Báo cáo khoa học: Expression of heme oxygenase-1 is repressed by interferon-c and induced by hypoxia in human retinal pigment epithelial cells

doi:10.1111/j.1432-1033.2004.04241.x

Eur. J. Biochem. 271, 3076–3084 (2004) (cid:1) FEBS 2004

Expression of heme oxygenase-1 is repressed by interferon-c and induced by hypoxia in human retinal pigment epithelial cells

Reiko Udono-Fujimori1, Kazuhiro Takahashi1, Kazuhisa Takeda1, Kazumichi Furuyama1, Kiriko Kaneko1, Shigeru Takahashi2, Makoto Tamai3 and Shigeki Shibahara1 1Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai; 2Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji; 3Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan

Bach1, a transcriptional repressor for the HO-1 gene. On the other hand, HO-1 and Bach1 mRNAs were induced by hypoxia in D407 cells but remained unchanged in ARPE-19 cells, suggesting that Bach1 is not a sole regulator for HO-1 expression. The hypoxia-mediated induction of HO-1 mRNA in D407 cells depends on gene transcription and protein synthesis, as judged by the effects of their inhibitors. The half-life of HO-1 mRNA did not change during hyp- oxia. Thus, hypoxia may increase transcription of the HO-1 gene through a certain protein factor in RPE cells. These results indicate that RPE cells maintain retinal homeostasis by repressing or inducing the expression of HO-1, depending on the microenvironment.

Keywords: heme oxygenase-1; hypoxia; interferon-c; oxida- tive stress; retinal pigment epithelium.

The retinal pigment epithelium (RPE) is essential for main- tenance of photoreceptors and normally functions under conditions enriched with reactive oxygen species. RPE therefore expresses various defense enzymes against oxida- tive stress, including heme oxygenase-1 (HO-1). HO-1 catalyzes heme breakdown to release iron, carbon monox- ide, and biliverdin, which is reduced to bilirubin, a potent radical scavenger. HO-1 expression is induced by various environmental factors, which has been established as a def- the ense mechanism. To explore the hypothesis that expression level of HO-1 is reduced in those RPE cells under certain conditions, we analyzed the effects of interferon-c and hypoxia, each of which represses the expression of HO-1 mRNA in other types of human cells. Expression levels of HO-1 mRNA were reduced by interferon-c in two human RPE cell lines, D407 and ARPE-19, which was consistently associated with the induction of mRNA for

The retinal pigment epithelium (RPE) forms a single cell layer located between the retinal photoreceptors and the vascular-rich choroids, thereby constituting the blood– retinal barrier. Thus, RPE normally functions under relatively high oxygen tensions in postnatal life. At the apical side, RPE contacts with outer segments of photo- receptors through its large numbers of villi, and is involved in phagocytosis of shed outer segments [1] and in uptake, processing, and transport of retinoids [2]. RPE also participates in absorption of light with its melanin granules. RPE is therefore essential for visual function and survival of the photoreceptors. Conversely, dysfunction of RPE may lead to loss of photoreceptors or retinal degeneration [3], which accounts for a major cause of aging-dependent visual impairment and blindness in the developed world.

Heme oxygenase is a rate-limiting enzyme in heme catabolism and cleaves heme to form biliverdin IXa, carbon monoxide, and iron [4]. Biliverdin IXa is immediately reduced to bilirubin IXa (bilirubin) during the last step of heme breakdown reaction [5]. There are two functional isozymes of heme oxygenase, heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) [6,7]. Expression of HO-1 mRNA is increased in human cells by the substrate heme [8], heavy metals [9–11], UV irradiation [10], and nitric oxide donors [12–14]. Because bilirubin functions as a natural radical scavenger [15,16], induction of HO-1 represents a protective response against oxidative stress. The physiolo- gical importance of HO-1 has been confirmed by the severe phenotypic alterations of the HO-1 deficient mice [17] and a patient with HO-1 deficiency [18]. In contrast, HO-2 is constitutively expressed and the expression levels of HO-2 mRNA are maintained within narrow ranges in human cells [12,13,19].

RPE expresses various enzymes that are important in protection against oxidative stress, including HO-1 [20,21], thereby coping with large amounts of oxygen radicals generated by light exposure and during active phagocytosis of shed outer segments. The pioneering study in the bovine ocular tissues has demonstrated the high activities of heme oxygenase and cytochrome P450-dependent monooxygen- ases in the ciliary body and the RPE [20]. Subsequent studies have shown that HO-1 is induced in rat retina by intense

Correspondence to S. Shibahara, Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Fax: + 81 22 7178118, Tel.: + 81 22 7178117, E-mail: shibahar@mail.tains.tohoku.ac.jp Abbreviations: HO, heme oxygenase; HRE, hypoxia-responsive ele- ment; IFN, interferon; IL, interleukin; RPE, retinal pigment epithe- lium; TGF, transforming growth factor; TNF, tumor necrosis factor. (Received 31 March 2004, revised 21 May 2004, accepted 2 June 2004)

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equilibrated with 5% CO2, 94% N2, and 1% O2 [40]. The cells were cultivated under normoxia or hypoxia for indicated hours, and harvested for extraction of RNA and protein. D407 cells were also incubated under normoxia or hypoxia with or without actinomycin D (1 lgÆmL)1) or cycloheximide (1 lgÆmL)1) for 12 or 24 h. The D407 cells were also incubated with the recombinant human TGF-b1 for the 12 h.

Northern blot analysis

visible light [22] and in cultured human RPE cells by transforming growth factor-b1 (TGF-b1) [23] and oxidative stresses [21,24,25]. These results support the notion that expression of HO-1 is important in the survival and maintenance of RPE in the adult retina. On the other hand, we have shown that HO-1 expression is reduced in human cell lines by treatment with interferon-c [26,27] or by hypoxia in primary cultures of human umbilical vein endothelial cells, coronary artery endothelial cells, and astrocytes [28], as well as in various human cell lines [27]. These results raise the possibility that some degree of reduction in the HO-1 expression is an important defense mechanism in human cells under certain conditions [29].

To explore the above hypothesis, we analyzed the effects of interferon-c and hypoxia on HO-1 expression in human RPE cells, both of which have been shown to repress HO-1 expression in other types of human cells. In addition, interferon-c induces the expression of major histocompat- ibility complex class II antigen on the cell surface of human RPE [30,31], and is involved in the pathophysiology of inflammatory ocular diseases, such as proliferative vitreo- retinopathy [32,33], cytomegalovirus retinitis [34] and toxo- plasma chorioretinitis [35]. Hypoxia has been suggested as a risk factor for diabetic retinopathy [36], which accounts for a common cause of blindness. However, there is no report concerning the effects of interferon-c or hypoxia on HO-1 expression in human RPE cells. Here we show the repression of HO-1 expression by interferon-c and the induction of HO-1 expression by hypoxia in human RPE cells.

Experimental procedures

Materials

Recombinant human interferon-c (IFN-c-1a, Imunomax-c) was a gift from Shionogi Co. (Osaka, Japan). Human tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) were obtained from PeproTech EC Ltd (London, UK).

Cell culture

Total RNA was extracted from cultured RPE cells and subjected to Northern blot analysis, as detailed previously [39,40]. The northern probes used for heme oxygenase mRNAs were the XhoI/XbaI fragment (nucleotide positions )64 to 923) derived from the human heme oxygenase-1 cDNA, pHHO1 [8], and the HinfI/HinfI fragment of human heme oxygenase-2 cDNA, pHHO2-1 [19]. The northern probe for human Bach1 mRNA was the Pst1 fragment of human Bach1 cDNA [41]. The human Nrf2 cDNA segment was prepared from total RNA of D407 cells by RT-PCR using a forward primer (5¢-AGTCAGAAACCAGT GGATCT-3¢) (GenBank accession number S74017; nucleo- tide positions 269–289) and a reverse primer (5¢-AGAT TCCACTGAGTGTTCTG-3¢) (nucleotide positions 1061– 1080). The human Nrf2 cDNA fragment of 810 basepairs was cloned into pBluescript KS, yielding a subclone, pBS- hNrf2. The EcoRI/NcoI fragment (vector/vector) of pBS- hNrf2 was used as a northern probe for Nrf2 mRNA. The expression of b-actin mRNA was examined as an internal control. The probe for b-actin mRNA was the SmaI/ScaI fragment (nucleotides 124–1050) of a human b-actin cDNA provided by T. Yamamoto (Tohoku University, Sendai, labeled with Japan). These DNA fragments were [a-32P]dCTP (Amersham Biosciences) by the random priming method and were used as hybridization probes. Total RNA (15 lg per sample) was electrophoresed on 1% agarose gels containing 2 M formaldehyde, transferred to nylon membranes filter (Zeta-probe membrane; Bio-Rad), and fixed with a UV-linker (Stratalinker 1800; Stratagene). The RNA blot was hybridized with each 32P-labeled probe, as described previously [39,40]. Radioactive signals were detected by exposing the filters to X-ray films (X-AR5; Kodak) or with a Bioimage Analyzer (BAS1500; Fuji Film Co. Ltd). The exposure time to X-ray films varied depend- ing on the experiments. The intensity of hybridization signals was determined by photo-stimulated luminescence with a Bioimage Analyzer.

The human RPE cell lines, ARPE-19 [37] and D407 [38], were provided by L. M. Hjelmeland (Department of Biological Chemistry, University of California, Davis, CA, USA) and R. C. Hunt (Department of Microbiology, University of South Carolina Medical School, Columbia, SC, USA), respectively. ARPE-19 cells were cultured in a 1 : 1 mixture of DMEM and nutrient mixture F12 contain- ing 10% fetal bovine serum, 2 mM L-glutamine, and antibi- otics (100 UÆmL)1 penicillin and 0.1 mgÆmL)1 streptomycin) [37]. D407 cells were cultured in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine and antibiotics (at the same amounts as above) [38]. Cells were cultured at 37 (cid:2)C under 5% CO2 and 95% air, unless otherwise indicated.

cytokines:

interferon-c

To examine the effects of cytokines on the expression of HO-1 mRNA, D407 and ARPE-19 cells were cultivated in fresh medium for 24 h and then exposed to the following (100 UÆmL)1), TNF-a three (20 ngÆmL)1) and IL-1b (10 ngÆmL)1), as described previ- ously [39]. The cells were incubated at 37 (cid:2)C for 24 h and were harvested for RNA extraction. For hypoxia experi- ments, human RPE cells were cultured in a chamber

Northern blot analysis for HO-1, TGF-b1 and b-actin was also performed with DIG Northern Starter Kit (Roche Diagnostics, Mannheim, Germany) according to the manu- facturer’s protocol. For preparing HO-1 RNA probe, the cDNA fragment (corresponding to nucleotides 81–878 of human HO-1 cDNA) (GenBank Accession Number X06985) was amplified by PCR using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), then cloned into pCR-bluntII-TOPO (Invitrogen, Carlsbad, CA, USA), and named pCR-hHO1. RT-PCR was performed for preparation of the human TGF-b1 cDNA. First strand cDNA was synthesized by ThermoscriptTM reverse tran- scriptase (Invitrogen) using mRNA from a human eryth- roleukemia cell line [42]. Then, a part of human TGF-b1

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Renilla luciferase (Promega). The fusion genes contained the firefly luciferase gene as a reporter under the control of the 5¢-flanking region of the human HO-1 gene. A promotorless construct (pGL3 Basic) was used as a control. Expression of reporter genes and pRL-TK was determined with the Dual-LuciferaseTM Reporter Assay System (Promega).

cDNA (corresponding to nucleotides 395–2159 of GenBank accession number NM_000660) was amplified using FastStart DNA polymerase (Roche Diagnostics) with GC-RICH solution, amplified fragment was cloned into pGEM-Teasy vector (Promega, Madison, WI, USA), and named pGEM-hTGFb1. SP6 RNA polymerase was used for transcription of RNA probe from pCR-hHO1.

Results

Western bolt analysis

Repression of HO-1 expression by interferon-c in the RPE cell lines

It has been reported that HO-1 expression was induced by oxidative stress in two human RPE cell lines, D407 and ARPE-19 [21,25], indicating that these RPE cells possess the defense system involving HO-1. These RPE cell lines therefore provide a good system to explore the hypothesis that expression of HO-1 is reduced as a defense mechanism under certain conditions. We have focused on interferon-c that has been shown to reduce the expression of HO-1 in human glioblastoma cells [26]. For comparison, we also analyzed the effects of the pro-inflammatory cytokines, TNF-a and IL-1b, both of which are also involved in the pathogenesis of proliferative vitreoretinopathy [32,33].

D407 human RPE cells were lysed in triple detergent lysis buffer containing 50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 lgÆL)1 phenyl- methylsulfonyl fluoride, 1 lgÆmL)1 aprotinin, 1 lgÆmL)1 Nonidet P40, and 0.5% sodium dexycholate. The cell lysates were centrifuged at 150 000 g for 10 min, and the superna- tant (10 lg of protein) was analyzed on a SDS-polyacryl- amide gel (10%). The proteins in the gel were treated with 20% methanol buffer containing 48 mM Tris, 39 mM glycine, and 0.037% SDS and electrophoretically trans- ferred to a poly(vinylidene difluoride) membrane (Immobi- lon-P, Millipore Corporation), which was pretreated with the same buffer. Expression of HO-1 was determined with anti-HO-1 Ig [19]. The specific immunocomplexes were detected with a Western blot kit (ECL Plus, Amersham Biosciences). Expression of a-tubulin was determined as an internal control with a-tubulin antibody (Neo Markers, Fremont, CA, USA).

HO-1 mRNA stability assays

D407 cells were incubated for 12 h in fresh medium under hypoxia or normoxia, followed by addition of actinomy- cin D (1 lgÆmL)1). The cells were further incubated for 1, 2.5, and 5 h after the addition of actinomycin D under hypoxia or normoxia and harvested at each time point for RNA extraction.

The dose–response and time-course studies showed that the maximum reduction was obtained with interferon-c at the dose of 100 UÆmL)1 and at 24 h of incubation (data not shown). The expression levels of HO-1 mRNA were decreased by the treatment with interferon-c, which was consistently associated with the induction of Bach1 mRNA (Fig. 1). Bach1 has been shown to function as a repressor of the HO-1 gene in mice [47] and in cultured human lung cancer cells [27]. The expression levels of HO-1 and Bach1 mRNAs are inversely regulated by interferon-c in both RPE cell lines. In contrast, TNF-a, IL-Ib, or their combination did not noticeably change the expression levels of HO-1 and Bach1 mRNAs in D407 cells, but each combination with interferon-c reduced the HO-1 expression and induced the Bach1 expression (Fig. 1A). In ARPE-19 cells, however, TNF-a or IL-Ib induced the expression of HO-1 mRNA but not Bach1 mRNA, and any combination with inter- feron-c decreased the HO-1 mRNA levels and increased Bach1 mRNA levels (Fig. 1B). Notably, the combination of TNF-a and IL-1b caused the concomitant induction of HO-1 and Bach1 mRNAs in ARPE-19 cells. The differen- tial effects of TNF-a and IL-1b in the two RPE cell lines may reflect the heterogeneity of RPE cells [31,48].

Effects of hypoxia on the expression of HO-1 in human RPE cell lines

Transient transfection assays D407 cells in six-well plates (1 · 106 cells per well) were transfected by the FuGENETM 6 protocol (Boehringer Mannheim). Reporter plasmids, pSV40 promoter-Epo HRE-Luc containing four copies of hypoxia-responsive element (HRE) and pSV40 promoter-Luc lacking HRE [43], were used as a positive and a negative control for hypoxic induction. Four constructs, phHOLUC40, phHO- LUC ()1976), phHOLUC ()981) and phHOLUC ()58) were reported previously [44]. The numbers indicate the nucleotide positions of the 5¢-ends of the growth region. The 4.5-kb PstI/Xho1 fragment of the human HO-1 gene [45,46] was inserted into the SmaI and XhoI site of pGL3-Basic vector (Promega), yielding phHOLUC45. pHHOLUC ()31), containing the fragment ()31 to +24), was prepared by inserting synthetic double-stranded oligonucleotides into the SmaI/XhoI site of pGL3-Basic vector and lacks the putative HRE site, CACGTG sequence ()44 to )39).

In contrast to interferon-c, hypoxia induced the expression levels of HO-1 mRNA in D407 cells and exerted no or only marginal effects in ARPE-19 cells (Fig. 2). In D407 RPE cells, the levels of HO-1 mRNA were increased by 6 h after exposure to hypoxia and reached the maximum by 12 h in which b-actin mRNA levels remained of hypoxia, unchanged (Fig. 2A). Likewise, hypoxia increased expres- sion of Bach1 mRNA, but decreased the mRNA levels of Nrf2, a transcriptional inducer of the HO-1 gene [49]. Thus, the expression levels of HO-1 and Bach1 mRNAs were concomitantly increased in D407 cells under hypoxia. In

D407 cells were transfected with each plasmid DNA, followed by 24-h incubation and harvested. The amounts of DNA used for transfection were 1 lg of a test fusion gene and 20 ng of an internal control, pRL-TK, containing the herpes simplex virus thymidine kinase promoter region upstream of

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ARPE-19 cells, hypoxia caused no noticeable changes in the expression levels of Bach1 mRNA and rather reduced Nrf2 mRNA (Fig. 2B).

Induction of HO-1 protein in D407 RPE cells by hypoxia

Fig. 1. Effects of cytokines on HO-1 mRNA expression in human RPE cells. Northern blot analyses of HO-1 mRNA and Bach1 mRNA in human RPE cells treated with the following cytokines: interferon-c (IFNc), TNF-a and IL-1b. D407 RPE cells (A) and ARPE-19 cells (B) were treated with one of these three cytokines, or a combination of two or three cytokines for 24 h. Each lane contains 15 lg total RNA. The lower gel image in each panel shows b-actin mRNA as an internal control. The data shown are from one of two independent experiments.

HO-1 protein levels were increased by hypoxia in D407 RPE cells by 12 h and 24 h, as judged by Western blot analysis (Fig. 3). In contrast, hypoxia had no noticeable effects on the expression levels of a-tubulin, an internal control.

Nature of the induction of HO-1 mRNA by hypoxia in D407 RPE cells

Fig. 2. Differential effects of hypoxia on expression of HO-1, Bach1, and Nrf2 mRNAs in human RPE cells. D407 (A) and ARPE19 (B) human RPE cells were cultivated under normoxia (20% O2; N) or hypoxia (1% O2; H) for the indicated time (h) and then harvested for RNA preparation. Shown are representative examples of Northern blot analyses. The lane labeled with (cid:1)0(cid:2) contained RNA prepared from the untreated cells. The lower gel image in each panel shows b-actin mRNA as an internal control.

Importantly, the hypoxia-mediated induction of HO-1 mRNA was prevented by the treatment with cycloheximide. Thus, RNA synthesis and new protein synthesis are

The time course study of HO-1 mRNA induction in D407 cells showed the relatively late peak of the induction at 12 h (Fig. 2A), compared to the effects of cadmium [46] or 12-O-tetradecanoylphorbol-13-acetate [50] with the maxi- mum induction by 3 h, each of which activates the transcription of the human HO-1 gene. Thus, the hypoxia- mediated induction of HO-1 expression may be a conse- quence of the production of a certain endogenous factor in D407 cells. We therefore studied the effects of actinomy- cin D, an inhibitor of transcription, and cycloheximide, an inhibitor of translation, on the hypoxia-mediated induction of HO-1 mRNA in D407 cells (Fig. 4). The expression levels of HO-1 and Bach1 mRNAs were reduced to the undetectable levels by the treatment with actinomycin D.

Fig. 3. Induction of HO-1 protein in D407 RPE cells by hypoxia. Western blot analysis of the HO-1 and a-tubulin protein. Each lane contained whole cell extracts prepared from D407 human RPE cells exposed to normoxia or hypoxia for the indicated number of hours. See Fig. 2 legend for key.

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required for the induction of HO-1 mRNA. Notably, the expression of Bach1 mRNA was increased by the treatment with cycloheximide under normoxia, and was further increased under hypoxia. These results suggest that the degradation of Bach1 mRNA may be enhanced by a protein factor with a short half-life, which is down-regulated by hypoxia.

Stability of HO-1 mRNA under hypoxia

Fig. 4. Inhibitory effects of cycloheximide or actinomycin D on expression of HO-1 mRNA in D407 RPE cells. D407 human RPE cells were incubated without or with actinomycin D (1 lgÆmL)1) or cycloheximide (1 lgÆmL)1) for the indicated time (h) under normoxia (N) or hypoxia (H). Middle and lower gel images show Bach1 mRNA expression for comparison and b-actin mRNA as an internal control.

We then analyzed the stability of HO-1 mRNA in D407 cells under hypoxia (Fig. 5), as it has been reported that hypoxia increased the stability of HO-1 mRNA in human dermal fibroblasts [51]. There was no significant difference in the half-life of HO-1 mRNA between under normoxia (mean ± SEM, 1.42 ± 0.12 h, n ¼ 3) and under hypoxia (1.63 ± 0.02 h). The short half-lives of HO-1 mRNA are consistent with the remarkable inhibitory effects of actino- mycin D (Fig. 4). These results suggest that the hypoxia- mediated induction of HO-1 mRNA expression is not due to the increased stability of HO-1 mRNA. It is also noteworthy that the maximum levels of HO-1 mRNA were significantly reduced after 5 h under hypoxia even in the absence of actinomycin D, indicating that the hypoxia- mediated induction of HO-1 expression represents an acute response in RPE cells.

Functional analysis of the HO-1 gene promoter in D407 RPE cells

the data suggest

All that hypoxia may increase the transcription of the HO-1 gene as a consequence of the production of a certain endogenous factor in D407 cells. On the other hand, we have been interested in the presence of a putative HRE sequence CACGTGA (positions )44 to )39) that overlaps the functional E-box in the human HO-1 gene promoter [11,45]. HRE is the binding site for hypoxia- inducible factor-1. We therefore performed transient expres- sion assays to analyze the effects of hypoxia on the promoter activity of the HO-1 gene in D407 cells (Fig. 6).

The basal promoter activity of phHOLUC45 was higher than that of phHOLUC40, which may be due to the presence of a Maf recognition element. Hypoxia did not influence the expression of any HO-1 constructs containing a putative HRE sequence CACGTGA, whereas the puta- tive HRE (the E-box motif) appears to be required for the basal promoter activity of the HO-1 gene. In contrast, hypoxia consistently increased the promoter activity of a construct, HRESV40, which contains four copies of HRE, but showed only marginal effects on the promoter activity of N-HRESV40, a negative control. Thus, the cis-acting elements located in the 5¢-upstream region of 4.5 kb are unable to confer the hypoxia-mediated induction or repres- sion on a reporter gene in D407 cells. Moreover, treatment with interferon-c exerted no noticeable effects on the

Fig. 5. Effect of hypoxia on the stability of HO-1 mRNA. (A) Northern blot analysis [conditions; normoxia + actinomycin D (N+AM-D), hypoxia (H), and hypoxia + actinomycin D (H+AM-D)]. D407 human RPE cells were incubated for 12 h under hypoxia or normoxia, and then further incubated for 1, 2.5, and 5 h after addition of actinomycin D (1 lgÆmL)1). Other conditions are the same as in Fig. 1. The data shown are from one of three independent experiments with similar results. (B) Relative expression levels of HO-1 mRNA. The intensities representing HO-1 mRNA at the time of addition of actinomycin D under each condition were considered to be 100%. The data shown are mean ± SEM (n ¼ 3).

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detectable levels, unlike the severe effect of actinomycin D (Fig. 4), as HO-1 is important in cell survival.

promoter activity of phHOLUC45 (data not shown), despite the presence of a Maf recognition element that is bound by Bach1-small Maf complexes [52].

Fig. 6. Effect of hypoxia on the human HO-1 gene promoter function. D407 human RPE cells were transfected with each reporter construct and incubated under normoxia or hypoxia. A putative HRE in the HO-1 gene promoter is marked with (cid:1)?(cid:2). The two constructs, shown near the bottom, represent positive and negative controls for hypoxia. Relative luciferase activity under normoxia or hypoxia is shown as the ratio to the normalized luciferase activity obtained with pGL3Basic under normoxia or hypoxia, respectively. The data are means ± SEM of three independent experi- ments.

Discussion

The RPE is a target cell infected by cytomegalovirus [54] and by the intracellular parasite Toxoplasma gondii [34]. Toxoplasmosis is a common protozoal infection in the developed world, and chorioretinitis is a major complication of ocular toxoplasmosis in infants, patients with acquired immune deficiency syndrome, and organ transplant recip- ients [35]. Incidentally, interferon-c at the concentration of 100 UÆmL)1 used in this study was shown to completely inhibit Toxoplasma gondii replication in cultured human RPE cells [35]. In this case, interferon-c has been shown to inhibit proliferation of parasites by inducing the expression of indoleamine 2,3-dioxygenase, a heme-containing enzyme that converts tryptophan to kynurenine, thereby depleting cellular tryptophan [35]. Likewise, the protective role of the interferon-c-induced indoleamine 2,3-dioxygenase has been shown in cytomegalovirus retinitis [34]. These results raise the intriguing possibility that the reduced HO-1 expression may result in the preservation of intracellular heme that is an essential component of indoleamine 2,3-dioxygenase.

A number of studies have shown the beneficial role of HO-1 induction, as described above, but the reduced expression of HO-1 has been largely ignored. We have hypothesized that a certain degree of reduction in the HO-1 expression levels may be beneficial under pathological conditions, such as infectious diseases or cancers, because the reduced HO-1 expression may result in the restriction of iron supply to pathogens or tumor cells or the preservation of intracellular heme that is an essential component of some defense enzymes [29]. In the former context, it has been reported that human pancreatic tumor cells over-expressing HO-1 show the aggressive properties when inoculated in immu- nodeficient mice, including the enhanced growth, angiogen- esis and lung metastasis [53], and treatment of these mice with a HO-1 inhibitor reduced the occurrence of lung metastasis [53]. These results provide the in vivo evidence suggesting the beneficial role of the reduced HO-1 activity. To explore the physiological significance of the reduced HO-1 expression, we analyzed the effect of cytokines on the expression of HO-1 in the RPE, which normally functions under the extreme conditions enriched with reactive oxygen species. We show that HO-1 expression is consistently reduced by interferon-c, even in RPE cells, which may reflect an important mechanism for the maintenance of the retinal homeostasis. It should be noted that the reduced expression levels of HO-1 mRNA are maintained at the

Hypoxia is a potent stimulus for neovascularization that is normally prevented in the adult retina. Therefore, hypoxia may reflect severe pathologic conditions for the RPE, such as retinal vascular occlusive diseases and retinal detachment, which may be followed by angiogenesis as seen in diabetic retinopathy and age-related macular degeneration. Here we show that hypoxia transiently induces the expression of HO-1 mRNA through an unknown protein factor in D407 cells but exerts no noticeable effects in ARPE-19 cells. Such differences in hypoxic responses may suggest the presence of the regulatory mechanism that maintains the HO-1

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expression levels within narrow ranges under hypoxia in RPE cells. Alternatively, the difference may simply reflect the regional variations in the differentiation state or proliferative capacity of RPE cells [31,48]. In fact, we have reported that hypoxia increases production of endothelin-1 in D407 cells but not in ARPE-19 cells, whereas hypoxia induces expres- sion of adrenomedullin in both RPE cell lines [40]. Moreover, two transcription factors, microphthalmia-associated tran- scription factor and OTX2, which are important in differ- entiation of RPE, are more abundantly expressed in ARPE- 19 cells than in D407 cells [55]. Thus, ARPE-19 cells may retain more differentiated properties, which is consistent with the previous report by other investigators [25].

It is noteworthy that HO-1 expression is not reduced by hypoxia in the two RPE cell lines, unlike other types of human cells [27,28]. To confirm these findings, we wish to perform experiments using primary cultures of human RPE, but are unable to obtain sufficient numbers of original RPE cells from donors. Instead, using a hypoxic chamber (10% oxygen) [60], we are working on a rodent model of high altitude retinopathy, although the interspecies difference has been well known in the regulation of HO-1 expression [29]. In summary, the present study has shown that interferon- c consistently reduces the expression of HO-1 mRNA in two types of human RPE cell lines, in which HO-1 mRNA is induced or remains unchanged under hypoxia. Thus, human RPE cells up-regulate or down-regulate the HO-1 expression through different pathways in a dynamic manner to cope with the changes in the retinal microenvironment.

Acknowledgements

Hypoxia up-regulates HO-1 expression in D407 RPE cells without affecting the half-life of HO-1 mRNA (Fig. 5), whereas other investigators have reported that hypoxia induces the expression of HO-1 in human dermal fibroblasts by increasing the half-life of HO-1 mRNA [51]. In human dermal fibroblasts, the induction was inhibited by cyclo- heximide and peaked by 10 h [51], which is similar to the properties of the HO-1 induction observed in D407 cells. These results suggest that hypoxia may induce HO-1 expression through newly synthesized protein factors but the induction mechanisms are different between RPE cells and skin fibroblasts. In addition to the well-known inter- species variations [29], the present study has shown the intercell differences in the hypoxic induction of human HO-1 gene expression.

We thank Dr R.C. Hunt for D407 RPE cells and Dr L.M. Hjelmeland for ARPE-19 RPE cells. We also thank Y. Fujii-Kuriyama and E. Ito for the HRE constructs and human Bach1 cDNA, respectively. This work was supported in part by Grants-in-Aid for Scientific Research (B), Scientific Research (C), for Exploratory Research, and for Priority Area from the Ministry of Education, Science, Sports, and Culture of Japan and by the 21st Century COE Program Special Research Grant (cid:1)the Center for Innovative Therapeutic Development for Common Diseases(cid:2) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported in part by the grants provided by Uehara Memorial Foundation.

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A question that remains to be answered is the identity of a protein factor that is responsible for the hypoxia-mediated induction of HO-1 expression in D407 RPE cells. Probably, many factors are induced by hypoxia at their protein levels in RPE cells. For example, we have shown the increased production of endothelin-1 and adrenomedullin in D407 RPE cells [40]. Furthermore, it has been reported that HO-1 expression is induced by TGF-b1 in human RPE cells [23], human renal epithelial cells [56], and A549 human lung cancer cells [57]. Thus, TGF-b1 is a candidate that may mediate the hypoxic induction of HO-1 in RPE cells. In fact, hypoxia increased the expression of TGF-b1 mRNA in D407 cells (data not shown), as reported in other human RPE cells [58]. However, hypoxia has been shown to reduce the production of TGF-b1 in human RPE cells [58]. Further studies, such as DNA microarray analysis, may help us to find such a factor.

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Bach1 is a heme-regulated transcriptional repressor for the HO-1 gene and plays an important role in the feedback the regulation of HO-1 expression [47,59]. However, expression of HO-1 and Bach1 mRNAs was concomitantly induced by the treatment with the combination of TNF-a and IL-1b in ARPE-19 cells, indicating that Bach1 is not a sole determinant for HO-1 mRNA expression in ARPE-19 cells. Likewise, hypoxia induced expression of HO-1 and Bach1 mRNAs in D407 cells, indicating that Bach1 may not be a key determinant for the hypoxia-mediated induction of HO-1 expression in D407 cells. In addition, hypoxia coordinately and rapidly induced expression of both HO-1 and Bach1 mRNAs in cultured rat and monkey cells, indicating that increased expression of Bach1 does not necessarily result in the inhibition of HO-1 transcription [27]. These results indicate that additional regulators, such as corepressors, may influence Bach1 activity.

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