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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 663-672
The Tat Protein of Human Immunodeficiency Virus Type-1 Promotes
Vascular Cell Growth and Locomotion by Engaging the  5 1 and
v3 Integrins and by Mobilizing Sequestered Basic Fibroblast
Growth Factor
By
Giovanni Barillari,
Cecilia Sgadari,
Valeria Fiorelli,
Felipe Samaniego,
Sandra Colombini,
Vittorio Manzari,
Andrea Modesti,
Bala C. Nair,
Aurelio Cafaro,
Michael Stürzl, and
Barbara Ensoli
From the Laboratory of Virology, Istituto Superiore di Sanità,
Rome, Italy; the Department of Experimental Medicine, University
"Tor Vergata," Rome, Italy; the Department of Allergy and
Clinical Immunology, University "La Sapienza," Rome, Italy; the
Institute of Human Virology, University of Maryland at Baltimore,
Baltimore, MD; Advanced BioScience Laboratories Inc, Kensington, MD;
the GSF-National Research Center for Environment and Health, Bavarian
Nordic Research Institute AS, Martinsried, Germany; and the Technical
University of Munich, Institute of Virology, Munich, Germany.
 |
ABSTRACT |
The Tat protein of human immunodeficiency virus type-1 (HIV-1) has
been shown to be released during acute infection of T cells by HIV-1
and to promote angiogenesis and Kaposi's sarcoma (KS) development in
infected individuals. In this study, we investigated the molecular
mechanisms responsible for the angiogenic effects of Tat. The results
shown herein indicate that two different Tat domains cooperate to
induce these effects by different pathways. The
arginine-glycine-aspartic acid (RGD) sequence present at the carboxyterminal of Tat mediates vascular cell migration and invasion by
binding to the  5 1 and v3 integrins. This interaction also provides endothelial cells with the adhesion signal they require to
grow in response to mitogens. At the same time, the Tat basic sequence
retrieves into a soluble form extracellular basic fibroblast growth
factor (bFGF) bound to heparan sulfate proteoglycans by competing for
heparin-binding sites. This soluble bFGF mediates Tat-induced vascular
cell growth. These effects resemble those of extracellular matrix
proteins, suggesting that Tat enhances angiogenesis and promotes KS
progression by a molecular mimicry of these molecules.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DURING ACUTE INFECTION of T cells by
human immunodeficiency virus type-1 (HIV-1), Tat, a transactivator of
viral gene expression,1 is released
extracellularly.2-4 In this form, Tat exerts activities
that have linked the protein to the pathogenesis of Kaposi's sarcoma
(KS), a disease of vascular origin that is very common and aggressive
in HIV-1-infected individuals (acquired immunodeficiency syndrome-KS
[AIDS-KS]) but mild and indolent in the absence of HIV
infection.5 In particular, Tat promotes the locomotion and
growth of spindle cells of endothelial origin derived from AIDS-KS
lesions (KS cells) and of normal endothelial cells,2,3,6-10
which are considered to be the precursors of KS cells.11
However, endothelial cells become responsive to the effects of Tat only
after activation with inflammatory cytokines (IC), such as
interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and
interferon- (IFN-).6-10 These IC are the same found to be increased in the lesions and blood of KS
patients.12-14
The requirement of other factors for Tat angiogenic effects is also
observed in vivo, because inoculation of Tat protein alone in nude mice
has little or no effect.15 In contrast, when Tat is
injected with IC or with suboptimal (non-lesion-forming) amounts of
basic fibroblast growth factor (bFGF), it promotes the development of
angioproliferative KS-like lesions in the inoculated
animals.9,15 It is of interest that bFGF production is
enhanced in KS cells or induced in endothelial cells by the same IC
that are required to promote endothelial cell responsiveness to
Tat.16-18
Tat has also been shown to bind F1K-1/KDR, one of the receptors for the
vascular endothelial growth factor (VEGF),19 suggesting an
additional mechanism for Tat to exert angiogenic activity. Both bFGF
and VEGF, two potent angiogenic factors,20,21 are highly
expressed in AIDS-KS lesions,15,22-25 where they synergize in promoting neoangiogenesis and edema.18,25
Tat can also promote KS and endothelial cell adhesion through the
binding of its arginine-glycine-aspartic acid (RGD) region to the
51 and v3 integrins.26 These receptors, which
bind the RGD sequence of extracellular matrix (ECM) proteins, such as
fibronectin (FN) and vitronectin (VN),27 are constitutively expressed by KS cells both in vitro and in primary
lesions,15,26 and their levels are increased in normal
endothelial cells by the same IC that induce bFGF expression and
cellular responsiveness to Tat.10,26 Other data indicated
that the basic region of Tat binds v5, an integrin that
recognizes similar sequences in VN.28
Altogether, these results indicated that Tat has properties similar to
both angiogenic factors and ECM proteins and that it requires the
cooperation of inflammatory or angiogenic cytokines to exert its
effects. However, they did not explain the mechanism(s) by which Tat
can promote angiogenesis and KS progression.
We report here that the angiogenic effects of Tat are mediated by two
domains of the protein. Specifically, the RGD region of Tat induces the
migration and invasion of KS and endothelial cells by binding to the
51 and v3 integrins. Additionally, the Tat basic sequence,
because of its affinity for heparin, releases preformed
extracellular-bound bFGF into a soluble form that mediates Tat-promoted
vascular cell growth.
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MATERIALS AND METHODS |
Reagents.
Recombinant HIV-1 Tat protein (from the IIIB isolate) was obtained and
handled as previously described.2-4,15 The peptides [11-24] Tat, [36-50] Tat, [46-60] Tat, [56-70] Tat, [65-80]
Tat, and [72-86] Tat were purchased from American Biotechnologies Inc (Cambridge, MA). The peptide [48-53] Tat, the FN cyclic peptide GRGDSP, and the mutated cyclic peptide GKGESP were purchased from Research Genetics (Huntsville, AL). Human recombinant IL-1 and IL-1, IL-2, IL-6, oncostatin M, TNF- and TNF-, IFN-, bFGF, and FN (from human plasma) were purchased from Boehringer Mannheim (Indianapolis, IN). VN (from human plasma), heparin (sodium salt, from
porcine intestinal mucosa), human collagen IV, and bovine serum
albumine (BSA) fraction V were from Sigma (St Louis, MO). Human
recombinant acidic FGF (aFGF) or endothelial cell growth supplement
(ECGS), an extract from bovine hypothalamus containing aFGF,29 were purchased from Collaborative Research Inc
(Bedford, MA). Human recombinant VEGF, anti-bFGF rabbit polyclonal
antibodies, and bFGF enzyme-linked immunosorbent assay (ELISA) kit were
from R&D Systems (Minneapolis, MN). The affinity-purified monoclonal antibodies (MoAbs) directed against CDw49e (5 chain of one of the FN
receptors), CD29 (1 chain of one of the FN receptors), CD51 (v
chain of one of the VN receptors), CD61 (3 chain of one of the VN
receptors; for review, see Hynes27), and the MoAb QBEND/10 directed against CD34, a marker expressed by both KS and
endothelial cells,30 were purchased from Amac Inc
(Westbrook, ME). MoAbs raised against the whole 51 or v3
integrins were purchased from Chemicon (Temecula, CA). MoAbs raised
against v5 were a gift from Dr E. Ruoslahti (La Jolla Cancer
Research Foundation, La Jolla, CA) or were purchased from Telios, Inc
(La Jolla, CA). The MoAb directed against factor VIII-related
antigen31 was from Dakopatt (Carpinteria, CA). The
sequence, synthesis, and purification of antisense bFGF or sense bFGF
phosphorothioate oligodeoxynucleotides (24 mers) directed against the
splice donor-acceptor site 1 of bFGF RNA have been previously
described.24 Cell culture media and media supplements were
purchased from GIBCO-BRL Life Technologies, Inc (Gaithersburg, MD).
Cell cultures.
Different strains of KS cells (AIDS-KS3, KS4, KS6, KS7, and KS8;
passage 6-12) were established and cultured as described previously.32 Three different strains of endothelial cell
derived from human umbilical vein (HUVE cells; passage 4-10) were
activated with combined IC (IC-HUVE cells), as reported
elsewhere.6-10,17
Migration, invasion, and growth assays.
The migration assays were performed in the Boyden chamber, as
previously described.8 Polycarbonate filters (12-µm pore; Nucleoprobe Inc, Cabin John, MD) were coated with type IV collagen or
recombinant Tat protein. The invasion assays were performed as the
migration assays, with the difference that the filters were coated
first with collagen IV and then with matrigel
(Collaborative Research), a reconstituted basement
membrane derived from a tumor cell line,33 to prevent the
migration of noninvasive cells.8 Growth assays were
performed by both the cell counting and the thymidine incorporation
methods, as described previously,2,3,6,7 with cells seeded
onto plates precoated with 1.5% gelatin or with recombinant HIV-1 Tat protein.
In the blocking experiments with antibodies or competitor peptides,
cells were seeded onto culture plates (for the growth assays) or
resuspended by trypsinization (for the migration and invasion assays)
and then preincubated on rotation in RPMI-0.01% BSA containing the
competitor peptides or antibodies at the indicated concentrations for
either 2 hours at 4°C or for 30 minutes at room temperature. Growth,
migration, and invasion assays were then performed as described above.
RNA analysis.
HUVE cells were incubated for 12 to 14 hours with Tat, [65-80] Tat,
bFGF, or their dilution buffer (phosphate-buffered saline [PBS]-0.1%
BSA). Total RNA was then extracted from the cells and subjected to
electrophoresis (10 µg for each lane) and Northern blot analysis. A
32[P]-labeled oligodeoxynucleotide corresponding to the
sequence +59 to +99 of collagenase IV cDNA (encoding the 72-kD form),
which detects a 3.4-kb transcript, was used as a probe, as previously described.15 The amount of RNA loaded in each lane was the
same as detected by ethidium bromide staining of the gels.
Measurement of extracellular soluble bFGF retrieved from cell- or
ECM-associated heparan sulfate proteoglycans (HSPG).
KS cells were incubated for 2 days with conditioned media from
activated T cells or with combined IC, which increase bFGF production
and release,16 or were cultured in the presence of exogenous bFGF. Cells were lifted nonenzymatically with a dissociation buffer (PBS-based chelating solution; GIBCO-BRL), washed with PBS, and
resuspended in RPMI. The plates were also rinsed with PBS. Cells or
plates were then treated for 20 minutes with the control buffer
(PBS-0.1% BSA), Tat, equimolar concentrations of Tat peptides, or
heparin. A limited trypsin digest of cells or plates was used to
retrieve the total bFGF bound to cells or ECM.16 Supernatants were centrifuged and tested for bFGF content by ELISA. To
avoid the loss of bFGF, all samples were handled in plastic ware
precoated with PBS-0.1% BSA.
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RESULTS |
Binding of Tat-RGD region to the 51 and
v3 integrins mediates vascular cell
migration, invasion, and collagenase IV activation induced by Tat.
To elucidate the mechanism(s) of Tat activity, initial studies were
focused on identifying the domains required for the effect of Tat on KS
and endothelial cell migration and invasion. This was performed by
using overlapping Tat peptides. These mapping studies indicated that
only the peptides containing the RGD region, namely [65-80] Tat and
[72-86] Tat, can induce the migration of KS and IC-activated HUVE
cells that was observed at concentrations equimolar to Tat (Fig
1A, left
panel). The migration induced by Tat-RGD region was dose-dependent (Fig
1A, right panel).
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| Fig 1.
Tat-promoted vascular cell locomotion and collagenase IV
activation are mediated by the binding of Tat-RGD region to 51
and v3. (A) shows the results of the migration assays with KS
cells ( ) and IC-HUVE cells ( ). bFGF (20 ng/mL) and Tat (20 ng/mL)
were used as the positive controls, whereas the peptide resuspension
buffer (PBS-0.1% BSA) was the negative control. In the left panel, Tat
peptides were used at concentrations equimolar to Tat. In the right
panel, Tat peptides were serially diluted to test whether their effect
was dose-dependent. The location of the peptides in Tat amino acid
sequence is shown on the top of the figure. (B) shows KS () and
IC-HUVE cell (), migration to Tat (20 ng/mL), [65-80] Tat (4 ng/mL), FN (30 µg/mL), or bFGF (20 ng/mL) after preincubation of the
cells with MoAbs (2 µg/mL each) directed against the and chains of 51 (anti-51), v3 (anti-v3), or
v5 (anti-v5). Immunohistochemical analyses indicated that
KS and HUVE cells express all these integrin chains26 (data
not shown). Antibodies directed against CD34 or factor VIII-related
antigen (antigens that are expressed by KS and IC-HUVE cells,
respectively)30,31 (data not shown) were used as controls
(CR-Ab). Polyclonal antibodies neutralizing the activity of bFGF
(anti-bFGF)22 were used to determine the role of this
cytokine in Tat-induced cell migration. The antibody dilution buffer
(PBS-0.1% BSA) was the negative control. For (A) and (B), results
(from 4 experiments, each in duplicate) refer to the number of migrated
cells/field (average of 5 fields/filter) and are expressed as the
percentage increase of cell migration over the number of cells migrated
toward buffer (0% increase), which was 20 (±2) cells/field for KS
cells and 15 (±1) cells/field for IC-HUVE cells. (C) shows KS () and IC-HUVE () cell
invasion to Tat or bFGF (20 ng/mL/each) after preincubation of the
cells with anti-51 and/or anti-v3 MoAbs (2 µg/mL) or with
buffer (PBS-0.1% BSA), as described in Materials and Methods. The
results shown are from 3 experiments, each in duplicate, and they are
relative to the number of invaded cells per field (average of 5 fields/filter). Data are expressed as the percentage increase of cell
invasion toward Tat or bFGF over the number of invaded cells in the
presence of buffer (0% increase), which was 10 (±2) cells/field for
KS cells and 9 (±1) cells/field for IC-HUVE cells. (D) shows the
Northern blot analysis of collagenase IV 72-kD gene expression in HUVE
cells incubated with Tat (10 ng/mL), [65-80] Tat (2 ng/mL), bFGF (1 µg/mL, positive control), or dilution buffer (PBS-0.1% BSA, negative
control). The amount of RNA loaded in the gels was always monitored by
ethidium bromide staining before Northern blotting. Repeated
experiments (4 times) gave similar or identical results. The Tat
concentration used is the most active in inducing collagenase IV 72-kD
mRNA expression.15 At all the concentrations tested, Tat
and [65-80] Tat were equally potent (data not shown). Preincubation
of HUVE cells with [72-86] Tat reproduced the results obtained with
[65-80] Tat (data not shown).
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Therefore, blocking experiments were performed with antibodies directed
against RGD-binding integrins expressed by KS and IC-HUVE cells, such
as 51, v3, and v5.26 As shown in Fig 1B,
KS and IC-HUVE cell migration to Tat or to the [65-80] Tat RGD
peptide was inhibited by either anti-5 and anti-1 antibodies or
anti-v and anti-3 antibodies but not by anti-v5
antibodies. In addition, inhibition was complete when both
anti-51 and anti-v3 antibodies were added together to the
cells. To the contrary, antibodies directed against other antigens
(CD34 and factor VIII-related antigen) expressed by KS and IC-HUVE
cells, respectively26,30,31 (data not shown), had no
effects on Tat-promoted cell migration.
Additional experiments performed with MoAbs directed against the whole
51 or v3 yielded similar results. Specifically, 70%, 75%,
and 90% inhibition of Tat-induced IC-HUVE cell migration was observed
with anti-51, anti-v3, or combined anti-51 and
anti-v3 antibodies, respectively. Moreover, the anti-51 or
anti-v3 antibodies inhibited FN-induced, but not bFGF-induced, cell migration (Fig 1B), indicating that the antibodies were
specific34 and nontoxic to the cells. Lastly, migration to
Tat was not affected by anti-bFGF antibodies (Fig 1B), indicating that
the chemiotactic effect of Tat is not due to the bFGF that is produced
by the cells.
Experiments were then performed to investigate the role of the
interaction between the Tat-RGD region and integrins in Tat-promoted cellular invasion. As for Tat-induced migration, anti-51 or anti-v3 antibodies inhibited KS and IC-HUVE cell invasion induced by Tat. Complete inhibition was again observed by the simultaneous addition of antibodies directed against both integrins (Fig 1C). The
effect of the antibodies was specific, because they did not inhibit
cellular invasion promoted by bFGF (Fig 1C). Consistent with these
data, the Tat RGD peptide also promoted collagenase IV 72-kD gene
expression at levels comparable with those induced by full-length Tat
(Fig 1D), which is known to activate collagenase expression during cell
invasion.15 Thus, Tat-promoted migration and invasion are
mediated by the binding of the RGD region of the protein to the
51 and v3 integrins. In contrast, v5, which is known
to bind the basic region of Tat,28 is not involved in these effects.
The basic and the RGD domains of Tat are both required for
Tat-induced growth of KS and endothelial cells.
Soluble Tat protein promotes KS and IC-HUVE cell
growth.2,3,6,7,10 To clarify the mechanism(s) of this Tat
effect, cell growth experiments were performed with the same Tat
peptides employed in the previous experiments. As shown in Fig
2A, the peptides containing the Tat basic
region, [46-60] and [48-53] Tat, induced the growth of both KS and
IC-HUVE cells, whereas the RGD-containing peptides, [65-80] and
[72-86] Tat, promoted only KS cell growth. In addition, both
[46-60] Tat and [65-80] Tat promoted KS cell growth in a
dose-dependent fashion (Fig 2A, right panel). Consistent with these
results, when the RGD and the basic Tat peptides were added together,
KS cell growth increased and reached levels similar to those observed
with the Tat protein. Differently from what was observed with KS cells,
the combination of [46-60] Tat and [65-80] Tat did not augment HUVE
cell proliferation induced by [46-60] Tat alone. Moreover, FN, an
RGD-containing molecule,27 promoted KS cell growth at
levels similar to those observed with RGD peptides but had no effect on
endothelial cells.
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| Fig 2.
Both the basic and RGD region mediate Tat-promoted
vascular cell growth. (A) shows the proliferative response of KS ()
and IC-HUVE () cells to Tat peptides, Tat (1 ng/mL), or FN (30 ng/mL). In the left panel, Tat peptides were used at concentrations
equimolar to Tat. In the right panel, Tat peptides were serially
diluted to determine the dose-dependency of their effect. (B) shows
peptide competition experiments of KS (left panel, ) and IC-HUVE
(right panel, ) cell growth. Cells were preincubated with an excess
of Tat peptides (5 µg/mL each) and then stimulated to growth with Tat
(1 ng/mL), as described above. Preincubation of the cells with buffer
was the negative control. For (A) and (B), experiments were performed
by cell counting. Data (from 3 experiments, each in duplicate) are
expressed as the percentage increase of cell growth over the number of
cells grown in the absence of mitogens (basal cell growth). This was 1 × 104 cells/well for KS cells and 1.2 × 104
cells/well for HUVE cells and was given a 0% increase value. Results
were also reproduced by the 3[H]-thymidine uptake
method (data not shown).
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Peptide competition experiments were then performed to clarify these
data. As shown in Fig 2B, Tat-promoted KS and endothelial cell growth
were inhibited only partially when the cells were preincubated with an
excess of [46-60] Tat or [65-80] Tat, and complete inhibition of KS
and HUVE cell growth was observed only when cells were preincubated
with an excess of the two peptides added together to the cells. Thus,
both the RGD and the basic region of Tat appear to participate in
Tat-promoted cell proliferation, suggesting that more than one pathway
is involved in this process. Therefore, the role of these two regions
of Tat on cell growth was further analyzed.
Tat-RGD region provides KS and endothelial cells with the adhesion
signal required for cell proliferation in response to mitogens.
To investigate the role of the RGD region and of the Tat-integrin
interaction in cellular growth, proliferative assays were performed
with Tat in the presence of integrin competitors, such as RGD
peptides,27 or with anti-51, anti-v3, or
anti-v5 antibodies. RGD peptides, but not the KGE-mutated
peptides that were used as controls, and anti-51 or anti-v3
antibodies, but not anti-v5 or control antibodies, blocked
Tat-induced KS and IC-HUVE cell growth (Fig
3, upper left panel). These results were
obtained at concentrations of antibodies or peptides that did not cause
cell detachment or affect basal cell growth (Fig 3, upper right panel).
Similarly, anti-51 or anti-v3 antibodies inhibited KS cell
growth induced by TNF-, a mitogen for these cells,6 and
HUVE cell proliferation induced by ECGS (Fig 3, lower left panel).
Thus, as with other growth factors, Tat-induced cell growth requires
integrin engagement.
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| Fig 3.
Tat RGD region provides vascular cells with the adhesion
signal required by the cells to respond to mitogens. KS () and
IC-HUVE () cells were preincubated with RGD or mutated KGE peptides
(1 µg/each) or with anti-51 and/or anti-v3 antibodies (4 µg/mL) and were then stimulated with Tat (1 ng/mL; upper left panel).
No effects of the competitor peptides or antibodies were observed on
cells grown in the absence of stimuli (upper right panel). Anti-CD34 or
anti-v5 MoAbs (4 µg/mL) were used as control antibodies (CR-Ab)
for KS cells. Anti-factor VIII-related antigen MoAbs (4 µg/mL) were
used as control antibodies (CR-Ab) for HUVE cells. TNF- (10 ng/mL)
or ECGS (45 µg/mL) were used as control mitogens (CR mitogen) for KS
and IC-HUVE cells, respectively (lower left panel). The preincubation
of the cells with antibody or peptide dilution buffer was the negative
control. In the upper panels and in the lower left panel, KS and
IC-HUVE cells were seeded onto gelatin-coated plates. In the lower
right panel, IC-HUVE cells were seeded onto plates coated with Tat (5 µg/mL), and then they were stimulated to grow with ECGS (45 µg/mL)
in the presence or absence of anti-51, -v3, or -v5
antibodies (4 µg/mL). Results (from 3 experiments, each in duplicate)
are expressed as the percentage increase of cell growth relative to the
growth of cells preincubated with buffer and stimulated with Tat,
TNF-, or ECGS (assumed as 100% of cell growth increase).
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This suggested that, by binding to 51 and v3, Tat may
provide endothelial cells with the same adhesion signal that is normally provided by ECM molecules and is required by the cells to
proliferate in response to mitogens.35,36 In fact,
anti-51 and anti-v3, but not anti-v5, antibodies
inhibited the proliferation of HUVE cells seeded on Tat-coated plates
and stimulated to grow with ECGS (Fig 3, lower right panel). Thus, the
interaction of Tat-RGD region with 51 and v3 provides the
adhesion signal required for cell growth in response to mitogens.
Tat basic region retrieves preformed HSPG-bound bFGF that
specifically triggers Tat-induced cell growth.
Further studies focused to elucidate the mechanism(s) by which Tat
basic region induces the growth of KS and endothelial cells. Basic
residues similar to those in Tat are also present in many growth
factors, and they bind HSPG associated with the cell membrane and the
ECM.37,38 For this reason, basic sequences can displace extracellular-bound angiogenic factors through a competitive effect for
heparin-binding sites.38,39 After their release from the cells, both Tat and bFGF bind to cell surface- and ECM-associated HSPG
through their basic region, which has a strong binding affinity for
heparin.4,37,40 The finding that the binding of Tat to heparin is competed out by bFGF4 suggested that Tat and
bFGF could compete for the same heparin-binding sites.
To determine this, KS cells were grown with IC that further increase
production and release of bFGF16 and then treated with scalar concentrations (from 0.1 ng/mL to 10 µg/mL) of Tat or Tat basic peptide. A gentle trypsin digest or heparin was used as a
positive control, because they are known to release HSPG-bound bFGF
into a soluble form.16,37 Soluble bFGF was then measured by
ELISA. As compared with trypsin treatment, angiogenic concentrations of
Tat or equimolar concentrations of [46-60] Tat basic peptide released
about 26% of cell-bound bFGF and 41% of ECM-bound bFGF produced by KS
cells (Fig
4A). These
levels of bFGF were similar to those retrieved by heparin (used to
evaluate the total retrievable bFGF).16,37 In contrast,
[56-70] Tat peptide (used as a control) was not capable of releasing
bFGF. The increase of soluble bFGF was detected 20 minutes after the
addition of Tat or heparin to the cells, and the levels remained
elevated for about 24 hours and returned to baseline after 48 hours
(data not shown). As observed for heparin, Tat was also able to
retrieve and maintain into a soluble form exogenous bFGF added to the
cells (Fig 4B). Because the addition of Tat did not increase bFGF mRNA
or intracellular bFGF content, as determined by Northern blot analysis
and ELISA, respectively (data not shown), the increase of extracellular
soluble bFGF by Tat is caused by the release of HSPG-bound
extracellular protein. Thus, the growth effect of Tat basic region
could be due to its capability of retrieving HSPG-bound bFGF produced
by KS and IC-activated endothelial cells. In fact, antisense oligomers directed against bFGF mRNA, previously shown to specifically inhibit bFGF expression24 but not control sense bFGF oligomers,
blocked Tat-promoted growth of KS cells that constitutively produce
bFGF (Fig 4C). This was associated with a reduction of intracellular- and extracellular-bound bFGF content as determined by ELISA after normalization to total protein content. In fact, intracellular bFGF
content was reduced upon antisense treatment from 9,606 µg/100 µg
of total protein to 3,840 µg/100 µg. Similarly, extracellular bound
bFGF was reduced from 1,162 to 770 pg/mL.
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| Fig 4.
Tat basic region retrieves sequestered bFGF into a
soluble form that mediates Tat-promoted vascular cell growth. (A) shows
the mean of bFGF levels (in picograms per milliliter) retrieved from
cells (left panel) or ECM (right panel). KS cells were lifted with a
cell dissociation buffer, and both the suspended cells or the plates
containing the ECM produced by the cells were incubated with Tat (25 ng/mL), [46-60] Tat (4 ng/mL), [56-70] Tat (4 ng/mL), heparin (30 µg/mL), or dilution buffer. A limited trypsin digest of suspended
cells or plates was used to retrieve the total bFGF bound to cells or
ECM.16 bFGF was then measured in the supernatants by ELISA.
(B) shows the mean values of bFGF (in picograms per milliliter)
maintained in a soluble form by Tat or Tat basic peptide. KS cells were
cultured in the presence of exogenous bFGF (1 ng/mL) and then incubated
for 20 minutes in control buffer (PBS-0.1% BSA), Tat (1 or 25 ng/mL),
[46-60] Tat (0.2 or 4 ng/mL), heparin (30 µg/mL), or Tat (25 ng/mL)
and heparin (30 µg/mL) combined sequentially. (C) The left panel shows proliferative assays
with KS cells cultured for 48 hours with 0.5 µmol/L antisense bFGF
(ASbFGF) or sense bFGF (SbFGF) oligomers.24 Tat (1 ng/mL)
and 3[H]-thymidine were then added to the cells, and
growth was monitored after 48 hours. Results (from 3 experiments, in 5 replicates) are expressed as the percentage increase of
3[H]-thymidine uptake after the addition of Tat as
compared with basal cell growth (0% increase, which was 1,874 ± 20
cpm). In the middle and right panels, anti-bFGF antibodies (10 µg/mL)
were added to KS cells before the addition of Tat (1 ng/mL) or
[48-53] Tat (0.1 ng/mL). The antibody buffer (PBS-0.1% BSA) was the
negative control. Results (from 4 experiments, each in duplicate) were
obtained by the cell counting method and expressed as the percentage
increase of cell growth over the number of KS cells grown in the
absence of mitogens (basal cell growth), which was 1 ± 104 cells/well. (D) shows proliferative assays with IC-HUVE
cells induced to proliferate with 1 ng/mL of Tat or 20 ng/mL of aFGF,
in the presence or absence of anti-bFGF antibodies (10 µg/mL). (E)
shows HUVE cell growth experiments with bFGF or VEGF (5 ng/mL each) in
the presence or absence of 1 ng/mL of Tat. For (D) and (E), results
from 4 experiments performed by the cell counting method refer to the
number of cells collected 4 to 5 days after the addition of bFGF, aFGF,
VEGF, or Tat. They are expressed as a percentage increase of cell
growth over the number of cells grown in the absence of mitogens (basal
cell growth).
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Consistent with this result, neutralizing antibodies directed against
bFGF22 blocked KS cell growth induced by Tat or by the Tat
basic peptide (Fig 4C). Anti-bFGF antibodies also blocked Tat-induced
growth of IC-HUVE cells that produce bFGF (Fig 4D). In contrast,
anti-bFGF antibodies did not block endothelial cell growth induced by
aFGF, which was used to control the antibody specificity (Fig 4D). In
agreement with its capability of maintaining exogenously added bFGF in
a soluble form, Tat enhanced bFGF-promoted proliferation of
nonactivated endothelial cells that do not produce bFGF (Fig 4E). In
contrast, Tat slightly inhibited the mitogenic effect of VEGF on
endothelial cells (Fig 4E). These results indicated that bFGF
specifically triggers Tat-induced vascular cell growth.
| |
DISCUSSION |
In this study, we have analyzed the mechanisms responsible for the
angiogenic, KS-promoting effect of Tat. The results indicate that KS
and endothelial cell migration and invasion induced by Tat are mediated
by the binding of its RGD region to the 51 and v3 integrins
(Fig 1). The RGD sequence of Tat is also sufficient to activate the
expression of collagenase IV 72-kD, which plays a key role in cell
invasion and angiogenesis.15,41,42 These data are
consistent with results obtained by others with RGD-containing molecules, such as FN or VN,34,41-43 and demonstrate that
Tat induces KS and endothelial cell locomotion by a molecular mimicry of ECM molecules. They are also in agreement with the fact that Tat
induces the synthesis and release of collagenase IV 92 kD in
monocytes44 and with the recent finding that Tat activates members of the focal adhesion kinase family that are induced by integrin triggering and that play a major role in cellular
migration.45,46
Differently from Tat-induced cell locomotion, the basic and RGD region
of Tat are both required for Tat-induced cell growth (Fig 2). It
appears that the interaction of Tat with 51 or v3 provides
KS and endothelial cells with the adhesion signal that is required for
their growth in response to mitogens. Consistent with this, endothelial
cells adhere to immobilized Tat in a fashion similar to FN or
VN,26 and under these conditions, the addition of exogenous
bFGF dramatically increases cell growth,15 as previously described for ECM molecules.35,36,47 The involvement of the RGD region in Tat-induced cell growth is also consistent with previous
data indicating that 51, which recognizes the RGD region of both
FN and Tat, mediates cellular growth promoted by FN in other
systems,48,49 and that the RGD region of Tat promotes in KS
cells the expression of the same mitogen-activated protein kinases that
are induced by integrin triggering.45
However, the adhesion signal is not sufficient to induce growth of
normal endothelial cells that also need the presence of an angiogenic
factor that triggers cell proliferation.50 However, Tat RGD
peptide or FN are mitogenic for KS cells (Fig 2). Because this does not
occur with normal cells (for review, see Levesque et
al51), these data suggest that KS cells may possess a
difference in intracellular signaling through integrins, as indicated
by previous studies with tumor cells.52
Differently from the Tat RGD peptide, full-length Tat protein or Tat
basic peptide are capable of promoting the growth of both KS and normal
endothelial cells, suggesting that Tat provides cells additionally with
a cell growth triggering signal.
Previous studies suggested that bFGF, which is a KS and endothelial
cell growth factor,20,22,24 may mediate Tat-induced vascular cell proliferation. Basic FGF, in fact, is present in all
systems in which Tat has a growth effect. Specifically, exogenous bFGF
is required to observe the angiogenic effect of Tat in
vivo.15 Moreover, bFGF is produced and released
extracellularly by KS cells that spontaneously proliferate in response
to Tat2,3,16,22 and by IC-activated endothelial cells that,
after this activation, proliferate with Tat.6,7,10,17 After
its release, bFGF binds the cell surface and the ECM-associated HSPG
remaining soluble only in a small fraction.16,37 The bound
bFGF fraction represents a localized storage of the growth factor that
is protected from proteolytic degradation and can be retrieved by
treatment of the cells with heparin, heparinase, or
trypsin.16,37
Several growth factors can bind HSPG through their basic residues (for
review, see Raines and Ross38). In contrast, other angiogenic molecules, such as the majority of VEGF isoforms, lack basic
residues and, therefore, do not bind the HSPG associated to the surface
of producer cells or to ECM, remaining soluble and
diffusible.21 Similarly to bFGF, Tat binds heparin through its basic sequence and can compete with bFGF for binding to
heparin.4 Heparin, in fact, can inhibit the mitogenic
effect of Tat (data not shown), as previously found for cellular
adhesion to immobilized Tat28 and transactivation of HIV-1
gene expression by extracellular Tat.4 Our data indicate
that the basic residues of Tat can displace preformed HSPG-bound bFGF
by competing for cell surface- and ECM-associated heparin binding sites
(Fig 4). This leads to an increase of the soluble fraction of bFGF at
levels promoting KS and endothelial cell growth, as shown by the
inhibition of Tat-induced cell growth by antisense bFGF oligomers
(reducing both the intracellular and extracellular content of bFGF) or
by neutralizing anti-bFGF antibodies (Fig 4). Consistent with its capability of maintaining exogenously added bFGF in a soluble and
highly diffusible form, Tat enhances endothelial cell proliferation promoted by bFGF. In contrast, Tat does not augment the growth effect
of VEGF on endothelial cells (Fig 4). Thus, although Tat binds the VEGF
receptor Flk-1/KDR,19,45 this does not lead to cell growth.
This is consistent with the finding that, differently from Tat or bFGF,
VEGF does not promote KS cell proliferation.18 Moreover,
Tat promotes the growth of IC-activated endothelial cells that produce
bFGF but not VEGF.18 Furthermore, in primary KS lesions,
the VEGF amounts are much higher than that of Tat,15,25 making unlikely an action (either activatory or inhibitory) of Tat on
the VEGF receptor. Thus, although both VEGF and bFGF are highly
expressed in AIDS-KS lesions, Tat synergizes with bFGF, and not with
VEGF, in promoting neoangiogenesis and, therefore, KS development and progression.
In conclusion, the results described herein demonstrate that the Tat
protein of HIV-1 is not directly angiogenic, but it enhances angiogenesis by mimicking the effects of ECM proteins on cell migration, invasion, and adhesion, and by mobilizing bFGF, a true angiogenic factor, which acts as the final mediator of Tat-induced KS
and endothelial cell growth. This explains why Tat needs exogenous bFGF
or factors promoting bFGF expression, such as IC, to exert its
angiogenic effect.
IC and bFGF are highly expressed in AIDS-KS lesions, where
extracellular Tat costains with the 51 and v3 integrins on both spindle cells and vessels.15 This suggests that the
mechanisms of Tat action described here are operative in vivo and that
integrin, bFGF, and Tat competitors should be considered as a
therapeutic strategy for AIDS-KS.
| |
ACKNOWLEDGMENT |
The authors thank Dr E. Ruoslahti for some of the anti-v5
antibodies and A. Lippa and F.M. Regini for editorial assistance.
| |
FOOTNOTES |
Submitted January 13, 1999; accepted March 24, 1999.
Supported by grants from the Associazione Italiana per la Ricerca sul
Cancro (AIRC), the Italian Ministry of Health (IX AIDS Project), the
Deutsche Forschungsgemeinschaft (DFG, SFB464), and the
Bundesministerium für Bildung und Forschung (BMBF, BioFuture Program).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Barbara Ensoli, MD, PhD, Director of
Research, Laboratory of Virology, Istituto Superiore di Sanità,
Viale Regina Elena 299, 00161 Rome, Italy; e-mail:
ensoli{at}virus1.net.iss.it.
| |
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[Abstract]
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D. Boettiger, L. Lynch, S. Blystone, and F. Huber
Distinct Ligand-binding Modes for Integrin alpha vbeta 3-Mediated Adhesion to Fibronectin versus Vitronectin
J. Biol. Chem.,
August 17, 2001;
276(34):
31684 - 31690.
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