Methamphetamine Induces AP-1 and NF-kB Binding and Transactivation in Human Brain

Cellular and molecular mechanisms of methamphet-amine (METH)-induced neurotoxicity may involve alter-ations of cellular redox status and induction of inflamma-tory genes in endothelial cells. To study these hypotheses, molecular signaling pathways of METH-induced inflammatory responses via activation of redox-sensitive transcription factors were investigated in hu-man brain microvascular endothelial cells (HBMEC). A dose-dependent depletion of total glutathione levels was detected in HBMEC exposed to METH. In addition, elec-trophoretic mobility shift assay (EMSA) showed signifi-cant increases in DNA binding activities of redox-responsive transcription factors, AP-1 and NF-kB, in HBMEC treated with METH. METH-mediated AP-1 or NF-kB activation was accompanied by induction of transactivation of AP-1 or NF-kB, as measured by dual luciferase assay using specific reporter plasmids. Be-cause NF-kB and AP-1 are known to regulate expression of inflammatory genes, expression of the gene encoding for tumor necrosis factor-a (TNF-a) was also studied in METH-treated HBMEC. A dose-dependent overexpres-sion of the TNF-a gene was observed in HBMEC treated with METH. The importance of AP-1 and NF-kB in METH-induced TNF-a gene was confirmed in functional promoter studies using constructs of the TNF-a pro-moter with mutated AP-1 or NF-kB sites. These results indicate that METH-induced disturbances in cellular re-dox status and activation of AP-1 and NF-kB can play critical roles in the signaling pathways leading to upregu-lation of inflammatory genes in human brain endothelia