Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules.

Abstract

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated lamininbinding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.Little is known about the cellular mechanisms that generate the complex patterns of axon growth seen in nervous system development. The search for factors that may control axonal growth and guidance in vivo has relied heavily on the study of neurite growth in vitro (1). While it has long been appreciated that cell-substratum interactions influence the growth of neurites (2, 3), only recently have candidates emerged for molecules that might serve such a function in vivo. Helfand et al. (4) and Collins (5) were the first to report that cultured heart cells produce a substance that, when adsorbed to a substratum, dramatically enhances neurite outgrowth by neurons plated on that substratum. Since then, many other cell types have been shown to release into conditioned medium (CM) ~ a substance with a similar effect on neurons (6-9). It is characteristic of these "neurite outgrowth-promoting factors" found in CM to adsorb preferentially to polycationic substrata (i.e., substrata coated with polylysine or polyorni-J Abbreviations used in this paper: BCE, bovine corneal endothelial; CM, conditioned medium (media); DME-0.I, Dulbecco's modified Eagle's medium containing 0. 1% glucose; EIA, enzyme-linked immunoassay; HeS, heparan sulfate; TBS, Tris-buffered saline. thine) and cause many types of neurons to extend neurites both sooner and more rapidly than they would on untreated polycationic substrata or other conventional culture substrata (e.g., plastic, collagen). Interestingly, neurite outgrowth-promoting factors do not replace trophic factors in supporting the long-term survival of neurons, and are unrelated to nerve growth factor (7,8).Recently, considerable efforts have been made to characterize the neurite outgrowth-promoting factors found in CM (10). They are produced by a large number of cell types (6,8), are prot...

Functional analysis of SIRPα in the growth cone

“…Thus, SIRP␣ seems to be necessary for the modulation of axonal growth by ECM molecules, such as laminin. As such it may provide at least a partial molecular explanation for the long-known observation that laminin promotes growth-factor-stimulated neurite outgrowth (Lander et al, 1985a;Lander et al, 1985b;Edgar et al, 1988;Goldberg et al, 2002;Liu et al, 2002). The interaction of SIRP␣ with CD47 (or other, so far unknown ligands) may further modulate growth factor responses.…”

“…It may suggest SIRP␣ involvement in growth control. Involvement of SIRP␣ in axonal growth control For a long time, cell adhesion has been known to affect neurite outgrowth (Edgar, 1985;Lander et al, 1985a;Lander et al, 1985b). At least in some cases, this seems to involve Rho A (Liu et al, 2002).…”

Extracellular Matrix Molecules And Their Receptors: Functions In Neural Development

“…Although it has been appreciated for several years that 1aminins from different sources have different structures and antigenic properties (cf. Lander et al 1985, Edgar et al 1988, only in the past year, with the discovery of new genes encoding LN homologues, has a definite advance been made in understanding the causes of this heterogeneity.…”

Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies.

“…In addition to purified EHS laminin, a considerable number of other neurite-promoting factors have been described, the activity of which appears to be due to laminin that is often complexed noncovalently with proteoglycan and with the protein entactin/nidogen (12,13,24,25). Antisera against Figure 1.…”