Synthesis and effects of basement membrane components in cultured rat Schwann cells

Schwann cells, the myelin-forming cells of the peripheral nervous system, are surrounded by a basement membrane. Whether cultured rat Schwann cells synthesize the basement membrane-specific components, laminin and collagen type IV, and whether these components influence the adhesion, morphology, and growth of these cells have been investigated. Both laminin and collagen type IV were detected in the cytoplasm of Schwann cells by immunofluorescence. After ascorbate treatment, laminin and collagen type IV were both found in an extracellular fibrillar matrix bound to the Schwann cell surface. Laminin was further localized on the Schwann cell surface by electron microscopy using gold immunolabeling. Anti-laminin IgG-labeled gold particles were scattered over the cell surface, and linear rows of particles and small aggregates were found along the cell edges and at points of contact with other cells. When added to the culture medium, laminin acted as a potent adhesion factor, stimulating Schwann cell adhesion as much as eightfold above control levels on type IV collagen. In the presence of laminin, the cells became stellate and by 24 hr had extended long, thin processes. Laminin also stimulated cell growth in a dose-dependent manner and anti-laminin IgG completely inhibited cell attachment and growth in the absence of exogenous laminin. Thus, cultured Schwann cells synthesize laminin and collagen type IV, two major components of basement membrane, and laminin may trigger Schwann cell differentiation in vivo during early stages of axon-Schwann cell interaction before myelination.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.     

Avian vasculogenesis and the distribution of collagens I, IV, laminin, and fibronectin in the heart primordia

The heart-forming regions of the early embryo are composed of splanchnic mesoderm, endoderm, and the associated ECM. The ECM of the heart-forming regions in stage 7-9 chicken embryos was examined using immunofluorescence. Affinity purified antibodies to chicken collagens type I and IV, chicken fibronectin, and mouse laminin were used as probes. We report that (1) the basement membrane of the endoderm contains immunoreactive laminin and collagen IV; (2) the nascent basement membrane of the heart splanchnic mesoderm contains immunoreactive laminin, but not type IV collagen, and (3) the prominent ECM between the splanchnic mesoderm and the endoderm (the primitive-heart ECM) contains collagen IV, collagen I, fibronectin, but not laminin. In addition, we describe microscopic observations on the spatial relationship of cardiogenic cells to the primitive-heart ECM and the endodermal basement membrane.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Localization of basement membrane glycoproteins in rat kidney during foetal development

The distribution of basement membrane glycoproteins (type IV collagen, laminin, fibronectin, and proteoglycans) was studied in foetal rat kidney by immunohistochemical techniques using polyclonal antibodies. From the first stages of nephron differentiation, all these glycoproteins were detectable by immunofluorescence in the tubular and glomerular basement membranes and in the mesangial matrix. As differentiation proceeded, labelling of glycoproteins progressively intensified, except for that of fibronectin, which gradually decreased in the glomerular basement membrane (GBM) and was barely observable at full differentiation. With immunoperoxidase staining in electron microscopy, all glycoproteins were seen to be widely dispersed in the spaces between the epithelial and endothelial glomerular cells so long as the GBM remained a loose structure. However, after it became a compact, 3-layered formation, type IV collagen and laminin were distributed throughout the GBM, whereas proteoglycans and anionic sites appeared as 2 rows of granules confined to the laminae rarae.     

The extracellular matrix of hydra is a porous sheet and contains type IV collagen

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 μm in diameter and about six pores per 100 μm² in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell–cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.     

Deposition of Basement Membrane Proteins in Attachment and Neurite Formation of Cultured Murine C-1300 Neuroblastoma Cells

The deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C-1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell-associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high-molecular weight proteins in the two cultures were markedly different. We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell–to–substratum interactions in C-1300 cell cultures.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Cells that emerge from embryonic explants produce fibers of type IV collagen

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.     

The influence of extracellular matrix components on the proliferation and migration of inner cell mass-derived parietal endodermal cells.

Reichert's membrane is a basement membrane deposited on the inner surfaces of rat and mouse trophectodermal (TE) cells beginning at the blastocyst stage of embryonic development that may play a role in the migration of the parietal endodermal (PE) cells to form an inner lining to the TE. The abilities of various glycoproteins present in Reichert's membrane to support PE cell migration and replication in vitro were examined by isolating inner cell masses (ICMs) from Day 5 rat blastocysts (Day 1 = day of vaginal plug) and culturing them (24-72 h) either on surfaces that had been precoated with collagen IV, fibronectin, or laminin or on thin (1-2-mm) gels of Matrigel (a tumor cell-derived basement membrane preparation) or type I collagen. Time-dependent changes in the area occupied by each ICM on the culture surface and the number of migrating cells per ICM were quantified by morphometric analysis. Type IV collagen, the basement membrane-specific collagen, supported ICM attachment and the outward migration (overall increase of approx. 60-fold in mean ICM area occupied on the culture surface) and proliferation (cell doublings following every 24 h of culture) of laminin-containing PE-like cells. These effects were not altered by the inclusion of exogenous fibronectin or laminin in the culture medium. Collagen IV coating concentrations as low as 0.16 micrograms/ml supported PE cell attachment and migration, and maximal responses were seen with a coating concentration of 0.63 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of laminin and collagens during avian neural crest development

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.     

Attachment of cells to basement membrane collagen type IV

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.     

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Laminin 5 Deposition Promotes Keratinocyte Motility

We examined the role of individual integrins in promoting human keratinocyte migration. In short-term assays on collagen type I- or fibronectin-coated substrates, migration was blocked by antibody to the α2 integrin and the α5 integrin, respectively. Unexpectedly, antibodies to integrin α3 also significantly inhibited cell locomotion on both ligands. Time-course immunofluorescence staining revealed that keratinocyte migration was accompanied by deposition of endogenous laminin 5. Since α3β1 is a known receptor for this ligand, this observation suggested that migrating keratinocytes use freshly deposited laminin 5 in locomotion. Indeed, further investigation showed that anti-laminin 5 blocking antibodies effectively inhibited keratinocyte motility on both collagen and fibronectin substrates. Furthermore, cell migration on laminin 5-coated substrates was blocked by both anti-α3 and anti-laminin 5 antibodies. Laminin 5 did not appear important in the initial attachment of keratinocytes, since adhesion of cells to collagen type I- or fibronectin-coated surfaces was not blocked by antibody to α3 integrin or to laminin 5, but could be inhibited by antibody to α2 or α5, respectively. Using anin vitrowound assay, blocking antibodies to α3 integrin and to laminin 5 also blocked reepithelization of the denuded monolayer. These results show that α3β1 integrin plays an important role in the migration of keratinocytes via their interaction with laminin 5. Furthermore, they suggest that cell migration is dependent not only on exogenous ligands but, importantly, on endogenously secreted laminin 5. Finally, the data are consistent with our earlier finding that laminin 5 is the first extracellular matrix component to be expressed and deposited by migrating keratinocytes during wound healingin vivo[1].