Cholecystitis is one of the most common gastrointestinal diseases. Inflammation induces the activation of proteases that can signal to cells by cleaving protease-activated receptors (PARs) to induce hemostasis, inflammation, pain, and repair. However, the distribution of PARs in the gallbladder is unknown, and their effects on gallbladder function have not been fully investigated. We localized immunoreactive PAR(1) and PAR(2) to the epithelium, muscle, and serosa of mouse gallbladder. mRNA transcripts corresponding to PAR(1) and PAR(2), but not PAR(4), were detected by RT-PCR and sequencing. Addition of thrombin and a PAR(1)-selective activating peptide (TFLLRN-NH(2)) to the serosal surface of mouse gallbladder mounted in an Ussing chamber stimulated an increase in short-circuit current in wild-type but not PAR(1) knockout mice. Similarly, serosally applied trypsin and PAR(2) activating peptide (SLIGRL-NH(2)) increased short-circuit current in wild-type but not PAR(2) knockout mice. Proteases and activating peptides strongly inhibited electrogenic responses to subsequent stimulation with the same agonist, indicating homologous desensitization. Removal of HCO(3)(-) ions from the serosal buffer reduced responses to thrombin and trypsin by >80%. Agonists of PAR(1) and PAR(2) increase intracellular Ca(2+) concentration in isolated and cultured gallbladder epithelial cells. The COX-2 inhibitor meloxicam and an inhibitor of CFTR prevented the stimulatory effect of PAR(1) but not PAR(2). Thus PAR(1) and PAR(2) are expressed in the epithelium of the mouse gallbladder, and serosally applied proteases cause a HCO(3)(-) secretion. The effects of PAR(1) but not PAR(2) depend on generation of prostaglandins and activation of CFTR. These mechanisms may markedly influence fluid and electrolyte secretion of the inflamed gallbladder when multiple proteases are generated.
"Experimentally, PARs can also be activated by synthetic peptides [referred to as an agonist peptide (AP)] that mimic the neo-amino terminus of the cleaved receptors. Specific PAR-APs are important tools for investigating the roles of PAR activation7. In a previous study, we observed that TF and PAR2 are highly expressed in the colon cancer cell line SW6208. "
[Show abstract][Hide abstract] ABSTRACT: Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent in green tea. The aim of this study is to investigate the effects of EGCG on proliferation and migration of the human colon cancer SW620 cells.
Proliferation and migration of SW620 cells were induced by the protease-activated receptor 2-agonist peptide (PAR2-AP, 100 μmol/L) or factor VIIa (10 nmol/L), and analyzed using MTT and Transwell assays, respectively. The cellular cytoskeleton was stained with rhodamine-conjugated phalloidin and examined with a laser scanning confocal fluorescence microscope. The expression of caspase-7, tissue factor (TF) and matrix metalloproteinase (MMP)-9 in the cells was examined using QT-PCR, ELISA and Western blot assays. The activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and nuclear factor-kappa B (NF-κB) signaling pathways was analyzed with Western blot.
Both PAR2-AP and factor VIIa promoted SW620 cell proliferation and migration, and caused cytoskeleton reorganization (increased filopodia and pseudopodia). Pretreatment with EGCG (25, 50, 75, and 100 μg/mL) dose-dependently blocked the cell proliferation and migration induced by PAR2-AP or factor VIIa. EGCG (100 μg/mL) prevented the cytoskeleton changes induced by PAR2-AP or factor VIIa. EGCG (100 μg/mL) counteracted the down-regulation of caspase-7 expression and up-regulation of TF and MMP-9 expression in the cells treated with PAR2-AP or factor VIIa. Furthermore, it blocked the activation of ERK1/2 and NF-κB (p65/RelA) induced by PAR2-AP or factor VIIa.
EGCG blocks the proliferation and migration of SW620 cells induced by PAR2-AP and factor VIIa via inhibition of the ERK1/2 and NF-κB pathways. The compound may serve as a preventive and therapeutic agent for colon cancers.
[Show abstract][Hide abstract] ABSTRACT: The tissue factor-factor VIIa (TF/VIIa) complex is believed to activate protease-activated receptor-2 (PAR2) and to trigger the malignant behavior of various types of cancer cells. In our previous study, it was demonstrated that TF and PAR2 were overexpressed in high metastatic potential colon cancer cells (SW620). Both PAR2 agonist (SLIGKV-NH2, PAR2-AP) and factor VIIa facilitated SW620 cell proliferation and migration. In the present study, the molecular mechanisms of TF/VIIa-induced SW620 cell proliferation and migration were investigated. It was found that factor VIIa (10 nM) significantly increased interleukin-8 (IL-8) expression at the mRNA and protein levels, enhanced TF mRNA expression and TF activity, and decreased caspase-7 expression at the mRNA and protein levels in the SW620 cells. These effects of factor VIIa were similar to those of PAR2-AP. All effects of factor VIIa were blocked by anti-TF and anti-PAR2 antibodies, but not by an isotype control antibody. Furthermore, both PAR2-AP and factor VIIa decreased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The results of this study suggest that the TF/VIIa complex regulates IL-8, TF and caspase-7 expression in SW620 cells via PAR2 activation, thereby promoting colon cancer cell proliferation and migration. The p38MAPK signal transduction pathway may negatively regulate these processes.
Molecular Medicine Reports 03/2010; 3(2):269-74. DOI:10.3892/mmr_00000250 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been almost a decade since the molecular cloning of all four members of the proteinase-activated receptor (PAR) family was completed. This unique family of G protein-coupled receptors (GPCRs) mediates specific cellular actions of various endogenous proteinases including thrombin, trypsin, tryptase, etc. and also certain exogenous enzymes. Increasing evidence has been clarifying the emerging roles played by PARs in health and disease. PARs, particularly PAR1 and PAR2, are distributed throughout the gastrointestinal (GI) tract, modulating various GI functions. One of the most important GI functions of PARs is regulation of exocrine secretion in the salivary glands, pancreas and GI mucosal epithelium. PARs also modulate motility of GI smooth muscle, involving multiple mechanisms. PAR2 appears to play dual roles in pancreatitis and related pain, being pro-inflammatory/pro-nociceptive and anti-inflammatory/anti-nociceptive. Similarly, dual roles for PAR1 and PAR2 have been demonstrated in mucosal inflammation/damage throughout the GI tract. There is also fundamental and clinical evidence for involvement of PAR2 in colonic pain. PARs are thus considered key molecules in regulation of GI functions and targets for development of drugs for treatment of various GI diseases.
British Journal of Pharmacology (2008) 153, S230–S240; doi:10.1038/sj.bjp.0707491; published online 12 November 2007
British Journal of Pharmacology 04/2008; 153 Suppl 1(S1):S230-40. DOI:10.1038/sj.bjp.0707491 · 4.84 Impact Factor
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