Opposite Effects of HIV-1 p17 Variants on PTEN Activation and Cell Growth in B Cells

The HIV-1 matrix protein p17 is a structural protein that can act in the extracellular environment to deregulate several functions of immune cells, through the interaction of its NH2-terminal region with a cellular surface receptor (p17R). The intracellular events triggered by p17/p17R interaction have been not completely characterized yet. In this study we analyze the signal transduction pathways induced by p17/p17R interaction and show that in Raji cells, a human B cell line stably expressing p17R on its surface, p17 induces a transient activation of the transcriptional factor AP-1. Moreover, it was found to upregulate pERK1/2 and downregulate pAkt, which are the major intracellular signalling components involved in AP-1 activation. These effects are mediated by the COOH-terminal region of p17, which displays the capability of keeping PTEN, a phosphatase that regulates the PI3K/Akt pathway, in an active state through the serin/threonin (Ser/Thr) kinase ROCK. Indeed, the COOH-terminal truncated form of p17 (p17Δ36) induced activation of the PI3K/Akt pathway by maintaining PTEN in an inactive phosphorylated form. Interestingly, we show that among different p17s, a variant derived from a Ugandan HIV-1 strain, named S75X, triggers an activation of PI3K/Akt signalling pathway, and leads to an increased B cell proliferation and malignant transformation. In summary, this study shows the role of the COOH-terminal region in modulating the p17 signalling pathways so highlighting the complexity of p17 binding to and signalling through its receptor(s). Moreover, it provides the first evidence on the presence of a p17 natural variant mimicking the p17Δ36-induced signalling in B cells and displaying the capacity of promoting B cell growth and tumorigenesis

The effects of p17 are mediated by ROCK-induced activation of PTEN.

<p>Cells, pre-treated with Rock inhibitor Y-27632 (25 µM) for 20 min (lane 4, 5, 6), were stimulated for 1 and 3 min with p17 (1 µg/ml) (lane 5, 6). Cells not stimulated were used as negative control (lane 4). Cells not treated with inhibitor (lane 1, 2, 3) were also stimulated for 1 and 3 min with p17 (1 µg/ml) (lane 2, 3). Western blot analysis of Raji lysates shows that Y-27632 completely abrogates p17-induced decrease of both Ser/Thr pPTEN (A) and pAkt (B), as verified by densitometric analysis and plotting of pPTEN/ERK1/2 and pAkt/ERK1/2. In the left panels, blots from one representative experiment of four with similar results are shown. In the right panels, values reported for Ser/Thr pPTEN (A) and pAkt (B) are the mean ± SD of four independent experiments.</p

Effects of p17, p17Δ36 and S75X on colony-forming ability of Raji cell line.

<p>Cells were plated in six-well plates and, after two days, medium was replaced by fresh medium with various concentrations, 0.05, 0.1 and 0.2 µg/ml of p17, p17Δ36 and S75X. Cells not stimulated were used as negative control. The cell growth was analyzed by using MTT. Data represent the average number of colonies ± SD from three independent experiments performed in triplicate. The statistical significance between control and treated cultures was calculated using one-way ANOVA performed separately for each concentration of p17 variants, across the three groups. Bonferroni's post test was used to compare data: ** P<0.01, *** P<0.001.</p

Binding of monomeric and trimeric p17 to p17R and induction of MCP-1 production.

<p>(A, B, C) Biotin-conjugated monomeric and trimeric p17 at 50 (black line), 200 (grey line) and 400 ng/ml (dotted line) was allowed to react with Raji (A), primary B cells (B) and H9 (C). Cells incubated with biotinylated GST were used as negative control (solid histogram). Binding of p17 to cells was detected by using APC-conjugated streptavidin. Data were analyzed using CELLQUEST Software and displayed as histograms. These results are representative of four different experiments with similar results. (D) Purified human monocytes were treated or not with GST, monomeric and trimeric p17 at a concentration of 1 µg/ml. Culture supernatants were collected 48 h after the stimulation of culture and analysed for the presence of MCP-1 by a standard quantitative ELISA. Bars represent the mean ± SD of four independent experiments performed in triplicate. Statistical analysis was performed by Wilcoxon matched pairs test. *** P<0.001, statistically different compared with GST.</p

Effects of p17 and p17Δ36 stimulation on ERK1/2, Akt and PTEN activity in Raji cells.

<p>(A, B) Cells were treated for 5 min with 0.05, 0.1, 0.5 and 1 µg/ml of p17 (A) and 0.05, 0.1, 0.5 and 1 µg/ml of p17Δ36 (B). Untreated cells were used as control. Western blot analysis of Raji lysates shows that p17 inhibits the activation of Akt and induces the activation of ERK1/2 (A), as shown by the respective phosphorylation state at any concentration tested, verified by densitometric analysis and plotting of the pAkt/ERK1/2 and pERK1/2/ERK1/2. On the contrary, p17Δ36 induces the activation of Akt and ERK1/2 (B), as shown by the increased phosphorylation at any concentration, verified by densitometric analysis and plotting of the pAkt/ERK1/2 and pERK1/2/ERK1/2. In the left panel blots from one representative experiment of four with similar results are shown. In the right panels, values reported for phosphorylation of Akt and ERK1/2 are the mean ± SD of four independent experiments. (C) Cells were treated for 5 min with 1 µg/ml of p17Δ36 (lane 2) and p17 (lane 3). Untreated cells were used as control (lane 1). Western blot analysis of Raji lysates shows that p17Δ36 induces an increase of Ser/Thr pPTEN in contrast to p17, as verified by densitometric analysis and plotting of the pPTEN/ERK1/2. In the left panel one representative blot of four with similar results is shown. In the right panel values reported for Ser/Thr pPTEN are the mean ± SD of four independent experiments.</p

Effects of p17, p17Δ36 and S75X on pERK1/2 and pAkt in Raji and B cells.

<p>(A) Cells were treated for 5 min with S75X concentrations of 0.05, 0.1, 0.5 and 1 µg/ml. Untreated cells were used as control. Western blot analysis of Raji lysates shows that S75X activates Akt and ERK1/2, as shown by the increased phosphorylation of both kinases at any concentration tested, verified by densitometric analysis and plotting of the pAkt/ERK1/2 and pERK1/2/ERK1/2. In the left panel, blots from one representative experiment of four with similar results are shown. In the right panel values reported for phosphorylation of Akt and ERK1/2 are the mean ± SD of four independent experiments. (B, C) Raji (B) and human primary B cells (C) were treated for 5 min with 0.1 µg/ml of p17, p17Δ36 and S75X. Untreated cells were used as control. Western blot analysis of lysates shows that p17 inhibits the activation of Akt and induces the activation of ERK1/2 either in Raji or human primary B cells, as verified by densitometric analysis and plotting of the pAkt/ERK1/2 and pERK1/2/ERK1/2. On the contrary, p17Δ36 and S75X induce the activation of Akt and ERK1/2 at any concentration tested, verified by densitometric analysis and plotting of the pAkt/ERK1/2 and pERK1/2/ERK1/2. In the left panels blots from one representative experiment of three with similar results are shown. In the right panels values reported for pAkt and p ERK1/2 are the mean ± SD of three independent experiments.</p

AP-1 activation in p17- and p17Δ36-treated Raji cells.

<p>(A, B) Nuclear extracts obtained from Raji cells collected after p17 and p17Δ36 treatments (1 µg/ml) were analysed for their binding activity to oligonucleotides specific for the transcription factor AP-1 and Oct-1. (A) Representative EMSA autoradiograms using nuclear extracts (10 µg per sample) from Raji cells collected at specific times 0.5, 1, 2 and 4 h after p17 and p17Δ36 treatment, and AP-1-specific radiolabeled oligonucleotides are shown. NS: p17-untreated cells. Protein-DNA complex specificity was confirmed by competition with an excess of unlabelled oligonucleotide probes. DNA-binding activity to Oct-1 was used as loading control. (B) The panel represents the densitometric analysis of changes in DNA-binding activity of AP-1 relative to Oct-1, expressed as fold of induction over p17-untreated cells. Ratios were expressed on a logarithmic scale in base 2. Bars represent the mean ± SD of four independent experiments. Statistical analysis was performed by two-way ANOVA. Bonferroni's post test was used to compare data: *** P<0.001.</p

Inhibition of p17 and p17Δ36 binding to its cellular receptor p17R.

<p>Biotin-conjugated p17 and p17Δ36 were allowed to react with Raji cells in the absence (A, D) or in presence of anti-p17 mAbs MBS-3 (B, E) or MK-18 (C, F). The interaction of biotinylated-p17 and -p17Δ36 with p17R was detected in Raji cells by using APC-conjugated streptavidin (grey histogram). Cells incubated with biotinylated GST were used as negative control (black histogram). Data were analyzed by using CELLQUEST software and displayed as histograms. The percentage of cells, which interact with p17 and p17Δ36 cells is given in the upper right corner of each panel. These results are representative of four different experiments with similar results.</p