RESUMEN
Integrins are transmembrane adhesion proteins that convey critical topobiological information and exert crucial signalling functions. In skin and hair follicle biology, beta1 integrins and their ligands are of particular interest. It is not yet known whether beta1 integrins play any role in the regulation of human hair growth and the expression pattern of beta1 integrin in the human pilosebaceous unit remains ill-defined. Here, we show that pilosebaceous immunoreactivity for beta1 integrin is most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath of human scalp hair follicles in situ and in vitro. Sites of beta1 integrin immunoreactivity co-express fibronectin and tenascin-C. Contrary to previous reports, beta1 integrin immunoreactivity in situ was not significantly upregulated in the human bulge region. Functionally, two beta1 integrin-activating antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp hair follicles in vitro and inhibited spontaneous hair follicle regression. This supports the concept that beta1 integrin-mediated signalling is also important in human hair growth control. The physiologically relevant organ culture assay employed here is a potential research tool for exploring whether targeted stimulation of beta1 integrin-mediated signalling is a suitable candidate for human hair loss management.
Asunto(s)
Folículo Piloso/metabolismo , Integrina beta1/metabolismo , Transducción de Señal/fisiología , Anticuerpos/farmacología , Bioensayo/métodos , Células Cultivadas , Tejido Conectivo/metabolismo , Tejido Conectivo/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Femenino , Fibronectinas/metabolismo , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Cabello/metabolismo , Folículo Piloso/efectos de los fármacos , Folículo Piloso/ultraestructura , Humanos , Inmunohistoquímica , Integrina beta1/efectos de los fármacos , Integrina beta1/genética , Ligandos , Técnicas de Cultivo de Órganos , Péptidos/farmacología , Tenascina/metabolismoRESUMEN
For many years the extracellular matrix was viewed as a benign scaffold for arranging cells within connective tissues, but it is now being redefined as a dynamic, mobile, and flexible key player in defining cellular behavior. Gene targeting, transgene expression, and spontaneous mutations of extracellular matrix proteins in mice have greatly accelerated our mechanistic view of the structural and instructive functions of the extracellular matrix in developmental and regenerative processes. This review summarizes the phenotypes of genetic mouse models carrying mutations in extracellular matrix proteins, with specific emphasis on recent advances. The application of reverse genetics has demonstrated the multifunctionality of matrix proteins in a biological context and, in addition, has brought a novel perspective to the understanding of human pathologies.
Asunto(s)
Matriz Extracelular/metabolismo , Mutación/genética , Animales , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Neovascularización Fisiológica , Transducción de SeñalAsunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Integrina beta1/fisiología , Animales , Cartílago/metabolismo , Adhesión Celular , Movimiento Celular , Células Madre Hematopoyéticas/citología , Humanos , Integrina beta1/metabolismo , Ratones , Músculos/embriología , Neoplasias/metabolismo , Piel/metabolismoRESUMEN
cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.
Asunto(s)
Cóclea/fisiología , Colágeno Tipo IX/fisiología , Audición/fisiología , Procolágeno/fisiología , Animales , Umbral Auditivo/fisiología , Northern Blotting/métodos , Cóclea/citología , Colágeno Tipo II/metabolismo , Colágeno Tipo IX/deficiencia , Pruebas Auditivas/métodos , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procolágeno/deficiencia , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The integrin family of extracellular matrix receptors regulates many aspects of cell life, in particular cell adhesion and migration. These two processes depend on organization of the actin cytoskeleton into adhesive and protrusive organelles in response to extracellular signals. Integrins are important switch points for the spatiotemporal control of actin-based motility in higher eukaryotes. Ligands of integrin cytoplasmic tails are central elements of signalling pathways involving small GTPases as well as protein and lipid kinases in the regulation of Factin crosslinking, actin treadmilling and de novo nucleation of actin filaments. We present an overview of common pathways and discuss recent evidence for their differential use by individual integrin receptors.
Asunto(s)
Actinas/metabolismo , Integrinas/metabolismo , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Modelos Biológicos , Unión Proteica , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5-9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells.
Asunto(s)
Aorta/embriología , Endotelio Vascular/embriología , Células Madre Hematopoyéticas/metabolismo , Mucinas/metabolismo , Animales , Agregación Celular , Diferenciación Celular , Embrión de Mamíferos/metabolismo , Epítopos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Inmunohistoquímica , Ratones/embriología , Mucinas/inmunología , SialomucinasRESUMEN
Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4-6, that flank a globular L4m module containing the cleavage site. Recombinant analysis of L4m, which includes an additional imperfect LE module essential for proper folding, demonstrated an unusual pattern of disulfide bonding. These connectivities prevented the release of gamma2LE1-3L4 m after BMP-1 cleavage which required in addition disulfide reshuffling by isomerases. The liberated segment bound through its L4 m module to heparin, nidogen-1, fibulin-1 and fibulin-2. A further heparin/sulfatide-binding site could be attributed to some arginine residues in module LE1. The gamma2LE4-6 segment remaining in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Heparina/metabolismo , Glicoproteínas de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Línea Celular , Disulfuros/química , Disulfuros/metabolismo , Esófago/química , Humanos , Sueros Inmunes/inmunología , Isomerismo , Ligandos , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Subunidades de Proteína , Conejos , Piel/química , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , KalininaRESUMEN
The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.
Asunto(s)
Epidermis/fisiología , Epidermis/ultraestructura , Glicoproteínas de Membrana/metabolismo , Envejecimiento , Alopecia/patología , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Diferenciación Celular , División Celular , Dermatitis/patología , Desmocolinas , Desmosomas/química , Desmosomas/metabolismo , Epidermis/patología , Párpados/patología , Marcación de Gen , Inmunohistoquímica , Integrina beta4 , Queratinas/metabolismo , Antígeno Ki-67/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Fenotipo , Isoformas de Proteínas , Recombinación Genética , Enfermedades de la Piel/patologíaRESUMEN
OBJECTIVE: The extracellular matrix (ECM) of hyaline cartilage contains an elaborated collagen fibrillar network, which is essential for the mechanical stability and the proper function of the tissue. Cartilage collagen fibrils consist of collagen II, the quantitatively minor collagens IX and XI, and several non-collagenous fibril-associated proteins. To understand the role some of these molecules in skeletal development, we have generated transgenic mouse strains harboring ablated genes for collagens II and IX, and matrilin-1. DESIGN: Mice lacking collagen II, collagen IX and matrilin-1 have been established earlier in our laboratory using standard techniques. To determine the consequences of the null mutations we used skeletal staining, histochemical and immunohistochemical assays, in situ hybridization and ultrastructural analysis. RESULTS: Transgenic mice deficient in collagen II (Col2a1-/-) die at birth and display a severely malformed skeleton characterized by abnormal endochondral ossification and impaired intervertebral disc development. Mice lacking collagen IX (Col9a1-/-) are viable and develop an osteoarthritis-like phenotype in knee joints between 9-12 months of age. To test the possibility that the reduction in collagen II content has an influence on the onset of degenerative changes of articular cartilage, we have generated mice, which are heterozygous for the collagen II null mutation and homozygous for the collagen IX null mutation. Col2a1+/- Col9a1-/- mice show no accelerated development of osteoarthritis compared with the collagen IX knockout animals. Finally, mice lacking matrilin-1, a non-collagenous glycoprotein that binds to both collagen fibrils and aggrecan, develop normally without detectable abnormalities in their skeleton. CONCLUSIONS: Our transgenic mouse strains carrying null mutations in genes encoding cartilage ECM proteins demonstrate that these proteins have different roles during skeletal development. Collagen II is important for cartilage formation, collagen IX for cartilage maintenance and matrilin-1 is redundant.
Asunto(s)
Cartílago Articular/crecimiento & desarrollo , Colágeno Tipo II/fisiología , Colágenos Asociados a Fibrillas/fisiología , Animales , Colágeno Tipo IX/fisiología , Proteínas de la Matriz Extracelular/fisiología , Miembro Anterior/anomalías , Glicoproteínas/fisiología , Miembro Posterior/anomalías , Proteínas Matrilinas , Ratones , Ratones Noqueados , Microscopía Electrónica , Adhesión en Parafina , Fenotipo , Adhesión en Plástico , Columna Vertebral/anomalíasRESUMEN
Type XIII collagen is a type II transmembrane protein found at many sites of cell adhesion in tissues. Homologous recombination was used to generate a transgenic mouse line (Col13a1(N/N)) that expresses N-terminally altered type XIII collagen molecules lacking the short cytosolic and transmembrane domains but retaining the large collagenous ectodomain. The mutant molecules were correctly transported to focal adhesions in cultured fibroblasts derived from the Col13a1(N/N) mice, but the cells showed decreased adhesion when plated on type IV collagen. These mice were viable and fertile, and in immunofluorescence stainings the mutant protein was located in adhesive tissue structures in the same manner as normal alpha1(XIII) chains. In immunoelectron microscopy of wild-type mice type XIII collagen was detected at the plasma membrane of skeletal muscle cells whereas in the mutant mice the protein was located in the adjacent extracellular matrix. Affected skeletal muscles showed abnormal myofibers with a fuzzy plasma membrane-basement membrane interphase along the muscle fiber and at the myotendinous junctions, disorganized myofilaments, and streaming of z-disks. The findings were progressive and the phenotype was aggravated by exercise. Thus type XIII collagen seems to participate in the linkage between muscle fiber and basement membrane, a function impaired by lack of the cytosolic and transmembrane domains.
Asunto(s)
Membrana Celular/metabolismo , Colágeno Tipo XIII/metabolismo , Citosol/metabolismo , Enfermedades Musculares/etiología , Estructura Terciaria de Proteína , Secuencia de Aminoácidos/genética , Animales , Adhesión Celular/fisiología , Células Cultivadas , Colágeno Tipo XIII/química , Colágeno Tipo XIII/genética , Progresión de la Enfermedad , Exones , Fibroblastos/fisiología , Eliminación de Gen , Ratones , Ratones Transgénicos , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Actividad Motora , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Enfermedades Musculares/patología , Enfermedades Musculares/fisiopatología , Recombinación GenéticaRESUMEN
The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Capilares/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Animales , Aorta/embriología , Aorta/patología , Northern Blotting , Southern Blotting , Embrión de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Vectores Genéticos , Homocigoto , Inmunohistoquímica , Riñón/embriología , Riñón/metabolismo , Riñón/patología , Pulmón/embriología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Transgénicos , Microscopía Electrónica , Microscopía Fluorescente , Modelos Genéticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.
Asunto(s)
Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Integrina beta1/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Factor Natriurético Atrial/farmacología , Canales de Calcio Tipo L/metabolismo , Compartimento Celular , Células Cultivadas , Citocalasina D/farmacología , Adhesiones Focales , Integrina beta1/genética , Isoproterenol/farmacología , Ratones , Antagonistas Muscarínicos/farmacología , Miocardio/citología , Óxido Nítrico/farmacología , Canales de Potasio/metabolismo , Transducción de SeñalRESUMEN
Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells. The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this review, we summarize the phenotypes of mice carrying mutant integrin genes and compare them with phenotypes of mice lacking the integrin ligands.
Asunto(s)
Integrinas/fisiología , Mutación , Animales , Inhibición de Migración Celular , Desarrollo Embrionario y Fetal/genética , Genes Letales , Hematopoyesis/genética , Hemostasis/genética , Integrinas/deficiencia , Integrinas/inmunología , Leucocitos/inmunología , Leucocitos/metabolismo , Ligandos , Ratones , Ratones Mutantes , Neovascularización Fisiológica/fisiología , Fenotipo , Transducción de Señal/inmunología , Transducción de Señal/fisiologíaRESUMEN
Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Proteínas de la Matriz Extracelular/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Encéfalo/embriología , Encéfalo/patología , Brevicano , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Hipocampo/fisiología , Lectinas Tipo C , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neurocano , Plasticidad Neuronal , Sinapsis/fisiología , Tenascina/genética , Regulación hacia ArribaRESUMEN
Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.
Asunto(s)
Anexina A7/metabolismo , Calcio/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Contracción Muscular/fisiología , Miocardio/metabolismo , Animales , Anexina A7/genética , Cafeína/farmacología , Cardiotónicos/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Colforsina/farmacología , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Femenino , Marcación de Gen , Glucosa/farmacología , Homeostasis , Hipoglucemiantes/farmacología , Immunoblotting , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Contracción Muscular/efectos de los fármacos , Miocardio/citología , Técnicas de Placa-Clamp , Tolbutamida/farmacologíaRESUMEN
Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha(2)beta(1) integrin. This finding suggested that clustering of alpha(2)beta(1) integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both alpha(2)beta(1) integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.
Asunto(s)
Integrinas/fisiología , Lectinas Tipo C , Lectinas/metabolismo , Agregación Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Venenos de Víboras , Animales , Sitios de Unión , Citometría de Flujo , Integrinas/metabolismo , Ligandos , Ratones , Peso Molecular , Receptores de ColágenoRESUMEN
Chondromodulin-I (CHM1) was identified recently as an angiogenesis inhibitor in cartilage. It is highly expressed in the avascular zones of cartilage but is absent in the late hypertrophic region, which is invaded by blood vessels during enchondral ossification. Blast searches with the C-terminal part of CHM1 in available databases led to the identification of human and mouse cDNAs encoding a new protein, Tendin, that shares high homology with CHM1. Based on computer predictions, Tendin is a type II transmembrane protein containing a putative proteinase cleavage and two glycosylation sites. Northern assays with mouse RNAs demonstrated strong expression of a 1.5-kb tendin transcript in the diaphragm, skeletal muscle, and the eye and low levels of expression in all other tissues investigated. In 17.5-day-old mouse embryos, in situ hybridization revealed high levels of tendin transcript in tendons and ligaments. Additional signals were detected in brain and spinal cord, liver, lung, bowels, thymus, and eye. Cartilage, where CHM1 is found, revealed low levels of tendin m-RNA. In adult mice, tendin is expressed in neurons of all brain regions and the spinal cord. The tendin gene is localized in the human Xq22 region, to which several human diseases have been mapped.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Ligamentos/fisiología , Proteínas de la Membrana/genética , Tendones/fisiología , Animales , Secuencia de Bases , ADN Complementario , Humanos , Hibridación in Situ , Ligamentos/química , Ligamentos/embriología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Tendones/química , Tendones/embriologíaRESUMEN
Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.
Asunto(s)
Plaquetas/metabolismo , Colágeno/metabolismo , Integrinas/metabolismo , Lectinas Tipo C , Adhesividad Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Adenosina Difosfato/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Tiempo de Sangría , Proteína C-Reactiva/farmacología , Coagulantes/farmacología , Colágeno/farmacología , Venenos de Crotálidos/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Integrina beta1/genética , Integrinas/deficiencia , Ratones , Ratones Noqueados , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Recuento de Plaquetas , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Receptores de Colágeno , Transducción de Señal/fisiología , Estrés Mecánico , Trombina/farmacología , Trombosis/genética , Trombosis/metabolismoRESUMEN
Embryonal stem (ES) cells that are homozygous null for the beta(1) integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/beta(1)-null chimeric mice. The failure of beta(1)-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, deepidermized human dermis (DED) we showed that epidermal basement membrane did not induce beta(1)-null ES cells to undergo keratinocyte differentiation and did not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also had no effect. However, when human dermal fibroblasts were incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increased dramatically, and small groups of keratin 14-positive cells differentiated from beta(1)-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and beta(1)-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFalpha all stimulated differentiation of keratin 14-positive beta(1)-null cells, and KGF and FGF10 were shown to be produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells were much less pronounced, suggesting that the concentrations of inducing factors already present in the medium were not limiting for wild-type cells. We conclude that the lack of beta(1) integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation.
Asunto(s)
Dermis/citología , Embrión de Mamíferos/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Sustancias de Crecimiento/fisiología , Integrina beta1/genética , Queratinocitos/citología , Células Madre/citología , Factor de Crecimiento Transformador alfa/metabolismo , Células 3T3 , Animales , Membrana Basal/metabolismo , Adhesión Celular , Diferenciación Celular , División Celular , Línea Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Humanos , Integrina beta1/fisiología , Queratina-14 , Queratinocitos/fisiología , Queratinas/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Proteínas Recombinantes/metabolismo , Células Madre/fisiologíaRESUMEN
Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell.