RESUMEN
BACKGROUND AND PURPOSE: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. EXPERIMENTAL APPROACH: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. KEY RESULTS: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. CONCLUSION AND IMPLICATIONS: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.
Asunto(s)
Quimotripsina , Mucosa Intestinal , Receptor PAR-1 , Receptor PAR-2 , Animales , Humanos , Ratones , Quimotripsina/metabolismo , Mucosa Intestinal/metabolismo , Ratones Endogámicos C57BL , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transducción de SeñalRESUMEN
(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.
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Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Ratones , Humanos , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Proteínas Nucleares , Factores de Transcripción/metabolismo , Aorta/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de Ciclo CelularRESUMEN
Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.
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Proteasas de Cisteína , Giardia lamblia , Mucinas , Receptor PAR-2 , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Expresión Génica , Giardia lamblia/enzimología , Giardia lamblia/genética , Células Caliciformes/metabolismo , Humanos , Mucinas/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismoRESUMEN
Vascular pathology is increased in diabetes because of reactive-oxygen-species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1-50 µM) protects tissue-intact and cultured vascular endothelial cells from hyperglycemia/ROS-induced dysfunction typified by reduced agonist-stimulated endothelium-dependent, nitric oxide-mediated vasorelaxation in response to muscarinic or proteinase-activated-receptor 2 agonists. Metformin not only attenuated hyperglycemia-induced ROS production in aorta-derived endothelial cell cultures but also prevented hyperglycemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues in accord with our observing a direct metformin-Nr4a1 interaction. Using in silico modeling of metformin-NR4A1 interactions, Nr4a1-mutagenesis, and a transfected human embryonic kidney 293T cell functional assay for metformin-activated Nr4a1, we identified two Nr4a1 prolines, P505/P549 (mouse sequences corresponding to human P501/P546), as key residues for enabling metformin to affect mitochondrial function. Our data indicate a critical role for Nr4a1 in metformin's endothelial-protective effects observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-I or complex-III oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations requiring the expression of Nr4a1 protect the vasculature from hyperglycemia-induced dysfunction in addition to metformin's action to enhance insulin action in patients with diabetes. SIGNIFICANCE STATEMENT: Metformin improves diabetic vasodilator function, having cardioprotective effects beyond glycemic control, but its mechanism to do so is unknown. We found that metformin at therapeutic concentrations (1-50µM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 µM) impairs vascular function. However, metformin's action requires the expression of the orphan nuclear receptor NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin preserves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.
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Endotelio Vascular/efectos de los fármacos , Hiperglucemia/prevención & control , Hipoglucemiantes/uso terapéutico , Metformina/uso terapéutico , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Células HEK293 , Humanos , Hiperglucemia/metabolismo , Hipoglucemiantes/farmacología , Masculino , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Estrés Oxidativo/fisiología , Vasodilatadores/farmacologíaRESUMEN
[This corrects the article DOI: 10.3389/fimmu.2020.629726.].
RESUMEN
Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.
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Artritis Psoriásica/inmunología , Señalización del Calcio/inmunología , Macrófagos/inmunología , Receptor PAR-2/inmunología , Triptasas/inmunología , Artritis Psoriásica/patología , Femenino , Humanos , Macrófagos/patología , MasculinoRESUMEN
We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr-/-mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr-/- mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr-/- or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr-/- mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr-/- mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr-/- mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr-/- mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Indoles/farmacología , Receptor X de Pregnano/agonistas , Receptor X de Pregnano/fisiología , Vasodilatación/efectos de los fármacos , Animales , Antibacterianos/farmacología , Células Cultivadas , Células Endoteliales/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Indoles/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microbiota/efectos de los fármacos , Microbiota/fisiología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptor X de Pregnano/genética , Vasodilatación/genéticaRESUMEN
This overview deals with mechanisms whereby hyperglycemia-induced oxidative stress compromises vascular endothelial function and provides a background for a recently published study illustrating the beneficial impact of endothelial sodium-glucose cotransporter 2 (SGLT2) inhibitors in attenuating hyperglycemia-induced vascular dysfunction in vitro. The data provide new insight that can possibly lead to improved drug therapy for people with type 2 diabetes. The working hypotheses that underpinned the experiments performed are provided, along with the findings of the study. For the causes of hyperglycemia-induced vascular endothelial dysfunction, the findings point to the key roles of: 1) functional endothelial SGLT2; 2) oxidative stress-induced signalling pathways including mammalian sarcoma virus kinase, the EGF receptor-kinase and protein kinase C; and 3) mitochondrial dysfunction triggered by hyperglycemia was mitigated by an SGLT2 inhibitor in the hyperglycemic mouse aorta vascular organ cultures. The overview sums up the approaches implicated by the study that can potentially counteract the detrimental impact of hyperglycemia on vascular function in people with diabetes, including the clinical use of SGLT2 inhibitors for those with type 2 diabetes already being treated, for example, with metformin, along with dietary supplementation with broccoli-derived sulforaphane and tetrahydrobiopterin. The caveats associated with the study for extending the findings from mice to humans are summarized, pointing to the need to validate the work using vascular tissues from humans. Suggestions for future clinical studies are made, including the assessment of the impact of the therapeutic strategies proposed on measurements of blood flow in subjects with diabetes.
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Enfermedades Cardiovasculares/tratamiento farmacológico , Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Hiperglucemia/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Transportador 2 de Sodio-Glucosa/química , Biomarcadores/análisis , Biopterinas/análogos & derivados , Biopterinas/uso terapéutico , Glucemia/análisis , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/metabolismo , Angiopatías Diabéticas/epidemiología , Angiopatías Diabéticas/metabolismo , Endotelio Vascular/patología , Humanos , Incidencia , Isotiocianatos/uso terapéutico , Pronóstico , SulfóxidosRESUMEN
Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR2) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/ß-arrestin signaling axis, identify the protease activity responsible for PAR2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/ß-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2-/- and ß-arrestin-2-/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR2-/- or ß-arrestin-2-/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR2 signaling and induce ß-arrestin-2-/--dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/ß-arrestin signaling axis. Thus, ß-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.
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Inflamación/metabolismo , Receptor PAR-2/metabolismo , Serina Proteasas/metabolismo , Transducción de Señal/fisiología , Arrestina beta 2/metabolismo , Alérgenos/metabolismo , Animales , Asma/metabolismo , Proteínas Bacterianas/metabolismo , Endopeptidasas/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Serina/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
Purpose: Myopia is a refractive disorder that degrades vision. It can be treated with atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, but the mechanism is unknown. Atropine may block α-adrenoceptors at concentrations ≥0.1 mM, and another potent myopia-inhibiting ligand, mamba toxin-3 (MT3), binds equally well to human mAChR M4 and α1A- and α2A-adrenoceptors. We hypothesized that mAChR antagonists could inhibit myopia via α2A-adrenoceptors, rather than mAChR M4. Methods: Human mAChR M4 (M4), chicken mAChR M4 (cM4), or human α2A-adrenergic receptor (hADRA2A) clones were cotransfected with CRE/promoter-luciferase (CRE-Luc; agonist-induced luminescence) and Renilla luciferase (RLuc; normalizing control) into human cells. Inhibition of normalized agonist-induced luminescence by antagonists (ATR: atropine; MT3; HIM: himbacine; PRZ: pirenzepine; TRP: tropicamide; OXY: oxyphenonium; QNB: 3-quinuclidinyl benzilate; DIC: dicyclomine; MEP: mepenzolate) was measured using the Dual-Glo Luciferase Assay System. Results: Relative inhibitory potencies of mAChR antagonists at mAChR M4/cM4, from most to least potent, were QNB > OXY ≥ ATR > MEP > HIM > DIC > PRZ > TRP. MT3 was 56× less potent at cM4 than at M4. Relative potencies of mAChR antagonists at hADRA2A, from most to least potent, were MT3 > HIM > ATR > OXY > PRZ > TRP > QNB > MEP; DIC did not antagonize. Conclusions: Muscarinic antagonists block hADRA2A signaling at concentrations comparable to those used to inhibit chick myopia (≥0.1 mM) in vivo. Relative potencies at hADRA2A, but not M4/cM4, correlate with reported abilities to inhibit chick form-deprivation myopia. mAChR antagonists might inhibit myopia via α2-adrenoceptors, instead of through the mAChR M4/cM4 receptor subtype.
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Atropina/farmacología , Antagonistas Muscarínicos/farmacología , Miopía/prevención & control , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Muscarínicos/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Animales , Proteína 9 Asociada a CRISPR , Carbacol/farmacología , Pollos , Agonistas Colinérgicos/farmacología , Clonidina/farmacología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ligandos , Receptor Muscarínico M3/genética , Receptor Muscarínico M4/metabolismo , TransfecciónRESUMEN
Hyperglycaemia is a major contributor to diabetic cardiovascular disease with hyperglycaemia-induced endothelial dysfunction recognized as the initiating cause. Coagulation pathway-regulated proteinase-activated receptors (PARs) that can regulate vascular tone in vivo cause eNOS-mediated endothelium-dependent vasodilation; but, the impact of hyperglycaemia on this vasodilatory action of PAR stimulation and the signalling pathways involved are unknown. We hypothesized that vascular sodium-glucose co-transporter 2 activity and hyperglycaemia-induced oxidative stress involving Src-kinase, EGF receptor-kinase, Rho-kinase and protein-kinase-C biochemical signalling pathways would compromise PAR2-mediated endothelium-dependent vasodilation. Using an organ culture approach, wherein murine aorta rings were maintained for 24â¯h at hyperglycaemic 25â¯mM versus euglycaemic 10â¯mM glucose, we observed severely blunted acetylcholine/muscarinic and PAR2-mediated endothelial eNOS/NO-dependent vasodilation. PEG-catalase, superoxide-dismutase, and NADPH-oxidase inhibition (VAS2870) and either SGLT2-inhibition (canagliflozin/dapagliflozin/empagliflozin) or antioxidant gene induction (sulforaphane), prevented the hyperglycaemia-induced impairment of PAR2-mediated vasodilation. Similarly, inhibition of Src-kinase, EGF receptor-kinase, protein kinase-C and Rho-kinase also preserved PAR2-mediated vasodilation in tissues cultured under hyperglycaemic conditions. Thus, intracellular hyperglycaemia, that can be prevented with an inhibitor of the SGLT2 cotransporter that was identified in the vascular tissue and tissue-derived cultured endothelial cells by qPCR, western blot and immunohistochemistry, leads to oxidative stress that compromises PAR2-mediated NOS-dependent vasodilation by an NAPDH oxidase/reactive-oxygen-species-triggered signalling pathway involving EGFR/Src/Rho-kinase and PKC. The data point to novel antioxidant therapeutic strategies including use of an SGLT2 inhibitor and sulforaphane to mitigate hyperglycaemia-induced endothelial dysfunction.
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Antioxidantes/farmacología , Aorta/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Receptor PAR-2/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Vasodilatación/efectos de los fármacos , Animales , Aorta/metabolismo , Aorta/patología , Aorta/fisiopatología , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Receptores ErbB/metabolismo , Hiperglucemia/sangre , Hiperglucemia/patología , Hiperglucemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Técnicas de Cultivo de Órganos , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Transportador 2 de Sodio-Glucosa/metabolismo , Quinasas Asociadas a rho/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
We propose that in the microenvironment of inflammatory tissues, including tumours, extracellular proteinases can modulate cell signalling in part by regulating proteinase-activated receptors (PARs). We have been exploring this mechanism in a variety of inflammation and tumour-related settings that include tumour-derived cultured cells from prostate and bladder cancer, as well as immune inflammatory cells that are involved in the pathology of inflammatory diseases including multiple sclerosis. Our work showed that proteinase signalling via the PARs affects prostate and bladder cancer-derived tumour cell behaviour and can regulate calcium signalling in human T-cell and macrophage-related inflammatory cells as well as in murine splenocytes. Further, we found that the tumour-derived prostate cancer cells and immune-related cells (Jurkat, THP1, mouse splenocytes) can produce PAR-regulating proteinases (including kallikreins: kallikrein-related peptidases), that can control tissue function by both a paracrine and autocrine mechanism. We suggest that this PAR-driven signalling process involving secreted microenvironment proteinases can play a key role in cancer and inflammatory diseases including multiple sclerosis.
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Inflamación/metabolismo , Péptido Hidrolasas/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Proteinasa-Activados/metabolismo , Microambiente Tumoral , Animales , Células Cultivadas , Humanos , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND AND PURPOSE: Previously, we demonstrated that exogenous heat shock protein 27 (HSP27/gene, HSPB1) treatment of human endothelial progenitor cells (EPCs) increases the synthesis and secretion of VEGF, improves EPC-migration/re-endothelialization and decreases neo-intima formation, suggesting a role for HSPB1 in regulating EPC function. We hypothesized that HSPB1 also affects mature endothelial cells (ECs) to alter EC-mediated vasoreactivity in vivo. Our work focused on endothelial NOS (eNOS)/NO-dependent relaxation induced by ACh and the coagulation pathway-activated receptor, proteinase-activated receptor 2 (PAR2). EXPERIMENTAL APPROACH: Aorta rings from male and female wild-type, HSPB1-null and HSPB1 overexpressing (HSPB1o/e) mice were contracted with phenylephrine, and NOS-dependent relaxation responses to ACh and PAR2 agonist, 2-furoyl-LIGRLO-NH2 , were measured without and with L-NAME and ODQ, either alone or in combination to block NO synthesis/action. Tissues from female HSPB1-null mice were treated in vitro with recombinant HSP27 and then used for bioassay as above. Furthermore, oestrogen-specific effects were evaluated using a bioassay of aorta isolated from ovariectomized mice. KEY RESULTS: Relative to males, HSPB1-null female mice exhibited an increased L-NAME-resistant relaxation induced by activation of either PAR2 or muscarinic ACh receptors that was blocked in the concurrent presence of both L-NAME and ODQ. mRNAs (qPCR) for eNOS and ODQ-sensitive guanylyl-cyclase were increased in females versus males. Treatment of isolated aorta tissue with HSPB1 improved tissue responsiveness in the presence of L-NAME. Ovariectomy did not affect NO sensitivity, supporting an oestrogen-independent role for HSPB1. CONCLUSIONS AND IMPLICATIONS: HSPB1 can regulate intact vascular endothelial function to affect NO-mediated vascular relaxation, especially in females.
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Inhibidores Enzimáticos/farmacología , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Receptor PAR-2/antagonistas & inhibidores , Receptores Muscarínicos/metabolismo , Vasodilatación/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/química , Femenino , Proteínas de Choque Térmico HSP27/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NG-Nitroarginina Metil Éster/química , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxadiazoles/química , Oxadiazoles/farmacología , Quinoxalinas/química , Quinoxalinas/farmacología , Receptor PAR-2/metabolismo , Relación Estructura-ActividadRESUMEN
Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and ß-arrestin interactions. By disrupting this PAR4 calcium/ß-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.
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Plaquetas/metabolismo , Receptores de Trombina/química , Receptores de Trombina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Transporte de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Trombosis/patología , beta-Arrestinas/metabolismoRESUMEN
Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringß-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.
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Señalización del Calcio , Sistema de Señalización de MAP Quinasas , Receptor PAR-2/agonistas , Trombina/metabolismo , Vasodilatación , Sustitución de Aminoácidos , Animales , Aorta , Arrestinas/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Endotelio Vascular/fisiología , Humanos , Técnicas In Vitro , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mutación , Oligopéptidos/farmacología , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteolisis , Conejos , Receptor PAR-2/química , Receptor PAR-2/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Vasodilatación/efectos de los fármacos , beta-ArrestinasRESUMEN
BACKGROUND AND PURPOSE: Transient receptor potential vanilloid-4 (TRPV4) is a calcium-permeant ion channel that is known to affect vascular function. The ability of TRPV4 to cause a vasoconstriction in blood vessels has not yet been mechanistically examined. Further in neuronal cells, TRPV4 signalling can be potentiated by GPCR activation. Thus, we studied the mechanisms underlying the vascular contractile action of TRPV4 and the GPCR-mediated potentiation of such vasoconstriction, both of which are as yet unappreciated aspects of TRPV4 function. EXPERIMENTAL APPROACH: The mechanisms of TRPV4-dependent regulation of vascular tone in isolated mouse aortae were studied using wire myography. TRPV4-dependent calcium signalling and prostanoid production was studied in cultured human umbilical vein endothelial cells (HUVECs). KEY RESULTS: In addition to the well-documented vasorelaxation response triggered by TRPV4 activation, we report here a TRPV4-triggered vasoconstriction in the mouse aorta that involves a COX-generated Tx receptor (TP) agonist that acts in a MAPK and Src kinase signalling dependent manner. This constriction is potentiated by activation of the GPCRs for angiotensin (AT1 receptors) or proteinases (PAR1 and PAR2) via transactivation of the EGF receptor and a process involving PKC. TRPV4-dependent vascular contraction can be blocked by COX inhibitors or with TP antagonists. Further, TRPV4 activation in HUVECs stimulated Tx release as detected by an elisa. CONCLUSION AND IMPLICATIONS: We conclude that the GPCR potentiation of TRPV4 action and TRPV4-dependent Tx receptor activation are important regulators of vascular function and could be therapeutically targeted in vascular diseases.
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Aorta/metabolismo , Receptores ErbB/fisiología , Receptores Acoplados a Proteínas G/fisiología , Canales Catiónicos TRPV/fisiología , Activación Transcripcional/fisiología , Vasoconstricción/fisiología , Animales , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Noqueados , Miografía , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Because angiotensin-II-mediated porcine coronary artery (PCA) vasoconstriction is blocked by protein tyrosine kinase (PYK) inhibitors, we hypothesized that proteinase-activated receptors (PARs), known to regulate vascular tension, like angiotensin-II, would also cause PCA contractions via PYK-dependent signalling pathways. EXPERIMENTAL APPROACH: Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1 /PAR2 -activating peptides, angiotensin-II, PGF2α , EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 (EGF-receptor kinase), AG1296 (PDGF receptor kinase), PP1 (Src kinase), U0126 and PD98059 (MEK/MAPKinase kinase), indomethacin/SC-560/NS-398 (COX-1/2) and L-NAME (NOS). KEY RESULTS: AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1 /PAR2 agonists, EGF and angiotensin-II, but not by PGF2α , the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1 /PAR2 -activating peptides and angiotensin-II. The EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs (PAR2 >> PAR1 ) and EGF itself, but not by angiotensin-II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α . CONCLUSION AND IMPLICATIONS: PAR1/2 -mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin-II and EGF. These signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm.
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Vasos Coronarios/enzimología , Proteínas Tirosina Quinasas/fisiología , Receptor PAR-1/fisiología , Receptor PAR-2/fisiología , Vasoconstricción/fisiología , Animales , Vasos Coronarios/efectos de los fármacos , Técnicas de Cultivo de Órganos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Porcinos , Vasoconstricción/efectos de los fármacosRESUMEN
Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.
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Péptidos/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal/genética , Secuencia de Aminoácidos , Animales , Implantación del Embrión , Femenino , Expresión Génica , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteolisis , Ratas , Receptor PAR-1/genética , Receptor PAR-2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gαi-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity.
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Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Elastasa de Leucocito/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Mieloblastina/metabolismo , Receptor PAR-1/metabolismo , Línea Celular Tumoral , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Células HEK293 , Humanos , Elastasa de Leucocito/genética , Mieloblastina/genética , Receptor PAR-1/genética , Trombina/genética , Trombina/metabolismoRESUMEN
We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.