| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B Activity by the Novel Quinone Derivative E33301
*
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama;
Department of Clinical Development Section, Eisai Co., Ltd., Tokyo;
Department of Anatomy, Nihon University School of Medicine, Tokyo; and
§
Second Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B consists of at least three steps:
degradation of IB
, translocation of NF-B into the nucleus, and
post-translational modification of NF-B (e.g., phosphorylation of
p65). In the present study, we found that a novel quinone derivative
E3330 selectively inhibited NF-B-mediated gene expression without
affecting any of these steps. E3330, when included in the culture
medium, suppressed NF-B DNA-binding activity in PMA-induced Jurkat
cell nuclear extracts, suggesting that the inhibition by E3330 of
NF-B-mediated gene expression was due to its ability to suppress
NF-B DNA-binding activity. Fractionation of the nuclear extracts by
column chromatography revealed that a nuclear factor enhanced NF-B
DNA-binding activity and that this enhancing activity was interrupted
after treatment with E3330. Moreover, a major polypeptide with a
molecular mass of 40 kDa was found to be in the highly purified
fraction containing the NF-B-enhancing activity and predominantly
bind E3330. Taken together, these results suggest that the NF-B
activity, after dissociation from IB, is enhanced by a nuclear
factor that is active irrespective of PMA treatment, and the nuclear
factor-mediated enhancement is selectively inhibited by E3330. Thus, we
conclude that E3330 may belong to a novel class of anti-NF-B
drugs. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B was originally described as a heterodimer complex
composed of p50 and p65 subunits (1, 2), but it is now known that three
other proteins, namely p52, c-Rel, and Rel B, can also participate in
dimer formation. All of these proteins are related by a stretch of
approximately 300 amino acids that is homologous to the
c-rel oncogene and to the Drosophila morphogen
dorsal (3, 4, 5, 6, 7, 8, 9, 10). DNA-binding dimers can be homodimers (except for Rel B)
and almost all possible heterodimers (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). Transcriptional
activation is chiefly due to p65, c-Rel, and Rel B (15, 16, 17, 18), although
under certain conditions p50 homodimers can also transactivate
(19).
Inducible NF-B is part of a signal transduction pathway, and its
activity seems to be regulated primarily at the post-translational
level. In most cells, NF-B is present in the cytoplasm in an
inactive form, complexed to an anchoring protein,
IB.3 Stimulation of cells
with a variety of agents, including PMA, cytokines such as TNF- and
IL-1, and oxidants and UV irradiation, leads to activation and
translocation of NF-B into the nucleus (reviewed in Refs. 20–22).
During activation, NF-B is released from IB through the
phosphorylation and degradation of IB (23, 24). Although the
dissociation step has been extensively studied, very little is known
about how NF-B is activated after dissociation from IB.
Phosphorylation of NF-B has recently been suggested to be involved
in NF-B activation, because phosphorylation of the p65 subunit of
NF-B increases its DNA-binding activity (25). Redox regulation is
another mode of regulation of NF-B activity; oxidation of NF-B
subunits in vitro abolishes their DNA-binding activity (26, 27, 28).
However, the physiologic significance of these findings still remains
obscure. On the other hand, several studies have indicated the presence
of cofactor-like activities for NF-B, such as HMG I(Y), Bcl-3, and
PC1 (29, 30, 31, 32, 33). The functional and physical associations of NF-B with
other factors are also considered to be important for the regulation of
the NF-B activity (34, 35, 36, 37).
The putative cellular target genes of NF-B are mainly involved in
inflammatory responses, immune and acute phase responses, lymphocyte
activation, and cell growth and differentiation (20). These genes
encode cell surface molecules such as IL-2R and MHC class I, a
number of cytokines including IL-2, IL-6, G-CSF, IFN-ß, and TNF-,
and cell adhesion molecules such as E-selectin, ICAM-1, and VCAM-1.
Moreover, NF-B is a critical transcription factor in regulating the
replication of HIV-1. Selective inhibition of NF-B, therefore, may
provide a rational approach for the treatment of a variety of human
diseases. Various reagents have already been proven effective in
inhibiting NF-B-inducible gene expression, and these reagents may be
classified into antioxidants and radical scavengers, protease
inhibitors, and proteosome inhibitors. For example, a potent
antioxidant, pyrrolidinedithiocarbamate, has been shown to inhibit the
degradation of IB and to suppress NF-B-inducible gene
expression (38, 39). This was also the case for sodium salicylate and
aspirin (40). Similar results were obtained when the protease
inhibitors tosylphenylalanine-chloromethyl ketone or
tosyllysine-chloromethyl ketone were used (23, 41). At this moment,
however, no reagents that suppress either nuclear translocation,
phosphorylation, or the transactivating activity of dissociated NF-B
have been reported.
A novel quinone derivative, E3330
((2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2-propenoic
acid), was reported to inhibit LPS-induced TNF- generation in human
monocytes, rat-resident and Propionibacterium-elicited
peritoneal macrophages, and rat Kupffer cells and spleen macrophages
(42). E3330 was also found to have a therapeutic effect in mice with
endotoxin-mediated hepatitis and in rats with galactosamine-induced
hepatitis, presumably as a result of E3330 inhibition of TNF-
generation (43, 44). Northern blot analysis indicated that the
inhibitory effect of E3330 on TNF- generation is due to inhibition
of mRNA biosynthesis and/or destabilization of mRNA. Because the gene
expression of TNF- is known to be regulated by NF-B (45, 46),
these results suggest that E3330 might suppress the transactivation
function of NF-B.
In the present study, we have examined the suppressive effect of E3330
on NF-B-mediated gene expression in PMA-induced Jurkat cells. We
show that E3330 inhibits NF-B DNA binding, most probably via an
interaction with a nuclear factor that activates NF-B activity.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Jurkat cells (TIB152; American Type Culture Collection,
Rockville, MD), a human T cell line, were grown in RPMI 1640 (Life
Technologies, Grand Island, NY) supplemented with 10% FCS (Moregate,
Melbourne, Australia). PMA, PHA, and A23187 were purchased from Sigma
Chemical Co. (St. Louis, MO). TNF- was purchased from Genzyme
(Cambridge, MA). IFN- was purchased from Fujisaki Institute
(Okayama, Japan) and IFN-
from Cellular Products Inc. (Buffalo, NY).
E3330 and [14C]E3330, obtained from Eisai (Tsukuba,
Japan), was dissolved in 100% ethanol at a concentration of 25 to 100
mM and added to culture medium at a final concentration of 25 to 100
µM. The final concentration of ethanol in each culture medium was
0.1% irrespective of the concentration of E3330.
Construction of reporter plasmids
Luciferase reporter plasmids were constructed containing the
luciferase gene under the control of promoters containing the
recognition sites for NF-B, Sp1, Oct, and AP-1. Oligonucleotides
corresponding to the recognition site of each of these factors were
synthesized using a DNA synthesizer (Applied Biosystems, Inc., Foster
City, CA). The sequences of these oligonucleotides were as follows:
5'-AAGGGACTTTCCGCTGGGGATTCCAG-3' (NF-B),
5'-GGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCT-3' (Sp1),
5'-TTGGGTAATTTGCATTTCTAAGAGCT-3' (Oct),
5'-CAGGTGTCTGACTCATGCTTTTTTAAGCT-3' (AP-1). The oligonucleotides were
annealed to their complementary oligonucleotides and ligated in tandem.
After having been blunt-ended, the tandemly ligated NF-B
oligonucleotide was inserted into the blunt-ended SacI site
of pUC119 and sequenced. The tandemly ligated Sp1 and AP-1
oligonucleotides were directly cloned into the SacI site in
the polylinker site of pUC119 and were then sequenced. After having
been blunt-ended, the tandemly ligated Oct oligonucleotide was inserted
into the SmaI site of pUC119 and sequenced.
The DNA fragment encompassing the HIV-1 sequence from -45 to +83, which contains the TATA box and the transcription start site of the viral genome, was amplified by PCR. The 5' and 3' primers, which contained the EcoRI and HindIII sites, respectively, were synthesized and used to amplify the HIV-1 DNA fragment by PCR. The EcoRI-HindIII fragment was inserted into the EcoRI-HindIII site of pUC119, and sequenced.
To construct reporter plasmids in which the expression of the
luciferase gene is under the control of the indicated transcription
factors, DNA fragments containing four copies of the NF-B site, six
copies of the Sp1 site, or five copies of the AP-1 site were prepared
by digestion with KpnI and EcoRI. Each of these
fragments, along with the HIV-1 DNA fragment containing terminal
EcoRI and HindIII sites, was inserted into the
KpnI-HindIII sites of PGV-B (Toyo Ink Mfg. Co.,
Ltd., Tokyo, Japan) containing the luciferase gene to construct
pNFkBHL, pSp1HL, and pAP1HL. The DNA fragment containing five copies of
the Oct site with terminal HincII and EcoRI sites
was inserted into the SmaI-HindIII sites of
PGV-B, along with the HIV-1 DNA fragment with terminal EcoRI
and HindIII sites to construct pOctHL.
To construct reporter plasmids in which the expression of the reporter gene is under the control of NFAT, GAS, or ISRE, oligonucleotides containing three copies of recognition site of these factors were synthesized. The sequences of these oligonucleotides were as follows: 5'-CGAGGAAAAACTGTTTCATAGAGGAAAAACTGTTTCATAGAGGAAAAACTGTTTCATAG-3' (NFAT), 5'-CGCTTTCCCGGAAATAGCTTTCCCGGAAATAGCTTTCCCGGAAATAG-3' (GAS), 5'-CGCAGTTTCACTTTCCCTAGCAGTTTCACTTTCCCTAGCAGTTTCACTTTCCCTAG-3' (ISRE). The oligonucleotides were annealed to their complementary oligonucleotides, resulting in production of the dsDNA fragments containing three copies of each recognition site with the KpnI and EcoRI sites at the ends. Each of these fragments, along with the HIV-1 DNA fragment containing terminal EcoRI and HindIII sites, was inserted into the KpnI-HindIII sites of PGV-B to construct pNFATHL, pGASHL, and pISREHL.
To construct pHIV-Luc and pHIV(
kB)-Luc, the DNA fragment
encompassing the HIV sequence from -670 to +83 was prepared from pCD12
(47) by digestion with HindIII. The fragment was inserted
into the HindIII site of PGV-B to construct pHIV-Luc. To
delete the NF-B site from the HIV promoter, two DNA fragments
encompassing the HIV sequences from -646 to -107 and from -80 to +29
were amplified by PCR. These fragments contained the XhoI
and EcoRI sites and the EcoRI and
BglII sites, respectively. After digestion with restriction
enzymes, two DNA fragments were inserted into the
XhoI-BglII sites of pHIV-Luc to construct
pHIV(kB)-Luc.
Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from Jurkat cells as described previously (48, 49). In brief, Jurkat cells were washed with PBS and resuspended in 4 packed cell volumes of a hypotonic lysis buffer (buffer A: 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT). After 20 min, cells were homogenized by 20 strokes with a loose-fitting Dounce homogenizer and then centrifuged for 6 min at 4,300 x g to separate the nuclei from the cytoplasmic fraction. Collected nuclei were washed with 5 packed cell volumes of buffer A. Washed nuclei were suspended in 1 packed cell volume of high salt buffer C (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) and homogenized again by 10 strokes of a loose-fitting Dounce homogenizer. The homogenate was incubated for 30 min at 4°C to extract protein from nuclei, then centrifuged for 30 min at 15,000 x g. The supernatant was dialyzed against buffer D (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) for 5 h at 4°C, and centrifuged for 20 min at 15,000 x g. The supernatant was quick-frozen in liquid nitrogen and stored at -80°C; it was used for experiments requiring nuclear extracts.
The cytoplasmic fraction was ultracentrifuged for 1 h at 150,000 x g. The supernatant was dialyzed against buffer D for 5 h at 4°C, then centrifuged for 20 min at 15,000 x g. The supernatant, quick-frozen in liquid nitrogen and stored at -80°C, was used for experiments requiring cytosolic fraction.
The DNA probes for EMSA were prepared as follows. The annealed
oligonucleotides described above, containing two NF-B sites, three
Sp1 sites, and one Oct site, were cloned into the polylinker site of
pUC119 and excised with EcoRI and HindIII. These
fragments were end labeled with the Klenow fragment and
[-32P]dATP and purified by agarose gel
electrophoresis. The binding reactions (10 µl) contained 4 µg of
nuclear extracts, 1 ng of the indicated DNA probe, 2 µg of
poly(dI-dC), 0.5 µg of ssDNA, and 20 µg of BSA in binding buffer
(20 mM HEPES, pH 7.9, 12% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA).
Reactions were initiated by the addition of nuclear extracts and
incubated at 30°C for 30 min before electrophoresis. Samples were
analyzed on native 4% polyacrylamide gels. Gels were dried and
autoradiographed. The Abs, specific for p65, p50, IB or Bcl-3,
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
When E3330 was used in EMSA, it was diluted into deionized water from
concentrated solutions in 100% ethanol and used at a final
concentration of 1 to 100 µM in the binding buffer. The final
concentration of ethanol in each binding reaction was 1% irrespective
of the concentration of E3330.
Transfections and luciferase assays
Transfections were performed by the electroporation method. Jurkat cells (1.25 x 107) were washed with RPMI 1640 and resuspended in 625 µl of RPMI 1640 containing 12.5 µg of reporter plasmid DNA. Electroporations were performed under conditions of 960 µF and 200 V using a Gene Pulser and a Gene Pulser Cuvette with 0.4 cm electrode gap (Bio-Rad, Hercules, CA). The cells were cultured in RPMI 1640 containing 10% FCS for 12 h. The cells were then centrifuged, divided into five 6-cm dishes, and incubated for 12 h before treatment with E3330. Two hours after E3330 treatment, the cells were treated with PMA. Cell extracts were prepared 12 h after treatment with PMA, and luciferase activities were measured using the Pica Gene detection kit (Toyo Ink Mfg.) and Lumat LB9501 (Berthold Japan K.K., Tokyo, Japan).
Immunoblotting
Nuclear extracts and cytosol fractions were prepared from Jurkat
cells as described previously (50), with a minor modification. In
brief, washed cells were resuspended in 400 µl of cold buffer A' (10
mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM
PMSF). The cells were allowed to swell on ice for 15 min, then 25 µl
of a 10% solution of Nonidet P-40 was added, and the mixture was
briefly vortexed for 10 s. The homogenate was centrifuged for
30 s in a microfuge. The supernatant containing the cytoplasmic
fraction was transferred into a fresh tube and centrifuged again at
100,000 x g for 1 h, and the supernatant was used
as cytosol. Cell disruption was confirmed by microscopic observation.
The nuclear pellet was resuspended in 50 µl of ice-cold buffer C' (20
mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM
PMSF) and gently vortexed at 4°C for 15 min. After centrifugation for
15 min at 4°C in a microfuge, the supernatant was recovered and used
as nuclear extract. Nuclear extracts (1 µg) and the cytoplasmic
fraction (6 µg) were fractionated by SDS-PAGE and electrotransferred
to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA).
The membrane was incubated with 5% nonfat milk overnight and incubated
with polyclonal Ab against IB or p65 (Santa Cruz Biotechnology)
for 2 h, then analyzed using the Amersham enhanced
chemiluminescense system (ECL; Amersham, Buckinghamshire, U.K.).
In vivo phosphate labeling and immunoprecipitation
Jurkat cells (107 cells) were washed once with phosphate-free RPMI 1640 and incubated for 2 h with 1 ml of phosphate-free RPMI 1640 in the presence or absence of E3330. The cells were then labeled with 1 mCi/ml [32P]H3PO4 (Amersham, Buckinghamshire, U.K.) for 30 min in the presence or absence of PMA. After labeling, the cells were washed with PBS and lysed in 1 ml of ice-cold RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM NaF, 10 mM Na3VO4) for 30 min on ice with occasional mixing. The lysates were centrifuged in a microcentrifuge for 20 min, and the supernatants were subsequently incubated with anti-p65 Ab coupled to protein A-Sepharose (1:1) (Pharmacia, Uppsala, Sweden) for 1 h at 4°C. The precipitated proteins, coupled to protein A beads, were washed three times with 1 ml of ice-cold RIPA buffer, boiled, and centrifuged. The supernatants were fractionated by SDS-PAGE. The gel was dried and exposed to an x-ray film (Kodak, Rochester, NY).
Two-dimensional phosphopeptide mapping
Two-dimensional phosphopeptide mapping was performed as described previously (51). The p65 band was identified by aligning the exposure with the gel. The band was cut out from the gel, and labeled p65 was eluted twice in 0.6 ml of elution buffer (50 mM NH4HCO3, 0.1% SDS, and 0.5% 2-ME). The eluted proteins were pooled and precipitated in the presence of 10 µg of BSA and 20% TCA. The proteins were then oxidized in the presence of performic acid, diluted in deionized water, frozen, and lyophilized. The pellets were resuspended in 50 µl of NH4HCO3 (pH 8.0), added with 10 µg of N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin, and incubated for 4 h at 37°C. Deionized water was added to the digests, frozen, and lyophilized. The pellet was dissolved in 400 µl of electrophoresis buffer (pH 1.9) and centrifuged. The supernatant was lyophilized. The pellet was resuspended in 10 µl of electrophoresis buffer (pH 1.9), centrifuged, and spotted on TLC plates. The first dimension was done by migrating the tryptic digests on a TLC plate in pH 1.9 electrophoresis buffer (formic acid, 25 ml; glacial acetic acid, 78 ml; deionized water, 897 ml). The second dimension was chromatographed using isobutyric acid buffer (isobutyric acid, 625 ml; n-butanol, 19 ml; pyridine, 48 ml; glacial acetic acid, 29 ml; deionized water, 279 ml). TLC plates were exposed to x-ray films for visualization.
Uptake and intracellular binding of [14C]E3330
Uptake and cellular distribution of E3330 were examined by incubating 8 x 107 Jurkat cells in 20 ml of complete media (RPMI 1640 + 10% FCS) containing 50 µM [14C]E3330 for 1 h. Jurkat cells were then washed twice with PBS and resuspended in 1 ml of buffer containing 20 mM Tris-HCl (pH 7.5) and 1 mM MgCl2. After 10 min at 4°C, the cell suspension was rapidly forced through a 25-gauge needle three times to disrupt the cells. A crude nuclear pellet was obtained by centrifugation at 2,000 x g for 10 min. The supernatant was centrifuged at 100,000 x g for 60 min in a Beckman TLA 100.3 ultracentrifuge (Beckman, Fullerton, CA) to separate the crude plasma membrane pellet from cytosol. The nuclear and membrane fractions were suspended in 1 ml of distilled water and homogenized by sonication. Aliquots (5, 25, and 125 µl) of each subcellular fraction were mixed with 10 ml of Aquasol (New England Nuclear, Boston, MA), and radioactivity was counted in a Beckman LS-9000 liquid scintillation counter.
Column chromatography
Nuclear extracts (4.5 ml) prepared from PMA-induced Jurkat cells
were loaded onto a phosphocellulose (P11) column, and bound proteins
were eluted stepwise with buffer D (containing 0.3 M, 0.5 M, and 1.0 M
KCl). Each fraction (20 ml) was dialyzed against HGKE buffer (20 mM
HEPES-NaOH (pH 7.9), 20% glycerol, 0.1 M KCl, and 0.2 mM EDTA). The
protein concentrations of nuclear extracts, and of 0.1 M, 0.3 M, 0.5 M,
and 1.0 M KCl fractions were 5.3, 0.34, 0.26, 0.17, and 0.09 mg/ml,
respectively. The majority of NF-B was recovered in the 0.3 M KCl
fraction, although a small amount of NF-B was eluted in the 0.1-M
KCl fraction. The dialyzed 0.5-M KCl fraction was further loaded onto a
DEAE Sepharose column. The flow-through fraction, which contained most
of the NF-B-enhancing activity, was applied directly onto a Mono S
column and eluted using a linear gradient of 0.1 to 1.0 M KCl in buffer
D. The peak fractions (0.6 to 0.8 M KCl) were pooled, loaded again onto
a Mono S column (Pharmacia) after dilution with buffer D without KCl,
and then eluted with 1.0 M KCl to concentrate the activity. The eluate
was loaded on a Superdex 75 column (Pharmacia) and fractionated by
running buffer D. The peak fractions were pooled and loaded again onto
a Mono S column to concentrate the activity, as described above for
E3330 ligand-western.
To test the NF-B-enhancing activity, each fraction after dialysis
against HGKE buffer was mixed with the 0.3 M KCl fraction and incubated
together with 10 µg of BSA at 37°C for 1 h in the presence or
absence of 10 to 100 µM E3330. After a further addition of 1 µg of
poly(dI-dC) and 1 ng of the indicated DNA probe, the mixture was
incubated at 25°C for 20 min, and protein-DNA complexes were resolved
on 4% polyacrylamide gels. The gel was dried and autoradiographed.
E3330 ligand-western
The fraction finally concentrated by Mono S column chromatography was resolved by SDS-PAGE and analyzed either by silver staining or E3330 ligand-western. Proteins fractionated by SDS-PAGE were electrotransferred to PVDF membrane. The protein blot was denatured and renatured by sequential treatment with buffer D' (20 mM HEPES, pH 7.9, 10% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) containing 0.01% Nonidet P-40 and 6 M guanidine-HCl for 15 min at room temperature; buffer D' containing 0.01% Nonidet P-40 and 4 M guanidine-HCl for 15 min at 4°C; buffer D' containing 0.01% Nonidet P-40 and 2 M guanidine-HCl for 15 min at 4°C; buffer D' containing 0.01% Nonidet P-40 and 1 M guanidine-HCl for 15 min at 4°C; buffer D' containing 0.01% Nonidet P-40 and 0.5 M guanidine-HCl for 15 min at 4°C; buffer D' containing 0.01% Nonidet P-40 and 0.25 M guanidine-HCl for 15 min at 4°C; buffer D' containing 0.01% Nonidet P-40 and 0.125 M guanidine-HCl for 15 min at 4°C; and buffer D' containing 0.01% Nonidet P-40 for 15 min at 4°C three times. The blot was incubated with buffer D' containing 0.01% Nonidet P-40 and [14C]E3330 for 12 h at 4°C and then washed four times with buffer D' containing 0.01% Nonidet P-40 for 10 min at 4°C, dried, and autoradiographed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B
To test for an inhibitory effect of E3330 on NF-B-mediated gene
expression, we transfected Jurkat cells with the pNFkBHL reporter
plasmid in which the expression of the luciferase gene is driven by
NF-B. After transfection, the cells were treated with the indicated
concentrations of E3330 for 2 h and induced for 12 h with
either PMA, PMA plus A23187, PMA plus PHA, or TNF-, and then
luciferase activity was measured (Fig. 1
A).
Among the inducers of NF-B that we tested, combined treatment with
PMA and A23187 gave the strongest stimulation of NF-B-mediated
expression of the reporter gene (Fig. 1A). However,
irrespective of the stimuli, E3330 clearly suppressed NF-B-mediated
transcription stimulation in a dose-dependent manner (Fig. 1A).
|
B-mediated transcription by analyzing the effect of E3330 on
the transcription from other promoters containing binding sites
for various transcription factors. For this purpose, the indicated
reporter plasmid was transfected into Jurkat cells instead of
pNFkBHL, and the cells were treated with E3330 and PMA as described
in the legend of Figure 1
B. In contrast to NF-B-mediated
transcription, the expression of luciferase activity under the control
of Sp1 or Oct was not affected by PMA induction, and E3330 had no
influence on this expression (Fig. 1B). We also
examined the inhibitory effect of E3330 on PMA-induced transcription
from the HIV promoter by using the pHIV-Luc reporter plasmid. In this
case, E3330 suppressed HIV promoter activity in a dose-dependent manner
(Fig. 1B). Transfection of the cells with
pHIV(kB)-Luc containing a deletion of the NF-B elements from the
HIV promoter resulted in a loss of both PMA induced expression and
suppression by E3330 (Fig. 1B).
Furthermore, we examined the effect of E3330 on other inducible
systems. Expression of the reporter gene under the control of AP-1 or
NFAT was induced by PMA plus A23187, and expression of STAT-mediated
reporter plasmids, pGASHL and pISREHL, was induced by IFN- and
IFN-, respectively. Figure 2 shows
that E3330 did not suppress but rather stimulated the AP-1-mediated
gene expression. The stimulation was not effective, but highly
reproducible. Although E3330 had a slightly suppressive effect on the
NFAT- or STAT-mediated gene expression, the suppression was not
effective as compared with that of NF-B-mediated gene expression
(Fig. 2). Thus, the results indicates that E3330 is a selective
inhibitor of NF-B-mediated stimulation of transcription.
|
B nor the
nuclear translocation of p65
The cytoplasmic inactive NF-B complex is known to be initially
activated via phosphorylation and degradation of IB, followed by
nuclear translocation of the active NF-B complex composed of p65 and
p50 subunits. To see whether E3330 has any influence on these
processes, the protein levels of both cytosolic IB and nuclear
p65 were monitored by Western blotting. As shown in Figure 3, treatment of Jurkat cells with PMA
resulted in a rapid disappearance of IB in the cytosolic fraction
and reciprocal accumulation of p65 in the nucleus. The addition of
E3330 did not apparently affect either the PMA-dependent disappearance
of IB or the nuclear accumulation of p65 (Fig. 3), indicating
that E3330 neither affects the degradation of IB nor the nuclear
translocation of NF-B. These results also suggest that E3330 does
not inhibit IB phosphorylation, since IB degradation
depends on its phosphorylation.
|
B reappeared in the cytosolic fraction 60 min after PMA
induction irrespective of E3330 treatment. However, the protein levels
of IB in the cells treated with both PMA and E3330 were
significantly reduced, compared with levels in the cells treated with
PMA alone (Fig. 3). This suggested that E3330 does not affect nuclear
translocation of the NF-B complex, but in some way reduces the
subsequent induction of IB synthesis. To confirm this point, we
examined the mRNA levels of IB using quantitative PCR. Total
cellular RNA was prepared from Jurkat cells after treatment with E3330
for 2 h and induction with PMA. After 60 min of PMA induction, the
levels of IB mRNA were reduced in the E3330-treated cells
compared with the untreated cells (data not shown). Since expression of
IB is positively regulated by various members of the NF-B
family (52, 53, 54), these results strongly suggest that E3330 suppresses
the transcriptional stimulatory activity of nuclear-translocated
NF-B, resulting in a reduction in IB synthesis.
Effect of E3330 on the DNA-binding activity of NF-B
To examine the effect of E3330 on the DNA-binding activity of
NF-B, we performed EMSA using nuclear extracts from Jurkat cells
treated with E3330 and induced with PMA. When the cells were treated
with PMA, NF-B DNA-binding activity was strongly induced (Fig. 4A). The treatment with
E3330 markedly impaired the induction of the DNA-binding activity of
NF-B in a dose-dependent manner (Fig. 4A). On the
other hand, the DNA-binding activity of either Sp1 or Oct was not
affected by E3330 treatment (Fig. 4A). These
protein-DNA complexes, indicated by arrowheads in Figure 4, were
confirmed to be specific for each transcription factor by competition
assays (data not shown). Ab supershift experiments indicated that these
protein-DNA complexes contained both p65 and p50 subunits (Fig. 4B).
|
We next studied the effect of E3330 on the phosphorylation of the
p65 subunit of NF-B. For this purpose, Jurkat cells were treated
with E3330, 32P-labeled orthophosphate was added to
the culture medium, and the cells were induced with PMA for 30 min as
described in the legend to Figure 5A. Then, cell extracts were
prepared, and the p65 subunit was immunoprecipitated as described in
Materials and Methods. Treatment with PMA significantly
increased the accumulation of radioactive phosphate in the p65 subunit
(Fig. 5A). E3330, however, did not affect the overall
phosphorylation level of p65, irrespective of PMA induction.
Two-dimensional phosphopeptide mapping further revealed that the
pattern of tryptic digestion of the phosphorylated p65 was almost
identical, irrespective of E3330 treatment (Fig. 5B).
This indicates that E3330 does not affect the phosphorylation process
of p65.
|
B DNA-binding activity and its partial
purification
We have shown above that E3330 neither affects the degradation of
IB, the nuclear translocation of p65, nor the phosphorylation of
p65. However, we clearly showed that E3330 impairs both the DNA-binding
and transcriptional stimulation activities of nuclear-translocated
NF-B. We hypothesized, therefore, that the presence of some nuclear
factor, required for NF-B activation, might be functionally targeted
by E3330. To investigate for the presence of this cofactor-like
activity, we fractionated PMA-stimulated Jurkat cell nuclear extracts
by phosphocellulose column chromatography. Each fraction was prepared
by stepwise elution with 0.1 M, 0.3 M, 0.5 M, and 1.0 M KCl buffer.
Western blot analysis showed that the majority of p65 eluted in the 0.3
M KCl fraction, and that a small amount of p65 eluted in the
flow-through fraction (Fig. 6A). On the other hand,
EMSA using a NF-B specific DNA probe showed that NF-B DNA-binding
activity was present exclusively in the 0.3 M KCl fraction, but the
activity was lower than that of the nuclear extracts (Fig. 6B). The absence of NF-B DNA-binding activity in
the flow-through fraction might be due to the presence of IB in
the fraction (data not shown). This hypothesis is supported by a
previous report (55) showing that the NF-B/IB complex was eluted
at 0.1 M KCl when HeLa cell cytosolic extracts were fractionated by
phosphocellulose column chromatography.
|
B in the 0.3 M KCl fraction, the 0.3 M KCl fraction was mixed
with other fractions and incubated together with E3330, as indicated in
the legend of Figure 6. This study showed that the NF-B DNA-binding
activity of the 0.3 M KCl fraction was enhanced by the addition of the
0.5 M KCl fraction (Fig. 6C). This enhancement,
however, was not observed on the addition of BSA or the heat-treated
0.5 M KCl fraction (Fig. 6D). Moreover, this
enhancement of NF-B DNA-binding activity was eliminated by
pretreatment of the 0.5 M KCl fraction with E3330 in a dose-dependent
manner (Fig. 6C, lanes 4–7). In contrast,
when E3330 was added after a 1-h incubation of a mixture of the 0.3 M
KCl and 0.5 M KCl fractions, E3330 had scarcely any effect on the
DNA-binding activity. (Fig. 6C, lanes 8–11). The
NF-B DNA-binding activity in the 0.3 M KCl fraction was not affected
by E3330 in the absence of the 0.5 M KCl fraction (Fig. 6C, lanes
12–15). The lower two bands resulting from intrinsic
binding activity in the 0.3 M KCl and 0.5 M KCl fractions were not
affected by E3330 (Fig. 6C). These results suggest
that E3330 does not affect NF-B DNA-binding activity by its direct
interaction with NF-B.
To test the dependence of the NF-B-enhancing activity on PMA
induction and its subcellular distribution, we prepared nuclear and
cytosolic fractions from PMA-treated or untreated Jurkat cells. All
these fractions were further fractionated by phosphocellulose column
chromatography, and each fraction were dialyzed against HGKE buffer.
EMSA showed that the majority of the NF-B-enhancing activity in the
0.5 M KCl fraction was found in the nuclear fraction, irrespective of
PMA treatment, suggesting that the enhancing activity was
constitutively active in Jurkat cell nuclei (data not shown).
To purify the NF-B-enhancing activity in the 0.5 M KCl fraction, we
conducted further purification of this fraction by using conventional
column chromatography, as described in Materials and
Methods. The final fraction concentrated using Mono S column after
Superdex 75 column chromatography contained one major polypeptide with
a molecular mass of 
40 kDa and several minor polypeptides (Fig. 7B). E3330
ligand-western assay, using [14C]E3330, showed that the
major polypeptide preferentially bound E3330 (Fig. 7B). The final fraction also stimulated the NF-B
DNA-binding activity; this enhancement was eliminated by pretreatment
with E3330 in a dose-dependent manner (Fig. 7C).
These results suggest that NF-B DNA-binding activity is positively
regulated via an interaction between NF-B and a nuclear factor, and
that this interaction is selectively inhibited by binding of E3330 with
the nuclear factor. Although it is not yet known that other nuclear
factors are involved in the enhancement of NF-B activity in addition
to the 40-kDa nuclear factor, further analyses are necessary to
elucidate this issue.
|
To examine the subcellular distribution of E3330, Jurkat cells
were incubated with [14C]E3330 for 1 h. The
cells were then fractionated into three fractions: nuclei, cytosol, and
plasma membrane. Radioactivity in each fraction was counted as
described in Materials and Methods. Table I shows that >50% of the
[14C]E3330 accumulated in the nuclear fraction. The
remaining radioactivity was distributed nearly equally between the
cytosol and plasma membrane fractions. This result indicates that the
subcellular distribution of E3330 was similar to that of the
NF-B-enhancing activity, supporting our suggestion described
above.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B has many wide-ranging effects that are controlled by a
complex regulatory network of inhibitors and coactivators (20, 21, 22).
Given the intimate connection between host defense reactions and
NF-B, this transcription factor and its associated regulators could
provide attractive targets for therapeutic intervention in a number of
diseases or pathologic conditions. In this line, a number of
anti-NF-B drugs have already been developed (23, 38, 39, 40, 41).
NF-B activity is induced by a variety of reagents including
cytokines, UV irradiation, dsRNA, and oxidative stress (20). Although
these stimuli modulate distinct intracellular signals (56, 57, 58, 59, 60, 61), all of
them have been shown to eventually target IB through a mechanism
involving the following sequence of events: phosphorylation of IB
at Ser32 and Ser36 residues (62, 63, 64),
phosphorylation-dependent multiubiquitination of IB at
Lys21 and Lys22 (65, 66, 67), and finally,
degradation of IB by the ubiquitin-dependent proteosome, which
results in the release of free NF-B (65). Almost all
anti-NF-B agents, e.g., antioxidants, protease inhibitors, and
proteosome inhibitors, target the dissociation and/or degradation of
IB (23, 38, 39, 40, 41). In contrast, our results strongly indicate that
E3330 does not affect IB, but rather, it influences NF-B
DNA-binding activity without affecting its translocation into the
nucleus (Figs. 3 and 4). Consistent with these results, E3330
suppressed NF-B-mediated reporter gene expression to a similar
extent in Jurkat cells, irrespective of whether PMA, TNF-, PMA plus
A23187, or PMA plus PHA was used to stimulate the cells (Fig. 1). E3330
may thus be classified as a novel type of anti-NF-B
drug.
We examined the specificity of E3330 inhibition by analyzing the effect
of E3330 on the transcription from the promoters under the control of
other transcription factors, Sp1, Oct, AP-1, NFAT, or STAT (Figs. 1B and 2). E3330 had no effect on Sp1, Oct, and AP-1. E3330
was not so effective in suppressing NFAT or STAT as compared with
NF-B (Fig. 2). In addition, we have previously showed that
glucocorticoid-inducible transcription was not inhibited by, but rather
was activated by E3330 (68). E3330, thus, selectively suppressed
NF-B-mediated transcription.
For efficient DNA binding, post-translational modification, especially
phosphorylation of the p65 subunit, has been shown to be essential
(25). E3330, however, did not significantly alter the PMA-induced
phosphorylation of p65 (Fig. 5). We, therefore, postulated the presence
of a cofactor-like activity for NF-B in the nucleus that would act
as the target of E3330 inhibition. This was supported by our finding
that the 0.5 M KCl fraction, which was free from NF-B, enhanced the
DNA-binding activity of NF-B. We propose that a nuclear factor
interacts with NF-B and that E3330 interferes with this interaction,
based on the following facts: 1) The majority of E3330 and the
NF-B-enhancing activity are localized in the nucleus; 2) the
pretreatment of the NF-B-enhancing activity with E3330 eliminated
the activity; and 3) E3330 bound a single polypeptide with a molecular
mass of 40 kDa in the highly purified fraction containing the
NF-B-enhancing activity.
Recently, several reports have addressed the transcriptional mechanism
of NF-B-mediated gene expression, revealing that several cofactors
are necessary to confer NF-B-mediated expression of particular
genes. E3330, therefore, might affect one of these or some other
unknown cofactor activities. In the case of the IFN-ß promoter, the
HMG I(Y) protein binds to the NF-B site and augments the ability of
NF-B to bind to this site, resulting in enhanced transcription from
the promoter, especially in response to virus infection (29). HMG I(Y)
has been suggested to contact the minor groove of the A/T-rich inner
region of the IFN-ß NF-B site (29). Since the NF-B site of HIV
long terminal repeat does not contain an A/T-rich inner core,
suppression of HIV promoter activity by E3330 seems unlikely to involve
HMG I(Y). Bcl-3, a member of the IB family, has recently been
suggested to activate transcription through NF-B sites. Two possible
mechanisms have been proposed; an indirect mechanism by which Bcl-3
antagonizes inhibitory p50 homodimers (30, 31); and a direct mechanism
by which Bcl-3 acts as an accessory factor, coupling with otherwise
inert p52 homodimers to form competent transactivators (32). Our
supershift experiments using Ab against Bcl-3, however, showed that
Bcl-3 was not involved in the NF-B-DNA complex formed in PMA-induced
Jurkat cell nuclear extracts (data not shown). We, therefore, consider
that our putative nuclear cofactor for NF-B is different from either
of these reported factors.
Recent studies also indicate that the transcriptional stimulation
activity of NF-B can be altered by interactions with other
transcription factors. It has been shown that NF-B physically and
functionally interacts with members of the bZip family including C/EBP
and AP-1 (34, 35). Other reports indicate that Sp1 acts synergistically
with NF-B to induce transcription of the HIV-1 long terminal repeat
(36, 37). Since the transcription activity of HIV-1 promoter was
suppressed by E3330, the interaction between NF-B and Sp1 could be
suppressed by E3330. However, E3330 clearly suppressed the enhanced
transcription from the promoter, which contained only the NF-B site
but not the Sp1 site (see Fig. 2). Therefore, it seems unlikely that
the interaction of NF-B with Sp1 is a main target of E3330.
Another possibility is suggested by the observed redox regulation of
NF-B DNA-binding activity (26, 27, 28). Since E3330 is a quinone
derivative, E3330 might take part in this reduction/oxidation
regulation of NF-B. In this respect, it would be interesting to see
whether our nuclear factor possesses reducing activity.
In conclusion, we show that E3330 is a novel type of NF-B inhibitor,
which suppresses the DNA-binding activity of NF-B by interfering
with its functional interaction with a nuclear factor. The molecular
cloning and characterization of this nuclear factor should reveal not
only how E3330 inhibits the activation of NF-B, but should also shed
light on the activation mechanism of NF-B.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hiroshi Handa, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226, Japan.
3 Abbreviations used in this paper: IB, inhibitor of nuclear factor B; NF-B, nuclear factor B; EMSA, electrophoretic mobility shift assay; Sp1, specificity protein 1; Oct, octamer-binding factor; AP-1, activator protein 1; NFAT, nuclear factor of activated T cells; [14C]E3330, 14C-labeled E3330; HGKE buffer, 20 mM HEPES-NaOH (pH 7.9), 20% glycerol, 0.1 M KCl, and 0.2 mM EDTA; PVDF, polyvinylidene difluoride; RLU, relative light unit; ISRE, interferon-stimulated response element; GAS, IFN- activation site.
Received for publication March 3, 1997. Accepted for publication September 16, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B is required for inhibition of NF-B by IB. Genes Dev. 3:1689.B by phosphorylation of its inhibitor IB. Nature 344:678.[Medline]
B enhancer binding protein that inhibits NF-B function. Cell 63:803.[Medline]
B: homology to rel and dorsal. Cell 62:1019.[Medline]
B is identical to factor KBF1 and homologous to the Rel oncogene product. Cell 62:1007.[Medline]
B p50. Cell 67:1075.[Medline]
B inhibition of the cloned p65 subunit of NF-B, a Rel-related polypeptide. Cell 64:961.[Medline]
B. Science 251:1490.B transcriptional activity. Genes Dev. 6:745.B subunit which stimulates HIV transcription in synergy with p65. Nature 352:733.[Medline]
B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene. EMBO J. 11:205.[Medline]
B sites. Proc. Natl. Acad. Sci. USA 88:3715.B subunits determine the specificity of transcriptional activation. Proc. Natl. Acad. Sci. USA 89:1529.B contacts DNA by a heterodimer of the p50 and p65 subunit. EMBO J. 10:1817.[Medline]
B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc. Natl. Acad. Sci. USA 89:1875.B p65 transcriptional activator and an alternatively spliced derivative. Mol. Cell. Biol. 12:444.B. EMBO J. 10:3805.[Medline]
B. Genes Dev. 6:775.B and Rel: participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143:1.[Medline]
B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
B. Annu. Rev. Cell Biol. 10:405.
B- is necessary for activation of transcription factor NF-B. Nature 365:182.[Medline]
B controls expression of inhibitor IB: evidence for an inducible autoregulatory pathway. Science 259:1912.B in vivo is regulated by multiple phosphorylations. EMBO J. 13:4597.[Medline]
B by reduction of a disulphide bond involving cystein 62. Nucleic Acids Res. 30:3821.
B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein. Mol. Cell. Biol. 12:3094.B binding activity by oxdation-reduction in vitro. Proc. Natl. Acad. Sci. USA 88:4328.B-dependent virus induction of the human IFN-ß gene. Cell 71:777.[Medline]
B-mediated transactivation by removing inhibiting p50 homodimers from select B sites. EMBO J. 12:3893.[Medline]
B-mediated inhibition. Nature 359:339.[Medline]
B motifs via association with DNA-binding p50B homodimers. Cell 72:729.[Medline]
B with TATA-binding protein, transcription factor IIB, and coactivators. J. Biol. Chem. 270:7219.B and C/EBP family members: a Rel domain-bZip interaction. Mol. Cell. Biol. 13:3964.B p65 and Fos/Jun transcription factors produces potentiated biological function. EMBO J. 12:3879.[Medline]
B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 12:3351.[Medline]
B activation in intact cells. J. Exp. Med. 175:1181.B transcription factor and HIV-1. EMBO J. 10:2247.[Medline]
B by sodium salicylate and aspirin. Science 265:956.B) and enhanced processing of the NF-B precursor p105 are obligatory steps in the activation of NF-B. Nucleic Acids Res. 21:5059. generation from monocytes and macrophages. Agents Actions 37:297.[Medline]
B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor a gene in primary macrophages. J. Exp. Med. 171:35.B-like motifs and of constitutive and inducible forms of NF-B. Mol. Cell. Biol. 10:1498.B transcription factor. Cell 53:211.[Medline]
B activity. Proc. Natl. Acad. Sci. USA 91:28.B-like gene is regulated by NF-B. EMBO J. 12:2773.[Medline]
B-/MAD3 inhibitor of NF-B: positive regulation by members of the rel/NF-B family. EMBO J. 12:5043.[Medline]
B in vitro. Genes Dev. 6:761.B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71:765.[Medline]
. Proc. Natl. Acad. Sci. USA 88:10009.B by tumor necrosis factor. J. Biol. Chem. 265:8339.B signal transduction pathway. Proc. Natl. Acad. Sci. USA 91:11527.B proteolysis by site-specific, signal induced phosphorylation. Science 267:1485.B on serines 32 and 36 controls IB proteolysis and NF-B activation in response to diverse stimuli. EMBO J. 14:2876.[Medline]
B in response to inducers of NF-B activity. Mol. Cell. Biol. 15:5339.[Abstract]
B to the ubiquitin-proteasome pathway. Genes Dev. 9:1586.B in vitro and in vivo requires the acidic C-terminal domain of the protein. Mol. Cell. Biol. 15:2413.[Abstract]
B requires site-specific ubiquitination. Proc. Natl. Acad. Sci. USA 92:11259.This article has been cited by other articles:
|
|
 |
|
  C. D. Curtis, D. L. Thorngren, Y. S. Ziegler, A. Sarkeshik, J. R. Yates, and A. M. Nardulli Apurinic/Apyrimidinic Endonuclease 1 Alters Estrogen Receptor Activity and Estrogen-Responsive Gene Expression Mol. Endocrinol., September 1, 2009; 23(9): 1346 - 1359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ando, S. Hirao, Y. Kabe, Y. Ogura, I. Sato, Y. Yamaguchi, T. Wada, and H. Handa A new APE1/Ref-1-dependent pathway leading to reduction of NF-{kappa}B and AP-1, and activation of their DNA-binding activity Nucleic Acids Res., August 1, 2008; 36(13): 4327 - 4336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shimizu, R. Ouchida, N. Yoshikawa, T. Hisada, H. Watanabe, K. Okamoto, M. Kusuhara, H. Handa, C. Morimoto, and H. Tanaka HEXIM1 forms a transcriptionally abortive complex with glucocorticoid receptor without involving 7SK RNA and positive transcription elongation factor b PNAS, June 14, 2005; 102(24): 8555 - 8560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Ma, K. Kinneer, J. Ye, and B. J. Chen Inhibition of Nuclear Factor {kappa}B by Phenolic Antioxidants: Interplay between Antioxidant Signaling and Inflammatory Cytokine Expression Mol. Pharmacol., August 1, 2003; 64(2): 211 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nishi, N. Shimizu, M. Hiramoto, I. Sato, Y. Yamaguchi, M. Hasegawa, S. Aizawa, H. Tanaka, K. Kataoka, H. Watanabe, et al. Spatial Redox Regulation of a Critical Cysteine Residue of NF-kappa B in Vivo J. Biol. Chem., November 8, 2002; 277(46): 44548 - 44556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nakamura, R. Ouchida, T. Kodama, T. Kawashima, Y. Makino, N. Yoshikawa, S. Watanabe, C. Morimoto, T. Kitamura, and H. Tanaka Cytokine Receptor Common beta Subunit-mediated STAT5 Activation Confers NF-kappa B Activation in Murine proB Cell Line Ba/F3 Cells J. Biol. Chem., February 15, 2002; 277(8): 6254 - 6265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yoshikawa, Y. Makino, K. Okamoto, C. Morimoto, I. Makino, and H. Tanaka Distinct Interaction of Cortivazol with the Ligand Binding Domain Confers Glucocorticoid Receptor Specificity. CORTIVAZOL IS A SPECIFIC LIGAND FOR THE GLUCOCORTICOID RECEPTOR J. Biol. Chem., February 8, 2002; 277(7): 5529 - 5540. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Miura, R. Ouchida, N. Yoshikawa, K. Okamoto, Y. Makino, T. Nakamura, C. Morimoto, I. Makino, and H. Tanaka Functional Modulation of the Glucocorticoid Receptor and Suppression of NF-kappa B-dependent Transcription by Ursodeoxycholic Acid J. Biol. Chem., December 7, 2001; 276(50): 47371 - 47378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hirota, M. Murata, Y. Sachi, H. Nakamura, J. Takeuchi, K. Mori, and J. Yodoi Distinct Roles of Thioredoxin in the Cytoplasm and in the Nucleus. A TWO-STEP MECHANISM OF REDOX REGULATION OF TRANSCRIPTION FACTOR NF-kappa B J. Biol. Chem., September 24, 1999; 274(39): 27891 - 27897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Martin, B. San-Antonio, and M. Fresno Regulation of Nuclear Factor kappa B Transactivation. IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND PROTEIN KINASE C zeta IN c-Rel ACTIVATION BY TUMOR NECROSIS FACTOR alpha J. Biol. Chem., May 4, 2001; 276(19): 15840 - 15849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Heiss, C. Herhaus, K. Klimo, H. Bartsch, and C. Gerhauser Nuclear Factor kappa B Is a Molecular Target for Sulforaphane-mediated Anti-inflammatory Mechanisms J. Biol. Chem., August 17, 2001; 276(34): 32008 - 32015. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Ma and K. Kinneer Chemoprotection by Phenolic Antioxidants. INHIBITION OF TUMOR NECROSIS FACTOR alpha INDUCTION IN MACROPHAGES J. Biol. Chem., January 18, 2002; 277(4): 2477 - 2484. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
