Transcriptional Regulation of Scar Gene Expression in Primary Astrocytes PAUL GRIS, 1,2 ALLYSON TIGHE, 1,3 DAVID LEVIN, 1 RAHUL SHARMA, 1 AND ARTHUR BROWN 1,2,3 * 1 The Spinal Cord Injury Team, BioTherapeutics Research Group, Robarts Research Institute, London, Ontario, Canada 2 Program in Neuroscience, The University of Western Ontario, London, Ontario, Canada 3 The Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada KEY WORDS CSPG; SOX9; xylosyltransferase; laminin; fibronectin ABSTRACT The failure of the adult injured spinal cord to support axo- nal regeneration is in part attributed to the glial scar. Reac- tive astrocytes constitute a major cellular component of the glial scar and are heterogeneous with respect to the extrac- ellular matrix proteins that they secrete. Astrocytes may produce antiregenerative molecules such as chondroitin sul- phate proteoglycans (CSPGs) or proregenerative molecules such as laminin and fibronectin. While many different CSPGs are expressed after spinal cord injury (SCI) they all rely on the same enzymes, xylosyltransferase-I and -II (XT- I, XT-II) and chondroitin 4-sulfotransferase (C4ST) to add the repulsive chondroitin sulfate side chains to their core proteins. We show that XT-I, XT-II, and C4ST are part of a CSPG biosynthetic gene (CBG) battery. Using primary astrocyte cultures and quantitative PCR we demonstrate that TGFb2, PDGF, and IL-6 induce the expression of CBGs, laminin and fibronectin by several-fold. We further show that over-expression of the transcription factor SOX9 also strongly induces the expression of CBGs but does not increase the expression of laminin or fibronectin. Corre- spondingly, SOX9 knock-down in primary astrocytes causes a decrease in CBG and an increase in laminin and fibronec- tin mRNA levels. Finally, we show that the in vivo expres- sion profiles of TGFb2, PDGF, IL-6, and SOX9 are consist- ent with their potential roles in differentially regulating CBGs, laminin and fibronectin gene expression in the injured spinal cord. This work suggests that SOX9 levels may be pivotal in determining the balance of pro- and anti- regenerative extracellular matrix molecules produced by astrocytes. V V C 2007 Wiley-Liss, Inc. INTRODUCTION The absence of axonal regeneration after spinal cord injury (SCI) has been attributed in part to the nonpermis- sive environment of the glial scar that inhibits axonal growth (Fawcett and Asher, 1999). Although macro- phages, microglia, oligodendrocytes, invading Schwann cells and meningeal fibroblasts contribute to the glial scar, astrocytes predominate (Fawcett and Asher, 1999). Reactive astrocytes in the injured CNS are heterogeneous with respect to their production of scar proteins (Fitch and Silver, 1997). Whereas in the majority of cases the extracellular matrix molecules (ECM) produced by astro- cytes have been shown to inhibit axonal regeneration (Bahr et al., 1995; Davies et al., 1999; McKeon et al., 1991; Reier and Houle, 1988), astrocytes also have been shown to secrete ECM molecules that promote axonal growth (McKeon et al., 1991). Thus astrocytes may pro- mote or inhibit regeneration after SCI depending upon the balance of growth-inhibiting and growth-promoting ECM molecules that they produce. Chondroitin sulfate proteoglycans (CSPGs) are prob- ably the most important of the inhibitory molecules pro- duced by reactive astrocytes (Eddleston and Mucke, 1993; Fawcett and Asher, 1999; Silver and Miller, 2004). In vivo and in vitro studies have shown that neurons cease to extend their axons into areas rich in CSPGs (Davies et al., 1997, 1999; McKeon et al., 1991; Zuo et al., 1998). CSPGs share a common structure comprising a central core protein with a number of chondroitin sulfate side chains (Morgenstern et al., 2002). Chondroitin sulfate side chain synthesis is initiated by the addition of xylose onto a serine moiety of the core protein. This rate-limiting step in the generation of chondroitin sulfate side chains function is carried out by the enzyme xylosyltransferase (XT) that exists in two isoforms encoded by two different genes XT-I and XT-II (Gotting et al., 2000; Kearns et al., 1991; Schwartz, 1977). These side chains are subse- quently sulfated by either chondroitin 4-sulfotransferase (C4ST) (Yamauchi et al., 2000) or chondroitin 6-sulfo- transferase (Fukuta et al., 1995) although, in astrocytes, C4ST predominates (Gallo and Bertolotto, 1990). Astrocytes can also produce an array of growth promot- ing molecules including laminin (Liesi and Silver, 1988), N-cadherin (Tomaselli et al., 1988), neural cell adhesion molecule (NCAM) (Neugebauer et al., 1988), and fibronec- tin (Matthiessen et al., 1989). Laminin and fibronectin have been shown to be good substrates for neurite exten- sion in in vitro models of axon growth (Costa et al., 2002; Fok-Seang et al., 1995; Hammarback et al., 1988; McKeon et al., 1991; Rogers et al., 1983, 1987). In vivo models dem- onstrate that sensory axon regeneration is dependent on astrocyte-associated fibronectin (Davies et al., 1997, 1999; Grant sponsors: Canadian Institutes of Health Research (CIHR), Krembil Foun- dation (Toronto). *Correspondence to: Dr. Arthur Brown, The Robarts Research Institute, 100 Perth Drive, London, Ontario, N6A 5K8 Canada. E-mail: abrown@robarts.ca Received 6 March 2007; Revised 7 May 2007; Accepted 1 June 2007 DOI 10.1002/glia.20537 Published online 27 June 2007 in Wiley InterScience (www.interscience. wiley.com). GLIA 55:1145–1155 (2007) V V C 2007 Wiley-Liss, Inc.