The glia maturation factor (GMF), which was discovered in our laboratory, is a highly conserved protein predominantly localized in astrocytes. GMF is an intracellular regulator of stress-related signal transduction. We now report that the overexpression of GMF in astrocytes leads to the destruction of primary oligodendrocytes by interactions between highly purified cultures of astrocytes, microglia, and oligodendrocytes. We infected astrocytes with a replication-defective adenovirus carrying the GMF cDNA. The overexpression of GMF caused the activation of p38 MAP kinase and transcription factor NF-jB, as well as the induction of granulocytemacrophage colony-stimulating factor (GM-CSF) mRNA and protein in astrocytes. Small interfering RNA-mediated GMF knockdown completely blocked the GMF-dependent activation of p38 mitogen-activated protein kinase (MAPK), NF-jB, and enhanced expression of GM-CSF by astrocytes. Inhibition of p38 MAPK or NF-jB by specific inhibitors prevented GM-CSF production. The cell-free conditioned medium from overexpressing GMF astrocytes contained 320 ± 33 pg/mL of GM-CSF, which was responsible for enhanced production and secretion of TNF-a, IL-1b, IL-6, and IP-10 by microglia. Presence of these inflammatory cytokines in the conditioned medium from microglia efficiently destroyed oligodendrocytes in culture. These results suggest that GMF-induced production of GM-CSF in astrocytes is depending on p38 MAPK and NF-jB activation. The GM-CSF-dependent expression and secretion of inflammatory cytokine/chemokine, TNF-a, IL-1b, IL-6, and IP-10, is cytotoxic to oligodendrocytes, the myelinforming cells in the central nervous system, and as well as neurons. Our results suggest a novel pathway of GMF-initiated cytotoxicity of brain cells, and implicate its involvement in inflammatory diseases such as multiple sclerosis.

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A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and rinally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of =17,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total known residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein.Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma line and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor f.The trophic function of the nervous system on peripheral organs was detected in an in vivo system as early as 1823 (1). The in vitro counterpart of this experiment was conducted in 1939 (2-4), showing the mitogenic effect of brain homogenates on cultured fibroblasts. However, it was not until 1972t that an autoregulatory role of brain-derived growth factors on brain cells was first demonstrated in this laboratory (5,6). This factor was named glia maturation factor (GMF), based on the bioassay system in which the activity was first observed. Since then this laboratory has engaged in an intensive search for the molecule responsible for this important function. The effort culminated in the isolation of a brain protein with a unique amino acid sequence documented below. MATERIALS AND METHODSPreliminary Purification of GMF-j3. The published procedure (7, 8) through the Sephadex G-75 step was followed with slight modifications. Briefly, four beef brains [1.0 kg (total wet weight)] were homogenized and centrifuged to obtain the crude extract. The ammonium sulfate precipitate between 45% and 70% saturation was dissolved in 100 ml of water and dialyzed for two 6-hr periods against 10 liters of water. The sample was adjusted to contain 0.02 M Tris HCl (pH 7.45) and applied to a DEAE-Sephacel column (2.5 x 37 cm). After eluting with 1.25 liters of a linear gradient of 0-0.3 M NaCl in the same buffer at 50 ml/hr, the fractions that showed mitogenic and morphologic activities on astrocytes were pooled (500 ml) and concentrated to 50 ml by Amicon PM10 filtration. The sample was applied to a Sephadex G-75 column (5 x 90 cm) and eluted with 0.15 M NaCl containing the above buffer at 40 ml/hr. The fractions active on astrocytes were pooled (500 ml) and used for the final steps of purification (see Results).Bioassay Production of Antibodies and Immunoassay. The mouse monoclonal antibody G2-0...

We previously demonstrated that glia maturation factor (GMF), a 17-kDa brain protein, can be phosphorylated in test tube by several protein kinases, and that endogenous GMF is rapidly phosphorylated upon stimulation of astrocytes by phorbol 12-myristate 13-acetate. We further observed that protein kinase A (PKA)-phosphorylated GMF is a potent inhibitor (IC50 = 3 nM) of the ERK1/ERK2 (p44/p42) subfamily of mitogen-activated protein (MAP) kinase. We now report that, by contrast, PKA-phosphorylated GMF strongly enhances the activity of a related but distinct subfamily of MAP kinase, the p38 MAP kinase, showing an increase of 60-fold over baseline and an EC50 of 7 nM. Non-phosphorylated GMF or GMF phosphorylated by other kinases exhibits only minimal effect. The intracellular interaction of PKA, GMF, and p38 is supported by the phosphorylation of GMF upon cellular stimulation by forskolin (blocked by PKA inhibitor) and by the co-immunoprecipitation of p38 with GMF from cell lysates. Withdrawal of nerve growth factor from PC12 leads to increased GMF phosphorylation with a time course similar to that reported for p38 activation. The results correlate well with a previous report that ERK and p38 carry out opposing functions and implicate GMF as a regulator of major cellular events.

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