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(Received for publication, March 18, 1996, and in revised form, June 3, 1996)
From the ABSTRACT The NF- INTRODUCTION Human monocytes and endothelial cells can be induced to express a
variety of genes involved in immune and inflammatory responses, cell
adhesion, blood coagulation, and fibrinolysis (1, 2). For example,
bacterial lipopolysaccharide (LPS)1
stimulates monocytic cells to rapidly and transiently express a defined
set of gene products including tumor necrosis factor The NF- cAMP induces the expression of numerous genes through the protein
kinase A (PKA)-mediated phosphorylation of CRE-binding factors,
including CRE-binding protein (CREB) (20). Elevation of intracellular
cAMP levels in monocytes and endothelial cells also inhibits the
induction of a distinct set of genes, including TNF This study examined inhibition of the transcriptional activation of the
TNF EXPERIMENTAL PROCEDURES LPS (Escherichia coli serotype
O111:B4) and forskolin were purchased from Calbiochem.
Bt2cAMP was purchased from Sigma. Human
recombinant TNF Human monocytic THP-1 cells were obtained from
the American Type Culture Collection (Rockville, MD) and cultured as
described (5). Primary cultures of human umbilical vein endothelial
cells (HUVECs) were obtained from collagenase-digested umbilical veins
or from Clonetics Corp. (San Diego, CA) and were cultured as described
(11). All experiments used HUVECs between passages 3 and 5.
Total RNA was isolated
from THP-1 cells using TRIZOL reagent (Life Technologies, Inc.) and
from HUVECs by phenol extraction as described (32). 10 µg of total
RNA was fractionated on a 1.2% agarose-formaldehyde gel and
transferred to a GeneScreen membrane (DuPont NEN). Membranes were
hybridized with various cDNA fragments labeled with
[ Nuclear run-on assays were performed as
described (33). Prehybridization, hybridization, and washing of the
filters were performed using a TNF pUHG10.3CAT contains the
chloramphenicol acetyltransferase (CAT) cDNA (21) and was kindly
provided by Dr. R. Hooft van Huijsduijnen (Glaxo Institute for
Molecular Biology, Geneva, Switzerland). pBasic-tet/VP16 was
constructed by inserting the cDNA for the tetracycline
repressor/VP16 fusion protein and the THP-1 cells (2 × 107) were transiently transfected using the DEAE-dextran
procedure (13). Cells were cultured for 24 h before exposure to
LPS (10 µg/ml) for 24 h in the presence or absence of
Bt2cAMP (1 mM). HUVECs (2 × 106) were transfected using DEAE-dextran as described (11).
Cells were cultured for 24 h before exposure to TNF THP-1 cells were stimulated for 1 h with LPS (10 µg/ml) in the presence or absence of Bt2cAMP (1 mM) or forskolin (50 µM). HUVECs were
stimulated with TNF I Confluent
monolayers of HUVECs in 10-cm tissue culture dishes were washed twice
with phosphate-free RPMI 1640 medium and incubated in phosphate-free
medium containing 200 mCi of 32PO4/ml (400-800
mCi/ml; ICN) for 1 h. Cells were stimulated with TNF RESULTS The effect of
elevated intracellular levels of cAMP on LPS induction of the TF and
TNF
TNF We examined the effect of Bt2cAMP
on LPS-induced TNF
The human TF promoter cloned upstream of the
luciferase reporter gene was used as a model system to examine
inhibition of promoter activity in THP-1 cells and HUVECs by
Bt2cAMP and forskolin. These studies indicated that
Bt2cAMP alone induced TF promoter activity (data not
shown), suggesting that these plasmids contain cryptic CRE sites. In
addition, cAMP and cAMP derivatives have been shown to interfere with
the luciferase assay (38). Therefore, we employed a two-plasmid
reporter system in which the first plasmid contained the human TF
promoter expressing a tet/VP16 transcription factor that transactivates
a promoter driving expression of the CAT reporter gene present on a
second plasmid, pUHG10.3CAT (21). This two-plasmid reporter system had
two advantages: (i) pUHG10.3CAT has been shown not to contain any
cryptic CRE sites that influence expression of CAT activity (21); and
(ii) it can be used to study weak promoters, such as the TF promoter,
because the promoter activity is amplified by expression of the
tet/VP16 transcription factor. TF promoter activity was increased by
4.3 ± 1.1-fold (mean ± S.E., n = 3) by
TNF
The NF-
Inhibition of NF-
The TF, E-selectin, and VCAM-1 genes are rapidly and transiently
expressed in endothelial cells (6, 11, 17). Binding of nuclear proteins
from HUVECs was examined 1 h after stimulation because
transcription of these genes is increased at this time (11, 17). TNF To confirm that elevated cAMP did not selectively inhibit the nuclear
translocation of p65, cytoplasmic and nuclear extracts from THP-1 cells
were examined by Western blotting using a p65-specific antibody. LPS
stimulation induced the nuclear translocation of p65 (Fig.
6A). Treatment of the cells with
Bt2cAMP did not affect the nuclear translocation of p65.
Similarly, preincubation of HUVECs for 20 min with forskolin (20 mM) had no effect on the TNF
To determine if elevated cAMP affected I A recent study showed that p65 is strongly phosphorylated
during the activation of NF-
The effect of increased levels of intracellular
cAMP and expression of the catalytic subunit of PKA on NF-
To determine if activation of PKA was required to inhibit
NF- DISCUSSION In this study, we demonstrated that activation of PKA by agents
that elevate cAMP in human monocytic and endothelial cells inhibited
the expression of a distinct set of NF- Our studies show that elevation of cAMP by both Bt2cAMP and
forskolin inhibited the functional activity of endogenous NF- A recent study using Jurkat T-cells suggested that cAMP inhibition of
interleukin-2 gene expression is due to a small change in the amount of
I Our study and those of others (21, 22, 25) have shown that elevated
cAMP in monocytes/macrophages and endothelial cells selectively
inhibits the induction of a distinct set of genes. In contrast,
elevated cAMP does not inhibit other inducible genes such as ICAM-1 and
interleukin-1 (21, 22, 25). Interestingly, induction of the ICAM-1 gene
in endothelial cells is regulated, at least in part, by NF- Elevated cAMP may also regulate the activity of other transcription
factors that are required for expression of the TNF PKA can modulate the activity of a number of intracellular signaling
pathways (47). In yeast, the PKA-dependent phosphorylation
of the transcription factor ADR1 inactivates its transcriptional
activity without altering its binding to DNA (48, 49). In our studies,
PKA may directly phosphorylate p65 or initiate a signaling cascade
involving other kinases that reduce the functional activity of
NF- Phorbol ester-dependent phosphorylation of the
TA2 transactivation domain of p65 enhances its
transactivating activity, suggesting that phorbol ester-activated
protein kinase C directly phosphorylates TA2 on serine
residues (40). We showed that forskolin did not modify the
TNF Pharmacologic agents that elevate cellular levels of cAMP, such as
pentoxifylline and rolipram, have been observed to inhibit LPS-induced
TNF FOOTNOTES Acknowledgments We acknowledge Dr. R. Hooft van Huijsduijnen
for providing pUHG10.3CAT, pUHG15.1, and polymerase chain reaction
primers; Dr. C. Kunsch for pCMVp65; Dr. G. Nabel for pRSVRelA(65)S276A;
Dr. M. Montminy for pPKA; Drs. M. Read and T. Collins (Brigham and
Women's Hospital, Boston, MA) for an ICAM-1 cDNA fragment; Dr. I. Verma for a c-fos cDNA fragment; Dr. A. Bierhaus
(Department of Pathology, Technical University of Dresden, Dresden,
Germany) for advice on Western blotting; Dr. E. Levin (Scripps Research
Institute, La Jolla, CA) for advice on phosphate labeling of
endothelial cells; M. Smith, H. McClary, and Y. Ko for technical
assistance; Drs. A. McLachlan and L. Curtiss for helpful suggestions;
Dr. T. S. Edgington for support; and J. Robertson for preparation of
the manuscript.
REFERENCES
Volume 271, Number 34,
Issue of August 23, 1996
pp. 20828-20835
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
B-mediated Transcription in
Human Monocytic Cells and Endothelial Cells*
§¶
,¶,,
and
Departments of Immunology and Vascular
Biology, Scripps Research Institute, La Jolla, California 92037, the
'' 
Department of Biology, Tanabe Research Laboratories, San
Diego, California 92121, and 
INSERM U294 and Service
d'Hematologie et d'Immunologie Biologiques, Chu Xavier Bichat, 46, rue Henri Huchard, 75877 Paris Cedex 18, France
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
B/Rel family of transcription factors
regulates the inducible expression of many genes in activated human
monocytes and endothelial cells. In this study, we examined the
molecular mechanism by which agents that elevate intracellular cAMP
inhibit the expression of the tumor necrosis factor 
(TNF),
tissue factor, endothelial leukocyte adhesion molecule-1, and vascular
cell adhesion molecule-1 genes. Both forskolin and dibutyryl cAMP,
which elevate intracellular cAMP by independent mechanisms, inhibited
TNF and tissue factor expression at the level of transcription.
Induction of NF-B-dependent gene expression in
transiently transfected human monocytic THP-1 cells and human umbilical
vein endothelial cells was inhibited by elevated cAMP and by
overexpression of the catalytic subunit of protein kinase A (PKA).
Elevated cAMP did not prevent nuclear translocation of p50/p65 and
c-Rel/p65 heterodimers, decrease nuclear translocation of p65, or
significantly modify TNF-induced phosphorylation of p65. Functional
studies demonstrated that transcriptional activation of a plasmid
containing multimerized B sites by p65 was inhibited by agents that
elevate cAMP and by overexpression of the catalytic subunit of PKA.
This study indicates that activation of PKA reduces the induction of a
distinct set of genes in monocytes and endothelial cells by inhibiting
NF-B-mediated transcription.
(TNF) (3)
and the transmembrane receptor tissue factor (TF) (4, 5). In
endothelial cells, LPS and cytokines induce the expression of various
adhesion molecules, including endothelial leukocyte adhesion molecule-1
(E-selectin), vascular cell adhesion molecule-1 (VCAM-1), and
intercellular adhesion molecule-1 (ICAM-1), which permit binding and
transmigration of leukocytes into sites of inflammation (6, 7, 8).
Activation of endothelial cells also induces TF expression, which
converts the normal anticoagulant surface to a procoagulant state
(9, 10, 11).
B/Rel family of transcription factors has been implicated in
the inducible expression of many genes in monocytes and endothelial
cells, including TNF, TF, E-selectin, and VCAM-1 (12, 13, 14, 15). The
NF-B/Rel family of transcription factors includes NFKB1 (p50), NFKB2
(p52), RelA (p65), RelB, and c-Rel (16). These factors can homo- and
heterodimerize to generate distinct transcription factors that regulate
expression of many different genes (16). The TNF, E-selectin, and
VCAM-1 genes are regulated by p50/p65 heterodimers (17), whereas the TF
gene is regulated by c-Rel/p65 heterodimers (18). In monocytes and
endothelial cells, LPS and cytokines induce the dissociation of the
inhibitory protein IB from pre-existing cytoplasmic NF-B·Rel
complexes, allowing the transcription factors to translocate to the
nucleus and initiate expression of target genes (19).
, TF, E-selectin,
and VCAM-1 (21, 22, 23, 24, 25). We (27) and others (26, 28) have shown that
pharmacologic agents that elevate intracellular levels of cAMP, such as
pentoxifylline and a prostacyclin analog, iloprost, inhibit LPS
induction of TF expression in human monocytes. In addition, agents such
as dibutyryl cAMP (Bt2cAMP), forskolin, and
isobutylmethylxanthine, which increase cAMP levels by independent
mechanisms, all inhibit induction of TF expression (29, 30). In
endothelial cells, elevation of cAMP and activation of PKA inhibit
cytokine induction of E-selectin, VCAM-1, and TF expression (21, 22,
31). Studies using both monocytes and endothelial cells indicate that
cAMP inhibits transcription of this distinct set of genes (21, 23, 25,
30).
, TF, E-selectin, and VCAM-1 genes in human monocytic and
endothelial cells by agents that elevate intracellular levels of
cAMP.
was obtained from Collaborative Biomedical Products
(Bedford, MA).
-32P]dCTP (>3000 Ci/mmol; ICN, Costa Mesa, CA) using
the Prime-It kit II (Stratagene, San Diego, CA). TNF and TF cDNA
fragments have been described previously (5). An E-selectin cDNA
fragment was obtained from R&D Systems (Minneapolis, MN), and a
1.3-kilobase VCAM-1 cDNA fragment was obtained by polymerase chain
reaction. A c-fos cDNA fragment was kindly provided by
Dr. I. Verma (Salk Institute, La Jolla, CA). Variations in RNA loadings
were assessed by reprobing filters with a housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase, obtained from Clontech (Palo
Alto, CA). Autoradiography was performed at 
70 °C using Kodak
X-Omat film.
cDNA fragment; a
glyceraldehyde-3-phosphate dehydrogenase cDNA fragment (Clontech);
and a vector control, pSP73 (Promega, Madison, WI). Radioactivity was
quantified using a PhosphorImager and ImageQuant software (Molecular
Dynamics, Inc., Sunnyvale, CA).
-globin intron/splice sequence
into the BamHI/PstI and NotI sites of
pBluescript, respectively. These DNA fragments were amplified by
polymerase chain reaction from pUHG15.1 as described (21) using primers
kindly provided by Dr. R. Hooft van Huijsduijnen. pTF-tet/VP16 was made
by inserting the human TF promoter (278 to +121 base pairs) into the
EcoRI/HindIII sites of pBasic-tet/VP16.
pSV40-tet/VP16 was created by cloning the minimal SV40 promoter from
the pGL2 promoter (Promega Corp.) into the
KpnI/HindIII sites of pBasic-tet/VP16.
p(B)4-tet/VP16 and p(B-1)4-tet/VP16
contain four copies of the TF-B site (34) and the murine Ig-B
site, respectively, cloned upstream of a minimal SV40 promoter in
pSV40-tet/VP16. pCMV-tet/VP16 (also known as pUHG15.1) (21) was used as
a positive control. Promoter activity was measured in cells
cotransfected with pBasic-tet/VP16 derivatives and pUHG10.3CAT (21).
pCMVp65 expresses p65 from a cytomegalovirus promoter and was kindly
provided by Dr. C. Kunsch (Human Genome Sciences, Rockville, MA).
pRSVRelA(65)S276A expresses p65 containing a mutation in the PKA
recognition site and was kindly provided by Dr. G. Nabel (Howard Hughes
Institute, Ann Arbor, MI). pPKA expresses the catalytic subunit of PKA
and was kindly provided by Dr. M. Montminy (Salk Institute).
(20 ng/ml)
for 24 h in the presence or absence of forskolin (50 µM). CAT activity was determined using a diffusion-based
assay (35). To assess variations in transfection efficiencies, cells
were transfected with the control plasmid pCMV (Clontech), which
expresses the LacZ gene. Levels of -galactosidase were
determined using the Galacto-Light assay system (Tropix Inc., Bedford,
MA) and exhibited <15% variation between samples.
(20 ng/ml) in the presence or absence of
forskolin (20-200 µM). Nuclear extract preparation and
electrophoretic mobility shift assay were performed as described (34,
36). Quantification of protein-DNA complexes was performed as described
above. Statistical analysis of data from three independent experiments
was performed using a paired Student's t test.
B was detected in cytoplasmic
extracts from THP-1 cells and HUVECs as described (11, 37) using a
1:500 dilution of an anti-IB antibody (Santa Cruz Biotechnology,
Santa Cruz, CA). Similarly, nuclear translocation of p65 was monitored
by Western blotting in THP-1 cells and HUVECs using a 1:1000 dilution
of an anti-p65 antibody (Santa Cruz Biotechnology).
(10 ng/ml)
for 1 h with or without a 20-min pretreatment with forskolin (20 mM). After incubation, the cells were washed, and p65 was
recovered by immunoprecipitation with an anti-p65 antibody using an
immunoprecipitation kit containing protein A beads (Boehringer
Mannheim). The precipitated proteins were washed several times, eluted
in sample buffer containing 25 mM dithiothreitol, separated
on 8-16% SDS-polyacrylamide gels (Novex), and visualized by
autoradiography.
Induction of a Distinct Set
of Genes in Human Monocytic and Endothelial Cells
genes in human monocytic THP-1 cells was investigated using
Bt2cAMP, a membrane-permeable analog of cAMP (30), or
forskolin, an activator of adenylyl cyclase (21). The reagent
concentration used in this study has been previously shown to increase
cAMP levels in monocytes/macrophages and HUVECs (21, 25). LPS
stimulation of THP-1 cells increased the steady-state levels of TF and
TNF mRNAs at 2 h (Fig. 1A). The
addition of Bt2cAMP immediately before LPS stimulation
abolished induction of TF and TNF mRNA expression. In addition,
Bt2cAMP reduced TF and TNF mRNA levels in
unstimulated cells. In contrast, Bt2cAMP induced expression
of c-fos mRNA in THP-1 cells, indicating that
Bt2cAMP activates PKA (Fig. 1A).
Fig. 1.
Elevated cAMP in THP-1 and HUVECs inhibits
the induction of TF, TNF, E-selectin, and VCAM-1 mRNA
expression. A, total RNA was extracted from THP-1 cells
exposed to LPS (10 µg/ml) for 2 h with or without
Bt2cAMP (dbcAMP; 1 mM).
B, total RNA was extracted from HUVECs exposed to TNF (20 ng/ml) for 1 h with or without a 20-min forskolin pretreatment
(Fsk; 100 µM). TF, TNF, E-selectin, and
VCAM-1 mRNA levels were determined by Northern blot analysis using
the appropriate radiolabeled human cDNA probes. Blots were
reprobed to determine glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) mRNA levels as a measure of RNA loading. Similar
results were observed in two independent experiments.
stimulation of HUVECs increased the steady-state levels of TF,
E-selectin, and VCAM-1 mRNAs at 1 h (Fig. 1B).
Pretreatment of HUVECs with forskolin before the addition of TNF
inhibited the induction of TF, E-selectin, and VCAM-1 mRNAs by
60 ± 9, 78 ± 8, and 73 ± 14%, respectively
(mean ± S.D., n = 3). In contrast, forskolin did
not inhibit TNF induction of ICAM-1 mRNA (data not shown),
consistent with previous studies (21, 22). These data indicate that
agents that increase intracellular levels of cAMP by two independent
mechanisms inhibit the induction of a distinct set of genes in human
monocytic and endothelial cells.
Gene Transcription
in Monocytic Cells
gene transcription in monocytic THP-1 cells using
nuclear run-on assays. LPS stimulation of THP-1 cells increased the
rate of transcription of the TNF gene by 11.7-fold at 1 h (Fig.
2). Treatment of the cells with Bt2cAMP
before the addition of LPS abolished the increase in the rate of
transcription of the TNF gene (Fig. 2). These results indicate that
cAMP inhibition of transcription may account for most, if not all, of
the observed inhibition of LPS-induced TNF mRNA expression.
Fig. 2.
Elevated cAMP inhibits LPS-induced TNF
gene transcription. Nuclei were isolated from unstimulated THP-1
cells, cells stimulated with LPS (10 µg/ml) for 1 h, and cells
treated with Bt2cAMP (dbcAMP; 1 mM)
before LPS stimulation. Labeled nuclear RNA levels were determined by
hybridization to glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) and TNF cDNAs and a vector control (pSP73).
The autoradiogram was exposed for 12 days at 80 °C with an
intensifier screen. Similar results were observed in two independent
experiments.
Induction of TF Promoter Activity in
Endothelial Cells
stimulation in transiently transfected HUVECs (Fig.
3). Treatment of the cells with forskolin before TNF
stimulation inhibited the induction of TF promoter activity by 44 ± 3% (mean ± S.E., n = 3). Forskolin alone did
not significantly affect TF promoter activity. In addition, TNF or
forskolin did not affect the level of CAT expression in cells
transfected with the promoterless control plasmid pBasic-tet/VP16 (Fig.
3). Similarly, Bt2cAMP (1 mM) strongly
inhibited LPS induction of TF promoter activity in transiently
transfected THP-1 cells (data not shown).
Fig. 3.
Elevated cAMP inhibits the induction of the
TF promoter in HUVECs. HUVECs were cotransfected with
pBasic-tet/VP16 (pBasic; 10 µg) and pUHG10.3CAT (10 µg)
or with pTF-tet/VP16 (pTF; 10 µg) and pUHG10.3CAT (10 µg) using DEAE-dextran. After 24 h, cells were stimulated with
TNF (20 ng/ml) with or without a 20-min pretreatment with forskolin
(Fsk; 50 µM) and incubated for a further
24 h before determining CAT activity. Results from three
independent experiments are shown. Transfection efficiencies were
assessed using pCMV and exhibited <15% variation between
samples.
B-mediated Transcription in Monocytic
and Endothelial Cells
B/Rel family of transcription
factors has been implicated in the inducible expression of the TF,
TNF, E-selectin, and VCAM-1 genes (12, 13, 14, 15). Elevated cAMP may
inhibit all of these genes by a common mechanism involving NF-B/Rel
proteins. To determine if elevation of cAMP inhibited NF-B-mediated
induction of gene transcription, THP-1 cells and HUVECs were
transfected with pSV40-tet/VP16 or p(B)4-tet/VP16, which
contains four B sites cloned upstream of the minimal SV40 promoter.
LPS stimulation of THP-1 cells transfected with
p(B)4-tet/VP16 resulted in a 5.0 ± 0.7-fold
(mean ± S.E., n = 5) induction of CAT activity
(Fig. 4A). Treatment of the THP-1 cells with
Bt2cAMP before LPS stimulation resulted in a 61 ± 5%
(mean ± S.E., n = 5) inhibition of LPS-induced
CAT activity. Similar results were observed using a plasmid containing
four copies of the murine Ig-B site (data not shown). No
induction of CAT activity was observed in THP-1 cells transfected with
pSV40-tet/VP16, which contains only the minimal SV40 promoter. In
HUVECs, TNF induced a 11.4 ± 2.3-fold (mean ± S.E.,
n = 5) induction of CAT activity in cells transfected
with p(B)4-tet/VP16, whereas no induction was observed
with cells transfected with pSV40-tet/VP16 (Fig. 4B).
Pretreatment of HUVECs with forskolin resulted in a 61 ± 10%
(mean ± S.E., n = 5) inhibition of TNF-induced
CAT activity (Fig. 4B). These data demonstrate that agents
that elevate cAMP strongly inhibit NF-B-mediated gene transcription
in human monocytic and endothelial cells.
Fig. 4.
Elevated cAMP inhibits NF-B-mediated gene
transcription in THP-1 cells and HUVECs. A, pSV40-tet/VP16
(pSV40; 5 µg) or p(B)4-tet/VP16
(p(B)4; 5 µg) was cotransfected with pUHG10.3CAT (5 µg) into THP-1 cells
using DEAE-dextran. After 24 h, cells were divided in four equal
portions and exposed to LPS (10 µg/ml) for 24 h in the presence
or absence of Bt2cAMP (dbcAMP; 1 mM)
as indicated. B, HUVECs were cotransfected with
pSV40-tet/VP16 (pSV40; 10 µg) or
p(B)4-tet/VP16
(p(B)4; 10 µg)
and pUHG10.3CAT (10 µg). After 24 h, cells were stimulated with
TNF (20 ng/ml) for 24 h with or without a 20-min pretreatment
with forskolin (Fsk; 50 µM). Transfection
efficiencies were assessed using pCMV and exhibited <15% variation
between samples. Results from five independent experiments are
shown.
B/Rel Proteins Is Not Affected by
Elevated cAMP
B-mediated transcription may be
due to a block in the nuclear translocation of NF-B/Rel proteins
and/or a reduction in the transcriptional activity of these proteins in
the nucleus. To examine the effect of an increase in intracellular
levels of cAMP on the nuclear translocation of these NF-B·Rel
complexes, electrophoretic mobility shift assays were performed using a
B site from the murine Ig enhancer, which binds p50/p65
heterodimers, and a B site from the human TF gene, which binds
c-Rel/p65 heterodimers (34). Previous competition and antibody
supershift experiments have established the protein composition of
these complexes (11, 34). Binding of nuclear proteins to these B
sites in THP-1 cells was examined 1 h after stimulation because
the maximal rate of transcription of the TF and TNF genes was
previously shown to occur 1 h after LPS stimulation (3, 5). LPS
stimulation of THP-1 cells for 1 h induced the nuclear
translocation of p50/p65 and c-Rel/p65 heterodimers that bound the
Ig-B and TF-B sites, respectively (Fig.
5A). No statistically significant differences
were observed between the levels of p50/65 (p < 0.49)
and c-Rel/p65 (p < 0.20) heterodimers in nuclear
extracts from cells treated with LPS in the presence and absence of
Bt2cAMP. Nuclear extracts from unstimulated THP-1 cells and
Bt2cAMP-treated cells showed no binding of NF-B·Rel
complexes (Fig. 5A).
Fig. 5.
cAMP does not inhibit the nuclear
translocation of NF-B/Rel proteins in THP-1 cells or HUVECs.
A, nuclear extracts were prepared from THP-1 cells incubated
for 1 h with LPS (10 µg/ml) in the presence or absence of
Bt2cAMP (dbcAMP; 1 mM) or forskolin
(Fsk; 50 µM). B, nuclear extracts
were prepared from HUVECs stimulated for 1 h with TNF (20 ng/ml) with or without a 20-min pretreatment with forskolin (100 µM). The presence of p50/p65 and c-Rel/p65 heterodimers
was determined by electrophoretic mobility shift assay using
radiolabeled oligonucleotides containing the murine Ig-B site and
TF-B site, respectively. The faster migrating band represents
nonspecific protein binding (34). Similar results were observed in two
independent experiments.
stimulation of HUVECs induced the nuclear translocation of p50/p65 and
c-Rel/p65 heterodimers (Fig. 5B). The addition of forskolin
to HUVECs before TNF stimulation had no effect on the nuclear
translocation of p50/p65 and c-Rel/p65 heterodimers (Fig.
5B). No statistically significant differences were observed
between the levels of p50/p65 (p < 0.44) and c-Rel/p65
(p < 0.14) in nuclear extracts from cells treated with
TNF in the presence and absence of forskolin. Forskolin alone did
not induce the nuclear translocation of NF-B·Rel complexes in
HUVECs (Fig. 5B). Similarly, forskolin did not inhibit the
nuclear translocation of p50/p65 induced by LPS stimulation of HUVECs
(data not shown). Antibody supershift experiments indicated that
elevated cAMP did not change the composition of the p50/p65 and
c-Rel/p65 heterodimeric complexes (data not shown). In addition,
antibody supershift experiments using an anti-CREB antibody excluded
the possibility that elevation of cAMP induced binding of CREB to the
NF-B·Rel complexes (data not shown). These results indicate that
Bt2cAMP and forskolin do not affect either the nuclear
translocation or DNA binding of NF-B·Rel complexes in monocytic
and endothelial cells.
-induced nuclear
translocation of p65 at 15 min (data not shown).
Fig. 6.
Elevated cAMP does not affect the nuclear
translocation of p65 or proteolysis of IB. A, nuclear
and cytoplasmic extracts were prepared from THP-1 cells treated with
LPS (10 µg/ml) for 1 h in the presence or absence of
Bt2cAMP (dbcAMP; 1 mM). p65 levels
in cytoplasmic and nuclear extracts were determined by Western blotting
using a 1:1000 dilution of a specific anti-p65 antibody. B,
cytosolic extracts were prepared from HUVECs pretreated with various
doses of forskolin (Fsk; 20-200 µM) for 20 min before the addition of TNF (20 ng/ml) for 15 min. IB
protein levels in cytoplasmic extracts were determined by Western
blotting using a 1:500 dilution of an anti-IB antibody.
B proteolysis, IB
levels in cytoplasmic extracts from HUVECs were analyzed by Western
blotting. TNF induced the proteolytic degradation of IB (Fig.
6B). Pretreatment of the cells with various doses of
forskolin (20-200 µM) did not significantly reduce the
proteolytic degradation of IB (Fig. 6B). Similarly,
Bt2cAMP (1 mM) did not affect the proteolytic
degradation of IB in THP-1 cells stimulated with LPS for 1 h
(data not shown).
-induced Phosphorylation of p65 Is Not Affected by Elevated
cAMP
B in vivo (39). More detailed
studies indicated that p65 contains two transactivation (TA) domains in
the carboxyl-terminal end (40). TA1 is constitutively
phosphorylated, whereas stimulation of cells induces phosphorylation of
TA2 and is correlated with increased transcriptional
activity (40). To determine if elevated cAMP inhibited the functional
activity of nuclear NF-B·Rel complexes by modifying the
phosphorylation of p65, we examined the phosphorylation of p65 in
TNF-stimulated HUVECs with or without forskolin. p65 was
immunoprecipitated from unlabeled HUVECs and detected by Western
blotting using an anti-p65 antibody to confirm selective recovery of
p65 (data not shown). In addition, immunoprecipitation of p65 was
prevented by the addition of the peptide (Santa Cruz Biotechnology)
that was used to raise the antibody (data not shown). TNF induced a
strong phosphorylation of p65 in HUVECs, which was not affected by
pretreatment of the cells with forskolin (Fig. 7). A low
constitutive phosphorylation of p65 was observed in unstimulated
HUVECs, which was not increased by treatment of the cells with
forskolin alone (Fig. 7). Additional phosphorylated proteins observed
in these studies may represent coimmunoprecipitation of other
NF-B/Rel proteins associated with p65 as shown previously (39).
These data indicate that activation of PKA does not modify the
TNF-induced phosphorylation of p65, although we cannot exclude the
possibility that forskolin changes the sites of phosphorylation of
p65.
Fig. 7.
TNF-induced phosphorylation of p65 in
HUVECs. Phosphate-labeled HUVECs were stimulated with TNF (10 ng/ml) for 1 h with or without a 20-min pretreatment with
forskolin (Fsk; 20 µM). Phosphorylated p65 was
recovered by immunoprecipitation and analyzed on an 8-16%
SDS-polyacrylamide gel, followed by autoradiography. Similar results
were observed in an independent experiment.
B-mediated Transcription Is Inhibited by Elevated cAMP and by
Activation of PKA
B-mediated
transcription was measured in HUVECs. Cells were cotransfected with
p(B)4-tet/VP16, pUHG10.3CAT, and pCMVp65. Expression of
p65 increased the transcriptional activity of
p(B)4-tet/VP16 by 22.1 ± 1.3-fold (mean ± S.E., n = 3) (Fig. 8A), but
did not affect the transcriptional activity of the control plasmid,
pSV40-tet/VP16 (data not shown). Forskolin reduced the level of p65
transactivation by 74 ± 10% (mean ± S.E.,
n = 3) (Fig. 8A). To exclude the possibility
that forskolin nonspecifically reduced transcription of the
cytomegalovirus promoter, which was used to express p65, we determined
CAT activity in the presence and absence of forskolin in HUVECs
transfected with pCMV-tet/VP16, which expresses the tet/VP16
transcription factor from the cytomegalovirus promoter. Forskolin did
not affect cytomegalovirus promoter activity as measured by the level
of CAT activity (Fig. 8A).
Fig. 8.
NF-B-mediated transcription and p65
transactivation activity are inhibited by elevated cAMP and PKA.
A, HUVECs were cotransfected with
p(B)4-tet/VP16
(p(B)4; 10 µg) and
pUHG10.3CAT (10 µg) or with p(B)4-tet/VP16 (10 µg),
pUHG10.3CAT (10 µg), and pCMVp65 (p65; 5 µg). The total
amount of plasmid in each transfection was equalized to 25 µg using
pUC18 DNA. Forskolin (Fsk; 50 µM) was added
24 h after transfection, and cells were incubated for a further
24 h before determining CAT activity. HUVECs were cotransfected
with pCMV-tet/VP16 (pCMV; 10 µg) and pUHG10.3 CAT (10 µg). After 24 h, cells were incubated in the presence and
absence of forskolin (50 µM) for a further 24 h.
Results from three independent experiments are shown. B,
HUVECs were cotransfected with p(B)4-tet/VP16
(p(B)4; 10 µg) and
pUHG10.3CAT (10 µg) with or without pPKA (PKA; 10 µg).
After 24 h, cells were stimulated with TNF (20 ng/ml) for
24 h. C, HUVECs were cotransfected with
p(B)4-tet/VP16
(p(B)4; 5 µg),
pUHG10.3CAT (5 µg), pCMVp65 (p65; 2 µg), and 5 or 10 µg of pPKA (PKA) as indicated. The total amount of plasmid
in each transfection was adjusted to 30 µg using pUC18 DNA. Similar
results were observed in two independent experiments.
B-mediated transcription, we examined the TNF induction of CAT
activity in HUVECs overexpressing the catalytic subunit of PKA. HUVECs
were cotransfected with p(B)4-tet/VP16, pUHG10.3CAT, and
pPKA, which expresses the catalytic subunit of PKA. Expression of PKA
inhibited the TNF induction of CAT activity by 69 ± 8%
(mean ± S.E., n = 3) (Fig. 8B). To
determine if activation of PKA inhibited p65 transactivation, HUVECs
were cotransfected with p(B)4-tet/VP16, pUHG10.3CAT, and
pCMVp65 in the presence and absence of pPKA. p65 transactivation was
inhibited in a dose-dependent manner by coexpression of the
catalytic subunit of PKA (Fig. 8C). Expression of maximal
levels of PKA inhibited p65 transactivation by 75 ± 10%
(mean ± S.E., n = 3). These data indicate that
activation of PKA either by an increase in intracellular levels of cAMP
or by expression of the catalytic subunit of PKA inhibits transcription
mediated by endogenous NF-B·Rel complexes or by overexpressed
p65.
B/Rel-regulated genes,
including TNF, TF, E-selectin, and VCAM-1. A common inhibitory
mechanism was identified that involved reduction in NF-B-mediated
transcription. Our studies demonstrated that nuclear translocation of
NF-B·Rel complexes was not affected by elevated cAMP. In addition,
forskolin did not modify the TNF-induced phosphorylation of p65.
However, transactivation by p65 was inhibited by activation of
endogenous PKA and by overexpression of the catalytic subunit of PKA.
These data indicate that activation of PKA reduced NF-B-mediated
transcription in human monocytic and endothelial cells.
B·Rel
complexes in human monocytic and endothelial cells. A recent study also
showed that forskolin inhibits NF-B-mediated transcription in HUVECs
(21). Inhibition of NF-B-mediated transcription may be due to a
block in the nuclear translocation of NF-B·Rel complexes and/or a
reduction in the transcriptional activity of these proteins in the
nucleus. Here, we showed that agents that elevate cAMP did not affect
the nuclear translocation of NF-B/Rel proteins or proteolytic
degradation of IB. Similarly, two recent reports indicated that
forskolin does not reduce the nuclear translocation of NF-B in human
promyelocytic HL-60 cells and HUVECs (21, 41). Forskolin and
Bt2cAMP did not alter the composition of the NF-B·Rel
complexes or induce the binding of CRE-binding protein to the
NF-B·Rel complexes (data not shown). Instead, we demonstrated that
elevation of cAMP or expression of the catalytic subunit of PKA
strongly inhibited transcription mediated by endogenous NF-B·Rel
complexes and overexpressed p65.
B, which selectively decreases the nuclear translocation of p65
(42). It should be noted that nuclear translocation of NF-B and p65
in Jurkat T-cells did not appear to be affected 40 min after
stimulation, which is similar to our studies using monocytic and
endothelial cells (Figs. 5 and 6), and only a small change was noted
2 h after stimulation (42). Functional studies using a chimeric
Gal4-p65 protein, which contains the transactivation domain of p65,
indicated that forskolin did not inhibit transactivation by Gal4-p65 in
human E14 T lymphoma cells (42). However, the use of Gal4-binding sites
and a chimeric protein in place of B-binding sites and wild-type p65
may not recapitulate cAMP-mediated inhibition. The use of different
cell types may account for these different inhibitory mechanisms.
Alternatively, costimulation of Jurkat cells and E14 T-cells with
phorbol ester and ionomycin rather than LPS or cytokines may explain
these differences.
B/Rel
proteins (17). However, inhibition of TNF-induced nuclear
translocation of NF-B in HUVECs by aspirin reduces VCAM-1 and
E-selectin expression without affecting ICAM-1 expression (43). In
addition, unlike VCAM-1, E-selectin, and TF, ICAM-1 is constitutively
expressed by HUVECs, and induction of ICAM-1 is delayed compared with
these other genes (17), suggesting that the ICAM-1 gene is regulated by
a different combination of transcription factors.
, TF, E-selectin,
and VCAM-1 genes. In monocytic THP-1 cells, Bt2cAMP
abolished LPS induction of TNF and TF mRNA expression and TNF
gene transcription, but only partially inhibited NF-B-mediated
transcription in transfected cells, suggesting the possibility that
cAMP may inhibit other transcription factors. Changes in the
composition of proteins binding to the CRE/ATF site in the E-selectin
promoter have been reported to contribute to the cAMP inhibition of the
E-selectin gene in bovine aortic endothelial cells (44). The TNF and
TF promoters contain AP-1 sites that contribute to the induction of
these genes (13, 45, 46). We cannot exclude the possibility that
elevated cAMP may, in part, reduce TNF and TF gene transcription by
modulating protein binding to these AP-1 sites and/or by inhibiting
AP-1 activity. Our preliminary studies indicate that cAMP increases
protein binding to both AP-1 sites in the TF promoter, but so far, no
changes in protein composition have been
detected.2 Further studies are required to
determine if cAMP inhibits expression of this distinct set of genes via
multiple mechanisms.
B·Rel complexes. Indeed, c-Rel, p65, and p50 all contain a
conserved serine in a consensus PKA recognition site
(X-Arg-Arg-X-Ser-X) near the
carboxyl-terminal end of the rel homology domain (50).
However, the role of this PKA site is unclear because it is not present
in all NF-B/Rel proteins. Mutation of serine 266 to alanine in c-Rel
reduces DNA binding and transactivation, suggesting that serine 266 contributes to protein dimerization (50). Similarly, mutation of serine
276 to alanine in p65 abolished its transactivation activity in HUVECs,
making it impossible to determine if elevated cAMP inhibited NF-B
activity by direct phosphorylation of the consensus PKA site of
p65.3
-induced phosphorylation of nuclear p65 in HUVECs. However,
changes in the phosphorylation pattern of p65 would not be detected in
this experiment. PKA may modulate NF-B activity by phosphorylating
cofactors that are required for transactivation. Alternatively, cAMP
may induce the synthesis or activation of transcriptional inhibitors of
NF-B-mediated transcription such as Bcl3 (51, 52) and p202, which
has recently been shown to inhibit NF-B enhancers (53). Finally,
activation of the PKA signaling pathway may inhibit NF-B-mediated
transcription by squelching coactivators.
and TF gene transcription in monocytes in vitro (27,
54, 55). In addition, pentoxifylline markedly inhibits increases in the
levels of TNF and TF in vivo in a chimpanzee model of
endotoxemia (56). Other drugs such as rolipram and amrinone suppress
T-cell activation in vitro and may be beneficial agents in
the treatment of autoimmune encephalomyelitis and acute cardiac
allographt rejection (57, 58). Thus, elevation of cellular cAMP
in vivo may reduce induction of gene expression and
inflammation associated with a variety of diseases.
§
Present address: INSERM U294 and Service d'Hematologie et
d'Immunologie Biologiques, Chu Xavier Bichat, 46, rue Henri Huchard,
75877 Paris Cedex 18, France.
¶
Contributed equally to this work.
Performed this work during the tenure of an Established
Investigatorship from the American Heart Association. To whom
correspondence should be addressed: Depts. of Immunology and
Vascular Biology, Scripps Research Inst., 10666 N. Torrey Pines Rd., La
Jolla, CA 92037. Tel.: 619-554-8594; Fax: 619-554-6146; E-mail:
nmackman{at}scripps.edu.
1
The abbreviations used are: LPS,
lipopolysaccharide; TNF, tumor necrosis factor ; TF, tissue
factor; E-selectin, endothelial leukocyte adhesion molecule-1; VCAM-1,
vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion
molecule-1; PKA, protein kinase A; CRE, cAMP response element;
Bt2cAMP, dibutyryl cAMP; HUVECs, human umbilical vein
endothelial cells; CAT, chloramphenicol acetyltransferase; TA,
transactivation; CREB, CRE-binding protein.
2
V. Ollivier and N. Mackman, unpublished
data.
3
G. C. N. Parry and N. Mackman, unpublished
data.
Supported by a scholarship from the Sanofi Association for
Thrombosis Research.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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P. C. Colombo, A. W. Ashton, S. Celaj, A. Talreja, J. E. Banchs, N. B. Dubois, M. Marinaccio, S. Malla, J. Lachmann, J. A. Ware, et al. Biopsy coupled to quantitative immunofluorescence: a new method to study the human vascular endothelium J Appl Physiol, March 1, 2002; 92(3): 1331 - 1338. [Abstract] [Full Text] [PDF]
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H. Isobe, K. Okajima, M. Uchiba, N. Harada, and H. Okabe Antithrombin prevents endotoxin-induced hypotension by inhibiting the induction of nitric oxide synthase in rats Blood, March 1, 2002; 99(5): 1638 - 1645. [Abstract] [Full Text] [PDF]
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 A. NAKAMURA, E. J. JOHNS, A. IMAIZUMI, Y. YANAGAWA, and T. KOHSAKA Activation of {beta}2-Adrenoceptor Prevents Shiga Toxin 2-Induced TNF-{alpha} Gene Transcription J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2288 - 2299. [Abstract] [Full Text] [PDF]
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N. D. Khoa, M. C. Montesinos, A. B. Reiss, D. Delano, N. Awadallah, and B. N. Cronstein Inflammatory Cytokines Regulate Function and Expression of Adenosine A2A Receptors in Human Monocytic THP-1 Cells J. Immunol., October 1, 2001; 167(7): 4026 - 4032. [Abstract] [Full Text] [PDF]
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J. S. Friedland, D. Constantin, T. C. Shaw, and E. Stylianou Regulation of interleukin-8 gene expression after phagocytosis of zymosan by human monocytic cells J. Leukoc. Biol., September 1, 2001; 70(3): 447 - 454. [Abstract] [Full Text] [PDF]
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R. Shenkar, H.-K. Yum, J. Arcaroli, J. Kupfner, and E. Abraham Interactions between CBP, NF-{kappa}B, and CREB in the lungs after hemorrhage and endotoxemia Am J Physiol Lung Cell Mol Physiol, August 1, 2001; 281(2): L418 - L426. [Abstract] [Full Text] [PDF]
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M. Goebeler, R. Gillitzer, K. Kilian, K. Utzel, E.-B. Brocker, U. R. Rapp, and S. Ludwig Multiple signaling pathways regulate NF-{kappa}B-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells Blood, January 1, 2001; 97(1): 46 - 55. [Abstract] [Full Text] [PDF]
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E. Speir, Z.-X. Yu, K. Takeda, V. J. Ferrans, and R. O. Cannon III Antioxidant Effect of Estrogen on Cytomegalovirus-Induced Gene Expression in Coronary Artery Smooth Muscle Cells Circulation, December 12, 2000; 102(24): 2990 - 2996. [Abstract] [Full Text] [PDF]
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A. Nakamura, E. J. Johns, A. Imaizumi, Y. Yanagawa, and T. Kohsaka {beta}2-Adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin Nephrol. Dial. Transplant., December 1, 2000; 15(12): 1928 - 1934. [Abstract] [Full Text] [PDF]
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R. D. Ye beta -Adrenergic agonists regulate NF-kappa B activation through multiple mechanisms Am J Physiol Lung Cell Mol Physiol, October 1, 2000; 279(4): L615 - L617. [Full Text] [PDF]
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P. Farmer and J. Pugin beta -Adrenergic agonists exert their "anti-inflammatory" effects in monocytic cells through the Ikappa B/NF-kappa B pathway Am J Physiol Lung Cell Mol Physiol, October 1, 2000; 279(4): L675 - L682. [Abstract] [Full Text] [PDF]
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N. Harada, K. Okajima, K. Murakami, S. Usune, C. Sato, K. Ohshima, and T. Katsuragi Adenosine and Selective A2A Receptor Agonists Reduce Ischemia/Reperfusion Injury of Rat Liver Mainly by Inhibiting Leukocyte Activation J. Pharmacol. Exp. Ther., September 1, 2000; 294(3): 1034 - 1042. [Abstract] [Full Text]
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A. L. True, A. Rahman, and A. B. Malik Activation of NF-kappa B induced by H2O2 and TNF-alpha and its effects on ICAM-1 expression in endothelial cells Am J Physiol Lung Cell Mol Physiol, August 1, 2000; 279(2): L302 - L311. [Abstract] [Full Text] [PDF]
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S. Komatsu, R. D. Berg, J. M. Russell, Y. Nimura, and D. N. Granger Enteric microflora contribute to constitutive ICAM-1 expression on vascular endothelial cells Am J Physiol Gastrointest Liver Physiol, July 1, 2000; 279(1): G186 - G191. [Abstract] [Full Text] [PDF]
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 W.-K. Kim, Y. Kan, D. Ganea, R. P. Hart, I. Gozes, and G. M. Jonakait Vasoactive Intestinal Peptide and Pituitary Adenylyl Cyclase-Activating Polypeptide Inhibit Tumor Necrosis Factor-alpha Production in Injured Spinal Cord and in Activated Microglia via a cAMP-Dependent Pathway J. Neurosci., May 15, 2000; 20(10): 3622 - 3630. [Abstract] [Full Text] [PDF]
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S. K. Manna, A. Mukhopadhyay, and B. B. Aggarwal Human Chorionic Gonadotropin Suppresses Activation of Nuclear Transcription Factor-kappa B and Activator Protein-1 Induced by Tumor Necrosis Factor J. Biol. Chem., April 28, 2000; 275(18): 13307 - 13314. [Abstract] [Full Text] [PDF]
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M. E. Pueyo, W. Gonzalez, A. Nicoletti, F. Savoie, J.-F. Arnal, and J.-B. Michel Angiotensin II Stimulates Endothelial Vascular Cell Adhesion Molecule-1 via Nuclear Factor-{kappa}B Activation Induced by Intracellular Oxidative Stress Arterioscler. Thromb. Vasc. Biol., March 1, 2000; 20(3): 645 - 651. [Abstract] [Full Text] [PDF]
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S. Hoshi, M. Goto, N. Koyama, K.-i. Nomoto, and H. Tanaka Regulation of Vascular Smooth Muscle Cell Proliferation by Nuclear Factor-kappa B and Its Inhibitor, I-kappa B J. Biol. Chem., January 14, 2000; 275(2): 883 - 889. [Abstract] [Full Text] [PDF]
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S. Wang, W. Wang, R. A. Wesley, and R. L. Danner A Sp1 Binding Site of the Tumor Necrosis Factor alpha Promoter Functions as a Nitric Oxide Response Element J. Biol. Chem., November 19, 1999; 274(47): 33190 - 33193. [Abstract] [Full Text] [PDF]
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M. Delgado and D. Ganea Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-activating Polypeptide Inhibit Interleukin-12 Transcription by Regulating Nuclear Factor kappa B and Ets Activation J. Biol. Chem., November 5, 1999; 274(45): 31930 - 31940. [Abstract] [Full Text] [PDF]
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 E. M. Boyle Jr, T. G. Canty Jr, E. N. Morgan, W. Yun, T. H. Pohlman, and E. D. Verrier Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription Ann. Thorac. Surg., November 1, 1999; 68(5): 1949 - 1953. [Abstract] [Full Text] [PDF]
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S. M. Jackson, F. Parhami, X.-P. Xi, J. A. Berliner, W. A. Hsueh, R. E. Law, and L. L. Demer Peroxisome Proliferator–Activated Receptor Activators Target Human Endothelial Cells to Inhibit Leukocyte–Endothelial Cell Interaction Arterioscler. Thromb. Vasc. Biol., September 1, 1999; 19(9): 2094 - 2104. [Abstract] [Full Text] [PDF]
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 M. D. Silverman, C. R. Waters, G. T. Hayman, J. Wigboldus, M. M. Samet, and P. I. Lelkes Tissue factor activity is increased in human endothelial cells cultured under elevated static pressure Am J Physiol Cell Physiol, August 1, 1999; 277(2): C233 - C242. [Abstract] [Full Text] [PDF]
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R. Shenkar and E. Abraham Mechanisms of Lung Neutrophil Activation After Hemorrhage or Endotoxemia: Roles of Reactive Oxygen Intermediates, NF-{kappa}B, and Cyclic AMP Response Element Binding Protein J. Immunol., July 15, 1999; 163(2): 954 - 962. [Abstract] [Full Text] [PDF]
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S.-Y. Jeong, S.-G. Ahn, J.-H. Lee, H.-S. Kim, J.-W. Kim, H. Rhim, S.-W. Jeong, and I.-K. Kim 3-Deazaadenosine, a S-Adenosylhomocysteine Hydrolase Inhibitor, Has Dual Effects on NF-kappa B Regulation. INHIBITION OF NF-kappa B TRANSCRIPTIONAL ACTIVITY AND PROMOTION OF Ikappa Balpha DEGRADATION J. Biol. Chem., July 2, 1999; 274(27): 18981 - 18988. [Abstract] [Full Text] [PDF]
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J. Anrather, V. Csizmadia, M. P. Soares, and H. Winkler Regulation of NF-kappa B RelA Phosphorylation and Transcriptional Activity by p21ras and Protein Kinase Czeta in Primary Endothelial Cells J. Biol. Chem., May 7, 1999; 274(19): 13594 - 13603. [Abstract] [Full Text] [PDF]
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B. Engelmann, S. Zieseniss, K. Brand, S. Page, A. Lentschat, A. J. Ulmer, and E. Gerlach Tissue Factor Expression of Human Monocytes Is Suppressed by Lysophosphatidylcholine Arterioscler. Thromb. Vasc. Biol., January 1, 1999; 19(1): 47 - 53. [Abstract] [Full Text] [PDF]
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M. Delgado, E. J. Munoz-Elias, Y. Kan, I. Gozes, M. Fridkin, D. E. Brenneman, R. P. Gomariz, and D. Ganea Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-activating Polypeptide Inhibit Tumor Necrosis Factor alpha Transcriptional Activation by Regulating Nuclear Factor-kB and cAMP Response Element-binding Protein/c-Jun J. Biol. Chem., November 20, 1998; 273(47): 31427 - 31436. [Abstract] [Full Text] [PDF]
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S. K. Manna and B. B. Aggarwal {alpha}-Melanocyte-Stimulating Hormone Inhibits the Nuclear Transcription Factor NF-{kappa}B Activation Induced by Various Inflammatory Agents J. Immunol., September 15, 1998; 161(6): 2873 - 2880. [Abstract] [Full Text] [PDF]
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P. Oeth, J. Yao, S.-T. Fan, and N. Mackman Retinoic Acid Selectively Inhibits Lipopolysaccharide Induction of Tissue Factor Gene Expression in Human Monocytes Blood, April 15, 1998; 91(8): 2857 - 2865. [Abstract] [Full Text] [PDF]
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 G. Krikun, F. Schatz, N. Mackman, S. Guller, and C. J. Lockwood Transcriptional Regulation of the Tissue Factor Gene by Progestins in Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., March 1, 1998; 83(3): 926 - 930. [Abstract] [Full Text]
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M. Ahmad, P. Theofanidis, and R. M. Medford Role of Activating Protein-1 in the Regulation of the Vascular Cell Adhesion Molecule-1 Gene Expression by Tumor Necrosis Factor-alpha J. Biol. Chem., February 20, 1998; 273(8): 4616 - 4621. [Abstract] [Full Text] [PDF]
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T. A. Bird, K. Schooley, S. K. Dower, H. Hagen, and G. D. Virca Activation of Nuclear Transcription Factor NF-kappa B by Interleukin-1 Is Accompanied by Casein Kinase II-mediated Phosphorylation of the p65 Subunit J. Biol. Chem., December 19, 1997; 272(51): 32606 - 32612. [Abstract] [Full Text] [PDF]
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U. R. Pendurthi, J. T. Williams, and L. V. M. Rao Inhibition of Tissue Factor Gene Activation in Cultured Endothelial Cells by Curcumin : Suppression of Activation of Transcription Factors Egr-1, AP-1, and NF-{kappa}B Arterioscler. Thromb. Vasc. Biol., December 1, 1997; 17(12): 3406 - 3413. [Abstract] [Full Text]
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