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(Received for publication, February 2, 1995; and in revised form, June 2, 1995) From the
Activation of CD4 positive T cells is a primary requirement for
human immunodeficiency virus (HIV) entry, efficient HIV replication,
and progression to AIDS. Utilizing CD4 positive T cell lines and
purified T cells from normal individuals, we have demonstrated that
native envelope glycoproteins of HIV, gp160, can induce activation of
transcription factor, activated protein-1 (AP-1). The stimulatory
effects of gp160 are mediated through the CD4 molecule, since treatment
of gp160 with soluble CD4-IgG abrogates its activity, and CD4 negative
T cell lines fail to be stimulated with gp160. Immunoprecipitation of the gp160-induced nuclear extracts with
polyclonal antibodies to Fos and Jun proteins indicates that AP-1
complex is comprised of members of these family of proteins. The
gp160-induced AP-1 complex is dependent upon protein tyrosine
phosphorylation and is protein synthesis-independent. This stimulation
can also be abolished by inhibitors of protein kinase C, but it is
unaffected by calcium channel blocker or cyclosporine A. This gp160
treatment adversely affects the functional capabilities of T cells;
pretreatment of CD4+ T cells with gp160 for 4 h at 37 °C
inhibited anti-CD3-induced interleukin-2 secretion. Effects similar to
gp160 were seen with anti-CD4 mAb. The aberrant activation of AP-1 by
gp160 in CD4 positive T cells could result in up-regulation of
cytokines containing AP-1 sites, e.g. interleukin-3 and
granulocyte macrophage colony-stimulating factor, and concurrently lead
to T cell unresponsiveness by inhibiting interleukin-2 secretion.
The CD4 molecule is the binding site of the human
immunodeficiency virus via the envelope glycoprotein,
gp160/gp120(1) . This interaction occurs at a specific region
at the external domain of the CD4 molecule. There have been conflicting
reports on the ability of gp160/gp120 to transduce biochemical signals
through the CD4 molecule on T cells. While increase in intracellular
calcium, hydrolysis of phosphatidyl inositol, and activation of
tyrosine kinases have been demonstrated by
some(2, 3, 4, 5) , others have
failed to observe these events(6, 7) . The
interaction of the CD4 molecule with the nonpolymorphic Utilizing
soluble envelope glycoproteins of HIV-1, gp160, we have previously
demonstrated that CD4-mediated signals result in biological effects.
These include up-regulation of CD40 ligand in CD4+ T cells,
resulting in polyclonal B cell differentiation(15) ; induction
of IL-3, IL-6, and granulocyte macrophage colony-stimulating factor
mRNA and cytokine secretion, which induce increased
myelopoiesis(16) ; and increased expression of Fas antigen on
CD4+ T cells, resulting in accelerated apoptosis in peripheral
blood mononuclear cells(17) . To delineate the nature of the
biochemical signals transduced through the CD4 molecule on T cells, we
have investigated the ability of gp160 and anti-CD4 mAbs to induce
activation of the transcription factor, activated protein 1 (AP-1). Physiological activation of T cells through the T cell receptor
results in the activation of AP-1(18) . AP-1 is a collection of
homodimeric and heterodimeric protein complexes of the c-fos and c-jun proto-oncogene products(19) . These
proteins interact with a common DNA binding site, the TPA-responsive
element (TGA(C/G)TCA) and activate gene transcription(20) . The
binding of AP-1 to the TPA-responsive element has been attributed to
post-translational modification of preexisting members of the Fos and
Jun family of proteins, involving phosphorylation and dephosphorylation
events(21) . Our results have demonstrated that the CD4-induced
signals transduced by gp160 or anti-CD4 mAb gp160 in T cells result in
activation of AP-1 by a mechanism that involves post-translational
activation of Fos and Jun family of proteins. Depression of
antigen-specific T cell responses is a relatively early feature of HIV
infection and precedes the quantitative decline of CD4+ T
cells(22) . Several investigators have clearly demonstrated the
inhibitory effects of gp120 on normal T cell functions (for review, see (23) ). The mechanism of gp120-mediated inhibition of T cell
responses involves inhibition of intracellular calcium mobilization,
hydrolysis of inositol phosphates, and activation of protein kinase C,
and kinase activity of
p56
Peripheral blood lymphocytes were purified by
Ficoll-Hypaque density gradient centrifugation. T cells were purified
from peripheral blood lymphocytes by rosetting 2 times with
neuraminidase-treated sheep red blood cells as described
earlier(15) .
Figure 1:
Stimulation of CD4 positive T cells
with gp160 induces AP-1 activation. Stimulation of E6-1 cells was
carried out by the addition of medium alone (lane1, unsti) or various concentrations of gp160 (lanes2, 3, and 4) and gp120 (lanes5, 6, and 7), or 1 µg/ml of
anti-CD4 mAb (Leu3a, lane8) or 50 ng/ml PMA (lane9), for 4 h at 37 °C. Lane10 comprised of competition of AP-1 binding by 10
Figure 2:
Kinetics of the gp160-induced AP-1
activation. E6-1 cells were stimulated with 1 mg/ml gp160 for
various time intervals indicated. AP-1 binding was analyzed by EMSA.
The lowerband shows nonspecific binding (ns).
Figure 3:
Pretreatment of gp160 with soluble CD4-IgG
or anti-gp160 antibodies abrogates its activity. E6-1 cells were
stimulated with medium alone (lane1, unsti)
or 1 µg/ml gp160 (lanes2-8) in the
presence of 10 or 1 µg/ml soluble CD4-IgG (Genentech, CA) (lanes3 and 4) or 1 µg/ml bovine serum
albumin, (lane5), 1:1000, 1:3000 dilution of
polyclonal goat anti-gp160 antibodies (lanes6 and 7), or 1:1000 dilution of normal goat serum (lane8). EMSA were performed as described under
``Materials and Methods.''
Figure 4:
The gp160-induced AP-1 complex contains
Fos and Jun family of proteins. Nuclear extracts were generated from
E6-1 cells either unstimulated (lane1) or
stimulated with 1 µg/ml gp160 for 4 h at 37 °C (lanes2-8). The gp160-induced nuclear extracts were
incubated with medium (lanes1 and 2), 1
µg (in 1 µl), or 0.1 µg (in 0.1 µl) of antibodies to
Fos and Jun proteins (which recognize all of the members of
the Fos/Jun family of proteins) or normal rabbit serum for 1 h at 4
°C. Immune complexes were immunoprecipitated with Protein
A-Sepharose beads (Pharmacia), and supernatants were analyzed for AP-1
binding. Results are representative of five separate
experiments.
In order to further demonstrate
that gp160-induced activation of AP-1 was mediated through the CD4
molecule, CD4+ and CD4 negative T cell lines were analyzed. Fig. 5shows that gp160 could stimulate AP-1 activation in
CD4+ H9 cells, Molt4 cells, but not in CD4 negative mutant Jurkat
T cells (JN). Here again, pretreatment of gp160 with soluble CD4
abrogated AP-1 activation in CD4+ T cells. All of these cells
could be effectively induce AP-1 activation upon stimulation with PMA.
These results demonstrate that the stimulatory activity of gp160 on
AP-1 activation is mediated through the CD4 molecule.
Figure 5:
CD4 positive T cell lines, but not CD4
negative T cell lines could be induced by gp160 to increase AP-1
activation. H9 cells were stimulated with medium alone (lane1, unsti), or with PMA, gp160, or gp120 (lanes2, 3, and 4) in the presence
of soluble CD4 IgG (lanes5, 6, and 7); E6-1 cells were stimulated with medium alone (lane8), PMA and gp160 (lanes9 and 10) as positive controls. CD4 positive Molt4 and CD4
negative JN (mutant CD4 negative Jurkat cells) were stimulated with
medium alone (lanes1 and 7, unsti), gp160, gp120 (lanes2 and 3 and lanes8 and 9) in the presence of
soluble CD4-IgG (lanes4 and 5 and lanes10 and 11) or PMA alone (lanes6 and 12).
Figure 6:
gp160 can induce CD8- but not
CD8+ peripheral blood T cells to induce AP-1 binding. Upper
panel, purified T cells were stimulated with medium alone (lane1, unsti), 1 µg/ml gp160 (lanes2 and 3), or gp120 (lanes4 and 5). CD4 and CD8 positive T cells were
separated by anti-CD8 mAb-conjugated magnetic beads (Dynal, Great Neck,
NY). Adherent cells were denoted CD8 positive and nonadherent cells as
CD4 positive. Nuclear extracts were assessed for AP-1 binding by EMSA.
The arrow indicates specific binding for AP-1, and the lower
band indicates nonspecific binding (ns). Lower panel,
gp160 induced AP-1 binding in CD4+ peripheral blood T cells can be
abrogated by soluble CD4- IgG. Purified T cells were stimulated
with medium alone (lane1, unsti) or 1
µg/ml gp160 (lanes2 and 3) or gp120 (lanes4 and 5) in the presence of soluble
CD4-IgG (lanes3 and 5); 50 ng/ml PMA (lane6). Nuclear extracts of CD4+ T cells were
analyzed for AP-1 binding by EMSA.
Figure 7:
The gp160-mediated AP-1 binding is
dependent on tyrosine phosphorylation, activation of protein kinase C,
but not on protein synthesis or increase in intracellular calcium or
CsA. E6- 1 cells were pretreated with various concentrations of
cyclosporine A (CsA), herbimycin A (HA), verapamil (ver), cycloheximide (CHX), or H7 and
stimulated with gp160 for 4 h at 37 °C. Electromobility shift
assays were performed as described under ``Materials and
Methods.''
We have demonstrated that the addition of gp160 to CD4+
T cells induces activation of transcription factor AP-1 by signals
transduced directly through the CD4 molecule. Signals transduced
through the CD4 molecule on T cells has been shown to play an important
role in regulating T cell functional responses mediated through the T
cell receptor(8) . Earlier studies had implicated that the
binding (adhesion) of the CD4 molecule with its natural ligand, MHC
class II molecule, participated in T cell activation by stabilizing the
T cell receptor (TCR)-MHC interactions (34) . In addition,
inhibition of T cell activation by anti-CD4 mAbs in MHC class II
independent systems suggested that inhibitory signals were transduced
through the CD4 molecule(35) . In contrast, recent experiments
have indicated that positive signals may be induced via the CD4
molecule, either by anti-CD4 mAbs (36) or by HIV envelope
glycoproteins,
gp160/gp120(2, 3, 4, 5) . Although
the CD4-induced signals have been shown to synergize with anti-CD3 or
anti-TCR mAb (37) the aberrant persistent activation through
this molecule on T cells, may contribute to the pathogenesis of
disease, e.g. in HIV infection(38) . Cellular
activation plays a central role in HIV infection(22) . Virus
internalization, syncitium formation, and proviral replication have
been shown to require cellular activation(39, 40) .
Several investigators have demonstrated that binding of gp160/gp120 to
CD4 molecules on the cell surface results in activation of biochemical
signals(2, 3, 4, 5) . We have
demonstrated that binding of gp160, (at concentrations found in
vivo,(32) ) to CD4 molecules on T cells can induce
biological events, e.g. up-regulation of CD40
ligand(15) , secretion of IL-6, IL-3, granulocyte macrophage
colony-stimulating factor, interferon In the
present study, we have demonstrated that gp160 can induce activation of
AP-1 in CD4+ T cells. The presence of Fos/Jun family of
proteins in the gp160-induced AP-1 complex was confirmed by abrogation
of AP-1 binding in immunoprecipitation experiments. Further studies are
needed to determine the involvement and functional role of individual
Fos/Jun components in the gp160-induced AP-1 complex. The stimulatory
effects of gp160 are mediated through the CD4 molecule, since
pretreatment of gp160 with soluble CD4- IgG abrogates its
activity. Furthermore, cell lines expressing the CD4 molecule (H9,
Molt4), but not CD4 negative cell line (JN), can be induced by gp160 to
activate AP-1. gp160 can also stimulate peripheral blood CD4 positive
cells, but not CD8 positive T cells to activate AP-1. Finally, the
stimulatory effects of gp160 can be mimicked by anti-CD4 mAb. These
results clearly demonstrate AP-1 activation by direct stimulation
through the CD4 molecule. Post-translational modifications upon T
cell activation involve activation of pre-existing Fos/Jun by
intracellular kinases and phosphatases(19) . The observation
that protein synthesis inhibitor, CHX failed to abrogate (and in fact
augments) the gp160-induced AP-1 binding at 4 h suggests that
post-translational modification of preexisting Fos and/or Jun induces
AP-1 binding at 4 h. Tyrosine phosphorylation inhibitor, herbimycin A,
abrogated the gp160-induced AP-1 binding. In resting cells, the
pre-existing Jun is phosphorylated on three sites at the C-terminal
domain next to its DNA binding domains (21) ; phosphorylated
states of these sites inhibits DNA binding(41) . The activation
of Jun requires dephosphorylation of these sites, possibly by a protein
kinase C-activated phosphatase(42) . Calcineurin, the
phosphatase that modulates NFAT activity(43) , is not involved
in c-Jun dephosphorylation, since AP-1 binding is unaffected by
treatment of cells with cyclosporine A. While intracellular calcium
channel blocker, verapamil, failed to block AP-1 binding, the
requirement of protein kinase C activation for the gp160-mediated AP-1
binding was demonstrated by the addition of inhibitors, H7 and
calphostin. Understanding the precise nature of the
post-transcriptional modification involving activation of the Ha-Ras
oncoprotein(44, 45) , mitogen-activated protein kinase
pathway(46) , or the newly described JNK and SAPK pathways (47, 48) may give an insight into the regulatory role
of CD4-mediated signals in T cell activation. AP-1 has been
demonstrated to be target for T cell clonal anergy, as demonstrated by
down-modulation of AP-1 binding and transactivation in anergized T cell
clones(49) . We and others have previously demonstrated that
pretreatment of CD4+ T cells with envelope proteins of HIV-1 can
induce unresponsiveness of T cells upon stimulation through
TCR Given
that the promoters of IL-2, IL-3, IL-6, granulocyte macrophage
colony-stimulating factor, TCR
Volume 270,
Number 33,
Issue of August 18, pp. 19364-19369, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
T Cells (*)
2 domain
of the MHC (
)class II molecule has been demonstrated to play
a vital role in activation of mature T cells and in T cell development
in the thymus(8) . Several studies have now demonstrated that
the CD4-MHC class II interaction is essential for effective signal
transduction, at low antigen concentrations, to increase the avidity,
in co-receptor-dependent systems(9, 10) . Studies
demonstrating the association of the src homologous tyrosine
kinase p56 and the putative p32 G-protein with
the cytoplasmic tail of the CD4 molecule have demonstrated that
biochemical signals can be transduced through the CD4
molecule(11, 12) . In this respect, exposure of
CD4+ T cells to anti-CD4 mAb or HIV gp120 has been shown to induce
activation of the Raf-1-related 110-kDa polypeptide, and
phosphatidylinositol 3- and phosphatidylinositol 4-kinases (13) and activation of NF-
B(14) .(24, 25, 26, 27, 28) .
The reduced proliferative responses were attributed to inhibition of
decreased IL-2 mRNA expression and IL-2 secretion(25) . In this
study, we have suggested that binding of envelope glycoproteins of HIV
to CD4+ T cells induces aberrant activation of the transcription
factor AP-1 (which plays a critical role in IL-2 gene transcription, (18) ) and results in inhibition anti-CD3 mAb-induced IL-2
secretion.
Envelope Glycoproteins
gp160 and gp120 were
purified from culture supernatants of a clone of HIV-infected Hut-78
cells, 6D4, as described earlier(28) . Briefly,
supernatant of cells grown in serum-free HB104 medium was concentrated
and passed through a lentil-lectin Sepharose column. Glycoproteins were
eluted with 400 mM
-methyl mannoside. gp160 was further
purified by affinity chromatography over anti-HIV mAb-Sepharose 4B column. The envelope glycoprotein preparations
were >95% pure and were not contaminated with endotoxins, as tested
by the Limulus amoebocyte lysate assay (E-TOXATE, Sigma).
Antibodies and Reagents
The following reagents and
resources were used: mAb to CD4 (Leu3a, IgG1; Becton Dickinson,
Mountainview, CA); mAb to CD3 (mAb 454, IgG2a, gift from Dr. N.
Chiorazzi, North Shore University Hospital, Manhasset, NY); nonimmune
mouse Ig (mIg; Chrompure IgG, Jackson ImmunoResearch, West Grove, PA). Polyclonal antibodies to Fos and Jun proteins used were as
follows; rabbit anti-c-Jun/AP1 and anti-c-Fos (Santa Cruz
Biotechologies, Santa Crus, CA) recognize all of the members of the
Fos/Jun family of proteins. Polyclonal rabbit anti-gp120 was developed by Advanced BioScience Labs Inc., Kensington, MD;
soluble CD4-IgG was a gift from Genentech, San Fransisco, CA.
Herbimycin A (Life Technologies, Inc.), cyclosporine A (Sandoz, East
Hanover, NJ), H-7, cycloheximide, 2-mercaptoethanol, verapamil, and
phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma.
Cells
CD4 positive clone of Jurkat T cells,
E6-1, obtained from the National Institutes of Health AIDS
Reference Reagent Program, Bethesda, MD, (donated by Dr. A. Weiss, (29) ) was maintained in RPMI 1640 medium (Whittaker)
supplemented with pennicillin and streptomycin and 10% fetal calf
serum. CD4 positive T cells H9, Molt4 were obtained from ATCC, Betheda,
MD. CD4 negative Jurkat T cells (JN) were mutant CD4 negative
(CD3+) cells by fluorescence-activated cell sorting analysis
following staining with fluorescein isothiocyanate-conjugated anti-CD3
mAb and anti-CD4-conjugated with phycoerythrin (Becton Dickinson,
Mountainview, CA).Immunomagnetic Separation of CD4+ and CD8+ T
Cells
Purified T cells stimulated with medium alone or with
gp160 for 4 h were incubated with anti-CD8 mAb conjugated
immunomagnetic beads (Dynal, Great Neck, NY) for 30 min at 4 °C on
a rotating shaker, as recommended by the manufacturer. The cells were
subjected to a magnetic field, and the unbound cells (designated CD8
negative, CD8- T cells) were carefully aspirated. The cells bound
to the beads were designated CD8 positive (CD8+) and were >90%
CD4+ as determined by flow cytometry. Nuclear proteins were
extracted as described below.Nuclear Extracts
Small scale nuclear extracts were
made from 2 10
unactivated or activated E6-1
cells as described previously(18) . Unless otherwise stated,
cells were stimulated with medium alone or various stimuli for 4 h at
37 °C. Cells were washed and resuspended in 10 mM Tris, pH
7.4, 10 mM NaCl, 3 mM MgCl
, 0.5 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride
and lysed by the addition of Nonidet P-40 to a final concentration of
0.5%. Nuclei were pelleted and washed in the same buffer without
Nonidet P-40, and nuclear proteins were extracted in buffer C (20
mM HEPES, pH 7.4, mM 0.42 NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 25% (v/v) glycerol, 0.01%
NaN
)(30) . After pelleting neclear debris, the
supernatant was removed and diluted with an equal volume of buffer D
(20 mM HEPES, pH 7.4, 50 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 0.01% NaN). This extract was
used directly in the electrophoretic mobility shift assay (EMSA). The
equivalence of the extracts was verified by protein estimation using
the BCA protein kit (Pierce).EMSA
Double-stranded oligonucleotides
corresponding to the consensus AP-1 binding sequence (5`-CGC TTG ATG
AGT CAG CGC GAA-3` (31) ) were obtained commercially (Promega,
Madison, WI) and end-labeled with [P]ATP
and polynucleotide kinase. In some experiments, the binding site of
AP-1 to the human IL-2 promoter (5`-AATTCCAAAGAGTCATCAGA-3`) was used
as a competitor(18) . For each binding reaction, 10,000 cpm
(0.2-0.5 ng) of end-labeled oligonucleotide was incubated for 30
min at room temperature with 5-8 µg of nuclear extract in the
presence of 3 mg of sheared poly(dI-dC) (Pharmacia Biotech Inc.). The
resulting DNA-protein complexes were analyzed by electrophoresis at 4
°C of 4% polyacrylamide gels. Unlabeled oligonucleotides used for
competitions were added to nuclear extracts and poly(dI-dC) prior to
the addition of labeled probe. Gels were dried on a gel drier (Bio-Rad)
and visualized by autoradiography. In some experiments,
nuclear extracts were incubated with antibodies to c-Jun, c-Fos, or
normal rabbit serum for 1 h on ice, followed by incubation with Protein
A-conjugated Sepharose (Pharmacia) for 30 min at 4 °C. After
centrifugation at 5000
g for 10 min, supernatants were
analyzed for AP-1 binding in EMSA, as described above.
IL-2 Secretion
CD4+ E6-1 cells,
CD4- JN cells, or purified CD4+ T cells were pretreated with
medium, various concentrations of gp120, or anti-CD4 mAb (mAb Leu3a)
followed by stimulation with anti-CD3 mAb (mAb 454) plus 10 ng/ml PMA
for 24 h. Culture supernatants were collected and analyzed for the
presence of IL-2 by commercial ELISA kit (R & D Systems,
Minneapolis, MN) according to the manufacturer's protocol.
gp160-induced AP-1 Activation in CD4+ T Cells
Fig. 1shows that the basal level of AP-1 activation in
the CD4+ E6-1 cells could be enhanced by stimulation with
gp160 at a concentration as low as 0.01 mg/ml. This concentration of
envelope proteins has been previously reported in the serum of
HIV-infected individuals(32) . Soluble gp120, anti-CD4 mAb (Leu3a) and PMA alone could also induce activation of AP-1 in
the CD4+ E6-1 cells. Specific binding was demonstrated by
abrogation of the AP-1 binding in the presence of excess unlabeled
oligonucleotides corresponding to the AP-1 site in the IL-2 promoter
(competitor, lane10). The kinetics of the induction
of the proteins was examined by using nuclear extracts from E6-1
cells stimulated for increasing amounts of time. The gp160-induced AP-1
binding was observed within 30 min, peaked at 4 h, persisted for 24 h
of stimulation (Fig. 2). The gp160-induced AP-1 binding
was specific, since it could be abrogated by pretreatment of gp160 with
goat anti-gp120 polyclonal antibody (Fig. 3, compare lane2 with lanes6 and 7); normal goat serum had no significant effect on the
stimulatory effect of gp160 (lane8). That
the constituents of the gp160-induced AP-1 complex comprises Jun and
Fos components was confirmed by abrogation of AP-1 binding by
immunoprecipitation of the gp160-stimulated nuclear extracts with
polyclonal antibodies to c-Jun and c-Fos (Fig. 4); even the basal level binding
of AP-1 was inhibited by the addition of these antibodies. Normal
rabbit serum had no significant effect on the gp160-induced AP-1
binding. These results indicate that gp160 can induce activation of
AP-1 in CD4+ T cells and that the AP-1 complex consists of Fos/Jun
family of proteins.
cold AP-1
oligonucleotides corresponding to the IL-2 AP-1 site. The hollowarrow indicates mobility of free probe, and the solidarrow indicates the position of specific AP-1 binding.
The result is a representative of at least five separate
experiments.
The gp160-induced Activation of AP-1 Was Mediated through
the CD4 Molecule
In order to demonstrate that the cell surface
molecule involved in the gp160-mediated stimulatory effects was CD4,
gp160 was first preincubated with soluble CD4- IgG for 30 min at
4 °C, prior to addition to the cell cultures. Fig. 3shows that the stimulatory activity of gp160 on
E6-1 cells could be abrogated by pretreatment of gp160 with
soluble CD4-IgG (compare lane2 with lanes3 and 4). An irrelevant protein, bovine serum
albumin, did not have any effect on the gp160-induced activation of
AP-1 (lane5).
Gp160 Induced AP-1 Activation in Peripheral Blood
CD4+ T Cells
In order to determine the ability of gp160 to
activate physiological cells and peripheral blood T cells, purified T
cells were first stimulated with gp160 for 4 h at 37 °C. CD4+
T cells were separated from CD8+ T cells by negative selection
using anti-CD8 mAb-conjugated magnetic beads; cells bound to the beads
were designated CD8+, and unbound T cells designated CD8-. Fig. 6shows that stimulation of CD8- T cells
(>90% CD4+ by flow cytometry) with gp160 induced AP-1
activation (upperrightpanel) no activation
of AP-1 occurred in the CD8+ T cells (upperleftpanel). The stimulatory effects of gp160 on CD8- T
cells could be abrogated by pretreatment of gp160 with soluble
CD4- IgG (lowerpanel). Soluble CD4- IgG
itself did not induce activation of AP-1 (data not shown).
Signal Requirements for the gp160-induced AP-1
Activation
In order to investigate the nature of the signals
involved in the activation of AP-1 by gp160, several pharmacological
inhibitors were utilized. Fig. 7shows that addition of
herbimycin A (HA, inhibitor of tyrosine phosphorylation), and H7 (inhibitor of protein kinase C) abrogated gp160-induced
AP-1 activation. A more specific inhibitor of protein kinase C
activity, calphostin (33) also inhibited the gp160-induced AP-1
binding (data not shown). Cyclosporine A (CsA) and the protein
synthesis inhibitor, cycloheximide (CHX) had no significant
effect on activation of AP-1, as did the calcium channel blocker,
verapamil (ver). These studies indicate that tyrosine
phosphorylation and activation of protein kinase C were involved in the
mechanism of the gp160-induced activation of AP-1 binding.
gp120 Inhibits IL-2 Secretion by CD4+ T
Cells
E6-1 cells or purified peripheral blood CD4+ T
cells were pretreated with 1 µg/ml gp120 or anti-CD4 mAb for 4 h at
37 °C (which induced AP-1 binding) and stimulated with anti-CD3 mAb
plus PMA for an additional 24 h at 37 °C, and culture supernatants
were analyzed for IL-2 secretion. Table I shows that gp120- or
anti-CD4 mAb-treated E6-1 cells and CD4+ peripheral blood T
cells were markedly inhibited in their ability to secrete IL-2 in
response to anti-CD3 mAb. Pretreatment of the CD4 negative T cells (JN)
cells with gp120 or anti-CD4 mAb, however, had no effect on anti-CD3
mAb plus PMA-induced IL-2 secretion.
, tumor necrosis factor
(16, 17) , and up-regulation of Fas antigen (17) on CD4 T cells. These observations
unequivocally demonstrate that gp160 can transduce signals through the
CD4 molecule in T cells culminating into biological events.
CD3
complex(23, 24, 25, 26) . It is
possible that the stimulatory effect of gp160 on AP-1 binding,
involving the repressive members of the Jun family, i.e. JunB(50, 51) , which may inhibit IL-2 gene
transcription. Our previous studies have demonstrated that pretreatment
of T cell clones with envelope glycoproteins or anti-CD4 mAb inhibited
IL-2 secretion at the transcriptional level(25, 26) .
In this study, we have shown that pretreatment of the CD4+ T cells
with gp160 or anti-CD4 mAb for 4 h at 37 °C (which results in
activation of AP-1 binding) inhibits anti-CD3 plus PMA-induced IL-2
secretion. Since the inhibition of IL-2 secretion by gp120 in these
experiments was independent of the presence of antigen presenting
cells, it can be hypothesized that signals mediated through the CD4
molecule in T cells, which results in activation of AP-1, may interfere
with signal transduction through the TCRCD3 complex., and the HIV long terminal repeat
contain AP-1 binding
sites(18, 52, 53, 54, 55, 56) ,
it is possible that the gp160-induced activation of AP-1 in T cells can
regulate the expression of these molecules. Since c-Jun and c-Fos have
also been implicated in the mechanism of
apoptosis(57, 58) , it is possible that gp160-induced
AP-1 activation may play a significant role in apoptosis mechanisms in
HIV infection. In conclusion, we have demonstrated that soluble
envelope glycoproteins of HIV-1, gp160, by binding to the CD4 molecule
on T cells, may transduce signals that result in aberrant activation of
AP-1. These effects by gp160, or by HIV itself in vivo, may
contribute to biological events, e.g. enhanced HIV
replication, hypergammaglobulinemia, increased cytokine secretion,
hypercellularity in bone marrow, apoptosis, and induction of T cell
unresponsiveness.
)
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 Y.-M. Yang, W. C. Hatch, Z.-Y. Liu, B. Du, and J. E. Groopman {beta}-Chemokine Induction of Activation Protein-1 and Cyclic AMP Responsive Element Activation in Human Myeloid Cells Cell Growth Differ., April 1, 2001; 12(4): 211 - 221. [Abstract] [Full Text]
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 T. H. Mogensen and S. R. Paludan Molecular Pathways in Virus-Induced Cytokine Production Microbiol. Mol. Biol. Rev., March 1, 2001; 65(1): 131 - 150. [Abstract] [Full Text] [PDF]
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 A. A. Kapasi, S. Fan, and P. C. Singhal Role of 14-3-3{epsilon}, c-Myc/Max, and Akt phosphorylation in HIV-1 gp 120-induced mesangial cell proliferation Am J Physiol Renal Physiol, February 1, 2001; 280(2): F333 - F342. [Abstract] [Full Text] [PDF]
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R. L. Hengel, B. M. Jones, M. S. Kennedy, M. R. Hubbard, and J. S. McDougal Markers of Lymphocyte Homing Distinguish CD4 T Cell Subsets That Turn Over in Response to HIV-1 Infection in Humans J. Immunol., September 15, 1999; 163(6): 3539 - 3548. [Abstract] [Full Text] [PDF]
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P. A. Klimiuk, J. J. Goronzy, and C. M. Weyand IL-16 as an Anti-Inflammatory Cytokine in Rheumatoid Synovitis J. Immunol., April 1, 1999; 162(7): 4293 - 4299. [Abstract] [Full Text] [PDF]
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W. Popik, J. E. Hesselgesser, and P. M. Pitha Binding of Human Immunodeficiency Virus Type 1 to CD4 and CXCR4 Receptors Differentially Regulates Expression of Inflammatory Genes and Activates the MEK/ERK Signaling Pathway J. Virol., August 1, 1998; 72(8): 6406 - 6413. [Abstract] [Full Text] [PDF]
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L. Briant, V. Robert-Hebmann, C. Acquaviva, A. Pelchen-Matthews, M. Marsh, and C. Devaux The Protein Tyrosine Kinase p56lck Is Required for Triggering NF-kappa B Activation upon Interaction of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 with Cell Surface CD4 J. Virol., July 1, 1998; 72(7): 6207 - 6214. [Abstract] [Full Text] [PDF]
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C. Guillerm, N. Coudronniere, V. Robert-Hebmann, and C. Devaux Delayed Human Immunodeficiency Virus Type 1-Induced Apoptosis in Cells Expressing Truncated Forms of CD4 J. Virol., March 1, 1998; 72(3): 1754 - 1761. [Abstract] [Full Text] [PDF]
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L. Briant, V. Robert-Hebmann, V. Sivan, A. Brunet, J. Pouyssegur, and C. Devaux Involvement of Extracellular Signal-Regulated Kinase Module in HIV-Mediated CD4 Signals Controlling Activation of Nuclear Factor-{kappa}B and AP-1 Transcription Factors J. Immunol., February 15, 1998; 160(4): 1875 - 1885. [Abstract] [Full Text] [PDF]
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 S. M. L. Tamma, N. Chirmule, H. Yagura, N. Oyaizu, V. Kalyanaraman, and S. Pahwa CD4 Cross-Linking (CD4XL) Induces RAS Activation and Tumor Necrosis Factor-alpha Secretion in CD4+ T Cells Blood, August 15, 1997; 90(4): 1588 - 1593. [Abstract] [Full Text] [PDF]
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 L. Briant, N. Signoret, M. Gaubin, V. Robert-Hebmann, X. Zhang, R. Murali, M. I. Greene, D. Piatier-Tonneau, and C. Devaux Transduction of Activation Signal That Follows HIV-1 Binding to CD4 and CD4 Dimerization Involves the Immunoglobulin CDR3-like Region in Domain 1 of CD4 J. Biol. Chem., August 1, 1997; 272(31): 19441 - 19450. [Abstract] [Full Text] [PDF]
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S. Bounou, N. Dumais, and M. J. Tremblay Attachment of Human Immunodeficiency Virus-1 (HIV-1) Particles Bearing Host-encoded B7-2 Proteins Leads to Nuclear Factor-kappa B- and Nuclear Factor of Activated T Cells-dependent Activation of HIV-1 Long Terminal Repeat Transcription J. Biol. Chem., February 23, 2001; 276(9): 6359 - 6369. [Abstract] [Full Text] [PDF]
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