We have investigated the regulation mechanism of chemical stress-induced hsp70 gene expression in murine L929 cells. Our data show that chemical treatments including sodium arsenite, cadmium chloride and sodium salicylate, induced significant synthesis of hsp70 and its mRNA. The induced hsp70 gene expression appears to be regulated at the transcriptional level. A factor (CHBF), which constitutively binds to the heat shock element (HSE) at 37°C, functions like a negative regulator and the heat-induced heat shock factor (HSF) acts as an activator. The chemical treatments that induce significant hsp70 synthesis activate HSF binding to HSE but also dissociate the HSE-CHBF complex. Some chemical treatments, e.g. IPTG, which fail to activate hsp70 gene transcription, still activate HSF binding to HSE. However, in this case, the HSE-CHBF complex remained like that of untreated control cells.
Stress response is nearly universal. Living organisms respond to the changes in their chemical, physical and biological environments by synthesizing a group of proteins called stress or heat shock proteins (Lindquist and Craig, 1988; Morimoto et al., 1990). Experimental evidence shows that these proteins, particularly, 70 kDa protein (hsp70), protect cells from stress-induced damages (Li et al., 1991, 1992; Liu et al., 1992; Pelham, 1982; Riabowol et al., 1988; Solomon et al., 1991).
The process of transcriptional regulation of hsp70 in eukaryotes is highly complex. It is believed that the activation of hsp70 gene transcription in eukaryotes during the stress response is mediated through a conserved DNA sequence, the heat shock elements (HSE) in the promoter of the heat shock genes (Amin et al., 1988; Pelham, 1982). It is reported that stress-induced binding of the pre-existing heat shock factor (HSF) to HSE activates hsp70 gene expression (Morimoto, 1993; Rabindran et al., 1993). However, in some circumstances, it has been reported that activated HSE-binding activity of HSF did not correlate directly with the transcription of hsp70 (Bruce et al., 1993; Hensold et al., 1990; Jurivich et al., 1992; Liu et al., 1993). Furthermore, human cells growing at 37°C constitutively produce hsp70, but no significant HSE-HSF-binding activity is observed (Kingston et al., 1987; Morimoto, 1993), suggesting that other mechanisms or factor(s) are required for the regulation of hsp70 gene expression. Several researchers observed that a 37°C constitutive HSE-binding factor (CHBF) exists in mammalian cells including HeLa cells (Mosser et al., 1988, 1990), in murine MEL cells (Hensold et al., 1990), and in rat Rat-1 cells. Liu et al. (1993) reported that the heat shock treatment dissociates CHBF from HSE in a dose-dependent manner. The dissociation appears to correlate with the transcription of the hsp70 gene in Rat-1 cells, suggesting that CHBF is involved in hsp70 transcriptional regulation as a negative regulator.
It has been known that chemical stress induces heat shock response (Lindquist et al., 1988). However, previous studies demonstrated that chemical treatments, such as sodium arsenite and cadmium chloride etc., clearly increased the synthesis of the constitutive form of hsp70 (hsc70) but failed to induce the synthesis of the inducible form of hsp70 (hsp70) in HA-1, Rat-1 and CHO cells (Laszlo and Li, 1985; Liu et al., 1993). Jurivich et al. (1992) also reported that salicylate treatment failed to induce hsp70 synthesis in HeLa cells. However, Lee et al. (1992) observed a significant synthesis of hsp70 in L929 cells undergoing arsenite-induced stress. These results led us to employ L929 cells to investigate the regulation mechanism of chemical stress-induced hsp70 gene expression. Specifically, we address the questions of whether the mechanism regulating hsp70 synthesis in cells undergoing chemical stress is the same or similar to that of heat shock stress, and whether the constitutive factor is involved in the regulation as a negative regulator.
Our data indicate that not only sodium arsenite treatment but also sodium salicylate or cadmium chloride treatments induce significant hsp70 synthesis in L929 cells. Chemically induced gene expression appears to be regulated at the transcriptional level by a similar mechanism to that of heat shock treatment. CHBF functions like a negative gene regulator. All chemical treatments that induce significant hsp70 synthesis dissociate the HSE-CHBF complex. In the chemical treatments that activate HSF but fail to induce hsp70 gene transcription, the HSE-CHBF complex remains intact.
MATERIALS AND METHODS
Cell culture and chemical stress treatments
Mouse fibrosarcoma L929 cells (Hepburn et al., 1988) were cultured in McCoy’s 5a medium (Cellgro) containing 26 mM sodium bicarbonate and 10% iron-supplemented calf serum (HyClone). The culture flasks or dishes containing cells were kept in a 37°C humidified incubator with a mixture of 95% air and 5% CO2. Sodium arsenite (ARS), cadmium chloride (CdCl2), sodium salicylate (SAL) and isopropyl β-D-thiogalactopyranoside (IPTG), which are reported to induce heat shock response (Li, 1983; Laszlo and Li, 1985; VanBo-gelan et al., 1990), were chosen to stress cells, in various amounts for indicated times. All the chemicals used in this experiment were purchased from Sigma Inc. (St Louis, MO). For heat shock treatment, flasks or dishes were sealed with parafilm and heated by total immersion in a circulating water bath maintained at the desired temperature within ± 0.05 deg. C.
Metabolic labeling and SDS-polyacrylamide gel electrophoresis
To examine the synthesis of hsp70 protein following chemical treatments, cells cultured in 35 mm dishes were incubated in methionine-free medium with 20 μCi/ml of Trans35S-Label (ICN, Trans35S-Label, methionine and cysteine; specific activity 1180 Ci/mmol) for 6–8 hours at 37°C after chemical treatments. Incorporation of 35S-labeled amino acid precursors was terminated by washing cells twice with ice-cold PBS and the proteins were precipitated with 10% trichloroacetic acid. The pellets were lysed in 1× SDS lysis buffer and an equal amount of radioactivity (counts per minute) was applied to a one-dimensional SDS-PAGE gel. Electrophoresis and autoradiography were carried out as described previously (Liu and Li, 1993). The location of hsp70 in SDS-PAGE was also identified by western blot analysis with antibody specifically against hsp70 and hsc70, according to the method described previously (Li et al., 1991).
Isolation of RNA and northern hybridization
Total cellular RNA was isolated with a RNA Zol TM kit (Biotecx Laboratories, Inc., Houston, Texas), according to the instructions of manufacturer. RNA (10 μ g/lane) was denatured with glyoxal/dimethyl sulfoxide and incubated at 5 5 °C for 1 hour, size-fractionated on a 1.2% agarose gel in 10 mM sodium phosphate, pH 7.4, and transferred onto a Hybond-N nylon membrane (Amersham) in 20× SSC (3 M sodium chloride, 0.3 M sodium citrate, pH 7). The blotted membranes were hybridized with the 2.3 kb BamHI-HindIII fragment of the human hsp70 gene (Liu et al., 1993), which was 32P-labeled with a random primer labeling kit according to the instructions of the manufacturer (Stratagene Inc.). After hybridization, the membranes were washed, dried and autoradiographed with Kodak X-OMAT film.
Preparation of whole cell extracts and analysis of the mobility of DNA-protein complexes by gel electrophoresis
Preparation of cell extracts and gel mobility shift assays were performed according to the methods previously described (Zimarino and Wu, 1987), with modification. Briefly, equal amounts of cellular proteins for each sample were incubated with a [32P]dCTP fill-in labeled double-stranded oligonucleotide containing the DNA sequence of the rat heat shock element (Liu et al., 1993), which is identical to the functional DNA sequence of mouse HSE (Hunt and Calderwood, 1990). The Sp1 oligonucleotide (see Baler et al., 1992) and the mutant double-stranded HSE oligonucleotide (CCCTAAAC-TATGTAAGATTATTTG) were used as the nonspecific binding competitors. The protein-bound and unbound oligonucleotides were separated electrophoretically on 4% native polyacrylamide gels in 0.5× TBE buffer (44.5 mM Tris, pH 8.0, 1 mM EDTA and 44.5 mM boric acid) for 3 hours at a constant 140 volts. The gels were dried and autoradiographed at −70°C with Kodak X-OMAT film and a Dupont Cranex lightning-plus intensifying screen. To trace clearly the weak constitutive HSE-binding activity of CHBF in L929 cells under stress, each cell lysate with 60–70 μ g total cellular proteins was used for the analysis of DNA-binding proteins.
To investigate the chemically induced synthesis of heat shock proteins, L929 cells were labeled for 8 hours with Trans35S-Label at 37°C after treatments with sodium arsenite (ARS), cadmium chloride (CdCl2), sodium salicylate (SAL) and isopropyl β-D-thiogalactopyranoside (IPTG). Our data show that hsp70 is undetectable in untreated cells (Fig. 1, lanes C). After cells were treated with 200 μ M ARS for 1–2 hours, significant induction of hsp70 synthesis was observed (Fig. 1B). Our data also show that treating cells with 200 μ M cadmium chloride for 3 hours induces significant hsp70 synthesis (Fig. 1A). The treatment with 5–30 mM sodium salicylate was reported to stimulate HSF binding to HSE but failed to activate hsp70 gene transcription in HeLa cells (Jurivich et al., 1992). When we performed an experiment to screen a wide range of chemical doses, we found that increasing the SAL concentration to 60–120 mM in culture medium induced a significant hsp70 synthesis (Fig. 1C). VanBogelan et al. (1990) reported that treating Escherichia coli cells with 5 mM IPTG induced the synthesis of hsp70. However, in a similar treatment with L929 cells, we did not detect a significant synthesis of either hsc70 or hsp70. In addition, the pattern of protein synthesis after IPTG treatment was very similar to that of untreated control cells. Even increasing the dose of IPTG up to 100 mM did not induce significant hsp70 synthesis (Fig. 1D). The induction of hsp70 by treatment with ARS was further confirmed by one-dimensional SDS-PAGE western blot analysis with monoclonal antibodies (SPA-820, StressGene Co.) specifically against the constitutive and inducible forms of hsp70. Fig. 2 clearly shows that treatment with 200 pM ARS for 1 hour induced significant hsp70 synthesis (lane 4), but 100 mM IPTG treatment for 1 hour failed to induce hsp70 synthesis (Fig. 2, lane 5).
To investigate the effects of chemical treatment on hsp70 mRNA synthesis and to determine if the translational regulation may contribute to the lack of hsp70 synthesis in cells after chemical treatment, we examined the accumulation of hsp70 mRNA at 37°C for various times following chemical treatment using northern hybridization analysis. Fig. 3 shows that there is only a low level of hsc70 mRNA at 37°C in unheated control cells (lane C). Fig. 3A shows that there is a significant accumulation of hsp70 mRNA in L929 cells after a 3 hour treatment with 200 μ M CdCl2. hsp70 mRNA accumulation increased gradually and reached its maximum level at 4 hours following treatment. A similar kinetics of hsp70 mRNA accumulation was also observed in cells treated with 200 μ M ARS for 1 hour (Fig. 3B).
Fig. 3C shows that treating cells with 120 mM SAL-induced hsp70 mRNA synthesis, but the kinetics of hsp70 mRNA accumulation is somehow different from that of CdCl2 or ARS treatment. In the treatment with sodium salicylate, the accumulation of hsp70 mRNA was detected from 1 to 2 hours after treatment, and then hsp70 mRNA decayed rapidly and disappeared at around 4 hours (Fig. 3C). Since the induction of hsp70 has been observed in cells treated with 120 mM SAL, we performed a study to exclude the possibility that the osmotic change in the medium stresses cells and induces hsp70 synthesis. The result shows that culturing cells in medium containing 120 mM sodium chloride for 1–8 hours did not induce hsp70 and its mRNA synthesis in L929 cells. Furthermore, Figs 1D and 3D show that IPTG treatment up to 100 mM did not induce synthesis of hsp70 and its mRNA. Therefore, induction of hsp70 synthesis by chemical treatment is unlikely due to the osmotic changes.
Since our data clearly show that chemically induced hsp70 synthesis is regulated at the transcriptional level, we investigated transcriptional regulation of hsp70 gene expression. This was accomplished by analyzing the HSE-binding activities of heat shock transcription factors in cells treated with chemicals. Fig. 4A shows the activity of transcriptional factor determined by gel mobility shift assays in cells treated with CdCl2. In untreated control cells, HSE-HSF binding was undetectable (lane C). The chemical treatment induced HSF binding to HSE in a dose-dependent manner; that is, higher doses of chemicals induced more HSF binding to HSE. However, when comparing the activation of HSF with hsp70 synthesis, the HSE-binding activity of HSF did not appear to correlate directly with hsp70 synthesis. For example, 100 μ M CdCl2 for 1 hour induced very weak hsp70 synthesis, although strong HSE-HSF binding was observed. Furthermore, 10–50 μ M ARS treatment for 1 hour significantly activated HSF binding to HSE, but hsp70 synthesis was undetectable (compare Figs 1B and 4B). More examples are shown in Fig. 5 that indicate that HSE-HSF binding does not correlate with hsp70 synthesis. Treatment with 10–100 mM IPTG did not induce hsp70 synthesis even though strong HSE-HSF-binding activity was observed (Fig. 5B). Similar results were also obtained in Chinese hamster fibroblasts HA-1 cells and rat fibroblasts Rat-1 cells treated with ARS or SAL (data not shown). These results clearly show that chemical stress-induced HSE-HSF binding alone is insufficient to activate hsp70 gene transcription and that other mechanisms might be necessary for the regulation of hsp70 gene expression.
Liu et al. (1993) reported that the heat shock-induced dissociation of the HSE-CHBF complex was heat dose-dependent and correlated with the transcription of the hsp70 gene in Rat-1 cells. Also, it has been observed that treatment with ARS or SAL induces HSF binding to HSE but has little effect on the HSE-CHBF complex. When L929 cells were used as a model system to investigate the regulation of hsp70 gene expression, a weak HSE-binding activity of CHBF at 37°C and easy dissociation of the HSE-CHBF complex were observed. When 60–70 μ g of total proteins for each sample was analyzed in gel mobility shift assays, the constitutive HSE-binding activity was clearly observed in cells at 37°C, the normal growth temperature for L929 cells (Fig. 4, lane C). Fig. 4 shows that increasing the chemical dose of ARS or CdCl2 gradually dissociates the HSE-HSF complex. When the stress treatments induced HSF binding to HSE and also dissociated the HSE-CHBF complex, a significant synthesis of hsp70 and its mRNA were observed (Fig. 4A and B). If the HSE-CHBF complex remained following treatment, even though a strong HSE-HSF-binding complex was observed, the accumulation of hsp70 and its mRNA was undetectable (compare Fig. 4 and Figs 1 and 2). Furthermore, we demonstrated that treatment with IPTG significantly activates HSF binding to HSE, but has little effect on the HSE-CHBF complex. No significant hsp70 or its mRNA synthesis was detected. Interestingly, the present observations are very similar to what we previously reported in cells under-going heat shock; that is, the constitutive HSE-binding factor (CHBF) functions as a negative regulator and HSF acts as an activator. Heat-induced stress and chemical-induced stress appear to share a common mechanism for regulation of hsp70 synthesis.
We also performed experiments to examine the specificity of the HSE-binding factors. Our data show that both the constitutive HSE-CHBF complex and the HSE-HSF complex in the extracts from cells undergoing chemical stress could be completely competed by a 50-fold or more excess of nonlabeled HSE oligonucleotide, but not by excess of the control oligonucleotide containing Sp1 or mutant HSE (data not shown). Therefore, the sequence specificity of the HSE-binding activity of CHBF and HSF has been confirmed.
In the present report, some of the findings on regulation of chemically induced hsp70 synthesis can be summarized as follows. Firstly, our data show that chemically induced gene expression appears to be regulated at the transcriptional level. Secondly, stress-induced rapid activation of heat shock factor (HSF) is a very common phenomenon when cells undergo stress or changes of culture condition. Some chemical treatments fail to induce hsp70 synthesis but still activate HSF. Stress-induced HSE-HSF binding alone is insufficient to stimulate hsp70 transcription. Thirdly, chemically induced hsp70 gene expression appears to be regulated by a similar mechanism to that of heat shock treatment, in which the constitutive HSE-binding factor (CHBF) functions as a negative regulator and HSF as an activator. Only treatments that activate HSF and dissociate the HSE-CHBF complex can induce significant hsp70 synthesis. Furthermore, the findings that the HSE-CHBF complex exists at a low level at 37°C and dissociates easily under stress suggest a possible reason why hsp70 synthesis can be easily induced in L929 cells.
Our data show that all of the tested chemical treatments activate HSF binding to HSE, but the binding of HSF to HSE alone does not necessarily result in hsp70 gene transcription. When the effects of heat shock and other stresses on hsp70 gene transcription and the HSE-binding activity of the transcriptional factors were examined, we were impressed by the close relationship between the dissociation of the HSE-CHBF complex and hsp70 synthesis. Actually, in all of the stresses tested, the high level of the HSE-CHBF complex always correlated with the lack of hsp70 gene expression, and the dissociation of the HSE-CHBF complex appeared to correlate with hsp70 synthesis. Only the stresses that induce HSF binding to HSE and also enhance the dissociation of the HSE-HSF complex are accompanied by significant synthesis of hsp70 and its mRNA. On the other hand, the weaker constitutive HSE-HSF-binding activity alone appears to be insufficient to start hsp70 gene transcription, as no detectable level of hsp70 was observed in L929 cells at 37°C. Our study strongly suggests the involvement of CHBF in hsp70 synthesis as a negative regulator.
Although we do not know exactly how HSF and CHBF work to regulate hsp70 gene expression at the transcriptional level, our studies still provide some clues to their role in hsp70 gene expression. The HSE-binding ability of both factors can be completely competed with an excess of non-labeled HSE oligonucleotide, suggesting that the binding sites for HSF and CHBF are similar or, at least, overlapping. The fact that the activation of HSF binding to HSE is much easier than the induction of dissociation of the HSE-CHBF complex in cells under stress indicates that they have different DNA binding activity. Indeed, Mosser et al. (1988) reported that the DNA binding sites for CHBF and HSF are similar but not identical, as determined by in vitro footprinting. It is likely that CHBF, as a negatively acting factor, constitutively binds to a sequence adjacent to or overlapping the DNA binding site for HSF and prevents the binding of HSF by steric hindrance in the treatments that fail to start hsp70 transcription. In the case of treatments that induce hsp70 synthesis, the stresses may directly induce the conformational change in CHBF, result in the release of CHBF from HSE and activate HSF binding to HSE. Alternately, hsp70 transcription may be activated by stress-induced protein denaturation, as we observed that the addition of denatured bovine albumin (BSA) to the reaction system resulted in the dissociation of the HSE-CHBF complex. In this case, the denatured proteins somehow affect the binding of CHBF to HSE and result in the release of CHBF from HSE.
HSF has been implicated as the key activator in the heat shock response (Abravaya et al., 1991; Rabindran et al., 1991; Sorger, 1991). As all heat shock protein genes have the heat shock element in their promoter region, it is likely that the heat-induced binding of HSF to HSE is a general activator for the heat shock response but not specific for activating hsp70 gene transcription. Zimarino and Wu (1987) reported that heat shock induces HSF binding to Drosophila HSE of hsp82. Others reported that heat induces the binding of HSF to the heat shock element of hsp70 (Kingston et al., 1987; Morimoto, 1993) and our group observed that heat shock induces HSE-binding activity by using the HSE of hsp27 as a probe (unpublished observation). Moreover, Westwood et al. (1991) reported that the transition to the induced state is accompanied by chromosomal redistribution of HSF to the heat-shock puff sites and HSF accumulation at over 150 additional chromosomal sites, suggesting that HSF has some unknown functions.
In this study, we observed that stress-induced HSE-HSF binding is a very common phenomenon when cells undergo stress or changes in culture conditions. The chemicals that we tested all induce the binding of HSF to HSE, even IPTG treatment, which failed to induce synthesis of hsp70 and its mRNA, activated HSF (Fig. 5B, upper bands). Moreover, some cell lines lack the heat shock response, even though stress-induced binding of HSF to HSE was detected (Hensold et al., 1990). Our data show that inducing HSF binding to HSE is much easier than inducing hsp70 synthesis in the cells examined. Also, it has been reported that other stress treatments such as radiation (Auger et al., 1992), oxidation (Bruce et al., 1993), hypoxia (Giaccia et al., 1992) and chemicals (Jurivich et al., 1992; Liu et al., 1993) all induce the binding of HSF to HSE but fail to induce hsp70 synthesis.
Although, our data show that strong HSE-HSF-binding activity alone is insufficient to stimulate transcription of the hsp70 gene, it is possible that transcriptional regulation of hsp70 synthesis requires further translational modification of HSF, such as phosphorylation, to stimulate hsp70 gene transcription. Indeed, it was revealed with gel mobility shift assays that phosphatase treatment of cell lysates altered the movement of HSF (Bruce et al., 1993; Larson et al., 1988; Sorger, 1987). However, direct evidence for involvement of phosphorylation or dephosphorylation of HSF in hsp70 gene expression is still lacking. Also, it is possible that the different treatments may induce different members of the HSF family and result in a distinct stress response, since the existence of multiple heat shock factor (HSF) genes in eukaryotes has been reported (Mosser et al., 1993; Nakai and Morimoto, 1993). However, Sarge et al. (1993) have identified HSF1 as the mediator of stress-induced stress gene transcription. They reported that HSF1 displays stress-induced DNA-binding activity, oligomerization and nuclear localization, while HSF2 does not. In our studies using prolonged native gel electrophoresis (Bruce et al., 1990), we have not observed different electrophoretic mobility of HSF in cells undergoing different treatments (data not shown).
We do not know exactly the relationship between HSF and CHBF. However, the accumulating data show that they are different factors, based on these findings; (1) polyclonal antibodies specifically recognize HSF but not CHBF in western blot analysis (Mosser et al., 1993; Nakai et al., 1993; Sarge et al., 1993); (2) the stress-induced activation of HSF does not rely on the dissociation of the HSE-CHBF complex and the two complexes can be detected in some treatments (Figs 4 and 5); (3) boiling-denatured BSA dissociated the HSE-CHBF complex but failed to activate HSF, suggesting different characteristics of HSE-binding to HSF and to CHBF (data not shown). Furthermore, Liu et al. (1993) previously reported that the kinetics of the recovery of the HSE-CHBF complex at 37°C was different from that of the HSE-HSF complex when cells were incubated at 37°C following heat shock.
It has been reported that arsenite or salicylate treatment activates HSF but fails to induce hsp70 synthesis in Rat-1, HA-1 and HeLa cells (Liu et al., 1993; Jurivich et al., 1992). In this study, we demonstrated that hsp70 synthesis can be induced not only with sodium arsenite treatment, but also by cadmium chloride or sodium salicylate treatment, in L929 cells. Previously, we observed that hsp70 synthesis can be induced by 41°C heat treatment in L929 cells. These results suggest that L929 cells have features that enable them to be easily induced to synthesize hsp70 under stress. When the HSE-binding activity of HSF was examined by gel mobility shift assays, no significant difference was observed between L929 and other cell lines. However, we observed a weak constitutive HSE-binding activity in L929 cells cultured at 37°C. Furthermore, our study shows that the treatments that fail to dissociate the HSE-CHBF complex in Rat-1 cells (Liu et al., 1993) dissociate the complex in L929 cells. The low level of HSE-HSF complex and its easy dissociation may be reasons why hsp70 synthesis in L929 cells is easily induced. However, we do not know, at present, whether the weak HSE-binding activity of CHBF in L929 cells is due to a small amount of CHBF or to the low HSE-binding ability of CHBF. Also, the translational modification of HSF may also involve the activation of hsp70 gene expression in L929 cells undergoing stress treatments.
This research was supported by NCI grants CA 48000 and, CA 44550, and William Beaumont Hospital Research Institute grant 93–09.