© 1997 American Heart Association, Inc.
Articles |
Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes
From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo (Japan).
Correspondence to Koh-ichiro Kinugawa, MD, The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7 to 3–1 Hongo, Bunkyo-ku, Tokyo, 113, Japan. E-mail kkinugawa{at}medsfgh.ucsf.edu
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Abstract Previous work has demonstrated that inducible NO synthase (iNOS) can be expressed in cardiac myocytes. In this study, we investigated transcriptional regulation of the iNOS gene in these cells. Lipopolysaccharide (LPS) induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. H-89, dexamethasone, herbimycin, genistein, staurosporine, or pyrrolidine dithiocarbamate (PDTC) attenuated the iNOS induction by LPS. Forskolin, interleukin (IL)-6, tumor necrosis factor (TNF)-
, or interferon (IFN)-
enhanced the
LPS-induced iNOS expression. Combined stimulation of IL-6 and TNF-
also induced iNOS. The 5'-upstream sequence of the rat iNOS gene
contains the nuclear factor-
B (NF-B) site, CAAT box, IFN-
activation site (GAS), and IFN regulatory factor (IRF) site. DNase I
footprinting assay revealed that the nuclear factors binding to these
elements were increased by LPS exposure. Transient transfection assay
suggested that these elements were indispensable for transcriptional
regulation of the iNOS induction. Electrophoretic mobility shift assay
revealed that LPS or TNF- increased binding activity for the NF-B
site. A slower-migrating complex binding to the CAAT box gave rise
after exposure to LPS or forskolin. Competition assay suggested that
this slower-migrating complex consisted of a heterodimer between a
member of CAAT box/enhancer binding (C/EBP) protein family and cAMP
responsive element binding protein (CREB). LPS or IL-6 increased
binding complexes for the IRF site, which was compatible with induction
of IRF-1. LPS, IL-6, or IFN- induced a novel binding complex for
GAS, which also existed in the 5'-flanking region of the IRF-1 gene.
These data suggest that (1) iNOS induction simultaneously
requires both NF-B activation and IRF-1 induction, and (2) the
heterodimer between C/EBP and CREB has synergistic effects on the iNOS
induction via the CAAT box.
Key Words: inducible nitric oxide synthase • transcriptional regulation • nuclear factor • cardiac myocyte • neonatal rat
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Introduction |
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As has been well defined, NO is an endothelium-derived vasorelaxing factor.1 In addition to its vasorelaxing character, accumulating results have demonstrated that NO directly affects myocardial function.2 NO is synthesized by NO synthases, which are classified into three isoforms, including neuronal NO synthase (NOS1), iNOS (NOS2), and endothelial NO synthase (NOS3).2 In cardiac myocytes, both NOS2 and NOS3 are expressed. We have demonstrated that IL-6 depressed myocardial function via cardiac NOS3 activation.3 We have also demonstrated that LPS induced NOS2 in cardiac myocytes and that the increased NO after NOS2 induction negatively modulated myocardial function.4 Endogenous NO produced by activated NOS3 or induced NOS2 depresses myocardial function possibly via an autocrine/paracrine mechanism.
Therefore, cardiac NO synthase can contribute to myocardial dysfunction
in various clinical settings, including graft rejection, reperfusion
injury, and congestive heart failure. Expression of iNOS5,6
as well as cytokines7–10 has been reported to be
augmented in these failing hearts, and the clinical importance of iNOS
induction is now increasing in cardiovascular medicine.
Various drugs can affect iNOS transcription, and multiple signal
transduction pathways may contribute to iNOS induction.2
The cloned 5'-flanking region of the murine iNOS gene contains multiple
consensus sites for transcription factors, including the NF-B site,
CAAT box, IRF site, GAS, TNF- responsive element, AP-1 site, and
IFN- response element.11 These regulatory elements have
important roles in iNOS induction in mice.12–14
Furthermore, the 5'-upstream sequence of iNOS genes has been cloned in
the rat,15 human,16,17 and
chicken.18 Species other than rodents have quite different
mechanisms in transcriptional regulation of the iNOS gene. Some
investigators have used the upstream fragment of the murine iNOS gene
for transfection to elucidate the regulation of iNOS induction in rat
cardiac myocytes.19 Although differences in the regulatory
mechanisms may be fewer between rodents than those between mice and
nonrodent species, we decided to investigate the transcriptional
regulation in the same species throughout the study.
Until now, iNOS induction in cardiac myocytes has mostly been examined in rats.2 We have also previously demonstrated a significant induction of iNOS after LPS exposure in rat cardiac myocytes.4 Therefore, we performed transfection studies with a cloned 5' region of the rat iNOS gene in rat cardiac myocytes. In addition, little evidence has been previously provided concerning nuclear factors, whose activation may be responsible for iNOS induction in cardiomyocytes. In the present study, we investigated how the transcription of the iNOS gene was regulated and what nuclear factors were essential in its induction.
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Materials and Methods |
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Preparation of Neonatal Rat Cardiac Myocyte Culture
Neonatal rat ventricular myocytes were isolated from 1-day-old Wistar rats as previously described.4,20 Neonatal rats were anesthesized with ether and killed with cervical dislocation. The cardiac cells were dissociated with collagenase (class II, Worthington Biochemical Co) and were preplated for 30 minutes. The nonadhered cells were cultured in media containing 6% fetal bovine serum and 0.1 mmol/L bromodeoxyuridine (Sigma Chemical Co). After a 3-day culture, media were exchanged for the serum-free media containing insulin-transferrin-selenium (Sigma). Over 90% of the cultured cells were spontaneously beating, and characterization of cell types by two-dimensional analysis of [Ca2+]i20 and immunocytochemistry with muscle-specific antibodies4 was consistent with >90% purity of myocytes in our cardiac myocyte culture. All experiments were performed at least 1 day after the exchange of the culture media.
RNA Preparation and Analysis
Total cellular RNA was isolated with the guanidinium
thiocyanate–phenol–chloroform extraction method and subjected to
Northern blot analysis. The cardiac iNOS probe was prepared as
previously described.4 The final wash was 0.1x SSC
containing 0.1% SDS at 65°C for 15 minutes. Relative amounts of the
iNOS mRNA species were determined by laser densitometry (LKB 2202 Ultra
Laser Densitometer, Pharmacia) and normalized by those of ß-actin.
The normalized scores of the LPS-stimulated group were arbitrarily set
at 100%.
Western Blot Analysis
Western blot analysis was performed as previously
described.4 The filter was incubated with polyclonal
antibody against synthetic oligopeptides corresponding to the
C-terminal of murine iNOS protein obtained from rabbit (Affinity
BioReagent) and subsequently with goat anti-rabbit immunoglobulin G
conjugated to horseradish peroxidase (Amersham). The blots were exposed
to x-ray films by the chemiluminescence method (ECL, Amersham).
Nitrite Assay and Cell Respiration Assay
Nitrite accumulation in the culture media was measured with a
Nitrate/Nitrite Assay Kit (Cayman Chemical Co). After removal of the
media, the myocytes were subsequently cultured in the fresh serum-free
media supplemented with 480 µmol/L (=200 µg/mL)
MTT (Sigma) for 4 hours. The results of MTT assay were expressed as
percentages of the control cells. Data of nitrite accumulation were
expressed as corrected values over the relative cellular viability of
the individual wells.
Cloning of the 5'-Flanking Region of the Rat iNOS Gene
Rat genomic DNA was prepared from the cultured neonatal rat
cardiac myocytes with a Genomic DNA Purification Kit (QIAGEN). We
amplified the rat genomic DNA using the upstream primer (primer 5–1,
5'-CAAAACACGAGGCTGAGCTGA-3') and the downstream primer (primer 6,
5'-CAGTCCCTTCACCAAGGTGG-3'), which were homologous to the 5'-upstream
sequence of the previously reported murine iNOS gene.11
We then obtained a 1111-bp fragment by PCR procedure and subcloned the fragment into the pCRII vector (TA Cloning kit, Invitrogen). The nucleotide sequence of the subcloned fragment was determined by a PRISM Ready Reaction DyeDeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems) and an automated DNA sequencer (model 373A, Applied Biosystems).
We also amplified the rat genomic DNA using the upstream primer (primer 3, 5'-GACAGAAAGCCAGAGAGCTCC-3') and the downstream primer (primer 4, 5'-GCAG-CCATCAGGTATTTATAC-3') and obtained a fragment of 337 bp.
After screening a rat genomic DNA library (Clontech) with the 337-bp
fragment as a probe, we obtained a positive clone. The insert size of
this EMBL3 clone was 
8 kb. An EcoRI fragment of this
clone was ligated into the pCRII vector, and the subcloned plasmid was
sequenced by the automated DNA sequencer as described above.
DNase I Footprinting Analysis
The 1111-bp insert obtained by PCR procedure was cut into 510-
and 600-bp fragments at the internal Xba I site. These two
fragments were again individually ligated into the pCRII vector. The
sequences of these plasmids were determined as described above. We
digested these plasmids of 10 pmol with HindIII and
labeled them with 32P. The labeled plasmids were again cut
with Xba I, and the resultant fragments of 570 or 660 bp
were used as probes for the footprinting analysis.
The labeled probe of 5x104 cpm was incubated with nuclear
extract of 5 µg at room temperature for 30 minutes. Nuclear
protein was extracted according to the previously described
method.21 The probe was subsequently incubated with 0.15 U
RNase-free DNase (Promega) at room temperature for exactly 1 minute. As
a size marker, DNA ladder (Marker 9, Nippon Gene) was also labeled with
32P. After nuclear protein was extracted, DNA solution was
loaded onto 6% urea-denaturing polyacrylamide gel. Dried gel
was exposed to a x-ray film with intensifying screens overnight.
Transient Transfection and CAT Assay
The promoter activity of the iNOS gene was analyzed on
the 1111-bp upstream sequence. The 1111-bp insert was ligated into the
pCAT basic vector (Promega). Most of deletion mutants were prepared
with a Kilo Sequence Deletion Kit (Takara Shuzo). The pCAT basic
vector, which was ligated with either the PCR fragment amplified with
primer 6 and 8 (5'-CTGTTTGTTCCTTCTCCCCTAA-3') or the Pst
I–Xba I fragment of 197 bp, was also used as a deletion
mutant. The sequence of these constructs was determined as described
above.
We transfected 5 µg of these constructs and 2 µg of pSV ß-gal (Promega) into myocytes in the presence of 40 µg transfectam (Promega). The efficiency of transient transfection was determined by ß-galactosidase activity, which was measured with a ß-galactosidase assay kit (Promega). The endogenous ß-galactosidase activity in the sample treated only with transfectam was subtracted from the whole ß-galactosidase activity of each sample. CAT activity was measured with a CAT assay kit (Promega). The radioactivity of the sample that did not contain any cellular extract was considered as background and was subtracted from the radioactivity of each sample. The subtracted radioactivity was normalized by protein content and the induced ß-galactosidase activity of each sample.
Synthetic Oligonucleotides and EMSA
Oligonucleotides for the NF-B site
(5'-GATCGAGGGGACTTTCCCTAGC-3'), AP-1 site
(5'-CTAGTGATGAGTCAGCCGGATC-3'), and CRE
(5'-GATTGGCTGACGTCAGA-GAGCT-3') were provided by a
GelShift Assay Kit (Stratagene). Oligonucleotides for
the CAAT box22
(5'-AGCTTCCATAGGTTACACAACTGGGATA-3'), IRF
site23 (5'-TCGAA-GTGAAAGTGAAAGTGAGACTCTAGA-3'),
and GAS24 (5'-GATCAGCTTCATTTCCCG-TAAATCCCTA-3')
were synthesized as previously described. Core recognition sequences
are in boldface.
Binding reactions were carried out with 5 µg of total nuclear
proteins in the presence of 100 µg/mL poly (dI-dC)
poly
(dI-dC) (Pharmacia) with the addition of 5x104 cpm of
32P-labeled oligonucleotide. In the
presence or absence of competitor, we incubated these products at
room temperature for 30 minutes. The mixtures were then electrophoresed
on 4% nondenaturing polyacrylamide gels in high ionic strength
buffer (50 mmol/L Tris, 380 mmol/L glycine, and
2 mmol/L EDTA, pH 8.5). Dried gels were exposed to x-ray
films overnight with intensifying screens.
Statistical Analysis
Values are expressed as mean±SEM. Comparison of the means of
the two groups was performed by the paired t test. A value
of P<.05 was considered to be statistically significant.
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Results |
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Induction of iNOS Is Related to Various Signal Transduction Pathways
We examined the effects of various interventions affecting intracellular signal transducing pathways on the iNOS mRNA induction. LPS (10 µg/mL) induced iNOS mRNA and protein in cardiac myocytes with marked increases in NO production in the media. We demonstrated that iNOS was induced almost exclusively in cardiac myocytes. The mRNA induction was maximal at 6 hours, whereas the protein induction was maximal at 24 hours. We compared the levels of iNOS induction after 6 hours at the mRNA level and 24 hours at the protein level. Although some of the pretreatments by themselves significantly affected cellular growth when drugs were applied for 24 hours, corrected nitrite accumulation after 24 hours by relative cell viability determined by MTT assay was coincident with the iNOS mRNA induction as well as the iNOS protein induction. We have summarized the levels of iNOS induction and NO production after various treatments in Tables 1
and 2. Representative blots
are shown in Figs 1 and 2.
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Forskolin (100 µmol/L) significantly enhanced the iNOS induction by LPS. Forskolin by itself did not induce iNOS. In contrast to the PKA activation, H-89 (Wako Pure Chemicals), a selective PKA inhibitor,25 substantially attenuated the iNOS induction by LPS. W-7 (10 µmol/L), a membrane-permeable calmodulin antagonist,26 did not affect the iNOS induction by LPS. In addition, extracellular Ca2+ chelation by EGTA (2 mmol/L), which completely ceased influx via the L-type Ca2+ channel, did not have any effects on the iNOS induction by LPS (data not shown).
Several reports have suggested that PKC has a crucial role in iNOS induction by IL-1ß.2,19,24 Staurosporine (1 µmol/L, Wako Pure Chemicals) or H-7 (10 µmol/L, Wako Pure Chemical, data not shown) also diminished the iNOS induction by LPS. On the other hand, calphostin C27 (250 nmol/L, Sigma), bisindolylmaleimide28 (70 nmol/L, Sigma), or PKC downregulation by 24-hour pretreatment with 2 µmol/L phorbol 12-myristate 13-acetate (Sigma, data not shown) had only marginal effects on it. Moreover, neither PKC activation by phorbol 12-myristate 13-acetate (200 nmol/L) nor angiotensin II (100 nmol/L, Sigma) affected the iNOS induction by LPS (data not shown). These results suggest that PKC does not contribute to the iNOS induction by LPS. The inhibitory effect of H-729 or staurosporine30 may be attributable to their nonspecific inhibition of protein kinases other than PKC. Herbimycin (2 µmol/L, Wako Pure Chemicals) or genistein (25 µmol/L, Sigma) significantly attenuated the iNOS induction by LPS. PD98059 (50 µmol/L, New England BioLabs), a MEK1 inhibitor, which inhibits the activation of p44/42 MAP-K,31 did not significantly affect the iNOS induction by LPS. Therefore, a tyrosine kinase other than MEK1 may be essential for the iNOS induction by LPS.
Pretreatment with dexamethasone (1 µmol/L, Sigma) or PDTC (100 µmol/L, Sigma) almost totally eliminated the iNOS induction by LPS. We also examined the role of protein synthesis in the iNOS induction. Cycloheximide (356 µmol/L [=100 µg/mL]) by itself did not induce iNOS in cardiac myocytes, and pretreatment with this drug significantly inhibited the iNOS induction.
Many reports have indicated that cytokines as well as LPS
induce iNOS in cardiac myocytes.2 We have also demonstrated
that IL-6 can induce iNOS in chick embryonic cardiac
myocytes.3 Among these cytokines, we examined the
effects of IL-6, TNF-, and IFN- on the iNOS induction in
cardiomyocytes. IL-6 (2000 U/mL, Ajinomoto Co Ltd), TNF-
(500 U/mL, Sigma), or IFN- (500 U/mL, Sigma) significantly enhanced
the iNOS induction by LPS. Combined administration with IL-6 and
TNF- significantly induced iNOS, and its induction was also enhanced
in the presence of forskolin.
Cloning of the 5'-Upstream Region of the Rat iNOS Gene
We analyzed the DNA fragments that were obtained by PCR
amplification and library screening. Eberhardt et al15
recently reported the sequence of the 5'-flanking region of the Wistar
rat iNOS gene, and the sequence that we obtained showed >99% homology
with their sequence. The nucleotide sequence data reported
in the present study will appear in the DDBJ, EMBL, and GenBank
nucleotide sequence databases with the accession number
D88768. Overall homology with the previously reported11
murine iNOS gene was 70.4%. There were two regions that had >85%
homology with the murine iNOS gene; those were -1025 to -645 and
-395 to +5. We found multiple consensus sites for various
transcription factors, mostly in the highly homologous regions. These
results may indicate that these two regions have important roles in
iNOS transcription.
Determination of Nuclear Factor Binding Sites by DNase I
Footprinting Assay
We performed a DNase I footprinting assay in order to determine
where nuclear factors could be bound to the 1-kb upstream region of
the rat iNOS gene. We obtained consistent findings in three
separate footprinting assays. We prepared probes of 570 bp and 660 bp
in size after cutting the fragment by HindIII and
Xba I. As shown in Fig 3, we
detected hypersensitive sites to DNase in both the probes after
incubation with nuclear extract. The hypersensitive sites were
localized at -880 (210-bp species in lanes 1 to 3 [Fig 3A])
and -75 (220-bp species in lanes 2 to 4 [Fig 3B]).
When the probes were incubated with the
nuclear extract from the control myocytes, the area between the
hypersensitive positions and the labeled HindIII ends (-75
to +40 and -990 to -880) was protected from digestion by DNase. These
results suggest that the TATA box and transcription start site (-38 to
+86), IRF site (-909 to -885), and GAS (-936 to -918 and -71 to
-62) are occupied by nuclear factors even in the control state. The
protected areas were diminished after coincubation with nonradiolabeled
probes of 570-bp (lanes 8 and 9 [Fig 3A]) or 660-bp (lanes 7 and 8
[Fig 3B]). When the probes were incubated with nuclear extract from
the LPS-treated myocytes, the protected area from DNase digestion was
expanded beyond that observed when incubated with the control samples.
The additional protected areas were localized at -880 to -680 (200-
to 400-bp species in lanes 2 and 3 [Fig 3A]) and -270 to -75 (220-
to 420-bp species in lanes 3 and 4 [Fig 3B]). These additional areas
were also specific, considering that preincubation with cold probes as
competitors eliminated these areas (lanes 8 and 9 [Fig 3A] and lanes
7 and 8 [Fig 3B]). We determined that the nuclear factors that are
bound to the NF-B site (-101 to -87), CAAT box (-163 to -155 and
-87 to -78), and the TNF responsive element (-122 to -111) were
increased after exposure to LPS. Several copies of IFN- response
element exist in the area between -885 and -685, and LPS may increase
the proteins bound to these sites. These DNA binding proteins induced
by LPS may cause additional conformational changes at the
DNase-hypersensitive sites and rather inhibit DNase digestion, which
results in decreases in hypersensitivity to DNase at -880 and -75, as
shown in lanes 2 and 3 [Fig 3A] and lanes 3 and 4 [Fig 3B].
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Assessment of Promoter Activity of the iNOS Gene
We prepared deletion mutants of the 5'-flanking region of the rat
iNOS gene. The structure of these mutants are shown in Fig 4. In the
myocytes transfected with the pCAT plasmid containing the 1111-bp
insert (construct 1), exposure to LPS (10 µg/mL) markedly
increased CAT enzymatic activity in these myocytes (Fig 5A). In the cells transfected with
construct 2, the induction of CAT was significantly decreased. In the
cells transfected with construct 3 or 4, a 4- to 5-fold loss of
inducibility of the CAT enzyme was observed. These results indicate the
important roles of the enhancer region that was localized between
-1025 and -893, including the distal NF-B site. In the myocytes
transfected with construct 5, LPS induced CAT to a lesser extent
compared with the myocytes transfected with construct 3 or 4. This may
suggest the significance of the CAAT box between -163 and -155 in the
transcription of iNOS mRNA. In the cells transfected with construct 6,
which did not have any promoter region, LPS did not induce CAT.
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P<.05 compared with construct 1+LPS (n=3). B,
Exposure to LPS (10 µg/mL) with forskolin (FSK, 100 µmol/L),
IL-6 (2000 U/mL), TNF-
Next, we examined effects of various interventions on the iNOS
transcription in the myocytes that were transfected with construct 1.
As shown in Fig 5B
, pretreatment with forskolin (100
µmol/L), IL-6 (2000 U/mL), TNF- (500 U/mL), or IFN- (500
U/mL) significantly enhanced the induction of CAT enzyme by LPS (10
µg/mL). In contrast, pretreatment with
dexamethasone (1 µmol/L) or herbimycin
(2 µmol/L) markedly attenuated the CAT enzyme activity
induced by LPS. These results suggest that alterations in the iNOS
transcription by these drugs are in part regulated by nuclear factors
that are bound to the 1-kb upstream region of the iNOS gene.
LPS Activates Multiple Nuclear Factors
To identify responsible pathways for the iNOS induction, nuclear
factor activation was examined by EMSA. Since our culture of cardiac
myocytes contains <5% nonmyocytes,4,20 almost all
the binding activities may be derived from cardiac myocytes. When
myocytes were treated with certain drugs for 24 hours, their viability
was significantly affected. However, in the case of EMSA, we used
nuclear extracts from the cells that were incubated for <6 hours. No
drugs that we applied changed cellular viability within 6 hours.
Therefore, we determined that EMSA was performed under equal conditions
for every treatment. We performed EMSA at least three times for each
treatment and obtained consistent findings.
Representative results of EMSA are shown in Figs 6 to 8. The effects of various drugs on transcription factors are summarized
in Tables 1 and 2.
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LPS treatment increased a binding activity for the NF-B site, which
might mean translocation of NF-B into the nucleus (Fig 6). No significant activity for the
NF-B site was observed at the basal condition, and these increases
did not require de novo protein synthesis. The maximal activation of
NF-B was attained at 6 hours, which might be coincident with the
time course of the iNOS mRNA induction by LPS.
Dexamethasone exerts functional antagonism against several
transcription factors, including NF-B.32,33
Dexamethasone treatment significantly attenuated the
NF-B binding activity for its consensus sequence, and this may
explain the marked inhibition by dexamethasone of the iNOS
induction. The PKA pathway was not involved in the NF-B activation,
whereas tyrosine kinase inhibition significantly attenuated it. As was
the case in the iNOS induction, a selective PKC inhibitor,
calphostin C, had a marginal effect on the NF-B activation, but
staurosporine markedly inhibited it. Pretreatment with PDTC
almost abolished the NF-B activation by LPS, suggesting that free
radical–mediated pathways may have a crucial role in the NF-B
activation. As has been well known,34 TNF-
activated NF-B. On the other hand, IL-6 did not
activate it at all.
As well as the NF-B site, the upstream sequence of the iNOS gene
contains the CAAT box that is bound to C/EBPs.35 One of the
C/EBPs, C/EBPß, which is also called NF-IL-6, is expressed in the
heart.35 As shown in Fig 7A, LPS induced the novel binding activity (B2), in addition to the
preexisting complex (B1). The induction of B2 was initiated at 2 hours
and lasted for 24 hours. Maximal induction was observed at 6 hours,
which was also coincident with the iNOS mRNA induction. Preincubation
with 100-fold cold oligonucleotides corresponding to
the CAAT box abolished the B1 and B2 activities, whereas preincubation
with cold CRE probe only eliminated B2 (Fig 7B). B2 also appeared after
exposure to forskolin. Incubation with polyclonal rabbit antibody
against the rat CREB protein (Upstate Biotechnology Inc) abolished the
B2 activity induced by LPS. EMSA for CRE also revealed that LPS induced
the CRE-binding activity containing CREB (Fig 7C, 7D, and 7E). These
results suggest that B1 consisted of a homodimer of C/EBP and that B2
was a heterodimer between C/EBP and CREB. The B2 activity induced by
LPS was significantly attenuated by tyrosine kinase inhibition, PKA
inhibition, or staurosporine pretreatment. Inhibition of
the B2 induction by staurosporine may be attributable to
its inhibitory effect on PKA.36 The appearance
of B2 required de novo protein synthesis. In contrast to LPS, TNF-
or IL-6 did not induce B2. The CRE-binding activity induced by LPS
appears to be regulated in the same manner as B2. Coincubation with
cold oligonucleotides corresponding to the CAAT box
significantly but not totally inhibited the CRE-binding activity
induced by LPS or forskolin (Fig 7D). The LPS-induced CRE-binding
activity was significantly supershifted after coincubation with
anti-CREB antibody (Fig 7E). This supershifted complex was specific for
CREB, considering that incubation with normal rabbit serum did not
change the binding pattern. These data suggest that the CRE-binding
activity induced by LPS consists of a homodimer of CREB and a
heterodimer between CREB and C/EBP and that the heterodimer between
CREB and C/EBP can be bound to CRE as well as the CAAT box.
The upstream region of the iNOS gene contains IRF sites, and recent
work has suggested that these elements are essential for iNOS
induction.13,37 We performed EMSA for the C13 oligomer (Fig 8A), which can be bound to IRF-1 or
IRF-2.23,38,39 Competition assay revealed that only B3 and
B4 were specific bindings for the IRF site. B3 was observed in the
nuclear extract of the control sample and increased after LPS exposure.
B4 was also induced by LPS. Induction of B4 required de novo protein
synthesis and was significantly attenuated by tyrosine kinase
inhibition, dexamethasone treatment, or
staurosporine exposure. Forskolin or TNF- did not affect
the binding pattern for the IRF site. IL-6 increased B3 with the
induction of B4.
The IRF-1 gene contains the GAS consensus site in its upstream
sequence, and transcription of IRF-1 is activated through
bindings of STAT proteins to GAS.40 GAS also exists in the
5'-flanking region of the iNOS gene. Binding activities (B5 and B6)
were already observed in the control condition, but a novel activity
(B7) was induced after exposure to LPS, IL-6, or IFN- for 30 minutes
(Fig 8B). TNF- or forskolin did not induce B7.
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Discussion |
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According to our data, induction of iNOS seems to require multiple nuclear factors, including NF-
B, C/EBP, CREB, and IRF-1. Among
various intracellular signal transduction pathways, activation of
tyrosine kinases appears to have a critical role in the iNOS induction
in cardiac myocytes.
Recently, 5'-flanking regions of iNOS genes have been cloned in the
mouse,11 rat,15 human,16,17 and
chicken.18 Transcriptional regulation of the iNOS genes has
also been examined in these species. The upstream region of the chicken
iNOS gene contains only the NF-B consensus site and CAAT box, and
LPS can upregulate iNOS transcription through these two sites without
any enhancer element.18 On the other hand, enhancer
elements located at -7 to -16 kb are requisite for the iNOS
transcription in human cell lines.17 In a murine
macrophage cell line, an 1-kb upstream sequence seems to be
sufficient for the iNOS transcription.11 Methylation
interference assay has recently suggested in vivo binding of nuclear
factors to the NF-B site, GAS, IRF-1 site, CAAT box, and Oct site in
the upstream region of the murine iNOS gene.41 We also
demonstrated in the present study that the 1-kb upstream region
of the rat iNOS gene had a crucial role in iNOS transcription.
cAMP-elevating agents accelerated the iNOS induction, although
forskolin by itself did not induce iNOS. Synergism on the iNOS
transcription by PKA activation has also been reported in other
studies.42,43 Generally,
Ser133-phosphorylated CREB is responsible
for transcriptional activation by PKA.44,45 CREB exists in
cardiac myocytes, and phosphorylated CREB is
translocated into the nucleus on PKA activation.46 However,
iNOS genes do not contain CRE in their 1-kb upstream region; thus,
transactivation of the iNOS transcription by the CREB homodimer may not
be probable. Recently, several reports have suggested that a
heterodimer between CREB family and C/EBP family proteins can act as a
transactivator through the CAAT box.47,48 The
transactivation seems to be maximal when CREB and C/EBP are both
phosphorylated.47,48 We have demonstrated
in the present study that CREB and one of the C/EBP family proteins
could form a heterodimer and be bound to the CAAT box in cardiac
myocytes. Attenuation of the binding complex by PKA inhibition suggests
that LPS may activate PKA, as has been reported.49
The LPS-induced CREB activation appears to require de novo protein
synthesis. The CREB gene contains CRE in its upstream region and is
transactivated by its own product, CREB.50 The
PKA activation by LPS initially phosphorylates CREB, and
the phosphorylated CREB may upregulate transcription of
CREB. On the other hand, tyrosine kinase inhibition also decreased the
CREB activation by LPS. This may suggest that LPS
phosphorylates CREB via pathways other than PKA activation,
although we cannot exclude the possibility that the PKA activation by
LPS needs tyrosine kinase activation in advance. CREB
phosphorylation via a PKA-independent pathway has been
reported in the case of nerve growth factor.51 All the
newly produced CREB may not be phosphorylated, and the
B2 induced by LPS may contain unphosphorylated CREB. If
cells are simultaneously stimulated with cAMP-elevating
agents, these induced CREBs can be phosphorylated, and
transactivation by the heterodimer through the CAAT box can be
augmented. Two copies of the CAAT boxes exist in the 5'-flanking region
of the rat iNOS gene, and our data suggest that the
phosphorylated heterodimer between CREB and C/EBP
transactivates the iNOS gene through the CAAT boxes. On the
other hand, combined stimulation with IL-6 and TNF- did not
activate CREB but induced low and significant levels of iNOS
mRNA. These data may indicate that CREB activation has a synergistic
effect but is not essential for the iNOS induction.
We observed a significant binding activity for the CAAT box even in the
control nuclear extract and determined that it consisted of a homodimer
of C/EBP. Akira et al35 also showed by EMSA that a
preexisting complex existed in the CAAT box of nonstimulated nuclear
extract.35 Even after LPS exposure, B1 activity was not
significantly changed. Moreover, various kinase inhibitors
did not affect B1 activity of the control or LPS-stimulated samples.
Therefore, even if C/EBP homodimer can transactivate the iNOS
gene through the CAAT box, its mechanism does not seem to depend on
protein synthesis, enhancement of translocation into nucleus, or
increases in DNA binding activity. Transactivation via C/EBP is
regulated by Thr235 phosphorylation, and a
recent study has shown that MAP-K phosphorylates C/EBPß
in vitro.52 Since LPS,53,54 IL-6,55
and TNF-56 activate MAP-K, it seems probable
that C/EBPß can be phosphorylated by these
cytokines.
Recent work has demonstrated that NF-B activation has a crucial role
in iNOS induction.11,14 Dexamethasone inhibits
the DNA binding activity of NF-B, although it is uncertain whether
the glucocorticoid receptor displaces the NF-B site33
(we consider it improbable because no novel binding activity was
observed in EMSA of the dexamethasone-treated sample) or
the glucocorticoid receptor physically combines with NF-B in the
nucleus.34 PDTC, a free radical scavenger, inhibits NF-B
release from I-B.57 We observed that
dexamethasone or PDTC attenuated the NF-B activation. In
these treatments, the NF-B activation was inhibited in parallel with
the attenuation of the iNOS induction. These data suggest that iNOS
induction is highly dependent on NF-B activation. Ishikawa et
al58 have reported that the NF-B activation by LPS is
dependent on tyrosine kinase and staurosporine-sensitive
protein kinase. We consistently observed that herbimycin or
staurosporine markedly decreased the NF-B activation by
LPS. Among cytokines, IL-1ß and TNF- activate
NF-B.34 Recently, several studies have reported that
PKC-dependent MAP-K activation has a crucial role in the iNOS induction
by IL-1ß.19,24 However, LPS can activate NF-B
through pathways independent of PKC.58 According to our
data, the iNOS induction by LPS does not require PKC or p44/42 MAP-K
activation. Activations of PKC and p44/42 MAP-K appear to be
unnecessary for iNOS induction in the context of NF-B activation.
Other members of MAP-K, such as p38, which the MEK1
inhibitor cannot affect, can be activated by
LPS,54 and these MAP-K members may play significant roles
in the NF-B activation and iNOS induction by LPS.
A recent study has demonstrated that IRF-1 is indispensable for iNOS induction.37 IRF acts as a transcription factor for the induction of interferons.23,38,39 On certain stimuli, IRF-1 is transcriptionally induced and is bound to its consensus sequence. Only IRF-1 but not IRF-2 can transactivate downstream genes. EMSA for the IRF site revealed that LPS or IL-6 induced a novel binding activity in addition to increases in the faster migrating complex. Three tandem C13 oligomers, which we used in EMSA, can be specifically bound to IRF-1 and IRF-2.38,39 We determined that LPS or IL-6 induces IRF family proteins, including IRF-1, whose induction is in parallel with the iNOS induction. Recently, Harroch et al40 also reported that IL-6 induced IRF-1 through STAT-dependent pathways. The IRF-1 gene contains GAS in its upstream sequence. Tyrosine phosphorylation of STAT1 or STAT3 induced by gp130-coupled cytokine receptors can translocate the STAT proteins into the nucleus and bind them to GAS. The bound STAT proteins can then transactivate the IRF-1 gene.59,60 We observed that tyrosine kinase inhibition significantly attenuates IRF-1 binding activity. Recent work has also demonstrated that staurosporine-sensitive serine/threonine phosphorylation is required in the binding of the STAT proteins to the GAS site of the IRF-1 gene.60 This may explain why the IRF-1 binding activity is inhibited by staurosporine.
We observed the induction of novel binding activities in EMSA for GAS
when cells were stimulated with LPS, IL-6, or IFN-. The upstream
sequence of the iNOS gene also contains GAS consensus sites. LPS, IL-6,
or IFN- may enhance transcription of the iNOS and IRF-1 genes
through activation of the STAT proteins. Recent studies have also
suggested that STAT1 activation is necessary for iNOS
induction.24,61
Combined stimulation with IL-6 and TNF- resulted in iNOS induction,
and EMSA revealed that NF-B and IRF-1 were both activated
but that CREB was not. IL-655 and TNF-56 may
phosphorylate C/EBP through MAP-K activation. Therefore,
iNOS induction minimally requires both NF-B and IRF-1 activation
and, possibly, C/EBP phosphorylation. The activation of
CREB may have only a synergistic effect on the iNOS induction.
We used LPS of 10 µg/mL throughout the present study, but even under the septic conditions, the serum concentration of LPS in vivo cannot attain such a high level. An LPS-binding protein, which exists in serum, enhances LPS-induced signal transduction when it forms a trimer with LPS and LPS receptors.62 Therefore, LPS-binding protein may be more relevant in the iNOS induction by LPS in vivo.
Cathecholamine or other inotropic agents that raise cAMP content in myocytes acutely relieve myocardial dysfunction, but iNOS induction may be upregulated by them. The upregulated iNOS may then cause sustained contractile depression. This mechanism might be responsible for the well-known finding that phosphodiesterase inhibitors have no beneficial effects on mortality of patients with congestive heart failure.63
One of the limitations of the present study is that we examined all signal transduction pathways leading to iNOS induction in neonatal rat cardiac myocytes. Further study is needed to determine whether similar findings can be obtained in adult cardiomyocytes.
In conclusion, iNOS induction is regulated by multiple signal transduction pathways in cardiac myocytes. Many studies are now disclosing significant roles of iNOS in various cardiovascular disorders. Understanding the precise mechanisms of myocardial iNOS induction may be helpful in the development of new therapeutic strategies for these cardiovascular diseases.
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Selected Abbreviations and Acronyms |
|---|
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|
Acknowledgments |
|---|
This study was supported by Grants-in-Aid for Scientific Research [No. (B)(2) 08457203 and (C) 07670755 to Dr Takahashi] of the Ministry of Education, Science, and Culture of Japan. The authors thank Ajinomoto Co Ltd for the kind gift of IL-6.
|
Footnotes |
|---|
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13–16, 1995, and published in abstract form (Circulation 1995; 92[suppl I]:I-572).
Received April 28, 1997; accepted September 2, 1997.
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References |
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V. Stangl, G. Baumann, K. Stangl, and S. B Felix Negative inotropic mediators released from the heart after myocardial ischaemia-reperfusion Cardiovasc Res, January 1, 2002; 53(1): 12 - 30. [Abstract] [Full Text] [PDF]
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 A. B. Gustafsson and L. L. Brunton beta -Adrenergic Stimulation of Rat Cardiac Fibroblasts Enhances Induction of Nitric-Oxide Synthase by Interleukin-1beta via Message Stabilization Mol. Pharmacol., April 13, 2001; 58(6): 1470 - 1478. [Abstract] [Full Text]
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B. Tantini, F. Flamigni, C. Pignatti, C. Stefanelli, M. Fattori, A. Facchini, E. Giordano, C. Clo, and C. M. Caldarera Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes Cardiovasc Res, February 1, 2001; 49(2): 408 - 416. [Abstract] [Full Text] [PDF]
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 F. S. Machado, G. A. Martins, J. C. S. Aliberti, F. L. A. C. Mestriner, F. Q. Cunha, and J. S. Silva Trypanosoma cruzi-Infected Cardiomyocytes Produce Chemokines and Cytokines That Trigger Potent Nitric Oxide-Dependent Trypanocidal Activity Circulation, December 12, 2000; 102(24): 3003 - 3008. [Abstract] [Full Text] [PDF]
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 M. Shimpo, U. Ikeda, Y. Maeda, K.-i. Ohya, Y. Murakami, and K. Shimada Effects of Aspirin-Like Drugs on Nitric Oxide Synthesis in Rat Vascular Smooth Muscle Cells Hypertension, May 1, 2000; 35(5): 1085 - 1091. [Abstract] [Full Text] [PDF]
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F. Jung, L. A. Palmer, N. Zhou, and R. A. Johns Hypoxic Regulation of Inducible Nitric Oxide Synthase via Hypoxia Inducible Factor-1 in Cardiac Myocytes Circ. Res., February 18, 2000; 86(3): 319 - 325. [Abstract] [Full Text] [PDF]
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W. Song, X. Lu, and Q. Feng Tumor necrosis factor-{alpha} induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes Cardiovasc Res, February 1, 2000; 45(3): 595 - 602. [Abstract] [Full Text] [PDF]
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Z. Guan, S. Y. Buckman, L. D. Springer, and A. R. Morrison Both p38alpha MAPK and JNK/SAPK Pathways Are Important for Induction of Nitric-oxide Synthase by Interleukin-1beta in Rat Glomerular Mesangial Cells J. Biol. Chem., December 17, 1999; 274(51): 36200 - 36206. [Abstract] [Full Text] [PDF]
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 E. GALEA and D. L. FEINSTEIN Regulation of the expression of the inflammatory nitric oxide synthase (NOS2) by cyclic AMP FASEB J, December 1, 1999; 13(15): 2125 - 2137. [Abstract] [Full Text]
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 M Cavicchi and B J R Whittle Potentiation of cytokine induced iNOS expression in the human intestinal epithelial cell line, DLD-1, by cyclic AMP Gut, September 1, 1999; 45(3): 367 - 374. [Abstract] [Full Text] [PDF]
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 J. A. Dye, K. B. Adler, J. H. Richards, and K. L. Dreher Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression Am J Physiol Lung Cell Mol Physiol, September 1, 1999; 277(3): L498 - L510. [Abstract] [Full Text] [PDF]
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 S. Yasuda and W. Y. W. Lew Angiotensin II exacerbates lipopolysaccharide-induced contractile depression in rabbit cardiac myocytes Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1442 - H1449. [Abstract] [Full Text] [PDF]
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 A. K. Gupta and B. C. Kone CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line Am J Physiol Renal Physiol, April 1, 1999; 276(4): F599 - F605. [Abstract] [Full Text] [PDF]
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 H. D. Battarbee, J. H. Zavecz, M. B. Grisham, R. E. Maloney, L. J. Chandler, J. W. Mercer Jr., and F. M. Cady Cardiac impairment and nitric oxide synthase activity in the chronic portal vein-stenosed rat Am J Physiol Gastrointest Liver Physiol, February 1, 1999; 276(2): G363 - G372. [Abstract] [Full Text] [PDF]
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M. C. LaPointe and E. Isenovic Interleukin-1ß Regulation of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Involves the p42/44 and p38 MAPK Signaling Pathways in Cardiac Myocytes Hypertension, January 1, 1999; 33(1): 276 - 282. [Abstract] [Full Text] [PDF]
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A. M. Diehl Roles of CCAAT/Enhancer-binding Proteins in Regulation of Liver Regenerative Growth J. Biol. Chem., November 20, 1998; 273(47): 30843 - 30846. [Abstract] [Full Text] [PDF]
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C. M. Larsen, K. A. W. Wadt, L. F. Juhl, H. U. Andersen, A. E. Karlsen, M. S.-S. Su, K. Seedorf, L. Shapiro, C. A. Dinarello, and T. Mandrup-Poulsen Interleukin-1beta -induced Rat Pancreatic Islet Nitric Oxide Synthesis Requires Both the p38 and Extracellular Signal-regulated Kinase 1/2 Mitogen-activated Protein Kinases J. Biol. Chem., June 12, 1998; 273(24): 15294 - 15300. [Abstract] [Full Text] [PDF]
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T. Shimizu, M. Yamato, Y. Isoi, T. Akutsu, T. Setomaru, K. Abe, A. Kikuchi, M. Umezu, and T. Okano Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces Circ. Res., February 22, 2002; 90 (3): e40 - e48. [Abstract] [Full Text] [PDF]
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