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Heat shock enhances transcriptional activation of the murine-inducible nitric oxide synthase gene¹

CHRISTOPHER E. P. GOLDRING^*,2, SYLVIE REVENEAU^23,, AURÉLIE CHANTOME, ALENA PANCE, CHRISTOPHE FLEURY, DAVID A HUME^§, DAVID SESTER^§, BERNARD MIGNOTTE and JEAN-FRANÇOIS JEANNIN

^* Department of Pharmacology, University of Liverpool, Liverpool L69 3BX, U.K.;
Cancer Immunotherapy Laboratory of the Ecole Pratique des Hautes Etudes and INSERM U-517, University of Bourgogne, Faculty of Medicine, 21000 Dijon, France;
Université Versailles/St.-Quentin, CNRS UPRES-A 8087, 78035 Versailles Cedex, France; and
^§ Institute for Molecular Bioscience, University of Queensland, Q4072, Australia

³Correspondence: Cancer Immunotherapy Laboratory of the Ecole Pratique des Hautes Etudes and INSERM U-517, University of Bourgogne, Faculty of medicine, 7 Boulevard Jeanne d’Arc, 21000 Dijon, France. E-mail: Sylvie.Reveneau{at}u-bourgogne.fr

SPECIFIC AIMS

Using the technique of in vivo footprinting by ligation-mediated polymerase chain reaction (PCR), we recently mapped changes in methylation sensitivity on guanine residues within, or adjacent to, many of the regulatory elements in the murine-inducible nitric oxide synthase (iNOS) gene that had previously been identified using in vitro techniques. We also identified hypomethylation at guanine residue(s) -898/899, which lies outside of any previously recognized element. We have now identified this region as a partial heat shock regulatory element adjacent to an E-box, and demonstrate that heat shock is able to modify NO synthesis via iNOS and that the heat shock element is implicated in the induction of lipopolysaccharide (LPS) -stimulated transcription of the iNOS gene.

PRINCIPAL FINDINGS

1. LPS and heat shock induce occupation of the HSE and E-box on the murine iNOS gene
We previously revealed, by in vivo footprinting, hypomethylation at guanine residues -898/899 in the murine iNOS distal enhancer DNA sequence upon exposure of RAW 264.7 macrophages to LPS. Since factor binding to this region had been so far unidentified, we used the Transfac Matrix database to search for putative known recognition elements. A possible site was detected at -902 to -898 corresponding to the sequence -⁹⁰²ATTCC^-898, which represents part of a putative heat shock element (HSE). A further possible match was also detected at -893 to -888, corresponding to ^-893CATGTG^-888, which is similar to the E-box consensus sequence, CACGTG. The E-box binds dimeric members of the basic-helix-loop-helix-leucine zipper family of proteins, such as the myc-max family and the upstream stimulating factor (USF) proteins. These factors can be activated during certain conditions of cellular stress and have indeed been found to play a role in the transcription of some heat shock proteins. Since both of these putative regulatory elements are implicated in the heat shock response, we conducted further in vivo footprinting experiments to examine whether they could also be occupied in vivo when macrophages are exposed to heat shock. Cells were exposed to LPS during and after the heat shock treatment, and a sample of cells exposed to LPS but receiving no heat shock treatment was also included. No methylation changes were seen in the in vivo control compared to the in vitro control (Fig. 1 ). Heat treatment of the cells reproducibly caused a hypomethylation at guanine -898/9 (within the putative partial HSE), similar to that induced by LPS treatment, as has previously been seen and shown again here (Fig. 1) . This is suggestive of inducible factor binding at this site. Exposure of the cells to LPS plus heat also resulted in a hypomethylation at -898/9, the degree of which was greater than that seen in the cells exposed to either LPS or heat alone. Furthermore, a hypermethylation was observed in the proximal guanines at -893/4 (of which one guanine is within the putative E-box and the other is just adjacent), which had not been detected in cells treated with only LPS or heat.

View larger version (65K): [in this window] [in a new window]   Figure 1. In vivo footprinting of the putative heat shock element within the iNOS enhancer noncoding strand. RAW 264.7 cells were exposed to 41°C, or left at 37°C, for 20 min in the presence or absence of LPS (100 ng/ml) and kept for 100 min at 37°C in the same medium. Genomic DNA was then immediately methylated and cellular viability was assessed using the trypan blue test. After genomic DNA extraction and piperidine treatment, LMPCR was carried out to amplify the fragments, which were then 32P-labeled and separated on a sequencing gel, prior to autoradiography. An in vitro methylated DNA control was also included. The numbers at the left of the figure indicate the position of the guanine fragment(s) relative to the iNOS transcriptional start site. The location of the enhancer B site is indicated. Open triangles indicate hypomethylation sites and the filled triangle represents the hypermethylation site.

2. Heat shock enhances iNOS gene expression by murine macrophages induced by LPS
Because of the likely occupation of the putative partial HSE within the iNOS enhancer, we attempted to determine whether heat shock is a potential inducer of macrophage iNOS transcription. We used the nuclear run-on assay, which measures the de novo synthesized mRNA of a given gene. We detected an 2.5-fold increase in the rate of transcription of the iNOS gene in cells exposed to a heat shock in the presence of LPS, compared to the rate of transcription in cells exposed to LPS alone. Transcription of the iNOS gene could not be detected in cells exposed to heat shock alone. These data have been further confirmed by reporter gene analysis. Heat shock induced a low basal luciferase activity in cells without LPS stimulation; combining heat shock and LPS stimulation, the induction was greater than with LPS alone. We then showed that this increase in the rate of transcription of the iNOS gene after stimulation by heat shock plus LPS was matched by an increase in iNOS protein and nitric oxide production.

3. Heat shock and LPS induce binding of USF1 and 2 proteins and HSF-1 on the iNOS promoter
Initially, we confirmed that LPS exposure and heat shock stimulated binding of one or more nuclear proteins to an oligonucleotide corresponding to E-box of the murine iNOS promoter. To determine if the binding was due to members of the USF family, we used antisera specific for USF1 and USF2, and found that both caused disappearance of the specific band shift, with the concurrent appearance of strong supershifts, implicating both of these proteins in the binding. HSF-1 binding was also investigated using an oligonucleotide that does not contain the E-box. In nuclear extracts of control cells, binding to this oligonucleotide was detected. This interaction was increased under conditions of LPS and LPS plus heat shock treatment. Heat shock treatment alone caused only a slight increase in the intensity of this band. The presence of an anti-HSF-1 antiserum greatly diminished the amount of specific complex formed.

4-Functional analysis of the identified sites
1Kb of the iNOS promoter were coupled to luciferase to study the functional activity of the HSEs and E-box sites (Fig. 2A ). As shown in Fig. 2B , transcriptional activity of the iNOS promoter increases upon LPS treatment in a dose-responsive manner, reaching a maximum of 2.5-fold induction with 100 ng/ml LPS. No significant difference in luciferase activity was seen when the E-box described here was mutated. However, mutation of the HSE sites abolished LPS activation, which was slightly detected only when the LPS dose was increased 10-fold (100 ng/ml). This indicates that this HSE site is implicated in iNOS transcriptional induction by LPS in macrophages. As described earlier, heat shock alone does not stimulate iNOS transcription in RAW 264.7 cells (Fig. 2B ). However, when accompanied by increasing concentrations of LPS, transcriptional activity is higher than with LPS alone (Fig. 2C ). When the E-box was mutated, no significant changes were detected in responseto the combined treatment of heat shock and LPS, confirming that this site is not important for this response. On the other hand, mutation of the HSE site abolished the effect of the combined treatment.SCHEMATIC

View larger version (22K): [in this window] [in a new window]   Figure 2. Reporter gene analysis of the wild-type and mutated iNOS promoter. The results are RLU/well (2x105 transfected cells) and represent the average of duplicate transfections, each assayed in duplicate and varying by no more than 20% of the mean. The same pattern of LPS activation and dependence upon heat shock was observed in an independent experiment using separate preparations of each of the plasmids. A) Sequences of the mutants used for the HSE or E-box. B) LPS responses obtained with each mutant. C) Combined LPS plus heat shock treatment.

View larger version (20K): [in this window] [in a new window]   Figure 3. No caption available.

CONCLUSIONS

The studies described here demonstrate the presence of two new candidate iNOS transcription regulatory elements and from their characterization, another agent, i.e. heat shock/stress, can be added to the list of iNOS (and macrophage NO) enhancers. However, these findings demonstrate that heat shock alone is not sufficient to induce iNOS expression. This is not surprising, since the mechanisms of high-output NO synthesis via the iNOS pathway are tightly controlled and, indeed, other sets of agents are known to act synergistically on iNOS transcription, e.g. interferon- and LPS. It is possible that for transcription of the iNOS gene to be initiated, a multiprotein assembly, i.e. an enhanceosome, must be set up on the enhancer and promoter, as has been postulated to be the case for other NF-B-induced genes. In this case, exposure of the cells to heat shock would not be expected to initiate this complete assembly. Although we observe in vitro that exposure of macrophages to heat shock augments USF1 and USF2 binding to the E-box and causes a slight increase in HSF-1 binding to the partial HSEs, in the in vivo genomic context heat shock only caused a hypomethylation at guanine -898/9 at a partial HSE, but did not lead to occupation of the E-box. Furthermore, heat shock does not lead to site occupation by other transcription factors, such as NF-B, necessary for iNOS transcription (see Fig. 1 ; compare the hypomethylations within the NF-B site in the LPS-treated cells, with the lack of hypomethylation in the heat shocked cells).

It is known there are a number of genes, apart from the classical heat shock genes, whose expression can be up-regulated by heat and whose regulatory regions contain HSEs and E-boxes. Nevertheless, there are reports of a diminished synthesis of cytokine-stimulated iNOS protein in cells previously exposed to heat shock, although these studies were carried out using either rat pulmonary artery smooth muscle cells or a human liver cell line. The latter study suggested a transcriptional effect, possibly via an inhibitory effect of the heat shock response on NF-B. We tested NF-B activity and found no differences attributable to exposure to heat shock (in EMSAs) in NF-B proteins activated by exposure of macrophages to LPS, binding to the B site in the enhancer or the promoter of the murine iNOS gene. This finding would appear to rule out a nonspecific effect of heat on NF-B activation in this model, and also reduces the possibility that the heat shock treatment of the cells merely accelerates the iNOS response to LPS.

There is clearly a physiological basis for an enhanced production of NO by macrophages exposed to heat concurrently with LPS. Gram-negative infections are associated with hyperthermia and often fever. Such elevations in temperature can initiate the heat shock response, so presumably a stimulation of iNOS gene expression and thus NO production by macrophages could be expected to occur in vivo. This could explain the well-known observation that fever decreases the growth and virulence of certain bacteria, since it has been reported by several groups that NO, in concert with other host molecules, has bactericidal properties. Furthermore, it would seem logical for the immune response to produce maximal levels of NO only under extreme circumstances such as this in order to combat a gram-negative infection, because of the potentially damaging effects of NO to nearby cells and tissues. It is not currently known if human macrophages can produce NO in response to heat shock/stress in combination with LPS or other agents, although the possibility exists for a USF/HSF1 involvement in the transcription of the human iNOS gene, since putative E-boxes and HSEs are present in the promoter region.

In conclusion, these data demonstrate the existence of two new regulatory elements, a HSE and an E-box in the iNOS gene, and that one of these (namely, the HSE) is functionally involved in the activation of iNOS transcription. These sites are occupied in vivo upon heat shock plus LPS exposure of murine macrophages concurrent with an increased rate of transcription of the iNOS gene, increased iNOS protein, and an increased output of NO.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.98-0509fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.98-0509fje

² The first two authors contributed equally to this work.

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