RESUMO
Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.
Assuntos
Cálcio/fisiologia , Receptor Muscarínico M3/fisiologia , Receptor PAR-2/fisiologia , Receptores Histamínicos H1/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células HT29 , Humanos , NF-kappa B/metabolismoRESUMO
OBJECTIVE: To investigate the relationship between single nucleotide polymorphisms (SNPs) of protease-activated receptor 2 (PAR-2) and the susceptibility to knee osteoarthritis (KOA) and synovial expression of inflammatory factors in the Chinese Han population. METHODS: Three hundred fifty KOA patients (KOA group) and 345 healthy volunteers (control group) were recruited for the study. Five milliliters of venous blood was taken from each subject to detect the PAR-2 rs1529505, rs631465, and rs2242991 locus genotypes. The expression of PAR-2 mRNA in the synovial tissue and the levels of matrix metalloproteinase (MMP-1), MMP-9, interleukin (IL)-6, and IL-1ß in the joint effusion were detected in 205 KOA patients who had undergone joint replacement surgery. RESULTS: The PAR-2 rs1529505 T allele and the rs2242991 G allele carriers had a higher risk of KOA (p < 0.001). The severity of KOA in patients with the PAR-2 rs1529505 and rs2242991 mutations were higher than in the wild-type controls (p < 0.05). The expression levels of the PAR-2 mRNA in wild types, heterozygotes, and homozygotes of the rs1529505 and rs2242991 loci increased in turn (p < 0.001). The levels of MMP-1, MMP-9, IL-6, and IL-1ß in the synovial fluid of the PAR-2 rs1529505 and rs2242991 locus mutants were distinctly higher than those with the wild-type alleles (p < 0.01). There was no correlation between the rs631465 SNP and susceptibility to KOA, severity of KOA, nor levels of PAR-2 mRNA, MMP-1, MMP-9, IL-6, and IL-1ß. CONCLUSIONS: The PAR-2 SNPs rs1529505 and rs2242991 are associated with the susceptibility to KOA. KOA is more severe in patients harboring the T and G alleles of these two SNPs, respectively. The levels of inflammatory factors in synovial tissue and blood are higher than those in wild-type patients.
Assuntos
Osteoartrite do Joelho/genética , Receptores Acoplados a Proteínas G/genética , Adulto , Idoso , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , China , Etnicidade/genética , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Inflamação/genética , Interleucina-1beta/genética , Interleucina-6/genética , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Osteoartrite do Joelho/fisiopatologia , Polimorfismo de Nucleotídeo Único/genética , Receptor PAR-2/genética , Receptor PAR-2/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Líquido Sinovial/metabolismoRESUMO
Compared to neutrophil chemoattractants, relatively little is known about the mechanism neutrophils use to respond to chemorepellents. We previously found that the soluble extracellular protein dipeptidyl peptidase IV (DPPIV) is a neutrophil chemorepellent. In this report, we show that an inhibitor of the protease activated receptor 2 (PAR2) blocks DPPIV-induced human neutrophil chemorepulsion, and that PAR2 agonists such as trypsin, tryptase, 2f-LIGRL, SLIGKV, and AC55541 induce human neutrophil chemorepulsion. Several PAR2 agonists in turn block the ability of the chemoattractant fMLP to attract neutrophils. Compared to neutrophils from male and female C57BL/6 mice, neutrophils from male and female mice lacking PAR2 are insensitive to the chemorepulsive effects of DPPIV or PAR2 agonists. Acute respiratory distress syndrome (ARDS) involves an insult-mediated influx of neutrophils into the lungs. In a mouse model of ARDS, aspiration of PAR2 agonists starting 24 h after an insult reduce neutrophil numbers in the bronchoalveolar lavage (BAL) fluid, as well as the post-BAL lung tissue. Together, these results indicate that the PAR2 receptor mediates DPPIV-induced chemorepulsion, and that PAR2 agonists might be useful to induce neutrophil chemorepulsion.
Assuntos
Dipeptidil Peptidase 4/farmacologia , Pulmão/imunologia , Neutrófilos/imunologia , Receptor PAR-2/fisiologia , Síndrome do Desconforto Respiratório/imunologia , Tripsina/farmacologia , Triptases/farmacologia , Animais , Células Cultivadas , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismoRESUMO
BACKGROUND AND AIMS: The function of interleukin (IL)-10-producing B cells (B10 cell) is compromised in patients with allergic diseases. Protease-activated receptor (PAR)-2 has immunoregulatory functions. This study aimed to elucidate the role of PAR2 in the suppression of IL-10 expression in peripheral B cells. METHODS: Peripheral blood B cells were collected from patients with allergic rhinitis (AR). A correlation between the expression of Bcl2-like protein 12 (Bcl2L12) and IL-10 in the B cells was analyzed. An AR mouse model was developed. RESULTS: We observed that the expression of IL-10 was lower in the peripheral B cells from patients with airway allergy. A negative correlation was identified between the expression of IL-10 and PAR2 in B cells. Activation of PAR2 of B cells increased the expression of Bcl2L12 and suppression of LPS-induced IL-10 expression, which were inhibited by knocking down the Bcl2L12 gene. Treating B cells from AR patients with Bcl2L12-shRNA-carrying liposomes reversed the capability of IL-10 expression and the immunosuppressive function. Administration of Bcl2L12 shRNA-carrying liposomes attenuated experimental AR in mice. CONCLUSIONS: Activation of PAR2 inhibits the expression of IL-10 in B cells, which can be reversed by treating B cells with Bcl2L12 shRNA-carrying liposomes. The data suggest that regulation of Bcl2L12 may be a novel approach in the treatment for AR.
Assuntos
Linfócitos B/metabolismo , Interleucina-10/metabolismo , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptor PAR-2/fisiologia , Rinite Alérgica/metabolismo , Regulação para Cima , Animais , Linfócitos B/enzimologia , Humanos , Camundongos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Rinite Alérgica/genéticaRESUMO
Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer, and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 µM) but not ERK1/2 phosphorylation (EC50 > 100 µM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 µM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing, and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.
Assuntos
Receptor PAR-2/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Cálcio/metabolismo , Cricetulus , Células HT29 , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor PAR-2/fisiologia , Relação Estrutura-Atividade , TransfecçãoRESUMO
Objective: To review the research progress on protease-activated receptor 2 (PAR-2) in the pathogenesis of osteoarthritis (OA). Methods: The relevant literature about the mechanism of PAR-2 in the occurrence and development of OA in recent years was extensively reviewed and comprehensively analyzed. Results: Abnormal activation of PAR-2 plays an important role in responses to occurrence and development of OA. Through regulating production and releasing of a variety of cytokines (such as inflammatory factors, metabolic factors, pain factors, etc.), the PAR-2 can involve in pathophysiological progression of OA articular cartilage, subchondral bone, and synovial membrane, as well as occurrence and transmission of pain. Conclusion: PAR-2 participation in the development of OA has been confirmed. However, since PAR-2 is complicated and widespread, it is necessary to study the specific role of PAR-2 and the interaction between various signal pathways in the progression of OA, and to elucidate the potential pathophysiological mechanisms of PAR-2 participating in the process of OA, in the hope of exploring the new targets for the effective control of OA.
Assuntos
Osteoartrite/metabolismo , Receptor PAR-2/fisiologia , Cartilagem Articular , Citocinas , Humanos , Membrana SinovialRESUMO
OBJECTIVE: The objective of this study was to evaluate whether an uncontrolled activation of mast cells and macrophages through protease-activated receptor-2 (PAR-2) during acute pancreatitis could develop lung injury. METHODS: Pancreatitis was induced in rats by intraductal infusion of sodium taurocholate. In a group of animals, PAR-2 antagonist or trypsin (TRP) inhibitor was intravenously administered before the pancreatitis induction. In additional groups, the animals were treated with PAR-2-activating peptide or pancreatic TRP. The myeloperoxidase (MPO) activity was measured to evaluate the progression of inflammation. RESULTS: Plasma from the animals with pancreatitis and pancreatic TRP induced the secretion of mast cells and alveolar macrophages as well as increased the density of PAR-2 in the plasma membrane. The treatment of alveolar macrophages with TRP, tryptase, as well as PAR-1- and PAR-2-activating peptide led to an increase in calcium-triggered exocytosis. Similar results were obtained in acinar cells. The intravenous injection of PAR-2-activating peptide and TRP induced an increase in MPO activity in the lung. The intravenous injection of PAR-2 antagonist or TRP inhibitor before the pancreatitis induction could prevent the increase in MPO activity in the pancreas and the lung. CONCLUSIONS: The TRP generated during acute pancreatitis could be involved in the progression of lung injury through the activation of PAR-2 in alveolar macrophages.
Assuntos
Pneumopatias/metabolismo , Pulmão/metabolismo , Pancreatite/metabolismo , Receptor PAR-2/metabolismo , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Doença Aguda , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Exocitose/efeitos dos fármacos , Imuno-Histoquímica , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Pneumopatias/fisiopatologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Microscopia Confocal , Oligopeptídeos/farmacologia , Pancreatite/induzido quimicamente , Pancreatite/fisiopatologia , Peroxidase/metabolismo , Ratos Wistar , Receptor PAR-2/fisiologia , Ácido Taurocólico , Tripsina/metabolismo , Tripsina/farmacologiaRESUMO
Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation.
Assuntos
Catepsinas/fisiologia , Receptor PAR-2/fisiologia , Sequência de Aminoácidos , Catepsinas/genética , Catepsinas/metabolismo , Células HeLa , Humanos , Queratinócitos/metabolismo , Mapeamento de Interação de Proteínas , Receptor PAR-2/química , Receptor PAR-2/genética , Transdução de SinaisRESUMO
Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.
Assuntos
Cisteína Proteases/farmacologia , Entamoeba histolytica/enzimologia , Interleucina-8/biossíntese , Mastócitos/efeitos dos fármacos , Proteínas de Protozoários/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Cisteína Proteases/metabolismo , Cisteína Proteases/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Entamoeba histolytica/patogenicidade , Exocitose , Humanos , Interleucina-8/genética , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Receptor PAR-2/fisiologia , VirulênciaRESUMO
Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.
Assuntos
Anemia Falciforme/complicações , Modelos Animais de Doenças , Fator Xa/metabolismo , Inflamação/etiologia , Trombina/metabolismo , Doenças Vasculares/etiologia , Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Anticoagulantes/farmacologia , Antitrombinas/farmacologia , Benzimidazóis/farmacologia , Transplante de Medula Óssea , Dabigatrana , Inibidores do Fator Xa , Feminino , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfolinas/farmacologia , Receptor PAR-1/fisiologia , Receptor PAR-2/fisiologia , Rivaroxabana , Tiofenos/farmacologia , Trombina/antagonistas & inibidores , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , beta-Alanina/análogos & derivados , beta-Alanina/farmacologiaRESUMO
Although the etiology of irritable bowel syndrome (IBS) remains unknown, clinical and laboratory observations suggest that within the broad and varying phenotype, that is, IBS, there may exist subgroups, which can be defined on the basis of a distinctive pathophysiological basis. Of these, postinfectious IBS is the best characterized; in IBS, in general, studies of inflammatory mediators and substances elaborated by cells involved in the intestinal immune response, such as proteases, suggest that some IBS sufferers can be differentiated on the basis of an aberrant immune response. Valdez-Morales and colleagues extend this concept by demonstrating the ability of supernatants of biopsy cultures from individuals with diarrhea-predominant IBS to enhance neuronal excitability-an effect that could well contribute to a clinical hallmark of IBS, namely, visceral hypersensitivity.
Assuntos
Colo/metabolismo , Constipação Intestinal/fisiopatologia , Diarreia/fisiopatologia , Gânglios Espinais/citologia , Síndrome do Intestino Irritável/fisiopatologia , Nociceptores/fisiologia , Receptor PAR-2/fisiologia , Animais , Feminino , Humanos , MasculinoRESUMO
Clinical and epidemiological studies support a connection between obesity and thrombosis, involving elevated expression of the prothrombotic molecules plasminogen activator inhibitor-1 and tissue factor (TF) and increased platelet activation. Cardiovascular diseases and metabolic syndrome-associated disorders, including obesity, insulin resistance, type 2 diabetes, and hepatic steatosis, involve inflammation elicited by infiltration and activation of immune cells, particularly macrophages, into adipose tissue. Although TF has been clearly linked to a procoagulant state in obesity, emerging genetic and pharmacologic evidence indicate that TF signaling via G protein-coupled protease-activated receptors (PAR2, PAR1) additionally drives multiple aspects of the metabolic syndrome. TF-PAR2 signaling in adipocytes contributes to diet-induced obesity by decreasing metabolism and energy expenditure, whereas TF-PAR2 signaling in hematopoietic and myeloid cells drives adipose tissue inflammation, hepatic steatosis, and insulin resistance. TF-initiated coagulation leading to thrombin-PAR1 signaling also contributes to diet-induced hepatic steatosis and inflammation in certain models. Thus, in obese patients, clinical markers of a prothrombotic state may indicate a risk for the development of complications of the metabolic syndrome. Furthermore, TF-induced signaling could provide new therapeutic targets for drug development at the intersection between obesity, inflammation, and thrombosis.
Assuntos
Inflamação/fisiopatologia , Obesidade/fisiopatologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Trombofilia/etiologia , Tromboplastina/fisiologia , Trombose/etiologia , Adipócitos/fisiologia , Tecido Adiposo/patologia , Animais , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Regulação da Expressão Gênica , Humanos , Inflamação/sangue , Inflamação/complicações , Resistência à Insulina/fisiologia , Macrófagos/fisiologia , Síndrome Metabólica/epidemiologia , Síndrome Metabólica/fisiopatologia , Camundongos , Camundongos Obesos , Modelos Biológicos , Obesidade/sangue , Obesidade/complicações , Receptor PAR-1/fisiologia , Receptor PAR-2/fisiologia , Fatores de Risco , Transdução de Sinais/fisiologia , Linfócitos T Reguladores/fisiologia , Trombofilia/fisiopatologia , Trombose/fisiopatologiaRESUMO
The tumor microenvironment is replete with proteinases. As a sensor of proteinases, proteinase activated receptor 2 (PAR2) plays critical roles in tumorigenesis. We showed that PAR2 and its activating proteinase were coexpressed in different colon cancer cell lines, including HT29. Inactivating proteinase or knockdown of PAR2 significantly not only reduced cell proliferation in vitro but also inhibited tumorigenicity of HT29 in vivo. In addition, activation of PAR2 promoted DNA synthesis and upregulated Cyclin D1 activity at both transcriptional and post-transcriptional levels. Further studies showed that miRNA-34a mediated PAR2-induced Cyclin D1 upregulation. Inhibition of miR-34a partially abolished the suppression of Cyclin D1 induced by PAR2 deficiency. In addition, we showed that TGF-ß contributed to the regulation of miR-34a by PAR2. Finally, in colorectal carcinoma samples, upregulation of PAR2 and downregulation of miR-34a were significantly correlated with grade and lymphomatic metastasis. Our findings provide the first evidence that miRNA mediates autocrine proteinase signaling-mediated cancer cell proliferation.
Assuntos
Proliferação de Células , Neoplasias do Colo/patologia , MicroRNAs/fisiologia , Receptor PAR-2/metabolismo , Transdução de Sinais/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Ciclina D1/metabolismo , Primers do DNA , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , Receptor PAR-2/genética , Receptor PAR-2/fisiologia , Transcrição Gênica , Regulação para CimaRESUMO
BACKGROUND: Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor suggested to play an important role in the proliferation and migration of tumor cells of epithelial origin. However, the role of PAR-2 in the setting of pancreatic cancer remains largely unexplored. OBJECTIVES: To understand the importance of PAR-2 in pancreatic cancer cell migration. METHODS AND RESULTS: The present study shows that PAR-2 does not affect pancreatic cancer cell proliferation but significantly induces the migration of pancreatic cancer cells in scratch assays. Interestingly, PAR-2 does not affect migration in a trans-well setting. This apparent discrepancy depends on extracellular ATP release in the scratch assays and the addition of exogenous (ATP)-induced PAR-2-dependent migration in trans-well assays, whereas a specific P2Y11 receptor antagonist prevents PAR-2-driven migration in scratch assays. In the scratch assays, inhibitors of Src, Rac, protein kinase C, mitogen-activated protein kinase kinase, p38, and epidermal growth factor (EGF) receptor blocked PAR-2-driven migration, whereas they did not affect fetal calf serum-driven wound closure. CONCLUSION: Taken together, PAR-2 activation drives pancreatic cancer cell migration via an EGF-Src-Rac-p38/mitogen-activated protein kinase kinase/EGF1/2 signaling pathway, which is facilitated by extracellular ATP. Targeting the PAR-2/ATP-driven signaling pathway may therefore limit cell migration, which could inhibit pancreatic cancer metastasis.
Assuntos
Trifosfato de Adenosina/farmacologia , Movimento Celular/fisiologia , Neoplasias Pancreáticas/patologia , Receptor PAR-2/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Humanos , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismoRESUMO
OBJECTIVES: This study examined whether mediators from biopsies of human irritable bowel syndrome (IBS) colons alter intrinsic excitability of colonic nociceptive dorsal root ganglion (DRG) neurons by a protease activated receptor 2 (PAR2)-mediated mechanism. METHODS: Colonic mucosal biopsies from IBS patients with constipation (IBS-C) or diarrhea (IBS-D) and from healthy controls were incubated in medium, and supernatants were collected. Small-diameter mouse colonic DRG neurons were incubated in supernatants overnight and perforated patch current-clamp recordings obtained. Measurements of rheobase and action potential discharge at twice rheobase were compared between IBS and controls to assess differences in intrinsic excitability. RESULTS: Supernatants from IBS-D patients elicited a marked increase in neuronal excitability compared with controls. These changes were consistent among individual patients but the relative contribution of rheobase and action potential discharge varied. In contrast, no differences in neuronal excitability were seen with IBS-C patient supernatants. The increased excitability seen with IBS-D supernatant was not observed in PAR2 knockout mice. A cysteine protease inhibitor, which had no effect on the pronociceptive actions of a serine protease, inhibited the proexcitatory actions of IBS-D supernatant. CONCLUSIONS: Soluble mediators from colonic biopsies from IBS-D but not IBS-C patients sensitized colonic nociceptive DRG neurons, suggesting differences between these two groups. PAR2 signaling plays a role in this action and this protease signaling pathway could provide novel biomarkers and therapeutic targets for treatment.
Assuntos
Colo/metabolismo , Constipação Intestinal/fisiopatologia , Diarreia/fisiopatologia , Gânglios Espinais/citologia , Síndrome do Intestino Irritável/fisiopatologia , Nociceptores/fisiologia , Receptor PAR-2/fisiologia , Potenciais de Ação/fisiologia , Adulto , Idoso , Animais , Biópsia , Estudos de Casos e Controles , Colo/inervação , Constipação Intestinal/etiologia , Diarreia/etiologia , Feminino , Humanos , Síndrome do Intestino Irritável/complicações , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Receptor PAR-2/genética , Adulto JovemRESUMO
Alimentary mucositis is a severe, dose-limiting, toxic side effect of cytotoxic chemotherapy and radiotherapy. Patients with mucositis often have reductions or breaks imposed on cytotoxic therapy, which may lead to reduced survival. Furthermore, there is an increased risk of infection and hospitalization, compounding the cost of treatment. There are currently limited therapeutic options for mucositis, and no effective prevention available. Mucin expression and secretion have been shown to be associated with mucositis. Furthermore, mucins exhibit protective effects on the alimentary tract through reducing mechanical and chemical stress, preventing bacterial overgrowth and penetration, and digestion of the mucosa. Additionally, a number of studies have implicated some key neurotransmitters in both mucositis and mucin secretion, suggesting that the enteric nervous system may also play a key role in the development of mucositis.
Assuntos
Antineoplásicos/efeitos adversos , Sistema Nervoso Entérico/fisiopatologia , Mucinas/metabolismo , Mucinas/fisiologia , Mucosite/fisiopatologia , Dinoprostona/fisiologia , Sistema Nervoso Entérico/efeitos dos fármacos , Humanos , Modelos Biológicos , Mucinas/efeitos dos fármacos , Mucosite/induzido quimicamente , Óxido Nítrico/fisiologia , Receptor PAR-2/fisiologia , Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
Tissue factor initiates the process of thrombosis and activates cell signaling through protease-activated receptor-2 (PAR-2). The aim of this study was to investigate the pathological role of PAR-2 signaling in pancreatic cancer. We first demonstrated that activated PAR-2 up-regulated the protein expression of both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, resulting in enhanced transcription of transforming growth factor-α (TGF-α). Down-regulation of HIFs-α by siRNA or YC-1, an HIF inhibitor, resulted in depleted levels of TGF-α protein. Furthermore, PAR-2, through integrin-linked kinase (ILK) signaling, including the p-AKT, promoted HIF protein expression. Diminishing ILK by siRNA decreased the levels of PAR-2-induced p-AKT, HIFs-α, and TGF-α; our results suggest that ILK is involved in the PAR-2-mediated TGF-α via an HIF-α-dependent pathway. Furthermore, the culture medium from PAR-2-treated pancreatic cancer cells enhanced human umbilical vein endothelial cell proliferation and tube formation, which was blocked by the MEK inhibitor, PD98059. We also found that activated PAR-2 enhanced tumor angiogenesis through the release of vascular endothelial growth factor-A (VEGF-A) from cancer cells, independent of the ILK/HIFs-α pathways. Consistent with microarray analysis, activated PAR-2 induced TGF-A and VEGF-A gene expression. In conclusion, the activation of PAR-2 signaling induced human pancreatic cancer progression through the induction of TGF-α expression by ILK/HIFs-α, as well as through MEK/VEGF-A-mediated angiogenesis, and it plays a role in the interaction between cancer progression and cancer-related thrombosis.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor PAR-2/fisiologia , Fator de Crescimento Transformador alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Flavonoides/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Indazóis/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neovascularização Patológica/enzimologia , Neovascularização Patológica/etiologia , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/fisiologia , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
AIMS: In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. METHODS AND RESULTS: PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. CONCLUSION: PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.
Assuntos
Circulação Colateral/fisiologia , Membro Posterior/irrigação sanguínea , Monócitos/fisiologia , Receptor PAR-1/fisiologia , Receptor PAR-2/fisiologia , Animais , Arteríolas/fisiologia , Diferenciação Celular , Modelos Animais de Doenças , Artéria Femoral , Isquemia , Lectinas Tipo C/imunologia , Ligadura , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Receptor PAR-1/deficiência , Receptores de Superfície Celular/imunologiaRESUMO
Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca(2+) concentration was measured using the Ca(2+)-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P<0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P<0.01), thymus and activation regulated chemokine (TARC/CCL17, P<0.01), and interleukin 8 (IL-8/CXCL8, P<0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P<0.05), GM-CSF (P<0.05), TARC/CCL17 (P<0.05), and IL-8/CXCL8 (P<0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca(2+) concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis.
Assuntos
Queratinócitos/imunologia , Receptor PAR-2/fisiologia , Animais , Cálcio/metabolismo , Quimiocinas/genética , Reações Cruzadas , Citocinas/genética , Dermatite Atópica/etiologia , Cães , Células HEK293 , Humanos , RNA Mensageiro/análise , Receptor PAR-2/agonistas , Tripsina/farmacologiaRESUMO
Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.