16th International AIDS Conference Toronto, Canada - August 13 - 18, 2006

HIV-1 TAT PROTEIN INDUCES COX-2 THROUGH A NFAT/AP-1 MEDIATED MECHANISM IN ASTROCYTOMA CELLS

Int Conf AIDS. 2006 Aug 13-18;16 Abstract No. ThAa0203

Blanco A.¹, Alvarez S.¹, Suñé C.², Fresno M.³, Muñoz-Fernández M.A.^1
1 Hospital Gregorio Marañón, Inmuno-Biología Molecular, Madrid, Spain, ² Centro Nacional de Biotecnología, Madrid, Spain, ³ Centro de Biología Molecular 'Severo Ochoa', Biología Molecular, Madrid, Spain

BACKGROUND: It has been described that viral protein Tat is an important mediator of neurotoxicity and a potential contributor to HIV-dementia. Moreover, induction of cyclooxygenase-2 (COX-2) has been involved in the pathology of several neurodegenerative diseases and described in the brain of HIV-1-infected people.

METHODS: We used the astrocytoma human cell line U-87. Detection of COX-2 mRNA and protein expression were carried out by RT-PCR, and Western-Blot. Transcriptional activity was measured using luciferase reporter gene assays in cells transiently transfected with reporter constructs containing various lengths of the human COX-2 promoter. The reporter plasmid pLTRxluc was used to confirm the extracellular pathway of this induction. We performed cotransfection experiments of COX-2 promoter construct with a dominant negative version of nuclear factor of activated T cells (NFAT) (dn-NFAT) and a dominant negative c-Jun expression plasmid (TAM-67). For EMSAs we used DNA probes of NFAT/AP-1 proximal site. Transient transfections with the COX-2 promoter construct containing either dNFAT, pNFAT or AP-1 sites mutated were used.

RESULTS: Tat was able to induce COX-2 mRNA and protein in U-87 as well as COX-2 promoter transcription, but did not active LTR expression in cells transiently transfected with LTR-luc. Sequential deletions of the promoter show that deletion of a distal Nuclear Factor-κB (NF-κB) site did not diminish Tat-dependent transcription. More importantly, transfection of NF-κB did not induce COX-2 transcription suggesting that NF-κB was not necessary to control Tat-mediated COX-2 transcription. However, Activating Protein-1 (AP-1), and NFAT-dependent transcriptions were induced by Tat. Transfection of TAM-67, and of a dn-NFAT, efficiently blocked the induction of COX-2 promoter. We confirmed these results by using specific inhibitors of NFAT and MAPK activation, as well as by EMSA assay.

CONCLUSIONS: COX-2 regulation by extracellular Tat in human U-87 astrocytoma cells is highly induced in a NFAT/AP-1 dependent way.

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2006-08-13
ThAa0203

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