Expression and Regulation of Heme Oxygenase Isozymes in the
Developing Mouse Cortex
HUI ZHAO, RONALD J. WONG, XUANDAI NGUYEN, FLORA KALISH, MASAMI MIZOBUCHI, HENDRIK J. VREMAN,
DAVID K. STEVENSON, AND CHRISTOPHER H. CONTAG
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305
ABSTRACT: Heme oxygenase (HO), the rate-limiting enzyme in
heme degradation, plays a role in neonatal jaundice. Understanding
the regulation of the developmental expression patterns of the two
HO isozymes, HO-1 and HO-2, is essential for targeting HO to
control pathologic jaundice, and uncovering the fundamental role that
they play in mammalian development. Here we characterized the
ontogeny of HO-1 and HO-2 expression in the developing mouse
cortex by in vivo bioluminescence imaging, quantitative RT-PCR,
and Western blot. HO-2, the predominant isoform in the adult cortex,
was relatively stable throughout all ages. HO-1 was observed to be
progressively down-regulated in an age-related manner. HO-1 ex-
pression in the adult cortex was also the lowest among the eight adult
tissues analyzed. Because there is a 283-bp CpG island region in the
HO-1 promoter, we hypothesized that methylation of the island is
responsible for the age-related HO-1 down-regulation in the cortex.
Methylation status was assessed using regular and quantitative meth-
ylation-specific PCR and the CpG island was found to be hypo-
methylated at all ages. Therefore, we conclude that HO-1 gene
expression in the cortex is developmentally-regulated and that meth-
ylation of the HO-1 CpG island is not associated with the down-
regulation of the gene. (Pediatr Res 60: 518–523, 2006)
cells and other hemoproteins (1). In this pathway, HO cleaves
the heme ring via oxidation at the ?-methene bridge to yield
equimolar quantities of biliverdin, carbon monoxide (CO),
and free iron. Biliverdin is subsequently converted to bilirubin
by biliverdin reductase. Among the many implications of HO
in development, it is the controlling step in bilirubin produc-
tion and has been identified as a target for prevention of
HO has many physiologic functions (2,3) and plays a key
role during development. It not only regulates cellular heme
and hemoprotein levels (e.g. cytochrome P450), but also all of
its heme degradation products are biologically active: the bile
pigments biliverdin and bilirubin are potent antioxidants (4);
CO generates cGMP and MAP kinase, and is shown to be a
potential vasodilator and smooth muscle relaxant (5); and free
eme oxygenase (HO) is the rate-limiting enzyme for the
degradation of heme derived from senescent red blood
iron regulates expression of various genes and hematopoiesis
(6,7). To date, two primary isoforms of HO have been iden-
tified in the human and rodent: the inducible HO-1, also
known as Hsp32, and the constitutive HO-2 (8,9). HO-3, a
third isoform cloned only from rats, has been reported to be a
processed pseudogene derived from HO-2 transcripts (10).
HO-1 and HO-2 have some protein similarities, but are en-
coded from different genes.
Expression of HO-1 is transcriptionally regulated by a
15-kb regulatory region, consisting of a basal promoter and
three enhancer regions (11). Several regulatory elements and
transcriptional factor binding sites have been reported to play
important roles in up-regulating HO-1, including metal re-
sponse elements (MREs), stress response elements (StREs),
AP-1, and NF-?B, etc. (12). However, only one transcription
factor, Bach1, has been demonstrated to have a repressor
effect on HO-1 expression (13). Modulation of HO-2 expres-
sion has only been observed in response to glucocorticoids
(14). Recently, HO-2 has been demonstrated to be part of a
calcium-sensitive potassium channel complex and functions
as an oxygen sensor (15).
DNA methylation is the major epigenetic modification that
can profoundly affect tissue-specific gene expression, X-chro-
mosome inactivation, genomic imprinting, immobilization of
mammalian transposons, and suppression of transcriptional
noise (16,17). In mammals, this modification occurs mainly on
cytosines in CpG dinucleotides located in CpG-rich DNA
fragments, the so-called “CpG islands.” These islands primar-
ily reside at the transcription start site of housekeeping genes
and are also associated with tissue-specific genes. In general,
it is believed that global demethylation occurs early in mam-
malian embryos after fertilization and until the blastocyst
stage. DNA methylation is then restored quickly after implan-
tation and maintained thereafter in somatic lineages (16).
These tissue-dependent and differentially-methylated regions
(T-DMR) are widely distributed and abundant in the normal
mouse genome and believed to be involved in the establish-
Received March 23, 2006; accepted July 4, 2006.
Correspondence: Hui Zhao, Ph.D., Research Associate, Department of Pediatrics,
Stanford University School of Medicine, 300 Pasteur Drive, Room S230, Stanford, CA
94305-5208; e-mail: firstname.lastname@example.org.
This work is supported by the National Institutes of Health grants HL58013 and
HL68703 and unrestricted funds from the Hess Research Fund, the Mary L. Johnson
Research Fund and Philips Medical Corp.
Abbreviations: BLI, bioluminescence imaging; HO, heme oxygenase; MSP,
methylation-specific PCR; Q-MSP, quantitative methylation-specific PCR
Copyright © 2006 International Pediatric Research Foundation, Inc.
Vol. 60, No. 5, 2006
Printed in U.S.A.
ment of cell- and tissue-specific gene expression (18,19).
However, accumulating evidence refute the idea of the very
restricted window of time when methylation occurs, showing
that either methylation or demethylation occurs at many ages.
In this study, we first characterized the developmental
pattern of HO-1 expression in the cortex of a luciferase
reporter mouse (HO-1-luc) using in vivo bioluminescence
imaging (BLI). We then systematically assessed the expres-
sion profiles of HO-1 and HO-2 in the developing cortex by
determining mRNA and protein. We further focused on the
mechanism of HO-1 down-regulation since HO-2 expression
was found relatively stable throughout ages, suggesting that
HO-2 was constitutively expressed. After identifying a CpG
island region in the HO-1 (but not in the HO-2) promoter, we
hypothesized that the methylation status of the CpG dinucle-
otides in this region was involved in the postnatal down-
regulation of HO-1 expression in the mouse cortex. We also
explored the role of Bach-1 in the regulation of HO-1 during
MATERIALS AND METHODS
Animals. The study was approved by the Institutional Animal Care and
Use Committee at Stanford University. All transgenic HO-1-luc mice, with
the transgene containing a 15-kb HO-1 promoter driving expression of the
reporter gene luciferase, were obtained from the Stanford Transgenic Animal
Facility (Stanford, CA). FVB mice were obtained from Charles River Labo-
ratories (Wilmington, MA).
Reagents. Luciferin (150 mg/mL, BioSynth, Naperville, IL) was dissolved
in water and filter-sterilized. Nembutal (50 mg/mL) was diluted 1:9 (5
mg/mL) for adults or 1:19 (2.5 mg/mL) for newborns up to 10g, with normal
In vivo bioluminescence imaging (BLI). Adult mice were anesthetized
using Nembutal (25–70 mg/kg BW) and imaged as previously described (20).
Because mice ?14d are virtually immobile, anesthesia was not required. Each
newborn was placed into a well of a six-well cell culture plate. All animals
were imaged using the In Vivo Imaging System (IVIS™, Xenogen Corp,
Alameda, CA) as previously described (21). Total photon emission from the
head region (representing the brain) was quantitated and plotted as fold
increase from adult (35d) levels.
Tissue preparation. Brains were immediately removed from animals,
cortex isolated and then divided as follows: 1) Whole portions were placed in
RNAlater (Qiagen, Germany) and stored at –20°C for total RNA; and 2) 100
? 2 mg tissue were diced and sonicated in nine volumes of buffer (22) and
stored at –80°C with protease inhibitors (Sigma Chemical Co., St. Louis,
MO) for Western blots. Protein concentrations were determined using the
Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA).
RT-PCR. Total RNA was extracted using the RNAeasy Mini Kit (Qiagen)
following the manufacturer’s instructions. HO-1, HO-2, and Bach1 mRNA
levels were quantified using Quanti-Tect SYBR Green RT-PCR kit (Qiagen),
performed in an iCycler iQ Real-Time Detection System (Bio-Rad) using
primers shown in Table 1. Standard mRNAs for HO-1, HO-2, and Bach1 were
synthesized from pRC-CMV-HO1 and pRC-CMV-HO2 using the Riboprobe
In Vitro Transcription System (Promega Corp., Madison, WI). The concen-
tration (g/?L) of the resulted standard RNA was measured by OD260and the
copy number was calculated by following formula:
Y (copies/?L) ? [X g/?L RNA/(transcription Length In Nucleotide ? 340)]
? 6.022 ? 1023.
Copy numbers of HO-1, HO-2, and Bach1 mRNA for each sample was
calculated from the standard curve and then normalized to ?-actin levels
(Figs. 2 and 6).
Western blot. 100 ?g of the sonicates was boiled for 10 min in protein
loading buffer and electrophoresed. Proteins were transferred to a PDVF
membrane (Bio-Rad) using a semidry transblotter. The membrane was probed
with HO-1 and HO-2 polyclonal antibodies (1:1000, Stressgen Corp., Victo-
ria, Canada) and protein levels quantitated by densitometry as previously
described (21). Coomassie blue staining and/or ?-actin immunoblotting were
performed to confirm equal loading of samples.
Identification of CpG island. A CpG island was identified using CpG Plot
program (http://www.ebi.ac.uk/emboss/cpgplot/) designed by EMBL-EBI
(European Bioinformatics Institute). DNA sequences (? 5kb) were screened
each time and plotted with the regions as defined CpG islands by the program.
In vitro methylation of the HO-1 BAC clone. BAC clone (rp21-411019)
containing the HO-1 gene was created from the RPCI mouse PAC library 21
(Children’s Hospital Oakland Research Institute, CHORI, Oakland, CA) and
its sequence created from the GeneBank AC005290. The BAC DNA was
tested to be unmethylated. To synthesize a positive control DNA for the
methylated CpG island, BAC DNA was treated with SssI methylase (New
England BioLab, Beverly, MA) and the completion of CpG methylation was
confirmed by digestion with BstUI.
Sodium bisulfite treatment of DNA and methylation-specific PCR (MSP).
Genomic DNA was isolated, treated with sodium bisulfite and followed by
methylation-specific PCR (MSP) and sequencing, as described (http://
www3.mdanderson.org/leukemia/methylation). In brief, genomic DNA was
isolated using the DNeasy Tissue Kit (Qiagen) and 2-?g were treated with
sodium bisulfite using the EZ DNA Methylation kit (ZYMO Research Corp.,
Orange, CA). Two ?L of resulting DNA was used as a template for the nested
MSP reaction. The first PCR step was: 95°C 15min; 94°C 30s, 53°C 30s, and
72°C 30s, 35 cycles; 72°C 5min. Program used for the second PCR reaction
was: 95°C 15min; 94°C 30s, 67°C 30s, and 72°C 45s, 35 cycles; 72°C 5min.
The products were then separated and analyzed by electrophoresis on 1.8%
Table 1. Primers used in this study
Accession # Primer SequencePCR Product
HO-1NM-010442(F) 5? CCTTCCCGAACATCGACAGCC 3?
(R) 5? GCAGCTCCTCAAACAGCTCAA 3?
(F) 5? GGAGGGGGTAGATGAGTCAGA 3
(R) 5? TCGGTCATGTGCTTCCTTGGT 3?
(F) 5? GGAGCAGGACTGTGAGGTGAA 3?
(R) 5? GGATTGGAAATCATTTCGTGAGA 3?
(F) 5? AAGGAGATTACTGCTCTGGCTCCTA 3?
(R) 5? ACTCATCGTACTCCTGCTTGCTGAT 3?
?-Actin NM-007393149 bp
MSP & Quantitative MSP
(F) 5? GAGTTATATGATTTATTTTTTTATAGGT 3?
(R) 5? CTCCATCACCAAACTAAACTAAT 3?
Methylated DNA (F) 5? CGGTTATTACGTGATTCGCG 3?
(R) 5? AACTCCGAACTATACTCGAAACG 3?
(F) 5? TTGTGTATTTAAAGGGTTGGTGTG 3?
(R) 5? CCAAACTATACTCAAAACAACTCTACACA 3?
Unmethylated DNA123 bp
HO EXPRESSION IN THE DEVELOPING CORTEX
Quantitative methylation-specific PCR (Q-MSP). The percentage of
methylated to unmethylated DNA was determined by Q-MSP using the
QuantiTect SYBR Green Real-time PCR Kit (Qiagen). A BAC plasmid,
treated or not treated with SssI methylase, was used to generate a standard
curve for methylated or unmethylated PCR, respectively. Dilution of the first
PCR product was amplified in real-time using MSP primer sets (Table 1).
PCR was run as follows: 95°C 15min; 94°C 15s, 60.5°C 30s, and 72°C 30s,
Calculations. All data are presented as mean ? SD. ANOVA analysis was
performed to determine significant differences as defined as p ? 0.05.
In vivo HO-1 transcription in the developing cortex. We
have observed using in vivo BLI that newborn mice have high
HO-1 transcriptional activity in contrast to that reported for
adult mice. To characterize the expression pattern, we as-
sessed brain HO-1 transcriptional activity in HO-1-luc mice
ranging from neonate to adult using BLI (Fig. 1). HO-1
transcription was highest (20-fold) in 1d-old mice, and then
progressively decreased to an average of 4-fold during 2–7d of
age, and finally falling to adult levels by 14d of age. Although
high individual variations of light emission were found, the
downward trend in HO-1 transcription was preserved among
all mice imaged.
Down-regulation of HO-1 in the developing cortex. To
investigate the mechanism of the developmental pattern of
HO-1 transcription, we analyzed the transcriptional profiles of
HO-1 and HO-2 mRNA in wild-type FVB mice, at embryonic
ages E14 and E18, 1d (neonate), 2 wks, and 6 wks (Fig. 2A).
HO-1 mRNA levels declined with age with the highest ex-
pression at E14 (7-fold, relative to 6-wk-old levels, p ?
0.0001), followed by a gradual decline during the perinatal
period (3-fold, p ? 0.02), and reaching the lowest levels at
adulthood. In contrast, HO-2 mRNA levels remained rela-
tively stable throughout all ages as expected and was the
predominant isoform at all ages (p ? 0.005, HO-1 versus
HO-2 for each age).
Down-regulation of cortical HO-1 protein was confirmed
by Western blot (Fig. 2B). Compared with adult levels, HO-1
protein was the highest (10-fold) at 1d of age, followed by a
gradual decrease, reaching lowest levels by day 28. Mean-
while, HO-2 protein levels were fairly constant over the age
range studied (data not shown) .
Expression of brain HO-1 and HO-2 among adult tissues.
To investigate whether the HO-1 expression profile during
brain development was unique to the developing cortex, we
assessed HO-1 expression in adult spleen, bone marrow,
kidney, liver, heart, testis, lung, and cortex. HO-1 was ex-
pressed variably among tissues – with the highest expression
found in the spleen and bone marrow. Minimal expression was
found in the cortex – more than 5-fold less than that of other
tissues (p ? 0.05), more specifically, 36-fold less than spleen
(p ? 0.0001) and 23.5-fold less than bone marrow (p ?
0.0001, Fig. 3A). HO-2 mRNA levels in all tissues were
relatively stable, except in the testis, which had 5- to 10-fold
higher levels than that of other tissues (p ? 0.0001).
When HO-1 protein levels were determined (Fig. 3B), no
protein was detected in the cortex. Spleen showed 2–3-fold
more HO-1 protein than that of liver, kidney, and testis, and
15-fold more than that of lung. The bands from heart samples
were slightly up-shifted, suggesting some post-translational
modifications of HO-1 protein in heart. HO-2 protein has a
different pattern, with the highest levels in adult testis fol-
lowed by adult brain (Fig. 3B).
When the relative ratio of HO-1 to HO-2 mRNA levels in
each tissues were compared (Table 2), HO-1 mRNA levels
were higher in the spleen (3.3-fold, p ? 0.05) and bone
marrow (1.6-fold, not significant) only. In contrast, HO-2 was
the predominant isozyme in the cortex (13.2-fold, p ? 0.01),
testis (10.7-fold, p ? 0.005), liver (1.9-fold, p ? 0.05), and
lung (1.5-fold, p ? 0.05).
Identification of a CpG island in HO-1 promoter. A 15-kb
upstream region of HO-1 coding sequence has been shown to
play a critical role in the transcriptional regulation of HO-1
(11). We identified a CpG-rich cluster of 283-bp in the
promoter and transcriptional start sites consisting of 25 CpG
dinucleotides located between 110 bp upstream and 172 bp
downstream of the transcriptional start site (Fig. 4). This DNA
sequence possesses all the properties of a target for methyl-
ation, which includes a composition of C and G of over 50%
and an observed/expected ratio of over 0.6 for a minimum of
200 bases. The region contains several putative transcriptional
factor binding sites, including Sp-1, C/EBP, AP-4 MYC/
MAX, TATA, and AP-2. Thirteen CpG dinucleotides are
located downstream of the transcriptional start site and among
them, three in Exon 1.
When we screened for the putative CpG rich cluster in the
upstream region of HO-2 coding sequence, no CpG islands
Hypomethylation of the HO-1 CpG island. To study the
possible involvement of DNA methylation in the down-
regulation of HO-1 expression, MSP was performed using two
sets of PCR primers, each of which can only recognize either
methylated or unmethylated DNA (Fig. 5A). Figure 5B shows
the presence of only unmethylated bands and the absence of
methylated bands in all tissues, which suggests that the HO-1
CpG island is hypomethylated and preserved throughout all
Figure 1. Transcriptional pattern of HO-1 expression in the developing
mouse cortex. In vivo HO-1 transcription levels in the developing mouse
cortex were assessed by BLI in mice aged 1–35d of age (n ? 3 for each age).
Representative whole body images are shown in the same scale (rainbow).
Photon emission was quantitated from the head region and normalized to day
35 levels. *p ? 0.001.
ZHAO ET AL.
tissues even in the developing cortex. After performing MSP
sequencing, we found that all of 25 CpG dinucleotides were
uniformly unmethylated (data not shown).
Because every tissue is a mixed population of different cell
types, it is virtually impossible to detect and quantify the small
percentage of the methylated DNA in the pool of mostly
unmethylated DNA using the traditional MSP method. There-
fore, we adapted a method called Q-MSP using real-time PCR
(23,24), a method that is very sensitive, capable of detecting 1
molecule of methylated DNA in 104of unmethylated DNA.
Using this methodology, we found that most samples con-
sisted of completely unmethylated DNA. Only embryonic
samples (E14 and E18 cortex, and E18 liver) contained a low
percentage of methylated DNA, ranging from only 0.1–15%
(Table 3). This low frequency of DNA methylation was not
found in any of the adult tissues studied.
Bach1 expression in the developing mouse cortex. To
explore another possible mechanism for the observed down-
regulation of HO-1 in the adult cortex, we investigated the role
of Bach1, the only identified repressor of HO-1 transcription
(25). We hypothesized that expression of Bach1 levels should
increase with increasing age. When Bach1 mRNA levels in
the developing cortex were measured using real-time RT-
PCR, we found that Bach1 expression was highest at age E14
and decreased progressively during development (Fig. 6).
Characterization of the developmental expression profiles
of HO isozymes is fundamental for the understanding of their
physiology functions and roles in age-related diseases. In this
study, we identified age-related expression patterns of HO
isozymes in the mouse cortex. HO-1 expression is high in
early fetal ages (E10 and 14), progressively decreases in
perinatal period, and then reaches lowest levels by adulthood;
while HO-2, the predominant isoform in brain, remains rela-
tively stable throughout development (Fig. 2). This ontogeny
of the mouse cortex is unique and distinct from the expression
profiles of other tissues, such as liver (26) and spleen. Al-
though HO-1 in the neonatal cortex has a similar mRNA
Figure 2. mRNA and protein levels of HO-1 and HO-2 in the developing
mouse cortex. (A) HO-1 (Œ) and HO-2 (e) mRNA levels (mean ? SD) in the
cortex from mice at ages E14, E18, newborn, 2 wks, and 6 wks (n ? 3 for
each age) were determined by real-time RT-PCR, expressed as copies/?g total
RNA, and normalized to ?-actin. (B) Western blots of HO-1 protein levels in
the developing mouse cortex. Each bar graph represents band intensities
compared with 35d levels. * p ? 0.0001 vs. all ages; † p ? 0.02 vs. 6 wk.
Figure 3. Tissue-specific expression HO-1 and HO-2 mRNA and protein
levels in adult mice. HO-1 (left) and HO-2 (right) mRNA (A) and protein (B)
levels from adult spleen (f), bone marrow ( ), kidney ( ), liver ( ), heart
RT-PCR and Western blot. * p ? 0.02, † p ? 0.0001 vs. all tissues; **p ?
0.025 vs. liver. Each bar graph represents the mean value of n ? 3 Western
??), testis (;), lung ( ), and cortex ( ) were determined by real-time
Table 2. Summary of HO isozyme mRNA expression in adult
Spleen Bone Marrow Kidney Liver Heart Testis Lung Cortex
1:1.9 1:1.6 1:10.7 1:1.5 1:13.2
Values are expressed as fold change relative to the cortex.
Figure 4. CpG island in the HO-1 promoter region. A 283-bp region con-
sisting of 25 CpG dinucleotides was identified in the HO-1 promoter, located
between 110-bp upstream and 172-bp downstream from the transcriptional
start site. DE1 and DE2, distal enhancers-1 and -2, respectively; PE, proximal
enhancer; and P, promoter.
HO EXPRESSION IN THE DEVELOPING CORTEX
expression level as the neonatal liver (data not shown), its
adult level is significantly less (87%) than that of the adult
liver. Moreover, cortical HO-1 is the lowest among all other
adult tissues tested suggesting that HO-1 expression is highly
suppressed during cortical maturation. Our data are consistent
with the findings of Bergeron et al. (27), which showed the
minimal HO-1 staining in adult rat brain endothelium, white
matter, and cortex by immunocytochemistry.
We speculated that suppression of HO-1 transcription in the
adult cortex might be controlled in a cell type- and region-
specific manner. Others have shown that in brains exposed to
stresses, such as hyperosmotic insult (28), heme administra-
tion (29), and ischemic injury (30), HO-1 expression is in-
duced only in astrocytes and microglia. HO-1 silencing phe-
nomenon was also reported in the study by Chauveau et al.
(31), in which HO-1 expression drastically decreased during
human and rat dendritic cell maturation induced in vitro as
well as in human tissue. They also found that prevention of
HO-1 silencing could inhibit LPS-induced dendritic cell phe-
notypic maturation and secretion of pro-inflammatory cyto-
kines, suggesting the necessity of HO-1 silencing in some cell
types for their function.
There are several mechanisms for gene silencing, one of
which is DNA methylation of CpG islands. Our data shows
that hypomethylation of the HO-1 CpG island in all tissues
tested, suggesting that methylation does not occur, even in the
less populated cells in the adult cortex. In addition, by se-
quencing this region, no
island (data not shown) were found, indicating that there is no
T-DMRs in HO-1 promoter. Therefore, we conclude that CpG
island methylation is not associated with HO-1 down-
regulation, and other mechanisms must be involved. To fur-
ther investigate other mechanisms, which may regulate this
down-regulation, we measured the expression of Bach1, the
only identified transcriptional repressor of HO-1 (25). We
found that Bach1 mRNA levels actually paralleled that of
HO-1 and therefore does not appear to silence HO-1 expres-
sion in the adult cortex (Fig. 6). We speculate that reduction of
transcriptional activators, such as Nrf-2, might be involved in
the down-regulation. Other possibilities might be that there is
a limited amount of heme in adult neuronal cells or induction
of multi-drug resistant protein (MDR) during cortical matura-
tion, which functions as a filter to block harmful agents from
In neonatal mice, an increase in HO-1 expression may be
the result of an abundance of heme, both as a substrate and
inducer of HO-1, released from senescing fetal erythrocytes,
which have a shortened life span during neonatal period. In
addition, Bergeron et al. (27) has shown that HO-1 expression
was cell-type specific in the brain, which suggests that HO-1
up-regulation may be due to the differentiation, proliferation
and redistribution of neuronal cells in the developing mouse
cortex. Moreover, the inducibility of HO-1 can also be influ-
enced by endocrine factors (26).
We have observed co-expression of both isozymes in all the
adult tissues collected, but their relative levels were quite
mCpG dinucleotides in the CpG
Figure 5. Methylation-specific PCR for assessing methylation patterns of
HO-1. (A) Primer design for MSP-PCR and methylation-specific sequencing.
Each filled circle represents a CpG dinucleotide and its position in the island.
The position and direction of primers for the nested PCR are shown with
arrows; (B) Methylation status in the (top panel) developing cortex: E14 and
E18 (n ? 6), neonate (n ? 3), 2 wk- and 6-wk-old mice (n ? 3), and in the
spleen, bone marrow, liver, kidney, testis, lung, and cortex of adult mice
(6-wk-old, n ? 3, middle panel) as assessed by MSP-PCR. Bottom panels
show validation of the method using control DNA: HO-1 PAC plasmid DNA
(unmethylated DNA, U) or the plasmid DNA treated with SssI methylase
(methylated DNA, M).
Table 3. Summary of quantitative measurements of methylated
and unmethylated CpG islands in the developing cortex
Percentages are shown as methylated to unmethylated.
Figure 6. Expression pattern of Bach1 in the developing mouse cortex.
Bach1 mRNA levels were determined by real-time RT-PCR in cortex of mice
at ages E14, E18, neonate, 2 wks, and 6 wks (n ? 3 for each age). Data were
calculated as copies/ng total RNA, normalized to ?-actin, and shown as fold
induction (mean? SD) from adult levels. *p ? 0.05.
ZHAO ET AL.
different. HO-2 expression exceeded that of HO-1 in most
organs, especially in the cortex (at all ages) and testis;
whereas, in the spleen and bone marrow, HO-1 was present
more abundantly than HO-2 (Table 2). Besides their function
in maintaining cellular homeostasis, each isozyme has its
unique tissue-specific role. In the cortex, the two isozymes
display distinct regional and cellular distributions with HO-1
present only in a few cell subtypes and HO-2 being very
widespread (32–35). The inducible HO-1 plays a mostly
protective role in the CNS, while the constitutively-expressed
HO-2 is important for maintaining neuronal function, proba-
bly by producing CO, a putative neurotransmitter (36,37).
High expression levels of HO-2 in the testis may be associated
with rodent male reproduction (38). HO-1 is expressed abun-
dantly in spleen and bone marrow, where senescent red blood
cells are sequestered and heme degraded. Moreover, HO-1
and its byproducts have anti-inflammatory and anti-apoptotic
properties, all which function in immune cell maturation and
Although HO-1 and HO-2 have been extensively studied,
the physiologic relevance of HO-1 and HO-2 and their recip-
rocal interrelationship is not quite fully understood. Our data
suggested that these two isozymes are regulated independently
in developing and adult tissues with unique physiologic roles.
Yet, their combined activities control tissue heme catabolism.
Therefore proper balance of the expression of both isozymes
is key to maintaining tissue integrity and homeostasis.
Acknowledgments. We thank Drs. Stacy Burns-Guydish
and Ichiro Morioka for their help with tissue sample collec-
tion. We also thank Drs. Weisheng Zhang and Aida Abate for
their critical reviews of the manuscript. We are indebted to Dr.
Kazuhiko Igarashi for providing us with the Bach1 antibody.
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HO EXPRESSION IN THE DEVELOPING CORTEX