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Departments of
* Pathology and
Pediatrics, Children’s Memorial Medical Center, Northwestern University Medical School, Chicago, Illinois 60614, USA
1Correspondence: Dept. of Pathology, Children’s Memorial Hospital, 2300 Children’s Plaza, Chicago, IL 60614, USA. E-mail: w-hsueh{at}nwu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. We examined a new mechanism of
intestinal iNOS regulation with respect to the role of nNOS and its
effect on NF-B. Young Sprague-Dawley rats were treated for 4 days
with 1) saline, 2) 7-nitroindazole (7-NI,
specific nNOS inhibitor), 3) 7-NI + pyrrolidine
dithiocarbamate (PDTC, NF-B inhibitor), or 4) PDTC.
Intestinal iNOS mRNA, NF-B activity, and the tissue content of the
regulatory IB
were examined. We found that 7-NI-treated animals
had higher intestinal NF-B (p50-p65) activity, lower IB
content, and increased intestinal iNOS mRNA, iNOS protein, and iNOS
activity compared with controls. All of these changes were abolished
when PDTC was given together with 7-NI. PDTC alone had no effect. 7-NI
induces a delayed increase in intestinal myeloperoxidase activity
(after elevation in NF-B and iNOS), which could be abrogated by
PDTC. We conclude that in normal rat small intestine, nNOS suppresses
the gene expression of iNOS through NF-B down-regulation and that
nNOS suppression leads to IB degradation, NF-B activation, and
iNOS expression.—Qu, X.-w., Wang, H., De Plaen, I. G., Rozenfeld, R.
A., Hsueh, W. Neuronal nitric oxide synthase (NOS) regulates the
expression of inducible NOS in rat small intestine via modulation of
nuclear factor kappa B.
Key Words: NO • gene regulation • transcription factor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2
). These isoforms,
although structurally related, differ from each other in their genetic
origin, anatomical distribution, ion dependence for activity, and
pathophysiological functions (1)
. nNOS and eNOS are
constitutively present in many cells and tissues, whereas iNOS is
usually not expressed except in the inflammatory state
(2)
. In the intestine, the constitutive NOS may have
important physiological functions such as maintaining the blood flow
(3
, 4)
and motility (5)
. The function of iNOS
in the gut is more complex. It is generally believed that iNOS is
involved in the defense mechanism against bacteria (2)
and
may mediate the inflammatory response (2)
when induced. We
have previously reported that although all 3 types of NOS are
constitutively present in rat small intestine, the predominant form
(>90%) is nNOS (6
, 7)
. Unlike most organs where no iNOS
is found in unstimulated conditions, normal intestine contains a low
level of iNOS that is ‘constitutively’ expressed; both iNOS protein
and iNOS activity can be detected, mostly in epithelial cells (8
, 7)
, although its contribution to the total tissue NOS activity
is usually less than 5% (7)
. The expression of iNOS in
many cells and tissues can be rapidly up-regulated after challenge with
bacterial lipopolysaccharide (LPS) and proinflammatory cytokines such
as interleukin 1, tumor necrosis factor (TNF-), or interferon
(IFN) (9)
. This regulation is presumably via the
activation of NF-B, a transcription factor involved in the
regulation of many proinflammatory cytokines and enzymes (reviewed in
ref 10
).
The transcription factor NF-B plays an important role in
inflammation by regulating the transcription of proinflammatory
cytokines (10)
, adhesion molecules (10)
, and
proinflammatory enzymes such as iNOS (10)
. NF-B is
constitutively active at a low level in some cells but most of the
NF-B is cytoplasmic, in an inactive state, and linked to a potent
inhibitor, IB (10)
. Upon stimulation by cytokines or
reactive oxygen species, NF-B dissociates from its inhibitor and is
translocated into the nucleus. There it binds specifically to the B
sites and initiates the transcription (10)
. NF-B
consists of homo- or heterodimers of subunits p50, p52, p65, RelB, and
c-Rel (10)
. The essential role of NF-B in iNOS
expression is suggested by the demonstration that deletion of the B
binding sites in the promoter region of iNOS renders the gene
unresponsive to cytokine stimulation (11)
. Due to the
potent proinflammatory role of iNOS, it is reasonable to assume that
iNOS is under tight control and is kept suppressed under normal
circumstances. However, its regulation in the physiological state is
poorly understood. It has been shown that exogenously applied NO
inhibits NF-B as well as expression of iNOS in human microglial
cells (12)
. In the present study, we examined the
hypothesis that in normal intestine, nNOS (the predominant NOS)
down-regulates iNOS and, conversely, nNOS inhibition induces iNOS
expression. We investigated the mechanism of this regulation with
respect to the role of transcription factor NF-B and its inhibitor,
IB.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 3) 7-NI +
pyrroldine dithiocarbamate (PDTC, an inhibitor of NF-B activation;
Sigma, St. Louis, Mo., 100 mg/ml in saline), 25 mg/kg, twice daily,
i.p. (14)
, and 4) PDTC only (25 mg/kg, twice
daily, i.p.). (Since preliminary experiments showed that DMSO, the
vehicle for 7-NI, has no effect on the parameters measured compared
with saline controls, only the saline control group is presented.) Each
animal was weighed at the time of injection (day 0) and
killed. At 1–4 day after the first drug injection, the animals were killed by an overdose of sodium pentobarbital and heart puncture. The procedure is in full compliance with the guidelines of the Institutional Animal Care and Use Committee. The small intestines were removed, rinsed with ice-cold saline containing dithiothreitol (DTT, 5.0 mM), minced with scissors, snap-frozen in liquid nitrogen, and stored in a -80°C freezer. Blood samples (0.5 ml/animal) were collected from some of the animals through a pyrogen-free catheter surgically implanted in the carotid artery and placed into sterilized, pyrogen-free centrifuge tubes (each containing 20 U heparin) before death. Plasma was then prepared from these blood samples by centrifugation at 15,000 x g for 5 min and stored in a -80°C freezer.
Determination of intestinal nuclear NF-B-DNA binding activity
The nuclear protein was extracted from intestinal tissue
following a published procedure (15)
. In short, the frozen
tissue sample was ground into fine powder with mortar/pestle in liquid
nitrogen and suspended in a ‘cushion buffer’ [10 mM HEPES containing
0.6% Nonidet-40, 150 mM NaCl, 1.0 mM EDTA, 0.5 mM phenylmethylsulfonyl
fluoride (PMSF), pH 7.9]. The suspension was then homogenized and
centrifuged at 1000 g, 4°C, for 2 min to remove large
tissue debris. Cell nuclei were harvested by centrifuging the
supernatant at 5000 g, 4°C, for 5 min and nuclear protein
was extracted with a hypertonic ‘lysing buffer’ [20 mM HEPES,
containing 25% (v/v) glycerol, 420 mM NaCl, 1.2 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and
leupeptin, aprotinin both at 0.5 µg/ml], followed by centrifugation.
The protein content of the nuclear extract was determined using a total
protein quantification kit (Sigma). The samples were stored in a
-80°C freezer before use.
The NF-B-DNA binding activity of the nuclear protein extract was
determined by measuring its binding to the
[32P]-labeled consensus oligo-polypeptide using
the polyacrylamide electrophoresis mobility shift assay (EMSA) as
described (16)
. Subunits forming the NF-B complex were
identified by a supershift procedure using mAb against the p65 or p50
subunit of NF-B (Santa Cruz Biotechnology, Santa Cruz, Calif.), as
described previously (16)
.
RNA extraction and reverse transcription-polymerase chain reaction
(RT-PCR)
Total RNA was extracted from intestinal tissue by
homogenizing the samples in a RNA-extracting agent, STAT-60 (TEL-TEST,
Friendswood, Tex.), together with chloroform treatment and isopropanol
precipitation, following the manufacturer’s instructions. The
extracted RNA was reversely transcribed at 37°C by using random
hexamer (pdN6) and Moloney murine leukemia virus reverse transcriptase
(Gibco-BRL, Grand Island, N.Y.). cDNA was amplified by PCR with
AmpliTaq DNA polymerase and specific primers flanking the corresponding
gene segments. The sequence of primers and the PCR thermocycle profiles
are as reported previously (17)
. The PCR products were
resolved by agarose gel electrophoresis and visualized by staining with
SYBR Green-1 (Molecular Probe, Eugene, Oreg.). iNOS transcription was
analyzed by semiquantitative RT-PCR. The fluorescence of the PCR
products of iNOS and ß-actin amplified from the same RT reaction was
analyzed using a STORM-860 Phosphor-imager; the iNOS/ß-actin ratio
was used as an index for the level of iNOS transcription for data
analysis.
Assay of iNOS activity
The activity of iNOS was determined by a
[14C] L-arginine conversion method, as
published (7)
. The iNOS activity is defined as the
difference between the EDTA containing reaction system and the reaction
system containing both EDTA and nonspecific NOS inhibitor
NG-monomethyl-L-arginine (CalBiochem) at 50 mM.
The protein content of the sample was determined using a Sigma total
protein kit.
Detection of iNOS and IB protein by immunoprecipitation and
Western blotting
Intestinal tissue lysate was prepared (18)
, and the
protein concentration was determined. After precleaning with protein-A
agarose, 1.0 ml tissue lysate (0.25 mg protein) was incubated with 30
µl anti-NOS mAb M-19 (Santa Cruz Biotechnology) at 4°C with gentle
shaking for 3 h, followed by incubation with excess (40 µl)
protein-A agarose beads for 2 h at 4°C. The bound immune complex
was eluted with Laemmli sample buffer (Bio-Rad Laboratories,
Hercules, Calif.) and boiling. After centrifugation, the supernatant
was loaded on a 7.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis for electrophoresis resolution. For the detection of
IB, tissue lysate/Laemmli buffer mixture was boiled and loaded
for electrophoresis. The resolved protein was transferred to a
nitrocellulose membrane, and the blot was detected with anti-iNOS mAb
M-19 or anti-IB mAb C-21 (Santa Cruz Biotechnology), horseradish
peroxidase-labeled goat anti-rabbit IgG antibody, and visualized with
an ECL system from Amersham.
Determination of intestinal myeloperoxidase activity and
histological examination
Myeloperoxidase (MPO, a marker enzyme for neutrophils) assay was
performed according to published methods (19)
. Briefly,
intestinal tissues were homogenized in 0.05 M potassium phosphate
buffer containing 0.5% hexadecyltrimethyl-ammonium bromide and EDTA (5
mM), and sonicated. An aliquot was mixed with substrate
(O-dianisidine HCl +
H2O2 in potassium phosphate
buffer) and the OD read at 460 nm. A standard curve was constructed
with serial dilutions of human MPO (Sigma).
Histological examination of the small intestine
Histological examination of the small intestine was performed to
assess intestinal injury and inflammation. Since no gross injury was
observed in the intestine in all animals receiving 7-NI, multiple
random section were removed, fixed in 10% formalin and processed for
microscopic examination. In addition, random sections were taken from
the liver, spleen, kidneys, heart, and lungs from 7-NI-treated animals
and processed for histology.
Determination of plasma LPS levels
The plasma level of bacterial LPS was determined using a
quantitative chromogenic version of the Limulus amebocyte lysate (LAL)
assay (20)
. The assay was performed using a LAL kit
(BioWhittaker Inc., Walkerville, Md.) and the procedure described in
the users’ manual from the manufacturer. Plasma samples were diluted
with LPS-free water at the ratio 1:9, and heated in 70°C water bath
for 10 min before the assay in order to suppress the endogenous
inhibitor of the LAL reaction.
Statistical analysis
One-way analysis of variance (with Bonferroni method used for
the post tests) was employed to analyze the data of multiple groups.
Tow-sided Student’s t test was used for the comparison of
any two single groups. Data are presented as mean ±
SE. The difference between groups was considered
significant when P<0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B
activation in small intestine
, 21)
(Fig. 1
, upper panel). As early as 1 day after the first injection, 7-NI
treatment significantly increased both the intestinal iNOS mRNA and
iNOS protein content, as revealed by RT-PCR and Western blot (Fig. 1)
.
The elevation in the iNOS protein content reached a peak at 4 days
after 7-NI treatment, with the protein content almost fivefold of that
of the controls.
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Parallel to the elevation in iNOS expression, there was an
up-regulation of the NF-B activity. NF-B is constitutively active
at a low level in rat small intestine as previously reported
(16)
. 7-NI treatment resulted in a significant enhancement
of the intestinal NF-B activity as soon as 1 day after the drug
administration (Fig. 2
, upper panel). Activation of NF-B was sustained throughout the
entire period (4 days) of 7-NI treatment. Supershift assays show that
the activated NF-B include both p65 and p50 subunits, probably as
both p50-p65 heterodimers and p50-p50 homodimers, since the NF-B
band was supershifted by anti-p50 mAb almost totally and by anti-p65
mAb partially (Fig. 2
, lower panel).
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PDTC suppressed 7-NI-induced NF-B activation and iNOS
expression in rat small intestine
PDTC, a NF-B inhibitor, was given to the animal together
with 7-NI in order to examine whether up-regulation of NF-B in the
intestine is causally related to iNOS expression. EMSA showed that
intestinal NF-B activity in animals treated with 7-NI plus PDTC was
much lower than that in animals receiving 7-NI alone (Fig. 3
, upper panel). PDTC also prevented 7-NI-induced iNOS expression
(Fig. 4
, upper panel), iNOS protein content (Fig. 3
, lower panel), and enzyme
activity (Fig. 4
, lower panel). PDTC itself showed no effect on either
intestinal NF-B activity (Fig. 3
, upper panel), intestinal iNOS gene
expression (Fig. 4
, upper panel), protein content (Fig. 3
, lower
panel), or enzyme activity (Fig. 4
, lower panel), compared with
controls (Fig. 3
, lower panel, and Fig. 4
).
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7-NI injection reduced the tissue content of IB in
the small intestine, which is reversed by PDTC
We examined the effect of 7-NI on the tissue content of IB,
which binds to NF-B and prevents its nuclear translocation. As shown
in Fig. 5
, animals that received 7-NI for 4 days had markedly lower intestinal
IB, which was reversed by the treatment with PDTC together with
7-NI. No significant difference was detected between the animals
treated with PDTC alone and controls.
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7-NI induces a delayed neutrophil sequestration in the small
intestine that is abrogated by PDTC; increase in MPO was much slower
than that of NF-B and iNOS expression
To rule out the possibility that increased iNOS expression in the
intestine was a result of an inflammatory response due to 7-NI
administration, we examined the change of intestinal MPO activity (an
index of neutrophil sequestration and inflammation) after 7-NI
injection. As shown in Fig. 6
, lower panel, animals that received 7-NI for 4 days had a marked
increase in intestinal MPO, which was abrogated by treatment with PDTC.
No significant difference was detected between the animals treated with
PDTC alone and sham controls. In contrast to intestinal NF-B and
iNOS expression (Figs. 1
and 2)
, which showed a significant elevation
on the first day after 7-NI administration, the rise in MPO activity
after 7-NI was much slower, showing virtually no change from baseline
on the first day and a barely 20% increase on the second day (Fig. 6
,
upper panel).
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No gross injury of the intestine was observed in any of the animals receiving 7-NI. Microscopic examination showed that on day 4, only 2 of 5 animals had very mild histological injury with focal loss of epithelial cells at the villous tip (not shown). Postmortem examination showed no pathology in other organs. The intestinal histology at 1 day after 7-NI is entirely unremarkable (n=6). There is no evidence of inflammation or neutrophil infiltration in any of the samples, suggesting that the increased MPO activity on day 4 was probably due to increased leukocyte rolling, margination, and adhesion rather than leukocyte infiltration of the tissue.
All animals tolerated 7-NI well. There were no signs of toxicity such as weight loss, diarrhea, or loss of activity for up to 4 days after 7-NI injection.
Plasma LPS levels are not elevated after 7-NI administration
LPS is a potent inducer of iNOS. It is possible that 7-NI induces
an inflammatory response in the intestine that results in mucosal
barrier breakdown, leading to bacterial translocation and LPS release.
To rule out the possibility that the increased iNOS expression is due
to bacteria/LPS entry, we examined the LPS level in the plasma in the
early stage after 7-NI treatment. As shown in Fig. 7
, there is no elevation in plasma LPS after 7-NI injection compared with
sham operated animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 4)
, preventing
leukocyte-endothelial adhesion (22)
, and protection
against injury (23
, 7)
. Our previous studies reveal nNOS
to be the predominant (>90%) cNOS in the intestine (6)
.
The main function of nNOS in the intestine is generally believed to be
mediation of the neuronal signal transmission in the NANC components of
the nervous system (24)
and regulating gut motility
(25
, 26)
. Since eNOS accounts for only a very small part
of intestinal NOS activity (6)
, it is possible that nNOS
in the intestine also functions as the protective eNOS. This is
suggested by our previous work showing an inverse relationship between
the degree of gut injury and nNOS activity after PAF injection
(7)
. The present study suggests another physiological
function of nNOS, i.e., to keep iNOS suppressed in the normal
condition, via the production of NO.
Nitric oxide has diverse physiological functions in the body. In
addition to its well-documented roles in the regulation of vasculature
tone, neurotransmission, and immune self-defense against microbes and
tumor cells (1
, 2)
, a growing number of studies have
recently implicated it in the regulation of gene expression,
particularly the expression of some adhesion molecules
(27)
and iNOS (28
, 29)
. The present study
adds one more role, i.e., gene regulation, to the vast and diverse
functions of this molecule. Although there has been some evidence
showing that NO from nNOS regulates iNOS expression in cultured
astrocytes (29)
, our study is the first to demonstrate its
role in gene regulation in vivo.
Our data suggest that, in the intestine, NO produced by nNOS
down-regulates iNOS via the suppression of NF-B activation. This is
supported by our findings that nNOS inhibition by 7-NI induces iNOS
expression and up-regulates NF-B activity, and that inhibition of
NF-B abolishes the effect of 7-NI. The detailed mechanism of this
regulation is unclear. However, it is unlikely that the iNOS induction
is a result of inflammatory response due to the injection of 7-NI. This
is supported by the following data. 1) The observed
activation of NF-B and iNOS expression preceded the inflammatory
response (indicated by the delayed neutrophil sequestration into the
intestine). 2) PDTC suppressed the 7-NI induced inflammatory
response, indicating that the development of the inflammatory response
is dependent on NF-B-dependent molecules such as proinflammatory
cytokines and adhesion molecules. In other words, inflammation is
consequent upon, but not preceding, NF-B activation. 3)
There is no elevation in plasma LPS levels after 7-NI injection,
indicating lack of bacteria/LPS translocation. 4)
Histological examination of the intestine showed no intestinal injury
on the first day after 7-NI injection. Although there was no sign of
intestinal injury or inflammation before iNOS change, preliminary
experiments showed that intestinal blood flow dropped to 
60% of
baseline level on the first day (data not shown). This degree of drop
in splanchnic flow is not necessarily pathological; it can be observed
in physiological conditions such as strenuous exercise (30
, 31)
. However, the possibility exists that the decreased flow
could result in change of redox status of the intestine, which may in
turn affect NF-B activity. An alternative explanation is that the
up-regulation of NF-B and subsequent iNOS gene expression after 7-NI
injection could be a direct effect of decreased NO. A similar
observation has been made in vitro in astrocyte culture.
Togashi et al. demonstrated that inhibition of NO production by
endogenous nNOS induces the activation of NF-B and subsequent iNOS
expression in these cells (29)
. It has also been
demonstrated that NO regulates iNOS expression by inhibiting the
binding of NF-B to DNA (32)
. That iNOS expression is
modulated by nNOS may be of special significance in organs such as
brain and small intestine, where nNOS is the predominant NOS form
(7)
.
Activation of NF-B is controlled by its cytoplasmic inhibitor,
IB, which binds NF-B and masks its nuclear translocation signal,
thus retaining it in the cytoplasm (33
, 34)
. Challenging
cells with cytokines or reactive oxygen radicals causes the
phosphorylation-ubiquitination and subsequent degradation of IB,
thus leading to the release of NF-B for its translocation to the
nucleus (35)
. In the present study, we showed that
modulation of IB is at least a part of the mechanism(s) whereby
NO regulates NF-B activity and iNOS expression in rat small
intestine. A similar observation has been made in endothelial cells
that exogenously applied NO inhibits NF-B (p50-p65) activation,
primarily through reducing the degradation or increasing the
transcription of IB (36)
. Further, NO has been
reported to inhibit NF-B-DNA binding affinity through
S-nitrosylation of the cysteine 62 residue of p50 (37)
.
The hypothetical model that nNOS suppresses iNOS expression through NO
modulation of NF-B activation makes good teleological sense. It is
generally believed that although a low level of NO is needed for
maintaining physiological function and cytoprotection, a large amount
of NO may be cytotoxic (38)
, especially in the presence of
ROS, when the toxic peroxynitrite is formed (39
, 40)
.
Hence NO production must be placed under stringent control. Under
normal conditions, iNOS (which produces massive amount of NO) is
unnecessary and should be contained. The NO-mediated suppression of
NF-B activity and subsequent down-regulation of the expression of
iNOS and other proinflammatory cytokines would serve this purpose very
well.
However, in a pathological situation such as inflammation, the demand
for NO production is changed. To initiate a successive inflammatory
response, the level of NO production has to be brought down first so
that leukocyte-endothelial adhesion, which is repressed by NO, may take
place (41
, 42)
. Indeed, many inflammatory mediators, such
as LPS, TNF, and IFN-, have been found to suppress the expression
(43
, 44)
or the activity (12
, 45
, 46)
of
cNOS. Our previous study also demonstrated that PAF, a potent
inflammatory mediator in the gut, reduced intestinal nNOS activity to
half of normal within 1 h (6)
. Such an inhibition of
cNOS activity would reduce the NO-dependent suppression of NF-B,
leading to NF-B-mediated transcription of proinflammatory cytokines
and adhesion molecules (27
, 47
48
49
50)
to facilitate the
inflammatory response. However, after successful mobilization of
leukocytes to the inflamed site, a large quantity of NO would soon be
needed for the bacteriocidal action (51
, 52)
. Thus, iNOS
would have to be ‘switched on’. The above-described cNOS-dependent,
NF-B-mediated regulation of iNOS expression could help to accomplish
this purpose.
Our results also suggest the existence of a NO-mediated negative
feedback loop that may have a profound effect on the regulation of the
inflammatory process, and cNOS and iNOS might ‘switch’ roles during
inflammation in order to meet the changed need of NO production.
Firstly, NO from iNOS would function to reduce the recruitment of
leukocytes into the inflamed region as well as to suppress free radical
production (53)
. Furthermore, NO could repress
NF-B-dependent transcription of iNOS and other pro-inflammatory
cytokines, thus helping to dissipate the inflammatory response. The
role of iNOS in the dissipation of inflammation is suggested by the
observed iNOS-dependent inhibition of adhesion molecule expression
(54)
and the exaggerated tissue damage in experimental
colitis in iNOS-deficient mice (55)
. Eventually, as iNOS
is suppressed, cNOS would resume the normal expression and activity,
and normalcy would be restored.
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ACKNOWLEDGMENTS |
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Received for publication March 22, 2000.
Revision received June 13, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B binding to DNA. Biochem. J. 322,609-613
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