RESUMEN
A considerable variety of basement membrane components, including in particular more than ten laminin isoforms and their novel alpha chains (alpha3, alpha4 and alpha5), has been characterized in recent studies. The functional properties of these components are increasingly being analyzed by recombinant technologies and by structural studies at atomic resolution, techniques which led to the elucidation of the nidogen-binding epitope on the laminin gamma1 chain. Novel insights into functions of basement membrane components have been obtained from gene-targeting experiments and studies of mutated genes identified in inherited disorders.
Asunto(s)
Laminina/química , Animales , Membrana Basal/metabolismo , Sitios de Unión , Epítopos/química , Humanos , Laminina/genética , Laminina/inmunología , Laminina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.
Asunto(s)
Anticuerpos/análisis , Enfermedad de Chagas/inmunología , Disacáridos/inmunología , Laminina/inmunología , Leishmaniasis/inmunología , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Epítopos , Galactosa/análogos & derivados , Galactosa/inmunología , Humanos , Macaca , Ratones , RadioinmunoensayoRESUMEN
Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.
Asunto(s)
Membrana Basal/metabolismo , Adhesión Celular , Colágeno/metabolismo , Animales , Anticuerpos/inmunología , Sitios de Unión , Línea Celular , Colágeno/inmunología , Fibronectinas/inmunología , Fibronectinas/metabolismo , Humanos , Laminina/inmunología , Laminina/metabolismo , Conformación ProteicaRESUMEN
Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.
Asunto(s)
Membrana Basal/ultraestructura , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Córnea/ultraestructura , Matriz Extracelular/ultraestructura , Glicosaminoglicanos/fisiología , Heparitina Sulfato/fisiología , Laminina/fisiología , Glicoproteínas de Membrana , Proteínas de la Membrana/fisiología , Proteoglicanos/fisiología , Animales , Sitios de Unión , Lámina Limitante Posterior/ultraestructura , Epitelio/ultraestructura , Epítopos , Técnica del Anticuerpo Fluorescente , Proteoglicanos de Heparán Sulfato , Inmunohistoquímica , Laminina/inmunología , Ratones , Microscopía Electrónica/métodosRESUMEN
Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite-promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.
Asunto(s)
Anticuerpos/inmunología , Axones/fisiología , Laminina/fisiología , Neurilemoma/metabolismo , Sarcoma Experimental/metabolismo , Animales , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Inmunoensayo , Laminina/inmunología , Teratoma , Células Tumorales CultivadasRESUMEN
We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.
Asunto(s)
Médula Renal/enzimología , Laminina/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Bovinos , Adhesión Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Técnicas Inmunológicas , Médula Renal/citología , Factores de Crecimiento Nervioso/farmacología , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Relación Estructura-ActividadRESUMEN
In previous studies rat hepatocytes have been shown to adhere to substrates composed of collagen or fibronectin. In the present communication, the basement membrane protein laminin is reported to mediated the attachment and spreading of hepatocytes. The cell attachment-mediating activity of laminin was compared with that of fibronectin. The activity of fibronectin was heat sensitive, whereas laminin retained its activity after boiling. On the other hand, reduction and alkylation or periodate oxidation of the proteins affected only the cell attachment activity of laminin. Preincubation of cells with soluble fibronectin inhibited initial cell attachment to fibronectin but not to laminin substrates, and, reversely, soluble laminin selectively inhibited cell attachment to laminin. These results suggest that attachment of cells to substrates of the two proteins involves different cellular receptors recognizing distinct and nonidentical structures in the proteins.
Asunto(s)
Adhesión Celular , Fibronectinas/fisiología , Glicoproteínas/fisiología , Animales , Movimiento Celular , Calor , Laminina , Hígado/citología , Masculino , Ácido Peryódico , RatasRESUMEN
Conversion of the nephrogenic mesenchyme into epithelial tubules requires an inductive stimulus from the ureter bud. Here we show with immunofluorescence techniques that the undifferentiated mesenchyme before induction expresses uniformly type I and type III collagens. Induction both in vivo and in vitro leads to a loss of these proteins and to the appearance of basement membrane components including type IV collagen. This change correlates both spatially and temporally with the determination of the mesenchyme and precedes and morphological events. During morphogenesis, type IV collagen concentrates at the borders of the developing tubular structures where, by electron microscopy, a thin, often discontinuous basal lamina was seen to cover the first pretubular cell aggregates. Subsequently, the differentiating tubules were surrounded by a well-developed basal lamina. No loss of the interstitial collagens was seen in the metanephric mesenchyme when brought into contact with noninducing tissues or when cultured alone. Similar observations were made with nonnephrogenic mesenchyme (salivary, lung) when exposed to various heterotypic tissues known to induce tubules in the nephrogenic mesenchyme. The sequential shift in the composition of the extracellular matrix from an interstitial, mesenchymal type to a differentiated, epithelial type is so far the first detectable response of the nephrogenic mesenchyme to the tubule-inducing signal.
Asunto(s)
Colágeno/metabolismo , Riñón/embriología , Procolágeno/metabolismo , Animales , Inducción Embrionaria , Espacio Extracelular/metabolismo , Riñón/citología , RatonesRESUMEN
It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.
Asunto(s)
Integrinas/fisiología , Riñón/crecimiento & desarrollo , Laminina/fisiología , Receptores Inmunológicos/fisiología , Animales , Anticuerpos , Anticuerpos Monoclonales , Adhesión Celular/fisiología , Diferenciación Celular , Epitelio/fisiología , Técnicas In Vitro , Integrinas/metabolismo , Riñón/embriología , Riñón/metabolismo , Túbulos Renales/embriología , Túbulos Renales/crecimiento & desarrollo , Laminina/metabolismo , Ratones , Receptores de LamininaRESUMEN
Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates. These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production.
Asunto(s)
Adhesión Celular , División Celular , Glicoproteínas/fisiología , Anticuerpos , Movimiento Celular , Células Cultivadas , Citoesqueleto/ultraestructura , Fibroblastos , Fibronectinas/metabolismo , Glicoproteínas/inmunología , Humanos , Laminina , Procolágeno/metabolismo , PielRESUMEN
Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN-collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.
Asunto(s)
Adhesión Celular , Colágeno/química , Placenta/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Complejo Antígeno-Anticuerpo , Pollos , Cromatografía de Afinidad , Colágeno/metabolismo , Colágeno/ultraestructura , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Placenta/metabolismo , Embarazo , Homología de Secuencia de AminoácidoRESUMEN
Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.
Asunto(s)
Colágeno , Matriz Extracelular/ultraestructura , Adenosina Trifosfato/farmacología , Animales , Embrión de Pollo , Técnica del Anticuerpo Fluorescente , Oro , Humanos , Microscopía Electrónica/métodos , Ratas , Tendones/ultraestructuraRESUMEN
Cells of the neural crest participate in a major class of cell migratory events during embryonic development. From indirect evidence, it has been suggested that fibronectin (FN) might be involved in these events. We have directly tested the role of FN in neural crest cell adhesion and migration using several in vitro model systems. Avian trunk neural crest cells adhered readily to purified plasma FN substrates and to extracellular matrices containing cellular FN. Their adhesion was inhibited by antibodies to a cell-binding fragment of FN. In contrast, these cells did not adhere to glass, type I collagen, or to bovine serum albumin in the absence of FN. Neural crest cell adhesion to laminin (LN) was significantly less than to FN; however, culturing of crest cells under conditions producing an epithelioid phenotype resulted in cells that could bind equally as well to LN as to FN. The migration of neural crest cells appeared to depend on both the substrate and the extent of cell interactions. Cells migrated substantially more rapidly on FN than on LN or type I collagen substrates; if provided a choice between stripes of FN and glass or LN, cells migrated preferentially on the FN. Migration was inhibited by antibodies against the cell-binding region of FN, and the inhibition could be reversed by a subsequent addition of exogenous FN. However, the migration on FN was random and displayed little persistence of direction unless cells were at high densities that permitted frequent contacts. The in vitro rate of migration of cells on FN-containing matrices was 50 microns/h, similar to their migration rates along the narrow regions of FN-containing extracellular matrix in migratory pathways in vivo. These results indicate that FN is important for neural crest cell adhesion and migration and that the high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are necessary for effective directional migration.
Asunto(s)
Adhesión Celular , Movimiento Celular , Fibronectinas/fisiología , Cresta Neural/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Embrión de Pollo , Espacio Extracelular/fisiología , Glicoproteínas/fisiología , LamininaRESUMEN
Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.
Asunto(s)
Cartílago/citología , Colágeno/biosíntesis , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Cartílago/metabolismo , Células Cultivadas , Técnica del Anticuerpo FluorescenteRESUMEN
We have obtained evidence that rat Schwann cells synthesize and secrete type IV procollagen. Metabolic labeling of primary cultures of Schwann cells plus neurons and analysis by SDS PAGE revealed the presence of a closely spaced pair of polypeptides in the medium of these cultures that (a) were susceptible to digestion by purified bacterial collagenase, (b) co-migrated with type IV procollagen secreted by rat parietal endoderm cells, and (c) were specifically immunoprecipitated by antibodies against mouse type IV collagen. Limited pepsin digestion of metabolically labeled medium or cell layers produced a pepsin-resistant fragment characteristic of pro-alpha 1(IV) chains. Removal of neuronal cell bodies from the cultures immediately before labeling did not reduce the amount of type IV procollagen detected in the medium. This indicated that Schwann cells, not neurons, were responsible for synthesis of type IV procollagen. We believe type IV procollagen is a major constituent of the Schwann-cell extracellular matrix based upon (a) its presence in a detergent-insoluble matrix preparation, (b) its presence in the cell layer of the cultures in a state in which it can be removed by brief treatment with bacterial collagenase or trypsin, and (c) positive immunofluorescence of Schwann cell-neuron cultures with anti-type-IV collagen antibodies. Secretion of type IV procollagen was substantially reduced when Schwann cells were maintained in the absence of neurons. This observation may account for the previously reported finding that Schwann cells assemble a basal lamina only when co-cultured with neurons (Bunge, M. B., A. K. Williams, and P. M. Wood, 1982, Dev. Biol., 92:449).
Asunto(s)
Colágeno/biosíntesis , Células de Schwann/metabolismo , Animales , Axones/metabolismo , Membrana Basal/metabolismo , Células Cultivadas , Colágeno/metabolismo , Espacio Extracelular/metabolismo , Neuronas/metabolismo , Procolágeno/biosíntesis , Procolágeno/metabolismo , Ratas , SolubilidadRESUMEN
A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115-residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de la Matriz Extracelular/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Clonación Molecular , Reacciones Cruzadas , ADN , Factor de Crecimiento Epidérmico/química , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Técnica del Anticuerpo Fluorescente , Ratones , Datos de Secuencia Molecular , Conformación Proteica , ARN Mensajero/análisis , Radioinmunoensayo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.
Asunto(s)
Integrinas/metabolismo , Laminina/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/fisiología , Mama/citología , Mama/metabolismo , Mama/ultraestructura , Adhesión Celular/fisiología , Línea Celular , Femenino , Humanos , Integrinas/inmunología , Integrinas/fisiología , Laminina/análisis , Laminina/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/ultraestructura , Melanoma/metabolismo , Melanoma/patología , Melanoma/ultraestructura , Ratones , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/ultraestructura , Fragmentos de Péptidos/fisiología , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Receptores de Laminina , Receptores de VitronectinaRESUMEN
A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.
Asunto(s)
Blastocisto/fisiología , Integrinas/fisiología , Células Madre/fisiología , Animales , Adhesión Celular/fisiología , Compartimento Celular , Quimiotaxis/fisiología , Células Clonales , Citoesqueleto/química , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Heterocigoto , Homocigoto , Integrina beta1 , Integrinas/deficiencia , Integrinas/genética , Laminina/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Morfogénesis/fisiología , Unión Proteica , TransfecciónRESUMEN
Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.
Asunto(s)
Cartílago/fisiología , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/fisiología , Proteoglicanos/fisiología , Animales , Membrana Basal/anomalías , Membrana Basal/crecimiento & desarrollo , Membrana Basal/fisiología , Calcificación Fisiológica/genética , Cartílago/anomalías , Cartílago/crecimiento & desarrollo , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Exostosis Múltiple Hereditaria/genética , Marcación de Gen , Genes Letales , Cardiopatías Congénitas/genética , Heparitina Sulfato/deficiencia , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Homocigoto , Ratones , Ratones Congénicos , Ratones Mutantes , Mutagénesis Insercional , Defectos del Tubo Neural/genética , Osificación Heterotópica/genética , Proteoglicanos/deficiencia , Proteoglicanos/genética , Proteoglicanos/metabolismo , Mapeo RestrictivoRESUMEN
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.