Motility Induced by Human Immunodeficiency Virus-1 Tat on Kaposi's Sarcoma Cells Requires Platelet-Activating Factor Synthesis
1999; Elsevier BV; Volume: 155; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65488-0
ISSN1525-2191
AutoresLuigi Biancone, Vincenzo Cantaluppi, Mariarosaria Boccellino, Benedetta Bussolati, Lorenzo Del Sorbo, Pier Giulio Conaldi, Adriana Albini, Antonio Toniolo, Giovanni Camussi,
Tópico(s)Platelet Disorders and Treatments
ResumoIn the present study, we evaluated whether motility of Kaposi's sarcoma (KS) spindle cells induced by HIV-1 Tat protein is dependent on the synthesis of platelet-activating factor (PAF). The results obtained indicate that Tat induced a dose-dependent synthesis of PAF from KS cells at a concentration as low as 0.1 ng/ml. PAF production started rapidly after Tat stimulation, peaking at 30 minutes and declining thereafter. Tat-induced cell migration was also a rapid event starting at 30 minutes. The motility was abrogated by addition of a panel of chemically unrelated PAF receptor antagonists (WEB 2170, CV 3988, CV 6209, and BN 52021), suggesting that the synthesized PAF mediates the motogenic effect of Tat. This effect was also present on cells plated on a type-I collagen-, fibronectin-, or basement membrane extract-coated surface. Expression of PAF receptor-specific mRNA was detected in KS cells. In addition, examination of the cytoskeletal organization showed that Tat-mediated KS cell redistribution of actin filaments and shape change was also inhibited by a PAF receptor antagonist. Moreover, PAF receptor blockade prevented the up-regulation of β1 integrin and the down-regulation of αvβ3 observed after stimulation of KS cells with Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in motility of KS cells. In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) spindle cells induced by HIV-1 Tat protein is dependent on the synthesis of platelet-activating factor (PAF). The results obtained indicate that Tat induced a dose-dependent synthesis of PAF from KS cells at a concentration as low as 0.1 ng/ml. PAF production started rapidly after Tat stimulation, peaking at 30 minutes and declining thereafter. Tat-induced cell migration was also a rapid event starting at 30 minutes. The motility was abrogated by addition of a panel of chemically unrelated PAF receptor antagonists (WEB 2170, CV 3988, CV 6209, and BN 52021), suggesting that the synthesized PAF mediates the motogenic effect of Tat. This effect was also present on cells plated on a type-I collagen-, fibronectin-, or basement membrane extract-coated surface. Expression of PAF receptor-specific mRNA was detected in KS cells. In addition, examination of the cytoskeletal organization showed that Tat-mediated KS cell redistribution of actin filaments and shape change was also inhibited by a PAF receptor antagonist. Moreover, PAF receptor blockade prevented the up-regulation of β1 integrin and the down-regulation of αvβ3 observed after stimulation of KS cells with Tat. In conclusion, the results of the present study indicate that Tat-induced PAF synthesis plays a critical role in triggering the events involved in motility of KS cells. Kaposi's sarcoma (KS) is one of the most common cancers affecting patients with human immunodeficiency virus-1 (HIV-1. infection. KS is a hemoangiosarcoma containing spindle-shaped cells, vascular smooth cells, endothelial cells, and fibroblasts.1Ensoli B Barillari G Gallo RC Cytokine and growth factors in the pathogenesis of AIDS-associated Kaposi's sarcoma.Immunol Rev. 1992; 127: 147-155Crossref PubMed Scopus (134) Google Scholar, 2Masood R Cai J Law R Gill P AIDS-associated Kaposi's sarcoma pathogenesis, clinical features, and treatment.J Opin Oncol. 1993; 5: 831-834Crossref PubMed Scopus (16) Google Scholar, 3Rabkin CS Janz S Lash A Coleman AE Musaba E Liotta L Biggar R Zhuang Z Monoclonal origin of multicentric Kaposi's sarcoma lesions.N Engl J Med. 1997; 336: 988-993Crossref PubMed Scopus (209) Google Scholar The growth and diffusion of KS have been ascribed to an imbalance in the network of soluble mediators caused by HIV-1 infection.4Ensoli B Barillari G Buonaguro L Gallo RC Molecular mechanisms in the pathogenesis of AIDS-associated Kaposi's sarcoma.Adv Exp Med Biol. 1991; 303: 27-38Crossref PubMed Scopus (23) Google Scholar We have recently observed that platelet-activating factor (PAF), produced in vitro by KS-derived spindle cells, induces and sustains in vivo angiogenesis in a murine model.5Bussolino F Arese M Montrucchio G Barra L Primo L Sanavio F Aglietta M Ghigo D Rola-Pleszczynski M Bosia A Albini A Camussi G Platelet-activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.J Clin Invest. 1995; 96: 940-952Crossref PubMed Scopus (92) Google Scholar Indeed, PAF is a phospholipid mediator of cell-to-cell communication that belongs to the structurally related family of acetylated phosphoglycerides.6Prescott SM Zimmerman GA McIntyre TM Platelet-activating factor.J Biol Chem. 1996; 268: 17381-17384Google Scholar Recently, it has been found that a mutation of a PAF-specific acetyl-hydrolase is the underlying defect of a congenital neurological disorder named Miller-Dieker lissencephaly, characterized by impaired migration of central neurons.7Hattori M Adachi H Tsujimoto M Arai H Inoue K Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase.Nature. 1994; 370: 216-218Crossref PubMed Scopus (456) Google Scholar Indeed, several lines of evidence provide support for a role of this agent in regulating cell contraction, migration, and adhesion.8Bussolino F Camussi G Aglietta M Braquet P Bosia A Pescarmona GP Sanavio F D'Urso M Marchisio PC Human endothelial cells are target for platelet-activating factor: platelet-activating factor induces changes in cytoskeleton structures.J Immunol. 1987; 139: 2439-2446PubMed Google Scholar, 9Camussi G Montrucchio G Lupia E De Martino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promeotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar A number of factors such as tumor necrosis factor-α (TNFα), hepatocyte growth factor (HGF), and interleukin-12, able to induce these events, were shown to act at least in part through the rapid synthesis of PAF.10Camussi G Turello E Bussolino F Baglioni C Tumor necrosis factor alters cytoskeletal organization and barrier function of endothelial cells.Int Arch Allergy Appl Immunol. 1991; 96: 84-91Crossref PubMed Scopus (48) Google Scholar, 11Camussi G Montrucchio G Lupia E Soldi R Comoglio PM Bussolino F Angiogenesis induced in vivo by hepatocyte growth factor is mediated by platelet-activating factor synthesis from macrophages.J Immunol. 1997; 158: 1302-1309PubMed Google Scholar, 12Bussolati B Mariano F Cignetti A Guarini A Cambi V Foà R Piccoli G Camussi G Platelet-activating factor synthesized by IL-12-stimulated PMN and NK cells mediates chemotaxis.J Immunol. 1998; 161: 1493-1500PubMed Google Scholar A number of cell surface structures were shown to interact with Tat. First, α5β1 and αvβ3 integrins may bind to Tat through its arginine-glycine-aspartic acid (RGD. sequence.13Albini A Benelli R Giunciuglio D Cai T Mariani G Ferrini S Noonan DM Identification of a novel domain of HIV tat involved in monocyte chemotaxis.J Biol Chem. 1998; 273: 15895-15900Crossref PubMed Scopus (105) Google Scholar Moreover, we found that HIV-1 Tat protein may interact with endothelial cells through binding to the mitogenic vascular endothelial growth factor-A (VEGF-A) receptor Flk-1.14Albini A Soldi R Giunciuglio G Giraudo E Benelli R Primo L Noonan D Salio M Camussi G Rockl W Bussolino F The angiogenesis induced by HIV-1 Tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.Nat Med. 1996; 2: 1371-1375Crossref PubMed Scopus (349) Google Scholar Furthermore, the chemokine receptors CCR2 and CCR3 may act as additional Tat receptors on monocytes.15Albini A Ferrini S Benelli R Sforzini S Giunciuglio D Aluigi MG Proudfoot AEI Alouani S Wells TNC Mariani G Rabin RL Farber JM Noonan DM HIV-1 Tat protein mimicry of chemokines.Proc Natl Acad Sci USA. 1998; 95: 13153-13158Crossref PubMed Scopus (246) Google Scholar Finally, it has been shown that HIV-1 Tat may interact with Flk-1 on KS 38 cells, activating a number of signal transduction pathways.16Ganju RK Munshi N Nair BC Liu ZY Gill P Groopman JE Human immunodeficiency virus Tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells.J Virol. 1998; 72: 6131-6137Crossref PubMed Google Scholar The aim of the present study was to evaluate whether HIV-1 Tat can stimulate the synthesis of PAF by KS cells and whether the newly synthesized PAF mediates the motogenic activity of Tat on these cells. Synthetic C16 PAF (1-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was obtained from Bachem Feinchemikalien (Bubendorf, Switzerland). CV 3988 was from Takeda Chemical Industries (Kyoto, Japan).15Albini A Ferrini S Benelli R Sforzini S Giunciuglio D Aluigi MG Proudfoot AEI Alouani S Wells TNC Mariani G Rabin RL Farber JM Noonan DM HIV-1 Tat protein mimicry of chemokines.Proc Natl Acad Sci USA. 1998; 95: 13153-13158Crossref PubMed Scopus (246) Google Scholar CV 6209 and BN 52021 were purchased from Biomol (Plymouth Meeting, PA). WEB 2170 was obtained from Boehringer Ingelheim KG, Germany.16Ganju RK Munshi N Nair BC Liu ZY Gill P Groopman JE Human immunodeficiency virus Tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells.J Virol. 1998; 72: 6131-6137Crossref PubMed Google Scholar Silica gel 60F254 thin-layer chromatography (TLC) plates were obtained from Merck (Darmstadt, Germany). mPorasil high-performance liquid chromatography (HPLC) columns were provided by Millipore Chromatographic Division (Waters, Milford, MA). RPMI 1640 medium was from GIBCO (Grand Island, NY) and bovine calf serum (BCS. was from Hyclone Lab (Logan, UT). Recombinant Tat was obtained from Intracell (London, UK). Polymyxin B, phospholipase A2, phospholipase A1, bovine serum albumin (BSA) fraction V (tested for not more than 1 ng endotoxin per mg), FMLP, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine were purchased from Sigma Chemical Company (St. Louis, MO). Rabbit polyclonal IgG anti-human flk-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). [3H]acetate ([3H]CH3CO2Na; 2.5 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). KS cell line derived from a HIV-1 patient and spontaneously immortalized was propagated as previously described.17Albini A Paglieri I Orengo G Carlone S Aluigi MG De Marchi R Matteucci C Mantovani A Carozzi F Donini S Benelli R The beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line.AIDS. 1997; 11: 713-721Crossref PubMed Scopus (103) Google Scholar In standard PAF synthesis assays, confluent KS cells maintained for 24 hours in DMEM without fetal calf serum (FCS) were stimulated in 1 ml Iscove's medium containing 0.25% BSA for various period of times with different doses of Tat. PAF released into the medium and cell associated was extracted and purified as previously described.12Bussolati B Mariano F Cignetti A Guarini A Cambi V Foà R Piccoli G Camussi G Platelet-activating factor synthesized by IL-12-stimulated PMN and NK cells mediates chemotaxis.J Immunol. 1998; 161: 1493-1500PubMed Google Scholar PAF extracted and purified by KS cells was quantified by bioassay on washed rabbit platelets.12Bussolati B Mariano F Cignetti A Guarini A Cambi V Foà R Piccoli G Camussi G Platelet-activating factor synthesized by IL-12-stimulated PMN and NK cells mediates chemotaxis.J Immunol. 1998; 161: 1493-1500PubMed Google Scholar PAF bioactivity, tested after extraction and purification by TLC and HPLC, was characterized by comparison with synthetic PAF according to the following criteria: i) induction of platelet aggregation by a pathway independent of both ADP- and arachidonic acid/thromboxane A2-mediated pathway; ii) specificity of platelet aggregation as inferred from the inhibitory effect of PAF receptor antagonist WEB 2170 (3 μmol/L); and iii) TLC and HPLC chromatographic behavior and physicochemical characteristics such as inactivation by base-catalyzed methanolysis or phospholipase A2 treatment and resistance to phospholipase A1 or treatment with weak base and acids.18McManus LM Woodard DS Deavers SI Pinckard RN Paf molecular heterogeneity.Lab Invest. 1993; 69: 639-650PubMed Google Scholar To study the incorporation of radioactive precursors, 5 × 105 KS cells were incubated in 1 ml RPMI 1640 for 30 minutes with 30 μCi [3H]acetate before stimulation.19Tufano MA Biancone L Rossano F Capasso C Baroni A DeMartino A Iorio L Silvestro L Camussi G Outer-membrane porins from Gram-negative bacteria stimulate platelet-activating factor biosynthesis by cultured human endothelial cells.Eur J Biochem. 1993; 214: 685-693Crossref PubMed Scopus (32) Google Scholar The cell pellets were extracted with formic acid added to lower the pH of the aqueous phase to 3.0 and lipids were fractionated by TLC on aluminum-sheet silica-gel plates (silica gel 60, F254, 0.2 mm thickness, Merck) using a solvent chloroform/methanol/acetic acid/water 50:25:8:4 by volume.19Tufano MA Biancone L Rossano F Capasso C Baroni A DeMartino A Iorio L Silvestro L Camussi G Outer-membrane porins from Gram-negative bacteria stimulate platelet-activating factor biosynthesis by cultured human endothelial cells.Eur J Biochem. 1993; 214: 685-693Crossref PubMed Scopus (32) Google Scholar The plates were cut into 1-cm sections and the radioactivity of each was measured. Radiolabeled C16-PAF was used as a standard. Cells (105/well) were plated and rested for 12 hours with medium M199 containing 1% FCS, then washed three times with PBS and incubated with RPMI and the agonist. Cell division did not start to any significant degree during the experiments. Cell migration was studied over a 20-hour period under a Nikon Diaphot inverted microscope with a 10× phase-contrast objective in an attached, hermetically sealed Plexiglas Nikon NP-2 incubator at 37°C. Cell migration was recorded using a JVC-1CCD video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging, Venice, Italy) and an IBM-compatible system equipped with a video card (Targa 2000, Truevision, Santa Clara, CA). Image analysis was performed by digital saving of images at intervals of 30 minutes. Migration tracks were generated by marking the position of nucleus of individual cells on each image. The net migratory speed (velocity straight line) was calculated by the MicroImage software based on the straight line distance between the starting and ending points divided by the time of observation. Migration of at least 30 cells was analyzed for each experimental condition. Values are given as mean ± SD. In selected experiments, KS cells were seeded on plates previously coated with 10 μg/ml bovine fibronectin (Sigma), bovine type-I collagen (Sigma), or reconstituted basement membrane (Matrigel, Sigma) overnight at 37°C. PAF receptor-specific mRNA was detected in total RNA extracted from cells by guanidinium thyocyanate phenol-chloroform precipitated with isopropanol. One microgram of RNA was treated with 6 U of RNase-free DNase for 1 hour at 37°C and then for 5 minutes at 94°C. Complementary DNA was obtained by using random hexamer primers (Perkin-Elmer Cetus, Norwalk, CT). Reverse transcription was carried out at 42°C for 60 minutes; in addition to 1 μg of RNA, the reaction mixture (20 μl) contained 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 5 mmol/L MgCl2, 1.0 mmol/L dNTPs, 20 U ribonuclease inhibitor, and 50 U of Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus). cDNA was then subjected to 35 cycles of amplification by the polymerase chain reaction in an automated DNA thermal cycler (Perkin-Elmer Cetus) by using these human PAF receptor mRNA-specific primer pairs: forward, 5′ CAC GGG CTC GAG ACC AAC ACA GTG CCC GAC AGT GCT 3′; reverse, 5′ CGC GGG ATC CCG GGT GAC CTG ATG TGC ATC ATT AAT 3′. The PCR reaction mixture (50 μl) contained 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 20 pmol of (+) and (−) primers, and 2 U thermostable DNA polymerase (Perkin-Elmer Cetus). Times and temperatures for denaturation, annealing, and extension were 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, respectively. Amplification product (262 bp) was analyzed in 2% agarose gels containing 0.5 μg/ml of ethidium bromide. As control, B16 cells (American Type Culture Collection, Manassas, VA) untransfected or transfected with human PAF receptor-specific cDNA (kindly provided by Dr. R. D. Ye, La Jolla, CA. were used. Cytoskeleton alterations were studied on fixed KS after permeabilization with 0.1% Triton X-100 in PBS and stained for F-actin with 2 μg/ml FITC-conjugated phalloidin (F-PHD; Sigma). In selected experiments apoptosis was evaluated by the XTT-based assay (Sigma). Briefly, cells were cultured in 96-well flat-bottomed microtiter plates (Falcon Labware, Oxnard, CA) at a concentration of 5 × 104 cells/well in RPMI. At different periods of time, cells were washed and incubated in serum-free Dulbecco's modified minimum essential medium containing 250 μg/ml XTT at 37°C. Apoptosis was also evaluated by cytofluorimetric analysis of DNA after propidimn iodide (PI) staining. Briefly, 106 cells were incubated for 4 hours at 4°C in 2 ml hypotonic solution containing 50 μg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100, and 20 μg/ml DNase-free RNase A. Cells with subdiploid DNA content (sub-G0/G1 peak) were considered apoptotic cells. All cultures were done in triplicate. After appropriate stimulation (at 37°C for 1, 4, or 12 hours. with Tat (10 ng/ml), Tat plus CV 3988 (3 μM), or vehicle alone, cells were detached from plates with 0.02% EDTA at 4°C, washed, resuspended in PBS containing 0.25% BSA, and incubated at 4°C for 30 minutes with containing 1 μg/ml of monoclonal anti-β1 or anti-avβ3 integrin antibodies (Chemicon, Temecula, CA) or with anti-PAF-R antibody (kind gift of Dr. M. Rola-Pleszczynski, Sherbrooke, PQ). As a second step reagent, FITC-conjugated anti-mouse IgG (Sigma) was used. Cells were analyzed on a FACS (Becton Dickinson, Mountain View, CA). All data are expressed as mean ± SD. Statistical analysis was performed by analysis of variance (ANOVA) with Neumann-Keul's multiple comparison test or Kolmogorov-Smirnov where appropriate. Unstimulated KS cells produced a small amount of PAF that in short-term experiments was detected associated with the cells. When stimulated with Tat, KS cells showed a rapid increase of PAF synthesis, which peaked at 15 minutes and decreased thereafter and was followed by a partial release into the medium (Figure 1A). As shown in Figure 1B, the effect of Tat on PAF synthesis was dose-dependent. Tat induced the synthesis of PAF at doses as low as 0.1 ng/ml. Using radioactive acetate as substrate for PAF synthesis, we found that PAF detected after stimulation with Tat was newly synthesized. The TLC analysis of lipid fractions extracted 15 minutes after addition of Tat to KS cells preincubated with [3H]-acetate demonstrated the presence of one main peak of radioactivity that co-migrated with synthetic [3H]-C16-PAF (Figure 1C). This peak was lower in the lipid fractions extracted from unstimulated KS cells. PAF-bioactive material extracted and purified from KS cells was insensitive to treatment with phospholipase A1 that cleaves the acyl- but not the alkyl-PAF.18McManus LM Woodard DS Deavers SI Pinckard RN Paf molecular heterogeneity.Lab Invest. 1993; 69: 639-650PubMed Google Scholar, 20Benveniste J LeCouedic JP Polonsky J Tence M Structural analysis of purified platelet activating factor by lipases.Nature. 1977; 269: 170-171Crossref PubMed Scopus (204) Google Scholar The amount of PAF-bioactive material treated with phospholipase A1 did not show a significant reduction of its biological activity (93 ± 1.9% recovered activity). To evaluate the efficiency of phospholipase A1 treatment 4 samples containing PAF-bioactive material were added with [14C]-acyl-PAF before treatment with phospholipase A1. The amount of [14C]-acyl-PAF hydrolyzed (recovered as a free fatty acid) was 82 ± 4.2%, whereas the biological activity was not significantly reduced (92 ± 2.8% recovered activity). PAF receptor-specific mRNA was detected by RT-PCR in total RNA extracted from KS cells. Analysis of mRNA from CHO cells transfected with human PAF-receptor specific cDNA but not untransfected cells displayed identical amplification product (Figure 2) to the one from KS cells thus providing additional control. Moreover, we evaluated by cytofluorimetric analysis whether Tat stimulation increased the surface expression of PAF receptor during experiments of cell motility. The results obtained indicated that the basal expression of PAF receptor was not enhanced by Tat at 4 and 12 hours (data not shown). The baseline migration rate of KS cells corresponding to the spontaneous motility of rested, unstimulated cells was first measured and found to remain steady for the whole period of observation, never exceeding 5 to 6 μm/hour speed (Figure 3, Figure 4). Incubation with Tat stimulated a marked dose-dependent acceleration of cell motility, peaking as early as 30 minutes after addition and maintaining a significantly higher speed compared to unstimulated KS cells for the time of observation (Figure 3, Figure 4). Similar enhancement of cell motility was observed after stimulation of KS cells with 10 ng/ml PAF (Figure 3A). Addition of the specific PAF receptor antagonists, WEB 2170,21Heuer HO Casals-Stenzel J Muacevic G Weber KH Pharmacologic activity of bepafant (WEB 2170), a new and selective hetrazepinoic antagonist of platelet-activating factor.J Pharmacol Exp Ther. 1990; 255: 962-970PubMed Google Scholar CV 3988,22Terashita Z Tsushima S Yoshioka S Namoto H Inada Y Nishikawa K CV 3988: a specific antagonist of platelet-activating factor (Paf-acether).Life Sci. 1983; 32: 1975-1982Crossref PubMed Scopus (330) Google Scholar CV 6209,23Terashita Z Imura Y Takatani M Tsushima S Nishikawa K CV-6209, a highly potent antagonist of platelet activating factor in vitro and in vivo.J Pharmacol Exp Ther. 1987; 242: 263-268PubMed Google Scholar or BN 52021,24Vercellotti GM Yin HQ Gustafson KS Nelson RD Jacob HS Platelet-activating factor primes neutrophil responses to agonists: role in promoting neutrophil-mediated endothelial damage.Blood. 1988; 71: 1100-1107Crossref PubMed Google Scholar which completely abrogated PAF-induced motility (not shown), also significantly inhibited motility stimulated by Tat (Figure 3, Figure 4). No significant difference in motility was observed among unstimulated cells and cells stimulated with Tat and treated with PAF receptor antagonist. Moreover, PAF receptor antagonist alone did not reduce the baseline migration of untreated KS cells (data not shown). These results suggest that the enhanced motility induced by Tat but not the spontaneous KS cell motility was PAF-dependent. As shown in Figure 5, we have also evaluated the role of different matrix substrates on Tat-induced KS cell motility. The results obtained indicate that Tat-induced motility was inhibited by the PAF receptor antagonist WEB 2170 not only on plastic but also on type 1 collagen (Figure 5A), fibronectin (Figure 5B), and reconstituted basement membrane (Matrigel; Figure 5C).Figure 4Micrographs representative of time-lapse analysis of KS cell motility performed by digital saving at 30-minute intervals. Migration tracks were generated by marking the position of the nucleus of individual cells of each image (see Materials and Methods). KS cells were incubated with vehicle alone (A), with 10 ng/ml Tat (B), or with Tat (10 ng/ml) in the presence of 3 μmol/L WEB2170 (C). Original magnification, ×120.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Evaluation of the role of different matrix substrates on Tat-induced KS cell motility. KS cells plated on type 1 collagen (A), fibronectin (B), and Matrigel (C) were stimulated with vehicle alone or with of Tat (10 ng/ml) or Tat plus PAF-receptor antagonists WEB2170 (3 μmol/L). Three experiments were performed with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The incubation of KS cells with Tat induced shape changes (Figure 6) and modified the normal distribution of actin-containing stress fibers (Figure 7). After exposure of KS cells to 10 ng/ml Tat for 1 hour at 37°C cells change shape (Figure 6D) and stress fibers tend to axially condense, retract, and appear to fuse (Figure 7B). Such alterations are observed also as a result of incubation with PAF (10 ng/ml; data not shown). Incubation with WEB 2170 as well as with CV 3988, CV 6209, or BN 52021 (data not shown. prevented both the change in cell shape (Figure 6F) and cytoskeleton changes (Figure 7C) induced by Tat. Tat-induced alterations were reversible after washing and culturing cells overnight (Figure 7B). In selected experiments to evaluate whether rounding up of the cells induced by Tat precluded apoptosis, we tested cell viability by XTT-based and subdiploid DNA content methods. Cell survival was 96 ± 5% on vehicle-treated cells and 98 ± 2% on 10 ng/ml Tat-treated cells for 12 hours. Cells with subdiploid DNA content accounted for 5 ± 3% of the vehicle-treated cells and 4 ± 3% of the 10 ng/ml Tat-treated cells. These results indicate that changes in cell shape and cytoskeleton were not associated with apoptosis.Figure 7Micrographs representative of F-actin distribution in fixed and permeabilized KS cells after incubation for 1 hour in the following experimental conditions: A, unstimulated KS cells (vehicle alone); B, KS cells stimulated with 10 ng/ml Tat; C, KS cells stimulated with 10 ng/ml Tat in the presence of 3 μmol/L WEB 2170; D, KS cells stimulated with Tat 10 ng/ml and then washed and cultured for 12 hours with RPMI supplemented with 10% FBS to evaluate reversal of cytoskeletal changes induced by Tat. Three experiments were performed with similar results. Original magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of PAF and Tat stimulation on integrin expression in the presence or absence of PAF receptor antagonist CV3988 was evaluated after 1, 4, and 12 hours. Two distinct trends of expression were observed for αvβ3 and β1 integrins. PAF as well as Tat stimulation induced a progressive down-regulation of αvβ3, which was already detectable at 4 hours (data not shown) and maximal at 12 hours (Figure 8, A and C). In contrast, both agonists induced up-regulation of β1, which was evident after 12 hours' stimulation (Figure 8, E and G). Treatment with the PAF receptor antagonist CV3988 abrogated both the down-regulation of αvβ3 (Figure 8, B and D) and the up-regulation of β1 (Figure 8, F and H) integrins induced by PAF and Tat. The results of the present study indicate that Tat induces synthesis of PAF by KS cells and that PAF, in turn, mediates the motogenic activity of Tat on these cells. PAF is a phospholipid mediator with multiple biological activities that has been shown to directly stimulate in vitro migration of endothelial cells and promote in vivo angiogenesis.9Camussi G Montrucchio G Lupia E De Martino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promeotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar Recently, we found that KS cells synthesize PAF after stimulation with cytokines and that PAF released in the supernatant of KS cells accounts, at least in part, for its angiogenic activity in vivo.5Bussolino F Arese M Montrucchio G Barra L Primo L Sanavio F Aglietta M Ghigo D Rola-Pleszczynski M Bosia A Albini A Camussi G Platelet-activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.J Clin Invest. 1995; 96: 940-952Crossref PubMed Scopus (92) Google Scholar Several studies indicate that Tat has a relevant role in the pathogenesis of Kaposi's sarcoma. Indeed, Tat transgenic mice develop Kaposi's sarcoma-like lesions.25Vogel J Hinrichs S Reynolds R Luviw J Jay G The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice.Nature. 1988; 335: 606-611Crossref PubMed Scopus (464) Google Scholar Tat may interact with cells either through α5β1 and αvβ3 integrins via its RGD sequence,13Albini A Benelli R Giunciuglio D Cai T Mariani G Ferrini S Noonan DM Identification of a novel domain of HIV tat involved in monocyte chemotaxis.J Biol Chem. 1998; 273: 15895-15900Crossref PubMed Scopus (105) Google Scholar through Flk- 1/KDR via its VEGF-like sequence,14Albini A Soldi R Giunciuglio G Giraudo E Benelli R Primo L Noonan D Salio M Camussi G Rockl W Bussolino F The angiogenesis induced by HIV-1 Tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.Nat Med. 1996; 2: 1371-1375Crossref PubMed Scopus (349) Google Scholar and/or through chemokine receptors CCR2 and CCR3.15Albini A Ferrini S Benelli R Sforzini S Giunciuglio D Aluigi MG Proudfoot AEI Alouani S Wells TNC Mariani G Rabin RL Farber JM Noonan DM HIV-1 Tat protein mimicry of chemokines.Proc Natl Acad Sci USA. 1998; 95: 13153-13158Crossref PubMed Scopus (246) Google Scholar Previous studies have shown that PAF mediates some of the biological properties of certain polypeptide mediators. In particular, PAF was found to mediate directional migration of endothelial cells induced by tumor necrosis factor10Camussi G Turello E Bussolino F Baglioni C Tumor necrosis factor alters cytoskeletal organization and barrier function of endothelial cells.Int Arch Allergy Appl Immunol. 1991; 96: 84-91Crossref PubMed Scopus (48) Google Scholar, 26Montrucchio G Lupia E Battaglia E Bussolino F Emanuelli G Camussi G Tumor-necrosis factor alpha-induced angiogenesis depends on “in situ” platelet-activating factor biosynthesis.J Exp Med. 1994; 180: 377-382Crossref PubMed Scopus (138) Google Scholar and hepatocyte growth factor.11Camussi G Montrucchio G Lupia E Soldi R Comoglio PM Bussolino F Angiogenesis induced in vivo by hepatocyte growth factor is mediated by platelet-activating factor synthesis from macrophages.J Immunol. 1997; 158: 1302-1309PubMed Google Scholar In the present study we demonstrate that a viral protein such as HIV-1 Tat induces synthesis of PAF by KS cells. PAF is newly synthesized, as shown by the incorporation of labeled acetate in the molecule of PAF and is released in the supernatant. Moreover, we found that PAF is instrumental in the motility of KS cells elicited by HIV-1 Tat. Indeed, in vitro KS cell migration induced by Tat was inhibited by a panel of chemically different PAF receptor antagonists. Therefore, one can envisage that PAF produced by KS cells has an autocrine and/or paracrine effect on the motility of these cells. The characteristics of the motility assay used in the present study exclude a gradient for both Tat and PAF, suggesting a chemokinetic effect. In this contest, PAF may act either as mediator of cell-to-cell communication involved in the amplification of the signal triggered by Tat or in the up-regulation of adhesion molecules involved in cell migration. Indeed, it has recently been shown that PAF up-regulates integrins and that the β1 integrins are critically involved in PAF-induced leukocyte locomotion in extravascular tissue.27Werr J Xie X Hedqvist P Ruoslahti E Lindbom L b1 integrins are critically involved in neutrophil locomotion in extravascular tissue in vivo.J Exp Med. 1998; 187: 2091-2096Crossref PubMed Scopus (138) Google Scholar The results of the present study indicate that Tat induced a PAF-dependent up-regulation of β1 integrins and down-regulation of αvβ3 integrins, because PAF mimics and a PAF receptor antagonist abrogates the events elicited by Tat. It is recognized that not only the activation, but also the modulation of surface concentration of integrins is instrumental for cell motility on extracellular matrix.28Palecek SP Loftus JC Ginsberg MH Lauffenburger DA Horwitz AF Integrin-ligand bind properties govern cell migration speed through cell-substratum adhesiveness.Nature. 1997; 385: 537-540Crossref PubMed Scopus (1198) Google Scholar In the present study, we have also evaluated the role of different matrix substrates on Tat-induced KS cell motility. The results obtained indicate that Tat-induce motility is PAF-dependent not only on plastic but also on more physiological substrates, such as type 1 collagen, fibronectin, and reconstituted basement membrane (Matrigel). Taken together, these results suggest that PAF synthesized by KS cells after stimulation with either HIV-1-derived Tat or cytokines may be instrumental in the infiltration of KS cells in tissues and in the interaction with the neoformed vessels that characterize this angiogenic tumor. In fact, PAF is able to trigger in vivo a neoangiogenic response by stimulating endothelial cell recruitment.9Camussi G Montrucchio G Lupia E De Martino A Perona L Arese M Vercellone A Toniolo A Bussolino F Platelet-activating factor directly stimulates in vitro migration of endothelial cells and promeotes in vivo angiogenesis by a heparin-dependent mechanism.J Immunol. 1995; 154: 6492-6501PubMed Google Scholar In conclusion, our observations that Tat stimulates the synthesis of PAF by KS cells and that a panel of specific PAF receptor antagonists inhibits the migration of KS cells triggered by Tat suggest that this phospholipid may act as a secondary mediator for the motility induced by this HIV-1 protein.
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