Enhanced nuclear factor-kappa B activation induced by tumour necrosis factor-alpha in stably tat-transfected cells is associated with the presence of cell-surface-bound Tat protein.
OBJECTIVE
An enhanced nuclear factor (NF)-kappa B activation in response to tumour necrosis factor (TNF)-alpha has been observed in stably tat-transfected cells. Recent experimental evidence suggests that Tat may autocrinously influence both cellular physiology and HIV-1 long terminal repeat-directed gene expression in Tat-producing cells. Therefore, the possible association of a Tat autocrinous loop with the enhanced NF-kappa B-binding activity induced by TNF-alpha in Tat-producing cells was studied by anti-Tat antibody blocking experiments.


METHODS
Permanently tat-transfected Jurkat cells, maintained either in the presence or absence of anti-Tat antibody, were studied for the presence of TNF-alpha-induced NF-kappa B-binding activity (quantified by electrophoretic mobility shift assays) and the presence of cell-surface-bound Tat (determined by flow cytometry and confocal microscopy of anti-Tat immunofluorescence-stained cell preparations.


RESULTS
The enhanced production of TNF-alpha-induced NF-kappa B binding activity exhibited by tat-transfected Jurkat cells was completely abolished in cell cultures maintained in the presence of anti-Tat antibody, thus indicating that the increased TNF-alpha-induced NF-kappa B binding activity observed in Jurkat-tat cells was dependent on the presence of Tat protein in an antibody-accessible location. In accordance with these findings, immunofluorescence-stained preparations of unfixed tat-transfected Jurkat cells showed the presence of cell-surface-bound Tat protein which was completely absent in cells incubated in the presence of anti-Tat antibodies.


CONCLUSIONS
This study demonstrates that the enhanced NF-kappa B activation exhibited by stably tat-transfected cells in response to TNF-alpha, is associated with cell surface interaction of extracellularly released Tat protein. These data add further evidence to the possible relevance of a Tat autocrinous loop in the physiology of Tat-producing cells and suggest that in HIV-1-infected cells Tat is likely to behave as a bifunctional molecule which not only acts from within facilitating NF-kappa B recruitment in the viral transcription complex, but may also act from without increasing the availability of activated NF-kappa B.
Ramazzotti
E
E
Institute of Microbiology, University of Bologna, St Orsola General Hospital, Italy.
Vignoli
M
M
Re
M C
MC
Furlini
G
G
La Placa
M
M
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015756:Genes, tat; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D054322:tat Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015696:Gene Products, tat; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D054322:tat Gene Products, Human Immunodeficiency Virus