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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 25, 2002 as doi:10.1096/fj.01-0450fje. |
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Cancer Immunotherapy Laboratory of the Ecole Pratique des Hautes Etudes and INSERM U-517, University of Bourgogne, Faculty of Medicine, 21000 Dijon, France
2Correspondence: EPHE, INSERM U-517, UFR Médecine, BP 87900, 21079 Dijon CEDEX, France. E-mail : apance{at}u-bourgogne.fr
SPECIFIC AIMS
Of the three isoforms of NO synthase in mammalian cells, it is the inducible nitric oxide synthase (iNOS or NOSII) that produces a large and continuous flux of NO that can result in cell cytotoxicity. Studying the transcriptional regulation of NOSII, we found a repressive region in the proximal promoter. Deletion of this region increases cytokine-stimulated transcriptional activation almost twofold compared with the full-length promoter. This region is capable of repressing transcription of a luciferase reporter gene driven by a basic viral promoter in several cell lines. An AP-1-like element present in the repressive region is involved in this effect, as demonstrated by deletional analysis. Two protein complexes that bind to this sequence are present in unstimulated cells; this element itself is capable of repressing a viral promoter, suggesting a role in the control of basal expression of the enzyme.
PRINCIPAL FINDINGS
It is known that a cytokine mix containing IL1-ß, IFN-
, and TNF-
induces NOSII expression in most cell types, producing high levels of NO. We confirmed the activation of NOSII in the HCT-8R cells by measuring nitrites and nitrates (NOx) accumulated in the medium after stimulation with these cytokines for 24 h. Production of NO by these cells is the result of NOSII expression, as shown by mRNA accumulation in a Northern blot and promoter activity, which are maximal after 6 h stimulation.
1. A region of the proximal NOSII promoter contains a repressor binding site
The NOSII promoter was analyzed by performing several random deletions within the proximal 7 kb region as depicted in Fig. 1
A. Deletion of the fragment from -351 to -1431 in the S1 construct provokes an almost twofold increase in luciferase activity vs. the full-length promoter (Fig. 1B
). The repressive role of this region was confirmed by its introduction into the pGL3 promoter luciferase vector, which contains an SV40 promoter controlling the luciferase gene. As shown in Fig. 2
C, the presence of this fragment causes a 50% decrease in luciferase activity with reference to the pGL3 promoter vector.
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Overlapping of the random deletions permitted us to localize the repressor between -351 and -632 bp. Examination of the sequence of this region showed the presence of an element with homology to AP-1 (Fig. 2A
), a family of transcription factors implicated in a negative regulation of NOSII.
2. The AP-1-like site is involved in transcriptional repression of NOSII
The sequence containing the AP-1-like site was used as a probe in gel shift mobility assays (EMSA), finding two complexes that bind to this element in unstimulated cells. These interactions seem to be specific since they are competed by the homologous sequence but not by unrelated nucleotides. The slow migrating complex increases slightly after stimulation, persisting at least until 8 h; it is partially competed by a consensus AP-1 sequence but not by a mutated one, confirming it contains proteins of this family of transcription factors. None of the components of these complexes was recognized by specific antibodies to the different AP1 proteins, indicating they may be novel members of the AP-1 family or unknown factors with affinity for AP-1-like sites.
We next deleted the AP-1-like site from the 7 kb promoter to examine its role in NOSII transcription. This modification, similar to the S1 deletion, significantly (P<0.001) increases transcriptional activity compared with the full-length promoter (Fig. 1B
). The implication of this element in the repressive effect was confirmed by deleting it from the S1 fragment introduced in the pGL3 promoter vector (Fig. 1C
), which provokes 25% recovery of activity. An oligonucleotide containing two of the AP-1-like sequences in tandem is capable of repressing the SV-40 promoter when introduced into the pGL3 promoter vector, confirming its direct participation in repression of NOSII transcription.
The effect of the repressor on each cytokine was studied separately and in different combinations to determine whether it affects one cytokine specifically. The full-length 7 kb promoter is most efficiently activated by the three cytokines, whereas the combination of IL1-ß plus IFN- induces 
50% of total activity. The S1 deletion permits an induction with these two cytokines similar to that obtained with the cytokine mix. Deletion of AP-1 induces an increase in activation with the two cytokines, but not to the levels obtained with the S1 deletion.
Since factors recognizing the AP-1-like sequence are constitutively present, we hypothesized that they might control the onset of NOSII transcriptional activation. Thus, a time course of transcriptional activation with this construct was performed. Even though the deletion increases transcriptional activity throughout stimulation, the activity of this construct follows the same kinetics as the full-length promoter.
CONCLUSION
In the present work, we describe the identification of a proximal region (S1) in the human NOSII promoter that contains a repressor, since transcriptional activity on cytokine stimulation increases when it is deleted. The repressive role of this fragment was confirmed by its ability to inhibit a viral promoter. The repressor region contains an AP-1-like site that we chose for further study, since earlier investigations showed that transfection of expression vectors for c-jun and c-fos inhibited NOSII promoter activity in transfected cells. This was shown at the mRNA level and using a 7 kb promoter region in a reporter gene even when no specific sequence was identified. The repressive effect of these components of the AP-1 family was detected at basal levels of transcription. The results coincide with the findings reported here: first, we find that the complexes recognizing the AP-1-like site are present in unstimulated cells; more importantly, introduction of the S1 region or AP-1-like site into the pGL3 promoter vector decreases activity at basal levels. We assayed specific anti c-fos and c-jun antibodies in our EMSAs, finding that the proteins reported here do not correspond to either factor.
The role of AP-1 in the regulation of NOSII expression is complex and somewhat controversial. Thus, two AP-1 sites at -5 kb have been deemed important by deletional analysis; components of the AP-1 family interacting with both sites were identified as jun D and Fra 2. Although our AP-1-like site has an intact half AP-1 consensus site (TGA), it differs from the sites described above by a 1 bp deletion. Therefore, it is likely that different dimers of AP-1 factors will bind to the activating sites and to the repressor site described here, as confirmed by a lack of reactivity with specific antibodies to Fra2 and JunD.
Several reports in the literature show repressive effects of AP-1 dimers on the transcription of various genes. For example, a newly discovered member of the AP-1 family, JDP-2, represses p53 transcription via a conserved, atypical AP-1 site in the p53 promoter. This novel factor, named jun-dimerizing protein (JDP2), is constitutively expressed in all cells and tissues analyzed. It was reported as the first c-jun repressor protein, and its properties are consistent with a role in maintaining low basal levels of AP-1 transcriptional activity in unstimulated cells. It is possible that the AP-1-like site identified here binds preferentially JDP2 dimers since it seems to function mainly at basal levels of transcription.
The role of the AP-1-like site in basal regulation is confirmed by the observation that the S1 region and the AP-1-like site itself decrease transcriptional activity of the pGL3 promoter vector in unstimulated conditions. The unresponsiveness of these constructs to cytokine stimulation could imply there are no relevant sites in the repressive region that would induce transcription. This agrees with findings reported so far showing that the indispensable elements for NOSII transcription are located upstream of -4 kb of the promoter. Indeed, when examining the effect of the repressive region on each cytokine separately, we were able to detect a higher activation only in response to IL1-ß and IFN- together vs. the full-length promoter. This is probably due to the more efficient induction by these two cytokines, as previously reported. It seems that various cytokines are needed to induce sufficiently the necessary factors in order to overcome the effect of the repressor. When the S1 region is deleted, activation achieved by this combination is similar to that obtained with the cytokine mix and almost twofold higher than that observed with the deletion of the AP-1-like site. This suggests the possibility that other elements within this region could be involved in the repressive effect.
These findings suggest that the factors with affinity for the AP-1-like site identified in the NOSII promoter play a role in the overall control of NOSII transcription, preventing its expression in the absence of stimulus and maintaining low basal levels of the enzyme. This would be consistent with our finding that the deletion of the AP-1-like site does not change the kinetics of transcription, but causes a general increase of transcriptional activity. The persistence of these factors after cytokine stimulation and the fact that in transient transfections the effect of the repressor is only detected as an increase of transcriptional activity on cytokine stimulation may ensure that the enzyme is expressed only in response to an adequate stimulus. The effect of this repressor was confirmed in other cell lines, including a colon and a breast cell line, showing that the repressor is generally present and active. This might constitute a mechanism to control overall levels of expression of this enzyme in order to avoid cytotoxic effects of NO under normal conditions. To our knowledge, this is the first report of a repressor controlling NOSII transcription; it is a finding that can have important implications for regulation of the enzyme.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0450fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0450fje 
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