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Endocrinology Vol. 149, No. 7 3267-3273
Copyright © 2008 by The Endocrine Society

3β-Hydroxysteroid-{Delta}24 Reductase Is a Hydrogen Peroxide Scavenger, Protecting Cells from Oxidative Stress-Induced Apoptosis

Xiuli Lu, Fukushi Kambe, Xia Cao, Yasuko Kozaki, Takahide Kaji, Takehisa Ishii and Hisao Seo

Department of Endocrinology (X.L., F.K., X.C., Y.K., H.S.), Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan; and Mitsubishi Pharma Corp. (T.K., T.I.), Yokohama 227-0033, Japan

Address all correspondence and requests for reprints to: Fukushi Kambe, M.D., Ph.D., Department of Endocrinology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: kambe{at}riem.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
3β-Hydroxysteroid-{Delta}24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H2O2) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24–/– mouse embryonic fibroblasts). H2O2 exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H2O2-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H2O2 was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H2O2-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H2O2, thereby protecting cells from oxidative stress-induced apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
HYDROGEN PEROXIDE (H2O2) is one of the main reactive oxygen species (ROS) in cells. H2O2 is constantly generated by normal aerobic metabolism. Because H2O2 is highly reactive to cell components, several enzymes exist to eliminate H2O2, such as catalase, glutathione peroxidase (GPx), and peroxiredoxin (Prx) (1, 2, 3). Although the low levels of H2O2 generated in response to some growth factors have been shown to serve as a second messenger for cell proliferation (4), the generation of excess H2O2 due to overproduction, impairment of the scavenging system, or administration of anticancer drugs elicits cytotoxic effects, which damage DNA, protein, and membrane lipids (5). In addition to such direct effects on cell components, H2O2 excess has been demonstrated to affect the intracellular signaling cascades that involve stress-activated protein kinases (SAPKs) (6, 7, 8).

MAPK cascades play an important role in regulation of cell viability, proliferation, senescence, and death. In mammals, there are three MAPKs: ERK, p38 MAPK, and c-Jun N-terminal kinase (JNK) (9). Whereas ERK mediates proliferative stimuli, p38 MAPK, and JNK are mainly involved in signal transduction for stress responses and apoptosis, especially when cells are under stress, so that these kinases are called SAPKs (8). Recent studies have shown that H2O2-induced apoptosis is associated with activation of p38 MAPK and JNK and that apoptosis signal-regulating kinase (ASK)-1 functions as an MAPK kinase kinase (10).

3β-Hydroxysteroid-{Delta}24 reductase (DHCR24; alias: hDiminuto/Seladin-1) is a 3β-hydroxysteroid-{Delta}24 reductase and catalyzes the final step in cholesterol biosynthesis, the conversion from desmosterol to cholesterol (11). The DHCR24 gene is expressed in all types of cells tested, and loss-of-function gene mutations cause a cholesterol-biosynthesis disorder, desmosterolosis (12). In addition to cholesterol-synthesizing activity, several biologically important activities of DHCR24 have been reported to date. DHCR24 interacts with p53 and increases its stability, thereby regulating cell growth and senescence (13). Furthermore, using patient specimens, we and others have reported that decreased DHCR24 expression is associated with apoptosis of adrenocortical cells in cortisol-producing adrenocortical adenoma (14) and with the neuronal cell death in the brain regions affected by Alzheimer’s disease (15). Conversely, increased expression is detected in melanoma derived from metastatic lesions, compared with the primary tumor (16). These studies suggest that DHCR24 possesses antiapoptotic activity toward many cell types.

However, the molecular basis that underlies the antiapoptotic activity of DHCR24 has not been fully elucidated. In a cell culture model, it has been shown that overexpression of DHCR24 by transfection of its expression plasmid protects cells from H2O2-induced cytotoxicity (15, 16). However, the physiological relevance of this phenomenon has not been evaluated in cells that normally express or lack DHCR24. In the present study, we investigated the effects of H2O2 on mouse embryonic fibroblasts (MEFs) prepared from DHCR24-knockout mice (DHCR24–/– MEFs). We provide evidence that DHCR24 is indispensable for protecting MEFs from H2O2-induced apoptosis and that DHCR24 possesses direct H2O2-scavenging activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture
Procedures for the preparation of DHCR24–/– MEFs have been described previously (17). Several lines of MEFs were established from mouse fetuses of gestational age 17–20 d and cultured in DMEM supplemented with 10% fetal bovine serum and nonessential amino acids. The MEFs of 10–20 passages were used for the experiments. PC12 cells (CRL-1721; American Type Culture Collection, Manassas, VA) were also cultured in DMEM supplemented with 5% fetal bovine serum and 10% horse serum. The cells were treated with 1 mM H2O2 or 0.2 µg/ml tunicamycin (TM; Sigma-Aldrich, St. Louis, MO) for various lengths of time. The cell images were obtained by a phase-contrast microscope (IMT-2; Olympus, Tokyo, Japan) equipped with a digital microscope camera (PDMC II; Polaroid, Waltham, MA).

Detection of apoptosis
Procedures for purification of fragmented genomic DNA from cell culture have been described previously (17). DNA fragmentation was also analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method using an in situ apoptosis kit (Takara, Otsu, Japan).

Western blot analysis
Procedures for preparation of whole-cell lysates and Western blot analysis have been described previously (18). In brief, whole-cell lysates (30 µg/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia, Piscataway, NJ). The blots were probed with the first antibodies as described below, followed by incubation with horseradish peroxidase-conjugated antirabbit or mouse IgG antibody. Rabbit antiphospho-ASK1 (T845), antiphospho-p38 MAPK (T180/Y182), and antiphospho-JNK (T183/Y185) were purchased from Cell Signaling (Beverly, MA). Mouse monoclonal anti-immunoglobulin heavy-chain binding protein (Bip)/GRP78 antibody was from BD Biosciences (Bedford, MA). Rabbit antiactin antibody was from Sigma-Aldrich. Mouse monoclonal anti-c-myc antibody was from Roche Diagnostics (Mannheim, Germany). Mouse monoclonal anti-histidine (His) antibody was from Amersham Biosciences (Arlington Heights, IL). The proteins were visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL). The images of the blotted membranes were obtained by an LAS-1000 lumino-image analyzer (Fuji Film, Tokyo, Japan), and densitometric analysis was performed using LAS-1000 software (Fuji Film).

Preparation of adenovirus expressing DHCR24
The entire coding sequence of human DHCR24 cDNA was amplified by PCR using sense primer 5'-GAATTCGCCACCATGGAGCCCGCCGTGTCGCTGGCC-3' and antisense primer 5'-CTCGAGGTGCCTGGCGGCCTTGCAGATCTTGTC-3' (EcoRI site, translation start site, and XhoI site are underlined). A Kozak sequence was introduced in the sense primer. The stop codon was deleted in the antisense primer. The amplified cDNA was cloned into the EcoRI-XhoI site of pcDNA3.1/myc-His-A plasmid (CLONTECH-Takara Bio, Otsu, Japan). The BamHI and PmeI fragment that contained DHCR24 cDNA and the sequence of myc-His tag in frame at the 3' end was excised out and cloned into the BglII-EcoRV site of pShuttle-CMV vector in AdEasy XL adenoviral vector system (Stratagene, La Jolla, CA). The recombinant adenovirus was prepared according to this system and named Ad-DHCR24-myc. The control adenovirus that expressed β-galactosidase was also constructed, and named Ad-LacZ.

Immunocytochemical analysis
Procedures for immunocytochemical analysis have been described previously (17, 18). After fixation and blocking, the cells were incubated with mouse monoclonal anti-c-myc antibody (Roche), followed by incubation with antimouse IgG antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). The counterstaining was performed using propidium iodide (PI). In some experiments, the cells were incubated with a mixture of mouse monoclonal anti-c-myc antibody and rabbit anticalreticulin antibody (Upstate, Lake Placid, NY) and then incubated with a mixture of antimouse IgG antibody conjugated with Alexa fluor-488 and antirabbit IgG antibody conjugated with Alexa fluor-568. Images were obtained using a confocal laser microscope (LSM510; Carl Zeiss, Jena, Germany) with the same set of optical parameters (17, 18) and were merged by Adobe Photoshop software (version 7.0.1; Adobe Systems, San Jose, CA).

Measurement of ROS production
Intracellular ROS were measured by a fluorescent dye technique (19). MEFs cultured on glass coverslips were treated for 10 min with 10 µM 2',7'-dichlorofluorescin diacetate (H2DCFDA; Molecular Probes) in PBS. The coverslips were placed in the chamber, which was mounted on the stage of an inverted microscope (Axiovert; Carl Zeiss) equipped with a confocal laser-scanning system (Oz; Noran Instruments, Middleton, WI), and the cells were exposed to 1 mM H2O2. Images were obtained every 10 min with excitation at 488 nm and emission greater than 500 nm with a long-pass barrier filter. The overall fluorescent intensities of individual cells were analyzed using Intervision 2D program (Noran).

Preparation of DHCR24 proteins
The plasmids for in vitro transcription and translation of DHCR24 and its mutants were constructed as follows. The entire coding sequence of human DHCR24 cDNA was amplified by PCR using sense primer 5'-GTCGACATGGAGCCCGCCGTGTCGCTGGCC-3' and antisense primer 5'-GTCGACTCAGTGCCTGGCGGCCTTGCAGAT-3' (SalI site is underlined), and cloned into the SalI site of pIVEX2.4a plasmid (Roche). This vector contained six His codons at the 5'-end of the cloning site. The cDNA for the mutant ORD (1–395) was made by deletion of cDNA encoding 396–516 amino acids, using a unique PstI site in DHCR24 cDNA. The cDNA for the mutant CTR (232–516) was amplified by PCR using sense primer 5'-GTCGACATCCCTGCCAAGAAGTACGTCAAG-3' and the same antisense primer described above and cloned into pIVEX2.4a. In vitro transcription and translation were performed using rapid translation system (RTS500 instrument; Roche) with GroE supplement (Roche). As a control protein, green fluorescence protein (GFP) with a His tag was also synthesized by RTS500. The proteins were then purified by Ni-Sepharose column (His SpinTrap; GE Healthcare, Uppsala, Sweden). After removing imidazole by a spin column filter (Millipore, Billerica, MA), H2O2-scavenging activity of the purified proteins was determined as described below. Protein purity was assessed by SDS-PAGE and Coomassie brilliant blue staining.

Measurement of H2O2-scavenging activity
H2O2-scavenging activities of the purified DHCR24 and its mutants were determined using a fluorescent catalase detection kit (Fluoro:Catalase; Cell Technology, Mountain View, CA). The kit uses a nonfluorescent substrate, 10-acetyl-3, 7-dihydroxyphenoxazine, which is converted to a fluorescent substance, resorufin, by H2O2 and determines the catalase activities in samples by measuring residual H2O2 after the incubation of samples and given H2O2.

Statistical analysis
Statistical analysis was performed by Student’s t test, and P < 0.05 was considered to be significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
DHCR24–/– MEFs are susceptible to H2O2-induced apoptosis
We first investigated the effect of oxidative stress on proliferation and apoptosis of DHCR24–/– MEFs. We previously reported that withdrawal of serum from culture medium rendered these cells susceptible to apoptosis (17) and that lack of cholesterol biosynthesis disrupted caveolae, cholesterol-enriched plasma membrane microdomains. Because caveolae contain functional growth factor receptors, their disruption impaired cell viability that resulted in apoptosis. In the present study, to avoid the influence of cholesterol depletion, DHCR24–/– MEFs were cultured in the presence of serum. Although, even under these conditions, cellular cholesterol levels were about half of those of wild-type MEFs and the desmosterol levels were comparable with the cholesterol levels (desmosterol was absent in wild-type MEFs), no remarkable influence of the altered sterol contents was observed on the proliferation of DHCR24–/– MEFs within 48 h (17).

To induce oxidative stress, we used a relatively high dose of H2O2 (1 mM) because a lower dose (0.2 mM) took more than 36 h to detect the cell death of DHCR24–/– MEFs. A number of DHCR24–/– MEFs were detached within 24 h after H2O2 exposure, whereas most of wild-type MEFs remained adherent (Fig. 1AGo). When analyzed quantitatively, significant loss of DHCR24–/– MEFs was detected at 9 h after H2O2. At 24 h, about half of the cells were lost. In contrast, wild-type MEFs were resistant to H2O2, although their proliferation appeared to stop, even in the presence of serum in the culture medium. At 24 h, fragmentation of genomic DNA was evident in DHCR24–/– MEFs (Fig. 1BGo), which suggested that the cell death was due to apoptosis. This was confirmed by TUNEL staining (Fig. 1CGo). Only a few TUNEL-positive cells were detected in wild-type MEFs, whereas most of the adherent DHCR24–/– MEFs were positively stained at 24 h. Similar results were obtained from other independent lines of wild-type and DHCR24–/– MEFs (data not shown). These results demonstrate that DHCR24–/– MEFs are highly vulnerable to H2O2-induced oxidative stress.


Figure 1
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  		FIG. 1. DHCR24–/– MEFs are susceptible to H2O2-induced apoptosis. Wild-type (WT) and DHCR24–/– MEFs were exposed to 1 mM H2O2. A, Cell images were obtained with a phase-contrast microscope at the indicated times after exposure. Bar, 100 µm. The number of adherent cells was scored after trypsinization and expressed as the percentage of the initial number. Mean ± SD (n = 5). *, P < 0.05 vs. the level in WT. B, At 24 h after H2O2 exposure, adherent and detached cells were collected, and fragmented genomic DNA was prepared and subjected to agarose gel electrophoresis. MW, Molecular weight marker. C, DNA fragmentation was also analyzed by the TUNEL method at 24 h after H2O2 exposure. Representative images are shown. Bar, 100 µm. The ratio of TUNEL-positive to adherent cells is shown in graphical form. Mean ± SD (n = 5). *, P < 0.05 vs. the level of WT. D, Whole-cell lysates were prepared at the indicated times after H2O2 exposure and subjected to Western blot analysis using antibodies against phospho-T845 ASK1, phospho-T180/Y182 p38 MAPK, phospho-T183/Y185 JNK, and actin. Representative images are shown. The phospho-kinase levels were normalized by the actin levels, and are expressed as a percentage of the maximal level in DHCR24–/– MEFs. Mean ± SD (n = 3). *, P < 0.05 vs. the WT level.

 
H2O2 induces sustained activation of ASK1, p38 MAPK, and JNK in DHCR24–/– MEFs
We studied the possible involvement of ASK1, p38 MAPK, and JNK in the apoptosis of DHCR24–/– MEFs. We determined the phosphorylation status of these kinases by Western blot analysis (Fig. 1DGo). Phosphorylation of T845, T180/Y182, and T183/Y185 are required for activation of ASK1, p38 MAPK, and JNK, respectively (20, 21, 22). Exposure of DHCR24–/– MEFs to H2O2 rapidly and markedly activated ASK1, p38 MAPK, and JNK within 30 min, and the activation persisted for 9 h. In contrast, activation of p38 MAPK and JNK was relatively weak in wild-type MEFs, and ASK1 activation was hardly detected. When analyzed quantitatively, the phosphorylation levels of these kinases in DHCR24–/– MEFs were significantly higher than those in wild-type MEFs at every time point after addition of H2O2.

It is known that transient and persistent activation of SAPKs results in different cell fate (23, 24). Early/transient activation of p38 MAPK/JNK is associated with cell survival, whereas late/sustained activation leads to apoptosis. It has also been shown that ASK1 is required for sustained activation of p38 MAPK/JNK and induction of apoptosis in response to H2O2 (10). Therefore, it was likely that the apoptosis of DHCR24–/– MEFs was triggered by the persistent activation of ASK1, p38 MAPK, and JNK. More importantly, our results suggest that DHCR24 acts upstream of the ASK1-SAPK cascade to prevent their persistent activation. In unstimulated cells, ASK1 is made inactive by binding with reduced thioredoxin (25). Upon oxidative stress, it is oxidized and dissociated from ASK1. The freed ASK1 is activated via its oligomer formation and autophosphorylation. The lack of ASK1 activation in wild-type MEFs suggests that DHCR24 has an antioxidative stress activity after H2O2 exposure.

Adenoviral delivery of DHCR24 protects DHCR24–/– MEFs from H2O2-induced apoptosis
Recombinant adenovirus that expresses full-length DHCR24 tagged with c-myc epitope at the C terminus (Ad-DHCR24-myc) was constructed for use in a complementation study. Immunocytochemical analysis showed the presence of DHCR24-myc in the cytoplasm of the infected DHCR24–/– MEFs, with greater than 90% infection efficiency (Fig. 2AGo). The abundant expression of DHCR24-myc was confirmed by Western blot analysis (Fig. 2BGo). Infected DHCR24–/– MEFs became resistant to H2O2 (Fig. 2CGo). For more than 36 h, the infected cells survived with only a small loss of cells (Fig. 2DGo). In contrast, DHCR24–/– MEFs infected with the control adenovirus expressing β-galactosidase (Ad-LacZ) were progressively lost after H2O2 exposure. The responses of p38 MAPK and JNK were measured at 3 h after H2O2 (Fig. 2EGo). Activation of both kinases was significantly attenuated in DHCR24–/– MEFs that expressed DHCR24-myc.


Figure 2
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  		FIG. 2. Adenoviral delivery of DHCR24 protects DHCR24–/– MEFs from H2O2-induced apoptosis. DHCR24–/– MEFs were infected with Ad-LacZ or Ad-DHCR24-myc. A, Immunocytochemical analysis was performed 48 h after infection, using mouse anti-myc antibody and antimouse IgG antibody conjugated with Alexa Fluor 488 (green fluorescence). PI staining was performed to identify the nucleus (red fluorescence). Images were obtained by confocal laser microscope. Bar, 100 µm. B, Expression of DHCR24-myc was demonstrated by Western blot analysis using anti-myc antibody. C, Infected cells were exposed to 1 mM H2O2, and cell images were obtained with a phase-contrast microscope at the indicated times after exposure. Bar, 100 µm. D, The number of adherent cells was counted and expressed as the percentage of the initial number. Mean ± SD (n = 5). *, P < 0.05 vs. levels of DHCR24–/– MEFs infected with Ad-LacZ. E, At 3 h after H2O2 exposure, phospho-p38 MAPK, phospho-JNK, and actin levels were determined by Western blot analysis. Phospho-kinase levels were normalized by the actin levels and are expressed as a percentage of the maximal level in DHCR24–/– MEFs infected with Ad-LacZ. The open and closed circles indicate Ad-LacZ- and Ad-DHCR24-myc-infected cells, respectively. Mean ± SD (n = 3). *, P < 0.05 vs. levels of DHCR24–/– MEFs infected with Ad-LacZ at 3 h.

 
Overexpression of DHCR24 protects PC12 cells from H2O2-induced apoptosis
Using Ad-DHCR24-myc, we also studied the antioxidative stress action of DHCR24 on pheochromocytoma PC12 cells. When apoptotic cells were determined by TUNEL staining at 24 h after H2O2 exposure, roughly 75% of PC12 cells infected with Ad-LacZ underwent apoptosis in response to H2O2, whereas cells infected with Ad-DHCR24-myc were resistant to apoptosis (Fig. 3AGo). Accordingly, H2O2 exposure induced the sustained activation of p38 MAPK and JNK in cells infected with Ad-LacZ (Fig. 3BGo). In contrast, kinase activation was markedly suppressed in cells infected with Ad-DHCR24-myc. These results were consistent with those obtained from DHCR24–/– MEFs.


Figure 3
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  		FIG. 3. DHCR24 overexpression suppresses H2O2-induced apoptosis of PC12 cells. PC12 cells infected with Ad-DHCR24-myc or Ad-LacZ for 48 h were exposed to 0.1 mM H2O2. A, At 24 h after H2O2 exposure, TUNEL staining was performed. PI staining was also done to detect the adherent cells. Representative images are shown. Bar, 100 µm. The ratio of TUNEL-positive to adherent cells is shown in graphical form. Mean ± SD (n = 5). *, P < 0.05 vs. levels of cells infected with Ad-LacZ. B, At 0, 0.5, 1, and 3 h after H2O2 exposure, the levels of DHCR24-myc, phospho-T180/Y182 p38 MAPK, phospho-T183/Y185 JNK, and actin were determined by Western blot analysis. Representative images are shown. Phospho-kinase levels were normalized by the actin levels and are expressed as the percentage of the maximal levels in the cells infected with Ad-LacZ. Mean ± SD (n = 3). *, P < 0.05 vs. levels in cells infected with Ad-LacZ.

 
DHCR24 scavenges ROS in MEFs treated with H2O2
To elucidate the anti-oxidative stress activity of DHCR24, we first measured intracellular ROS levels in DHCR24–/– MEFs after H2O2 exposure, using a cell-permeant indicator, H2DCFDA. Representative cell images at 30 min after H2O2 exposure are presented in Fig. 4AGo. H2O2 markedly increased ROS levels in most DHCR24–/– MEFs, whereas much weaker ROS generation was observed in wild-type MEFs. Expression of DHCR24-myc in DHCR24–/– MEFs markedly attenuated H2O2-dependent ROS production, the levels being comparable with those in wild-type MEFs. The time course of ROS production is presented in Fig. 4BGo. Sustained ROS production was observed in DHCR24–/– MEFs, whereas this was markedly suppressed by DHCR24-myc expression. These results demonstrate that DHCR24 possesses ROS-scavenging activity.


Figure 4
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  		FIG. 4. DHCR24 scavenges intracellular ROS generated on H2O2 exposure. Wild-type (WT), DHCR24–/– MEFs, and DHCR24–/– MEFs infected with Ad-LacZ or Ad-DHCR24-myc for 48 h were treated for 10 min with 10 µM H2DCFDA, a cell-permeant indicator for ROS, and were then exposed to 1 mM H2O2. Cell images were obtained with confocal laser microscope every 10 min after exposure. A, Representative images at 10 and 30 min after H2O2 exposure are presented. Intracellular ROS levels are indicated by pseudocolor. Bar, 100 µm. B, Time courses of ROS production are depicted. Changes in overall fluorescent intensity of individual cells were followed by sequential images, and the values were expressed the intensity at 10 min as 1. Mean ± SD (n = 40). *, P < 0.05 vs. the levels of DHCR24–/– MEFs.

 
DHCR24 protects DHCR24–/– MEFs from TM-induced apoptosis
Intracellular localization of DHCR24-myc was examined immunocytochemically (Fig. 5AGo). Most of fluorescent signals from DHCR24-myc merged with those from calreticulin, a calcium-binding protein in endoplasmic reticulum (ER), which is consistent with a previous report (15). ER localization of DHCR24 suggested its site-specific, ROS-scavenging activity. We thus studied a protective role of DHCR24 in ER stress-induced apoptosis because ROS generation has been reported in ER stress (26, 27). Newly synthesized secretory and membrane-associated proteins undergo disulfide-bond formation and isomerization in ER, which leads to correctly folded and assembled proteins. In various pathological conditions, this ER function is perturbed, and unfolded and misfolded proteins accumulate in ER, which results in ER stress. Excessive production of disulfide bonds causes overgeneration of ROS which accelerates this stress.


Figure 5
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  		FIG. 5. DHCR24 attenuates ER stress-induced cell death by eliminating intracellular ROS. A, DHCR24–/– MEFs were infected with Ad-DHCR24-myc. Immunocytochemical analysis was performed 48 h after infection, using mouse anti-myc antibody and antimouse IgG antibody conjugated with Alexa Fluor 488 (green fluorescence) and rabbit anticalreticulin antibody and antirabbit IgG antibody conjugated with Alexa Fluor 568 (red fluorescence). Bar, 50 µm. B, Wild-type (WT), DHCR24–/– MEFs, and DHCR24–/– MEFs infected with Ad-DHCR24-myc were treated with 0.2 µg/ml TM for the indicated times. The number of adherent cells was scored and expressed as a percentage of the initial number. Mean ± SD (n = 5). *, P < 0.05 vs. the levels at time 0; #, P < 0.05 vs. the levels of DHCR24–/– MEFs. C, Whole-cell lysates were prepared at the indicated times after TM and subjected to Western blot analysis using antibodies against Bip, phospho-p38 MAPK, actin, and myc. Representative images are shown. D, Cells were treated with or without TM for 24 h and then incubated for 10 min with H2DCFDA. Cell images were obtained with confocal laser microscope. Intracellular ROS levels are indicated by pseudocolor as shown in Fig. 3AGo. Bar, 100 µm.

 
We used TM, an inhibitor of N-linked glycosylation, to induce ER stress. Treatment of DHCR24–/– MEFs with low-dose TM (0.2 µg/ml) induced marked cell death at 48 h (Fig. 5BGo). In contrast, wild-type MEFs and Ad-DHCR24-myc-infected DHCR24–/– MEFs were resistant to TM. Western blot analysis revealed that, in the three cells, expression of Bip, a major ER chaperone, was increased with similar kinetics, which suggested that a similar intensity of ER stress occurred in these cells (Fig. 5CGo). However, sustained activation of p38 MAPK was observed only in DHCR24–/– MEFs. When ROS production was examined using H2DCFDA (Fig. 5DGo), marked ROS production was observed only in DHCR24–/– MEFs.

To cope with ER stress, mammalian cells trigger an adaptive response, termed unfolded protein response, which includes induction of Bip expression. When the stress is prolonged or adaptation fails, ER activates proapoptotic pathways that include p38 MAPK activation (26). Our results showed that TM induced marked ROS production in DHCR24–/– MEFs, which was associated with sustained p38 MAPK activation and cell death, whereas TM had less effect on cells infected with Ad-DHCR24-myc and on wild-type MEFs. It was therefore strongly suggested that DHCR24 exerts anti-ER stress activity, at least in part, by eliminating intracellular ROS.

DHCR24 scavenges H2O2 in vitro
We next examined whether DHCR24 could directly decompose H2O2. We synthesized His-tagged DHCR24 and its mutants in Escherichia coli and purified them by Ni column (Fig. 6AGo). WT (1–516) is wild-type DHCR24. The C-terminal deletion mutant, ORD (1–395), includes the entire putative oxidoreductive domain (ORD) (11). The N-terminal deletion mutant, C-terminal region (CTR; 232–516), lacks the ORD, but it contains the entire CTR, which has no similarity to known protein domains. In our pilot experiments of in vitro translation without GroE chaperones, a large proportion of synthesized DHCR24 and its mutants formed insoluble aggregates. This was obvious in wild-type DHCR24 and ORD (1–395) rather than CTR (232–516). Because ORD exhibits higher hydrophobicity and contains putative transmembrane domains, the presence of ORD might accelerate aggregate formation. GroE chaperones are involved in proper folding of various cytoplasmic proteins in E. coli (28). Application of GroE chaperones to in vitro translation much improved solubility of DHCR24 and its mutants. Substantial amounts of proteins were purified by Ni column (Fig. 6BGo). Interestingly, it was found that some GroE chaperone was copurified by Ni column (Fig. 6BGo, closed arrowhead), which indicated its tight interaction with DHCR24. Western blot analysis showed that all purified proteins were recognized by anti-His antibody (Fig. 6CGo), which confirmed the purification of WT (1–516), the size of which was identical with that of the copurified GroE chaperone. When applied to the H2O2-scavenging assay, WT (1–516) exhibited significant scavenging activity, and heat denaturation led to loss of the activity, which suggested enzymatic decomposition of H2O2 by DHCR24 (Fig. 6DGo). Whereas ORD (1–395) preserved the activity, CTR (232–516) lost it, which indicated the important role of ORD in H2O2 decomposition. The absence of scavenging activity of CTR (232–516) indicated that the GroE chaperone itself had no scavenging activity. Together, these results demonstrate that DHCR24 has direct H2O2-scavenging activity.


Figure 6
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  		FIG. 6. DHCR24 scavenges H2O2 in vitro. A, Human DHCR24 consists of 516 amino acids. Putative ORD is mapped within the N-terminal half-region (amino acids 1–231). The C-terminal region (CTR, amino acids 232–516) does not contain known functional domains. His-tagged, wild-type DHCR24 [WT (1–516)], its C-terminal deletion mutant [ORD (1–395)], its N-terminal deletion mutant [CTR (232–516)], and GFP were synthesized in vitro in the presence of GroE chaperones and purified by Ni column. B, Purified proteins were subjected to SDS-PAGE, and image of the gel stained with Coomassie Brilliant Blue is presented. Closed arrowhead indicates the band of GroE chaperone that was copurified with DHCR24 and its mutants. Its position is identical with that of wild-type DHCR24. C, Image of Western blot analysis using anti-His antibody is shown. This confirmed the purification of wild-type DHCR24. D, Purified proteins (10 pmol) were incubated with 40 µM H2O2 for 30 min at 25 C, and the amounts of residual H2O2 were determined. Heat-denatured (hd) WT was made by incubating WT (1–516) at 96 C for 5 min. The H2O2-scavenging activity is expressed as H2O2 moles decomposed per second per mole protein. Mean ± SD (n = 3). *, P < 0.05 vs. the levels of GFP; #, P < 0.05 vs. the levels of WT.

 
DHCR24 is an ER-resident, H2O2-scavenging enzyme
Catalase is known to be one of the enzymes with the highest turnover rate. In our assay conditions, the H2O2-scavenging activity of catalase was 7.3 ± 1.4 x 103 mol/sec·mol protein, whereas that of DHCR24 was 1.3 ± 0.5 x 103, which suggested that DHCR24 has a severalfold lower activity than catalase. To date, three H2O2-scavenging enzymes have been identified: catalase, GPx, and Prx. Interestingly, they have been shown to considerably vary in their intracellular localization, cellular content, and catalytic activity (1, 2, 3, 29, 30). Catalase is exclusively localized to a particular organelle, the peroxisome, whereas the major isoforms of GPx and Prx are mainly present in the cytosol. DHCR24 was shown to be localized to the ER. The different intracellular localization suggests that each enzyme plays a distinct role by eliminating H2O2 in an organelle-specific manner.

In this study, we have described a previously unknown physiological role for DHCR24. For the first time, DHCR24 is demonstrated to possess H2O2-scavenging activity, thereby protecting cells from H2O2- and ER stress-induced apoptosis. ER stress plays a key role in the pathogenesis of neurodegenerative disorders, diabetes, ischemia, tumor growth, and immune response (31). Further research on the role of DHCR24 in ER homeostasis will enhance our understanding of the molecular mechanisms of ER stress and provide novel therapeutic strategies.


   Acknowledgments
 
We thank Quark Biotech Inc. and Lexicon Genetics for the generous gift of DHCR24-knockout mice.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research and a Center of Excellence grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 13, 2008

Abbreviations: ASK, Apoptosis signal-regulating kinase; Bip, immunoglobulin heavy-chain binding protein; CTR, C-terminal region; DHCR24, 3β-hydroxysteroid-{Delta}24 reductase; ER, endoplasmic reticulum; GFP, green fluorescence protein; GPx, glutathione peroxidase; H2DCFDA, 2',7'-dichlorofluorescin diacetate; His, histidine; JNK, c-Jun N-terminal kinase; MEF, mouse embryonic fibroblast; ORD, oxidoreductive domain; PI, propidium iodide; Prx, peroxiredoxin; ROS, reactive oxygen species; SAPK, stress-activated protein kinase; TM, tunicamycin; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling.

Received January 8, 2008.

Accepted for publication March 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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