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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.
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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
To develop new degrader molecules from an existing protein ligand a linkage vector must be identified and then joined with a suitable E3 ligase without disrupting binding to the respective targets. This is typically achieved through empirically evaluating the degradation efficacy of a series of synthetic degraders. Our strategy for determining optimal linkage sites utilises biotinylated protein ligands, linked via potential conjugation sites of an inhibitor to confirm whether target protein is maintained after forming a conjugate. This method provides low-cost, qualitative evidence that the addition of a linker moiety at a specific position can be tolerated, guiding further optimisation. We demonstrate the application of this method through the exploration of linkage vectors on A-485, a known ligand of p300/CBP, and found a conjugation site through a urea moiety. Pomalidomide was then conjugated through this site with several different linkers and cell viability and degradation were assessed for this library using a myeloma cell line, MM1.S. Compound 18i, with a PEG4 linker, was found to be the most effective p300 degrader and linker length greater than 10 atoms afforded enhanced degradation.
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Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell (EC) gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified kallikrein-related peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial ECs, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured ECs. Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs, as determined by NFκB activation, expression of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1, and monocyte adhesion. Furthermore, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.
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Aterosclerosis/genética , Células Endoteliales/fisiología , Regulación de la Expresión Génica , Inflamación/genética , Calicreínas/genética , Animales , Aterosclerosis/fisiopatología , Inflamación/fisiopatología , Calicreínas/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
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.].
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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
The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-ß type I receptor ALK5, but the molecular details of this process have remained unclear. Here, we hypothesized that RAC1B-mediated suppression of activin receptor-like kinase 5 (ALK5) involves proteinase-activated receptor 2 (PAR2), a G protein-coupled receptor encoded by F2RL1 that is crucial for sustaining ALK5 expression. We found in pancreatic carcinoma Panc1 cells that PAR2 is upregulated by TGF-ß1 in an ALK5-dependent manner and that siRNA-mediated knockdown of RAC1B increased both basal and TGF-ß1-induced expression of PAR2. Further, the simultaneous knockdown of PAR2 and RAC1B rescued Panc1 cells from a RAC1B knockdown-induced increase in ALK5 abundance and the ALK5-mediated increase in TGF-ß1-induced migratory activity. Conversely, Panc1 cells with stable ectopic expression of RAC1B displayed reduced ALK5 expression, an impaired upregulation of PAR2, and a reduced migratory responsiveness to TGF-ß1 stimulation. However, these effects could be reversed by ectopic overexpression of PAR2. Moreover, the knockdown of PAR2 alone in Panc1 cells and HaCaT keratinocytes phenocopied RAC1B's ability to suppress ALK5 abundance and TGF-ß1-induced chemokinesis and growth inhibition. Lastly, we found that the RAC1B knockdown-induced increase in TGF-ß1-induced PAR2 mRNA expression was sensitive to pharmacological inhibition of MEK-ERK signaling. Our data show that in pancreatic and skin epithelial cells, downregulation of ALK5 activity by RAC1B is secondary to suppression of F2RL1/PAR2 expression. Since F2RL1 itself is a TGF-ß target gene and its upregulation by TGF-ß1 is mediated by ALK5 and MEK-ERK signaling, we suggest the existence of a feed-forward signaling loop involving ALK5 and PAR2 that is efficiently suppressed by RAC1B to restrict TGF-ß-driven cell motility and growth inhibition.
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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.
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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
The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-ß1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-ß type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of TGFBR1 to induction by TGF-ß1 stimulation. RAC1B KO also increased TGF-ß1-induced C-terminal SMAD3 phosphorylation, SMAD3 transcriptional activity, growth inhibition, and cell migration. The KD of ALK5 expression by RNA interference or inactivation of the ALK5 kinase activity by dominant-negative interference or ATP-competitive inhibition rescued the cells from the RAC1B KD/KO-mediated increase in TGF-ß1-induced cell migration, whereas the ectopic expression of kinase-active ALK5 mimicked this RAC1B KD/KO effect. We conclude that RAC1B downregulates the abundance of ALK5 and SMAD3 signaling, thereby attenuating TGF-ß/SMAD3-driven cellular responses, such as growth inhibition and cell motility.
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HIV-associated neurocognitive disorders (HAND) is a syndrome defined by neurocognitive deficits that are driven by viral neurotoxins, cytokines, free radicals, and proteases expressed in the brain. This neurological disease has also been linked to activation of Protease-Activated Receptors 1 and 2 (PAR1,2). These receptors are highly expressed in the central nervous system and are upregulated in HAND. Secretory basic-amino-acid-specific Proprotein Convertases (PCs), which cleave precursor proteins at basic residues, are also induced in HAND. They are vital for many biological processes including HIV-1 entry into cells. The cytoprotective role of Furin, PC5, and PACE4 has been linked to the presence of a potential PC-cleavage site R41XXXXR46↓ in PAR1. Furthermore, Furin binds PAR1 and both are trapped in the trans-Golgi-network (TGN) as inactive proteins, likely due to the intermediary trafficking role of phospho-Furin acidic cluster sorting protein 1 (PACS1). Nothing is known about PAR2 and its possible recognition by PCs at its putative R31XXXXR36↓ processing site. The present study implicates PACS1 in the retrograde trafficking of PAR1 to the TGN and demonstrates that the cytosolic extreme C-terminal tail of PAR1 contains an acidic phosphorylatable PACS1-sensitive domain. We further show the requirement of Asn47 in PAR1 for its Furin-dependent TGN localization. Our data revealed that Furin is the only convertase that efficiently cleaves PAR2 at Arg36↓. N-glycosylation of PAR2 at Asn30 reduces the efficacy, but enhances selectivity of the Furin cleavage. Finally, in co-cultures comprised of human neuroblastoma SK-N-SH cells (stably expressing PAR1/2 and/or Furin) and HIV-1-infected primary macrophages, we demonstrate that the expression of Furin enhances neuronal cell viability in the context of PAR1- or PAR2-induced neuronal cytotoxicity. The present study provides insights into early stages of HIV-1 induced neuronal injury and the protective role of Furin in neurons co-expressing PAR1 and/or PAR2, as observed in HAND.
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Furina/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Inflamación/virología , Trastornos Neurocognitivos/virología , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Animales , Células CHO , Cricetulus , Células HEK293 , Infecciones por VIH/virología , Humanos , Inflamación/metabolismo , Trastornos Neurocognitivos/metabolismo , TransfecciónRESUMEN
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
Transforming growth factor-ß (TGF-ß), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF-ß1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF-ß receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether Gq-calcium signaling is a prerequisite for PAR2 to enhance TGF-ß signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF-ß1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF-ß1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-Gq-calcium signaling arm failed to suppress TGF-ß1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF-ß1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF-ß1 synergy may involve TGF-ß1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF-ß's prooncogenic function, inhibiting PAR2-Gq-calcium signaling alone would not be sufficient to achieve this effect.
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Señalización del Calcio/fisiología , Movimiento Celular/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células HEK293 , Humanos , Oligopéptidos/farmacología , Receptor PAR-2 , Receptor Tipo I de Factor de Crecimiento Transformador betaRESUMEN
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
UNLABELLED: Plasmodium falciparum malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy. IMPORTANCE: Plasmodium falciparum malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.
Asunto(s)
Adhesión Celular , Interacciones Huésped-Patógeno , Plasmodium falciparum/fisiología , Proteolisis , Proteínas Protozoarias/metabolismo , Trombina/metabolismo , Células Cultivadas , Células Endoteliales/fisiología , Eritrocitos/parasitología , HumanosRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-ß and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-ß and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-ß1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro. Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2-/- mice displayed a greatly attenuated sensitivity to TGF-ß1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-ß type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-ß1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-ß1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-ß-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Movimiento Celular/genética , Neoplasias Pancreáticas/patología , Proteínas Serina-Treonina Quinasas/genética , Receptores Acoplados a Proteínas G/fisiología , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor PAR-2 , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/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.