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HIV-1-Tat Protein Activates Phosphatidylinositol 3-Kinase/ AKT-dependent Survival Pathways in Kaposi's Sarcoma Cells*

  • Maria Chiara Deregibus
    Affiliations
    From the Cattedra di Nefrologia, Dipartimento di Medicina Interna Università di Torino, Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126
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  • Vincenzo Cantaluppi
    Affiliations
    From the Cattedra di Nefrologia, Dipartimento di Medicina Interna Università di Torino, Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126
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  • Sophie Doublier
    Footnotes
    Affiliations
    From the Cattedra di Nefrologia, Dipartimento di Medicina Interna Università di Torino, Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126
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  • Maria Felice Brizzi
    Affiliations
    From the Cattedra di Nefrologia, Dipartimento di Medicina Interna Università di Torino, Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126
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  • Ilaria Deambrosis
    Affiliations
    From the Cattedra di Nefrologia, Dipartimento di Medicina Interna Università di Torino, Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126
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  • Adriana Albini
    Affiliations
    Istituto Nazionale per la Ricerca sul Cancro, Genova 16100, Italy
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  • Giovanni Camussi
    Correspondence
    To whom correspondence should be addressed: Cattedra di Nefrologia, Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, Torino 10126, Italy. Tel.: 39-011-6336708; Fax: 39-011-6631184;
    Affiliations
    From the Cattedra di Nefrologia, Dipartimento di Medicina Interna Università di Torino, Centro Ricerca Medicina Sperimentale (CeRMS), Torino 10126
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  • Author Footnotes
    * This work was supported in part by the Associazione Italiana per la Ricerca sul Cancro (to G. C. and M. F. B.), the National Research Council, Targeted Project “Biotechnology,” MIUR COFIN 01, Istituto Superiore di Sanità AIDS Grant 30C.11 (to G. C.), and AIDS Grant 40C4 (to A. A).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Recipient of a Marie Curie individual fellowship from the European Community.
Open AccessPublished:May 06, 2002DOI:https://doi.org/10.1074/jbc.M200921200
      In this study we found that Tat protected vincristine-treated Kaposi's sarcoma cells from apoptosis and from down-regulation of several anti-apoptotic genes such asAKT-1, AKT-2, BCL2,BCL-XL, and insulin-like growth factor I and induced thede novo expression of the interleukin-3 gene. Moreover, we found that Tat enhanced phosphorylation of AKT and BAD proteins. The inhibition of phosphatidylinositol 3-kinase with two unrelated pharmacological inhibitors, wortmannin and LY294002, abrogated both the anti-apoptotic effect and the phosphorylation of AKT induced by Tat. After treatment with Tat, the AKT enzymatic activity showed a biphasic increase: an early activation (15 min), independent from protein synthesis; and a delayed activation (24 h), which was significantly decreased upon blockage of protein synthesis. Experiments with a function blocking anti-vascular endothelial cell growth factor receptor-2 antibody suggested that both the early and delayed AKT activation and the protection from apoptosis were triggered by the interaction of Tat with vascular endothelial cell growth factor receptor-2. Moreover, experiments with function-blocking antibodies directed against insulin-like growth factor I/insulin-like growth factor I receptor or interleukin-3 indicated their involvement in the delayed activation of AKT and their contribution to the anti-apoptotic effect of Tat on vincristine-treated Kaposi's sarcoma cells.
      KS
      Kaposi's sarcoma
      HIV-1
      human immunodeficiency virus, type 1
      IGF-I
      insulin-like growth factor I
      IGF-IR
      IGF-I receptor
      IL-3
      interleukin-3
      PI3K
      phosphatidylinositol 3-kinase
      PBS
      phosphate-buffered saline
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      TUNEL
      terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling
      HHV8
      human herpesvirus 8
      VEGF
      vascular endothelial growth factor
      MOPS
      4-morpholinepropanesulfonic acid
      Kaposi's sarcoma (KS)1is a hemangiosarcoma containing spindle-shaped cells, fibroblasts, inflammatory cells, vascular endothelial cells, and smooth muscle cells characterized by the presence of an intense neoangiogenesis (
      • Ensoli B.
      • Barillari G.
      • Salahuddin S.Z.
      • Gallo R.C.
      • Wong-Staal F.
      ,
      • Gallo R.C.
      ,
      • Rabkin C.S.
      • Janz S.
      • Lash A.
      • Coleman A.E.
      • Musaba E.
      • Liotta L.
      • Biggar R.J.
      • Zhuang Z.
      ). KS is frequently associated with immune-depressed conditions, including HIV-1 infection and long term post-transplantation therapy (
      • Mitsuyasu R.T.
      ). All forms of KS are associated with infection by human herpesvirus 8 (HHV8) (
      • Chang Y.
      • Cesarman E.
      • Pessin M.S.
      • Lee F.
      • Culpepper J.
      • Knowles D.M.
      • Moore P.S.
      ,
      • Boshoff C.
      • Weiss R.A.
      ). AIDS-associated KS is particularly aggressive, and it is one of the principal neoplasms in regions of Africa affected by both high endemic HHV8 and epidemic HIV infection (
      • Chokumonga E.
      • Levy L.M.
      • Bassett M.T.
      • Mauchaza B.G.
      • Thomas D.B.
      • Parkin D.M.
      ).
      Several studies (
      • Albini A.
      • Benelli R.
      • Presta M.
      ,
      • Ensoli B.
      • Barillari G.
      • Buonaguro L.
      • Gallo R.C.
      ,
      • Ensoli B.
      • Barillari G.
      • Gallo R.C.
      ) suggest a primary role for the protein encoded by the tat gene of HIV-1 in the induction and development of Kaposi's sarcoma in patients affected by AIDS. HIV-1-tatgene transgenic mice have been shown to develop Kaposi's sarcoma-like skin lesions (
      • Ensoli B.
      • Barillari G.
      • Salahuddin S.Z.
      • Gallo R.C.
      • Wong-Staal F.
      ,
      • Vogel J.
      • Hinrichs S.H.
      • Reynolds R.K.
      • Luciw P.A.
      • Jay G.
      ,
      • Corallini A.
      • Altavilla G.
      • Pozzi L.
      • Bignozzi F.
      • Negrini M.
      • Rimessi P.
      • Gualandi F.
      • Barbanti-Brodano G.
      ,
      • Samaniego F.
      • Markham P.D.
      • Gendelman R.
      • Gallo R.C.
      • Ensoli B.
      ). Furthermore, Tat has been shown to activate the vascular endothelial growth factor (VEGF) receptor 2/KDR (VEGFR2) on endothelial (
      • Albini A.
      • Soldi R.
      • Giunciuglio D.
      • Girando E.
      • Benelli R.
      • Primo L.
      • Noonan D.
      • Salio M.
      • Camussi G.
      • Rockl W.
      • Bussolino F.
      ) and KS cells (
      • Ganju R.K.
      • Munsi N.
      • Nair B.C.
      • Liu Z.Y.
      • Gill P.
      • Groopman J.E.
      ,
      • Morini M.
      • Benelli R.
      • Giungiuglio D.
      • Carlone S.
      • Arena G.
      • Noonan D.M.
      • Albini A.
      ) and to stimulate angiogenesis (
      • Albini A.
      • Fontanini G.
      • Masiello L.
      • Tacchettin C.
      • Bigini D.
      • Luzzi P.
      • Noonan D.M.
      • Stetler-Stevenson W.G.
      ). It has been also shown that Tat, VEGF, and basic fibroblast growth factor synergized in the induction of KS (
      • Ensoli B.
      • Gendelman R.
      • Markham P.
      • Fiorelli V.
      • Colombini S.
      • Raffeld M.
      • Cafaro A.
      • Chang H.K.
      • Brady J.N.
      • Gallo R.C.
      ).
      The growth and diffusion of KS have been ascribed not only to a dysregulation of cellular proliferation but also to a resistance against apoptotic signals derived from the activation of endogenous or exogenous execution death programs. Tat has been shown to prevent apoptosis of different cell lines of lymphoid, epithelial, and neuronal origin (
      • Zauli G.
      • Gibellini D.
      • Dilani D.
      • Mazzoni M.
      • Borgatti P., La
      • Placa M.
      • Capitani S.
      ). However, Tat does not have an univocal role in modulating apoptosis in lymphocytes as it decreases apoptosis of HIV-1-infected T-cells in peripheral blood and induces apoptosis of uninfected T-cells (
      • McCloskey T.W.
      • Ott M.
      • Tribale E.
      • Khan S.A.
      • Teichberg S.
      • Paul M.O.
      • Pahwa S.
      • Verdin E.
      • Chirmule N.
      ) through a Fas-dependent mechanism (
      • Westendorp M.O.
      • Frank R.
      • Ochsenbauer C.
      • Stricker K.
      • Dhein J.
      • Walczak H.
      • Debatin K.M.
      • Krammer P.H.
      ). Recently, we found that Tat is a survival factor for KS and endothelial cells (
      • Cantaluppi V.
      • Biancone L.
      • Boccellino M.
      • Doublier S.
      • Benelli R.
      • Carlone S.
      • Albini A.
      • Camussi G.
      ), suggesting a putative role in tumor development in a microenvironment characterized by immune system impairment.
      The aim of this study was to investigate the molecular mechanisms through which HIV-1-Tat protein inhibits vincristine-induced apoptosis of KS cells. The analysis of the expression of genes involved in modulation of apoptosis prompted us to focus on the role of phosphatidylinositol 3-kinase (PI3K)-dependent activation of AKT.

      RESULTS

      Immunohistochemistry using antibodies directed against factor VIII, P1H12 (an endothelial cell marker), vimentin, desmin, SMC α-actin, VEGFR1, and VEGFR2 on primary KS cell cultures gave a staining pattern similar to that reported previously for KS-imm spindle cells described by Albini et al. (
      • Albini A.
      • Paglieri I.
      • Orengo G.
      • Carlone S.
      • Aluigi M.G.
      • DeMarchi R.
      • Matteucci C.
      • Mantovani A.
      • Carozzi F.
      • Donini S.
      • Benelli R.
      ) (TableI). Typical strong positive vimentin staining was observed on KS cells. KS cells usually express a mixed endothelial-macrophage-mesenchymal cell phenotype. Primary KS and KS-imm cell cultures were both positive for myeloperoxidase, a macrophage marker, and for the endothelial cell marker CD145 detected by the anti-P1H12 monoclonal antibody (
      • St Croix B.
      • Rago C.
      • Velculescu G.
      • Traverso G.
      • Romans K.E.
      • Montgomery E.
      • Lal A.
      • Riggins G.J.
      • Lengauer C.
      • Vogelstein B.
      • Kinzler K.W.
      ). Moreover, both cell lines were positive for VEGFR2 but not for VEGFR1.
      Table ICharacterization of KS cell primary culture
      MarkersHUVECKS cellsKS imm
      Vimentin+++++++++
      P1H12
      P1H12 monoclonal antibody detects the CD146 of an endothelial cell marker (32).
      ++++++++
      FVIII+++/−
      Desmin+/−
      SMC α-actin+++
      Myeloperoxidase++
      VEGFR-1++
      VEGFR-2+++++
      1-a P1H12 monoclonal antibody detects the CD146 of an endothelial cell marker (
      • St Croix B.
      • Rago C.
      • Velculescu G.
      • Traverso G.
      • Romans K.E.
      • Montgomery E.
      • Lal A.
      • Riggins G.J.
      • Lengauer C.
      • Vogelstein B.
      • Kinzler K.W.
      ).
      As reported previously (
      • Cantaluppi V.
      • Biancone L.
      • Boccellino M.
      • Doublier S.
      • Benelli R.
      • Carlone S.
      • Albini A.
      • Camussi G.
      ), HIV-1-Tat acted as a survival factor for KS cells and prevented vincristine-induced apoptosis of KS-imm. Similar results were obtained in KS cell primary culture exposed to vincristine (0.25 μg/ml) in the presence of Tat (10 ng/ml) (Fig.1).
      Figure thumbnail gr1
      Figure 1Comparison of the anti-apoptotic effect of Tat on primary KS and KS-imm cell cultures. The percentage of apoptotic cells subjected to the TUNEL assay after 24 h of incubation with vehicle alone or in the presence of 0.25 μg/ml vincristine or 0.25 μg/ml vincristine plus 10 ng/ml Tat is shown. Primary KS cells, black column; KS-imm cells, open column. Data are expressed as mean ± 1 S.D. from three different experiments.
      Gene array technology was used to investigate the effect of vincristine and Tat on the expression of genes that modulate apoptosis by KS cells (Table II and Fig. 2). Fig. 2 A shows gene expression in unstimulated cells. Hybridization of RNA extracted from KS cells treated with vincristine was associated with a loss of expression of some anti-apoptotic genes such as AKT-1 andAKT-2, BCL2, BCL-X, MCL-1and IGF-I, and with enhanced expression of pro-apoptotic genes such as BIK (Fig. 2 C). After addition of Tat to vincristine-treated KS cells, we observed the reappearance of expression of the anti-apoptotic genes AKT-1and AKT-2, BCL-2, BCL-X, andIGF-I and the de novo expression ofIL-3 gene (Fig. 2 D). Stimulation of KS cells with Tat alone, in addition to the genes mentioned above, induced the expression of BAX, BCL-6, andBAG-1 (Fig. 2 B). No significant variation in the expression of the housekeeping genes (β-actin and GAPDH) was observed in the different experimental conditions. The bacterial plasmid pUC18 was always negative.
      Figure thumbnail gr2
      Figure 2Gene expression of KS cells after different stimuli. A, gene expression under basal conditions;B, effect of 24 h of treatment of KS cells with 10 ng/ml Tat; C, after 24 h of treatment with 0.25 μg/ml vincristine; D, after 24 h of treatment with 0.25 μg/ml vincristine in the presence of 10 ng/ml Tat. Total RNA extracted from KS cells subjected to different experimental conditions was used as template for biotinylated cDNA probe synthesis as described under “Experimental Procedures.” Probes were hybridized to gene array containing duplicate spots of 23 genes of the human BCL-2 family and apoptosis regulatory network. Two housekeeping genes, β-actin and GAPDH , were used for data normalization. The bacterial plasmid pUC18 was used as a negative control. The various genes and their position on the grid are reported in . Note in C the disappearance of several genes including those coding for AKT-1 and AKT-2 (grid position 1C-D and 1E-F), BCL-2 (grid position 4A-B), BCL-XL (grid position5A-B), IGF-1 (grid position 7C-D), andMCL-1 (grid position 8A-B) anti-apoptotic genes. With the exception of MCL-1, all these genes were re-expressed, and the IL-3 gene (grid position7E-F) was expressed de novo when vincristine-treated KS cells were stimulated with Tat (D). These data are representative of three experiments.
      The observation that Tat abrogated the effect of vincristine onAKT gene expression prompted us to test whether AKT was involved in the anti-apoptotic effect of Tat. It has been shown that the anti-apoptotic effect of AKT depends on PI3K activation (
      • Zhang L.
      • Zhou W.
      • Velculescu V.E.
      • Kern S.E.
      • Hruban R.H.
      • Hamilton S.R.
      • Vogelstein B.
      • Kinzler K.W.
      ,
      • Talapatra S.
      • Thompson C.B.
      ,
      • Zhou H.
      • Xin-Ming L.
      • Meinkoth J.
      • Pittman R.N.
      ). Thus, we evaluated the effect of two unrelated PI3K pharmacological inhibitors, wortmannin and LY294002 (
      • Brunet A.
      • Datta S.R.
      • Greenberg M.E.
      ). As shown in Fig.3 vincristine-induced apoptosis of KS cells was inhibited by Tat. Pretreatment with wortmannin (0.1 μm) and LY294002 (10 μm) completely abrogated the anti-apoptotic effect of Tat. Fig.4 shows that vincristine slightly decreased the expression of AKT protein consistently with the down-regulation of AKT gene expression observed by gene array analysis. However, vincristine mainly impaired the ability of AKT to undergo phosphorylation (P-AKT). Treatment with Tat not only restored the expression of AKT protein but also its phosphorylation. When PI3K was inhibited by wortmannin (0.1 μm) and LY294002 (10 μm), Tat-induced phosphorylation of AKT was significantly prevented. These compounds alone had no significant effect on AKT kinase activity in serum-starved cells but inhibited AKT activity induced by Tat (Fig.5 A). Phosphorylation of BAD at serine 136 is one of the potential mechanisms through which AKT inhibits apoptosis (
      • Del Peso L.
      • Gonzales-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ,
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ). We therefore studied whether phosphorylation of BAD paralleled that of AKT. As shown in Fig.5 B, vincristine induced a concomitant reduction of P-AKT and P-BAD. Tat treatment of KS cells incubated with vincristine restored both AKT and BAD phosphorylation. No significant changes in BAD protein expression were observed under the different experimental conditions.
      Figure thumbnail gr3
      Figure 3Effect of PI3K pharmacological inhibitors on the anti-apoptotic effect of Tat. A, DNA fragmentation was detected after DNA extraction and electrophoresis on 2% agarose gel. B, the percentage of apoptotic KS cells subjected to the TUNEL assay after 24 h of incubation with different stimuli (see “Experimental Procedures”). KS cells were incubated in vehicle either alone (row 1) or in the presence of 0.25 μg/ml vincristine (row 2), 0.25 μg/ml vincristine plus 10 ng/ml Tat (row 3), 0.25 μg/ml vincristine plus 10 ng/ml Tat and 0.1 μm wortmannin (row 4), vincristine plus 10 ng/ml Tat and 10 μm LY294002 (row 5), 0.1 μmwortmannin alone (row 6), or 10 μm LY294002 (row 7). Data are expressed as mean ± 1 S.D. of three different experiments.
      Figure thumbnail gr4
      Figure 4Effect of treatment with Tat and PI3K pharmacological inhibitors on AKT in KS cells incubated with vincristine. Cell lysates (30 μg of protein) were immunoblotted with anti-P-AKT, AKT, or β-actin antibodies. A, densitometric analysis; B, representative immunoblot of P-AKT expression (A, black column) and AKT expression (A, open column). Row 1, control KS cells; row 2, KS cells treated with 0.25 μg/ml vincristine; row 3, KS cells treated with 0.25 μg/ml vincristine plus 10 ng/ml Tat; row 4, KS cells treated with 0.25 μg/ml vincristine plus 10 ng/ml Tat and 0.1 μm wortmannin; row 5, KS cells treated with vincristine plus 10 ng/ml Tat and 10 μmLY294002. A, data are expressed as mean ± 1 S.D. of three different experiments.
      Figure thumbnail gr5
      Figure 5Effect of Tat on AKT kinase activity and on AKT and BAD phosphorylation. A, effect of wortmannin and LY294002 on AKT kinase activity. Kinase reactions and Western blot analyses were performed on anti-AKT immunoprecipitates obtained from KS cell lysates, as described under “Experimental Procedures.” Serum-starved KS cells, unstimulated or stimulated with 10 ng/ml Tat (for 15 min), were preincubated for 30 min with or without 0.1 μm wortmannin or 10 μm LY294002. The autoradiogram corresponds to a representative experiment repeated twice. 32P-Labeled products ( 32 P-H2B) as well as specific bands detected by the anti-AKT antibody are indicated. B, concomitant phosphorylation of AKT and BAD in KS cells treated with Tat. Cell lysates (30 μg of proteins) were divided into 4 aliquots and immunoblotted with anti-P-AKT, AKT, P-BAD, BAD, and β-actin antibodies. Row 1, control KS cells; row 2, KS cells treated with 0.25 μg/ml vincristine; row 3, KS cells treated with 0.25 μg/ml vincristine plus 10 ng/ml Tat. Data are representative of three experiments.
      To evaluate whether Tat directly induced AKT enzymatic activity, AKT kinase activity was measured after AKT immunoprecipitation from KS cell lysates at different times of incubation. In parallel, phosphorylation of recombinant BAD used as substrate for AKT (
      • Del Peso L.
      • Gonzales-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ,
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ,
      • Zauli G.
      • Milani D.
      • Mirandola P.
      • Mazzoni M.
      • Secchiero P.
      • Miscia S.
      • Capitani S.
      ) was evaluated on the same KS cell lysates. As shown in Fig.6, two waves of AKT kinase activity and BAD phosphorylation were observed after incubation of KS cells with Tat. Tat induced an early increase in AKT activity that peaked at 15 min and decreased after 60 min. This early AKT activation was not inhibited by cycloheximide, an inhibitor of protein synthesis (data not shown). In addition, Tat induced a delayed AKT activation that was detectable after 24 h; this activation was significantly decreased by cycloheximide, indicating that a Tat-induced synthesis of secondary mediators may contribute to this AKT activation. Previous studies (
      • Cantaluppi V.
      • Biancone L.
      • Boccellino M.
      • Doublier S.
      • Benelli R.
      • Carlone S.
      • Albini A.
      • Camussi G.
      ) demonstrated that Tat stimulates KS cells through VEGFR-2. As shown in Fig. 7 A, pretreatment of KS cells with a function blocking anti-VEGFR-2 antibody significantly reduced the survival effect of Tat on vincristine-treated KS cells. However, when the anti-VEGFR-2 antibody was added 1 h after Tat stimulation, the inhibitory effect on Tat-induced survival was less effective. These results suggest that the effects of the anti-VEGFR-2 blocking antibody were mainly due to interference of Tat interactions with VEGFR-2 rather than any indirect effects such as Tat-induced synthesis of VEGF. As shown in Fig. 8, the anti-VEGFR-2-blocking antibody also inhibited both the early and late enhancement of BAD phosphorylation induced by Tat.
      Figure thumbnail gr6
      Figure 6Time course of AKT kinase activity and BAD phosphorylation assay after stimulation of KS cells with 10 ng/ml Tat. Kinase reactions and Western blot analysis were performed on anti-AKT immunoprecipitates from the corresponding lysates, as described under “Experimental Procedures.” AKT activity was also assessed as BAD phosphorylation by KS cell lysates using recombinant BAD-agarose as substrate, and P-BAD was detected by Western blot analysis (see “Experimental Procedures”). Where indicated, cells were treated for 24 h with Tat in the presence of 0.1 mg/ml cycloheximide (Cyx) to inhibit protein synthesis.A, open column, AKT kinase activity;shaded column, AKT activity detected using recombinant BAD as substrate for phosphorylation. Data are expressed as fold increase in respect to unstimulated cells (mean ± 1 S.D. of three different experiments). B, the autoradiogram corresponds to a representative experiment. Similar results were obtained in three independent experiments. 32P-Labeled products ( 32 P-H2B) as well as specific bands detected by the anti-AKT antibody are indicated. The Western blot analysis of P-BAD is representative of three independent experiments of phosphorylation of recombinant BAD (3.3 μg) by KS cell lysates (1 mg of protein) obtained after 15, 30, and 60 min and 12 and 24 h of incubation with Tat and after 24 h of incubation with Tat and cycloheximide.
      Figure thumbnail gr7
      Figure 7Effect of anti-VEGFR-2, IGF-I/IGF-IR, and IL-3 blocking antibodies on Tat-induced survival of KS cells treated with vincristine for 24 h by the XTT-based assay (see “Experimental Procedures”). A, KS cells were untreated (alone) or incubated with 0.25 μg/ml vincristine, with 0.25 μg/ml vincristine in combination with 10 ng/ml Tat, or with 10 ng/ml VEGF. Where indicated cell treated with vincristine plus Tat were preincubated 10 min before the beginning of the experiment (anti-VEGFR2 pre) or were incubated 1 h after the beginning of the experiment (anti-VEGFR2 post) with blocking antibody anti-VEGFR-2 (10 μg/ml). B, KS cells were untreated (alone) or incubated with 0.25 μg/ml vincristine, or with 0.25 μg/ml vincristine in combination with 10 ng/ml Tat, or with 10 ng/ml IGF-1, or with 20 ng/ml IL-3. Where indicated cells treated with vincristine plus Tat were preincubated 10 min before the beginning of the experiment with a mixture containing 10 μg/ml anti-IGF-I and 10 μg/ml anti-IGF-IR blocking antibodies or with anti-IL-3 blocking antibodies (10 μg/ml). Data are expressed as mean ± 1 S.D. of O.D. variation of three different experiments.
      Figure thumbnail gr8
      Figure 8Effect of anti-VEGFR-2 blocking antibody on Tat-mediated activation of AKT detected as phosphorylation of recombinant BAD. AKT-induced phosphorylation of BAD was measured in KS cell lysates at different times of incubation with 10 ng/ml Tat using recombinant BAD as substrate (see “Experimental Procedures”). A representative experiment of phosphorylation of recombinant BAD (3.3 μg) by KS cell lysates (1 mg of protein) was obtained after 15 and 30 min and 24 h of incubation with Tat. Where indicated, 10 μg/ml anti-VEGFR-2 antibody was added 10 min before stimulation with Tat. The densitometric analysis was performed, and data are expressed as fold induction over the unstimulated control. Data are representative of three independent experiments.
      Because gene array analysis indicated that Tat was able to triggerIGF-I gene expression, we investigated whether IGF-I may contribute to the anti-apoptotic effect of Tat in KS cells. IGF-I is known to induce resistance to apoptosis in several cell types via the activation of AKT (
      • Weihua W.
      • Lee W.L., Wu, Y.Y.
      • Chen D.
      • Liu T.J.
      • Jang A.
      • Sharma P.M.
      • Wang P.H.
      ). We observed that the delayed activation of AKT-dependent BAD phosphorylation (Fig.9) and the survival effect of Tat (Fig.7 B) were significantly reduced after treatment of KS cells with a combination of anti-IGF-I and anti-IGF-IR antibodies. These results suggested that the synthesis of IGF-I triggered by Tat contributed to the delayed AKT activation and to the resistance to apoptosis. Indeed, IGF-I significantly prevented vincristine-induced KS cell death. Moreover, Tat was shown to induce the expression of the gene coding for IL-3, a cytokine known to activate AKT (
      • Del Peso L.
      • Gonzales-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ). An anti-IL-3 function-blocking antibody also reduced the survival effect of Tat on vincristine-treated cells (Fig.7 B) and the delayed activation of AKT-dependent BAD phosphorylation (Fig. 9). The inhibitory effect on delayed Tat-induced AKT-dependent BAD phosphorylation was particularly evident when cells were treated with anti-IGF-I/anti-IGF-IR and anti-IL-3 blocking antibodies. In contrast, no effect was observed on the early activation of AKT induced by Tat (data not shown).
      Figure thumbnail gr9
      Figure 9Effect of anti-IGF-I/anti-IGF-IR and anti-IL-3 blocking antibodies on Tat-mediated activation of AKT detected as phosphorylation of recombinant BAD. AKT-induced phosphorylation of BAD was measured in KS cell lysates after 24 h of incubation in the following conditions: row 1, unstimulated control; row 2, 0.25 μg/ml vincristine;row 3, 0.25 μg/ml vincristine plus 10 ng/ml Tat;row 4, vincristine plus 10 ng/ml IGF-I; row 5, vincristine plus 10 ng/ml Tat plus 10 μg/ml anti-IGF-I and 10 μg/ml anti-IGF-IR antibodies; row 6, vincristine plus 10 ng/ml IGF-I plus 10 μg/ml anti-IGF-I and 10 μg/ml anti-IGF-IR antibodies; row 7, vincristine plus 10 ng/ml Tat plus 10 μg/ml anti-IL-3 blocking antibodies; row 8, vincristine plus IL-3 (20 μg/ml); row 9, vincristine plus 20 ng/ml IL-3 plus 10 μg/ml anti-IL-3 blocking antibodies; row 10, vincristine plus 10 ng/ml Tat plus 10 μg/ml anti-IGF-I and 10 μg/ml anti-IGF-IR antibodies plus 10 μg/ml anti-IL-3 blocking antibody. The densitometric analysis was performed, and the data, expressed as fold induction over unstimulated control, are representative of three independent experiments.

      DISCUSSION

      KS tumorigenesis seems to depend not only on the dysregulation of cellular proliferation but also on resistance to apoptotic signals. Indeed, KS cells are resistant to Fas-mediated apoptosis despite the expression of Fas on their surface (
      • Mori S.
      • Murakami-Mori K.
      • Jewett A.
      • Nakamura S.
      • Bonavida B.
      ). Moreover, KS cells derived from AIDS patients are often resistant to chemotherapy drugs (
      • Lee F.C.
      • Mitsuyasu R.T.
      ). We demonstrated recently (
      • Cantaluppi V.
      • Biancone L.
      • Boccellino M.
      • Doublier S.
      • Benelli R.
      • Carlone S.
      • Albini A.
      • Camussi G.
      ) that Tat can increase survival of vincristine-treated KS cells. This increased survival mainly depended on an anti-apoptotic effect of Tat that was detectable at concentrations of Tat well below those observed in AIDS patients (
      • Masood R.
      • Cai J.
      • Law R.
      • Gill P.
      ). The mechanisms by which Tat induces resistance to apoptosis remain largely unknown. However, because KS cells express KDR/VEGFR-2 and Tat is able to bind to and activate endothelial (
      • Albini A.
      • Soldi R.
      • Giunciuglio D.
      • Girando E.
      • Benelli R.
      • Primo L.
      • Noonan D.
      • Salio M.
      • Camussi G.
      • Rockl W.
      • Bussolino F.
      ) and KS (
      • Ganju R.K.
      • Munsi N.
      • Nair B.C.
      • Liu Z.Y.
      • Gill P.
      • Groopman J.E.
      ,
      • Morini M.
      • Benelli R.
      • Giungiuglio D.
      • Carlone S.
      • Arena G.
      • Noonan D.M.
      • Albini A.
      ) cells expressing the KDR/VEGFR-2 receptor, KDR/VEGFR-2 activation may transduce the anti-apoptotic signal of Tat.
      We therefore investigated the molecular mechanisms involved in the anti-apoptotic effect of Tat on vincristine-treated KS cells.
      Gene expression analysis by gene array technology demonstrated that vincristine treatment induced a down-regulation of several anti-apoptotic genes, including AKT-1 and AKT-2,BCL2, BCL-XL, and IGF-I and enhanced the expression of pro-apoptotic genes such asBIK. Tat treatment abrogated the effect of vincristine on the anti-apoptotic genes AKT-1 andAKT-2, BCL-2, BCL-XL, andIGF-I and induced the de novo expression ofIL-3 in vincristine-treated KS cells. These results are consistent with an enhanced transcription of several genes induced by Tat, which includes not only anti-apoptotic genes but also pro-apoptotic genes such as BAX and BAG-1. However, the final effect appears to be protection from apoptosis.
      It is known that AKT regulates cell survival and apoptosis at a post-mitochondrial level (
      • Zhou H.
      • Xin-Ming L.
      • Meinkoth J.
      • Pittman R.N.
      ). On the basis of the observation that vincristine down-regulated and that Tat restored AKT gene expression, we investigated whether AKT mediated the anti-apoptotic effect of Tat on KS cells.
      The PI3K/AKT is one of the central pathways involved in survival signaling (
      • Talapatra S.
      • Thompson C.B.
      ). Several receptors, including those for VEGF (
      • Gerber H.P., Mc
      • Murtrey A.
      • Kowalskj J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ), IGF-I (
      • Weihua W.
      • Lee W.L., Wu, Y.Y.
      • Chen D.
      • Liu T.J.
      • Jang A.
      • Sharma P.M.
      • Wang P.H.
      ), and IL-3 (
      • Del Peso L.
      • Gonzales-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ), transmit survival signals through these pathways. PI3K activation catalyzes the transfer of a phosphate group from ATP to the D3 position of phosphatidylinositol, thus generating 3′-phosphatidylinositol phosphates (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ). These latter compounds serve as binding sites for proteins that possess pleckstrin homology domains such as AKT. The binding of AKT to 3′-phosphatidylinositol phosphate results in its translocation from cytosol to plasma membrane and phosphorylation of threonine 308 and serine 473 residues. Phosphorylation of threonine 308 and membrane localization depend on the activation of a phosphatidylinositol-dependent kinase-1, which also contains a pleckstrin homology domain (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ).
      Inhibition of PI3K with two unrelated pharmacological inhibitors, wortmannin and LY294002, abrogated AKT phosphorylation and the protective effect of Tat on vincristine-induced apoptosis of KS cells, suggesting that the PI3K/AKT pathway is involved in Tat-mediated anti-apoptotic effect.
      Several studies (
      • Zhang L.
      • Zhou W.
      • Velculescu V.E.
      • Kern S.E.
      • Hruban R.H.
      • Hamilton S.R.
      • Vogelstein B.
      • Kinzler K.W.
      ,
      • Talapatra S.
      • Thompson C.B.
      ,
      • Zhou H.
      • Xin-Ming L.
      • Meinkoth J.
      • Pittman R.N.
      ) have shown that AKT is the major effector of PI3K survival signaling. However, the mechanism by which AKT suppresses death is incompletely known. Phosphorylation of BAD at serine 136 is one of the potential mechanisms involved in AKT-dependent survival (
      • Del Peso L.
      • Gonzales-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ). Non-phosphorylated BAD binds to BCL-XL in the mitochondrial membrane thereby preventing BCL-XL from promoting cell survival. When phosphorylated, BAD is released from BCL-XL, which can then prevent apoptosis by inhibiting the release of cytochromec and is sequestered in the cytoplasm by 14-3-3 survival factors. In the present study we observed that Tat induces concomitant phosphorylation of BAD and AKT suggesting the involvement of this pathway in Tat-induced KS cells survival. Indeed, the inhibitors of PI3K/AKT pathway also abrogated BAD phosphorylation (
      • Talapatra S.
      • Thompson C.B.
      ). The AKT-dependent regulation of cell metabolism may also contribute to the anti-apoptotic effect on KS cells (
      • Plas D.R.
      • Talapatra S.
      • Edinger A.L.
      • Rathmell J.C.
      • Thompson C.B.
      ,
      • Barthel A.
      • Okino S.T.
      • Liao J.
      • Nakatani K., Li, J.
      • Whitlock J.P., Jr.
      • Roth A.
      ).
      AKT-mediated cell survival may also depend on its phosphorylation and inactivation of Forkhead transcription factor FKHRL1 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ,
      • Kops G.J.
      • de Ruiter N.D., De
      • Vries-Smits A.M.
      • Powell D.R.
      • Bos J.L.
      • Burgering B.M.
      ,
      • Biggs W.H.I.
      • Meisenhelder J.
      • Hunter T.
      • Cavenee W.K.
      • Arden K.C.
      ). In the absence of phosphorylation, FKHRL1 migrates to the nucleus leading to the transcription and the surface expression of FasL that in turn mediates a cell death cascade (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). However, the involvement of this pathway in the anti-apoptotic effect of Tat is unlikely, as we have demonstrated previously (
      • Cantaluppi V.
      • Biancone L.
      • Boccellino M.
      • Doublier S.
      • Benelli R.
      • Carlone S.
      • Albini A.
      • Camussi G.
      ) that Tat did not affect Fas/FasL expression by KS cells.
      HIV-1-Tat protein has been shown to down-regulate cAMP-response element-binding protein transcription factor expression in PC12 neuronal cells through PI3K/AKT/cyclic nucleoside phosphodiesterase pathway (
      • Zauli G.
      • Milani D.
      • Mirandola P.
      • Mazzoni M.
      • Secchiero P.
      • Miscia S.
      • Capitani S.
      ). Moreover, the PI3K/AKT pathway was found to be activated by Tat in T-lymphoblastoid Jurkat cells (
      • Borgatti P.
      • Zauli G.
      • Colamussi M.L.
      • Gibellini D.
      • Previati M.
      • Cantley L.L.
      • Capitani S.
      ).
      In the present study we demonstrated that Tat induced two waves of AKT activity in KS cells. The early activation of AKT was independent of protein synthesis, suggesting a direct activation of PI3K/AKT pathway by Tat. This early AKT activation was inhibited by anti-VEGFR-2 antibodies more efficiently when added before Tat stimulation than when added 1 h after. These results are consistent with the recent observation (
      • Morini M.
      • Benelli R.
      • Giungiuglio D.
      • Carlone S.
      • Arena G.
      • Noonan D.M.
      • Albini A.
      ) that the stimulation of KS cells by Tat mainly occurs through the VEGFR-2. The delayed AKT activation detectable at 24 h was significantly decreased by cycloheximide, an inhibitor of protein synthesis, suggesting that a Tat-induced synthesis of secondary mediators may contribute to AKT activation. One could speculate that Tat could stimulate the synthesis of VEGF, which in turn mediates the anti-apoptotic effect of Tat. However, because anti-VEGFR2 blocking antibodies only partially inhibited the survival effect of Tat when added 1 h after Tat stimulation, it is reasonable to assume that beside VEGF other mediators could be involved in mediating the Tat effect.
      Because gene array analysis indicated that Tat was able to triggerIGF-I and IL-3 gene expression, we investigated whether these cytokines may contribute to the anti-apoptotic effect of Tat in KS cells. IGF-I is known to induce resistance to apoptosis in several cell types via the activation of AKT (
      • Weihua W.
      • Lee W.L., Wu, Y.Y.
      • Chen D.
      • Liu T.J.
      • Jang A.
      • Sharma P.M.
      • Wang P.H.
      ). IL-3 also mediates an anti-apoptotic effect on lymphocytes by the PI3K/AKT pathway (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S., Fu, H.
      • Gotoh Y.
      • Greenberg M.E.
      ). We found that IGF-1 and, to a less extent, IL-3 inhibit apoptosis of KS cells induced by vincristine. Moreover, the treatment of KS cells with anti-IGF-I receptor and anti-IL-3 blocking antibodies significantly reduced the anti-apoptotic effect of Tat and the delayed activation of AKT. These results suggested that the synthesis of IGF-I and IL-3 triggered by Tat might contribute to the resistance to apoptosis induced by Tat.
      It has been described that Tat has opposite effects on apoptosis not only on different cell types but also on the same cell type under different conditions. Indeed, Tat induces apoptosis of uninfected T-cells (
      • McCloskey T.W.
      • Ott M.
      • Tribale E.
      • Khan S.A.
      • Teichberg S.
      • Paul M.O.
      • Pahwa S.
      • Verdin E.
      • Chirmule N.
      ) through a Fas-dependent mechanism (
      • Westendorp M.O.
      • Frank R.
      • Ochsenbauer C.
      • Stricker K.
      • Dhein J.
      • Walczak H.
      • Debatin K.M.
      • Krammer P.H.
      ), whereas in peripheral blood HIV-1-infected T-cells prevent apoptosis (
      • McCloskey T.W.
      • Ott M.
      • Tribale E.
      • Khan S.A.
      • Teichberg S.
      • Paul M.O.
      • Pahwa S.
      • Verdin E.
      • Chirmule N.
      ). Moreover, Tat was shown to prevent apoptosis of different tumor cell lines of lymphoid, epithelial, and neuronal origin (
      • Zauli G.
      • Gibellini D.
      • Dilani D.
      • Mazzoni M.
      • Borgatti P., La
      • Placa M.
      • Capitani S.
      ,
      • Cantaluppi V.
      • Biancone L.
      • Boccellino M.
      • Doublier S.
      • Benelli R.
      • Carlone S.
      • Albini A.
      • Camussi G.
      ), suggesting its putative role in tumoral development in a microenvironment characterized by immune system impairment. We observed that Tat not only enhances the transcription of anti-apoptotic but also of some pro-apoptotic genes such as BAX andBAG-1. Moreover, Tat is known to induce production of several cytokines and growth factors that may influence the sensitivity to apoptosis. Therefore, the different effects on apoptosis elicited by Tat may depend not only on a differential activation of pro- and anti-apoptotic genes but also on cytokines and growth factors released in the cell microenvironment. In this contest, stimuli that activate the AKT-dependent pathway may contribute to the anti-apoptotic effect of Tat.
      Recently, it has been shown (
      • Montaner S.
      • Sodhi A.
      • Pece S.
      • Mesri E.A.
      • Gutkind J.S.
      ) that the HHV8 G protein-coupled receptor also promotes endothelial cell survival through an AKT-dependent pathway. Several lines of evidence indicate a role of HHV8 in the pathogenesis of KS. Endothelial cells in early KS lesions are infected with HHV8, and HHV8 has been shown to be able to infect endothelial cells in vitro, inducing a spindle-shape morphology and increasing the proliferative lifespan in all the cells in culture, despite the indication that only a limited portion appeared to be infected (
      • Flore O.
      • Rafii S.
      • Ely S.
      • O'Leary J.J.
      • Hyjek E.M.
      • Cesarman E.
      ,
      • Moses A.V.
      • Fish K.N.
      • Ruhl R.
      • Smith P.P.
      • Strussenberg J.G.
      • Zhu L.
      • Chandran B.
      • Nelson J.A.
      ). This suggests that paracrine effects induced by HHV8 infection are able to phenotypically modify neighboring cells (
      • Cesarman E.
      • Mesri E.A.
      • Gershengorn M.C.
      ). Therefore, in AIDS patients the HIV-1-Tat protein may contribute to HHV8 infection with the activation of the PI3K/AKT pathway in sensitive cells concurring to the pathogenesis of KS.
      In conclusion, the results of the present study indicate that the HIV-1-Tat protein acts as a survival factor for KS via a PI3K/AKT pathway. This pathway is mainly triggered by the stimulation of KS cells through VEGFR-2, which leads to the transcription of several anti-apoptotic genes including IGF-I and IL-3. These cytokines may then contribute with Tat to the activation of PI3K/AKT-mediated survival signals.

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