©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sodium Salicylate and Yeast Heat Shock Gene Transcription (*)

Charles Giardina , John T. Lis (§)

From the (1) Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The anti-inflammatory drug sodium salicylate modulates the activity of specific transcription factors in humans. Here we show that salicylate and sorbate, another organic acid, stimulate DNA binding by yeast heat shock transcription factor (HSF) in vivo. Surprisingly, salicylate inhibits heat shock gene transcription even in cells induced by a prior heat shock. This inhibition of transcription occurs at a step after HSF and transcription factor IID binding but before promoter melting by RNA polymerase. Salicylate appears to generate a tight binding but activation-impotent HSF by cytoplasmic acidification, since inhibiting proton efflux from cells triggers this same DNA binding and inhibition of heat shock gene expression.


INTRODUCTION

Salicylates are widely prescribed anti-inflammatory drugs, yet their mechanism of action is not well understood. It has recently been found that salicylates affect the activity of two human transcription factors: heat shock factor (HSF)() and nuclear factor-B (NF-B) (1, 2) . During inflammation, an increased tissue temperature and other forms of physiological stress induce transcription of the heat shock genes (1, 3, 4) . The ability of salicylate to activate the DNA binding activity of HSF could therefore be contributing to its pharmacological effectiveness. Salicylate has also been shown to inhibit the activation of NF-B (2) . NF-B is responsible for activating a spectrum of genes involved in the inflammatory response, including various cytokines and cell adhesion molecules (5, 6, 7) . In addition, HIV-1 transcription requires NF-B, and salicylate's inhibition of HIV-1 replication may be a result of its ability to inhibit NF-B activation (5, 8, 9, 10) . Salicylate also plays an important role in plant systemic acquired resistance, where it serves to activate the pathogenesis-related genes (11, 12, 13) .

Here we show that sodium salicylate can inhibit heat shock gene transcription in yeast. The inhibition of heat shock gene transcription appears to be specific and is probably mediated through the formation of an activation-impotent, tight DNA-binding HSF. By comparison with other chemical agents with similar effects, we provide evidence that salicylate affects HSF and heat shock gene transcription by lowering the cellular pH.


EXPERIMENTAL PROCEDURES

Yeast Growth and in Vivo DMS Footprinting

The yeast strain YPH102 was grown in YEPD medium to an optical density of 1.5 and treated as described in the text. For heat-shocked samples, yeast were transferred to a flask prewarmed to 39 °C for 15 min. 10 ml of culture was pelleted and resuspended in 150 µl of fresh medium (either at room temperature or at 39 °C for the heat shock samples). This suspension is treated with 1 µl of DMS for 1 min. 50 µl was then withdrawn, and the reaction was terminated by dispersing in 550 µl of sorbitol stop buffer (0.9 M sorbitol, 0.1 M Tris-HCl (pH 8.0), 0.1 M EDTA, 40 mM -mercaptoethanol). Spheroplast preparation and DNA isolation was performed as described (14) . The sites of DMS modification were cleaved by treating with 1 M piperidine for 30 min at 88 °C. After piperidine treatment, DNA was dried, resuspended in 50 ml of 0.3 M sodium acetate, ethanol-precipitated, and washed with 70% ethanol. Usually, about one-fourth of this DNA was analyzed by ligation-mediated polymerase chain reaction as described (14) . Primers to view DMS reactivity were: primer 1, GGTTGGTATTAAGATGAGAATTAACC; and primer 2, TAACCGCTCATAAAACCATGCGCGT.

RNA Preparation and Primer Extension

A 5-ml culture of yeast was treated as described in the text. Cells were pelleted, washed with water, and resuspended in 400 µl of 10 mM Tris-Cl, pH 7.5, 10 mM EDTA, 0.5% SDS. This suspension was extracted with water-equilibrated phenol at 65 °C over the course of 1 h. Samples were then chilled on ice and centrifuged for 5 min. The aqueous phase was extracted once more with phenol and then twice with chloroform. RNA was then ethanol-precipitated and resuspended in water. 30 µg of RNA was mixed with 0.2 pmol of end-labeled primer, ethanol-precipitated, washed, resuspended in 11 µl of water, and heated to 75 °C for 5 min. Samples were cooled on ice, and 9 µl of reaction mixture was added to give the final concentrations: 1 first strand buffer (Life Technologies, Inc.); 10 mM dithiothreitol; 2 mM dNTPs; 1 unit/µl RNasin (Promega); 5 units/µl SuperScript II reverse transcriptase (Life Technologies, Inc.). This reaction was incubated for 1 h at 45 °C. DNA was then ethanol-precipitated and run on a 5% polyacrylamide-urea sequencing gel. The primers were: for HSP and HSC82, CCAATTGCTTTGGATCAGACAAAG; for ACT1, GCTGATGTAGTAGAAGATCCTATTC; for GAL1, TTTGCGCTAGAATTGAACTCAGGTAC.

KMnO Footprinting

Cultures were treated as described in the text. 5 ml of culture was pelleted and resuspended in 50 µl of fresh media either at room temperature (24 °C) or at 39 °C when heat shocked. This suspension was treated with 3.5 µl of 0.35 M KMnO for 1 min. Reaction was stopped by adding 550 µl of sorbitol stop buffer. DNA was prepared and treated like the DMS-treated samples above. KMnO modification of the promoter was observed using ligation-mediated polymerase chain reaction and the following primers: primer 1, CCAATTGCTTTGGATCAGACAAAG; and primer 2, CAAAGATTTGTATCTAATTTTATCCAACGC.

DES Treatment

DES (Sigma) was dissolved to 10 mM in methanol and added to the medium dropwise with constant agitation. For control cells, methanol was added to give a final concentration equal to that used for the most concentrated DES treatment. After a 45-min incubation at room temperature, DMS footprinting was performed.


RESULTS AND DISCUSSION

We recently demonstrated that, similar to human cells, heat shock stimulates the DNA binding activity of yeast HSF in vivo(16) .() We were curious as to whether sodium salicylate could also stimulate the DNA binding activity of yeast HSF, as it can in human cells, since yeast would provide a very tractable system to investigate salicylate's effect on transcription factor activity. Fig. 1shows a DMS footprint over the HSP82 heat shock elements (HSEs) in cells treated with 0 to 30 mM sodium salicylate. At 0 mM salicylate, under non-heat shock conditions (the NHSlane of Fig. 1 ), HSF binding to the strong HSE of this promoter, HSE1, is revealed by the protection of G residues at -161, -162, and -171 (compare the NHSlane to the DNAlane). Sodium salicylate increases HSF binding to the promoter as revealed by a slight increase in protection of the G residues in HSE1, and, more dramatically, by the formation of a hyper-reactive site at -210 in a weaker HSE of this promoter, HSE3 (this hyper-reactive site is also formed when DMS footprinting is performed in vitro with high concentrations of purified HSF). The HSF binding induced by sodium salicylate is actually greater than that found in heat-shocked cells (Fig. 1). The salicylate concentration required to stimulate yeast HSF binding is similar to that needed to affect HSF and NF-B in human cells, which is also similar to that required for its anti-inflammatory activity (1, 2, 17, 18) .


Figure 1: Sodium salicylate stimulates HSF binding to the HSP82 promoter in yeast. Yeast were treated with 0, 3, 10, and 30 mM sodium salicylate for 10 min before performing DMS footprinting, as indicated. Also shown is the DMS modification pattern on purified DNA ( DNA) and DNA in cells undergoing a 39 °C heat shock ( HS). The locations of the strong HSE1 and weak HSE2 and HSE3 are indicated, and the numbering is relative to the transcriptional start site. A long and a short exposure of the same gel is shown to display clearly all G residues.



While both heat shock and salicylate stimulate HSF DNA binding, we found that, unlike heat shock, salicylate is a potent inhibitor of heat-activated HSP82 transcription in yeast. Fig. 2 A shows the levels of HSP82 RNA (and RNA from its cognate gene, HSC82) quantified by primer extension. Sodium salicylate strongly inhibits the heat induction of both these genes. This is unlike the situation in humans where salicylate is non-inhibitory and possibly potentiates the stress response (1) . The inhibition of yeast heat-activated transcription is stable, lasting for at least 2 h (Fig. 2 B). This effect on transcription is remarkably specific, however, since salicylate does not interfere with galactose induction of the GAL1 gene nor does it affect the steady-state levels of ACT1 RNA (Fig. 2 A) or dramatically alter cell growth rates (not shown). As would be expected for an inhibitor of heat shock gene transcription, salicylate inhibited the acquisition of thermotolerance to a 52 °C heat shock conferred by a 39 °C heat shock pretreatment (not shown) (19) . It is not clear how the hyperactivation of HSF's DNA binding activity inhibits heat-induced transcription in yeast. Perhaps the salicylate stimulation of HSF DNA binding interferes with another HSF modification step, such as phosphorylation (1, 20) , leaving the promoter occupied only by an inactive form of this transcription factor.


Figure 2: A, the effect of salicylate on HSP82, HSC82, GAL1 and ACT1 gene transcription. In lanes 1-4, RNA was prepared from yeast heat shocked for 30 min at 39 °C ( HSlanes) and non-heat-shocked yeast ( NHSlanes). In the sodium salicylate ( NaSa) + lanes, cultures contained 30 mM sodium salicylate. In lanes 5-8, yeast were grown with raffinose as the carbon source after which cells were transferred to fresh raffinose ( RAFlanes) or fresh galactose media ( GALlanes). RNA was quantified by primer extension using primers for HSC82, HSP82, ACT1 and GAL1.B, salicylate's inhibition of heat shock gene transcription is stable. RNA was prepared from control yeast ( lane1), yeast grown at room temperature in the presence of 30 mM sodium salicylate ( lanes 2-4), or yeast grown at 39 °C in the presence ( lanes 5-7) or absence ( lanes 8-10) of 30 mM sodium salicylate. In lanes5-10, RNA was prepared 30, 60, or 120 min after initiation of heat shock, as indicated, with salicylate treatment in lanes 5-7 initiated 10 min before heat shock. Lanes 2-4 are the same as lanes5-7except the cells were maintained at room temperature for the times shown. Primer extension of isolated RNA was performed as above.



To gain some insight into which step in the transcription cycle is blocked by salicylate, we examined the KMnO modification pattern of the HSP82 promoter. KMnO reactivity with DNA increases upon DNA melting, so this reagent is useful for detecting RNA polymerase open complexes (14) . In addition, a reduction in the TATA box's reactivity with KMnO is diagnostic of transcription factor IID binding (21) . Salicylate was found to inhibit promoter melting (note the hyper-reactive sites at -55 and those close to the transcription start site are not detectable in the presence of salicylate) but not TATA protection (Fig. 3). These findings suggest that salicylate is inhibiting transcription at a step after transcription factor IID binding but before polymerase open complex formation. Salicylate even inhibits promoter melting when added 10 min after heat shock, a time sufficient to fully activate transcription of heat shock genes (Fig. 3) (22) .


Figure 3: Salicylate inhibits the heat shock-induced melting of the HSP82 promoter, but not TBP binding. In lanes 2-5, yeast were heat shocked ( HS lanes) or maintained at room temperature ( NHS lanes) in the presence of 30 mM sodium salicylate ( NaSa) where indicated. In these lanes, salicylate treatment was initiated 10 min prior to heat shocking. Lane 1 shows the KMnO modification reaction on purified DNA. After a 10-min heat shock, KMnO footprinting was performed. Lanes 6-9 are the same as lanes 2-5 except salicylate treatment was initiated 10 min after heat shock. 7.5 min after addition of salicylate to the indicated samples, KMnO footprinting was performed. The arrow indicates the position of the transcriptional start site, and the numbering is relative to this start site. The TATA box is labeled.



The effect of salicylate on heat shock gene transcription observed here is similar to that recently found for other weak organic acids (23) . For example, sorbate, a commonly used food preservative, also blocks the activation of heat shock gene transcription in yeast. This inhibition is most potent when the treatment is performed in low pH medium (23) . Low pH increases the concentration of protonated acid in the medium, which is probably the form that most efficiently crosses the membrane. We found that at pH 4.5 or 5.5, sorbate also stimulated the DNA binding activity of HSF (Fig. 4 A). The pH of the medium had a similar effect on the stimulation of HSF DNA binding by salicylate (Fig. 4 A). The effects of sorbate and salicylate on yeast heat shock gene transcription and yeast HSF are similar, indicating that these acids may be working through a common mechanism.


Figure 4: A, sodium sorbate and sodium salicylate both stimulate HSF DNA binding in yeast most efficiently in low pH media. Cells were incubated in YEPD media with pH of 4.5, 5.5, or 6.5 plus or minus 10 mM sodium sorbate or sodium salicylate, as indicated. After 10 min, DMS footprinting was performed as in Fig. 1. In all other experiments the media pH was 5.6 at the time of DMS treatment. B, DES stimulates the DNA binding activity of yeast HSF. Yeast were incubated for 45 min at room temperature with 0, 25, 50, or 100 µM DES, as indicated. DMS footprinting was then performed as in Fig. 1.



One well characterized activity of acetylsalicylate (aspirin) is its ability to inhibit prostaglandin synthesis (18, 24) . However, sodium salicylate is a poor inhibitor of prostaglandin synthesis so it is unlikely that this is the mechanism by which it stimulates the DNA binding of yeast HSF (18) . Since sodium salicylate affects yeast heat shock gene transcription and HSF DNA binding in a manner similar to that of the structurally unrelated organic acid, sodium sorbate, it could be influencing transcription by acidifying the cellular cytoplasm (23) . Organic acids diffuse through the membrane in their protonated form and dissociate in the higher pH of the cytoplasm (25) . Evidence that organic acids inhibit heat shock gene transcription by cytoplasmic acidification comes from the finding that a number of other chemical agents and mutations that decrease cytoplasmic pH also inhibit heat shock gene transcription. For example, inhibition of the plasma membrane proton pump, either by diethylstilbestrol (DES) or by mutation, also inhibits the heat shock gene expression (23, 26) .

To test if cytoplasmic acidification is the condition that activates the binding of HSF to DNA, we examined HSF binding to the HSP82 promoter in yeast cells treated with DES. DES-treated cells were indeed found to have hyperactivated HSF binding as seen by DMS footprinting (Fig. 4 B). Therefore, lowering cytoplasmic pH by a means that does not require addition of organic acids leads to the same activation of HSF binding as seen in salicylate-treated cells. Interestingly, human monomeric HSF in in vitro extracts from uninduced cells can also be activated to trimerize and thereby acquire tight DNA binding activity by simply lowering the pH to between 5.8 and 6.4 (15) . We speculate that a low pH favors formation of the DNA-binding trimer, but this trimer is locked in a conformation that cannot activate transcription. Although this inactive trimer does not respond to heat shock under the conditions tested here, i.e. growth in rich medium with glucose, we have obtained preliminary evidence that under glucose-derepressing conditions (growth in galactose), heat-induced transcription can occur in the presence of salicylate (not shown). Therefore, under glucose-derepressing conditions, yeast appear to respond to salicylate more like human cells (1) .

Although our analysis here is limited to the effect of salicylate on yeast HSF and heat shock gene transcription, it is possible that salicylate may inhibit NF-B activation in humans by reducing cellular pH (2) . In addition, this activity of salicylate could be part of its signaling activity in plants. In this regard, establishing yeast as a model system should facilitate the study of salicylate's effects on eukaryotic cell physiology and gene regulation.


FOOTNOTES

*
This study was supported supported by National Institutes of Health Grant GM25232 (to J. T. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Section of Biochemistry, Molecular and Cell Biology, Biotechnology Bldg., Cornell University, Ithaca, NY 14853. Tel.: 607-255-2442; Fax: 607-255-2428.

The abbreviations used are: HSF, heat shock factor; NF-B, nuclear factor-B; HIV-1, human immunodeficiency virus, type 1; DMS, dimethyl sulfate; HSE, heat shock element; NHS, nonheat shock; DES, diethylstilbestrol.

Giardina, C., and Lis, J. T. (1995) Mol. Cell. Biol. 15, in press.


ACKNOWLEDGEMENTS

We thank T. O'Brien, L. Shopland, and H. Shi for critically reading this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.