Cell cycle regulation of human interleukin-8 gene expression by the human immunodeficiency virus type 1 Tat protein.
The human immunodeficiency virus type 1 (HIV-1) Tat protein has been reported to transactivate several cellular genes, including the potent chemotactic factor interleukin-8 (IL-8). Consistent with these in vitro assays, elevated levels of IL-8 protein are found in the serum of HIV-infected individuals. We now extend these observations by demonstrating that Tat induction of IL-8 is linked to the cell cycle. Cells that constitutively express the Tat(1-86) protein (eTat) and control cells (pCEP) were reversibly blocked at the G(1)/S border with hydroxyurea or thymidine. The cells were subsequently released, and IL-8 expression was monitored by RNase protection assays and enzyme-linked immunosorbent assay (ELISA). RNase protection assays demonstrated that IL-8 mRNA expression is transiently induced, approximately fourfold, as the Tat-expressing cells enter S phase. Consistent with the RNase protection assay, an increase in IL-8 protein was observed in the cell supernatant using an IL-8 ELISA. Similar experiments were performed following a reversible block at the G(2)/M border with nocodazole and release into G(1). Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G(1). Using gel shift as well as an immobilized DNA binding assay, we demonstrate that the increase in IL-8 gene expression correlates with a specific increase in p65 NF-kappa B binding activity only in the nucleus of the Tat-expressing cells. Moreover, the CREB-binding protein coactivator is present in the complex in the Tat cell line. Finally, we demonstrate that the presence of the proteasome inhibitor MG-132 inhibits the induction of NF-kappa B binding, as well as IL-8 expression, supporting the role of NF-kappa B.
Mahieux
R
R
Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Lambert
P F
PF
Agbottah
E
E
Halanski
M A
MA
Deng
L
L
Kashanchi
F
F
Brady
J N
JN
D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics*; D007976:Leupeptins / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016196:S Phase; D014158:Transcription, Genetic; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D016209:Interleukin-8; D007976:Leupeptins; D016328:NF-kappa B; D054322:tat Gene Products, Human Immunodeficiency Virus; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D006918:Hydroxyurea