Differentiation of axon-related Schwann cells in vitro: II. Control of myelin formation by basal lamina.

Several recent observations suggest that Schwann cell (SC) differentiation, including myelin formation, is dependent upon the development of basal lamina which characteristically surrounds each axon-SC unit in peripheral nerve. This dependence can be tested in a neuron-SC culture system developed in our laboratory in which SC differentiation, including basal lamina formation and myelination, is faithfully reproduced. The use of serum-free, defined medium (DM) with this culture system allows axon-driven SC proliferation but not basal lamina formation or myelination. We previously demonstrated that ascorbic acid, in the presence of a nondialyzable serum factor(s), stimulates basal lamina assembly and myelin formation with similar dose-response relationships (Eldridge et al., 1987). We hypothesized that ascorbic acid acts to promote SC myelination indirectly, by enabling the assembly of basal lamina. We now provide support for this hypothesis by demonstrating the following. (1) Pepsin-resistant triple-helical collagen molecules were produced only by SCs grown in the presence of ascorbic acid, suggesting that triple-helical type IV collagen may mediate the effect of ascorbic acid on basal lamina formation. (2) The formation of myelin by oligodendrocytes, which myelinate axons in the CNS without the concomitant deposition of basal lamina, was little affected by ascorbic acid, suggesting that the biosynthesis and assembly of myelin per se does not require ascorbic acid. (3) The provision of exogenous basal lamina matrix to SCs grown with neurons in DM without ascorbic acid promoted control levels of myelination (and basal lamina formation); the provision of exogenous fibrillar collagen matrix did not. (4) Purified laminin promoted control levels of myelination in the absence of ascorbic acid, but purified type IV collagen and heparan sulfate proteoglycan (HSPG) did not. Laminin caused SCs to assemble basal lamina-like structures that contained not only laminin but also HSPG and non-triple-helical type IV collagen. Thus, several types of experiments demonstrate that SC myelin formation can be controlled by regulating the ability of the SC to assemble basal lamina, illustrating that acquisition of basal lamina is a crucial prefatory step for further SC differentiation.

Structural analysis and expression of the human thrombospondin gene promoter.

We have isolated a 34-kilobase pair genomic thrombospondin clone and determined the sequence of 2033 base pairs of the 5'-flanking region, the first entirely untranslated exon and the first intron. A number of interesting regulatory motifs were identified. The transcription start site, determined by primer extension and S1 nuclease analysis, was shown to be 20-30 bases 3' of a TATA-like sequence, TTTAAAA. We have also shown that a thrombospondin promoter-bovine growth hormone fusion gene is transcribed in transiently transfected cells. We conclude that the genomic clone contains the transcriptional signals of the thrombospondin gene and will therefore be useful in the analysis of cis-acting elements that regulate the expression of this gene.

Effects of cis-4-hydroxy-L-proline, and inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells.

The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [3H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.

Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation.

Rat Schwann cells cultured with dorsal root ganglion neurons in a serum-free defined medium fail to ensheathe or myelinate axons or assemble basal laminae. Replacement of defined medium with medium that contains human placental serum (HPS) and chick embryo extract (EE) results in both basal lamina and myelin formation. In the present study, the individual effects of HPS and EE on basal lamina assembly and on myelin formation by Schwann cells cultured with neurons have been examined. Some batches of HPS were unable to promote myelin formation in the absence of EE, as assessed by quantitative evaluation of cultures stained with Sudan black; such HPS also failed to promote basal lamina assembly, as assessed by immunofluorescence using antibodies against laminin, type IV collagen, and heparan sulfate proteoglycan. The addition of EE or L-ascorbic acid with such HPS led to the formation of large quantities of myelin and to the assembly of basal laminae. Pretreatment of EE with ascorbic acid oxidase abolished the EE activity, whereas trypsin did not. Other batches of HPS were found to promote both basal lamina and myelin formation in the absence of either EE or ascorbic acid. Ascorbic acid oxidase treatment or dialysis of these batches of HPS abolished their ability to promote Schwann cell differentiation, whereas the subsequent addition of ascorbic acid restored that ability. Ascorbic acid in the absence of serum was relatively ineffective in promoting either basal lamina or myelin formation. Fetal bovine serum was as effective as HPS in allowing ascorbic acid (and several analogs but not other reducing agents) to manifest its ability to promote Schwann cell differentiation. We suggest that ascorbic acid promotes Schwann cell myelin formation by enabling the Schwann cell to assemble a basal lamina, which is required for complete differentiation.

Basal lamina-associated heparan sulphate proteoglycan in the rat PNS: characterization and localization using monoclonal antibodies.

Cultured rat Schwann cells produce a basal lamina (BL)-associated heparan sulphate proteoglycan (HSPG). The HSPG has an apparent molecular weight of greater than 450 kD, is sensitive to both heparinase and heparitinase and contains a core protein of approximately 400 kD. Two independently derived monoclonal antibodies, B3 and C17, recognize this HSPG. Using B3 and C17, we found that this HSPG, or immunologically related material, is present in BLs throughout the body and in a small number of connective tissue sites without a formed BL. In the PNS it is present in BLs of Schwann cell-axon units, in synaptic and extrasynaptic portions of muscle fibre BL, and in the BLs of satellite cells that ensheath neurons in sympathetic and sensory ganglia. This HSPG is not detectable in the neuropil of the brain and spinal cord. Neurons, Schwann cells and fibroblasts cultured alone do not assemble a BL or accumulate immunocytochemically detectable amounts of this HSPG, but it is present in BLs assembled in myotube and in Schwann cell-neuron cultures. Thus, this HSPG is a component of most, if not all, BLs in the PNS.

Linkage between axonal ensheathment and basal lamina production by Schwann cells.

The availability of several methods for the preparation of SCs free of other cell types has allowed recent experimentation providing new insights into the capacity of SCs to synthesize, release, and organize extracellular matrix materials, particularly those of the basal lamina. When these SC populations are combined in tissue culture with pure populations of neurons capable of directing SC function (without fibroblasts), new aspects of interrelationships between these cell types have come to light. In this brief chapter we review the results from this experimental approach during the last decade, and suggest the implications these observations have for interpreting known differences in SC functional expression in various body regions as well as for understanding certain disease processes. Of particular note is the discovery of an apparently essential linkage between the function of the SCs in organizing and relating to basal lamina and their ability to ensheathe and myelinate axons. It now appears that SC functional expression requires an alliance not only with the nerve fiber but also with the ECM through the production and organization of a basal lamina.

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).