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Journal of Virology, January 2000, p. 344-353, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Specific Molecular Structures of
Human Immunodeficiency Virus Type 1 Tat Relevant for Its Biological
Effects on Vascular Endothelial Cells
Stefania
Mitola,1,2
Raffaella
Soldi,1,2
Ilaria
Zanon,1,2
Luca
Barra,1,2
Maria Ines
Gutierrez,3
Ben
Berkhout,4
Mauro
Giacca,3 and
Federico
Bussolino1,2,*
Institute for Cancer Research and Treatment
(I.R.C.C.), 10060 Candiolo,1 Department
of Genetics, Biology and Biochemistry, School of Medicine, University
of Torino, 10100 Turin,2 and Molecular
Medicine Laboratory, International Centre for Genetic Engineering and
Biotechnology, 34102 Trieste,3 Italy, and
Department of Human Retrovirology, University of Amsterdam,
Academic Medical Center, 1100 DE Amsterdam, The
Netherlands4
Received 28 May 1999/Accepted 17 September 1999
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ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) Tat transactivates
viral genes and is released by infected cells, acting as a soluble
mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type
2 (VEGFR-2) and integrins. A structure-activity relationship study was
performed by functional analysis of Tat substitution and deletion
variants to define the Tat determinants necessary for EC activation.
Variants were made (i) in the basic and (ii) in the cysteine-rich
domains and (iii) in the C-terminal region containing the RGD sequence
required for integrin recognition. Our results led to the following
conclusions. (i) Besides a high-affinity binding site corresponding to
VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the
cysteine-rich variants bind only to the low-affinity binding sites and
do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they
have a reduced ability to activate EC in vitro, and they lack
angiogenic activity. (iii) Mutants with mutations in the C-terminal
region are partially defective for in vitro biological activities and
in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In
conclusion, regions encoded by the first exon of tat are
necessary and sufficient for activation of VEGFR-2. However, the
C-terminal region, most probably through RGD-mediated integrin
engagement, is indispensable for full activation of an in vitro and in
vivo angiogenic program.
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INTRODUCTION |
Tat is one of the regulatory
proteins of human immunodeficiency virus type 1 (HIV-1). The protein is
composed of 86 to 104 amino acids (aa) (according to the viral isolate)
encoded by two exons. In the portion encoded by the first exon (72 amino acids) four distinct regions can be recognized (N-terminal,
cysteine rich, core, and basic). The second exon encodes the C-terminal region, containing a RGD sequence (31). Tat plays an
essential role in viral replication by up-regulating viral gene
expression in infected cells by increasing the rates of transcriptional
initiation and elongation by DNA polymerase II (31). Tat
also modulates the expression of cellular genes involved in cell
survival and proliferation or in coding for cytokines (11, 12, 23,
29, 37, 41, 54, 63, 66, 69). This newly acquired cell phenotype
will contribute to the pathogenesis of specific diseases associate with
HIV infection. Besides its intracellular effects, Tat may alter
cellular behavior when it is released by infected cells in the
microenvironment (10, 15). Tat easily enters different cell
types contributing to the transactivation of the HIV-1 long terminal
repeat (LTR) promoter in latently infected cells (20, 39).
Alternatively, it acts as soluble mediator affecting the physiologic
functions of cells of the immune (30, 35, 43, 49, 66) and
nervous (33, 42, 48) systems. Moreover, it influences the
apoptotic program in T cells (26, 36, 41, 65, 70) and in
neurons (47, 59), thus favoring the progression of AIDS and
the associated brain damage.
However, one of the most relevant targets for Tat is the vascular
system, where it activates a proinflammatory and angiogenic program.
Tat can up-regulate the expression of endothelial cell (EC) adhesion
molecules (13, 28), resulting in leukocyte extravasion, which is essential for the homing of infected lymphomononuclear cells
into lymphoid organs and for the tissue injury characteristic of some
features of disease progression. Alone or combined with inflammatory
cytokines, Tat induces EC to proliferate, release proteolytic enzymes,
and migrate and is fully angiogenic in vivo (1, 3-5, 18).
These features could be relevant to the chronic inflammatory damages
characteristic of several AIDS-associated diseases (17).
Furthermore, the ability of Tat to enter EC during the cell cycle could
favor HIV-1 replication in some EC areas which are virus reservoirs
(44, 45). Finally, Tat participates in the progression of
Kaposi's sarcoma, both as a growth factor for spindle cells which
represent the core of the tumor and as a means of sustaining its
vascularization (14, 16). Furthermore, Tat transgenic mice
generated by using either the HIV LTR (63) or the BK
polyomavirus promoter (11) develop Kaposi's sarcoma-like lesions and tumors of different histotypes, supporting the pathogenetic role of Tat in Kaposi's sarcoma and in the vascularization of neoplasms associated with AIDS.
The molecular mechanisms leading to these broad and pleiotropic
activities are largely unknown. By the use of peptides spanning specific domain of the Tat structure or of functional blocking antibodies, some investigators demonstrated that Tat binding to integrin through the RGD sequence near the C terminus (7,
62) is relevant for the activation of lymphocytes
(70), EC (5, 13), monocytes (6, 35),
and neuronal cells (48). Through its N-terminal structure,
Tat interacts with dipeptidyl peptidase IV, located on the T cell
surface, and suppresses antigen-induced cell activation (26,
68). Additionally, Tat binds to and activates the tyrosine kinase
receptors encoded by the KDR and Flt-1 genes in
EC and Kaposi's sarcoma cells (4, 21) and monocytes
(43), respectively.
We initiated a study of the structure-activity relationship of Tat to
identify functionally important domains that are responsible for the
activation of the angiogenic program in vascular EC. We report here
that cysteine-rich and basic domains are relevant for functional
activation of VEGFR-2 whereas the C-terminal region does not directly
participate in the receptor activation but is required for full cell activation.
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MATERIALS AND METHODS |
Cells.
Human EC from umbilical cord veins, prepared and
characterized as previously described (9), were grown in
M199 (Gibco, Grand Island, N.Y.) supplemented with 20% fetal calf
serum (FCS) (Irvine, Santa Ana, Calif.), EC growth factor (100 µg/ml)
(Sigma Chemical Co., St. Louis, Mo.), and porcine heparin (100 µg/ml) (Sigma). They were used at passage II and grown on a plastic surface coated with porcine gelatin (Sigma), unless specified.
Tat molecules.
Recombinant wild-type HIV-1 Tat of 86 (Tat86) and 101 (Tat101) amino acids were
expressed in Escherichia coli as maltose-binding protein
(MBP) or glutathione S-transferase (GST) fusion proteins. They are referred to below as Tat (MBP-Tat) and *Tat (GST-Tat), respectively. Mutant constructs were obtained by a recombinant PCR
procedure with overlapping oligonucleotides corresponding to the
mutated sequences, and the specific mutations were verified by DNA
sequencing. To obtain Tat mutants, a PstI-BamHI
cDNA fragment of Tat86 containing the coding region of both
exons was subcloned in the pALTER-Ex1 vector (Promega, Madison, Wis.).
Site-directed mutagenesis was carried out with the Altered Sites
mutagenesis kit (Promega) by using mutant oligonucleotides to introduce
specific mutations (Tat D80E, 5' CCC GAG GGG AAC CGA CAG GCC 3'; Tat
R78K/D80E, 5' CCC GAG GGG AAC CGA CAG CC 3' and 5' ACC TCC CAA TCC AAA
GGG GAA CCG AC 3'; Tat R49G/K50I, 5' CTC CTA TGG CGG GAT CAA GCG GAG AC
3'; Tat R49G/K50I/R52L/R53I, 5' CTC CTA TGG CGG GAT CAA GCG GAG AC and
5' CGG GAT CAA GCT AAT ACA GCG ACG AAG 3'). The mutated cDNAs (317-bp
EcoRI-BamHI fragments) were subcloned into the
pMAL-c2 vector (New England Biolabs, Beverly, Mass.) and expressed as specified by the manufacturer. Tat86 and its mutants were
purified to homogeneity from bacterial cell lysates by affinity
chromatography on amylose resin, as specified by New England Biolabs,
and used as fusion proteins.
*Tat86 and its mutated derivatives including the product of
the first exon, *Tat72, a nonconservative mutant with
mutations in basic [*Tat R(49,52,53,55,56,57)A] and cysteine-rich
[*Tat C(22,25,27)A] regions, were produced as previously described
(40), as well as *Tat101 (61).
Recombinant fusion proteins were purified by glutathione-Sepharose
affinity chromatography (Sigma) (53). The purified MBP and
GST fusion proteins gave a unique band after sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%
polyacrylamide) and silver staining. Tat86,
*Tat86, and *Tat101 and their mutants were
lipopolysaccharide free, as assessed by the Limulus assay
(Sigma). Tat and *Tat were able to induce transcriptional activation of
the HIV-1 LTR in HL3T1 cells that contain the bacterial gene of
chloramphenicol acetyltransferase (CAT) directed by the HIV-1 LTR
(67). Tat86 and *Tat101 dose
dependently stimulated CAT expression. Briefly, Tat molecules were
added to confluent HL3T1 cells in a 100-mm-diameter dish containing
prewarmed phosphate-buffered saline (PBS). Cells were immediately
scraped from the plastic surface, resuspended in fresh medium, and
centrifuged. The cells were again plated in a CO2
incubator, and a CAT assay was performed after 6 h as described
previously (67). Under our experimental conditions, just
scraping of the cells in absence of Tat molecules had no effect on
LTR-directed CAT gene expression (125 ± 56 cpm of
[3H]acetylated chloramphenicol [n = 3]). However, CAT gene expression was markedly elevated in cells
that received Tat86 at 0.5 µg/ml (423 ± 86 cpm of
[3H]acetylated chloramphenicol), 1 µg/ml (2,345 ± 167 cpm of [3H]acetylated chloramphenicol), or 5 µg/ml
(6,543 ± 567 cpm of [3H]acetylated chloramphenicol)
or *Tat86 at 0.5 µg/ml (512 ± 34 cpm of
[3H]acetylated chloramphenicol), 1 µg/ml (3,145 ± 343 cpm of [3H]acetylated chloramphenicol), or 5 µg/ml
(7,009 ± 671 cpm of [3H]acetylated chloramphenicol).
Tat molecules were stored at
80°C in aliquots of 5 µg/10 µl of
PBS containing 0.1% human serum albumin (HSA; Farma Biagini,
Lucca,
Italy) and 1 mM dithiothreitol
(Sigma).
Iodination of Tat molecules and binding studies.
MBP, GST,
Tat86, *Tat86, Tat R78K/D80E, Tat
R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2-µg samples) were
dissolved in 200 µl of 20 mM sodium phosphate buffer (pH 7.4) without
dithiothreitol and transferred in iodogen-coated tubes (50 µg/ml)
(Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins
were iodinated (5 min at 4°C) with 0.2 mCi of 125I
(Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). A
20-µl volume of 20 mM phosphate buffer (pH 7.2) containing 1% HSA,
0.4 M NaCl, and 0.1%
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS;
Pierce) was added, and the reaction products were separated on
Sephadex-G10. The specific activities of the tracers were as follows:
MBP, 1 µCi/118 fmol; GST, 1 µCi/134 fmol; Tat86, 1 µCi/108 fmol; *Tat86, 1 µCi/100 fmol; Tat R78K/D80E, 1 µCi/128 fmol; Tat R49G/K50I/R52L/R53I, 1 µCi/112 fmol; and *Tat
C(22,25,27)A, 1 µCi/103 fmol. [125I]Tat86
and [125I]*Tat86 retained their biological
activity on Kaposi's sarcoma cells (8).
For binding studies, adherent EC on 24-well plates were incubated for
90 min at 22°C in 200 µl of M199 containing 20 mM HEPES
(pH 7.4),
0.1% HSA, 0.2 U of heparin, 100 µg of soybean trypsin
inhibitor per
ml, bacitracin (binding buffer), and increasing
concentrations of
iodinated Tat molecules or control proteins
in the presence of 100-fold
excess of unlabeled proteins. After
washes in buffer binding, the cells
were extracted with 2% SDS
in PBS. Specific binding (calculated by
subtracting from the total
the cpm bound after incubation with a
100-excess of unlabelled
ligand) was approximately 80%. Curve
displacement binding was
obtained by incubating cells with 0.05 nM
[
125I]Tat and processed as described above. Kinetic
parameters were
estimated with the Ligand program (Elsevier-Biosoft,
Cambridge,
United
Kingdom).
Immunoprecipitation and immunoblotting.
Confluent EC
(107 cells/150-cm2 dish) were made quiescent by
20 h of starvation in M199 containing 0.5% FCS and 0.1% HSA,
preincubated for 15 min at 37°C with 1 mM
Na3VO4, and then stimulated as detailed in
Results in the presence of heparin (1 U/ml). The cells were lysed in a
50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1% Triton
X-100, and protease and phosphatase inhibitors (pepstatin, 50 µg/ml;
leupeptin, 50 µg/ml; aprotinin, 10 µg/ml; phenylmethylsulfonyl fluoride, 1 mM; soybean trypsin inhibitor, 500 µg/ml;
ZnCl2, 100 µM, Na3VO4, 1 mM
[Sigma]). After centrifugation (20 min at 10,000 × g), supernatants were precleared by incubation for 1 h with protein A-Sepharose or with anti-mouse immunoglobulin-agarose (Sigma).
Samples (1 mg of protein) were incubated with rabbit anti-VEGFR-2
polyclonal antibody (no. C-1158; Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif.) or antiphosphotyrosine monoclonal antibody (MAb) (clone
G410; Upstate Biotechnology Inc., Lake Placid, N.Y.) (5 to 10 µg/ml)
for 1 h at 4°C, and immune complexes were recovered on protein
A-Sepharose or anti-mouse Ig-agarose. Immunoprecipitates were washed
four times with lysis buffer, twice with the same buffer without Triton
X-100, and once with Tris-buffered saline. Proteins were solubilized
under reducing conditions, separated by SDS-PAGE (8 or 10%
polyacrylamide), transferred to Immobilon-P sheets (Millipore, Bedford,
Mass.), and probed with antiphosphotyrosine MAb or with anti-VEGFR-2
antibody. The enhanced chemiluminescence technique (Amersham Pharmacia
Biotech) was used for detection.
PI 3-kinase assay.
A phosphoinositide 3-kinase (PI 3-kinase)
assay was performed directly on antiphosphotyrosine immunoprecipitates
exactly as described previously (58). Briefly,
immunoprecipitates were incubated with 40 µM ATP, 50 to 100 µCi of
[
-32P]ATP (Amersham), and a presonicated mixture of
phosphatidylinositol-4,5-bisphosphate and phosphatidylserine (final
concentration of both lipids, 50 µg/ml [Sigma]) in 25 mM HEPES (pH
7.4)-and 1 mM EGTA. The reaction was stopped after a 10-min incubation
at room temperature by the addition of 1 volume of 1 M HCl and 2 volumes of chloroform-methanol (1:1). The lipids in the organic phase
were separated by thin-layer chromatography (Silica Gel 60; Merck,
Darmstadt, Germany) in 1-propanol-2 M acetic acid (65:35, vol/vol) and
visualized by autoradiography.
Migration, proliferation, and adhesion assays.
EC motility
was studied by using a modified Boyden chamber technique exactly as
previously described (9).
To evaluate EC proliferation, 2 × 10
3 cells were
plated in M199 containing 20% FCS in 96-well plates (Falcon, Becton
Dickinson
Labware, Bedford, Mass.) coated with gelatin. After 24 h, the
medium was removed and replaced with M199 containing 2.5% FCS.
Tat molecules were added on days 0, 2, and 4, and the cell number
was
estimated on day 6 as previously described (
9).
To study EC adhesion to immobilized Tat molecules, 96-well polysterene
plates (Falcon) were coated overnight at 4°C with Tat
molecules or
control proteins (10 µg/well), washed, and then incubated
for an
additional 2 h at room temperature with 1% HSA in PBS.
Cells were
detached in cold PBS containing 2 mM EGTA, washed twice
in M199
containing 1% FCS, and plated (5 × 10
4/0.1 ml) in
the adhesion assay. After a 1-h incubation at 37°C,
the plates were
extensively washed in M199 containing 1% FCS,
fixed, and stained with
crystal violet (
9). The absorbance
was read at 540 nm in a
microtiter plate spectrophotometer (EL340;
Bio-Tek Instruments,
Highland Park, Vt.).
In vivo angiogenesis.
Matrigel (Becton Dickinson)
supplemented with 10 U of heparin per ml was mixed with Tat molecules
or control proteins and injected (0.7 ml) into the subcutaneous tissue
of BALB/c male mice (Charles River, Conago, Italy) along the peritoneal
midline. After 5 days, the mice were killed and gels were processed for histology, morphometric analysis, and hemoglobin content determination measured with a Drabkin reagent kit (Sigma) as previously described (8).
Statistical methods.
One-way analysis of variance (ANOVA)
and the Student-Neuman-Keuls test were used to test the difference
within the experimental blocks of each biological assay. Statistical
analyses were performed with STATISTICA for Windows, version 4.5 (StatSoft, Tulsa, Okla.).
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RESULTS |
Effect of Tat and Tat mutants on biological functions of EC.
It has been reported that soluble Tat induces the activation of
vascular endothelium and of Kaposi's sarcoma cells through the
engagement of VEGFR-2 (4, 21) and the integrin system (5, 13, 16). Furthermore, Tat mimics the extracellular matrix protein and favors cell adhesion through the amino acid sequence
RGD (7) or the basic domain (62, 64). To examine the Tat regions involved in EC activation, a set of Tat mutants was
constructed (Fig. 1), expressed as fusion
proteins in E. coli, and studied for their ability to induce
cell migration, proliferation, and adhesion.
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FIG. 1.
Domain structure of the HIV-1 Tat protein and mutations
introduced. The arbitrary domain structure is that of Kuppuswamy et al.
(34). The number of mutated amino acids is indicated.
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First we studied the effect of mutations in the basic region. Tat
R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A
had
a reduced effect in terms of migration in the Boyden chamber
(Fig.
2) and proliferation (Fig.
3) of human EC compared to wild-type
molecules (Tat
86 or *Tat
101) (
P < 0.005), with Tat R49G/K50I being
more active than Tat
R49G/K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A
(
P < 0.005). Finally, the adhesion of EC to Tat
R49G/K50I/R52L/R53I
and *Tat R(49,52,53,55,56,57)A was markedly reduced
with respect
to the adhesion to Tat
86 or Tat R49G/K50I
(
P < 0.005) (Fig.
4).
These data suggest that the basic domain is relevant for all the
EC
biological activities considered.
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FIG. 2.
Effect of Tat molecules on the migration of EC. The
migration of EC across a 5-µm-pore-size polycarbonate filter in
response to Tat molecules (20 ng/ml) or vehicle was evaluated by using
a Boyden chamber. At the end of the incubation (37°C for 6 h),
the filters were removed and stained and five high-power oil immersion
fields were counted (400× magnification). Results (mean and standard
deviation) of one experiment (performed in triplicate) representative
of at least three independent experiments are shown. Data were analyzed
by ANOVA (F = 41.11) and the Student-Newman-Keuls test.
* indicates P < 0.05 within Tat-stimulated EC and
unstimulated or control protein-stimulated cells; § indicates
P < 0.005 within wild-type Tat molecule-stimulated EC
and Tat mutant-stimulated cells.
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FIG. 3.
Effect of Tat molecules on EC proliferation. A total of
2 × 103 EC were plated in 96-well plates and grown
for 12 h in M199 containing 20% FCS. After 24 h, the medium
was removed and replaced with M199 containing 2.5% FCS. Tat molecules
(20 ng/ml) were added on days 0, 2, and 4, and the cell number was
estimated on day 6. Cells were fixed and stained with crystal violet,
and the absorbance was read at 540 nm. Results (mean and standard
deviation) of one experiment (performed in quadruplicate)
representative of at least four independent experiments are shown. Data
were analyzed by ANOVA (F = 86.31) and the
Student-Newman-Keuls test. * indicates P < 0.05
within Tat molecule-stimulated EC and unstimulated or control
protein-stimulated cells; § indicates P < 0.005
within wild-type Tat molecule-stimulated EC and Tat mutant-stimulated
cells.
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FIG. 4.
Effect of immobilized Tat molecules on adhesion of
endothelial cells. A plastic surface was coated overnight at 4°C with
Tat molecules or control proteins (10 µg/well) and then saturated
with 1% HSA. Suspended cells (5 × 104/0.1 ml) were
seeded, and after a 1-h incubation at 37°C, plates were extensively
washed in M199 containing 1% FCS, fixed, and stained with crystal
violet. The optical density (O.D.) was read at 540 nm in a microtiter
plate spectrophotometer. Results (mean and standard deviation) of one
experiment (performed in quadruplicate) representative of at least four
independent experiments are shown. Data were analyzed by ANOVA
(F = 71.16) and the Student-Newman-Keuls test. * indicates P < 0.05 within Tat molecule-stimulated EC
and unstimulated or control protein-stimulated cells; § indicates
P < 0.005 within wild-type Tat molecule-stimulated EC
and Tat mutant-stimulated cells.
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Analysis of the mutants with mutations in the RGD sequence indicated
that this sequence is also necessary for full activation
of the
migration (Fig.
2) and proliferation (Fig.
3) of EC and
is required for
the adhesion process (Fig.
4). Tat R78K/D80E showed
reduced
proliferation and migration activities compared to Tat
86 (
P < 0.005). Tat D80E had an effect similar to
Tat
86, indicating
that a unique mutation is insufficient to
impair the activities
of Tat
86 on EC. Deletion of the
sequence encoded by exon 2 (*Tat
72)
resulted in a molecule
with biological activities (migration and
proliferation) similar to
those of Tat R78K/D80E (Fig.
2 and
3).
Similarly, *Tat
72
and Tat R78K/D80E were not permissive for EC
adhesion (Fig.
4).
*Tat C(22,25,27)A, the mutant with the mutation in the cysteine-rich
region, was a weak activator of EC migration and proliferation
and
showed little impairment of the adhesive property (Fig.
2 to
4). This
suggests that the cysteine-rich region of Tat is required
for migration
and proliferation of EC rather than for their adhesion
to the
extracellular
matrix.
In an effort to better define the activation of EC by the different Tat
domains, the migration of EC was triggered by different
concentrations
of Tat
86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E,
and *Tat
C(22,25,27)A. All concentrations of the mutants tested
(5 to 100 ng/ml)
showed a reduced activity compared to Tat
86.
However, the
highest concentrations of Tat R78K/D80E (50 to 100
ng/ml) tended to
reach the activity of Tat
86 (Fig.
5).
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FIG. 5.
Dose-dependent effect of Tat86 ( ), Tat
R49G/K50I/R52L/R53I ( ), TatR78K/D80E ( ), and *Tat C(22,25,27)A
( ) on EC migration. The cell migration was studied by the Boyden
chamber technique as described in the legend to Fig. 1. The data
obtained with *Tat86 have not been reported as
superimposable on those obtained with Tat86. Results (mean
and standard deviation) of one experiment (performed in triplicate) of
two are shown.
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MBP and GST, the two proteins fused to the different Tat molecules
used, did not promote migration, proliferation, or adhesion
at any
concentration tested (0.1 to 200 ng/ml) (Fig.
2 to
4).
In vivo effect of Tat and Tat mutants in an angiogenesis
model.
The capacity of Tat to induce migration and proliferation
of EC has as in vivo counterpart, i.e., the formation of new blood vessels in rabbit and mouse models (3, 4). Analysis of the effects of Tat mutants in a murine angiogenesis assay is reported in
Table 1. Progressive mutations in the
basic region or in the RGD sequence were associated with an increasing
loss of angiogenic activity compared to the effect of wild-type
molecules (Tat86 or *Tat101). The deletion of
the amino acids encoded by exon 2 of tat led to negligible
angiogenic activity. Also, the mutation in the cysteine-rich region did
not promote angiogenesis. Overall, the in vivo data clearly indicate
that the angiogenic program elicited by Tat is dependent on the
molecular integrity of its basic and cysteine-rich domains as well as
that of the exon 2 product.
Binding of Tat and Tat mutants to EC.
We have previously
demonstrated that EC express high-affinity binding sites for Tat,
identified as VEGFR-2 (4). In this study we used a large
amount of [125I]Tat86 with high specific
activity, which permitted us to study also the presence of low-affinity
binding sites on EC. The binding of Tat was evaluated by using
saturation binding curves and by competitive displacement of
[125I]Tat86. The direct ligand binding curves
suggested that Tat binding approached saturability without reaching
full saturation. Scatchard analysis suggested the presence of two
classes of binding sites. Besides a high-affinity binding site
(Kd = 15.7 ± 5.3 pM;
Bmax = 31.9 ± 7.2 fmol [n = 3]), a low-affinity (Kd = 8.5 ± 3.2 nM; Bmax = 2.6 ± 1.4 pmol
[n = 3]) and high-capacity binding site was also
observed (Fig. 6; Table
2). The competitive displacement experiments of [125I]Tat86 with increasing
amounts of cold ligand confirmed the presence of two binding sites
(Table 2).
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FIG. 6.
Specific binding at equilibrium and Scatchard plot of
[125I]Tat86 (A and B),
[125I]Tat R78K/D80E (C and D), [125I] Tat
R49G/K50I/R52L/R53I (E and F), and [125I] *Tat
C(22,25,27)A (G and H) to EC. Monolayers (105 cells) were
incubated for 90 min at 22°C with the indicated concentrations of
iodinated molecules in the presence of a 100-fold excess of cold
ligands. Results of one experiment of three are shown.
Tat86: Kd (site 1) = 12.9 pM;
Bmax (site 1) = 28.9 fmol;
Kd (site 2) = 4.21 nM;
Bmax (site 2) = 1.32 pmol. Tat R78K/D80E:
Kd (site 1) = 23.8 pM;
Bmax (site 1) = 38.4 fmol;
Kd (site 2) = 13.2 nM;
Bmax (site 2) = 1.50 pmol. Tat
R49G/K50I/R52L/R53I: Kd (site 1) = 4.58 nM;
Bmax (site 1) = 3.51 pmol. *Tat
C(22,25,27)A: Kd (site 1) = 1.15 nM;
Bmax (site 1) = 1.39 pmol.
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By using Tat mutants, we examined the regions of Tat required for
binding to EC. Saturation binding curves of [
125I]Tat
R49G/K50I/R52L/R53I and of [
125I]*Tat C(22,25,27)A
(Fig.
6) as well competitive displacement
of
[
125I]Tat
86 with the two unlabeled peptides
(Table
2) indicated that
these mutants bound EC only with low affinity.
The number of low-affinity
sites bound by [
125I]Tat
R49G/K50I/R52L/R53I and [
125I]*Tat C(22,25,27)A was
increased compared to that bound by
[
125I]Tat
86 (Table
2). The same experimental
approaches showed that
Tat R78K/D80E bound endothelium in almost the
same manner as did
Tat
86 (Fig.
6; Table
2).
[
125I]MBP and [
125I]GST did not show any
specific binding to EC, and the unlabeled
proteins were unable to
displace [
125I]Tat (data not
shown).
Effect of Tat and Tat mutants on VEGFR-2 phosphorylation and signal
transduction.
Figure 7 shows the
tyrosine phosphorylation of VEGFR-2 immunoprecipitated from EC
stimulated with Tat86 and different Tat variants.
Tat86, *Tat72, Tat R78K/D80E, and TatD80E
induced the phosphorylation of VEGFR-2. Moreover, we consistently
observed that Tat R78K/D80E phosphorylated the receptor more
efficiently than Tat86 did. In contrast, mutants with
mutations in the basic domain [(Tat R49G/K50I, Tat
R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A] showed a reduced
ability to phosphorylate the receptor. This feature depends on the
number of basic residues mutated, with Tat R49G/K50I being more
active than Tat R49G/K50I/R52L/R53I and *Tat
R(49,52,53,55,56,57)A. These data confirm that the binding and
activation of VEGFR-2 are mediated by the basic domain, while the RGD
sequence seems to be not involved in receptor phosphorylation.
Mutations in the cysteine-rich region [*Tat C(22,25,27)A]
also abrogated the ability of Tat to promote VEGFR-2 phosphorylation.
MBP and GST did not phosphorylate VEGFR-2 (Fig. 7).
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 7.
Effect of Tat molecules on tyrosine phosphorylation of
VEGFR-2. Quiescent, confluent EC were preincubated for 15 min at 37°C
with 1 mM Na3VO4 and then stimulated with Tat
molecules (20 ng/ml) for 10 min. The cells were lysed and
immunoprecipitated with anti-VEGFR-2 antibody. The immunoprecipitate
was analyzed by SDS-PAGE followed by immunoblotting with
antiphosphotyrosine MAb. Subsequently, the blots were reprobed with
anti-VEGFR-2 antibody. Immunoreactive bands were detected by enhanced
chemiluminescence. The results are representative of three similar
experiments.
|
|
It has been recently demonstrated that PI 3-kinase activation is
associated with the signals elicited by VEGFR-2 activation
elicited by
VEGF-A and Tat
86 (
25,
58). To support the notion
that the basic domain of Tat
86 is involved in the
signalling pathways
downstream of VEGFR-2, we tested the activity of PI
3-kinase in
EC stimulated with Tat R49G/K50I/R52L/R53I and Tat
R78K/D80E.
The results reported in Fig.
8
show that Tat R49G/K50I/R52L/R53I
activated PI 3-kinase activity to a
lesser extent than Tat
86 or
Tat R78K/D80E did.
View larger version (56K):
[in this window]
[in a new window]
|
FIG. 8.
Effect of Tat molecules on PI 3-kinase in endothelial
cells. Quiescent, confluent ECs were stimulated with Tat molecules (20 ng/ml) for 15 min at 37°C. The PI 3-kinase assay was performed on
immune complexes done with MAb antiphosphotyrosine (anti-PY) antibodies
from lysates of EC in the presence of 40 µM ATP, 50 µCi of
[-32P]ATP, and 50 µg of a presonicated mixture of
phosphatidylinositol-4,5-bisphosphate and phosphatidylserine per ml in
25 mM HEPES (pH 7.4)-1 mM EGTA. The extracted lipids were separated by
thin-layer chromatography and visualized by autoradiography.
PIP3, phosphatidylinositol-3,4-bisphosphate; Prot-A, a PI
3-kinase assay done on protein A alone. Spots corresponding to
PIP3 were recovered and counted (n = 3):
control, 420 ± 132 cpm; Tat86, 1,342 ± 231; Tat
R78K/D80E, 1,280 ± 280; Tat R49G/K50I/R52L/R53I, 750 ± 201. I.P., immunoprecipitate.
|
|
| |
DISCUSSION |
HIV-1 Tat is essential for viral replication in infected cells and
acts as a soluble mediator in the microenvironment. Several studies on
the structure-activity relationship of Tat have determined the domains
involved in gene transcription. The basic domain (aa 49 to 57) mediates
the binding of Tat to trans-activation element RNA (22,
27), directs it to the nucleus (27, 52), and is
essential for the recruitment of the p300/CBP transcriptional coactivator (40). A cluster of acidic residues (Glu2, Asp5, and Glu9) in the proline-rich domain, the cysteine-rich domain (aa 22 to 37), and the core domains (aa 32 to 47) all contribute to
LTR-directed transcriptional activation (22, 38, 51, 52,
56). More recently, it has been reported that the C-terminal domain encoded by exon 2 also makes a small contribution to viral replication (60).
In this study we have analyzed Tat mutants for binding to and
activation of EC, which are among the more relevant extracellular targets for this viral protein (1, 3-5, 13, 18, 28).
In a previous work (4), we detected only a high-affinity
binding site corresponding to VEGFR-2, as demonstrated by the ability
of VEGF-A to displace [125I]Tat. However, it has been
recently demonstrated that Tat binds to heparan sulfate (3,
53) and to integrins (7, 62) that both have features
of low-affinity, high-capacity binding sites. Therefore, we have
revised these binding studies by using larger amounts of
[125I]Tat than in our previous study, labeled at high
efficiency. Under these experimental conditions, we detected two
binding sites for Tat on the surface of EC. The presence of MBP did not
interfere with the binding, because it did not show any specific
binding to the cells and did not displace [125I]Tat. The
Kd of the first was in the picomolar range, and
that of the second was in the nanomolar range. We can hypothesize that this low-affinity site reflects the binding of Tat to membrane glycosaminoglycans and/or to integrins.
To elucidate the functional importance of the basic domain of Tat, two,
four, or six nonconservative mutations were made. Variants with a
double mutations exhibited only a slightly reduced activity compared to
Tat86 or Tat101 in term of migration,
proliferation, and adhesion of EC and of angiogenesis induction.
However, the variants with four or six substitutions showed a marked
decrease in the activities studied. Because Tat signals inside the
cells through the tyrosine kinase VEGFR-2 (4, 21, 58), we
investigated the tyrosine phosphorylation of VEGFR-2. Tat
R49G/K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A failed to activate
the receptor. Tat R49G/K50I induced VEGFR-2 phosphorylation but to a
lesser extent. The variants with two or four mutations did not activate PI 3-kinase, which is downstream of Tat86 and
VEGF-A-activated VEGFR-2 (58). These results are in
agreement with the similar observations obtained with a peptide
encompassing the basic domain, which mimics the effect of
Tat86 on VEGFR-2 (4, 21). The variant with four
mutations did not retain the ability to bind to the high-affinity sites
on EC, which correspond to VEGFR-2 (4). Taken together with
the previous data, this suggests that the Tat basic domain is crucial
for the binding and activation of VEGFR-2. The relevance of the
positively charged amino acids in performing these functions is
consistent with the observation that the charged residues R82, K84, and
H86 of VEGF-A are important for VEGFR-2 recognition (32).
To ascertain whether the RGD sequence is required for Tat binding or
activity, mutants with single (D80E) and double (R78K/D80E) mutations
and a variant truncated after the residue 72 (Tat72) were
obtained. Tat R78K/D80E and Tat72 had reduced biological activity in vitro and in vivo, which is consistent with previous reports showing that 
v
3-,
v5-, and
51-integrin participate in Tat-induced
activation of EC (5, 13) as well as of other cell types
(6, 7, 35, 48, 62, 70). Moreover, the RGD sequence and the
C-terminal domain are not required for VEGFR-2 activation, as shown by
the ability of Tat R78K/D80E and Tat72 to induce VEGFR-2
phosphorylation. Furthermore, the variant with the double mutations
activated PI 3-kinase activity and retained the ability to bind both
low- and high-affinity Tat binding sites on the EC membrane. In our
experiments, we have observed that the activity of Tat R78K/D80E on
VEGFR-2 phosphorylation was greater than that elicited by
Tat86. The explanation of this result is lacking, but it
will be interesting to determine the role of VEGFR-2 regulatory
pathways associated with integrins. For instance, we have recently
shown that 3-integrin is associated with
VEGF-A-stimulated VEGFR-2 and that a MAb against this integrin inhibits
the activation of the receptor (58). Alternatively, this
variant can assume a spatial conformation more favorable for receptor
activation, independent of the interaction with integrins. Therefore,
the activation of the biological activities of EC related to
angiogenesis most probably requires the engagement of VEGFR-2 as well
as integrins by two specific molecular determinants of Tat: the basic
domain and the product of exon 2 containing the RGD motif. Many of the integrin-induced signalling pathways are also normally activated by
binding of soluble growth factors to their receptors, which suggests
the existence of coordinate mechanisms between integrins and growth
factors in the control of cell functions, and there is increasing
evidence that growth factors can induce an appropriate cellular
response only when the target cells express defined sets of integrins
(24, 55). Normally, growth factors do not directly bind to
and activate integrin. The data reported here and those showing that
anti-v3 or anti-VEGFR-2 neutralizing
antibodies (4, 5, 58) inhibit the EC response to Tat suggest
that this viral protein is a unique example of a molecule which turns on intracellular signals by direct activation of both receptor and
integrin systems. Furthermore, besides playing a direct role in
integrin activation, the C terminus region of Tat could regulate VEGFR-2 through the presence of other determinants relevant for the
engagement of neuropilin-1, a coreceptor of this tyrosine kinase
receptor (57).
*Tat C(22,25,27)A was used to study the role of the cysteine-rich
region of Tat in EC activation. This variant was less potent than
Tat86 or Tat101 in terms of migration,
proliferation, adhesion, and in vivo angiogenesis, bound only the
low-affinity binding site, and showed low efficiency in VEGFR-2
phosphorylation. Because it has been suggested that Tat forms a dimer
bridging cysteine-rich regions from each monomer (19) and
that two cysteine residues are pivotal for the VEGF-A dimerization and
the subsequent binding to and activation of endothelium (46,
50), it could be hypothesized that the active form of Tat on the
endothelium has a dimeric structure. The relevance of this domain has
also been emphasized by Albini et al., who demonstrated its role in the
migration of monocytes (2).
Except for Tat D80E, all mutants studied showed a reduced ability to
activate EC functions, but their effects are only partially overlapping. For example, mutants with mutations in the RGD sequence have reduced biological activities but fully activated VEGFR-2 phosphorylation. Tat R49G/K50I promoted cell adhesion as
Tat86, but its migratory and proliferative capacities were
consistently lower than those of Tat86. Mutants with
mutations in basic and in cysteine-rich domains had similar kinetic
binding parameters, which differed from those of TatR78K/E80D.
Furthermore, Tat E80D was similar to Tat86 or
*Tat101 in all assays performed. Taken together, these
observations exclude the possibility that the effects of the Tat
mutants are caused merely by an unfolded protein, as also reported for
other studies performed with Tat mutants (22, 27, 38, 40, 51, 52,
56, 60).
In conclusion, this study demonstrates that the product of the first
exon of tat is crucial for the activation of VEGFR-2 and for
induction of the activation of an angiogenic program in EC. However, to
obtain full activation of this program, Tat also requires the
C-terminal region containing the RGD sequence. The C-terminal region
could influence the VEGFR-2 response by interacting with integrin
subunits or with neuropilin-1 (57), which modulate the
functions of this tyrosine kinase receptor, or with other unidentified
coreceptors. Further studies of the hypothesized role of Tat
dimerization by intermolecular cysteine bridge formation should make it
possible to define the receptor-ligand interaction more precisely.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the European Community
(Biomed-2 Project BMHL-CT96-0669), Italian Association for Cancer
Research (A.I.R.C.), Istituto Superiore di Sanità (Programma nazionale sull'AIDS-Patogenesi, immunità e vaccino per l'AIDS; Program on Tumor Therapy), Centro Nazionale delle Ricerche (Progetto Finalizzato Biotecnologie), and Ministero dell' Università e della Ricerca Scientifica e Tecnologica (60% and Programmi di Ricerca
di Rilevante Interesse Nazionale-1998). S.M. and I.Z. are supported by
grants from FIRC and from the "Gigi Ghirotti" foundation, respectively.
| |
ADDENDUM IN PROOF |
Boykins et al. (R. A. Boykins, R. Mahieux, U. T. Shankavaram, Y. S. Gho, S. F. Lee, I. K. Hewlett, L. M. Wahl, H. K. Kleiman, J. N. Brady, K. M. Yamada, and S. Dhawan, J. Immunol. 163:15-20, 1999) recently reported that a peptide containing six cysteine residues
(from amino acids 21 to 40 of the Tat molecule) is angiogenic in
chicken chorioallantoic membranes.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: I.R.C.C., Strada
Provinciale 142, Km 3.95, 10060 Candiolo (Turin), Italy. Phone:
39-011-9933347. Fax: 39-011-9933524. E-mail:
fbussoli{at}mail.ircc.unito.it.
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Journal of Virology, January 2000, p. 344-353, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
