Nitric oxide (NO) (
)is a short-lived bioactive
molecule participating in the physiology and/or pathophysiology of many
organ systems(1) . The expression of the inducible isoform of
nitric oxide synthase (NOS II or iNOS) is regulated mainly at the
transcriptional level(2) . Inflammatory stimuli such as
bacterial lipopolysaccharide (LPS) and cytokines induce the expression
of this enzyme in many cell types. Interestingly, in some cells, agents
other than cytokines are efficacious inducers of NOS II expression. For
example, in rat mesangial cells, cAMP-elevating agents stimulate NOS II
expression(3) . Phorbol ester induction of NOS II has been
reported for rat peritoneal macrophages(4) . In murine BALB 3T3
fibroblasts, NOS II is expressed in response to forskolin, dibutyryl
cAMP, or tetradecanoylphorbol-13-acetate (TPA)(5) .
Analyses
of the cloned murine NOS II promoter (6, 7, 8) have revealed the presence of
numerous consensus sequences for the binding of transcription factors.
Of these potentially relevant transcription factors, nuclear
factor-
B (NF-
B) (6, 9) and interferon
regulatory factor (10, 11) have been shown to be
functionally important for NOS II induction. The molecular mechanisms
utilized by other second messenger pathways are still unclear. In rat
mesangial cells, the inhibitor of NF-
B activation, pyrrolidine
dithiocarbamate (PDTC), blocked NOS II expression induced by
interleukin-1
(IL-1
), but not the expression stimulated by
8-bromo-cAMP, suggesting two different induction pathways(12) .
In the current study, we attempted to induce NOS II expression in
3T3 fibroblasts via four different second messenger pathways, namely
receptor tyrosine kinase, protein kinase C, protein kinase A, and
protein kinase G. We characterized the induction processes with
modulators of NOS II induction such as transforming growth
factor-
1 (TGF-
1), dexamethasone, PDTC, and
3,4-dichloroisocoumarin (DCI). The experiments indicate that all NOS
II-inducing second messenger pathways are modulated in the same way and
all converge in the activation of NF-
B as an essential
transcription factor.
MATERIALS AND METHODS
Reagents
Mouse INF-
, human TNF-
and
human TGF-
1 were purchased from Genzyme. LPS (Escherichia coli 026:B6), PDTC, DCI, dexamethasone, forskolin, 8-bromo-cAMP,
8-bromo-cGMP, isobutylmethylxanthine (IBMX) and TPA were purchased from
Sigma. Isotopes were obtained from Amersham Corp. Restrictions enzymes,
polynucleotide kinase, Taq polymerase, S1 nuclease, T3 RNA
polymerase, dNTPs, and oligo(dT) primer were purchased from Pharmacia
Biotech Inc. RNase ONE(TM) was obtained from Promega. Superscript
reverse transcriptase and DNase I were obtained from Life Technologies,
Inc.
Cell Culture
Murine BALB 3T3 fibroblasts and RAW
264.7 macrophages (both ATCC) were grown in Dulbecco's modified
Eagle's medium (DMEM, Life Technologies, Inc.) with 10% fetal
bovine serum, 2 mML-glutamine, penicillin, and
streptomycin. For induction, confluent 3T3 cells were cultured for 16 h
in DMEM with only 0.5% fetal calf serum and then incubated for
3-18 h (in DMEM with 0.5% fetal calf serum) with one of the
following agents: INF-
(100 units/ml), TNF-
(10 ng/ml), LPS
(1 µg/ml), TPA (50 ng/ml), forskolin (100 µM),
8-bromo-cAMP (1 mM), IBMX (250 µM), or
8-bromo-cGMP (1 mM). In some experiments the following
modulators of NOS II induction were added with one of the inducing
agents: dexamethasone (5 µM), TGF-
1(2 ng/ml), PDTC
(100 µM), or DCI (50 µM).
NOS II Protein Preparation and Western
Blotting
3T3 cells (untreated and induced for 18 h with
TNF-
, 10 ng/ml; LPS, 1 µg/ml; TPA, 50 ng/ml; or forskolin, 100
µM) were homogenized on ice as described previously for
brain tissue or endothelial cells(13, 14) .
Homogenates were centrifuged at 100,000
g for 1 h, and
the soluble (cytosolic) fraction was partially purified on
2`,5`-ADP-Sepharose(13, 14) . The eluates from the
affinity columns were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (7.5% gels)(15) .
The proteins were transferred to nitrocellulose membranes (Schleicher
& Schuell) by electroblotting (Bio-Rad). All subsequent steps were
performed at room temperature. The blots were blocked in Blotto (3%
non-fat dried milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 45 min. The blots were incubated for 1 h
with a monoclonal anti-NOS II antibody (1 µg/ml, Transduction
Laboratories, Lexington, KY) in Tris-buffered saline/Tween (TBS/T: 10
mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20)
containing 50 mg/liter gelatin. The blots were washed twice with TBS/T
(7 min each) and then incubated for 30 min with horseradish
peroxidase-conjugated goat anti-mouse IgG diluted 1:2000 in TBS/T with
50 mg/liter gelatin. The blots were washed three times (5 min each) in
TBS/T, followed by one wash (5 min) in TBS alone. The immunocomplexes
were developed using an enhanced horseradish peroxidase/luminol
chemiluminescence reaction (DuPont NEN) according to the
manufacturer's instructions.
Cloning of a Murine NOS II and a Murine
-Actin cDNA
Fragment
Total RNA was isolated by guanidinium
isothiocyanate/phenol/chloroform extraction (16) from RAW 264.7
cells induced with 1 µg/ml LPS for 16 h. Two µg of this RNA
were annealed with 0.5 µg of oligo(dT) primer (Pharmacia) and
reverse-transcribed with Superscript reverse transcriptase (Life
Technologies, Inc.) following the manufacture's instructions.
Reverse transcriptase-generated cDNA encoding for murine NOS II and
murine
-actin were amplified using PCR. Oligonucleotide primers
for NOS II and
-actin were: GACAAGAGGCTGCCCCCC (sense), and
GCTGGGAGTCATGGAGCCG (antisense); GTGGGCCGCTCTAGGCACCAA (sense), and
CTCTTTGATGTCACGCACGATTTC (antisense), respectively. They generated PCR
fragments corresponding to the murine NOS II cDNA (17) (positions 2612-3170) and murine
-actin (18) (positions 25-564). PCR was performed in 100 µl
of Taq polymerase buffer (Pharmacia), containing 0.2 mM dNTPs, 1.5 mM MgCl
2 units of Taq polymerase, 50 pmol oligonucleotide primers, and reverse
transcriptase products (0.10 of the reverse transcriptase reaction).
After a initial denaturation step of 95 °C for 5 min, 30 cycles
were performed (1 min at 95 °C, 1 min at 60 °C, and 1 min at 72
°C). The final extension period at 72 °C was 10 min. Amplified
cDNA fragments (NOS II, 559 base pairs;
-actin, 540 base pairs)
were cloned into the EcoRV site of pCR-Script (Stratagene)
using the Sure Clone ligation kit (Pharmacia), generating the cDNA
clones pCR_NOS II_mouse and pCR_
-actin_mouse. DNA sequences of the
cloned PCR products were determined from plasmid templates using the
dideoxy chain termination method with the T7 sequencing kit
(Pharmacia).
Preparation of DNA and Antisense RNA Probes
To
generate radiolabeled DNA probes for S1 nuclease protection analysis,
the cDNA clones pCR_NOS II_mouse and pCR_
-actin_mouse were
restricted with NcoI or BglII, dephosporylated (with
calf intestinal alkaline phosphatase, Boehringer Mannheim), extracted
with phenol/chloroform, and concentrated by ethanol precipitation.
Fifty to one hundred ng of this DNA were labeled with
[
-
P]ATP using polynucleotide kinase
(Pharmacia). The radiolabeled DNA was separated from unincorporated
radioactivity using NucTrap probe purification columns (Stratagene). To
generate radiolabeled antisense RNA probes for RNase protection assays,
the cDNA clones pCR_NOS II_mouse and pCR_
-actin_mouse were
linearized with NcoI or BstEII, extracted with
phenol/chloroform, and concentrated by ethanol precipitation. One half
of a microgram of this DNA was in vitro transcribed using T3
RNA polymerase (Pharmacia) and [
-
P]UTP.
After a 1-h incubation, the template DNA was degraded with DNase I for
15 min. The radiolabeled RNA was purified using NucTrap probe
purification columns (Stratagene).
S1 Nuclease Protection Analyses and RNase Protection
Analyses
S1 nuclease protection analyses were performed as
described(19, 20) . Briefly, after a denaturation step
at 85 °C for 30 min, 20 µg of total RNA isolated by the
guanidinium isothiocyanate/phenol/chloroform extraction method (16) were hybridized at 52 °C for 16 h with 75,000 cpm
labeled NOS II DNA probe and 30,000 cpm labeled
-actin DNA probe
in hybridization buffer (40 mM Pipes, pH 6.4, 400 mM NaCl, 1 mM EDTA, 80% formamide) in a volume of 30 µl.
The S1 nuclease digestion was started by adding 310 µl of digesting
buffer (280 mM NaCl, 4.5 mM Zn(CH
COO
)
, pH 4.5, 30
µg/ml denatured salmon sperm DNA, and 300 units/ml S1 nuclease).
After 20 min at 37 °C, the reaction was stopped by adding 65 µl
of stop-buffer (2.5 M NH
-acetate, 50 mM EDTA), followed by a phenol/chloroform extraction. The reaction
products were concentrated by ethanol precipitation and analyzed by
electrophoresis in denaturing urea-polyacrylamide gels (8 M urea, 6% polyacrylamide gel electrophoresis). The electrophoresis
buffer was 1
TBE (1.08% Tris, pH 8.3, 0.55% boric acid, and 20
mM EDTA). The gels were electrophoresed for 2-3 h,
dried, and exposed to x-ray films. The protected DNA fragments of NOS
II and
-actin were 380 and 150 nucleotides, respectively. RNase
protection assays were performed with RNase ONE(TM) according to the
manufacturer's instructions (Promega). Briefly, following
denaturation, 20 µg of total RNA (prepared as described above) were
hybridized with 100,000 cpm labeled NOS II antisense RNA probe and
10,000 cpm labeled
-actin antisense RNA probe at 51 °C for 16
h in a volume of 30 µl. Then the mixture was digested with 5 units
of RNase ONE(TM) for 1 h at room temperature in 300 µl. The
reaction was stopped with 1% SDS, and the samples were concentrated and
electrophoresed as described for the S1 nuclease protection analysis.
The protected RNA fragments of NOS II and
-actin were 184 and 108
nucleotides, respectively.
Electrophoretic Mobility Shift Assay
(EMSA)
NF-
B binding activity in the nuclei of control 3T3
fibroblast- or RAW 264.7 cells, or cells incubated for 3 h with one of
the NOS II-inducing agents mentioned above were determined by EMSA
using the Promega gel shift assay system. Nuclear proteins were
extracted from the cells by detergent lysis(21) . Ten µg of
nuclear protein were incubated with 17.5 fmol of
P-labeled
double-stranded oligonucleotide containing a motif for NF-
B
binding (5`-AGTTGAGGGGACTTTCCCAGGC-3`). In some experiments, 1.75 pmol
of an oligonucleotide with the putative NF-
B binding sequence of
the murine NOS II promoter (5`-CAACTGGGGACTCTCCCTTTG-3`) were added.
The DNA-protein complexes were analyzed on 5% polyacrylamide gels
(electrophoresis buffer: 6.7 mM Tris/HCl, pH 7.5, 3.3 mM sodium acetate, 1 mM EDTA), dried, and autoradiographed.
Measurement of NO Production by
Chemiluminescence
Confluent 3T3 fibroblasts were cultured for 18
h in DMEM containing 10% fetal bovine serum. Control cells received no
additions to the medium; other cells were incubated with LPS (1
µg/ml), TNF-
(10 ng/ml), TPA (50 ng/ml), or forskolin (100
µM). After 18 h, the cell supernatants were collected and
aliquots were deproteinized with 2 volumes of ethanol. Following
centrifugation, 200 µl of the supernatant were injected into a
collection chamber containing 100 mM KI in 10 mM sulfuric acid. This strong reducing environment converts
NO
(and nitrosyl compounds) back to NO. A
constant stream of N
gas carried the NO into a nitric oxide
analyzer (Sievers, Boulder, CO) where the NO was reacted with ozone,
resulting in the emission of light. The light emission is proportional
to the NO formed; standard amounts of NO
were used for calibration.
RESULTS AND DISCUSSION
Stimulation of Different Second Messenger Pathways
Induced NOS II mRNA Expression
In 3T3 cells, NOS II mRNA was
markedly induced with IFN-
(100 units/ml) or TNF-
(10 ng/ml) (Fig. 1). NOS II expression was also enhanced with TPA (50
ng/ml) or the cAMP-elevating agents forskolin (100 µM) or
8-bromo-cAMP (1 mM) (Fig. 2). In contrast, 8-bromo-cGMP
(1 mM) was ineffective as a stimulator of NOS II induction (Fig. 2). The phosphodiesterase inhibitor IBMX (250
µM) also produced a marked induction of NOS II mRNA in 3T3
cells (Fig. 3). This can be explained by the increase in cAMP,
but not cGMP (cf. Fig. 2). LPS (up to 1 µg/ml)
showed little efficacy in inducing NOS II mRNA (Fig. 3). Thus
the stimulation of the receptor tyrosine kinase pathway (by INF-
,
TNF-
, and possibly LPS), the stimulation of the protein kinase C
pathway (by TPA), and the stimulation of the protein kinase A pathway
(by forskolin, 8-bromo-cAMP, and IBMX) all induced the transcription of
NOS II mRNA in 3T3 fibroblasts.
Figure 1:
Double S1 nuclease protection analysis
using cDNA probes for murine NOS II and
-actin (for
standardization). RNAs were prepared from untreated 3T3 fibroblasts
(control, Co) and 3T3 cells induced with human tumor necrosis
factor-
(10 ng/ml, TNF-
) or murine interferon-
(100 units/ml, IFN-
). T, tRNA control; M, molecular weight markers (pGl
-Basic, Promega,
restricted with HinfI). The gel is representative of three
experiments with similar results. Densitometric analysis demonstrated
that the NOS II signal in the TNF-
lane (after correction
by the
-actin signal) was 1033% of the NOS II signal in the
control (Co) lane, and the NOS II signal in the IFN-
lane was 440% of the NOS II signal in the control (Co)
lane.
Figure 2:
S1
nuclease protection analysis using cDNA probes for murine NOS II and
-actin (for standardization). RNAs were prepared from untreated
control 3T3 cells (Co) and cells stimulated with the following
agents: the protein kinase C stimulator tetradecanoylphorbol-13-acetate
(50 ng/ml, TPA), the adenylyl cyclase-stimulating agent
forskolin (100 µM, Forsk), 8-bromo-cAMP (1
mM, cAMP), or 8-bromo-cGMP (1 mM, cGMP). T, tRNA control; M, molecular weight
markers (pGl
-Basic restricted with HinfI). The gel
is representative of three experiments with similar results.
Densitometric analysis demonstrated that the NOS II signal in the TPA lane (after correction by the
-actin signal) was 460%
of the NOS II signal in the control (Co) lane; the NOS II
signal in the Forsk lane was 570% of the NOS II signal in the
control (Co) lane, and the NOS II signal in the cAMP lane was 1270% of the NOS II signal in the control (Co) lane.
The NOS II signal in the cGMP lane was not different (53%)
from the signal in the control (Co) lane
(100%).
Figure 3:
S1
nuclease protection analysis using cDNA probes for murine NOS II and
-actin (for standardization). RNAs were obtained from 3T3 cells
stimulated with various agents in the absence and presence of
dexamethasone (5 µM). The following stimulating agents
were used: bacterial lipopolysaccharide (1 µg/ml, LPS),
and LPS in the presence of dexamethasone (Dex);
tetradecanoylphorbol-13-acetate (50 ng/ml, TPA) and TPA in the
presence of dexamethasone (Dex); the phosphodiesterase
inhibitor isobutylmethylxanthine (250 µM, IBMX)
and IBMX in the presence of dexamethasone (Dex). T,
tRNA control; M, molecular weight markers
(pGl
-Basic restricted with HinfI). The gel is
representative of three experiments with similar results. Densitometric
analysis demonstrated that the NOS II signal in the LPS + Dex
lane (after correction by the
-actin signal) was 40% of the
NOS II signal in the LPS lane; the NOS II signal in the TPA + Dex lane was 28% of the NOS II signal in the TPA lane, and the NOS II signal in the IBMX + Dex
lane was 15% of the NOS II signal in the IBMX
lane.
Because double protected bands for
NOS II mRNA were seen in some of the S1 nuclease analyses, RNase
protection assays were performed on the same RNAs (using antisense RNA
probes derived from the same NOS II cDNA fragment). The RNase
protection assays yielded single protected bands and quantitatively
similar results as obtained in the S1 nuclease protection assays (data
not shown). Therefore, the double bands seen in the S1 assays are
unlikely to represent two different NOS II mRNAs.
Small amounts of
NOS II mRNA were detected even in the absence of cytokines or
stimulants (cf. Fig. 1and Fig. 2). The same
phenomenon has been described previously for murine 3T3 cells (5) and human DLD-1 epithelial cells(22) . It may
either represent a low constitutive expression of this isoform or an
autocrine/paracrine induction of these cells by endogenous
cytokines(22) .
In 3T3 cells, INF-
alone produced a
marked induction of NOS II mRNA (Fig. 1). There is controversy
as to whether INF-
alone can induce NOS II in RAW 264.7
macrophages. While this has been described by some authors(8) ,
others only see an effect of INF-
in the presence of
LPS(6, 7) . In the current experiments, polymyxin B
(10 µg/ml), an inhibitor of the induction of murine cells by
LPS(23) , did not prevent the NOS II-inducing action
of INF-
(100 units/ml), TNF-
(10 ng/ml), TPA (50 ng/ml), or
forskolin (100 µM) (n = 4, data not
shown), suggesting that INF-
alone is an effective NOS II inducer
in 3T3 cells.
The signal transduction pathways effective in inducing
NOS II expression vary considerably between cell types (and probably
species). In many cells, stimulation of the protein kinase C pathway
has little or no effect on NOS II induction by itself, but potentiates
cytokine induction(24, 25, 26) . In 3T3
fibroblasts, it is an efficacious inducing pathway by itself.
Stimulators of the protein kinase A pathway alone have been shown to
promote NOS II expression in vascular smooth muscle cells and rat
mesangial cells(12, 27, 28) . On the other
hand, protein kinase A activation seems to inhibit NOS II induction in
rat RINm5F insulinoma cells(26) . Thus the NOS II-inducing
mechanisms seem to be cell-specific, and the stimulation pattern
observed in the present study (Fig. 1Fig. 2Fig. 3)
is unique to 3T3 cells.
Stimulation of Different Second Messenger Pathways in 3T3
Cells Increased NOS II Protein Expression
Similar to the NOS II
mRNA expression induced by the various signal transduction pathways,
expression of NOS II immunoreactive protein was stimulated by TNF-
(10 ng/ml), LPS (1 µg/ml), TPA (50 ng/ml), or forskolin (100
µM) (Fig. 4). Noninduced 3T3 cells showed no NOS II
immunoreactivity in Western blots (n = 3, not shown).
Figure 4:
Western blot analysis of the soluble
(cytosolic) fraction from 3T3 fibroblasts. Protein samples were
prepared from 3T3 cells as described under ``Materials and
Methods,'' separated on SDS-polyacrylamide gels (7.5%), and
transferred to nitrocellulose membranes. A anti-NOS II antibody was
used for detection. 3T3 cells were induced with different agents before
protein was prepared: tumor necrosis factor-
(10 ng/ml, TNF-
), bacterial lipopolysaccharide (1 µg/ml, LPS), tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), or forskolin (100 µM, Forsk). The
blot is representative of three experiments with similar results. The
double (or triple) bands detected by the antibody probably correspond
to the double or triple bands seen by Stuehr et al.(54) for the purified NOS II protein from
macrophages.
Stimulation of Different Second Messenger Pathways in 3T3
Cells Enhanced NO
Production
Incubation of 3T3 fibroblasts with TNF-
,
TPA, or forskolin markedly enhanced the NO
content in the supernatant of the cells (Fig. 5). LPS was
a much weaker stimulant of 3T3 cell NO
production (Fig. 5). This indicates that NOS II protein and
activity is also induced via the receptor tyrosine kinase, protein
kinase C, and protein kinase A pathways.
Figure 5:
Chemiluminescence determination of
NO
in the supernatant of 3T3 cells as an
indicator of NO production. The cells were kept for 18 h in culture
medium alone (control, Co) or were incubated with
bacterial lipopolysaccharide (1 µg/ml, LPS), tumor
necrosis factor-
(10 ng/ml, TNF-
),
tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), or forskolin
(100 µM, Forsk).
Stimulation of Three Different Second Messenger Pathways
in 3T3 Cells Induced Proteins with NF-
B Binding
Activity
NF-
B is a multisubunit transcription factor that
can rapidly activate the expression of genes involved in immune and
acute phase responses(29) . NF-
B is composed mainly of
proteins with molecular weights of 50 kDa (p50) and 65 kDa (p65). Both
types of proteins share significant homology with the proto-oncogene
c-rel(30, 31, 32) . The proteins
p50, p65, and c-Rel can interact with each other and, following
activation, bind the NF-
B response element as homo- or
heterodimers (33) (consensus sequence:
GGGRNNYYCC)(34) . In its unstimulated form, NF-
B is
present in the cytosol bound to the inhibitory protein I-kB. After
induction of cells by a variety of agents, NF-
B is released from
I-kB and translocated to the nucleus. Agents that have been described
as NF-
B activators include mitogens, cytokines, and LPS, TPA, and
cAMP(29, 35) . EMSA experiments shown in Fig. 6demonstrated that nuclear extracts of untreated 3T3 cells
contained low concentrations of proteins that bind an oligonucleotide
containing the NF-
B response element. Incubation of 3T3 cells
either with TPA (50 ng/ml), TNF (10 ng/ml) or 8-bromo-cAMP (1
mM) markedly increased the NF-
B binding activity (Fig. 6). In 3T3 fibroblasts, TNF-
was the most efficacious
inducer of NF-
B binding activity tested. TPA and cAMP-elevating
agents (8-bromo-cAMP or forskolin) were less efficacious in inducing
NF-
B binding activity; there were no significant differences in
efficacy between these two classes of agents. This parallels the NOS II
mRNA and NOS II protein expression as well as the NOS activity
stimulated by these compounds. The protein-DNA interaction was totally
prevented in all cases with an excess of unlabeled double-stranded
oligonucleotide containing the NF-
B site of the murine NOS II
promoter ( Fig. 6and data not shown). These data suggest that,
in 3T3 cells, the receptor tyrosine kinase pathway, the protein kinase
A pathway, and the protein kinase C pathway stimulate the activation of
transcription factor NF-
B. While cytokines such as TNF-
can
activate NF-
B in most cell types, there is a marked inter-cell
variability for the protein kinase A and C pathways. For example, in
murine RAW264.7 cells, neither the protein kinase A pathway nor the
protein kinase C pathway are able to stimulate this transcription
factor; they even inhibit NF-
B-dependent reporter gene expression
in response to LPS(36) . In human Jurkat T cells, the protein
kinase C pathway, but not the protein kinase A pathway activates
NF-
B(37) . Conversely, in murine J774 macrophages,
activators of protein kinase A are effective stimulators of NF-
B,
whereas protein kinase C activators failed to stimulate this
transcription factor(38) .
Figure 6:
EMSA using a 5`-end-labeled consensus
oligonucleotide for NF-
B binding (O) and nuclear extracts
from 3T3 fibroblasts (3T3) and murine RAW 264.7 macrophages as
positive controls (RAW). 3T3 cells were incubated for 3 h with
medium alone (negative control, Co), tumor necrosis
factor-
(10 ng/ml, TNF-
),
tetradecanoylphorbol-13-acetate (50 ng/ml, TPA), or
8-bromo-cAMP (1 mM, cAMP). Densitometric analyses of
the gels demonstrated that TPA and 8-bromo-cAMP were about
equieffective in stimulation NF-
B binding activity, whereas
TNF-
induced the largest increase in NF-
B binding activity.
Nuclear extracts from LPS-induced RAW 264.7 macrophages are known to
contain NF-
B binding activity (9) and were used as
positive controls (RAW). RAW 264.7 macrophages were induced
with bacterial lipopolysaccharide (1 µg/ml, LPS), and the
same nuclear protein extract from RAW 264.7 cells was tested in the
presence of a 100-fold excess of an oligonucleotide containing the
NF-
B binding site of the murine NOS II promoter (competition
experiment, LPS comp.). The gels are representative of three
experiments yielding similar results.
Effect of Dexamethasone on NOS II mRNA
Expression
Glucocorticoids such as dexamethasone have been known
for some years to inhibit cytokine induction of NOS II activity in various cell types (such as endothelial cells, macrophages, and
smooth muscle
cells(39, 40, 41, 42) ). More
recently, this inhibition has also been demonstrated at the mRNA
level in several cell types(5, 43, 44) .
In a recent communication, Kunz et al.(45) demonstrated in rat mesangial cells that
dexamethasone prevented the induction of NOS II activity in
response to IL-1
and dibutyryl cAMP. Interestingly, NOS II mRNA levels were only reduced when dibutyryl cAMP was used as
the inducing agent, but not after IL-1
. Consequently, these
authors postulated that dexamethasone acts at different levels,
depending on the stimulus used to suppress NOS II induction in rat
mesangial cells(45) . In the current study we examined the
effect of dexamethasone (5 µM) on NOS II mRNA expression
in 3T3 cells. We found that the steroid was equally effective against
inductions produced by LPS, TPA or IBMX (Fig. 3). Also the NOS
II mRNA inductions in response to TNF-
(10 ng/ml) or INF-
(100 units/ml) were markedly inhibited by dexamethasone (5
µM) (n = 3, not shown).
Effect of TGF-
1 on NOS II mRNA
Expression
TGF-
1 is an inhibitor of NOS II induction in
mouse macrophages and rat vascular smooth muscle
cells(42, 43, 46, 47, 48) .
On the other hand, in 3T3 cells and in bovine retinal pigmented
epithelial cells, TGF-
1 has been described as a stimulator of
cytokine-induced NOS II mRNA induction (43, 49) . Also
in the current experiments, TGF-
1 (2 ng/ml) potentiated NOS II
mRNA production irrespective of the second messenger pathway used for
induction (Fig. 7).
Figure 7:
S1 nuclease protection analysis using cDNA
probes for murine NOS II and
-actin (for standardization). RNAs
were obtained from 3T3 cells stimulated with various agents in the
absence and presence of transforming growth factor-
1 (2 ng/ml, TGF-
1). TGF-
1 alone did not produced any NOS II
induction. The following stimulating agents were used: bacterial
lipopolysaccharide (1 µg/ml, LPS) and LPS in the presence
of TGF-
1; tetradecanoylphorbol-13-acetate (50 ng/ml, TPA)
and TPA in the presence of TGF-
1; the phosphodiesterase inhibitor
isobutylmethylxanthine (250 µM, IBMX) and IBMX in
the presence of TGF-
1. T, tRNA control; M,
molecular weight markers (pGl
-Basic restricted with HinfI). The gel is representative of three experiments with
similar results. Densitometric analysis demonstrated that the NOS II
signal in the LPS + TGF-
1 lane (after correction by
the
-actin signal) was 130% of the NOS II signal in the LPS
lane; the NOS II signal in the TPA + TGF-
1 lane was 230% of the NOS II signal in the TPA lane, and the NOS II
signal in the IBMX + TGF-
1 lane was 330% of the NOS
II signal in the IBMX lane.
Inhibition of NF-
B Activation Blocks NOS II mRNA
Induction
The activation of NF-
B can be blocked by thiol
compounds such as PDTC or diethyldithiocarbamate, which leave the DNA
binding activity of other transcription factors (e.g. SP1,
Oct, and CREB) unaffected(50) . PDTC or diethyldithiocarbamate
have been shown to prevent the induction of NOS II in LPS-induced
murine macrophages (9, 51) and rat alveolar
macrophages(52) . Eberhardt et al.(12) reported that PDTC inhibits the induction of NOS II
expression in response to IL-1
, but not to dibutyryl cAMP. They
concluded that in rat mesangial cells cAMP-stimulated NOS II expression
is activated through a transcription factor different from NF-
B.
In the current series of experiments in 3T3 cells, PDTC prevented the
induction of the NOS II mRNA expression in response to all inducing
compounds used (Fig. 8). Also DCI, a serine protease inhibitor,
which blocks NF-
B activation by inhibiting proteolytic degradation
of I-
B(53) , blocked (by over 90%) NOS II mRNA expression
induced by INF-
(100 units/ml), TNF-
(10 ng/ml), TPA (50
ng/ml), and forskolin (100 µM) (n = 3,
data not shown). This confirms the results of our EMSA experiments and
indicates that in 3T3 fibroblasts NF-
B is essential for NOS II
induction in response to different second messengers. Interestingly,
the inhibition of NOS II induction by dexamethasone (described above)
is likely to reflect its ability to inactivate NF-
B(20) .
Figure 8:
S1 nuclease protection analysis using cDNA
probes for murine NOS II and
-actin (for standardization). RNA
were obtained from unstimulated 3T3 fibroblasts (Co) and 3T3
cells stimulated with various agents in the absence and presence of
pyrrolidine dithiocarbamate (100 µM, PDTC). The
following stimulators were used: the adenylyl cyclase-stimulating agent
forskolin (100 µM, Forsk) and forskolin in the
presence of PDTC; interferon-
(100 units/ml, IFN-
)
and IFN-
in the presence of PDTC; tetradecanoylphorbol-13-acetate
(50 ng/ml, TPA) and TPA in the presence of PDTC. M, molecular
weight markers (pGl2-Basic restricted with HinfI). The gel is
representative of four experiments with similar results. Densitometric
analysis demonstrated that the NOS II signal In the Forsk + PDTC lane (after correction by the
-actin signal) was 28% of
the NOS II signal in the Forsk lane; the NOS II signal in the IFN-
+ PDTC lane was 5% of the NOS II signal in the IFN-
lane, and the NOS II signal in the TPA + PDTC lane was 12% of the NOS II signal in the TPA
lane.
In conclusion, our data demonstrate that in 3T3 cells at least three
different signal transduction pathways can stimulated NOS II mRNA
expression, namely the cytokine/receptor tyrosine kinase pathway, the
cAMP/protein kinase A pathway, and the protein kinase C pathway. All
these pathways seem to converge in the activation of the essential
transcription factor NF-
B, which increases the transcription of
the NOS II gene.