Malignant glioma is a consistently fatal brain cancer. The tumor invades the surrounding tissue, limiting complete surgical removal and thereby initiating recurrence. Identifying molecules critical for glioma invasion is essential to develop targeted, effective therapies. The expression of astrocyte elevated gene-1 (AEG-1) increases in malignant glioma and AEG-1 regulates in vitro invasion and migration of malignant glioma cells by activating the nuclear factor-KB (NF-KB) signaling pathway. The present studies elucidate the domains of AEG-1 important for mediating its function. Serial NH 2 -terminal and COOH-terminal deletion mutants were constructed and functional analysis revealed that the NH 2 -terminal 71 amino acids were essential for invasion, migration, and NF-KB-activating properties of AEG-1. The p65-interaction domain was identified between amino acids 101 to 205, indicating that p65 interaction alone is not sufficient to mediate AEG-1 function. Coimmunoprecipitation assays revealed that AEG-1 interacts with cyclic AMPresponsive element binding protein-binding protein (CBP), indicating that it might act as a bridging factor between NF-KB, CBP, and the basal transcription machinery. Chromatin immunoprecipitation assays showed that AEG-1 is associated with the NF-KB binding element in the interleukin-8 promoter. Thus, AEG-1 might function as a coactivator for NF-KB, consequently augmenting expression of genes necessary for invasion of glioma cells. In these contexts, AEG-1 represents a viable potential target for the therapy of malignant glioma.

It is well established that Ha-ras and c-myc genes collaborate in promoting transformation, tumor progression, and metastasis. However, the precise mechanism underlying this cooperation remains unclear. In the present study, we document that astrocyte elevated gene-1 (AEG-1) is a downstream target molecule of Ha-ras and c-myc, mediating their tumor-promoting effects. AEG-1 expression is elevated in diverse neoplastic states, it cooperates with Ha-ras to promote transformation, and its overexpression augments invasion of transformed cells, demonstrating its functional involvement in Ha-ras-mediated tumorigenesis. We now document that AEG-1 expression is markedly induced by oncogenic Ha-ras, activating the phosphatidylinositol 3-kinase signaling pathway that augments binding of c-Myc to key E-box elements in the AEG-1 promoter, thereby regulating AEG-1 transcription. In addition, Ha-ras-mediated colony formation is inhibited by AEG-1 siRNA. This is a demonstration that Ha-ras activation of a tumorpromoting gene is regulated directly by c-Myc DNA binding via phosphatidylinositol 3-kinase signaling, thus revealing a previously uncharacterized mechanism of Ha-ras-mediated oncogenesis through AEG-1.tumor-promoting gene ͉ signaling pathway ͉ transcription T he ras protooncogene is a small GTP͞GDP-binding protein that plays a critical role in cell growth control as a central component of mitogenic signaling (1). Ras activation initiates a complex array of signal transduction pathways including the Raf͞MAPK (ERK) pathway, primarily involved in plasmamembrane-to-nucleus signaling crucial for mitogen-induced cell proliferation (2, 3); the phosphatidylinositol 3-kinase (PI3K)͞ AKT pathway, which is involved in cell survival signaling (4); the Rac͞Rho pathway, involved in cytoskeletal remodeling (5); and Rac͞JNK and Rac͞p38 pathways, both of which appear to be involved in cell stress responses, growth inhibition, and apoptotic signaling (6-8). Activation of Ras signaling pathways is essential for cells to exit a quiescent state and pass through the G 1 phase of the cell cycle (9). Under normal conditions, the action of Ras and other members of the Ras pathway are stringently regulated during the cell cycle and under different growth conditions (10). In a tumor cell, the oncogenic activation of ras is a consequence of point mutations that either impair GTPase activity or enhance GTP-binding affinity, resulting in a highly active proliferative signal (1). In addition, it is possible that the downstream protein targets of that signal transduction pathway might be expressed abnormally. Ras mutations are found in a wide variety of human cancers (11). Therefore, aberrant Ras signaling represents a nodal pathway regulating tumor-cell growth and providing a potential target for cancer therapy (12, 13).We recently reported the cloning and functional characterization of an HIV-1-inducible gene, astrocyte elevated gene-1 (AEG-1), which is induced in primary human fetal astrocytes infected with HIV-1 or treated with gp120 or TNF-␣ (14-1...

Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

Glutamate is an essential neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters primarily expressed in astroglial cells. Dysfunction of EAAT2 is implicated in acute and chronic neurological disorders, including stroke/ischemia, temporal lobe epilepsy, amyotrophic lateral sclerosis, Alzheimer disease, human immunodeficiency virus 1-associated dementia, and growth of malignant gliomas. Ceftriaxone, one of the ␤-lactam antibiotics, is a stimulator of EAAT2 expression with neuroprotective effects in both in vitro and in vivo models based in part on its ability to inhibit neuronal cell death by glutamate excitotoxicity. Based on this consideration and its lack of toxicity, ceftriaxone has potential to manipulate glutamate transmission and ameliorate neurotoxicity. We investigated the mechanism by which ceftriaxone enhances EAAT2 expression in primary human fetal astrocytes (PHFA). Ceftriaxone elevated EAAT2 transcription in PHFA through the nuclear factor-B (NF-B) signaling pathway. The antibiotic promoted nuclear translocation of p65 and activation of NF-B. The specific NF-B binding site at the ؊272 position of the EAAT2 promoter was responsible for ceftriaxone-mediated EAAT2 induction. In addition, ceftriaxone increased glutamate uptake, a primary function of EAAT2, and EAAT2 small interference RNA completely inhibited ceftriaxone-induced glutamate uptake activity in PHFA. Taken together, our data indicate that ceftriaxone is a potent modulator of glutamate transport in PHFA through NF-Bmediated EAAT2 promoter activation. These findings suggest a mechanism for ceftriaxone modulation of glutamate transport and for its potential effects on ameliorating specific neurodegenerative diseases through modulation of extracellular glutamate.Glutamate plays a central role in brain physiology and pathology (1). It is the major mediator of excitatory signal transduction in the mammalian central nervous system and is implicated in most aspects of normal brain function, including cognition, memory, and learning (2). Glutamate exerts its signaling role by acting on glutamate receptors located on the surface of target cells. Accordingly, it is the glutamate concentration in the surrounding extracellular fluid that determines the extent of receptor stimulation. It is clinically relevant that the extracellular glutamate concentration is maintained at a low level, because excessive activation of glutamate receptors is harmful and glutamate is a potent neurotoxin at high concentration (2). The brain contains large quantities of glutamate, but only a small fraction of this glutamate is normally present in the extracellular fluid. Glutamate is constantly being released from cells and is continually being removed from the extracellular fluid (3). Glutamate transporters, also known as excitatory amino acid transporters, regulate this removal of glutamate from the extracellular fluid (3). Five excitatory amino acid transporter (EAAT) 4 cDNAs have been identified an...