HUMAN IMMUNODEFICIENCY VIRUS-1 TAT STIMULATES TRANSCRIPTION OF THE TRANSFORMING-GROWTH FACTOR-ALPHA GENE IN AN EPIDERMAL GROWTH FACTOR-DEPENDENT MANNER

The human immunodeficiency virus 1 (HIV-1) tat gene encodes a protein of critical importance for viral transcription. In addition, Tat has b een shown capable of entering cells, stimulating cell proliferation, a nd altering host cell gene expression. We examined the effect of Tat o n the expression of the transforming growth factor alpha (TGF-alpha) g ene in MDA468 human breast carcinoma cells. We showed that these cells were capable of supporting the activation of the HIV-1 long terminal repeat by Tat. Then, in cotransfection assays, in which the TGF-alpha promoter was linked to a luciferase reporter gene and the tat gene was expressed under the control of the SV40 early promoter, we showed tha t tat gene expression increased TGF-alpha-luciferase reporter function but only in cells stimulated with epidermal growth factor (EGF). The effects of tat and EGF were dose dependent. To confirm these cotransfe ction data, Tat was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) and purified on glutathione-agaro se. GST-Tat was introduced into the MDA468 cells either in the presenc e of chloroquine or by scrape loading. The biological activity of GST- Tat was tested on cells that had been stablely transfected with the HI V-1 long terminal repeat linked to luciferase as a reporter. GST-Tat w as then introduced into the cells, and the level of TGF-alpha mRNA was determined. As in the cotransfection assays, GST-Tat had a minor effe ct on the TGF-alpha mRNA when applied alone to the cells. However, in combination with EGF, GST-Tat exposure resulted in a marked increase i n the cellular content of the TGF-alpha mRNA. These results indicate t hat HIV-1 Tat increases the TGF-alpha response to EGF in MDA468 cells. Since TGF-alpha is believed to be under autocrine regulation in these cells, Tat may augment this autocrine loop.