From the Curriculum in Genetics and Molecular
Biology, the
Department of Biology, and the
§ Lineberger Comprehensive Cancer Center, University of
North Carolina, Chapel Hill, North Carolina 27599-7295
Received for publication, January 31, 2001, and in revised form, April 3, 2001
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ABSTRACT |
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The sedative and anti-nausea drug thalidomide,
which causes birth defects in humans, has been shown to have both
anti-inflammatory and anti-oncogenic properties. The anti-inflammatory
effect of thalidomide is associated with suppression of cytokine
expression and the anti-oncogenic effect with inhibition of
angiogenesis. It is presently unclear whether the teratogenic
properties of thalidomide are connected in any way to the beneficial,
anti-disease characteristics of this drug. The transcription factor
NF- Thalidomide was distributed in the late 1950s as a sedative and an
anti-nausea medication for first trimester pregnancy (1). Prenatal use
of the drug, however, produced severe developmental defects to the
human fetus, including limb deformities (2). Investigation into the
mechanism of action by thalidomide demonstrated that it acts as a
teratogen and not as a mutagen (3, 4). The teratogenic properties of
thalidomide are poorly understood but have been proposed to involve the
production of reactive oxygen species leading to subsequent DNA damage
(5). Although the correlation of birth defects with use of thalidomide
led to its removal from the market, continued clinical use established
that thalidomide possesses immunomodulatory and anti-angiogenic
properties, both of which are currently under investigation (6-9).
Diseases such as erythema nodosum leprosum (10-12), rheumatoid
arthritis (13-16), and cancer (17-19) are currently being treated
with thalidomide, although its mechanism of action remains unclear. As
an immunomodulator (20, 21), thalidomide has been shown to suppress
lipopolysaccharide-induced production of
TNF NF- Cell Culture--
Human Jurkat T cell lymphocytes were
maintained in RPMI 1640 plus 10% fetal bovine serum and antibiotics.
The human vascular endothelial cell line EA.hy926 (a gift from Cora
Jean S. Edgell, University of North Carolina, Chapel Hill) was
maintained in Dulbecco's modified Eagle's medium H supplemented with
10% fetal bovine serum, 1× hypoxanthine/aminopterin/thymidine medium
supplement (Sigma) and antibiotics.
Cell Treatment--
Cells were treated with 10 (Jurkat) or 5 ng/ml (EA.hy926) human recombinant TNF Nuclear and Cytoplasmic Extracts--
Cells were passaged
24 h prior to treatment in 10 ml of medium in 100-mm dishes
(EA.hy926) or 25-cm2 flasks (Jurkat) at a density of 2 × 106 (EA.hy926) or 1 × 106 cells/ml
(Jurkat). Post-treatment, the cells were harvested by scraping
(EA.hy926) or by centrifugation (Jurkat), washed 2 times with
phosphate-buffered saline, and lysed, on ice, in 3 pellet volumes of
cytoplasmic extraction buffer (10 mM HEPES, pH 7.6, 60 mM KCl, 1 mM EDTA, 0.2 (EA.hy926) or 0.075%
(Jurkat) Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2.5 µg/ml each of
aprotinin, leupeptin, and pepstatin). Nuclei were pelleted and washed
once in 100 µl of cytoplasmic extraction buffer without Nonidet P-40
and repelleted. The supernatant was added to the cytoplasmic samples.
The nuclear pellet was resuspended and lysed in 2 pellet volumes of
nuclear extraction buffer (20 mM Tris, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
25% glycerol, 2.5 µg/ml each of aprotinin, leupeptin, and
pepstatin). The final salt concentration was adjusted to 400 mM with NaCl. The cytoplasmic and nuclear extracts were
cleared by centrifugation, and supernatants were transferred to new
tubes. A final concentration of 20% glycerol was added to the
cytoplasmic extracts. Both nuclear and cytoplasmic extracts were
assayed for protein concentrations using a Bio-Rad protein assay dye
that incorporates the Bradford method. All extracts were stored at
Electrophoretic Mobility Shift Assays (EMSA)--
Equal amounts
of nuclear extract (5 µg of protein) were incubated for 15 min at
room temperature with a 32P-labeled DNA probe containing an
NF- Reporter Gene Assay--
Transient transfections were performed
using Superfect (Qiagen Inc., Valencia, CA) and 5 µg of a luciferase
reporter construct containing 3 tandem wild-type or mutated
NF- Northern Analysis--
RNA was isolated using the RNeasy Total
RNA Kit as recommended by the manufacturer (Qiagen). Samples were run
on a formaldehyde-agarose gel and transferred overnight to nylon
filter. Cross-linking of the RNA to the nylon filter was done using a
UV cross-linker (Stratagene, La Jolla, CA). Membranes were probed for
IL-8 mRNA expression and GAPDH mRNA (loading control) using
randomly labeled probe (Amersham Pharmacia Biotech) at 68 °C in
Quickhyb (Stratagene) solution as recommended by the manufacturer.
Washes were performed twice in 2× SSC, 0.1% SDS for 15 min at
42 °C, and once with 0.1× SSC, 0.1% SDS for 5 min at 60 °C.
Filters were exposed to film overnight.
Ribonuclease Protection Assay--
EA.hy926 endothelial cells
were treated for 60 min with TNF Western Blot Analysis--
50 µg of cytoplasmic extracts were
fractionated on a 10% SDS-PAGE gel and transferred to a nitrocellulose
membrane (Schleicher & Schuell). Membranes were blocked and probed for
I Kinase Assay--
Cells were either not treated or treated with
TNF Quantitative Analysis--
Autoradiographs were captured and
stored for analysis using a gel capturing system that utilizes the NIH
Image 1.61 software. Gel captures were quantified using volume
quantitation and local median background correction using Molecular
Dynamics ImagequaNTTM software program.
Thalidomide Blocks NF-
To determine whether thalidomide could inhibit the induction of NF- Suppression of NF- Inhibition of IL-8 Gene Expression in Endothelial Cells by
Thalidomide--
Thalidomide is being used as a cancer therapy partly
based on its ability to inhibit neovascularization (26, 27). Angiogenic factors such as IL-8 are transcriptionally regulated by NF-
Since thalidomide can inhibit activation of an NF-
NF- Thalidomide Inhibits IKK Activity--
We have demonstrated that
thalidomide can inhibit NF-
To investigate the loss of I
The ability of thalidomide to inhibit IKK activity likely explains the
suppression of NF-B has been shown to be a key regulator of inflammatory genes such
as tumor necrosis factor-
and interleukin-8. Inhibition of NF-
B is associated with reduced inflammation in animal models, such as those
for rheumatoid arthritis. We show here that thalidomide can block
NF-
B activation through a mechanism that involves the inhibition of
activity of the I
B kinase. Consistent with the observed inhibition
of NF-
B, thalidomide blocked the cytokine-induced expression of
NF-
B-regulated genes such as those encoding
interleukin-8, TRAF1, and
c-IAP2. These data indicate that the therapeutic potential for thalidomide may be based on its ability to block NF-
B activation through suppression of I
B kinase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1 (22, 23) and IL-12
(24, 25), two cytokines critical for the induction of cellular immune
responses. The anti-angiogenic properties of thalidomide have been
demonstrated by its ability to inhibit growth factor-induced
neovascularization in rabbit (26) and mouse corneal assays (27).
Neovascularization occurs through a process that requires the induction
of a number of cellular genes including IL-8 (28, 29).
Transcriptional up-regulation of IL-8, as well TNF
and IL-12, can occur through activation of the
transcription factor NF-
B (30).
B is a DNA-binding factor, originally identified as a
regulator of immunoglobulin
light chain gene expression (31), that
functions as a dimer of subunits of a family of ubiquitously expressed
transcription factors (32, 33). Five mammalian members of the family
have been identified as follows: p50/NF-
B1, p65/RelA, c-Rel, RelB,
and p52/NF-
B2. Although numerous homodimeric and heterodimeric forms
of this factor have been identified, classic NF-
B is composed of the
p50-p65 heterodimer. In unstimulated cells, the majority of NF-
B is
localized to the cytoplasm where it is tightly bound to the inhibitory
proteins of the I
B family. Specifically, I
B
is a key molecular
target involved in the regulation of NF-
B transcription factors
during inflammatory responses. Upon stimulation by extracellular
inducers of NF-
B, such as TNF
or IL-1
(30), I
B
is
rapidly phosphorylated by the I
B kinase (IKK) complex on serine
residues 32 and 36 (34). This phosphorylation leads to the
ubiquitination and subsequent degradation of I
B
by the proteasome
followed by nuclear translocation of NF-
B. Once NF-
B enters the
nucleus, it can positively regulate the expression of genes involved in
the immune and inflammatory response, such as IL-8,
IL-12, and TNF
(35). Additionally, NF-
B is
now known to be a critical regulator of the oncogenic process through its ability to regulate genes involved in cell growth, suppression of
apoptosis, and metastasis (36-38). We demonstrate here that NF-
B
DNA binding is inhibited by thalidomide through a mechanism that
involves the suppression of IKK activity. Consistent with its ability
to block NF-
B binding, we show that thalidomide also inhibits the
expression of IL-8 message as well as other
NF-
B-dependent genes. Our data provide a molecular
mechanism to potentially explain the anti-inflammatory and
anti-oncogenic properties of thalidomide.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(Promega) diluted in
phosphate-buffered saline. Thalidomide (generously provided by the
Celgene Corp., Warren, NJ) was resuspended in dimethyl sulfoxide
(Me2SO) and used at a final concentration of 10 or
40 µg/ml. IL-1
(Promega) was used at a concentration of 10 ng/ml.
70 °C until analyzed.
B-binding site from the class I MHC promoter
(5'-CAGGGCTGGGGATTCCCCATCTCCACAGTTTCACTTC-3') or an
Oct-1-binding site (5'-TGTCGAATGCAAATCACTAGAA-3') in
binding buffer (10 mM Tris, pH 7.7, 10% glycerol, 1 mM dithiothreitol, 1 mM EDTA) plus 2 µg of
poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech). The final salt
concentration was adjusted to 50-100 mM using NaCl.
Complexes were separated on a 5% non-denaturing polyacrylamide gel,
dried, and autoradiographed. For supershift assays, nuclear extracts
were preincubated for 15 min with 1-2 µl of rabbit polyclonal
antibodies raised against the NF-
B family subunits p65 (Rockland
Co., Gilbertsville, PA) or p50 (Santa Cruz Biotechnology, Inc. Santa
Cruz, CA) prior to the addition of the DNA probe.
B-binding sites from the promoter region of the class I MHC
promoter (a gift from Bill Sugden, University of Wisconsin, Madison).
24 h post-transfection, the cells were either not treated or
treated with TNF
with or without thalidomide for specified times.
Cells were harvested by centrifugation, washed with phosphate-buffered
saline, and lysed with 1× Reporter Lysis Buffer (Promega) according to
the manufacturer's instructions. Luciferase assays were performed in
duplicate using equal amounts of protein with 200 µM
D-luciferin (Sigma) as the substrate (39). Relative light
units were measured with an AutoLumat LB953 luminometer (Berthold
Analytical Instruments, Inc, Nashua, NH) and standardized to light
units obtained from transfections performed with salmon sperm DNA.
(5 ng/ml) alone or in the presence
of thalidomide (40 µg/ml) or Me2SO (40 µl). Cells were
scraped, and total RNA was harvested using Trizol solution (Life
Technologies, Inc.). Using a custom made RiboquantTM
Multiprobe RNase Protection Assay System (Pharmingen) containing a
template of the NF-
B-responsive genes as follows: TRAF1,
TRAF2, A1/Bfl-1, c-IAP2, IL-8, and IL-2R
,
RNAs were hybridized overnight and subjected to RNase treatment as
recommended by the manufacturer. Annealed protected RNA products were
fractionated by SDS-PAGE and analyzed by autoradiography.
B
degradation using an antibody specific for I
B
(Santa
Cruz Biotechnology). To control for loading, the membranes were
stripped and reprobed using an antibody for
-tubulin (Sigma).
or TNF
plus thalidomide for specified times and harvested by
centrifugation in ice-cold phosphate-buffered saline containing
phosphatase inhibitors (40). Equal amounts (500 µg) of whole cell
extracts were immunoprecipitated using an antibody against the
subunit of IKK (a gift of Dr. F. Mercurio, Signal Pharmaceuticals, San
Diego, CA). Kinase activity was determined by incubating the
immunoprecipitates with 4 µg of GST-I
B
(amino acids 1-54)
wild-type substrate or a mutated form of I
B
(S32T,S36T) in the
presence of [
-32P]ATP, as described previously (40).
The immunoprecipitates were subjected to SDS-PAGE, dried, and
visualized by autoradiography.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
B Induction by the Inflammatory Cytokines
TNF
and IL-1
--
Previous reports have provided evidence that
thalidomide can inhibit the DNA binding activity of NF-
B (41, 42).
However, these reports did not address whether the observed inhibition of DNA binding affected NF-
B transcriptional activity. Furthermore, the mechanism by which thalidomide suppresses NF-
B remains unknown. To gain insight into these unanswered questions, we first assayed NF-
B DNA binding activity in Jurkat T cells treated with both an
inflammatory cytokine and thalidomide. We examined the potential effects of thalidomide treatment on NF-
B activation by stimulating cells with 10 ng/ml TNF
for 10 min in the presence or absence of
thalidomide (Fig. 1A). As
expected, NF-
B was induced within 10 min after TNF
treatment as
seen by increased DNA binding activity (Fig. 1A, compare
lanes 1 and 2). Based on the average of five independent experiments, TNF
-induced binding of NF-
B was
inhibited by 62% when thalidomide was added to the cells
simultaneously with cytokine treatment (Fig. 1A, lanes 6 and
7) as measured by volume quantitation (see "Materials and
Methods"). Treatment of cells with thalidomide (Fig. 1A, lanes
4 and 5) or Me2SO alone (lane 3)
had no effect on basal NF-
B DNA binding activity. Thalidomide treatment also had no inhibitory effect on the binding of a second transcription factor, Oct-1 (Fig. 1B). Furthermore, direct
addition of thalidomide to TNF
-treated nuclear extracts did not
affect the binding of NF-
B to the DNA (data not shown). These data
indicate that suppression of NF-
B binding by thalidomide is not due
to interference between the transcription factor and the DNA, but rather that thalidomide acts specifically to inhibit TNF
-induced NF-
B activity and not as a general transcription factor inhibitor. Supershift assays indicate that the major NF-
B complex induced by
TNF
stimulation was the p50-p65 heterodimer (Fig.
1C).
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Fig. 1.
Thalidomide inhibits DNA binding activity of
the p50/p65 NF- B heterodimer in the presence
of the inducers TNF
and
IL-1
. A, Jurkat T lymphocytes
were either not treated (lane 1) or treated with 10 ng/ml
TNF
(lane 2), Me2SO (DMSO)
(lane 3), thalidomide (Thal) alone at 40 µg/ml
(lane 4), or 10 µg/ml (lane 5), or thalidomide
plus TNF
for 10 min (lanes 6 and 7). Cells
were harvested, and nuclear extracts were prepared and analyzed by
EMSA. The data shown are representative of five independent
experiments. B, cells were treated as in A, and
EMSA was repeated with nuclear extracts incubated with a DNA probe
containing either an NF-
B-binding site (lanes 1-3) or an
Oct-1-binding site (lanes 4-6). C, nuclear
extracts were preincubated with antibodies (Ab) raised
against various NF-
B subunits and analyzed by EMSA. D,
cells were either untreated (lane 1) or treated with IL-1
(10 ng/ml) in the presence (lanes 3, 5, and 7) or
absence (lanes 2, 4, and 6) of thalidomide at the
indicated time points and analyzed by EMSA. Supershift analysis was
performed using the p65 (lane 8) or the p50 (lane
9) antibodies.
B
by other stimuli, we tested its effect on NF-
B activation by the
inflammatory cytokine IL-1
. Jurkat cells were treated with IL-1
in the absence or presence of thalidomide, and nuclear extracts were
prepared from cells harvested at the specified time points (Fig.
1D). As seen with TNF
-thalidomide co-treatment, the
presence of thalidomide at the time of IL-1
induction suppressed activation of NF-
B. We found both heterodimer and homodimer forms of
NF-
B were inhibited at 10 and 20 min (Fig. 1D, lanes 5 and 7) post-induction but not at 5 min (Fig. 1D,
compare lanes 2 and 3). Supershift analysis
exhibits authentic NF-
B complexes being induced following IL-1
treatment (Fig. 1D, lanes 8 and 9).
Overall, these data indicate that thalidomide can block NF-
B
activation by cytokines that utilize distinct upstream signaling
pathways, suggesting that the mechanism of suppression by thalidomide
acts at a downstream site that is common to both the IL-1
and TNF
signaling cascades.
B Transcriptional Activity by
Thalidomide--
To determine if thalidomide inhibited NF-
B
transcriptional activity, we performed transient assays using a
reporter plasmid containing multiple wild-type or mutated
NF-
B-binding sites upstream of a luciferase gene. Jurkat cells were
transfected with the reporter and were then treated with TNF
in the
presence or absence of thalidomide for the times indicated (Fig.
2). Treatment of cells with thalidomide
at the time of TNF
induction led to a 1.7-fold increase in
transcriptional activity of the wild-type reporter as compared with a
6-fold increase with TNF
alone (Fig. 2A). The ability of
thalidomide to inhibit the transcriptional induction by TNF
was
transient, however, as suppression was partially lost at 9 h. This
can be explained by the fact that thalidomide has a 4.5-h half-life in
aqueous solutions (42). Thalidomide did not block the activity of a
reporter containing mutated NF-
B sites, indicating that the
inhibition does not block general transcriptional responses (Fig.
2B). These results demonstrate that thalidomide not only
acts to inhibit NF-
B DNA binding activity but also inhibits the
ability of NF-
B to activate gene expression.
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Fig. 2.
Thalidomide inhibits
TNF -induced NF-
B
transcriptional activation. Jurkat cells were transiently
transfected using a reporter plasmid containing three wild-type
(A) or mutant (B) NF-
B-binding sites from the
class I MHC promoter upstream of a luciferase gene. 24 h
post-transfection, the cells were either not treated or treated with
TNF
alone or in the presence of thalidomide for the indicated times.
Whole cell lysates were prepared as described under "Materials and
Methods" and analyzed for luciferase activity. The data are
representative of three independent experiments performed in
duplicate.
B, and
TNF
-dependent angiogenesis requires the activation of
NF-
B for the expression of IL-8 (28). We were interested
in evaluating the effect of thalidomide on NF-
B in endothelial
cells. To do this we assayed binding activity by utilizing EA.hy926
endothelial cells, a clonal cell line derived from the human umbilical
vein endothelial cells (43). We treated these cells with TNF
(5 ng/ml) in the absence or presence of thalidomide and analyzed nuclear
extracts by EMSA (Fig. 3A). As
expected, TNF
treatment led to an increase in NF-
B DNA binding
activity at 10 min (Fig. 3A, lane 2) as compared with the
untreated control (Fig. 3A, lane 1). As seen previously with
Jurkat T cells, thalidomide treatment of EA.hy926 cells at the time of
induction inhibited this increase (Fig. 3A, lane 3). The
major complex observed in TNF
activation of endothelial cells is the
p50-p65 heterodimer as observed by the supershifted complexes (Fig.
3A, lanes 4 and 5). These results demonstrate
that thalidomide is an effective inhibitor of inducible NF-
B
activity in endothelial cells. The ability of thalidomide to inhibit
NF-
B activation in Jurkat cells as well as endothelial cells
indicates that the inhibitory action is not cell type-specific.
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Fig. 3.
TNF -induced
transcriptional up-regulation of IL-8 is suppressed by
thalidomide treatment in endothelial cells. EA.hy926 endothelial
cells were not treated or treated with TNF
alone (5 ng/ml) or
simultaneously with thalidomide (40 µg/ml) for the specified times.
A, cells were harvested at 10 min, and nuclear extracts were
prepared and analyzed by EMSA for activation of NF-
B. B,
cells were harvested 1 h post-treatment, and total RNA was isolated and
analyzed by Northern analysis for IL-8 expression
(upper panel). The data shown are representative of two
independent experiments. GAPDH was used as a loading control
(lower panel). Ab, antibody.
B reporter gene,
we sought to determine if it could inhibit the transcriptional activation of an endogenous NF-
B-regulated gene. Endothelial cells
were treated with TNF
, with or without thalidomide exposure, and RNA
was isolated for Northern blot analysis (Fig. 3B). TNF
treatment alone (lanes 3, 5, 7, and 9) increased
the expression of IL-8 mRNA as compared with untreated cells (Fig.
3B, lanes 1 and 2). However, thalidomide
treatment inhibited the expression of IL-8 mRNA by 56% (as
measured by volume quantitation) at 1 h (Fig. 3B,
lane 4). Treatment of the cells for longer time points inhibited the TNF
-induced activation of gene expression by ~90% (lanes 6, 8 and 10) as compared with TNF
alone. Therefore, the ability of thalidomide to inhibit the binding
activity of NF-
B, as seen by EMSA, correlates well with the
inhibition of NF-
B-regulated endogenous gene expression at the 1-h
time point but results in an even greater inhibition of gene expression
at the 3-6-h time points. Development of angiogenesis in certain
models is dependent on the expression of IL-8 (28, 44).
Since IL-8 is transcriptionally regulated by NF-
B and
inhibited by thalidomide, we propose that one mechanism whereby
thalidomide may function as an anti-angiogenic factor is through the
suppression of NF-
B-regulated expression of IL-8 and
potentially other angiogenic factors.
B regulates genes involved in inflammatory responses as well as
genes associated with the inhibition of apoptosis. The ability of
thalidomide to function as an anti-inflammatory agent is likely due, in
part, to its ability to block the induction of inflammatory gene
expression through the inhibition of NF-
B. To test the effect
thalidomide exposure has on the regulation of other
NF-
B-dependent genes, we implemented a ribonuclease protection assay. By using an ribonuclease protection assay template specific for several NF-
B-regulated genes (Fig.
4), we examined the effect of thalidomide
treatment on NF-
B-dependent gene expression. RNA was
harvested from untreated EA.hy926 endothelial cells, cells treated with
TNF
, and cells co-treated with TNF
and thalidomide or
Me2SO. The RNA was hybridized to the radioactively labeled, in vitro transcribed DNA template. As expected, TNF
induced the expression of several NF-
B-regulated genes (Fig.
4A, lane 2). Consistent with the ability of thalidomide to
inhibit NF-
B DNA binding activity, thalidomide not only has the
ability to inhibit TNF
-induced expression of IL-8 but can
also inhibit the inducible expression of other NF-
B-regulated genes
such as TRAF1, TRAF2, A1/Bfl-1, c-IAP2, and IL-2R
(Fig. 4A, lane 3). Treatment of cells with
Me2SO in the presence of TNF
has no effect on gene
expression (Fig. 4A, lane 4). These data indicate that
thalidomide blocks the TNF
-induced expression of several
NF-
B-regulated genes.
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Fig. 4.
Thalidomide suppresses the activation of many
NF- B-regulated genes in the presence of
TNF
. EA.hy926 cells were treated for 60 min with TNF
(5 ng/ml) alone or in the presence of thalidomide (40 µg/ml) or Me2SO (DMSO) (40 µl). The cells
were harvested by scraping, and total RNA was isolated as described
under "Materials and Methods." A, the RNA was incubated
with the in vitro transcribed DNA template and analyzed by
autoradiography. Loading control genes are represented as
L32 and GAPDH. B, autoradiography at a
longer exposure time. A representative sample of NF-
B-regulated
genes reveal 42% inhibition of gene expression in the presence of
thalidomide for c-IAP2 and 66% inhibition for A1/Bfl-1.
B activation in response to different
cytokines that utilize distinct upstream pathways. This suggests that
the inhibitory action of thalidomide on NF-
B binding lies downstream
of the cytokine-receptor interaction and recruitment of associated
factors but upstream of the induction of NF-
B nuclear translocation.
Both TNF
and IL-1
signal NF-
B activation through the induction
of IKK (30, 45-48). IKK activation results in the phosphorylation of
I
B
on serine residues 32 and 36, which ultimately leads to the
degradation of this inhibitor (49-52). Since thalidomide is capable of
inhibiting DNA binding activity, as well as inhibiting the
transcription potential of NF-
B, we performed Western blot analysis
to determine what effect thalidomide has on the regulation of the
NF-
B inhibitory protein, I
B
. Cytoplasmic extracts from cells
treated with TNF
alone or in combination with thalidomide were
examined for the presence of I
B
(Fig.
5). Although untreated cells contain high levels of I
B
(lane 1), TNF
treatment resulted in
the degradation of 85% (measured by volume quantitation) of the
protein by 30 min following cytokine exposure (Fig. 5, lanes
2-4). This degradation was followed by an almost complete
resynthesis of I
B
by 60 min (Fig. 5, lane 5). However,
when thalidomide was present at the time of induction, I
B
degradation is suppressed to 45% by 30 min, leaving 65% of the
protein intact even in the presence of a potent NF-
B activator (Fig.
5, lanes 6-8). These data are consistent with the previous
data on the ability of thalidomide to inhibit NF-
B binding as
measured by EMSA. Moreover, no resynthesis of I
B
occurs at 60 min
in the presence of thalidomide (Fig. 5, lane 9), unlike
TNF
treatment alone. Since I
B
is transcriptionally regulated
by NF-
B, these data support the finding that thalidomide inhibits
the transcriptional activity of NF-
B.
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Fig. 5.
Degradation of
I B
is inhibited by
thalidomide. Jurkat cells were either not treated or treated with
TNF
in the presence or absence of thalidomide (Thal) for
the indicated time points. Cells were harvested by centrifugation, and
cytoplasmic extract proteins were separated a 10% SDS-PAGE gel and
transferred to nitrocellulose. The membrane was analyzed for
degradation of I
B
with an anti-I
B
antibody (upper
panel). The membrane was stripped and reprobed for expression of
-tubulin to control for loading (lower panel). The data
presented are representative of two independent experiments.
B
degradation in the presence of
thalidomide, we examined IKK activity following drug treatment. By
using antibodies against the
subunit of the IKK complex, IKK was
immunoprecipitated from extracts of cells that had been untreated or
treated with TNF
or co-treated with thalidomide and TNF
.
Immunoprecipitates were assayed for kinase activity by incubating with
a GST-I
B
(amino acids 1-54) fusion protein in the presence of
[
-32P]ATP, electrophoresed on a polyacrylamide gel,
and analyzed by autoradiography. TNF
treatment of cells leads to an
increase in kinase activity by 5 min that continued through the 20-min time period. Quantitative analysis of TNF
-induced IKK activity demonstrated that thalidomide inhibited this increase in activity by
53% at the 10- (Fig. 6A, lane
5) and 20-min time points (lane 7) as compared with
TNF
alone (lanes 4 and 6). However,
simultaneous treatment of cells with TNF
and thalidomide did not
block the induction of IKK activity at 5 min (Fig. 6A, lane
3). In contrast, addition of thalidomide to cells for 30 min prior
to TNF
stimulation inhibited IKK activity at 5 min following
cytokine exposure (Fig. 6B, lane 3). This indicates that the
lack of inhibition at the 5-min time point under simultaneous treatment
conditions likely represents a lag period for the
inhibitory activity of thalidomide. The lack of phosphorylation seen on
a mutant GST-I
B
(S32T,S36T) substrate indicated that the
phosphorylation activity of the kinase is specific for serine residues
32 and 36 (data not shown).
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Fig. 6.
Thalidomide inhibits IKK activity. Cells
were either not treated or treated with TNF alone or simultaneously
with thalidomide at 40 µg/ml for the specified times. Whole cell
extracts were prepared, and 500 µg of protein was immunoprecipitated
with an anti-IKK
antibody. A, immunoprecipitates were
incubated with wild-type GST-I
B
fusion protein in the presence of
[
-32P]ATP. The immunoprecipitates were run on a 10%
SDS-PAGE gel, dried, and exposed to film. The data are representative
of two independent experiments. B, cells were treated with
thalidomide for 30 min prior to TNF
stimulation. Whole cell extracts
were harvested and analyzed by in vitro kinase assay as
stated in A.
B DNA binding and transcriptional activity. We
propose that the inhibition of NF-
B by thalidomide also explains the
anti-inflammatory and anti-oncogenic properties of thalidomide. It is
possible there may be other mechanisms associated with thalidomide
action that can block NF-
B activity on other levels. Thalidomide may
inhibit another factor or signaling pathway that may be involved in
inflammatory or oncogenic potential. For example, thalidomide has also
been reported to block Sp1 DNA binding activity (41), and it has been
proposed that Sp1 and NF-
B can synergistically regulate
transcription of certain genes (53). Thus, thalidomide may inhibit the
expression of genes that are regulated by both Sp1 and NF-
B. It is
certain that thalidomide can inhibit the ability of NF-
B to bind to
the DNA. Our data demonstrate that thalidomide also functions to block
the transcriptional activity of NF-
B. Additionally, we show that
thalidomide inhibits the phosphorylation of I
B
by altering IKK
activity. In summary, the data indicate that NF-
B is a molecular
target for thalidomide action, potentially serving as a unifying theme
to explain the ability of thalidomide to suppress inflammatory
responses as well as inhibit angiogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Sandy Westerheide and Raquel Sitcheran for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by NCI Grants CA72771 and CA75080 (to A. S. B.) from the National Institutes of Health and American Cancer Society Grant PF9903801 (to D. C. G.).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.
This work is dedicated to the memory of Mary Ellen Keifer.
¶ Supported by Predoctoral Training Grant NRSA in Genetics T32 GM07092 from the National Institutes of Health.
** To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7295. Tel.: 919-966-3652; Fax: 919-966-0444; E-mail: jhall@med.unc.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M100938200
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor-
;
IKK, I
B kinase;
IL, interleukin;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
MHC, major histocompatibility
complex;
EMSA, electrophoretic mobility shift assays.
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