Osteopontin is upregulated during in vivo demyelination and remyelination and enhances myelin formation in vitro

Abstract

We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN^−/− mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.

Introduction

Myelination is a complex process in the vertebrate central nervous system, involving migration, proliferation, and differentiation of oligodendrocyte progenitor cells followed by highly complex interactions between the processes of mature oligodendrocytes and the axonal tracts of neurons Baumann and Pham-Dinh, 2001, Lubetzki and Stankoff, 2000, Orentas and Miller, 1998, Vabnick and Shrager, 1998. Several growth factors, notably insulin-like growth factor 1 (IGF-1), platelet-derived growth factor AA (PDGF-AA), leukemia inhibitory factor (LIF), and basic fibroblast-derived growth factor (bFGF), control the proliferation and survival of oligodendrocytes and their precursors Mayer et al., 1994, McKinnon et al., 1990, Raff et al., 1988. However, mechanisms underlying how the myelin sheath is formed around an axon are still unknown.

The adult nervous system retains remyelinative capacity in the absence of extrinsic stimuli Franklin, 2002, Wolswijk, 1998. Following toxin-induced or autoimmune-mediated myelin damage, oligodendrocyte progenitors, expressing markers such as the chondroitin sulfate proteoglycan NG2, and the platelet-derived growth factor alpha receptor (PDGFαR) cause remyelinating oligodendrocytes Chang et al., 2000, Dubois-Dalcq and Armstrong, 1990, Nishiyama, 1998, Nishiyama, 2001, Nishiyama et al., 1999, Watanabe et al., 2002. The development of oligodendrocyte precursors (OLPs) is therefore a key mechanism in the process of remyelination Gensert and Goldman, 1997, Levine and Reynolds, 1999, Levine et al., 2001, Redwine and Armstrong, 1998. In vivo, the subventricular zone (SVZ) was recently shown to generate oligodendrocytes in response to demyelination Decker et al., 2002, Picard-Riera et al., 2002.

Oral administration to rodents of the organic compound cuprizone (bis-cyclohexanone oxalyldihydrazone), a copper chelator toxic to oligodendrocytes, results in demyelination of specific areas within the corpus callosum, followed by spontaneous remyelination upon termination of treatment Blakemore, 1972, Blakemore, 1973, Ludwin, 1978, Ludwin, 1979, Matsushima and Morell, 2001. Cuprizone-induced CNS tissue damage thus constitutes a model for studying remyelination in vivo Arnett et al., 2001, Mason et al., 2000b, Mason et al., 2001, McMahon et al., 2001.

In the current study, microarray analysis of cuprizone-treated frontal brain tissue was used to identify temporal changes in the levels of mRNA during the demyelination and remyelination process. One of the genes that exhibited considerable changes in expression during the remyelination phase was osteopontin (opn) (Oldberg et al., 1986), also known as early T-lymphocyte activation protein 1 (Eta-1) (Ashkar et al., 2000) or secreted phosphoprotein 1 (Spp1) (Sodek et al., 2000). OPN is a secreted glycoprotein with pleiotropic developmental and immunological functions (Denhardt et al., 2001). In vitro, OPN regulates adhesion, proliferation, migration, differentiation, and survival of various cell types (Sodek et al., 2000). However, the precise function of the protein in the CNS remains unknown. OPN is a member of the matricellular class of secreted glycoproteins, including osteonectin/SPARC, thrombospondins 1 and 2, tenascins C and X, and vitronectin, since it contains an Arg-Gly-Asp (RGD) cell adhesion motif mediating interactions with several receptors, such as the α_vβ₃ integrin Helluin et al., 2000, Horton, 1997.

Here, we demonstrate the in vitro capacity of OPN protein to induce proliferation of oligodendrocyte-derived cell lines Jung et al., 1995, Louis et al., 1992 and also increase myelination in primary mixed cortical cultures (Lubetzki et al., 1993). We show immunohistochemical evaluation of OPN expression during de- and remyelination in vivo, suggesting a role for OPN in the process of myelination and remyelination. Our data suggest that OPN, secreted by astrocytes and microglial cells during developmental myelination and demyelination, is a novel stimulator of myelination and remyelination.