Differential Expression of hsp70 Stress Proteins in Human Endothelial Cells Exposed to Heat Shock and Hydrogen Peroxide L. Jornot, M. E. Mirault, and A. F. Junod Respiratory Division, University Hospital, Geneva, Switzerland, and Unite d'Ontogenese et de Genetique moleculaire, Centre de Recherche du Centre Hospitalier de l'Universite Laval, Quebec, Canada
The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45° C), exposure to hydrogen peroxide (HzOz) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (02 plus HzOz), as well as exposure to 95% Os, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45° C; (2) treatment with a 5-mM HzO z bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95 % Oz. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and HzOz-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to HzOz treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.
Heat shock proteins (hsps) are synthesized in most, if not all, cells in response to an increase of temperature above normal physiologic level or following exposure to many seemingly unrelated stimuli, including amino acid analogs, heavy metals, iodoacetamide, oxidizing agents, ethanol, recovery from anoxia, inhibitors of energy metabolism, and infection by adenovirus 5 or simian virus 40 (1, 2). This response is characterized by a rapid increase in transcription of a small set of genes and the preferential translation of these mRNAs into hsps. The major hsps are a set of highly conserved proteins having molecular masses of about 28, 70, and 82 to 90 kD. The most abundant and best studied subset of them is the 70-kD protein family. It is now established that multiple
(Received in original form November 8, 1990 and in revisedform February 21, 1991) Address correspondence to: Alain F. Junod, M.D., Respiratory Division, Hopital Cantonal Universitaire de Geneve, 24, rue Micheli-du-Crest, 1211 Geneve 4, Switzerland. Abbreviations: dithiothreitol, OTT; fetal calf serum, FCS; heat shock cognate, hsc; heat shock factor, HSF; heat shock protein, hsp; human umbilical vein endothelial cells, HUVEC; hypoxanthine HX; hydrogen peroxide, HzOz; sodium dodecyl sulfate, SDS; uridine triphosphate, UTP; xanthine oxidase, XO. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 265-275, 1991
genes encode several distinct "70-kD" hsp members that have slightly different molecular weight and/or charges but are structurally and immunologically related (3-5). Some of these forms are typically heat-inducible (hsp70), and their expression is stimulated manyfold after heat shock treatment. Besides these hsp70 genes, the family contains additional members that are abundantly expressed in the absence of stress (constitutive), often referred to as heat shock cognate (hsc70) genes, and others encoding a 78-kD glucoseregulated protein (grp78). Structural analysis of these genes has revealed a remarkably high degree of conservation from bacteria to humans. There is both direct and indirect evidence obtained in different species that hsp70 proteins are necessary in helping cells and organisms survive thermal damage. Although the mechanisms of action of hsp70 and related proteins still remain largely a matter of speculation, there is now compelling genetic and biochemical evidence that these proteins belong to a family of ATP-dependent "unfoldases" implicated in protein assembly/dissociation of multimeric complexes, translocation, and import/export processes across membranes (6). It was also suggested that these proteins could play an important role in cell growth and differentiation (1, 2). Recently, a number of reports have suggested that oxidative stress and heat shock responses overlap. For example,
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it was shown in bacteria that the synthesis of several proteins including an hsp70 analog (dnaK in Escherichia coli) is induced by both heat shock and exposure to hydrogen peroxide (HzOz) but that distinct transcription factors positively regulate the expression of heat shock and oxidative stress proteins (7). It was also found that a variety of oxidants as well as heat shock in prokaryotes induce the rapid accumulation of AppppA and other related adenylate dinucleotides (8). These so-called "alarmones" were proposed to be putative signals to modulate cellular gene expression and metabolism to adapt to oxidative stress. Further links between heat shock and oxidative stress may include the finding that depletion of endogenous glutathione can increase susceptibility to killing by heat (9) and that antioxidant enzymes levels can be enhanced by exposure to elevated temperature (10-13). In previous studies done in our laboratory on the effects of hyperoxia and oxygen metabolites generated by hypoxanthine (HX)-xanthine oxidase (XO) on protein synthesis in endothelial cells (14, 15), we did not detect any induction of hsp synthesis. Instead, we found a marked decrease in overall protein synthesis, which was associated with translational defects. In the present study, we therefore explored the possibility that hsp mRNAs could be induced by oxidative stress, but that their translation into corresponding hsps is defective or repressed. We looked in particular at the effects of HzO z, which was shown to induce heat shock puffs in Drosophila melanogasterpolytene nuclei (16, 17). We have analyzed the expression of the major 70-kD hsps in human endothelial cells exposed to heat shock and oxidative conditions, both at the mRNA level by Northern blot analysis and at the protein level by one- and two-dimensional gel analysis of pulselabeled proteins and by immunologic analysis. We found that hsp70 synthesis in response to elevated temperatures results from the combined increase in mRNA synthesis and selective translation into corresponding hsps. On the other hand, we found that the oxidizing agent HzOz, as well as the oxygen metabolites generated extracellularly by the enzyme system HX-XO, induced a dose-dependent transcription of hsp70 genes, whereas hyperoxia did not. However, the magnitude of the oxidant-induced response was considerably lower than that induced by heat stress, and the hsp70 proteins were hardly detectable. Nuclear run-on transcription analysis showed that the effect of heat shock and HzOz on hsp70 gene expression could be ascribed to a transcriptional induction. Furthermore, using two-dimensional gel electrophoresis analysis and a monoclonal antibody specific for human hsp70, we demonstrate that heat stress induced a series of at least five structurally related proteins in the 70-kD molecular mass area, whereas HzO z induced essentially one acidic isoform.
Materials and Methods Materials All chemicals were of analytical grade and came from Merck (Darmstadt, Germany) and Fluka (Buchs, Switzerland). Catalase (EC 1.11.1.6) from bovine liver, xanthine oxidase (EC 1.1.3.22) grade III from buttermilk, oligo(dT) cellulose, and the anti-mouse IgG antibody labeled with peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). [a-3ZP]uridine triphosphate (UTP) (800 mCi/mmol),
[a-3ZP]cytidine triphosphate (3,000 Ci/mmol) , and L-(3sS]methionine (1,000 Ci/mmol), the Multiprime DNA labeling kit, Hyperfilm", and the monoclonal anti-human 72173-kD hsp antibody were from Amersham (Buckinghamshire, UK). The SP6 polymerase transcription kit, the plasmid vector pSPTl9, and the restriction enzymes were obtained from Pharmacia LKB (Uppsala, Sweden). Biodyne transfer membranes were purchased from Pall (Glen Cove, NY), and nitrocellulose membranes were from Schleicher and Schuell (Feldbach, Switzerland). RPMI 1640 culture medium and fetal calf serum (FCS) were from GIBCO (Renfrewshire, Scotland). Endothelial cell growth supplement was obtained from Serva (Heidelberg, Germany). Cell Culture Human endothelial cells were obtained from umbilical cord veins (HUVEC) using 0.1% collagenase in Krebs-Ringer bicarbonate buffer (18). The cells were plated in gelatin-coated (0.1%) petri dishes and grown in RPMI 1640 supplemented with 25 mM Hepes, 50 U/ml penicillin, 50 ~g/ml streptomycin, 10% FCS, 30 ~g/ml endothelial cell growth supplement, and 90 ~g/ml heparin. The medium was replaced every second day. In this study, cells from second passages were used. Endothelial cells were characterized using morphologic (phase-contrast microscopy) and functional (measurement of the angiotensin-converting enzyme activity) criteria as previously described (14). Experimental Conditions All experiments were performed on confluent cells from second passages. Exposure to heat shock. The petri dishes containing the cells were heated in a waterbath for 30 min at 43, 44, or 45° C in RPMI 1640 supplemented with 10% FCS. The culture medium was preheated at 37° C, and its temperature reached that of the waterbath after ~5 min. After exposure to heat, the cells were allowed to recover in the 37° C incubator for the times indicated in the figures. Exposure to hyperoxia. The cells were exposed to 95 % O, for 5 days in a humidified chamber, as described in reference 14. Exposure to H202 • HzO z diluted in RPMI medium was added to the cells at the final concentrations of 1 and 5 mM. The cells were incubated at 37° C for 20 min. Catalase was then added (200 U/ml), the medium was removed, and the cells were washed 3 times with RPMI and allowed to recover in RPMI with 10% FCS at 37° C for various times, as indicated in the legends of the figures. Exposure to the HX-XO system. The cells were incubated with 15, 30, and 50 mU/ml XO in the presence of2 mM HX in Krebs-Ringer buffer (pH 7.4) supplemented with 25 mM Hepes for 1 h at 37° C. After the treatment, the cells were washed thoroughly with RPMl and incubated in RPMI with 10% FCS at 37° C for different periods of recovery, as indicated in the legends of the figures. Control cells were incubated with 2 mM HX in Krebs-Ringer buffer. RNA Extraction and Northern Blot Analysis Total cellular RNA was prepared by the guanidine thiocyanate method of Chirgwin and colleagues (19). Briefly, RNA was isolated by homogenization in a 4-M solution of the pro-
Jornot, Mirault, and Junod: Stress Proteins in Response to Heat Shock and Oxidants
tein denaturant guanidinium thiocyanate containing 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was then isolated free of protein and DNA through 5.7 M cesium chloride in 0.1 M EDTA and precipitated overnight with ethanol. Polyt.A):' RNA was obtained from equal amounts oftotal RNA by passing over an oligo(dT)cellulose column, according to the method of Maniatis and associates (20). For Northern analysis, total RNA or poly(A)+ RNA fractions were denatured and fractionated by electrophoresis on 1% agarose gels in 5 % formaldehyde, as described by Maniatis and associates (20). After electrophoresis, the RNA was transferred onto a Biodyne membrane according to the specifications of the manufacturer in 20x SSC (l x SSC: 0.15 M NaCI, 0.015 M Na-citrate [pH 7]). The membranes were baked for 2 h at 80° C, prehybridized for at least 4 to 5 h at 55° C in sx SSC, 10 mM NaP04 , 50% formamide, 5 x Denhart's (1 x Denhart's: 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 0.1% sodium dodecyl sulfate (SDS), and 250 J.tg/ml salmon sperm, and hybridized overnight in the same buffer at 65° C with a [32P]UTP labeled anti-sense RNA transcript (1 to 2 x 106 cpm/ml). This RNA probe was prepared from a pSPT19 vector, using an SP6 polymerase transcription kit from Pharmacia. This plasmid contains a 1-kb Bgl II fragment derived from a genomic DNA clone coding for human hsp70 inserted into the BamHl site of pSPT19 (21). The DNA sequence of this hsp70 gene was determined (Dworniczak and Mirault, unpublished observation) and found to correspond to that of another clone independently isolated by others (22). We have shown that the Bgl II fragment probe was specific for heat shock-inducible hsp70 mRNA and did not hybridize to hsc70 mRNA under stringent hybridization conditions (21). For analysis of hsc70 mRNA, the membranes were hybridized with a DNA probe labeled using the Multiprime labeling kit from Amersham in the same buffer described above, at 42° C for 12 to 16 h. This probe was an isolated rv1-kb EcoR I restriction fragment derived from an entire human hsc70 gene (21). After hybridization, the membranes were washed sequentially in 2x SSC, and 0.2x SSC containing 0.1% SDS at 65 to 70° C. They were then subjected to autoradiography on Hyperfilm (Amersham) at -70° C using intensifying screens. The autoradiographs were scanned by a densitometer (Bio-Rad VD620). The density of the hybridization signal was expressed as arbitrary units. Labeling of Proteins and Gel Electrophoresis L-[3sS]methionine (1,000 Ci/mmol) was added to the cell cultures during the last hour of the recovery period to a final concentration of 20 J.tCi/ml and 50 J.tCi/ml for analysis by one-dimensional and two-dimensional electrophoresis, respectively. After the labeling period, the medium was removed and the cells were washed with cold Krebs-Ringer buffer. For analysis by one-dimensional gel electrophoresis, the cells were solubilized by the addition of sample buffer (40 mM Tris [pH 6.8],10% glycerol, 5% SDS, and 250 mM dithiothreitol [DTT]). The samples were boiled for 2 min and stored at -20° C until analysis. For analysis by twodimensional gel electrophoresis, the cells were harvested in 10% SDS and 150 mM DTT, boiled for 2 min and stored at
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- 20° C. Prior to electrophoresis, the samples were diluted 1:4 with sample buffer containing 65 mM DTT, 65 mM (3[(3-cholamidopropyl)dimethylammonio]l-propane sulfonate) (CHAPS), 9 M urea, and 5% ampholytes, pH 3 to 10. One-dimensional gel electrophoresis. The labeled proteins were resolved by SDS polyacrylamide gel electrophoresis in slab gels with 10% aery lamide, as described by Laemmli (23). After staining with Coomassie blue, the gels were dried and autoradiographed at -70° C using Hyperfilm and intensifying screens. Two-dimensional gel electrophoresis. The procedures were as described by O'Farrell (24). Briefly, proteins were focused in the first dimension under reducing conditions using 5.5% ampholytes (1.1%, pH 5 to 7; 4.4%, pH 3 to 10) and separated in the second dimension on 10% acrylamide slab gels in the presence of 10% SDS. Celllysates containing equal amounts of trichloroacetic acid-precipitable radioactivity ( rv3 x 106 cpm) were applied to each gel of each experimental series. After electrophoresis, the proteins were transferred onto nitrocellulose membranes for immunoblot analysis. Western Blot Analysis After transfer, the nitrocellulose membranes were incubated overnight at 4° C with a monoclonal anti-human 72/73-kD hsp antibody (1:1,000) in 10 mM Tris-HCI (pH 7.4), 0.15 M NaCI, and 5 % nonfat dry milk. The primary antibody was washed away, and peroxidase-conjugated anti-mouse was applied (1:100) for 2 h at room temperature. Again, the blots were washed, and the immunoreactive proteins were visualized by exposure to 4-chloro-1-naphtol (60 mg/100 ml) in 10 mM Tris (pH 6.8) containing 20 % methanol and 3 mM H202. The nitrocellulose membranes were dried and exposed to Hyperfilm at -70° C. Nuclear Run-on Transcription Assays hsp70 gene transcription rates were determined by quantitative hybridization of 32P-Iabeled RNA made in nuclei from control cells, cells exposed to 45° C for 30 min, and cells treated with 5 mM H 202 for 20 min, to excess hsp70 DNA, as described in reference 25. Briefly, cells were washed with ice-cold phosphate-buffered saline (pH 7.4) and scraped with a rubber policeman in phosphate-buffered saline. The cells were then collected by centrifugation (800 X g) and resuspended in cold lysis buffer (0.32 M sucrose, 3 mM csci, 2 mM Mg-acetate, 10 mM Tris-HCI [pH 8], 1 mM DTT, 0.1% Triton X-100) and homogenized with 10 to 15 strokes in a Dounce homogenizer. The nuclei were isolated and pelleted through a layer of 2 M sucrose in 10 mM TrisHCI (pH 8), 5 mM Mg-acetate, 1 mM DTT by centrifugation for 45 min at 20,000 rpm in a Beckman SW50.1 rotor. The nuclear pellet was resuspended in 200 J.tl of glycerol storage buffer (50 mM Tris-HCI [pH 8.3], 40% glycerol, 5 mM MgCI2, 0.1 mM EDTA), quick-frozen in liquid nitrogen, and stored at -70° C until analysis. Nuclear transcripts were labeled with [32P]UTP and extracted as described in reference 26. Equal amounts of labeled RNA were used for hybridization to an excess of hsp70 DNA (8 J.tg DNA of the PST19 subclone described above) and, as a control, plasmid PST19 bound to Biodyne membrane using a slot-blot apparatus. Filters were prehybridized for 24 h and hybridized for
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4S0C
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Time of recovery (hours)
Figure 1. Time course of induction of hsp70 mRNA by heat shock . Cells were heat-shocked for 30 min at 43, 44, and 45° C, then allowed to recover at 37° C for 20 min and 1, 2, 4, 6, and 8 h. For each time point, 5 JIg of total RNA was analyzed by Northern hybridization as described in MATERIALS AND METHODS. Shown are a typical RNA blot (inset) and semiquantitative densitometric data from three individual experiments (mean ± SEM). The autoradiographic exposure time was 2 h.
the mRNA accumulation was slightly delayed, maximal at 4 h and sharply decreased by 6 and 8 h. Upon exposure to 45° C, the response was more delayed and more intense: the hsp70 mRNA level, barely detectable after 20 min of recovery, was maximal by 4 to 6 h and then declined significantly to reach, by 8 h, a quantity still comparable to the maximum observed after a 43° C heat shock . The corresponding patterns of protein synthesis are shown in Figure 2 . The pSS]methionine-labeled proteins synthesized in cells under normal conditions (37° C) or after a 30-min exposure to 43, 44, and 45° C, after recovery periods of2, 4,6, and 8 h at 37° C, were analyzed by one-dimensional gel electrophoresis. As can be seen, HUVEC responded to heat shock by inducing the synthesis of major stress proteins of about 70 kD (hsp70), and to a much lesser extent, the hsp90 protein doublet. That the prominent 70-kD band is, in fact, the 70-kD stress proteins was confirmed by Western blot analysis using a specific monoclonal antibody (data not shown). The time course and temperature dependence of hsp70 synthesis appear to closely reflect the mRNA levels observed in Figure 1. Thus, cells exposed to a 43° C heat shock showed almost immediate synthesis of hsp70, but the expression ceased by 6 h of recovery. In cells heated at 44°C, the synthesis of hsp70 was much higher by 2 h, remained high within 4 to 6 h, and eventually ceased by 8 h. After a 45° C shock, overall protein synthesis first declined during the first 2 h then recovered by 4 h, while hsp70 synthesis was vigorous for up to 8 h after heat shock.
c 2
3 days at 65° C. The relative amount of bound RNA was determined by densitometry of the autoradiographs. hsp70 mRNA Stability hsp70 mRNA half-life was determined from an actinomycin D decay curve. Immediately after the treatments, the cells were washed and allowed to recover at 37° C in the presence of 10 I-'g/ml of actinomycin D to block RNA transcription. The cells were lysed 2, 4, 6, and 8 h after addition of the drug, and total RNA was extracted and analyzed by Northern blot as described above.
Results Time Course of Induction of hsp70 mRNA and Proteins by Heat Shock The Northern blot shown in Figure 1 displays the effect of a 30-min exposure to 43, 44, and 45° Con hsp70 mRNA induction in HUVEC. The time course of expression of hsp70 mRNA appeared to be differentially regulated as a function of temperature. Densitometric analysis of hybridizations from three separate experiments clearly shows different kinetics of hsp70 mRNA accumulation in response to graded increase in temperature. Upon exposure to 43° C, the induction of hsp70 transcripts was almost maximal during the first 20 min after exposure, clearly reached a maximum within 1 h, and gradually declined later on. After exposure to 44° C,
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The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45° C), exposure to hydrogen peroxide (HzOz) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (02 plus HzOz), as well as exposure to 95% Os, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45° C; (2) treatment with a 5-mM HzO z bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95 % Oz. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and HzOz-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to HzOz treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.
Heat shock proteins (hsps) are synthesized in most, if not all, cells in response to an increase of temperature above normal physiologic level or following exposure to many seemingly unrelated stimuli, including amino acid analogs, heavy metals, iodoacetamide, oxidizing agents, ethanol, recovery from anoxia, inhibitors of energy metabolism, and infection by adenovirus 5 or simian virus 40 (1, 2). This response is characterized by a rapid increase in transcription of a small set of genes and the preferential translation of these mRNAs into hsps. The major hsps are a set of highly conserved proteins having molecular masses of about 28, 70, and 82 to 90 kD. The most abundant and best studied subset of them is the 70-kD protein family. It is now established that multiple
(Received in original form November 8, 1990 and in revisedform February 21, 1991) Address correspondence to: Alain F. Junod, M.D., Respiratory Division, Hopital Cantonal Universitaire de Geneve, 24, rue Micheli-du-Crest, 1211 Geneve 4, Switzerland. Abbreviations: dithiothreitol, OTT; fetal calf serum, FCS; heat shock cognate, hsc; heat shock factor, HSF; heat shock protein, hsp; human umbilical vein endothelial cells, HUVEC; hypoxanthine HX; hydrogen peroxide, HzOz; sodium dodecyl sulfate, SDS; uridine triphosphate, UTP; xanthine oxidase, XO. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 265-275, 1991
genes encode several distinct "70-kD" hsp members that have slightly different molecular weight and/or charges but are structurally and immunologically related (3-5). Some of these forms are typically heat-inducible (hsp70), and their expression is stimulated manyfold after heat shock treatment. Besides these hsp70 genes, the family contains additional members that are abundantly expressed in the absence of stress (constitutive), often referred to as heat shock cognate (hsc70) genes, and others encoding a 78-kD glucoseregulated protein (grp78). Structural analysis of these genes has revealed a remarkably high degree of conservation from bacteria to humans. There is both direct and indirect evidence obtained in different species that hsp70 proteins are necessary in helping cells and organisms survive thermal damage. Although the mechanisms of action of hsp70 and related proteins still remain largely a matter of speculation, there is now compelling genetic and biochemical evidence that these proteins belong to a family of ATP-dependent "unfoldases" implicated in protein assembly/dissociation of multimeric complexes, translocation, and import/export processes across membranes (6). It was also suggested that these proteins could play an important role in cell growth and differentiation (1, 2). Recently, a number of reports have suggested that oxidative stress and heat shock responses overlap. For example,
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it was shown in bacteria that the synthesis of several proteins including an hsp70 analog (dnaK in Escherichia coli) is induced by both heat shock and exposure to hydrogen peroxide (HzOz) but that distinct transcription factors positively regulate the expression of heat shock and oxidative stress proteins (7). It was also found that a variety of oxidants as well as heat shock in prokaryotes induce the rapid accumulation of AppppA and other related adenylate dinucleotides (8). These so-called "alarmones" were proposed to be putative signals to modulate cellular gene expression and metabolism to adapt to oxidative stress. Further links between heat shock and oxidative stress may include the finding that depletion of endogenous glutathione can increase susceptibility to killing by heat (9) and that antioxidant enzymes levels can be enhanced by exposure to elevated temperature (10-13). In previous studies done in our laboratory on the effects of hyperoxia and oxygen metabolites generated by hypoxanthine (HX)-xanthine oxidase (XO) on protein synthesis in endothelial cells (14, 15), we did not detect any induction of hsp synthesis. Instead, we found a marked decrease in overall protein synthesis, which was associated with translational defects. In the present study, we therefore explored the possibility that hsp mRNAs could be induced by oxidative stress, but that their translation into corresponding hsps is defective or repressed. We looked in particular at the effects of HzO z, which was shown to induce heat shock puffs in Drosophila melanogasterpolytene nuclei (16, 17). We have analyzed the expression of the major 70-kD hsps in human endothelial cells exposed to heat shock and oxidative conditions, both at the mRNA level by Northern blot analysis and at the protein level by one- and two-dimensional gel analysis of pulselabeled proteins and by immunologic analysis. We found that hsp70 synthesis in response to elevated temperatures results from the combined increase in mRNA synthesis and selective translation into corresponding hsps. On the other hand, we found that the oxidizing agent HzOz, as well as the oxygen metabolites generated extracellularly by the enzyme system HX-XO, induced a dose-dependent transcription of hsp70 genes, whereas hyperoxia did not. However, the magnitude of the oxidant-induced response was considerably lower than that induced by heat stress, and the hsp70 proteins were hardly detectable. Nuclear run-on transcription analysis showed that the effect of heat shock and HzOz on hsp70 gene expression could be ascribed to a transcriptional induction. Furthermore, using two-dimensional gel electrophoresis analysis and a monoclonal antibody specific for human hsp70, we demonstrate that heat stress induced a series of at least five structurally related proteins in the 70-kD molecular mass area, whereas HzO z induced essentially one acidic isoform.
Materials and Methods Materials All chemicals were of analytical grade and came from Merck (Darmstadt, Germany) and Fluka (Buchs, Switzerland). Catalase (EC 1.11.1.6) from bovine liver, xanthine oxidase (EC 1.1.3.22) grade III from buttermilk, oligo(dT) cellulose, and the anti-mouse IgG antibody labeled with peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). [a-3ZP]uridine triphosphate (UTP) (800 mCi/mmol),
[a-3ZP]cytidine triphosphate (3,000 Ci/mmol) , and L-(3sS]methionine (1,000 Ci/mmol), the Multiprime DNA labeling kit, Hyperfilm", and the monoclonal anti-human 72173-kD hsp antibody were from Amersham (Buckinghamshire, UK). The SP6 polymerase transcription kit, the plasmid vector pSPTl9, and the restriction enzymes were obtained from Pharmacia LKB (Uppsala, Sweden). Biodyne transfer membranes were purchased from Pall (Glen Cove, NY), and nitrocellulose membranes were from Schleicher and Schuell (Feldbach, Switzerland). RPMI 1640 culture medium and fetal calf serum (FCS) were from GIBCO (Renfrewshire, Scotland). Endothelial cell growth supplement was obtained from Serva (Heidelberg, Germany). Cell Culture Human endothelial cells were obtained from umbilical cord veins (HUVEC) using 0.1% collagenase in Krebs-Ringer bicarbonate buffer (18). The cells were plated in gelatin-coated (0.1%) petri dishes and grown in RPMI 1640 supplemented with 25 mM Hepes, 50 U/ml penicillin, 50 ~g/ml streptomycin, 10% FCS, 30 ~g/ml endothelial cell growth supplement, and 90 ~g/ml heparin. The medium was replaced every second day. In this study, cells from second passages were used. Endothelial cells were characterized using morphologic (phase-contrast microscopy) and functional (measurement of the angiotensin-converting enzyme activity) criteria as previously described (14). Experimental Conditions All experiments were performed on confluent cells from second passages. Exposure to heat shock. The petri dishes containing the cells were heated in a waterbath for 30 min at 43, 44, or 45° C in RPMI 1640 supplemented with 10% FCS. The culture medium was preheated at 37° C, and its temperature reached that of the waterbath after ~5 min. After exposure to heat, the cells were allowed to recover in the 37° C incubator for the times indicated in the figures. Exposure to hyperoxia. The cells were exposed to 95 % O, for 5 days in a humidified chamber, as described in reference 14. Exposure to H202 • HzO z diluted in RPMI medium was added to the cells at the final concentrations of 1 and 5 mM. The cells were incubated at 37° C for 20 min. Catalase was then added (200 U/ml), the medium was removed, and the cells were washed 3 times with RPMI and allowed to recover in RPMI with 10% FCS at 37° C for various times, as indicated in the legends of the figures. Exposure to the HX-XO system. The cells were incubated with 15, 30, and 50 mU/ml XO in the presence of2 mM HX in Krebs-Ringer buffer (pH 7.4) supplemented with 25 mM Hepes for 1 h at 37° C. After the treatment, the cells were washed thoroughly with RPMl and incubated in RPMI with 10% FCS at 37° C for different periods of recovery, as indicated in the legends of the figures. Control cells were incubated with 2 mM HX in Krebs-Ringer buffer. RNA Extraction and Northern Blot Analysis Total cellular RNA was prepared by the guanidine thiocyanate method of Chirgwin and colleagues (19). Briefly, RNA was isolated by homogenization in a 4-M solution of the pro-
Jornot, Mirault, and Junod: Stress Proteins in Response to Heat Shock and Oxidants
tein denaturant guanidinium thiocyanate containing 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was then isolated free of protein and DNA through 5.7 M cesium chloride in 0.1 M EDTA and precipitated overnight with ethanol. Polyt.A):' RNA was obtained from equal amounts oftotal RNA by passing over an oligo(dT)cellulose column, according to the method of Maniatis and associates (20). For Northern analysis, total RNA or poly(A)+ RNA fractions were denatured and fractionated by electrophoresis on 1% agarose gels in 5 % formaldehyde, as described by Maniatis and associates (20). After electrophoresis, the RNA was transferred onto a Biodyne membrane according to the specifications of the manufacturer in 20x SSC (l x SSC: 0.15 M NaCI, 0.015 M Na-citrate [pH 7]). The membranes were baked for 2 h at 80° C, prehybridized for at least 4 to 5 h at 55° C in sx SSC, 10 mM NaP04 , 50% formamide, 5 x Denhart's (1 x Denhart's: 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 0.1% sodium dodecyl sulfate (SDS), and 250 J.tg/ml salmon sperm, and hybridized overnight in the same buffer at 65° C with a [32P]UTP labeled anti-sense RNA transcript (1 to 2 x 106 cpm/ml). This RNA probe was prepared from a pSPT19 vector, using an SP6 polymerase transcription kit from Pharmacia. This plasmid contains a 1-kb Bgl II fragment derived from a genomic DNA clone coding for human hsp70 inserted into the BamHl site of pSPT19 (21). The DNA sequence of this hsp70 gene was determined (Dworniczak and Mirault, unpublished observation) and found to correspond to that of another clone independently isolated by others (22). We have shown that the Bgl II fragment probe was specific for heat shock-inducible hsp70 mRNA and did not hybridize to hsc70 mRNA under stringent hybridization conditions (21). For analysis of hsc70 mRNA, the membranes were hybridized with a DNA probe labeled using the Multiprime labeling kit from Amersham in the same buffer described above, at 42° C for 12 to 16 h. This probe was an isolated rv1-kb EcoR I restriction fragment derived from an entire human hsc70 gene (21). After hybridization, the membranes were washed sequentially in 2x SSC, and 0.2x SSC containing 0.1% SDS at 65 to 70° C. They were then subjected to autoradiography on Hyperfilm (Amersham) at -70° C using intensifying screens. The autoradiographs were scanned by a densitometer (Bio-Rad VD620). The density of the hybridization signal was expressed as arbitrary units. Labeling of Proteins and Gel Electrophoresis L-[3sS]methionine (1,000 Ci/mmol) was added to the cell cultures during the last hour of the recovery period to a final concentration of 20 J.tCi/ml and 50 J.tCi/ml for analysis by one-dimensional and two-dimensional electrophoresis, respectively. After the labeling period, the medium was removed and the cells were washed with cold Krebs-Ringer buffer. For analysis by one-dimensional gel electrophoresis, the cells were solubilized by the addition of sample buffer (40 mM Tris [pH 6.8],10% glycerol, 5% SDS, and 250 mM dithiothreitol [DTT]). The samples were boiled for 2 min and stored at -20° C until analysis. For analysis by twodimensional gel electrophoresis, the cells were harvested in 10% SDS and 150 mM DTT, boiled for 2 min and stored at
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- 20° C. Prior to electrophoresis, the samples were diluted 1:4 with sample buffer containing 65 mM DTT, 65 mM (3[(3-cholamidopropyl)dimethylammonio]l-propane sulfonate) (CHAPS), 9 M urea, and 5% ampholytes, pH 3 to 10. One-dimensional gel electrophoresis. The labeled proteins were resolved by SDS polyacrylamide gel electrophoresis in slab gels with 10% aery lamide, as described by Laemmli (23). After staining with Coomassie blue, the gels were dried and autoradiographed at -70° C using Hyperfilm and intensifying screens. Two-dimensional gel electrophoresis. The procedures were as described by O'Farrell (24). Briefly, proteins were focused in the first dimension under reducing conditions using 5.5% ampholytes (1.1%, pH 5 to 7; 4.4%, pH 3 to 10) and separated in the second dimension on 10% acrylamide slab gels in the presence of 10% SDS. Celllysates containing equal amounts of trichloroacetic acid-precipitable radioactivity ( rv3 x 106 cpm) were applied to each gel of each experimental series. After electrophoresis, the proteins were transferred onto nitrocellulose membranes for immunoblot analysis. Western Blot Analysis After transfer, the nitrocellulose membranes were incubated overnight at 4° C with a monoclonal anti-human 72/73-kD hsp antibody (1:1,000) in 10 mM Tris-HCI (pH 7.4), 0.15 M NaCI, and 5 % nonfat dry milk. The primary antibody was washed away, and peroxidase-conjugated anti-mouse was applied (1:100) for 2 h at room temperature. Again, the blots were washed, and the immunoreactive proteins were visualized by exposure to 4-chloro-1-naphtol (60 mg/100 ml) in 10 mM Tris (pH 6.8) containing 20 % methanol and 3 mM H202. The nitrocellulose membranes were dried and exposed to Hyperfilm at -70° C. Nuclear Run-on Transcription Assays hsp70 gene transcription rates were determined by quantitative hybridization of 32P-Iabeled RNA made in nuclei from control cells, cells exposed to 45° C for 30 min, and cells treated with 5 mM H 202 for 20 min, to excess hsp70 DNA, as described in reference 25. Briefly, cells were washed with ice-cold phosphate-buffered saline (pH 7.4) and scraped with a rubber policeman in phosphate-buffered saline. The cells were then collected by centrifugation (800 X g) and resuspended in cold lysis buffer (0.32 M sucrose, 3 mM csci, 2 mM Mg-acetate, 10 mM Tris-HCI [pH 8], 1 mM DTT, 0.1% Triton X-100) and homogenized with 10 to 15 strokes in a Dounce homogenizer. The nuclei were isolated and pelleted through a layer of 2 M sucrose in 10 mM TrisHCI (pH 8), 5 mM Mg-acetate, 1 mM DTT by centrifugation for 45 min at 20,000 rpm in a Beckman SW50.1 rotor. The nuclear pellet was resuspended in 200 J.tl of glycerol storage buffer (50 mM Tris-HCI [pH 8.3], 40% glycerol, 5 mM MgCI2, 0.1 mM EDTA), quick-frozen in liquid nitrogen, and stored at -70° C until analysis. Nuclear transcripts were labeled with [32P]UTP and extracted as described in reference 26. Equal amounts of labeled RNA were used for hybridization to an excess of hsp70 DNA (8 J.tg DNA of the PST19 subclone described above) and, as a control, plasmid PST19 bound to Biodyne membrane using a slot-blot apparatus. Filters were prehybridized for 24 h and hybridized for
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
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Figure 1. Time course of induction of hsp70 mRNA by heat shock . Cells were heat-shocked for 30 min at 43, 44, and 45° C, then allowed to recover at 37° C for 20 min and 1, 2, 4, 6, and 8 h. For each time point, 5 JIg of total RNA was analyzed by Northern hybridization as described in MATERIALS AND METHODS. Shown are a typical RNA blot (inset) and semiquantitative densitometric data from three individual experiments (mean ± SEM). The autoradiographic exposure time was 2 h.
the mRNA accumulation was slightly delayed, maximal at 4 h and sharply decreased by 6 and 8 h. Upon exposure to 45° C, the response was more delayed and more intense: the hsp70 mRNA level, barely detectable after 20 min of recovery, was maximal by 4 to 6 h and then declined significantly to reach, by 8 h, a quantity still comparable to the maximum observed after a 43° C heat shock . The corresponding patterns of protein synthesis are shown in Figure 2 . The pSS]methionine-labeled proteins synthesized in cells under normal conditions (37° C) or after a 30-min exposure to 43, 44, and 45° C, after recovery periods of2, 4,6, and 8 h at 37° C, were analyzed by one-dimensional gel electrophoresis. As can be seen, HUVEC responded to heat shock by inducing the synthesis of major stress proteins of about 70 kD (hsp70), and to a much lesser extent, the hsp90 protein doublet. That the prominent 70-kD band is, in fact, the 70-kD stress proteins was confirmed by Western blot analysis using a specific monoclonal antibody (data not shown). The time course and temperature dependence of hsp70 synthesis appear to closely reflect the mRNA levels observed in Figure 1. Thus, cells exposed to a 43° C heat shock showed almost immediate synthesis of hsp70, but the expression ceased by 6 h of recovery. In cells heated at 44°C, the synthesis of hsp70 was much higher by 2 h, remained high within 4 to 6 h, and eventually ceased by 8 h. After a 45° C shock, overall protein synthesis first declined during the first 2 h then recovered by 4 h, while hsp70 synthesis was vigorous for up to 8 h after heat shock.
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3 days at 65° C. The relative amount of bound RNA was determined by densitometry of the autoradiographs. hsp70 mRNA Stability hsp70 mRNA half-life was determined from an actinomycin D decay curve. Immediately after the treatments, the cells were washed and allowed to recover at 37° C in the presence of 10 I-'g/ml of actinomycin D to block RNA transcription. The cells were lysed 2, 4, 6, and 8 h after addition of the drug, and total RNA was extracted and analyzed by Northern blot as described above.
Results Time Course of Induction of hsp70 mRNA and Proteins by Heat Shock The Northern blot shown in Figure 1 displays the effect of a 30-min exposure to 43, 44, and 45° Con hsp70 mRNA induction in HUVEC. The time course of expression of hsp70 mRNA appeared to be differentially regulated as a function of temperature. Densitometric analysis of hybridizations from three separate experiments clearly shows different kinetics of hsp70 mRNA accumulation in response to graded increase in temperature. Upon exposure to 43° C, the induction of hsp70 transcripts was almost maximal during the first 20 min after exposure, clearly reached a maximum within 1 h, and gradually declined later on. After exposure to 44° C,
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