From the Unité 571 du CNRS,
Bâtiment 430, Université Paris-Sud, F-91405 Orsay Cedex
and § Unité de Biologie des Rétrovirus, Institut
Pasteur, 28 rue du Dr. Roux,
F-75724 Paris Cedex 15, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the human lymphoblastoid T cell line
JJhan-5.1, stably transfected with a human immunodeficiency virus-1
long terminal repeat luciferase vector, the level of luciferase
activity is dependent on activation of the nuclear factor B
(NF-
B) transcription factor. Tumor necrosis factor-induced
luciferase activity was not modified in JJhan-5.1 cells co-cultivated
with murine adenocarcinoma EMT-6 cells but was strongly decreased when
nitric oxide (NO) synthase 2 expression was induced in these cells. Two
NO synthase inhibitors counteracted this inhibitory effect. Tumor
necrosis factor-
binding to JJhan-5.1 cells was not modified after
incubation with EMT-6 cells. Viability and protein synthesis in
JJhan-5.1 cells were also unchanged. Induction of NF-
B DNA binding
activity was inhibited when EMT-6 cells expressed NO synthase 2 activity. Aminoguanidine, which completely abolished nitrite
production, prevented this inhibition. NF-
B activation was also
strongly inhibited by S-nitrosoglutathione but was
marginally affected by
N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine. Taken together, these results indicated that NO-related species, released by EMT-6 effector cells and probably different from NO itself,
inhibited NF-
B activation in human lymphoblastoid target cells.
Consequently, transcriptional activity of a long terminal repeat-driven
luciferase gene construct was markedly diminished.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nitric oxide is a diffusible, cell-permeable reactive molecule
synthesized by three P-450-type NO synthase
(NOS)1 isoenzymes, NOS 1, 2, and 3. NOS 1 and 3, usually constitutive, are transiently activated by
Ca2+/calmodulin binding and were initially characterized in
neuronal and vascular endothelial cells, respectively (reviewed in
Refs. 1 and 2). The cytokine-inducible NOS 2 can be expressed in a wide
range of cell types and tissues and, once induced, generates a
sustained flux of NO that has cytotoxic consequences for tumor cells
and pathogens (3, 4). For instance, nitric oxide and related reactive
nitrogen species have antiviral properties, most frequently shown for
DNA viruses but also, in vivo, for the RNA Coxsackie B3
virus and the Friend leukemia murine retrovirus (5-8). At a molecular
level, the antiviral mechanisms of nitrogen oxides are only partially
understood. Viral DNA synthesis requires efficient deoxyribonucleotide
synthesis. It has been proposed that inhibition of viral ribonucleotide
reductase might partially explain the inhibition of DNA virus
replication (6, 7). This mechanism might also be relevant to
retroviruses, as reverse transcriptase and ribonucleotide reductase
inhibitors synergize against HIV infection (9). NO can also affect the
viral life cycle by altering intracellular signals required for viral
replication, including transcription factors. The transcription factor
Zta, which mediates the switch from latent to lytic infection in
Epstein-Barr virus-infected cells, is down-regulated by NO (10). Two
C- nitroso compounds have also been shown to inhibit HIV-1
infectivity by ejecting zinc from a zinc finger transcription factor
(11). The importance of NO production in HIV-1 infection has been
already established. It has been proposed that activation of neuronal
NOS 1 activity by the gp120 envelope protein might explain the
neurotoxicity of the virus (12). There is also evidence that gp120 and
gp41 protein determinants can induce NOS 2 activity in human glial cultures and that retroviral infection induces NOS 2 expression in
human monocytes (13, 14). These results have suggested a detrimental
role for NO in HIV-associated pathology. However, analysis of nitrite
serum levels in HIV-1-infected, seronegative children has led to the
conclusion that NO might be involved in limiting the infection (15).
Moreover, the onset of functional NOS 2 expression in HIV-infected
monocyte cultures was correlated with a sudden drop in reverse
transcriptase activity (13), a result that could be interpreted as a
negative effect of NO on retroviral replication. The long terminal
repeat (LTR) sequence of HIV-1 contains positive and negative
transcriptional regulatory elements that control the expression of the
integrated proviral genome (reviewed in Ref. 16). Among these, two
NF-B binding sites constitute the viral enhancer (16, 17). NF-
B
is a p65(RelA)/p50 heterodimeric transcription factor belonging to the
NF-
B/Rel family, retained inactive in the cytoplasm by association
with inhibitory molecules (I-
B) (reviewed in Ref. 18).
Phosphorylation of I-
B and, in most cases, subsequent proteolysis
enables translocation of NF-
B to the nucleus, where its binding to
cognate DNA sequences transregulates gene expression. In different
models, NF-
B activation has been shown to be inhibited by nitrogen
oxides (19-23), although in others, including human peripheral blood
mononuclear cells, NO activated the NF-
B signaling pathway (24-26).
Since viral components are able to trigger NO production in host cells,
it was of interest to determine whether NO might influence the
NF-
B-dependent transcriptional activity of the HIV-1
LTR, either positively or negatively. Bioactive species derived from
NOS activities include NO, N2O3, nitrosothiols, dinitrosyl-iron complexes and peroxynitrite, and probably other species
as well, each exhibiting a specific reactivity (1). This complexity
cannot be reproduced by using chemical NO precursors, although these
molecules have been used extensively in previous studies and have
therefore contributed most of our current knowledge about NF-
B
modulation by nitrogen oxides. The role of endogenously produced NO has
been examined in a few models, involving apparently constitutive NOS 1 or 3 activities (21, 23, 27). So far, the effect of NOS 2 products on
NF-
B induction in target cells has not been evaluated.
In the present work, we used murine EMT-6 cells activated for NOS 2 expression as effector cells capable of producing physiological concentrations of NO-related species over a prolonged period (28), to
test the hypothesis that NO might modulate HIV-1 LTR activity through
NF-B function. We chose a human lymphoblastoid target cell line
transfected with a luciferase reporter gene controlled by the HIV-1 LTR
promoter sequence.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Cultures and Reagents--
The JJhan-5.1 clone was derived
from the J. Jhan cell line (itself derived from Jurkat cells) stably
co-transfected with a LTR-Luc plasmid carrying the luciferase reporter
gene under the control of the U3R (BglII-HindIII
fragment) of HIV-1 LTR and the pSV2-TK-Neo vector carrying the G418
resistance gene under the control of the SV40 early promoter (29).
JJhan-5.1 and J. Jhan human lymphoblastoid T cell lines, as well as the
EMT-6 murine mammary adenocarcinoma cell line (28), were grown in RPMI
1640 medium (Life Technologies, Inc., Cergy-Pontoise, France),
supplemented with antibiotics, 5% heat-inactivated fetal calf serum,
300 mg/ml L-glutamine, and 25 mM HEPES, pH 7.4. G418 (Life Technologies, Inc.) was added in JJhan-5.1 cultures at 400 µg/ml and removed 24 h before experiments. Murine recombinant
IFN- (specific activity, 1 × 107 units/mg) was
provided by Dr. Adolf (Ernst-Boehringer Institut für Arzneimittel
Forschung, Vienna, Austria). Human recombinant TNF-
(specific
activity, 1.6 × 106 units/mg) was a gift from Dr.
Bousseau (Rhône-Poulenc Rorer, Vitry-sur-Seine, France). LPS
from Salmonella enteritidis, TPA, PHA, and
L-NAME were purchased from Sigma. Carboxy-PTIO
(2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) was
from France Biochem (Meudon, France). Human 125I-labeled
TNF-
(1.2 MBq/µg, 1 µg/ml) was purchased from Life Science
Products (Les Ulis, France). Plastic cell culture flasks and dishes
were from CML (Nemours, France). GSNO and DETA-NO were purchased from
Alexis Corp. (San Diego, CA) and RBI (Natick, MA), respectively.
Stimulation of EMT-6 Cells--
EMT-6 cells (3 × 106) were seeded in a 75-cm2 culture flask,
cultured for 24 h, and stimulated overnight with 40 units/ml
murine IFN- and 100 ng/ml LPS for NOS 2 induction (28). When
mentioned, L-NAME or aminoguanidine were added at the same
time and at a concentration of 2 mM.
Exposure to NOS 2 Activity and Luciferase Induction in JJhan-5.1
Cells--
JJhan-5.1 cells (2 × 107) were
co-cultured in 20 ml for up to 4 h with subconfluent EMT-6 cells
activated for NOS 2 expression, as described above. Thereafter,
non-adherent JJhan-5.1 cells were harvested, centrifuged, and adjusted
to 4 × 106 cells/ml. The supernatant was subsequently
assayed for nitrite content. JJhan-5.1 cells (2 × 106) were exposed in duplicate to 500 units/ml
TNF- or to 0.5 µM TPA and 5 µg/ml PHA, for 4-6 h.
Cell lysates were assayed for luciferase activity using a
luciferase assay system (Promega) following the manufacturer's
instructions. Results are expressed as the percentage shown in Equation 1.
![]() |
(Eq. 1) |
Whole Cell Extracts for EMSA--
JJhan-5.1 cells co-cultured
with EMT-6 cells or exposed to GSNO or DETA-NO for 4 h were
harvested and then stimulated with TNF- for 30 min (2 × 106 cells/well/500 µl). The cells were then collected,
rinsed twice with ice-cold phosphate-buffered saline, and resuspended
in 3 volumes of 10 mM HEPES buffer, pH 7.9, containing 0.1 mM EDTA, 0.4 M NaCl, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 µg/ml leupeptin, 4 µg/ml aprotinin, 1.5 µg/ml pepstatin, 1 µg/ml chymostatin, and 2 µg/ml antipain (30). Cells were frozen in liquid N2 and
then thawed and centrifuged at 100,000 × g for 10 min.
The supernatant, consisting of whole cell extract, was used for
EMSA.
Electrophoretic Mobility Shift Assay (EMSA)--
Binding
reactions were prepared in 10 µl by mixing 10 µg of protein extract
with 2 µl of binding buffer containing 20% Ficoll, 100 mM HEPES, pH 7.5, 300 mM KCl, 10 mM
dithiothreitol, 0.05% Nonidet P-40, 0.5 mg/ml bovine serum albumin,
and 10 mg/ml salmon sperm DNA (30). Finally, 10,000 cpm of
32P-radiolabeled (Klenow fragment, Appligene)
double-stranded oligonucleotide was added, and the mixture was
incubated at 4 °C for 20 min. Samples were loaded on a 5%
polyacrylamide gel electrophoresis and run at 10 V/cm for 2 h in
0.5 × TBE buffer. After electrophoresis, gels were dried and
exposed to Fuji XR films at 70 °C. The sequence of the
B
oligonucleotide, corresponding to a HIV-1 site, was as follows.
![]() |
![]() |
TNF Binding Assay--
The assay was performed following the
method of Baglioni et al. (31). J. Jhan cells were
co-cultured for 4 h with activated EMT-6 cells, as described
above. They were adjusted to 20 × 106 cells/ml and
incubated for 6 h at 4 °C in 100 µl of culture medium containing 0.1-5 nM human 125I-labeled
TNF-. Another set of similar samples was incubated with a 40-fold
excess of cold TNF-
to determine the nonspecific binding. The
incubation mixture was then transferred over 400 µl of a silicon oil
phase (Rhodorsil, Rhône Poulenc Rorer) and centrifuged for 10 min
at 10,000 × g. The cell pellet was carefully collected
and lysed with 0.5 ml of 0.1% Triton X-100. Radioactivity in the
lysate and in the aqueous phase was determined by liquid scintillation
counting. Calculation of the TNF binding dissociation constant
(Kd) and the number of binding sites was done using
Multifit 2.0 software (Day Computing, Cambridge, UK).
Nitrite Assay-- Nitrite concentrations in cell culture supernatants were measured with the Griess reagent, as described previously (32).
Cell Viability Assay-- The viability of JJhan-5.1 cells exposed to stimulated EMT-6 cells for 4 h was assayed, using the trypan blue exclusion test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
JJhan-5.1 cells that have been stably transfected with an HIV-1
LTR-driven luciferase reporter gene express constitutive luciferase activity at a low level, and this activity is considerably enhanced by
NF-B activators such as TNF-
. To explore a possible effect of
nitrogen oxides on HIV-1 LTR activation, JJhan-5.1 cells were exposed
to NO produced enzymatically by a cellular NOS 2 activity. EMT-6 cells,
stimulated for NOS 2 expression by IFN-
and LPS (here referred to as
stimulated EMT-6 cells), were found to be a convenient
source of NO since, unlike macrophages, they do not release TNF-
when stimulated with LPS (not shown). Since inhibitors of NF-
B
activation such as antioxidants are more active when added as a
pretreatment, JJhan-5.1 cells were first co-cultured with EMT-6 cells
and then cultured alone for luciferase expression. These sequential
incubations also prevented artifactual effects of nitrogen oxides on
luciferase expression and activity. Control experiments undertaken at
the beginning of this work had shown that JJhan-5.1 cell viability was
not altered by co-culture with EMT-6 cells. In particular, protein
synthesis in the T cell line was not significantly affected by prior
co-culture with EMT-6 cells. Incorporation into trichloroacetic
acid-precipitable material was 99.7 ± 4.8 and 96.9 ± 16.3%
in JJhan-5.1 cells previously incubated for 4 h with unstimulated
or stimulated EMT-6 cells, respectively, as compared with JJhan-5.1
cells cultured alone (mean ± S.E., n = 4).
In the absence of NOS 2 induction, the constitutive and TNF-induced luciferase activities were not significantly modified when JJhan-5.1 cells were co-cultured for 4 h with EMT-6 cells (Table I). The low constitutive luciferase activity of JJhan-5.1 cells was also unchanged upon co-culture with stimulated EMT-6 cells. In these experiments, nitrite production, taken as an index of NOS 2 activity in EMT-6 cells, was as high as in macrophage cultures, ranging from 2 to 4 µM per h. Therefore, in this model, a sustained release of nitrogen oxides by NOS 2 activity did not modify the basal activity of the LTR promoter.
|
In contrast, the TNF-induced NF-B activation of the LTR promoter in
JJhan-5.1 cells was inhibited 73% by stimulated EMT-6 cells (Table I).
The inhibition increased with time, reaching 0, 23, 78, and 90% when
JJhan 5.1 cells were cultured with stimulated EMT-6 cells for 1, 2, 4, and 6 h, respectively (mean of triplicates in one experiment, S.D.
<5%). Two NOS inhibitors prevented this inhibition. The best
protection against stimulated EMT-6 cells was achieved with
aminoguanidine, which inhibited nitrite production more efficiently
than L-NAME (Table I). Inhibitors alone had no effect on
luciferase expression. A nitric oxide scavenger, carboxy-PTIO, also
prevented the decrease in luciferase level (data not shown).
Compilation of different experiments revealed a strong correlation
between NOS 2 activity, measured as nitrite release, and inhibition of
TNF-dependent luciferase induction in JJhan-5.1 target
cells (Fig. 1). EMT-6 cells culture
supernatants were not inhibitory (data not shown). Taken together,
these results indicated that physiologic concentrations of NO (or a
related nitrogen oxide) impaired the induction of a LTR-controlled
luciferase reporter gene in response to TNF-
. TNF-
binding to
JJhan-5.1 cells was not modified after incubation with EMT-6 cells. The receptor affinity (Kd = 1.39 ± 0.28 and
1.35 ± 0.35 nM) and the number of sites per cell
(972 ± 111 and 990 ± 110) were similar in JJhan-5.1 cells
previously cultured alone or with stimulated EMT-6 cells, respectively
(mean ± S.E., n = 2). Thus, the decrease in
inducible luciferase expression mediated by stimulated EMT-6 cells did
not result from a defect in TNF-
binding to JJhan-5.1 cells.
|
Like TNF-, an association of 0.5 µM TPA and 5 µg/ml
PHA induces luciferase expression in JJhan-5.1 cells. When these cells were co-cultured with EMT-6 cells stimulated for NO production, subsequent stimulation with TPA and PHA was strongly inhibited, resulting in luciferase expression of 20.9 ± 5.9%, as compared with JJhan-5.1 cells alone. The presence of 2 mM
L-NAME partially prevented this inhibition (percent
luciferase = 49.8 ± 7.04; mean ± S.E.,
n = 4; p < 0.01 compared with cells
without inhibitor). These results suggested that NO inhibited a common
step in the signaling pathways triggered by TNF-
and TPA/PHA,
leading to HIV-1 LTR activation. Since the activity of the viral
promoter is dependent on NF-
B activation, whether it is induced by
TNF-
or by TPA/PHA, it was postulated that NO might inhibit the
LTR-driven luciferase expression via the inhibition of NF-
B
activation.
To address this question, electrophoretic mobility shift assays were
performed with whole cell extracts from JJhan-5.1 cells. As described
previously for the J.Jhan parental clone (29), stimulation with TNF-
induced in JJhan-5.1 cells a DNA binding activity toward a
B
oligonucleotide sequence (Fig. 2).
Supershift experiments with antibodies against p50, p65(RelA),
and c-Rel subunits of NF-
B confirmed the predominance of
p50/p65(RelA) dimer in the
B binding proteins (not shown).
Co-culture of JJhan-5.1 cells for 4 h with stimulated EMT-6 cells
followed by the addition of TNF-
strongly inhibited the appearance
of the NF-
B binding activity (Fig. 2). Unstimulated EMT-6 cells,
which did not express NOS 2 activity, did not modify the response of
JJhan-5.1 cells to TNF-
. The NOS 2 inhibitors aminoguanidine and
L-NAME prevented the effect of stimulated EMT-6 cells on
NF-
B activation. Their efficacy (aminoguanidine
L-NAME) was directly related to their ability to inhibit
nitrite synthesis by EMT-6 cells (Fig. 2). It was therefore concluded
from these experiments that NO-related species released by EMT-6 cells
inhibited NF-
B activation induced by TNF-
and also by TPA/PHA,
thereby inhibiting the TNF-induced LTR transactivation.
|
In an attempt to characterize the NO-derived species involved in
inhibition of NF-B activation, we investigated the effects of two
different chemical precursors of nitrogen oxides, GSNO and DETA-NO.
DETA-NO releases mostly the radical ·NO molecule and exhibits a
long half-life (t1/2 = 20 h at 37 °C) that
minimizes the formation of autoxidation products like
N2O3 (33). GSNO is an
S-nitrosothiol, and as such can be considered as a precursor
of NO+, NO, and even NO
-like species,
depending on its redox environment. As shown in Fig.
3, GSNO strongly inhibited the activation
of NF-
B in JJhan-5.1 cells stimulated with TNF-
. Maximal
inhibition occurred between 94 and 187 µM, corresponding
to 13 and 32 µM nitrite produced in the medium. On the
basis of nitrite production, taken as a rough estimate of total
nitrogen oxide output, GSNO was as effective as EMT-6 cells (compare
Fig. 2, lane 3, with Fig. 3, lane 4). In
contrast, DETA-NO was a very weak inhibitor of NF-
B activation, even
at a concentration of 2 mM (Fig.
4). Since the low pH of the Griess assay
accelerated DETA-NO breakdown, measurement of nitrite production was
not possible here. Yet the amount of total NO released was estimated
from the half-life of DETA-NO, after we had checked that the presence
of JJhan-5.1 cells had not modified the value previously reported (not
shown). Nitrite and nitrate (NOx
) are the stable end
products of NO oxidation. An estimation of the concentration of
NOx
produced within 4 h was therefore calculated (Fig. 4). Even if one assumes a low
nitrite:nitrate ratio in
NOx
(e.g. 1:4),
2 mM DETA-NO, which caused less than 50% inhibition of
NF-
B activation, would have generated as much as 130 µM nitrite. This is in sharp contrast with GSNO, which
displayed a similar inhibitory effect toward NF-
B activation with a
10-fold lower nitrite production (Fig. 3 lane 3). Thus, it
seems that the nitrogen oxide species produced from DETA-NO breakdown,
and identified as NO itself by others, is only marginally involved in
inhibition of NF-
B activation, under our experimental conditions.
Accordingly, spermine-NO, which belongs to the same class of "pure"
NO donors as DETA-NO but exhibits a shorter half-life
(t1/2 = 39 min) (33), did not inhibit NF-
B
activation by more than 50%, up to 500 µM (data not
shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study demonstrates that a NOS 2 product inhibits the
HIV-1 LTR promoter activity induced by TNF- or TPA/PHA, in a human T
cell line. This inhibition is correlated with a large decrease in
NF-
B activation. In contrast, NOS 2 activity did not affect the
basal luciferase level. Oxidants like hydrogen peroxide have been
reported to down-regulate TNF-
binding (34). Strong oxidants derived
from NO, including peroxynitrite, can be produced in living cells. It
was therefore important to determine whether TNF-
binding to
JJhan-5.1 cells was not modified after co-culture with stimulated EMT-6
cells. Our results indicate that, under our conditions, no reduction in
TNF-
receptor affinity or density could be detected. NOS 2 activity
also inhibited the induction of luciferase activity by TPA and PHA, a
stimulus that did not involve binding to TNF receptors. Both signaling
pathways converge on NF-
B activation, supporting the hypothesis that
inhibition of NF-
B accounted for the decrease in induction of the
LTR luciferase gene.
Previous findings concerning the modulation of NF-B activation by NO
are controversial. Early studies indicated that the chemical NO donors
sodium nitroprusside (SNP) and
S-nitroso-N-acetylpenicillamine (SNAP) were
capable of activating NF-
B in human peripheral blood mononuclear
cells (24). This was correlated with an enhancement of protein tyrosine
phosphatase activity and with an increase in the kinase activity of
p56lck. The same authors demonstrated an enhancement of GTPase
activity of the guanine nucleotide-binding protein p21ras after
exposure to NO, accompanied by S-nitrosylation of this protein (25, 26, 35). These data led to the hypothesis that NO alone,
by inducing the S-nitrosylation of p21ras, activated
the G protein which, in turn, perhaps through a protein tyrosine
phosphatase/kinase signaling pathway involving p56lck, and
possibly mitogen-activated protein kinases, ultimately activated NF-
B. Our results did not corroborate this activation pathway. By
using the JJhan-5.1 cell line and EMT-6 cells capable of producing sustained levels of nitrogen oxides, we never observed significant activation of the NF-
B transcription factor or induction of the NF-
B-dependent HIV-1 LTR promoter activity by NOS 2 activity. On the contrary, our findings indicate an inhibition of
NF-
B activation by nitrogen oxides. Other studies in different
models have reported an inhibitory effect of chemical NO donors (SNP or
GSNO) on NF-
B activation (19-21). An explanation for these conflicting results, compared with those of Lander's group (24-26), might be related to the different fluxes of NO used in these
experiments. Lander and co-workers (24- 26) found that micromolar or
submicromolar concentrations of pharmacological sources of NO (NO gas,
SNP, and SNAP) were sufficient to activate NF-
B in gel shift assays (24, 26). On the other hand, we used high output murine NOS 2 activity
to generate NO under physiological conditions, at a mean rate of 2-4
µM NO2
/h. In other
reports, inhibitory concentrations of NO donors were frequently 100 times higher than the micromolar amounts shown to activate NF-
B
per se (19-21). Therefore, it may be that low amounts of NO
would activate NF-
B by themselves in some cell types, whereas high
fluxes of NO would be inhibitory.
Our model takes into account the formation of NO-related species such
as nitrosothiols, peroxynitrite, and dinitrosyl iron complexes, which
would be expected to be produced at the same time as NO in
NOS-expressing cells. Although numerous studies have made use of
exogenous organo precursors of NO (SNP, SNAP, GSNO, and sydnonimines),
only a few experiments have been undertaken to delineate the role of NO
produced endogenously by NOS activity. NOS inhibitors activated NF-B
in unstimulated endothelial cells (but much so less than TNF-
),
suggesting the involvement of NOS 3 activity in NF-
B regulation (21,
23). Inhibition of NOS 2 activity induced by LPS or silica in the mouse
macrophage cell line RAW 264.7 resulted in an autocrine enhancement of
NF-
B activation in the same cells, consistent with a negative
feedback of NO on nos 2 gene transcription (36). Thus our
experiments demonstrate for the first time a direct inhibitory effect
of NO-related species generated by NOS 2 activity on NF-
B
activation, in a paracrine model comprising distinct effector and
target cells. In this respect, our results indicate that different
NO-related species might greatly differ in their ability to inhibit
NF-
B activation. On a concentration basis, the nitrosothiol GSNO is
much more efficient than DETA-NO. If the comparison is made on the
basis of the estimated rate of nitrogen oxide production, DETA-NO is
about 10 times less efficient than GSNO. Since DETA-NO releases mostly
the NO radical (33), it thus seems likely that NO itself was not or was
only marginally involved in the inhibition of NF-
B activation. The
fact that a nitrosothiol like GSNO could mimic the inhibition of
NF-
B activation observed with stimulated EMT-6 cells suggests that
the relevant inhibitory nitrogen oxide in the co-culture model might
also be a nitrosothiol, or at least a nitrosating species.
Interestingly, nitrosothiols have been detected in vivo and
in cell cultures in vitro (37-39). Moreover, GSNO and SNAP
have also been successfully used by other authors (19-21, 23) to
inhibit the transcriptional activation of NF-
B-dependent
genes. Finally, Peng et al. (27) demonstrated stabilization
of IkB-
and transcriptional induction of the IkB-
gene by GSNO,
providing a molecular support for the deficient activation of NF-
B
in the presence of NO-derived molecules. SNP and SNAP also directly
inhibited the DNA binding activity of NF-
B, probably via the
nitrosation of a cysteine residue critical for DNA recognition,
identified as Cys-62 in the p50 subunit (22). Thus, our experiments
showing the inhibition of NF-
B activation by a physiological source
of nitrogen oxides, which is stimulated EMT-6 cells, further validate
a posteriori those experiments that used
S-nitroso derivatives as chemical substitutes for NO
synthase activity.
The relevance of the present findings to HIV-1 pathology has to be
considered. Our results revealed a significant inhibition of
NF-B-dependent LTR transactivation by a high flux of NO
generated continuously for 2-4 h. The main cell targets of HIV viruses
are lymphocytes and macrophages. Since NF-
B activity is a major
requirement for inducing HIV LTR activity in circulating lymphocytes
and in monocytes/macrophages (40, 41), the negative effect of NO on
this activation might have important implications in reducing viral
replication. The inhibition required a sustained production of nitrogen
oxides, produced by inducible, high output NOS 2 activity. Human
lymphocytes have been shown to release only low amounts of NO (10, 42).
Induction of NOS 2 in human monocytes/macrophages is controversial
(reviewed in Ref. 43). Although there is some evidence that macrophages
can produce NO at a high level under favorable conditions, NOS 2 activity in HIV-infected monocytes is weak (13). In the human brain,
which is dramatically affected by viral toxicity, induction of NOS 2 activity was not observed in microglia, the brain macrophages (44, 45).
In contrast, cytokine-activated human astrocytes were reported to
express this NOS isoform (44, 45). The activity of fetal astrocytes was high, similar to the murine counterpart (45). NOS 2 activity is
expressed in human astrocytes in response to viral membrane components
or to inflammatory cytokines such as interleukin-1 and TNF-
,
released during HIV-1 infection (14, 45, 46). Astrocytes activated for
NOS 2 expression might thus inhibit the NF-
B-dependent
HIV-LTR transactivation in other glial cells, in a paracrine manner.
The antiviral effect of NOS 2 might limit the reactivation of the HIV-1
provirus, in the same way as it blocks the activation of the latent
Epstein-Barr virus genome (10), thereby contributing to a latent HIV-1
infection in glia (14) and participating at the same time in HIV-1
pathology through NO-induced neurotoxicity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Béatrice Wolfersgerger for excellent technical assistance, Philippe Benech for helpful advice, and Gillian Barratt for assistance in the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant 6555 from the Association pour la Recherohe contre le Cancer and Grant ACC-SV 5 from the Ministère de l'Enseignement Supérieur et de la Recherche.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: ERS CNRS 571, Bât 430, UPS Orsay, F-91405 Orsay Cedex, France. Tel.: 33 01 6915 7972; Fax: 33 01 6985 3715; E-mail: michel.lepoivre{at}bbmpc.u-psud.fr.
1
The abbreviations used are: NOS, nitric oxide
synthase; DETA-NO,
N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine; GSNO, S-nitrosoglutathione; HIV, human immunodeficiency
virus; IFN, interferon; LPS, lipopolysaccharide; LTR, long terminal
repeat; NF-B, nuclear factor
B; SNAP,
S-nitroso-N-acetyl-DL-penicillamine; SNP, sodium nitroprusside; TNF, tumor necrosis factor; TPA,
12-O-tetradecanoylphorbol-13-acetate; EMSA, electrophoretic
mobility shift assay; PHA, phytohemagglutinin; L-NAME,
N
-nitro-L-arginine methyl ester.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|