Proliferative activity of extracellular HIV-1 Tat protein in human epithelial cells: expression profile of pathogenetically relevant genes

Abstract

Background: Tat is being tested as a component of HIV vaccines. Tat activity has been mainly investigated on cells of lymphoid/hematopoietic lineages. HIV-1, however, is known to infect many different cells of both solid organs and mucosal surfaces. The activity of two-exon (aa 1-101) and synthetic (aa 1-86) Tat was studied on mammary and amniotic epithelial cells cultured under low serum conditions.

Results: small concentrations of Tat (100 ng/ml) stimulated cell proliferation. Tat antibodies neutralized the mitogenic Tat activity. Changes of gene expression in Tat-treated cells were evaluated by RT-PCR and gene-array methods. Within 4 hours of treatment, exposure to Tat is followed by up-regulation of some cell cycle-associated genes (transcription factors, cyclin/cdk complexes, genes of apoptotic pathways) and of genes relevant to HIV pathogenesis [chemokine receptors (CXCR4, CCR3), chemotactic cytokines (SDF-1, RANTES, SCYC1, SCYE1), IL6 family cytokines, inflammatory cytokines, factors of the TGF-beta family (TGFb, BMP-1, BMP-2)]. Up-regulation of anti-inflammatory cytokines (IL-10, IL-19, IL-20), a hallmark of other persistent viral infections, was a remarkable feature of Tat-treated epithelial cell lines.

Conclusion: extracellular Tat is mitogenic for mammary and amniotic epithelial cells and stimulates the expression of genes of pathogenetic interest in HIV infection. These effects may favor virus replication and may facilitate the mother-to-child transmission of virus.

Figure 1

Growth of mammary epithelial cells exposed to different doses of Tat_101aa. Cells were cultured in low-serum medium. Growth was measured by the XTT assay after 48 h of Tat treatment. Each bar represents the average of 3 different tests + SD. *, P < 0.05, as compared with untreated cell cultures.

Figure 2

Monolayers of MCF-7 and MEC-1 cells cultured in low-serum medium and treated or not with extracellular Tat_101aa (100 ng/ml for 36 hours). Increased numbers of cells can be observed in Tat-treated cultures as compared to untreated control cultures. Phase contrast; microscopic fields taken with a 10× objective.

Figure 3

Neutralization by anti-Tat antibodies of the proliferative response to Tat of five different human epithelial cell lines cultured in low-serum medium. Cells were either left untreated (open bars) or exposed to 100 ng/ml Tat_86aa (closed bars) at time 0. Cells were counted by microscopy at day-0, -1 and -2 after plating. Antibody tMAb-B (1 μg/ml) was mixed with Tat before treatment (red bars) or given alone (green bars). Bars represent the mean of three wells. Experiments on AV-3 cells (bottom panel) have also demonstrated the Tat-neutralizing activity of a rabbit polyclonal anti-Tat antibody: anti-Tat plus Tat (yellow bars) and anti-Tat alone (gray bars). Results are expressed as number of cells/well. Each bar represents the average of 3 different tests. Standard deviations of the mean are not reported, but were within 12% of the mean.

Figure 4

Semiquantitative RT-PCR of VEGF family genes in MCF-7 cells cultured in low-serum medium and exposed for different times to extracellular Tat_86aa. Increased expression of the VEGF receptor-2 (Flk-1/KDR) and VEGF isoform 165. To a lower extent, the VEGF isoform-121 was also up-regulated. The GAPDH signal was used as a control. MW, DNA molecular weight ladder.