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

Expression of heme oxygenase-1 is repressed by interferon-c and
induced by hypoxia in human retinal pigment epithelial cells
Reiko Udono-Fujimori
1
, Kazuhiro Takahashi
1
, Kazuhisa Takeda
1
, Kazumichi Furuyama
1
, Kiriko Kaneko
1
,
Shigeru Takahashi
2
, Makoto Tamai
3
and Shigeki Shibahara
1
1
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai;
2
Laboratory of
Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji;
3
Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan
The retinal pigment e pithelium ( RPE) is essential for main-
tenance of photoreceptors and normally functions under
conditions enriched with reactive oxygen species. RPE
therefore expresses various defense e nzymes 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 s cavenger. HO-1 expression is i nduced by various
environmental factors, which has b een established as a def-
ense mechanism. To explore the hypothesis that the
expression level of HO-1 is reduced in those RPE cells under
certain conditions, we a nalyzed the effects of i nterferon-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
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 D 407 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 tran scription 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 stre ss; retinal pigment epithelium.
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 i ts 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 f or visual function and s urvival of
the photoreceptors. Conversely, dysfunction of RPE may
lead to loss of photoreceptors or retinal degeneration [3],
which accounts for a m ajor 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 I Xa, 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], inductio n 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, H O-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 b y light exposure and during a ctive 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 M olecular 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:
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 3 1 March 2004, revised 21 May 2004,
accepted 2 J une 2004)
Eur. J. Biochem. 271, 3076–3084 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04241.x
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 s urvival 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 treatmen t 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 h ypothesis, 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], c ytomegalovirus retinitis [34] and toxo-
plasma chorioretinitis [35]. Hypoxia has been suggested as a
risk factor for diabetic retinopathy [36], which a ccounts for a
common cause of blindness. However, t here 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
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 m
ML
-glutamine, a nd antibi-
otics (100 UÆmL
)1
penicillin and 0 .1 mgÆmL
)1
streptomycin)
[37]. D407 cells were cultured in DMEM containing 10%
fetal bovin e s erum, 2 m
ML
-glutamine and antibiotics (at the
same amounts as above) [38]. Cells were cultured at 37 °C
under 5% CO
2
and 95% air, unless otherwise indicated.
To examine the effects of cytokines on the expression of
HO-1 mRNA, D407 a nd ARPE-19 cells were cultivated in
fresh medium for 24 h and then exposed to the following
three cytokines: interferon-c (100 UÆmL
)1
), TNF-a
(20 ngÆmL
)1
)andIL-1b (10 ngÆmL
)1
), as described previ-

ously [39]. The cells were incubated at 37 °C for 24 h and
were harvested for RNA extraction. For hypoxia experi-
ments, human RPE cells were cultured in a chamber
equilibrated with 5% CO
2
,94%N
2
,and1%O
2
[40]. The
cells were cultivated under normoxia or hypoxia for
indicated hours, and harvested for extraction o f RNA and
protein. D407 cells were also incubated under normoxia o r
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
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 c loned into pBluescript KS, yielding a subclone, p BS-
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 p robe 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,
Japan). These DNA fragments were labeled with
[a-
32
P]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, tran sferred to
nylon membranes filter (Zeta-probe membrane; Bio-Rad),
and fixed with a UV-linker (Stratalinker 1800; Stratagene).
The RNA blot was hybridized w ith each
32
P-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 A nalyzer (BAS1500; Fu ji 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.
Northern blot analysis for HO-1, TGF-b1andb-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 Thermoscript
TM
reverse tran-
scriptase (Invitrogen) using mRNA from a human eryth-
roleukemia cell line [42]. Then, a part of human TGF-b1
Ó FEBS 2004 Dynamic regulation of HO-1 expression in RPE (Eur. J. Biochem. 271) 3077
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.
Western bolt analysis

D407 human RPE cells were lysed in triple detergent lysis
buffer containing 50 m
M
Tris/HCl (pH 8.0), 150 m
M
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 m
M
Tris, 39 m
M
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 specifi c 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 i ncubated for 1,
2.5, and 5 h after the addition of actinomycin D under
hypoxia or normoxia and harvested at each time p oint for
RNA extraction.
Transient transfection assays
D407 cells in six-well plates (1 · 10
6
cells per well) were
transfected by the FuGENE
TM
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 positio ns of the 5¢-ends of the growth region. The
4.5-kb PstI/Xho1 f ragment of the human HO-1 gene [45,46]
was inserted into the SmaIandXhoIsiteofpGL3-Basic
vector (Promega), yielding phHOLUC45. pHHOLUC
()31), containing the f ragment ()31 to +24), was prepared
by inserting synthetic double-stranded oligonucleotides into
the SmaI/XhoIsiteofpGL3-Basicvectorandlacksthe

putative HRE site, CACGTG sequence ()44 to )39).
D407 cells were transfected with each plasmid DNA,
followed by 24-h incubation and harvested. The amounts of
DNA usedfor t ransfection were 1 lg of a test fusiongene and
20 ng of an internal control, pRL-TK, containing the herpes
simplex virus thymidine kinase promoter region upstream of
Renilla luciferase (Promega). The fusion genes contained the
firefly luciferase gene a s 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-Luciferase
TM
Reporter Assay System (Promega).
Results
Repression of HO-1 expression by interferon-c
in the RPE cell lines
It has been reported t hat 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 posse ss
the de fense system involving HO-1. These RPE cell lines
therefore provide a good system to explore the hypothesis
that expression of HO-1 is re duced 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 e ffects of the pro-inflammatory cytokines,
TNF-a and I L-1b, both of which are also involved in the
pathogenesis of proliferative vitreoretinopathy [32,33].
The dose–response and time-course studies showed that

the maximum reduction was obtained with interferon-c at
thedoseof100UÆmL
)1
and at 24 h of incubation (data not
shown). T he expression levels of HO-1 mRNA were
decreased by the treatment with interferon-c,whichwas
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. I n contrast, T NF-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 d ifferen-
tial effects of TNF-a and IL-1b in the two RPE cell lines
may reflect the hetero geneity of R PE cells [31,48].
Effects of hypoxia on the expression of HO-1 in human
RPE cell lines
In contrast to interferon-c, hypoxia indu ced 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 increase d by 6 h after

exposure to hypoxia and reached the maximum by 12 h
of hypoxia, in which b-actin mRNA levels remained
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
3078 R. Udono-Fujimori et al. (Eur. J. Biochem. 271) Ó FEBS 2004
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
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
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 c admium [46] or
12-O-tetradecanoylphorbol-13-acetate [50] with the maxi-
mum induction by 3 h, e ach of which activates the
transcription of the human HO-1 gene. Thus, the hypoxia-
mediated indu ction 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.
Importantly, the hypoxia-mediated induction of HO-1
mRNA was prevented by the treatment with cycloheximide.
Thus, RNA synthesis and new protein synthesis are
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 un der norm oxia (20% O
2
;N)or
hypoxia (1% O
2
; H) for the indicated time (h) and then harvested for
RNA preparation. Shown are representative examples of Northern
blot analyses. The lane labe led with Ô0Õ contained RNA prepared from
the untreated cells. The lower gel image in each panel s hows b-actin
mRNA as an internal control.
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 c ell extrac ts prepared fro m D407 hu man R PE ce lls
exposed to norm oxia o r hypoxia for the indicated number of hours.
See Fig. 2 legend for key.
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
humanRPEcellstreatedwiththefoll owing c ytokines: 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 2 4 h. Each lane contains 15 lgtotalRNA.The
lower gel image in each panel shows b-actin mRNA as an internal

control. Th e d ata shown are from one of t wo in dep endent experiments.
Ó FEBS 2004 Dynamic regulation of HO-1 expression in RPE (Eur. J. Biochem. 271) 3079
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
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 t he maximum levels o f HO-1 mRNA w ere
significantly reduced after 5 h under hypoxia even in the
absence of a ctinomycin 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

All the data suggest 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 th e 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 h ypoxia-
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 t he expression o f any HO-1 constructs containing
a putative HRE sequence CACGTGA, whereas the p uta-
tive HRE (the E-box motif) appears to be required for the
basal promoter activity of th e 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 t o confer the hypoxia-mediated induction or repres-
sion on a reporter gene in D407 cells. M oreover, treatment
with interferon-c exerted no noticeable effects on the
Fig. 4. Inhibitory effects of cycloheximide or actinomycin D o n
expression of HO-1 mRNA in D407 RPE cells. D407 human RPE cells
were incubated with out or with actinomycin D (1 lgÆmL
)1
)or

cycloheximide (1 lgÆmL
)1
) for the indicated time (h) under normoxia
(N) o r hypoxia (H). Middle and lower gel images show Bach1 mRNA
expression for comparison and b-actin mRNA as an internal control.
Fig. 5. Effect of hypoxia on the stability of H O-1 mRNA. (A) N orthern
blot analysis [conditions; normoxia + actinomycin D (N+AM-D),
hypoxia (H), and hypo xia + actinomycin D (H+AM-D)]. D4 07
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 a re from one o f three indepe ndent exp eriments
with similar results. (B) Relative expression levels of HO-1 mRNA.
The intensities representing H O-1 mRNA at the time of addition of
actinomycin D un der e ach co ndition were con sidered to be 100% . Th e
data shown are mean ± S EM (n ¼ 3).
3080 R. Udono-Fujimori et al. (Eur. J. Biochem. 271) Ó FEBS 2004
promoter activity of phHOLUC45 (data not shown),
despite the presence of a Maf recognition element that is
bound by Bach1-small Maf complexes [52].
Discussion
A number of studies have shown the beneficial role of HO-1
induction, as described above, but the red uced 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, i ncluding 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 o f 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,eveninRPEcells,whichmay
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
detectable levels, unlike the severe effect of actinomycin D
(Fig. 4), as HO-1 is important in cell s urvival.
The R PE 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 c oncentration of
100 UÆmL
)1

used in this study was shown to completely
inhibit Toxoplasma gondii replication in cultured human
RPE cells [35]. I n this case , interferon-c has been shown to
inhibit proliferation of parasites by inducin g 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]. T hese 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.
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 a ngiogenesis as s een in d iabetic
retinopathy and age-related macular degeneration. Here we
show that hypoxia transiently induces the expression of
HO-1 mRNA through an unknown p rotein 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
Fig. 6. Effect of hypoxia on the human HO-1 g ene promote r function. D407 human RPE cells were transfected with each reporter c onstruct and
incubated under normoxia or hypoxia. A putative HRE in the HO-1 gene promoter is marked with Ô?Õ. The two constructs, shown n ear the b ottom,
represent positive and negative controls for hypoxia. Relative luciferase activity under normoxia or hypoxia is shown as the ratio t o the normalized
luciferase activity obtained with pG L3Basic under normoxia or hypoxia, respectively. T he data are means ± S EM of three independent experi-
ments.
Ó FEBS 2004 Dynamic regulation of HO-1 expression in RPE (Eur. J. Biochem. 271) 3081
expression levels within narrow ranges under hypoxia in

RPE c ells. Alternative ly, the diffe ren ce may s imp ly r eflect 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 bothRPE cell lines [40]. M oreover,
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 m ore differentiated properties, which is consistent with
the previous report by other investigators [25].
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]. I n 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 i nduce 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 h ypoxic induction of human
HO-1 gene expression.
A question that remains to be answered is the i dentity of a
protein factor that is responsible for the hypoxia-mediated
induction of HO-1 expression in D407 RPE cells. Probably,

many factors a re 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], a nd 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-b1mRNAin
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]. F urther
studies, such as DNA microarray analysis, may help us to
find such a factor.
Bach1 is a heme-regulated transcriptional repressor for
the HO-1 gene and plays an important role in the feedback
regulation of HO-1 expression [47,59]. However, the
expression of HO-1 and Bach1 mRNAs w as 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 e xpression 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.
It is noteworthy that HO-1 expression is not reduced by
hypoxia in th e two RPE cell lines, unlike other types of
human cells [27,28]. To confi rm 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 w orking on a rodent model of high
altitude retinopathy, although the interspecies difference has
been well known in th e 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 t ypes 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
We thank Dr R.C. Hunt for D 407 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 Rese arch
(B), Scientific Research (C), f or 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 Resea rch Gra nt
Ôthe Center for Innovative Therapeutic Developm ent for Common
DiseasesÕ from the Ministry of Education, Science, Sports, and Culture
of Jap an. This work was also sup portedinpartbythegrantsprovided
by Uehara Memorial Foundation.
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