Phenylarsine oxide inhibits heat shock protein 70 induction in cultured guinea pig gastric mucosal cells

Kazuhito Rokutan, Mami Miyoshi, Shigetada Teshima, Tomoko Kawai, Tsukasa Kawahara, and Kyoichi Kishi

Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Phenylarsine oxide (PAO) forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP) but not by dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). PAO at 2 µM or higher inhibited heat shock protein 70 (HSP70) induction within minutes in cultured guinea pig gastric mucosal cells exposed to heat (43°C) for 30 min. PAO did not affect the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1) induced by heat stress, but it completely blocked the binding activity of HSF1 to the heat shock element (HSE), leading to the block of expression of HSP70 mRNA and accumulation of HSP70 in the cells. These inhibitions were completely reversed with 2 µM DMP but not with 0.1 mM DTT or 1 mM 2-ME, suggesting specific interactions between PAO and vicinal dithiol-containing molecules. Thioredoxin (Trx) reversed the inhibition of the binding activity of HSF1 in whole cell extracts prepared from PAO-treated, heat-stressed cells. Our results suggest that PAO may react with vicinal-containing molecules including Trx and specifically block the interaction between HSF1 and HSE.

heat shock response; heat shock factor 1; heat shock element; vicinal dithiol; thioredoxin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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EUKARYOTIC AND PROKARYOTIC cells, exposed to temperatures above the physiological level, exhibit the heat shock response by inducing a set of highly conserved proteins referred to as heat shock proteins (HSPs). In mammalian cells, the heat shock response is mediated by activation of specific preexisting transregulatory proteins, the heat shock factors (HSFs). The mammalian HSF gene family includes four distinct HSF genes (HSF1-4) (36, 37, 39, 43, 45), which have been molecularly identified in chicken (HSF1-3), mouse (HSF1 and 2), and human (HSF1, 2, and 4). HSF1 is now identified as the mediator of stress-induced transcription of HSP genes (42, 45, 47). The promoter regions of HSP genes contain a motif known as the heat shock element (HSE), to which HSF1 binds and promotes their expressions (see Refs. 28 and 31 for reviews). Upon exposure to various forms of stress, HSF1 undergoes oligomerization from a non-DNA-binding monomer to a DNA-binding trimer and translocates into the nucleus to interact with HSE (2, 28, 31, 42). The mechanisms regulating the DNA binding and transcriptional activity of HSF1 have been extensively studied. Recently, it has been shown that hyperphosphorylation of HSF1 may be required for the transcriptional activation but not for the DNA-binding activity (8, 25). Heat stress is known to stimulate several protein kinases, such as the mitogen-activated protein kinase (MAPK) (26), protein kinase C (PKC) (40, 52), and c-Jun NH2-terminal kinase (1). PKC and possibly other undefined kinases have been suggested to catalyze the hyperphosphorylation of HSF1 (9, 19), leading to full transcriptional competence, whereas phosphorylation of HSF1 by MAPK or glycogen synthase kinase 3 was reported to rather repress transcriptional activation by HSF1 (7).

In this study, we examined the effect of a unique compound, phenylarsine oxide (PAO), on the HSP70 induction in cultured guinea pig gastric mucosal cells, and we found that treatment with PAO at micromolar concentrations completely inhibited the induction of HSP70 by heat shock. This trivalent arsenical compound is known to react with two thiol groups of closely spaced protein cysteinyl residues (vicinal dithiols) to form stable dithioarsine rings (48). The complex cannot be decomposed by monothiols, such as 2-mercaptoethanol (2-ME), but in the presence of low-molecular-weight dithiols the binding is competitively reversed. British anti-Lewisite, 2,3-dimercaptopropanol (DMP), effectively reverses the protein-PAO complex to form the more stable five-membered ring; 1,4-dithiothreitol (DTT), which forms an unstable seven-membered ring with PAO, is much less effective in this regard than DMP (49).

Frost and Lane (12) first reported that PAO specifically inhibited insulin-dependent hexose uptake in 3T3-L1 adipocytes, and subsequent studies showed that part of this inhibition may be the result of interaction with protein tyrosine phosphatase (PTPase) (3, 4, 11). In addition, PAO is known to modify intracellular signal transduction pathways by interacting with vicinal dithiol-containing molecules, resulting in the inhibition of several important functions, including platelet activation (50), hepatocyte endocytosis (15), CD45 phosphatase activity in T cells (14), and the respiratory burst of human neutrophils (5, 27).

Numerous chemicals are known to induce HSPs, whereas only a few agents are known to selectively inhibit the HSP induction. Quercetin, a bioflavonoid, was first introduced as an inhibitor of the HSP induction (22). A subsequent study showed that treatment with this compound at over 50 µM for 12 h decreased the HSF1 level and consequently inhibited the heat shock response (34). Genistein at 100 µM was also reported to inhibit the HSP70 induction by heat shock, possibly by blocking a step that occurs after the binding of the HSF1 but before the initiation of transcription (38). Compared with these compounds, PAO inhibited HSP70 induction by heat stress more rapidly (within 5 min) and at a lower concentration (2 µM). Furthermore, we found that PAO exerted its inhibitory action specifically by blocking the DNA-binding activity of HSF1.


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Reagents and media. RPMI 1640 and Eagle's minimal essential medium (MEM) were purchased from Nissui Pharmaceutical, Tokyo, Japan. Methionine-free RPMI 1640 was obtained from GIBCO BRL, Life Technologies, Grand Island, NY. 35S-protein labeling mix (>1,000 Ci/mmol) containing >= 77% L-methionine and >= 18% L-cysteine was from Dupont New England Nuclear, Boston, MA. [gamma -32P]ATP (>5,000 Ci/mmol), [alpha -32P]dCTP (>3,000 Ci/mmol), a random-primer kit, T4 polynucleotide kinase, nylon transfer membranes (Hybond-N Plus), an enhanced chemiluminescence (ECL) Western blot detection kit, and a reduced form of recombinant Escherichia coli thioredoxin (Trx) were purchased from Amersham, Little Chalfont, UK. Acid guanidinium thiocyanate-phenol-chloroform mixture was obtained from Nippon Gene, Toyama, Japan. cDNA clones for human HSP70 (ATCC 57090) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ATCC 57090) were purchased from American Type Culture Collection, Rockville, MD. A rabbit polyclonal antibody against bovine HSP70 was a gift from Dr. Hideaki Itoh (University of Akita, Akita, Japan). A rabbit polyclonal antibody against HSF1 was purchased from StressGen Biotechnologies, Victoria, BC, Canada. A monoclonal antibody against phosphotyrosine (clone Py54) was from Oncogene Science, Uniondale, NY.

Preparation and culture of gastric mucosal cells. The present study was approved by the Animal Care Committee of the University of Tokushima. Male guinea pigs weighing ~200 g were purchased from Japan SLC, Shizuoka, Japan. Gastric mucosal cells were isolated aseptically from fundic glands and cultured for 2 days in RPMI 1640 containing 10% FCS, 2 mM glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin in 5% CO2-95% air, as described previously (18, 41, 51). The guinea pig gastric cell preparation was already characterized by cytochemical and immunocytochemical techniques in a previous report (51). The majority of cultured cells (89 ± 3%: mean ± SD; n = 6) contained large galactose oxidase-Schiff reaction-positive granules characteristic of pit cells. Less than 1% of the cells contained granules positive for paradoxical concanavalin-A reaction, which is relatively specific for mucous neck cells. Parietal cells made up 4-5% of the total cells. Cell viability was assessed by lactate dehydrogenase release, as described previously (18, 41).

Protein labeling and analysis of synthesized proteins. Heat treatment of cells was done exactly as described previously (41). Immediately after exposing to heat shock at 43°C for 30 min, cells in 35-mm-diameter culture dishes were washed twice with MEM prewarmed at 37°C and incubated for 1 h in methionine-free RPMI 1640 containing 10% dialyzed FCS and 50 µM L-[35S]methionine (16 µCi/ml). After labeling, synthesized proteins (1 × 105 cpm of 35S per lane) were separated by SDS-PAGE and analyzed by autoradiography, as previously described (18, 41).

Detection of HSP70 and HSF1 by immunoblot analysis. The level of HSP70 was measured by immunoblot analysis with the antibody against a stress-inducible HSP70 as previously described (18, 41).

For examining subcellular localization of HSF1, cytosol and nuclear fractions were prepared by the method of Schreiber et al. (44) and subjected to immunoblot analysis. Cells were washed with ice-cold PBS, harvested with a rubber policeman, and collected in microcentrifuge tubes. The cells, pelleted by centrifugation at 900 g for 10 min at 4°C, were suspended in 10 mM HEPES buffer, pH 7.9, containing 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1 µg/ml leupeptin. The cells were allowed to swell on ice for 10 min, and then they were lysed by adding Nonidet P-40 at a final concentration of 0.6% (vol/vol). Lysis was completed by vortexing for 10 s. The sample was centrifuged at 13,000 g for 30 s in a microcentrifuge at 4°C. The supernatant was used as the cytosol fraction. The resulting pellets were resuspended in 20 mM HEPES, pH 7.9, containing 25% (vol/vol) glycerol, 0.42 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, and 1 µg/ml leupeptin and agitated on ice for 20 min. The supernatant, obtained by centrifugation at 13,000 g for 20 min at 4°C was used as the nuclear extract. These cytosol and nuclear extracts were separated in SDS/8%-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After nonspecific binding sites were blocked with purified milk casein at a final concentration of 4%, the membrane was incubated with an antibody against HSP70 or HSF1 for 1 h at room temperature. After being washed with PBS containing 0.2% Tween-20, bound antibodies were detected with an ECL detection kit.

Immunoprecipitation and analysis of tyrosine phosphorylation of HSF1. Before and after exposure to heat shock for the indicated times in the absence or presence of PAO, cells were washed three times with ice-cold PBS, and 500 µl of ice-cold lysis buffer, consisting of 10 mM Tris · HCl (pH 7.8), 0.5% (vol/vol) Nonidet P-40, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 0.1 mM sodium orthovanadate, 0.1% bovine serum albumin, 1 mM EDTA, 0.5 mM PMSF, and 1 µg/ml leupeptin, was added. The plates were scraped, and cell lysates were transferred to microcentrifuge tubes. After centrifugation at 18,000 g for 30 min at 4°C, the supernatant (500 µg protein) was precleaned by mixing with protein G-Sepharose beads (30 µl/500 µl supernatant) on an orbital shaker for 2 h at 4°C, and then it was centrifuged at 200 g for 30 s. The supernatant was incubated with an antibody against HSF1 (3 µg protein) for 2 h at 4°C, and then it was incubated for 1 h with 30 µl of protein G-Sepharose beads (50% slurry). The mixture was centrifuged at 200 g for 30 s, and the sedimented beads were washed four times with lysis buffer lacking bovine serum albumin. After one further wash with 50 mM Tris · HCl, pH 6.8, bound proteins were eluted by boiling in 15 µl of 2× Laemmli buffer for 5 min. Immunoprecipitates were separated by SDS/8%-PAGE and transferred to a PVDF membrane. The amounts of HSF1 and its tyrosine-phosphorylated form were determined by immunoblot analysis, using monoclonal antibodies against HSF1 and phosphotyrosine, respectively.

Northern blot analysis. Total RNA was isolated with an acid guanidinium thiocyanate-phenol-chloroform mixture (6). Total RNA was subjected to electrophoresis (10 µg RNA per lane) in 1% agarose gels containing 0.6 M formaldehyde and transferred to nylon membrane filters (Amersham). Northern hybridization with a cDNA probe for human HSP70 or GAPDH was performed, as previously described (18, 41). Bound probes were analyzed using a Fujix Bio-Analyzer BAS-2000 (Fuji Photo Film, Tokyo, Japan) and autoradiographed by exposure to a Fuji RX-R film for an appropriate time.

Gel mobility shift assay. Whole cell extracts were prepared based on the method of Mosser et al. (33) and used for gel mobility shift assay. Cultured cells in 35-mm-diameter culture dishes were harvested with a rubber policeman and collected in microcentrifuge tubes. After centrifugation at 5,000 rpm for 5 s in a microcentrifuge, pelleted cells were rapidly frozen in liquid nitrogen. The frozen pellets were suspended in 20 mM HEPES (pH 7.9) containing 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.1 mM DTT, and 1 µg/ml leupeptin. The mixture was gently pipetted with a glass pipette and stood on ice for 15 min. Whole cell extracts were obtained by ultracentrifugation of the mixture at 100,000 g for 5 min at 4°C. The supernatants were dialyzed against binding reaction buffer, consisting of 10 mM Tris · HCl (pH 7.8), 50 mM NaCl, 1 mM EDTA, 0.5 mM PMSF, 0.1 mM DTT, 1 µg/ml leupeptin, and 5% (vol/vol) glycerol, using an oscillatory microdialysis system (Bio-Tech International, Bellenue, WA). The dialyzed samples were stored at -80°C. In some experiments, DTT was omitted during the preparation and dialysis of whole cell extracts.

Gel mobility shift assay was performed using a synthetic double-stranded HSE oligonucleotide (5'-GATCTCGGCTGGAATATTCCCGACCTGGCAGCCGA-3'), as described previously (18, 41). The binding reaction was carried out for 20 min at 25°C, and the HSF1-HSE complex was detected by electrophoresis using a 4% polyacrylamide gel, as described previously (18, 41).


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Effect of PAO on protein synthesis. PAO is known to rapidly permeate into the cell interior within minutes and to interact with vicinal dithiol-containing proteins (13). The effect of PAO on protein synthesis in gastric mucosal cells was examined by metabolic labeling with L-[35S]methionine. PAO at 0.1 µM or lower did not change the rate of overall protein synthesis. However, 1 µM or higher concentrations of PAO decreased the rate of overall protein synthesis in a dose-dependent manner: the rate decreased to 84 ± 12% (mean ± SD, n = 4) of the control value with 1 µM PAO, 76 ± 12% (n = 4) with 2 µM PAO, 53 ± 14% (n = 4) with 5 µM PAO, and 35 ± 11% (n = 4) with 10 µM PAO. Heat stress itself decreased the rate to 69 ± 8% (n = 5) of the control value. In the presence of PAO, heat stress further reduced the rate to 55 ± 10% (n = 4) of the control value at 1 µM PAO, 41 ± 11% (n = 4) at 2 µM PAO, 23 ± 11% (n = 4) at 5 µM PAO, and 10 ± 3% (n = 4) at 10 µM PAO.

PAO at different concentrations (0.01 to 10 µM) was added to cultured gastric mucosal cells, and 5 min later, these cells were exposed to heat shock at 43°C for 30 min. During the 1-h recovery period, these cells were incubated with L-[35S]methionine for 1 h. Labeled proteins were separated by SDS-PAGE, and the proteins synthesized were detected by autoradiography (Fig. 1). Lanes were loaded on an equal counts-per-minute basis. This method reveals the relative rate of synthesis and not the absolute amount of synthesis. As shown in Fig. 1, lane 3, heat stress induced a 72-kDa protein corresponding to HSP70 in untreated control cells (18, 41). When 1 µM or higher concentrations of PAO were added and included during the heat stress and metabolic pulse labeling, PAO inhibited the HSP70 synthesis, and 2 µM PAO completely blocked it (lane 7). When PAO was removed by washing prior to heat shock, the inhibition did not continue during the pulse labeling period (lane 8). On the other hand, when PAO was removed after heat stress, the rate of HSP70 synthesis was still suppressed to 25 ± 3% (n = 3) of the control value (lane 9). When 2 µM PAO was first added to the PAO-untreated, heat-stressed cells, it could inhibit the HSP70 synthesis by 70 ± 5% (n = 3) (lane 10). These labeling experiments showed that PAO rapidly inhibited the synthesis of HSP70, but this inhibition was roughly estimated to be completely reversed during 1-1.5 h after removal of PAO.


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Fig. 1.   Effect of phenylarsine oxide (PAO) on protein synthesis in gastric mucosal cells. After cultured gastric mucosal cells were untreated or treated with PAO at the indicated concentrations for 5 min, they were exposed to heat at 43°C for 30 min and then pulse-labeled with L-[35S]methionine (16 µCi/ml) for 1 h in the absence (lane 3) or presence of the same concentrations of PAO (lanes 4-7), as described in MATERIALS AND METHODS. Untreated and unstressed cells (lane 1) and PAO-treated and unstressed cells (lane 2) were also radiolabeled in the same manner. Cells were treated with 2 µM PAO only for 5 min before heat stress (lane 8), 30 min during heat shock (lane 9), or 1 h after finishing heat stress (lane 10). These cells were also radiolabeled. After labeling, an equal amount of 35S (1 × 105 cpm per lane) was separated in SDS/10%-PAGE. The gel was stained with Coomassie brilliant blue R-250, dried, and exposed to Fuji RX-R film for an appropriate time. The molecular mass standards are shown on right. The results are representative of those obtained in 4 separate experiments.

Effects of DMP or 2-ME on inhibition of HSP70 synthesis by PAO. The pattern of protein synthesis was examined in detail by exposure of the gels for longer periods. Treatment of cells with 2 µM PAO reduced the rate of overall protein synthesis by about 25%; however, PAO at this concentration completely blocked the HSP70 synthesis without affecting the pattern of individual protein synthesis (Fig. 2, lane 3), suggesting that the inhibition of HSP70 induction by PAO was not related to the reduction of overall protein synthesis. In addition to HSP70, PAO occasionally inhibited the syntheses of several other proteins after heat stress (lane 6). PAO is believed to exert its actions by the formation of heterocyclic covalent adducts between vicinal dithiols on proteins. To confirm this, we tested whether DMP reversed the PAO-induced inhibition of HSP70 induction. Treatment of cells with 2 µM DMP alone did not change the pattern of individual protein synthesis before (lane 2) or after heat stress (lane 5), compared with that of untreated control (lane 1) or heat-stressed cells (lane 4), respectively. When 2 µM DMP was added after treatment with 2 µM PAO for 5 min, DMP completely reversed the PAO-induced inhibition of HSP70 synthesis (lane 7), whereas 2-ME at 1 mM did not reverse the inhibition (lane 8).


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Fig. 2.   Effect of 2,3-dimercaptopropanol (DMP) or 2-mercaptoethanol (2-ME) on PAO-induced inhibition of heat shock protein 70 (HSP70) synthesis. Cultured cells were untreated or treated with 2 µM DMP or 2 µM PAO for 5 min. These cells were unexposed (lanes 1-3) or exposed to heat at 43°C for 30 min (lanes 4-6). The PAO-pretreated cells were incubated with 2 µM DMP (lane 7) or 1 mM 2-ME (lane 8) for 5 min and exposed to heat stress at 43°C for 30 min. All of these cells were radiolabeled with L-[35S]methionine (16 µCi/ml) for 1 h, and synthesized proteins were analyzed as described in the legend to Fig. 1. The molecular mass standards are shown on right. The results are representative of those obtained in 4 separate experiments.

Western blot analysis with the anti-HSP70 antibody also revealed that PAO at 2 µM or higher completely inhibited the HSP70 accumulation when PAO was included during the heat treatment and recovery period (1.5 h), and the inhibition with 2 µM PAO was completely reversed with 2 µM DMP but not by 1 mM 2-ME (data not shown).

Effects of PAO on HSP70 mRNA expression. The PAO effect continued for at least 60 min even after PAO was removed by washing. To avoid extracellular interactions between PAO and the reducing agents, cells were always washed three times with RPMI 1640 after treatment with PAO, DMP, or 2-ME in the following experiments. Northern blot analysis with a cDNA probe for the human HSP70 shows that HSP70 mRNA was scarcely detected in untreated control cells (Fig. 3, lane 1). Heat stress rapidly increased the HSP70 mRNA level within 10 min (lanes 2-4). Pretreatment with 2 µM PAO for 5 min prior to heat exposure continued to inhibit the transcript expression during the experimental period (30 min) (lanes 5-8). Treatment with 1 mM 2-ME did not change the heat stress-induced expression of the HSP70 mRNA (lanes 21-24) and did not affect the PAO-induced inhibition of the transcript expression (lanes 13-16). When cells were treated with 2 µM DMP for 5 min prior to heat treatment, DMP partially suppressed the HSP70 expression (lanes 17-20); the amount of HSP70 mRNA, standardized by that of GAPDH mRNA, decreased to 67 ± 12% (mean ± SD, n = 4) of the control value at 20 min. However, 2 µM DMP could completely restore the HSP70 mRNA expression in PAO-pretreated cells (lanes 9-12).


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Fig. 3.   Effect of PAO on HSP70 mRNA expression by heat stress. Cultured cells were untreated (lanes 1-4) or treated for 5 min at 37°C with 2 µM PAO (lanes 5-8), 2 µM DMP (lanes 17-20), or 1 mM 2-ME (lanes 21-24) and then washed three times with RPMI 1640. These cells were exposed to heat at 43°C for the indicated times. DMP at 2 µM (lanes 9-12) or 1 mM 2-ME (lanes 13-16) was added to the PAO-pretreated and washed cells. After incubation for 5 min at 37°C, cells were washed again with RPMI 1640 and subjected to heat treatment for the indicated times. Total RNA was extracted, and samples of 10 µg RNA were subjected to Northern blot analysis with a cDNA for human HSP70 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described in MATERIALS AND METHODS. These results are representative of those obtained in 4 separate experiments.

Effect of PAO on activation of HSF1 in cells and whole cell extracts exposed to heat stress. The heat shock response is mediated by activation of HSF1. After exposure of cultured cells to heat stress at 43°C, whole cell extracts were prepared, and the activation of HSF1 was examined by gel mobility shift assay with a HSE oligonucleotide. In these experiments, cells were always washed after treatment with PAO, DMP, or 2-ME. As shown in Fig. 4A, lanes 2-4, an HSF1-HSE complex was produced within 5 min as already reported in a previous report (41). When cells pretreated with 2 µM PAO for 5 min were exposed to heat stress, the HSE-binding activity was not detected (lanes 6-8). This inhibition was again reversed by 2 µM DMP (lanes 9-12) but not by 1 mM 2-ME (lanes 13-16), suggesting that distinct vicinal dithiol-containing molecules may be involved in the activation of HSF1. During the preparation of whole cell extract and the binding reaction, 0.1 mM DTT was always included; however, the presence of DTT could not reverse the PAO-induced inhibition of the HSE-binding activity.


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Fig. 4.   Heat shock element (HSE)-binding activity in gastric mucosal cells. A: control cells (lanes 1-4) and cells pretreated with 2 µM PAO for 5 min (lanes 5-8) were washed three times with RPMI 1640 and exposed to heat at 43°C for the indicated times. After the PAO-pretreated cells were washed, they were incubated with 2 µM DMP (lanes 9-12) or 1 mM 2-ME (lanes 13-16) for 5 min. After being washed, these cells were subjected to heat stress for the indicated times. Whole cell proteins were extracted, and gel mobility shift assay was performed with a 32P-labeled HSE oligonucleotide, as described previously (18, 41). An HSF1-HSE complex is indicated as "HSF1." "NS" denotes a nonspecific DNA-protein complex. The results are representative of those obtained in 5 separate experiments. B: whole cell proteins were prepared from untreated and unstressed cells (lanes 1 and 3-6). These extracts were exposed to heat at 43°C for 30 min in the absence (lane 3) or presence of PAO at the indicated concentrations (lanes 4-6). In these cases, 1,4-dithiothreitol (DTT) was omitted during the preparation and heat treatment. These samples were subjected to the binding reaction for 20 min at 25°C in the presence of 0.1 mM DTT. The HSE-binding activities were detected as described above and compared with that in heat-stressed cells (lane 2). The results are confirmed in 3 separate experiments. HSF, heat shock factor.

We further tested whether PAO directly interfered with the activation of HSF1. For this purpose, whole cell extract was prepared from untreated control cells, and HSF1 was activated in vitro by exposure to heat at 43°C for 30 min. When DTT is already present in the extracts, externally added PAO interacts with DTT (27). To avoid this chemical interaction, DTT was omitted during the preparation of whole cell extracts and their exposure to heat treatment. DTT (0.1 mM) was first added after the heat treatment was finished, and the binding reaction was done in the presence of 0.1 mM DTT. As shown in Fig. 4B, heat treatment of the whole cell extracts produced an apparent HSE-binding activity (Fig. 4B, lane 3), although the intensity was lower than that in cells exposed to heat shock (lane 2). Inclusion of PAO during the exposure of whole cell extract to heat stress inhibited the production of the HSE-binding activity in a dose-dependent manner, and 10 µM PAO completely blocked the formation of a HSF1-HSE complex (lane 6). Thus PAO appeared to directly inhibit the activation of HSF1 by heat stress.

Effects of PAO on nuclear translocation and phosphorylation of HSF1. HSF1 is present mainly in the cytoplasm as a monomer in normal, unstressed cells. Upon exposure to heat stress, it rapidly trimerizes, acquires DNA-binding activity, is transported into the nucleus and phosphorylated, and becomes transcriptionally competent (2, 8, 9, 25, 31, 42). Before heat stress, HSF1 was detected mainly in the cytosol of control cells (Fig. 5A, lanes 1 and 2). After heat stress began, the HSF1 level in the cytosol gradually decreased (lanes 3-5), and, concomitantly, a phosphorylated form of HSF1 with a molecular mass of 85 kDa appeared in the nuclear fraction (lanes 8-10). The level of phosphorylated HSF1, indicated as "p-HSF1" in Fig. 5, time-dependently increased. The 85-kDa band disappeared when nuclear extracts from heat-shocked cells were treated with potato acid phosphatase, and as a consequence, the amount of 67-kDa HSF1 increased (Fig. 5C). Thus the 85-kDa protein was confirmed to be a phosphorylated HSF1. As shown in Fig. 5B, the HSF1 level in the cytosol of PAO-treated cells was similar to that in control cells (Fig. 5B, lanes 1 and 2). Furthermore, PAO did not affect the heat stress-induced nuclear translocation and phosphorylation of HSF1 (Fig. 5B, lanes 8-10).


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Fig. 5.   Nuclear translocation and phosphorylation of HSF1. Untreated cells (A) and PAO-pretreated cells (B) were exposed to heat stress at 43°C for the indicated times. Cytosol and nuclear fractions were prepared, as described in the MATERIALS AND METHODS. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblot analysis with an antibody against HSF1 was performed as described in MATERIALS AND METHODS. C: the nuclear fraction was prepared from cells exposed to heat at 43°C for 30 min and was incubated at 37°C for 60 min in the absence (lane 1) or presence of 1 U of potato acid phosphatase (lane 2). These samples were separated by SDS/8%-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting with an antibody against HSF1, as described in MATERIALS AND METHODS. p-HSF1, phosphorylated HSF1; n-HSF1, native (unphosphorylated) HSF1. Similar results were confirmed in 3 separate experiments.

Effects of PAO, DMP, and Trx on HSE-binding reaction of HSF1. The results shown in Figs. 4 and 5 raised the possibility that PAO might specifically block the interaction between activated HSF1 and HSE oligonucleotide. To address this issue, whole cell extract containing activated HSF1 was prepared from heat-stressed cells without DTT. This extract was treated with different concentrations of PAO at 25°C for 5 min and then subjected to the HSE-binding reaction in the presence of 0.1 mM DTT. As shown in Fig. 6A, lanes 2-5, the HSE-binding ability decreased by increasing the concentration of PAO, and 10 µM PAO completely inhibited the formation of a HSF1-HSE complex, showing that PAO could directly inhibit the binding activity of the activated HSF1. Alternatively, cells pretreated with 2 µM PAO were exposed to heat shock, and whole cell extract was prepared from these cells. Treatment of this extract with 10 µM DMP for 5 min at 25°C completely restored the binding activity (lane 7), also demonstrating that PAO blocked the binding reaction of activated HSF1, possibly by interacting with vicinal dithiol-containing molecule(s).


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Fig. 6.   Effects of PAO, DMP, and thioredoxin (Trx) on HSE-binding activity of HSF1. A: whole cell extracts were prepared without DTT from unstressed control cells (lane 1), heat-shocked cells (lanes 2-5), and PAO-treated and heat-shocked cells (lanes 6 and 7). These extracts were untreated (lanes 1, 2, and 6) or treated at 25°C for 30 min with different concentrations of PAO (lanes 3-5) or 10 µM DMP (lane 7) prior to the binding reaction in the presence of DTT. The HSE-binding activity was detected as described in the legend to Fig. 5. Similar results were obtained in 3 separate experiments. B: whole cell proteins were extracted from unstressed control cells (lanes 1 and 2) and PAO-treated and heat-stressed cells (lanes 3-5). The extracts were incubated at 25°C for 30 min in the absence of Trx (lanes 1 and 3) or presence of Trx at 5 µM (lane 4) or 10 µM (lanes 2 and 5) prior to the binding reaction. The binding activity was detected as described above. Similar results were obtained in 3 separate experiments.

There are a number of vicinal dithiol-containing molecules in cells that may directly or indirectly modulate the DNA-binding activity of HSF1. We focused on a vicinal dithiol-containing molecule, Trx, that might exert the DMP-like activity in cells. It has been shown that Trx is involved in the reduction/oxidation (redox) regulation of DNA-binding activity of several transcription factors, such as nuclear factor-kappa B (NF-kappa B) (17, 30). To examine the effects of Trx, whole cell extracts were prepared from control cells or PAO-treated and heat-stressed cells. These extracts were incubated with 5 or 10 µM Trx for 20 min at 25°C and then subjected to gel mobility shift assay (Fig. 6B). Trx alone did not produce the HSE-binding activity in whole cell extract from unstressed control cells (Fig. 6B, lane 2), but it effectively restored the binding ability of HSF1 in the extracts from PAO-treated and heat-shocked cells (lanes 4 and 5), similar to the effect of DMP (Fig. 6A, lane 7), suggesting that Trx might reverse the PAO-induced inhibition of binding activity of HSF1.

Effect of PAO on tyrosine phosphorylation of HSF1. PAO is well known to inhibit PTPase activity by interacting with vicinal dithiols of this enzyme (3, 4, 11, 12). It was also reported that heat stress induces tyrosine phosphorylation of several undefined proteins (29). PAO might disturb the regulatory balance of PTPase activity and tyrosine phosphorylation; thereby, it interferes with the DNA binding. To test this possibility, we examined whether PAO enhanced the heat-initiated protein tyrosine phosphorylation in cultured cells by Western blot analysis with an antiphosphotyrosine antibody. We could not obtain any consistent change in protein tyrosine phosphorylation after heat stress in the presence or absence of PAO (data not shown). Next, HSF1 was isolated from untreated or PAO-treated cells before and after exposure to heat stress by immunoprecipitation with an anti-HSF1 antibody, and tyrosine phosphorylation of HSF1 was studied (Fig. 7A). In immunoprecipitates from both control cells and PAO-treated cells, an antibody against HSF1 recognized a 67-kDa HSF1 before heat stress and the phosphorylated 85-kDa and the 67-kDa HSF1s after heat stress (Fig. 7B). The 67-kDa HSF1 and the phosphorylated 85-kDa HSF1 were not tyrosine phosphorylated (Fig. 7A, lanes 1-4).


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Fig. 7.   Effect of PAO on tyrosine phosphorylation of HSF1. Whole cell proteins were prepared from control cells and cells treated with 2 µM PAO before (lanes 1 and 3) and 30 min after heat stress (lanes 2 and 4), and immunoprecipitation with an anti-HSF1 antibody was performed, as described in MATERIALS AND METHODS. The immunoprecipitates were separated by SDS/8%-PAGE and transferred to PVDF membranes. Immunoblotting was performed with the use of anti-phosphotyrosine antibody (A) or anti-HSF1 antibody (B). Similar results were confirmed in a separate experiment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PAO is a trivalent arsenical compound that permeates into the cell interior within 1 min and reacts with multiple target molecules that contain vicinal dithiols, including phosphatases, kinases, glutathione transferases, Trx, steroid and thyroid hormone receptors, and many others. Among these interactions, PAO is frequently used as a potent inhibitor for PTPase. In a previous report (27), we showed that PAO not only inhibited PTPase activity but also blocked the stimulus-induced activation of PKC in human neutrophils. PKC activators stimulate the heat shock response, and a PKC inhibitor, staurosporine, is known to inhibit HSP70 and HSP28 mRNA expressions in HT 29 cells (19). At the same time, it was reported that heat shock induced protein tyrosine phosphorylation (29), and an inhibitor of tyrosine kinases, genistein, blocked transcription of the HSP70 gene without affecting the DNA binding of HSF1 (38), suggesting that protein tyrosine phosphorylation might be involved in the HSP70 gene expression. These results led us to consider that PAO might modify the heat shock response by changing the regulatory balance of protein phosphorylation.

Upon exposure to heat stress, HSF1 undergoes multistep activation. Under normal conditions, HSF1 is present mainly in the cytoplasm as a monomer. HSF1 consists of a conserved amino-terminal DNA-binding domain, multiple arrays of hydrophobic heptad repeats, and a carboxy-terminal transcription activation domain (16). Monomers appear to form a fold-back structure that masks the DNA-binding domain and the transcription activation domain, through interactions between leucine zippers (10, 28, 53). Heat shock causes HSF1 to unfold and expose the nuclear localization signal and the trimerization region, leading to formation of active HSE-binding trimers. Recently, several lines of evidence have suggested that the active HES-binding trimer may require hyperphosphorylation for unmasking the transcription activation domain that is located toward the carboxy-terminal end of each monomer and for becoming a transactivator of the HSP genes (8, 25, 46).

We report here that PAO could have inhibited transcription of the HSP70 gene in gastric mucosal cells. Quercetin is also known as a inhibitor of the HSP induction (22). This compound at higher concentrations (over 50 µM) was shown to inhibit the induction by slowly downregulating the HSF1 expression (34). In contrast, PAO at 2 µM completely blocked the HSP70 induction within minutes and did not change the HSF1 level. PAO did not induce tyrosine phosphorylation of HSF1 and other intracellular proteins (Fig. 7 and data not shown). We examined the effects of PAO on the multistep activation processes of HSF1, and we found that PAO did not block the trimerization (indirectly shown by nuclear translocation), nuclear translocation, and phosphorylation, but it completely inhibited the binding to HSE.

The PAO-induced inhibitions of DNA-binding activity of HSF1, expression of the HSP70 transcript, and HSP70 synthesis were completely reversed by a dithiol reagent DMP at the same concentration, but 2-ME and DTT did not, even at 500- and 50-fold molar excesses, respectively. These results suggested that PAO may exert its unique action by interacting with a specific dithiol-containing molecule(s) that plays a critical role in the activation of HSF1. In vitro binding experiments more clearly demonstrated this unique inhibitory action of PAO: inclusion of PAO during the binding reaction interfered with the binding of activated HSF1 to HSE, and PAO also blocked the acquisition of the HSE-binding activity by heat treatment of whole cell extracts from unstressed control cells. Alternatively, DMP restored the HSE-binding activity in whole cell extracts prepared from PAO-treated and heat-exposed cells. These results suggest that PAO may specifically interfere with the reaction between the active HSE-binding trimer and HSE.

It should be noted that the inhibition of heat shock response by PAO was reversible. When PAO was removed by washing, gastric mucosal cells could restore the heat stress response within 1 to 1.5 h, suggesting that the cells may possess endogenous reducing systems, like DMP, which can reduce the dithioarsine complex. The cell-free experiments demonstrated that Trx was a possible enzyme catalyzing this reduction. Trx contains the conserved two redox-active cysteine residues in the active site. Trx, maintained in the reduced state by Trx reductase and NADPH (20), functions as a hydrogen donor for reducing protein disulfides (21). Trx is known to regulate the DNA-binding activity of NF-kappa B by reducing a conserved cysteine residue in position 62 on the DNA-binding domain (17, 30). It has been shown that, in response to various stresses, Trx is induced and translocates from the cytosol into the nucleus, where it regulates protein-protein or protein-nucleic acid interactions through the redox state of protein cysteine residues (35).

Huang et al. (23) reported that high concentrations of DTT or 2-ME inhibited the activation of HSF1 in heat-stressed cells, suggesting that alteration of the intracellular redox state may trigger the activation of HSF1. Jacquier-Sarlin and Polla (24) showed that the HSF1 DNA-binding activity in heat-treated cells was inhibited by incubating whole cell proteins with H2O2, and this inhibition was restored by incubation with Trx. They suggested that the oxidized HSF1 is unable to bind HSE, whereas Trx may effectively reduce the oxidized cysteine residue(s) in the DNA-binding domain and restore the DNA-binding activity (24). We also reported that intracellular glutathione may be involved in the activation of HSF1 and suggested that the reversible oxidation of cysteine sulfhydryls by glutathione (S-thiolation/dethiolation) may be account for this redox regulation (41). In fact, Trx effectively reduces the disulfide formation (20, 21). However, the PAO-induced inhibition appeared not to be due to the S-thiolation of HSF1, since DTT at 0.1 mM, included during the preparation and binding reaction, must reduce the S-thiolation, whereas it did not reverse the PAO-induced inhibition. Thus PAO appeared to exert its inhibitory action specifically through formation of dithioarsine complex with vicinal-containing molecule(s). Mammalian HSF1s do not have vicinal dithiol-containing sequences, and there is no evidence that HSF1 is a direct target molecule against PAO. However, it has recently been shown that the heat shock response is regulated by complex cross talks between a family of heat shock factors, molecular chaperons, and negative regulators (see Ref. 32 for a review). It is possible to speculate that PAO might react with two thiols of closely spaced cysteine residues in association with the protein-protein interactions among these regulators.

The present study does not completely reveal the biochemical reactions of PAO leading to inhibition of the HSF1 activation. However, we introduce a potent and unique inhibitor of the heat shock response that may be a useful tool for studying the regulatory mechanism of the activation of HSF1 and possibly other redox-sensitive transcription factors.


    ACKNOWLEDGEMENTS

This work was supported by Grant-in-Aid 106704799 for Scientific Research from The Ministry of Education, Science and Culture (to K. Rokutan).


    FOOTNOTES

Address for reprint requests and other correspondence: K. Rokutan, Dept. of Nutrition, School of Medicine, The Univ. of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan (E-mail: rokutan{at}nutr.med.tokushima-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 January 2000; accepted in final form 23 June 2000.


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