Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cytokine-stimulated inducible nitric oxide
synthase (iNOS) gene expression is dependent on nuclear factor-B
(NF-
B) activation and is suppressed by glucocorticoids (GC). In this
study we examined the molecular mechanisms of GC inhibition of iNOS
expression in rat hepatocytes. Combinations of tumor necrosis
factor-
, interleukin-1
, and interferon-
(cytokine mixture CM)
induced high levels of iNOS mRNA and NO synthesis. The synthetic GC
dexamethasone markedly repressed iNOS mRNA and protein expression, and
nuclear run-on assays showed that this inhibition was occurring at the
level of transcription. In addition, transfection studies showed that CM-stimulated activity of a 1.6-kb murine iNOS promoter fragment linked
upstream of luciferase was suppressed by dexamethasone. Electromobility
shift assays demonstrated that CM induced the appearance of an NF-
B
complex composed of p50 and p65 subunits; the addition of dexamethasone
markedly decreased this band shift. I-
B
expression was decreased
by CM and upregulated in the presence of dexamethasone. Subsequently,
nuclear p65 levels were decreased by dexamethasone compared with
CM-treated cells. Thus GC repress NF-
B DNA-binding activity in rat
hepatocytes in part through the upregulation of its inhibitor
I-
B
. These data indicate that one mechanism by which GC
block iNOS expression is through the inhibition of NF-
B activation
resulting in decreased iNOS transcription.
inducible nitric oxide synthase; nuclear factor-B; tumor
necrosis-
; interleukin-1
; interferon-
; glucocorticoids; gene
regulation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NITRIC OXIDE (NO) synthesized by the inducible form of NO synthase (iNOS) plays a role in numerous inflammatory and immune processes, such as sepsis, hemorrhagic shock, transplant rejection, and autoimmune diseases (5, 20). Although some epithelial cells have been shown to express iNOS constitutively (1), most cells express iNOS mRNA and protein only after exposure to specific stimulating agents. In these cells cytokines, viruses, or lipopolysaccharide (LPS) stimulate iNOS expression in a widespread fashion, leading to the production of large amounts of NO. NO exerts cytoprotective and beneficial actions (e.g., antitumor, antimicrobial, and anti-atherogenic effects); however, overproduction of NO can have cytotoxic or other detrimental effects (e.g., septic shock, apoptosis, or direct cellular injury). iNOS expression therefore must be tightly regulated to achieve the maximal benefit of NO while avoiding toxicity.
Previously, we reported that tumor necrosis factor- (TNF-
),
interleukin-1
(IL-1
), interferon-
(IFN-
), and LPS can act synergistically to induce iNOS gene expression in rodent and human hepatocytes, whereas glucocorticoids (GC) inhibit iNOS expression (9,
10). This increase in cytokine-induced hepatic iNOS mRNA levels is due
in part to the transcriptional activation of the iNOS gene (7, 8). The
cis-regulatory elements required for the activation of the murine iNOS promoter have been identified and
shown to reside within 1 kb of the transcriptional start site of the
iNOS gene (18, 31). In contrast, the proximal 1 kb of the
5'-flanking region of the human iNOS gene is not
sufficient to activate the iNOS promoter. We recently demonstrated that
cytokine-responsive elements upstream of
3.8 kb are required for
activation of the human iNOS promoter (7). Notwithstanding these
intrinsic and important differences between the human and murine iNOS
promoters, many studies have now shown that cytokine activation of the
iNOS gene is dependent on the transcription factor nuclear factor-
B (NF-
B) (11, 30). NF-
B in its inactive state resides in the cytoplasm bound by its inhibitor, I-
B
. Activation of NF-
B by a
wide variety of inflammatory stimuli leads to degradation of I-
B
either through a ubiquitin-proteosome pathway or proteosome-independent pathway (12). This allows NF-
B to translocate to the nucleus and
bind to the promoter of its target genes. Numerous inflammatory and
immunomodulatory genes including cytokines, cell adhesion molecules, and major histocompatibility complex proteins are known to
be regulated by NF-
B.
Despite extensive clinical use of GC as immunosuppressive agents, we
have only begun to understand the molecular mechanisms by which these
drugs exert their anti-inflammatory actions. It is known that GC bind
to steroid hormone receptors, leading to activated GC receptors that
then bind to either GC response elements (GRE) or negative GRE,
resulting in transcriptional activation or repression of genes,
respectively. The majority of genes that are downregulated by steroids,
however, lack negative GRE in their promoters. Furthermore, GC have
been shown to inhibit the transcription factor AP-1 through
protein-protein interactions (13), and yet only a few of the genes
affected by GC contain functional binding sites for AP-1 in their
promoters. Recently, several researchers have demonstrated that GC are
able to suppress NF-B through cross-coupling mechanisms (26) or
through the induction of I-
B
(2, 25); it now appears that NF-
B
inhibition may be the predominant mechanism by which proinflammatory
genes are suppressed by GC. However, the precise mechanism by which GC
downregulate iNOS expression is unknown. In this study, we explored the
molecular mechanisms underlying GC downregulation of iNOS gene
expression in rat hepatocytes. We demonstrate that GC upregulate
I-
B
expression and decrease nuclear p65 translocation, thus
inhibiting iNOS gene transcription and promoter activation by
decreasing NF-
B DNA binding activity.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
Murine recombinant TNF- was purchased from Genzyme (Cambridge, MA),
human recombinant IL-1
was generously provided by Craig Reynolds
(National Cancer Institute), and murine recombinant IFN-
was
obtained from GIBCO BRL (Grand Island, NY). Dexamethasone and
pyrrolidine dithiocarbamate (PDTC) were from Sigma (St. Louis, MO), and
T4 polynucleotide kinase and lipofectin were purchased from United
States Biochemical (Cleveland, OH) and GIBCO BRL, respectively.
Cell culture.
Rat hepatocytes were isolated from male Sprague-Dawley rats weighing
200-250 g (Harlan Sprague Dawley, Madison, WI), using a modified
in situ collagenase (type IV, Sigma) perfusion technique as previously
described (10). Hepatocytes (5 × 106) were plated onto 100-mm
gelatin-coated cell culture dishes (Corning, Corning, NY) and
maintained at 37°C, 95% air-5%
CO2 in William's E (GIBCO)
supplemented with L-arginine
(0.50 mM), insulin (106 M),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (15 mM), L-glutamine,
penicillin, streptomycin, and 10% low endotoxin calf serum (Hyclone
Laboratories, Logan, UT). After an 18-h incubation hepatocytes were
treated with a cytokine mixture (CM) of TNF-
(500 U/ml), IL-1
(200 U/ml), and IFN-
(100 U/ml) or different combinations of these
cytokines in the presence or absence of dexamethasone
(10
9 to
10
5 M).
Nitrite plus nitrate assay. Culture supernatants were collected 24 h after cytokine treatment and assayed for nitrite plus nitrate, the stable end-products of NO oxidation, using an automated procedure based on the Griess reaction (9).
Northern blot analysis.
The iNOS probe used was a 2.3-kb BamH I fragment from
the human hepatocyte iNOS cDNA (11). The I-B
cDNA probe was
generously provided by Gary Nabel (Univ. of Michigan). RNA extraction,
Northern blot analysis, and autoradiography were performed as described previously (10). After being probed for iNOS or I-
B
, membranes were stripped with boiling 5 mM EDTA and 0.1% sodium dodecyl sulfate (SDS) and rehybridized with a probe for 18S ribosomal RNA. Relative mRNA levels were quantitated by PhosphorImager scanning with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
SDS-polyacrylamide gel electrophoresis and Western blotting.
Cytosolic and nuclear proteins were prepared as described (6, 14) and
quantitated with bicinchoninic acid protein assay reagent (Pierce
Chemical, Rockford, IL). Western blot analysis was carried out by
separating proteins (75 µg) with 8% SDS-polyacrylamide gel
electrophoresis and electrophoretically transferring the products to
nitrocellulose membranes (Schleicher & Schuell, Keene, NH) (14).
Nonspecific binding to the membrane was blocked by 5% nonfat dry milk
in phosphate-buffered saline-Tween overnight at 4°C. Blots were
washed in phosphate-buffered saline-Tween and then incubated for 1 h
with either monoclonal mouse anti-iNOS antibody (1:5,000 dilution,
Transduction Laboratories, Lexington, KY), polyclonal rabbit
anti-I-B
antibody (1:2,000 dilution; Rockland, Gilbertsville,
PA), and polyclonal rabbit anti-p65 antibody (1:1,000 dilution;
Rockland). The secondary antibody used was a peroxidase-conjugated goat
anti-mouse immunoglobulin G in a 1:1,000 dilution (Schleicher & Schuell). Membranes were developed with the enhanced
chemiluminescence-detection system (DuPont-NEN, Boston, MA) and exposed
to film.
Nuclear run-on assay. Hepatocytes were stimulated with CM with or without dexamethasone for 4 h. Nuclei were purified as previously reported (7, 8). Linearized plasmid containing full-length human iNOS, glyceraldehyde-3-phosphate dehydrogenase, and glutaminase cDNA were immobilized and crosslinked on a GeneScreen membrane (9). Hybridizations were carried out as described, and results were quantitated with the PhosphorImager (7, 8).
Promoter analysis.
A deletional construct containing 1.6 kb of the 5'-flanking
region of the murine iNOS gene ligated upstream of the reporter gene
luciferase (gift of Charles Lowenstein, Johns Hopkins Univ.) was
transiently transfected into hepatocytes using liposomes as described
(6). Serum-free transfections were carried out in six-well plates
(Corning) using 1 µg of DNA and 10 µg of lipofectin for 6 h. After
an overnight recovery hepatocytes were treated with cytokines for 6 h
in the presence or absence of dexamethasone (106 M) or the NF-
B
inhibitor PDTC (100 µM), after which luciferase activity was measured
using a commercially available assay kit (Promega, Madison, WI) (6).
Electromobility shift assays.
Hepatocytes were treated with cytokines in the presence or absence of
dexamethasone (106 M) or
PDTC (100 µM) for 1 h. Preparation of nuclear extracts and
electromobility shift assays were carried out essentially as described
(6). The oligonucleotide used was derived from the murine
iNOS promoter (positions
90 to
71) (18) and contained a
functional NF-
B element (italized):
CAACTGGGGACTCTCCCTTTG. A mutant
NF-
B oligonucleotide was also used as a mutant competitor: AAGCTGGGTCCACTCCCTTTG. Nuclear
extracts (10 µg) were incubated with ~0.5 ng (~40,000 counts/min)
of 32P-end-labeled oligonucleotide
(T4 polynucleotide kinase) for 30 min at room temperature. In other
experiments nuclear extracts were incubated with excess unlabeled
NF-
B oligomers or antibodies against the different subunits of
NF-
B (Santa Cruz Biotechnology and kind gift of Nancy Rice, National
Cancer Institute) for 15 min before the addition of labeled probe.
DNA-protein complexes were electrophoretically resolved on a 5%
nondenaturing polyacrylamide gel, which was then dried and subjected to
autoradiography.
Statistical analysis. The significance of differences was determined by analysis of variance (ANOVA) using the Statview statistics program (Abacus Concepts, Berkeley, CA). Statistical significance was established at P < 0.01.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dexamethasone inhibits cytokine-induced iNOS mRNA and protein
synthesis.
To investigate the effects of GC on iNOS mRNA and NO production in rat
hepatocytes, combinations of TNF-, IFN-
, and IL-1
were added
to cultures in the presence or absence of the synthetic GC
dexamethasone. Consistent with our previous results (8, 10)
combinations of these cytokines (CM) or IL-1
alone induced iNOS mRNA
expression (Fig.
1A,
top) and NO production as measured by nitrite and nitrate release (Fig.
1B). The addition of dexamethasone significantly inhibited iNOS mRNA levels (Fig.
1A,
bottom) and NO production
(~50-75% maximum inhibition) in all groups. As shown in Fig.
2, the inhibitory effect on CM-induced NO
synthesis was concentration dependent, with an ~60% decrease in
nitrite and nitrate release at the highest concentration of
dexamethasone (10
5 M).
Western blot analysis showed expression of iNOS protein with CM
stimulation (Fig. 3), and similar to the
decrease in mRNA expression, protein levels were also lower in the
presence of dexamethasone. These data therefore indicate that
cytokine-induced NO synthesis in hepatocytes is inhibited by GC at the
mRNA and protein levels.
|
|
|
Dexamethasone suppresses iNOS gene transcription and promoter activation. To examine whether the suppressive effects of GC occurred at the level of transcription, nuclear run-on assays were performed. Shown in Fig. 4 is a representative nuclear run-on experiment looking at the effects of CM with or without dexamethasone on iNOS gene transcription. An average of several experiments indicates that CM stimulation increases iNOS transcription 8- to 10-fold in rat hepatocytes, and the addition of dexamethasone results in a 50% inhibition of iNOS transcription.
|
|
NF-B DNA binding activity is suppressed by
dexamethasone.
Several studies in other cell types have shown that GC inhibit NF-
B
activation (2, 25, 26). To investigate the effects of GC on the
DNA-binding activity of NF-
B in hepatocytes, electromobility shift
assays were carried out. A single NF-
B complex appeared on CM
stimulation of hepatocytes (Fig.
6A).
Both dexamethasone and PDTC partially prevented the appearance of this
shifted band, indicating that the repressor effect of dexamethasone on
cytokine-induced iNOS gene transcription is mediated in part by
downregulating NF-
B activity. The specificity of the binding was
confirmed by cold competition with wild-type and mutant probes (Fig.
6B). Immunodepletion supershift
studies with antibodies revealed that the complex was composed of p50
and p65 subunits. Antibodies against C-rel had no effect (data not
shown).
|
I-B
expression is upregulated by
cytokines and dexamethasone.
Dexamethasone has been shown to upregulate the synthesis of I-
B
in monocytes and lymphocytes (2, 25) but not in primary endothelial
cells or A549/8 cells (3, 15). To analyze the effects of GC on
I-
B
expression in rat hepatocytes, we performed Northern blot
analysis for I-
B
mRNA. Control cells showed basal expression of
I-
B
mRNA, and this was decreased by the addition of CM. When
dexamethasone was added alone or with CM there was a two- to threefold
upregulation in I-
B
mRNA (Fig. 7).
Consistent with the mRNA findings Western blot analysis revealed basal
I-
B
protein levels that were also decreased by CM and preserved
by dexamethasone (Fig. 8). Thus GC
positively affect I-
B
mRNA and protein expression in rat
hepatocytes.
|
|
Nuclear p65 levels are decreased by dexamethasone.
To discern whether the increase in I-B
expression resulted in a
decrease in p65 nuclear translocation cytosolic and nuclear proteins
levels were assayed by Western blot analysis. These studies revealed
that nuclear p65 levels were increased in CM-treated hepatocytes and
were decreased when dexamethasone was added to CM (Fig.
9), suggesting that p65 is sequestered by
the newly formed I-
B
in the cytoplasm, thereby preventing nuclear
translocation of NF-
B. Western blot analysis for cytosolic p65
protein showed basal expression in control hepatocytes; both CM alone
or CM and dexamethasone increased cytosolic p65 protein levels (data
not shown). The lack of a reciprocal change in cytosolic p65 for the CM
and dexamethasone group may be due to the already increased level of
cytosolic p65 expression and the fact that the cytosolic content
reflects the steady state between synthesis and degradation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GC inhibit the expression of many cytokine and immunomodulatory genes, including iNOS (10, 15, 16, 23). In this study we characterized some of the mechanisms involved in GC inhibition of iNOS gene expression in rat hepatocytes. The synthetic GC dexamethasone markedly suppressed cytokine-stimulated iNOS expression and NO synthesis in rat hepatocytes (Figs. 1 and 2). Because iNOS mRNA levels were reduced, it was not surprising that cytokine-induced iNOS protein levels were also decreased by dexamethasone (Fig. 3). Nuclear run-on analysis showed that dexamethasone-mediated iNOS repression in rat hepatocytes occurred at the level of transcription (Fig. 4). The transcriptional suppression by GC was confirmed by reporter gene analysis of a 1.6-kb murine iNOS promoter fragment (Fig. 5). The ~40% suppression of luciferase activity that we measured was less than the 60-75% decrease in mRNA levels, suggesting that dexamethasone may have posttranscriptional effects on iNOS expression in hepatocytes. Although we did not explore the possibility of posttranslational effects, others have shown that GC prolong iNOS mRNA half-life in other cell types (16, 23), and we are currently investigating the possibility that dexamethasone enhances iNOS mRNA stability. A recent study in rat mesangial cells showed that steroids reduced iNOS translational rates and also increased the degradation of iNOS protein (16). Simmons et al. (28) demonstrated an additional regulatory mechanism whereby dexamethasone decreases NO production by limiting L-arginine and tetrahydrobiopterin (BH4) availability in cardiac microvascular endothelial cells.
The transcription factor NF-B is required for LPS and cytokine
induction of the murine iNOS promoter (11, 30), and we have also
reported that iNOS gene expression is dependent on NF-
B in rat
hepatocytes (6). The addition of the antioxidant PDTC resulted in an
almost complete abrogation of CM-induced luciferase activity,
suggesting that NF-
B is required for iNOS promoter activation in rat
hepatocytes (Fig. 5). Electromobility shift assays demonstrated that
NF-
B DNA binding activity was suppressed by dexamethasone (Fig. 6).
Two major mechanisms appear to be at work in GC repression of NF-
B.
The first involves the upregulation of I-
B
expression by
dexamethasone (Figs. 7 and 8), resulting in a reduction of the amount
of NF-
B that translocates to the nucleus (Fig. 9). This is in
agreement with the findings of Scheinman et al. (25) and Auphan et al.
(2) who reported that dexamethasone increased I-
B
expression in
HeLa and Jurkat cells, respectively. The second mechanism involves
cross-coupling and physical association between the activated GC
receptor and NF-
B, leading to the inhibition of DNA transactivation
(24, 26). The relative importance of each mechanism appears to be
cell-type dependent. For instance, I-
B
levels in endothelial
cells and lung epithelial cells do not seem to be influenced
significantly by dexamethasone (3, 15) compared with monocytes and
lymphocytes (2, 25). Our studies show that I-
B
mRNA expression
was upregulated by dexamethasone (Fig. 7) and that I-
B
protein
levels are stabilized (Fig. 8). This suggests that the decrease in the
DNA-binding activity of NF-
B in rat hepatocytes is due in part to
the prevention of NF-
B translocation to the nucleus. The fact that
nuclear p65 levels were diminished in our study confirms that newly
formed I-
B
decreases the nuclear translocation of p65, resulting
in a reduction in NF-
B-DNA binding activity.
In summary, dexamethasone inhibits iNOS gene transcription in rat
hepatocytes by increasing I-B
expression and decreasing NF-
B
activity. This documents the importance of NF-
B in the regulation of
iNOS gene expression and suggests that targeting NF-
B activation may
be a more specific way of modulating NO production.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Debra Williams for excellent technical assistance, Charles
J. Lowenstein for providing the murine macrophage iNOS promoter, and
Gary Nabel for the I-B
probe.
![]() |
FOOTNOTES |
---|
M. E. de Vera and B. S. Taylor contributed equally to the work in this study.
This work was presented in part at the 30th Annual Meeting of the Association for Academic Surgery, Chicago, IL, November 14, 1996.
This study was supported by National Institute of General Medical Sciences Grants GM-52021, GM-44100, and GM-37753
Address for reprint requests: D. A. Geller, Dept. of Surgery, 497 Scaife Hall, Pittsburgh, PA 15261.
Received 11 March 1997; accepted in final form 22 August 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Asano, K.,
C. B. Chee,
B. Gaston,
C. M. Lilly,
C. Gerard,
J. M. Drazen,
and
J. S. Stamler.
Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells.
Proc. Natl. Acad. Sci. USA
91:
10089-10093,
1994
2.
Auphan, N.,
J. A. DiDonato,
C. Rosette,
A. Helmberg,
and
M. Karin.
Immunosuppression by glucocorticoids: inhibition of NF-B activity through induction of I
B synthesis.
Science
270:
286-290,
1995[Abstract].
3.
Brostjan, C.,
J. Anrather,
V. Csizmadia,
D. Stroka,
M. Soares,
F. H. Bach,
and
H. Winkler.
Glucocorticoid-mediated repression of NFB activity in endothelial cells does not involve induction of I-
B
synthesis.
J. Biol. Chem.
271:
19612-19616,
1996
4.
Chartrain, N. A.,
D. A. Geller,
P. P. Koty,
N. F. Sitrin,
A. K. Nussler,
E. P. Hoffman,
T. R. Billiar,
N. I. Hutchinson,
and
J. S. Mudgett.
Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene.
J. Biol. Chem.
269:
6765-6772,
1994
5.
De Vera, M. E.,
D. A. Geller,
and
T. R. Billiar.
Hepatic inducible nitric oxide synthase: regulation and function.
Biochem. Soc. Trans.
23:
1008-1013,
1995[Medline].
6.
De Vera, M. E.,
Y. M. Kim,
H. R. Wong,
T. R. Billiar,
and
D. A. Geller.
Heat shock response inhibits cytokine-inducible nitric oxide synthase expression in rat hepatocytes.
Hepatology
24:
1238-1245,
1996[Medline].
7.
De Vera, M. E.,
R. A. Shapiro,
A. K. Nussler,
J. S. Mudgett,
R. L. Simmons,
S. M. Morris,
T. R. Billiar,
and
D. A. Geller.
Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.
Proc. Natl. Acad. Sci. USA
93:
1054-1059,
1996
8.
Geller, D. A.,
M. E. de Vera,
D. A. Russell,
R. A. Shapiro,
A. K. Nussler,
R. L. Simmons,
and
T. R. Billiar.
A central role for interleukin-1 in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase: IL-1
induces hepatic nitric oxide synthesis.
J. Immunol.
155:
4890-4898,
1995[Abstract].
9.
Geller, D. A.,
C. J. Lowenstein,
R. A. Shapiro,
A. K. Nussler,
M. Di Silvio,
S. C. Wang,
D. K. Nakayama,
R. L. Simmons,
S. H. Snyder,
and
T. R. Billiar.
Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes.
Proc. Natl. Acad. Sci. USA
90:
3491-3495,
1993[Abstract].
10.
Geller, D. A.,
A. K. Nussler,
M. Di Silvio,
C. J. Lowenstein,
R. A. Shapiro,
S. C. Wang,
R. L. Simmons,
and
T. R. Billiar.
Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA
90:
522-526,
1993[Abstract].
11.
Griscavage, J. M.,
S. Wilk,
and
L. J. Ignarro.
Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-B.
Proc. Natl. Acad. Sci. USA
93:
3308-3312,
1996
12.
Imbert, V.,
R. A. Rupec,
A. Livolsi,
H. L. Pahl,
E. Traenker,
C. Mueller-Dieckman,
D. Farahifar,
B. Rossi,
P. Auberger,
P. A. Baeuerle,
and
J. Peyron.
Tyrosine phosphorylation of IB-
activates NF-
B without proteolytic degradation of I
B-
.
Cell
86:
787-798,
1996[Medline].
13.
Jonat, C.,
H. J. Rahmsdorf,
K.-K. Park,
A. Cato,
S. Gebel,
H. Ponta,
and
P. Herrlich.
Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone.
Cell
62:
1189-1204,
1990[Medline].
14.
Kim, Y. M.,
M. E. de Vera,
S. C. Watkins,
and
T. R. Billiar.
Nitric oxide protects cultured rat hepatocytes from TNF--induced apoptosis by inducing heat shock protein 70 expression.
J. Biol. Chem.
272:
1402-1411,
1997
15.
Kleinert, H.,
C. Euchenhofer,
I. Ihrig-Biedert,
and
U. Förstermann.
Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-B.
Mol. Pharmacol.
49:
15-21,
1996[Abstract].
16.
Kunz, D.,
G. Walker,
W. Eberhardt,
and
J. Pfeilschifter.
Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.
Proc. Natl. Acad. Sci. USA
93:
255-259,
1996
17.
Lee, S. W.,
A.-P. Tsou,
H. Chan,
J. Thomas,
K. Petrie,
E. M. Eugui,
and
A. C. Allison.
Glucocorticoids selectively inhibit the transcription of the interleukin 1 gene and decrease the stability of interleukin 1
mRNA.
Proc. Natl. Acad. Sci. USA
85:
1204-1208,
1988[Abstract].
18.
Lowenstein, C. J.,
E. W. Alley,
P. Raval,
A. M. Snowman,
S. H. Snyder,
S. W. Russell,
and
W. J. Murphy.
Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon- and lipopolysaccharide.
Proc. Natl. Acad. Sci. USA
90:
9730-9734,
1993[Abstract].
19.
Martin, E.,
C. Nathan,
and
Q.-W. Xie.
Role of interferon regulatory factor 1 in induction of nitric oxide synthase.
J. Exp. Med.
180:
977-984,
1994[Abstract].
20.
Nathan, C.
Nitric oxide as a secretory product of mammalian cells.
FASEB J.
6:
3051-3064,
1992
21.
Nussler, A. K.,
M. Di Silvio,
T. R. Billiar,
R. A. Hoffman,
D. A. Geller,
R. Selby,
J. Madariaga,
and
R. L. Simmons.
Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin.
J. Exp. Med.
176:
261-264,
1992[Abstract].
22.
Peng, H.-B.,
P. Libby,
and
J. K. Liao.
Induction and stabilization of I-B
by nitric oxide mediates inhibition of NF-
B.
J. Biol. Chem.
270:
14214-14219,
1995
23.
Perella, J. A.,
M. Yoshizumi,
Z. Fen,
J.-C. Tsai,
C. M. Hsieh,
S. Kourembanas,
and
M. E. Lee.
Transforming growth factor-1 but not dexamethasone, down-regulates nitric-oxide synthase mRNA after its induction by interleukin-1
in rat smooth muscle cells.
J. Biol. Chem.
269:
14595-14600,
1994
24.
Ray, A.,
and
K. E. Prefontaine.
Physical association and functional antagonism between the p65 subunit of transcription factor NF-B and the glucocorticoid receptor.
Proc. Natl. Acad. Sci. USA
91:
752-756,
1994[Abstract].
25.
Scheinman, R. I.,
P. C. Cogswell,
A. K. Lofquist,
and
A. S. Baldwin, Jr.
Role of transcriptional activation of I-B
in mediation of immunosuppression by glucocorticoids.
Science
270:
283-286,
1995[Abstract].
26.
Scheinman, R. I.,
A. Gualberto,
C. M. Jewell,
J. A. Cidlowski,
and
A. S. Baldwin, Jr.
Characterization of mechanisms involved in transrepression of NF-B by activated glucocorticoid receptors.
Mol. Cell. Biol.
15:
943-953,
1995[Abstract].
27.
Sherman, M. P.,
E. E. Aeberhard,
V. Z. Wong,
J. M. Griscavage,
and
L. J. Ignarro.
Pyrrolidine dithiocarbamate inhibits induction of nitric oxide synthase activity in rat alveolar macrophages.
Biochem. Biophys. Res. Commun.
191:
1301-1308,
1993[Medline].
28.
Simmons, W. W.,
D. Ungureanu-Longrois,
G. K. Smith,
T. W. Smith,
and
R. A. Kelly.
Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and L-arginine transport.
J. Biol. Chem.
271:
23928-23937,
1996
29.
Spink, J.,
J. Cohen,
and
T. J. Evans.
The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase: activation by nuclear factor-B.
J. Biol. Chem.
270:
29541-29547,
1995
30.
Xie, Q.-W.,
Y. Kashiwabara,
and
C. Nathan.
Role of transcription factor NF-B/Rel in induction of nitric oxide synthase.
J. Biol. Chem.
269:
4705-4708,
1994
31.
Xie, Q.-W.,
R. Whisnant,
and
C. Nathan.
Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon- and bacterial lipopolysaccharide.
J. Exp. Med.
177:
1779-1784,
1993[Abstract].