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Regulation of heme oxygenase-1 expression by dopamine in cultured C6 glioma and primary astrocytes
Molecular Brain Research 73 Ž1999. 50–59 www.elsevier.comrlocaterbres
Research report
Regulation of heme oxygenase-1 expression by dopamine in cultured C6 glioma and primary astrocytes Jeffrey Schmidt a , Kirsten Mertz b
b,1
, James I. Morgan
b,)
a Section of Pediatric Critical Care, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN, 38105-2794, USA Department of DeÕelopmental Neurobiology, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN, 38105-2794, USA
Accepted 20 July 1999
Abstract Heme oxygenase-1 ŽHO-1. is an inducible enzyme involved in heme catabolism, tissue iron homeostasis and the cellular response to oxidative stress. Elevated HO-1 expression in astrocytes has been observed in association with abnormal iron deposition and increased oxidative stress in Parkinson’s disease ŽPD.. Since HO-1 could contribute to these aspects of PD pathobiology we have investigated its regulation in cultured astrocytes and C6 glioma cells. Here we report that dopamine is a potent inducer of HO-1. This induction is not mediated by a classical dopamine receptor and is not mimicked by a range of catecholamines and dopamine metabolites. When the time-course of HO-1 expression was compared between dopamine and hemin, the latter induced the gene immediately while the former did so with a lag. This suggested two distinct signal transduction pathways. However, cycloheximide blocked both hemin- and dopamine-induced HO-1 expression, suggesting that both pathways may involve proteins with short half-lives. Ascorbic acid blocked dopamine induction of HO-1 but had no effect on hemin-induced expression. This suggested that dopamine may signal upstream of the unstable protein by producing pro-oxidant metabolites or byproducts. Inhibition of monoamine oxidases A or B or catechol-O-methyl transferase did not block HO-1 induction by dopamine, indicating that these enzymes were not converting dopamine to an active metabolite. These results suggest that dopamine, released or secreted from affected neurons, may trigger HO-1 expression in neighboring astrocytes. HO-1 and its metabolites could then contribute to the oxidative stress and iron deposition associated with PD. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Stimulus-transcription coupling; Receptor; Cycloheximide; Lactacystin; Ascorbic acid; Catecholamine
1. Introduction The symptoms of Parkinson’s disease ŽPD. are produced by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta ŽSNpc. w16x. The brains of PD victims also show evidence of oxidative stress, metabolic compromise, iron deposition and astrocytosis in the SN w6,8,17–20,38,40,44,55x. However, the etiological relevance of these associated processes to the neuronal loss remains a matter of debate. Similarly, much indirect evidence points to dopamine or products of dopam ine m etabolism as contributing to PD w9,13,14,29,42x. However, a specific pathogenic role for this catecholamine in PD has not been proven. Thus, there )
Corresponding author. Fax: q 1-901-495-3143; E-mail: [email protected] 1 Present address: Anatomisches Institut, Anatomie and Zellbiologie, Uni Bonn, Nussallee 10, D-53115 Bonn, Germany.
remains a quest for a pathobiological process that can accommodate the various cellular and biochemical findings in PD. It was reported recently that expression of heme oxygenase-1 ŽHO-1. is elevated in astrocytes within the substantia nigra of PD victims w46x. Heme oxygenases are the rate limiting enzymes of heme degradation and play critical roles in cellular heme and iron homeostasis w26,27,39,48– 50x. Three heme oxygenase isoforms have been described w27,28x. HO-1 is an inducible gene and its expression has been linked to the cellular oxidative stress response w27x. In addition, HO-1 is induced by heat shock, heme and heme metabolites, inflammatory cytokines and heavy metals w26,27x. HO-1 null mice exhibit excessive tissue iron deposition, reduced resistance to oxidative stress and chronic inflammation w39x. In contrast, mice that overexpress HO-1 exhibit reduced characteristics of oxidative damage w27x. Furthermore, pre-induction of HO-1 Žpreconditioning. confers increased resistance to oxidative stressors in a number
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 3 1 - 4
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of paradigms w1,2,4,22,32,34,51–53x. Thus, HO-1 is generally thought to provide protection against oxidative stress. Given the expression of HO-1 in PD brain and the parallels between its function and the associated pathological findings in PD Že.g., increased oxidative damage and deposition of iron., this enzyme could contribute to the pathobiology of this disorder. Therefore, we have attempted to identify candidate molecules that trigger HO-1 expression in astrocytes. Dopamine is known to produce oxidative damage and generate neurotoxic products w9,13,14,29,42x. Therefore, we have explored the possibility that this catecholamine can mediate HO-1 transcription in astrocytes. This report establishes that dopamine can induce HO-1 in the C6 glioma and RAW 264.7 macrophage cell lines as well as primary astrocytes in culture. Furthermore, this induction exhibits a high degree of structural specificity and does not appear to be mediated by a conventional receptor pathway. Like hemin, the classical substrate inducer of HO-1 expression w27x, dopamine, appears to signal via a protein with a short half-life. However, the time-course of HO-1 induction by dopamine is delayed compared to hemin. Moreover, unlike hemin, dopamine induction of HO-1 appears to involve a pro-oxidant species; indicating that the upstream signaling pathways for these two agents are distinct. These results are discussed in the framework of a stimulus-transcription coupling pathway for dopamine that may have relevance to the pathogenesis of PD. While this manuscript was in preparation, a report appeared that also demonstrated a number of these responses to dopamine in primary astrocyte culture w45x.
2. Materials and methods 2.1. Materials Rat C6 glioma cells were obtained from American Type Culture Collection ŽRochester, NY.. Mouse RAW264.7 cells were obtained from Dr. J. Shenep. C57Blr6 J mice were purchased from Charles River Laboratories ŽWillmington, MA. and maintained within the Animal Resource Center at St. Jude Children’s Research Hospital. Dulbecco’s modified Eagle’s medium ŽDMEM. was obtained from BioWhittaker ŽWalkersville, MD.. Fetal bovine serum ŽFBS. was obtained from Harlan ŽIndianapolis, IN. and horse serum ŽHS. was purchased from HyClone Laboratories ŽLogan, UT.. All other culture media and ancillary tissue culture reagents were purchased from Gibco BRL ŽLife Technologies, Burlington, Ontario, Canada.. All culture flasks and filter bottles were purchased from Fisher Scientific ŽSpringfield, NJ.. Dopamine, L-dopa, norepinepherine, GBR-12909, amoxapine, SCH-23390, SKF38393, spiperone, pergolide, clorgyline, deprenyl, DOPAC, and HVA were purchased from RBI Ždivision of Sigma, Natick, MA.. Hemin, ascorbic acid, hydrogen peroxide,
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ovomucoid albumin, bovine serum albumin, cycloheximide, and 4-methoxy tyramine were purchased from the Sigma ŽSt. Louis, MO.. 3,5-dinitrocatechol was purchased from Tocris Cookson, ŽBristol, UK.. Lactacystin was purchased from Kamiya Biochemical ŽSeattle, WA.. 2.2. Rat C6 glioma cell cultures Cell monolayers were grown in 75 cm2 flasks in Ham’s F-10 medium, 15% heat-inactivated FBS, and 1% penicillinrstreptomycin at 378C in 5% CO 2 for 2–3 days. For experimentation, cells were detached by trypsinization and seeded into 25 cm2 flasks and grown to densities of 1–5 = 10 7 cellsrflask for use. 2.3. Mouse RAW 264.7 macrophage cell cultures Cell monolayers were grown to 90% confluence in 75 cm2 flasks in DMEM, 10% heat-inactivated FBS, at 378C in 5% CO 2 over 2–3 days. For experimentation, cells were split and seeded into 25 cm2 flasks in fresh medium, and grown to 5 = 10 7 cellsrflask over 1–2 days. 2.4. Mouse primary astrocyte cultures Primary astrocyte cultures were prepared from the cerebral cortices and midbrain of neonatal ŽP0–P3. C57Blr6 J mice. Cells were plated in Corning 25 cm2 flasks for 1–2 weeks using a modified method adapted from Cardozo w5x. The plating medium consisted of BME, NaHCO 3 , BSA, ovomucoid albumin, glucose, CMA, HEPES, and 15% FBS. Astroglia were fed with a medium consisting of BME, glucose, glutamine, penicillinrstreptomycin, CMA, 2% rat serum, 15% FBS and 5% horse serum and grown to confluence Ž1 = 10 7 cells.. Neurons and microglia were removed by addition of trypsin and shaking on the second and fourth days after plating. 2.5. RNA isolation and northern analysis Cells were treated with various agents andror appropriate solvents. At appropriate times, total RNA was isolated from cell monolayers using the RNeasy Mini Kit ŽQiagen, Valencia, CA. per the manufacturer’s protocol. Optical densities were determined on all samples for RNA quantitation. Five-microgram total RNA were electrophoresed on 0.8% agarose gels and were transferred electrophoretically to Hybond nylon membranes ŽAmersham, Arlington Heights, IL.. RNA was analyzed by Northern blotting according to previously described procedures except where otherwise noted w30x. Ethidium bromide staining andror rehybridization with a probe to G3PDH were used to control for RNA loading and transfer. A mouse HO-1 cDNA probe Žbases 75–944; encoding initiator methionine to stop codon. was cloned and sequenced by standard techniques. This Sac1rBamH1 fragment was subcloned
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into pBluescript SKII q and used for Northern blot analysis. The cDNA probes were labeled with Ž a-32 P. dCTP ŽAmersham. using the Megaprime random primer labeling system ŽAmersham, RPN 1606. and purified over Sephadex G-50 columns ŽBoehringer Mannheim, Indianapolis, IN.. Membranes were prehybridized and hybridized with the QuikHyb hybridization buffer ŽStratagene, La Jolla, CA. at 688C. Hybridization with labeled probe was calculated to give 2–3 = 10 6 c.p.m.rml of buffer. Membranes were exposed overnight to Kodak Biomax film ŽEastman Kodak, Rochester, NY..
3. Results HO-1 is known to be induced by a range of agents, including oxidant stress, heavy metals, heme and heme metabolites, heat shock and inflammatory cytokines w26,27x. Since dopamine has been implicated in PD and can produce reactive oxygen species, we examined whether it could trigger HO-1 mRNA expression in culture. Three cell types were used: primary mouse midbrain astrocytes, rat C6 glioma Žwhich have some characteristics of astrocytes. and the murine RAW264.7 monocytic cell line Žwhich have properties of macrophages and serve as representatives of a non-astrocytic cell line.. In all cases the effects of dopamine were compared with those of hemin, a classical inducer of HO-1. For these experiments, Northern blot analysis was used to monitor HO-1 expression levels. Immunoblotting was also used in both dose–response and time course analyses and confirmed that HO-1 mRNA mirrored HO-1 protein levels Ždata not shown.. Application of hemin or dopamine triggered expression of HO-1 mRNA in primary astrocytes ŽFig. 1A., C6 glioma ŽFig. 1B. and RAW264.7 cells ŽFig. 1C.. Thus, dopamine elicited a robust induction of HO-1 in astrocytes, astrocyte-like cells and an unrelated cell type. To charac-
Fig. 2. Dopamine dose-dependently induces HO-1 mRNA in rat C6 glioma cells. Cultures were exposed to dopamine ŽDA. at the indicated concentrations for 24 h. Subsequently, HO-1 mRNA levels were determined by Northern blot analysis.
terize this response further, a dopamine dose–response analysis was carried out for C6 cells ŽFig. 2.. Low micromolar and even submicromolar concentrations of dopamine elicited expression of HO-1 ŽFig. 2 and data not shown.. While these are still relatively high concentrations of dopamine, it is quite conceivable that these levels are achieved locally in the brain as a consequence of neuronal damage or dopamine dumping. Although dopamine is reported to be toxic to cultured neurons w29x, it is well tolerated by the cells used in this study. Indeed, we routinely applied high micromolar and even low millimolar concentrations of the catecholamine for 1–2 days without obvious signs of cell death Žsee Figs. 1 and 4 and data not shown..
Fig. 1. Hemin and dopamine induce HO-1 mRNA expression in primary mouse astrocyte cultures ŽPanel A., cultured rat C6 glioma cells ŽPanel B., and cultured mouse RAW macrophages ŽPanel C.. Cell cultures were treated with either hemin or dopamine ŽDA. at the indicated doses and 24 h later total RNA was extracted and analyzed for HO-1 expression by Northern blot analysis.
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expression in C6 cells ŽFig. 3A.. A number of catecholamines are taken up by selective transport proteins. Therefore, it was possible that dopamine triggered gene expression subsequent to its uptake into a target cell. To address this possibility, C6 cells were pretreated with either the dopamine uptake inhibitor, GBR-12909 ŽFig. 3B. or the catecholamine uptake blocker, amoxapine ŽFig. 3C.. Neither inhibitor attenuated dopamine-induced HO-1 expression ŽFig. 3B,C.; indicating that these transporters were not involved in the stimulus-transcription coupling pathway utilized by dopamine. As dopamine did not appear to act via its conventional signaling pathway, we next examined whether a dopamine precursor or metabolite might be the active agent. Neither the dopamine precursor, L-dihydroxyphenylalanine ŽL-
Fig. 3. Dopamine induction of HO-1 is not mediated by conventional dopamine receptors or catecholamine transporters. Panel A: C6 cells were treated for 24 h with either a D1 ŽSKF-38393: SKF. or D2 Žpergolide. agonist. Twenty-four hours later HO-1 expression was assessed by Northern blot analysis. Neither agonist induced HO-1 expression. C6 cells were also pretreated for 1 h with either D1 ŽSCH-23390: SCH. or D2 Žspiperone. antagonists. Subsequently, the cells were exposed to dopamine ŽDA. for 24 h and HO-1 expression measured by Northern blot. Neither antagonist blocked dopamine-induced HO-1 expression. Panel B: C6 cells were pretreated for 1 h with the dopamine uptake inhibitor, GBR-12909 ŽGBR.. Subsequently, cells were treated with dopamine and HO-1 mRNA levels determined 24 h later. The uptake inhibitor did not attenuate dopamine-induced HO-1 expression. Panel C: C6 cells were pretreated for 1 h with the catecholamine uptake blocker, amoxapine ŽAMOX.. Subsequently, cells were treated with dopamine and HO-1 mRNA levels determined 24 h later. Amoxapine had no effect upon dopamine-induced HO-1 expression.
Activation of gene transcription frequently accompanies engagement of a receptor by its ligand w31x. To establish whether dopamine induced HO-1 expression via a classical receptor a pharmacological analysis was performed with various dopamine receptor agonists and antagonists. Pretreatment of C6 cells with either SCH-23390 ŽD1 antagonist. or spiperone ŽD2 antagonist. failed to attenuate HO-1 induction by dopamine ŽFig. 3A.. This suggested that dopamine was not acting via D1 or D2 receptors. This conclusion was strengthened by the finding that neither D1 ŽSKF-38393. nor D2 Žpergolide. agonists activated HO-1
Fig. 4. Dopamine precursors and metabolites do not mediate HO-1 induction. C6 cells were treated for 24 h with one of dihydroxyphenylalanine ŽL-DOPA., norepinephrine ŽNOREPI., dihydroxyphenylacetic acid ŽDOPAC., 4-methoxy tyramine Ž4-MT., or homovanillic acid ŽHVA. at the indicated doses. Dopamine ŽDA. was also tested for comparative purposes. Subsequently, HO-1 expression was measured by Northern blot. Only DOPAC produced a small increase in HO-1 mRNA levels at the highest doses tested.
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Fig. 5. MAO and COMT inhibitors do not influence dopamine-induced HO-1 expression. Panel A: C6 cells were pretreated with the MAO-B inhibitor, deprenyl for 1 h prior to addition of dopamine ŽDA.. HO-1 mRNA was measured 24 h later by Northern blot. Deprenyl, did not block dopamine-induced HO-1 expression. Panel B: C6 cells were pretreated with the COMT inhibitor, 3,5-dinitrocatechol ŽDNC. for 1 h prior to addition of dopamine ŽDA.. HO-1 expression was measured 24 h later by Northern blot. Note that dopamine-induced HO-1 expression was not attenuated by the COMT blocker.
DOPA. nor the sequential metabolite of dopamine, norepinephrine, induced HO-1 expression ŽFig. 4.. Thus, of the biologically active catecholamines, only dopamine induced HO-1. In addition, L-DOPA, which is the front-line support therapy for PD, is inactive. Dopamine is degraded by two principal enzymatic routes involving monoamine oxidases ŽMAO. and catechol-o-methyl transferase ŽCOMT.. Products of these pathways include 4-methoxytyramine Ž4-MT., dihydroxyphenylacetic acid ŽDOPAC. and homovanilic acid ŽHVA.. Of these agents, only DOPAC showed any activity ŽFig. 4.. However, the dose response curve for DOPAC revealed that it was 10–100fold less potent than dopamine establishing that it cannot be the biologically relevant metabolite that mediates HO-1
induction Žcompare Fig. 2 with Fig. 4.. Since MAOs and COMT may potentially generate other dopamine metabolites and as there may be byproducts of these reactions Že.g., reactive oxygen species. that may be causing induction of HO-1, C6 cells were treated with inhibitors of MAOs Ždeprenyl and clorgyline; Fig. 5A. or COMT Ž3,5dinitrocatechol; Fig. 5B. prior to administration of dopamine. None of these inhibitors, or combination of inhibitors, attenuated dopamine-induced HO-1 expression ŽFig. 5 and data not shown.. Therefore, it is unlikely that a direct or indirect product of the catabolism of dopamine by these three enzymes is necessary for HO-1 induction. As the upstream signaling pathway of dopamine to HO-1 gene expression appeared to be atypical, further experiments were performed to shed light on the molecular components of its transcription coupling pathway. When the time-courses of HO-1 expression triggered by hemin and dopamine were compared, hemin elicited an almost immediate increase in HO-1 mRNA levels while dopamine-induced HO-1 expression was delayed by several hours ŽFig. 6.. This suggested that there were differences in the signal transduction pathways that coupled hemin and dopamine to HO-1 transcription. It has been reported that HO-1 has the properties of an immediate-early gene w37x. To establish whether hemin and dopamine elicited HO-1 expression via an immediate-early response, C6 cells were pretreated with cycloheximide and then exposed to either the HO-1 substrate or the catecholamine. Classically, IEGs are superinduced by presentation of a stimulus in the presence of a protein synthesis inhibitor w7x. However, cycloheximide completely blocked the induction of HO-1 by both hemin and dopamine ŽFig. 7A.. Indeed, cycloheximide consistently suppressed basal HO-1 expression ŽFig. 7A and data not shown.. Thus, neither hemin nor dopamine elicit HO-1 induction via an immediate-early gene response. Moreover, since hemin can elevate HO-1 expression within 30 min Ždata not shown. and this is
Fig. 6. Hemin and dopamine elicit distinct time courses of HO-1 mRNA expression in C6 cells. C6 cells were treated with hemin Ž50 mM. or dopamine Ž500 mM. for the indicated times whereupon the cultures were harvested and total RNA isolated. Subsequently, HO-1 mRNA levels were determined by Northern blot.
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Fig. 7. Cycloheximide, lactacystin and ascorbate influence basal and stimulated HO-1 expression in C6 cells. Panel A: C6 cells were pretreated with cycloheximide ŽCyclohex. for 1 h. Subsequently, cohorts of carrier or cycloheximide-exposed cells were treated with dopamine ŽDA. or hemin and incubated for a further 8 h. Subsequently, HO-1 mRNA levels were determined by Northern blot. Note that cycloheximide reduced basal as well as dopamine- and hemin-induced HO-1 mRNA levels. Panel B: C6 cells were treated with the indicated doses of lactacystin ŽLacta. for 24 h. Subsequently, HO-1 mRNA levels were measured by Northern blot. The proteosome inhibitor, lactacystin produced a dose-dependent increase in HO-1 mRNA. Panel C: C6 cells were pretreated with ascorbic acid ŽAscorb. for 1 h. Subsequently, the cells were exposed to hemin or dopamine ŽDA. and HO-1 mRNA levels determined 24 h later by Northern blot. Ascorbate blocked dopamine but not hemin induction of HO-1 mRNA.
completely blocked by pretreatment ŽFig. 7. or even cotreatment Ždata not shown. with cycloheximide, a proteinŽs. with a relatively short half-life must participate in both basal and stimulated expression of HO-1. To test this hypothesis, C6 cells were treated with lactacystin, an inhibitor of proteasome-dependent proteolysis w36x. Lactacystin caused a dose-dependent induction of HO-1 ŽFig. 7B., indicating that simply blocking degradation of the intrinsic proteinŽs. is sufficient to induce HO-1 expression. Therefore, dopamine and hemin may act via the same or distinct labile proteins. However, there is a delay for dopamine to activate this downstream event. While we could not identify a dopamine metabolite that was an obvious candidate for the inductive signal, the delay of induction may nevertheless be indicative of a
requirement for the processing of dopamine. This putative processing does not occur spontaneously in vitro since aging dopamine in aqueous solution does not eliminate the lag period Ždata not shown.. This suggests that dopamine is actively metabolized Ži.e., requires cells., although this metabolism is not mediated by MAOs or COMT ŽFig. 5.. It is known that dopamine can be oxidized by a variety of other enzymes including xanthine oxidase, lipoxygenase and tyrosinase w10,21,41x. Therefore, we assessed whether antioxidants affected dopamine’s ability to induce HO-1. Treatment of C6 cells with ascorbic acid blocked dopamine induction of HO-1 while having no effect on hemin-induced HO-1 expression ŽFig. 7C.. This suggested that the oxidation of dopamine or one of its metabolites was central to its inducing activity. Moreover, this requirement
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Fig. 8. Hydrogen peroxide is a weak inducer of HO-1 expression compared to dopamine. Panel A: Primary mouse astrocytes were exposed to dopamine ŽDA. or hydrogen peroxide ŽH 2 O 2 . and HO-1 levels measured 24 h later by Northern blot analysis. Hydrogen peroxide is a weaker inducer of HO-1 than dopamine in cultured astrocytes. Panel B: C6 cells were exposed to hydrogen peroxide ŽH 2 O 2 . at the indicated doses for 24 h and HO-1 mRNA was then measured by Northern blot analysis. Hydrogen peroxide is a marginal inducer of HO-1 in C6 cells.
did not exist for hemin. Dopamine can be metabolized to yield peroxides w12x. However, it is unlikely that hydrogen peroxide is the mediator of dopamine activity as this superoxide is a much weaker inducer of HO-1 than dopamine in both astrocytes ŽFig. 8A. and C6 cells ŽFig. 8B. at near toxic concentrations.
4. Discussion As elevated HO-1 expression in astrocytes has been associated with PD w46x, we set out to identify molecules that might be the endogenous signals for this transcriptional response. Here we show that dopamine can trigger expression of HO-1 in astrocytes and astrocyte-like cells at concentrations that may be attained locally during pathological insults in vivo ŽFigs. 1 and 2.. This finding is consistent with another recent study in primary cultured astrocytes w45x. However, the response to dopamine is not astrocyte-specific as it is also observed in a monocytic cell line in culture ŽFig. 1.. Pharmacological analysis reveals that the pathway through which dopamine activates HO-1 transcription is not blocked by dopamine receptor antagonists and is not mimicked by D1 or D2 agonists ŽFigs. 3 and 4., confirming findings in the study of Schipper et al. w45x. In addition, it is shown here that dopamine and catechol uptake inhibitors do not attenuate HO-1 induction by dopamine ŽFig. 3B and C.. Although dopamine does not appear to activate HO-1 expression via a classical dopamine receptor its signal transduction pathway does have some mechanistic similarities with that of hemin. The observation that even relatively brief pretreatment with cycloheximide blocks HO-1 induction by hemin and dopamine Žand depresses basal HO-1 expression; Fig. 7A. indicates that the transcriptioncoupling pathways of these agents Žas well as basal transcription. probably involve a proteinŽs. with a short halflife. The latter contention is also supported by the observa-
tion that lactacystin, a proteasome inhibitor, induces HO-1 expression in C6 cells ŽFig. 7B.. Together, the lactacystin and cycloheximide results suggest that a proteinŽs. that is rapidly degraded via the proteasome is required for HO-1 induction by dopamine and hemin. Potential mediators include, Hif1-alpha and p53, both of which are known to undergo rapid, proteasome-mediated degradation w3x. Moreover, a Hif-1 response element has been identified in the HO-1 promoter w23x. HO-1 has been suggested to be an IEG w37x. Typically, IEG induction is rapid, transient and protein synthesis-independent w7x. Although, hemin does induce HO-1 rapidly, dopamine only activates expression after a delay of several hours ŽFig. 6.. Moreover, elevated HO-1 expression persists for days in response to both hemin and dopamine ŽFig. 6.. Finally, induction of HO-1 by both agents is blocked by cycloheximide ŽFig. 7A.. Therefore, HO-1 does not behave as an IEG in C6 cells in response to either hemin or dopamine. Although hemin and dopamine possibly share a common downstream transcriptional mechanism, the timecourses of HO-1 induction indicate that the upstream elements of the pathways are distinct ŽFig. 6.. The characteristic lag in HO-1 expression seen with dopamine ŽFig. 6. suggests that it activates transcription via an indirect mechanism. A number of general possibilities exist to account for this delay. First, dopamine may alter intracellular heme biosynthesis or heme homeostasis. In this scenario, elevated intracellular heme would ultimately mediate the dopamine signal transduction pathway. This possibility is presently under study. A second possibility is that dopamine must first induce or repress expression of another gene whose product then either stimulates or inhibits expression of HO-1, respectively. While this mechanism cannot be formally excluded, the lag time is probably insufficient to accommodate it. A third potential mechanism involves the metabolism of dopamine. In this scenario, dopamine is not active per se but rather one of its
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metabolites or byproducts of its metabolism provides the stimulus for HO-1 induction. Although a number of dopamine metabolites were inactive and inhibition of the principal enzymes in dopamine catabolism had no effect on dopamine-induced HO-1 expression, this possibility still cannot be excluded. This is because dopamine may be metabolized by other enzymes. Indeed, the finding that ascorbic acid blocks dopamine-induced HO-1 expression ŽFig. 7C. suggests that oxidation of dopamine or a dopamine metabolite Žor a byproduct of dopamine metabolism with pro-oxidant activity. is essential for HO-1 induction. This finding corroborates observations in primary astrocytes w45x. As hemin induction of HO-1 is insensitive to ascorbic acid, the target for this antioxidant must be upstream in the dopamine signal transduction cascade. This finding also serves to discriminate two stimulus-transcription coupling pathways that may converge on a final common element. Like Schipper et al. w45x, we considered the possibility that generation of hydrogen peroxide from dopamine may be the critical mediator. However, we feel this is unlikely in astrocytes as hydrogen peroxide is a weak inducer when compared to dopamine ŽFig. 8A.. Moreover, in C6 cells hydrogen peroxide is at best a marginal inducer of HO-1 even at near toxic doses ŽFig. 8B.. Present studies are aimed at identifying the ascorbic acid-sensitive step in the dopamine signaling pathway. Dopamine has long been thought to contribute to neurodegenerative disorders such as PD and this catecholamine has been shown to be neurotoxic both in culture and in vivo w9,13,14,29,42x. This toxicity has been attributed to the iron catalyzed oxidation of dopamine to quinones and the production of superoxide and peroxynitrite. The dopamine quinones can react with sulfhydryl groups of cysteine in proteins and glutathione resulting in compromised protein function and cellular reducing capacity w11,13,15x. The free radicals can directly cause oxidative damage to macromolecules w12x. HO-1 may contribute to this process in a number of ways. First, degradation of heme by HO-1 will liberate iron, which could then potentially contribute to oxidative damage and catalyze further oxidation of dopamine to neurotoxic intermediates w12,45x. Indeed, the presence of excessive iron deposition in the substantia nigra of PD victims may be attributable to dysregulation of HO-1 expression w45,46x. Second, HO-1 is a component of an oxidative stress response program that is generally believed to confer protection against oxidative insults w1,2,22,32,34,51x. This belief is based upon preconditioning experiments and analysis of mice that either lack HO-1 or overexpress the gene. Thus, HO-1 may function to minimize damage caused by ongoing oxidative stress. It is not completely clear how HO-1 confers this protection although this may be mediated by it reducing the levels of heme-bound forms of critical proteins such as those involved in cellular respiration Že.g., cytochromes. or signal transduction pathways Že.g., guanylyl cyclase.. In addition,
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heme-bound cytochrome c is essential for some cell suicide pathways w24x and its degradation by HO-1 would be predicted to subvert this program. Furthermore, the products of HO-1 activity, biliverdin and bilirubin are known to be potent antioxidants w25,33,47,54x and exogenously administered carbon monoxide has recently been shown to confer protection against oxidative stress w35x. In this general scenario, activation of the HO-1 pathway in the PD brain may be a beneficial response providing resistance to oxidative stress and attenuating cell death. A third possibility is that excessive production of HO-1 may be a maladaptive response. For example, HO-1 metabolism of heme liberates carbon monoxide, which will compromise cellular respiration. In addition, HO-1 produces biliverdin, which is effectively converted to bilirubin. In the context of kernicterus, the latter is known to be capable of causing neural damage w43x. Thus, rather than provide protection, HO-1 expression may exacerbate pathological processes. Pharmacological modulation of HO-1 expression, whether through induction or blockade of transcription pathways, may offer novel therapeutic approaches in the treatment of PD. Future studies will be directed at dissecting these possibilities in animal models of this disease.
Acknowledgements We thank Dr. Angeles Fernandez-Gonzales for helpful discussion and Nichola Wigle and Carol Jacks for assistance in preparing the figures and manuscript. This work was supported in part by the National Institutes of Health Cancer Center Support CORE Grant P30 CA 21765 and the American Lebanese Syrian Associated Charities ŽALSAC..
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Research report
Regulation of heme oxygenase-1 expression by dopamine in cultured C6 glioma and primary astrocytes Jeffrey Schmidt a , Kirsten Mertz b
b,1
, James I. Morgan
b,)
a Section of Pediatric Critical Care, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN, 38105-2794, USA Department of DeÕelopmental Neurobiology, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN, 38105-2794, USA
Accepted 20 July 1999
Abstract Heme oxygenase-1 ŽHO-1. is an inducible enzyme involved in heme catabolism, tissue iron homeostasis and the cellular response to oxidative stress. Elevated HO-1 expression in astrocytes has been observed in association with abnormal iron deposition and increased oxidative stress in Parkinson’s disease ŽPD.. Since HO-1 could contribute to these aspects of PD pathobiology we have investigated its regulation in cultured astrocytes and C6 glioma cells. Here we report that dopamine is a potent inducer of HO-1. This induction is not mediated by a classical dopamine receptor and is not mimicked by a range of catecholamines and dopamine metabolites. When the time-course of HO-1 expression was compared between dopamine and hemin, the latter induced the gene immediately while the former did so with a lag. This suggested two distinct signal transduction pathways. However, cycloheximide blocked both hemin- and dopamine-induced HO-1 expression, suggesting that both pathways may involve proteins with short half-lives. Ascorbic acid blocked dopamine induction of HO-1 but had no effect on hemin-induced expression. This suggested that dopamine may signal upstream of the unstable protein by producing pro-oxidant metabolites or byproducts. Inhibition of monoamine oxidases A or B or catechol-O-methyl transferase did not block HO-1 induction by dopamine, indicating that these enzymes were not converting dopamine to an active metabolite. These results suggest that dopamine, released or secreted from affected neurons, may trigger HO-1 expression in neighboring astrocytes. HO-1 and its metabolites could then contribute to the oxidative stress and iron deposition associated with PD. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Stimulus-transcription coupling; Receptor; Cycloheximide; Lactacystin; Ascorbic acid; Catecholamine
1. Introduction The symptoms of Parkinson’s disease ŽPD. are produced by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta ŽSNpc. w16x. The brains of PD victims also show evidence of oxidative stress, metabolic compromise, iron deposition and astrocytosis in the SN w6,8,17–20,38,40,44,55x. However, the etiological relevance of these associated processes to the neuronal loss remains a matter of debate. Similarly, much indirect evidence points to dopamine or products of dopam ine m etabolism as contributing to PD w9,13,14,29,42x. However, a specific pathogenic role for this catecholamine in PD has not been proven. Thus, there )
Corresponding author. Fax: q 1-901-495-3143; E-mail: [email protected] 1 Present address: Anatomisches Institut, Anatomie and Zellbiologie, Uni Bonn, Nussallee 10, D-53115 Bonn, Germany.
remains a quest for a pathobiological process that can accommodate the various cellular and biochemical findings in PD. It was reported recently that expression of heme oxygenase-1 ŽHO-1. is elevated in astrocytes within the substantia nigra of PD victims w46x. Heme oxygenases are the rate limiting enzymes of heme degradation and play critical roles in cellular heme and iron homeostasis w26,27,39,48– 50x. Three heme oxygenase isoforms have been described w27,28x. HO-1 is an inducible gene and its expression has been linked to the cellular oxidative stress response w27x. In addition, HO-1 is induced by heat shock, heme and heme metabolites, inflammatory cytokines and heavy metals w26,27x. HO-1 null mice exhibit excessive tissue iron deposition, reduced resistance to oxidative stress and chronic inflammation w39x. In contrast, mice that overexpress HO-1 exhibit reduced characteristics of oxidative damage w27x. Furthermore, pre-induction of HO-1 Žpreconditioning. confers increased resistance to oxidative stressors in a number
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 3 1 - 4
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of paradigms w1,2,4,22,32,34,51–53x. Thus, HO-1 is generally thought to provide protection against oxidative stress. Given the expression of HO-1 in PD brain and the parallels between its function and the associated pathological findings in PD Že.g., increased oxidative damage and deposition of iron., this enzyme could contribute to the pathobiology of this disorder. Therefore, we have attempted to identify candidate molecules that trigger HO-1 expression in astrocytes. Dopamine is known to produce oxidative damage and generate neurotoxic products w9,13,14,29,42x. Therefore, we have explored the possibility that this catecholamine can mediate HO-1 transcription in astrocytes. This report establishes that dopamine can induce HO-1 in the C6 glioma and RAW 264.7 macrophage cell lines as well as primary astrocytes in culture. Furthermore, this induction exhibits a high degree of structural specificity and does not appear to be mediated by a conventional receptor pathway. Like hemin, the classical substrate inducer of HO-1 expression w27x, dopamine, appears to signal via a protein with a short half-life. However, the time-course of HO-1 induction by dopamine is delayed compared to hemin. Moreover, unlike hemin, dopamine induction of HO-1 appears to involve a pro-oxidant species; indicating that the upstream signaling pathways for these two agents are distinct. These results are discussed in the framework of a stimulus-transcription coupling pathway for dopamine that may have relevance to the pathogenesis of PD. While this manuscript was in preparation, a report appeared that also demonstrated a number of these responses to dopamine in primary astrocyte culture w45x.
2. Materials and methods 2.1. Materials Rat C6 glioma cells were obtained from American Type Culture Collection ŽRochester, NY.. Mouse RAW264.7 cells were obtained from Dr. J. Shenep. C57Blr6 J mice were purchased from Charles River Laboratories ŽWillmington, MA. and maintained within the Animal Resource Center at St. Jude Children’s Research Hospital. Dulbecco’s modified Eagle’s medium ŽDMEM. was obtained from BioWhittaker ŽWalkersville, MD.. Fetal bovine serum ŽFBS. was obtained from Harlan ŽIndianapolis, IN. and horse serum ŽHS. was purchased from HyClone Laboratories ŽLogan, UT.. All other culture media and ancillary tissue culture reagents were purchased from Gibco BRL ŽLife Technologies, Burlington, Ontario, Canada.. All culture flasks and filter bottles were purchased from Fisher Scientific ŽSpringfield, NJ.. Dopamine, L-dopa, norepinepherine, GBR-12909, amoxapine, SCH-23390, SKF38393, spiperone, pergolide, clorgyline, deprenyl, DOPAC, and HVA were purchased from RBI Ždivision of Sigma, Natick, MA.. Hemin, ascorbic acid, hydrogen peroxide,
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ovomucoid albumin, bovine serum albumin, cycloheximide, and 4-methoxy tyramine were purchased from the Sigma ŽSt. Louis, MO.. 3,5-dinitrocatechol was purchased from Tocris Cookson, ŽBristol, UK.. Lactacystin was purchased from Kamiya Biochemical ŽSeattle, WA.. 2.2. Rat C6 glioma cell cultures Cell monolayers were grown in 75 cm2 flasks in Ham’s F-10 medium, 15% heat-inactivated FBS, and 1% penicillinrstreptomycin at 378C in 5% CO 2 for 2–3 days. For experimentation, cells were detached by trypsinization and seeded into 25 cm2 flasks and grown to densities of 1–5 = 10 7 cellsrflask for use. 2.3. Mouse RAW 264.7 macrophage cell cultures Cell monolayers were grown to 90% confluence in 75 cm2 flasks in DMEM, 10% heat-inactivated FBS, at 378C in 5% CO 2 over 2–3 days. For experimentation, cells were split and seeded into 25 cm2 flasks in fresh medium, and grown to 5 = 10 7 cellsrflask over 1–2 days. 2.4. Mouse primary astrocyte cultures Primary astrocyte cultures were prepared from the cerebral cortices and midbrain of neonatal ŽP0–P3. C57Blr6 J mice. Cells were plated in Corning 25 cm2 flasks for 1–2 weeks using a modified method adapted from Cardozo w5x. The plating medium consisted of BME, NaHCO 3 , BSA, ovomucoid albumin, glucose, CMA, HEPES, and 15% FBS. Astroglia were fed with a medium consisting of BME, glucose, glutamine, penicillinrstreptomycin, CMA, 2% rat serum, 15% FBS and 5% horse serum and grown to confluence Ž1 = 10 7 cells.. Neurons and microglia were removed by addition of trypsin and shaking on the second and fourth days after plating. 2.5. RNA isolation and northern analysis Cells were treated with various agents andror appropriate solvents. At appropriate times, total RNA was isolated from cell monolayers using the RNeasy Mini Kit ŽQiagen, Valencia, CA. per the manufacturer’s protocol. Optical densities were determined on all samples for RNA quantitation. Five-microgram total RNA were electrophoresed on 0.8% agarose gels and were transferred electrophoretically to Hybond nylon membranes ŽAmersham, Arlington Heights, IL.. RNA was analyzed by Northern blotting according to previously described procedures except where otherwise noted w30x. Ethidium bromide staining andror rehybridization with a probe to G3PDH were used to control for RNA loading and transfer. A mouse HO-1 cDNA probe Žbases 75–944; encoding initiator methionine to stop codon. was cloned and sequenced by standard techniques. This Sac1rBamH1 fragment was subcloned
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into pBluescript SKII q and used for Northern blot analysis. The cDNA probes were labeled with Ž a-32 P. dCTP ŽAmersham. using the Megaprime random primer labeling system ŽAmersham, RPN 1606. and purified over Sephadex G-50 columns ŽBoehringer Mannheim, Indianapolis, IN.. Membranes were prehybridized and hybridized with the QuikHyb hybridization buffer ŽStratagene, La Jolla, CA. at 688C. Hybridization with labeled probe was calculated to give 2–3 = 10 6 c.p.m.rml of buffer. Membranes were exposed overnight to Kodak Biomax film ŽEastman Kodak, Rochester, NY..
3. Results HO-1 is known to be induced by a range of agents, including oxidant stress, heavy metals, heme and heme metabolites, heat shock and inflammatory cytokines w26,27x. Since dopamine has been implicated in PD and can produce reactive oxygen species, we examined whether it could trigger HO-1 mRNA expression in culture. Three cell types were used: primary mouse midbrain astrocytes, rat C6 glioma Žwhich have some characteristics of astrocytes. and the murine RAW264.7 monocytic cell line Žwhich have properties of macrophages and serve as representatives of a non-astrocytic cell line.. In all cases the effects of dopamine were compared with those of hemin, a classical inducer of HO-1. For these experiments, Northern blot analysis was used to monitor HO-1 expression levels. Immunoblotting was also used in both dose–response and time course analyses and confirmed that HO-1 mRNA mirrored HO-1 protein levels Ždata not shown.. Application of hemin or dopamine triggered expression of HO-1 mRNA in primary astrocytes ŽFig. 1A., C6 glioma ŽFig. 1B. and RAW264.7 cells ŽFig. 1C.. Thus, dopamine elicited a robust induction of HO-1 in astrocytes, astrocyte-like cells and an unrelated cell type. To charac-
Fig. 2. Dopamine dose-dependently induces HO-1 mRNA in rat C6 glioma cells. Cultures were exposed to dopamine ŽDA. at the indicated concentrations for 24 h. Subsequently, HO-1 mRNA levels were determined by Northern blot analysis.
terize this response further, a dopamine dose–response analysis was carried out for C6 cells ŽFig. 2.. Low micromolar and even submicromolar concentrations of dopamine elicited expression of HO-1 ŽFig. 2 and data not shown.. While these are still relatively high concentrations of dopamine, it is quite conceivable that these levels are achieved locally in the brain as a consequence of neuronal damage or dopamine dumping. Although dopamine is reported to be toxic to cultured neurons w29x, it is well tolerated by the cells used in this study. Indeed, we routinely applied high micromolar and even low millimolar concentrations of the catecholamine for 1–2 days without obvious signs of cell death Žsee Figs. 1 and 4 and data not shown..
Fig. 1. Hemin and dopamine induce HO-1 mRNA expression in primary mouse astrocyte cultures ŽPanel A., cultured rat C6 glioma cells ŽPanel B., and cultured mouse RAW macrophages ŽPanel C.. Cell cultures were treated with either hemin or dopamine ŽDA. at the indicated doses and 24 h later total RNA was extracted and analyzed for HO-1 expression by Northern blot analysis.
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expression in C6 cells ŽFig. 3A.. A number of catecholamines are taken up by selective transport proteins. Therefore, it was possible that dopamine triggered gene expression subsequent to its uptake into a target cell. To address this possibility, C6 cells were pretreated with either the dopamine uptake inhibitor, GBR-12909 ŽFig. 3B. or the catecholamine uptake blocker, amoxapine ŽFig. 3C.. Neither inhibitor attenuated dopamine-induced HO-1 expression ŽFig. 3B,C.; indicating that these transporters were not involved in the stimulus-transcription coupling pathway utilized by dopamine. As dopamine did not appear to act via its conventional signaling pathway, we next examined whether a dopamine precursor or metabolite might be the active agent. Neither the dopamine precursor, L-dihydroxyphenylalanine ŽL-
Fig. 3. Dopamine induction of HO-1 is not mediated by conventional dopamine receptors or catecholamine transporters. Panel A: C6 cells were treated for 24 h with either a D1 ŽSKF-38393: SKF. or D2 Žpergolide. agonist. Twenty-four hours later HO-1 expression was assessed by Northern blot analysis. Neither agonist induced HO-1 expression. C6 cells were also pretreated for 1 h with either D1 ŽSCH-23390: SCH. or D2 Žspiperone. antagonists. Subsequently, the cells were exposed to dopamine ŽDA. for 24 h and HO-1 expression measured by Northern blot. Neither antagonist blocked dopamine-induced HO-1 expression. Panel B: C6 cells were pretreated for 1 h with the dopamine uptake inhibitor, GBR-12909 ŽGBR.. Subsequently, cells were treated with dopamine and HO-1 mRNA levels determined 24 h later. The uptake inhibitor did not attenuate dopamine-induced HO-1 expression. Panel C: C6 cells were pretreated for 1 h with the catecholamine uptake blocker, amoxapine ŽAMOX.. Subsequently, cells were treated with dopamine and HO-1 mRNA levels determined 24 h later. Amoxapine had no effect upon dopamine-induced HO-1 expression.
Activation of gene transcription frequently accompanies engagement of a receptor by its ligand w31x. To establish whether dopamine induced HO-1 expression via a classical receptor a pharmacological analysis was performed with various dopamine receptor agonists and antagonists. Pretreatment of C6 cells with either SCH-23390 ŽD1 antagonist. or spiperone ŽD2 antagonist. failed to attenuate HO-1 induction by dopamine ŽFig. 3A.. This suggested that dopamine was not acting via D1 or D2 receptors. This conclusion was strengthened by the finding that neither D1 ŽSKF-38393. nor D2 Žpergolide. agonists activated HO-1
Fig. 4. Dopamine precursors and metabolites do not mediate HO-1 induction. C6 cells were treated for 24 h with one of dihydroxyphenylalanine ŽL-DOPA., norepinephrine ŽNOREPI., dihydroxyphenylacetic acid ŽDOPAC., 4-methoxy tyramine Ž4-MT., or homovanillic acid ŽHVA. at the indicated doses. Dopamine ŽDA. was also tested for comparative purposes. Subsequently, HO-1 expression was measured by Northern blot. Only DOPAC produced a small increase in HO-1 mRNA levels at the highest doses tested.
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Fig. 5. MAO and COMT inhibitors do not influence dopamine-induced HO-1 expression. Panel A: C6 cells were pretreated with the MAO-B inhibitor, deprenyl for 1 h prior to addition of dopamine ŽDA.. HO-1 mRNA was measured 24 h later by Northern blot. Deprenyl, did not block dopamine-induced HO-1 expression. Panel B: C6 cells were pretreated with the COMT inhibitor, 3,5-dinitrocatechol ŽDNC. for 1 h prior to addition of dopamine ŽDA.. HO-1 expression was measured 24 h later by Northern blot. Note that dopamine-induced HO-1 expression was not attenuated by the COMT blocker.
DOPA. nor the sequential metabolite of dopamine, norepinephrine, induced HO-1 expression ŽFig. 4.. Thus, of the biologically active catecholamines, only dopamine induced HO-1. In addition, L-DOPA, which is the front-line support therapy for PD, is inactive. Dopamine is degraded by two principal enzymatic routes involving monoamine oxidases ŽMAO. and catechol-o-methyl transferase ŽCOMT.. Products of these pathways include 4-methoxytyramine Ž4-MT., dihydroxyphenylacetic acid ŽDOPAC. and homovanilic acid ŽHVA.. Of these agents, only DOPAC showed any activity ŽFig. 4.. However, the dose response curve for DOPAC revealed that it was 10–100fold less potent than dopamine establishing that it cannot be the biologically relevant metabolite that mediates HO-1
induction Žcompare Fig. 2 with Fig. 4.. Since MAOs and COMT may potentially generate other dopamine metabolites and as there may be byproducts of these reactions Že.g., reactive oxygen species. that may be causing induction of HO-1, C6 cells were treated with inhibitors of MAOs Ždeprenyl and clorgyline; Fig. 5A. or COMT Ž3,5dinitrocatechol; Fig. 5B. prior to administration of dopamine. None of these inhibitors, or combination of inhibitors, attenuated dopamine-induced HO-1 expression ŽFig. 5 and data not shown.. Therefore, it is unlikely that a direct or indirect product of the catabolism of dopamine by these three enzymes is necessary for HO-1 induction. As the upstream signaling pathway of dopamine to HO-1 gene expression appeared to be atypical, further experiments were performed to shed light on the molecular components of its transcription coupling pathway. When the time-courses of HO-1 expression triggered by hemin and dopamine were compared, hemin elicited an almost immediate increase in HO-1 mRNA levels while dopamine-induced HO-1 expression was delayed by several hours ŽFig. 6.. This suggested that there were differences in the signal transduction pathways that coupled hemin and dopamine to HO-1 transcription. It has been reported that HO-1 has the properties of an immediate-early gene w37x. To establish whether hemin and dopamine elicited HO-1 expression via an immediate-early response, C6 cells were pretreated with cycloheximide and then exposed to either the HO-1 substrate or the catecholamine. Classically, IEGs are superinduced by presentation of a stimulus in the presence of a protein synthesis inhibitor w7x. However, cycloheximide completely blocked the induction of HO-1 by both hemin and dopamine ŽFig. 7A.. Indeed, cycloheximide consistently suppressed basal HO-1 expression ŽFig. 7A and data not shown.. Thus, neither hemin nor dopamine elicit HO-1 induction via an immediate-early gene response. Moreover, since hemin can elevate HO-1 expression within 30 min Ždata not shown. and this is
Fig. 6. Hemin and dopamine elicit distinct time courses of HO-1 mRNA expression in C6 cells. C6 cells were treated with hemin Ž50 mM. or dopamine Ž500 mM. for the indicated times whereupon the cultures were harvested and total RNA isolated. Subsequently, HO-1 mRNA levels were determined by Northern blot.
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Fig. 7. Cycloheximide, lactacystin and ascorbate influence basal and stimulated HO-1 expression in C6 cells. Panel A: C6 cells were pretreated with cycloheximide ŽCyclohex. for 1 h. Subsequently, cohorts of carrier or cycloheximide-exposed cells were treated with dopamine ŽDA. or hemin and incubated for a further 8 h. Subsequently, HO-1 mRNA levels were determined by Northern blot. Note that cycloheximide reduced basal as well as dopamine- and hemin-induced HO-1 mRNA levels. Panel B: C6 cells were treated with the indicated doses of lactacystin ŽLacta. for 24 h. Subsequently, HO-1 mRNA levels were measured by Northern blot. The proteosome inhibitor, lactacystin produced a dose-dependent increase in HO-1 mRNA. Panel C: C6 cells were pretreated with ascorbic acid ŽAscorb. for 1 h. Subsequently, the cells were exposed to hemin or dopamine ŽDA. and HO-1 mRNA levels determined 24 h later by Northern blot. Ascorbate blocked dopamine but not hemin induction of HO-1 mRNA.
completely blocked by pretreatment ŽFig. 7. or even cotreatment Ždata not shown. with cycloheximide, a proteinŽs. with a relatively short half-life must participate in both basal and stimulated expression of HO-1. To test this hypothesis, C6 cells were treated with lactacystin, an inhibitor of proteasome-dependent proteolysis w36x. Lactacystin caused a dose-dependent induction of HO-1 ŽFig. 7B., indicating that simply blocking degradation of the intrinsic proteinŽs. is sufficient to induce HO-1 expression. Therefore, dopamine and hemin may act via the same or distinct labile proteins. However, there is a delay for dopamine to activate this downstream event. While we could not identify a dopamine metabolite that was an obvious candidate for the inductive signal, the delay of induction may nevertheless be indicative of a
requirement for the processing of dopamine. This putative processing does not occur spontaneously in vitro since aging dopamine in aqueous solution does not eliminate the lag period Ždata not shown.. This suggests that dopamine is actively metabolized Ži.e., requires cells., although this metabolism is not mediated by MAOs or COMT ŽFig. 5.. It is known that dopamine can be oxidized by a variety of other enzymes including xanthine oxidase, lipoxygenase and tyrosinase w10,21,41x. Therefore, we assessed whether antioxidants affected dopamine’s ability to induce HO-1. Treatment of C6 cells with ascorbic acid blocked dopamine induction of HO-1 while having no effect on hemin-induced HO-1 expression ŽFig. 7C.. This suggested that the oxidation of dopamine or one of its metabolites was central to its inducing activity. Moreover, this requirement
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J. Schmidt et al.r Molecular Brain Research 73 (1999) 50–59
Fig. 8. Hydrogen peroxide is a weak inducer of HO-1 expression compared to dopamine. Panel A: Primary mouse astrocytes were exposed to dopamine ŽDA. or hydrogen peroxide ŽH 2 O 2 . and HO-1 levels measured 24 h later by Northern blot analysis. Hydrogen peroxide is a weaker inducer of HO-1 than dopamine in cultured astrocytes. Panel B: C6 cells were exposed to hydrogen peroxide ŽH 2 O 2 . at the indicated doses for 24 h and HO-1 mRNA was then measured by Northern blot analysis. Hydrogen peroxide is a marginal inducer of HO-1 in C6 cells.
did not exist for hemin. Dopamine can be metabolized to yield peroxides w12x. However, it is unlikely that hydrogen peroxide is the mediator of dopamine activity as this superoxide is a much weaker inducer of HO-1 than dopamine in both astrocytes ŽFig. 8A. and C6 cells ŽFig. 8B. at near toxic concentrations.
4. Discussion As elevated HO-1 expression in astrocytes has been associated with PD w46x, we set out to identify molecules that might be the endogenous signals for this transcriptional response. Here we show that dopamine can trigger expression of HO-1 in astrocytes and astrocyte-like cells at concentrations that may be attained locally during pathological insults in vivo ŽFigs. 1 and 2.. This finding is consistent with another recent study in primary cultured astrocytes w45x. However, the response to dopamine is not astrocyte-specific as it is also observed in a monocytic cell line in culture ŽFig. 1.. Pharmacological analysis reveals that the pathway through which dopamine activates HO-1 transcription is not blocked by dopamine receptor antagonists and is not mimicked by D1 or D2 agonists ŽFigs. 3 and 4., confirming findings in the study of Schipper et al. w45x. In addition, it is shown here that dopamine and catechol uptake inhibitors do not attenuate HO-1 induction by dopamine ŽFig. 3B and C.. Although dopamine does not appear to activate HO-1 expression via a classical dopamine receptor its signal transduction pathway does have some mechanistic similarities with that of hemin. The observation that even relatively brief pretreatment with cycloheximide blocks HO-1 induction by hemin and dopamine Žand depresses basal HO-1 expression; Fig. 7A. indicates that the transcriptioncoupling pathways of these agents Žas well as basal transcription. probably involve a proteinŽs. with a short halflife. The latter contention is also supported by the observa-
tion that lactacystin, a proteasome inhibitor, induces HO-1 expression in C6 cells ŽFig. 7B.. Together, the lactacystin and cycloheximide results suggest that a proteinŽs. that is rapidly degraded via the proteasome is required for HO-1 induction by dopamine and hemin. Potential mediators include, Hif1-alpha and p53, both of which are known to undergo rapid, proteasome-mediated degradation w3x. Moreover, a Hif-1 response element has been identified in the HO-1 promoter w23x. HO-1 has been suggested to be an IEG w37x. Typically, IEG induction is rapid, transient and protein synthesis-independent w7x. Although, hemin does induce HO-1 rapidly, dopamine only activates expression after a delay of several hours ŽFig. 6.. Moreover, elevated HO-1 expression persists for days in response to both hemin and dopamine ŽFig. 6.. Finally, induction of HO-1 by both agents is blocked by cycloheximide ŽFig. 7A.. Therefore, HO-1 does not behave as an IEG in C6 cells in response to either hemin or dopamine. Although hemin and dopamine possibly share a common downstream transcriptional mechanism, the timecourses of HO-1 induction indicate that the upstream elements of the pathways are distinct ŽFig. 6.. The characteristic lag in HO-1 expression seen with dopamine ŽFig. 6. suggests that it activates transcription via an indirect mechanism. A number of general possibilities exist to account for this delay. First, dopamine may alter intracellular heme biosynthesis or heme homeostasis. In this scenario, elevated intracellular heme would ultimately mediate the dopamine signal transduction pathway. This possibility is presently under study. A second possibility is that dopamine must first induce or repress expression of another gene whose product then either stimulates or inhibits expression of HO-1, respectively. While this mechanism cannot be formally excluded, the lag time is probably insufficient to accommodate it. A third potential mechanism involves the metabolism of dopamine. In this scenario, dopamine is not active per se but rather one of its
J. Schmidt et al.r Molecular Brain Research 73 (1999) 50–59
metabolites or byproducts of its metabolism provides the stimulus for HO-1 induction. Although a number of dopamine metabolites were inactive and inhibition of the principal enzymes in dopamine catabolism had no effect on dopamine-induced HO-1 expression, this possibility still cannot be excluded. This is because dopamine may be metabolized by other enzymes. Indeed, the finding that ascorbic acid blocks dopamine-induced HO-1 expression ŽFig. 7C. suggests that oxidation of dopamine or a dopamine metabolite Žor a byproduct of dopamine metabolism with pro-oxidant activity. is essential for HO-1 induction. This finding corroborates observations in primary astrocytes w45x. As hemin induction of HO-1 is insensitive to ascorbic acid, the target for this antioxidant must be upstream in the dopamine signal transduction cascade. This finding also serves to discriminate two stimulus-transcription coupling pathways that may converge on a final common element. Like Schipper et al. w45x, we considered the possibility that generation of hydrogen peroxide from dopamine may be the critical mediator. However, we feel this is unlikely in astrocytes as hydrogen peroxide is a weak inducer when compared to dopamine ŽFig. 8A.. Moreover, in C6 cells hydrogen peroxide is at best a marginal inducer of HO-1 even at near toxic doses ŽFig. 8B.. Present studies are aimed at identifying the ascorbic acid-sensitive step in the dopamine signaling pathway. Dopamine has long been thought to contribute to neurodegenerative disorders such as PD and this catecholamine has been shown to be neurotoxic both in culture and in vivo w9,13,14,29,42x. This toxicity has been attributed to the iron catalyzed oxidation of dopamine to quinones and the production of superoxide and peroxynitrite. The dopamine quinones can react with sulfhydryl groups of cysteine in proteins and glutathione resulting in compromised protein function and cellular reducing capacity w11,13,15x. The free radicals can directly cause oxidative damage to macromolecules w12x. HO-1 may contribute to this process in a number of ways. First, degradation of heme by HO-1 will liberate iron, which could then potentially contribute to oxidative damage and catalyze further oxidation of dopamine to neurotoxic intermediates w12,45x. Indeed, the presence of excessive iron deposition in the substantia nigra of PD victims may be attributable to dysregulation of HO-1 expression w45,46x. Second, HO-1 is a component of an oxidative stress response program that is generally believed to confer protection against oxidative insults w1,2,22,32,34,51x. This belief is based upon preconditioning experiments and analysis of mice that either lack HO-1 or overexpress the gene. Thus, HO-1 may function to minimize damage caused by ongoing oxidative stress. It is not completely clear how HO-1 confers this protection although this may be mediated by it reducing the levels of heme-bound forms of critical proteins such as those involved in cellular respiration Že.g., cytochromes. or signal transduction pathways Že.g., guanylyl cyclase.. In addition,
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heme-bound cytochrome c is essential for some cell suicide pathways w24x and its degradation by HO-1 would be predicted to subvert this program. Furthermore, the products of HO-1 activity, biliverdin and bilirubin are known to be potent antioxidants w25,33,47,54x and exogenously administered carbon monoxide has recently been shown to confer protection against oxidative stress w35x. In this general scenario, activation of the HO-1 pathway in the PD brain may be a beneficial response providing resistance to oxidative stress and attenuating cell death. A third possibility is that excessive production of HO-1 may be a maladaptive response. For example, HO-1 metabolism of heme liberates carbon monoxide, which will compromise cellular respiration. In addition, HO-1 produces biliverdin, which is effectively converted to bilirubin. In the context of kernicterus, the latter is known to be capable of causing neural damage w43x. Thus, rather than provide protection, HO-1 expression may exacerbate pathological processes. Pharmacological modulation of HO-1 expression, whether through induction or blockade of transcription pathways, may offer novel therapeutic approaches in the treatment of PD. Future studies will be directed at dissecting these possibilities in animal models of this disease.
Acknowledgements We thank Dr. Angeles Fernandez-Gonzales for helpful discussion and Nichola Wigle and Carol Jacks for assistance in preparing the figures and manuscript. This work was supported in part by the National Institutes of Health Cancer Center Support CORE Grant P30 CA 21765 and the American Lebanese Syrian Associated Charities ŽALSAC..
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