Abstract. A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences . The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain . The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues . Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence .
Cultured rat Schwann cells grown in association with sensory neurons when labeled with [3H]leucine, [3 H]glucosamine, or [31 S]methionine release labeled polypeptides into the culture medium . Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of >20 polypeptides with molecular weights ranging from 15,000 to >250,000 . Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for -40% of the leucine or methionine radioactivity in medium polypeptides . Schwann cells grown in a serum-free defined medium, in which Schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides than cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides . Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE . This change in the pattern of polypeptides released by Schwann cells is accompanied by changes in the association between Schwann cells and axons. An early step in the establishment of normal axon-Schwann cell relations appears to be an inward migration of Schwann cells into axonal bundles and spreading of Schwann cells along neurites . These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by Schwann cells may be important for the development of normal axonal ensheathment .The development of peripheral nerves requires the cooperative activity of three different cell types: peripheral neurons, Schwann cells, and fibroblasts . The study of peripheral nerve development has been aided greatly by the recent development of tissue culture techniques that allow one to obtain and culture separately or in combination each of these three cell types (21). With this tissue culture system it has been possible to show, for example, that sensory axons are mitogenic for Schwann cells (16,22) and that this mitogen is probably a neurite membrane protein (17,18) . It has also been demonstrated that Schwann cells synthesize and secrete several collagen types and that the basal lamina that covers the axon-Schwann cell unit is synthesized by Schwann cells in the absence of fibroblasts (5) proline analogue cis-hydroxyproline (which inhibits collagen hydroxylation and secretion) causes abnormal Schwann cell behavior : the basal lamina coverage becomes patchy and incomplete, and in addition, abnormal and incomplete ensheatment of the axons by Schwann cells occurs (8) . Culturing of Schwann cells and neurons in a serum-free defined medium developed by Bottenstein and Sato for growth of neuro...
Abstract. Schwann cells synthesize two heparan sulfate proteoglycans, one that is a component of the Schwann cell basement membrane and a smaller one that is an integral component of the Schwann cell plasma membrane. To determine the functions of these molecules, Schwann cell-nerve cell cultures were grown in medium containing a specific inhibitor of proteoglycan biosynthesis, 4-methylumbelliferyl-13-Dxyloside. Treatment with 1 mM 13-D-xyloside caused a 90% reduction in the accumulation of ssSO4-1abeled proteoglycans in the cell layer of the cultures. Gel filtration analysis revealed that both the basement membrane and plasma membrane proteoglycans were affected. Inhibition of proteoglycan biosynthesis was accompanied by an inhibition of laminin deposition into extracellular matrix as determined by immunostaining of cultures and by immunoblotting of cellassociated proteins. This occurred even though there was no decrease in the amount of laminin detected in the medium of 13-D-xyloside-treated cultures. Deposition of collagen type IV was similarly affected. In addition, there was no myelin produced in 13-D-xyloside treated cultures. However, when 13-xyloside-treated cultures were supplied with exogenous basement membrane, Schwann cells produced numerous myelin segments. These results indicate that Schwann cell proteoglycans play an essential role in basement membrane assembly, and that the integral plasma membrane proteoglycan is not required for the basement membrane to exert its effects on Schwann cell differentiation.T HE extracellular matrix (ECM) 1 plays an essential role in the development of many tissues, affecting such cellular functions as adhesion, migration, cell shape, proliferation, and gene expression (reviewed by Hay [14]). The ability to purify several of the major molecular constituents of the ECM has made it possible to study their properties in detail, and is beginning to provide an understanding of ECM action at the molecular level.An example of a cell whose development is dependent upon ECM contact is the Schwann cell. The principal function of Schwann cells is to provide myelin and unmyelinated ensheathment for peripheral nerve fibers. In addition, Schwann cells synthesize a portion of the peripheral nerve connective tissue matrix, including the basement membrane that surrounds each Schwann cell axon unit (4-8, 11, 28, 29). Experiments performed with primary tissue cultures of Schwann cells and nerve cells have demonstrated that contact with this basement membrane by Schwann cells is essential for their ensheathment and myelination of axons (6,10,30).These findings have led to investigations into the biochemical composition of the Schwann cell ECM, with the goal of understanding the mechanism of its effect on Schwann cell differentiation. These experiments have established that 1. Abbreviations used in this paper: ECM, extracellular matrix; HS, heparan sulfate; PG, proteoglycan.Schwann cells synthesize several collagen types (5), including collagen type IV (8), the basement membrane glycopro...
Summary The syndecans, a family of cell-surface heparan sulphate proteoglycans, have been proposed to mediate cellular interactions with extracellular effector molecules, such as growth factors and components of the extracellular matrix, during critical phases of development.Transcripts of all four syndecans are expressed at varying levels in the developing rat intestine and in a series of immature rat intestinal epithelial cell lines. In addition, we report the novel finding that, in the intestinal epithelial cell lines, expression of syndecan-1 transcript is up-regulated by transformation with activated H-ras. This is in contrast to other cell lines in which ras transformation is associated with a decrease in syndecan-1 levels. The observed increase in the syndecan-1 occurs as a result of increased transcription and can be correlated with the degree of transformation of the IEC-18 cells. Transformation is also associated with a decrease in apparent molecular weight and increased shedding of the proteoglycan into the culture medium. Increased shedding of syndecan-1 into the culture medium after transformation with H-ras may contribute to the disruption of proteoglycan interactions with the extracellular matrix, leading to alterations in cell adhesion and organization.Keywords: proteoglycan; syndecan; ras transformation; intestinal epithelial cell In the rat intestine, dramatic changes over the last few days of gestation result in the reorganization of a tubular structure into the mature crypt-villus structure in which the mesenchyme is lined by a simple columnar epithelium (Moog, 1979;Trier and Moxey, 1979;Madara et al, 1981). Underlying this morphogenesis and the maintenance of the adult crypt-villus structure are reciprocal interactions between the epithelium and mesenchyme (Kedinger et al, 1986) that involve recognition phenomena between cell-surface molecules and components of the pericellular environment. Membrane-bound proteoglycans contribute to the regulation of cell behaviour through interactions with extracellular matrix components or by acting as co-receptors for biologically active peptides (Klagsbrun and Baird, 1991;Rapraeger et al, 1991;Yayon et al, 1991;Aviezer et al, 1994).Two gene families encoding cell-surface heparan sulphate proteoglycans (HSPG) have been identified on the basis of sequence similarities between their core proteins: the glypicanrelated integral membrane proteoglycans (PGs) and syndecan-like integral membrane PGs (David, 1993). We have reported that OCI-5, a glypican-related HSPG (Filmus et al, 1995), is involved in intestinal development (Filmus et al, 1988). Although syndecans-1 and -4 have been detected in the adult intestine (Kim et al, 1994), the involvement of the syndecan family in intestinal development or differentiation is largely unknown. The role of syndecan-1 in epithelial-mesenchymal interactions during morphogenesis of a variety of tissues raises the possibility of a similar role in the developing intestine. The recent cloning of the four members of the syndecan...
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