombin receptors.     

DJ-1 may contribute to metastasis of non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients?? tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (P?=?0.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (P?=?0.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (P?=?0.039). DJ-1 might contribute to the metastasis of NSCLC.     

Protease-activated receptor-1 down-regulation: a mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of beta(2)-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.     

Predictive value of protease-activated receptor-2 (PAR2) in cervical cancer metastasis

Metastasis is the primary cause of an unfavourable prognosis in patients with malignant cancer. Over the last decade, the role of proteinases in the tumour microenvironment has attracted increasing attention. As a sensor of proteinases, proteinase-activated receptor 2 (PAR₂) plays crucial roles in the metastatic progression of cervical cancer. In the present study, the expression of PAR₂ in multiple types of cancer was analysed by Gene Expression Profiling Interactive Analysis (GEPIA). Kaplan-Meier plotter was used to calculate the correlation between survival and the levels of PAR₂, Grb-associated binding protein 2(Gab2) and miR-125b. Immunohistochemistry (IHC) was performed to examine PAR₂ expression in a tissue microarray (TMA) of CESCs. Empower Stats was used to assess the predictive value of PAR₂ in the metastatic potential of CESC. We found that PAR₂ up-regulation was observed in multiple types of cancer. Moreover, PAR₂ expression was positively correlated with the clinicopathologic characteristics of CESC. miR-125b and its target Gab2, which are strongly associated with PAR₂-induced cell migration, are well-characterized as predictors of the prognostic value of CESC. Most importantly, the Cancer Genome Atlas (TCGA) data set analysis showed that the area under the curve (AUC) of the PAR₂ model was significantly greater than that of the traditional model (0.833 vs 0.790, P < .05), demonstrating the predictive value of PAR₂ in CESC metastasis. Our results suggest that PAR₂ may serve as a prognostic factor for metastasis in CESC patients.     

A specific cell surface glycoconjugate controlling cell motility: evidence by functional monoclonal antibodies that inhibit cell motility and tumor cell metastasis

The biochemical basis of cell motility has been viewed as a complex process involving cell surface membrane proteins, integrin receptors, growth factors and their receptors, and cytoskeletal components [Rosen & Goldberg (1989) In Vitro 25, 1079]. The possible involvement of glycoconjugates at the cell surface in controlling cell motility has not been systematically investigated. We addressed this question using functional monoclonal antibodies (MAbs), which inhibit cell motility and the metastatic potential of tumor cells, as probes. Two such MAbs, derived from two independent processes of immunization and selection, were found to directed to a common specific carbohydrate structure, Fuc alpha 1----2Gal beta 1----R. MAb MIA-15-5 was established after immunization of mice with small cell lung carcinoma line PC7 and selected on the basis of inhibition of U937 and HEL cell migration. MAb MIA-22-20 was established after immunization with lung adenocarcinoma line MAC-10 and selected on the basis of inhibition of MAC-10 cell migration. These two MAbs were both IgM and were consistently reactive with the Fuc alpha 1----2Gal beta 1----R structure, regardless of the identity of the R group. Various other anti-H MAbs, specific to carrier isotype, did not affect cell motility. MAb MIA-15-5 reacted with 30-40% of high-metastatic variant BL6 of mouse melanoma B16 line but with only less than 5% of low-metastatic variant F1. Metastatic deposition to lung after injection of BL6 cells was inhibited if MAb MIA-15-5 was injected within 3 h but was not inhibited by injection of other anti-H antibodies under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR₂), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR₂ regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR₂ agonist stimulated a transient increase in [Ca²⁺]_i. TRPV4 expression led to a markedly sustained increase in [Ca²⁺]_i. Removal of extracellular Ca²⁺ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A₂ and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR₂ generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR₂-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR₂ was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR₂ signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR₂-stimulated neurogenic inflammation. Thus, PAR₂ activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR₂.     

Mechanisms for modulation of mouse gastrointestinal motility by proteinase-activated receptor (PAR)-1 and -2 in vitro

Proteinase-activated receptor (PAR)-1 or -2 modulates gastrointestinal transit in vivo. To clarify the underlying mechanisms, we characterized contraction/relaxation caused by TFLLR-NH2 and SLIGRL-NH2, PAR-1- and -2-activating peptides, respectively, in gastric and small intestinal (duodenal, jejunal and ileal) smooth muscle isolated from wild-type and PAR-2-knockout mice. Either SLIGRL-NH2 or TFLLR-NH2 caused both relaxation and contraction in the gastrointestinal preparations from wild-type animals. Apamin, a K+ channel inhibitor, tended to enhance the peptide-evoked contraction in some of the gastrointestinal preparations, whereas it inhibited relaxation responses to either peptide completely in the stomach, but only partially in the small intestine. Indomethacin reduced the contraction caused by SLIGRL-NH2 or TFLLR-NH2 in both gastric and ileal preparations, but unaffected apamin-insensitive relaxant effect of either peptide in ileal preparations. Repeated treatment with capsaicin suppressed the contractile effect of either peptide in the stomach, but not clearly in the ileum, whereas it enhanced the apamin-insensitive relaxant effect in ileal preparations. In any gastrointestinal preparations from PAR-2-knockout mice, SLIGRL-NH2 produced no responses. Thus, the inhibitory component in tension modulation by PAR-1 and -2 involves both apamin-sensitive and -insensitive mechanisms in the small intestine, but is predominantly attributable to the former mechanism in the stomach. The excitatory component in the PAR-1 and -2 modulation may be mediated, in part, by activation of capsaicin-sensitive sensory nerves and/or endogenous prostaglandin formation. Our study thus clarifies the multiple mechanisms for gastrointestinal motility modulation by PAR-1 and -2, and also provides ultimate evidence for involvement of PAR-2.     

Overexpression of DTL enhances cell motility and promotes tumor metastasis in cervical adenocarcinoma by inducing RAC1-JNK-FOXO1 axis

Cervical adenocarcinoma is an important disease that affects young women and it has a high mortality and poor prognosis. Denticleless E3 ubiquitin protein ligase homolog (DTL) gene with oncogenic function has been evaluated in several cancers. Through this study, we aimed to clarify the clinical and molecular characteristics of cervical adenocarcinoma involving overexpression of DTL and elucidate its molecular mechanism. Bioinformatics analysis was performed through multiple databases. RNA sequencing was used to obtain differentially expressed genes after DTL was overexpressed in cells. The role of DTL in cervical adenocarcinoma was explored through in vitro and in vivo experiments. We found that DTL has an unfavorable prognostic implication for patients with cervical adenocarcinoma. Overexpression of DTL induced the migration and invasion of tumor cells in vitro and promoted intra-pulmonary metastasis in vivo. In addition, DTL activated JNK through RAC1 and upregulated FOXO1 to induce epithelial–mesenchymal transition, and the migration and invasion of tumor cells. Therefore, we conclude that overexpression of DTL enhanced cell motility and promoted tumor metastasis of cervical adenocarcinoma by regulating the RAC1-JNK-FOXO1 axis. These results suggest that DTL may become a potential therapeutic target for antitumor metastasis of cervical adenocarcinoma.Subject terms: Cancer epigenetics, Cervical cancer     

Protease-activated receptor-1 (hPar1), a survival factor eliciting tumor progression

Although ample evidence point to the central involvement of protease activated receptor-1 (PAR1) in tumor progression, little is known about the fate of the tumor when hPar1 is being silenced. We observed that hPar1 antisense clones exhibit low PAR1 levels, attenuated cell proliferation and invasion in vitro, and tumor formation in vivo. These clones showed noticeably reduced paxillin phosphorylation compared with the parental A375SM cells, whereas no change in the integrin levels was noticed. Antisense clones injected into the mice resulted in very few and only occasional small tumors, whereas advanced and vascularized tumors were observed in A375SM cells. The antisense-derived tumor sections expressed active caspase-3, increased terminal deoxynucleotidyl transferase-mediated nick-end labeling staining, and a markedly reduced proliferating cell nuclear antigen level compared with A375SM cell-derived tissue sections. Likewise, ablation of the hPar1 gene in a tetracycline-inducible hPar1 system leads to apoptosis in immature blood vessels, whereas mature vessels were unaffected. The activation of PAR1-induced pAkt/protein kinase B abrogated serum-deprived Bim(EL) induction and also markedly inhibited Bax levels. On the other hand, small interfering RNA silencing of the hPar1 gene induced the expression of Bim(EL), a direct substrate of Akt/protein kinase B and also induced expression of active caspase-9 and caspase-3. These results altogether identify PAR1 as a survival factor that protects cells from undergoing apoptosis. We conclude that whereas PAR1 gene expression correlates with tumor progression, its neutralization effectively initiates an apoptotic pathway leading at least in part to significantly reduced tumor formation.     

A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR(2)F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe(240)) and 0.084 (Ser(240)) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR(2)F240S displayed a significant reduction in sensitivity toward trypsin ( approximately 3.7-fold) and the PAR2-activating peptides, SLIGKV-NH(2) ( approximately 2.5-fold) and SLIGRL-NH(2) ( approximately 2.8-fold), but an increased sensitivity toward the selective PAR2 agonist, trans-cinnamoyl-LIGRLO-NH(2) ( approximately 4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH(2) ( approximately 7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH(2) and a PAR4-derived peptide, trans-cinnamoyl-YPGKF-NH(2), were selective PAR(2)F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR(2)F240S, suggesting that Phe(240) is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.