1Department of Medicine, Division of Nephrology, Hypertension, and Transplantation, and 2Department of Neuroscience, University of Florida, Gainesville, Florida 32610
Submitted 4 April 2003 ; accepted in final form 21 May 2003
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ABSTRACT |
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gene transcription; renal proximal tubule cells; endothelial cells; heme proteins; molecular regulation
HO opens the heme ring, producing equimolar quantities of biliverdin, iron,
and carbon monoxide (CO). Biliverdin is subsequently converted to bilirubin by
biliverdin reductase. Recent studies have highlighted the important biological
effects of the HO reaction product(s) that possesses antioxidant,
anti-inflammatory, and antiapoptotic functions (reviewed in Ref.
21). Two isoforms of HO have
been characterized: an inducible enzyme, HO-1, and a constitutive isoform,
HO-2 (28). A putative isozyme,
HO-3, isolated from rat brain and sharing 90% homology with HO-2, has
also been described (30). HO-1
and HO-2 are products of different genes and share
40% amino acid
homology (28). We and others
demonstrated that induction of HO-1 by chemical inducers
(2,
32) or selective
overexpression (1,
41) is cytoprotective both in
vitro and in vivo, findings that have been further substantiated by studies in
HO-1 knockout mice and a patient with HO-1 deficiency
(34,
41,
48).
The mechanisms underlying HO-1 induction are complex and tightly regulated at the transcriptional level. Adding to the complexity, regulation of the HO-1 gene is species and cell specific. The promoter of the rat HO-1 gene, for instance, has a heat shock responsive element that is not functional in the human HO-1 gene (50). A GT repeat region identified at position -258/-198 in the human HO-1 promoter is absent in the mouse HO-1 gene (49). Interestingly, length polymorphisms of this GT repeat correlate with disease development in atherosclerosis (16), emphysema (49), and vascular restenosis (18) in humans.
The human HO-1 gene, located on chromosome 22q12
(26), has five exons and spans
14 kb. A potential cadmium response element (TGCTAGATTT) has been
identified between -4.5 and -4.0 kb of the 5'-flanking region
(44) of the human HO-1 gene;
however, the cadmium response element in the mouse HO-1 gene is located
downstream of this sequence
(13). A threefold increase in
luciferase activity was reported with a -4.5-kb human HO-1 construct in
response to cadmium, with only a minimal response to hemin, sodium arsenite,
or cobalt protoporphyrin in HeLa cells
(44). We previously showed
that the identical 4.5-kb human HO-1 gene promoter fragment responds, in part,
to both heme and cadmium with a two- to threefold increase in reporter gene
activity compared with
20- to 30-fold increase in endogenous HO-1 mRNA in
human aortic endothelial and renal proximal tubular cells
(4). Furthermore, this 4.5-kb
promoter fragment does not respond to other stimuli such as
13-hydroperoxyoctadecadienoic acid (13-HPODE), hydrogen peroxide
(H2O2), and hyperoxia that directly increase de novo
HO-1 gene transcription (4,
19). We report here the
identification of an enhancer region internal to the human HO-1 gene that,
together with the 4.5-kb promoter, recapitulates levels of heme and cadmium
induction observed using steady-state Northern analysis of the endogenous
gene.
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MATERIALS AND METHODS |
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Cell culture. Human renal proximal tubule cells (HPTC) (Clonetics,
Walkersville, MD) were grown in renal epithelial basal medium supplemented
with 5% FBS, gentamicin (50 µg/ml), amphotericin B (50 µg/ml), insulin
(5 µg/ml), transferrin (10 µg/ml), triiodothyronine (6.5 ng/ml),
hydrocortisone (0.5 µg/ml), epinephrine (0.5 µg/ml), and human epidermal
growth factor (10 ng/ml). These cells are positive for
-glutamyltranspeptidase, an enzyme marker for proximal tubule cells.
The presence of microvilli, abundant mitochondria, lysosomes, and endocytotic
vacuoles, morphological features of proximal tubule cells, were confirmed by
electron microscopy in these cells. Human aortic endothelial cells (HAEC)
[derived from segments of human aorta obtained from heart transplant donors
and previously characterized by positive staining for factor VIII-related
antigen and acetylated LDL
(46)] were grown in
endothelial basal medium (Clonetics), supplemented with 10% FBS, gentamicin
(50 µg/ml), amphotericin B (50 µg/ml), hydrocortisone (1 µg/ml),
human epidermal growth factor (10 ng/ml), and bovine brain extract (6
µg/ml) at 37°C in 95% air-5% CO2. Studies were performed
over a range of no more than six to eight passages. Sixteen hours before
treatment with HO-1 inducers, growth media was changed to a 0.5 and 1%
FBS-containing media for HPTC and HAEC, respectively.
Preparation of HO-1 inducers. Hemin, CdCl2, and
13-HPODE were prepared fresh daily. A stock solution of hemin (1 mM) was
prepared using 10 mM NaOH. CdCl2 (5 mM stock solution) and
H2O2 (10 mM stock) were prepared in water and sterile
filtered. 13-HPODE was prepared as previously described
(4). Lyophilized
TGF-1 was dissolved in 4 mM HCl containing 0.1% bovine serum
albumin to obtain a 2 ng/µl stock solution, aliquoted, and stored at
-80°C.
Cloning and characterization of a human HO-1 genomic clone. A 4.5-kb fragment of the 5'-flanking region of the human HO-1 gene, including the transcription initiation site, was generated by long-range PCR (PerkinElmer, Foster City, CA) using human genomic DNA as a template, as described previously (4). To obtain additional genomic sequences for the human HO-1 gene, two single-copy probes were designed, one from the -4.5/-4.0-kb region and one from an internal exon, and a commercially available human P1 bacteriophage library (Genome Systems, St. Louis, MO) was screened. Two P1 clones (P1 11715 and 11716) were identified by both probes. These clones were characterized by restriction map and sequence analysis, and the presence of the entire human HO-1 gene, as well as >15 kb of the 5'-flanking region, was verified in P1 11716. More recently, the efforts of the human genome project resulted in the sequencing of a bacterial artificial chromosome (BAC) clone, containing portions of chromosome 22 (accession no. Z82244 [GenBank] ). With the use of this BAC clone, sequences of the human HO-1 gene promoter fragments were confirmed, and larger fragments, further upstream, were generated by long-range PCR and restriction digestion (see Fig. 2).
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Plasmid construction. Five'-flanking promoter fragments of
the human HO-1 gene, extending from -4.0, -4.5, and -9.1 kb to +80-bp
position, were generated using oligonucleotide primers
(Table 1) specific to the HO-1
gene by long-range PCR (PerkinElmer). The 4.0- and 4.5-kb PCR products were
ligated into TA cloning vectors (Invitrogen, Carlsbad, CA), and the 9.1-kb PCR
product was cloned into pCR-XLTOPO (Invitrogen). BamHI sites were
incorporated in the primers for the 4.0- and 4.5-kb fragments and
SalI sites were incorporated in the primers for the 9.1-kb fragment
to enable subcloning into a promoterless human growth hormone (hGH) reporter
gene, generating pHOGH/4.0, pHOGH/4.5, and pHOGH/9.1. A 155-bp HO-1 promoter
hGH plasmid was generated by deletion of a 4.345-kb HO-1 promoter fragment
using NdeI followed by religation, resulting in the formation of
pHOGH/155. The 4.5-kb promoter fragment was also cloned into the
BglII site in a luciferase reporter (pGL3) generating pHOGL3/4.5. The
9.1-kb promoter fragment (with SalI sites on each end) was cloned
into the XhoI site of pGL3, generating pHOGL3/9.1. A 3.5-kb HO-1
promoter reporter vector was generated by cutting the pHOGL3/9.1 with
NheI, which cuts HO-1 at 3.5 kb 5' from the start site and
cuts pGL3 once 5' from the XhoI site, and religating to
generate pHOGL3/3.5.
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The entire +1- to +12.5-kb region of the human HO-1 gene (including all exons and introns) was generated using long-range PCR with oligonucleotide primers specific to the HO-1 gene (Table 1). The 12.5-kb PCR product was cloned into pCR-XL-TOPO and then subcloned into pHOGH/4.5, pHOGL3/4.5, or pHOGL3/3.5, generating pHOGH/4.5/+12.5, pHOGL3/4.5/+12.5, and pHOGL3/3.5/+12.5. SalI sites were included at each end of the 12.5-kb fragment to enable cloning into the SalI site of the hGH and luciferase vectors. We obtained both 5'-3' and 3'-5' orientations of the +12.5-kb fragment in the hGH vector containing the -4.5-kb HO-1 promoter region [pHOGH/4.5/+12.5 and pHOGH/4.5/+12.5 (3'-5'), respectively]. The +12.5-kb fragment was also cloned into the HindIII site of an hGH vector containing a heterologous herpes virus thymidine kinase (TK) minimal promoter to generate pHOTKGH/+12.5. All constructs were verified for orientation by sequencing and restriction analysis.
Transfection of the reporter gene. HPTC and HAEC were transiently
transfected with the reporter gene vectors by the DEAE-dextran method using a
batch transfection protocol
(4). The DEAE-dextran method
was optimized to achieve reproducible transfection efficiency (20-25%) in
HPTC and HAEC as assessed by cotransfection with pcDNA3.1/Lac-z (Invitrogen).
The transfection protocol for HPTC was as follows: 60-70% confluent cells in
150-mm tissue culture plates were washed once with HBSS and once with
Tris-buffered saline solution (TBS). A 1.0-ml solution containing equimolar
amounts of the plasmid(s) (normalized to 8.1 µg of pHOGH/4.5) and
DEAE-dextran (10 mg/ml) was added per plate and rocked for 1 h at room
temperature. The cells were shocked for 1 min at room temperature with 10%
DMSO in TBS and then washed once with HBSS. The cells were incubated in
complete media (18 ml/plate) containing chloroquine diphosphate (100 µM) to
inhibit lysosomal degradation of the DNA. After 4-h incubation at 37°C in
5% CO2-room air, the cells were washed twice with HBSS and
incubated in normal growth media overnight.
The transfection protocol for HAEC was as follows: 60-70% confluent cells in 150-mm tissue culture plates were washed once with PBS. Complete media containing 10% NuSerum (BD Biosciences, San Jose, CA) was added to the plates. The transfection solution, 1.5 ml DEAE-dextran solution (10 mg/ml) containing equimolar amounts of the plasmid(s) (normalized to 8.1 µg of pHOGH/4.5), was added to each dish dropwise and then gently swirled to ensure that the DNA/DEAE-dextran was uniformly applied to the cells. Chloroquine diphosphate (100 µM) was then added to the medium. Cells were incubated for 4 h at 37°C in 5% CO2-room air and then shocked for 1 min at room temperature with 10% DMSO in PBS, washed twice with PBS, and incubated in growth media containing 10% FBS overnight.
At twenty-four hours posttransfection, the cells (both cell types) were passaged from 150-mm dishes into several 100-mm dishes (for hGH assays) or 12-well trays (for luciferase assays) to ensure equal transfection efficiency between experimental treatments. Cotransfection with pcDNA3.1/Lac-z demonstrated equal amounts of DNA in each of the wells. After the cells were allowed to recover for 24 h, they were treated with stimulus or vehicle (control). Sixteen to seventy-two hours following stimulus exposure, cells were evaluated for hGH mRNA content by Northern analysis, levels of secreted hGH protein in the culture media, as described previously (4), and luciferase activity was monitored using the luciferase reporter assay system (Promega) according to the manufacturer's instructions. Luminescence was measured with a Sirius Luminometer (Berthold Detection Systems, Pforzheim, Germany).
RNA isolation, molecular probes, and Northern analysis. Total
cellular RNA was isolated by the method of Chomczynski and Sacchi
(17). Samples were
electrophoresed on a 1% agarose formaldehyde gel and electrotransferred to a
charged nylon hybridization membrane. The cDNA probes for hGH, human HO-1, and
human GAPDH were radiolabeled with [-32P]dATP using a random
primer labeling kit, according to the instructions of the manufacturer
(Invitrogen), and purified over a G-50 column (Amersham Pharmacia Biotech,
Piscataway, NJ). Membranes were hybridized overnight at 60°C, washed in a
high-stringency buffer (0.04 M sodium phosphate, 1 mM EDTA, 1% SDS) at
65°C, and subjected to autoradiography. To quantitate expression levels,
autoradiographs were scanned on a Hewlett-Packard ScanJet 4C using DeskScan II
software and densitometry was performed using National Institutes of Health
Image 1.63 software. Experiments were internally controlled by hybridization
with GAPDH, normalized, and expressed as a percentage of maximal expression.
All experiments were repeated with at least two to three independent RNA
preparations to show reproducibility.
Statistical analysis. Data are expressed as means ± SE. Statistical analyses were performed using ANOVA and the Student-Newman-Keuls test. All results are considered significant at P < 0.05.
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RESULTS |
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We previously demonstrated the importance of de novo transcription by
nuclear run-on analysis in HAEC and HPTC treated with hemin (5 µM) and
observed increased HO-1 gene transcription
(4). To evaluate the role of
message stability in the induction of HO-1 mRNA following stimulation with
hemin, the half-life of HO-1 mRNA was measured. Confluent HPTC were exposed to
hemin (5 µM) for 4 h and washed with HBSS followed by the addition of fresh
media containing actinomycin D (4 µM) with or without additional hemin. As
shown in Fig. 1D, the
half-life of HO-1 mRNA was similar (4 h) with and without additional
hemin treatment, suggesting that message stability is not involved. These
findings are consistent with previous studies in other cell types
(10) and enable us to use
these human primary cultured cells for further studies to evaluate the
transcriptional activation of the human HO-1 gene by hemin.
Identification of the enhancer region internal to the human HO-1
gene. To identify regulatory elements in the human HO-1 gene that mediate
its transcription following stimulation with hemin, multiple promoter
fragments (155 bp, 4.0 kb, 4.5 kb, and 9.1 kb) were generated and incorporated
into promoterless reporter vectors as shown in
Fig. 2 and described in
MATERIALS AND METHODS. Growth hormone mRNA levels, measured by
Northern analysis, were used as a direct measure of reporter gene
transcription rates. HAEC transfected with hGH vectors containing -155-bp or
-4.0-kb promoter constructs demonstrated no hGH mRNA induction in response to
hemin (data not shown) and the -4.5- and -9.1-kb constructs resulted in a
modest hemin-mediated induction in hGH mRNA (6.7- and
9.9-fold,
respectively), as shown in Fig.
3A.
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Because levels of reporter gene activity did not correlate with
steady-state Northern levels of HO-1 induction with hemin, we hypothesized
that additional regulatory sequences might exist within the HO-1 gene.
Therefore, a construct containing the 12.5-kb region internal to the HO-1 gene
with the 4.5-kb HO-1 promoter (pHOGH/4.5/+12.5) was generated. Unfortunately,
we were unable to subclone the 12.5-kb fragment with the 9.1-kb HO-1 promoter.
The construct, pHOGH/4.5/+12.5, demonstrated significant hemin- as well as
CdCl2-inducible hGH mRNA in HAEC (16.1- and 32.4-fold,
respectively; Fig.
3B). Similar results were observed in HPTC: pHOGH/4.5
yielded a 6.6- and 4.3-fold increase in response to hemin and
CdCl2, respectively, and pHOGH/4.5/+12.5 resulted in a 27- and
18-fold increase in response to hemin and CdCl2, respectively, over
control (untreated cells) transfected with pHOGH/4.5
(Fig. 3C). These
results indicate the presence of sequences internal to the HO-1 gene that
potentially function as an enhancer. Interestingly, the putative enhancer
region does not function for all known inducers of HO-1 as pHOGH/4.5/+12.5
failed to respond to TGF-1 (2 ng/ml),
H2O2 (200 µM), or 13-HPODE (25 µM)
(Fig. 3B).
Reporter activity, as measured by hGH protein or luciferase activity,
yielded results similar to hGH mRNA measurements. No promoter activity was
detected in cells transfected with hGH constructs containing -155-bp or
-4.0-kb promoter fragments and treated with hemin (5 µM)
(Fig. 4A). Cells
transfected with vectors containing the -4.5- or -9.1-kb promoter constructs
and exposed to hemin (5 µM) demonstrated a 3.6- and 4.5-fold, respectively,
increase in hGH protein over untreated cells
(Fig. 4A). The
construct containing the 12.5-kb region internal to the HO-1 gene, in
conjunction with the -4.5-kb HO-1 promoter region, demonstrated elevated basal
levels as well as significant hemin-inducible hGH protein levels,
5.1-fold over untreated cells (Fig.
4A). Luciferase reporter vectors containing HO-1 gene
fragments were generated to confirm the results with another reporter gene as
well as ease of cloning and simplicity of the assay. Luciferase/HO-1 vectors
yielded results similar to hGH reporter vectors in both HAEC and HPTC
(Fig. 4, B and
C, respectively).
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Orientation-independent effects of the HO-1 enhancer region. To determine whether the +12.5-kb fragment functions as a true enhancer, both orientations (5'-3' and 3'-5') were cloned into pHOGH/4.5 to give pHOGH/4.5/+12.5 and pHOGH/4.5/+12.5 (3'-5'). Similar levels of heme-mediated induction, as measured by Northern analysis of hGH mRNA, were observed, regardless of orientation (Fig. 5). In addition, to determine whether the enhancer could function with a heterologous promoter, the +12.5-kb fragment was cloned into a hGH vector containing a TK promoter (pHOTKGH/+12.5), which is a 200-bp minimal, TATA-containing promoter. Transient transfection of the 12.5-kb fragment in conjunction with a TK promoter failed to respond to hemin, as assessed by Northern analysis of hGH mRNA (data not shown), suggesting that the enhancer only functions with the HO-1 promoter.
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To further define the relevant sequences in the HO-1 promoter region that are required for enhancer function, the +12.5-kb enhancer fragment was evaluated with a 3.5-kb promoter of the human HO-1 gene pHOGL3/3.5/+12.5. As shown in Fig. 6, the +12.5-kb fragment requires the 4.5-kb HO-1 promoter fragment to function, because the 3.5-kb promoter with the +12.5-kb enhancer did not demonstrate any basal or hemin-inducible activity. The results demonstrate that enhancer sequences internal to the human HO-1 gene (within 12.5 kb), in conjunction with regions between -3.5 and -4.5 kb of the HO-1 promoter, mediate transcriptional activation of the human HO-1 gene by hemin and the enhancer is likely composed of a complex set of interacting elements.
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DISCUSSION |
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Thus far, studies on mammalian HO-1 gene regulation have focused mainly on the mouse HO-1 gene (5-13), where multiple inducer-specific stress response elements have been localized within 10 kb of the 5'-flanking region. Two distal promoter regions, E1 and E2, at -4.0 and -10 kb, respectively, are required for induction of the mouse HO-1 gene in response to heme, heavy metals, H2O2, and sodium arsenite (5-7, 13). It was proposed that activation of the mouse HO-1 gene by most stimuli is mediated exclusively via the E1 and/or E2 regions. These regions contain consensus binding sites for the NF-E2-related factor Nrf2, and overexpression studies coupled with mutations in these binding sites have implicated the involvement of Nrf2 in the induction of the mouse HO-1 gene (12, 20).
To test whether promoter elements in the 5'-flanking region of the
human HO-1 gene can analogously control heme-mediated induction, we initially
analyzed several human HO-1 promoter constructs up to -9.1 kb, which contain
sequences potentially similar to the E1 and E2 regions
in the mouse gene. The 4.5- and 9.1-kb promoter fragments demonstrated a
modest induction with heme; however, unlike the E1 and
E2 regions of the mouse gene, these constructs did not completely
recapitulate steady-state Northern levels of HO-1 induction. In addition,
unlike the mouse promoter, we also found that the human HO-1 promoter
constructs showed no response to other known HO-1 stimuli, including 13-HPODE,
hyperoxia, curcumin, and TGF-1, in human cultured cells
(4,
19, and unpublished
observations).
In our efforts to mimic endogenous, stimulus-dependent transcription
levels, we hypothesized that additional regulatory element(s) were necessary
for hemeand cadmium-mediated induction of the human HO-1 gene. To identify
other relevant regulatory sequences, we tested the entire +12.5-kb HO-1 gene,
including introns and exons, in conjunction with the -4.5-kb human HO-1
promoter and observed significant hemeand cadmium-dependent induction of
reporter activity. Furthermore, this enhancer region was specific for heme and
cadmium and did not function for other known HO-1 stimuli, such as
TGF-1, H2O2, or 13-HPODE. The large
+12.5-kb fragment, in part, satisfied the characteristics of a true enhancer
region by functioning in an orientation-independent manner. However, when the
HO-1 promoter was replaced with a heterologous TK promoter, no induction by
heme was observed, thus demonstrating that this region functions as a
gene-specific enhancer. This is substantiated by our findings that the
enhancer region requires a portion of the HO-1 promoter to mimic endogenous
levels of induction. Specifically, we demonstrated that a region between -4.5
and -3.5 kb is required for enhancer function. Our data also indicated the
importance of an additional region between -4.5 and -9.1 kb for heme-mediated
HO-1 promoter activity. However, we were unable to evaluate the +12.5-kb
enhancer in the context of the -9.1-kb promoter construct due to our inability
to successfully subclone both of these large fragments into a single plasmid.
Studies to further delineate the regulatory sequences within the enhancer
region are currently in progress. As these regions become more well defined,
constructs containing the relevant sequences within the enhancer will be
evaluated with the -9.1-kb HO-1 promoter.
Regulatory sequences functioning as enhancers have been identified within
introns of several genes, including manganese superoxide dismutase,
platelet-derived growth factor A, and alcohol dehydrogenase-1
(15,
37,
45). We performed sequence
comparisons with the 12.5-kb enhancer region and did not identify any
sequences that are linked to known heme or cadmium response elements. Alam and
Den (8) previously reported
that the region analogous to our enhancer region containing the entire protein
coding region of the mouse HO-1 gene coupled to the mouse HO-1 promoter did
not respond to heme or cadmium in rodent cells. Two possibilities could
account for the lack of response. On the one hand, the limited size (3.0
kb) of the mouse HO-1 promoter used in these studies may account for the
negative results (8). On the
other hand, perhaps it is a reflection of the differential regulation of the
mouse vs. human HO-1 genes.
Our observations indicate that the human HO-1 gene requires regulatory
sequences that differ from those previously implicated for the mouse gene.
Other interspecies differences in HO-1 gene regulation have also been noted.
For example, the human HO-1 promoter contains a GT repeat region that is
absent in the mouse HO-1 gene
(49). Deletion of this repeat
region results in a significant elevation of basal HO-1 promoter activity
(unpublished observations), similar to previously reported results using HO-1
promoter constructs with shorter GT repeats
(49), suggesting that this
human-specific region may function as a negative regulator of HO-1 gene
expression. Studies have also demonstrated that HO-1 is induced by hypoxia in
rat, bovine, mouse, and monkey cells but is rather repressed in human cells
(25,
27). Similarly, HO-1 has been
popularized as heat shock protein 32 based on its inducibility by heat shock
in rodent cells (38,
39). However, heat shock does
not induce HO-1 in human cells
(35). Although cytokines
induce HO-1 in rodent cells, previous studies have reported that
interferon- decreases human HO-1 gene expression
(43). Furthermore, a putative
cadmium response element has been identified in the human HO-1 promoter
(44); however, the cadmium
response element in the mouse HO-1 gene is immediately 3' to this region
and is an Nrf2 consensus sequence
(13). Collectively, these
observations suggest that the underlying molecular mechanisms that regulate
the human HO-1 gene are different from those described for mouse HO-1 gene
expression.
Recent studies have demonstrated that the expression of the mouse HO-1 gene is regulated through antagonism of transcription activators and the repressor Bach1 (42). Under normal physiological conditions, HO-1 expression is repressed by Bach1, and increased heme levels displace Bach1 from the E1 and E2 regions, allowing activators to bind to the regulatory sequences (42). The authors suggest that the transcriptional regulation of the mouse HO-1 gene involves a direct sensing of heme levels by Bach1, generating a feedback loop by the substrate, as in the lac operon. Studies to explore the involvement of Bach1 in human HO-1 gene regulation will be of interest.
In summary, our data provide evidence that at least two regions in the human HO-1 promoter, one between -4.5 and -4.0 and another between -9.1 and -4.5, are required for activation of the HO-1 gene by heme. Most importantly, we identified a novel transcriptional enhancer internal to the human HO-1 gene that, in conjunction with the HO-1 promoter, recapitulates a steady-state level expression of HO-1 by heme and cadmium. Studies on regulation of the human HO-1 gene are biologically relevant because of the well-documented beneficial effects of HO-1 activity that include degradation of heme (a toxic prooxidant), generation of bilirubin (an antioxidant), coinduction of ferritin (an intracellular repository for iron), and the antiapoptotic and anti-inflammatory effects of CO (36). We are continuing our investigation of the molecular mechanisms involved in the regulation of the human HO-1 gene in an effort to fine tune endogenous HO-1 gene expression to ultimately optimize its potential as a therapeutic modality.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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