Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1997;81:911-921

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kinugawa, K.-i.
Right arrow Articles by Takahashi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kinugawa, K.-i.
Right arrow Articles by Takahashi, T.
(Circulation Research. 1997;81:911-921.)
© 1997 American Heart Association, Inc.

Articles

Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes

Koh-ichiro Kinugawa, Tatsuya Shimizu, Atsushi Yao, Osami Kohmoto, Takashi Serizawa, , Toshiyuki Takahashi

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


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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)-{alpha}, or interferon (IFN)-{gamma} enhanced the LPS-induced iNOS expression. Combined stimulation of IL-6 and TNF-{alpha} also induced iNOS. The 5'-upstream sequence of the rat iNOS gene contains the nuclear factor-{kappa}B (NF-{kappa}B) site, CAAT box, IFN-{gamma} 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-{alpha} increased binding activity for the NF-{kappa}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-{gamma} 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-{kappa}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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-{kappa}B site, CAAT box, IRF site, GAS, TNF-{alpha} responsive element, AP-1 site, and IFN-{gamma} 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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {approx}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 {approx}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 {approx}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-{kappa}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 {approx}5 µg of total nuclear proteins in the presence of 100 µg/mL poly (dI-dC){bullet}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 {approx}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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1Down and 2Down. Representative blots are shown in Figs 1Down and 2Down.


View this table:
[in this window]
[in a new window]
  		Table 1. Inhibitory Mechanisms of Various Drugs on iNOS Induction by LPS


View this table:
[in this window]
[in a new window]
  		Table 2. Nuclear Factor Requirements for iNOS Induction



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Effects of various interventions on iNOS mRNA induction. Forskolin (FSK, 100 µmol/L) significantly enhanced the LPS (10 µg/mL, 6 hours)–induced iNOS induction. FSK by itself did not induce iNOS mRNA. Cytokines, including IL-6 (2000 U/mL), TNF-{alpha} (200 U/mL), and IFN-{gamma} (500 U/mL), had a synergistic effect on the iNOS induction by LPS. IL-6 or TNF-{alpha} by itself or combination with FSK did not induce iNOS, but treatment with both IL-6 and TNF-{alpha} significantly induced iNOS mRNA, and FSK also enhanced the induction. H-89 (10 µmol/L), herbimycin A (Herb, 2 µmol/L), genistein (Gen, 25 µmol/mL), or dexamethasone (DEX, 1 µmol/L) significantly inhibited iNOS induction by LPS. PDTC (100 µmol/L) also attenuated iNOS induction. Staurosporine (ST, 1 µmol/L) inhibited iNOS induction, whereas calphostin C (Cal, 250 nmol/L), bisindolylmaleimide (BIS, 70 nmol/mL), and PD98059 (PD, 50 µmol/mL) did not affect it. Inhibition of protein synthesis by cycloheximide (CHX, 356 µmol/L [=100 µg/mL]) itself did not induce iNOS and significantly depressed the iNOS induction by LPS. C indicates control. Three separate analyses showed consistent results.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Effects of various interventions on iNOS protein induction. Forskolin (FSK, 100 µmol/L) accelerated induction of iNOS protein after exposure to LPS (10 µg/mL) for 24 hours. IL-6 (2000 U/mL), TNF-{alpha} (500 U/mL), and IFN-{gamma} (500 U/mL) had synergistic effects on the iNOS induction by LPS. A single application of IL-6 or TNF-{alpha} did not induce iNOS, but combined exposure to both the cytokines for 24 hours significantly induced iNOS. H-89 (10 µmol/L) significantly inhibited the induction of iNOS by LPS (24 hours). Herbimycin (Herb, 2 µmol/L), genistein (Gen, 25 µmol/mL), and dexamethasone (DEX, 1 µmol/L) markedly attenuated iNOS induction by LPS (24 hours). Bisindolylmaleimide (BIS, 70 nmol/mL) and PD98059 (PD, 50 µmol/mL) did not affect iNOS induction by LPS. C indicates control. Three separate analyses showed consistent results.

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-{alpha}, and IFN-{gamma} on the iNOS induction in cardiomyocytes. IL-6 (2000 U/mL, Ajinomoto Co Ltd), TNF-{alpha} (500 U/mL, Sigma), or IFN-{gamma} (500 U/mL, Sigma) significantly enhanced the iNOS induction by LPS. Combined administration with IL-6 and TNF-{alpha} 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 {approx}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 3Down, we detected hypersensitive sites to DNase in both the probes after incubation with nuclear extract. The hypersensitive sites were localized at {approx}-880 ({approx}210-bp species in lanes 1 to 3 [Fig 3ADown]) and -75 ({approx}220-bp species in lanes 2 to 4 [Fig 3BDown]). 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 3ADown]) or 660-bp (lanes 7 and 8 [Fig 3BDown]). 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 3ADown]) and -270 to -75 (220- to 420-bp species in lanes 3 and 4 [Fig 3BDown]). These additional areas were also specific, considering that preincubation with cold probes as competitors eliminated these areas (lanes 8 and 9 [Fig 3ADown] and lanes 7 and 8 [Fig 3BDown]). We determined that the nuclear factors that are bound to the NF-{kappa}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-{gamma} 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 3ADown] and lanes 3 and 4 [Fig 3BDown].



View larger version (54K):
[in this window]
[in a new window]
  		Figure 3. Determination of nuclear factor binding sites by DNase I footprinting assay. We performed footprinting analyses using probes of 570 bp (panel A) and 660 bp (panel B). Labeled DNA ladder for size determination is shown in lanes 5 and 6 (panel A) and lanes 1 and 9 (panel B). The undigested probes are indicated as Xba I in the figure. Without nuclear extract, digestion patterns of the probes are shown in lanes 4 and 7 (panel A) and lanes 5 and 6 (panel B). These probes were incubated with nuclear extract from the control myocytes (lane 1 [panel A] and lane 2 [panel B]), the cells exposed to LPS (10 µg/mL) for 2 hours (lane 2 [panel A] and lane 3 [panel B]), and the cells exposed to LPS for 6 hours (lane 3 [panel A] and lane 4 [panel B]). As competitors, cold probes of 10-fold (lane 8 [panel A] and lane 7 [panel B]) or 100-fold (lane 9 [panel A] and lane 8 [panel B]) molar excess were incubated with nuclear extract from the LPS (6 hours)–stimulated cells. With the competitors, the digestion pattern was almost restored, but the hypersensitive site remained digested. Fuzzy bands in lane 9 (panel A) and lane 8 (panel B) may be due to overload of DNA.

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 4Down. 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 5ADown). 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-{kappa}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.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. Structure of deletion mutants of the iNOS promoter region. Construct 1 contains the full length of the cloned 5'-flanking region of the rat iNOS gene. Constructs 2 to 6 have various deleted inserts of the 5' region.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Assessment of promoter activity by transient transfection assay. A, Compared with nontreated control (open bars), CAT enzymatic activity normalized by ß-galactosidase activity (CAT/ß-gal) was markedly increased after application of LPS (10 µg/mL, solid bars) in the myocytes transfected with construct 1 containing a 1111-bp insert. In the cells transfected with the deletion mutants, CAT activities were significantly decreased. Values are expressed as mean±SEM. *P<.01 compared with construct 1+LPS; {dagger}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-{alpha} (500 U/mL), or IFN-{gamma} (500 U/mL) significantly increased the CAT activities in the cells transfected with construct 1 compared with exposure solely to LPS. On the other hand, pretreatment with dexamethasone (DEX, 1 µmol/L) or herbimycin (Herb, 2 µmol/L) significantly attenuated the induction of CAT after application of LPS in the myocytes transfected with construct 1. C indicates control. Values are expressed as mean±SEM. *P<.01 compared with LPS; {dagger}P<.05 compared with LPS (n=3).

Next, we examined effects of various interventions on the iNOS transcription in the myocytes that were transfected with construct 1. As shown in Fig 5BUp, pretreatment with forskolin (100 µmol/L), IL-6 (2000 U/mL), TNF-{alpha} (500 U/mL), or IFN-{gamma} (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 {approx}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 8DownDownDown. The effects of various drugs on transcription factors are summarized in Tables 1Up and 2Up.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 6. EMSA for the NF-{kappa}B site. A, LPS (10 µg/mL) augmented binding activity to the NF-{kappa}B consensus sequence. Its activation was observed after 30 minutes of exposure to LPS and was maximal at 6 hours. Nonspecific binding activity was not observed. B, TNF-{alpha} alone (500 U/mL, 6 hours) activated NF-{kappa}B. In contrast, IL-6 (2000 U/mL, 6 hours) did not activate NF-{kappa}B. C indicates control; Comp, competitor; and FSK, forskolin.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 7. EMSA for the CAAT box (panels A and B) and CRE (panels C, D, and E). A, In addition to a preexisting complex (B1), a slower-migrating complex (B2) gave rise after exposure to LPS (10 µg/mL) for >2 hours in EMSA for the CAAT box. Maximal induction of B2 was observed at 6 hours. B, Preincubation with the 100-fold cold probe corresponding to the CAAT box abolished both B1 and B2, whereas preincubation with cold symmetrical CRE probe (100-fold) eliminated only B2. Forskolin (FSK, 100 µmol/L, 6 hours) also induced B2, which competed with the cold CRE probe. Preincubation with polyclonal antibody against CREB (Ab) abolished the B2 activity induced by LPS. C, LPS (10 µg/mL) time-dependently increased the binding activity for CRE. D, Forskolin (FSK, 100 µmol/L, 6 hours) also induced the CRE-binding activity. Competition assay suggested that the increased binding activity by LPS or FSK was CRE specific. Coincubation with the cold oligonucleotides corresponding to the CAAT box significantly decreased the CRE-binding activity induced by LPS or FSK. E, We observed a significant supershifted complex (SC) after incubation with Ab. C indicates control; NS, nonspecific bindings; FP, free probe; RS, normal rabbit serum; and Comp, competitor.



View larger version (61K):
[in this window]
[in a new window]
  		Figure 8. EMSA for the IRF site (panel A) and GAS (panel B). A, From the results of the competition assay using a nonradiolabeled oligonucleotide corresponding to the IRF site, two specific binding activities were identified in EMSA for the IRF site. One faster binding activity (B3) was observed in the control sample (C); after LPS (10 µg/mL) exposure for 6 hours, B3 was increased with the induction of a slower binding activity (B4). B, Two binding activities (B5 and B6) for GAS were observed in the control sample (C), but a novel binding activity (B7) was induced after exposure to LPS (10 µg/mL), IL-6 (2000 U/mL), or IFN-{gamma} (500 U/mL) for 30 minutes. Forskolin (FSK, 100 µmol/L, 30 minutes) and TNF-{alpha} (500 U/mL, 30 minutes) did not induce B7. B5, B6, and B7 binding activities were specific, considering the results of the competition assay with a cold probe corresponding to the GAS site. NS indicates nonspecific bindings; FP, free probe.

LPS treatment increased a binding activity for the NF-{kappa}B site, which might mean translocation of NF-{kappa}B into the nucleus (Fig 6Up). No significant activity for the NF-{kappa}B site was observed at the basal condition, and these increases did not require de novo protein synthesis. The maximal activation of NF-{kappa}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-{kappa}B.32,33 Dexamethasone treatment significantly attenuated the NF-{kappa}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-{kappa}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-{kappa}B activation, but staurosporine markedly inhibited it. Pretreatment with PDTC almost abolished the NF-{kappa}B activation by LPS, suggesting that free radical–mediated pathways may have a crucial role in the NF-{kappa}B activation. As has been well known,34 TNF-{alpha} activated NF-{kappa}B. On the other hand, IL-6 did not activate it at all.

As well as the NF-{kappa}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 7AUp, 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 7BUp). 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 7CUp, 7DUp, and 7EUp). 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-{alpha} 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 7DUp). The LPS-induced CRE-binding activity was significantly supershifted after coincubation with anti-CREB antibody (Fig 7EUp). 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 8AUp), 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-{alpha} 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-{gamma} for 30 minutes (Fig 8BUp). TNF-{alpha} or forskolin did not induce B7.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
According to our data, induction of iNOS seems to require multiple nuclear factors, including NF-{kappa}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-{kappa}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 {approx}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-{kappa}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 {approx}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 {approx}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-{alpha} 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-{alpha}56 activate MAP-K, it seems probable that C/EBPß can be phosphorylated by these cytokines.

Recent work has demonstrated that NF-{kappa}B activation has a crucial role in iNOS induction.11,14 Dexamethasone inhibits the DNA binding activity of NF-{kappa}B, although it is uncertain whether the glucocorticoid receptor displaces the NF-{kappa}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-{kappa}B in the nucleus.34 PDTC, a free radical scavenger, inhibits NF-{kappa}B release from I-{kappa}B.57 We observed that dexamethasone or PDTC attenuated the NF-{kappa}B activation. In these treatments, the NF-{kappa}B activation was inhibited in parallel with the attenuation of the iNOS induction. These data suggest that iNOS induction is highly dependent on NF-{kappa}B activation. Ishikawa et al58 have reported that the NF-{kappa}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-{kappa}B activation by LPS. Among cytokines, IL-1ß and TNF-{alpha} activate NF-{kappa}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-{kappa}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-{kappa}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-{kappa}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-{gamma}. The upstream sequence of the iNOS gene also contains GAS consensus sites. LPS, IL-6, or IFN-{gamma} 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-{alpha} resulted in iNOS induction, and EMSA revealed that NF-{kappa}B and IRF-1 were both activated but that CREB was not. IL-655 and TNF-{alpha}56 may phosphorylate C/EBP through MAP-K activation. Therefore, iNOS induction minimally requires both NF-{kappa}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.


*    Selected Abbreviations and Acronyms
 
AP-1 = activator protein-1
CAT = chloramphenicol acetyltransferase
C/EBP = CAAT box/enhancer binding protein
CRE = cAMP responsive element
CREB = CRE binding protein
EMSA = electrophoretic mobility shift assay
GAS = IFN-{gamma} activation site
H-7 = 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride
H-89 = N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide
IFN = interferon
IL = interleukin
iNOS = inducible NO synthase
IRF = interferon regulatory factor
LPS = lipopolysaccharide
MAP-K = mitogen-activated protein kinase
MEK = MAP-K/extracellular signal–regulated kinase (ERK) kinase
NF-{kappa}B = nuclear factor-{kappa}B
NOS1, NOS2, NOS3 = NO synthase isoforms 1, 2, and 3
PCR = polymerase chain reaction
PDTC = pyrrolidine dithiocarbamate
PKA = cAMP-dependent protein kinase
PKC = protein kinase C
STAT = signal transducers and activators of transcription
TNF = tumor necrosis factor
W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride


*    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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109–142.[Medline] [Order article via Infotrieve]
  2. Kelly RA, Balligand J-L, Smith TW. Nitric oxide and cardiac function. Circ Res. 1996;79:363–380.[Free Full Text]
  3. Kinugawa K, Takahashi T, Kohmoto O, Yao A, Aoyagi T, Momomura S, Hirata Y, Serizawa T. Nitric oxide–mediated effects of interleukin-6 on [Ca2+]i and cell contraction in cultured chick ventricular myocytes. Circ Res. 1994;75:285–295.[Abstract/Free Full Text]
  4. Kinugawa K, Kohmoto O, Yao A, Serizawa T, Takahashi T. Cardiac inducible nitric oxide synthase negatively modulates myocardial function in cultured rat myocytes. Am J Physiol. 1997;272:H35–H47.[Abstract/Free Full Text]
  5. Haywood GA, Tsao PS, von der Leyen HE, Mann MJ, Keeling PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR, Bishopric NH, Cooke JP, McKenna WJ, Fowler MB. Expression of inducible nitric oxide synthase in human heart failure. Circulation. 1996;93:1087–1094.[Abstract/Free Full Text]
  6. Lewis NP, Tsao PS, Rickenbacher PR, Xue C, Johns RA, Haywood GA, von der Leyen H, Trindade PT, Cooke JP, Hunt SA, Billingham MA, Valantine HA, Fowler MB. Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle. Circulation. 1996;93:720–729.[Abstract/Free Full Text]
  7. Satoh M, Tamura G, Segawa I, Tashiro A, Hiramori K, Satodate R. Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy. Virchows Arch. 1996;427:503–509.[Medline] [Order article via Infotrieve]
  8. Kukielka GL, Smith CW, Manning AM, Youker KA, Michael LH, Entman MH. Induction of interleukin-6 synthesis in the myocardium: potential role in postreperfusion inflammatory injury. Circulation. 1995;92:1866–1875.[Abstract/Free Full Text]
  9. Neumann FJ, Ott I, Gawaz M, Richardt G, Holzapfel H, Jochum M, Schomig A. Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. Circulation. 1995;92:748–755.[Abstract/Free Full Text]
  10. Deng MC, Erren M, Kammerling L, Gunther F, Kerber S, Fahrenkamp A, Assmann G, Breithardt G, Scheld HH. The relation of interleukin-6, tumor necrosis factor-alpha, IL-2, and IL-2 receptor levels to cellular rejection, allograft dysfunction, and clinical events early after cardiac transplantation. Transplantation. 1995;60:1118–1124.[Medline] [Order article via Infotrieve]
  11. Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, Murphy WJ. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon {gamma} and lipopolysaccharide. Proc Natl Acad Sci U S A. 1993;90:9730–9734.[Abstract/Free Full Text]
  12. Xie Q-W, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon {gamma} and bacterial lipopolysaccharide. J Exp Med. 1993;177:1779–1784.[Abstract/Free Full Text]
  13. Martin E, Nathan C, Xie Q-W. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J Exp Med. 1994;180:977–984.[Abstract/Free Full Text]
  14. Xie Q-W, Kashiwabara Y, Nathan C. Role of transcription factor NF-{kappa}B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994;269:4705–4708.[Abstract/Free Full Text]
  15. Eberhardt W, Kunz D, Hummel R, Pfeilschifter J. Molecular cloning of the rat inducible nitric oxide synthase gene promoter. Biochem Biophys Res Commun. 1996;223:752–756.[Medline] [Order article via Infotrieve]
  16. Chartrain NA, Geller DA, Koty PP, Sitrin NF, Nussler AK, Hoffman EP, Billiar TB, Hutchinson NI, Mudgett JS. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J Biol Chem. 1994;269:6765–6772.[Abstract/Free Full Text]
  17. De Vera ME, Shapiro RA, Nussler AK, Mudgett JS, Simmons RL Jr, Morris MS, Billiar TR, Geller DA. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc Natl Acad Sci U S A. 1996;93:1054–1059.[Abstract/Free Full Text]
  18. Lin AW, Chang CC, McCormick CC. Molecular cloning and expression of an avian macrophage nitric-oxide synthase cDNA and the analysis of the genomic 5'-flanking region. J Biol Chem. 1996;271:11911–11919.[Abstract/Free Full Text]
  19. LaPointe MC, Sitkins JR. Mechanisms of interleukin-1beta regulation of nitric oxide synthesis in cardiac myocytes. Hypertension. 1996;27:709–714.[Abstract/Free Full Text]
  20. Kinugawa K, Takahashi T, Kohmoto O, Yao A, Ikenouchi H, Serizawa T. Ca2+-growth coupling in angiotensin II-induced hypertrophy in cultured rat cardiac cells. Cardiovasc Res. 1995;30:419–431.[Medline] [Order article via Infotrieve]
  21. Yao A, Takahashi T, Aoyagi T, Kinugawa K, Kohmoto O, Sugiura S, Serizawa T. Immediate-early gene induction and MAP kinase activation during recovery from metabolic inhibition in cultured cardiac myocytes. J Clin Invest. 1995;96:69–77.
  22. Betts JC, Cheshire JK, Akira S, Kishimoto T, Woo P. The role of NF-{kappa}B and NF-IL6 transactivating factors in the synergistic activation of human serum amyloid A gene expression. J Biol Chem. 1993;268:25624–25631.[Abstract/Free Full Text]
  23. Fujita T, Shibuya H, Hotta H, Yamanishi K, Taniguchi T. Interferon-ß gene regulation: tandemly repeated sequences of a synthetic 6 bp oligomer function as a virus-inducible enhancer. Cell. 1987;49:357–367.[Medline] [Order article via Infotrieve]
  24. Singh K, Balligand J-L, Fischer TA, Smith TW, Kelly RA. Regulation of cytokine-inducible nitric oxide synthase in cardiac myocytes and microvascular endothelial cells: role of extracellular signal-regulated kinases (ERK1/ERK2) and STAT1{alpha}. J Biol Chem. 1996;271:1–7.[Free Full Text]
  25. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito N, Toshioka T, Hidaka H. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem. 1990;265:4924–4927.
  26. Hidaka H, Sasaki Y, Tanaka T, Endo T, Ohno S, Fujii Y, Nagata T. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981;78:4354–4357.[Abstract/Free Full Text]
  27. Kobayashi E, Nakano H, Morimoto M, Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibition of protein kinase C. Biochem Biophys Res Commun. 1989;159:548–553.[Medline] [Order article via Infotrieve]
  28. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991;266:15771–15781.[Abstract/Free Full Text]
  29. Kawamoto S, Hidaka H. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. Biochem Biophys Res Commun. 1984;125:258–264.[Medline] [Order article via Infotrieve]
  30. Fallon J. Staurosporine inhibits a tyrosine protein kinase in human hepatoma cell membranes. Biochem Biophys Res Commun. 1990;170:1191–1196.[Medline] [Order article via Infotrieve]
  31. Pang L, Sawada T, Decker SJ, Saltiel AR. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995;270:13585–13588.[Abstract/Free Full Text]
  32. Mukaida N, Morita M, Ishikawa Y, Rice N, Okamoto S, Kasahara T, Matsushima K. Novel mechanism of glucocorticoid-mediated gene repression. J Biol Chem. 1994;269:13289–13296.[Abstract/Free Full Text]
  33. Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcriptional factor NF-{kappa}B and the glucocorticoid receptor. Proc Natl Acad Sci U S A. 1994;91:752–756.[Abstract/Free Full Text]
  34. Schütze S, Machleidt T, Krönke M. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J Leukoc Biol. 1994;56:533–541.[Abstract]
  35. Akira S, Ishii H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima, Hirano T, Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990;9:1897–1906.[Medline] [Order article via Infotrieve]
  36. Persaud SJ, Jones PM, Howell SL. Staurosporine inhibits protein kinases activated by Ca2+ and cyclic AMP in addition to inhibiting protein kinase C in rat islets of Langerhans. Mol Cell Endocrinol. 1993;94:55–60.[Medline] [Order article via Infotrieve]
  37. Kamijo R, Harada H, Matsuyama T, Bosland M, Gerecitano J, Shapiro D, Le J, Koh SI, Kimura T, Green SJ, Mak TW, Taniguchi T, Vilcek J. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science. 1994;263:1612–1615.[Abstract/Free Full Text]
  38. Miyamoto M, Fujita T, Kimura Y, Maruyama M, Harada H, Sudo Y, Miyata T, Taniguchi T. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-ß gene regulatory elements. Cell. 1988;54:903–913.[Medline] [Order article via Infotrieve]
  39. Harada H, Willison K, Sakakibara J, Miyamoto M, Fujita T, Taniguchi T. Absence of the type I IFN system in EC cells: transcriptional activator (IRF-1) and repressor (IRF-2) genes are developmentally regulated. Cell. 1990;63:303–312.[Medline] [Order article via Infotrieve]
  40. Harroch S, Revel M, Chebath J. Induction by interleukin-6 of interferon regulatory factor 1 (IRF-1) gene response element pIRE and cell type-dependent control of IRF-1 binding to DNA. EMBO J. 1994;13:1942–1949.[Medline] [Order article via Infotrieve]
  41. Goldring CEP, Reveneau S, Algarte M, Jeannin J-F. In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL6. Nucleic Acids Res. 1996;24:1682–1687.[Abstract/Free Full Text]
  42. Oddis CV, Simmons RL, Hattler BG, Finkel MS. cAMP enhances inducible nitric oxide synthase mRNA stability in cardiac myocytes. Am J Physiol. 1995;269:H2044–H2050.[Abstract/Free Full Text]
  43. Koide M, Kawahara Y, Nakayama I, Tsuda T, Yokoyama M. Cyclic AMP-elevating agents induce an inducible type of nitric oxide synthase in cultured vascular smooth muscle cells. J Biol Chem. 1993;268:24959–24966.[Abstract/Free Full Text]
  44. Yamamoto KK, Gonzalez GA, Biggs WH III, Montminy MR. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature. 1988;334:494–498.[Medline] [Order article via Infotrieve]
  45. Nichols M, Weih F, Schmid W, DeVack C, Kowenz-Leutz E, Luckow B, Boshart M, Schütz G. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription. EMBO J. 1992;11:3337–3346.[Medline] [Order article via Infotrieve]
  46. Goldspink PH, Russell B. The cAMP response element binding protein is expressed and phosphorylated in cardiac myocytes. Circ Res. 1994;74:1042–1049.[Abstract/Free Full Text]
  47. Vallejo M, Ron D, Miller CP, Habener JF. C/ATF, a member of the activating transcription factor family of DNA-binding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response elements. Proc Natl Acad Sci U S A. 1993;90:4679–4683.[Abstract/Free Full Text]
  48. Tsukada J, Saito K, Waterman WR, Webb AC, Auron PE. Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1ß gene. Mol Cell Biol. 1994;14:7285–7297.[Abstract/Free Full Text]
  49. Fujihara M, Muroi M, Muroi Y, Ito N, Suzuki T. Mechanism of lipopolysaccharide-triggered junB activation in a mouse macrophage-like cell line (J774). J Biol Chem. 1993;268:14898–14905.[Abstract/Free Full Text]
  50. Meyer TE, Waeber G, Lin J, Beckmann W, Habener JF. The promoter of the gene encoding 3', 5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription. Endocrinology. 1993;132:779–780.
  51. Ginty DD, Bonni A, G