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J Biol Chem, Vol. 274, Issue 27, 18981-18988, July 2, 1999
B Regulation
B TRANSCRIPTIONAL ACTIVITY AND PROMOTION OF
IB
DEGRADATION*
,,,,, and§¶
From the Department of Biochemistry,
§ Research Institute of Molecular Genetics, College of
Medicine, The Catholic University of Korea, Seoul 137-701, Korea
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Previously we reported that 3-deazaadenosine
(DZA), a potent inhibitor and substrate for
S-adenosylhomocysteine hydrolase inhibits bacterial
lipopolysaccharide-induced transcription of tumor necrosis factor- Nuclear factor- Activation of NF- 3-Deazaadenosine (DZA) was developed to be one of the most potent
inhibitors of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) (18). This agent binds to S-adenosylhomocysteine hydrolase
resulting in the accumulation of S-adenosylhomocysteine and
S-adenosylmethionine, and serves as a substrate for the
enzyme resulting in the huge accumulation of
3-deazaadenosylhomocysteine (DZA-Hcy) in cultured cells (18, 19),
especially in liver tissue (20). DZA exerts a number of interesting
biological properties, such as anti-human immunodeficiency virus (HIV)
activity (21, 22), immunosuppressive and anti-inflammatory effects (23,
24), inhibition of lymphocyte-mediated cytolysis (25), inhibition of
cytokine expression including TNF- Previously, we reported that DZA inhibits LPS-induced TNF- Chemicals--
DZA, 3-deaza-(±)-aristeromycin (DZAri), and
DZA-Hcy were donated by Dr. Chiang of the Walter Reed Army Institute of
Research, Washington, D. C. Hcy-thiolactone and LPS
(Escherichia coli, No. 0127 B-8) were purchased
from Sigma. Recombinant glutathione S-transferase (GST)
fusion protein of human TNF- Cell Culture--
Mouse macrophage RAW 264.7, human monocytic
THP-1, and SV40-transformed African green monkey kidney COS-7 cells
were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). PBMC were isolated from defibrinated blood using
Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) by
density-gradient separation, followed by adherence to tissue culture
dish for 2 h at 37 °C. Nonadherent cells were removed by
washing the monolayer four times with phosphate-buffered saline. All
cells were cultured in RPMI 1640 medium supplemented with 20 mM HEPES, 25 mM sodium bicarbonate, 50 µg/ml
gentamicin, and 10% heat-inactivated (56 °C for 30 min) fetal
bovine serum (Hyclone Laboratories Inc., Logan, UT) at 37 °C in an
atmosphere of 5% CO2.
Northern Blot Analysis--
Total cellular RNA was isolated from
cells at 2 h after LPS (1 µg/ml) stimulation according to a
previously described method (31). Five µg of total RNA was separated
on a 1% agarose gel containing 2.2 M formaldehyde.
Northern blot analysis was performed as described (26). All of the
digoxigenin (Roche Molecular Biochemicals, Mannheim, Germany)-labeled
probes were prepared from each specific primer and cDNA fragments
of amplimer sets (CLONTECH, Palo Alto, CA) by
polymerase chain reaction.
Preparation of Cytoplasmic Fraction and Nuclear
Extract--
After RAW 264.7 cells were stimulated with LPS (1 µg/ml) for 1 h and COS-7 cells were stimulated with recombinant
GST-human TNF- EMSA--
Five µg of nuclear protein and 1 µg of poly(dI-dC)
(Roche Molecular Biochemicals) per reaction were incubated for 15 min
at room temperature with NF- Western Blot Analysis--
Ten µg of proteins were subjected
to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for NF- Plasmids and Transient Transfection Analysis--
Plasmid J16
containing two copies of the wild-type NF- In Vivo Labeling and Immunoprecipitation--
Confluent
monolayers of RAW 264.7 cells in 10-cm tissue culture dishes were
washed twice with phosphate-free Dulbecco's minimum essential medium
and incubated in phosphate-free Dulbecco's minimal essential medium
for 1 h. Media was replaced by fresh phosphate-free Dulbecco's
minimal essential medium containing 100 µCi of
32Pi per ml (ICN, Costa Mesa, CA), and the
cells were incubated for 2 h. Cells were pretreated with or
without DZA (100 µM) for 1 h, and stimulated by the
addition of LPS (1 µg/ml). After incubation for 1 h, the cells
were washed, and p65 was recovered by immunoprecipitation with anti-p65
(C-20, Santa Cruz Biotechnology) and protein A-Sepharose (Amersham
Pharmacia Biotech). The immunopellets were fractionated by SDS-PAGE,
and the gel was stained with Coomassie Brilliant Blue R-250 to confirm
that the equal amount of protein was loaded in each wall. Dried gel was
subjected to autoradiography.
IKK Assay--
RAW 264.7 cells were treated with 100 µM DZA for various time intervals, and subjected to IKK
assay. IKK activity was measured as described (34), using recombinant
protein of GST-human I LPS-induced Expression of TNF- DNA Binding Activity of NF- Nuclear Translocation of NF- LPS-induced NF- LPS-induced Phosphorylation of p65 Is Inhibited by DZA--
To
determine if DZA might inhibit the functional activation of NF- TNF-induced NF- DZA Induces Proteolytic Degradation of I DZA-induced Proteolytic Degradation of I In this study, we demonstrated the dual effects of DZA on the
regulation of NF-
and interleukin-1
in mouse macrophage RAW 264.7 cells. In this
study, we demonstrate the effects of DZA on nuclear factor-B
(NF-B) regulation. DZA inhibits the transcriptional activity of
NF-B through the hindrance of p65 (Rel-A) phosphorylation without
reduction of its nuclear translocation and DNA binding activity. The
inhibitory effect of DZA on NF-B transcriptional activity is
potentiated by the addition of homocysteine. Taken together, DZA
promotes the proteolytic degradation of IB, but not IB,
resulting in an increase of DNA binding activity of NF-B in the
nucleus in the absence of its transcriptional activity in RAW 264.7 cells. The reduction of IB by DZA is neither involved in IB
kinase complex activation nor modulated by the addition of
homocysteine. This study strongly suggests that DZA may be a potent
drug for the treatment of diseases in which NF-B plays a central
pathogenic role, as well as a useful tool for studying the regulation
and physiological functions of NF-B.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
(NF-B),1 a ubiquitous
transcription factor, is a pivotal regulator of immune responses,
inflammation, cell proliferation, oncogenesis, and apoptosis (1-3). A
prototype member of the Rel family, hetero- or homodimeric NF-B, is
made from monomers that have a highly conserved approximately 300-amino acid amino-terminal domain, which is called the Rel homology domain, which functions in DNA binding, nuclear translocation, formation of
dimers, and IB binding. Most of the dimeric NF-B complexes are
stored in the cytoplasm of unstimulated cells as inactive complexes
through interactions with a group of inhibitory proteins called IB.
Recently, the vertebrate IB family such as IB, IB,
Bcl-3, precursor proteins p105 and p100, and IB
were reported (4,
5). Inactive cytoplasmic NF-B can be activated by stimulation of
cells with a broad range of NF-B-inducing agents including bacterial
lipopolysaccharide (LPS) and tumor necrosis factor- (TNF-).
Despite differences among these stimuli, one of their common targets is
the cytoplasmic NF-B·IB complexes. These signals known to
activate NF-B result in phosphorylation, subsequent ubiquitination,
and proteasome-mediated degradation of the IB proteins, allowing
NF-B to translocate into the nucleus, bind to specific B sites
and thereby activate target genes such as various cytokines, cell
adhesion molecules, acute-phase proteins, and immunoreceptors (6).
Researchers have long searched to identify IB kinase which
phosphorylates the IB proteins to initiate the activation cascade of
NF-B. Recently, IB kinase (IKK) and IB kinase (IKK) were reported as essential kinases for NF-B activation
downstream of TNF- and interleukin-1 (IL-1) receptors (7-9).
B could be inhibited through diverse mechanisms by
manifold compounds at distinct positions in the activation cascade. One
group of NF-B inhibitors which share the property of being
anti-oxidative, includes N-acetyl-L-cysteine
(10), acetylsalicylic acid (11), and pyrrolidine dithiocarbamate (12). Some inhibitors interrupt the induced degradation of IB proteins by
the inhibition of 26 S protease (13), or by increase of IB synthesis (14, 15). Another group of inhibitors hinders the transcriptional activity of NF-B already bound to DNA. This group includes SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (16), and elevated intracellular cyclic AMP (cAMP) (17).
and IL-1 (26), inhibition of
cell adhesion molecule expression (27, 28), and induction of apoptosis
in human and mouse leukemia cells (29, 30). Although the wide variety
of biological properties underscores that DZA is to be an effective
drug for treatment of many human diseases, the action mechanism of DZA
is not yet fully understood.
and
IL-1 transcription in mouse macrophage RAW264.7 cells (26). In this
study, elementary experiments confirming DZA inhibition of TNF-
transcription in human monocytic macrophage THP-1 cells and human
peripheral blood monocytes (PBMC) encouraged us to investigate the
effect of DZA on NF-B activation with the intention of understanding the cellular mechanism of DZA. DZA potently inhibited the
transcriptional activity of NF-B through the hindrance of p65
phosphorylation without reduction of nuclear translocation and DNA
binding activity of NF-B. In RAW 264.7 cells, DZA promoted the
proteolytic degradation of IB resulting in an increase of DNA
binding activity of NF-B in the nucleus. These results strongly
suggest that DZA may serve as a potent drug for the treatment of
diseases in which NF-B plays an important pathogenic role, as well
as a useful tool for studying the regulation and physiological
functions of NF-B.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and GST human IB were kindly provided by Dr. Dae-Myung Jue in our department. Unless specified otherwise, all reagents were purchased from Sigma.
protein (50 ng/ml) for 1 h, cells were washed
twice with phosphate-buffered saline. Cytosolic fraction and nuclear
extract for Western blot analysis and electrophoretic mobility shift
assay (EMSA) were prepared as described by Dignam et al.
(32). Concentration of protein was determined using Coomassie Plus
Protein Assay Reagent (Pierce).
B consensus sequence (Santa Cruz
Biotechnology, Santa Cruz, CA), which was 3'-end labeled with
32P, in binding buffer (20 mM HEPES, pH 7.6, 1 mM EDTA, 10 mM
(NH4)2SO4, 1 mM DTT,
0.2% (w/v) Tween 20, 30 mM KCl). After the binding
reaction, samples were analyzed by electrophoresis on a 6% native
polyacrylamide gel that was run in 0.5× Tris borate-EDTA (TBE) buffer,
pH 8.0, and then the dried gel was subjected to autoradiography. For
competition, 50-fold unlabeled NF-B or AP-1 consensus sequence
(Santa Cruz Biotechnology) were used.
B
proteins or 12.5% SDS-PAGE for IB proteins, and transferred to a
nitrocellulose membrane in transfer buffer (25 mM Tris
base, 193 mM glycine, 20% methanol) at 500 mA for 4 h. Western blot analysis was performed as described (26). All primary
antibodies used in this study (anti-p65 (C-20), anti-p50 (NLS),
anti-c-Rel (N), anti-IB/MAD-3 (C-21), and anti-IB (C-20))
were purchased from Santa Cruz Biotechnology.
B-binding site, and J32
containing two copies of the mutant NF-B-binding site, upstream of a
truncated c-fos promoter which was linked to the
chloramphenicol acetyltransferase (CAT) gene (33), were kindly provided
by Dr. David Baltimore of California Institute of Technology, Pasadena,
CA. pSVL65 expressing p65 of NF-B from a SV40 promoter was kindly
donated by Dr. Mahnhoon Park of Mogam Biotechnology Research Institute,
Yongin, Kyunggi-do, Korea. pSVL (Amersham Pharmacia Biotech) was used
as a control taking the place of pSVL65. To assess variations in
transfection efficiencies, a control plasmid pCMV
(CLONTECH) which expresses the LacZ gene was used. Transfection of cells was performed using FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals) according to a
procedure recommended by the manufacturer. Cells transfected with
either J16 or J32 were cultured for 18 h, and then exposed to LPS
or TNF- in the presence or absence of DZA alone or DZA with Hcy together. For the pSVL65 transfection, cells were exposed to 100 µM DZA at 6 h after transfection, and further
incubated for 18 h. Induction of CAT expression was determined
using CAT enzyme-linked immunosorbent assay (Roche Molecular
Biochemicals) according to the manufacturer's instructions, and
standardized to constitutive levels of -galactosidase activity.
B as a substrate. A polyclonal antibody to
IKK (M-280) was obtained from Santa Cruz Biotechnology.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mRNA Is Inhibited by
DZA--
Transcription of TNF- and IL-1 was inhibited by DZA in
a dose-dependent manner in RAW 264.7 cells stimulated by
LPS (26). To investigate the effects of DZA on the expression of
TNF- in other types of cells, THP-1 cells and PBMC were pretreated
with increasing concentrations of DZA for 1 h, and stimulated by
the addition of LPS at a final concentration of 1 µg/ml. As shown in
Fig. 1, LPS stimulation increased the
steady-state levels of TNF- mRNA as early as 2 h. Treatment
of DZA inhibited the LPS-induced expression of TNF- mRNA dose
dependently in both THP-1 cells and PBMC in the same manner as in RAW
264.7 cells. Expression of -actin mRNA as a control was not
affected by DZA in any of the cells. These results show that DZA
efficiently inhibits the LPS-induced expression of TNF- mRNA and
this potency is not only restricted to murine macrophage cells.
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Fig. 1.
LPS-induced expression of
TNF- mRNA is inhibited by DZA. Cells
were pretreated with DZA at the indicated dosages (µM)
for 1 h before stimulation with LPS. At 2 h after addition of
LPS (1 µg/ml), total RNAs were extracted and Northern blot analysis
was performed. Data illustrated are from a single experiment and are
representative of a total of three separate experiments.
B Is Increased by DZA--
Since the
activation of transcription factor NF-B has been shown to be
indispensable for TNF- expression induced by LPS (35), we examined
the effect of DZA on DNA binding activity of NF-B in the nucleus of
RAW 264.7 cells by EMSA, using 32P-labeled NF-B specific
oligonucleotides. An inducible protein-DNA complex was observed in the
nuclear extracts from LPS-stimulated RAW 264.7 (Fig.
2A). Unexpectedly, cells
pretreated with DZA revealed no significant decrease of the LPS-induced
DNA binding activity of NF-B, even though expression of the TNF-
gene was potently inhibited by exposure to DZA. Moreover, DNA binding
activity was potentiated in nuclear extracts dose dependently by DZA,
irrespective of LPS stimulation. In competition experiments, a 50-fold
amount of unlabeled NF-B-specific oligonucleotide absolutely
inhibited typical binding activities, but the same amount of unlabeled
AP-1 oligonucleotide failed to inhibit binding activities, confirming their specificities (Fig. 2B). These results suggest that
the inhibition of TNF- gene expression by DZA occurred without
down-regulation of NF-B DNA binding activity, and that DZA increases
DNA binding activity of NF-B in RAW 264.7 cells. Since the presence
of a reducing agent such as DTT in preparation of the nuclear extract and the execution of EMSA could mask lost DNA binding activity of
NF-B, we repeated the EMSA assay without DTT (Fig. 2C).
Removal of DTT from the assay could not modulate each of the DNA
binding activities which were presented by DTT in an assay with DTT,
indicating that the DZA inhibition of TNF- gene expression is not
caused by a loss of NF-B DNA binding activity.
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Fig. 2.
DNA binding activity of
NF- B is increased by DZA. A,
dose-response effect of DZA in RAW 264.7 cells. The cells were
pretreated with or without DZA at the indicated concentrations
(µM) for 1 h, and then stimulated by the addition of
LPS (1 µg/ml) or not stimulated. Nuclear extracts were prepared at
1 h after stimulation. 5 µg of each nuclear protein were
subjected to a DNA binding reaction with 32P-end-labeled
NF-B consensus sequence, and then DNA-protein complexes were
separated by native polyacrylamide gel electrophoresis. B,
competition experiments in RAW 264.7 cells using 50-fold cold NF-B
binding oligonucleotide or AP-1. C, removal of DTT in EMSA.
Preparation of nuclear extracts in RAW 264.7 cells and DNA binding
reactions were accomplished without DTT. Data illustrated are from a
single experiment and are representative of a total of three separate
experiments.
B Is Increased by DZA--
We
examined the effects of DZA on the translocation of p65 (Rel-A), p50
(NF-B1), and c-Rel into the nucleus of RAW 264.7 cells stimulated
with or without LPS using Western blot analysis. LPS stimulation
increased protein levels of each NF-B subunit in the nucleus.
Treatment of DZA at a concentration of 100 µM induced an
increase of Rel family proteins in the nucleus of RAW 264.7 cells
regardless of LPS stimulation (Fig.
3A). These results are in
exact agreement with the results in Fig. 2A, which shows that DZA induces nuclear translocation of NF-B and potentiates its
LPS-induced translocation in RAW 264.7 cells. We next established the
modulation of p65 in the nucleus of RAW 264.7 cells by DZA at either
various concentrations or various time intervals. Similar to the
results in Fig. 2A, DZA at 100 µM
concentration increased the levels of p65 in the nucleus regardless of
LPS stimulation (Fig. 3B). Fig. 3C shows the
modulation of p65 level in the nucleus by treatment of DZA at various
time intervals. p65 in the nucleus increased remarkably at 60 and 120 min after treatment with DZA, even though there was a slight decrease
at 15 min. The total amount of cellular p65 was not modulated by
DZA, whereas the amount of cytoplasmic p65 decreased
proportionally to the increase of nuclear p65 by DZA as measured in a
Western blot analysis of total cellular extracts and cytoplasmic
fractions (data not shown). These results demonstrate that DZA
increases the amount of p65 nuclear translocation in RAW 264.7 cells,
and that the increase of p65 in the nucleus by DZA is caused only by
nuclear translocation, and not by the enhancement of p65
expression.
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Fig. 3.
Nuclear translocation of
NF- B is increased by DZA. A,
the effect of DZA in RAW 264.7 cells. The cells were pretreated with or
without 100 µM DZA for 1 h, and then stimulated by
the addition of LPS (1 µg/ml). After 1 h, nuclear extracts were
prepared. 10 µg of each nuclear protein was fractionated by SDS-PAGE,
and then subjected to Western blot analysis with specific antibodies
against p65, p50, c-Rel, respectively. B, dose-response
effect of DZA. RAW 264.7 cells were pretreated with DZA at the
indicated concentrations (µM) for 1 h, and then
stimulated by the addition of LPS (1 µg/ml) or not stimulated. After
1 h, nuclear extracts were prepared. 10 µg of each nuclear
protein was separated by SDS-PAGE, and then Western blot analysis with
specific antibody against p65 was performed. C,
time-response effect of DZA. RAW 264.7 cells were treated with 100 µM DZA for the indicated times. At the end of the times,
nuclear extracts were prepared. 10 µg of each nuclear protein was
separated by SDS-PAGE, and then Western blot analysis with specific
antibody against p65 was performed. Data illustrated are from a single
experiment and are representative of a total of three separate
experiments.
B Transcriptional Activity Is Inhibited by DZA,
and This Inhibitory Effect Is Augmented by the Addition of
Hcy--
Since DZA is known to inhibit the expression of TNF-
mRNA without a diminution of NF-B nuclear translocation and DNA
binding activity, we proved the effects of DZA on the NF-B
transcriptional activity by transient transfection experiments of RAW
264.7 cells using reporter gene constructs carrying two copies of the
wild-type (J16) NF-B binding sequence or mutant (J32) NF-B
binding sequence in front of the CAT gene, which have been shown to
specifically respond to NF-B activation (33). LPS stimulation of
cells transfected with J16 resulted in a 4.7-fold induction of CAT
expression (Fig. 4A), whereas
no induction of CAT expression by LPS was observed in cells transfected
with J32. Treatment of the cells with 100 µM DZA for
1 h before LPS stimulation resulted in a drastic inhibition of
LPS-induced CAT expression. LPS-induced transcriptional activity of
NF-B was dose dependently inhibited by DZA (Fig. 4B).
These results strongly suggest that DZA potently inhibits NF-B
transcriptional activity in RAW 264.7 cells stimulated by LPS, even
though it more intensely provokes the nuclear translocation and DNA
binding activity of NF-B. The effect of Hcy on DZA inhibition of
NF-B transcriptional activity was examined. There was a tendency to inhibit NF-B transcriptional activity more than the inhibition observed by DZA alone (Fig. 4B). These results imply that
DZA inhibition of NF-B transcriptional activity is involved in the accumulation of DZA-Hcy in RAW 264.7 cells. To determine the effect of
DZA on CAT expression induced by expression of p65, RAW 264.7 cells
were co-transfected with CAT-reporter plasmids and pSVL65 that express
large amounts of p65. As a control, cells were transfected with pSVL
substituting for pSVL65. After 6 h of transfection, one group of
cells was exposed for 18 h to 100 µM DZA and the other was not (Fig. 4C). Expression of p65 induced a
5.9-fold increase of CAT protein, driven from J16 under the absence of DZA treatment, but did not induce CAT protein expression from J32. The
expression of CAT protein was completely inhibited from the cells
transfected with pSVL65 when DZA was treated into cells at the
concentration of 100 µM, although the induction of CAT expression in the cells transfected with pSVL 65 was higher than that
in the cells only stimulated with LPS. The production of p65 was not
affected by DZA as monitored by Western blot analysis using anti-p65
(data not shown). These results, together with the results in Fig. 4,
A and B, showing DZA inhibition of LPS-induced NF-B transcriptional activity, suggest that DZA directly inhibits the transcriptional activity of p65 but not at the signal cascade of
LPS-induced NF-B activation. To elucidate whether any of the observed effects could be due to DZA-Hcy, cells to be exposed to 100 µM DZA for 1 h before LPS stimulation were
pretreated with a more specific and potent inhibitor of
S-adenosylhomocysteine hydrolase, DZAri, to block the
intracellular accumulation of DZA-Hcy (Fig. 4D). Treatment
of DZAri almost completely abrogated the inhibition of NF-B
transcriptional activity in cells treated with 100 µM
DZA, indicating a central role for DZA-Hcy in the DZA inhibition of
NF-B transcriptional activity. No inhibition of NF-B
transcriptional activity was observed in cells treated with DZAri
alone. To investigate whether exogenous DZA-Hcy is capable of
inhibition, cells were exposed to 100 µM DZA-Hcy for 1 h before LPS stimulation. As shown in Fig. 4D,
exogenous DZA-Hcy failed to inhibit NF-B transcriptional activity in
the cells stimulated with LPS, demonstrating that DZA-Hcy is incapable
of inhibition when given exogenously.
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Fig. 4.
LPS-induced NF- B
transcriptional activity is inhibited by DZA in RAW 264.7 cells.
The cells were transiently transfected with reporter gene constructs
carrying two copies of the wild type (J16, 
) NF-B binding
sequence or mutant type (J32, 
) NF-B binding sequence in front of
the CAT gene. A, the effect of DZA on NF-B
transcriptional activity. At 18 h after transfection with J16
() or J32 (), cells were treated with or without 100 µM DZA for 1 h before stimulation. After 18 h
of stimulation by the addition of LPS (1 µg/ml), cells were lyzed to
determine the expression of CAT. B, the effect of Hcy on the
inhibitory effect of DZA. At 18 h after transfection with J16,
cells were treated for 1 h with or without DZA at the indicated
dosages (µM), in the absence () or presence () of
Hcy (100 µM). After 18 h of stimulation by the
addition of LPS (1 µg/ml), cells were lysed to determine the
expression of CAT. C, the effect of DZA in the cells
transfected with pSVL65 expressing p65. The cells were co-transfected
with pSVL65 and J16 (), or with pSVL65 and J32 (). As a control,
cells were transfected with pSVL substituting pSVL65. At 6 h after
transfection, cells were treated with or without 100 µM
DZA, and incubated for a further 18 h. And then cells were lysed
to determine the expression of CAT. D, the effect of DZA
derivatives. At 18 h after transfection with J16, cells were
treated for 1 h with 100 µM DZA or 100 µM DZA-Hcy before stimulation. For the DZAri treatment,
100 µM DZAri was given 1 h before DZA treatment.
After 18 h of stimulation by the addition of LPS (1 µg/ml),
cells were lysed to determine the expression of CAT. Determination of
CAT expression was performed using CAT enzyme-linked immunosorbent
assay kit. Values are mean ± S.D. (n = 3).
B by
modifying phosphorylation of p65, we examined the phosphorylation of
p65 in LPS-stimulated RAW 264.7 cells in the presence or absence of
DZA. As shown in Fig. 5, LPS induced a strong phosphorylation of p65. In cells pretreated with 100 µM DZA, however, LPS-induced phosphorylation of p65 was
markedly reduced. A low constitutive phosphorylation of p65 was
observed in unstimulated cells, which was reduced by treatment of the
cells with DZA alone. These results strongly suggest that DZA inhibits the transcriptional activity of NF-B by the hindrance of p65 phosphorylation required for the functional activation of NF-B.
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Fig. 5.
LPS-induced phosphorylation of p65 is
inhibited by DZA. Phosphate-labeled RAW 264.7 cells were
pretreated with or without DZA (100 µM) for 1 h, and
stimulated with the addition of LPS (1 µg/ml). After incubation for
1 h, p65 was recovered by immunoprecipitation using specific
antibody and fractionated by SDS-PAGE, followed by autoradiography.
Similar results were obtained in an independent experiment.
B Transcriptional Activity Is Inhibited by
DZA--
We tried to determine if DZA could inhibit NF-B
transcriptional activity without affecting nuclear translocation in
non-hematopoietic cells stimulated by other inducers of NF-B.
TNF- stimulation of COS-7 cells prominently induced the nuclear
translocation of NF-B and the DNA binding activity of NF-B, which
was not observed in unstimulated cells (Fig.
6A). The addition of 100 µM DZA to COS-7 cells before TNF- stimulation had no
effect on nuclear translocation or DNA binding activity of NF-B,
suggesting that DZA did not affect TNF--induced nuclear
translocation of NF-B in COS-7 cells. DZA alone did not induce the
nuclear translocation of NF-B in the cells. In transient
transfection experiments of COS-7 cells using reporter gene constructs
(J16 or J32), TNF- stimulation of cells transfected with J16
resulted in CAT expression that was 2.6-fold greater (Fig.
6B). No induction of CAT expression by TNF- was observed
in cells transfected with J32. Treatment of these cells with 100 µM DZA for 1 h before TNF- stimulation resulted
in complete inhibition of CAT protein expression. These results reveal
that DZA is a common and potent inhibitor of NF-B transcriptional
activity induced by distinct stimuli. The effect of Hcy on DZA
inhibition of NF-B transcriptional activity was examined. As shown
in Fig. 6C, DZA dose dependently inhibited the
transcriptional activity of NF-B in cells stimulated by TNF-. Combination of DZA and Hcy more potently inhibited NF-B
transcriptional activity than inhibition observed by DZA alone. These
results show that DZA inhibition of NF-B transcriptional activity is involved in the accumulation of DZA-Hcy in COS-7 cells stimulated with
TNF. Cells co-transfected with pSVL65 and CAT-reporter plasmid (J16
or J32) showed a 3.0-fold expression of CAT protein driven from J16,
but not from J32 (Fig. 6D). 100 µM DZA
strongly inhibited the induction of CAT protein in cells expressing
p65, suggesting that DZA directly inhibits the transcriptional activity
of p65.
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Fig. 6.
TNF-induced NF- B
transcriptional activity is inhibited by DZA in COS-7 cells.
A, the effect of DZA on NF-B binding activity. The cells
were pretreated with or without 100 µM DZA for 1 h
before stimulation, and then stimulated by the addition of recombinant
GST-human TNF- (50 ng/ml). Nuclear extracts were prepared at 1 h after stimulation. 5 µg of each nuclear protein were subjected to a
DNA binding reaction with 32P-end-labeled NF-B consensus
sequence, and then DNA-protein complexes were separated by native
polyacrylamide gel electrophoresis. B, the effect of DZA on
NF-B transcriptional activity. The cells were transiently
transfected with reporter gene constructs carrying two copies of the
wild type (J16, ) or mutant type (J32, ) NF-B binding sequence
in front of the CAT gene. At 18 h after transfection, cells were
treated with or without 100 µM DZA for 1 h before
stimulation. After 18 h of stimulation by the addition of TNF-
(50 ng/ml), cells were lysed to determine the expression of CAT.
C, the effect of Hcy on the inhibitory effect of DZA. At
18 h after transfection with J16, cells were treated for 1 h
with or without DZA at the indicated dosages (µM), in the
absence () or presence () of Hcy (100 µM). After
18 h of stimulation by the addition of TNF- (50 ng/ml), cells
were lysed to determine the expression of CAT. D, the effect
of DZA in the cells transfected with pSVL65 expressing p65. The cells
were co-transfected with pSVL65 and J16 (), or with pSVL65 and J32
(). As a control, cells were transfected with pSVL substituting
pSVL65. At 6 h after transfection, cells were treated with or
without 100 µM DZA, and incubated for a further 18 h. And then cells were lysed to determine the expression of CAT. Determination of CAT expression was performed
using CAT enzyme-linked immunosorbent assay kit. Values are mean ± S.D. (n = 3).
B, but Not
IB--
Translocation of NF-B into the nucleus is linked to
proteolytic degradation of IB proteins (1), and as we have described above, DZA increases NF-B nuclear translocation in RAW 264.7 cells.
To test whether DZA promotes proteolytic degradation of IB proteins,
RAW 264.7 cells were pretreated with DZA following stimulation with or
without LPS. As shown in Fig.
7A, the level of IB
protein was notably reduced by treatment of DZA alone for 2 h at a
concentration of 100 µM. The IB protein was fully recovered at 1 h after LPS stimulation without DZA, since IB is autoregulated through the activation of NF-B (3, 36). Notably,
DZA pretreatment before LPS prevented synthesis of IB which was
recovered when treated with LPS alone, indicating that DZA interfered
with the synthesis of IB because the transcriptional activity of
NF-B was inhibited by DZA. Resynthesis of IB after stimulation
of LPS was more potently prevented by the addition of Hcy plus DZA
(data not shown). We also examined the effect of DZA on IB levels
in RAW 264.7 cells at various time intervals. IB levels were
reduced from their level at 60 min to their level at 120 min after
treatment with DZA, and this reduction was continued to 240 min (Fig.
7B). Thus, these results demonstrate that DZA promotes the
degradation of IB and prevents its resynthesis. Furthermore,
these results reasonably support that DZA increases nuclear
translocation and DNA binding activity of NF-B in RAW 264.7 cells. A
different IB isoform, IB is one of the major regulators of
NF-B activity, whose kinetics is slower than that of IB in
response to NF-B inducers such as LPS (6). We investigated the
effect of DZA on IB in RAW 264.7 cells (Fig. 7, C and
D). Stimulation of LPS for 1 h without pretreatment of
DZA led to the disappearance of IB, which would normally exist in
Western blot analysis using unstimulated RAW 264.7 cells (Fig.
7C). The disappearance of IB was also observed in
cells pretreated for 1 h with DZA at increasing concentrations
following stimulation with LPS. Interestingly, treatment of 100 µM DZA alone for 2 h at several concentrations could
not reduce the level of IB (Fig. 7C), and IB
remained continuously until 4 h after exposure of 100 µM DZA (Fig. 7D), implying that DZA cannot
lead to IB down-regulation, and the down-regulation of IB
is not involved in enhanced nuclear translocation of NF-B, mediated
by DZA in RAW 264.7 cells.
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Fig. 7.
DZA induces proteolytic degradation of
I B.
A, dose-response effect of DZA on IB in RAW 264.7 cells. The cells were pretreated with DZA at the indicated
concentrations (µM) for 1 h, and then stimulated by
the addition of LPS (1 µg/ml) or not stimulated. After 1 h,
cytosolic fractions were prepared. 10 µg of each protein was
separated by SDS-PAGE, and then Western blot analysis with specific
antibody against IB was performed. B, time-response
effect of DZA on IB. RAW 264.7 cells were treated with 100 µM DZA for the indicated times. At the end of the times,
cytosolic fractions were prepared. 10 µg of each protein was
separated by SDS-PAGE, and then Western blot analysis with specific
antibody against IB was performed. C, dose-response
effect of DZA on IB in RAW 264.7 cells. The cells were pretreated
with DZA at the indicated concentrations (µM) for 1 h, and then stimulated by the addition of LPS (1 µg/ml) or not
stimulated. After 1 h, cytosolic fractions were prepared. 10 µg
of each protein was separated by SDS-PAGE, and then Western blot
analysis with specific antibody against IB was performed.
D, time-response effect of DZA on IB. RAW 264.7 cells
were treated with 100 µM DZA for the indicated times. At
the end of the times, cytosolic fractions were prepared. 10 µg of
each protein was separated by SDS-PAGE, and then Western blot analysis
with specific antibody against IB was performed. Data illustrated
are from a single experiment and are representative of a total of three
separate experiments.
B Is Neither
Modulated by Hcy nor Involved in IKK Complex Activation--
The
addition of 100 µM Hcy potentiated the inhibitory effect
of DZA on NF-B transcriptional activity. To investigate whether Hcy
can potentiate the effect of DZA on proteolytic degradation of IB
in RAW 264.7 cells, cells were treated with increasing concentrations
of DZA in the presence or absence of 100 µM Hcy. As shown
in Fig. 8A, additional
treatment of Hcy could not modulate the proteolytic effect of DZA on
IB. The treatment of Hcy alone could not reduce IB at all.
In addition, the synergic effect of Hcy was not observed in cells
treated with 100 µM DZA for increasing times (Fig.
8B). These results indicate that DZA-induced proteolytic degradation of IB in RAW 264.7 cells is dissociated from the accumulation of DZA-Hcy. Recently, it was reported that IKK complexes consisting of IKK and IKK control phosphorylation of IB
proteins in cytokine-induced NF-B activation pathway (7-9). We
tried to determine if IKK complexes can participate in the
down-regulation of IB by DZA in RAW 264.7 cells. As shown in Fig.
8C, treatment of cells with LPS for 15 min severely
activated IKK complexes leading to excessive phosphorylation of
recombinant IB protein. However, exposure with 100 µM DZA from 15 to 240 min never activated IKK complexes,
showing that DZA-induced proteolytic degradation of IB in RAW
264.7 cells is independent of activation of IKK complexes.
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Fig. 8.
DZA-induced proteolytic degradation of
I B is neither
modulated by Hcy nor involved in IKK complex activation.
A, RAW 264.7 cells were treated with increasing
concentrations of DZA for 2 h in the presence or absence of 100 µM Hcy, and then cytosolic fractions were prepared. 10 µg of each protein was subjected to Western blot analysis using
specific antibody against IB. B, the cells were
treated for the indicated times with 100 µM DZA, in the
absence or presence of Hcy (100 µM). At the end of the
times, cytosolic fractions were prepared. 10 µg of each protein was
separated by SDS-PAGE, and then Western blot analysis with specific
antibody against IB was performed. C, the cells were
treated with 100 µM DZA or LPS (1 µg/ml) for the
indicated times. At the end of the times, total cell lysates were
prepared, and IKK complexes were recovered by immunoprecipitation.
IKK assay was accomplished using recombinant protein of
GST-IB as a substrate. Similar results were obtained in an
independent experiment.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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B, inhibition of NF-B transcriptional activity, and promotion of IB degradation. A hypothetical diagram of the effects of DZA is illustrated in Fig. 9.
There have been many reports that described some interesting properties
of DZA, including the inhibitory effect on the expression of cytokines
and cell adhesion molecules, the induction of apoptosis and an
anti-HIV effect. The involvement of NF-B in the expression of
numerous cytokines and adhesion molecules which promote several
diseases is well known (1). Our previous study (26) and this paper shows that DZA potently inhibited the transcription of cytokines such
as TNF- and IL-1. Additionally, other researchers reported that
the adherence of cells was inhibited through the suppression of
adhesion molecule expression by DZA (27, 28). Even though they provided
for the possibility of DZA as a potent therapeutic agent for diseases
in which these cytokines and adhesion molecules play a central
pathogenic role, the mechanism has not been elucidated. Since these
cytokines and adherence molecules are included in specific target genes
of NF-B, this study successfully explains how DZA inhibits the
expression of cytokines and adhesion molecules.
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Fig. 9.
Hypothetical diagram illustrating the effects
of DZA on NF- B regulation.
Beg et al. (37) reported that a phenotype of the
p65
/ mice died during embryonic development through
massive apoptosis of hepatocytes, suggesting that NF-B might play an
important role in protecting cells from apoptosis, instead of promoting
growth of cells. Additionally, recent reports (38-41) have described a role for NF-B in blocking apoptosis induced by TNF. Interestingly, this protective function of NF-B was apparent not only against TNF
but also against cells treated with ionizing radiation or chemotherapeutic agents (39). On the other hand, the role of the Rel
proteins in oncogenic transformation is quite well established, and it
is now clear that Rel proteins malignantly transform and immortalize
cells by inducing expression of specific genes. These findings suggest
the possibility that some of the target genes for Rel-mediated
oncogenesis and for NF-B-mediated blockage of apoptosis may be
identical. Thus, it is strongly suggested that agents which inhibit
NF-B transcriptional activity may have both direct anti-cancer
effects and effects that facilitate apoptosis in tumor cells. Recently,
we reported that DZA induced apoptosis in lymphocytic mouse leukemia
L1210 cells (30). The report showed that even though DZA raised the
NF-B binding activity during apoptosis induction, c-myc
transcription that is a downstream target gene of NF-B (1, 42) was
markedly reduced at the early stage of apoptosis induction. Similarly,
the reduction of c-myc transcription in DZA-induced
apoptosis was also observed in human promyelocytic leukemia HL-60 cells
by other researchers (43, 44). Collectively, it is reasonable that DZA
inhibition of NF-B transcriptional activity may entirely serve as a
potent inducing factor or may participate at least in part of the
induction of apoptosis by DZA in tumor cells. Finally, these findings
point to DZA as a useful chemotherapeutic agent which may prevent
malignant transformation of normal cells into tumor cells and induce
apoptosis of tumor cells through the inhibition of NF-B
transcriptional activity.
NF-B, whose enhancer elements are located in HIV-long terminal
repeats, is a key molecule of HIV replication for the pathogenesis of
AIDS (45), suggesting that NF-B may be a target for the therapy of
AIDS. Previously, other researchers described that DZA may have an
anti-HIV effect (21, 22), but the mechanism of this effect has not been
understood. The finding of DZA inhibition of NF-B transcriptional
activity described in this study explains how DZA can exert an anti-HIV
effect as described by other researchers. The inhibition of NF-B
transcriptional activity by DZA suggests that DZA may inhibit the
replication of HIV, implying that DZA may be a useful drug for the
therapeutic treatment of AIDS.
Naumann and Scheidereit (46) described that p65 is strongly
phosphorylated during the activation of NF-B in vivo. A
recent study demonstrated that the transcriptional activity of NF-B is regulated through phosphorylation of p65 by cAMP-independent activation of a catalytic subunit of protein kinase A (PKAc) associated with IB proteins by a mechanism which signals caused degradation of
IB proteins results in the activation of PKAc and subsequent phosphorylation of p65 (47). In this study, we showed that DZA disturbed the phosphorylation of p65 induced by LPS. So, we suggest a
possibility that accumulated cellular DZA-Hcy, caused by treatment of
cells with DZA, might inhibit the activation of PKAc associated with
IB proteins, or interrupt the PKAc-mediated phosphorylation of p65
directly or indirectly. SB203580, a specific inhibitor of p38
mitogen-activated protein kinase, and elevated intracellular cAMP has
been represented as an inhibitor of NF-B, which inhibit NF-B
transcriptional activity already bound to DNA in the nucleus (16, 17).
Even though, DZA and these inhibitors share a similarity that all of
them inhibit NF-B transcriptional activity without affecting its
nuclear translocation and DNA binding activity, there is a definite
difference in the mechanism of NF-B inhibition. In contrast to
SB203580 and elevated cellular cAMP, DZA inhibits the phosphorylation
of p65.
This study indicates that DZA inhibits NF-B transcriptional activity
through a hindrance of p65 phosphorylation by the accumulation of
DZA-Hcy in cells. Nevertheless, we cannot totally rule out that DZA
independent of cellular DZA-Hcy accumulation, may have interfered with
NF-B transcriptional activity. In the report by Backlund et
al. (48), treatment with 100 µM DZA resulted in
significant accumulation of DZA-Hcy in RAW 264.7 cells. The addition of
Hcy to DZA increased the accumulation of DZA-Hcy to about 5 times the
level caused by DZA alone. In contrast to a greatly increased
accumulation of DZA-Hcy by the addition of Hcy to DZA treatment in the
cells, we observed a slightly potentiated inhibition of NF-B
transcriptional activity by the addition of Hcy to DZA.
We described that DZA promotes the proteolytic degradation of IB,
but not IB in RAW 264.7 cells, leading to an increase of nuclear
translocation and DNA binding activity. Increased DNA binding activity
of NF-B in the nucleus by DZA was also observed in DZA-induced
apoptosis in L1210 cells (30), and DZA might reduce IB in this
case. However, a DZA-mediated increase of NF-B DNA binding activity
in the nucleus was not discernable in COS-7 cells, suggesting that
DZA-mediated IB degradation is cell-type specific. A number of
kinases have been suggested to phosphorylate IB, and recently a
high molecular mass, approximately 700 kDa, kinase complex termed IKK
(7-9) was identified. However, DZA mediates IB degradation
through an IKK-independent pathway and DZA can stimulate other kinase
pathways. We conclude this because IKK was not activated at all by DZA
while it was intensely activated by LPS in this study. One notable fact
is that DZA stimulates proteolytic degradation of IB but not
IB. Among kinases which have been reported to induce degradation
of IB proteins, including IKK, none can selectively differentiate
between IB and IB. Thus, we expect that DZA might be a
valuable probe to identify kinases that function with only
IB.
In conclusion, we demonstrate that DZA has dual effects on NF-B
regulation. One is an inhibitory effect on NF-B transcriptional activity already bound to target DNA, and another is the promotion of
proteolytic degradation of IB. Although the wide variety of
biological properties observed with DZA has emphasized that DZA is to
be an effective drug for treatment of human diseases including
inflammation, infections, and tumors, the mechanism and target
molecules of DZA's action have never been elucidated. This study is
the first to demonstrate that NF-B is a specific target molecule of
DZA and suggests that DZA may be a potent drug for the treatment of
diseases in which NF-B plays an important pathogenic role, as well
as a useful tool for studying the regulation and physiological
functions of NF-B.
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ACKNOWLEDGEMENT |
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We appreciate Dr. David Baltimore, California Institute of Technology, Pasadena, CA, for the kind donations of J16 and J32.
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FOOTNOTES |
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* This work was supported in part by funding from the 1995 and 1997 Basic Research Promotion Fund of the Ministry of Education, Korea, and the Korea Science and Engineering Foundation (KOSEF), Cancer Research Center at Seoul National University, Grant 97K4-0401-00-01-3.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: Dept. of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea. Tel.: 82-2-590-1175; Fax: 82-2-596-4435; E-mail: ikkim{at}cmc.cuk.ac.kr.
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ABBREVIATIONS |
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The abbreviations used are:
NF-B, nuclear
factor-B;
DZA, 3-deazaadenosine;
Hcy, homocysteine;
LPS, lipopolysaccharide;
TNF, tumor necrosis factor;
IKK, IB kinase;
PBMC, human peripheral blood monocytes;
DZA-Hcy, 3-deazaadenosylhomocysteine;
IL-1, interleukin-1;
DZAri, 3-deaza-(±)-aristeromycin;
EMSA, electrophoretic mobility shift assay;
CAT, chloramphenicol acetyltransferase;
PKAc, catalytic subunit of
protein kinase A;
GST, glutathione S-transferase;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
HIV, human
immunodeficiency virus.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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