RESUMEN
Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2ß1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFß1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2ß1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFß1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2ß1 signaling, ahead of establishing Peptide 5 as a potential intervention.
Asunto(s)
Colágeno Tipo I/metabolismo , Conexina 43/metabolismo , Túbulos Renales Proximales/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Adhesión Celular , Línea Celular , Células Cultivadas , Colágeno Tipo I/fisiología , Conexina 43/fisiología , Conexinas/metabolismo , Citocinas , Células Epiteliales/metabolismo , Humanos , Integrina alfa2beta1/metabolismo , Integrina alfa2beta1/fisiología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
This study was designed to investigate (i) extracellular matrix to specify adhesive substrates to human dura mater cell (hDMC); (ii) the alteration on adhesion-related molecules in hDMC; and (iii) secreted matrix metalloproteinases (MMPs) linked with extracellular matrix remodeling after exposure to inflammation. The hDMC was cultured from human dura mater tissue, and the studies were performed with hDMC after co-culturing with macrophage like THP-1 cells (MÏ). The adhesion of co-cultured hDMC through collagen I increased 6.4-fold and through collagen IV increased 5.0-fold compared with the adhesion of naïve cells (p < 0.001). Integrin subtype α2 ß1 expression was increased 6.3-fold (p < 0.001) and α1 expression was decreased 2.0-fold (p < 0.001) in the co-cultured cells compared with the naïve cells. Co-culturing induced significant increases in MMP-1 (13.9-fold, p < 0.01), MMP-3 (7.6-fold, p < 0.01), and VEGF (VEGF: 3.8-fold, p < 0.05) expression and decreases in MMP-9 (0.1-fold, p < 0.01) compared with the sum of naïve hDMC and MÏ values. Increased hDMC adhesion under inflammatory conditions is caused by an increased cellular affinity for collagen I and IV mediated by increased hDMC levels of integrin subtype α2 ß1 and environmental MMP-1, -3 and decreased MMP-9. Selective integrin subtype α2 ß1 inhibition assay showed 37.8% and 35.7% reduction in adhesion of co-cultured hDMC to collagen I (p < 0.001) and IV (p = 0.057), respectively. The present study provides insight into the pathological conditions related to dura mater adhesion in inflammation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-11, 2019.
Asunto(s)
Adhesión Celular , Duramadre/citología , Matriz Extracelular/fisiología , Inflamación/fisiopatología , Metaloproteinasas de la Matriz/metabolismo , Adulto , Anciano , Técnicas de Cocultivo , Colágeno Tipo I/fisiología , Colágeno Tipo IV/fisiología , Duramadre/enzimología , Duramadre/fisiopatología , Femenino , Humanos , Integrina alfa2beta1/fisiología , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Células THP-1RESUMEN
Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of ß1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1ß1-, α2ß1- and α10ß1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.
Asunto(s)
Condrocitos/fisiología , Colágenos Fibrilares/química , Cadenas alfa de Integrinas/química , Integrina alfa1beta1/química , Integrina alfa2beta1/química , Animales , Cartílago Articular/citología , Bovinos , Adhesión Celular , Células Cultivadas , Embrión de Pollo , Receptores con Dominio Discoidina/fisiología , Colágenos Fibrilares/fisiología , Humanos , Proteínas Inmovilizadas/química , Cadenas alfa de Integrinas/fisiología , Integrina alfa1beta1/fisiología , Integrina alfa2beta1/fisiología , Unión ProteicaRESUMEN
Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2ß1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.
Asunto(s)
Plaquetas/efectos de los fármacos , Colágeno/farmacología , Activación Plaquetaria/fisiología , Proteína-Lisina 6-Oxidasa/fisiología , Trombofilia/enzimología , Animales , Plaquetas/citología , Traumatismos de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/etiología , Integrina alfa2beta1/fisiología , Megacariocitos/enzimología , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/farmacología , Adhesividad Plaquetaria/genética , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/genética , Factor Plaquetario 4/genética , Regiones Promotoras Genéticas , Proteína-Lisina 6-Oxidasa/genética , Ratas , Trombofilia/genéticaRESUMEN
Th17 cells are critical effectors in inflammation and tissue damage such as bone erosion, but the mechanisms regulating their activation in this process are not fully understood. In this study, we considered the cooperation between cytokine receptors and integrin pathways in Th17-osteoclast function. We found that human Th17 cells coexpress IL-7R and the collagen-binding integrin α2ß1 (CD49b), and IL-7 increases their adhesion to collagen via α2ß1 integrin. In addition, coengagement of the two receptors in human Th17 cells cooperatively enhanced their IL-17 production and their osteoclastogenic function. The functional cooperation between IL-7R and α2ß1 integrin involves activation of the JAK/PI3K/AKT (protein kinase B) and MAPK/ERK pathways. We also showed that IL-7-induced bone loss in vivo is associated with Th17 cell expansion. Moreover, blockade of α2ß1 integrin with a neutralizing mAb inhibited IL-7-induced bone loss and osteoclast numbers by reducing Th17 cell numbers in the bone marrow and reducing the production of IL-17 and the receptor activator of NF-κB ligand. Thus, the cooperation between IL-7R and α2ß1 integrin can represent an important pathogenic pathway in Th17-osteoclast function associated with inflammatory diseases.
Asunto(s)
Resorción Ósea/etiología , Integrina alfa2beta1/fisiología , Receptores de Interleucina-7/fisiología , Células Th17/fisiología , Adhesión Celular , Polaridad Celular , Colágeno/farmacología , Humanos , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas , Osteoclastos/fisiología , Osteogénesis , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
Phosphatidylinositol 3-kinaseß (PI3Kß) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kß activation downstream of integrin αIIbß3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kß upon recruitment of the two main platelet collagen receptors, integrin α2ß1 and GPVI. PI3Kß-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2ß1, but occurred normally upon GPVI ligation. Integrin α2ß1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kß activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KßKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kß activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kß inhibitor TGX-221. These results outline important differences in the regulation of PI3Kß by GPVI and integrin α2ß1 and suggest that inhibition of Pyk2 may target PI3Kß activation in a selective context of platelet stimulation.
Asunto(s)
Quinasa 2 de Adhesión Focal/fisiología , Integrina alfa2beta1/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Animales , Células Cultivadas , Activación Enzimática , Humanos , Ratones , Ratones Noqueados , Transducción de SeñalRESUMEN
Silencing of α2ß1 integrin expression significantly promoted anchorage-dependent apoptosis (anoikis) and drastically reduced clonal activity of MCF-7 human breast carcinoma cells. Depletion of α2ß1 enhanced the production of apoptotic protein p53 and of inhibitor of cyclin-dependent protein kinases, p27, while downregulating antiapoptotic protein Bcl-2 and multifunctional protein cMyc. Blocking the expression of α2ß1 had no effect on activity of protein kinase Akt, but it sharply increased the kinase activity of Erk1/2. Pharmacological inhibition of Erk1/2 had a minor effect on anoikis of control cells, while it reduced anoikis of cells with downregulated α2ß1 to the level of control cells. The data show for the first time that integrin α2ß1 is implicated in the protection of tumor cells from anoikis through a mechanism based on the inhibition of protein kinase Erk.
Asunto(s)
Anoicis , Neoplasias de la Mama/metabolismo , Integrina alfa2beta1/fisiología , Neoplasias de la Mama/enzimología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Células MCF-7 , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The α2ß1 integrin, also known as VLA-2, GPIa-IIa, CD49b, was first identified as an extracellular matrix receptor for collagens and/or laminins [55, 56]. It is now recognized that the α2ß1 integrin serves as a receptor for many matrix and nonmatrix molecules [35, 79, 128]. Extensive analyses have clearly elucidated the α2 I domain structural motifs required for ligand binding, and also defined distinct conformations that lead to inactive, partially active or highly active ligand binding [3, 37, 66, 123, 136, 137, 140]. The mechanisms by which the α2ß1 integrin plays a critical role in platelet function and homeostasis have been carefully defined via in vitro and in vivo experiments [76, 104, 117, 125]. Genetic and epidemiologic studies have confirmed human physiology and disease states mediated by this receptor in immunity, cancer, and development [6, 20, 21, 32, 43, 90]. The role of the α2ß1 integrin in these multiple complex biologic processes will be discussed in the chapter.
Asunto(s)
Integrina alfa2beta1/fisiología , Animales , Hemostasis , Humanos , Inmunidad Innata , Integrina alfa2beta1/química , Neovascularización Fisiológica , Estructura Terciaria de Proteína , Transducción de Señal , Cicatrización de HeridasRESUMEN
Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2ß1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2ß1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2ß1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2ß1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2ß1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor-related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2ß1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/terapia , Artritis Reumatoide/metabolismo , Integrina alfa2beta1/fisiología , Osteólisis/prevención & control , Receptores de Colágeno/fisiología , Células Th17/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Cartílago Articular/patología , Colágeno/farmacología , Cricetinae , Regulación hacia Abajo , Femenino , Humanos , Inflamación , Integrina alfa2beta1/antagonistas & inhibidores , Interleucina-17/sangre , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos DBA , FN-kappa B/fisiología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Osteoclastos/patología , Osteólisis/etiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Ligando RANK/sangre , Receptores de Colágeno/antagonistas & inhibidores , Transducción de Señal , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Células Th17/fisiologíaRESUMEN
BACKGROUND: Keloid disease is a fibroproliferative disorder, with an ill-defined treatment that is characterized by excessive extracellular matrix deposition. Mechanical tension promotes deposition of extracellular matrix and overexpression of tension-related proteins, which is associated with keloid disease. The aim of this study was to investigate the effect of tension-related proteins on extracellular matrix steady-state synthesis in primary keloid fibroblasts. METHODS: Keloid fibroblasts (n = 10) and normal skin (n = 4) fibroblast cultures were established from passages 0 to 3. A panel of 21 tension-related genes from microarray data were assessed at mRNA (quantitative reverse-transcriptase polymerase chain reaction) and protein (in-cell Western blotting) levels. Three genes were significantly altered in keloid tissue and fibroblasts, and their functional role was assessed using siRNA knockdown. RESULTS: Hsp27, α2ß1-integrin, and PAI-2 were significantly up-regulated (p < 0.05)in keloid tissue and fibroblasts compared with normal skin. Hsp27, α2ß1-integrin, and PAI-2 expression was inhibited by RNA interference. Both the mRNA and protein levels of Hsp27, α2ß1-integrin, and PAI-2 significantly decreased (p < 0.05) in keloid fibroblasts at 48 hours after transfection. After down-regulation of Hsp27, α2ß1-integrin, and PAI-2, the expression of intracellular extracellular matrix was significantly reduced (p < 0.05). Water-soluble tetrazolium salt-1 assay showed that transfection of Hsp27, α2ß1-integrin, and PAI-2 did not influence the viability/metabolic activity of keloid fibroblasts. CONCLUSIONS: This study demonstrates overexpression of key tension-related proteins in keloid tissue and keloid fibroblasts. Knockdown of Hsp27, PAI-2, and α2ß1-integrin by RNA interference attenuates the expression of mRNA and protein levels and certain other extracellular matrix molecules.
Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP27/fisiología , Integrina alfa2beta1/fisiología , Queloide/metabolismo , Inhibidor 2 de Activador Plasminogénico/fisiología , Regulación hacia Arriba , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK α2ß1 develop normally, are fertile, and like their systemic α2ß1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet α2ß1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of α2ß1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of α2 integrin in selected cell types.
Asunto(s)
Integrina alfa2beta1/fisiología , Megacariocitos/metabolismo , Recuento de Plaquetas , Animales , Secuencia de Bases , Western Blotting , Adhesión Celular , Colágeno Tipo I/metabolismo , Cartilla de ADN , Técnicas de Inactivación de Genes , Integrina alfa2beta1/genética , Megacariocitos/citología , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin α2ß1 stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we sought to determine whether α2ß1 integrin is a potential mediator of skeletal metastasis. To examine whether α2ß1 integrin mediates PCa metastasis, α2 integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaPcol PCa cells. We document that the over-expression of α2 cDNA stimulated whereas α2 shRNA inhibited the ability of transduced cells to bind to or migrate towards collagen in vitro. Correspondingly, α2 integrin knock-down reduced the tumor burden of intra-osseous tumors compared to control-transduced cells. To investigate the clinical significance of α2ß1 expression in PCa, α2ß1 protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that α2ß1 protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that α2ß1 contributes to the selective metastasis to the bone. Taken together, these data support that α2ß1 integrin is needed for the efficient metastasis of PCa cells to the skeleton.
Asunto(s)
Neoplasias Óseas/secundario , Integrina alfa2beta1/fisiología , Neoplasias de la Próstata/patología , Secuencia de Bases , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Humanos , Inmunohistoquímica , Masculino , Neoplasias de la Próstata/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Guided cell migration is important in tissue development, repair, and engineering. We have previously demonstrated that applied electric fields (EFs) enhanced and directed ligament fibroblast migration and collagen production, depending on EF parameters. Electrical stimulation is widely used for the treatment of pain and to promote wound healing. In orthopaedic practices, applied EFs promote bone healing and ligament repair in vivo. In the current study, stimulation waveforms used in physical therapy for promoting tissue repair were adapted to examine their effects on ACL fibroblast migration. Using different waveform and field strengths, we discovered a decoupling of cell motility and directionality, which suggests disparate mechanisms. Integrin, a major extracellular matrix receptor, polarized in response to applied EFs and controlled cell directionality and signaling. Furthermore, we demonstrated that RhoA is a mediator between integrin aggregation and directed cell migration. Polarization is essential in directed cell migration and our study establishes an outside-in signaling mechanism for EF-induced cell directionality.
Asunto(s)
Ligamento Cruzado Anterior/citología , Movimiento Celular/fisiología , Integrina alfa2beta1/fisiología , Cicatrización de Heridas/fisiología , Proteína de Unión al GTP rhoA/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología , Estimulación Eléctrica , Fibroblastos/fisiología , Porcinos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
UNLABELLED: Asthma development may be driven by T helper lymphocytes with eosinophils playing the role of major effector cells. Recruitment of the inflammatory cells from blood to the airways is mediated by adhesive molecules, e.g. selectins and integrins. The most important in cell trafficking are integrins containing α(4) and ß(2) subunits. We hypothesized that also collagen receptors: α(1)ß(1) and α(2)ß(1), may be involved in cell migration to the inflammatory site in asthma. The aim of the study was to determine whether the inhibition of α(1)ß(1) or α(2)ß(1) integrins, affects transmigration of eosinophils and peripheral blood mononuclear cells (PBMC) through human microvascular endothelial cells monolayer (HMVEC) seeded on collagen IV coated wells in moderate persistent atopic asthmatics. METHODS: PBMC from 9 asthmatics were separated by gradient centrifugation followed by negative magnetic separation of eosinophils. Snake venom derived anti-adhesive proteins: viperistatin and VP12 (potent and selective inhibitors of α(1)ß(1) and α(2)ß(1) integrins, respectively) as well as VLO4 (a non-selective inhibitor of α(4)ß(1), α(5)ß(1) and α(v)ß(3) - used as a positive control), were used for inhibition studies. All anti-adhesive proteins studied, inhibited eosinophils, but only VLO4 affected PBMC transmigration through HMVEC. In bronchial asthma both collagen receptors α(1)ß(1) and α(2)ß(1) are likely to be involved in eosinophil transmigration to the inflammatory site. The role of α(2)ß(1) on lymphocytes is probably different. As the α(2)ß(1) integrin has been described as a stimulator of collagen accumulation, it might be, at least in part, responsible for asthma airway remodelling.
Asunto(s)
Eosinófilos/fisiología , Integrina alfa1beta1/fisiología , Integrina alfa2beta1/fisiología , Migración Transendotelial y Transepitelial/fisiología , Adulto , Línea Celular , Células Cultivadas , Células Endoteliales/fisiología , Eosinófilos/efectos de los fármacos , Femenino , Humanos , Integrina alfa1beta1/antagonistas & inhibidores , Integrina alfa2beta1/antagonistas & inhibidores , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Masculino , Microvasos/fisiología , Venenos de Serpiente/farmacología , Migración Transendotelial y Transepitelial/efectos de los fármacosRESUMEN
Vascular morphogenesis is a key process for development, reproduction, and pathogenesis. Thus understanding the mechanisms of this process is of pathophysiological importance. Despite the fact that collagen I is the most abundant and potent promorphogenic molecule known, the molecular mechanisms by which this protein regulates endothelial cell tube morphogenesis are still unclear. Here we provide strong evidence that collagen I induces tube morphogenesis by inhibiting glycogen synthase kinase 3ß (GSK3ß). Further mechanistic studies revealed that GSK3ß activity is regulated by protein kinase D (PKD). PKD inhibited GSK3ß activity, which was required for collagen I-induced endothelial tube morphogenesis. We also found that GSK3ß regulated trafficking of integrin α(2)ß(1) in a Rab11-dependent manner. Taken together, our studies highlight the important role of PKD in the regulation of collagen I-induced vascular morphogenesis and show that it is mediated by the modulation of GSK3ß activity and integrin α(2)ß(1) trafficking.
Asunto(s)
Células Endoteliales/enzimología , Glucógeno Sintasa Quinasa 3/fisiología , Integrina alfa2beta1/fisiología , Morfogénesis/fisiología , Proteína Quinasa C/fisiología , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Transporte de Proteínas/fisiología , Transducción de SeñalRESUMEN
At sites of vascular injury, collagen-mediated platelet adhesion and activation have long been known as one of the first events in platelet-dependent thrombus formation. Studying patients with bleeding disorders that are caused by defective platelet adhesion to collagen resulted in the identification of several platelet collagen receptors, with glycoprotein VI and integrin α2ß1 being the most important ones. Subsequent development of specific collagen receptor knockout mice and various inhibitors of platelet binding to collagen have further proven the role of these receptors in haemostasis and thrombosis. The search for clinically applicable inhibitors for use as antithrombotic drug has led to the identification of inhibitory antibodies, soluble receptor fragments, peptides, collagen-mimetics and proteins from snake venoms or haematophagous animals. In experimental settings, these inhibitors have a good antithrombotic effect, with little prolongation of bleeding times, suggesting a larger therapeutic window than currently available antiplatelet drugs. However, at present, none of the collagen receptor blockers are in clinical development yet.
Asunto(s)
Colágeno/antagonistas & inhibidores , Integrina alfa2beta1/antagonistas & inhibidores , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Humanos , Integrina alfa2beta1/química , Integrina alfa2beta1/fisiología , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/fisiología , Trombosis/tratamiento farmacológicoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α(2)ß(1) integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3ß and high levels of active ß-catenin in the nucleus. Knockdown of ß-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α(2)ß(1) integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3ß and active ß-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α(2)ß(1) integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the ß-catenin pathway.
Asunto(s)
Fibroblastos/patología , Fibrosis Pulmonar Idiopática/metabolismo , Integrina alfa2beta1/fisiología , beta Catenina/metabolismo , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Activación Enzimática , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Fibrosis Pulmonar Idiopática/patología , Integrina alfa2beta1/antagonistas & inhibidores , Integrina alfa2beta1/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Fosforilación , Proteína Fosfatasa 2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages, known as pili, have been recently described in streptococcal pathogens, including GBS. The pilus tip adhesin, PilA, contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the host receptor and the contribution of PilA in central nervous system (CNS) disease pathogenesis are unknown. Here we show that PilA binds collagen, which promotes GBS interaction with the α2ß1 integrin resulting in activation of host chemokine expression and neutrophil recruitment during infection. Mice infected with the PilA-deficient mutant exhibit delayed mortality, a decrease in neutrophil infiltration and bacterial CNS dissemination. We find that PilA-mediated virulence is dependent on neutrophil influx as neutrophil depletion results in a decrease in BBB permeability and GBS-BBB penetration. Our results suggest that the bacterial pilus, specifically the PilA adhesin, has a dual role in immune activation and bacterial entry into the CNS.
Asunto(s)
Barrera Hematoencefálica , Fimbrias Bacterianas , Integrina alfa2beta1/fisiología , Streptococcus agalactiae/fisiología , Animales , Adhesión Bacteriana , Quimiocinas/inmunología , Quimiotaxis de Leucocito , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Interleucina-8/metabolismo , Meningitis Bacterianas/inmunología , Meningitis Bacterianas/microbiología , Ratones , Neutrófilos/inmunología , Transducción de Señal , Streptococcus agalactiae/inmunologíaRESUMEN
Platelets play a central role in maintaining hemostasis mainly by binding to subendothelial collagen exposed upon vascular injury, thereby initiating thrombus formation. Platelets can bind directly to the exposed collagen through two major receptors i.e. the integrin a2b1 and glycoprotein (GP) VI. However, under high shear conditions the GPIb-V-IX receptor complex and its main ligand von Willebrand Factor are additionally needed for firm platelet adhesion to the vessel wall. In this review, we summarize the current knowledge on the individual roles and structure-function relationships of these main platelet adhesion receptors.
Asunto(s)
Colágeno/fisiología , Adhesividad Plaquetaria/fisiología , Endotelio Vascular/fisiología , Hemostasis/fisiología , Humanos , Integrina alfa2beta1/química , Integrina alfa2beta1/fisiología , Modelos Moleculares , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/fisiología , Estructura Terciaria de Proteína , Trombosis/sangre , Trombosis/etiología , Trombosis/fisiopatología , Factor de von Willebrand/química , Factor de von Willebrand/fisiologíaRESUMEN
Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that ß1 integrins may stimulate metastasis of a number of cancers, expression of the ß1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2ß1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2ß1 integrin in normal breast epithelium and loss of α2ß1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or ß1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2ß1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.