| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Cattedra di Nefrologia,*
Dipartimento di
Medicina Interna, Università di Torino, Torino; the Istituto
Nazionale per la Ricerca sul Cancro,
Genova;
and the Cattedra di Microbiologia,
Dipartimento di Scienze Cliniche e Biologiche, Università
dell’Insubria, Varese, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 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.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(TNF), 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.10-12
A number of cell surface structures were
shown to interact with Tat. First, 5ß1 and vß3 integrins may
bind to Tat through its arginine-glycine-aspartic acid (RGD)
sequence.13
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-1.14
Furthermore, the chemokine receptors CCR2 and CCR3
may act as additional Tat receptors on monocytes.15
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.16
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.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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).15 CV 6209 and BN 52021 were purchased from Biomol (Plymouth Meeting, PA). WEB 2170 was obtained from Boehringer Ingelheim KG, Germany.16 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).
In Vitro PAF Synthesis by KS Cells
KS cell line derived from a HIV-1 patient and spontaneously immortalized was propagated as previously described.17 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.12 PAF extracted and purified by KS cells was quantified by bioassay on washed rabbit platelets.12 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.18
To study the incorporation of radioactive precursors, 5 x 105 KS cells were incubated in 1 ml RPMI 1640 for 30 minutes with 30 µCi [3H]acetate before stimulation.19 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.19 The plates were cut into 1-cm sections and the radioactivity of each was measured. Radiolabeled C16-PAF was used as a standard.
In Vitro KS Cell Migration
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 10x 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 mRNA Expression
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.
Cell Cytoskeleton Studies
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).
Apoptosis Assays
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 x 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.
Cytofluorimetric Analysis
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-ß1 or anti-avß3 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).
Statistical Analysis
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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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.18,20
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 (Figures 3A and 4A)
.
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 (Figures 3A and 4B)
. 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 2170,21
CV 3988,22
CV
6209,23
or BN 52021,24
which completely
abrogated PAF-induced motility (not shown), also significantly
inhibited motility stimulated by Tat (Figures 3B and 4C)
. 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
).
|
|
|
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.
|
|
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 vß3 and ß1 integrins. PAF as well as Tat
stimulation induced a progressive down-regulation of vß3, 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 ß1, 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 vß3 (Figure 8, B and D)
and the
up-regulation of ß1 (Figure 8, F and H)
integrins induced by PAF and
Tat.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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.25
Tat may interact with
cells either through 5ß1 and vß3 integrins via its RGD
sequence,13
through Flk- 1/KDR via its VEGF-like
sequence,14
and/or through chemokine receptors CCR2 and
CCR3.15
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 factor10,26
and hepatocyte growth
factor.11
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 ß1 integrins are critically
involved in PAF-induced leukocyte locomotion in extravascular
tissue.27
The results of the present study indicate that
Tat induced a PAF-dependent up-regulation of ß1 integrins and
down-regulation of vß3 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.28
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.9 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.
|
Footnotes |
|---|
Supported by Istituto Superiore di Sanità (ISS, AIDS Grant no. 30B.10), the "Associazione Italiana per la Ricerca sul Cancro" and the National Research Council (CNR), Targeted Project "Biotechnology" (to G. C.).
Accepted for publication July 25, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Doublier, M. Ceretto, E. Lupia, S. Bravo, B. Bussolati, and G. Camussi The Proangiogenic Phenotype of Tumor-Derived Endothelial Cells is Reverted by the Overexpression of Platelet-Activating Factor Acetylhydrolase Clin. Cancer Res., October 1, 2007; 13(19): 5710 - 5718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Boccellino, G. Camussi, A. Giovane, L. Ferro, V. Calderaro, C. Balestrieri, and L. Quagliuolo Platelet-Activating Factor Regulates Cadherin-Catenin Adhesion System Expression and {beta}-Catenin Phosphorylation during Kaposi's Sarcoma Cell Motility Am. J. Pathol., May 1, 2005; 166(5): 1515 - 1522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hudry-Clergeon, D. Stengel, E. Ninio, and I. Vilgrain Platelet-activating factor increases VE-cadherin tyrosine phosphorylation in mouse endothelial cells and its association with the PtdIns3'-kinase FASEB J, April 1, 2005; 19(6): 512 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Biancone, V. Cantaluppi, L. Del Sorbo, S. Russo, L. W. Tjoelker, and G. Camussi Platelet-activating Factor Inactivation by Local Expression of Platelet-activating Factor Acetyl-Hydrolase Modifies Tumor Vascularization and Growth Clin. Cancer Res., September 15, 2003; 9(11): 4214 - 4220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Conaldi, A. Bottelli, A. Baj, C. Serra, L. Fiore, G. Federico, B. Bussolati, and G. Camussi Human Immunodeficiency Virus-1 Tat Induces Hyperproliferation and Dysregulation of Renal Glomerular Epithelial Cells Am. J. Pathol., July 1, 2002; 161(1): 53 - 61. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Cassoni, A. Sapino, S. Deaglio, B. Bussolati, M. Volante, L. Munaron, A. Albini, A. Torrisi, and G. Bussolati Oxytocin Is a Growth Factor for Kaposi's Sarcoma Cells: Evidence of Endocrine-Immunological Cross-Talk Cancer Res., April 1, 2002; 62(8): 2406 - 2413. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, cell-associated PAF; 
, released
PAF) or with vehicle alone
(
, cell-associated PAF; 
, released
PAF). Numbers are the mean ± SD of three
individual experiments. B shows the effect of different doses
of Tat on PAF synthesis by KS cells and of thrombin
(2 U/ml) used as positive
control. The numbers are the mean ± SD of five individual
experiments. C shows the incorporation of radiolabeled acetate
in the newly synthesized PAF after stimulation of KS cells with Tat.
Representative TLC analysis of radiolabeled lipids extracted from
5 x 10
