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.     

Isolation and characteristics of the extracellular matrix of cultured cells

Sodium deoxycholate extraction was used to isolate extracellular matrix from various cultured cells: human and murine embryonic fibroblasts, epithelial lines of mouse (MPTR), rat (IAR 2 and IAR 20), pig (SPEV) and cow (FBT). Protein composition of the matrix was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence. The matrix morphology was investigated by scanning electron microscopy. In cell lines FBT and MPTR the major component of the matrix was laminin, whereas in other lines and fibroblasts it was fibronectin. The matrix of the majority of lines had a fibrillar structure, and the fibrils usually formed networks. MPTR cells had a punctate matrix composed of laminin and collagen type IV, densely covering the substratum. The treatment of the matrix by hyaluronidase and/or DNAase I did not influence its protein composition. The isolated matrix of different structure and composition may serve a biological substratum in studies of normal tumor cell behavior in tissue culture.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

Formation of basement membranes in the embryonic kidney: an immunohistological study

Specific antibodies to laminin, type IV collagen, basement-membrane proteoglycan, and fibronectin have been used in immunofluorescence microscopy to study the development of basement membranes of the embryonic kidney. Kidney tubules are known to form from the nephrogenic mesenchyme as a result of an inductive tissue interaction. This involves a change in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses fibronectin but no detectable laminin, type IV collagen, or basement-membrane proteoglycan. During the inductive interaction, basement-membrane specific components (laminin, type IV collagen, basement membrane proteoglycan) become detectable in the induced area, whereas fibronectin is lost. While the differentiation to epithelial cells of the kidney requires an inductive interaction, the development of the vasculature seems to involve an ingrowth of cells which throughout development deposits basement-membrane specific components, as well as fibronectin. These cells form the endothelium and possibly also the mesangium of the glomerulus, and contribute to the formation of the glomerular basement membrane. An analysis of differentiation of the kidney mesenchyme in vitro in the absence of circulation supports these conclusions. Because a continuity with vasculature is required for glomerular endothelial cell differentiation, it is possible that these cells are derived from outside vasculature.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.     

Deposition of extracellular matrix along the pathways of migrating fibroblasts

Summary Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin- or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development.     

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.     

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.     

Stromal assemblies containing collagen types IV and VI and fibronectin in the developing embryonic avian cornea

The morphogenesis of type IV collagen-containing structures in the stromal matrix of the developing avian cornea was investigated using immunofluorescence and immunoelectron microscopic histochemistry. Two forms of type IV collagen-containing structures were seen; these differed in their probable origin, structure, molecular composition, and developmental fate. The major form of stromal type IV collagen-containing material, termed "strings," was observed only after swelling of the primary stroma and the onset of mesenchymal invasion. These strings are presumed to be products of the stromal cells. In immunofluorescence histochemistry they appeared as linear segments of type IV collagen-specific immunoreactivity. In immunoelectron microscopy, they appeared initially as electron-dense sausages of variable length and orientation. They frequently were associated with cell surfaces and, in fortuitous sections, appeared to connect adjacent cells. The strings also contained type VI collagen and fibronectin, but very little, if any, of the basement membrane components laminin and heparin sulfate proteoglycan (HSPG). As the stroma continued to expand in thickness, more of these structures were observed in a radial orientation, becoming quite long and less tortuous. Later in development, as stromal condensation proceeded, they disappeared. We suggest that the strings function to stabilize the stromal matrix, and perhaps to limit the rate and/or extent of stromal expansion, during a phase of rapid swelling and matrix deposition. The other form of type IV collagen-containing stromal material appeared as irregularly shaped plaques of basement membrane-like material identical to those previously described in mature corneas. These are likely derived from the corneal endothelial cells. They contained other basement membrane-associated components (laminin, HSPG) and fibronectin, but not type VI collagen. This material persists in mature corneas as sparse irregular stromal plaques and as matrix in the interface between Descemet's membrane and the corneal stroma.     

Expression of fibronectin receptor (integrin) in the uterus of rats in relation to the estrous cycle

Summary The expression and localization of fibronectin receptor (integrin), fibronectin, laminin and collagen type IV in the endometrium of the rat uterus during each period of the estrous cycle were investigated by immunofluorescent microscopy. Fibronectin receptor was observed at the epithelial cells of the endometrium and at vascular endothelial cells. At proestrus, when epithelial cells actively migrate, fibronectin receptor was observed at the basal and lateral epithelial cell surfaces. During estrus, fibronectin receptor had begun to disappear, little fibronectin receptor was observed at metestrus or diestrus. No prominent changes in the localization of fibronectin (seen at the vascular endothelial cells and in the stroma) or of laminin and collagen type IV (seen at the muscles and at the basement membranes of the epithelial and vascular endothelial cells) were observed in relation to the estrous cycle. Thus, uterine epithelial cells, like epithelial cells of the healing cornea, increase their expression of fibronectin receptor during active migration, probably facilitating their attachment to stromal fibronectin. This fibronectin-fibronectin receptor mechanism may underlie epithelial repair, whether the defect results from physiological processes or from an insult.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices

The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes. These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate. The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer. The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM). The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably. However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel. This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells. These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.     

Expression of fibronectin receptor (integrin) in the uterus of rats in relation to the estrous cycle.

The expression and localization of fibronectin receptor (integrin), fibronectin, laminin and collagen type IV in the endometrium of the rat uterus during each period of the estrous cycle were investigated by immunofluorescent microscopy. Fibronectin receptor was observed at the epithelial cells of the endometrium and at vascular endothelial cells. At proestrus, when epithelial cells actively migrate, fibronectin receptor was observed at the basal and lateral epithelial cell surfaces. During estrus, fibronectin receptor had begun to disappear, little fibronectin receptor was observed at metestrus or diestrus. No prominent changes in the localization of fibronectin (seen at the vascular endothelial cells and in the stroma) or of laminin and collagen type IV (seen at the muscles and at the basement membranes of the epithelial and vascular endothelial cells) were observed in relation to the estrous cycle. Thus, uterine epithelial cells, like epithelial cells of the healing cornea, increase their expression of fibronectin receptor during active migration, probably facilitating their attachment to stromal fibronectin. This fibronectin-fibronectin receptor mechanism may underlie epithelial repair, whether the defect results from physiological processes or from an insult.     

Extracellular matrix components cooperate to activate phosphatidyl inositol-4-phosphate 5-kinase

Interaction of mesangial cells with extracellular matrix proteins can provide a means to modify cellular anchorage and traction through an interaction of integrins with activation of the actin cytoskeleton. We investigated intracellular signalling of matrix components fibronectin and laminin in mesangial cells in monolayer and 3-dimensional configurations to show a fibronectin-dependent activation of phosphatidylinositol-4-phosphate 5-kinase (up to threefold), which is augmented by a laminin-dependent increase in phospholipase D activity. Functional responsiveness to fibronectin and laminin addition was seen in the contraction of free-floating 3-dimensional mesangial cell-embedded collagen gels, a well-defined system reflecting coupling of extracellular matrix-cell events to activation of the actin cytoskeleton. Activation of phosphatidylinositol-4-phosphate 5-kinase and contraction of mesangial cell-embedded collagen gels in response to fibronectin and laminin were inhibited by pretreatment of mesangial cells with lovastatin and restored by isoprenoid augmentation with geranylgeraniol, supporting a role for the ras-related protein Rho in this process.     

Basal lamina glycoproteins laminin and type IV collagen are assembled into a fine-fibered matrix in cultures of a teratocarcinoma-derived endodermal cell line

Extracellular matrix glycoproteins synthesized and deposited by a mouse teratocarcinoma-derived endodermal cell line (PYS-2) in culture were analysed by metabolic labelling and immunochemical methods, and the matrix structure was studied by immunofluorescence and electron microscopy. PYS-2 cells secreted two major high-molecular weight glycoproteins, laminin and type IV collagen, which were deposited in apparently unprocessed form under the cells into a lamellar matrix composed of a loose network of fine fibrils and attached dense grains. The cells did not synthesize detectable amounts of fibronectin, but the matrix was found to bind fibronectin from the culture medium. The matrix structure was sensitive to bacterial collagenase indicating a role for type IV collagen in matrix integrity. The PYS-2 matrix which contains defined basal lamina glycoproteins provides possibilities for in vitro studies on the organization of deposited basal lamina components.     

The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel), and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel, laminin and type IV collagen, were also examined. Tissue-type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.     

Congenital nephrosis of the Finnish type (CNF): matrix components of the glomerular basement membranes and of cultured mesangial cells

Summary Congenital nephrosis of the Finnish type (CNF) is a hereditary renal disease of unknown aetiology manifested by massive proteinuria of the newborn and unresponsive to any treatment. In this study kidney samples and cultured glomerular mesangial cells from five patients with CNF were studied by indirect immunofluorescence microscopy for the presence and location of major basement membrane matrix (GBM) components. Histological changes of glomeruli ranging from mild thickening of basement membranes to total obliteration and sclerosis were seen. Notably, thickening of the subepithelial layer of Bowman's capsules was regularly seen along with hypercellularity at the juxtaglomerular areas. The matrix components studied (laminin, plasma- and cellular fibronectin, type IV collagen, including the NC-1, alpha-1 and alpha-3 chains, heparan sulphate proteoglycan (HSPG) core protein, thrombospondin) were characteristically seen within the glomeruli. Local thickenings alternating with total loss of epitopes along the GBM were seen, especially with anti-type IV collagen and anti-HSPG antibodies. Sera from CNF patients after transplantation failed to show antibodies against GBM structures in immunofluorescence microscopy, suggesting that no missing epitopes of GBM are introduced with the transplant kidney. Cultured mesangial cells of CNF glomeruli also showed continued in vitro production of the matrix components and their incorporation into the matrix underneath the cell layer.