Interferon-g Inhibits Cell Cycle Exit in Differentiating Oligodendrocyte Progenitor Cells LI-JIN CHEW, 1 * WILLIAM C. KING, 2 ANN KENNEDY, 1 AND VITTORIO GALLO 1 1 Center for Neuroscience Research, Children’s Research Institute, Children’s National Medical Center, Washington, DC 2 Center for Cancer Research and Immunology, Children’s National Medical Center, Washington, DC KEY WORDS cytokine; myelin development; proliferation; survival; gene expression ABSTRACT The developmental processes of the oligodendrocyte pro- genitor cell (OPC) lineage that are targeted by interferon-g (IFN-g) were studied in primary rat OPC cultures. Under conditions of thyroid hormone-mediated oligodendrocyte dif- ferentiation, IFN-g produced a dose-dependent apoptotic response in OPCs. The lowest dose tested (15 ng/ml or 75 U/ml) was nonapoptotic, but activated detectable STAT1 DNA-binding. At this dose, IFN-g reduced the percentage of mature O1 1 cells and increased the percentage of imma- ture A2B5 1 OPCs. This was observed without significant change in total cell number and cytotoxicity, and was accompanied by an increase in BrdU-labeled A2B5 1 and O4 1 cells. FACS analysis confirmed a lack of apoptotic sub- G1 cells and revealed a greater percentage of S- and G2/M- phase OPCs with IFN-g treatment. Dual immunostaining with Ki-67 and Olig2 showed a smaller percentage of Olig2 1 cells in G 0 phase in IFN-g-treated OPCs, indicating loss of G1 control. Instead, increased levels and phosphory- lation of the checkpoint protein p34cdc2 by IFN- suggested increased partial arrest in G2. IFN-g not only sustained expression of PCNA and the G1-S regulators retinoblas- toma protein, cyclin D1, cyclin E, and cdk2, but also decreased p27 levels. In addition to changes in cell prolif- eration and differentiation, IFN-g attenuated myelin basic protein (MBP) expression significantly, which was asso- ciated with decreased expression of both MBP and Sox10 RNAs. These findings indicate that IFN-g not only main- tains cell cycle activity that could predispose OPCs to apop- tosis, but also overrides G 1 –G 0 signals leading to thyroid hormone-mediated terminal differentiation and myelin gene expression. V V C 2005 Wiley-Liss, Inc. INTRODUCTION Remyelination by oligodendrocyte progenitor cells (OPCs) requires the recapitulation of events that nor- mally occur during development: OPCs proliferate, migrate to the demyelinated lesion, and differentiate into myelin forming cells upon contacting axons (Frank- lin and Blakemore, 1997). Terminal differentiation of cells is often associated with a transition from a prolif- erative to a quiescent state, from which differentiated cells do not reenter cell division (Halevy et al., 1995; Yan and Ziff, 1995). Oligodendrocyte lineage progression is regulated by an intrinsic program that is modulated by extracellular signals. In vitro, the combined applica- tion of the growth factors basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) inhi- bits oligodendrocyte differentiation through maintaining continuous cell division (Bogler et al., 1990; McKinnon et al., 1990), a phenomenon that may involve increased sensitivity to glutamate receptor modulation (Gallo et al., 1994; Chew et al., 1997). Growth factor withdra- wal and thyroid hormone treatment, on the other hand, result in cessation of cell division and accelerated differ- entiation into oligodendrocytes (Collarini et al., 1992; Tang et al., 1998). The importance of thyroid hormone in oligodendrocyte differentiation was demonstrated in mice deficient in thyroid hormone receptor a1, whose oligodendrocyte progenitor cells (OPCs) were unable to stop dividing and differentiate in response to thyroid hormone in culture (Billon et al., 2002). Interferon-g (IFN-g) has been implicated as an impor- tant factor in the immune-mediated demyelinating dis- ease multiple sclerosis. Elevated T-cell secretion of IFN-g (Strunk et al., 2000) is promoted by neurons and glia in inflamed CNS (Chabas et al., 2001), and gene targets of IFN-g-receptor signaling pathways are increased in MS lesions (Lock et al., 2002). Experimentally, IFN-g has been shown to exhibit pathological effects on oligodendrocytes in vitro and myelination in vivo. Cultured oligodendro- cytes treated with IFN-g undergo apoptosis or necrosis (Vartanian et al., 1995; Baerwald and Popko, 1998), or dis- play metabolic perturbations (Turnley et al., 1991). Mye- lin gene expression is also decreased by IFN-g treatment (Agresti et al., 1996; Baerwald and Popko, 1998). When IFN-g is overexpressed in the CNS, a shaking phenotype is observed, which is correlated with myelin abnormalities (Corbin et al., 1996; Horwitz et al., 1997). Death-related pathways initiated by IFN-g have been studied in both CG4 cells and primary OPCs (Andrews et al., 1998), but IFN-g can also inhibit myelin gene expression and OPC differentiation without overt cell death (Agresti et al., 1996). IFN-g-induced MHC I, IRF-1 activation, and STAT1 binding have been demonstrated (Agresti et al., 1996, 1998; Andrews et al., 1998) in OPCs, *Correspondence to: Li-Jin Chew, Center for Neuroscience Research, Rm. 5340, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010. E-mail: lchew@cnmcresearch.org Received 6 December 2004; Accepted 5 April 2005 DOI 10.1002/glia.20232 Published online 26 May 2005 in Wiley InterScience (www.interscience.wiley. com). V V C 2005 Wiley-Liss, Inc. GLIA 52:127–143 (2005)