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-κB
- Olga Zelenaia1,1,
- Brian D. Schlag1,1,
- Gordon E. Gochenauer1,
- Raquelli Ganel4,
- Wei Song3,
- Jacqueline S. Beesley2,
- Judith B. Grinspan2,
- Jeffrey D. Rothstein4 and
- Michael B. Robinson1
- Departments of 1Pediatrics and Pharmacology (O.Z., B.D.S., G.E.G., M.B.R), 2Department of Neurology (J.B.G., J.S.B.), 3Department of Neuroscience (W.S.), Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania; and 4Department of Neurology, The Johns Hopkins University, Baltimore Maryland (R.G., J.D.R)
Abstract
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-α] 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 EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-κB. 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-κB. 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.
Footnotes
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Send reprint requests to: Dr. Michael B. Robinson, 502N Abramson Pediatric Research Building, 34th and Civic Center Blvd., Philadelphia, PA 19104-4318. E-mail: Robinson{at}pharm.med.upenn.edu
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↵1 O.Z. and B.D.S. contributed equally to the present study.
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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.).
- Abbreviations:
- CNS
- central nervous system
- EGFR
- epidermal growth factor receptor
- TGF-α
- transforming growth factor-α
- 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
- PLCγ
- phospholipase Cγ
- MAP
- mitogen-activated protein
- MEK
- MAP kinase kinase
- Erk
- extracellular signal receptor-activated kinase
- PKC
- protein kinase C
- NF-κB
- nuclear factor-κB
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- Received November 11, 1999.
- Accepted January 3, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
