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-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) 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-kappaB. 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-kappaB. 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.

Recent studies have shown that N 6 ,2 H -O-dibutyryladenosine 3 H :5 H cyclic monophosphate (dbcAMP) increases the expression of speci®c subtypes of Na 1 -dependent glutamate transporters in cultured astrocytes. Our group also found that treatment of astrocytes with dbcAMP for several days increases the Na 1 -independent accumulation of L-[ 3 H]glutamate. In this study, the properties of this Na 1 -independent accumulation were characterized, and the mechanism by which dbcAMP up-regulates this process was investigated. This accumulation was markedly reduced in the absence of Cl 2 and was also inhibited by several anion-exchange inhibitors, including 4,4 H -diisothiocyanatostilbene-2,2 H -disulfonic acid, 4,4 H -dinitrostilbene-2,2 H -disulfonic acid and 4-acetamido-4 Hisothiocyanatostilbene-2,2 H -disulfonic acid, suggesting that this activity is mediated by a Cl 2 -dependent transporter. In addition, this activity was inhibited by micromolar concentrations of several inhibitors of another Cl 2 -dependent (Na 1 -independent) transport activity frequently referred to as system x c 2 (L-cystine, L-a-aminoadipate, L-homocysteate, quisqualate, b-N-oxalyl-l-a,b-diaminopropionate, ibotenate). This activity was competitively inhibited by several phenylglycine derivatives previously characterized as inhibitors of metabotropic glutamate receptor activation. The concentration-dependence for Na 1 -independent, Cl 2 -dependent L-[ 3 H]glutamate uptake activity was compared for dbcAMP-treated and untreated astrocytes. Treatment with dbcAMP increased the V max of this Cl 2 -dependent transport activity by sixfold but had no effect on the K m value. System x c 2 requires two subunits, xCT and 4F2hc/CD98, to reconstitute functional activity. We found that dbcAMP caused a twofold increase in the levels of xCT mRNA and a sevenfold increase in the levels of 4F2hc/ CD98 protein. This study indicates that dbcAMP up-regulates Cl 2 -dependent L-[ 3 H]glutamate transport activity in astrocytes and suggests that this effect is related to increased expression of both subunits of system x c 2 . Because this activity is thought to be important for the synthesis of glutathione and protection from oxidant injury, understanding the regulation of system x c 2 may provide alternate approaches to limit this form of injury. Keywords: carboxyphenylglycine, CD98, cystine, 4F2hc, glutamate transport, system x c 2 .Effective clearance of glutamate, a major excitatory amino acid in the mammalian CNS, from the synaptic cleft is necessary to maintain ef®cient synaptic transmission. It is thought that the main mechanism of the removal of glutamate is a family of ®ve recently cloned high-af®nity Na 1 -dependent transporters (for review, see Danbolt 1994;Sims and Robinson 1999). The transporters GLT-1 and GLAST are primarily found in astrocytes, EAAC1 and EAAT4 are predominantly found in neurons and EAAT5 is found only in retina. These transporters are responsible for preventing the accumulation of excitotoxic levels of glutamate as well as limitin...