Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711
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ABSTRACT |
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Interleukin-1 (IL-1
), tumor necrosis
factor-
(TNF-
), and lipopolysaccharide (LPS) were examined for
their ability to regulate the activity and protein levels of inducible
nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle
cells. Treatment with these agents resulted in a time-dependent
increase in NOS II activity. After 48 h, NOS II activity, measured as
L-[3H]citrulline
production, was increased 24.3 ± 6.9 pmol · min
1 · mg
protein
1 by 1 nM IL-1
and 3.2 ± 1.1 pmol · min
1 · mg
protein
1 by 1 nM TNF-
,
and increased synergistically by a combination of the two (51.8 ± 7.3 pmol · min
1 · mg
protein
1). Measurement of
NOS II activity as nitrite production yielded similar results: IL-1
,
27.2 ± 1.2; TNF-
, 1.6 ± 0.1; and IL-1
+ TNF-
, 46.8 ± 3.2 pmol · min
1 · mg
protein
1 above basal. LPS
(10 µg/ml) had a small but significant effect at 48 h that was only
additive with that of IL-1
. The increase in NOS II activity induced
by IL-1
and TNF-
was inhibited 73-86% by transforming
growth factor-
1 (TGF-
1). The NOS isoform induced by IL-1
and
TNF-
was identified as NOS II by Western immunoblot analysis and
confirmed by its 66-97% inhibition by 100 µM
S-methylisothiourea, a selective NOS
II inhibitor, and its
Ca2+-independent activity. We
conclude that the cytokines IL-1
and TNF-
act independently and
synergistically to stimulate NOS II expression and enzymatic activity
in rat colonic smooth muscle through a mechanism sensitive to
inhibition by TGF-
1.
nitric oxide; inducible nitric oxide synthase; lipopolysaccharide; transforming growth factor-; interleukin-1
; tumor necrosis
factor-
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INTRODUCTION |
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THREE DISTINCT ISOFORMS of nitric oxide synthase (NOS I, II, and III), the enzyme responsible for production of nitric oxide (NO), have been identified (2, 19, 23). The NOS I and NOS III isoforms are constitutively expressed, and the expression of the NOS II isoform is inducible. NOS I is expressed by neurons of the central and peripheral nervous system (2) and by fast muscle fibers of skeletal muscle (12). Within the gastrointestinal tract, NOS I is expressed by nerves of the myenteric plexus (21). NOS III is expressed by endothelial cells (23), smooth muscle cells (26), epithelial cells of the bronchial tree (24), and pyramidal cells of the hippocampus (5). Within the gastrointestinal tract, NOS III is expressed by smooth muscle cells (26) and by the interstitial cells of Cajal (33). The activity of NOS I and NOS III is regulated by the level of cytosolic Ca2+. NOS II may be expressed in macrophages (8) as well as in a number of cell types throughout the body. Within the gastrointestinal tract NOS II expression can be induced in epithelial cells (27) and in cells of the muscularis propria (30). In contrast to NOS I and NOS III, NOS II, once expressed, is present in much greater amounts and is continuously active due to the tight binding of calmodulin even at basal levels of cytosolic Ca2+. These properties result in the production of much greater amounts of NO by NOS II, typically within the micromolar range, compared with NOS I and NOS III (3).
NOS I is found in nerves of the myenteric plexus, where NO acts as an inhibitory neurotransmitter and facilitates vasoactive intestinal polypeptide (VIP) release from nerve terminals (11). NOS III is found within the smooth muscle cells (11, 26), where it is activated by VIP and pituitary adenylate cyclase-activating polypeptide, leading to generation of NO and muscle relaxation (11, 16), and in the interstitial cells of Cajal (33) located at the boundary between muscle and nerve, which are responsible for electrical slow wave activity in the gastrointestinal tract. The role of NOS III in the interstitial cells of Cajal is not clear.
NOS II expression is induced in the intestine in ulcerative colitis and
Crohn's disease (1, 15). NOS II expression has been induced in in vivo
models of colitis in animals after treatment with agents such as
2,4,6-trinitrobenzenesulfonic acid, lipopolysaccharide (LPS), or
peptidoglycan-polysaccharide (10, 14, 27, 31). NOS II expression and
activity in these settings is induced in mucosal cells, tissue
macrophages, the myenteric plexus, and the muscularis propria. NOS II
induction results in muscle hyperplasia and decreased contractility,
effects that are inhibited by NOS inhibitors (10, 31). These studies
were performed in whole thicknesses of intestine or in muscle strips
where, in addition to muscle cells, other cell types such as epithelial
and inflammatory cells, neurons, and glia may contribute to the
observed events. NOS II expression can be induced directly in cultured
vascular, pulmonary, and uterine smooth muscle cells by exposure to the cytokines interleukin-1 (IL-1
) and tumor necrosis factor-
(TNF-
) or LPS (17-20). The ability of cytokines to
cause induction of NOS II expression in gastrointestinal smooth muscle
cells has not been demonstrated directly.
In the present study IL-1, TNF-
, and LPS from
Escherichia coli serotype O55:B5 were
examined for their ability to directly induce NOS II expression and NO
generation in smooth muscle cells cultured from the rat colon. The
results show that IL-1
, TNF-
, and LPS induce NOS II expression
and increase NO production by cultured colonic smooth muscle cells in a
time-dependent fashion. The effects of IL-1
and TNF-
are
synergistic. Cytokine-induced NOS II expression occurs through a
TGF-
1-sensitive mechanism.
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METHODS |
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Preparation of isolated muscle cells from rat colon. Muscle cells were isolated separately from the longitudinal and circular muscle layers of the colon of male Sprague-Dawley rats by adaptation of methods described previously (11, 13, 16). Briefly, the colon was cut into 2-cm segments, and the longitudinal layer was removed by tangential stroking. The segments were opened longitudinally, and the mucosa was removed by blunt dissection. The remaining circular muscle segments were incubated at 31°C in 25 ml of medium containing 0.15% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. The medium consisted of (in mM) 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 14 glucose, and 2.1% Eagle's essential amino acid mixture. After a 60-min incubation, the partially digested muscle strips were washed free of enzymes and incubated in enzyme-free medium for 30 min to allow the cells to disperse spontaneously.
Cell culture of rat colonic muscle cells. Primary cultures of rat colonic muscle cells were initiated and maintained by modification of previously described methods (13). Cells dispersed from the circular muscle layer were harvested by filtration through 500-µm Nitex mesh and then centrifuged at 150 g for 5 min. Cells were resuspended and washed twice by centrifugation at 150 g for 5 min and resuspension in Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamicin, and 2.5 µg/ml amphotericin B. After the final washing, cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (DMEM-10) and the same antibiotics. The cells were plated at a concentration of 5 × 105 cells/ml as determined by counting in a hemacytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 was replaced every 3 days until the cells reached confluence.
Primary cultures of colonic muscle cells were passaged on reaching confluence by first washing three times with phosphate-buffered saline (PBS). After the PBS was removed, cells were treated for 2 min with 0.05% trypsin and 0.53 mM EDTA. The trypsin activity was neutralized by addition of a fourfold excess of DMEM-10. The resulting cell suspension was centrifuged at 350 g for 10 min at 4°C. The pellet was washed twice by centrifugation at 350 g and resuspension in HBSS. The final cell pellet was resuspended in DMEM-10 at a concentration of 2.5 × 106 cells/ml and plated in either 100-mm dishes or 24- or six-well plates, depending on the experiment to be performed. The medium was changed after 24 h and then every 3 days thereafter. All subsequent studies were performed in cultured muscle cells in first passage on day 7, when cells attained confluence. Previous studies (13) have shown that muscle cells treated in this manner yield cultures of smooth muscle cells, with 97 ± 2% of cells expressing phenotypic markers characteristic of smooth muscle cells when examined by immunofluorescence. The cultures are essentially devoid of nonmuscle, i.e., neural, epithelial, and inflammatory cells.Induction of NOS activity.
Confluent muscle cell cultures were washed three times with PBS and
then incubated in phenol red-free DMEM-10 in the presence of 1 nM
IL-1, 1 nM TNF-
, and 10 µg/ml LPS alone and in combination for
various time periods of 0, 1, 2, 4, 6, 12, 24, or 48 h. The effect of
inhibitors was examined after a 1-h preincubation with inhibitor
followed by treatment with IL-1
, TNF-
, or LPS.
Measurement of NO formation. NOS activity was measured in cultures of muscle cells loaded with L-[3H]arginine and expressed as the amount of L-[3H]citrulline produced, by modification of previously described techniques (11, 16). L-[3H]citrulline and NO are produced in equimolar amounts by the action of NOS on L-[3H]arginine. Muscle cells growing in six-well dishes were treated with cytokines as described above. The medium was aspirated, and the cells were washed three times with 2 ml of HBSS. The cells were then incubated for 30 min at 37°C with L-arginine-free Earle's balanced salt solution (EBSS). This medium was aspirated, and the cells were incubated at 37°C for various time periods for up to 30 min in 0.75 ml EBSS containing 5 µM L-arginine and 2 µCi/ml L-[3H]arginine (sp act 44.2 Ci/mmol). The reaction was terminated, and the cells were extracted by addition of 0.75 ml of 10% tricholoracetic acid at 4°C. The extracts were centrifuged at 10,000 g for 15 min at 4°C, and 1-ml aliquots of the resulting supernatant were removed and extracted three times with 2 ml of water-saturated diethyl ether. Aliquots (0.5 ml) of the resulting aqueous phase were removed and neutralized by addition of 2 ml of 20 mM HEPES (pH 6) solution. L-[3H]citrulline was separated from unconverted L-[3H]arginine by flow-through chromatography in Dowex (AG50W-X8) columns. The amount of L-[3H]citrulline was taken to represent the stoichiometric production of NO. Results were expressed as the increase in NOS activity in picomoles L-[3H]citrulline per minute per milligram protein above basal levels in untreated control cultures.
In some experiments the Ca2+ dependence of the conversion of L-[3H]arginine to L-[3H]citrulline was investigated in cells incubated in Ca2+-free EBSS containing the chelating agent ethylene glycol-bis(Measurement of total nitrite production. NO was also measured from total nitrite formation with the use of the Greiss reaction, by modification of the techniques of Green et al. (9). Briefly, cultures of muscle cells growing in six-well plates were washed free of phenol red and incubated with cytokines as described above. At various time points the culture medium was aspirated and centrifuged at 10,000 g for 5 min to remove cellular debris. A 1-ml aliquot was removed and added to a 1-ml aliquot of Greiss reagent. Greiss reagent was prepared fresh daily by mixing equal volumes of a 0.1% solution of N-(1-naphthyl)ethylenediamine dihydrochloride and 1% solution of sulfanilamide in 5% phosphoric acid. Absorbance was measured at 545 nm, and nitrite concentration was calculated from a standard curve of sodium nitrite. The response was linear over the range 0.1 to 100 µM nitrite. The amount of nitrite was taken to represent the production of NO (9). Results were expressed as the increase in NOS activity in picomoles nitrite per minute per milligram protein above basal levels in untreated control cultures.
In some experiments, the effect of selective inhibitors of NOS activity, L-NAME (1 mM) and SMT (10 µM), on nitrite production was examined by addition of inhibitor 1 h before the addition of cytokine or LPS. Although inhibitors of NOS interfere with the analysis of nitrite by the Greiss reaction in some systems, no changes in the nitrite standard curve were observed in the presence of 1 mM L-NAME or 100 µM SMT in the present study.Western blotting of NOS II.
The expression of NOS II protein was examined by Western blot analysis
in cultured cells and in cells freshly dispersed from the circular
layer of rat colon. Cultured but not freshly dispersed cells were
treated for 48 h with 1 nM IL-1 alone and in combination with 1 nM
TNF-
. The freshly dispersed muscle cells or cultured muscle cells
growing in 100-mm plates were washed three times with ice-cold PBS, and
whole cell lysates were prepared by adding 1.5 ml of boiling sample
buffer to the cells. The resulting cell lysates were boiled for an
additional 5 min and then placed on ice. Samples were sonicated to
shear DNA and then centrifuged at 12,000 g at 4°C for 15 min. The resulting
supernatant was heated to 95°C for 5 min. Proteins were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on
4-12% gradient acrylamide gels in aliquots (20 µl) containing
equal amounts of protein (30 µg). The separated proteins were
electrotransferred to polyvinylidene difluoride membranes. After
transfer the membranes were incubated in blocking buffer consisting of
PBS, pH 7.6, and containing 10% milk protein for 1 h at room
temperature. The membranes were then incubated for 2 h at room
temperature in blocking solution containing a 1:1,000 dilution of a
polyclonal immunoglobulin G (IgG) antibody raised in rabbits to a
synthetic peptide derived from the COOH-terminal end of the murine
macrophage NOS, amino acids 1131-1144. This antibody reacts fully
with rat NOS II protein but has no cross-reactivity with either NOS I
or NOS III protein. Membranes were washed three times with PBS
containing 0.1% Tween 20 and 1% milk protein. Membranes were
incubated for 1 h in blocking buffer containing a 1:2,000 dilution of a
horseradish peroxidase-conjugated antibody to rabbit IgG raised in
goat. The membranes were washed an additional three times in PBS
containing 0.3% Tween 20 and 1% milk protein. The protein bands were
visualized by enhanced chemiluminescence.
Statistical and densitometric analysis. Values are means ± SE of n experiments, where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired values as appropriate. Densitometric analysis was performed using computerized densitometry and ImageQuant NT software (Molecular Dynamics). Densitometric values for protein bands were determined in areas of equal size and are reported in arbitrary units above background values.
Materials.
Recombinant human IL-1 and TNF-
and human TGF-
1 were obtained
from Collaborative Biomedical Products (Bedford, MA); horseradish peroxidase-conjugated goat anti-rabbit IgG was obtained from Amersham (Arlington Heights, IL); rabbit polyclonal antibody to inducible NOS
was obtained from Affinity Bioreagents (Neshanic Station, NJ);
L-[3H]arginine
(sp act 44.2 Ci/mmol) was obtained from DuPont-NEN (Boston, MA);
Bio-Rad protein reagent was obtained from Bio-Rad Laboratories
(Hercules, CA); DMEM, HBSS, and EBSS were obtained from Mediatech
(Herndon, VA); fetal bovine serum was obtained from BioWhittaker
(Walkersville, MD); collagenase (CLS type II) and soybean trypsin
inhibitor were obtained from Worthington Biochemicals (Freehold, NJ);
HEPES was obtained from Research Organics (Cleveland, OH); and culture
plasticware was obtained from Corning (Corning, NY). LPS from
E. coli serotype O55:B5,
cyclohexamide, and all other chemicals and reagents were obtained from
Sigma Chemical (St. Louis, MO).
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RESULTS |
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Stimulation of NOS activity by TNF-,
IL-1
, and LPS.
Confluent cultures of rat colonic muscle cells were treated for 0, 1, 2, 4, 6, 12, 24, and 48 h with TNF-
(1 nM), IL-1
(1 nM), or a
combination of both cytokines. Basal nitrite production in cultured
muscle cells remained constant in the absence of cytokines (range:
1.6 ± 0.6 to 4.1 ± 2.5 pmol
nitrite · min
1 · mg
protein
1). IL-1
caused
a time-dependent increase in NOS activity (Fig. 1A)
that became significant after 6 h (2.7 ± 1.1 pmol · min
1 · mg
protein
1 above basal;
P < 0.05) and was sustained for 48 h
(27.2 ± 1.6 pmol · min
1 · mg
protein
1 above basal;
P < 0.001). TNF-
caused a small
increase in NOS activity (Fig. 1A)
that became significant after 48-h incubation (1.6 ± 0.1 pmol · min
1 · mg
protein
1 above basal;
P < 0.01). Addition of a combination
of 1 nM IL-1
and 1 nM TNF-
caused a significant increase in NOS
activity over that observed with IL-1
alone (24 h: 31.6 ± 3.5 vs. 23.9 ± 2.5 pmol · min
1 · mg
protein
1 above basal;
P < 0.05; 48 h: 46.8 ± 3.2 vs.
27.2 ± 1.0 pmol · min
1 · mg
protein
1 above basal;
P < 0.005) (Figs.
1A and
2A). The
increase after 48 h was synergistic and was significantly greater than
the sum of the responses to IL-1
and TNF-
(sum: 28.4 ± 1.1 pmol · min
1 · mg
protein
1 above basal;
combination: 46.8 ± 3.2 pmol · min
1 · mg
protein
1 above basal;
P < 0.001). LPS (10 µg/ml) caused
a small increase in NOS activity after 48 h (2.6 ± 0.5 pmol · min
1 · mg
protein
1 above basal;
P < 0.01) (Fig.
2A). Unlike TNF-
, the combination of LPS and IL-1
was additive rather than synergistic after 48-h incubation (sum: 29.8 ± 1.2 pmol · min
1 · mg
protein
1 above basal;
combination: 31.3 ± 1.6 pmol · min
1 · mg
protein
1 above basal) (Fig.
2A).
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Inhibition of NOS activity by TGF-1.
In other cell types, including vascular and pulmonary smooth muscle,
TGF-
1 inhibits cytokine-stimulated NOS II activity (7, 8, 22).
Previous studies have shown that cultured intestinal smooth muscle
cells secrete TGF-
1 in a time-dependent fashion (13). Concentrations
of TGF-
1 are low early in culture and at confluence (after 7 days in
culture), increasing 10-fold in postconfluent cells (after 14 days in
culture). To determine whether TGF-
1 might inhibit NOS activity
stimulated by cytokines in these cells, confluent cultures were treated
for 48 h with 1 nM IL-1
, 1 nM TNF-
, or a combination of both
cytokines in the presence or absence of 1 nM TGF-
1. In the presence
of TGF-
1 the ability of IL-1
, TNF-
, or LPS to increase NOS
activity measured by nitrite production was significantly inhibited
(range: 75 ± 13% to 86 ± 6% inhibition) (Fig.
3).
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Characterization of NOS II protein by Western blot analysis.
The expression of NOS II in both freshly isolated and cultured muscle
cells was investigated further by Western blot analysis. The NOS II
specific antibody identified a protein band migrating with an apparent
molecular mass of ~130 kDa, the known molecular size of authentic rat
NOS II protein. In control experiments a band of ~130-kDa molecular
mass was identified by the NOS II specific antibody in cell lysates
derived from interferon-- and LPS-stimulated RAW264.7 macrophages,
which are known to express NOS II protein (data not shown). In freshly
isolated colonic smooth muscle cells, NOS II protein was essentially
undetectable (Fig.
4A).
NOS II protein was detectable in untreated confluent muscle cells in culture. Treatment with either 1 nM IL-1
or a combination of 1 nM
TNF-
and IL-1
caused an increase in the levels of NOS II protein
detected (Fig. 4A).
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Biochemical identification of the cytokine-stimulated NOS isoform.
Two biochemical approaches were used to identify the NOS isoform
stimulated by treatment of muscle cells with IL-1 and TNF-
. The
first entailed use of the NOS antagonists
L-NAME and SMT (25) and the
second used the characteristics of
Ca2+ dependence of NOS activity
(3).
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DISCUSSION |
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This study shows that IL-1, TNF-
, and LPS increase NOS II protein
levels and stimulate NOS II activity in cultured smooth muscle cells of
the rat colon. The effects of IL-1
and TNF-
on NOS II activity
were synergistic, whereas the effects of IL-1
and LPS were only
additive. The increase in NOS II activity stimulated by IL-1
and
TNF-
involved new protein synthesis and could be inhibited by
cyclohexamide. TGF-
1 inhibited the increase in NOS II activity
stimulated by IL-1
, TNF-
, and LPS.
The identity of the NOS II protein induced by IL-1, TNF-
, and LPS
was determined by Western blot analysis, and the increase in expression
was evaluated by densitometry. The identification of NOS II was
supported by the fact that NOS activity measured either as total
nitrite production or from the formation of
L-[3H]citrulline
was virtually abolished by the preferential NOS II inhibitor SMT and
was strongly inhibited by
L-NAME. NOS activity measured
from the formation of
L-[3H]citrulline
was also Ca2+ independent,
consistent with the known characteristics of NOS II.
The mechanism by which TGF-1 inhibited cytokine- and LPS-induced NOS
II activity was not examined in the present study. Similar inhibition
by TGF-
1 was also noted in other cell types, for example, smooth
muscle cell cultures of rat aortic (20) and pulmonary artery (7) and
cultures of RAW267.4 macrophages (8). In TGF-
1 null
[TGF-
1(
/
)] mice, basal unstimulated NOS II
activity and NO production were significantly higher in heart, kidney, and peritoneal macrophages than in control [TGF-
1(+/+)
and TGF-
1(+/
)] littermates (29). Inhibition of
cytokine-stimulated NOS II activity by TGF-
1 was attributed to three
general mechanisms: decreased NOS II mRNA stability, reduced NOS II
translation, and increased degradation of NOS II protein (7, 20, 29,
32). The effects of TGF-
1 on colonic smooth muscle likely represent
similar processes.
Although NOS II protein expression could not be detected in freshly
isolated muscle cells from the rat colon, NOS II protein was detectable
in cells after growth in culture. This may be attributed to the
presence of stimulatory cytokines in the serum used in the culture
medium. The expression of NOS II in the cultured muscle cells could
also be affected by the ability of intestinal smooth muscle cells in
culture to produce TGF-1. Our previous studies have shown that
TGF-
1 production by rapidly proliferating smooth muscle cells is
low, increases twofold in confluent cultures (1 wk in culture), and
increases 10-fold in postconfluent cultures (2 wk in culture) (13). The
spontaneous increase in NOS II expression observed in the current study
therefore occurred during a period of low but increasing endogenous
TGF-
1 production. This suggests that the level of NOS II expression
represents the sum of stimulatory (cytokines such as IL-1
and
TNF-
) and inhibitory (TGF-
1) stimuli whereby endogenous TGF-
1
may have acted to attenuate the expression of NOS II.
Several mechanisms may contribute to the synergy between IL-1 and
TNF-
on NOS II expression: activation of cell surface receptors and
generation of second messengers and/or endocytosis of the
cytokine and activation of nuclear receptors. Both mechanisms could
affect transcriptional regulation of the NOS II gene in smooth muscle
cells. IL-1
receptors on the cell surface are coupled to generation
of adenosine 3',5'-cyclic monophosphate (cAMP), and two
copies of the cAMP response element have been delineated in the rat NOS
II promotor region (6). TNF-
also interacts with cell surface
receptors, which can generate a variety of intracellular second
messengers, including cyclic nucleotides and prostaglandins. Activation
of nuclear receptors after receptor-mediated endocytosis of cytokines
has been identified in a variety of cells (4). The rat NOS II gene has
sites within its promotor region that are putative IL-1
-responsive
sites [two nuclear factor-
B (NF-
B) binding sites and two
TNF-
response elements] (6). The potential for activation of
multiple regions of the rat NOS II promoter by IL-1
and TNF-
may
explain the synergistic effects of these cytokines in the present
study. Similarly, the LPS-mediated effects on transcriptional
regulation of the rat NOS II gene promoter have been shown to rely on
the presence of NF-
B binding motifs in the promoter region, the same
two NF-
B sites in the promoter region with which IL-1
interacts
(6, 32). Translational and posttranslational mechanisms likely also
play a role in the regulation of NOS II expression, as seen with
TGF-
1 (7, 20, 29, 32).
Inflammatory bowel disease in humans is associated with increased NOS
II expression in the muscular layers (1, 15). Enterocolitis experimentally induced in rats by agents such as
2,4,6-trinitrobenzenesulfonic acid, acetic acid, and LPS or by
intestinal parasites is associated with the release of cytokines and is
accompanied by an increase in NOS II expression in various tissue
layers, including muscle (10, 14, 27, 30, 31). The present study
corroborates these findings and demonstrates the ability of two
cytokines, IL-1 and TNF-
, to act synergistically in inducing NOS
II expression directly in smooth muscle cells. Conceivably in colitis
the various cytokines that are released could also act synergistically
to increase NOS II expression. Inhibitory factors such as TGF-
1 could act to attenuate such expression.
In summary, the cytokines IL-1 and TNF-
act independently and
synergistically to stimulate NOS II expression and enzymatic activity
in rat colonic smooth muscle through a mechanism sensitive to
inhibition by TGF-
1. Similar mechanisms may be involved in inflammatory bowel disease, where the level of NOS II expression might
be the net effect of both stimulation and inhibition by various
cytokines produced during inflammation.
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ACKNOWLEDGEMENTS |
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I thank Toni L. Bushman for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49691.
Address reprint requests to PO Box 980711, Division of Gastroenterology, Virginia Commonwealth Univ./MCV, Richmond, VA 23298-0711.
Received 5 March 1997; accepted in final form 13 October 1997.
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