GLIA 5:300-305 (1992)
Heme Oxygenase Is a Heat Shock Protein and PEST Protein in Rat Astroglial Cells BARNEY E. DWYER,1,3,5ROBERT N. NISHIMURA,".3JEAN DE VELLIS*5.6 AND TADASHI YOSHIDA7 'Molecular Neurobiology Laboratory, and ' I n Vitro Remyelination Laboratory, Veterans Affairs Medical Center, Sepulveda, California 91343; Departments of "Neurology and *Anatomy and Psychiatry, "Brain Research Institute, and 'Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, California; and 7Department of Molecular and Pathological Biochemistry, Yamagata University School of Medicine, Yamagata, Japan
KEY WORDS
Stress protein, Cancer, Brain, Oxidative injury
ABSTRACT Cultured rat forebrain astrocytes contained significant amounts of immunostainable heme oxygenase-1 (HO-1) isozyme, whereas HO-1 was undetectable in spontaneously transformed r a t astroglial cells (ATs). HO-1 was inducible in both cell types by heat shock and by submicromolar amounts of H202.Inhibition of RNA synthesis with actinomycin D or protein synthesis with cycloheximide resulted in the rapid loss of immunostainable heme oxygenase in astrocytes. Analysis of the primary structure of heme oxygenase suggests that it is a PEST protein, i.e., targeted for rapid turnover. 0 1992 Wiley-Liss, Inc.
INTRODUCTION Heme oxygenase (EC 1.14.99.3) (HO) catalyzes the rate-limiting step in heme degradation, yielding biliverdin, which is subsequently degraded to bilirubin (Maines, 1984). HO has been resolved into two components, designated HO-1 and HO-2, appearing in several tissues including brain (Trakshel et al., 1988). HO-1 and HO-2 appear to be products of different genes (Cruse and Maines, 1988). They differ immunologically and physically and show species diversity (Trakshel and Maines, 1989). HO is also a heat shock protein. The rat HO gene contains a functional heat shock element consensus sequence in its promoter region (Shibahara et al., 1987). It is inducible by a variety of stressful conditions including hyperthermia in human HEP 3B hepatoma cells (Mitani et al., 19891, by near ultraviolet radiation, hydrogen peroxide (H,O,), and sodium arsenite in human skin fibroblasts (Keyse and Tyrrell, 1987, 1989), by sodium arsenite and cadmium ions in HeLa and HL60 cells (Taketani et al., 19891, and by hemin in human macrophages and glioma cells (Yoshida et al., 1988; see Stocker 1990 for review). Previously we showed that rat astrocytes exposed to micromolar concentrations of H20, synthesized a 30/34 kDa protein (Nishimura et al., 1988a); we tentatively identified it a s HO (Dwyer et al., 1990). The induction of HO 0 1992 Wiley-Liss, Inc
in brain tissue, a nonhematopoietic organ that is not a site of heme catabolism vis-a-vis the spleen, suggested that the enzyme played a n important role in the metabolism of cellular hemo-proteins, which include cytochromes, peroxidase, and catalase. The fact that HO was a heat shock protein suggested that its induction after stress may protect cells from injury. We have further characterized stress-inducible HO in rat astroglial cells and show that HO undergoes rapid turnover under some conditions. MATERIALS AND METHODS Tissue Culture Spontaneously transformed rat astroglial cells (ATs) and rat forebrain astrocytes were used in this study. ATs are a cell line derived from a subpopulation of rat cortical glial cells that spontaneously transformed in culture (Bressler and de Vellis, 1985). Frozen ATs were
Received April 6,1991; accepted September 27,1991.
A preliminary report was presented at the 21st Annual Meeting of the American Society for Neurochemistry held in Phoenix, Arizona on March 4-9, 1990. Address reprint requests to Dr. Barney E. Dwyer, Molecular Neurobiology Laboratory (111N-l), Veterans Affairs Medical Center, 16111 Plummer S t , Sepulveda, CA 91343.
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were prepared a s previously described (Dwyer et al., 1991). Slot blots were used for quantitation of heme oxygenase RNA. Oligonucleotides were prepared using the Model 390 PCR Mate (Applied Biosystems, Foster City, CAI and purified before use on Applied Biosystems OPC columns using the manufacturer's protocol. Oligo 1 (3'-CTT GTG TTT CTG GTC TCA GGG AGT GTC TGT-5') is antisense to nucleotides 798-827 in rat HO-1 cDNA (Shibahara et al., 1985). Oligo 2 (3'-TCG TTA AGT TCG TCA AGA TGG CGC GGT CCT-5') is Heat Shock antisense to nucleotides 584-613 in rat HO-2 cDNA Fifteen to 30 min before heat shock, ATs or astrocytes (Rotenberg and Maines, 1990). Longer probes that conwere switched to fresh, serum-free methionine-defi- tain these sequences were used successfully to distincient DMEM medium prewarmed to 37°C. Cultures guish between the two forms of HO in rat brain (Sun et were immersed in a 45°C water bath for 10 or 20 min al., 1990). A search of Gen Bank (re1 64) for potential after which L35S]methionine (30 p,Ci/ml; Translabel, sequences to which these probes might hybridize reICN, Costa Mesa, CA) was added and the incubation vealed none that would be stable under our hybridization conditions. Oligonucleotides were end-labelled as was continued a t 37°C for 4 h. follows: oligonucleotide (10 pmol) was incubated a t 37°C in buffer (50 mM Tris HCl, pH 7.6, 10 mM magneHydrogen Peroxide Exposure sium chloride, 5 mM dithioerythritol, 0.1 mM spermiATs or astrocytes were switched to fresh, serum-free dine, 0.1 mM EDTA) containing 150 p,Ci [y-32PlATP D F and diluted H,O, was added to achieve a concen- (ICN, Costa Mesa, CAI and 10 units of T4 kinase. tration of 0.5 pM. Cells were incubated with H,O, for Probe-specific activities were >lo6 cpdpmol. For hy30 or 60 min. The medium was removed and replaced bridization, RNA blots were heat sealed in plastic bags with methionine-deficient DMEM, [35S]methionine containing hybridization buffer [6x SSC, pH 7.0, 5~ was added (30 p,Ci/ml), and the cells were incubated at Denhardt's solution, 50 mM sodium phosphate, 0.15% sodium pyrophosphate, 0.1% sodium dodecyl sulfate 37°C for 4 h. (SDS), and 0.1 mg/ml salmon sperm DNA1 where lOOx Denhardt's = 2% (w/v) polyvinylpyrrolidone, 2% (w/v) Protein Analysis BSA, 2% (w/v) ficoll, and 20x SSC = 3 M sodium chloPolyacrylamide gel electrophoresis and autoradiog- ride, 0.3 M sodium citrate, pH 7.0. Blots were preincuraphy were performed a s previously described (Nish- bated for 2 h a t 42"C, then 32P-labelled oligonucleotide imura et al., 1988b). Western blots were prepared using was injected into the bag, and the incubation was conthe procedure of Towbin et al. (1979) as described by tinued for 12-15 h a t 42°C with gentle shaking. NonhyNishimura et al. (198813). Blots were immunostained bridized probe was removed by three 20 min washes a t using a lyophilized IgG preparation containing HO an- room temperature with 2 x SSC containing 0.1% SDS tibodies raised in rabbit against HO purified from the and three high-stringency 20min washes with the liver of bromobenzene-treated rats (Ishizawa et al., same buffer a t 60°C. Predicted melting temperatures 1983; Yoshida and E(lkuchi, 1979). We conclude the an- (Tm) calculated from: Tm = 16.6(log[Nafl) + 81.5 + tibody is directed against HO-1 since HO-1 is the pre- 0.41(%G + C) - 675 (number of bases in probe) for dominant isozyme of HO in normal liver and its activity oligo-1 and oligo-2 were 72.2"C and 73.5"C, respecis increased 100-fold by bromobenzene treatment tively, using the "a+] of the wash buffer for the calcu(Maines et al., 1986). Liver HO-2 was not affected by lation. The stringency was verified experimentally. bromobenzene and likely represents a very small frac- Washes a t 70°C under the conditions described above tion of total HO in this preparation. Furthermore, the removed most of the radioactivity specifically bound. antibody recognized only one major band in rat liver (Ishizawa et al., 1983; Yoshida and Sato, 1989) and others have failed to detect cross-reactivity between RESULTS their HO-1 and HO-2 antibodies in several tissues Response of Astrocytes and ATs to Stress (Maines et al., 1986; Sun et al., 1990; Trakshel et al., Astrocytes and transformed ATs heated to 45°C for 1986). The antibody [diluted 1:lOO with bovine serum albumin (BSA)-saline] was applied for 1 h a t 4°C and 10 or 20 min showed a dose-dependent increase in rathe presence of HO was visualized using the rabbit IgG dioactivity associated with a 30/34 kDa band (Figs. 1, lanes a-c, 2, lanes a-c); the relative labelling of the Vecta stain kit (Vector Labs, Burlingame, CA). 30/34 kDa protein estimated by densitometry was increased over two-fold in astrocytes and over sevenfold RNA Analysis in ATs after a 45"C, 20 min heat shock. Increased incorTotal cellular RNA was extracted (Chomczynski and poration of 135Slmethionine was also noted in bands Sacchi, 1987) and Northern blots and RNA slot blots that correspond to the major 68, 70, 89, and 97 kDa
thawed and then passaged in Dulbecco's modified essential medium (DMEMYHam's F-12 ( D F , 1/1, v/v) containing 10% calf serum (HyClone Laboratories, Logan, UT). Cells were plated a t a density of 105/25 cm2 and used when confluent. Secondary astrocytes passaged from primary rat forebrain astrocytes (Nishimura et al., 198813) were maintained in D/F containing 5% calf serum.
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Fig. 1. Autoradiography of I”SJ-labelled astrocyte proteins after heat shock and H,O,-treatment. Cultures were labeled with [”’Slmethionine for 4 h after the end of heat shock or H,O,-exposure. The relative incorporation of L”’S1methionine into the 30/34 kDa heme oxygenase (HO) band (indicated in brackets below) was estimated by densitometry from the peak area of the optical density trace. Area measurements were normalized to control cells, which were given the value 1.0. Lane a: Control astrocytes (1.0). Astrocytes after a heat shock of lane b 45°C for 10 min (1.5),or lane c 45°C for 20 min (2.1). Astrocytes treated with H,O, for lane e 30 min (2.4)or lane f 1 h (2.6). Astrocytes pretreated with actinomycin D (0.2 (*g/ml)for 10 min before heat shock lane d a t 45°C for 20 rnin (0.25)or H,O, lane g for 1 h (0.25).Size markers in the margin indicate the area of the gel to which heat shock proteins with apparent molecular weights of 68,70,89,and 97 kDa migrate. Equal cpms were applied to each lane.
heat shock proteins previously described in heatshocked rat astrocytes and ATs (Dwyer et al., 1991; Nishimura et al., 198813, 1991). Exposure of astrocytes and ATs to 0.5 FM H,O, for 30 min or 1 h also increased [35S]methionine labelling of the 30134 kDa band (Figs. le,f vs. l a and 2e,f vs. 2a). In both cell types the relative labelling was increased two to threefold. However, we note that we may be underestimating the magnitude of the increase ih HO labelling in ATs. The 30134 kDa band may be “contaminated by a n additional protein(s) since radiolabelling in this region was seen in controls in spite of the lack of HO immunostaining (see below).
HO Immunostaining We identified the 30134 kDa band as HO. The antibody stained a single major band that corresponded exactly to the band of increased radioactivity from H,O,-treated astrocytes (Fig. 3).As additional controls, duplicate blots of identical samples were immunostained for lactate dehydrogenase and glycerol phosphate dehydrogenase, two putative stress proteins of similar molecular size. Neither antibody recognized the 30134 kDa band. In contrast to control astrocytes HO was not detectable in control ATs (Fig. 4A, lane 1vs. B, lane 1).Heat shock increased immunostainable HO in astrocytes (Fig. 4A, lanes 2, 3) and ATs (Fig. 4B, lanes
Fig. 2. Autoradiography of L”S1-labelled proteins from transformed rat astroglial cells (ATs) after heat shock and H,O, treatment. Cultures were labeled with I”S]methionine for 4 h after the end of heat shock or H,O, treatment. The relative incorporation of [35Slmethionine is indicated in brackets (see legend to Fig. 1).Lane a: Control ATs (1.0).ATs after a heat shock of lane b at 45°C for 10 min (4.4)or lane c at 45°C for 20 min (7.6).ATs treated with H,O, for lane e 30 min (3.2) or lane f 1h (2.7).ATs pretreated with actinomycin D (0.2 pg/ml) for 10 min before heat shock lane d at 45°C for 20 min (1.3)or H,O, (lane g ) for 1 h (2.8).Equal cpms were applied to each lane.
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Fig. 3. Western blot of H,O,-treated astrocytes. Rat forebrain astrocytes were incubated for 2 h with 1 (*M H,O, and labelled with [““Slmethioninefor 3 h Left: Autoradiograph of transblotted astrocyte proteins labelled with [”Slmethionine. Lane 1, control; lane 2, H,O,treated. The 30/34 kDa protein band is indicated by arrows. Right: Lower portions of the same blot from lanes containing identical samples were excised and immunostained with antibodies to heme oxygenase (HO), lactate dehydrogenase (LDH), and glycerol phosphate dehydrogenase (GPDH). Lane 1, control; lane 2, H,O,-treated. The heme oxygenase antibody was described in Materials and Methods. Rabbit IgG against rat skeletal muscle GPDH was kindly provided by Dr. Shalani Kumar (UCLA School of Medicine). Rabbit polyclonal antibody against the porcine H4 isozyme of lactate dehydrogenase was purchased from Ventex Laboratories (Portland, ME). Antibodies were used at 1:100 dilutions.
2, 3). The increase in HO was much more dramatic for ATs since control astrocytes contain significant amounts of HO. However, visual inspection of the Western blots suggests that final HO levels achieved are roughly equivalent in both cell types (Fig. 4A, lane 3 vs. B, lane 3). H,02 exposure also increased the accumulation of HO in astrocytes (Fig. 4A, lanes 5, 6 vs. lane 1)
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Fig. 4. Immunostained Western blot of heat-shocked and H,O,treated astrocytes and transformed astroglial cells (ATs). Each sample is from the corresponding experiment in Figures 1 and 2. Control astrocytes (A, lane 1)and ATs (B, lane 1).Astrocytes (A, lanes 2 , 3 ) or ATs (B, lanes 2 , 3 ) harvested 4 h after a 45°C 10 min heat shock (A, lane 2; B, lane 21, or after a 45°C 20 min heat shock (A, lane 3; B, lane 3). Astrocytes (A, lanes 5 , 6 ) or ATs (B, lanes 5 , 6 ) treated with H,O, for 30 rnin (A, lane 5; B, lane 5) or 1 h (A, lane 6; B, lane 6) and harvested 4 h later. Astrocytes (A, lanes 4, 7) or ATs (B, lanes 4, 7) pretreated with actinomycin D (0.2 pg/ml) for 10 min before a 45°C 20 min heat shock (A, lane 4; B, lane 4) or 1 hr with H,O, (A, lane 7; B, lane 7) and harvested 4 hr later. One hundred micrograms of protein were applied to each lane.
and ATs (Fig. 4B, lanes 5, 6 vs. lane 1).The most dramatic increase was seen in ATs but the levels attained were less than after heat shock. Transcriptional Regulation Oligo-1 (which recognizes HO-1 mRNA) hybridized to a single band of approximately 1.7 kb in size in both astrocytes and ATs (Fig. 5). We failed to detect RNA transcripts corresponding to HO-2 with oligo-2 and for that reason the remainder of this study is focused on HO-1. Slot blots were used to quantiate HO-1 mRNA. Heat shock increased the relative level of HO-1 mRNA in a time-dependent manner in both astrocytes and ATs (Fig. 61, approximating the magnitude of increased HO labelling with ["Slmethionine. This correlation held for H,O,-treated ATs but not for H,O,-treated astrocytes, in which the two- to threefold increase in L3?3]methionine noted in Figure 1 occurred in the presence of relatively unchanged levels of HO-1 mRNA (Fig. 7). Since the data suggested that newly synthesized HO mRNA may be preferentially used, we examined the effects of actinomycin D on HO synthesis. A 10 min preincubation with actinomycin D (0.2 kglml) before heat shock and H,O, treatment reduced labelling of the 30134 kDa band by about 75% in astrocytes (Fig. 1, lanes d, g) and blocked the stress-induced accumulation of HO (Fig. 4A, lanes 4, 7). (In fact, HO levels were below those expected of nonstressed control cells-see below). [35S]methionine labelling of HSP-68 was also abolished and labelling of HSP-70, HSP-89, and HSP-97 was markedly reduced (Fig. 1,lanes d, g). Actinomycin D blocked the accumulation of immunostainable HO in ATs after heat shock of H,O, treatment (Fig. 4B, lanes 4, 7) as well as the heat shock- and H,O,induced increased in HO synthesis in ATs. However, in the case of ATs, actinomycin D did not reduce ["Slmethionine incorporation into the 30134 kDa band below control levels (Fig. 2, lanes d, g). The ability of actinomycin D to block HO accumulation in the presence of
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Fig. 5. Northern blot of RNA from heat-shocked and H,O,-treated astrocytes and transformed astroglial cells (ATs). Ten micrograms of total cellular RNA was applied to each lane. A Heat shock. Control astrocytes (lane a) and ATs (lane d). Astrocytes heat shocked (45°C at 20 min) and harvested at 30 min (lane b) or 2 h (lane c ) later. ATs heat shocked (45°C at 20 min) and harvested 30 min (lane e ) or 2 h (lane f) later. B: H,O,-treatment. Control astrocytes (lane g ) and ATs (lanej). Astrocytes exposed to 0.5 pM H,O, for 1 h and harvested 30 min (lane h) or 2 h (lane i) later. ATs exposed to 0.5 pM H,O, for 1 h and harvested 30 min (lane k)or 2 h (lane 1) later. Membranes were hybridized with a "P-labelled oligonucleotide probe specific for heme oxygenase-1 isozyme. Arrowheads mark the gel origin ( 0 )and end (E). A single 1.7 kb transcript inducible by heat shock or H,O, was detected (arrow). A 0.24-9.5 kilobase RNA ladder (Gibco-BRL)was run along with the samples as a standard from which transcript size was estimated.
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2 Fig. 6 . Heme oxygenase-l mRNA content after heat shock in astrocytes and transformed rat astroglial cells (ATs). Total cellular RNA was blotted in decreasing amounts (2.0 pg, 1.0 pg, 0.5 pg, and 0.25 pg, from left to right) for each sample (1A-C and 2A-C). RNA blots were hybridized to "P-labelled oligo-1 (recognizing HO-1) and the relative content of HO-1 mRNA was estimated by densitometry. Peak area under the optical density trace was plotted against ng RNA blotted, and a linear regression analysis was performed. The relative amounts of HO RNA in each sample were estimated from the slope of the regression line. HO mRNA measurements were normalized to control cells, which were given the value 1.0. Relative HO-1 mRNA content is indicated in brackets after each experimental treatment. 1 A Control astrocytes (1.0). Astrocytes heat shocked (45°C for 20 min) and harvested after (1B) 20 min (1.9) and (1C) 2 h (7.0). 2 A Control ATs (1.0). ATs heat shocked (45°C for 20 min) and harvested after (2Bf20 min (3.3)and ( 2 C ) 2 h (12.0).
continued labelling of the 30134 kDa band offers additional evidence that the 30134 kDa band in ATs contains additional proteids). A most surprising finding in this study was that immunostainable HO rapidly disappeared in actinomycin D-treated astrocytes (Fig. 4A, lanes 4, 7 vs. lane 11,suggesting rapid turnover of this protein. To confirm this finding, nonstressed astrocytes were incubated with either actinomycin D or the pro-
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sponsive HO-1 isozyme is continuously synthesized while still retaining its inducibility. However, it would 1 follow from this line of reasoning that ATs grown under nearly identical conditions are not stressed in culture since control ATs did not have detectable levels of HO-1. Another possibility is that the HO-1 promoter in 2 the nonstressed astrocyte is “leaky” and allows low levels of transcription of the HO-1 gene. In this regard, we Fig. 7. Heme oxygenase-l mRNA content after H,O, treatment in have noted that the levels of HSP-68 mRNA are very astrocytes and transformed rat astroglial cells (ATs). Total cellular RNA was blotted in decreasing amounts (2.0 pg, 1.0 kg, 0.5 pg, and low in nonstressed astrocytes yet immunostainable 0.25 pg, from left to right) for each sample (IA-C and 2A-C) and HSP-68 accumulates a s cells age in culture (Nishimura hybridized to ”P-labelled oligo-1. The relative content of HO-1 mRNA et al., 1991). While we conclude that HO-1 is present was estimated as described in the legend to Figure 6 and is indicated in brackets after each experimental treatment. 1 A Control astrocytes constitutively in astrocytes, we can not rule out the (1.0). Astrocytes exposed to 0.5 pM H,O, for I h and harvested after (IB) 20 min (0.95) and (1C) 2 h (1.1).2 A Control ATs (1.0) ATs ex- presence of HO-2 although we were unable to detect its mRNA. HO-2 has kinetic properties similar to HO-1 posed to 0.5 kM H,O, for 1 h and harvested after (2B) 20 min (5.1)and (2C) 2 h (37). (Maines et al., 1986; Trakshel e t al., 1986) but it is noninducible by a variety of stimuli. For this reason it is less likely to be detected as a band of increased radioactivity after electrophoresis and it is unlikely to be detected on Western blots since no cross-reactivity would be expected between our antibody and HO-2. Efforts a b c d are under way to obtain antibodies that can distinguish between the two forms of heme oxygenase. Fig. 8. Immunostained Western blot of actinomycin D- and cycloOur data suggest that HO-1 accumulation is highly heximide-treated astrocytes. Lane a: Control astrocytes. Astrocytes treated with actinomycin D (0.2 pg/ml) for 30 min (lane b) or 70 min regulated in rat astroglial cells. Compared to astro(lane c), washed with fresh DMEM, and incubated in DMEM for an cytes, ATs accumulate relatively greater levels of HO-1 additional 4 h. Lane d: Astrocytes switched to DMEM and incubated with cycloheximide (1mM) for 4 h. Blots were performed as described after heat shock and HO synthesis is proportionately in Materials and Methods. One hundred micrograms of protein were greater. However, the final level to which HO-1 accuapplied to each lane. mulated in heat-shocked astrocytes and ATs was similar, suggesting that after certain levels of HO-1 are reached regulatory influence may be exerted to stop its tein synthesis inhibitor cycloheximide (Fig. 8). Both compounds resulted in the rapid disappearance of im- synthesis. Transcriptional control of HO synthesis is munostainable HO, suggesting that this protein is se- suggested by stress-induced increases in HO-1 mRNA and by actinomycin D inhibition of HO synthesis. This lectively targeted for rapid degradation. is consistent with the conclusion that heme oxygenase is regulated by transcriptional mechanisms in heatshocked rat glioma cells (Shibahara et al., 1987). HowDISCUSSION ever, control is likely to be complex. Regulation of Rat brain astrocytes contain detectable levels of mRNA levels through degradation pathways, translaHO-1 in contrast to spontaneously transformed, tumor- tional regulation, and regulation of protein turnover igenic ATs, in which HO-1 was not detectable. HO-1 is may be equally important. Inhibition of RNA or protein synthesis resulted in the also a stress-inducible protein in both cell types. These conclusions are based on antibody specificity and ap- rapid disappearance of immunostainable HO, suggestproximate molecular size of the protein. Although HO-2 ing that this protein is targeted for rapid degradation is reported to be the predominant form of HO in brain under some conditions although the degradative path(Sun et al., 1990; Trakshel et al., 1988), we were unable way is uncertain. Using the PEST-FIND computer proto confirm its presence in our astroglial cells. If our gram (kindly provided by Dr. M. Rechsteiner) t o anaantibody recognized both HO-1 and HO-2 we would lyze the amino acid sequence of HO-1 derived from the have expected both to be detected on Western blots published HO-1 cDNA sequence (Shibahara et al., since the estimated molecular size of rat liver HO-1 and 19851, we found heme oxygenase to contain a possible HO-2 [30 and 36 kD, respectively (Trakshel and PEST sequence (PEST-FIND score = 3.84) in the carMaines, 1989)l would be easily resolvable by electro- boxyl terminal portion of the protein (amino acids 239phoresis. Furthermore, HO-2 mRNA was not detect- 254). PEST sequences are structural motifs that are able although previous studies suggest it should be the present in a high percentage of rapidly degraded enmore abundant mRNA in brain (Sun et al., 1990; Trak- zymes and regulatory proteins including ornithine deshe1 et al., 1988). It is not likely that HO-1 mRNAwould carboxylase and the cellular proto-oncogenes c-fos and have been missed in whole brain studies (Sun et al., c-myc (Rogers et al., 1986). To our knowledge this is the 1990) since astrocytes constitute a significant propor- first description of heme oxygenase a s a PEST protein. tion of brain cells. One explanation is that astrocytes in In addition, the primary structure of HO-1 contains the culture are continually stressed so that the stress-re- sequence -F -R Q (- Phe A r g Gln-amino acids C
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234-2381, which may target proteins for enhanced degradation by lysosomes during serum withdrawal (Chiang and Dice, 1988). We conclude from our data that HO is a stress protein in rat astrocytes and in transformed rat astroglial cells. It is inducible by heat shock, H,O,, and lead (unpublished data). Secondly, HO is a very sensitive indicator of oxidative stress in some cells. It is induced by submicromolar amounts of H,O, in the range known to produce adverse biological effects (Fligiel et al., 1984) a t a time when there was only weak induction of the major inducible heat shock protein, HSP-68 (see also Nishimura et al., 1988a). We predict that HO will protect stressed cells. One possible mechanism is by elevating intracellular bilirubin, which is a n antioxidant (Stocker et al., 1987).On the other hand, the enzymatic degradation of heme by HO liberates Fe (111,which can promote cell injury by the generation of reactive oxygen species in the Fenton reaction, a mechanism proposed for the iron-dependent inhibition of brain Na -K' -ATPase by hemoglobin (Levere et al., 1989). Our results show that heme oxygenase is relatively abundant in astrocytes compared to transformed astroglial cells. On this basis we predict that astrocytes and ATs will show differential sensitivity to various types of injury and that they will prove useful in understanding the role of HO in mechanisms of cell injury.
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Keyse, S.M. and Tyrrell, R.M. (1987) Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts. J . Biol. Chem., 26214821-14825. Keyse, S.M. and Tyrrell, R.M. (1989) Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Nutl. Acad. Sci. U.S.A., 86:99-103. Levere, R.D., Escalante, B., Schwartzman, M.L., and Abraham, N.G. (1989) Role of heme oxygenase in heme-mediated inhibition of rat brain Na ' -K ' -ATPase: Protection by tin-protoporpbyrin. Neurochem. Res., 142361-864. Maines, M.D. (1984) New developments in the regulation of heme metabolism and their implications. CRC Crit. Reu. Toxicol., 12:241314. Maines, M.D., Trakshel, G.M., and Kutty, R.K. (1986) Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J . Biol. Chem., 261:411-419. Mitani, K., Fujita, H., Sassa, S., and Kappas, A. (19891 Heat shock induction of heme osygenase mRNA in human HEP 3B hepatoma cells. Biochem. Biophys. Res. Commun., 165:437441. Nishimura, R.N., Dwyer, B.E., Cole, R., and de Vellis, J. (1988a) Induction of the 68/72 kDa heat-shock protein during hydrogen peroxide toxicity. In: NATO A S I Series, Vol. 22, Cellular and Molecular Aspects of Neural Development and Regeneration. A. Gorio, J. de Vellis. B. Haber. and J.R. Perez-Polo. eds. Surinner-Verlan. New York, pp, 227-231. Nishimura, R.N., Dwyer, B.E., Welch, W.J., Cole, R., de Vellis, J., and Liotta, K. (198813)The induction of the maior heat-stress protein in purified rat glial cells. J . Neurosci. Res., 2O:lX-lS. Nishimura, R.N., Dwyer, B.E., Clegg, K., Cole, R., and de Vellis, J. (1991) Comparison of the heat shock response in culturcd cortical neurons and astrocytes. Mol. Brain Res., 9 : 3 9 4 5 . Rogers, S., Wells, R., and Rechsteiner, M. (1986)Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science, 234:364-368. Rotenberg, M.O. and Maines, M.D. (1990) Isolation, characterization, and exoression in Escherichia coli of a cDNA encoding. rat, heme oxygen'ase-2 J Bzol Chem , 265 7501-7506 Shibahara, S , Muller, R , Taguchi, H , and Yoshida, T (1985) Cloning and expression of cDNA for rat heme oxygenase Proc Nut1 Acad Sci. U.S.A., 82:7865-7869. Shibahara, S., Muller, R.M., and Taguchi, H. (1987) Transcriptional control of rat heme oxygenase by heat shock. J . Bid. Chem., 262:12889-12892. Stocker, R. (1990) Induction of haem oxygenase as a defence against oxidative stress. Free Rad. Res. Commun., 9:lOl-112. Stocker, R., Yamamoto, Y., McDonagh, A.F., Glazer, A.N., and Ames, B.N. (1987) Bilirubin is an antioxidant of possible physiological importance. Science, 235: 1043-1046. Sun, Y., Rotenberg, M.O., and Maines, M.D. (1990) Developmental expression of heme oxygenase isozymes in rat brain. Two HO-2 mRNAs are detected. J . B i d Chem., 265:8212-8217. Taketani, S., Kohno, H., Yoshinaga, T., and Tokunaga, R. (1989) The h u m a n 32-kDa stress protein induced by exposure to arsenite and cadmium ions is heme oxygenase. FEBS Lett., 245:173-176. Towbin, H., Staehlin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76:4350-4354. Trakshel, G.M. and Maines, M.D. (1989) Multiplicity of heme oxygenase isozymes. HO-1 and HO-2 are different molecular species in rat and rabbit. J . Biol. Chem., 264:1323-1328. Trakshel, G.M., Kutty, R.K., and Maines, M.D. (1986)Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible form. J . Biol. Chem., 261:1113111137. Trakshel, G.M., Kutty, R.K., and Maines, M.D. (1988) Resolution of the rat brain heme oxygenase activity: Absence of a detectable amount of the inducible form (HO-1). Arch. Biochem. Bioph,ys., 260:732-739. Yoshida, T. and Kikuchi, G. (1979)Purification and properties of heme oxygenase from rat liver microsomes. J . Biol. Chem., 254:44874491. Yoshida, T. and Sato, M. (1989) Posttranslational and direct integration of heme oxygentix into microsomes. Biochem. Biophys. Res. Commun., 163:1086-1092. .Yoshida. T., Biro, P., Cohen. T.. Muller, R.M., and Shibahara, S. (1988) Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur. J . Biochem.. 171:457-461. U
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ACKNOWLEDGMENTS
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This work was supported by the research service of the Department of Veterans Affairs. The authors would like to acknowledge the excellent technical assistance of Shauna McClure, Joan Kobrine, Greg Wang, and Ruth Cole. The help provided by Dr. Shigeki Shibahara to obtain antibody to heme oxygenase is greatly appreciated. REFERENCES Bresslcr, J.P. and de Vellis, J. (1985) Neoplastic transformation of newborn rat astrocytes in culture. Brain Res., 3 4 8 2 - 2 7 . Chiang, H. and Dice, J.F. (1988) Peptide sequences that target proteins for enhanced degradation during serum withdrawal. J . Biol. Chem., 263:6797-6805. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162:156-159. Cruse, I. and Maines, M.D. (1988) Evidence suggesting that the two forms of heme oxygenase are products of different genes. J . Biol. Chern., 263:3348-3353. Dwyer, B.E., Nishimura, R.N., and de Vellis, J. (1990)Tentative identification of a 30-34 kDa stress protein a s heme oxygenase. Am. Soc. Neurochem. Ahstr., 20:128. Dwyer, B.E., Nishimura, R.N., de Vellis, J., and Clegg, K.B. (19911 Regulation of heat shock protein synthesis in rat astrocytes. J . Neurosci. Res., 28:352-358. Fligiel, S.E.G., Lee, E.C., McCoy, J.P., Johnson, K.J., and Varani, J. (19841Protein degradation following treatment with hydrogen peroxide. A m . J . Pathol., 115:418425. Ishizawa, S., Yoshida, T., and Kikuchi, G. (1983) Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues a s well as the induced enzymes. J . Biol. Chem., 258:4220-4225.
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Heme Oxygenase Is a Heat Shock Protein and PEST Protein in Rat Astroglial Cells BARNEY E. DWYER,1,3,5ROBERT N. NISHIMURA,".3JEAN DE VELLIS*5.6 AND TADASHI YOSHIDA7 'Molecular Neurobiology Laboratory, and ' I n Vitro Remyelination Laboratory, Veterans Affairs Medical Center, Sepulveda, California 91343; Departments of "Neurology and *Anatomy and Psychiatry, "Brain Research Institute, and 'Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, California; and 7Department of Molecular and Pathological Biochemistry, Yamagata University School of Medicine, Yamagata, Japan
KEY WORDS
Stress protein, Cancer, Brain, Oxidative injury
ABSTRACT Cultured rat forebrain astrocytes contained significant amounts of immunostainable heme oxygenase-1 (HO-1) isozyme, whereas HO-1 was undetectable in spontaneously transformed r a t astroglial cells (ATs). HO-1 was inducible in both cell types by heat shock and by submicromolar amounts of H202.Inhibition of RNA synthesis with actinomycin D or protein synthesis with cycloheximide resulted in the rapid loss of immunostainable heme oxygenase in astrocytes. Analysis of the primary structure of heme oxygenase suggests that it is a PEST protein, i.e., targeted for rapid turnover. 0 1992 Wiley-Liss, Inc.
INTRODUCTION Heme oxygenase (EC 1.14.99.3) (HO) catalyzes the rate-limiting step in heme degradation, yielding biliverdin, which is subsequently degraded to bilirubin (Maines, 1984). HO has been resolved into two components, designated HO-1 and HO-2, appearing in several tissues including brain (Trakshel et al., 1988). HO-1 and HO-2 appear to be products of different genes (Cruse and Maines, 1988). They differ immunologically and physically and show species diversity (Trakshel and Maines, 1989). HO is also a heat shock protein. The rat HO gene contains a functional heat shock element consensus sequence in its promoter region (Shibahara et al., 1987). It is inducible by a variety of stressful conditions including hyperthermia in human HEP 3B hepatoma cells (Mitani et al., 19891, by near ultraviolet radiation, hydrogen peroxide (H,O,), and sodium arsenite in human skin fibroblasts (Keyse and Tyrrell, 1987, 1989), by sodium arsenite and cadmium ions in HeLa and HL60 cells (Taketani et al., 19891, and by hemin in human macrophages and glioma cells (Yoshida et al., 1988; see Stocker 1990 for review). Previously we showed that rat astrocytes exposed to micromolar concentrations of H20, synthesized a 30/34 kDa protein (Nishimura et al., 1988a); we tentatively identified it a s HO (Dwyer et al., 1990). The induction of HO 0 1992 Wiley-Liss, Inc
in brain tissue, a nonhematopoietic organ that is not a site of heme catabolism vis-a-vis the spleen, suggested that the enzyme played a n important role in the metabolism of cellular hemo-proteins, which include cytochromes, peroxidase, and catalase. The fact that HO was a heat shock protein suggested that its induction after stress may protect cells from injury. We have further characterized stress-inducible HO in rat astroglial cells and show that HO undergoes rapid turnover under some conditions. MATERIALS AND METHODS Tissue Culture Spontaneously transformed rat astroglial cells (ATs) and rat forebrain astrocytes were used in this study. ATs are a cell line derived from a subpopulation of rat cortical glial cells that spontaneously transformed in culture (Bressler and de Vellis, 1985). Frozen ATs were
Received April 6,1991; accepted September 27,1991.
A preliminary report was presented at the 21st Annual Meeting of the American Society for Neurochemistry held in Phoenix, Arizona on March 4-9, 1990. Address reprint requests to Dr. Barney E. Dwyer, Molecular Neurobiology Laboratory (111N-l), Veterans Affairs Medical Center, 16111 Plummer S t , Sepulveda, CA 91343.
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were prepared a s previously described (Dwyer et al., 1991). Slot blots were used for quantitation of heme oxygenase RNA. Oligonucleotides were prepared using the Model 390 PCR Mate (Applied Biosystems, Foster City, CAI and purified before use on Applied Biosystems OPC columns using the manufacturer's protocol. Oligo 1 (3'-CTT GTG TTT CTG GTC TCA GGG AGT GTC TGT-5') is antisense to nucleotides 798-827 in rat HO-1 cDNA (Shibahara et al., 1985). Oligo 2 (3'-TCG TTA AGT TCG TCA AGA TGG CGC GGT CCT-5') is Heat Shock antisense to nucleotides 584-613 in rat HO-2 cDNA Fifteen to 30 min before heat shock, ATs or astrocytes (Rotenberg and Maines, 1990). Longer probes that conwere switched to fresh, serum-free methionine-defi- tain these sequences were used successfully to distincient DMEM medium prewarmed to 37°C. Cultures guish between the two forms of HO in rat brain (Sun et were immersed in a 45°C water bath for 10 or 20 min al., 1990). A search of Gen Bank (re1 64) for potential after which L35S]methionine (30 p,Ci/ml; Translabel, sequences to which these probes might hybridize reICN, Costa Mesa, CA) was added and the incubation vealed none that would be stable under our hybridization conditions. Oligonucleotides were end-labelled as was continued a t 37°C for 4 h. follows: oligonucleotide (10 pmol) was incubated a t 37°C in buffer (50 mM Tris HCl, pH 7.6, 10 mM magneHydrogen Peroxide Exposure sium chloride, 5 mM dithioerythritol, 0.1 mM spermiATs or astrocytes were switched to fresh, serum-free dine, 0.1 mM EDTA) containing 150 p,Ci [y-32PlATP D F and diluted H,O, was added to achieve a concen- (ICN, Costa Mesa, CAI and 10 units of T4 kinase. tration of 0.5 pM. Cells were incubated with H,O, for Probe-specific activities were >lo6 cpdpmol. For hy30 or 60 min. The medium was removed and replaced bridization, RNA blots were heat sealed in plastic bags with methionine-deficient DMEM, [35S]methionine containing hybridization buffer [6x SSC, pH 7.0, 5~ was added (30 p,Ci/ml), and the cells were incubated at Denhardt's solution, 50 mM sodium phosphate, 0.15% sodium pyrophosphate, 0.1% sodium dodecyl sulfate 37°C for 4 h. (SDS), and 0.1 mg/ml salmon sperm DNA1 where lOOx Denhardt's = 2% (w/v) polyvinylpyrrolidone, 2% (w/v) Protein Analysis BSA, 2% (w/v) ficoll, and 20x SSC = 3 M sodium chloPolyacrylamide gel electrophoresis and autoradiog- ride, 0.3 M sodium citrate, pH 7.0. Blots were preincuraphy were performed a s previously described (Nish- bated for 2 h a t 42"C, then 32P-labelled oligonucleotide imura et al., 1988b). Western blots were prepared using was injected into the bag, and the incubation was conthe procedure of Towbin et al. (1979) as described by tinued for 12-15 h a t 42°C with gentle shaking. NonhyNishimura et al. (198813). Blots were immunostained bridized probe was removed by three 20 min washes a t using a lyophilized IgG preparation containing HO an- room temperature with 2 x SSC containing 0.1% SDS tibodies raised in rabbit against HO purified from the and three high-stringency 20min washes with the liver of bromobenzene-treated rats (Ishizawa et al., same buffer a t 60°C. Predicted melting temperatures 1983; Yoshida and E(lkuchi, 1979). We conclude the an- (Tm) calculated from: Tm = 16.6(log[Nafl) + 81.5 + tibody is directed against HO-1 since HO-1 is the pre- 0.41(%G + C) - 675 (number of bases in probe) for dominant isozyme of HO in normal liver and its activity oligo-1 and oligo-2 were 72.2"C and 73.5"C, respecis increased 100-fold by bromobenzene treatment tively, using the "a+] of the wash buffer for the calcu(Maines et al., 1986). Liver HO-2 was not affected by lation. The stringency was verified experimentally. bromobenzene and likely represents a very small frac- Washes a t 70°C under the conditions described above tion of total HO in this preparation. Furthermore, the removed most of the radioactivity specifically bound. antibody recognized only one major band in rat liver (Ishizawa et al., 1983; Yoshida and Sato, 1989) and others have failed to detect cross-reactivity between RESULTS their HO-1 and HO-2 antibodies in several tissues Response of Astrocytes and ATs to Stress (Maines et al., 1986; Sun et al., 1990; Trakshel et al., Astrocytes and transformed ATs heated to 45°C for 1986). The antibody [diluted 1:lOO with bovine serum albumin (BSA)-saline] was applied for 1 h a t 4°C and 10 or 20 min showed a dose-dependent increase in rathe presence of HO was visualized using the rabbit IgG dioactivity associated with a 30/34 kDa band (Figs. 1, lanes a-c, 2, lanes a-c); the relative labelling of the Vecta stain kit (Vector Labs, Burlingame, CA). 30/34 kDa protein estimated by densitometry was increased over two-fold in astrocytes and over sevenfold RNA Analysis in ATs after a 45"C, 20 min heat shock. Increased incorTotal cellular RNA was extracted (Chomczynski and poration of 135Slmethionine was also noted in bands Sacchi, 1987) and Northern blots and RNA slot blots that correspond to the major 68, 70, 89, and 97 kDa
thawed and then passaged in Dulbecco's modified essential medium (DMEMYHam's F-12 ( D F , 1/1, v/v) containing 10% calf serum (HyClone Laboratories, Logan, UT). Cells were plated a t a density of 105/25 cm2 and used when confluent. Secondary astrocytes passaged from primary rat forebrain astrocytes (Nishimura et al., 198813) were maintained in D/F containing 5% calf serum.
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Fig. 1. Autoradiography of I”SJ-labelled astrocyte proteins after heat shock and H,O,-treatment. Cultures were labeled with [”’Slmethionine for 4 h after the end of heat shock or H,O,-exposure. The relative incorporation of L”’S1methionine into the 30/34 kDa heme oxygenase (HO) band (indicated in brackets below) was estimated by densitometry from the peak area of the optical density trace. Area measurements were normalized to control cells, which were given the value 1.0. Lane a: Control astrocytes (1.0). Astrocytes after a heat shock of lane b 45°C for 10 min (1.5),or lane c 45°C for 20 min (2.1). Astrocytes treated with H,O, for lane e 30 min (2.4)or lane f 1 h (2.6). Astrocytes pretreated with actinomycin D (0.2 (*g/ml)for 10 min before heat shock lane d a t 45°C for 20 rnin (0.25)or H,O, lane g for 1 h (0.25).Size markers in the margin indicate the area of the gel to which heat shock proteins with apparent molecular weights of 68,70,89,and 97 kDa migrate. Equal cpms were applied to each lane.
heat shock proteins previously described in heatshocked rat astrocytes and ATs (Dwyer et al., 1991; Nishimura et al., 198813, 1991). Exposure of astrocytes and ATs to 0.5 FM H,O, for 30 min or 1 h also increased [35S]methionine labelling of the 30134 kDa band (Figs. le,f vs. l a and 2e,f vs. 2a). In both cell types the relative labelling was increased two to threefold. However, we note that we may be underestimating the magnitude of the increase ih HO labelling in ATs. The 30134 kDa band may be “contaminated by a n additional protein(s) since radiolabelling in this region was seen in controls in spite of the lack of HO immunostaining (see below).
HO Immunostaining We identified the 30134 kDa band as HO. The antibody stained a single major band that corresponded exactly to the band of increased radioactivity from H,O,-treated astrocytes (Fig. 3).As additional controls, duplicate blots of identical samples were immunostained for lactate dehydrogenase and glycerol phosphate dehydrogenase, two putative stress proteins of similar molecular size. Neither antibody recognized the 30134 kDa band. In contrast to control astrocytes HO was not detectable in control ATs (Fig. 4A, lane 1vs. B, lane 1).Heat shock increased immunostainable HO in astrocytes (Fig. 4A, lanes 2, 3) and ATs (Fig. 4B, lanes
Fig. 2. Autoradiography of L”S1-labelled proteins from transformed rat astroglial cells (ATs) after heat shock and H,O, treatment. Cultures were labeled with I”S]methionine for 4 h after the end of heat shock or H,O, treatment. The relative incorporation of [35Slmethionine is indicated in brackets (see legend to Fig. 1).Lane a: Control ATs (1.0).ATs after a heat shock of lane b at 45°C for 10 min (4.4)or lane c at 45°C for 20 min (7.6).ATs treated with H,O, for lane e 30 min (3.2) or lane f 1h (2.7).ATs pretreated with actinomycin D (0.2 pg/ml) for 10 min before heat shock lane d at 45°C for 20 min (1.3)or H,O, (lane g ) for 1 h (2.8).Equal cpms were applied to each lane.
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Fig. 3. Western blot of H,O,-treated astrocytes. Rat forebrain astrocytes were incubated for 2 h with 1 (*M H,O, and labelled with [““Slmethioninefor 3 h Left: Autoradiograph of transblotted astrocyte proteins labelled with [”Slmethionine. Lane 1, control; lane 2, H,O,treated. The 30/34 kDa protein band is indicated by arrows. Right: Lower portions of the same blot from lanes containing identical samples were excised and immunostained with antibodies to heme oxygenase (HO), lactate dehydrogenase (LDH), and glycerol phosphate dehydrogenase (GPDH). Lane 1, control; lane 2, H,O,-treated. The heme oxygenase antibody was described in Materials and Methods. Rabbit IgG against rat skeletal muscle GPDH was kindly provided by Dr. Shalani Kumar (UCLA School of Medicine). Rabbit polyclonal antibody against the porcine H4 isozyme of lactate dehydrogenase was purchased from Ventex Laboratories (Portland, ME). Antibodies were used at 1:100 dilutions.
2, 3). The increase in HO was much more dramatic for ATs since control astrocytes contain significant amounts of HO. However, visual inspection of the Western blots suggests that final HO levels achieved are roughly equivalent in both cell types (Fig. 4A, lane 3 vs. B, lane 3). H,02 exposure also increased the accumulation of HO in astrocytes (Fig. 4A, lanes 5, 6 vs. lane 1)
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Fig. 4. Immunostained Western blot of heat-shocked and H,O,treated astrocytes and transformed astroglial cells (ATs). Each sample is from the corresponding experiment in Figures 1 and 2. Control astrocytes (A, lane 1)and ATs (B, lane 1).Astrocytes (A, lanes 2 , 3 ) or ATs (B, lanes 2 , 3 ) harvested 4 h after a 45°C 10 min heat shock (A, lane 2; B, lane 21, or after a 45°C 20 min heat shock (A, lane 3; B, lane 3). Astrocytes (A, lanes 5 , 6 ) or ATs (B, lanes 5 , 6 ) treated with H,O, for 30 rnin (A, lane 5; B, lane 5) or 1 h (A, lane 6; B, lane 6) and harvested 4 h later. Astrocytes (A, lanes 4, 7) or ATs (B, lanes 4, 7) pretreated with actinomycin D (0.2 pg/ml) for 10 min before a 45°C 20 min heat shock (A, lane 4; B, lane 4) or 1 hr with H,O, (A, lane 7; B, lane 7) and harvested 4 hr later. One hundred micrograms of protein were applied to each lane.
and ATs (Fig. 4B, lanes 5, 6 vs. lane 1).The most dramatic increase was seen in ATs but the levels attained were less than after heat shock. Transcriptional Regulation Oligo-1 (which recognizes HO-1 mRNA) hybridized to a single band of approximately 1.7 kb in size in both astrocytes and ATs (Fig. 5). We failed to detect RNA transcripts corresponding to HO-2 with oligo-2 and for that reason the remainder of this study is focused on HO-1. Slot blots were used to quantiate HO-1 mRNA. Heat shock increased the relative level of HO-1 mRNA in a time-dependent manner in both astrocytes and ATs (Fig. 61, approximating the magnitude of increased HO labelling with ["Slmethionine. This correlation held for H,O,-treated ATs but not for H,O,-treated astrocytes, in which the two- to threefold increase in L3?3]methionine noted in Figure 1 occurred in the presence of relatively unchanged levels of HO-1 mRNA (Fig. 7). Since the data suggested that newly synthesized HO mRNA may be preferentially used, we examined the effects of actinomycin D on HO synthesis. A 10 min preincubation with actinomycin D (0.2 kglml) before heat shock and H,O, treatment reduced labelling of the 30134 kDa band by about 75% in astrocytes (Fig. 1, lanes d, g) and blocked the stress-induced accumulation of HO (Fig. 4A, lanes 4, 7). (In fact, HO levels were below those expected of nonstressed control cells-see below). [35S]methionine labelling of HSP-68 was also abolished and labelling of HSP-70, HSP-89, and HSP-97 was markedly reduced (Fig. 1,lanes d, g). Actinomycin D blocked the accumulation of immunostainable HO in ATs after heat shock of H,O, treatment (Fig. 4B, lanes 4, 7) as well as the heat shock- and H,O,induced increased in HO synthesis in ATs. However, in the case of ATs, actinomycin D did not reduce ["Slmethionine incorporation into the 30134 kDa band below control levels (Fig. 2, lanes d, g). The ability of actinomycin D to block HO accumulation in the presence of
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Fig. 5. Northern blot of RNA from heat-shocked and H,O,-treated astrocytes and transformed astroglial cells (ATs). Ten micrograms of total cellular RNA was applied to each lane. A Heat shock. Control astrocytes (lane a) and ATs (lane d). Astrocytes heat shocked (45°C at 20 min) and harvested at 30 min (lane b) or 2 h (lane c ) later. ATs heat shocked (45°C at 20 min) and harvested 30 min (lane e ) or 2 h (lane f) later. B: H,O,-treatment. Control astrocytes (lane g ) and ATs (lanej). Astrocytes exposed to 0.5 pM H,O, for 1 h and harvested 30 min (lane h) or 2 h (lane i) later. ATs exposed to 0.5 pM H,O, for 1 h and harvested 30 min (lane k)or 2 h (lane 1) later. Membranes were hybridized with a "P-labelled oligonucleotide probe specific for heme oxygenase-1 isozyme. Arrowheads mark the gel origin ( 0 )and end (E). A single 1.7 kb transcript inducible by heat shock or H,O, was detected (arrow). A 0.24-9.5 kilobase RNA ladder (Gibco-BRL)was run along with the samples as a standard from which transcript size was estimated.
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2 Fig. 6 . Heme oxygenase-l mRNA content after heat shock in astrocytes and transformed rat astroglial cells (ATs). Total cellular RNA was blotted in decreasing amounts (2.0 pg, 1.0 pg, 0.5 pg, and 0.25 pg, from left to right) for each sample (1A-C and 2A-C). RNA blots were hybridized to "P-labelled oligo-1 (recognizing HO-1) and the relative content of HO-1 mRNA was estimated by densitometry. Peak area under the optical density trace was plotted against ng RNA blotted, and a linear regression analysis was performed. The relative amounts of HO RNA in each sample were estimated from the slope of the regression line. HO mRNA measurements were normalized to control cells, which were given the value 1.0. Relative HO-1 mRNA content is indicated in brackets after each experimental treatment. 1 A Control astrocytes (1.0). Astrocytes heat shocked (45°C for 20 min) and harvested after (1B) 20 min (1.9) and (1C) 2 h (7.0). 2 A Control ATs (1.0). ATs heat shocked (45°C for 20 min) and harvested after (2Bf20 min (3.3)and ( 2 C ) 2 h (12.0).
continued labelling of the 30134 kDa band offers additional evidence that the 30134 kDa band in ATs contains additional proteids). A most surprising finding in this study was that immunostainable HO rapidly disappeared in actinomycin D-treated astrocytes (Fig. 4A, lanes 4, 7 vs. lane 11,suggesting rapid turnover of this protein. To confirm this finding, nonstressed astrocytes were incubated with either actinomycin D or the pro-
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sponsive HO-1 isozyme is continuously synthesized while still retaining its inducibility. However, it would 1 follow from this line of reasoning that ATs grown under nearly identical conditions are not stressed in culture since control ATs did not have detectable levels of HO-1. Another possibility is that the HO-1 promoter in 2 the nonstressed astrocyte is “leaky” and allows low levels of transcription of the HO-1 gene. In this regard, we Fig. 7. Heme oxygenase-l mRNA content after H,O, treatment in have noted that the levels of HSP-68 mRNA are very astrocytes and transformed rat astroglial cells (ATs). Total cellular RNA was blotted in decreasing amounts (2.0 pg, 1.0 kg, 0.5 pg, and low in nonstressed astrocytes yet immunostainable 0.25 pg, from left to right) for each sample (IA-C and 2A-C) and HSP-68 accumulates a s cells age in culture (Nishimura hybridized to ”P-labelled oligo-1. The relative content of HO-1 mRNA et al., 1991). While we conclude that HO-1 is present was estimated as described in the legend to Figure 6 and is indicated in brackets after each experimental treatment. 1 A Control astrocytes constitutively in astrocytes, we can not rule out the (1.0). Astrocytes exposed to 0.5 pM H,O, for I h and harvested after (IB) 20 min (0.95) and (1C) 2 h (1.1).2 A Control ATs (1.0) ATs ex- presence of HO-2 although we were unable to detect its mRNA. HO-2 has kinetic properties similar to HO-1 posed to 0.5 kM H,O, for 1 h and harvested after (2B) 20 min (5.1)and (2C) 2 h (37). (Maines et al., 1986; Trakshel e t al., 1986) but it is noninducible by a variety of stimuli. For this reason it is less likely to be detected as a band of increased radioactivity after electrophoresis and it is unlikely to be detected on Western blots since no cross-reactivity would be expected between our antibody and HO-2. Efforts a b c d are under way to obtain antibodies that can distinguish between the two forms of heme oxygenase. Fig. 8. Immunostained Western blot of actinomycin D- and cycloOur data suggest that HO-1 accumulation is highly heximide-treated astrocytes. Lane a: Control astrocytes. Astrocytes treated with actinomycin D (0.2 pg/ml) for 30 min (lane b) or 70 min regulated in rat astroglial cells. Compared to astro(lane c), washed with fresh DMEM, and incubated in DMEM for an cytes, ATs accumulate relatively greater levels of HO-1 additional 4 h. Lane d: Astrocytes switched to DMEM and incubated with cycloheximide (1mM) for 4 h. Blots were performed as described after heat shock and HO synthesis is proportionately in Materials and Methods. One hundred micrograms of protein were greater. However, the final level to which HO-1 accuapplied to each lane. mulated in heat-shocked astrocytes and ATs was similar, suggesting that after certain levels of HO-1 are reached regulatory influence may be exerted to stop its tein synthesis inhibitor cycloheximide (Fig. 8). Both compounds resulted in the rapid disappearance of im- synthesis. Transcriptional control of HO synthesis is munostainable HO, suggesting that this protein is se- suggested by stress-induced increases in HO-1 mRNA and by actinomycin D inhibition of HO synthesis. This lectively targeted for rapid degradation. is consistent with the conclusion that heme oxygenase is regulated by transcriptional mechanisms in heatshocked rat glioma cells (Shibahara et al., 1987). HowDISCUSSION ever, control is likely to be complex. Regulation of Rat brain astrocytes contain detectable levels of mRNA levels through degradation pathways, translaHO-1 in contrast to spontaneously transformed, tumor- tional regulation, and regulation of protein turnover igenic ATs, in which HO-1 was not detectable. HO-1 is may be equally important. Inhibition of RNA or protein synthesis resulted in the also a stress-inducible protein in both cell types. These conclusions are based on antibody specificity and ap- rapid disappearance of immunostainable HO, suggestproximate molecular size of the protein. Although HO-2 ing that this protein is targeted for rapid degradation is reported to be the predominant form of HO in brain under some conditions although the degradative path(Sun et al., 1990; Trakshel et al., 1988), we were unable way is uncertain. Using the PEST-FIND computer proto confirm its presence in our astroglial cells. If our gram (kindly provided by Dr. M. Rechsteiner) t o anaantibody recognized both HO-1 and HO-2 we would lyze the amino acid sequence of HO-1 derived from the have expected both to be detected on Western blots published HO-1 cDNA sequence (Shibahara et al., since the estimated molecular size of rat liver HO-1 and 19851, we found heme oxygenase to contain a possible HO-2 [30 and 36 kD, respectively (Trakshel and PEST sequence (PEST-FIND score = 3.84) in the carMaines, 1989)l would be easily resolvable by electro- boxyl terminal portion of the protein (amino acids 239phoresis. Furthermore, HO-2 mRNA was not detect- 254). PEST sequences are structural motifs that are able although previous studies suggest it should be the present in a high percentage of rapidly degraded enmore abundant mRNA in brain (Sun et al., 1990; Trak- zymes and regulatory proteins including ornithine deshe1 et al., 1988). It is not likely that HO-1 mRNAwould carboxylase and the cellular proto-oncogenes c-fos and have been missed in whole brain studies (Sun et al., c-myc (Rogers et al., 1986). To our knowledge this is the 1990) since astrocytes constitute a significant propor- first description of heme oxygenase a s a PEST protein. tion of brain cells. One explanation is that astrocytes in In addition, the primary structure of HO-1 contains the culture are continually stressed so that the stress-re- sequence -F -R Q (- Phe A r g Gln-amino acids C
HEME OXYGENASE IS A GLIAL STRESS PROTEIN
234-2381, which may target proteins for enhanced degradation by lysosomes during serum withdrawal (Chiang and Dice, 1988). We conclude from our data that HO is a stress protein in rat astrocytes and in transformed rat astroglial cells. It is inducible by heat shock, H,O,, and lead (unpublished data). Secondly, HO is a very sensitive indicator of oxidative stress in some cells. It is induced by submicromolar amounts of H,O, in the range known to produce adverse biological effects (Fligiel et al., 1984) a t a time when there was only weak induction of the major inducible heat shock protein, HSP-68 (see also Nishimura et al., 1988a). We predict that HO will protect stressed cells. One possible mechanism is by elevating intracellular bilirubin, which is a n antioxidant (Stocker et al., 1987).On the other hand, the enzymatic degradation of heme by HO liberates Fe (111,which can promote cell injury by the generation of reactive oxygen species in the Fenton reaction, a mechanism proposed for the iron-dependent inhibition of brain Na -K' -ATPase by hemoglobin (Levere et al., 1989). Our results show that heme oxygenase is relatively abundant in astrocytes compared to transformed astroglial cells. On this basis we predict that astrocytes and ATs will show differential sensitivity to various types of injury and that they will prove useful in understanding the role of HO in mechanisms of cell injury.
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ACKNOWLEDGMENTS
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This work was supported by the research service of the Department of Veterans Affairs. The authors would like to acknowledge the excellent technical assistance of Shauna McClure, Joan Kobrine, Greg Wang, and Ruth Cole. The help provided by Dr. Shigeki Shibahara to obtain antibody to heme oxygenase is greatly appreciated. REFERENCES Bresslcr, J.P. and de Vellis, J. (1985) Neoplastic transformation of newborn rat astrocytes in culture. Brain Res., 3 4 8 2 - 2 7 . Chiang, H. and Dice, J.F. (1988) Peptide sequences that target proteins for enhanced degradation during serum withdrawal. J . Biol. Chem., 263:6797-6805. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162:156-159. Cruse, I. and Maines, M.D. (1988) Evidence suggesting that the two forms of heme oxygenase are products of different genes. J . Biol. Chern., 263:3348-3353. Dwyer, B.E., Nishimura, R.N., and de Vellis, J. (1990)Tentative identification of a 30-34 kDa stress protein a s heme oxygenase. Am. Soc. Neurochem. Ahstr., 20:128. Dwyer, B.E., Nishimura, R.N., de Vellis, J., and Clegg, K.B. (19911 Regulation of heat shock protein synthesis in rat astrocytes. J . Neurosci. Res., 28:352-358. Fligiel, S.E.G., Lee, E.C., McCoy, J.P., Johnson, K.J., and Varani, J. (19841Protein degradation following treatment with hydrogen peroxide. A m . J . Pathol., 115:418425. Ishizawa, S., Yoshida, T., and Kikuchi, G. (1983) Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues a s well as the induced enzymes. J . Biol. Chem., 258:4220-4225.
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