Axonal Regulation of Schwann Cell Integrin Expression Suggests a Role for ct 6 4 in Myelination

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, /31 and/34, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of fll but virtually no f14 or MAG, When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of/34 and MAG increased dramatically in myelinating cells, whereas fll levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, f14 levels also increase during the period of myelination in sharp contrast to/31 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, #4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with/34 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly,/~4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express #1 in association with the od and c~6 subunits, while myelinating Schwann cells express ~6~4 and possibly t~l/31. B4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of/34 during Schwarm cell myelination and suggest that ot6~4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina. URISG development, Schwann cells ensheathe or myelinate nerve fibers in the continuous presence of basal lamina (Webster et al., 1973). The basal lamina is deposited on the outer surface of the Schwann cell-axon unit and is required for normal ensheathment or myelination of axons (Bunge and Bunge, 1986). This requirement has been most clearly demonstrated by in vitro studies in which purified populations of Schwann cells were cocultured with neurons under conditions that either promote or inhibit the formation of the basal lamina. Such studies have demonstrated that Schwann cells do not assemble a basal lamina when cultured in media lacking ascorbic acid, which is needed for the formation of triple-helical collagen (Eldridge et al., 1987), or in the presence of cis-4hydroxy-L-proline, a biosynthetic inhibitor of collagen forAddress all correspondence to Dr. James L. Salzer, Department of Cell Biology, New York University Medical School, 550 First Avenue, New York, NY 10016. marion (Eldridge et al., 1988). Under such conditions Schwann cells fail to ensheathe or myelinate neurites normally. Reversal of the basal lamina defect in these cultures by adding ascorbic acid or removing the biosynthetic inhibitor respectively, or by adding exogenous extracellular matrix materials (Carey et al., 1986), resulted in a rapid correction of the ensheathment defect and formation of normal myelin sheaths. Additional evidence supporting a requirement for the basal lamina during Schwann cell myelination has come from studies of the dystrophic mouse mutant. In this spontaneously occurring mouse mutant, defects in Schwann cell ensheathment and myelination are present in the proximal spinal roots (Bradley and Jenkison, 1973; Stifling, 1975) and have been correlated with associated defects in the Schwann cell basal lamina (Madrid et al., 1975). While the precise molecular defect in these animals is not known, these studies provide correlative support for the importance of the basal lamina for Schwann cell function. © The Rockefeller University Press, 0021-9525/93/12/1223/14 $2.00 The Journal of Cell Biology, Volume 123, Number 5, December 1993 1223-1236 1223 on O cber 0, 2017 jcb.rress. org D ow nladed fom Although these findings indicate that interactions between the Schwann cell and the extracellular matrix are critical for Schwann cell ensheathment and myelination, little is known about how the basal lamina regulates Schwann cell function. Despite the failure of Schwann cells to ensheathe or myelinate in the absence of a basal lamina, axons are still able to induce the proliferation of Schwann cells (Moya et al., 1980) and their expression of several myelin-related components, notably galactose cerebroside, the myelin-associated glycoprotein (MAG), t and P0 (Owens and Bunge, 1989; Brunden and Brown, 1990). Interestingly, in the absence of a basal lamina, MAG, which is normally present in the inner turn of the myelin sheath where it is believed to promote adhesion to the axon (Trapp and Quarles, 1982), is instead diffusely present at the Schwann cell surface, not just in proximity to the axon (Owens and Bunge, 1989). These results suggest that, while the basal lamina is not required for myelin protein synthesis, it is required for assembly of the myelin sheath which, in turn, may depend on the ability of the Schwann cell to establish an appropriate cellular polarity, with adaxonal and abaxonal surfaces (Bunge et al., 1986). In addition, the presence of the basal lamina could provide critical mechanical support to the Schwann cell as it extends processes around the axon during ensheathment or myelination. This is consistent with observations that the outer surface of the Schwann cell, which is in contact with the basal lamina, remains essentially stationary whereas the inner loop progressively circumnavigates around the axon during myelination (Bunge et al., 1989). It is also possible that, as in other systems, the extracellular matrix may have a role in transmembrane signaling of Schwann cells via extracellular matrix receptors (Hynes, 1992; Juliano and Haskill, 1993). The requirement for a basal lamina suggests that integrins have a critical role in Schwann cell function. Integrins are heterodimeric receptors, consisting of an ct and 13 subunit, that mediate the interactions of cells with extraceUular matrices and other cells (Buck and Horwitz, 1987; Hynes, 1987; Ruoslahti and Pierschbacher, 1987; Reichardt and Tomaselli, 1991). Limited studies have been done to date on the integrins expressed by Schwann cells. These studies suggest that the integrins al/31, c~2/31, ct5/31, and et6/34 are expressed by some Schwann cells in peripheral nerves (Sonnenberg et al., 1990; Toyota et al., 1990; Hsiao et al., 1991; Lefcort et al., 1992; Jaakkola et al., 1993). However, very little is known about the regulation of their expression during Schwann cell differentiation, their distribution in peripheral nerve, or their function during myelination. To initiate studies on the mechanisms by which the basal lamina regulates Schwann cell myelination, and the role of integrins in mediating these interactions, we have characterized, in an in vitro myelination system, the expression of the /31 and/34 integrins. Culturing Schwann cells alone or with neurons has allowed us to investigate the role of the axon in regulating Schwann cell integrin expression in the absence of other factors, including perineurial cells, that potentially complicate similar analyses in vivo. We have found that 1. Abbreviations used in this paper: DRG, dorsal root ganglion; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; BrDU, 5 -bromo-2'-deoxyuridine. the o~6B1 laminin receptor is constitutively expressed by Schwann cells, even in the absence of axonal contact, whereas expression by Schwarm cells of another basement membrane receptor, a6/34, is axonally regulated and dramatically increases at the onset of myelination in vitro. Immunolocalization studies suggest that ot6/34 may be present in the uncompacted membranes of the nascent myelin sheath, whereas in mature myelin, it is expressed at the outer Schwann cell membrane and in some Schmidt-Lanterman clefts. These results suggest that ot6/34 is likely to have an important role in promoting Schwann cell myelination via its interactions with the basal lamina. Materials and Methods