Effects of 18-kDa translocator protein knockdown on gene expression of glutamate receptors, transporters, and metabolism, and on cell viability affected by glutamate.

OBJECTIVE: Previously, several important roles for glutamate have been described for the biology of primary brain tumors. For example, glutamate has been suggested to promote glioma cell proliferation by the activation of the 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) subtype of glutamate receptors. In the present study, we determined the potential regulatory roles of the 18-kDa translocator protein (TSPO) in the glutamatergic system in relation to cell death of brain tumor cells through knockdown of the TSPO by genetic manipulation. MATERIALS AND METHODS: With microarray analysis and validation of gene expression of particular genes using real-time PCR, we found effects because of small inhibitory RNA knockdown of the TSPO in human U118MG glioblastoma cells on gene expression of glutamate receptors, glutamate transporters, and enzymes for glutamate metabolism. We also applied antisense RNA to silence TSPO in rat C6 glioblastoma cells and assayed the effects on DNA fragmentation, indicative of apoptosis, because of glutamate exposure. RESULTS: In particular, the effects of TSPO silencing in human U118MG cells related to glutamate metabolism indicate a net effect of a reduction in glutamate levels, which may potentially protect the cells in question from cell death. The TSPO knockdown in C6 cells showed that TSPO is required for the induction of apoptosis because of glutamate exposure. CONCLUSION: These findings show that interactions between the TSPO and the glutamatergic system may play a role in tumor development of glioblastoma cells. This may also have implications for our understanding of the involvement of the TSPO in secondary brain damage and neurodegenerative diseases.

Golgi localization determinants in ArfGAP1 and in new tissue-specific ArfGAP1 isoforms.

The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulum-Golgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204-214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244-2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue in-frame insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.