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Catalytically inactive heme oxygenase-2 mutant is cytoprotective
Free Radical Biology & Medicine 39 (2005) 558 – 564 www.elsevier.com/locate/freeradbiomed
Original Contribution
Catalytically inactive heme oxygenase-2 mutant is cytoprotective Yun-Sook Kim, Sylvain Dore´* Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, ACCM Dept., Neuro. Res. Div., 720 Rutland Ave., Ross Research Bldg. 365, Baltimore, MD 21205, USA Received 19 November 2004; revised 10 March 2005; accepted 10 April 2005 Available online 28 April 2005
Abstract Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45Aoverexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself. D 2005 Elsevier Inc. All rights reserved. Keywords: Hemin; Bilirubin; Carbon monoxide; Hydrogen peroxide; Iron
Introduction Heme oxygenase (HO) catalyzes heme (iron-protoporphyrin IX) degradation into iron, carbon monoxide, and biliverdin/bilirubin. Heme is essential for the function of all aerobic cells. It is at the core of numerous hemoproteins (such as myoglobin, catalase, glutathione peroxidase, cytochrome, soluble guanylate cyclase, superoxide dismutase, and nitric oxide synthases) and plays a key role in controlling numerous cell functions. Cellular heme levels are
Abbreviations: CPR, cytochrome P450 reductase; DNPH, 2,4-dinitrophenylhydrazine; HEK, human embryonic kidney; HO, heme oxygenase; MTT, 3-(4,5-dimethylthiazol-2yl)2,5-diphenyl tetrazolium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; PBS, phosphatebuffered saline; WT, wildtype. * Corresponding author. Fax: +1 410 955 7271. E-mail address: [email protected] (S. Dore´). URL: www.hopkinsmedicine.edu/dorelab (S. Dore´). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.04.009
tightly controlled, as heme can generate reactive oxygen species in the redox reaction of heme with oxygen, which is achieved by a fine balance between heme biosynthesis and catabolism by HO. Free heme is considerably more toxic than heme present within proteins. These reactions that cause free radical production lead to increased membranar peroxidation and damage to proteins and DNA [1 – 3]. Two catalytically active heme oxygenase isozymes have been reported: HO-1, an inducible form, and HO-2, which is constitutively expressed. HO, unlike nitric oxide synthase, does not have the reductase activity within its structure; consequently, it needs NADPHcytochrome P450 reductase (CPR), as an electron donor, for its oxidative cleavage. The first step of HO reaction is to oxidize heme into a-meso-hydroxyheme, which subsequently reacts with oxygen to generate verdoheme and carbon monoxide. Finally, the verdoheme, in a reaction with CPR and oxygen, is converted to biliverdin and ferrous iron (Fe2+) [4].
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The homology of the amino acid sequences of HO-1 and HO-2 is approximately 40% [5]. Histidine 25 in human HO-1 and histidine 45 in human HO-2 are important amino acids in the active site structure [6,7]. However, HO-1 and HO-2 may perform distinct protective functions against tissue injury. Their synthesis and activities are differentially regulated in mammalian cells [8]. HO-1, a heat shock protein, is induced by most factors, while HO-2, under normal conditions, accounts for almost all HO activity in the brain. It is now generally accepted that HO plays a crucial role in controlling cellular homeostasis and acting as a physiologic regulator. We have demonstrated the neuroprotective role of HO-2 [9]. By comparing wildtype (WT) mice with HO-2 knockout (HO-2 / ) mice, we showed that, after subjection to a middle cerebral artery occlusion (MCAO) stroke model, infarct size was approximately twice greater in knockout mice [10,11]. Similar results were reproduced using WT mice with HO inhibitor. We also provided evidence for the antiapoptotic role of HO-2 by showing an increase in the number of in situ, end-labeled, apoptotic-like cells in HO-2 / brain after stroke, by showing increased apoptotic-like cell death in cerebellar granular cell cultures in HO-2 / under serumdeprivation conditions, and by showing reduced DNA ladder profiles in cells overexpressing HO-2 [11,12]. HO-2 is highly expressed in brain, suggesting a diversity of function far beyond the heme degradation, especially considering the ongoing debate regarding whether a significant intracellular pool of free heme exists within cells that is capable of generating sufficient amounts of the bioactive metabolites, which should account for all HO-2 cytoprotective action. Here, in order to acquire a better understanding of the cellular role/function of HO-2 protein, we addressed whether the catalytic activity of HO-2 is crucial against induced toxicity. We tested the cytoprotective role of wildtype HO-2 (HO-2WT) versus a catalytically inactive mutant (HO-2H45A), using a point mutation of histidine into alanine (HO-2H45A), against both heme- and hydrogen peroxide (H2O2)-induced toxicity. As expected, cells transfected with the inactive HO-2H45A mutant were not protected against hemin-induced toxicity. Surprisingly, we observed that after transfection with the inactive HO-2H45A mutant, cells were protected against H2O2-induced toxicity. Together, the data show that overexpression of an inactive HO-2 mutant renders cells more resistant to H2O2 and prevents cell death. These results also support the concept of the array of actions involved in the HO-2 cytoprotective role.
Materials and methods Materials All chemicals, unless stated otherwise, were purchased from Sigma Chemical Co. (St. Louis, MO).
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Cell cultures Human embryonic kidney (HEK293) cells were maintained, as described before, in Dulbecco’s modified Eagle medium (GIBCO, Carlsbad, CA) with 10% fetal bovine serum, penicillin (100 U/ml; GIBCO), and streptomycin (100 Ag/ml; GIBCO) in a humidified atmosphere of 95% air/5% CO2 at 37-C [13]. Plasmid constructions of HO-2WT or mutant HO-2H45A and transfection To generate the myc-tagged HO-2 expression construct, the human HO-2 cDNA was polymerase chain reaction (PCR)-amplified, and the PCR fragments were subcloned into EcoRI/NotI sites of pcDNA3.1 (Invitrogen, Carlsbad, CA). To generate catalytically inactive HO-2, histidine 45 was replaced with alanine using the QuickChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). The sequences of all constructs were confirmed by nucleotide sequencing with the deoxy-chain-termination method. To express HO-2WT or HO-2H45A proteins transiently, stably transfected HEK293 cells overexpressing human cytochrome P450 reductase (CPR) were transfected with pcDNA3.1-HO-2WT or pcDNA3.1-HO-2H45A construct using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After the transfection, cells were grown for another 45 h before the indicated treatment. Hemin and hydrogen peroxide (H2O2) treatment Hemin was freshly dissolved in 100 mM NaOH at a concentration of 100 mM stock solution and then diluted to the final concentrations (300 and 400 AM). H2O2 was freshly diluted in the culture medium at a concentration of 100 mM stock solution and directly added to the transiently transfected cells at the desired final concentrations (200 and 300 AM). All experiments were performed under dim light to prevent heme pigment photogradation. HO assay The HO activity was measured as previously described, with some modifications [10]. Transfected cells were harvested by a cell scraper in ice-cold phosphate-buffered saline (PBS) with protease inhibitor cocktail (Roche Biosciences, Palo Alto, CA), suspended in the lysis buffer (PBS, pH 7.4, protease inhibitor cocktail, 0.1 mM phenylmethyl sulfonyl fluoride), and sonicated by an Ultrasonic Cell Disruptor (MedSonic, Inc., Farmingdale, NY). Membrane fractions from cells (5 Ag) were incubated with NADPH (1 mM) and 55Fe-heme (Perkin Elmer Life Sciences, Boston, MA) for 6 min at 30-C. The assay was performed with or without the HO-specific inhibitor protoporphyrin IX (SnPPIX) at a final concentration of 5 AM. Reactions were terminated by the addition of phenol:chloroform:isoamyl
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alcohol (25:24:1), and uncleaved heme and biliverdin were removed from liberated 55Fe. 55Fe was counted in the aqueous phase. The data are expressed as percentage of activity to mock control. We performed three separate experiments.
groups were analyzed, and significance was analyzed by Student’s t test; p < 0.05 were considered statistically significant.
Results Western blotting Total cell extracts (30 Ag) were prepared and separated on 12% Bis-Tris gel (Invitrogen). The gel was then transferred electrophoretically to the nitrocellulose membrane (Invitrogen). The membranes were blocked with 5% skim milk in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and incubated with a 1:2000 dilution of polyclonal antibodies against HO-1, HO-2, or CPR (all purchased from Stressgen Bioreagents, Victoria, BC), and/or anti-actin (purchased from Sigma) for 1 h at room temperature, with constant shaking. The membranes were then washed and probed with horseradish peroxidaseconjugated goat anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ) at a dilution of 1:5000. Chemiluminescence detection was performed with the Amersham ECL detection kit according to the manufacturer’s instructions. MTT assay To measure the cell viability, the media were replaced with fresh media containing 0.5 mg/ml MTT (3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) after treatment [14]. After return to the incubator for 2 h, 0.1 N HCl and 0.1% Triton X-100 in anhydrous isopropanol were added in each well to dissolve the formazan precipitates. Cell viability was determined by measuring the absorbance at 570 nm. Detection of protein oxidation Oxidatively modified protein was measured after an 18-h treatment with 300 AM of H2O2 using the Oxybloti kit (Chemicon, Inc., Temecula, CA) [15]. After culture medium was aspirated, cells were washed and then harvested in 100 Al of lysis buffer (PBS, pH 7.4, 0.1 mM phenylmethyl sulfonyl fluoride, and 1 protease inhibitor cocktail; Roche, Indianapolis, IN). Carbonyl groups were derivatized with 2,4-dinitrophenylhydrazine (DNPH), according to the manufacturer’s instructions. Proteins were separated on a 12% Bis-Tris gel and transferred onto a nitrocellulose membrane. The DNP-derivatized proteins were detected with rabbit antiDNP (1:500) followed by goat anti-rabbit IgG (1:500). Immunoreactive proteins were visualized using the ECL detection kit. Statistical analyses Data in the present study are expressed as means T SE of measurements. Differences in the mean values among the
To investigate the cytoprotective role of HO-2 and the importance of its catalytic activity, we first transfected human epithelial kidney (HEK293) cells with the pcDNA3.1-hHO2WT (HO-2WT) or the pcDNA3.1-hHO-2H45A (HO-2H45A) in which the histidine 45 was replaced by alanine located within the catalytic site [7]. To ensure that the electron donor of the HO reaction, i.e., cytochrome P450 reductase (CPR), would not be a limiting factor [16], we used stable HEK293 cells that overexpressed CPR. Using these cells, we completed transfection with the HO-2WT and HO-2H45A; as a mock control, the empty vector was used. As shown in Fig. 1, we confirmed overexpression levels of HO-2WT or HO-2H45A proteins by Western blot analysis. We quantified the overexpression levels of HO-2WT and HO-2H45A by densitometry (depicted on the histogram) and observed no significant differences in HO-2 overexpression levels between HO-2WT and HO-2H45A cells. Basal expression levels of HO-1 and CPR proteins were not changed after transfection and were not affected by the presence of HO-2WT and HO-2H45A. In addition to loading 30 Ag of protein, as quantified using the protein assay, we confirmed that equal amounts of protein were loaded and transferred in each sample lane using the anti-actin antibody (Fig. 1). It has been previously demonstrated that the histidine 45 in hHO-2 is in the proximal ligand of the heme and is an essential site for the heme degradation activity of HO-2 [7]. We measured the HO activity using cell extracts of each of the transfected cells with and without the presence of the HO inhibitor SnPPIX (5 AM). As shown in Table 1, the HO
Fig. 1. Detection of overexpression of HO-2WT and HO-2H45A in HEK293CPR cells. Using stable overexpression of cytochrome P450 reductase in HEK293 cells (HEK293-CPR), transfection with plasmids encoding human HO-2WT or HO-2H45A was performed; cells were then harvested, lysed, and separated by 12% Bis-Tris gel. Overexpression of HO-2WT and HO-2H45A protein levels was detected by Western blotting with specific anti-HO-2 polyclonal antibodies. CPR, HO-1, and actin levels were also detected using polyclonal antibodies. The same amount of proteins was loaded into each lane, and efficacy of gel transfer was confirmed by similar detectable expression levels of actin within all lanes.
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Table 1 HO activity in HEK293-CPR cells transfected with mock, HO-2WT, and HO-2H45A HO activity +SnPPIX Mock HO-2WT HO-2H45A
100.0 T 0.0 120.8 T 13.7 112.2 T 12.6
SnPPIX 168.1 T 10.4 390.5 T 16.7* 197.0 T 13.5ns
HO activity was measured in the presence or in the absence of the HOspecific inhibitor, SnPPIX (5 AM). HO activity was measured and indicated as a percentage of the mock-transfected cells. The experiment was conducted in triplicate and replicated three times. ns, nonsignificant in comparison with mock-control cells. * p values of <0.05 were considered statistically significant.
activity of HO-2WT -transfected cells was significantly increased compared to mock-transfected cell lines, whereas the HO-specific activity of the catalytically inactive HO2H45A-transfected cells was not significantly different from the mock transfectant. Heme participates in crucial regulatory cellular functions mainly because it is at the core of numerous proteins with enzymatic actions. Considering that heme is very reactive and has powerful prooxidative properties, its intracellular free levels must be kept to a minimum [17]. The metabolism of heme is catalyzed by HO enzymatic action. To understand cytoprotective effects of catalytic activity of HO-2, we first treated the transfected cells with 300 or 400 AM of freshly prepared hemin. We monitored cell viability using the MTT assay after an 18-h application of hemin and compared the survival of mock, HO-2WT -, and HO-2H45Atransfected cells (Figs. 2– 4). As we expected, HO-2WT transfected cells were protected against hemin-induced cytotoxicity, while the mutant HO-2H45A-transfected cells became more sensitive to hemin. These mutant catalytically inactive HO-2H45A cells were also not significantly different from the cells transfected with the empty vector (mock-
Fig. 3. Resistance to H2O2-induced cytotoxicity in HO-2WT- and HO2H45A-transfected HEK293-CPR cells. Cells transfected with either HO2WT or HO-2H45A were treated with or 300 AM H2O2 for 18 h. Cell viability was measured using the MTT assay and expressed as a percentage of mock controls. *p < 0.05 from mock-control cells (n = 4).
control cells). The catalytic activity of HO-2 appears to contribute to the prevention of substantial damage induced after exogenous treatment with hemin. To better understand the cytoprotective effects of HO-2WT versus HO-2H45A, we treated the cells with 200 or 300 AM of hydrogen peroxide (H2O2) and measured cell viability by the MTT assay after 18 h of incubation. Comparison of the cell viability among mock-, HO-2WT -, and HO-2H45A-transfected cells indicated that HO-2WT and HO-2H45A cells were two and three times more resistant than the mock-control cells. There were no distinctive differences between HO-2WT and HO-2H45A in terms of protecting cell survival against H2O2-induced cytotoxicity. In addition, the cytoprotective effects of HO-2WT and HO-2H45A cells in response to H2O2-induced cytotoxicity was tested by monitoring the levels of oxidatively modified proteins. Densitometry analysis indicated that the levels of oxidatively modified proteins in the HO-2WT and HO-2H45A cells were less than those of the mock-control cells. No significant differences were observed between HO-2WT and HO-2H45A cells, indicating that overexpression of an inactive HO-2H45A mutant resulted in reduction in protein damage by H2O2.
Discussion
Fig. 2. Resistance to hemin-induced cytotoxicity in the HO-2WT -transfected HEK293-CPR cells. Cells transfected with either HO-2WT or HO-2H45A were treated with 300 or 400 AM hemin for 18 h. Cell viability was measured using the MTT assay and expressed as a percentaage of mock controls. *p < 0.05 from mock-control cells (n = 4).
In this study, we have found differences in the cytoprotective effects of heme oxygenase-2 based on its catalytic activity in response to hemin- and hydrogen peroxideinduced toxicity. As expected, human HEK293-cytochrome P450 reductase cells transiently transfected with catalytically inactive human HO-2H45A mutant exhibit greater sensitivity to hemin-induced toxicity as compared to the HO-2WT-transfected cells. Transfection of the HO-2WT causes HEK293-CPR cells to become more resistant to
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Fig. 4. Detection of oxidatively modified proteins in control mock-, HO2WT-, and HO-2H45A-transfected HEK293-CPR cells in response to H2O2 toxicity. The transfected cells were treated with 300 AM H2O2 for 18 h. The protein extracts were reacted with 2,4-dinitrophenylhydrazine (DNPH). The oxidatively modified proteins were revealed by Western blotting with antiDNP antibody. Actin was used to detect the same amount of protein loading in each lane, and molecular weight markers (kDa) are indicated on the left. The histograms show the quantification by densitometry of the experiments. The experiments were duplicated with similar results. The value of 100% represents the carbonyl levels in the vehicle-treated HEK293-CPR cells.
oxidative stress by H2O2 as compared to control; surprisingly, the catalytically inactive HO-2H45A offers similar protection, suggesting that even the catalytically inactive HO-2H45A mutant is cytoprotective. Hori et al. conducted similar work transfecting a catalytically inactive rat HO-1 mutant into a human lymphoma cell line [18], and they revealed comparable results, although they postulated upregulation of antioxidant systems. However, here, we are instead discussing the possibility that direct interactions of HO with other proteins lead to a cascade of several intracellular events, resulting in cytoprotection. Heme plays an important role among various cells and tissues because of its incorporation into hemoproteins. Consequently, heme levels are tightly regulated by control of heme synthesis and degradation. Under normal conditions, the amount of free heme within a cell is kept
minimal. Free heme is very reactive and is a potent prooxidant, causing tissue and cell damage. When heme is released from heme-containing proteins, it is generally not recycled and must be rapidly degraded. The main enzyme for its degradation is HO. Recently, work has focused on the biologic and physiologic effects of the heme metabolites of HO reaction. HO catalyzes the degradation of the heme (iron-protoporphyrinIX) into iron, carbon monoxide (CO), and biliverdin, the latter being rapidly reduced into bilirubin by biliverdin reductase. We and others have reported that biliverdin by itself could be a potent antioxidant [10,19,20]. Bilirubin, generated from the action of biliverdin reductase, is also considered to be a radical scavenger. In addition, a small but potentially physiologically significant portion of bilirubin can then be recycled into biliverdin [10,21]. Iron liberated from the opening of the protoporphyrin ring is then free and able to generate reactive oxygen species through different reactions, such as the classic Fenton reaction. The Fenton reaction, which involves H2O2 and a ferrous iron catalyst, has been known as an oxidizing reaction. The peroxide is broken down into a hydroxide ion, and a hydroxyl free radical is the major oxidizing species [22]. Under normal conditions, the amount of ferritin, a high-capacity iron-storage protein, has been estimated to be enough to bind the amount of iron generated by turnover of physiologic concentrations of heme by HO [23]. Carbon monoxide is also derived from the HO reaction [24,25]. It is a gaseous molecule that can move throughout intracellular and extracellular compartments and has been suggested to modulate the activity of guanylyl cyclase. One important distinction of CO versus nitric oxide (NO) is that CO has a significantly longer half-life than NO. Among other actions of CO, it has been reported to have vasodilatory, antiproliferative, antiapoptotic, and antiinflammatory properties [26,27]. Modulation of HO activity, through either changes in protein expression levels or posttranslational modification [28], has led to a general consensus that, under physiologic conditions (not supraphysiologic), the HO enzymes lead to cytoprotection. Based on the previous observation by Ishikawa et al. that human HO-2H45A, in which histidine 45 is replaced with alanine, abolishes the enzymatic activity [7], we took advantage of this mutant and tested its intrinsic cellular properties. Here, we report that increased expression of the inactive HO-2H45A mutant is cytoprotective against H2O2induced toxicity. This result suggests that not only can the metabolites of heme degradation be protective, but the HO protein itself might also be protective. This challenging observation is also supported by the ongoing debate concerning the source of heme. The general consensus is that the intracellular free heme level is kept minimal and that most actions attributed to the heme metabolites are normally protective at high nanomolar and low micromolar concentrations. Heme levels can rapidly rise from degradation of heme-containing proteins. However, as only one molecule of heme is released per hemoprotein, it is unlikely that
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micromolar levels of free heme can be generated through this process. Heme de novo synthesis is also tightly regulated to respond to cellular demand for the generation of new hemoproteins. Consequently, we assume that the HO protein itself is likely to interact with other proteins and affect diverse signaling pathways leading to cytoprotection. Further work in this regard is warranted. Until now, only a few examples of such protein interactors with HO have been reported. We have previously observed that amyloid precursor proteins and amyloid precursor-like proteins would interact with HO proteins and affect HO activity [13]. Moreover, the catalytic activity of HO-2 increases as a result of protein phosphorylation, which would be mediated by kinases, such as protein kinase C (PKC) and CKII kinase [10,28]. Recently, it was suggested that HO-1 is found associated with caveolin in mouse mesangial cells [29] and rat endothelial cells [30], and it was proposed that such interaction decreased HO-1 enzymatic activity. We have previously reported that differences between HO-1 and HO-2 subcellular localization could also discriminate between different cellular outcomes [15]. Additionally, the group led by Dr. Dennery showed that HO-1 and HO-2 proteins interact with each other and proposed that this interaction would serve to limit HO activity [31]. They were also the first to suggest that such interaction could promote nonenzymatic functions of HO. Under our experimental conditions, in which HO activity was protective and a basal amount of HO-1 was present in our cells, transfection of the mutant HO-2H45A should have decreased total HO activity and cell survival; however, what we observed was that mutant HO-2H45A was cytoprotective in response to H2O2-induced cytotoxicity. In summary, human HEK293-CPR cells transfected with inactive hHO-2H45A are less resistant against hemin-induced cytotoxicity, as expected; however, this HO-2H45A inactive mutant astonishingly protects cells against induced free radical damage. We propose that HO-2H45A, by binding with known and still unknown protein interactors, allows the cells to become more resistant to cellular-induced injury. Further experiments are necessary to elucidate the HO-2 cytoprotective mechanisms. This investigation could lead to new approaches toward a better understanding of the physiologic and cellular defense mechanisms of HO-2 activity and/or of the HO-2 protein itself mediating the potential antioxidant, antiinflammatory, and antiapoptotic effects.
Acknowledgments This work was supported by NIH Grants NS046400, AG022971, AA014911, and AT001836 (S.D.), and a postdoctoral fellowship from the Mid-Atlantic American Heart and Stroke Association (Y.S.K.). We thank Darren F. Boehning, PhD, for providing the HO-2H45A plasmid, Tzipora Sofare, MA, for editorial assistance, and Raymond C. Koehler, PhD, for helpful discussions.
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Original Contribution
Catalytically inactive heme oxygenase-2 mutant is cytoprotective Yun-Sook Kim, Sylvain Dore´* Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, ACCM Dept., Neuro. Res. Div., 720 Rutland Ave., Ross Research Bldg. 365, Baltimore, MD 21205, USA Received 19 November 2004; revised 10 March 2005; accepted 10 April 2005 Available online 28 April 2005
Abstract Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45Aoverexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself. D 2005 Elsevier Inc. All rights reserved. Keywords: Hemin; Bilirubin; Carbon monoxide; Hydrogen peroxide; Iron
Introduction Heme oxygenase (HO) catalyzes heme (iron-protoporphyrin IX) degradation into iron, carbon monoxide, and biliverdin/bilirubin. Heme is essential for the function of all aerobic cells. It is at the core of numerous hemoproteins (such as myoglobin, catalase, glutathione peroxidase, cytochrome, soluble guanylate cyclase, superoxide dismutase, and nitric oxide synthases) and plays a key role in controlling numerous cell functions. Cellular heme levels are
Abbreviations: CPR, cytochrome P450 reductase; DNPH, 2,4-dinitrophenylhydrazine; HEK, human embryonic kidney; HO, heme oxygenase; MTT, 3-(4,5-dimethylthiazol-2yl)2,5-diphenyl tetrazolium bromide; NADPH, nicotinamide adenine dinucleotide phosphate; PBS, phosphatebuffered saline; WT, wildtype. * Corresponding author. Fax: +1 410 955 7271. E-mail address: [email protected] (S. Dore´). URL: www.hopkinsmedicine.edu/dorelab (S. Dore´). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.04.009
tightly controlled, as heme can generate reactive oxygen species in the redox reaction of heme with oxygen, which is achieved by a fine balance between heme biosynthesis and catabolism by HO. Free heme is considerably more toxic than heme present within proteins. These reactions that cause free radical production lead to increased membranar peroxidation and damage to proteins and DNA [1 – 3]. Two catalytically active heme oxygenase isozymes have been reported: HO-1, an inducible form, and HO-2, which is constitutively expressed. HO, unlike nitric oxide synthase, does not have the reductase activity within its structure; consequently, it needs NADPHcytochrome P450 reductase (CPR), as an electron donor, for its oxidative cleavage. The first step of HO reaction is to oxidize heme into a-meso-hydroxyheme, which subsequently reacts with oxygen to generate verdoheme and carbon monoxide. Finally, the verdoheme, in a reaction with CPR and oxygen, is converted to biliverdin and ferrous iron (Fe2+) [4].
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The homology of the amino acid sequences of HO-1 and HO-2 is approximately 40% [5]. Histidine 25 in human HO-1 and histidine 45 in human HO-2 are important amino acids in the active site structure [6,7]. However, HO-1 and HO-2 may perform distinct protective functions against tissue injury. Their synthesis and activities are differentially regulated in mammalian cells [8]. HO-1, a heat shock protein, is induced by most factors, while HO-2, under normal conditions, accounts for almost all HO activity in the brain. It is now generally accepted that HO plays a crucial role in controlling cellular homeostasis and acting as a physiologic regulator. We have demonstrated the neuroprotective role of HO-2 [9]. By comparing wildtype (WT) mice with HO-2 knockout (HO-2 / ) mice, we showed that, after subjection to a middle cerebral artery occlusion (MCAO) stroke model, infarct size was approximately twice greater in knockout mice [10,11]. Similar results were reproduced using WT mice with HO inhibitor. We also provided evidence for the antiapoptotic role of HO-2 by showing an increase in the number of in situ, end-labeled, apoptotic-like cells in HO-2 / brain after stroke, by showing increased apoptotic-like cell death in cerebellar granular cell cultures in HO-2 / under serumdeprivation conditions, and by showing reduced DNA ladder profiles in cells overexpressing HO-2 [11,12]. HO-2 is highly expressed in brain, suggesting a diversity of function far beyond the heme degradation, especially considering the ongoing debate regarding whether a significant intracellular pool of free heme exists within cells that is capable of generating sufficient amounts of the bioactive metabolites, which should account for all HO-2 cytoprotective action. Here, in order to acquire a better understanding of the cellular role/function of HO-2 protein, we addressed whether the catalytic activity of HO-2 is crucial against induced toxicity. We tested the cytoprotective role of wildtype HO-2 (HO-2WT) versus a catalytically inactive mutant (HO-2H45A), using a point mutation of histidine into alanine (HO-2H45A), against both heme- and hydrogen peroxide (H2O2)-induced toxicity. As expected, cells transfected with the inactive HO-2H45A mutant were not protected against hemin-induced toxicity. Surprisingly, we observed that after transfection with the inactive HO-2H45A mutant, cells were protected against H2O2-induced toxicity. Together, the data show that overexpression of an inactive HO-2 mutant renders cells more resistant to H2O2 and prevents cell death. These results also support the concept of the array of actions involved in the HO-2 cytoprotective role.
Materials and methods Materials All chemicals, unless stated otherwise, were purchased from Sigma Chemical Co. (St. Louis, MO).
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Cell cultures Human embryonic kidney (HEK293) cells were maintained, as described before, in Dulbecco’s modified Eagle medium (GIBCO, Carlsbad, CA) with 10% fetal bovine serum, penicillin (100 U/ml; GIBCO), and streptomycin (100 Ag/ml; GIBCO) in a humidified atmosphere of 95% air/5% CO2 at 37-C [13]. Plasmid constructions of HO-2WT or mutant HO-2H45A and transfection To generate the myc-tagged HO-2 expression construct, the human HO-2 cDNA was polymerase chain reaction (PCR)-amplified, and the PCR fragments were subcloned into EcoRI/NotI sites of pcDNA3.1 (Invitrogen, Carlsbad, CA). To generate catalytically inactive HO-2, histidine 45 was replaced with alanine using the QuickChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). The sequences of all constructs were confirmed by nucleotide sequencing with the deoxy-chain-termination method. To express HO-2WT or HO-2H45A proteins transiently, stably transfected HEK293 cells overexpressing human cytochrome P450 reductase (CPR) were transfected with pcDNA3.1-HO-2WT or pcDNA3.1-HO-2H45A construct using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After the transfection, cells were grown for another 45 h before the indicated treatment. Hemin and hydrogen peroxide (H2O2) treatment Hemin was freshly dissolved in 100 mM NaOH at a concentration of 100 mM stock solution and then diluted to the final concentrations (300 and 400 AM). H2O2 was freshly diluted in the culture medium at a concentration of 100 mM stock solution and directly added to the transiently transfected cells at the desired final concentrations (200 and 300 AM). All experiments were performed under dim light to prevent heme pigment photogradation. HO assay The HO activity was measured as previously described, with some modifications [10]. Transfected cells were harvested by a cell scraper in ice-cold phosphate-buffered saline (PBS) with protease inhibitor cocktail (Roche Biosciences, Palo Alto, CA), suspended in the lysis buffer (PBS, pH 7.4, protease inhibitor cocktail, 0.1 mM phenylmethyl sulfonyl fluoride), and sonicated by an Ultrasonic Cell Disruptor (MedSonic, Inc., Farmingdale, NY). Membrane fractions from cells (5 Ag) were incubated with NADPH (1 mM) and 55Fe-heme (Perkin Elmer Life Sciences, Boston, MA) for 6 min at 30-C. The assay was performed with or without the HO-specific inhibitor protoporphyrin IX (SnPPIX) at a final concentration of 5 AM. Reactions were terminated by the addition of phenol:chloroform:isoamyl
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alcohol (25:24:1), and uncleaved heme and biliverdin were removed from liberated 55Fe. 55Fe was counted in the aqueous phase. The data are expressed as percentage of activity to mock control. We performed three separate experiments.
groups were analyzed, and significance was analyzed by Student’s t test; p < 0.05 were considered statistically significant.
Results Western blotting Total cell extracts (30 Ag) were prepared and separated on 12% Bis-Tris gel (Invitrogen). The gel was then transferred electrophoretically to the nitrocellulose membrane (Invitrogen). The membranes were blocked with 5% skim milk in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and incubated with a 1:2000 dilution of polyclonal antibodies against HO-1, HO-2, or CPR (all purchased from Stressgen Bioreagents, Victoria, BC), and/or anti-actin (purchased from Sigma) for 1 h at room temperature, with constant shaking. The membranes were then washed and probed with horseradish peroxidaseconjugated goat anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ) at a dilution of 1:5000. Chemiluminescence detection was performed with the Amersham ECL detection kit according to the manufacturer’s instructions. MTT assay To measure the cell viability, the media were replaced with fresh media containing 0.5 mg/ml MTT (3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) after treatment [14]. After return to the incubator for 2 h, 0.1 N HCl and 0.1% Triton X-100 in anhydrous isopropanol were added in each well to dissolve the formazan precipitates. Cell viability was determined by measuring the absorbance at 570 nm. Detection of protein oxidation Oxidatively modified protein was measured after an 18-h treatment with 300 AM of H2O2 using the Oxybloti kit (Chemicon, Inc., Temecula, CA) [15]. After culture medium was aspirated, cells were washed and then harvested in 100 Al of lysis buffer (PBS, pH 7.4, 0.1 mM phenylmethyl sulfonyl fluoride, and 1 protease inhibitor cocktail; Roche, Indianapolis, IN). Carbonyl groups were derivatized with 2,4-dinitrophenylhydrazine (DNPH), according to the manufacturer’s instructions. Proteins were separated on a 12% Bis-Tris gel and transferred onto a nitrocellulose membrane. The DNP-derivatized proteins were detected with rabbit antiDNP (1:500) followed by goat anti-rabbit IgG (1:500). Immunoreactive proteins were visualized using the ECL detection kit. Statistical analyses Data in the present study are expressed as means T SE of measurements. Differences in the mean values among the
To investigate the cytoprotective role of HO-2 and the importance of its catalytic activity, we first transfected human epithelial kidney (HEK293) cells with the pcDNA3.1-hHO2WT (HO-2WT) or the pcDNA3.1-hHO-2H45A (HO-2H45A) in which the histidine 45 was replaced by alanine located within the catalytic site [7]. To ensure that the electron donor of the HO reaction, i.e., cytochrome P450 reductase (CPR), would not be a limiting factor [16], we used stable HEK293 cells that overexpressed CPR. Using these cells, we completed transfection with the HO-2WT and HO-2H45A; as a mock control, the empty vector was used. As shown in Fig. 1, we confirmed overexpression levels of HO-2WT or HO-2H45A proteins by Western blot analysis. We quantified the overexpression levels of HO-2WT and HO-2H45A by densitometry (depicted on the histogram) and observed no significant differences in HO-2 overexpression levels between HO-2WT and HO-2H45A cells. Basal expression levels of HO-1 and CPR proteins were not changed after transfection and were not affected by the presence of HO-2WT and HO-2H45A. In addition to loading 30 Ag of protein, as quantified using the protein assay, we confirmed that equal amounts of protein were loaded and transferred in each sample lane using the anti-actin antibody (Fig. 1). It has been previously demonstrated that the histidine 45 in hHO-2 is in the proximal ligand of the heme and is an essential site for the heme degradation activity of HO-2 [7]. We measured the HO activity using cell extracts of each of the transfected cells with and without the presence of the HO inhibitor SnPPIX (5 AM). As shown in Table 1, the HO
Fig. 1. Detection of overexpression of HO-2WT and HO-2H45A in HEK293CPR cells. Using stable overexpression of cytochrome P450 reductase in HEK293 cells (HEK293-CPR), transfection with plasmids encoding human HO-2WT or HO-2H45A was performed; cells were then harvested, lysed, and separated by 12% Bis-Tris gel. Overexpression of HO-2WT and HO-2H45A protein levels was detected by Western blotting with specific anti-HO-2 polyclonal antibodies. CPR, HO-1, and actin levels were also detected using polyclonal antibodies. The same amount of proteins was loaded into each lane, and efficacy of gel transfer was confirmed by similar detectable expression levels of actin within all lanes.
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Table 1 HO activity in HEK293-CPR cells transfected with mock, HO-2WT, and HO-2H45A HO activity +SnPPIX Mock HO-2WT HO-2H45A
100.0 T 0.0 120.8 T 13.7 112.2 T 12.6
SnPPIX 168.1 T 10.4 390.5 T 16.7* 197.0 T 13.5ns
HO activity was measured in the presence or in the absence of the HOspecific inhibitor, SnPPIX (5 AM). HO activity was measured and indicated as a percentage of the mock-transfected cells. The experiment was conducted in triplicate and replicated three times. ns, nonsignificant in comparison with mock-control cells. * p values of <0.05 were considered statistically significant.
activity of HO-2WT -transfected cells was significantly increased compared to mock-transfected cell lines, whereas the HO-specific activity of the catalytically inactive HO2H45A-transfected cells was not significantly different from the mock transfectant. Heme participates in crucial regulatory cellular functions mainly because it is at the core of numerous proteins with enzymatic actions. Considering that heme is very reactive and has powerful prooxidative properties, its intracellular free levels must be kept to a minimum [17]. The metabolism of heme is catalyzed by HO enzymatic action. To understand cytoprotective effects of catalytic activity of HO-2, we first treated the transfected cells with 300 or 400 AM of freshly prepared hemin. We monitored cell viability using the MTT assay after an 18-h application of hemin and compared the survival of mock, HO-2WT -, and HO-2H45Atransfected cells (Figs. 2– 4). As we expected, HO-2WT transfected cells were protected against hemin-induced cytotoxicity, while the mutant HO-2H45A-transfected cells became more sensitive to hemin. These mutant catalytically inactive HO-2H45A cells were also not significantly different from the cells transfected with the empty vector (mock-
Fig. 3. Resistance to H2O2-induced cytotoxicity in HO-2WT- and HO2H45A-transfected HEK293-CPR cells. Cells transfected with either HO2WT or HO-2H45A were treated with or 300 AM H2O2 for 18 h. Cell viability was measured using the MTT assay and expressed as a percentage of mock controls. *p < 0.05 from mock-control cells (n = 4).
control cells). The catalytic activity of HO-2 appears to contribute to the prevention of substantial damage induced after exogenous treatment with hemin. To better understand the cytoprotective effects of HO-2WT versus HO-2H45A, we treated the cells with 200 or 300 AM of hydrogen peroxide (H2O2) and measured cell viability by the MTT assay after 18 h of incubation. Comparison of the cell viability among mock-, HO-2WT -, and HO-2H45A-transfected cells indicated that HO-2WT and HO-2H45A cells were two and three times more resistant than the mock-control cells. There were no distinctive differences between HO-2WT and HO-2H45A in terms of protecting cell survival against H2O2-induced cytotoxicity. In addition, the cytoprotective effects of HO-2WT and HO-2H45A cells in response to H2O2-induced cytotoxicity was tested by monitoring the levels of oxidatively modified proteins. Densitometry analysis indicated that the levels of oxidatively modified proteins in the HO-2WT and HO-2H45A cells were less than those of the mock-control cells. No significant differences were observed between HO-2WT and HO-2H45A cells, indicating that overexpression of an inactive HO-2H45A mutant resulted in reduction in protein damage by H2O2.
Discussion
Fig. 2. Resistance to hemin-induced cytotoxicity in the HO-2WT -transfected HEK293-CPR cells. Cells transfected with either HO-2WT or HO-2H45A were treated with 300 or 400 AM hemin for 18 h. Cell viability was measured using the MTT assay and expressed as a percentaage of mock controls. *p < 0.05 from mock-control cells (n = 4).
In this study, we have found differences in the cytoprotective effects of heme oxygenase-2 based on its catalytic activity in response to hemin- and hydrogen peroxideinduced toxicity. As expected, human HEK293-cytochrome P450 reductase cells transiently transfected with catalytically inactive human HO-2H45A mutant exhibit greater sensitivity to hemin-induced toxicity as compared to the HO-2WT-transfected cells. Transfection of the HO-2WT causes HEK293-CPR cells to become more resistant to
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Fig. 4. Detection of oxidatively modified proteins in control mock-, HO2WT-, and HO-2H45A-transfected HEK293-CPR cells in response to H2O2 toxicity. The transfected cells were treated with 300 AM H2O2 for 18 h. The protein extracts were reacted with 2,4-dinitrophenylhydrazine (DNPH). The oxidatively modified proteins were revealed by Western blotting with antiDNP antibody. Actin was used to detect the same amount of protein loading in each lane, and molecular weight markers (kDa) are indicated on the left. The histograms show the quantification by densitometry of the experiments. The experiments were duplicated with similar results. The value of 100% represents the carbonyl levels in the vehicle-treated HEK293-CPR cells.
oxidative stress by H2O2 as compared to control; surprisingly, the catalytically inactive HO-2H45A offers similar protection, suggesting that even the catalytically inactive HO-2H45A mutant is cytoprotective. Hori et al. conducted similar work transfecting a catalytically inactive rat HO-1 mutant into a human lymphoma cell line [18], and they revealed comparable results, although they postulated upregulation of antioxidant systems. However, here, we are instead discussing the possibility that direct interactions of HO with other proteins lead to a cascade of several intracellular events, resulting in cytoprotection. Heme plays an important role among various cells and tissues because of its incorporation into hemoproteins. Consequently, heme levels are tightly regulated by control of heme synthesis and degradation. Under normal conditions, the amount of free heme within a cell is kept
minimal. Free heme is very reactive and is a potent prooxidant, causing tissue and cell damage. When heme is released from heme-containing proteins, it is generally not recycled and must be rapidly degraded. The main enzyme for its degradation is HO. Recently, work has focused on the biologic and physiologic effects of the heme metabolites of HO reaction. HO catalyzes the degradation of the heme (iron-protoporphyrinIX) into iron, carbon monoxide (CO), and biliverdin, the latter being rapidly reduced into bilirubin by biliverdin reductase. We and others have reported that biliverdin by itself could be a potent antioxidant [10,19,20]. Bilirubin, generated from the action of biliverdin reductase, is also considered to be a radical scavenger. In addition, a small but potentially physiologically significant portion of bilirubin can then be recycled into biliverdin [10,21]. Iron liberated from the opening of the protoporphyrin ring is then free and able to generate reactive oxygen species through different reactions, such as the classic Fenton reaction. The Fenton reaction, which involves H2O2 and a ferrous iron catalyst, has been known as an oxidizing reaction. The peroxide is broken down into a hydroxide ion, and a hydroxyl free radical is the major oxidizing species [22]. Under normal conditions, the amount of ferritin, a high-capacity iron-storage protein, has been estimated to be enough to bind the amount of iron generated by turnover of physiologic concentrations of heme by HO [23]. Carbon monoxide is also derived from the HO reaction [24,25]. It is a gaseous molecule that can move throughout intracellular and extracellular compartments and has been suggested to modulate the activity of guanylyl cyclase. One important distinction of CO versus nitric oxide (NO) is that CO has a significantly longer half-life than NO. Among other actions of CO, it has been reported to have vasodilatory, antiproliferative, antiapoptotic, and antiinflammatory properties [26,27]. Modulation of HO activity, through either changes in protein expression levels or posttranslational modification [28], has led to a general consensus that, under physiologic conditions (not supraphysiologic), the HO enzymes lead to cytoprotection. Based on the previous observation by Ishikawa et al. that human HO-2H45A, in which histidine 45 is replaced with alanine, abolishes the enzymatic activity [7], we took advantage of this mutant and tested its intrinsic cellular properties. Here, we report that increased expression of the inactive HO-2H45A mutant is cytoprotective against H2O2induced toxicity. This result suggests that not only can the metabolites of heme degradation be protective, but the HO protein itself might also be protective. This challenging observation is also supported by the ongoing debate concerning the source of heme. The general consensus is that the intracellular free heme level is kept minimal and that most actions attributed to the heme metabolites are normally protective at high nanomolar and low micromolar concentrations. Heme levels can rapidly rise from degradation of heme-containing proteins. However, as only one molecule of heme is released per hemoprotein, it is unlikely that
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micromolar levels of free heme can be generated through this process. Heme de novo synthesis is also tightly regulated to respond to cellular demand for the generation of new hemoproteins. Consequently, we assume that the HO protein itself is likely to interact with other proteins and affect diverse signaling pathways leading to cytoprotection. Further work in this regard is warranted. Until now, only a few examples of such protein interactors with HO have been reported. We have previously observed that amyloid precursor proteins and amyloid precursor-like proteins would interact with HO proteins and affect HO activity [13]. Moreover, the catalytic activity of HO-2 increases as a result of protein phosphorylation, which would be mediated by kinases, such as protein kinase C (PKC) and CKII kinase [10,28]. Recently, it was suggested that HO-1 is found associated with caveolin in mouse mesangial cells [29] and rat endothelial cells [30], and it was proposed that such interaction decreased HO-1 enzymatic activity. We have previously reported that differences between HO-1 and HO-2 subcellular localization could also discriminate between different cellular outcomes [15]. Additionally, the group led by Dr. Dennery showed that HO-1 and HO-2 proteins interact with each other and proposed that this interaction would serve to limit HO activity [31]. They were also the first to suggest that such interaction could promote nonenzymatic functions of HO. Under our experimental conditions, in which HO activity was protective and a basal amount of HO-1 was present in our cells, transfection of the mutant HO-2H45A should have decreased total HO activity and cell survival; however, what we observed was that mutant HO-2H45A was cytoprotective in response to H2O2-induced cytotoxicity. In summary, human HEK293-CPR cells transfected with inactive hHO-2H45A are less resistant against hemin-induced cytotoxicity, as expected; however, this HO-2H45A inactive mutant astonishingly protects cells against induced free radical damage. We propose that HO-2H45A, by binding with known and still unknown protein interactors, allows the cells to become more resistant to cellular-induced injury. Further experiments are necessary to elucidate the HO-2 cytoprotective mechanisms. This investigation could lead to new approaches toward a better understanding of the physiologic and cellular defense mechanisms of HO-2 activity and/or of the HO-2 protein itself mediating the potential antioxidant, antiinflammatory, and antiapoptotic effects.
Acknowledgments This work was supported by NIH Grants NS046400, AG022971, AA014911, and AT001836 (S.D.), and a postdoctoral fellowship from the Mid-Atlantic American Heart and Stroke Association (Y.S.K.). We thank Darren F. Boehning, PhD, for providing the HO-2H45A plasmid, Tzipora Sofare, MA, for editorial assistance, and Raymond C. Koehler, PhD, for helpful discussions.
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