Identification of a collagen potentiated neurite promoting factor isolated from C6 glioma cells

The C6 glial cell line has been used as a model cell system for the investigation of new glial produced neurotrophic and neurotropic molecules. By using the C6 cell line grown in a defined medium on collagen, this laboratory has isolated a distinct neurite promoting factor (NPF) that is potentiated by the presence of collagen (CPNPF). We have observed that C6 cells cultured in a defined medium on collagen (rat type-I) slowed their growth rate and expressed an astrocytic- or oligodendrocytic-like morphology. CPNPF, at this state of purity, appears to be a distinct NPF which induces neurite outgrowth (neurites of 1 or more somal diameters) in PC12 cells. These neurite promotion effects, however, appear to support the neuron morphology for only a short period (4 days) of time without the presence of neurotrophic factor (NTF). The neurite promoting activity is ineffective in inducing neurite outgrowth using mouse neuroblastoma cells (neuro-2a). CPNPF appears to be a heat stable protein whose activity does not depend on the presence of intact collagen, heparin sulfate proteoglycan (HSPG), or chondroitin sulfate proteoglycan (CSPG). Exposure to dissociative conditions results in a loss of neurite promoting activity. CPNPF is not a glycoprotein that contains an accessible alpha-D-mannopyranosyl, alpha-D-glucopyranosyl, or a sterically related residue (hydroxyl groups in the C-3,4, and 5 positions). Although these residues are not present on all glycoproteins, it does indicate that CPNPF is most likely not a glycoprotein. CPNPF activity is not blocked by neutralizing antibodies directed toward NGF, beta-FGF, IL-1 beta, IL-6, TGF-beta 2, TGF-beta 1.2, TGF-beta 3, TGF-beta 5, or EGF. CPNPF appears to either be oligomeric protein or a complex of proteins. On the basis of indirect evidence, it does not appear to be glial derived protease nexin-I. The alteration in morphology of the C6 glial cell line by serum-free conditions in the presence of collagen may have induced the production of a potentially new NPF not seen by previous investigators.