Impact of extracellular HIV-TAT on the regulation of EDF-1 levels in human endothelial cells.
EDF-1 has been isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 (HIV-) Tat, a viral protein known to function as a cytokine in the activation of endothelial cells. Here we provide the molecular evidence that the inhibition of EDF-1 mRNA is transcriptionally regulated in human endothelial cells. Indeed, HIV-Tat inhibits the luciferase activity of endothelial cells transiently transfected with a construct containing 2300 bp of EDF-1 promoter cloned upstream of a luciferase reporter system. The decrease of EDF-1 RNA, however, does not translate into any alteration at the protein level, even when the cells are exposed to MG132, a proteasome inhibitor. Analogously, no modulation of the total amounts of EDF-1 by HIV-Tat has been observed in the presence of pro-inflammatory cytokine interleukin 1beta, which induces endothelial responsiveness to the in vitro effects of HIV-Tat. We have previously shown that EDF-1 is cytosolic and can be translocated to the nucleus upon activation of protein kinases A and C. In response to HIV-Tat, EDF-1 is mainly in the cytosol. Since cytosolic EDF-1 binds and sequesters calmodulin, an important regulator of endothelial nitric oxide synthase, these results might explain why we do not observe any induction of nitric oxide in endothelial cells exposed to HIV-Tat.
Department of Preclinical Sciences LITA-Vialba, Università di Milano, Via GB Grassi 74, Milan, Italy.
M
M
Mariotti
F
F
Bolognese
S
S
Ferrè
M
M
Leidi
J A M
JA
Maier
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000494:pharmacology*
D000199:Actins; D002148:Calmodulin-Binding Proteins; C115911:EDF1 protein, human; D012333:RNA, Messenger; D054322:tat Gene Products, Human Immunodeficiency Virus