 |
INTRODUCTION |
Inflammation-mediated tissue destruction results from recruitment
and extravasation of inflammatory cells at some tissue site, usually
because of injury or infection. Host-pathogen interactions trigger the
inflammatory response, and the balance between the pro- and
anti-inflammatory forces will determine the outcome of the infection.
The vascular endothelium is an essential component of this balance and
contributes to the separation between vascular and interstitial spaces.
At areas of inflammation, neutrophils are recruited by a combination of
adhesion molecule up-regulation and chemokine secretion. The leukocytes
in turn activate their own inflammatory programs of increased reactive
oxygen species generation and cytokine secretion. Endothelial cells
respond in kind by becoming further activated. Accordingly, diseases
that affect the endothelium result in marked vasculopathies and failure of the endothelial barrier.
In HIV1 infection there is a
diffuse endothelial cell involvement, not often recognized, that
includes increased susceptibility to inflammatory cytokines, increased
adhesion molecule expression, and increased neutrophil and mononuclear
cell adhesion. Cardiovascular complications, such as myocarditis with
infiltrating neutrophils and mononuclear cells are often observed, even
in the absence of infectious pathogens (1, 2). Even though HIV may
enter endothelial cells via transcytosis (3), productive infection is
very hard to achieve unless the cells are proliferating in the presence
of cytokines (4). On the other hand, transgenic mice carrying a
replication-defective provirus develop smooth muscle hypertrophy as
well as adventitial infiltration by T-lymphocytes. This vascular
remodeling leads to narrowing of the blood vessels of different sizes
with the resultant ischemia in organs such as brain, heart, kidney,
pancreas, and spleen (5). In view of these observations, the question
remains as to what is causing this inflammation and vasculopathy when
there is no obvious infectious or opportunistic pathogen.
The HIV-1 Tat protein is an early viral protein of 101 amino acids when
isolated from primary HIV isolates, or of 86 amino acids when isolated
from the laboratory strain HBX2, and which still retains full activity
(6). Expression of Tat is critical for productive HIV infection. The
tat gene consists of 2 coding exons. The first one, which
encodes 72 amino acids, is sufficient to activate HIV LTR-mediated gene
expression in co-transfection promoter-reporter assays (7). However, in
the context of viral infection, where the integrated provirus is
subject to chromatin influences, the second exon is required for trans
activation of the LTR (8). Exon 1 contains the cysteine-rich
trans-activation domain, the core, and an arginine-rich
motif that is responsible for Tat-TAR RNA interactions (9-11).
The arginine-rich motif acts as a nuclear localization signal as well
(12, 13) and is responsible for NF-
B activation in HeLa cells (14).
The second exon codes for the remaining amino acids. Tat acts by
binding to a region located at the 5'-end of the viral transcript. This
region, called TAR, forms a stable stem-loop structure having a high
affinity for Tat (7, 11). The NH2-terminal
trans-activation domain, when tethered to TAR, strongly
interacts with cyclin T1, which is a component of transcription
elongation factor 
(7, 15). The cyclin-dependent kinase
9 that is part of this complex then phosphorylates the
carboxyl-terminal domain of RNA polymerase II. This phosphorylation
facilitates the elongation step by preventing premature termination and
ensures production of the full-length viral transcript (16-18). The
end result is a significant increase in the production of viral
proteins, essential for productive infection.
Tat can be secreted from infected cells and circulates in the
bloodstream of infected individuals. From the circulation, Tat enters
uninfected cells and once internalized, it alters cellular physiology
by positively or negatively affecting gene expression. For example, Tat
activates transforming growth factor- expression in human
chondrocytes (19), TNF expression in mononuclear cells (20), IL-8
secretion in endothelial cells (21), and T cell lines following CD3-
and CD28-mediated co-stimulation (22). Tat increases IL-1 production
in monocytic cells, and IL-6 protein and mRNA in astrocytes, both
effects independent of TNF-
production (23). Tat also increases FAS
and FAS-ligand transcription, presumably via NF-B (24), and induces
IL-10 in peripheral blood monocytes (25). Microglia exposed to Tat
induce nitric-oxide synthase and NO production, and this induction is
also dependent upon NF-B (26). On the other hand, negative effects
include repression of major histocompatibility class-I gene promoter
activity (27) and of the important antioxidant enzyme
maganese-superoxide dismutase (28). Interestingly, a deregulation of
cytokine expression and/or secretion is a hallmark of HIV infection
(29-32).
These studies indicate that Tat may be having some of its positive
transcriptional effects through NF-B activation. Indeed, Tat
activates the transcription factor NF-B in HeLa cells. This activation is mediated by PKR, the double-stranded
RNA-dependent protein kinase, which phosphorylates IB
leading to its degradation (14). Subsequent NF-B translocation into
the nucleus increases expression of a cascade of inflammatory genes.
Tat introduced via liposomes results in nuclear translocation of
NF-B (34) and induction of CD69 gene transcription in an
erythroleukemia cell line (35) and interleukin-8 secretion in a T-cell
line (22). Extracellular Tat is associated with an increase in both NF-B binding and protein kinase C activity in primary fetal human astrocytes (36). On the other hand, Tat does not activate the NF-B
responsive reporter construct, (PRDII)4-CAT, but can
synergize with NF-B in the activation of both HIV-derived and
non-HIV-derived promoters (37). Tat induces matrix metalloproteinase-9
in monocytes through protein-tyrosine phosphatase-mediated activation
of NF-B (38). In Jurkat T cells, Tat-mediated activation of NF-B
is dependent upon activation of the T cell-specific tyrosine kinase p56lck (33). Thus, Tat-mediated NF-B activation may
be via multiple signal transduction pathways.
The adhesion molecule E-selectin is induced at inflammatory sites where
it exhibits restricted and tightly regulated expression in endothelial
cells. E-selectin up-regulation is accomplished via increased cytokines
such as TNF, and mediated in part by two closely apposed NF-B sites
(39). Early studies demonstrated that as part of the immune activation
seen in AIDS patients, soluble E-selectin levels were elevated (40,
41). Furthermore, Tat increases E-selectin expression in human
umbilical vein endothelial cells (42).
Because endothelial cell adhesiveness for neutrophils consists of an
interplay between adhesion molecules and circulating neutrophils, and
since Tat has been demonstrated to increase E-selectin expression in
normal endothelial cells and activate NF-B in some cells, we
examined whether the Tat-mediated increases in E-selectin expression
require NF-B. Here we demonstrate for the first time that this
Tat-mediated up-regulation of E-selectin requires NF-B and the
synthesis of new macromolecules. Furthermore, consistent with previous
reports of Tat-mediated NF-B activation in HeLa cells, we
demonstrate that the basic domain of Tat is necessary for this induction.
| |
EXPERIMENTAL PROCEDURES |
Materials and
Reagents--
Isopropyl-1-thio--D-galactopyranoside
(IPTG), antibiotics, cycloheximide, actinomycin D, cell dissociation
buffer, IGEPAL (Nonidet P-40), salts, bovine heart
cAMP-dependent protein kinase, and buffers were purchased
from Sigma. GSH-Sepharose and ECL chemiluminescence kit were from
Amersham Bioscience; tumor necrosis factor- (TNF) used for all the
present studies was obtained from Pepro Tech, Inc.; fluorescein
isothiocyanate-conjugated Cd62E (clone 1.2B6) was obtained from
Research Diagnostics Inc.; antibodies against p65, p52, RelB, Sp3, and
actin were obtained from Santa Cruz Biotechnology; antibodies against
cRel were obtained from Rockland, and p50 from Geneka Biotechnology
Inc. Endothelial cell culture medium was EGM-2 (EGM-2 Bullet kit,
BioWhittaker, San Diego, CA), supplemented with 2% fetal calf serum.
Serum-free Opti-MEM and neomycin were purchased from Invitrogen
(Gaithersburg, MD). Bradford reagent for protein determination was
purchased from Bio-Rad. The Tat antibody was kindly provided by the
AIDS Reference and Research Reagent Program and was originally
contributed by Dr. Bryan Cullen (43). Superfect reagent for
transfection was purchased from Qiagen and used according to
manufacturer's specifications.
Plasmids--
Plasmid GST-Tat (GST-Tat 1 86R TK) contains the
2-exon 86-amino acid wild-type tat gene cloned into pGEX2TK
(Amersham Bioscience); pGST-Tat 1 48
TK is truncated after amino
acid 48 and contains a functional activation domain; pGST-Tat 1 48
C22G contains a truncation after amino acid 48, and a non-functional
activation domain because there is a point mutation at cysteine 22. All
of these GST fusion vectors were obtained from the NIH AIDS Research and Reference Reagent Program and contributed by Dr. Andrew P. Rice.
Parental vector pGEX-2TK was purchased from Amersham Bioscience. This
vector is designed for inducible expression of genes as fusions with
the Schistosoma japonicum glutathione
S-transferase (GST). Because the fusion constructs are under
the control of the tac promoter, induction with IPTG leads
to high levels of expression. Affinity chromatography with
GSH-Sepharose followed by thrombin cleavage is used to purify the
protein of interest. Plasmid pMT/V5-His C (Invitrogen) is a
Drosophila expression vector that carries the fly
metallothionein promoter, which allows for CuSO4 induction of cloned genes. The HIV-1 86-amino acid Tat was cloned into the multiple cloning site of this vector to generate the plasmid
pMT-tat.2 Plasmid
pELAM-Luc (pE-Luc), kindly provided by Dr. Tom McIntyre, contains
sequences 840 base pairs upstream of the transcriptional start site of
the human E-selectin gene driving firefly luciferase transcription.
Control plasmid pGL3B containing the promoterless luciferase gene was
obtained from Promega Biotechnology. Plasmid pCoHygro, obtained from
Invitrogen, containing a hygromycin resistance gene under the control
of the Drosophila copia promoter, was used for the
generation of stable S2-tat cells. Plasmid pHOOK-3
(Invitrogen), an expression vector carrying the cytomegalovirus
promoter, was used to create a pHOOK-tat expression vector
for transient transfections of mammalian cells.
Cell Culture and Stimulation--
HUVEC were obtained from
BioWhittaker and maintained in EGM-2. Cultures were maintained at
37 °C in a 6.5% CO2 humidified atmosphere. Adhesion
molecule induction via NF-B declines as passage number increases and
is sensitive to growth state, therefore, expanded cells were used at
passages 2-3 and cells at 3 days postconfluence were used for all the
experiments. HeLa and HeLa-tat cells were obtained through
the NIH AIDS Research and Reference Reagent Program and contributed by
Drs. W. Haseltine and E. Terwilliger. These cells were cultured in
Opti-MEM with 3.75% fetal calf serum in the absence of antibiotics.
The HeLa-tat cells were grown in the presence of 800 µg/ml
neomycin. Schneider 2 (S2) Drosophila cells were purchased
from Invitrogen and cultured in complete Drosophila Expression System medium according to the manufacturer's instructions. For transfection and selection of stable S2-tat cells, S2
cells were grown in 6-well culture dishes to a density of 1 × 106 cells, collected, washed, and co-transfected with
pCoHygro/pMT-tat via
Ca3PO4. Cells were plated and allowed to
recover for 24 h in complete Drosophila Expression
System medium. Stable clones were selected by growth in 300 µg/ml
hygromycin for at least 2 months. Transgene expression was induced by
24 h incubation in the presence of 500 µM
CuSO4.
Transient Transfection and Luciferase Assays--
HeLa or
HeLa-tat cells were seeded at a density of 5 × 105 cells/well on 6-well tissue culture dishes and grown
until 60% confluence. Cells were washed with sterile PBS and incubated
in the presence of 2 µg of pE-Luc or pGL3B in Superfect reagent for
18 h. Cells were lysed in passive lysis buffer (Promega luciferase
transfection kit) according to the manufacturer, lysate collected, and
total protein determined. Aliquots of 20 µl were assayed for light
emission with a plate-reader luminometer. In a separate series of
experiments, HUVEC were seeded in 6-well tissue culture dishes and
grown to 60% confluence. Transfection and luciferase assays were
performed as described above, except that the cells were co-transfected with equimolar amounts of either pHOOK-3/pE-Luc or
pHOOK-tat/pE-Luc.
Cytoplasmic and Nuclear Protein Extractions--
For nuclear and
cytoplasmic protein extraction, confluent HUVEC cultures grown on 10-cm
tissue culture dishes were washed with sterile PBS and fed 5 ml of
serum-free Opti-MEM containing 20 ng/ml recombinant human TNF, 500 ng/ml recombinant Tat, or the indicated control medium for 1 h.
After addition of 5 ml of EGM-2 containing 2% fetal calf serum, cells
were incubated for the indicated times. Therefore, no serum was present
during the first hour of incubation, and only 1% during the remainder.
Cells were harvested by rinsing twice with PBS (calcium and
magnesium-free), followed by incubation in a non-enzymatic cell
dissociation buffer for 5 min at 37 °C. Cells were detached by
scraping, transferred to microcentrifuge tubes, and pelleted at
5,000 × g for 1 min. The supernatant was removed,
cells resuspended in 1 ml of cold hypotonic buffer (10 mM
HEPES, 1.5 mM MgCl2, and 10 mM KCl
adjusted to pH 7.9 with KOH) containing 0.2 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 0.3 µg/ml
leupeptin, and 0.5 mM DTT, and washed by centrifugation at
2,000 × g for 5 min at 4 °C. The pellet was resuspended in 5 times the packed cell volume of cold hypotonic buffer
with protease inhibitors as described above and allowed to swell on ice
for 20 min. IGEPAL was then added to 0.1% and the swollen cells
incubated an additional 5 min on ice followed by homogenization with 20 strokes of a microtube pestle homogenizer (Fisher Scientific). Cell
lysis and nuclear integrity were verified by trypan blue exclusion
analysis. Nuclei were pelleted at 13,000 × g for 5 min
at 4 °C. The supernatant containing the cytoplasmic fraction was
transferred to another tube and stored at 
80 °C until further use.
The nuclei were resuspended in 3-4 times the packed nuclear volumes of
cold hypotonic buffer with protease inhibitors as above, washed once by
centrifugation at 13,000 × g at 4 °C, and
resuspended gently in one-half packed nuclear volume of cold low salt
buffer (20 mM HEPES, 0.2 mM EDTA, 25%
glycerol, 1.5 mM MgCl2, 20 mM KCl,
pH 7.9). One-quarter packed nuclear volume of cold hypertonic buffer
(10 mM HEPES, 0.1 mM EDTA, 50 mM
KCl, 300 mM NaCl, 10% glycerol, 1.5 mM
MgCl2 at pH 7.9) was added in a dropwise manner to prevent
lysis of the nuclei. Nuclear proteins were extracted at 4 °C for 30 min, with a gently rotating motion. The nuclear protein extract was
clarified by centrifugation at 13,000 × g for 20 min
at 4 °C. Nuclear and cytoplasmic protein concentrations were
determined by the Bradford reagent microassay protocol using bovine
serum albumin as a standard. To assess the purity of the fractions and
to test our cell fractionation technique, 15 µg of nuclear or
cytoplasmic proteins extracted from HUVEC, HeLa, or HeLa-tat
cells were immunoblotted with actin or Sp3 antibodies.
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
proteins were diluted in a 2:1 mixture of low salt:hypertonic buffer to
a concentration of 0.75 µg/µl and 2.5 µg of this protein were
used in the binding reaction. This procedure ensured that the salt as
well as the protein concentration was the same in all the reactions.
The oligonucleotide used contained the human E-selectin B site 3 (39) flanked by additional E-selectin-specific sequences: 5'-
GCCATTGGGGATTTCCTCTTT-3' (B site underlined). Complementary oligos (Invitrogen) were annealed and end-labeled with
[
-32P]ATP and T4 polynucleotide kinase (Promega).
Labeled oligos were separated from unincorporated nucleotides using a
Sephadex G-25 spin column (5 Prime 
3 Prime). The labeled probe was
diluted to 8-12 fmol/µl (~10,000 cpm) and 1 µl/binding reaction
was used. The binding reaction also contained 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM,
Tris-HCl, pH 7.5, 0.05 mg/ml poly(dI-dC)-poly(dI-dC), and 2.5 µg of
nuclear protein (3.3 µl of 0.75 µg/ml dilution) in a final volume
of 9 µl. For competition studies, 50-fold molar excess of cold
competitor B or an irrelevant oligonucleotide was included. The
reaction was mixed by gentle rocking and incubated at room temperature
for 20 min. After binding, 1 µl of 10 × EMSA buffer (250 mM Tris-HCl, pH 7.5, 0.2% bromphenol blue, and 40%
glycerol) was added to each reaction and loaded onto a 4%
polyacrylamide gel (40:1 acrylamide:bis-acrylamide) containing 1 × TGE buffer (25 mM Tris-HCl, pH 7.5, 190 mM
glycine, 1 mM EDTA, pH 8.3) and 10% glycerol. The protein
complexes were separated at 12 mA/gel for ~2 h. Gels were dried
in vacuo and exposed to Bio-Max X-Ray (Kodak) film. Multiple
exposures of the film were obtained to ensure that the signal was
within the linear range of the film.
Antibody Supershift Assays--
After 20 min incubation of the
oligonucleotide and the nuclear protein extract, 1 µg of antibodies
against p50, p65, RelB, cRel, or p52 was added and the mixture
incubated for an additional 15 min before electrophoresis.
Purification of Wild-type and Truncated Tat Proteins--
The
HIV-1 Tat protein was purified in our laboratory as described (44),
using affinity chromatography. The truncated Tat mutants and the
parental GST were purified using the same method. Expression from these
vectors results in GST-fusion proteins, linked by a thrombin-sensitive
peptide. Host Escherichia coli cells were grown to
A590 = 0.5-0.7 in 500 ml of culture volume. Induction of fusion proteins was achieved by growth in the presence of
0.1 mM IPTG for 2.5 h. Cells were collected by
centrifugation at 14,000 × g, the pellet was
resuspended in 4 ml of EBC-DTT buffer (50 mM Tris-HCl, pH
8.0, 120 mM NaCl, 0.1% IGEPAL, and 5 mM DTT) and sonicated for 1 min twice, with a 1-min incubation on ice between
sonications. The homogenate was clarified by centrifugation in
microtubes at 13,000 × g for 15 min at 4 °C. The
supernatant was collected and frozen at 80 °C. For GST-Tat
purification, all of the buffers were degassed to minimize the exposure
of Tat to oxygen. This treatment consistently gave biologically active Tat preparations. Glutathione-Sepharose beads (GSH-Sepharose, Amersham
Bioscience) were equilibrated in EBC-DTT buffer by washing twice with 1 ml of EBC-DTT buffer followed by resuspension in the same buffer to
create a 50% slurry. Two-hundred fifty microliters of the 50% slurry
were added to the bacterial supernatant containing the induced GST
fusion protein and the mixture incubated for 30 min at 4 °C with
constant rocking. After washing the beads extensively with 12 volumes
of EBC-DTT twice and 5 volumes of thrombin cleavage buffer (50 mM Tris, pH 7.6, 20 mM KCl, and 1 mM DTT) once, the Tat peptide was cleaved by incubation
with 12 units of thrombin A in 200 µl of thrombin cleavage buffer for
1.5-3 h at room temperature with constant rocking. To collect the
cleaved Tat, the sample was incubated at 37 °C for 3 min and then
centrifuged at 13,000 × g for 30 s at room
temperature and the supernatant containing the pure Tat transferred to
a clean vial. To maximize the yield, the Sepharose beads were
resuspended in 50 µl of thrombin cleavage buffer, heated to 37 °C
for 3 min, and centrifuged at 13,000 × g again. This
elution was performed a total of three times and the final supernatants
pooled. The pure Tat preparation was divided into 100-µl aliquots,
flash frozen, and stored in liquid nitrogen. To determine Tat
biological activity, the pure protein was electroporated into HeLa
cells in the presence of the indicator plasmid pLUCA41, kindly provided
by Dr. Gail Harrison, which contained the HIV LTR controlling firefly
luciferase expression. Active Tat preparations consistently activated
LTR-driven luciferase expression at least 5,000-fold. Bacterially
expressed GST extracts purified as described for the GST-Tat fusion
proteins were used as control. Presence of endotoxin was assessed by an
E-TOXATE assay from Sigma.
Adenovirus Transduction--
An adenovirus vector containing an
IB mutant (S32A/S36A) super-repressor insert (AdIBSR) or a
green fluorescent protein (AdGFP) insert was used. The recombinant
protein contained an HA epitope tag to facilitate detection of the
transduced construct. Cells were grown to 100% confluence, medium
removed, and replaced with serum-free medium containing the virus at a
multiplicity of transduction of 30 plaque forming units/cell. After an
overnight incubation, monolayers were washed and exposed to Tat or TNF
for 6 h. The cells were collected and processed for nuclear
protein extraction or for E-selectin cell surface expression.
Western Blot Analysis--
Cytoplasmic or nuclear proteins were
resolved by SDS-polyacrylamide gel electrophoresis in 5-15% gradient
pre-cast gels, electroblotted in a buffer containing 25 mM
Tris, 192 mM glycine, and 10% methanol onto a
polyvinylidene difluoride membrane (Bio-Rad), stained with antibodies
against p65, actin, or Sp3 and a horseradish peroxidase-coupled secondary antibody (ECL, Amersham Bioscience), and detected on film.
Molecular weight standards on an adjacent lane were the Benchmark
standards from Bio-Rad. Autoradiographs were quantitated by
densitometry. Membranes were stripped and equal loading of proteins
assessed by colloidal gold staining (Bio-Rad). For all p65 immunoblots,
0.5 µg of protein (nuclear, cytoplasmic, or whole cell) was loaded
per well; for actin and Sp3 immunoblots, 15 µg of protein (nuclear or
cytoplasmic) were loaded per well.
Cell Surface E-selectin Expression--
Cells were grown on
6-well tissue culture dishes until confluent, and incubated for 3 additional days. Then, cells were incubated with serum-free Opti-MEM
supplemented with 500 ng/ml Tat ± Tat antibody, 500 ng/ml
truncated Tat mutants, 20 ng/ml TNF, 500 ng/ml GST control extracts,
500 ng/ml boiled Tat or with serum-free media alone. After 1 h, 1 ml of EGM-2 with 2% serum was added and the cells incubated for an
additional 5 h. Cells were incubated for 30 min with 5 µg/ml
cycloheximide or 2 µg/ml actinomycin D, prior to addition of Tat or
TNF for a total 6-h incubation. For endotoxin exposure, cells were
exposed to 500 ng/ml E. coli lipopolysaccharide (055:B5,
Sigma) for 6 h in the presence or absence of serum. Cells were
exposed for 5 min to cell dissociation buffer (Sigma) at 37 °C after
washing with calcium and magnesium-free PBS. Cells were harvested by
scraping followed by centrifugation at 600 × g for 5 min. The pellet was resuspended in 400 µl of FACS binding buffer (1%
fetal calf serum, 0.2% NaN3 in PBS), and 200-µl aliquots transferred to round-bottom 96-well plates. Cells were centrifuged at
200 × g for 5 min at 4 °C, supernatant removed by
gentle tapping against the sink, and pellets resuspended in 100 µl of
binding buffer containing 5 ng/µl fluorescein
isothiocyanate-conjugated anti-human E-selectin (mouse monoclonal,
clone 1.2B6, IgG1). In control experiments, an IgG isotype control
antibody was used. After incubation on ice for 15 min, cells were
washed twice by centrifugation with binding buffer. After the last
wash, the cells were resuspended in 700 µl of binding buffer and
analyzed on a FACSscalibur fluorescence cytometer (Becton Dickinson).
In experiments with the AdGFP, secondary antibody used was conjugated
to biotin, and detection was via cytochrome-streptavidin-mediated
fluorescence. For quantitation of E-selectin expression, 10,000 cells
were acquired per experimental set. Voltage and gain parameters were
established and gates assigned during the acquisition of unstained
cells. In the analysis, regions M1 and M2 were gated and set according to the fluorescence of cells exposed to medium alone. Region M1 was
determined to be of low fluorescence intensity and consistently contained cells with mean fluorescence intensities less than 10. Region
M2 was determined to be of high fluorescence intensity and consistently
contained cells with mean fluorescence intensities higher than 50. Region M1 was therefore set between 0 and 10 and region M2 between 10 and 104. Cells having mean fluorescence intensities less
than 10 were considered negative for E-selectin. Cells with mean
fluorescence intensities higher than 10 were considered positive for
E-selectin. The results are therefore expressed as the percentage of
cells in region M2. For all experiments, TNF treatment of HUVEC was used as the positive control. This exposure allowed us to control for
differences in source, passage number, and growth state of the HUVEC
used in each particular experiment.
Statistical Analyses--
With the exception of autoradiographs
and Western blot analyses, all results shown represent mean ± S.E. Results were analyzed by one-way ANOVA.
| |
RESULTS |
Purity of the Tat Preparation--
Recombinant Tat protein was
purified from bacteria (E. coli SURE cells) harboring a
pGST-tat fusion construct under the control of the
tac promoter. Induction with IPTG increased expression of
the fusion protein (Fig. 1A,
lane 2). After binding to GSH-Sepharose beads, the fused Tat
was cleaved with thrombin and subsequently isolated. Western blot
analyses with a Tat polyclonal antibody confirmed the presence of Tat
in the final preparation (Fig. 1A, lane 4). There
was some GST-tat in the uninduced bacterial extracts (Fig.
1A, lane 1) possibly due to leakiness of the
tac promoter, even in the absence of IPTG. A significant
amount of GST-tat was detected in the flow-through (Fig.
1A, lane 3); this effect is more evident when the
GSH-Sepharose beads have been in storage for prolonged periods of time
(not shown). When newly purchased beads or beads whose reduced
glutathione is regenerated in the presence of DTT are used, the amount
of GST-tat in the flow-through is considerably reduced (data
not shown). A representative colloidal gold-stained membrane of a Tat
preparation is shown in Fig. 1B, left panel. The
right panel shows a different Tat preparation. The band
denoted by the open arrow represents a 70-kDa bacterial chaperonin, the product of the E. coli gene dnaK, which
co-purifies with recombinant proteins (45). This association can be
disrupted by incubation of the lysate in the presence of ATP and
MgSO4, prior to affinity purification (45). Presence of
this protein is not responsible for the Tat effects observed since
control GST extracts do not activate NF-kB translocation or E-selectin expression (discussed later). Furthermore, extracts where this protein
was absent retained Tat activity (Fig. 1B, Tat
2).
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Fig. 1.
Purity of the Tat preparation.
Recombinant Tat protein was purified from E. coli SURE cells
harboring a pGST-tat fusion construct under the control of
the tac promoter. A, Western blot analyses of 50 ng of the total bacterial extract before (uninduced) and after
induction (induced) with IPTG, the flow-through (FT) after
binding the fusion protein to GSH-Sepharose and the final Tat eluate.
The proteins were resolved by SDS-PAGE, transferred to polyvinylidene
difluoride, and pure Tat detected using a polyclonal antibody. The
arrows point to Tat, the arrowhead points to
GST-Tat. B, colloidal gold stained membrane with 650 ng of
the total bacterial extract (lysate), FT, and two different Tat
preparations. The arrows point to Tat, the
arrowhead points to GST and the open arrow points
to a 70-kDa bacterial chaperonin that frequently co-purifies with
recombinant proteins. C, in vitro phosphorylation
of the purified bacterial products from the pGST or pGST-tat
plasmids with protein kinase from bovine heart. D, Western
blot analyses of purified recombinant Tat (rTat) compared with Tat
expressed in S2-tat cells after CuSO4 induction.
pMT refers to S2 cells stably transfected with the empty
expression vector. NS, non-specific.
|
|
The pGST-Tat fusion construct includes a cardiac muscle
cAMP-dependent protein kinase recognition sequence
downstream of the thrombin cleavage site, which allows the detection of
cleaved fusion products by phosphorylation in vitro. To
further establish the purity of the Tat preparation, the purified
product as well as extracts from cells harboring the control parental
plasmid (pGST) were phosphorylated in vitro with bovine
heart cAMP-dependent protein kinase and
[-32P]ATP for 30 min on ice. The results shown in Fig.
1C demonstrate that only the extract purified from the
GST-Tat-harboring bacteria contains a phosphorylated protein of the
correct molecular weight.
Finally, we compared bacterially purified Tat (rTat, Fig.
1D) to Tat expressed in a Drosophila cell line
engineered to stably produce Tat (S2-tat) upon
CuSO4 induction. The Tat-specific band was only detected in
the induced S2 cell cultures and not in uninduced cells or in cells
transfected with the parental pMT vector. The upper band seen in all
the S2 extracts is a nonspecific product. In our hands, bacterially
expressed Tat always migrates as 3 separate bands on denaturing gels.
Presumably, the reducing environment of the bacterial host (SURE cells,
Stratagene) is responsible for this observed behavior. These cells have
been engineered with mutations in several recombination and DNA repair
genes. Nevertheless, the Tat purified from bacteria co-migrated with
the Tat present in the induced S2 cells. Taken in toto,
these results demonstrate that the bacterially purified Tat is ~95%
pure and comparable with Tat expressed in eukaryotic expression systems.
Tat Up-regulates E-selectin Expression in Human Endothelial
Cells--
Tat is secreted into the extracellular compartment and once
taken up by target cells can affect the expression of cellular genes.
To establish whether Tat may affect adhesion molecule expression in
endothelial cells, we exposed HUVEC to recombinant Tat purified from
bacteria. Confluent monolayers were exposed to culture medium alone,
Tat, TNF, or GST extracts for 6 h and E-selectin expression measured by FACS. Histograms of cell counts versus
fluorescence intensity are shown in Fig.
2A. Neither culture medium
alone nor control extracts from bacteria harboring the parental GST
vector induced E-selectin expression. Cells grown in the presence of 500 ng/ml Tat show an increase in the percentage of cells in region M2
(E-selectin positive) from 4 to 93% when compared with cells grown in
medium alone. This concentration of Tat was deemed optimal because
higher concentrations (between 500 ng/ml and 1 µg/ml) did not induce
further, and lower concentrations (between 50 and 400 ng/ml) were not
as efficient. A similar fluorescence shift was observed in TNF-treated
cells. The kinetics of E-selectin up-regulation showed a similar
pattern for both Tat and TNF, with TNF inducing an increase in
expression in 20% of the population by 2 h, while Tat reached a
level of 4% at this time point. Both agents stimulated expression
maximally by 5 h (Fig. 2B). Treatments for a total of
6 h were therefore chosen for all of the subsequent experiments.
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Fig. 2.
Effects of Tat on HUVEC E-selectin
expression. Confluent, HUVEC monolayers were exposed to Tat, TNF,
GST, or cell culture medium alone. After exposure, cells were collected
and processed for detection of surface E-selectin by FACS as described
under "Experimental Procedures." 10,000 cells per experimental set
were acquired. Regions M1 and M2 were set according to the fluorescence
of the cells exposed to medium alone. Region M1 is of low fluorescence
intensity (background fluorescence) while region M2 is of high
fluorescence intensity and considered positive for E-selectin
induction. A, cells were incubated in the presence of the
indicated agents for a total of 6 h. Percentage of cells in region
M2 with medium alone was 4%, with GST, 11%, with Tat, 93%, with TNF,
95%. B, kinetics of E-selectin surface expression;  ,
medium alone;  , 500 ng/ml Tat;  , 20 ng/ml TNF. At 2 h, 20%
of the cells with TNF, while 4% of the cells with Tat or media alone
were positive for E-selectin. Results expressed as mean ± S.E.,
n = 6. C, summary of E-selectin expression
in response to the indicated agents for 6 h. 48 TK and 48
C22G are truncated Tat products with or without a functional activation
domain, respectively, while anti-Tat refers to polyclonal Tat
antiserum.
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To address which functional domains of Tat may be responsible for the
E-selectin up-regulation, truncation mutants with or without a
functional activation domain were tested. Fig. 2C shows that
neither the truncated Tat product with a functional activation domain
(48TK), nor the truncated product with a non-functional activation
domain (48C22G) activated E-selectin expression. Tat boiled for 10 min, purified GST extracts or culture medium alone also failed to
significantly induce E-selectin expression. These treatments increased
E-selectin expression to less than 20%, compared with an average of
87% ± 1.5 for active Tat. Thus, the difference between the
Tat-mediated changes and the control treatments is due to Tat activity.
Tat that had been incubated in the presence of a Tat antibody
(anti-Tat) for 30 min prior to treatment resulted in only 23%
E-selectin expression, demonstrating the specificity of the effect.
Endotoxin treatment for 6 h did not increase E-selectin, regardless of whether serum was present or not (data not shown). TNF,
used as a positive control, induced E-selectin to levels comparable
with Tat.
To test whether the E-selectin promoter is responsive to Tat, HeLa, or
HeLa-tat cells were transfected with a plasmid containing upstream regulatory sequences of the human E-selectin promoter driving
expression of luciferase (pE-Luc, Fig.
3A). The promoterless luciferase vector pGL3B was used as a control. Luciferase expression was increased ~6-fold compared with the pGL3B alone in the
HeLa-tat, but not in the HeLa cells. When endothelial cells
were co-transfected with a Tat-expressing plasmid and pE-Luc, there was
a 15-fold increase in luciferase expression when compared with
co-transfection with empty vector (pHOOK) and the pE-Luc. These results
suggest that the presence of Tat is necessary for the induction,
regardless of whether the cells are stably expressing the Tat protein
or whether the tat gene is introduced with the reporter
construct.
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Fig. 3.
Effects of Tat on an E-selectin
promoter-reporter construct. A, HeLa or HeLa-tat
cells were transfected with plasmid pE-Luc, containing upstream
regulatory sequences of the human E-selectin promoter driving
expression of luciferase or with the promoterless luciferase vector,
pGL3B. Light emission was detected with a luminometer and expressed as
luciferase light units. B, HUVEC were co-transfected with a
mammalian Tat-expression vector (pHOOK-tat) and pE-Luc and
luciferase activity was measured. pHOOK is the empty vector.
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Tat Stimulates NF-B Translocation in Endothelial Cells--
We
compared the ability of Tat or TNF to up-regulate B family members
by using an oligonucleotide containing the b site 3 of the human
E-selectin promoter in gel shift assays with nuclear proteins extracted
from HUVEC. After 20 min incubation with Tat there was very little
NF-B activation, while incubation with TNF strongly resulted in
considerable translocation at this time point (Fig.
4A). Neither medium alone nor
boiled Tat had any effect on B translocation. The composition of the
nucleoprotein complexes was investigated by antibody supershift
analyses. The slower migrating complexes contained p65/p50 heterodimers
(top arrow), while the faster migrating ones contained p50
homodimers (bottom arrow). When the extracts were incubated
with the RelB antibody, nucleoprotein complexes disappeared in the
TNF-treated, but not in the Tat-treated cells, suggesting the presence
of RelB in some of the complexes. At 6 h, Tat or TNF strongly
induced B binding to the oligonucleotide (Fig. 4B).
Furthermore, cells incubated with medium alone showed an increased
binding, suggesting that the addition of serum after the first hour of
incubation may be stimulatory to NF-B. The c-Rel antibody resulted
in a weakly supershifted product in the TNF, but not in the Tat-treated
samples, suggesting that there are qualitative differences between Tat
and TNF-stimulated complexes. In a separate experiment, gel shift
assays were performed with nuclear proteins from cells exposed to
control GST extracts for 6 h (Fig. 4B). NF-B
translocation was not detected in these extracts.
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Fig. 4.
Tat-dependent activation of
NF-B. Confluent HUVEC monolayers were
incubated with Tat, TNF, cell culture medium alone, or boiled Tat
(bTat). Nuclear extracts of cells exposed to the control GST
preparation for 6 h are shown in panel B. After
exposure to these agents for 20 min (panel A) or 6 h
(panel B), the cells were collected and nuclear proteins
extracted as described under "Experimental Procedures." The nuclear
extracts were assayed for the presence of active NF-B complexes by
EMSA using an oligonucleotide containing the proximal B site of the
human E-selectin promoter. Arrows point to two visible
NF-B complexes. The identity of the protein in the complexes was
analyzed by supershifting with 1 µg of p65, p50, p52, cRel, or RelB
specific antibodies. Results shown are representative of five
independent experiments.
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Western blot analyses with 0.5 µg of either cytoplasmic or nuclear
proteins corroborated that TNF-mediated translocation of p65 is
observed by 20 min, while Tat resulted in a delayed response that was
first detected at 2 h (Fig.
5A). Again, increased p65 expression in the cytoplasm of cells exposed to medium after 2 h
probably is a reflection of the re-introduction of serum into the
system at 1 h. Interestingly, at later time points, steady-state levels of nuclear p65 in TNF-treated extracts were higher than in
Tat-treated extracts. Boiled Tat had no effect at any of the time
points examined. To exclude the possibility that the nuclear proteins
may have been contaminated with cytoplasmic proteins during the
extraction, 15 µg of nuclear or cytoplasmic proteins from cells
exposed to the indicated treatments were immunoblotted with an actin
antibody (Fig. 5B). Levels of immunoreactive actin were
readily evident in the cytoplasm, but very little in the nucleus,
regardless of treatment. As an additional control for cross-contamination, HeLa or HeLa-tat cells were
fractionated using the same procedure as for the endothelial cells, and
15 µg of nuclear or cytoplasmic proteins subjected to Sp3
immunoblotting. Fig. 5C shows that Sp3 is only found in the
nucleus, but not the cytoplasm of the cells. Furthermore, immunoblots
for p65 were performed with only 0.5 µg of protein per well, while to
detect Sp3 or actin, 15 µg of proteins were required. Thus, it was
not possible to detect both p65 and actin on the same membrane. The fact that there is very little detectable actin in the nucleus, or Sp3
in the cytoplasm at these protein amounts makes the possibility of
cross-contamination unlikely. Independently prepared extracts have
yielded exactly the same results, validating our laboratories' cell
fractionation technique (data not shown). These results indicate that
the presence of p65 in the nucleus of Tat or TNF-treated cells, but not
cells exposed to media alone, is due to translocation and not
contamination. In contrast, when whole cell extracts were subjected to
p65 immunoblot analyses, the intensity of all the p65 bands was the
same for Tat or TNF at all time points tested (Fig.
6). These results demonstrate that
neither Tat nor TNF resulted in increased expression of p65 during the
exposure suggesting that the NF-B-mediated effects are due to its
translocation and not to increased expression.
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Fig. 5.
Kinetics of nuclear translocation of
NF-B p65 in response to Tat. Confluent
HUVEC were exposed to Tat, TNF, cell culture medium or boiled Tat
(bTat) for the indicated times. Cells were collected and nuclear
proteins extracted as described under "Experimental Procedures."
A, 0.5-µg aliquots of cytoplasmic (CF) and
nuclear fractions (NF) were analyzed for nuclear
translocation of p65 by Western blotting. Results shown are
representative of three separate experiments. B, 15 µg of
HUVEC CF and NF were analyzed for -actin. C, 15 µg of
CF and NF from HeLa or HeLa-tat cells were probed for the
nuclear transcription factor, Sp3 by Western blot.
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Fig. 6.
Expression levels of p65 after Tat
exposure. Confluent HUVEC exposed to Tat, TNF, or cell culture
medium for the indicated times were collected and whole cell proteins
extracted as described under "Experimental Procedures." Whole cell
lysates (0.5 µg) were analyzed for p65 expression levels via Western
blots. Results are representative of three separate experiments.
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Tat-mediated E-selectin Up-regulation Requires New Macromolecule
Synthesis--
To determine whether Tat-mediated E-selectin
up-regulation requires transcription or translation, cells were
pretreated with either actinomycin D or cycloheximide for 30 min prior
to exposure to Tat followed by measurements of E-selectin expression.
60% of cells were positive for E-selectin after exposure to Tat (Fig. 7). Actinomycin D or cycloheximide
treatment inhibited this up-regulation almost completely, demonstrating
that the Tat-mediated effects require the synthesis of new
macromolecules. Neither actinomycin D nor cycloheximide resulted in
endothelial cell apoptosis, as measured by annexin V staining (data not
shown).
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Fig. 7.
Effects of cycloheximide or actinomycin D on
Tat-stimulated E-selectin expression. Confluent HUVEC were exposed
to Tat for 6 h after a 30-min preincubation in the presence or
absence of either 5 µg/ml cycloheximide (CHX) or 2 µg/ml
actinomycin D (ActD). After exposure, the cells were
collected and processed for E-selectin measurements via FACS as
described under "Experimental Procedures." Regions M1 and M2 were
defined as described in the legend to Fig. 2. Results expressed as
mean ± S.E., n = 4.
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Tat-mediated E-selectin Up-regulation Requires NF-B
Activation--
Translocation of NF-B requires phosphorylation and
subsequent degradation of the inhibitory cytoplasmic IB.
Phosphorylation is therefore an essential component of this activation.
HUVEC were transduced with an adenovirus vector coding for a
recombinant IB super-repressor protein. This construct contains two
site-directed mutations at serines 32 and 36 preventing their
phosphorylation. The result is a constitutively inactive NF-B. To
control for any effects due to adenovirus transduction itself, an
adenovirus containing a GFP insert was used. EMSA analyses demonstrated
that the expression of this super-repressor completely abolished Tat or
TNF-mediated NF-B binding to the E-selectin B oligo (Fig. 8). The IB super-repressor adenovirus
completely inhibited p65 translocation to the nucleus in response to
either Tat or TNF (Fig. 9), and abolished
Tat-mediated E-selectin up-regulation (Fig.
10, A and B). A
recombinant adenovirus-GFP control had no effect on E-selectin
expression. Examination of GFP fluorescence indicated that close to
100% of the cells had been transduced with the virus (not shown).
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Fig. 8.
Inhibition of NF-B
activation by
AdIBSR.
Confluent HUVEC were transduced with an adenovirus construct encoding a
super-repressor (AdIBSR) form of IB at a multiplicity of
transduction = 30 plaque forming units/cell. 24 h after
transduction, cells were exposed to Tat, TNF, or cell culture medium
for 6 h. After exposure, the cells were collected and nuclear
proteins extracted as described under "Experimental Procedures."
The nuclear extracts were assayed for the presence of active NF-B
complexes by EMSA. The top arrow points to the shifted
complexes.
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Fig. 9.
Inhibition of NF-B
p65 translocation by
AdIBSR. After
transduction and exposure as described in Fig. 8, cells were collected,
fractionated, and nuclear proteins assayed for evidence of p65 nuclear
translocation by Western blot. The top panel shows p65 in
the CF, while the bottom panel shows p65 in the NF.
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Fig. 10.
Inhibition of Tat-mediated E-selectin
expression by
AdIBSR. After
transduction and exposure as described in the legend to Fig. 8, cells
were collected and processed for E-selectin surface expression
measurements via FACS. Since GFP fluorescence would interfere with
fluorescein isothiocyanate fluorescence, E-selectin expression in the
cells transduced with the control adenovirus (AdGFP) was
measured via streptavidin-biotin/cychrome fluorescence. Panel
A shows representative histograms. Panel B shows the
average ± S.E., n = 4.
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DISCUSSION |
Abnormalities in leukocyte-endothelial cell interactions have been
reported in HIV infection. For example, infection of monocytes results
in elevation of adhesion molecule LFA-1 (46) and a marked increase in
adherence to human capillary endothelial cell monolayers derived from
brain, lung, and skin.
E-selectin, one of the adhesion molecules activated in HIV infection,
is tightly regulated by multiple NF-B sites in its promoter (47),
and is transcriptionally silent until its rapid induction by
inflammatory cytokines or reactive oxygen species (48). Previous
reports have demonstrated that Tat increases E-selectin expression, a
phenomenon that is augmented in the presence of TNF (49). Furthermore,
studies have demonstrated increased monocyte adhesion to endothelium
exposed to Tat (50). Because E-selectin up-regulation in response to
cytokines requires an intact B site in the promoter (48, 51), the
possibility that Tat-mediated E-selectin up-regulation also requires
NF-B activation was investigated in these studies.
The closely apposed B sites in the promoter of the human E-selectin
gene are essential for maximal expression in response to TNF (39).
Instead of using a B consensus oligonucleotide in the gel-shift
studies described here, we chose to test an oligonucleotide containing
the E-selectin B site 3, located within 99 to 80 bases from the
transcriptional start site. NF-B binds preferentially to sites 2 and
3 and mutations within these sites are more specific at inhibiting
cytokine-mediated E-selectin expression (39). TNF activates
translocation and binding of p65/p50 heterodimers to this site 3 (51).
Likewise, Tat activated this heterodimer in endothelial cells, albeit
with delayed kinetics. Translocation of p65 and binding to the
E-selectin oligonucleotide were observed as early as 20 min after
initiation of TNF exposure, when only 20% of the cells were positive
for E-selectin after 2 h. In contrast, Tat-dependent
translocation and binding of p65 after 20 min and E-selectin expression
after 2 h were undetectable, suggesting that these agents activate
NF-B via different mechanisms. Indeed, Tat-mediated activation of
NF-B in lymphocytes is dependent upon a functional p56lck
tyrosine kinase, while TNF-mediated activation is not (33). However,
E-selectin levels were comparable after treatment with Tat or TNF for
5 h. The delayed response after Tat may be a reflection of the
fact that TNF recruits other factors to the E-selectin promoter,
forming higher order structures that include the high mobility group
protein Y(I), and ATF2/c-Jun heterodimers and cAMP-response element-binding proteins (52, 53). Even though Tat activates NF-B
translocation and binding to the site 3 of the E-selectin promoter,
there may be a delay in the recruitment of other unknown accessory
proteins to the E-selectin promoter, also delaying transcription of the
E-selectin gene. In fact, other investigators have demonstrated that
the rate-limiting step in E-selectin expression is transcription initiation (54). Therefore, anything that delays recruitment of factors
to the transcriptional initiation complex will delay expression of the
gene. Tat induces the expression of the adhesion molecules vascular
cell adhesion molecule 1, intercellular adhesion molecule 1, the
chemokine monocyte chemoattractant protein 1, and the cytokine IL-6.
The expression of these genes is partly regulated by NF-B. The
pattern of induction in response to Tat differed from that to TNF, with
higher peak levels that occurred earlier in response to Tat (55). In
these studies, Tat and TNF activated comparable levels of NF-B,
consistent with our present findings.
To map the region of Tat responsible for the E-selectin up-regulation,
truncated mutants with/without a functional activation domain were
tested along with the wild-type 86-amino acid Tat. All mutants were
tested as recombinant proteins. Even though the activation domain was
present in the mutants, amino acids beyond residue 48 have been deleted
and therefore the basic domain (amino acids 49 to 58) as well as the
region encoded by the second exon are absent. None of the mutants
retained the ability to induce E-selectin, suggesting that
carboxyl-terminal amino acids are important. Interestingly, Demarchi
et al. (14) have demonstrated that Tat-mediated NF-B
activation in HL3T1 cells requires the integrity of the basic domain of
Tat. Thus, deleting this basic domain would abolish Tat-mediated
NF-B activation and E-selectin up-regulation.
The family of NF-B transcription factors includes c-Rel,
RelB, p52, p50, and p65. NF-B is constitutively bound to the
inhibitory IB in the cytoplasm of non-B cells. Upon stimulation,
IB is phosphorylated, ubiquitinated, and degraded by the proteasome (56). This releases NF-B, which translocates to the nucleus and
activates transcription of specific genes. The diverse stimuli that
result in IB phosphorylation are still being investigated, but the
downstream effects are fairly well known. In the present study, either
TNF or Tat resulted in NF-B activation by IB phosphorylation because the B super-repressing adenovirus resulted in complete inhibition of NF-B translocation, particularly p65. The inhibition was also reflected in decreased binding of p65 to the E-selectin oligonucleotide and in decreased E-selectin expression. This effect was
specific to NF-B and not an artifact of adenovirus transduction because the control adenovirus-GFP had no effect. This control adenovirus also had no effect on p65 translocation as measured via
Western blot or gel shift analyses (not shown).
Our experiments have corroborated the ability of extracellular Tat to
affect cellular physiology. Tat in the culture medium could be binding
to a receptor whose activation and subsequent signal transduction may
result in NF-B translocation. In fact, the transfection studies with
E-selectin luciferase reporter constructs (Fig. 3) suggest that the
presence of a tat gene in the nucleus also activates the
E-selectin promoter. The reporter construct used for these studies
contains 840 base pairs upstream of the transcriptional start site of
the human E-selectin gene, which includes all 3 B sites. At this
time, we cannot exclude the contribution of B sites other than site
3 to the Tat-responsiveness of this promoter. Interestingly, E-selectin
promoter-driven luciferase expression was much higher in the
endothelial cells, maybe reflecting the restricted expression of the
E-selectin gene to these specific cells. When Tat is introduced via
liposomes into HL3T1 cells, the interferon-inducible protein kinase is
activated, which in turn phosphorylates IB and results in NF-B
translocation (14). This activation is independent of CDC42 and RAC,
two pathways shown to be indispensable for TNF-mediated E-selectin
transcription (51). Thus, different pathways are activated in response
to Tat or TNF. Nevertheless, kinetics of E-selectin expression are consistent with previous reports in HUVEC, where peak levels are detected by 4 h, returning to baseline by 24 h after TNF
induction (57, 58). On the other hand, even without transfection, Tat is able to enter cells, since its positively charged protein
transduction domain stimulates cellular uptake (59, 60) and its RGD
domain is responsible for binding to cell surface integrins (10). The RGD and basic domains of Tat stimulate and modulate the VEGF receptor Flk-1/KDR and components of the focal adhesion kinase in Kaposi's sarcoma cells (61, 62).
The Tat concentration required to up-regulate E-selectin was optimal at
500 ng/ml. A small percentage of HIV-infected patients have detectable
serum levels of Tat that are in the nanogram/ml range (63, 64).
However, this may be an underestimation of the actual levels because
Tat may be sequestered in lymphoid tissues, for example (64). When pure
Tat preparations were analyzed via Western blots, additional
Tat-specific bands were detected. Because Tat has 7 cysteine residues
in tandem (65, 66), these bands probably represent complexes formed by
intra- and intermolecular disulfide bridges, which are difficult to
dissociate, even in the presence of the denaturing and reducing
conditions of the gel. These Tat complexes may be inactive and
therefore, the actual concentration of active Tat may be considerably
lower than the protein concentration would suggest. These additional
Tat bands were not present in the S2-tat cell extracts
possibly because the reducing environments in the S2 and the E. coli cells are different. Nevertheless, the Tat expressed in the
S2 cells co-migrated with one of the Tat-specific bands seen in bacteria.
The up-regulation of E-selectin also requires new macromolecular
synthesis, as demonstrated by the inhibitory effect of cycloheximide or
actinomycin D. Several reports have suggested that cycloheximide in
combination with TNF treatment induces apoptosis in endothelial cells
(67). However, we did not detect any evidence of apoptosis in the time
frame of exposure to TNF (not shown). Because growth state and passage
number markedly affect cytokine-mediated adhesion molecule expression
in endothelial cells, the experiments were performed in cells 3 days
post-confluence. Studies that report increased apoptosis in
response to protein synthesis inhibitors and TNF have been performed on
exponentially growing endothelial cells. Interestingly, agents such as
endostatin have a pro-apoptotic effect selective for proliferating
endothelial cells (68, 69). Furthermore, in the present studies, the
inhibitors were added prior to the exposure to Tat or TNF, and
therefore, it is unlikely that increased apoptosis would have been
detected (67). Whether Tat is having a growth-inducing effect in these
cells is unlikely, since cells were quiescent and non-proliferating. In
other systems, and depending on the cell type, Tat may induce apoptosis
(63, 70) or act as a growth factor (71).
Recombinant Tat purified from a bacterial expression system may have
some level of lipopolysaccharide contamination, potentially stimulating
endothelial cells to produce TNF, and in an autocrine fashion, induce
E-selectin expression. However, IL-1 production in monocytic cells,
and IL-6 protein and mRNA in astrocytes, are Tat-mediated
NF-B-mediated effects that are independent of TNF- production
(23). Furthermore, endotoxin contamination is unlikely because boiled
Tat or GST control extracts had only a minor effect (<20%) on
activation of these pathways and a Tat-specific antibody successfully
inhibited Tat-mediated E-selectin up-regulation (Fig. 2C).
GST extracts alone had no effect when NF-B translocation was
assessed in endothelial cells (Fig. 4B). Therefore, any
co-purified bacterial proteins or endotoxin responsible for the
observed effects would have been present in these extracts. Endothelial
cells are able to secrete TNF in response to lipopolysaccharide, but
these effects are observed after prolonged incubation. In addition, endotoxin-mediated effects on HUVEC require the presence of
lipopolysaccharide-binding protein and soluble CD14, both present in
serum (72, 73). In the present studies, there was no serum during the
first hour of incubation with Tat, and the incubations were for no
longer than 6 h, again arguing against endotoxin contamination.
Endothelial cells play a central role in the inflammatory
process by secreting chemoattractants that recruit circulating
leukocytes to the site of injury. Thus, mechanisms that recruit
neutrophils or mononuclear cells to the interstitium will contribute to
endothelial cell injury and failure. A major route of HIV entry into
the brain is proposed to be through a virus-induced up-regulation of
E-selectin and V-CAM-1 expression. The infected monocytes bring HIV and
Tat in contact with the endothelium, activate surrounding microglia, and prime the surrounding endothelial cells (74). Because selectins mediate rolling, which is the initial step in leukocyte adhesion to
endothelial cells (75), increased expression of this molecule would
result in enhanced interactions between the endothelium and circulating
leukocytes. By taking advantage of the host inflammatory response, Tat
allows the virus to escape the vascular space and invade interstitial
spaces. Macrophages and monocytes, as viral reservoirs, secrete Tat, in
conjunction with cytokines and oxidants, which brings these molecules
near the by-stander endothelial cells. Given the long list of immune
system mediators whose expression is increased by Tat-mediated NF-B
activation, it is not surprising that HIV-infected patients exhibit
exuberant inflammatory responses. In conclusion, these studies have
linked the ability of Tat to activate NF-B and E-selectin with the
potential to modulate the interactions between leukocytes and the
endothelium and reset the balance between pro- and anti-inflammatory forces.
B-dependent Mechanism*
,
, and
TOP
ABSTRACT
INTRODUCTION
