Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and Hep
Trang 1the increased expression of heme oxygenase-1 in human cell lines
Yuanying Ding1, Yong Z Zhang1, Kazumichi Furuyama1, Kazuhiro Ogawa2*, Kazuhiko Igarashi3 and 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, Tohoku University Graduate School of Medicine, Sendai, Japan
Heme is an invaluable molecule that is essential for life
and is involved in many cellular processes that sense
or use oxygen The intracellular concentration of heme
is maintained by the rate of its synthesis and
degrada-tion [1] Many enzymes and their regulators are
responsible for heme synthesis [1,2] On the other
hand, heme degradation is mediated by two
structur-ally related isozymes, HO-1 and HO-2, to generate
biliverdin IXa, carbon monoxide (CO), and ferrous iron [3] Biliverdin IXa is immediately reduced to bili-rubin IXa HO-1 has attracted particular attention, because its expression is induced by its substrate, heme, in animals [4,5] and in primary cultures of macrophages [6–9] It has therefore provided a good example of substrate-mediated induction of an enzyme
in mammals [10,11]
Keywords
cancer cell lines; heme homeostasis; heme
oxygenase; isozymes; short interfering RNA
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, 13-1 Takara-machi,
Kanazawa 920-8640, Japan
(Received 28 July 2006, revised 3 October
2006, accepted 4 October 2006)
doi:10.1111/j.1742-4658.2006.05526.x
Intracellular heme concentrations are maintained in part by heme degrada-tion, which is catalyzed by heme oxygenase Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2 Recent studies have identified HO-2 as a potential oxygen sensor To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expres-sion profiles of HO-2 and the biochemical consequences of HO-2 knock-down with specific short interfering RNA (siRNA) in human cells Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells In contrast, knockdown of HO-1 expression did not noticeably influ-ence HO-2 expression HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellu-lar heme homeostasis These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2
Abbreviations
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO, heme oxygenase; MARE, Maf recognition element; SA, succinylacetone; SnPP, Sn-protoporphyrin.
Trang 2On the other hand, it has been reported that
expres-sion of HO-1 is decreased in several types of human
cell under various conditions, such as hypoxia [12,13]
and treatment with interferon-c [14,15] or
desferriox-amine, an iron chelator [12] Likewise, the expression
of HO-2 is decreased in the placental tissues of
abnor-mal pregnancies [16,17] and in cultured human
tropho-blast cells [18] We have recently shown that the
expression levels of HO-1 and HO-2 are decreased in
several human cell lines under hypoxia [19] In mice,
expression of HO-1 and HO-2 proteins is decreased in
decidua and placenta during Th1-mediated abortion
[20] Moreover, expression of HO-1 and HO-2 proteins
is transiently decreased in the liver, but increased in
the heart during acclimatization of mice to normobaric
hypoxia [21] These results suggest that expression of
HO-1 and HO-2 is regulated in a complex manner to
maintain intracellular heme concentrations or the
availability of free heme for various hemoproteins
Free heme is defined as either heme that is newly
syn-thesized but not yet bound to hemoproteins or heme
that has been released from hemoproteins [22]
HO-2 contains the cysteine and proline (CP) motifs
[23], whereas HO-1 lacks a cysteine residue [24,25]
Each CP motif of HO-2 may function as a
heme-bind-ing site [26], suggestheme-bind-ing that HO-2 may sequester heme
to maintain the intracellular heme concentrations or
ameliorate heme-mediated oxidative stress Moreover,
unlike the severe phenotypes of HO-1-deficient mice
(HO-1–⁄ –), including prenatal lethality [27], HO-2–⁄ –
mice are fertile and survive for at least 1 year under
basal conditions [28], but show ejaculatory
abnormalit-ies [29] and high susceptibility to oxygen toxicity [30]
Recent studies of our group [31] and other
investiga-tors [32] have shown that HO-2 functions as a
poten-tial oxygen sensor
In the present study, we show that HO-2
knock-down is associated with the induction of HO-1
expres-sion in human cancer cell lines HO-2 knockdown may
mimic a certain physiological change that is important
in the maintenance of cellular heme homeostasis We
provide evidence that HO-2 may modulate the
expres-sion level of HO-1 by affecting HO-1 mRNA stability
and intracellular heme concentration
Results
Expression profiles of HO-1 and HO-2 in various
human cell types
We initially analyzed the expression profiles of HO-1
and HO-2 in eight human cell lines by northern and
western blot analyses (Fig 1A,B) Expression of HO-1
mRNA was detected in five of these cell lines, but hardly at all in the other three (K562 erythroleukemia, Jurkat T cell, and H146 small cell lung cancer), in which HO-1 protein was also undetectable In contrast, HO-2 mRNA and protein were both expressed in all eight cell lines (Fig 1A,B) We also measured the heme content of these cell lines: it was about twofold higher
in YN-1 and K562 erythroleukemia cells than in the other cell types (Fig 1C) Higher heme content may reflect hemoglobin production in YN-1 and K562 cells [33,34] In fact, the population of hemoglobin-positive cells was about 4.4% in YN-1 cells and 4.9% in K562 cells under basal conditions [19] Otherwise, there was
A
B
C
Fig 1 Expression profiles of HO-1 and HO-2 in various human cell lines (A) Total RNA and proteins were prepared from the indicated cell lines and subjected to northern blot analysis and (B) western blot analysis (A) Northern blot analysis Each lane contains 15 lg total RNA The bottom panel shows the expression of 18S rRNA as
an internal control Note that the blot was exposed to the film for the longest time (1 min) to detect the low expression levels of HO-1 in some cell lines (B) Western blot analysis Each lane contains 20 lg protein The same filter was reused for b-actin expression as an internal control (C) Cellular heme contents Cells were cultured for 48 h, and harvested for the measurement of heme content (ng ⁄ 10 6 cells).
Trang 3no apparent correlation between the expression levels
of HO-1 and HO-2 and cellular heme content The
cel-lular heme content represents the sum of free heme
and bound heme of various hemoproteins
Role of heme metabolism in cellular heme
content
To evaluate the contribution of heme synthesis and
degradation to cellular heme content, we treated
YN-1 erythroleukemia, HeLa cervical cancer, and
HepG2 hepatoma cells for 48 h with succinylacetone
(SA) or Sn-protoporphyrin (SnPP) (Fig 2) These
dis-tinctive cell lines were chosen because both HO-1 and
HO-2 are expressed at detectable concentrations
(Fig 1) [19] SA is a specific inhibitor of
d-aminolevu-linic acid dehydratase, the second enzyme of the heme
biosynthetic pathway SnPP is a competitive inhibitor
of HO activity [35] The heme content of all three cell
lines was significantly decreased after treatment with
SA, but increased after treatment with SnPP
(Fig 2A,B) Thus, an appropriate balance between
heme synthesis and heme degradation is responsible
for maintenance of cellular heme contents More importantly, these results indicate that measurement
of heme content is useful for evaluation of heme dynamics in cultured cells
Regulatory role of free heme in expression
of HO-1
To explore the role of free heme in HO-1 and HO-2 expression, we treated HeLa and HepG2 cells with
SA and determined the expression levels of HO-1 and HO-2 (Fig 3) HO-1 mRNA expression was signifi-cantly reduced in HeLa and HepG2 cells after treat-ment with SA for 6 h, whereas HO-2 mRNA expression was not noticeably changed by SA treat-ment (Fig 3A,C) Western blot analysis revealed that treatment with SA reduced the expression of HO-1 protein in HeLa and HepG2 cells, but did not change the HO-2 protein concentration (Fig 3B,D) These results suggest that a certain threshold concentration
of free heme may determine the basal expression levels
of HO-1
Effects of HO-1 or HO-2 short interfering RNA (siRNA) on the expression of HO-1
To explore the functional significance of HO-1 and HO-2, we selectively reduced the expression of HO-1
or HO-2 mRNA with each siRNA HeLa and HepG2 cells were transfected with siRNA targeted to HO-1, HO-2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and total RNA was extracted from cells after 24 h incubation and subjected to northern blot analysis (Fig 4A,C) HO-1 siRNA decreased HO-1 mRNA expression levels by 60%, but exerted no noticeable effects on HO-2 and GAPDH mRNA con-centrations in HeLa cells (Fig 4A) Likewise, HO-2 siRNA and GAPDH siRNA specifically decreased the expression of HO-2 mRNA and GAPDH mRNA by more than 90%, respectively Unexpectedly, treatment
of HeLa cells with HO-2 siRNA induced the expres-sion of HO-1 mRNA and protein without affecting the concentration of GAPDH mRNA and b-actin (Fig 4A,B) Likewise, the selective reduction of HO-2 mRNA with HO-2 siRNA induced the expression of HO-1 mRNA and protein in HepG2 cells, but did not change the concentrations of GAPDH mRNA and b-actin (Fig 4C,D) Consistent with HO-1 mRNA expression, expression of HO-1 protein was also induced by the treatment with HO-2 siRNA in HepG2 cells These results indicate that the down-regulation of HO-2 expression is associated with induction of HO-1 expression
Fig 2 Effects of SA and SnPP on cellular heme content in human
cell lines YN-1 erythroleukemia, HeLa cervical cancer, and HepG2
hepatoma cells were treated with 5 m M SA (A) or 50 l M SnPP (B)
for 48 h, and the cellular heme contents were measured Heme
contents are shown as ng ⁄ 10 6
cells The data are mean ± SEM from three independent experiments *P < 0.05, **P < 0.01.
Trang 4We then confirmed the effects of HO-2 knockdown
using another HO-2 siRNA with a different sequence
(HO-2 siRNA1) [32] and a scrambled HO-2 siRNA
(siHO-2-R) in HeLa and HepG2 cells (Fig 5) HO-2
siRNA1 efficiently decreased the expression of HO-2
mRNA and protein and induced the expression of
HO-1 mRNA and protein In contrast, the scrambled
HO-2 siRNA did not affect the expression of HO-1
and HO-2 mRNAs and proteins in HeLa and HepG2
cells (Fig 5) Thus, the induction of HO-1 is due to
the selective repression of HO-2 expression achieved
with HO-2 siRNA
Knockdown of HO-2 expression causes time-dependent induction of HO-1 mRNA expression
We then performed a time course study to confirm the effects of HO-2 siRNA on the expression of HO-1 mRNA in HeLa cells (Fig 6A) HO-2 siRNA efficiently reduced the expression of HO-2 mRNA at 6 h, which was further decreased at 12 h In contrast, expression of HO-1 mRNA was time-dependently increased, reaching
a maximum at 24 h (Fig 6A,B) HO-2 siRNA had not noticeably changed the concentrations of GAPDH
A
B
C
D
Fig 3 Effects of SA on HO-1 and HO-2 expression levels in human cell lines HeLa cells (A and B) and HepG2 cells (C and D) were left untreated or treated with SA (5 m M ) for the indicated time and harvested The upper panels in (A) and (C) show the northern blot analysis of HO-1 and HO-2 mRNA in HeLa (A) and HepG2 cells (C) Each lane contains 15 lg total RNA The expression of 18S rRNA is also shown as
an internal control The data presented are from one of three independent experiments The lower panels in (A) and (C) show relative expression levels of HO-1 and HO-2 mRNA The intensity of the signals representing HO-1 or HO-2 mRNA was normalized with respect to the intensity of the 18S rRNA signal The ratio of each normalized value to the control value in untreated cells at 6 h is shown as the relative expression level of HO-1 or HO-2 mRNA Asterisks represent significant differences compared with the control at 6 h (*P < 0.05,
**P < 0.01) (B and D) Western blot analysis of HO-1 and HO-2 proteins in HeLa (B) and HepG2 cells (D) Each lane contains 10 lg protein The relative expression levels are shown in the lower panels To normalize the expression levels, the same filter was reused for b-actin monoclonal antibody The intensity representing HO-1 or HO-2 protein was normalized with respect to the intensity for the b-actin signal The data are mean ± SEM from three independent experiments *P < 0.05.
Trang 5mRNA by 24 h It should be noted that HO-2 siRNA
causes late-onset induction of HO-1 mRNA In this
con-text, we have reported that maximum induction of HO-1
mRNA expression was detected within 3 h by treatment with cadmium in HeLa cells, which is due to increased transcription of the HO-1 gene [36] Thus, HO-2
D B
Fig 4 Knockdown of HO-1 or HO-2 expression by each siRNA in HeLa and HepG2 cells HeLa cells (A and B) and HepG2 cells (C and D) were treated for 24 h with siHO-1, siHO-2 or siGAPDH as described in Experimental procedures Cells treated with Lipofectamine 2000 transfection reagent alone were included as a control siGAPDH was also used as a control for transfection with siRNA Other methods are same as in Fig 3 (A and C) Northern blot analysis of HO-1, HO-2 and GAPDH mRNAs The intensity of the signals representing HO-1 or HO-2 mRNA was normalized with respect to the intensity of the 18S rRNA signal The ratio of each normalized value to the value in untreated cells is shown as the relative expression level of HO-1 or HO-2 mRNA (**P < 0.01) (B and D) Western blot analysis of HO-1 and HO-2 proteins The intensity representing HO-1 or HO-2 protein was normalized with respect to the intensity of the b-actin signal The ratio
of each normalized value to the control value in siRNA-untreated cells (control) is shown as the relative expression level of HO-1 or HO-2 protein (*P < 0.05, **P < 0.01) The data are mean ± SEM from three independent experiments.
Trang 6knockdown may evoke a certain metabolic change,
which in turn induces HO-1 mRNA expression
HO-2 knockdown increases stability of HO-1
mRNA
Consequently, we analyzed the stability of HO-1
mRNA in HeLa cells treated with HO-2 siRNA In
this series of experiments, HeLa cells were left
untrans-fected or transuntrans-fected with the indicated siRNA, and
cultured for 12 h before addition of actinomycin D
The half-life of HO-1 mRNA was about 3 h in
untransfected and siGAPDH-treated HeLa cells
(Fig 7A,B), which is in good agreement with the
half-life of HO-1 mRNA determined in HeLa cells [37]
Interestingly, the half-life of HO-1 mRNA was
pro-longed to about 7 h in HeLa cells transfected with
siHO-2 Thus, the induction of HO-1 mRNA with
HO-2 knockdown may be in part due to the increased
stability of HO-1 mRNA
Effects of HO-2 knockdown on HO-1 and HO-2
promoter activities
To assess the biochemical consequences of HO-2
knockdown, we analyzed whether HO-2 siRNA affects
the promoter activity of the human HO-1 gene, using reporter constructs (Fig 8) The 4.5-kb promoter region of the HO-1 gene, carried by phHOLUC45, has been shown to be responsive to cadmium [36] and sodium nitroprusside [38], but unresponsive to hemin [9,36] We also included model constructs of pRBGP2 and pRBGP4, containing three copies of the Maf recognition element (MARE) and three copies of the mutated MARE, respectively [39] Co-transfection with siHO-2 significantly increased the promoter activity of phHOLUC45, which contains the MARE, but showed
no effect on the promoter activity of phHOLUC40, lacking the MARE, the HO-2 gene promoter of phHO2()1494) and phHO2()663), or a control pro-moter of pGL3-Basic Likewise, transfection with siHO-2 significantly increased the pRBGP2 promoter activity but not pRBGP4 Knockdown of HO-1 with siHO-1 tended to increase the HO-1 promoter activity, but the degree of activation was not statistically signifi-cant Neither siHO-1 nor siGAPDH significantly influ-enced the promoter activities of the HO-2 gene, pRBGP2 or pRBGP4 We also analyzed the effects of hemin treatment on the HO-1 gene promoter activity (Fig 8B), showing that hemin significantly increased the expression of pRBGP2 but not the expression of phHOLUC45 and pRBGP4 These results suggest that
Fig 5 Induction of HO-1 mRNA expression with knockdown of HO-2 in HeLa and HepG2 cells Cells were treated for 24 h with each of two siRNAs against HO-2 (siHO-2 and HO-2siRNA1), scrambled siHO-2 (scramble) or siGAPDH Other methods are the same as in Fig 4 Expression levels of HO-1, HO-2 and GAPDH mRNAs were determined in HeLa cells (A) and HepG2 cells (C) by northern blot analysis HO-1 and HO-2 proteins were determined in HeLa cells (B) and HepG2 cells (D) by western blot analysis The data are mean ± SEM from three independent experiments *P < 0.05, **P < 0.01.
Trang 7HO-2 knockdown may transactivate the promoter of
phHOLUC45 through a heme-independent mechanism
Moreover, HO-2 knockdown may cause a metabolic
change similar to that evoked by cadmium [36] or
sodium nitroprusside [38], each of which activated the
expression of a reporter gene under the regulation of
the 4.5-kb HO-1 gene promoter It should be noted,
however, that heme activates the HO-1 gene promoter
[40], but the relevant cis-acting element is not present
in the 4.5-kb promoter region It is therefore
conceiv-able that HO-2 knockdown may induce HO-1
expres-sion through not only the heme-independent
mechanism but also the heme-dependent mechanism
Heme accumulation caused by knockdown of
HO-1 or HO-2 expression
To explore the biological implication of the
knock-down experiments and to evaluate the relative
contri-bution of HO-1 and HO-2 to the total amount of
heme degradation, we measured the heme content of
HeLa cells and HepG2 cells after transfection with
HO-1 or HO-2 siRNA There were no significant chan-ges in heme content in HeLa cells (Fig 9A) and HepG2 cells (Fig 9B), which were transfected with each HO siRNA Thus, heme content may be main-tained by a compensatory mechanism, or the changes
in heme content may be below the detectable limit of the assay method used Accordingly, we treated HeLa and HepG2 cells for 12 h with 1 lm hemin, a subopti-mal concentration for the induction of HO-1 mRNA, and then measured cellular heme content It should be noted that hemin at this concentration does not notice-ably induce HO-1 expression in HeLa and HepG2 cells (data not shown) In HeLa cells that were left untrans-fected (control) or transuntrans-fected with siGAPDH or siHO-1, the heme content was twofold higher after treatment with hemin (Fig 9A), whereas the heme content remained unchanged in HepG2 cells treated with hemin (Fig 9B) Thus, heme may be more effi-ciently incorporated into HeLa cells than HepG2 cells
or the incorporated heme may exceed the capacity of heme degradation mediated by HO-1 and HO-2 in HeLa cells In fact, HO activity was detected in
A
Fig 6 Time-dependent induction of HO-1 mRNA expression with HO-2 knockdown (A) Northern blot analysis HeLa cells were cultured for the indicated time (0, 6, 12, 24 and 48 h) after transfection with HO-2 or GAPDH siRNA Total RNA was extracted and subjected to northern blot analysis Each lane contains 10 lg total RNA Relative expression levels of HO-1 (B) and HO-2 (C) mRNAs are shown The intensity rep-resenting HO-1 or HO-2 mRNA was normalized with respect to the intensity of 18 S rRNA The ratio of each normalized value to the control value at 0 time (0 h) is shown as the relative expression level of HO-1 or HO-2 mRNA The data are mean ± SEM from three independent experiments *P < 0.05, **P < 0.01.
Trang 8HepG2 cells, but not in HeLa cells [19] Moreover,
heme content was further increased twofold in
hemin-treated HeLa cells transfected with siHO-2 (Fig 9A),
even though HO-2 knockdown with siHO-2 induced
HO-1 expression (see Figs 4 and 6) On the other
hand, knockdown of either HO-1 or HO-2 expression
resulted in the accumulation of heme in the
hemin-treated HepG2 cells Taken together with the
siHO-2-mediated induction of HO-1 expression, these results
suggest that HO-2 rather than HO-1 may play the
pre-dominant role in heme degradation in cultured human
cells
Discussion
We have shown that the selective knockdown of HO-2
expression with each of two different siRNAs is
consis-tently associated with increased expression of HO-1
mRNA and protein Moreover, we provide evidence
that at least three mechanisms may account for the siHO-2-mediated induction of HO-1 expression: increased stability of HO-1 mRNA and heme-depend-ent and heme-independheme-depend-ent transcriptional regulation of the HO-1 gene These metabolic consequences of HO-2 knockdown suggest a regulatory role for HO-2 in the co-ordinated expression of HO-1 and HO-2 We there-fore propose that HO-2 may modulate the expression
of HO-1
Incidentally, the three cell lines with low HO-1 expression, K562, Jurkat, and H146, were maintained
in suspension culture (Fig 1), although HO-1 is expressed in two other cell lines, YN-1 and KG1, that were also maintained in suspension culture These results suggest that the cellular microenvironment, such as cell attachment, may influence the expression
of HO-1 The dominant expression of HO-2 protein, in comparison with the low expression of HO-1 protein,
in H146 small cell lung cancer cells is of particular interest because small cell lung cancer is derived from the airway neuroepithelial body [41], which functions
as an oxygen-sensing organ in the lung The neuroepit-helial body is responsible for ventilation-perfusion matching, which may be impaired in HO-2–⁄ – mice [31]
It is noteworthy that HO-2 knockdown increased the heme content of the hemin-treated HeLa and HepG2 cells (Fig 9A,B), despite the induction of HO-1 expression (Figs 4 and 6) These results suggest that the increased HO-1 expression may be insufficient
to compensate for a certain degree of reduction in HO-2 protein Taken together with the ubiquitous expression profile of HO-2 in the cell lines examined (Fig 1), we suggest that HO-2 may be a key enzyme responsible for maintaining cellular heme concen-trations In this context, HO-2 contains at least two copies of the CP motif, which may be bound by heme but is not present in HO-1 [23,26] Thus, the down-regulation of HO-2 may transiently increase the cel-lular free heme concentration, which in turn increases the expression of HO-1 mRNA
The induction of HO-1 expression mediated by HO-2 knockdown may account for the phenotypic dif-ferences between HO-1–⁄ – mice and HO-2–⁄ – mice Unlike the partial lethality of HO-1–⁄ – [27], HO-2–⁄ – mice are able to survive for at least a year under basal conditions [28] These mice can probably compensate for the loss of HO-2 by increasing the expression of HO-1, which is supported by the following observa-tions First, HO-2–⁄ – mice show no noticeable changes
or only a marginal decrease in arterial carboxyhemo-globin, a marker of overall heme degradation [27,28] Secondly, no differences in heme concentration were
A
B
Fig 7 HO-2 knockdown increases the stability of HO-1 mRNA (A)
Northern blot analysis HeLa cells, which were left untransfected
(Control) or transfected with the indicated siRNA, were cultured for
12 h, and then treated with actinomycin D (AMD) (1 lgÆmL)1) for
the indicated time (h) Each lane contains 15 lg total RNA The lane
labeled 0 h contained RNA prepared from cells harvested just
before the addition of AMD (0 h) (B) Relative expression levels of
HO-1 mRNA The intensity representing HO-1 mRNA was
normal-ized with respect to the intensity of 18S rRNA The intensity
repre-senting HO-1 mRNA at the time of addition of AMD (0 h) under
each condition was considered to be 1 One representative of two
independent experiments with similar results is shown.
Trang 9detected in tissue homogenates prepared from multiple
tissues of HO-2–⁄ – mice [42] Thirdly, HO-1 protein is
indeed over-expressed in the lung [30] and pulmonary
venous myocardium [31] of HO-2–⁄ – mice Taken
together with our proposal that HO-2 down-regulates
HO-1 expression, these results suggest that heme
homeostasis is maintained in HO-2–⁄ – mice through
appropriate resetting of HO-1 expression In contrast
with HO-1 expression, expression of HO-2 mRNA and
protein was not increased in several human cell lines
examined [15,19,37] Such a mode of regulation of
HO-2 expression may account for the severe phenotype
of HO-1–⁄ – mice [27] In particular, the compensation
achieved by HO-2 is not sufficient in the
HO-1-enriched organs, such as spleen, liver, and bone
mar-row In fact, HO-1–⁄ – mice suffer from severe anemia
and iron deposits [27] Likewise, human HO-1
defici-ency is characterized by hypobilirubinemia, persistent
hemolytic anemia, and iron deposits in the liver [43]
HO-2 knockdown increased the transient expression
of phHOLUC45 through the enhancer region of the human HO-1 gene, located between )4.5 kb and )4 kb The increased expression of phHOLUC45 sug-gests that the cellular microenvironment generated by HO-2 knockdown may mimic the metabolic change evoked by cadmium [36] or sodium nitroprusside [38], each of which activates the expression of a reporter gene under the regulation of the 4.5-kb HO-1 gene promoter This region contains the cadmium-respon-sive element [36] and the MARE [44,45], but lacks the element required for full activation by hemin [9,36,40] It is the MARE site that is bound by Nrf2,
a transcription activator, or Bach1, a transcription repressor, each of which functions as a heterodimer with a member of the Maf family [46] Bach1 is a heme-responsive repressor, and its repression activity
is lost when Bach1 is bound by heme, which in turn leads to transcriptional activation of the HO-1 gene
A
B
Fig 8 Knockdown of HO-2 expression increases the HO-1 promoter activity HeLa cells were transiently transfected with each reporter con-struct (3 lg), then incubated for 24 h, and either re-transfected with each siRNA (A) or treated with 50 l M hemin (B), as described in Experi-mental procedures After 24 h of incubation, luciferase activity was measured The test promoters analyzed are shown on the left Relative luciferase activity is shown as the ratio to the normalized luciferase activity obtained with pGL3Basic; the normalized luciferase activity used was that in cells treated with siGAPDH in (A) and that in cells untreated with hemin in (B) The data are mean ± SEM from three independ-ent experimindepend-ents **P < 0.01.
Trang 10through the MARE [45–49] The increased
concentra-tion of endogenous heme may facilitate the binding
of Nrf2, instead of Bach1, to the MARE to activate
the MARE-dependent promoter, as reported for the
mouse HO-1 gene [47] However, in contrast with the
mouse HO-1 gene, the human HO-1 gene promoter is
under complex regulation [9,40,50] In fact, both
knockdown of HO-2 and hemin treatment resulted in
activation of the MARE-dependent promoter,
pRBGP2, whereas hemin did not increase the
tran-sient expression of phHOLUC45 (Fig 8B) It has
been reported that hemin induces HO-1 expression in
HeLa cells [36,51], and hemin activates transcription
of the human HO-1 gene [40] Taken together with
the findings that HO-2 knockdown tends to cause
heme accumulation, we suggest that HO-2 may
modulate transcription of the HO-1 gene through
both heme-dependent and heme-independent
mecha-nisms
In summary, HO-2 may determine the expression level of HO-1 by affecting HO-1 mRNA stability and transcription of the HO-1 gene This study also reveals
an important regulatory role for HO-2 in the co-ordi-nated expression of HO-1 and HO-2 and the mainten-ance of cellular heme concentrations
Experimental procedures
Materials Hemin and 4,6-dioxoheptanoic acid (SA) were purchased from Sigma Chemical (St Louis, MO, USA) SnPP was from Porphyrin Products (Logan, UT, USA)
Cell cultures Human cell lines used were HeLa cervical carcinoma cells, HepG2 hepatoma cells, K562 and YN-1 erythroleukemia cells, Jurkat T-lymphocyte cells, KG1 myeloid cells, H146 small cell lung cancer cells, and HMV-II melanoma cells H146 small cell lung cancer cells were obtained from ATCC (HTB-173) and cultured in RPMI-1640 medium HMV-II melanoma cells were obtained from Riken Cell Bank and cultured in nutrient mixture Ham’s F12 med-ium HeLa and HepG2 cells were maintained in Dul-becco’s modified Eagle’s medium (Sigma) YN-1 cells were maintained in Iscove’s modified Dulbecco’s medium (Sigma), and K562, KG1 and Jurkat cells were maintained in RPMI-1640 medium (Sigma) Each medium contained 10% heat-inactivated fetal bovine serum, penicillin G (100 UÆmL)1), and streptomycin sulfate (100 lgÆmL)1) Cells were incubated at 37C under 5% CO2⁄ 95% room air, unless otherwise specified HepG2, HeLa and YN-1 cells were treated with 50 lm SnPP or 5 mm SA for up to
48 h SnPP was freshly prepared and added immediately
to the culture medium The culture dishes were placed in the incubator
Northern and western blot analyses Total RNAs and proteins were extracted from cells, and subjected to northern and western blot analyses [15,19] HO-1 and HO-2 RNA probes were transcribed by SP6 RNA polymerase from pCR-hHO1, carrying the human HO-1 cDNA fragment (positions 81–878), and pCR-hHO2, carrying the human HO-2 cDNA fragment (posi-tions 85–939), as described previously [19] mRNA signals were detected with the DIG Northern Starter Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol In western blot analysis, the sig-nals of proteins were detected with the ECL Plus Western Blot Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s protocol The antibody
Fig 9 Heme accumulation after knockdown of HO-1 or HO-2
expression HeLa cells (A) and HepG2 cells (B) were cultured for
12 h after transfection with siHO1, siHO2, or siGAPDH, then
trea-ted with 1 l M hemin for 12 h, and harvested for the measurement
of heme content Cells were treated for 24 h with Lipofectamine
2000 transfection reagent alone (control) The data are mean ± SEM
from three independent experiments **P < 0.01.