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Cardiovascular Research 1999 42(1):104-112; doi:10.1016/S0008-6363(98)00285-5
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Copyright © 1999, European Society of Cardiology

Induction of interleukin (IL)-6 by hypoxia is mediated by nuclear factor (NF)-{kappa}B and NF-IL6 in cardiac myocytes

Hideo Matsuia, Yoshiji Iharaa, Yasushi Fujioa, Keita Kunisadaa, Sizuo Akirab, Tadamitsu Kishimotoa and Keiko Yamauchi-Takiharaa,*

aDepartment of Medicine III, Osaka University Medical School, Suita, Osaka 565-0871, Japan
bDepartment of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8131, Japan

* Corresponding author. Tel.: +81-6-879-3835; fax: +81-6-879-3839; e-mail: takihara@imed3.med.osaka-u.ac.jp

Received 11 June 1998; accepted 9 September 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: We have previously reported that interleukin (IL)-6 is induced by hypoxic stimulation in cardiac myocytes. In the present study, we examined the induction of potent transcription factors of IL-6, nuclear factor (NF)-{kappa}B and NF-IL6, in cardiac myocytes subjected to hypoxia. Methods: Five different lengths of IL-6 promoter-luciferase reporter plasmids and three mutant plasmids, in which the binding sites of NF-{kappa}B and/or NF-IL6 were disrupted, were transfected into neonatal rat cardiac myocytes. Luciferase activities after hypoxic stimulation were measured. Electrophoretic mobility shift assays were performed using oligonucleotides containing the binding site for NF-{kappa}B or NF-IL6 as a probe. Results: Hypoxic stimulation for 4 h increased luciferase activity by 5.7 fold in –179/+12-luciferase reporter plasmid, whereas no significant increase was observed in –60/+12-luciferase plasmid. Decrease in luciferase activity was more prominent when the NF-{kappa}B binding site was disrupted rather than when the NF-IL6 binding site was disrupted. Moreover, when both sites were disrupted, luciferase activity increased only by 1.5 fold. Electrophoretic mobility shift assays demonstrated enhanced binding activity to oligonucleotides containing the NF-{kappa}B binding site in hypoxic cardiac myocytes, which displayed a supershift with antibody to its subunit, p50 or p65. The binding activity to the NF-IL6 probe also enhanced and displayed a supershift with antibody to NF-IL6. Conclusions: Although hypoxic stimulation induced NF-{kappa}B and NF-IL6 in cardiac myocytes, NF-{kappa}B may be the primary positive regulator of transcriptional activation of the IL-6 gene in the context of hypoxia.

KEYWORDS Cardiac myocytes; Hypoxia; Interleukin-6; Gene expression; Nuclear factor-{kappa}B; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The expression of a growing number of genes has been shown to be induced by hypoxic stimulation in cardiac myocytes and vascular endothelial cells [1–4]. However, the molecular basis for the induction of these genes has not been elucidated in cardiac myocytes. Interleukin (IL)-6 is a multifunctional cytokine that affects various cell types. The pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and IL-1β, elicit IL-6 synthesis in a wide range of target cells [5, 6]. We recently reported that hypoxic stimulation induced the expression of IL-6 mRNA and increased IL-6 production in cardiac myocytes [1]. IL-6 and other pro-inflammatory cytokines are reported to be involved in the pathogenesis of myocardial injury in myocarditis [7]and ischemic heart disease [8]. However, recent studies have addressed novel facets of IL-6 activity in myocardial injury; the role of this cytokine as an anti-inflammatory agent. IL-6 suppressed the generation of IL-1 and TNF in mice stimulated with lipopolysaccharide [9]and exerted beneficial effects on viral myocarditis in mice [10].

The transcriptional activation of the IL-6 gene has been reported to depend on the 14-bp palindromic sequence, located at position –150, and a nuclear factor for IL-6 expression (NF-IL6) bound to this site [11]. NF-IL6 activates the IL-6 gene and is also responsible for the regulation of the genes encoding other inflammatory cytokines, acute-phase proteins, albumin, c-fos, and viral proteins [12]. Moreover, the promoter region of the IL-6 gene has a binding site for the transcription factor, nuclear factor (NF)-{kappa}B, in addition to the NF-IL6-binding site. Transcription factors, NF-IL6 and NF-{kappa}B, are known to synergistically activate the transcription of inflammatory cytokines [13]. NF-{kappa}B is another well-characterized transcription factor that plays an important role in inflammatory responses and cell growth regulation, and is considered to be involved in the expression of many inducible cellular genes that encode cytokines and acute-phase proteins [14]. Recently, in vivo transfer of a double-stranded oligonucleotide containing the NF-{kappa}B cis element was reported to reduce the extent of myocardial infarction [15].

In the present study, we elucidated the molecular mechanisms of hypoxia-induced transcriptional activation of the IL-6 gene. Activation of DNA-binding proteins, both NF-{kappa}B and NF-IL6, during hypoxia could potentially regulate expression of the gene products relevant to ischemia, including IL-8, angiotensinogen, intracellular adhesion molecule-1 (ICAM-1) and granulocyte colony-stimulating factor (G-CSF) [16–19]. These findings indicate that activation of NF-IL6 and NF-{kappa}B in cardiac myocytes under hypoxic stimulation has complex consequences affecting both pro- and anti-inflammatory cytokines.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Medium-199 (M-199), newborn calf serum (NCS; Life Technologies, Gaithersburg, MD, USA), and bromodeoxyuridine (Sigma Chemical, St. Louis, MO, USA) were used for myocardial cell culture. Human recombinant IL-1β (100 µg/ml in 20 mM of phosphate buffer), kindly provided by Ohtsuka Pharmaceutical, (Tokushima, Japan) was used in this study.

2.2 Cell culture
Primary cultures of cardiac myocytes were prepared from 1-day-old neonatal rat hearts as described previously [1]. Cultures were enriched for myocardial cells by preplating for 60 min, and then plated at a concentration of 1.2x105 cells per ml onto 35-mm plastic dishes and cultured at 37°C in 95% air/5% CO2. The cells were maintained with M-199 supplemented with 10% NCS and 0.1 mM bromodeoxyuridine for 24 h. Using this method, we routinely obtained enriched cultures containing more than 95% myocytes as assayed by immunofluorescence staining with anti-sarcomeric {alpha}-actinin antibody (clone EA-53, Sigma Chemical).

Highly enriched cultures of non-myocardial cells (NMC) were prepared by two passages of the cells that adhered to the culture dishes during preplating. This culture yielded a preparation of the cells consisting of more than 95% fibroblasts and were used for the experiments at a concentration of 1.8x105 cells per ml.

On the day of the experiment, the medium was changed again. For the hypoxic stimulation, the cells were cultured in a 95% N2/5% CO2 atmosphere as described previously [1].

2.3 Northern blotting
Cardiac myocytes were exposed to hypoxia or IL-1β for 30 or 60 min. Total RNA was prepared by the acid guanidinium thiocyanate–phenol–chloroform method [20]. Twenty µg of total RNA was size-fractionated by 1.2% formaldehyde–agarose gel electrophoresis, and blotted onto a nylon membrane (Hybond N+; Amersham International PLC, Little Chalfont, United Kingdom). A EcoRI/BglII-digested 0.6-kb fragment from murine IL-6 cDNA [1]was used as a probe. Hybridization was performed as described previously [1].

2.4 Plasmid construction
We employed the cytomegalovirus (CMV)-p65 expression vector, in which p65 cDNA was under the control of the CMV enhancer/promoter, and the elongation factor (EF)-NF-IL6 expression vector, in which NF-IL6 cDNA was under the control of the EF-1{alpha} enhancer/promoter for the NF-{kappa}B (p65) and NF-IL6 transfection study.

Five different lengths of the IL-6 gene promoter, from positions –840 to +12, –417 to +12, –179 to +12, –140 to +12, or –60 to +12 were prepared by polymerase chain reaction (PCR) method. PCR was performed using human genomic DNA (100 ng) as a template. Oligonucleotide primers were designed according to the sequences as reported previously [21]. The IL-6 promoter-luciferase plasmids were constructed by inserting these PCR products into the luciferase reporter gene and expressed as p840, p417, p179, p140 or p60, respectively. Thereafter, three kinds of mutant IL-6 promoter-luciferase plasmids were also constructed. In one, the NF-{kappa}B-binding site was disrupted by converting GGGATTTTCC to AATATTTTCC (pm{kappa}B), and in another, the NF-IL6-binding site was disrupted by converting ACATTGCACAATCT to ACACTACAAACTCT (pmIL6) [13]. The plasmid disrupted both sites: pm{kappa}B/IL6 was constructed by using the site-directed mutagenesis kit (LA PCR in vitro mutagenesis kit; Takara, Otsu, Japan) as described previously [22]. Each plasmid was confirmed by sequencing using an automatic sequence analyzer (ABI PRISM 310; Applied Biosystems, Foster City, CA, USA).

2.5 Luciferase assays
Transient transfection of cultured cardiac myocytes was performed using Transfectam (BioSepra, Marlborough, MA, USA), according to the manufacturer’s instructions. Within 16–24 h after plating, cultured cardiac myocytes were incubated with a transfection mixture with 3 µg per dish of reporter plasmids and 1 µg per dish of RSV-β-galactosidase expression vector (pCH110) as an internal control plasmid. After 2 h, the same volume of M-199 with 10% NCS was added and the cells were incubated for another 8 h. Cardiac myocytes were maintained with M-199 with 10% NCS for 2 days and exposed to hypoxia or IL-1β for 4 h. Total cell lysates were collected and luciferase activity was measured by luminometer using Pica Gene luciferase assay system (Toyo Ink, Tokyo, Japan). β-galactosidase activities were obtained from the same samples by measuring O.D. at 420 nm.

2.6 Electrophoretic gel mobility shift assays (EMSAs)
Cardiac myocytes were harvested after 1-h stimulation of hypoxia or IL-1β using 0.5 ml of hypotonic buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM phenylmethyl sulfonyl fluoride (PMSF). After 10 min of incubation on ice, 55 µl of 10% NP-40 was added to the lysates and mixed by vortex. Thereafter, the lysates were centrifuged at 10 000 rpm for 10 min and the precipitants were resuspended by 50 µl of hypertonic buffer containing 10 mM HEPES, 420 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM PMSF. They were mixed by vortex and centrifuged at 15 000 rpm for 30 min. The supernatants were used as crude nuclear extracts.

Complementary 22-bp oligonucleotides containing the NF-{kappa}B-binding site (Gel Shift Competition Oligonucleotides B; STRATAGENE, La Jolla, CA, USA) are as follows: 5'-GATCGAGGGGACTTTCCCTAGC-3' and 5'-GCTAGGGAAAGTCCCCTCGATC-3'. Complementary 24-bp oligonucleotides representing a portion of the IL-6 promoter containing the putative NF-IL6-binding site, were prepared as follows [23]: 5'-CGACGTCACATTGTGCAATCTTAA-3' and 5'-TTAAGATTGCACAATGTGACGTCG-3'. Oligonucleotides were annealed and 5'-end labeled with [{gamma}-32P]ATP with T4 polynucleotide kinase (Takara, Otsu, Japan) using standard procedures. Binding reactions were performed by pre-incubating 5 µg of crude nuclear extracts, 4 µg poly(dI-dC)poly(dI-dC) (Pharmacia Biotech, Uppsala, Sweden) and double-stranded 32P-labeled oligonucleotides (1x105 cpm/assay) in 7.5 mM HEPES, pH 7.6; 0.9 mM EDTA; 3.6 mM MgCl2, 10% glycerol; 4.7 mM DTT for 30 min at room temperature.

Samples were loaded directly on to non-denaturing 4% polyacrylamide/bisacrylamide gel prepared in 50 mM Tris, 380 mM glycine and 2 mM EDTA. Electrophoresis was performed at 20 mA for 1–2 h at room temperature. For the competition studies, a 100-fold molar excess of one of the above unlabelled probes was employed. The oligonucleotide probes used for mutant NF-IL6 were prepared as follows: 5'-CGACGTCACACTATGAACTCTTAA-3' and 5'-TTAAGAGTTCATAGTGTGACGTCG-3'. Oligonucleotide probes used for mutant NF-{kappa}B were prepared as follows: 5'-GATCGAGAATACTTTCCCTAGC-3' and 5'-GCTAGGGAAAGTATTCTCGATC-3'.

Supershift experiments were performed as described above except that 1 µg of antibody to NF-{kappa}B; p50 (NF-{kappa}B1) or p65 (RelA), or C/EBP-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the binding mixture before adding the labeled probe.

2.7 Statistical analysis
Statistical analyses were performed using Student’s t-test. A value of P less than 0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Hypoxic or IL-1β stimulation induces IL-6 mRNA expression in cardiac myocytes
The initial experiments were performed to examine the expression of IL-6 mRNA by cardiac myocytes. Neonatal rat cardiac myocytes were cultured under hypoxic or IL-1β (10 ng/ml) stimulation for the time indicated. As shown in Fig. 1, unstimulated cardiac myocytes lacked detectable IL-6 mRNA expression by Northern blot analysis (lane 1). However, IL-1β stimulation induced expression of IL-6 mRNA as early as 30 min (lane 2) and reached a maximal level at 60 min (lane 3). Hypoxic stimulation also induced the expression of IL-6 mRNA in cardiac myocytes (lanes 4 and 5). Ethidium bromide-stained 18S ribosomal RNA bands are shown to indicate equal loading of total RNA (Fig. 1; lower panel).


Figure 1
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  		Fig. 1 Expression of IL-6 mRNA in cardiac myocytes. Cultured neonatal rat cardiac myocytes (1.2x105 cells per ml) were stimulated with IL-1β (10 ng/ml) or hypoxia. Total RNA was isolated after 30 or 60 min of stimulation, separated by 1.2% formaldehyde–agarose gel electrophoresis, and blotted onto a nylon membrane (Hybond N+). Murine IL-6 cDNA fragment was used for hybridization (upper panel). Ethidium bromide-stained 18S ribosomal RNA bands are shown to indicate equal loading of RNA (lower panel). Data are representative of three separate experiments.

 
3.2 NF-{kappa}B and NF-IL6 cooperatively activate the IL-6 promoter
To study the effects of NF-{kappa}B and NF-IL6 on the transcriptional activation of the IL-6 gene in cardiac myocytes, we performed transient cotransfection assays using the IL-6 promoter (–179/+12)-luciferase reporter plasmid and recombinant NF-{kappa}B (p65) and NF-IL6 proteins. The expression vectors for p65 (1 µg/dish) and/or NF-IL6 (0.05 µg/dish) were cotransfected with the IL-6 promoter-luciferase plasmid and β-galactosidase gene into cultured cardiac myocytes. As shown in Fig. 2, transfection of p65 or NF-IL6 expression vector alone increased luciferase activity 4.7 and 2.7 fold, respectively, in our system. When the expression vectors for both p65 and NF-IL6 were cotransfected simultaneously, a 6.3-fold increase in luciferase activity was observed. This result showed that NF-{kappa}B and NF-IL6 cooperatively activate the IL-6 promoter in cardiac myocytes, which was consistent with the previous study in other cell systems [13].


Figure 2
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  		Fig. 2 Activation of the IL-6 gene promoter by cotransfection of expression vectors for NF-{kappa}B (p65) and/or NF-IL6 in cardiac myocytes. Cultured neonatal rat cardiac myocytes (1.2x105 cells per ml) were cotransfected with the IL-6 promoter (–179/+12)-luciferase plasmid (p179), β-galactosidase and expression vectors for NF-{kappa}B (p65; 1.0 µg/dish) and/or NF-IL6 (0.05 µg/dish). Cellular extracts were prepared 48 h after the transfection and luciferase activity was examined. Transfection efficiencies were normalized by β-galactosidase activity. One unit of relative activity represents the luciferase activity obtained from cardiac myocytes transfected with the IL-6 promoter-luciferase plasmid only. Values are given as means±SEM. The experiments were repeated three times, and similar results were obtained. * P<0.05 for neither p65 nor NF-IL6 expression vector.

 
3.3 Effects of disruption of either the NF-{kappa}B or NF-IL6 site on IL-1β-induced activation of the IL-6 gene
Previous studies with deletion mutants in other cell systems demonstrated that both NF-{kappa}B- and NF-IL6-binding sites in the promoter region of the IL-6 gene were essential for transcriptional activation of the IL-6 gene [13]. To examine whether these two sites are important for transcriptional activation of the IL-6 gene by IL-1β stimulation in cardiac myocytes, cultured cardiac myocytes were transfected with –179/+12-luciferase reporter plasmids (p179) and stimulated with IL-1β.

As shown in Fig. 3, a 12-fold increase in promoter activity was observed when cardiac myocytes were stimulated with 1 ng/ml of IL-1β for 4 h. The promoter region from –179 to +12 contains both NF-IL6 and NF-{kappa}B binding sites. To confirm the significance of these binding sites for IL-1β-induced IL-6 expression in cardiac myocytes, either the NF-{kappa}B (pm{kappa}B) or NF-IL6 (pmIL6) binding site was disrupted by multiple nucleotide sequence substitutions. The luciferase activity in IL-1β-stimulated cardiac myocytes transfected with either the pm{kappa}B or pmIL6 increased 3.1 or 9 fold, respectively (Fig. 3). Although there was no significant differences in the luciferase activity observed in p179 and pmIL6, augmented luciferase activity after IL-1β stimulation was significantly attenuated in pm{kappa}B. These results indicate that the disruption of the NF-{kappa}B binding site drastically impaired promoter activity and that NF-{kappa}B might be critical for transcriptional activation of the IL-6 gene in cardiac myocytes stimulated with IL-1β.


Figure 3
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  		Fig. 3 Effects of disruption of either the NF-{kappa}B or NF-IL6 site on the IL-1β-induced activation of the IL-6 gene. Cultured cardiac myocytes were transfected with IL-6 promoter-luciferase plasmid (p179, pm{kappa}B, or pmIL6) and stimulated with 1 ng/ml IL-1β for 4 h. The luciferase activity was normalized by β-galactosidase activity. Values are given as means±SEM of three samples. Similar results were obtained in three independent experiments. * P<0.05 for IL-1β (–); {dagger} P<0.05 for p179 IL-1β (+).

 
3.4 IL-6 gene promoter contains hypoxia-responsive element
To investigate the transcriptional activation of the IL-6 gene under hypoxic stimulation, five different lengths of IL-6 promoter-luciferase plasmids were constructed and transfected into cardiac myocytes and luciferase activity was measured with or without hypoxic stimulation. As shown in Fig. 4, the –840/+12-luciferase plasmid (p840) presented enhanced promoter activity (5.4 fold) after hypoxic stimulation. The deletion constructs; –417/+12-luciferase plasmid (p417), –179/+12-luciferase plasmid (p179) and –140/+12-luciferase plasmid (p140) also presented significantly augmented activation after hypoxia. Because of the significantly increased basal activity in p140 (P<0.05 vs. p840), the augmented promoter activity in p140 was decreased to 2.6 fold after hypoxic stimulation. The –60/+12-luciferase plasmid (p60) presented significantly low basal activity and the activity was unaffected after hypoxic stimulation. These data suggest that the –179/-60 region, containing NF-{kappa}B and NF-IL6 binding sites, is involved in the hypoxia induced transcriptional activation of the IL-6 gene.


Figure 4
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  		Fig. 4 IL-6 gene promoter contains hypoxia-responsive element. Cultured cardiac myocytes were transfected with five different lengths of IL-6 promoter (–840/+12, –417/+12, –179/+12, –140/+12 or –60/+12)-luciferase plasmids (p840, p417, p179, p140 or p60, respectively) and incubated under hypoxia (95% N2/5% CO2) for 4 h. Luciferase assay was performed as described in Fig. 2. Values are given as means±SEM of five samples. Similar results were obtained in three independent experiments. * P<0.05 vs hypoxia (–); {dagger} P<0.05 vs p840 hypoxia (–).

 
3.5 Effects of disruption of either the NF-{kappa}B or NF-IL6 site on hypoxia-induced activation of the IL-6 gene
We next examined the effects of disruption of either the NF-IL6 or NF-{kappa}B binding site on hypoxia induced activation of the IL-6 gene in cardiac myocytes. As shown in Fig. 5, a 5.7-fold increase in luciferase activity was observed when cardiac myocytes were subjected to hypoxic stimulation for 4 h (p179). To confirm the significance of these binding sites for hypoxia-induced IL-6 expression in cardiac myocytes, either pm{kappa}B or pmIL6 was transfected. The increase in promoter activity obtained after hypoxic stimulation was attenuated to 2.9 or 4.1 fold with pm{kappa}B or pmIL6, respectively. Moreover, when both the NF-IL6 and NF-{kappa}B binding sites were disrupted (pm{kappa}B/IL6), hypoxic stimulation increased promoter activity only 1.5 fold. The augmented promoter activity after hypoxic stimulation was significantly deteriorated in pm{kappa}B and pm{kappa}B/IL6 (P<0.05 vs. p179). These results indicate that NF-{kappa}B binding site is important for transcriptional activation of the IL-6 gene in hypoxic cardiac myocytes.


Figure 5
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  		Fig. 5 Effects of disruption of NF-{kappa}B and/or NF-IL6 site on the hypoxia-induced activation of the IL-6 gene. Cultured cardiac myocytes were transfected with the IL-6 promoter luciferase plasmid (p179, pm{kappa}B, pmIL6 or pm{kappa}B/IL6) and incubated under hypoxia (95% N2/5% CO2) for 4 h. The luciferase assay was performed as described in Fig. 2. Values are given as means±SEM of five samples. Similar results were obtained in three independent experiments. * P<0.05 hypoxia (–); {dagger} P<0.05 vs p179 hypoxia (+).

 
3.6 NF-{kappa}B and NF-IL6 are activated in hypoxic cardiac myocytes
To confirm the results obtained from the above transient transfection assays, EMSAs and supershift assays were performed. EMSAs demonstrated enhanced binding activity using an NF-{kappa}B motif as a labeled probe and extracts from cardiac myocytes exposed to IL-1β or hypoxia (Fig. 6, lanes 1–5; arrowhead). IL-1β (1 ng/ml) induced more enhanced activity than that observed by hypoxic stimulation. Competition experiments showed that a 100-fold molar excess of the unlabelled NF-{kappa}B motif completely blocked the appearance of the IL-1β- or hypoxia-induced binding complex (Fig. 6, lanes 2 and 6), whereas excess unlabelled mutant probe showed no effect (Fig. 6, lane 3). As proteins interact with NF-{kappa}B motifs, supershift assays were performed. The addition of antibody to p50 or p65 resulted in the appearance of a more slowly migrating supershift bands (Fig. 6, lanes 7 and 8; arrows) with attenuation of the major, more rapidly migrating band induced by hypoxia.


Figure 6
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  		Fig. 6 Hypoxia-mediated enhancement of nuclear binding activity for NF-{kappa}B motif in cardiac myocytes. Cultured cardiac myocytes were stimulated with 1 ng/ml IL-1β (lanes 1–3) or hypoxia (lanes 5–8) for 1 h. Crude nuclear extracts were incubated in the presence of a 100-fold molar excess of the unlabelled probe (lane 2 and 6) or the unlabelled mutant probe (lane 3), 1 µg of anti-p50 (NF-{kappa}B1) antibody (lane 7) or 1 µg of p65 (Rel A) antibody (lane 8). Electrophoretic gel mobility shift assays were performed. These experiments were repeated three times with identical results. Arrowhead: A induction of DNA-binding activity to NF-{kappa}B motif in cardiac myocytes stimulated with IL-1β or hypoxia.

 
Incubation of an NF-IL6 motif as a labeled probe with nuclear extracts of cardiac myocytes exposed to hypoxia or IL-1β showed an induction of DNA-binding activity compared with the control (Fig. 7, lanes 1–3; arrowhead). Competition studies with the excess unlabelled NF-IL6 motif probe completely prevented enhancement of the hypoxia-induced band (Fig. 7, lane 4), whereas excess unlabelled mutant probe showed no effect (Fig. 7, lane 5). Supershift assay using antibody to C/EBP β (NF-IL6) demonstrated that NF-IL6 contributed to the hypoxia-induced activation of NF-IL6 binding activity (Fig. 7, lane 6; arrowhead). The probe for NF-IL6 motif formed an unknown complex (Fig. 7; open arrowhead), which was unchanged with or without stimulation.


Figure 7
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  		Fig. 7 Hypoxia-mediated enhancement of nuclear binding activity for NF-IL6 motif in cardiac myocytes. Cultured cardiac myocytes were stimulated with 1 ng/ml IL-1β (lanes 2) or hypoxia (lanes 3–6) for 1 h. Crude nuclear extracts were incubated in the presence of a 100-fold molar excess of the unlabelled probe (lane 4), unlabelled mutant NF-IL6 probe (lane 5) or 1 µg of anti-C/EBP-β (NF-IL6) antibody (lane 6). Electrophoretic gel mobility shift assays were performed. These experiments were repeated three times with identical results. Arrowhead: An induction of DNA-binding activity to NF-IL6 motif in cardiac myocytes stimulated with IL-1β or hypoxia. Open arrowhead: unknown band detected in the samples either with or without stimulation.

 
EMSAs were also performed by incubating these probes with nuclear extracts from NMC (Fig. 8). Although, nuclear extracts from IL-1β-stimulated NMC induced the activation of both transcription factors; NF-{kappa}B (Fig. 8A, lane 3; arrow) and NF-IL6 (Fig. 8B, lane 3; arrow), hypoxic stimulation showed no effect. These results indicated that hypoxia-induced activation of NF-{kappa}B and NF-IL6 occurred mainly in cardiac myocytes, not in NMC isolated from the heart.


Figure 8
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  		Fig. 8 NF-{kappa}B motif (A) or NF-IL6 motif (B) binds to the nuclear extracts from non-myocardial cells. Non myocardial cells (NMC) were stimulated with hypoxia (lane 2) or 1 ng/ml IL-1β (lanes 3) for 1 h. Crude nuclear extracts were incubated with NF-{kappa}B motif (A) or NF-IL6 motif (B) as in Figs. 6 and 7Go. These experiments were repeated three times with identical results. Arrow: An induction of DNA-binding activity to NF-{kappa}B and NF-IL6 motifs in IL-1β-stimulated NMC. Open arrowhead: unknown band detected in the samples either with or without stimulation.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The comparison of nucleotides between mouse and human IL-6 genes revealed that the sequence within the first 300-bp of the 5'-flanking region was highly conserved [21]. We reported the presence of cis-acting regulatory elements, including NF-{kappa}B- and NF-IL6-binding sites in the first 180-bp sequence of the 5'-flanking region of the IL-6 gene [12]. A previous study demonstrated that NF-{kappa}B and NF-IL6 revealed a central role in the transcriptional regulation of the IL-6 gene expression [13]. However, the molecular mechanisms underlying transcriptional activation of the IL-6 gene in cardiac myocytes has not been examined yet. In the present study, we investigated the trans-acting factors that induce transcriptional upregulation of the IL-6 gene in hypoxic cardiac myocytes.

IL-1β is reported to induce upregulation of inducible nitric oxide synthase [24]and cardiac myocyte growth [25]and a well-known stimulus for IL-6 production in many other cells. Transient transfection assays using p179 and two kinds of mutant IL-6 promoter-luciferase plasmids (pm{kappa}B and pmIL6) presented that IL-1β augmented the IL-6 gene promoter activity in cardiac myocytes and that NF-{kappa}B plays a major role in the induction of the IL-6 gene in cardiac myocytes. The results obtained from EMSAs demonstrated that IL-1β apparently augmented the DNA-binding activity to NF-{kappa}B in cardiac myocytes.

Exposure of cardiac myocytes to hypoxia increases the transcription and release of the IL-6 gene product [1]. Although there have been many studies concerning hemodynamic or physiological mechanisms under ischemic or hypoxic conditions, little is known about the molecular mechanism underlying the response of cardiac myocytes to hypoxia, including signal transduction and consequent regulation of the target genes. Hypoxia-induced expression of IL-6 is not limited to cardiac myocytes, but is also observed in vascular smooth muscle cells [26]and endothelial cells [3]. Recently, Yan and colleagues reported that hypoxia-induced IL-6 expression in endothelial cells was mediated by the activation of NF-IL6 [3]and transgenic mice bearing the NF-IL6 site showed promoted expression of the transgene in the heart after hypoxic stimulation [27]. On the contrary, IL-8 gene induction in endothelial cells after hypoxic stimulation is reported to associate with the increased binding activity in the nuclear extract for the NF-{kappa}B site [4].

In the present study, we demonstrated that IL-6 gene expression is induced by hypoxic stress mainly through the activation of NF-{kappa}B in cardiac myocytes. Transfection assays using five kinds of deletion mutations clearly showed that the –140/–60 region is involved in hypoxia-induced transcriptional activation and using three kinds of mutations represented the significance of the NF-{kappa}B binding site in the activation of the IL-6 gene under hypoxic stress. Although NF-IL6 binding activity was enhanced by hypoxic stimulation (Fig. 7), NF-IL6 might not be the primary positive regulator of the hypoxia-induced IL-6 mRNA expression.

The supershift assays demonstrated that the proteins which are activated by hypoxic stimulation and which interacted with the NF-{kappa}B motif consisted of p50 and p65. These results are consistent with the previous report that NF-{kappa}B activated by tumor necrosis factor (TNF) consisted of p50 and p65 in endothelial cells [4]. Many stimuli are known to activate NF-{kappa}B. Recently, the tumor necrosis receptor associated factor (TRAF) family is reported to mediate the activation of NF-{kappa}B by TNF or IL-1β [28, 29]. Another report provided the evidence that hypoxic activation of NF-{kappa}B is mediated by Ras and Raf signaling pathways but not by mitogen-activated protein (MAP) kinase in NIH3T3 cells [30]. In cardiac myocytes, hypoxic stress is reported to activate the Src family tyrosine kinase, Ras, Raf-1, MAP kinase kinase and MAP kinase [31–33]. However, the intracellular mechanism of activation of NF-{kappa}B in cardiac myocytes is not well understood.

The supershift assay using anti-C/EBP β (NF-IL6) demonstrated that the DNA–protein complex which was induced by hypoxic stress was proved to be NF-IL6. The activation of NF-IL6 was reported to be mediated by MAP kinase. As hypoxia is known to activate Ras/MAP kinase cascade, the activation of NF-IL6 under hypoxic condition in cardiac myocytes might be mediated by MAP kinase [32, 33].

The unresponsiveness of the fibroblasts to hypoxic stress was observed in the activation of transcription factors, NF-{kappa}B and NF-IL6 (Fig. 8). Cell-specificities of gene regulation by hypoxic stimulation were also reported previously [2, 34]. The findings in the present study could explain the mechanisms of our previous results that the expression of IL-6 mRNA was induced by the hypoxic stress in cardiac myocytes, but not induced in the fibroblasts [1].

Because of the low expression level of IL-6 receptor in cardiac myocytes, IL-6 derived from cardiac myocytes might act on other cells, such as fibroblasts or lymphocytes in paracrine mechanism. IL-6 is reported to suppress inflammation with the inhibition of the production of IL-1β or TNF [9]. Especially in experimental viral myocarditis, IL-6 administration was reported to decrease viral titer, viral replication and myocardial injury associated with reduced titer of TNF in serum [10]. A recent study using IL-6-deficient mice also revealed that endogenous IL-6 plays an anti-inflammatory role in acute inflammatory responses by controlling the level of pro-inflammatory cytokines, but not anti-inflammatory cytokines [35]. There are many other genes than IL-6 that have both NF-{kappa}B and NF-IL6 binding sites in promoter region, such as IL-8, angiotensinogen, inducible nitric oxide synthase, ICAM-1 and G-CSF. Activation of NF-{kappa}B and NF-IL6 under hypoxic stress would regulate these genes, which might modulate pathophysiological consequences in the ischemic heart. These results are the first step in confirming NF-IL6 and NF-{kappa}B as transcription factors that induce gene expression under hypoxic conditions in cardiac myocytes.

In conclusion, we demonstrated that hypoxic stimulation induced transcription factors, both NF-{kappa}B and NF-IL6 in cardiac myocytes, and NF-{kappa}B played crucial role in the trans-activation of the IL-6 gene in the context of hypoxia.

Time for primary reveiw 21 days.


   Acknowledgements
 
This study was supported by a Grant-in-Aid for Scientific Research of Priority Areas from the Japanese Ministry of Education, Science and Culture and grants from the Study Group of Molecular Cardiology, The Cell Science Research Foundation and Japan Heart Foundation: Pfizer Pharmaceutical Grant for Research on Coronary Artery Disease. The authors wish to thank Ms. Y. Yamaguchi and Ms. M. Katayama for excellent secretarial assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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