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 been shown capable of entering cells, stimulating cell proliferation, and altering host cell gene expression. We examined the effect of Tat on the expression of the transforming growth factor alpha (TGF-alpha) gene 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 that 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 cotransfection data, Tat was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) and purified on glutathione-agarose. GST-Tat was introduced into the MDA468 cells either in the presence of chloroquine or by scrape loading. The biological activity of GST-Tat was tested on cells that had been stablely transfected with the HIV-1 long terminal repeat linked to luciferase as a reporter. GST-Tat was then introduced into the cells, and the level of TGF-alpha mRNA was determined.(ABSTRACT TRUNCATED AT 250 WORDS)
Nabell
L M
LM
Department of Medicine, University of Alabama at Birmingham 35294.
Raja
R H
RH
Sayeski
P P
PP
Paterson
A J
AJ
Kudlow
J E
JE
D004815:Epidermal Growth Factor / Q000235:genetics*; D005260:Female; D015696:Gene Products, tat / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D011905:Genes, ras / Q000235:genetics / Q000502:physiology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*; D016211:Transforming Growth Factor alpha / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012333:RNA, Messenger; D016211:Transforming Growth Factor alpha; D054322:tat Gene Products, Human Immunodeficiency Virus; D004815:Epidermal Growth Factor; D008156:Luciferases