RESUMEN
Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNγ) is centrally involved in intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFNγ to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFNγ-induced signalling cascade. AQP3 promoter regions involved in the response to IFNγ were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFNγ to assess for changes in AQP3 mRNA expression. IFNγ decreased AQP3 mRNA expression in HT-29 cells in a time- and concentration-dependent manner and reduced functional AQP3 protein expression (decreased 3H-labelled glycerol uptake). IFNγ also reduced AQP3 expression in enteroids derived from human colonic biopsies. Knockdown of STAT1 partially prevented the IFNγ-induced downregulation of AQP3 expression, whereas STAT3 and Sp3 knockdowns resulted in increased baseline expression of AQP3 but did not alter IFNγ-induced downregulation. Constitutive transcription of AQP3 is downregulated by IFNγ as demonstrated using the luciferase reporter system, with Sp3 bound to the AQP3 promoter as shown by chromatin immunoprecipitation. AQP3 constitutive transcription in intestinal epithelial cells is downregulated by IFNγ. This response requires STAT1 that is postulated to drive the downregulation of AQP3 expression through increased acetylation or decreased deacetylation the AQP3 promoter, ultimately resulting in decreased constitutive transcription of AQP3. KEY MESSAGES: ⢠IFNγ suppresses the expression of AQP3 in intestinal epithelial cells. ⢠Proximal AQP3 promoter elements are sufficient to drive constitutive expression and mediate the IFNγ-induced downregulation of AQP3 mRNA expression. ⢠IFNγ-induced suppression of AQP3 is dependent upon STAT1 expression, but not STAT3, Sp1, or Sp3.
Asunto(s)
Acuaporina 3/genética , Interferón gamma/farmacología , Mucosa Intestinal/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Excessive accumulation of neutrophils and their uncontrolled death by necrosis at the site of inflammation exacerbates inflammatory responses and leads to self-amplifying tissue injury and loss of organ function, as exemplified in a variety of respiratory diseases. In homeostasis, neutrophils are inactivated by apoptosis, and non phlogistically removed by neighboring macrophages in a process known as efferocytosis, which promotes the resolution of inflammation. The present study assessed the potential anti-inflammatory and pro-resolution benefits of tylvalosin, a recently developed broad-spectrum veterinary macrolide derived from tylosin. Recent findings indicate that tylvalosin may modulate inflammation by suppressing NF-κB activation. Neutrophils and monocyte-derived macrophages were isolated from fresh blood samples obtained from 12- to 22-week-old pigs. Leukocytes exposed to vehicle or to tylvalosin (0.1, 1.0, or 10 µg/mL; 0.096-9.6 µM) were assessed at various time points for apoptosis, necrosis, efferocytosis, and changes in the production of cytokines and lipid mediators. The findings indicate that tylvalosin increases porcine neutrophil and macrophage apoptosis in a concentration- and time-dependent manner, without altering levels of necrosis or reactive oxygen species production. Importantly, tylvalosin increased the release of pro-resolving Lipoxin A4 (LXA4) and Resolvin D1 (RvD 1 ) while inhibiting the production of pro-inflammatory Leukotriene B4 (LTB4) in Ca2+ ionophore-stimulated porcine neutrophils. Tylvalosin increased neutrophil phospholipase C activity, an enzyme involved in releasing arachidonic acid from membrane stores. Tylvalosin also inhibited pro-inflammatory chemokine (C-X-C motif) ligand 8 (CXCL-8, also known as Interleukin-8) and interleukin-1 alpha (IL-1α) protein secretion in bacterial lipopolysaccharide-stimulated macrophages. Together, these data illustrate that tylvalosin has potent immunomodulatory effects in porcine leukocytes in addition to its antimicrobial properties.
RESUMEN
Inflammatory diseases of the gut are associated with altered electrolyte and water transport, leading to the development of diarrhea. Epithelially expressed aquaporins (AQPs) are downregulated in inflammation, although the mechanisms involved are not known. We hypothesized that AQP3 expression in intestinal epithelial cells is altered in intestinal inflammation and that these changes are driven by tumor necrosis factor (TNF) α Human colonic adenocarcinoma (HT-29) cells were treated with TNFα to investigate signaling mechanisms in vitro. AQP3 expression was assessed by real-time PCR and radiolabeled glycerol uptake, with select inhibitors and a luciferase reporter construct used to further elucidate intracellular signaling. AQP3 expression was downregulated in HT-29 cells treated with TNFα Luciferase reporter construct experiments revealed that TNFα downregulated constitutive transcriptional activity of the AQP3 promoter, and inhibition of MEK/ERK and nuclear factor κB (NF-κB) signaling prevented the decrease in AQP3 mRNA expression. Constitutive AQP3 expression was suppressed by specificity protein (Sp) 3, and knockdown of this transcription factor bound to the AQP3 promoter was able to partially prevent the TNFα-induced downregulation of AQP3. TNFα signals through MEK/ERK and NF-κB to enhance the negative transcriptional control of AQP3 expression exerted by Sp3. Similar mechanisms regulate numerous ion channels, suggesting a common mechanism by which both ion and water transport are altered in inflammation.
Asunto(s)
Acuaporina 3/metabolismo , Enterocitos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Acuaporina 3/genética , Enterocitos/efectos de los fármacos , Células HT29 , Humanos , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción Sp3/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
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.
Asunto(s)
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
The accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4 (LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4 [LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytes in vitro and in Mannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammation in vivo and characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4 and prostaglandin E2 (PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2 (PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4 in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Dinoprostona/antagonistas & inhibidores , Disacáridos/farmacología , Compuestos Heterocíclicos/farmacología , Leucotrieno B4/antagonistas & inhibidores , Lipoxinas/agonistas , Fosfolipasas A2/metabolismo , Neumonía/tratamiento farmacológico , Animales , Líquido del Lavado Bronquioalveolar/química , Calcimicina/farmacología , Bovinos , Dinoprostona/biosíntesis , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/biosíntesis , Lipoxinas/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Material Particulado , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/patología , Cultivo Primario de Células , ZimosanRESUMEN
The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow-derived macrophages, but not dendritic cells, to process and present the I-A(b)-immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-A(b) binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-A(b) mice that were deficient in the p47(phox) or gp91(phox) subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4(+) T cells in the CNS, despite eliciting a normal primary CD4(+) T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4(+) T cell-driven autoimmune disease processes.
Asunto(s)
Células de la Médula Ósea/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/inmunología , NADPH Oxidasas/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Células de la Médula Ósea/patología , Linfocitos T CD4-Positivos , Catepsina L/genética , Catepsina L/inmunología , Catepsinas/genética , Catepsinas/inmunología , Línea Celular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Epítopos de Linfocito T/genética , Antígenos de Histocompatibilidad Clase II/genética , Macrófagos/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/inmunología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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 compared signalling pathways such as calcium transients, MAPK activation, ß-arrestin interactions and receptor internalization triggered by kallikrein-related peptidases (KLKs) 8 and 14 in human and rat proteinase-activated receptor (PAR)2-expressing human embryonic kidney (HEK) and Kirsten transformed rat kidney (KNRK) cells. Further, we analysed processing by KLK8 vs. KLK14 of synthetic human and rat PAR2-derived sequences representing the cleavage-activation domain of PAR2. Our data show that like KLK14, KLK8 can unmask a PAR2 receptor-activating sequence from a peptide precursor. However, whilst KLK8, like KLK14, can signal in rat-PAR2-expressing KNRK cells, this enzyme cannot signal via human PAR2 in HEK or KNRK cells, but rather, disarms HEK PAR1. Thus, KLK8 and KLK14 can signal differentially via the PARs to affect tissue function.
Asunto(s)
Calicreínas/metabolismo , Receptor PAR-2/metabolismo , Transducción de Señal , Animales , Células HEK293 , Humanos , Transporte de Proteínas , RatasRESUMEN
Proteinase-activated receptor (PAR)(2), a G protein-coupled receptor activated by serine proteinases, has been implicated in both intestinal inflammation and epithelial proliferation. Cyclooxygenase (COX)-2 is overexpressed in the gut during inflammation as well as in colon cancer. We hypothesized that PAR(2) drives COX-2 expression in intestinal epithelial cells. Treatment of Caco-2 colon cancer cells with the PAR(2)-activating peptide 2-furoyl-LIGRLO-NH(2) (2fLI), but not by its reverse-sequence PAR(2)-inactive peptide, for 3 h led to an increase in intracellular COX-2 protein expression accompanied by a COX-2-dependent increase in prostaglandin E(2) production. 2fLI treatment for 30 min significantly increased metalloproteinase activity in the culture supernatant. Increased epidermal growth factor receptor (EGFR) phosphorylation was observed in cell lysates following 40 min of treatment with 2fLI. The broad-spectrum metalloproteinase inhibitor marimastat inhibited both COX-2 expression and EGFR phosphorylation. The EGFR tyrosine kinase inhibitor PD153035 also abolished 2fLI-induced COX-2 expression. Although PAR(2) activation increased ERK MAPK phosphorylation, neither ERK pathway inhibitors nor a p38 MAPK inhibitor affected 2fLI-induced COX-2 expression. However, inhibition of either Src tyrosine kinase signaling by PP2, Rho kinase signaling by Y27632, or phosphatidylinositol 3 (PI3) kinase signaling by LY294002 prevented 2fLI-induced COX-2 expression. Trypsin increased COX-2 expression through PAR(2) in Caco-2 cells and in an EGFR-dependent manner in the noncancerous intestinal epithelial cell-6 cell line. In conclusion, PAR(2) activation drives COX-2 expression in Caco-2 cells via metalloproteinase-dependent EGFR transactivation and activation of Src, Rho, and PI3 kinase signaling. Our findings provide a mechanism whereby PAR(2) can participate in the progression from chronic inflammation to cancer in the intestine.
Asunto(s)
Ciclooxigenasa 2/biosíntesis , Células Epiteliales/metabolismo , Receptores ErbB/fisiología , Mucosa Intestinal/metabolismo , Receptor PAR-2/fisiología , Anfirregulina , Western Blotting , Células CACO-2 , Línea Celular , Cromatografía Líquida de Alta Presión , Ciclooxigenasa 2/genética , Dinoprostona/metabolismo , Familia de Proteínas EGF , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/genética , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/citología , Metaloproteinasas de la Matriz/biosíntesis , Fosfatidilinositol 3-Quinasa/fisiología , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/fisiología , Activación Transcripcional/efectos de los fármacos , Tripsina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasas Asociadas a rho/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.
Asunto(s)
Receptores Proteinasa-Activados/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Silenciador del Gen/efectos de los fármacos , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Ratas , Receptores Proteinasa-Activados/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/química , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ß-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.
Asunto(s)
Señalización del Calcio/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Receptor PAR-2/agonistas , Receptor PAR-2/metabolismo , Animales , Arrestinas/genética , Arrestinas/metabolismo , Señalización del Calcio/efectos de los fármacos , Catepsina G/genética , Catepsina G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Elastasa de Leucocito , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mieloblastina/genética , Mieloblastina/metabolismo , Péptidos/farmacología , Estructura Terciaria de Proteína , Ratas , Receptor PAR-2/genética , beta-ArrestinasRESUMEN
Epithelial permeability to ions and larger molecules in the gut is essential for fluid balance, and its dysregulation contributes to intestinal pathology. We investigated the effect of digestive serine proteases on epithelial paracellular permeability. Trypsin, chymotrypsin, and elastase elicited sustained increases in transepithelial resistance (R(TE)) in polarized monolayers of three intestinal epithelial cell lines. This effect was reflected by decreases in paracellular conductances of Na+ and Cl- and a concomitant decrease in permeability to 3,000 molecular weight dextran. The enzyme activities of the proteases were required, yet activators of known protease-activated receptors (PARs) did not reproduce the effect of these proteases on R(TE). PKCzeta isoform-specific inhibitor significantly reduced the trypsin-induced increase in R(TE) whereas PKCzeta activity was increased in cells treated with trypsin and chymotrypsin compared with control cells; this activity was reduced to control levels in the presence of PKCzeta-specific inhibitor. Ca2+ chelators and pharmacological inhibitors of cell signaling support the role for PKCzeta in the protease-induced effect. Finally, we showed that treatment with the serine proteases increased occludin immunostaining and zonula occludin-1 coimmunoprecipitation with occludin in the detergent-insoluble fraction of cell lysates, and these increases were ablated by pretreatment with PKCzeta-specific inhibitor. This finding indicates increased insertion of occludin into the cell junctional complex. These data demonstrate a role for serine proteases in the facilitation of epithelial barrier function through a mechanism that is independent of PARs and is mediated by activation of PKCzeta.
Asunto(s)
Cloruros/metabolismo , Células Epiteliales/enzimología , Intestinos/enzimología , Proteína Quinasa C/metabolismo , Serina Endopeptidasas/metabolismo , Sodio/metabolismo , Uniones Estrechas/enzimología , Animales , Células CACO-2 , Polaridad Celular , Quimotripsina/metabolismo , Dextranos/metabolismo , Perros , Regulación hacia Abajo , Impedancia Eléctrica , Activación Enzimática , Células Epiteliales/efectos de los fármacos , Humanos , Intestinos/efectos de los fármacos , Transporte Iónico , Cinética , Proteínas de la Membrana/metabolismo , Ocludina , Elastasa Pancreática/metabolismo , Permeabilidad , Fosfoproteínas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Transducción de Señal , Trombina/metabolismo , Uniones Estrechas/efectos de los fármacos , Tripsina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
We constructed a random library of hexapeptides displayed on the surface of bacteriophage T7 to determine the substrate specificity of proteinases. The phage-displayed library was subjected to repeated rounds of biopanning with native implantation serine proteinase and recombinant human kallikrein-related peptidase 6 (KLK6) followed by selection and identification of putative substrates. For both enzymes, the results obtained demonstrate a preference for arginine and lysine at multiple positions in the recognition cleavage motif, confirming their previously reported trypsin-like substrate specificity. In the case of KLK6, there is also a pronounced presence of tryptophan within the cleaved peptide sequences, indicating its potential dual substrate specificity, acting as both a trypsin and chymotrypsin-like enzyme.
Asunto(s)
Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Bacteriófago T7/genética , Cromatografía Líquida de Alta Presión , Humanos , Ratones , Biblioteca de Péptidos , Serina Endopeptidasas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , VolumetríaRESUMEN
The proteinase-activated receptor-2 (PAR2)-activating peptide with an N-terminal furoyl group modification, 2-furoyl-LIGRLO-NH2 (2fLI), was derivatized via its free ornithine amino group to yield [3H]propionyl-2fLI and Alexa Fluor 594-2fLI that were used as receptor probes for ligand binding assays and receptor visualization both for cultured cells in vitro and for colonic epithelial cells in vivo. The binding of the radiolabeled and fluorescent PAR2 probes was shown to be present in PAR2-transfected Kirsten normal rat kidney cells, but not in vector-alone-transfected cells, and was abolished by pretreatment of cells with saturating concentrations of receptor-selective PAR2 peptide agonists such as SLIGRL-NH2 and the parent agonist 2fLI but not by reverse-sequence peptides such as 2-furoyl-OLRGIL-NH2 that cannot activate PAR2. The relative orders of potencies for a series of PAR2 peptide agonists to compete for the binding of [3H]propionyl-2fLI (2fLI >> SLIGRL-NH2 approximately= trans-cinnamoyl-LIGRLO-NH2 > SLIGKV-NH2 > SLIGKT-NH2) mirrored qualitatively their relative potencies for PAR2-mediated calcium signaling in the same cells or for vasorelaxation in a rat aorta vascular assay. In the vascular assay, the potency of Alexa Fluor 594-2fLI was the same as 2fLI. We conclude that ornithine-derivatized 2fLI peptides are conveniently synthesized PAR2 probes that will be of value for future studies of receptor binding and visualization.