Dominant-negative effect of the c-fos family gene products on inducible NO synthase expression in macrophages

Seiji Okada1,2, Shintaro Obata1, Masahiko Hatano1 and Takeshi Tokuhisa1

1 Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan 2 Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan

Correspondence to: T. Tokuhisa; E-mail: tokuhisa{at}med.m.chiba-u.ac.jp
Transmitting editor: T. Watanabe


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Activation of murine peritoneal macrophages or the macrophage cell line RAW264 with IFN-{gamma} and bacterial lipopolysaccharide promotes a transient up-regulation of c-fos family gene expression following inducible NO synthase (iNOS) production. Since introduction of a double mutation into the two AP-1-binding sites in the iNOS promoter region reduced the promoter activity to 25% of the authentic one in activated RAW264 cells, the induced c-Fos/AP-1 may promote iNOS expression in activated macrophages. Surprisingly, overexpression of c-fos in activated macrophages completely suppressed the production of iNOS, but not that of IL-6 and IL-1ß. The regulatory effect was also observed by overexpression of c-fos, c-jun or fosB on the promoter activity as deduced from transfection experiments. However, the mutation of AP-1-binding sites in the promoter region did not abrogate the regulatory effect of c-fos and the effect of c-fos was diminished by co-transfection with c-jun, but not with fosB, suggesting no relation between the regulatory effect and a c-Fos/AP-1 complex. Expression of NF-IL6 (C/EBPß), whose gene product can make a non-functional heterodimer with c-Fos family proteins, was transiently induced in activated macrophages. Overexpression of NF-IL6 in activated RAW264 cells augmented iNOS promoter activity and reduced the regulatory effect of c-fos overexpression. Thus, overproduction of c-Fos family proteins acts as a dominant-negative-type regulator on iNOS expression in activated macrophages.

Keywords: monocyte/macrophage, NO, cellular activation, transcription factor


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
High-output NO production from activated macrophages represents a major mechanism for macrophage cytotoxicity against a variety of microbes (13). Activated macrophages also induce cytokine productions such as tumor necrosis factor (TNF)-{alpha}, IFN-{gamma}, IL-1ß and IL-6, and those cytokines favor the amplification of NO production (4). Despite its beneficial role in host defense, sustained NO production can be deleterious to the host. The diversity of the pathologic conditions by NO is based on the existence of different signals and implies the involvement of different transcriptional activators to control inducible NO synthase (iNOS) transcription. Indeed, iNOS induced by cytokines and/or inflammatory stimuli has been implicated in experimental arthritis (5), inflammatory bowel disease (6), hypotension associated with septic shock (7) and other types of tissue injury (13). Therefore, the proper regulation of iNOS production during an inflammatory process is critical for host defense.

For murine macrophages, IFN-{gamma} and lipopolysaccharide (LPS) represent the strongest stimuli (3,8), and the most potent combinations of synergizing stimuli for induction of iNOS expression (9). The murine iNOS promoter, a 1749-bp fragment from the 5'-flanking region of the iNOS gene, contains numerous consensus binding sequences for known transcription factors induced by stimulation with IFN-{gamma} or LPS (10,11). These include two copies each of NF-{kappa}B, AP-1 and TNF response elements. Many of these response elements are located in two clusters of the promoter region: region I (ranging from +10 to –300 bp upstream of the TATA box) and region II (–1100 to –800 bp). Region II is supposed to be primarily important for IFN-{gamma}-mediated induction of iNOS because it contains binding sites for IFN regulatory factor-1, signal transducers and activators of transcription 1, and NF-{kappa}B (10). On the other hand, region I has been shown to be the principal target region for LPS-mediated iNOS induction since it contains three NF-IL6-binding sites, one TNF response element and one NF-{kappa}B-binding site.

A number of groups have demonstrated the importance of this proximal promoter region for iNOS expression (1013). Since introduction of a double mutation into the NF-IL6-binding site (–153/-142) of an iNOS promoter construct resulted in a reduction of the promoter activity by ~90% after stimulation with IFN-{gamma} and LPS (12), this NF-IL6-binding site may be important for maintaining a high transcriptional rate of the iNOS gene. However, expression of iNOS and pro-inflammatory cytokines by NF-IL6-deficient macrophages is unexpectedly comparable to that observed with wild-type macrophages (14). NF-IL6 (C/EBPß), a member of the basic leucine zipper family of transcription factors, exhibits homology with C/EBP{alpha} and C/EBP{delta} (15). All three of the C/EBP factors are expressed by macrophages, and the DNA-binding activity of NF-IL6 and C/EBP{delta} is increased by treatment with LPS, while that of C/EBP{alpha} is decreased (16). Expression of any of these transcription factors is sufficient to confer the LPS-inducible expression of IL-6 (16). Therefore, it is likely that the lack of NF-IL6 is compensated for by the induction of C/EBP{delta} upon LPS treatment.

When macrophages are activated with IFN-{gamma} and LPS, expression of the immediate-early genes is transiently induced within 1 h after stimulation. These immediate-early gene products may transduce initial signals from cell-surface receptors to the iNOS gene. The immediate-early gene c-fos and its family genes are also induced in macrophages activated with IFN-{gamma} and LPS. Products of the c-fos family genes (c-fos, fosB, fra-1 and fra-2) can make a heterodimer with products of the jun family (c-jun, junB and junD) to compose the nuclear transcription factor AP-1 (1719). Although the iNOS promoter region contains two copies of the AP-1 response element (11), the role of c-fos family gene products in activation of the iNOS gene has not been determined. Here we show that introduction of a double mutation into the two AP-1-binding sites in the iNOS promoter region reduced the promoter activity to 25% of the authentic one, suggesting a role for the induced c-Fos/AP-1 in iNOS expression in activated macrophages. However, overexpression of c-fos in activated macrophages resulted in the specific down-regulation of iNOS expression and the regulatory mechanism of iNOS expression was not due to overproduction of a c-Fos/AP-1 complex. Since c-Fos family proteins can make a non-functional heterodimer with a member of the basic leucine zipper family protein (20), a part of the regulatory mechanisms by c-fos overexpression may be due to a dominant-negative-type regulation of a member of the basic leucine zipper family protein including C/EBP family proteins. We discuss the dominant-negative-type regulation of iNOS expression by c-fos overexpression in activated macrophages.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice, a cell line and reagents
C57BL/6CrSlc mice were purchased from Japan SLC (Hamamatsu, Japan). Transgenic mice carrying the mouse c-fos gene under the control of the Mx gene promoter (Mx-c-fos) and c-fos-deficient mice (21,22) were maintained by heterozygous mating in our animal facilities. A murine macrophage cell line RAW264 was obtained from RIKEN Cell Bank (Tsukuba, Japan). Recombinant murine IFN-{gamma} (sp. act. 1.0 x 107 U/mg) was purchased from Genzyme (Cambridge, MA). LPS (from Escherichia coli, serotype 0111:B4) was purchased from Sigma (St Louis, MO).

Macrophage cell culture
Macrophages from mice were prepared from resident peritoneal cells or bone marrow cells. Peritoneal cells were harvested without elicitation and cultured in six-well tissue culture plates for 2 h at 37°C in 5% CO2. Bone marrow-derived macrophages were generated by culturing non-adherent bone marrow cells with 200 U/ml rIL-3 for 10 days. After the culture, non-adherent cells were removed by extensive washing, and adherent cells were harvested by tripsinization and used as macrophages (8). Macrophages were cultured in RPMI 1640 medium (Sigma) supplemented with 100 µg/ml streptomycin sulfate (Wako, Osaka, Japan), 100 U/ml penicillin G potassium (Banyu, Tokyo, Japan) and 10% (v/v) heat-inactivated FCS (Sigma).

Assay for NO synthesis and cytokine productions
Synthesis of NO by activated macrophages was measured by the assay for nitrite (NO2) in culture supernatants, as previously described (4). Briefly, 100 µl of culture supernatants was mixed with an equal volume of the Griess reagent [0.5% sulfanilamide, 0.05% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4] in a 96-well tissue culture plate for 10 min at room temperature. The absorbance of samples was measured on a plate reader (Bio-Rad, Richmond, CA) at 570 nm. The nitrite concentration was calculated using sodium nitrite as a standard. Cytokines released into culture supernatants were detected by the Cytokine Immunoassay Kit (Biosource, Camarillo, CA) according to the manufacturer’s protocol.

Measurement of H2O2 production by a flow cytometry
H2O2 was measured by a method previously described with modification (23). Briefly, peritoneal macrophages were stimulated with IFN-{gamma} (20 U/ml), LPS (10 µg/ml) and phorbol myristate acetate (100 ng/ml) (Sigma) for 24 h. 2'7'-Dihydrodichlorofluorescein diacetate (DCFH-DA, 20 µM; Molecular Probes) was added to the culture and incubated for 15 min. The DCFH-DA fluorescence was analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA).

Northern blot analysis
Total RNA was isolated from macrophages using the TRIzol RNA isolation reagent (Life Technologies, Grand Island, NY). mRNA was detected by Northern blot as previously described (8). Briefly, total RNA (10 µg) was loaded on a 1.0% agarose gel and transferred to a nylon membrane (Boehringer Mannheim, Mannheim, Germany). The filter was hybridized with a digoxigenin (DIG)-labeled probe overnight at 50°C. The probe on the filter was detected with sheep anti-DIG antibody conjugated with alkaline phosphatase (Boehringer Mannheim). Murine iNOS cDNA (24), murine c-fos, fosB, c-jun, junB, junD, fra-1 and fra-2 (gifts from Dr E. F. Wagner, Vienna, Austria), and murine NF-IL6 (a gift from Dr S. Akira, Osaka, Japan) were subcloned into pGEM vectors and labeled by DIG, using PCR with T7 and SP6 primers, then used as a probe.

RT-PCR analysis
Total RNA was reverse transcribed using Superscript (Life Technologies) and oligo(dT) (Pharmacia, Piscataway, NJ), in a final volume of 20 µl, and 1 µl of cDNAs was used for PCR. After an initial 5-min incubation at 94°C, the 30 cycles of PCR were carried out using the following conditions: iNOS, IL-1ß, IL-6, TNF-{alpha} and IFN-{gamma} cDNAs; denaturation at 94°C for 40 s, annealing at 55°C for 50 s and polymerization at 72°C for 60 s; G3PDH cDNA; denaturation at 94°C for 40 s, annealing at 60°C for 50 s and polymerization at 72°C for 60 s. PCR primers for the cDNA amplification were as follows: iNOS primers, 5'-TCTCATCAGTTCTATGGCCC-3' and 5'-GGGAGTAGACAAGGTACAAC-3'; IL-1ß primers, 5'-TTGACGGACCCCAAAAGATG-3' and 5'-GAGAAGGTGCTCATGTCCTCA-3'; IL-6 primers, 5'-GTTCTCTGGGAAATCGTGGA-3' and 5'-TGTACTCCAGGTAGCTATGG-3'; TNF-{alpha} primers, 5'-TCTCATCAGTTCTATGGCCC-3' and 5'-GGGAGTAGACAAGGTACAAC-3'; IFN-{gamma} primers, 5'-AGCGGCTGACTGAACTCAGATTGTAG-3' and 5'-GTCACAGTTTTCAGCTGTATAGGG-3'; and G3PDH primers, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGTAGGCCATGAGGTCCACCAC-3'.

Plasmid construction and reporter gene analysis
A 1749-bp HincII fragment corresponding to the 5'-flanking region of murine iNOS fused to a luciferase reporter gene (BNXH-iNOS-Luciferase) was provided by Dr C. K. Glass (San Diego, CA). Point mutations of the AP-1-binding site from –1069 to –1063 (5'-TGACTTA-3' to 5'-TGCATTA-3') and from –488 to –482 (5'-TGACTTA-3' to 5'-TGCATTA-3') were introduced by the Altered Site In Vitro Mutagenesis System (Promega, Madison, WI) according to the manufacturer’s protocol, and verified by sequencing.

The murine c-fos, c-jun, fosB and NF-IL6 cDNAs were subcloned into pcDNA3 expression vectors. RAW264 cells were transiently transfected by a lipofection method using FuGene6 (Roche Molecular Biochemicals). Plasmid (total 2 µg) preincubated with 3 µl of FuGene6 in 100 µl of OPTI-MEM (Life Technologies) for 15 min at room temperature was added to 3 x 105 cells/ml in six-well plates and incubated at 37°C for 24 h. Cells were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml) for 8 h, washed once with PBS, and subjected to determination of luciferase activity by the Dual luciferase system (Promega) according to the manufacturer’s instruction. Firefly luciferase activity was corrected upon co-transfection of cells with a Renilla luciferase vector pRL-TK (Promega).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Regulatory effect of c-fos overexpression on iNOS expression in activated macrophages
Expression of the c-fos and jun family genes and the iNOS gene in a macrophage cell line RAW264 cells and peritoneal macrophages after stimulation with IFN-{gamma} and LPS was examined by Northern blot. As shown in Fig. 1, c-fos mRNA was expressed in RAW264 cells without stimulation as described (25) and expression of c-fos was clearly up-regulated within 30 min after stimulation. The expression was down-regulated 1 h after stimulation and then slightly up-regulated 6 h after stimulation. In contrast, expression of iNOS mRNA was induced 2 h after stimulation and the amount of mRNA increased until 8 h after stimulation. Expression of fosB and c-jun was induced within 30 min and down-regulated 2 h after stimulation, which is a typical expression pattern as an immediate-early gene. Expression of junB, junD, fra-1 and fra-2 was also induced within 30 min, and down-regulated 2–8 h after stimulation. Expression of c-fos and iNOS in activated peritoneal macrophages was similar to that in activated RAW264 cells (data not shown).



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Fig. 1. Expression of iNOS and c-fos family genes in the RAW264 macrophage cell line. RAW264 cells were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml). Expression of the iNOS gene, and the c-fos and jun family genes was analyzed by Northern blot. The data including 28S and 18S, ribosomal RNAs, indicate the amount of RNA loaded.

 
Since expression of the exogenous c-fos gene was strongly induced in LPS-activated lymphocytes from transgenic mice carrying the c-fos gene under the control of the Mx promoter (Mx-c-fos) (26), iNOS expression was examined in peritoneal macrophages from Mx-c-fos mice after stimulation with IFN-{gamma} and LPS by Northern blot (Fig. 2A). When expression of the exogenous c-fos was strongly induced in Mx-c-fos macrophages after stimulation, expression of iNOS mRNA was suppressed in activated Mx-c-fos macrophages until 4 h after stimulation. The suppression was confirmed by measuring NO2 activity in culture supernatants by the Griess method (Fig. 2B). NO2 was detected in the culture supernatants of control macrophages within 8 h after stimulation and the level increased until 48 h after stimulation. However, NO2 activity was not detected in the culture supernatants of Mx-c-fos macrophages until 48 h after stimulation.



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Fig. 2. Expression of iNOS mRNA and NO production by activated macrophages with c-fos overexpression. Peritoneal macrophages from Mx-c-fos and control mice were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml). (A) Expression of iNOS and c-fos genes in the macrophages was analyzed by Northern blot. The data including 28S and 18S, ribosomal RNAs, indicate the amount of RNA loaded. (B) NO production in the culture supernatants was analyzed by the Griess method. Results are the mean ± SD from triplicate cultures.

 
Expression of iNOS, IL-1ß, IL-6, IFN-{gamma} and TNF-{alpha} was analyzed in Mx-c-fos macrophages 24 h after stimulation with IFN-{gamma} and LPS by RT-PCR (Fig. 3A). Expression of iNOS was not detected in Mx-c-fos macrophages after stimulation. Since expression of IL-1ß, IL-6 and IFN-{gamma} was induced in both Mx-c-fos and control macrophages after stimulation, overexpression of c-fos specifically regulated the iNOS expression. Expression of TNF-{alpha} was detected in both control and Mx-c-fos macrophages before stimulation, and induced in them after stimulation. However, the amount of TNF-{alpha} in activated Mx-c-fos macrophages was lower than that in control macrophages. This reduction of TNF-{alpha} production was confirmed at the level of protein in culture supernatants (Fig. 3B). The level of TNF-{alpha}, but not that of IL-6 and IL-1ß, produced by Mx-c-fos macrophages was 100-fold lower than that by control macrophages. Activity of H2O2 production in activated Mx-c-fos macrophages was also analyzed on a flow cytometry with DCFH-DA (Fig. 3C). The activity was induced in both Mx-c-fos and control macrophages. Thus, overexpression of c-fos clearly reduced expression of iNOS and TNF-{alpha}, but not that of other genes examined in activated macrophages.



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Fig. 3. Production of inflammatory cytokines and superoxide by activated macrophages with c-fos overexpression. Peritoneal macrophages from Mx-c-fos and control mice were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml) for 24 h. (A) Expression of cytokine mRNAs in the macrophages was analyzed by RT-PCR. Triangles indicate the 3-fold dilution of cDNAs. M: DNA size marker. G3PDH indicates the amount of cDNA loaded. (B) Cytokine production in the culture supernatants was analyzed by the cytokine immunoassay. Results are the mean ± SD from triplicate cultures. (C) Superoxide production in the macrophages was analyzed by a flow cytometry. Bold and thin lines indicate the expression of DCFH-DA in the macrophages with and without stimulation respectively.

 
The relation between loss of the c-fos gene and expression of iNOS mRNA was further examined using c-fos-deficient mice. Peritoneal macrophages from c-fos-deficient mice were stimulated with IFN-{gamma} and LPS, and expression of iNOS mRNA 4 h after stimulation (Fig. 4A) and the level of NO2 in culture supernatants 48 h after stimulation (Fig. 4B) were measured by Northern blot and the Griess method respectively. However, these responses were not augmented at all by the absence of the c-fos gene. Since c-fos family genes such as fosB, fra-1 and fra-2 were also induced in activated macrophages, these results suggest the functional redundancy of c-Fos family proteins.



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Fig. 4. Expression of iNOS mRNA and NO production by activated c-fos-deficient macrophages. Bone marrow-derived macrophages from c-fos-deficient (–/–) and littermate control (+/–) mice were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml). (A) Expression of iNOS mRNA in the macrophages 4 h after stimulation was analyzed by Northern blot. ß-Actin mRNAs indicate the amount of RNA loaded. (B) NO production in the culture supernatants 48 h after stimulation was analyzed by the Griess method. Results are the mean ± SD from triplicate cultures.

 
Regulatory effect of c-fos overexpression on iNOS promoter activity
Regulatory mechanisms of iNOS expression by c-fos overexpression were analyzed using the luciferase reporter gene with the iNOS promoter. The reporter gene was transiently transfected into RAW264 cells, and luciferase activity in these transfectants was analyzed 24 h after stimulation with IFN-{gamma} and LPS (Fig. 5A). The activity was strongly induced by the stimulation. When the c-fos gene was co-transfected with the reporter gene into RAW264 cells, the activity was reduced, repeating the regulatory effect of c-fos overexpression in Mx-c-fos macrophages.



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Fig. 5. Regulatory effect of c-fos family genes on iNOS promoter activity. The reporter gene with the iNOS promoter was co-transfected with various genes into RAW264 cells. These transfected cells were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml). Relative luciferase activity in these cells was determined 24 h after stimulation. Results are the mean ± SD from three independent experiments. (A) The reporter gene was co-transfected with the c-fos expression vector (c-fos: +) or the control vector (c-fos: –). (B) The iNOS promoter with the mutation of the AP-1-binding sites was co-transfected with the c-fos expression vector (c-fos) or the control vector (pcDNA3). (C) The reporter gene was co-transfected with the indicated amounts of c-fos, c-jun, fosB or control (pcDNA3) expression vectors into RAW264 cells.

 
In order to examine whether the regulatory effect is due to c-Fos/AP-1 or not, we mutated two copies of the AP-1 response element in the iNOS promoter. These mutated reporter genes were transfected into RAW264 cells with or without the exogenous c-fos gene (Fig. 5B). These transfectants were stimulated with IFN-{gamma} and LPS, and the luciferase activity in these transfectants was measured 24 h after stimulation. When the AP-1 response element on the proximal side, but not on the distal side, was mutated, the luciferase activity was reduced by 50% of the original promoter. The mutation of both sides attenuated the activity to 25% of the original. These results suggest that AP-1 is the important transcription factor for iNOS expression. However, overexpression of c-fos negatively regulated transcriptional activity of the iNOS promoter without the AP-1 response elements.

We further examined the regulatory effect of AP-1 on iNOS expression in RAW264 cells co-transfected with the reporter gene and various combinations of the c-fos, c-jun or fosB gene (Fig. 5C). These transfectants were stimulated with IFN-{gamma} and LPS, and the luciferase activity in these transfectants was measured 24 h after stimulation. Overexpression of c-jun or fosB without c-fos also suppressed the promoter activity, suggesting that overexpression of any c-fos or jun family genes reduces the promoter activity. To examine the relation of AP-1 to the regulatory effect, various amounts of c-fos and c-jun genes were overexpressed in activated RAW264 cells. However, co-transfection of c-fos and c-jun into RAW264 cells did not reveal the regulatory effect, suggesting that the regulatory effect of c-fos overexpression is not due to overproduction of a c-Fos/AP-1 complex. Furthermore, the regulatory effect was augmented by co-transfection of c-fos and fosB into RAW264 cells. These results strongly suggest dominant-negative-type regulation by the c-Fos family proteins.

Dominant-negative-type regulatory effect of c-Fos on NF-IL6 activity in activated macrophages
Since the c-Fos family proteins can make a heterodimer with a member of the basic leucine zipper family protein (20), a part of the dominant-negative-type regulatory effect of c-fos overexpression may be due to diminished activity of the basic leucine zipper family proteins such as NF-IL6, which are important for iNOS expression (12), by making a heterodimer. To examine the possibility, expression of NF-IL6 was examined in activated macrophages by Northern blot. As shown in Fig. 6(A), expression of NF-IL6 mRNA was up-regulated in RAW264 cells within 30 min after stimulation with IFN-{gamma} and LPS, down-regulated 2 h after stimulation, and then up-regulated 4 h after stimulation. This expression pattern is similar to c-fos expression.



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Fig. 6. Regulatory effect of c-Fos on NF-IL6 activity. (A) Expression of NF-IL6 and c-fos mRNAs in RAW264 cells activated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml) was analyzed by Northern blot. G3PDH indicates the amount of cDNA loaded. (B) RAW264 cells were transiently transfected with the reporter gene with the iNOS promoter and the indicated amounts of NF-IL6, c-fos or control (pcDNA3) vectors. These cells were stimulated with IFN-{gamma} (20 U/ml) and LPS (10 µg/ml). Relative luciferase activity in these cells was determined 24 h after stimulation. Results are the mean ± SD from three independent experiments.

 
To examine effect of NF-IL6 overexpression on the regulatory effect of c-fos overexpression, these genes were co-transfected with the luciferase reporter gene with the iNOS promoter into RAW264 cells (Fig. 6B). The luciferase activity in the transfectants was measured 24 h after stimulation with IFN-{gamma} and LPS. Transfection of NF-IL6 without c-fos augmented the promoter activity to twice as much as the control level in activated RAW264 cells. Transfection of c-fos without NF-IL6 reduced the promoter activity by a dose-dependent manner. However, this reduction in the promoter activity by c-fos overexpression was clearly diminished by co-transfection of NF-IL6. These results suggest the dominant-negative-type effect of c-fos overexpression on NF-IL6 activity.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The c-fos and jun family genes are transiently up-regulated in macrophages 30 min after stimulation, and iNOS expression is detected in the macrophages 2 h after stimulation. Furthermore, the iNOS promoter region contains two copies of the AP-1 response element (11). When RAW264 cells were transiently transfected with luciferase reporter plasmids containing the 1.7-kb murine iNOS promoter with the mutation of AP-1-binding sites, the mutation of the proximal or both AP-1-binding sites reduced the activity to 50 or 25% respectively. Thus, the induced c-Fos/AP-1 may play a positive role in iNOS expression as an immediate-early gene product. Expression of the c-fos family genes is slightly up-regulated again in activated macrophages 8 h after stimulation. Since iNOS expression is continuously high in activated macrophages until 24 h after stimulation (data not shown), this second wave of c-fos family gene expression may also contribute to maintenance of iNOS expression. These results suggest that a transient up-regulation of c-Fos/AP-1 is required for full activation of the murine iNOS promoter.

Transcriptional regulation of human iNOS (hiNOS) is controlled by a 8.3-kb 5'-flanking region of the hiNOS gene (27). The region contains two copies of the AP-1 response element, and full activation of the hiNOS promoter by stimulation of cytokines (i.e. TNF-{alpha}, IL-1ß or IFN-{gamma}) also requires both downstream and upstream AP-1-binding sites (27). Thus, c-Fos/AP-1 is required for full activation of the promoter of both murine and human iNOS genes.

Overexpression of the c-fos family genes, however, suppressed iNOS expression in activated macrophages. The regulatory mechanisms of c-fos overexpression cannot be explained by overproduction of c-Fos/AP-1, because deletion of AP-1-binding regions in the promoter cannot diminish the regulatory effect of c-fos overexpression. Furthermore, co-transfection of c-fos and fosB augmented the regulatory activity of c-fos, whereas co-transfection of c-fos and c-jun attenuated the regulatory activity. c-Fos dimerizes with Jun family proteins, but not with c-Fos family proteins, to make AP-1, indicating no relation between the regulatory effect and c-Fos/AP-1. These results prompted us to speculate on a dominant-negative-type of regulation by c-fos overexpression. Since c-Fos can dimerize with a member of the basic leucine zipper family protein and this heterodimer loses the binding activity to its consensus DNA-binding motif (20), overproduction of c-Fos may act as a dominant-negative-type regulator on the activity of a member of the basic leucine zipper family protein.

The NF-IL6 gene, which is a member of the basic leucine zipper family gene, is also induced in activated macrophages as an immediate-early gene and kinetics of the expression were similar to that of c-fos. Transfection of NF-IL6 augmented iNOS promoter activity in activated RAW264 cells, suggesting the enhancer activity of NF-IL6 on iNOS expression. Since NF-IL6 can make a heterodimer with c-Fos family proteins to lose its DNA-binding activity (20), overproduction of c-Fos was suggested to diminish the activity of NF-IL6 in activated macrophages by heterodimer formation. Indeed, overexpression of NF-IL6 attenuated the regulatory effect of c-fos overexpression on iNOS promoter activity. Thus, a part of the regulatory mechanisms of c-fos overexpression could be explained by attenuation of NF-IL6 activity, probably by making a heterodimer with c-Fos.

NF-IL6 (C/EBPß) is a member of the C/EBP family (15), and transcriptional activity of NF-IL6, C/EBP{alpha} and C/EBP{delta} is redundant with regard to expression of IL-6 in activated macrophages (16). The functional redundancy of NF-IL6 in iNOS expression is also suggested by the evidence that iNOS expression by NF-IL6-deficient macrophages is comparable to that observed by wild-type macrophages (14). c-Fos can dimerize three of the C/EBP family proteins to lose their DNA-binding activity, suggesting that overexpression of c-fos may be able to suppress the activity of C/EBP family proteins in activated macrophages. CHOP, a member of the C/EBP family, contains two prolines substituting for two residues in the basic region that are critical for binding to the consensus DNA sequence and CHOP inhibits the DNA-binding activity of other C/EBP family proteins by forming a heterodimer (28). Thus, Fos and Jun family proteins like CHOP can be negative modulators of the activity of C/EBP family proteins in activated macrophages. However, a mutation into the NF-IL6-binding site of the iNOS promoter cannot completely abrogate the promoter activity in activated macrophages (12), although overexpression of c-fos completely suppressed iNOS production in activated macrophages. Thus, the regulatory mechanisms by c-fos overexpression cannot be explained only by attenuation of the activity of C/EBP family proteins. Further study is required to elucidate the regulatory mechanism of iNOS expression by c-fos overexpression in activated macrophages.

Overexpression of c-fos also suppressed the induction of TNF-{alpha} expression in activated macrophages. The promoter region of the human TNF-{alpha} gene possesses binding sites for NF-{kappa}B, NF-IL6 and c-Jun/AP-1 (2931), and each of these sites has been shown to be capable of contributing to the activation of the TNF-{alpha} promoter in macrophages (3033). Although NF-{kappa}B and NF-IL6 interact through the Rel domain and the basic leucine zipper domain to synergistically activate expression of other cytokine genes including IL-6, IL-8 and IL-12 p40 (3438), no synergy between NF-{kappa}B and NF-IL6 or c-Jun was observed in activation of the TNF-{alpha} gene (32). c-Jun enhances NF-IL6-induced activation of the TNF-{alpha} gene in macrophages (39). These results suggest that the regulatory mechanism of TNF-{alpha} expression by overexpression of c-fos is different from that of iNOS expression.

In summary, expression of c-fos family genes was induced in macrophages activated with IFN-{gamma} and LPS, and the induced c-Fos/AP-1 may promote iNOS expression in activated macrophages. However, overexpression of the c-fos family genes completely inhibited iNOS expression in activated macrophages. This regulation is not due to overproduction of c-Fos/AP-1, but due to the dominant-negative effect of the c-Fos family and a part of the effect may be to regulate the activity of a member of the basic leucine zipper family protein such as NF-IL6.


    Acknowledgements
 
We thank Dr C. K. Glass for murine iNOS fused to a luciferase reporter gene (BNXH-iNOS-Luciferase), Dr E. F. Wagner for AP-1 gene family cDNAs, Dr S. Akira for murine NF-IL6 gene, H. Satake for her skillful technical assistance and K. Ujiie for her secretarial assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan.


    Abbreviations
 
DCFH-DA—2'7'-dihydrodichlorofluorescein diacetate

DIG—digoxigenin

INOS—inducible NO synthase

LPS—lipopolysaccharide

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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