From the Laboratoire du Stress Cellulaire, Centre de
Génétique Moléculaire et Cellulaire, CNRS-UMR 5534, Université Claude Bernard Lyon-I, 69622 Villeurbanne Cedex, France
We report here that amino acid analogs, which
activate hsp70 promoter, are powerful transcriptional activators of
human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR), an
activation which was impaired when the two
B sites present in the
LTR were mutated or deleted. Amino acid analogs also stimulated the
transcription of a
B-controlled reporter gene. Upon treatment with
amino acid analogs, the two NF-
B subunits (p65 and p50), which are
characterized by a relatively long half-life, redistributed into the
nucleus where they bound to
B elements. This phenomenon, which began to be detectable after 1 h of treatment, was concomitant with the
degradation of the short lived inhibitory subunit I
B-
by the
proteasome. However, contrasting with other NF-
B inducers that
trigger I
B-
degradation through a phosphorylation step, amino
acid analogs did not change I
B-
isoform composition.
Antioxidant conditions inhibited amino acid analog stimulatory action
toward NF-
B. This suggests that aberrant protein conformation
probably generates a pro-oxidant state that is necessary for I
B-
proteolysis by the proteasome. Moreover, this activation of NF-
B
appeared different from that mediated by endoplasmic reticulum overload as it was not inhibited by calcium chelation.
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INTRODUCTION |
HIV-11 infection is
characterized by a phase of clinical latency in the course of which the
rate of replication of the virus is important, notably in lymphoid
organs (1, 2). Other cells that are infected in a latent manner
necessitate a reactivation to generate viral production. Stimuli that
mediate such reactivation include cytokines, phorbol esters, tumor
promoters, protein kinase inhibitors (3), co-infection by other viruses
(4, 5), cadmium, arsenite (6), oxidative conditions (7), or thermal stress (8-10). These stimuli regulate the transcription of the HIV-1
provirus through the modulation of the complex eukaryotic promoter
localized in the long terminal repeat (LTR). This promoter contains
binding sites for many transcription factors, including NF-
B, SP1,
USF, and AP1 (11, 12). In the case of oxidative stress mediated by
either hydrogen peroxide or tumor necrosis factor-
(TNF-
), the
transcriptional activation of HIV-1 LTR was clearly shown to depend on
NF-
B (7). In contrast, HIV-1 LTR activation by protein kinase
inhibitors has not yet been demonstrated to require any specific
transcription factor (3). Similarly, despite striking
similarities between the kinetics of thermal activation of the LTR and
the heat shock promoter (HSE), the molecular mechanism leading to the
activation of HIV-1 LTR by heat shock is still not understood (9,
10).
Heat shock, which induces the accumulation of misfolded or damaged
proteins, results in the preferential expression of heat shock or
stress proteins (hsp), which contribute in helping the cell to recover
from thermal damage (13-18). Other conditions or agents that interfere
with protein folding as well as denatured proteins are usually stress
protein inducers. They include denatured bovine serum albumin, mutated
actin, heavy metals, arsenite, and amino acid analogs (18-21). The
latter agents are structural analogs of natural amino acids and are
therefore rapidly incorporated into newly synthesized proteins
(19, 22). Hence, they induce irreversible aberrant protein
conformations that lead to the induction of stress proteins, such as
hsp70, and glucose-regulated proteins, i.e. Grp78/Bip (17,
23-25).
NF-
B belongs to a family of inducible transcription factors, the
Rel/NF-
B family (26-28). In addition to being a major enhancer of
the HIV-1 LTR promoter, NF-
B regulates the expression of numerous cellular genes, particularly those involved in the immune and inflammatory responses (29-32). NF-
B activation is under the
control of the I
B family of inhibitory subunits. The most studied
I
B protein is I
B-
which associates in the cytoplasm with
NF-
B dimer (33-36). This interaction masks the nuclear localization signal of NF-
B (37, 38) and therefore inhibits the nuclear translocation of this dimeric factor. NF-
B is activated by a large
number of signals, which include inflammatory cytokines, phorbol esters
(phorbol 12-myristate 13-acetate) (39), pathogenic agents (32), UV
irradiations (40), oxidative stress (7), or protein accumulation in the
endoplasmic reticulum (41). In activated cells, the cytoplasmic
NF-
B-I
B-
complex is disrupted, and NF-
B is then allowed to
migrate into the nucleus where it can bind to
B elements (42). The
NF-
B-I
B-
complex is disrupted through I
B-
phosphorylation at the level of serines 32 and 36 (43-47), which
triggers the ubiquitination of this protein and its specific
degradation by the proteasome (44, 48-50). This pathway is inhibited
by antioxidant drugs (7) or by the overexpression of detoxifiant
enzymes such as catalase (51) or glutathione peroxidase (52),
suggesting that crucial redox events or intracellular reactive oxygen
species are involved in NF-
B activation. Recently, an alternative
mechanism of NF-
B activation has been reported to occur in Jurkat T
cells stimulated with pervanadate. In this case, tyrosine
phosphorylation of I
B-
activates NF-
B without proteolytic
degradation of I
B-
(53). However, redox modulation of this
phenomenon, (ROS are potent inhibitors of tyrosine phosphatases (54))
is still a matter of debate (53, 55, 56).
Independently of its rapid breakdown induced by several stimuli
and compared with NF-
B protein subunits, I
B-
is inherently unstable and undergoes a continuous turnover. This phenomenon does not
require the prior phosphorylation or ubiquitination of I
B-
but
still appears mediated by the proteasome (57).
Since HIV-1 LTR is transcriptionally activated by several cellular
stress, including heat shock (6, 8-10), we tested whether the
accumulation of aberrantly conformed proteins could trigger the
activity of this promoter. To this end, the inducible activity of amino
acid analogs have been tested. We demonstrate here that azetidine or
canavanine, which mimic proline and arginine, respectively, are
powerful transcriptional activators of HIV-1 LTR. This stimulation, which necessitated the
B sites present in the LTR, was impaired by
antioxidizing conditions. In contrast, hsp70 promoter activation by
amino acid analogs was stimulated by antioxidant conditions. Concomitantly with HIV-1 LTR activation, we show that amino acid analogs induced the proteasome-mediated degradation of the inhibitory subunit I
B-
without a prior change in its isoform distribution. Hence, aberrant protein conformation appears to generate pro-oxidant conditions that trigger the degradation of short lived and probably misfolded proteins, such as I
B-
, and subsequently the activation of NF-
B, a phenomenon that results in HIV-1 transcriptional
stimulation.
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MATERIALS AND METHODS |
Cell Cultures--
HeLa cells were grown at 37 °C in the
presence of 5% CO2 in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 5% fetal calf serum. The human breast
carcinoma T47D cell derivatives T47D-Hygro and the corresponding cell
line T47D-GPx that overexpress selenoglutathione peroxidase have
already been described (52, 58, 59). They were grown at 37 °C in the
presence of 5% CO2 in Hepes-buffered RPMI medium
(Sigma Chimie, Saint-Quentin Fallavier, France) supplemented with
10% fetal calf serum (Life Technologies, Inc., France), 0.1 µM fresh sodium selenite, 2 mM
L-glutamine, 0.5 µg/ml insulin, 100 units/ml penicillin,
and 0.1 mg/ml streptomycin. Prior to amino acid analog treatments,
cells were incubated 30 min in DMEM or RPMI medium supplemented with 5 or 10% dialyzed fetal calf serum. The proteasome inhibitor
N-Cbz-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) (Sigma Chimie, Saint-Quentin Fallavier, France), which had been dissolved in Me2SO at a concentration of 60 mM, was added 50 min prior to stimulation with amino
acid analogs, at the final concentration of 60 µM.
Reagents and Plasmids--
Actinomycin D, azetidine
(L-azetidine-2-carboxylic acid), canavanine, pyrrolidine
dithiocarbamate (PDTC), PSI, hydrogen peroxide, and type B gelatin were
from Sigma Chimie (Saint Quentin Fallavier, France). The calcium
chelator
[1,2-bis-(O-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid tetra(acetoxy-methyl)ester] (BAPTA-AM) was from Calbiochem (Meudon, France). Anti-p65/RelA, anti-p50, and anti-I
B
/MAD-3 were
from Santa Cruz Biotechnology (Santa Cruz, CA). pLTR-cat, p17-cat-neo,
and pSV2-cat plasmids were described elsewhere (10). p2x
B-37TKcat
vector, which is made of two artificially constructed
B sites in
front of a cat gene, has already been described (60). pCMV
is a plasmid from CLONTECH (Palo Alto, CA)
that contains the
-galactosidase gene under the control of the
cytomegalovirus promoter. pLTR-PstI is identical to pLTR-cat
except that the first two bases of the two consensus
B sequences
present in the LTR were mutated. This mutant was produced by megaprimer
polymerase chain reaction mutagenesis (61). Primer for the entire HIV-1 LTR were upstream primer (5
-GGGCTCGAGACCTAGAAAAACATGG-3
),
containing a XhoI site (underlined) and downstream (reverse) primer
(5
-GGGTCTAGAAGCTTTATTGAGGCTTAAGCAG-3
), containing
sites for XbaI and HindIII. The mutagenic
primer
(5
-TTGCTACAACAGACTTCCGCTGCAGACTTTCCAG-3
) created a PstI site between the two mutated NF-
B
consensus binding sites. Mutated bases are in boldface type. A first
round of amplification with the mutagenic and down stream primers was
used to amplify the 3
region of the HIV-1 LTR. The resulting fragment
which was mutated in both NF-
B sites was gel purified and used as
downstream primer in a subsequent reaction to amplify the entire HIV-1
LTR. The final polymerase chain reaction product of 0.7 kilobase pair was gel purified and the presence of the PstI site verified
by digestion. The fragment was then digested with
XhoI-XbaI and ligated into the pLTR-cat vector
from which the wild type HIV-1 LTR had been removed by
XhoI-XbaI digestion. Mutant plasmids which
contained the full-length sequence with PstI and
HindIII sites were sequenced to confirm the mutated sites
and the absence of other mutations.
Transfection, CAT, and
-Galactosidase Assays--
HeLa cells
were seeded out 12 h before transfection at a density of 3 × 106 cells/100-mm dishes. Transfection experiments were
performed as described by Kretz-Remy and Arrigo (10). Briefly, 8 µg
of the desired cat-containing plasmid described above were
added to a transfection buffer containing 24 µl of Transfectam
reagent (Promega, France) and then mixed with the 2-ml culture medium. Ten hours after transfection, cells were trypsinized and replated into
four to six 60-mm dishes. Twelve hours later, cells were submitted to
various amino acid analog treatments and were allowed to recover for
24 h prior to harvesting. Transfected cells were lysed, and 50 µg of total cellular proteins were analyzed using the Boehringer
CAT-ELISA test according to the manufacturer's instructions. The
percentage of cells expressing
-galactosidase was monitored by
5-bromo-chloro-3-indolyl
-D-galactosidase staining (62).
Indirect Immunofluorescence Analysis--
HeLa cells growing on
glass coverslips coated with 0.1% of type B gelatin were submitted to
various treatments with amino acid analogs. They were then rinsed with
PBS at 37 °C and fixed for 90 s in cold methanol; anti-p65/RelA
antibody was diluted 1:100 in PBS containing 0.1% bovine serum
albumin. Goat anti-rabbit immunoglobulin coupled to isothiocyanate was
used as a second antibody (Organon Teknica-Cappel, Fresnes, France).
The stained cells were examined and photographed with a Zeiss Axioskop
photomicroscope. Fluorescent images were recorded onto Tri-X Pan
(Eastman Kodak Co.) film.
Electrophoretic Mobility Shift Assays--
Extraction of
DNA-binding proteins and binding conditions have been previously
described (10). Briefly, 10 µg of protein from nuclear extracts were
incubated with a 20,000 cpm (Cerenkov) 32P-labeled
B DNA
probe in the presence of 4 µg of poly(dI-dC) (Pharmacia) and 1 µl
of 10 × BB buffer. Reaction was for 15 min at room temperature
following the addition of the 32P-labeled
B probe. The
double-strand oligonucleotide used to detect the DNA binding activity
of NF-
B was as described previously (52, 63). For the competition
experiments, 10 or 40 ng of cold
B probe were added to the binding
mix including proteins just before the incubation with the
32P-labeled
B probe. Supershift experiments were also
performed by adding, 30 min before the incubation with the
32P-labeled
B probe, 2 µg of an antibody raised
against the p65/RelA subunit of NF-
B to the binding mix including
proteins. Native 4% acrylamide gels were used to analyze the samples.
Autoradiographs of the gels were recorded onto BioMax MR films (Eastman
Kodak Co.).
Radiolabeling and Cellular Extraction--
HeLa cells were pulse
labeled wih [35S]methionine and -cysteine
(Tran35S-label, 1504 Ci/mmol, ICN, Costa Mesa, CA) for
different periods of time in methionine- and cysteine-free DMEM (ICN)
containing 5% dialyzed fetal calf serum. Thereafter, cells were rinsed
with PBS and scrapped off the dish. Before cellular extraction,
aliquots were withdrawn for determination of protein concentration. For cellular extraction, 1.5 × 106 HeLa or T47D cells
grown in 60-mm cell culture dishes and submitted to various amino acid
analog treatments were washed with cold PBS. The cells were then
scraped from the dishes and pelleted for 5 min at 1000 × g. The cellular pellet was then lysed and boiled in Laemmli
sample buffer for 5 min.
Gel Electrophoresis, Immunoblotting, and
Immunoprecipitation--
One- and two-dimensional gel electrophoresis
and immunoblots were performed as already described using gels that
contained 10% (45, 47, 69-71) or 12.5-15% (52) acrylamide. The
isoelectrofocusing gels were made up with 60% of pH 4-6 and 40% of
pH 3-10 Ampholines (Sigma Chimie, Saint-Quentin Fallavier, France).
Isoelectrofocusing sample buffer contained pH 6-8 Ampholine.
Immunoblots were probed with p65/RelA, p50, or I
B-
/MAD-3 antisera
as primary antibodies and were revealed with the ECL kit from Amersham
Corp. (United Kingdom). The duration of exposure was calculated to be
in the linear response of the film. For immunoprecipitation under
native conditions, a constant number of radiolabeled cells was
analyzed. [35S]Methionine-labeled cells were rinsed in
PBS, lysed in radioimmune precipitation buffer (50 mM Tris,
pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS) and clarified for 10 min at 10,000 × g. Immunoprecipitation was performed by incubating cell
lysates with anti-I
B-
or preimmune serum for 3 h on ice before incubation of 1 h in the presence of protein A-Sepharose CL4B (Sigma Chimie, Saint-Quentin Fallavier, France). Thereafter, the
protein A-immunocomplexes were centrifuged for 15 s at 10,000 × g, washed several times with radioimmune precipitation
buffer, and boiled in SDS sample buffer. After removal of protein
A-Sepharose by centrifugation, samples were analyzed by SDS-PAGE and
autoradiography.
 |
RESULTS |
Amino Acid Analogs Azetidine and Canavanine Activate HIV-1
LTR--
The transcriptional activity of HIV-1 LTR was tested in HeLa
cells transiently transfected with pLTR-cat, a plasmid that contains the cat gene under the control of the HIV-1 LTR (see
"Materials and Methods"). Parallel experiments were also performed
with p17-cat-neo plasmid that contains the cat gene under
the control of the promoter of the human hsp70 gene. Transfection
efficiency at about 60%, tested by using the
-galactosidase
gene-bearing pCMV
plasmid, was similar in all experiments (not
shown). Fig. 1A shows the pattern of activation of pLTR-cat and p17-cat-neo plasmids in HeLa
cells exposed for 6 h to increasing concentrations of the proline
analog, azetidine. The level of CAT polypeptide was quantified after a
24-h recovery period in a medium devoid of azetidine by CAT-ELISA
testing (see "Materials and Methods"). A gradual increase of CAT
polypeptide driven by pLTR-cat or p17-cat-neo plasmids was observed
following treatment with increasing doses of azetidine. Of interest,
the intensity of LTR activation was about 4-fold that of the hsp70
promoter. The reverse was observed when cells were exposed to heat
shock (Fig. 1A). In addition, following exposure to 15 mM azetidine for different time periods, the kinetics of activation of HIV-1 LTR peaked after 6 h of treatment (Fig.
1B). As seen in Fig. 1, C and D,
similar kinetics of activation of HIV-1 LTR and hsp70 promoter were
also observed when canavanine, an arginine analog, was used instead of
azetidine. In this case, however, the difference in the inducibility of
the LTR versus hsp70 promoter was only 2.5-fold instead of
the 4-fold induced by azetidine. Control experiments were performed
using the pSV2-cat plasmid that contains the cat gene under
the control of the constitutive early promoter of SV40 virus. In this
case, no change in the level of CAT polypeptide was induced by
azetidine, canavanine (Fig. 1, E and F) or heat
(not shown) (10). Hence, amino acid analogs act as potent activators of
HIV-1 LTR.

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Fig. 1.
Kinetics of activation of HIV-1 LTR by
treatment with amino acid analogs azetidine and canavanine.
A and B, effect of azetidine treatments on HIV-1 LTR
and hsp70 (HS) promoter-dependent gene expression.
HeLa cells, transiently transfected with a HIV-1 LTR-dependent (pLTR-cat, black
columns) reporter cat construct, were treated with
increasing concentrations of azetidine (5-15 mM) for
6 h (A) or with azetidine 15 mM during
various time periods (1-24 h) (B) and allowed to recover
24 h before being harvested. In A, the kinetic of
activation of a HS-dependent reporter cat construct (p17-cat-neo, hatched columns) by
azetidine is also represented. A positive control (HS
control) was also performed by submitting the transfected HeLa
cells to a 43 °C heat-shock treatment for 90 min followed by a 24-h
recovery period. C and D, as in A and
B, but in this case, transfected HeLa cells were treated
with canavanine. E and F, effect of azetidine and
canavanine treatments on HeLa cells transiently transfected with
a SV40 promoter driven reporter cat construct.
HeLa cells, transiently transfected with pSV2-cat plasmid (gray
columns), were treated for 6 h with the indicated
concentrations of azetidine (E) or canavanine (F) and allowed to recover for 24 h in the absence of analogs before being analyzed. The level of cytoplasmic CAT enzyme was quantified by
ELISA as described under "Materials and Methods." The degree of
activation was calculated by dividing the CAT concentration of the
different samples by the CAT concentration of the standard nonstressed cells. The histograms shown are one
representation of three independent and identical experiments;
S.D. are presented (n = 3).
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We next investigated whether the amino acid analog activation of HIV-1
LTR resulted in transcriptional events. This was assessed by analyzing
the effect of actinomycin D (0.5 µg/ml) on the production of the CAT
polypeptide driven by HIV-1 LTR. To this end, transfected HeLa cells
were treated for 6 h with increasing concentrations of either
azetidine (Fig. 2A) or
canavanine (Fig. 2B) and then allowed to recover for 3 h in a medium devoid of amino acid analogs. Actinomycin D was added 10 min prior to the beginning of the treatment with the analogs and the
recovery period was of only 3 h to minimize the cytotoxic effect
induced by actinomycin D. As seen in Fig. 2, A and
B, actinomycin D abolished CAT polypeptide production induced by azetidine or canavanine, as compared with the level of this
protein measured in cells not treated with actinomycin D (Fig. 2,
A and B, plot C). Taken together,
these results suggest that the activation of HIV-1 LTR by amino acid
analogs is specific and transcriptionally regulated.

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Fig. 2.
Effect of actinomycin D on HIV-1 LTR
activation by amino acid analog treatments. HeLa cells transfected
with the HIV-1 LTR-dependent reporter cat
construct were incubated for 10 min with actinomycin D at 0.5 µg/ml
before being treated for 6 h with the indicated concentrations of
azetidine (A) or canavanine (B). After 3 h
of recovery in the absence of amino acid analogs, the level of CAT
accumulated was determined as described under "Materials and
Methods." In A and B, plot C
represents the activation of pLTR-cat by 15 mM of either
azetidine or canavanine in the absence of actinomycin D. Determination
of CAT concentration and presentation of the results are as in Fig. 1.
The histograms shown are representative of three independent and
identical experiments; S.D. are presented (n = 3). In
this experiment, the level of CAT was analyzed 3 h (instead of
24 h) after the different treatments to minimize the inhibitory
effect of actinomycin D on the recovery of normal cellular functions
after the drug treatments.
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Transcriptional Activation of HIV-1 LTR by Amino Acid Analogs
Requires NF-
B Binding Sites--
We next investigated whether the
two
B sites present in HIV-1 LTR were necessary for the activation
of this promoter by amino acid analogs. To test this hypothesis, we
analyzed the inducibility of an HIV-1 LTR that contains the two
B
sites mutated in the two first guanine residues of the consensus
NF-
B binding site (see "Materials and Methods"). Mutation of
these highly conserved bases has been described to inhibit NF-
B
binding and
B transcriptional activation induced by UV light or
phorbol esters (64, 65). Following transient transfection with
wild-type (pLTR-cat) or mutant (pLTR-PstI) LTR, HeLa cells
were treated with increasing concentrations of either azetidine (Fig.
3A) or canavanine (Fig. 3B). After 24 h of recovery, the level of CAT produced
was analyzed, as described above, by CAT-ELISA. Fig. 3, A
and B, shows that the replacement of the two first guanine
residues in the two
B sites of HIV-1 LTR slightly diminished the
basal activity of the promoter and completely abrogated its activation
by either azetidine or canavanine. The same result was obtained when
the entire consensus
B sites were deleted (not shown) by using (
B) HIV LTR-cat plasmid (66). Hence,
B sites appear necessary for
amino acid analog-mediated transcriptional activation of HIV-1 LTR.
This observation was strengthened by the fact that, in transfected HeLa
cells, the p2x
B-37TKcat vector, which contains two artificially constructed
B sites in front of a cat gene (see
"Materials and Methods") (60), was activated by amino acid analogs
with an intensity that was almost equal to that of the wild-type LTR
promoter (Fig. 3C). These observations therefore suggest
that the activation of HIV-1 LTR by amino acid analogs is mediated by
the two
B sites present in this promoter and that amino acid analogs
activate a transcription factor that mediate
B-dependent
gene expression.

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Fig. 3.
Transcriptional activation of HIV-1 LTR by
amino acid analogs requires NF- B binding sites. HeLa cells,
transiently transfected with a wild-type (pLTR-cat,
black columns) or NF- B mutated (pLTR-PstI,
gray columns) HIV-1 LTR-dependent reporter cat construct, were treated for 6 h with the indicated
concentrations of either azetidine (A) or canavanine
(B). The cells were then allowed to recover for 24 h
before being harvested. C, HeLa cells transiently
transfected with a B-dependent cat construct
(p2x B-37TKcat) were submitted to various concentrations of azetidine
(gray plots) or canavanine (hatched plots) for
6 h and allowed to recover for 24 h before being harvested.
Determination of CAT concentration and presentation of the results are
as in Fig. 1. The histograms shown are representative of three
identical experiments; S.D. are presented (n = 3).
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Amino Acid Analogs Are Powerful Activators of NF-
B Transcription
Factor--
To investigate whether the activation of HIV-1 LTR or
isolated
B promoter by amino acid analogs correlated with NF-
B
activation, the nuclear redistribution of NF-
B subunits as well as
the in vitro DNA binding of this factor were analyzed. It is
seen in the immunofluorescence analysis presented in Fig.
4 that the p65/RelA subunit of NF-
B
redistributed into the nucleus in HeLa cells treated with 15 mM of azetidine. This phenomenon, which was already detectable after 1 h of treatment, gradually increased until
6 h of incubation with azetidine. Moreover, the nuclear
redistribution of p65/RelA was reversible since it was no longer
observed in cells allowed to recover for 4 h in the absence of
azetidine. A similar observation was made when HeLa cells were treated
with canavanine or when the other subunit of NF-
B, p50, was analyzed (not shown). In vitro DNA binding of NF-
B was
investigated by electrophoretic mobility shift assays. To this end,
nuclear extracts were prepared from HeLa cells either left untreated or
exposed for 6 h to 5 or 15 mM of azetidine or
canavanine. DNA binding assays were performed using a DNA probe
encompassing the
B motif (see "Materials and Methods"). It is
seen in Fig. 5 that azetidine or
canavanine induced the formation of a specific protein/
B
oligonucleotide complex that was not observed in untreated control
cells. Competition experiments performed with an excess of
nonradioactive
B oligonucleotide competed effectively for complex
binding. Moreover, a supershifted band was observed when the reaction
mixture was incubated with an antibody that recognizes the p65/RelA
subunit of NF-
B, demonstrating that the complex binds specifically
to the
B site and contains the p65 protein. Hence, treatments of
HeLa cells by amino acid analogs induce the specific binding of NF-
B
to "
B" oligonucleotide.

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Fig. 4.
Immunofluorescence analysis of p65 cellular
locale in amino acid analog-treated Hela cells. HeLa cells were
either left untreated (A), treated with 15 mM
azetidine during 1 (B), 2 (C), 4 (D),
or 6 (E) h, or treated for 6 h with 15 mM
azetidine and allowed to recover for 4 h in the absence of this
analog (F). The cells were then fixed with cold methanol
before being processed for indirect immunofluorescence analysis using
anti-p65/RelA antibody as described under "Materials and Methods."
Note the reversible translocation of p65/RelA to the nucleus induced by
azetidine treatment. Bar, 10 µm.
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Fig. 5.
Effect of amino acid analog treatments on the
DNA binding activity of NF- B. HeLa cells were either left
untreated (0) or treated for 6 h with 5 or 15 mM of azetidine or canavanine. Nuclear extracts were
prepared and equal amounts (10 µg) of nuclear proteins were incubated
with a 32P-labeled oligonucleotide encompassing the B
motif as described under "Materials and Methods." Comp.,
competition experiments: B-100 or -400 competition performed with
either 10 or 40 ng of unlabeled B probe added to the binding
mixture. In this case, extracts were made from HeLa cell treated for
6 h with 15 mM azetidine or canavanine. Ab,
supershift performed by adding an antiserum recognizing the p65/RelA
subunit of NF- B to the binding mixture of similarly treated HeLa
cells. Samples were analyzed on native 4% polyacrylamide gel. An
autoradiograph of the gel is presented. Arrows denote the
position of the nonspecific (ns), specific, and supershifted
complexes and that of the free probe.
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Amino Acid Analogs Activate NF-
B through I
B-
Degradation
by the Proteasome but without Changing I
B-
Isoform
Distribution--
Amino acid analogs, which mimic natural amino acids,
are incorporated into nascent polypeptides and therefore induce an
irreversible abnormal conformation of newly synthesized proteins that
could result in their rapid degradation (17, 19). We then investigated whether treatments of HeLa cells by amino acid analogs altered the
level of the p65/RelA, p50, and I
B-
subunits of NF-
B. It is
seen in the immunoblot analysis presented in Fig.
6, that amino acid analogs did not modify
the electrophoretic mobility and cellular content of p65/Rel A (Fig.
6A) or p50 (Fig. 6B). In contrast, treatments of
HeLa cells by 15 mM of azetidine or canavanine altered I
B-
stability. Immunoblots probed with an antibody specific for
I
B-
/MAD-3 (Fig. 6, C and D) show that
azetidine (C) or canavanine (D) induced a drastic
decrease of I
B-
level which began to be detectable after 60 min
of treatment.

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Fig. 6.
Effects of amino acid analog treatments on
the electrophoretic mobility and stability of the p65/RelA, p50, and
I B- subunits of NF- B. A and B, HeLa
cells were treated with 15 mM azetidine or canavanine for
1, 2, 4, or 6 h, or were kept untreated (c). C and D, HeLa cells were treated with azetidine
(C) or canavanine (D) during various time ranging
from 5 min to 6 h. Control untreated cells (0) or
control untreated cells kept in DMEM containing dialyzed fetal calf
serum (dFCS/0) are presented. Equal amounts of total cellular extracts were separated by a 12.5% (p65 and
p50) or a 15% (I B- ) SDS-polyacrylamide gel
electrophoresis. The cellular contents of NF- B subunits p65/RelA
(A), p50 (B), and I B- (C and
D) were analyzed in immunoblots probed with antibodies that recognize specifically these proteins and revealed by ECL. Note that
amino acid analogs did not alter the cellular content of p50 and
p65/RelA, which contrasts with the induction of decreased levels of
I B- by these treatments.
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I
B-
is characterized by a short half-life (35, 67) which, in HeLa
cells, was estimated by pulse-chase experiments followed by
immunoprecipitation to be about 30-40 min (data not shown). Hence,
experiments were performed to test whether the rapid disappearance of
I
B-
observed above did not simply result from protein synthesis inhibition induced by amino acid analogs. To this end, HeLa cells were
labeled with [35S]methionine/cysteine prior to and at
different time periods during the treatment. It is seen in Fig.
7A, that, in our conditions of
treatment with azetidine, the overall pattern of protein synthesis was
not significantly altered, excepted for the enhanced synthesis of two
high molecular weight proteins which are probably hsp70 and Grp78.
These two proteins displayed different kinetics of activation; Grp78
expression was transient and peaked after 2 h of treatment while
hsp70 was expressed later. A similar result was observed when cells
were treated with canavanine (not shown). The level of I
B-
synthesis was next analyzed by immunoprecipitation of extracts of HeLa
cells treated for 60 min with 15 mM of azetidine or
canavanine and labeled with [35S]methionine/cysteine for
the last 40 min of the treatment (see "Materials and Methods"). As
seen in Fig. 7B, I
B-
was still synthesized in the
presence of azetidine or canavanine, hence suggesting that these
analogs were incorporated into this protein. However, in this case, the
level of [35S]methionine/cysteine incorporation in
I
B-
appeared less intense than in cells not treated with the
analogs. This phenomenon probably reflects the stimulated degradation
of I
B-
in amino acid analog treated cells, since it is rather
improbable that analogs will discriminate between the translation of
specific mRNAs.

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Fig. 7.
Effect of amino acid analogs on global and
I B- protein synthesis. A, HeLa cells were either left
untreated or treated with 15 mM of azetidine for 0.5-6 h.
Cells were labeled with [35S]methionine and
[35S]cysteine for the last 30 min of azetidine
treatments, and the pattern of protein synthesis was analyzed by
SDS-PAGE. An autoradiogram of the gel is presented. The positions of
the standard proteins of known molecular masses expressed in
kilodaltons are indicated on the left. The position of
migration of hsp70 and Grp78 is indicated by empty and
full arrowheads, respectively. B, HeLa cells were either left untreated or treated for 60 min with 15 mM
azetidine or canavanine. [35S]Methionine/cysteine was
added to the culture medium during the last 40 min of the treatment.
Cells were then processed for immunoprecipitation using either
nonimmune (p) or anti-I B- (i) antibody as
described under "Materials and Methods." An autoradiogram of the
gel is presented. Note that the pattern of global protein synthesis is not altered by azetidine and that I B- is still synthesized in the
presence of azetidine and canavanine.
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The possibility of an involvement of the proteasome pathway in the
degradation of I
B-
, in amino acid analog-treated cells, was
tested by studying the effect of a proteasome inhibitor on I
B-
stability, during azetidine treatments. It is seen in Fig. 8 that a 50-min preincubation of HeLa
cells with 60 µM of the specific inhibitor of the
proteasome chymotrypsin-like activity (PSI) (44, 68) completely
abolished I
B-
degradation induced by azetidine. A similar effect
was observed in cells treated with canavanine. This result therefore
demonstrates that the chymotrypsin-like activity of the proteasome is
probably responsible for the degradation of I
B-
in amino acid
analog-treated HeLa cells.

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Fig. 8.
Effect of a proteasome inhibitor on the
stability of I B- in HeLa cells treated with azetidine. HeLa
cells were either left untreated (control) or pretreated
with 60 µM of a specific inhibitor (PSI) of
the chymotrypsin-like activity of the proteasome followed by incubation
with 15 mM of azetidine for the indicated periods of time.
Equal amounts of total cellular proteins were separated on 15%
SDS-PAGE, and the cellular content of I B- was investigated by
immunoblot analysis using an antibody specific to this protein, as
described under "Materials and Methods."
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Several reports have described the rapid appearance, in
SDS-polyacrylamide gel containing 10% acrylamide, of a slow migrating phosphorylated form of I
B-
that precedes the degradation of this
protein in response to TNF-
(44, 45, 47, 52, 69-71). This transient
phosphorylation of I
B-
controls the degradation of this protein
by the proteasome (44). The hypothetical phosphorylation of I
B-
by amino acid analogs was therefore investigated. It is seen in Fig.
9 that azetidine treatments did not
induce the appearance of a slow migrating phospho-isoform of I
B-
even if cells were incubated with 60 µM of the proteasome
inhibitor PSI. By contrast, a 5-min treatment of HeLa cells with
hydrogen peroxide did induce the appearance of a slow migrating
I
B-
phospho-isoform which was rapidly degraded except if cells
were treated with PSI, a phenomenon which was already reported by
others (44, 45, 47, 69-71). Changes in I
B-
isoform composition
in response to azetidine or canavanine treatments was further analyzed
by two-dimensional immunoblot. This technique allows I
B-
to be resolved into two major isoforms; the acidic "b" phospho-isoform and the more basic "a" unphosphorylated isoform (52). Azetidine (Fig. 10A) or canavanine
(not shown) treatments were not found to change the isoform pattern of
I
B-
, hence, suggesting that this protein is not phosphorylated in
response to treatment with these compounds. In contrast, a five minute
treatment with 250 µM H2O2
drastically increased the level of the b isoform and concomitantly decreased the level of the a isoform (Fig. 10B) confirming
that a large fraction of I
B-
is rapidly phosphorylated in
response to hydrogen peroxide. These results, therefore strongly
suggest that, in HeLa cells, amino acid analogs activate NF-
B, via
I
B-
degradation by the proteasome, but apparently without the
crucial step of I
B-
phosphorylation.

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Fig. 9.
One-dimensional immunoblot analysis of
I B- isoforms in HeLa cells exposed to azetidine or hydrogen
peroxide. HeLa cells were treated for various time periods with
250 µM hydrogen peroxide or 15 mM azetidine
in the presence or not of 60 µM proteasome inhibitor PSI.
Cells were harvested, and equal amounts of total cellular proteins were
separated in SDS-PAGE containing 10% acrylamide as described by
Traenckner et al. (44). The cellular contents of I B-
isoforms was analyzed in immunoblot probed with I B- antibody and
revealed by ECL as described under "Materials and Methods." Note
the absence of any slow migrating I B- phospho-isoform after amino
acid analog treatments even in the presence of proteasome inhibitor, in
contrast to the rapid appearance of I B- phospho-isoform (P-I B- ) after H2O2
treatment.
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Fig. 10.
Two-dimensional immunoblot analysis of
I B- isoforms in Hela cells exposed to azetidine or hydrogen
peroxide. Total proteins of HeLa cells, treated or not with 15 mM azetidine (A) or 250 µM
hydrogen peroxide (B) during various time periods were analyzed in two-dimensional immunoblots probed with anti-I B- antibody and revealed by ECL as described under "Materials and Methods." As indicated, analysis was performed before (c)
and after 15, 30, 60, 180, or 360 min of treatment with azetidine or
before (c) and after 5, 15, 30, 45, or 60 min of treatment with hydrogen peroxide. The more acidic with apparent higher molecular weight I B- phospho-isoform is indicated as the b
isoform. The less acidic and faster migrating I B- isoform is
indicated as the a isoform. Note that azetidine treatments
did not change the isoform pattern of I B- , in contrast to the
rapid appearance of I B- b phospho-isoform induced by hydrogen
peroxide.
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The Antioxidant PDTC Strongly Decreases the Transcriptional
Activation of HIV-1 LTR by Amino Acid Analogs while the Reverse Is
Observed in Case of Human hsp70 Promoter--
Most NF-
B inducers
have been reported to be inhibited by antioxidants, such as PDTC,
implying that intracellular redox or reactive oxygen species modulate
the signal transduction that results in NF-
B activation (7, 72). We
therefore investigated whether PDTC could modulate HIV-1 LTR
transcriptional activation by amino acid analogs. To this end, HeLa
cells, transiently transfected with pLTR-cat plasmid were first
incubated for 1 h with 100 µM of PDTC before being
exposed or not for 6 h to increasing concentrations of azetidine
or canavanine. 24 h after these treatments, the amount of CAT
produced was estimated by CAT-ELISA testing (see "Materials and
Methods"). Fig. 11, A and
B, shows that PDTC drastically inhibited the activation of
the HIV-1 LTR by amino acid analogs. Similar results were observed when
cells were pretreated for 1 h with 30 mM of an other
antioxidant drug N-acetylcysteine (not shown). Antioxidants
also very efficiently blocked the amino acid analog-mediated transcriptional activation of the p2x
B-37TKcat vector which contains two
B sites in front of a cat gene (not shown). Control
experiments were also performed in which HeLa cells were transiently
transfected with p17-cat-neo plasmid. In this case, the activation of
human hsp70 promoter by amino acid analogs was not decreased by PDTC treatments, but in contrast drastically increased (Fig.
11C). The same observations were made when cells were
pretreated with N-acetylcysteine (not shown). The amino acid
analog activation of NF-
B and/or its binding to
B sites, present
in HIV-1 LTR, appear therefore likely to be dependent on an
intracellular pro-oxidant state induced by these compounds.

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Fig. 11.
Effect of the antioxidant PDTC on
HIV-1 LTR activation by amino acid analogs. HeLa cells transiently
transfected with an HIV-1 LTR (A and
B)-dependent reporter cat construct
were either left untreated or pretreated for 1 h with 100 µM PDTC. Subsequently, cells were treated for 6 h
with the indicated concentrations of azetidine (A) or
canavanine (B). Cells were then allowed to recover for
24 h before being harvested. C, control experiment
performed with HeLa cells transiently transfected with hsp70
promoter-dependent reporter cat construct
(p17-cat-neo). Azetidine or canavanine treatments in
presence or not of PDTC were performed as in A and B. Determination of CAT concentration and presentation of
the results are as in Fig. 1. The histograms shown are representative of three identical experiments; S.D. are presented (n = 3).
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Antioxidants or Glutathione Peroxidase Overexpression Inhibit
I
B-
Degradation Induced by Amino Acid Analogs--
Since
antioxidants were able to counteract the activation of HIV-1 LTR by
amino acid analogs, we investigated whether, in these conditions, the
stability of I
B-
was also modulated. As described above, HeLa
cells were treated or not with 100 µM PDTC prior to their
exposure to 15 mM of azetidine for various time periods.
Whole cell extracts were then analyzed in immunoblots probed with an
antibody specific for I
B-
/MAD-3. Fig.
12A shows that PDTC
completely abolished the azetidine-mediated degradation of I
B-
. A
similar observation was made when cells were treated with canavanine
instead of azetidine. To exclude the possibility of a nonspecific
effect of PDTC, we analyzed I
B-
stability in human T47D cell
transfectants that overexpress the detoxifiant enzyme selenoglutathione
peroxidase. These cells feature low reactive oxygen species levels
(73). Moreover, activation of NF-
B by oxidative stress was strongly
decreased in these cells through an inhibition of I
B-
phosphorylation and degradation (52). It is seen in Fig. 12B
that, in control T47D cells treated with 15 mM azetidine,
I
B-
is degraded while selenoglutathione peroxidase overexpression
abolished this effect. This observation correlated with the fact that
the activation of HIV-1 LTR by amino acid analogs was also inhibited in
these cells (not shown). These results therefore suggest that, in cells
treated by amino acid analogs, a pro-oxidant status is a prerequisite
for inducing NF-
B activation through I
B-
degradation by the
proteasome.

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Fig. 12.
Inhibition of amino acid analog-induced
I B- degradation by treatment with PDTC or glutathione peroxidase
overexpression. A, HeLa cells were either left untreated or
pretreated for 1 h with 100 µM PDTC. Subsequently,
cells were treated during various time periods ranging from 15 min to
6 h with 15 mM azetidine. Equal amounts of total
cellular proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and the cellular content of I B- was investigated by immunoblot analysis using an antibody specific to this protein. B, same as in A, but in this case, control
(Hygro) or glutathione peroxidase overexpressing
(GPx) human carcinoma T47D cells (see "Materials and
Methods") (52) were exposed to azetidine treatments. Note the
inhibition of amino acid analog-mediated I B- degradation in
HeLa cells treated with PDTC as well as in T47D cells
overexpressing glutathione peroxidase.
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We next investigated whether the oxidative stress generated by amino
acid analogs originated from the accumulation of mal-folded proteins in
the endoplasmic reticulum (ER). Indeed, a protein overload of the ER is
known to induce pro-oxidative conditions that trigger NF-
B
activation (41). This overload releases Ca2+ from the ER
which then activates ROS producing enzymes such as cyclooxygenase
and/or lipooxygenases (41, 74, 75). Since this ER overload activation
of NF-
B is inhibited by the Ca2+ chelator BAPTA-AM (75),
we therefore incubated HeLa cells for 1 h with 20 µM
of BAPTA-AM before amino acid analog treatment. This pretreatment was
not found to prevent I
B-
-induced degradation (data not shown),
hence suggesting that the oxidative stress generated by amino acid
analogs was not due to an accumulation of mal-folded proteins, mediated
by these coumpounds, in the ER.
 |
DISCUSSION |
Amino Acid Analogs Activate HIV-1 LTR through a
B-dependent Mechanism--
HIV-1 LTR is
transcriptionally activated by several cellular stress including heat
shock (6, 8-10). However, the molecular mechanisms underlying the
thermal activation of HIV-1 LTR are still a matter of speculation.
Indeed, heat stress, and to a larger scale, fever, are very complex
stimuli that involve juxtaposition of multiple events and among them
the induction of unfolded and denatured proteins (17). To test whether
the accumulation of aberrantly conformed proteins could be a signal for
HIV-1 LTR activation, we studied the effect of amino acid analog
treatments on HIV-1 LTR. Amino acid analogs induce irreversible protein
conformation and are potent activators of the heat shock promoter and
of the unfolded protein response pathway (19, 41, 76, 77). We show here
that azetidine and canavanine were about four times more powerful to
activate HIV-1 LTR than the heat shock promoter. This difference in
activation was reversed when cells were exposed to heat shock.
Moreover, we have observed that the activation of HIV-1 LTR by amino
acid analogs depended on the two
B sites located in the LTR and that
a
B driven reporter gene was strongly activated by these analogs.
These results therefore indicate that amino acid analogs are new
members of the already impressive list of
B-mediated transcriptional
activators. Besides, these findings suggest that accumulation of
mal-folded proteins in the cell triggers
B-mediated transcription
and that pathologies interfering with protein folding are probably
inducers of
B-dependent gene expression.
Amino Acid Analogs Activate the Transcription Factor
NF-
B--
We have shown that treatments of HeLa cells with amino
acid analogs induced the specific DNA binding and transcriptional
activity of NF-
B. A similar activation was observed in an other
human cell line (T47D cells). Since amino acid analogs can induce
abnormal conformation of the proteins in which they are incorporated,
it can be hypothesized that the accumulation of misfolded proteins represents a stimulus that trigger NF-
B activation. This hypothesis is strengthened by the fact that puromycin, an analog of aminoacyl tRNA
which induces a premature release of polypeptide chains from the
translation machinery, therefore creating abnormal polypeptides inside
the cell, can induce
B-dependent transcriptional
activity and NF-
B
activation.2
Amino Acid Analogs Induce I
B-
Degradation without Any Prior
Modification of the Distribution of Its Isoforms--
We report here
that amino acid analogs activate NF-
B through I
B-
degradation.
This breakdown of I
B-
level is not induced by an inhibition of
global protein synthesis nor of I
B-
synthesis, since we showed
that, after 60 min of treatment with 15 mM azetidine, global protein synthesis was not altered and I
B-
was still
synthesized. On the other hand, at that time of azetidine treatment,
I
B-
level was drastically decreased, suggesting that the
degradation of I
B-
was enhanced during the amino acid analog
treatment. Stimulation-induced breakdown of I
B-
or basal turnover
of this inhibitory subunit are both mediated by the proteasome (57). We
show here that a specific proteasome inhibitor (PSI) completely abrogated I
B-
degradation after azetidine treatment, suggesting an important role played by this protease. I
B-
level was even slightly enhanced in the presence of this inhibitor, confirming the
role of the proteasome in I
B-
basal turnover (57). Moreover, this
degradation of I
B-
is a slow phenomenon, which begins to be
detectable after 1 h of treatment with 15 mM azetidine
or canavanine, and is not preceded by a change in I
B-
isoform
distribution, as shown by one- or two-dimensional immunoblot analysis.
This suggests that amino acid analogs do not trigger I
B-
phosphorylation prior to its degradation. This phenomenon contrasts
with the TNF-
- or H2O2-mediated rapid
phosphorylation and subsequent degradation of I
B-
that has been
reported by many authors (45, 47, 52, 69). Hence, one could hypothesize
that I
B-
breakdown in the presence of amino acid analogs may
result of an unbalanced basal turnover of this protein instead of an
induced phosphorylation-ubiquitination-mediated breakdown. Indeed,
I
B-
basal turnover does not appear to require any ubiquitine
conjugation or phosphorylation event, but is still dependent on the
proteasome (57).
The 60 min required to observe degradation of I
B-
by the
proteasome probably reflects the time needed to incorporate amino acid
analogs into proteins that have a relatively short half-life. Since
amino acid analogs can induce abnormal protein conformation, the
gradual accumulation of misfolded proteins may be a signal for
I
B-
degradation. On the other hand, I
B-
is rich in proline and arginine (78) and has a short half-life of about 30-40 min (35,
67) (this study). Consequently, the azetidine or canavanine treatments
used in this study probably poison I
B-
and generate aberrant
conformation of this polypeptide. This phenomenon may directly
stimulate the basal degradation of I
B-
by the proteasome. In
comparison, the half-life of the two NF-
B subunits, p65 and p50, is
very long (between 8 and 24 h) (35). Thus, although p65 and p50
are rich in arginine and proline (79, 80), their very long half-lives
suggest that, in our experimental conditions, only a small fraction of
these proteins is altered by amino acid analogs. These observations
suggest that NF-
B activation by amino acid analogs is not a
consequence of amino acid analog-poisoned p50 and p65 but rather
results from the direct or indirect action of amino acid analogs on the
short lived protein I
B-
. Since NF-
B activation appears to be
time-correlated with I
B-
degradation, it is difficult to decide
whether or not amino acid analogs trigger the release of I
B-
from
the NF-
B-I
B-
complex before I
B-
is degraded.
NF-
B Activation by Amino Acid Analogs Is
Redox-dependent--
Like most NF-
B inducers tested so
far, the transcriptional activation of
B-driven genes by canavanine
or azetidine was inhibited by the antioxidant PDTC. A similar
observation was made when the experiment was carried out in T47D cells
that overexpress glutathione peroxidase. Our experiments therefore
suggest that amino acid analogs induce an intracellular oxidative
stress that is essential for triggering NF-
B activation. In
contrast, antioxidant conditions drastically enhanced the
transcriptional activation of hsp70 promoter by amino acid analogs.
Hence, while
B and hsp70 promoters are activated by several common
inducers, the maximal inducibility of these two promoters appears
inversely regulated by intracellular redox. It is not known how amino
acid analogs generate oxidative stress. However, these compounds are
known to trigger the synthesis of the ER protein chaperone Grp78/Bip
and other glucose-related proteins (17, 23-25) through the unfolded
protein response pathway (41, 76, 77). Indeed, in our conditions, the
accumulation of Grp78, which reflects that of misfolded proteins in the
ER, was transient and peaked after 2 h of treatment with the
analogs, indicating that HeLa cells were under such an ER stress.
Nevertheless, the unfolded protein response signal transduction pathway
does not seem to involve the generation of reactive oxygen species (81-84). Hence, we tested whether an other type of ER stress caused by
ER overload, which is known to generate oxidative stress, the so-called
redox-dependent NF-
B ER to nucleus stress pathway (41), could be responsible for the pro-oxidative status induced by amino acid
analogs. This ER overload results in a Ca2+ release from
the ER that activates ROS producing enzymes such as cyclooxygenase
and/or lipooxygenase and subsequently NF-
B (41, 74). Preincubation
of HeLa cells with the intracellular Ca2+ chelator
BAPTA-AM, which abolishes NF-
B induction by ER overload (75), was
not found to prevent I
B-
induced degradation by amino acid
analogs. Hence, the oxidative stress generated by amino acid analogs,
which is necessary for I
B-
degradation by the proteasome, is
probably not due to the effect mediated by amino acid analogs in the
ER.
The Proteolytic Degradation of I
B-
Requires Pro-oxidant
Conditions--
In the case of NF-
B activation by hydrogen peroxide
or TNF-
, I
B-
phosphorylation at serines 32 and 36 precedes
(69-71) and is necessary for the rapid degradation of this protein by the proteasome (44, 45, 48-50). In this system of activation, I
B-
phosphorylation and degradation are abolished by antioxidants (42, 69) or the overexpression of the detoxifiant enzyme glutathione peroxidase (52). Here we show that the degradation of I
B-
triggered by amino acid analogs occurs in the absence of any
modification of I
B-
isoform distribution but still requires
pro-oxidant conditions. Indeed, treatment with PDTC and overexpression
of glutathione peroxidase abolish I
B-
degradation mediated by
amino acid analogs, whereas no phosphorylation of this protein could be
detected. Hence, on one hand, glutathione peroxidase overactivity can
inhibit the phosphorylation-independent I
B-
proteolysis by amino
acid analogs, and on the other hand this enzyme also abolishes the phosphorylation-dependent degradation of I
B-
(52).
These apparently conflicting observations suggest that, in addition to
phosphorylation, the proteolytic degradation of I
B-
per
se, by the proteasome, is probably also redox-modulated. In this
respect, it is interesting to mention that, in unstressed T47D cells,
glutathione peroxidase overexpression increased the I
B-
half-life
by a factor of two.2 This observation favors the hypothesis
that the basal turnover of this protein is also under the control of
intracellular redox. It is, however, not yet known whether the activity
of the I
B-
protease is regulated by redox. In this respect, it is
interesting to note that the proteasome contains a phosphorylated
subunit (85, 86) whose level of phosphorylation may represent a target that could be modulated by intracellular redox.
We thank P. A. Baeuerle (Freiburg
University, Germany, and Tularik Inc., San Francisco, CA) for the kind
gift of p2x
B-37TKcat vector and Dominique Guillet for excellent
technical assistance.