Journal of Neuroscience Research 28:352-358 (1991)
Regulation of Heat Shock Protein Synthesis in Rat Astrocytes B.E. Dwyer, R.N. Nishimura, J. de Vellis, and K.B. Clegg Molecular Neurobiology Laboratory (B .E.D.), In Vitro Remyelination Laboratory (R.N.N.), and Hypertension Research Laboratory (K.B .C.), VA Medical Center, Sepulveda, California; Departments of Neurology (B.E.D., R.N.N.), Anatomy and Psychiatry (J.deV.), the Brain Research Institute (B.E.D., J.deV.), and Mental Retardation Research Center (J.deV.), UCLA School of Medicine, Los Angeles, California
Rat forebrain astrocytes synthesize heat shock proteins with molecular weights 97,89,70,68, and 30-34 kilodaltons. The stress inducible 68-kDa heat shock protein (HSP-68) was vigorously expressed by astrocytes in culture after a 45"C, 20 min heat shock. HSP68 synthesis was poorly inducible by a second heat shock given 16 hr after the initial heat shock. Decreased [3sS]methionine incorporation into HSP-68 correlated with low levels of HSP-68 mRNA present after the second heat shock. The data suggest that control of HSP-68 mRNA levels by transcriptional/ posttranscriptional mechanisms is a major site for regulation of HSP-68 synthesis.
kDa heat shock protein (HSP-68) which is synthesized in brain after a variety of stimuli (Clark and Brown, 1985; Cosgrove and Brown, 1983; Nowak, 1985; Dienel et al., 1986; Brown et al., 1989; Dwyer et al., 1989; Gower et al., 1989; Uney et al., 1988). The function of the 70 kDa HSPs is not well understood but evidence is accumulating for their essential role in the recovery of cells after acute stress: the microinjection into rat embryo fibroblasts of antibodies to the 70 kDa heat shock proteins impaired the ability of these cells to survive a brief heat shock, apparently by interfering with heat shock protein function (Riabowol et al., 1988). A Chinese hamster ovary cell line, selected to overexpress the 5'-promoter region of the HSP70 gene, thereby titrating out heat Key words: gene expression, gene regulation, brain shock transcription factor, failed to synthesize HSP-68 injury, glial cells and displayed increased thermosensitivity during heat shock (Johnston and Kucey, 1988). Additionally heat shock has been shown to protect retinal photoreceptor INTRODUCTION cells from light-induced injury (Barbe et al., 1988). PhoThe heat shock response refers to the activation of toreceptor protection was correlated with the synthesis of a set of cellular genes and synthesis of heat shock pro- HSP-68 in this study. In relative terms, CNS astrocytes teins (HSPs) after exposure to diverse and potentially are much more tolerant of many kinds of stress than are harmful stimuli including supraphysiological tempera- CNS neurons. Rat brain astrocytes in culture are capable ture, heavy metals, and amino acid analogs (Lindquist and Craig, 1988). The major HSPs commonly noted are 68-, 70-, and 89-, and 110 kilodalton (kDa) proteins Received April 2, 1990; revised July 12, 1990; accepted July 24, (Subjeck and Shyy, 1986).' The 70 kDa family of heat 1990. shock proteins includes a constitutively synthesized 70 Address reprint requests to Dr. Barney E. Dwyer, Molecular NeurokDa member. This protein was shown to be abundant in biology Lab (1 11N-I), VA Medical Center, 16111 Plummer St., brain and was identified as a clathrin-uncoating ATPase Sepulveda, CA 91343. (Chappell et al. 1986; de Waegh and Brady, 1989). It Acknowledgements: Supported by the United Cerebral Palsy Foundamay also be identical to the microtubule-associated pro- tions, Inc., and by the Research Service of the Department of Veterans tein, beta-internexin (Green and Liem, 1989). A second Affairs. The authors would like to acknowledge the excellent technical member of this family is the highly stress-inducible 68 assistance of Jimmy Sison, Greg Wang, and Ruth Cole. 'In accordance with the recommendation of Subjeck and Shyy (1986) we refer to the 70 kDa family of heat shock proteins as HSP-68, the major inducible heat shock protein, and HSP-70, the constitutively synthesized member. Heat shock proteins with molecular mass 83-90 kDa are designated HSP-89, and those with molecular mass 105-1 12 kDa are designated HSP-I 10.
0 1991 Wiley-Liss, Inc.
A preliminary report was presented at the 20th Annual Meeting of the American Society for Neurochemistry held at Chicago, IL, in March 1989. Abbreviations: HSPs-Heat shock proteins, DMEM-Dulbecco's Modified Eagle's Medium, PBS-phosphate-buffered saline, SDSsodium dodecylsulfate, BSA-bovine serum albumin, HSTF-heat shock transcription factor.
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of expressing a vigorous heat shock response (Nishimura et al., 1988) in contrast to cultured cortical neurons (Nishimura et al., 1987, 1990). This also appears true for cerebellar astrocytes and granule cells (Marini et al., 1990). We have undertaken studies to elucidate how the heat shock response is regulated in brain cells. We show here that HSP-68 synthesis in heat-shocked rat brain astrocytes reflects HSP-68 mRNA levels. The possible role of HSP-68 or other cycloheximide-sensitive proteins in regulating HSP-68 induction is discussed.
in an equal volume of O.06M Tris, pH 6.8, 5% 2-mercaptoethanol, 2% SDS, and 10% glycerol by heating at 100°C for 3 min. Electrophoresis was carried out on 12% polyacrylamide slab gels with 4% stacking gels utilizing the discontinuous buffer system of Laemmli (1970). Dried gels were exposed to Kodak X-Omat R (XAR-5) film for up to 2 weeks for autoradiography.
Heat Shock and Protein Labelling Fifteen to 30 minutes before heat shock astrocytes were switched to fresh, serum-free DMEM prewarmed to 37°C. Heat shock was initiated by immersing culture dishes in a waterbath at 45°C ? 0.3”C for up to 20 min as specified in the text. Cells to be labelled were immediately switched to 3 ml of methionine-deficient DMEM prewarmed to 37°C; 100 pCi [35S]methionine (Translabel, ICN Radiochemicals, Irvine, CA) was added and the incubation was continued at 37°C. Under these conditions [3sS]methionine incorporation was linear in control cultures for at least 3 hr. The average standard deviation was 17% of the mean for the 12 cultures used. In double heat shock -experiments heat-shocked astrocytes in DMEM/F12 were returned to the 37°C incubator for 16 hr at which time they were given a second heat shock (45°C for 10 min) and were labelled as described above. Radioactive labelling was terminated by removing the radioactive medium and washing the cells twice with ice-cold PBS. Protein was precipitated with 10% trichloroacetic acid (w/v) containing 0.1% unlabelled L-methionine. Cells were scraped from the flask and the acidinsoluble protein was pelleted and dissolved in 0.1% sodium dodecylsulfate (SDS). Aliquots were solubilized
RNA Preparation Total astrocyte RNA was extracted with guanidinium isothiocyanate-phenol-chloroform (Chomczynski and Sacchi, 1987). Astrocytes ( 106-107 cells) were trypsinized to remove them from their flask, suspended in 5 ml of ice-cold PBS, and pelleted by centrifugation at 800g for 5 min. Packed cells were extracted with 1 ml of GIT buffer [4M guanidinium isothiocyanate, 0.025M sodium citrate, pH 7.0, 0.5% (w/v) sodium laurylsarcosine, 0.1M 2-mercaptoethanol]. To the extract was added 0.1 volume of 3 M sodium acetate, pH 4.0, 0.2 volumes of chloroform/isoamyl alcohol (24/1), and 1 volume of water-saturated phenol, and the mixture was vigorously shaken. Phase separation was achieved by centrifugation at 4°C and the RNA-containing upper phase was removed and diluted with an equal volume of 2propanol. RNA was precipitated at -20°C and pelleted by centrifugation. The pellet was dissolved in a small volume of GIT buffer and RNA was reprecipitated at -20°C after the addition of an equal volume of 2-propanol. The RNA pellet was washed three times with 80% ethanol and stored at -80°C until used. RNA substantially free of protein contamination (A260/280 > 1.70) was typically obtained.
Western Blotting and Immunostaining Western blot analysis was performed as described by Towbin et al. (1979). Briefly, unstained gels were soaked in blotting buffer (0.025M Tris, pH 8.3, 0.192M MATERIALS AND METHODS glycine, 20% methanol). Proteins were transferred onto Astrocyte Culture nitrocellulose membranes by applying a current of 0.3 A Purified cultures of forebrain astrocytes were pre- for 16-18 hr in a Bio-Rad blot apparatus. Membranes pared from newborn rat by the method of McCarthy and were immersed overnight at room temperature in BSAde Vellis (1980) except mechanical dissociation of fore- saline [O.OlM Tris-HCI pH 7.4,0. 155M NaCl, 3% (w/v) brain was utilized (Lu et al., 1980). Mixed glial cultures bovine serum albumin] and immunostained with a mouse were grown in 75 cm2 flasks. At 8-10 days in vitro monoclonal antibody, C-92, to the inducible HeLa 72 oligodendrocytes were shaken from the mixed cultures. kDa stress protein (kindly provided by Dr. William J. Astrocytes were washed with 0.02% EDTA in Ca+*- Welch of the Univ. Calif., San Francisco, CA). This and Mgt2-free PBS (0.1M sodium phosphate, pH 7.0) monoclonal antibody which has been characterized by and dissociated by trypsinization. Astrocytes were re- Welch and Feramisco (1984) reacts specifically with plated at a density of 1 X lo6 cells/25 cm2 flask and HSP-68 in rat astrocytes (Nishimura et al., 1988). Antigrown in Dulbecco’s modified Eagle’s medium body was diluted 1:100 with BSA-saline and was applied (DMEM):Ham’s F12 (1:l) containing 10% fetal calf se- overnight at 4°C. The presence of HSP-68 was visualized rum (v/v) for 7-21 days until used (15-29 days in vitro). by an immunoperoxidase procedure by using the IgG Astrocytes prepared in this way were 98% pure by GFAP Mouse Vecta-Stain Kit (Vector Laboratories, Burlinantibody immunostaining (Nishimura et al., 1988). game, CA).
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32P-RiboprobePreparation
Hybridization Nitrocellulose blots were sealed in a plastic bag A cloned DNA fragment of the major inducible with hybridization buffer (0.1 in1 of per square cm of human heat shock protein gene (plasmid pH 2.3, Wu et of 50% (v/v) formamide, membrane) which consisted al., 1985) spanning the entire coding region was kindly 5 X SSC, 0.05M sodium phosphate, pH 7.4, 0.1 mg/ml provided by Dr. Richard Morimoto (Northwestern Univ., Evanston, IL). The Hind III-Bam H1 fragment salmon sperm DNA (Sigma Chem. Co.), 0.2% SDS, and was cut from the original vector and directionally 5 X Denhardt's solution (100 X Denhardt's = 2% (w/v) subcloned into plasmid pT7/T3-19, a dual promoter polyvinylpyrrolidone, 2% (w/v) bovine serum albumin, vector (Bethesda Research Laboratories, Gaithersburg, and 2% (w/v) Ficoll). Membranes were prehybridized by MD). The plasmid was grown in E. coli (DH5 alpha) incubation at 60°C for 2-3 hr. '*P-riboprobe (2 X lo6 and purified on a cesium chloride gradient. pH 2.3 is cpm per/ ml) was then injected and the bag was resealed homologous to a 2.6 kilobase mRNA induced in and incubated at 60°C for 12 hr. Membranes were seheat-shocked HeLa cells, which has been shown by in quentially washed with 2X SSC containing 0.1% SDS vitro translation and hybridization-selection to code for a (three changes) at 37"C, 2 X SSC containing RNase A (1 70 kDa protein not present in control cells (Wu et al., pg/ml) at room temperature for 15 min, and again with 1985). 32P-labelled riboprobe homologous to HSP-68 2 x SSC containing 0.1% SDS (three times) at room was prepared by using the Riboprobe Gemini system temperature. The membranes were air dried and exposed (Promega Biological Research Products, Madison, WI) to Kodak X-OMAT AR-5 film for autoradiography. For and purified by NENSORB column chromatography a series of eight standards hybridized to 32P-riboprobe (New England Nuclear, Boston, MA). Unlabelled and exposed to X-ray film under the conditions described HSP-68 mRNA was prepared by transcribing the strand here the standard deviation of the mean as a percent of complementary to the probe and used as a standard the mean for each of the standards was: 0.05 ng (14.5%), 0.1 ng (7.1%), 0.5 ng (5.1%), 0.5 ng (4.4%), and 2.5 ng ladder for RNA blot experiments. (3.3%). Northern Blot Analysis RNA was separated on 1.4% agarose gels prepared in 10 mM sodium phosphate, pH 7.0, by using a BioRad mini subcell. RNA was dissolved in denaturing buffer (1M glyoxal, 50% DMSO, 10 mM sodium phosphate, pH 7.0) and heated for 1 hr at 50°C. Glycerol and bromophenol blue were added to a final concentration of 10% and 0.08%. Gels were run in 10 mM sodium phosphate, pH 7.0, in a cold-room, at 30 V for 45 min and at 75 V until the bromophenol blue migrated of the gel (about 3 hr). Buffer was changed every 45 min. Lanes containing standards were cut from the gel and stained with acridine orange (30 pg/ml). RNA on the remaining gel was transferred to a nitrocellulose membrane by using 10 X SSC (20 X SSC = 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0). RNA slot blots were used to quantitate HSP-68 mRNA. Total RNA (4.4 pg) was diluted to 55 p1 with sterile water, 33 pl of 20 x SSC, and 22 p1 of 37% formaldehyde solution and heated at 65°C for 15 min. Duplicate 2 pg samples of RNA (50 pl) were blotted under vacuum onto nitrocellulose membranes (previously wetted with 20 X SSC) placed over a strip of wetted Whatman 3MM chromatography paper in a BRL blotting apparatus. HSP-68 RNA standards (0.05-2.5 ng) were included on each blot. Each well was washed with 50 pl of 20 X SSC before samples were applied and ~ after samples were apagain with 50 p1 of 2 0 SSC plied.
RESULTS Heat Shock Response of Astrocytes Rat cortical astrocytes heated at 45°C for 10 rnin dramatically induced synthesis of the 68 kDa heat shock protein, HSP-68 (Fig. lb). The relative incorporation of [35S]methionine into several other proteins approximately 97, 89, 70, and 30-34 kDa in size is also shown (Fig. lb). However, astrocytes given a second heat shock 16 hr after the first heat shock did not respond in the same way. Astrocyte cultures were heat shocked at 45°C for 10 rnin (Fig. lc,d), 15 rnin (Fig. le,f), or 20 min (Fig. 1g,h) and returned to 37°C. Sixteen hours later some of the cultures were kept at 37°C (Fig. Ic,e,g,) while others were given a second, 45"C, 10 min heat shock (Fig. ld,f,h). Both groups were labelled with [35S]methionine for 3 hr. HSP-68 synthesis after the second heat shock (Fig. Id,f,h) did not reach maximal response compared to a single heat shock (Fig. lb) when the initial heat shock was 10 or 15 rnin (Fig. ld,f) and was barely detectable when the initial heat shock was 20 min (Fig. lh). This could not be accounted for by decreased [35S]methionine incorporation into astrocytes after the second heat shock. [35S]methionine incorporation into the 70-, 89-, and 97 kDa proteins also appeared to be increased after a second heat shock except possibly when the initial heat shock was was 20 rnin (Fig. lg,h). However, in this case [35S]methionine incorporation into both
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-97 -89 70 "68
-
-3w34
-2.8 -2.5
4E
Fig. 1. Autoradiography of [35S]-labelled astrocyte proteins after heat shock. [35S]methionine incorporation in astrocytes was described in Methods. a: Control. h: Astrocytes labelled for 3 hr after heat shock (45"C, 10 rnin). Double heat shock: Astrocytes were given a second heat shock (45"C, 10 min) 16 hr after an initial 45°C heat shock lasting 10 rnin (d), 15 min Fig. 2 . Northern blot of mRNA from heat-shocked astrocytes. (0, and 20 rnin (h) and were labelled for 3 hours. Additional Ten micrograms of total cellular RNA from control and heatcultures were given a single 45°C heat shock lasting 10 min (c), shocked astrocytes (45"C, 20 min, cells harvested 3 hr later) 15 min ( e ) , and 20 rnin (g) and were labelled 16 hr later for 3 was separated by agarose gel electrophoresis and blotted onto hr. Equal cpm were applied to each lane. Incorporation of nitrocellulose membranes as described in Methods. Mem[35S]methionine ( c p d p g X 7.19, 10.4, 7.36, 6.70, branes were hybridized with a 32P-labelled riboprobe specific 5.10,6.84, 6.91, 5.33 in a-h respectively. Heat shock proteins for mRNA encoding the inducible 68 kDa heat shock protein, 97, 89,70,68, and 30-34 kDa in size are indicated by X~-CWS.HSP-68. Arrowheads mark the gel origin (0)and end (E). Two heat-inducible transcripts, 2.5 and 2.8 kilobases in size, respectively, were detected. A 0.24-9.5 kilobase RNA ladder protein bands appeared greater than in controls (Fig. (BRL) was run along with samples as a standard from which transcript size was estimated. lg,h vs. a).
Transcriptional Regulation We investigated HSP-68 further to determine whether the transcriptional pattern followed the translational pattern. HSP-68 mRNA was measured by using a "P-labelled riboprobe derived from cloned cDNA for the human, inducible heat shock protein. Northern blot analysis revealed that this probe detects two bands, 2.8 and 2.5 kilobases in size, which are inducible by heat and absent in control astrocytes (Fig. 2). Two stress-induc-
ible HSP-68 transcripts have been reported previously in rodent tissue (Lowe and Moran, 1986; Moalic et al., 1989; Brown and Rush, 1990). These bands most likely represent HSP-68 transcripts with different lengths of 3'-termini poly (A) tails (Hunt and Morimoto, 1985). Because there was no significant hybridization to any other RNA species under our hybridization conditions, RNA slot-blots were used in the remaining exper-
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a b c d
e
f
g
h
i
Fig. 3. HSP-68 mRNA content after heat shock. Astrocytes were given a single 45"C, 20 rnin heat shock and harvested at various times afterwards. Total cellular RNA (2 pg) was blotted and hybridized to a "P-riboprobe specific for HSP-68 as described in Methods. HSP-68 mRNA was estimated from autoradiographs by comparison of sample optical densities with those obtained from standard HSP-68 mRNA (lanes e-i)
blotted at the same time: (e) 0.05 ng, (f)0.1 ng, (g) 0.5 ng, (h) 1.0 ng, and (i) 2.5 ng. HSP-68 mRNA content: (a) control astrocytes,
Regulation of Heat Shock Protein Synthesis in Rat Astrocytes B.E. Dwyer, R.N. Nishimura, J. de Vellis, and K.B. Clegg Molecular Neurobiology Laboratory (B .E.D.), In Vitro Remyelination Laboratory (R.N.N.), and Hypertension Research Laboratory (K.B .C.), VA Medical Center, Sepulveda, California; Departments of Neurology (B.E.D., R.N.N.), Anatomy and Psychiatry (J.deV.), the Brain Research Institute (B.E.D., J.deV.), and Mental Retardation Research Center (J.deV.), UCLA School of Medicine, Los Angeles, California
Rat forebrain astrocytes synthesize heat shock proteins with molecular weights 97,89,70,68, and 30-34 kilodaltons. The stress inducible 68-kDa heat shock protein (HSP-68) was vigorously expressed by astrocytes in culture after a 45"C, 20 min heat shock. HSP68 synthesis was poorly inducible by a second heat shock given 16 hr after the initial heat shock. Decreased [3sS]methionine incorporation into HSP-68 correlated with low levels of HSP-68 mRNA present after the second heat shock. The data suggest that control of HSP-68 mRNA levels by transcriptional/ posttranscriptional mechanisms is a major site for regulation of HSP-68 synthesis.
kDa heat shock protein (HSP-68) which is synthesized in brain after a variety of stimuli (Clark and Brown, 1985; Cosgrove and Brown, 1983; Nowak, 1985; Dienel et al., 1986; Brown et al., 1989; Dwyer et al., 1989; Gower et al., 1989; Uney et al., 1988). The function of the 70 kDa HSPs is not well understood but evidence is accumulating for their essential role in the recovery of cells after acute stress: the microinjection into rat embryo fibroblasts of antibodies to the 70 kDa heat shock proteins impaired the ability of these cells to survive a brief heat shock, apparently by interfering with heat shock protein function (Riabowol et al., 1988). A Chinese hamster ovary cell line, selected to overexpress the 5'-promoter region of the HSP70 gene, thereby titrating out heat Key words: gene expression, gene regulation, brain shock transcription factor, failed to synthesize HSP-68 injury, glial cells and displayed increased thermosensitivity during heat shock (Johnston and Kucey, 1988). Additionally heat shock has been shown to protect retinal photoreceptor INTRODUCTION cells from light-induced injury (Barbe et al., 1988). PhoThe heat shock response refers to the activation of toreceptor protection was correlated with the synthesis of a set of cellular genes and synthesis of heat shock pro- HSP-68 in this study. In relative terms, CNS astrocytes teins (HSPs) after exposure to diverse and potentially are much more tolerant of many kinds of stress than are harmful stimuli including supraphysiological tempera- CNS neurons. Rat brain astrocytes in culture are capable ture, heavy metals, and amino acid analogs (Lindquist and Craig, 1988). The major HSPs commonly noted are 68-, 70-, and 89-, and 110 kilodalton (kDa) proteins Received April 2, 1990; revised July 12, 1990; accepted July 24, (Subjeck and Shyy, 1986).' The 70 kDa family of heat 1990. shock proteins includes a constitutively synthesized 70 Address reprint requests to Dr. Barney E. Dwyer, Molecular NeurokDa member. This protein was shown to be abundant in biology Lab (1 11N-I), VA Medical Center, 16111 Plummer St., brain and was identified as a clathrin-uncoating ATPase Sepulveda, CA 91343. (Chappell et al. 1986; de Waegh and Brady, 1989). It Acknowledgements: Supported by the United Cerebral Palsy Foundamay also be identical to the microtubule-associated pro- tions, Inc., and by the Research Service of the Department of Veterans tein, beta-internexin (Green and Liem, 1989). A second Affairs. The authors would like to acknowledge the excellent technical member of this family is the highly stress-inducible 68 assistance of Jimmy Sison, Greg Wang, and Ruth Cole. 'In accordance with the recommendation of Subjeck and Shyy (1986) we refer to the 70 kDa family of heat shock proteins as HSP-68, the major inducible heat shock protein, and HSP-70, the constitutively synthesized member. Heat shock proteins with molecular mass 83-90 kDa are designated HSP-89, and those with molecular mass 105-1 12 kDa are designated HSP-I 10.
0 1991 Wiley-Liss, Inc.
A preliminary report was presented at the 20th Annual Meeting of the American Society for Neurochemistry held at Chicago, IL, in March 1989. Abbreviations: HSPs-Heat shock proteins, DMEM-Dulbecco's Modified Eagle's Medium, PBS-phosphate-buffered saline, SDSsodium dodecylsulfate, BSA-bovine serum albumin, HSTF-heat shock transcription factor.
Heat Shock in Astrocytes
353
of expressing a vigorous heat shock response (Nishimura et al., 1988) in contrast to cultured cortical neurons (Nishimura et al., 1987, 1990). This also appears true for cerebellar astrocytes and granule cells (Marini et al., 1990). We have undertaken studies to elucidate how the heat shock response is regulated in brain cells. We show here that HSP-68 synthesis in heat-shocked rat brain astrocytes reflects HSP-68 mRNA levels. The possible role of HSP-68 or other cycloheximide-sensitive proteins in regulating HSP-68 induction is discussed.
in an equal volume of O.06M Tris, pH 6.8, 5% 2-mercaptoethanol, 2% SDS, and 10% glycerol by heating at 100°C for 3 min. Electrophoresis was carried out on 12% polyacrylamide slab gels with 4% stacking gels utilizing the discontinuous buffer system of Laemmli (1970). Dried gels were exposed to Kodak X-Omat R (XAR-5) film for up to 2 weeks for autoradiography.
Heat Shock and Protein Labelling Fifteen to 30 minutes before heat shock astrocytes were switched to fresh, serum-free DMEM prewarmed to 37°C. Heat shock was initiated by immersing culture dishes in a waterbath at 45°C ? 0.3”C for up to 20 min as specified in the text. Cells to be labelled were immediately switched to 3 ml of methionine-deficient DMEM prewarmed to 37°C; 100 pCi [35S]methionine (Translabel, ICN Radiochemicals, Irvine, CA) was added and the incubation was continued at 37°C. Under these conditions [3sS]methionine incorporation was linear in control cultures for at least 3 hr. The average standard deviation was 17% of the mean for the 12 cultures used. In double heat shock -experiments heat-shocked astrocytes in DMEM/F12 were returned to the 37°C incubator for 16 hr at which time they were given a second heat shock (45°C for 10 min) and were labelled as described above. Radioactive labelling was terminated by removing the radioactive medium and washing the cells twice with ice-cold PBS. Protein was precipitated with 10% trichloroacetic acid (w/v) containing 0.1% unlabelled L-methionine. Cells were scraped from the flask and the acidinsoluble protein was pelleted and dissolved in 0.1% sodium dodecylsulfate (SDS). Aliquots were solubilized
RNA Preparation Total astrocyte RNA was extracted with guanidinium isothiocyanate-phenol-chloroform (Chomczynski and Sacchi, 1987). Astrocytes ( 106-107 cells) were trypsinized to remove them from their flask, suspended in 5 ml of ice-cold PBS, and pelleted by centrifugation at 800g for 5 min. Packed cells were extracted with 1 ml of GIT buffer [4M guanidinium isothiocyanate, 0.025M sodium citrate, pH 7.0, 0.5% (w/v) sodium laurylsarcosine, 0.1M 2-mercaptoethanol]. To the extract was added 0.1 volume of 3 M sodium acetate, pH 4.0, 0.2 volumes of chloroform/isoamyl alcohol (24/1), and 1 volume of water-saturated phenol, and the mixture was vigorously shaken. Phase separation was achieved by centrifugation at 4°C and the RNA-containing upper phase was removed and diluted with an equal volume of 2propanol. RNA was precipitated at -20°C and pelleted by centrifugation. The pellet was dissolved in a small volume of GIT buffer and RNA was reprecipitated at -20°C after the addition of an equal volume of 2-propanol. The RNA pellet was washed three times with 80% ethanol and stored at -80°C until used. RNA substantially free of protein contamination (A260/280 > 1.70) was typically obtained.
Western Blotting and Immunostaining Western blot analysis was performed as described by Towbin et al. (1979). Briefly, unstained gels were soaked in blotting buffer (0.025M Tris, pH 8.3, 0.192M MATERIALS AND METHODS glycine, 20% methanol). Proteins were transferred onto Astrocyte Culture nitrocellulose membranes by applying a current of 0.3 A Purified cultures of forebrain astrocytes were pre- for 16-18 hr in a Bio-Rad blot apparatus. Membranes pared from newborn rat by the method of McCarthy and were immersed overnight at room temperature in BSAde Vellis (1980) except mechanical dissociation of fore- saline [O.OlM Tris-HCI pH 7.4,0. 155M NaCl, 3% (w/v) brain was utilized (Lu et al., 1980). Mixed glial cultures bovine serum albumin] and immunostained with a mouse were grown in 75 cm2 flasks. At 8-10 days in vitro monoclonal antibody, C-92, to the inducible HeLa 72 oligodendrocytes were shaken from the mixed cultures. kDa stress protein (kindly provided by Dr. William J. Astrocytes were washed with 0.02% EDTA in Ca+*- Welch of the Univ. Calif., San Francisco, CA). This and Mgt2-free PBS (0.1M sodium phosphate, pH 7.0) monoclonal antibody which has been characterized by and dissociated by trypsinization. Astrocytes were re- Welch and Feramisco (1984) reacts specifically with plated at a density of 1 X lo6 cells/25 cm2 flask and HSP-68 in rat astrocytes (Nishimura et al., 1988). Antigrown in Dulbecco’s modified Eagle’s medium body was diluted 1:100 with BSA-saline and was applied (DMEM):Ham’s F12 (1:l) containing 10% fetal calf se- overnight at 4°C. The presence of HSP-68 was visualized rum (v/v) for 7-21 days until used (15-29 days in vitro). by an immunoperoxidase procedure by using the IgG Astrocytes prepared in this way were 98% pure by GFAP Mouse Vecta-Stain Kit (Vector Laboratories, Burlinantibody immunostaining (Nishimura et al., 1988). game, CA).
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32P-RiboprobePreparation
Hybridization Nitrocellulose blots were sealed in a plastic bag A cloned DNA fragment of the major inducible with hybridization buffer (0.1 in1 of per square cm of human heat shock protein gene (plasmid pH 2.3, Wu et of 50% (v/v) formamide, membrane) which consisted al., 1985) spanning the entire coding region was kindly 5 X SSC, 0.05M sodium phosphate, pH 7.4, 0.1 mg/ml provided by Dr. Richard Morimoto (Northwestern Univ., Evanston, IL). The Hind III-Bam H1 fragment salmon sperm DNA (Sigma Chem. Co.), 0.2% SDS, and was cut from the original vector and directionally 5 X Denhardt's solution (100 X Denhardt's = 2% (w/v) subcloned into plasmid pT7/T3-19, a dual promoter polyvinylpyrrolidone, 2% (w/v) bovine serum albumin, vector (Bethesda Research Laboratories, Gaithersburg, and 2% (w/v) Ficoll). Membranes were prehybridized by MD). The plasmid was grown in E. coli (DH5 alpha) incubation at 60°C for 2-3 hr. '*P-riboprobe (2 X lo6 and purified on a cesium chloride gradient. pH 2.3 is cpm per/ ml) was then injected and the bag was resealed homologous to a 2.6 kilobase mRNA induced in and incubated at 60°C for 12 hr. Membranes were seheat-shocked HeLa cells, which has been shown by in quentially washed with 2X SSC containing 0.1% SDS vitro translation and hybridization-selection to code for a (three changes) at 37"C, 2 X SSC containing RNase A (1 70 kDa protein not present in control cells (Wu et al., pg/ml) at room temperature for 15 min, and again with 1985). 32P-labelled riboprobe homologous to HSP-68 2 x SSC containing 0.1% SDS (three times) at room was prepared by using the Riboprobe Gemini system temperature. The membranes were air dried and exposed (Promega Biological Research Products, Madison, WI) to Kodak X-OMAT AR-5 film for autoradiography. For and purified by NENSORB column chromatography a series of eight standards hybridized to 32P-riboprobe (New England Nuclear, Boston, MA). Unlabelled and exposed to X-ray film under the conditions described HSP-68 mRNA was prepared by transcribing the strand here the standard deviation of the mean as a percent of complementary to the probe and used as a standard the mean for each of the standards was: 0.05 ng (14.5%), 0.1 ng (7.1%), 0.5 ng (5.1%), 0.5 ng (4.4%), and 2.5 ng ladder for RNA blot experiments. (3.3%). Northern Blot Analysis RNA was separated on 1.4% agarose gels prepared in 10 mM sodium phosphate, pH 7.0, by using a BioRad mini subcell. RNA was dissolved in denaturing buffer (1M glyoxal, 50% DMSO, 10 mM sodium phosphate, pH 7.0) and heated for 1 hr at 50°C. Glycerol and bromophenol blue were added to a final concentration of 10% and 0.08%. Gels were run in 10 mM sodium phosphate, pH 7.0, in a cold-room, at 30 V for 45 min and at 75 V until the bromophenol blue migrated of the gel (about 3 hr). Buffer was changed every 45 min. Lanes containing standards were cut from the gel and stained with acridine orange (30 pg/ml). RNA on the remaining gel was transferred to a nitrocellulose membrane by using 10 X SSC (20 X SSC = 3 M sodium chloride, 0.3 M sodium citrate, pH 7.0). RNA slot blots were used to quantitate HSP-68 mRNA. Total RNA (4.4 pg) was diluted to 55 p1 with sterile water, 33 pl of 20 x SSC, and 22 p1 of 37% formaldehyde solution and heated at 65°C for 15 min. Duplicate 2 pg samples of RNA (50 pl) were blotted under vacuum onto nitrocellulose membranes (previously wetted with 20 X SSC) placed over a strip of wetted Whatman 3MM chromatography paper in a BRL blotting apparatus. HSP-68 RNA standards (0.05-2.5 ng) were included on each blot. Each well was washed with 50 pl of 20 X SSC before samples were applied and ~ after samples were apagain with 50 p1 of 2 0 SSC plied.
RESULTS Heat Shock Response of Astrocytes Rat cortical astrocytes heated at 45°C for 10 rnin dramatically induced synthesis of the 68 kDa heat shock protein, HSP-68 (Fig. lb). The relative incorporation of [35S]methionine into several other proteins approximately 97, 89, 70, and 30-34 kDa in size is also shown (Fig. lb). However, astrocytes given a second heat shock 16 hr after the first heat shock did not respond in the same way. Astrocyte cultures were heat shocked at 45°C for 10 rnin (Fig. lc,d), 15 rnin (Fig. le,f), or 20 min (Fig. 1g,h) and returned to 37°C. Sixteen hours later some of the cultures were kept at 37°C (Fig. Ic,e,g,) while others were given a second, 45"C, 10 min heat shock (Fig. ld,f,h). Both groups were labelled with [35S]methionine for 3 hr. HSP-68 synthesis after the second heat shock (Fig. Id,f,h) did not reach maximal response compared to a single heat shock (Fig. lb) when the initial heat shock was 10 or 15 rnin (Fig. ld,f) and was barely detectable when the initial heat shock was 20 min (Fig. lh). This could not be accounted for by decreased [35S]methionine incorporation into astrocytes after the second heat shock. [35S]methionine incorporation into the 70-, 89-, and 97 kDa proteins also appeared to be increased after a second heat shock except possibly when the initial heat shock was was 20 rnin (Fig. lg,h). However, in this case [35S]methionine incorporation into both
Heat Shock in Astrocytes
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Fig. 1. Autoradiography of [35S]-labelled astrocyte proteins after heat shock. [35S]methionine incorporation in astrocytes was described in Methods. a: Control. h: Astrocytes labelled for 3 hr after heat shock (45"C, 10 rnin). Double heat shock: Astrocytes were given a second heat shock (45"C, 10 min) 16 hr after an initial 45°C heat shock lasting 10 rnin (d), 15 min Fig. 2 . Northern blot of mRNA from heat-shocked astrocytes. (0, and 20 rnin (h) and were labelled for 3 hours. Additional Ten micrograms of total cellular RNA from control and heatcultures were given a single 45°C heat shock lasting 10 min (c), shocked astrocytes (45"C, 20 min, cells harvested 3 hr later) 15 min ( e ) , and 20 rnin (g) and were labelled 16 hr later for 3 was separated by agarose gel electrophoresis and blotted onto hr. Equal cpm were applied to each lane. Incorporation of nitrocellulose membranes as described in Methods. Mem[35S]methionine ( c p d p g X 7.19, 10.4, 7.36, 6.70, branes were hybridized with a 32P-labelled riboprobe specific 5.10,6.84, 6.91, 5.33 in a-h respectively. Heat shock proteins for mRNA encoding the inducible 68 kDa heat shock protein, 97, 89,70,68, and 30-34 kDa in size are indicated by X~-CWS.HSP-68. Arrowheads mark the gel origin (0)and end (E). Two heat-inducible transcripts, 2.5 and 2.8 kilobases in size, respectively, were detected. A 0.24-9.5 kilobase RNA ladder protein bands appeared greater than in controls (Fig. (BRL) was run along with samples as a standard from which transcript size was estimated. lg,h vs. a).
Transcriptional Regulation We investigated HSP-68 further to determine whether the transcriptional pattern followed the translational pattern. HSP-68 mRNA was measured by using a "P-labelled riboprobe derived from cloned cDNA for the human, inducible heat shock protein. Northern blot analysis revealed that this probe detects two bands, 2.8 and 2.5 kilobases in size, which are inducible by heat and absent in control astrocytes (Fig. 2). Two stress-induc-
ible HSP-68 transcripts have been reported previously in rodent tissue (Lowe and Moran, 1986; Moalic et al., 1989; Brown and Rush, 1990). These bands most likely represent HSP-68 transcripts with different lengths of 3'-termini poly (A) tails (Hunt and Morimoto, 1985). Because there was no significant hybridization to any other RNA species under our hybridization conditions, RNA slot-blots were used in the remaining exper-
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Fig. 3. HSP-68 mRNA content after heat shock. Astrocytes were given a single 45"C, 20 rnin heat shock and harvested at various times afterwards. Total cellular RNA (2 pg) was blotted and hybridized to a "P-riboprobe specific for HSP-68 as described in Methods. HSP-68 mRNA was estimated from autoradiographs by comparison of sample optical densities with those obtained from standard HSP-68 mRNA (lanes e-i)
blotted at the same time: (e) 0.05 ng, (f)0.1 ng, (g) 0.5 ng, (h) 1.0 ng, and (i) 2.5 ng. HSP-68 mRNA content: (a) control astrocytes,
