Here we show that hypoxia 1% oxygen reduces the expression levels of heme oxyge-nase-2 mRNA and protein after 48 h of incubation in human cell lines, inclu-ding Jurkat T-lymphocytes, YN-
Trang 1various types of human cell lines
A possible strategy for the maintenance of intracellular heme level Yongzhao Zhang1, Kazumichi Furuyama1, Kiriko Kaneko1, Yuanying Ding1, Kazuhiro Ogawa2,*, Miki Yoshizawa1, Masaki Kawamura1, Kazuhisa Takeda1, Tadashi Yoshida3and Shigeki Shibahara1
1 Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Japan
2 Department of Molecular Pharmacology, Tohoku University School of Medicine, Sendai, Japan
3 Department of Biochemistry, Yamagata University School of Medicine, Yamagata, Japan
Heme oxygenase (HO) is the rate-limiting enzyme in
heme catabolism and cleaves heme to release iron,
car-bon monoxide and biliverdin at the expense of
molecu-lar oxygen and NADPH [1,2] HO consists of two
structurally related isozymes, HO-1 and HO-2 [3–5]
Characteristically, human HO-1 contains no cysteine
residue [6], whereas HO-2 contains at least two copies of
a potential heme-binding site, consisting of the cysteine and proline (CP motif) [7,8] Importantly, these CP motifs are not involved in heme breakdown reactions [8], suggesting that HO-2 may sequester heme to main-tain the intracellular heme level In addition, expression
of HO-1 mRNA is induced by various stimuli, such as hemin and nitric oxide donors, in which expression of
Keywords
erythroid cells; heme oxygenase-1; heme
oxygenase-2; hemoglobin; hypoxia
Correspondence
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 22 717 8118
Tel: +81 22 717 8117
E-mail: shibahar@mail.tains.tohoku.ac.jp
*Present address
Department of Molecular Pharmacology,
Kanazawa University Graduate School of
Medical Science, Kanazawa, Japan
(Received 26 January 2006, revised 5 May
2006, accepted 15 May 2006)
doi:10.1111/j.1742-4658.2006.05319.x
Heme oxygenase consists of two structurally related isozymes, heme oxyge-nase-1 and and heme oxygenase-2, each of which cleaves heme to form bili-verdin, iron and carbon monoxide Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas lit-tle is known about the regulation of heme oxygenase-2 expression Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxyge-nase-2 mRNA and protein after 48 h of incubation in human cell lines, inclu-ding Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxyge-nase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life
of heme oxygenase-1 mRNA (9.5 h) Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia Thus, the reduced expression of heme oxygenase-2 may represent
an important adaptation to hypoxia in certain cell types, which may contrib-ute to the maintenance of the intracellular heme level
Abbreviations
HO, heme oxygenase; HRE, hypoxia response element; MARE, Maf recognition element.
Trang 2HO-2 mRNA is largely unchanged [9–12] Notably,
hypoxia decreases the expression of HO-1 in several
types of human cell [13,14], but conversely induces it in
cultured human dermal fibroblasts [15] and a retinal
pig-ment epithelial cell line [16] Thus, expression of HO-1
mRNA is differentially regulated in human cells by
hyp-oxia, depending on cell type On the other hand, recent
reports have shown that expression of HO-2 is decreased
in the placental tissues of abnormal pregnancies [17,18]
and in cultured human trophoblast cells [19] However,
little is known about the regulation of HO-2 expression
Using HO-2-deficient mice [20], we have shown that
the mice lacking HO-2 exhibit hypoxemia with normal
arterial CO2 tension (Paco2) and attenuated hypoxic
ventilatory responses with normal hypercapnic
ventila-tory responses [21], which led us to propose a novel
function of HO-2 as an oxygen sensor Subsequently,
it has been shown that HO-2 interacts with the
a-sub-unit of a large-conductance, calcium-sensitive
potas-sium channel (the BK channel) and may function as
an oxygen sensor for the BK channel [22] Taken
together, these results suggest that hypoxia may inhibit
the BK channel activity in the carotid body through
HO-2, which ultimately enhances ventilation
Clinically, hypoxia represents a decrease in O2
pres-sure in inspired gas and causes hypoxemia, which is a
hemodynamic stress and could lead to pulmonary
hypertension [23,24] Hypoxemia is a common
mani-festation of various diseases, such as chronic
obstruct-ive pulmonary disease [25], and is also seen in the
HO-2-deficient mice [21] Moreover, we have shown
that the expression levels of HO-2 protein were
decreased by about 40% in the mouse liver after
7 days of normobaric hypoxia (10% oxygen) and
returned to the basal level after 14 days of hypoxia
[26] It is therefore of significance to study the
regula-tion of HO-2 expression in human cells under hypoxia
In the present study, we have analyzed the effect of
hypoxia on the expression levels of HO-1 and HO-2 in
various types of human cell line, including
erythrole-ukemia and hepatoma cells We have shown that
hyp-oxia reduces the expression of HO-2 in five out of six
cell lines examined We suggest that the reduced
expression of HO-2 represents an important response
during acclimatization to hypoxia
Results
Effects of hypoxia on HO-1 and HO-2 expression
in human cell lines
We initially analyzed the effects of hypoxia on the
expression of HO-1 and HO-2 in human cell lines of
bone marrow origin, including KG1 myeloid cells, Jur-kat T-lymphocytes, and K562 and YN-1 erythroid cells YN-1 cells were established from the peripheral blood of a patient with chronic myelogenous leukemia
in blastic crisis [27] Each cell line was incubated for
48 h under normoxia or hypoxia (5% or 1% oxygen) HO-1 mRNA expression was substantially increased under 1% oxygen in KG1 and YN-1 cells, whereas HO-1 mRNA was undetectable in Jurkat and K562 cells (Fig 1A) In contrast, expression of HO-2 mRNA was detected in these four cell lines and decreased by hypoxia (1% oxygen) in Jurkat, K562 and YN-1 cells, but remained unchanged in KG1 cells Under hypoxia, expression levels of b-actin mRNA were unchanged
We next measured the levels of HO-1 and HO-2 pro-teins by western blot analysis (Fig 1B) The expression levels of HO-1 protein remained unchanged in KG1 cells and decreased by about 20% in YN-1 cells after
A
B
Fig 1 Effects of hypoxia on expression of heme oxygenase (HO)-1 and HO-2 in human cell lines of bone marrow origin (A) Northern blot analysis of HO-1 and HO-2 mRNA KG1 myeloid cells, Jurkat T-lymphocytes and K562 and YN-1 erythroleukemia cells were cul-tured under normoxia (N) or hypoxia (H) (5% or 1% oxygen) for
48 h, and harvested Total RNA was extracted from each cell line, and then subjected to northern blot analysis Each lane contains
15 lg of total RNA The bottom panel shows the expression of b-actin mRNA as an internal control (B) Western blot analysis The indicated cells were harvested after cultivation under normoxia (N)
or hypoxia (1% oxygen) for 48 h The cell extracts were prepared for western blot analysis of HO-1 and HO-2 The lane labeled 0 h contained cell extracts prepared from untreated cells harvested just before starting the experiment Each lane contains 20 lg of protein.
To normalize the expression levels, the same filter was reused for a-tubulin monoclonal antibody Note that HO-1 mRNA and protein were undetectable in Jurkat and K562 cells under the conditions used.
Trang 348 h of hypoxia (1% oxygen), despite the increased
expression of HO-1 mRNA in these two cell lines
Consistent with the HO-1 mRNA level, HO-1 protein
was undetectable in Jurkat and K562 cells The HO-2
protein levels were noticeably decreased under hypoxia
in Jurkat, K562 and YN-1 cells, in which a-tubulin
protein levels were not changed The expression levels
of HO-2 protein were decreased by about 26% under
hypoxia in both K562 and YN-1 cells Thus, hypoxia
consistently decreased the expression levels of HO-2
mRNA and protein in Jurkat T-lymphocytes and
YN-1 and K562 erythroid cell lines
To further analyze the effects of hypoxia on the
expression of HO-1 and HO-2, we performed similar
analyses in two human cancer cell lines, HeLa cervical
cancer and HepG2 hepatoma cells Hypoxia (1%
oxy-gen) decreased the expression levels of HO-1 and
HO-2 mRNA after 48 h of incubation in the two cell
lines (Fig 2A) Likewise, hypoxia decreased the levels
of HO-1 and HO-2 proteins by more than 60% and
30%, respectively, in HeLa cells, and by 90% and
20%, respectively, in HepG2 cells (Fig 2B) Taken
together, these results indicate that hypoxia reduces
the expression levels of HO-2 mRNA and protein in
five out of six cell lines, with the exception of KG1
myeloid cells
Hypoxia decreases the expression levels of HO-2 protein in YN-1 and K562 cells
To confirm the hypoxia-mediated reduction of HO-2 expression, we performed a time-course study in YN-1 and K562 erythroleukemia cell lines In YN-1 cells, the expression levels of HO-1 and HO-2 proteins were sig-nificantly reduced after 48 h of hypoxia (1% oxygen) (Fig 3A,B) In K562 cells, hypoxia reduced HO-2 pro-tein after 48 h (Fig 3C,D), although the expression of HO-1 protein remained undetectable
Hypoxia decreases HO activity in YN-1, K562 and HepG2 cells
We next measured HO activity in YN-1, K562, HeLa and HepG2 cells exposed to hypoxia for 48 h, when the expression levels of HO-2 protein were significantly decreased (Figs 2 and 3) HO activity was determined
in the microsomal fraction, prepared from normoxia
or hypoxia-exposed cells, as described in Experimental procedures It should be noted that sufficient amounts
of purified biliverdin reductase and cytochrome P450 reductase were added to the reaction mixture to meas-ure the full HO activity HO activity was decreased in YN-1, K562 and HepG2 cells by hypoxia (Fig 4) In contrast, HO activity was undetectable in HeLa cells treated under normoxia or hypoxia, which is consistent with our previous report that HO activity was unde-tectable in HeLa cells [9] Thus, HO activity is not pro-portional to the expression levels of HO-1 and HO-2 proteins, detected by western blot analysis
Opposite effects of hypoxia on expression of HO-1 and HO-2 mRNA in YN-1 cells
To explore the mechanism of the hypoxia-mediated reduction of HO-1 and HO-2 protein levels, we ana-lyzed the effects of hypoxia on the expression of HO-1 and HO-2 mRNA in YN-1 erythroleukemia cells, which express detectable levels of both HO-1 and HO-2 mRNA (Fig 1A) The expression levels of HO-1 mRNA were induced after 48 h of hypoxia (1% oxy-gen) (Fig 5A,B), whereas HO-2 mRNA levels were significantly decreased (Fig 5A,C) In contrast, under mild hypoxia (5% oxygen), the changes in HO-1 and HO-2 mRNA levels were not statistically significant Thus, hypoxia (1% oxygen) decreased the expression levels of HO-1 protein in YN-1 cells (Fig 3A), despite the increased expression of HO-1 mRNA (Fig 5A)
On the other hand, the expression levels of HO-2 mRNA and protein were both reduced under hypoxia (1% oxygen) (Figs 3A and 5A)
A
B
Fig 2 Decreased expression of heme oxygenase (HO)-1 and HO-2
under hypoxia in human cancer cells (A) Northern blot analysis of
HO-1 and HO-2 mRNA HeLa cervical cancer and HepG2 hepatoma
cells were cultured under normoxia (N) or hypoxia (H: 1% oxygen)
for 48 h, and harvested Each lane contains 15 lg of total RNA.
The bottom panel shows the expression of 28S rRNA as an internal
control (B) Western blot analysis HeLa and HepG2 cells were
har-vested after cultivation under normoxia (N) or hypoxia (1% oxygen)
for 48 h The cell extracts were prepared for western blot analysis
of HO-1 and HO-2 Each lane contains 20 lg of protein To
normal-ize the expression levels, the same filter was reused for a-tubulin
monoclonal antibody.
Trang 4Stability of HO-1 and HO-2 mRNA under hypoxia
We then analyzed the stability of HO-1 and HO-2
mRNA in YN-1 cells under hypoxia (1% oxygen) In
this series of experiments, YN-1 cells were precultured
for 12 h under normoxia or hypoxia before addition of
actinomycin D The half-life of HO-1 mRNA in YN-1
cells was about 9.5 h under normoxia, and remained
unchanged under hypoxia (Fig 6A,B) In contrast, the half-life of HO-2 mRNA was about 12 h under norm-oxia (Fig 6A,C), and was shortened to 6 h under hyp-oxia The decreased levels of HO-2 mRNA may be in part due to the enhanced degradation of the HO-2 mRNA
Functional analysis of the HO-1 and HO-2 gene promoters under hypoxia
To address the question of whether hypoxia influences the promoter activity of the human HO-1 or HO-2 gene, we performed transient expression assays Prior
to the functional analysis of the HO-2 gene promoter,
we determined its transcription initiation site by 5¢-RACE, and this indicated that transcription is initiated from multiple sites (Fig 7A) The most 5¢-upstream initiation site was identified as the G resi-due at position + 1, which is located 139 bp upstream
of the start ATG codon in exon 2 The presence of the exon 1 sequence was also confirmed by RT-PCR ana-lysis of YN-1 RNA The 5¢-flanking sequence lacks a consensus TATA box but contains several sequence motifs for binding of transcription factors, such as Sp1 Incidentally, the HO-2 gene and the gene enco-ding HSCARG of unknown function (GenBank
acces-Fig 3 Time-dependent effects of hypoxia on heme oxygenase (HO)-2 protein levels in two erythroid cell lines YN-1 (A, B) and K562 cells (C, D) were cultured under normoxia (N) or hypoxia (1% oxygen) for the indicated numbers of hours, and the cell extracts were subjected to western blot analysis (A, C) Other conditions are described in Fig 1B The intensities of the signals in (A) and (C) were quantified, and the intensity representing HO-1 or HO-2 protein was normalized with respect to the intensity for the a-tubulin signal Shown are the relative expression levels of HO-1 and HO-2 proteins in YN-1 cells (B) and those of HO-2 protein in K562 cells (D) The intensity representing HO-1
or HO-2 protein at the 0 time (0 h) is considered to be 100% The ratio of each normalized value to the 0 time value (indicated by 0) is shown as the relative expression level of HO-1 or HO-2 protein Asterisks represent statistically significant differences compared to 0 h:
*P < 0.05; **P < 0.01.
a e H 2 G p H 2 5 K 1 -N Y
0
3
.
0
6
.
0
9
.
0
2
.
1
N
% 1 H
Fig 4 Hypoxia decreases the heme oxygenase (HO) activity in
YN-1, K562 and HepG2 cells.YN-1, K562, HepG2 and HeLa cells
were cultured for 48 h under normoxia or hypoxia, and then
harves-ted The microsome fraction was prepared and used for the assay
of HO activity The data are means ± SEM of three independent
experiments Note that the HO activity was undetectable in YN-1
cells exposed to hypoxia and in HeLa cells exposed to normoxia or
hypoxia (shown as ND).
Trang 5sion number AAG09721) are located adjacently in a
head-to-head orientation, and their transcription start
sites are 1.5 kb apart We therefore analyzed the
promoter function of the 1.5 kb 5¢-flanking region of
the HO-2 gene in the present study
YN-1 cells were transfected with each construct of the
HO-1and HO-2 gene promoters (Fig 7B) The reporter
plasmids used for the HO-1 gene included phHOLUC45
with a Maf recognition element (MARE) and
phHO-LUC40 without MARE [16], and those for the HO-2
gene, phHO2(-1492), phHO2(-663), and phHO2(-25)
The HO-1 gene promoter contains a putative
hypoxia-responsive element (HRE) sequence CACGTGA
(posi-tions ) 44 to ) 39) that overlaps the functional E-box
[14,16,28] Hypoxia did not change the expression of
phHOLUC45, phHOLUC40 or phHOLUC(-58) in
YN-1 cells (Fig 7B), despite the fact that a putative
HRE sequence is present in phHOLUC45 and
phHO-LUC40 Likewise, hypoxia did not influence the
expres-sion of HO-2 promoter constructs in YN-1 cells
(Fig 7B) 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 NHRESV40, a
negat-ive control for hypoxic induction
Hypoxia increases cellular heme contents in human cell lines
To explore the implication for the reduced expression levels of HO-1 and HO-2 proteins under hypoxia, we studied whether hypoxia influences the cellular heme contents in YN-1, HepG2 and HeLa cells (Fig 8) Heme contents were measured in each cell line after incubation under normoxia or hypoxia for 48 h Heme contents were increased in the three cell lines after
48 h of culture under hypoxia (Fig 8) The degree of increase was small but statistically significant
Discussion
We have hypothesized that a certain degree of reduc-tion in heme degradareduc-tion is probably important in the preservation of intracellular heme, an essential compo-nent of various hemoproteins [5] The present study has shown that hypoxia consistently reduces the expression levels of HO-2 mRNA and protein in five out of six human cell lines: Jurkat, YN-1, K562, HeLa and HepG2 In this context, Newby et al [29] des-cribed decreased levels of HO-2 protein in placentas
of women who reside at high altitude and are thus
A
Fig 5 Differential effects of hypoxia on heme oxygenase (HO)-1 and HO-2 mRNA expression in YN-1 cells (A) Northern blot analysis YN-1 cells were harvested after cultivation under normoxia (N) or hypoxia (5% or 1% oxygen) for the indicated numbers of hours Each lane con-tains 15 lg of total RNA The lane labeled 0 h contained RNA prepared from untreated cells harvested just before starting the experiment.
At the bottom of each panel, 28S rRNA of each sample was visualized by ethidium bromide staining The data are from one of three inde-pendent experiments with similar results (B, C) Relative expression levels of HO-1 and HO-2 mRNA The intensities of the signals in (A) were quantified, and the intensity representing HO-1 or HO-2 mRNA was normalized with respect to the intensity for the 28S RNA signal The intensity representing HO-1 or HO-2 mRNA at the 0 time (0 h) is considered to be 100% The ratio of each normalized value to the
0 time value (indicated by 0 h) is shown as the relative expression level of HO-1 or HO-2 mRNA Asterisks represent statistically significant differences compared to 0 h: *P < 0.05; **P < 0.01.
Trang 6exposed to chronic hypoxia Moreover, we have shown
that the expression levels of HO-2 protein were
transi-ently decreased in the mouse liver after 7 days of
normobaric hypoxia [26] These results suggest that the
reduced expression of HO-2 protein may be an
import-ant hypoxic response in certain cell types
Hypoxia exerted differential effects on the expression
of HO-1, depending on the cell line (Figs 1 and 2) It
should be noted that HO-1 protein levels remained
unchanged in KG1 myeloid cells and were significantly
reduced in YN-1 erythroleukemia cells after 48 h of
hypoxia (1% oxygen), despite the increased level of
HO-1 mRNA These results suggest that certain
mech-anisms, such as the active degradation of HO-1
pro-tein, might ensure constant or reduced expression
levels of HO-1 protein in these cell lines under
hypo-xia In contrast, the expression levels of HO-1 mRNA
and protein were consistently decreased in HeLa and
HepG2 cells Interestingly, hypoxia tends to increase
the cellular heme contents in YN-1, HepG2 and HeLa
cells, which might be a consequence of reduced heme
degradation and⁄ or enhanced heme synthesis [30,31]
In the present study, we focused on YN-1 erythrole-ukemia cells to investigate the hypoxia-mediated reduc-tion of HO-2 expression It is tempting to speculate that the decreased heme degradation may contribute in part
to the maintenance of the heme supply for hemoglobin production in erythroid cells In fact, it has been repor-ted that chemically induced erythroid differentiation is associated with a reduction of HO-1 expression in MEL mouse erythroleukemia cells [32] Moreover, heme indu-ces the expression of the a-globin gene in K562 human erythroleukemia cells [33] and the number of hemoglo-bin-producing cells in YN-1 cells [27,34] Conversely, the deficiency of heme in erythroid cells causes differen-tiation arrest in mice [35] These results indicate that heme is essential for differentiation of erythroid cells In this context, our preliminary data suggest that treatment for 48 h under hypoxia may increase the proportion
of hemoglobin-positive YN-1 cells (from 4.4 ± 0.3% under normoxia to 9.6 ± 1.0% under hypoxia) and K562 cells (from 4.9 ± 0.7% to 6.9 ± 0.5%); this was measured by staining cells with o-dianisidine This method was based on the peroxidase activity of
hemo-A
Fig 6 Effects of hypoxia on the stability of heme oxygenase (HO)-1 and HO-2 mRNAs (A) Northern blot analysis YN-1 cells were cultured for 12 h under normoxia or hypoxia (1% oxygen), and then treated with or without actinomycin D (AMD) (1 lgÆmL)1) for the indicated num-ber of hours Each lane contains 15 lg of total RNA The lane labeled 0 h contained RNA prepared from precultured cells harvested just before the addition of actinomycin D (0 h) (B, C) Relative expression levels of HO-1 and HO-2 mRNA under normoxia or hypoxia The inten-sity representing HO-1 or HO-2 mRNA was normalized with respect to the inteninten-sity of b-actin mRNA The inteninten-sity representing HO-1 or HO-2 mRNA at the time of addition of actinomycin D (0 h) under each condition is considered to be 100%.
Trang 7globin [36] However, the increase in the
hemoglobin-positive cells could be a result of a hypoxia-mediated
increase of transferrin receptors [37] and⁄ or
erythroid-specific 5-aminolevulinate synthase [30] Further
experi-ments are required to address the role of heme degrada-tion in the populadegrada-tion of hemoglobin-positive cells
To the best of our knowledge, there has been no report on the promoter function of the human HO-2
Fig 7 Characterization of the human heme oxygenase (HO)-2 gene promoter (A) Identification of the transcription start sites The upstream region of the HO-2 gene is shown Exon 1 encodes the untranslated region, and exon 2 encodes the protein-coding region (closed box), including the ATG translation–initiation codon The nucleotide sequences of the proximal promoter and exon 1 are shown Major transcription start sites, identified by 5¢-RACE, are indicated in bold Residue 1 represents the 5¢ end of exon 1 A newly identified 5¢-untranslated region
is shown as a stippled box, and underlined is the exon 1 region (from 58 to 98), based on the NCBI Blast database (accession number NT_010552) The 5¢ end of intron 1 is also shown in lower case (B) Promoter activities of HO-1 and HO-2 genes under hypoxia YN-1 cells were transfected with each reporter construct and then incubated under normoxia or hypoxia for 48 h The two constructs, named HRESV40 and N-HRESV40, 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 pGL3-basic under normoxia or hypoxia, respectively The data are means ± SEM of three independent experiments; **P < 0.01.
Trang 8gene We have identified the multiple transcription
initi-ation sites of the HO-2 gene and confirmed that the
HO-2 gene promoter is juxtaposed to the HSCARG
gene in the opposite direction Thus, the HO-2 gene and
the HSCARG gene share a common promoter region,
known as a bidirectional promoter The bidirectional
promoters are sometimes found in mammalian genes,
and belong to the family of TATA-less and GC-rich
promoters [38,39] Such features are consistent with the
HO-2gene promoter In the present study, hypoxia did
not influence the expression of a reporter gene, carrying
the 1.5 kb bidirectional promoter region Taken
together with the shortened half-life of HO-2 mRNA
under hypoxia, we suggest that the reduced expression
of HO-2 mRNA may be achieved at least in part by
post-transcriptional mechanisms, such as enhanced
deg-radation of HO-2 mRNA However, functional studies
with further upstream regions or downstream regions
including a large intron 1 of the HO-2 gene are required
In summary, the present study has suggested that
the reduced expression of HO-2 protein may
contrib-ute to the maintenance of intracellular heme level in
certain human cell types under hypoxia
Experimental procedures
Cell culture and hypoxia study
The human cell lines used were KG1 myeloid cells, K562
erythroid cells, and Jurkat T-lymphocyte cells and were
maintained in RPMI-1640 medium (Sigma, St Louis, MO,
USA) YN-1 human erythroid cells [27,34] were maintained
in Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma)
HeLa and HepG2 cells were maintained in DMEM Each
medium contains 10% heat-inactivated FBS, penicillin G
hypoxia experiments, cells were cultured for the indicated
[13] In some experiments, cells were incubated under mild
harves-ted for total RNA extraction and protein extraction
Northern blot analysis Total RNA was extracted from cultured cells and subjected
to northern blot analysis, as detailed previously [16] The signals for HO-1, HO-2 and b-actin mRNA were detected with the DIG Northern Starter Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s pro-tocol For preparation of HO-1 and HO-2 RNA probes, the human HO-1 cDNA of positions 81–878 [6] (GenBank accession number X06985) and the human HO-2 cDNA fragment (nucleotide positions 85–939) [7,9] (GenBank accession number P30519) were amplified by PCR using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), and then cloned into pCR-bluntII-TOPO (Invitro-gen, Carlsbad, CA, USA), and named pCR-hHO1 and pCR-hHO2, respectively SP6 RNA polymerase was used for transcription of RNA probe from pCR-hHO1 and pCR-hHO2
Western blot analysis Harvested cells were lysed in the lysis buffer (20 mm Hepes
phe-nylmethylsulfonyl fluoride), as detailed previously [9,16] The cell lysates were centrifuged at 15 000 g for 10 min (KUBOTA RA-50J1 fix-angle rotor, KUBOTA, Tokyo, Japan), and the supernatant (10 mg of protein) was ana-lyzed on a 10% SDS-polyacrylamide gel The proteins in the gel were electrophoretically transferred to a polyvinylid-ene difluoride membrane (Immobilon-P, Millipore Corpora-tion, Billerica, MA, USA) The membranes were treated for
from Shigeru Taketani) [40] or anti-HO-2 antibody (Stress-gene Canada, Victoria, Canada) HO-1 and HO-2 proteins were detected with ECL Plus western blot kit (Amersham Biosciences, Piscataway, NJ, USA) Expression of a-tubulin was examined as an internal control using a-tubulin mono-clonal antibody (NeoMarkers, Fremont, CA, USA)
Assay for HO catalytic activity
incuba-ted for 48 h under normoxia or hypoxia (1% oxygen), and harvested for the assay of HO activity, as described previ-ously [2,41,42] Cells were suspended in 2 mL of 20 mm
Fig 8 Increased heme contents under hypoxia in YN-1, HepG2
and HeLa cells YN-1, HepG2 and HeLa cells were cultured under
normoxia or hypoxia (1% oxygen) for 48 h, and cellular heme
con-tents were measured, as described in Experimental procedures.
The data are from three independent experiments Asterisks
repre-sent statistically significant differences compared to normoxia
con-trol: *P < 0.05; **P < 0.01.
Trang 9potassium phosphate buffer (KPB) (pH 7.4) containing
134 mm KCl, and disrupted by sonication The microsome
fraction was prepared by two steps of centrifugation and
was suspended by sonication in 150 lL of 50 mm KPB
con-taining 0.1% Triton X-100 Each sample (300 lg of
pro-tein) was added to the standard reaction mixture of
200 lL, which contained 0.1 m KPB (pH 7.4), 15 lm
and 15 lg of cytochrome P450 reductase After 2 min of
of 10 lL of NADPH (8.4 lgÆmL) or 10 lL of water as a
blank mixture The reaction mixture was incubated at
used to measure the absorbance at 468 nm The amounts
of bilirubin formed in the reaction system were calculated
Effects of actinomycin D on the expression of
HO-1 and HO-2 mRNA
To study the effects of hypoxia on the stability of HO-1
and HO-2 mRNA, YN-1 cells were incubated for 12 h in
fresh medium under normoxia or hypoxia, followed by
(Calbiochem-Behring, La Jolla, CA) [16,44] The cells were further
incu-bated for 2, 6 or 12 h under normoxia or hypoxia, and then
harvested at each time point for RNA extraction
Identification of 5¢ end of HO-2 mRNA and the
promoter region of the HO-2 gene
To amplify the 5¢ end of HO-2 cDNA, nested PCR was
carried out using the BD Marathon-Ready human testis
cDNA library (BD Biosciences Clontech, Palo Alto, CA,
(Epicen-tre, Madison, WI) Human testis cDNA was used, because
HO-2 protein is enriched in the testis [21,45] The sense
PCR primers were an adapter primer and its nested
(down-stream) primer, which is located upstream of the cDNA,
and the antisense primers for HO-2 cDNA were a
gene-spe-cific primer-1, 5¢-CAGGTCCAGGGCGTTCATCCTGGC
CCGG-3¢, located in exon-4, and its nested (upstream)
pri-mer, 5¢-CCCCCCGAGAGATCCCCCATGTAGCGGG-3¢,
located in exon-4 The two steps of PCR were performed
according to the supplier’s protocol The nested PCR
used to identify potential cis-elements in the 5¢-flanking region of the HO-2 gene
Luciferase reporter constructs The 1.5 kb 5¢-flanking region of the HO-2 gene was ampli-fied by PCR using human genomic DNA as a template and
a primer set designed from a published sequence (GenBank accession number P30519) (sense, 5¢-AGATCTATCCCTT GAGGCCTTGTCCGCTTG-3¢; antisense, 5¢-AAGCTTG
CC GCAGGTCGCTGTCGCCTG-3¢; these contain a BglII site and a HindIII site, respectively) The genomic fragment
vec-tor (Promega, Madison, WI, USA) containing luciferase as
a reporter gene The cloned 1.5 kb promoter region was used as a template to generate deletions in the HO-2 pro-moter All PCR products (1494, 663 and 25 bp) were puri-fied and subcloned in the pGL3-basic vector, yielding phHO2(-1492), phHO2(-663), and phHO2(-25) The identity
of each construct was confirmed by sequencing
The human HO-1 gene–luciferase constructs, phHO-LUC45 [16], phHOLUC40, and phHOLUC(-58) [47], carry the 4.5 kb, 4.0 kb and 58 bp fragments of the human HO-1 gene [10,48], respectively Reporter plasmids, HRESV40 containing four copies of HRE and NHRESV40 lacking HRE [49], were used as a positive and a negative control for hypoxia, respectively
Transient transfection assays Transfection was performed with DMRIE-C reagent (Invi-trogen), following the supplier’s protocol YN-1 cells
) were cotransfected with each promoter–reporter fusion plasmid (0.784 lg) and pRL-TK vector (16 ng) (Promega), and incubated for 4.5 h Then, the transfected YN-1 cells were incubated for 48 h under normoxia or hyp-oxia (1% oxygen), and harvested Luciferase activity was measured with a luminometer using the Dual Luciferase Assay System (Promega), as detailed previously [14,16] A promoterless construct, pGL3-basic, was used as a control The data are means ± SEM of three independent experi-ments with each plasmid DNA preparation
Fluorometric assay of heme
determined as described previously [50] Cell suspensions
Eppendorf 5415 R benchtop refrigerated centrifuge (rotor
Ham-burg, Germany), and 0.5 mL of 2 m oxalic acid was added
to the pellet The mixtures were shaken vigorously and
without heating were used as a blank for measurement of
Trang 10endogenous porphyrins After cooling, fluorescence was
determined in an RF-5300PC spectrofluorometer (Shimadzu
Corp., Kyoto, Japan) The excitation wavelength was
400 nm, and the fluorescence emission was determined at
662 nm Hemin solutions, containing 0, 1, 10, 50 or 100 ng
of hemin, were prepared in 0.5 mL of oxalic acid, and used
cells were used to deter-mine heme contents
Acknowledgements
We thank S Taketani for anti-HO-1 and Y
Fujii-Kuriyama for the HRE constructs This study was
supported by Grants-in-aid for Scientific Research (B),
for Scientific Research on Priority Areas, and by the
21st Century COE Program Special Research Grant,
the Center for Innovative Therapeutic Development
for Common Diseases, from the Ministry of
Educa-tion, Science, Sports, and Culture of Japan This study
was also supported by a grant provided by the Uehara
Memorial Foundation
References
1 Tenhunen R, Marver HS & Schmid R (1968) The
enzy-matic conversion of heme to bilirubin by microsomal
heme oxygenase Proc Natl Acad Sci USA 61, 748–755
2 Yoshida T & Kikuchi G (1978) Features of the reaction
of heme degradation catalyzed by the reconstituted
microsomal heme oxygenase system J Biol Chem 253,
4230–4236
3 Shibahara S, Muller R, Taguchi H & Yoshida T (1985)
Cloning and expression of cDNA for rat heme
oxyge-nase Proc Natl Acad Sci USA 82, 7865–7869
4 Maines MD, Trakshel GM & Kutty RK (1986)
Char-acterization of two constitutive forms of rat liver
microsomal heme oxygenase Only one molecular
spe-cies of the enzyme is inducible J Biol Chem 261,
411–419
5 Shibahara S (2003) The heme oxygenase dilemma in
cellular homeostasis: new insights for the feedback
regu-lation of heme catabolism Tohoku J Exp Med 200,
167–186
6 Yoshida T, Biro P, Cohen T, Muller RM & Shibahara
S (1988) Human heme oxygenase cDNA and induction
of its mRNA by hemin Eur J Biochem 171, 457–461
7 Ishikawa K, Takeuchi N, Takahashi S, Matera KM,
Sato M, Shibahara S, Rousseau DL, Ikeda-Saito M &
Yoshida T (1995) Heme oxygenase-2 Properties of the
heme complex of the purified tryptic fragment of
recom-binant human heme oxygenase-2 J Biol Chem 270,
6345–6350
8 McCoubrey WK, Huang TJ & Maines MD (1997)
Heme oxygenase-2 is a hemoprotein and binds heme
through heme regulatory motifs that are not involved in heme catalysis J Biol Chem 272, 12568–12574
9 Shibahara S, Yoshizawa M, Suzuki H, Takeda K, Meguro K & Endo K (1993) Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation J Biochem (Tokyo) 113, 214–218
10 Takeda K, Ishizawa S, Sato M, Yoshida T & Shibahara
S (1994) Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene J Biol Chem 269, 22858– 22867
11 Takahashi K, Hara E, Suzuki H, Sasano H & Shiba-hara S (1996) Expression of heme oxygenase isozyme mRNAs in the human brain and induction of heme oxygenase-1 by nitric oxide donors J Neurochem 67, 482–489
12 Takahashi K, Hara E, Ogawa K, Kimura D, Fujita H
& Shibahara S (1997) Possible implications of the induc-tion of human heme oxygenase-1 by nitric oxide donors
J Biochem (Tokyo) 121, 1162–1168
13 Nakayama M, Takahashi K, Kitamuro T, Yasumoto
K, Katayose D, Shirato K, Fujii-Kuriyama Y & Shiba-hara S (2000) Repression of heme oxygenase-1 by hypoxia in vascular endothelial cells Biochem Biophys Res Commun 271, 665–671
14 Kitamuro T, Takahashi K, Ogawa K, Udono-Fujimori
R, Takeda K, Furuyama K, Nakayama M, Sun J, Fuj-ita H, Hida W et al (2003) Bach1 functions as a hypo-xia-inducible repressor for the heme oxygenase-1 gene in human cells J Biol Chem 278, 9125–9133
15 Panchenko MV, Farber HW & Korn JH (2000) Induc-tion of heme oxygenase-1 by hypoxia and free radicals
in human dermal fibroblasts Am J Physiol Cell Physiol
278, C92–C101
16 Udono-Fujimori R, Takahashi K, Takeda K, Furuyama
K, Kaneko K, Takahashi S, Tamai M & Shibahara S (2004) Expression of heme oxygenase-1 is repressed by interferon-gamma and induced by hypoxia in human retinal pigment epithelial cells Eur J Biochem 271, 3076–3084
17 Zenclussen AC, Lim E, Knoeller S, Knackstedt M, Her-twig K, Hagen E, Klapp BF & Arck PC (2003) Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies Am J Reprod Immunol
50, 66–76
18 Lash GE, McLaughlin BE, MacDonald-Goodfellow
SK, Smith GN, Brien JF, Marks GS, Nakatsu K & Graham CH (2003) Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta Am J Physiol Heart Circ Physiol 284, H160–H167
19 Appleton SD, Marks GS, Nakatsu K, Brien JF, Smith
GN, Graham CH & Lash GE (2003) Effects of hypoxia
on heme oxygenase expression in human chorionic villi