Synthesis of basement membrane components by differentiated thyroid cells

Morphological studies indicate that basement membrane formation or maintenance can be achieved in cultures of thyroid cells. In the present investigation we have studied the biosynthesis of this extracellular matrix by differentiated porcine thyroid cells in culture. They were prepared by two procedures: (1) thyroid cells isolated by dispase digestion of the thyroid gland were maintained in serum-free medium on poly(L-lysine) coated dishes; (2) thyroid follicles released by collagenase treatment of the gland were isolated by differential filtration and cultured in suspension on agarose-coated dishes. In both cases, functional follicular-like structures were obtained as shown by their ability to organify Na125I and to respond to thyrotropin stimulation (250 microU/ml). After incubating the cells with radiolabeled proline or methionine, collagen synthesis was observed with the two types of culture, as shown by the formation of radioactive hydroxyproline and by the synthesis of peptides with electrophoretic properties identical to those of authentic collagen molecules and susceptible to collagenase. Besides variable amounts of type I and type III collagen-like peptides, significant proportions of labeled peptides migrated with type IV collagen chains and were precipitated by anti-type IV collagen antibody; thyrotropin had no significant effect either on the total collagen synthesis or on the relative amounts of the different collagen peptides. When thyroid cells were incubated with [35S]sulfate, a labeled glycosaminoglycan with chromatographic properties analogous to that of heparan sulfate could be obtained in both culture conditions; here again, no effect of thyrotropin was observed. The ability of differentiated porcine thyroid cells to synthesize basement membrane was suggested by their production of type IV collagen and heparan sulfate, two of its potential components. Thyrotropin, which drastically enhanced the functional property of the cells, did not seem to regulate this synthesis.     

Studies on cultured rat Schwann cells

Summary Cultured rat Schwann cells do not exhibit the ring-like changes in cell shape previously reported to be induced in the Schwann cell line RN22 by elevation of intracellular cyclic AMP. They do, however, undergo different shape changes on treatment with cholera toxin or low serum concentration. Furthermore, DNA synthesis in the cell line is inhibited by treatment with cholera toxin and unaffected by bovine pituitary extract, though both of these agents stimulate DNA synthesis in normal Schwann cells. Our results, therefore, do not support the hypothesis that elevation of intracellular cyclic AMP is a positive signal for myelination by the Schwann cell. Moreover, they illustrate the need for caution in drawing conclusions about normal cells of the nervous system from studies on neural cell lines. Paper I in this series is reference 2.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

A calcium-activated cation-selective channel in rat cultured Schwann cells

Calcium-activated channels, in the plasma membrane of rat cultured Schwann cells were studied in isolated 'inside-out' membrane patches. With identical (150 mM NaCl) solutions on either side of the membrane, a single channel conductance of 32 pS was calculated for inward current; the conductance was somewhat less for outward current. The channel is about equally permeable to sodium and potassium ions, but is not detectably permeable to either chloride or calcium. Under our experimental conditions the channel is activated by high (more than 10(-4) M) concentrations of calcium and is sensitive to voltage, channel activity increasing with membrane depolarization.     

Inositol uptake by cultured isolated rat Schwann cells

The uptake of radiolabeled myo-inositol by Schwann cells isolated from the sciatic nerve of 2–4 day old rats was found to occur by a saturable, sodium-dependent phlorizin-inhibited mechanism with an estimated K_m of 30μM. The system was inhibited by galactose and glucose but not by galactitol. At high concentrations of myo-inositol, a diffusion-like process appeared to be functional. The characteristics of the saturable system are very similar to those of myo-inositol uptake by the endoneural fascicle preparation of sciatic nerve.     

Assays for cholinergic properties in cultured rat Schwann cells

Cultured rat Schwann cells did not contain detectable levels of choline-acetyltransferase (less than 0.5 pmol ACh min-1 mg-1 of protein) or of acetylcholinesterase and nicotinic acetylcholine receptors. After adding Schwann cells to primary rat myotube cultures, the level of cholineacetyl-transferase in the co-cultures increased after three weeks to as high as 5 pmol ACh min-1 mg-1. The activity appearing in co-cultures sedimented at approximately 4S, and was inhibited 50% by 4(1-napthylvinyl)pyridine in the concentration range of 10-50 microM. After treatment of co-cultures with anti rat neural antigen-1 (RAN-1) and complement, 70-80% of the activity was lost, suggesting that it is induced in the Schwann cells. Attempts to obtain the effect by exposure of Schwann cells to medium conditioned by the myotube cultures, or by co-culture with fibroblasts gave levels of activity at or below the limit of detection.     

Cerebellar granule cells contain a membrane mitogen for cultured Schwann cells

Proliferation of Schwann cells is one of the first events that occurs after contact with a growing axon. To further define the distribution and properties of this axonal mitogen, we have (a) cocultured cerebellar granule cells, which lack glial ensheathment in vivo with Schwann cells; and (b) exposed Schwann cell cultures to isolated granule cell membranes. Schwann cells cocultured with granule cells had a 30-fold increase in the labeling index over Schwann cells cultured alone, suggesting that the mitogen is located on the granule cell surface. Inhibition of granule cell proteoglycan synthesis caused a decrease in the granule cells' ability to stimulate Schwann cell proliferation. Membranes isolated from cerebellar granule cells when added to Schwann cell cultures caused a 45-fold stimulation in [3H]thymidine incorporation. The granule cell mitogenic signal was heat and trypsin sensitive and did not require lysosomal processing by Schwann cells to elicit its proliferative effect. The ability of granule cells and their isolated membranes to stimulate Schwann cell proliferation suggests that the mitogenic signal for Schwann cells is a ubiquitous factor present on all axons regardless of their ultimate state of glial ensheathment.     

A proteome map of primary cultured rat Schwann cells

Background

Schwann cells (SCs) are the principal glial cells of the peripheral nervous system with a wide range of biological functions. SCs play a key role in peripheral nerve regeneration and are involved in several hereditary peripheral neuropathies. The objective of this study was to gain new insight into the whole protein composition of SCs.

Results

Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS) was performed to identify the protein expressions in primary cultured SCs of rats. We identified a total of 1,232 proteins, which were categorized into 20 functional classes. We also used quantitative real time RT-PCR and Western blot analysis to validate some of proteomics-identified proteins.

Conclusion

We showed for the first time the proteome map of SCs. Our data could serve as a reference library to provide basic information for understanding SC biology.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts

Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.     

Synthesis of the glomerular basement membrane in the rat kidney

To study the origin and the formation of the glomerular basement membrane, autoradiographic investigations with³H-proline and³H-leucine have been performed in ultrathin and semithin sections of the glomeruli of 42 male rats. The results of this study indicate that, of the three cell types of the glomerulus, the epithelial cells (=podocytes) synthesize the proline-rich scleroproteins of the glomerular basement membrane. Our autoradiographic studies have yielded no evidence for participation of the endothelial or mesangial cells in the formation of the basement membrane. The mesangial cells appear to be responsible for the synthesis of the mesangial matrix only.     

Biosynthesis of type IV collagen by cultured rat Schwann cells

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).     

Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane

Summary The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system qualitative changes in the expression of intermediate filament proteins.     

Studies on cultured rat Schwann cells. II. Comparison with a rat Schwann cell line.

Cultured rat Schwann cells do not exhibit the ring-like changes in cell shape previously reported to be induced in the Schwann cell line RN22 by elevation of intracellular cyclic AMP. They do, however, undergo different shape changes on treatment with cholera toxin or low serum concentration. Furthermore, DNA synthesis in the cell line is inhibited by treatment with cholera toxin and unaffected by bovine pituitary extract, though both of these agents stimulate DNA synthesis in normal Schwann cells. Our results, therefore, do not support the hypothesis that elevation of intracellular cyclic AMP is a positive signal for myelination by the Schwann cell. Moreover, they illustrate the need for caution in drawing conclusions about normal cells of the nervous system from studies on neural cell lines.     

Intracellular accumulation of basement membrane components during morphogenesis of rat submandibular gland

The distribution of two basement membrane (BM) components, laminin (LN) and type IV collagen (COLL IV), during acino-tubular morphogenesis of rat submandibular gland was examined immunohistochemically to determine the role of BM in the development of acino-tubular structures. On day 14 of gestation, LN could be found only in the BM separating an undifferentiated cell cluster of gland epithelium from surrounding mesenchyme. However, during a short period through days 15 to 17, LN was detected not only in the BM but also in intracellular vesicles of the cells of the terminal cluster. Immunoelectron microscopy showed the intracellular immunoreactive sites to be rough endoplasmic reticulum, indicating that active LN synthesis occurs in the cells of the terminal cluster. Intracellular immunostaining of LN disappeared completely on day 19 with the development of simple epithelium from the cell cluster, even though BM remained reactive. COLL IV also was accumulated in the intracellular vesicles of terminal cluster cells on day 16 of gestation but not on day 19. These results indicate that synthesis of certain BM components is transiently stimulated in gland epithelium before the formation of simple epithelial structure, and that these components are significantly involved in morphogenesis of the submandibular gland.     

Ganglioside stimulation of nerve growth factor synthesis in cultured rat Schwann cells

To investigate effects of gangliosides on nerve growth factor (NGF) synthesis/secretion by Schwann cells, we obtained Schwann cells from dorsal sensory ganglia of one-day old Wistar rats and cultured them with various concentrations of a mixed ganglioside comprising GM1, GD1a, GD1b, and GT1b. NGF synthesis was evaluated by the measurement of NGF concentration in the conditioned medium using an enzyme immunoassay. In the continuous presence of 10(-3) M gangliosides, the NGF concentration in the medium showed a four fold increase on the 4th day, and it then decreased by the 8th day. The present results indicate that gangliosides promote the production/synthesis of NGF by Schwann cells.     

Schwann cells,basement lamina,and collagen in developing rat dorsal root gangliain vitro

Observations on developing rat dorsal root gangliain vitro indicated that the formation of a basement lamina associated with the Schwann cells did not occur until after migration had ceased and the cells had become committed to an axon or another Schwann cell. The presence of a basement lamina was therefore characteristic only of committed Schwann cellsin vitro. It also appeared that Schwann cells produced collagen following formation of the basement lamina.