Epidermal Growth Factor Receptor Agonists Increase Expression of Glutamate Transporter GLT-1 in Astrocytes through Pathways Dependent on Phosphatidylinositol 3-Kinase and Transcription Factor NF-kB

The glial glutamate transporter GLT-1 may be the predominant Na-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-a] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the Vmax for Na -dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGFand dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury. The acidic amino acid glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS). Low extracellular concentrations of glutamate, a prerequisite for the high signal-to-noise ratio of synaptic communication, are maintained by a family of Na-dependent transporters (for review, see Sims and Robinson, 1999). These proteins, which rapidly clear glutamate from the synaptic cleft, are essential for signal termination, neurotransmitter recycling, and prevention of excitotoxicity. Five highaffinity subtypes, identified by molecular cloning, are differentially expressed throughout the CNS. EAAC1 (EAAT3) and EAAT4 are localized predominantly in neurons, and EAAT5 is enriched in retinal tissue, whereas GLAST (EAAT1) and GLT-1 (EAAT2) are generally expressed in astrocytes (for review, see Sims and Robinson, 1999). Both in vivo and in vitro studies have provided compelling evidence that transport into astrocytes is the predominant route for clearance of extracellular glutamate and for limiting excitotoxicity. Several reports indicate that in neuronal culture models, Na-dependent transport into astrocytes attenuates glutamate toxicity (for review, see Robinson and This study was supported by Grants NS29868 and HD26979 (to M.B.R), NS33958 (to J.D.R.), NS36465 (to M.B.R. and J.D.R.), and NS34017 (to J.B.G.). 1 O.Z. and B.D.S. contributed equally to the present study. ABBREVIATIONS: CNS, central nervous system; EGFR, epidermal growth factor receptor; TGF-a, transforming growth factor-a; dbcAMP, dibutyryl-cAMP; NCM, neuron-conditioned medium; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; PDTC, pyrrolidinedithiocarbamate; ECL, enhanced chemiluminescence; NGF, nerve growth factor; PDGF, platelet-derived growth factor; Bis II, bisindolylmaleimide II; bFGF, basic fibroblast growth factor; TBS, Tris-buffered saline; TGT, TBS containing 5% normal goat serum and 0.1% Triton X-100; GFP, green fluorescent protein; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PLCg, phospholipase Cg; MAP, mitogen-activated protein; MEK, MAP kinase kinase; Erk, extracellular signal receptor-activated kinase; PKC, protein kinase C; NF-kB, nuclear factor-kB. 0026-895X/00/040667-12$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:667–678 (2000). 667 at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from Dowd, 1997). Furthermore, gene deletion, gene knockdown, and pharmacological studies indicate that the GLT-1 subtype may contribute up to 90% of total transport in the forebrain (for review, see Robinson, 1999). Transport deficiency and down-regulation of GLT-1 and/or GLAST are associated with neurodegenerative disorders such as amyotrophic lateral sclerosis, epilepsy, hypoxia/ischemia, and head trauma (for review, see Sims and Robinson, 1999). Therefore, up-regulation of glial transporters may be a promising strategy for the treatment and/or prevention of neurodegeneration accompanying CNS insults. GLT-1, a major glial transporter, would be an appropriate target for such a strategy. Although the genes have been identified for these transporters, the promoter elements have not been characterized, and the mechanisms regulating their expression remain unclear. There is evidence to suggest that induction of GLT-1 is associated with astrocyte differentiation, but very little is known about the mechanisms involved (Swanson et al., 1997; Schlag et al., 1998). In vivo, the expression of GLT-1 changes dramatically during development with low levels in the early postnatal period and a rapid increase during synaptogenesis (for review, see Sims and Robinson, 1999). In contrast to mature astrocytes in vivo, primary astrocytes in culture express essentially no GLT-1 protein and thus can be used as a model system to identify the molecular mechanisms controlling transporter expression. cAMP analogs and coculturing with neurons stimulate expression of GLT-1 in these astrocytes (Gegelashvili et al., 1997; Swanson et al., 1997; Schlag et al., 1998). In both cases, induction of GLT-1 is associated with differentiation of astrocytes. The effects of coculturing with neurons can be at least partially attributed to the release of a secreted molecule. Although both neurons and astrocytes in cocultures release various types of molecules, epidermal growth factor receptor (EGFR) agonists have been strongly implicated in the regulation of proliferation and differentiation of astrocytes in vitro and in vivo (see Discussion). In the present study, we demonstrate that growth factors that act through EGFR [EGF and transforming growth factor-a (TGFa)] induce expression of GLT-1 in primary astrocytes in culture. The effects of cell permeable inhibitors of various signaling pathways activated by either dibutyryl-cAMP (dbcAMP) or EGF were examined. Although some of these inhibitors selectively blocked the effects of either dbcAMP or EGF, others blocked the effects of both EGF and dbcAMP. Similarly, some of the same inhibitors blocked the neuron-conditioned medium (NCM)-mediated induction of GLT-1. These studies suggest that dbcAMP, EGF, and NCM induce GLT-1 expression through activation of the same signaling pathways. Experimental Procedures Materials. Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT); all other cell culture reagents were from Gibco-BRL (Gaithersberg, MD). Anti-glial fibrillary acidic protein (GFAP) antibody, poly-D-lysine, antiactin antibody, dbcAMP, pyrrolidinedithiocarbamate (PDTC), and porcine pancreas insulin were obtained from Sigma Chemical Co. (St. Louis, MO). L-[H]glutamate and [a-P]deoxycytidine 59-triphosphate were obtained from DuPont/ NEN (Boston, MA). Donkey anti-rabbit horseradish peroxidase IgG, rainbow molecular weight markers, Hybond N1, and enhanced chemiluminescence (ECL) kits were purchased from Amersham (Arlington Heights, IL). Immobilon P membrane was from Millipore (Bedford, MA). Dihydrokainate was purchased from Genosys (The Woodlands, TX). 7S Nerve growth factor (mouse submaxillary gland) (NGF), human recombinant platelet-derived growth factor (BB homodimer) (PDGF), genistein, PD98059, tyrphostin A25, wortmannin, bisindolylmaleimide II (Bis II), and KT5720 were purchased from Calbiochem (La Jolla, CA). Mouse recombinant EGF, basic fibroblast growth factor (bFGF) and TGF-a were obtained from Collaborative Biomedical Products (Bedford, MA). LY294002 was obtained from Biomol (Plymouth Meeting, PA). All growth factors and PDTC were dissolved in sterile deionized water. All inhibitors were dissolved in dimethyl sulfoxide. GenePorter transfection reagent was purchased from Gene Therapy Systems (San Diego, CA). Immumount was purchased from Shandon (Pittsburgh, PA). Anti-mouse IgG and IgM-fluorescein and anti-rabbit IgG-rhodamine conjugates were obtained from Jackson ImmunoResearch (West Grove, PA). A2B5 antibody was made as previously described (for original citation, see Grinspan et al., 1996). Rabbit complement was purchased from ICN Biomedicals (Aurora, OH) or from Accurate Chemical & Scientific Corp. (Westbury, NY). The GLT-1 cDNA in pBluescript SKwas the generous gift of Dr. B. Kanner. The GLAST cDNA was generated by reverse transcription-polymerase chain reaction with specific primers and cloned into pBluescript SK-. Cell Culture. Astrocyte cultures were prepared from the cortices of neonatal rats (1–3 days old) as previously described (Schlag et al., 1998) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS, 10% Hams F-12, and 0.24% penicillin/streptomycin (10,000 U/ml penicillin, 10,000 mg/ml streptomycin). Cells were plated at a uniform density of 2.5 3 10 cells/ml (3 3 10 cells/cm) onto 10-cm or 12-well sterile polystyrene dishes. The cultures were maintained in a 5% CO2 incubator at 37°C and fed with a complete medium exchange twice a week for 14 days. At 14 days in vitro, cultures reach confluency. Approximately 95% of the cells in these cultures are astrocytes based on expression of cellspecific immunohistochemical markers (for original citation, see Schlag et al., 1998). To kill contaminating oligodendrocyte precursors (A2B5-positive cells), these cultures were washed once with HEPES-buffered saline solution, and then incubated in Dulbecco’s modified Eagle’s medium (2 ml/10-cm dish or 500 ml/well in a 12-well plate) with A2B5 hybridoma supernatant (diluted 1:50) and rabbit complement (diluted 1:20) for 45 min at 37°C and 5% CO2. The optimal concentration of A2B5 antibody and complement required for the complete elimination of A2B5/GLT-1 positive cells was determined in preliminary experiments. The cultures were washed 3 times with HEPES-buffered saline solution, incubated for 24 h in standard culture medium and then treated. Cells were fed with a complete medium exchange and fresh drug or vehicle every 3 to 4