1 Arthritis Center and Section of Rheumatology and 2 Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H2O2 and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H2O2 and CCCP on HO-1 mRNA. Addition of Fe2+, Fe3+, or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H2O2 or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.
stress response; messenger ribonucleic acid stability; iron depletion; scleroderma
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INTRODUCTION |
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HYPOXIA IS KNOWN TO modulate a number of cellular metabolic functions, including what has been called the stress response. One of the stress response proteins is heme oxygenase-1 (HO-1; EC 1.14.99.3) (33, 50), a rate-limiting enzyme in heme catabolism. It is responsible for converting a potential oxidant, heme, into a potential antioxidant, biliverdin (49), gaseous carbon monoxide, and free Fe2+. HO-1 is stimulated by hypoxia in certain cell types, including vascular smooth muscle cells, astrocytes, cardiomyocytes, and Chinese hamster ovary (CHO) cells (14, 37, 38, 51). Hypoxic induction of HO-1 in CHO cells is thought to be mediated by hypoxia-inducible factor-1 (HIF-1) (31). However, other mechanisms could be operative as well, since hypoxia induces HO-1 mRNA in a mutant CHO cell line defective in HIF-1 (54). The physiological role of HO-1 induction in hypoxia is still under investigation. It has been proposed that carbon monoxide resulting from HO-1 activity in hypoxic vascular smooth muscle cells plays a role in the regulation of the vessel tone via activation of soluble guanylyl cyclase (37). In the brain, carbon monoxide produced by another isoform of heme oxygenase, HO-2, as well as by HO-1, was implicated in the role of neurotransmitter, acting by a mechanism similar to NO (35, 47).
HO-1 is also induced by H2O2, ultraviolet (UV) irradiation, sulfhydryl reagents, divalent metal ions, and other oxidative agents known to stimulate free radical formation and/or GSH depletion in the cell (1, 3, 6, 34). HO-1 induction by H2O2 and UV irradiation is attenuated by the addition of cellular antioxidants (GSH, vitamin E), which suggests that free radicals generated by H2O2 and UV damage may have a signaling role in overexpression of HO-1 (3). There is a large body of evidence suggesting that increased HO-1 activity is beneficial for the cell and that its induction correlates with prolonged cells survival under environmental stress (11, 21, 40, 43, 48, 53). Thus embryonic cells from HO-1 knockout mice are significantly less resistant to the cytotoxicity induced by H2O2 than the wild-type controls. In vivo, HO-1 induction was found to inhibit inflammation and correlated with prolonged survival of cardiac xenografts. Recently, Hancock et al. (21) directly demonstrated a protective role for HO-1 in the prevention of chronic rejection in a mouse cardiac allograft model. Prior selective stimulation of HO-1 by cobalt protoporphyrin blocked induction of an inflammation marker, E-selectin, by oxidant or by alloantibody in cultured endothelial cells. Although the overall antioxidant and protective effect of HO-1 induction is well accepted, the precise mechanism of this process still remains unclear.
Tissue hypoxia is thought to play a role in systemic sclerosis (SSc; scleroderma), a disease characterized by dermal microvascular injury and decreased perfusion of tissues. Silverstein et al. (46) measured tissue oxygen pressure and found that sclerodermatous skin is hypoxic compared with the uninvolved skin of SSc patients or that of control individuals. In normal primary skin fibroblasts, hypoxic conditions induce collagen mRNA, suggesting that low oxygen pressure may contribute to the increased fibrogenic properties of SSc fibroblasts and to the pathogenesis of the disease (15). It is thus likely that hypoxia affects metabolism of SSc fibroblasts in vivo.
In the present study, we describe novel findings concerning the mechanism of HO-1 mRNA induction by hypoxia in skin fibroblasts. We demonstrate that free radicals are not involved in HO-1 induction by hypoxia, unlike the case in oxidative stress. Iron depletion by a specific iron chelator, desferrioxamine mesylate (DFAM), completely prevented hypoxic induction of HO-1, suggesting a role for an iron-dependent component in regulation of HO-1 mRNA by hypoxia. Treatment of cells by hypoxia resulted in strong stabilization of HO-1 mRNA, suggesting that specific posttranscriptional regulatory mechanisms are operative in hypoxia.
We show that normal and SSc skin fibroblasts respond to hypoxia by induction of the stress defense molecule HO-1. Fibroblasts from lesional SSc skin demonstrated greater induction of HO-1 mRNA than nonlesional SSc fibroblasts or normal fibroblasts. This suggests that chronic hypoxia, in vivo, may lead to long-term alterations in metabolism of stress response proteins.
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EXPERIMENTAL PROCEDURES |
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Cells and reagents. Normal human dermal fibroblast cell lines were obtained by punch biopsy from the forearm or upper arm of healthy donors. SSc fibroblast strains were derived from biopsies of areas of clinically involved (lesional) and uninvolved (nonlesional) skin from patients with SSc as previously described (25). Mean ages of patients and controls were 35 (range 27-47) and 31 yr (range 22-51), respectively. All patients were diagnosed with the diffuse form of SSc, according to the criteria of the American College of Rheumatology (2). Normal and SSc fibroblasts were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) and were passaged every 7-10 days. Experiments were performed on cells that were three to nine passages from primary cultures.
DFAM, ferric ammonium citrate [Fe(III)], ferrous sulfate [Fe(II)], carbonyl cyanide m-chlorophenylhydrazone (CCCP), reduced GSH, (±)-Northern blot.
Total RNA was extracted from confluent fibroblast cultures using a
guanidinium thiocyanate-phenol-chloroform single-step method (7)
(TRIzol; GIBCO). Equal amounts of RNA were loaded on
agarose-formaldehyde gels and capillary transferred to MSI nylon
membrane (MSI, Westboro, MA). The membranes were prehybridized for 2 h
at 42°C in a solution containing 50% formamide, 5× SSC
(1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0),
5× Denhardt's reagent, 0.5% SDS, and 100 mg/ml denatured salmon
sperm DNA and were hybridized overnight in a solution containing 50%
formamide, 5× SSC, 10% dextran sulfate, 0.5% SDS, and
32P-labeled cDNA probe generated
using a random primer labeling kit (GIBCO). After hybridization,
membranes were washed (1× SSC and 0.1% SDS at 55°C) and
exposed (X-OMAT film, Kodak, Rochester, NY). pBluescript vector
containing a 900-bp human HO-1 cDNA fragment was purchased from Genome
Systems (St. Louis, MO) and used to prepare a probe for hybridization.
Mouse -actin cDNA was kindly provided by Dr. B. Kream
(University of Connecticut Health Center, Farmington, CT). To correct
for differences in RNA loading, the membranes were stripped and
rehybridized with
-32P-labeled
oligonucleotide probe complementary to 18S ribosomal RNA. The intensity
of the bands on autoradiography was measured using laser scanning
densitometry, unless otherwise indicated. The HO-1 mRNA levels in
treated cells were normalized to control untreated samples and
expressed as multiples of controls. All experiments were performed at
least twice.
Hypoxia. Cells (106) were plated in 35-mm tissue culture dishes (Corning) in 2 ml of medium. After 24 h, cells were rinsed with PBS and conditioned for 24 h in DMEM supplemented with 0.5% FBS. For hypoxia experiments, dishes were placed in a sealed, humidified modular chamber (Billups-Rothenberg, Del Mar, CA) and flushed for 10 min with 5% CO2-95% N2. Normoxic cells were grown in a humidified tissue culture incubator in 95% air-5% CO2. Under the conditions of culture, hypoxia produces a PO2 of 20-25 mmHg in the medium and normoxia produces a PO2 of 140 mmHg in the medium (19, 20).
Analysis of HO-1 mRNA decay rates.
Cells were cultured and treated with hypoxia, ferric ammonium citrate,
holo-transferrin, or
H2O2
as described above (see also Figs. 4-6). The RNA polymerase II
inhibitor DRB (20 µg/ml) was added to the medium and incubated for
the indicated times. Total RNA was isolated at intervals and was
subjected to Northern blotting. Membranes were successively probed with
HO-1, -actin, and 18S ribosomal RNA probes. Signals were quantitated
using scanning laser densitometry and normalized to the 18S ribosomal
RNA signal. Relative mRNA levels remaining were plotted against time
elapsed on semilogarithmic coordinates. HO-1 mRNA half-lives were
calculated after linear regression analysis (Microsoft Excel).
Measurement of free radicals in intact cells. A fluorescent probe, chloromethyl-2,7-dichlorofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR), was used to assess the levels of free radicals and H2O2 in fibroblast cells. DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus enabling measurement of intracellular free radicals and H2O2. Fibroblasts were plated in 12-well plates at a concentration of 2 × 105 cells/well and allowed to adhere for 20-24 h in DMEM with 10% FBS. After 18-20 h of conditioning in DMEM supplemented with 0.5% FBS, cells were rinsed twice with PBS and incubated with 5 µM DCFDA in 0.5 ml of PBS for 1 h at 37°C. In some instances, GSH was added to the cells for the last 15 min of incubation. Cells were then rinsed twice with PBS to wash off the excess dye, and fresh PBS (with GSH, as indicated) was added. Cells were exposed to hypoxia, 0.25 mM H2O2, or 0.03 mM CCCP for various periods. Fluorescence was measured at appropriate time points by using a CytoFluor 2300 fluorescence plate reader (excitation 485 nm, emission 530 nm). The fluorescence of the hypoxic samples was measured immediately after unsealing of the chamber. Duplicate samples were present in every experiment; duplicate readings did not vary by more than 5%. Each experiment was repeated at least three times.
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RESULTS |
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Hypoxia transiently elevates HO-1 mRNA expression in
normal skin fibroblasts.
We assessed the expression of HO-1 mRNA in normal fibroblasts cultured
under hypoxic conditions. As shown in Fig.
1, skin fibroblasts constitutively express
low basal levels of HO-1 mRNA (indicated as 0 h of hypoxia). Exposure
of cells to hypoxia induced HO-1 mRNA in a time-dependent manner.
Induction was detectable at 5 h, peaked after 10 h of hypoxia, and
decreased to the basal level of expression by 24 h. Thus skin
fibroblasts are able to respond to lower oxygen tension by expressing
increased amounts of HO-1 mRNA.
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Induction of HO-1 mRNA by hypoxia is unaffected by antioxidants.
We hypothesized that hypoxic stress may be accompanied by the
generation of free radicals, which in turn induce HO-1. If so, free
radical scavengers should inhibit HO-1 induction by hypoxia. We
therefore examined whether the HO-1 response to hypoxia is affected by
the thiol antioxidant GSH. Induction of HO-1 mRNA by hypoxia was not
attenuated in the presence of GSH (Fig.
2A). As
a control, to ensure the effectiveness of this free radical scavenger
in our cell system, we examined the GSH effect on HO-1 induction by
known free radical-generating agents,
H2O2
and CCCP. H2O2
induced HO-1 mRNA in fibroblasts (Fig.
2B), as previously reported (28).
CCCP, a potent uncoupler of mitochondrial oxidative phosphorylation and
deenergizer of membrane potential, also acts as an oxidant. Addition of
CCCP also increased HO-1 mRNA. The induction of HO-1 mRNA by both
H2O2
and CCCP was abrogated by the presence of GSH (Fig. 2). We also
examined the effect of other antioxidants on induction of HO-1 by
hypoxia. Neither
N-acetyl-L-cysteine nor vitamin E affected induction of HO-1 mRNA by hypoxia; in contrast, these antioxidants attenuated the induction of HO-1 mRNA by either H2O2
or CCCP (data not shown). These results suggest that, in normal skin
fibroblasts, free radicals are not involved in induction of HO-1 mRNA
by hypoxia.
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Hypoxic conditions do not increase the rate of free radical
generation by skin fibroblasts.
To assess directly whether hypoxia affects the level of free radical
production in skin fibroblasts, we used a fluorescent probe, DCFDA,
that has been used in a number of cell types to measure the level of
free radicals. DCFDA is passively transported into the intact cell and
modified by cellular esterases; the modified probe reacts with
intracellular free radicals and
H2O2.
In cultured fibroblasts, there was a time-dependent increase in the
fluorescence signal with addition of either
H2O2
(Fig.
3A)
or CCCP (Fig. 3B). CCCP-dependent
induction of fluorescence was completely abrogated by GSH, and the
H2O2-dependent
signal was significantly attenuated by the presence of GSH. Hypoxia,
however, not only failed to induce fluorescence but caused slow,
moderate decreases of the baseline signal, suggesting a lack of net
free radical generation (Fig. 3C).
Reoxygenation of the hypoxic sample resulted in a slow increase of
fluorescence (Fig. 3C) to a level
approaching the baseline signal. Addition of
H2O2
(Fig. 3C) or CCCP (not shown) to
fibroblasts that had been hypoxic for 2 h induced the fluorescence
signal in a manner comparable to that seen in nonhypoxic cells,
indicating that DCFDA was still within the cells and that its ability
to react with free radicals was not altered during hypoxia. Thus data
presented in Figs. 2 and 3 suggest that hypoxic induction of HO-1 mRNA
does not involve free radicals; moreover, hypoxia does not lead to free
radical generation in skin fibroblasts.
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Effect of iron depletion on HO-1 mRNA induction by hypoxia.
There is a growing body of evidence that intracellular iron may be
involved in stress signaling (13, 41, 44, 52). We hypothesized that
iron might be involved in the induction of HO-1 mRNA by hypoxia. The
cell-permeant iron complexon DFAM has been used in a number of cell
systems; it is able to specifically deplete intracellular iron (39).
Induction of HO-1 mRNA by hypoxia was abrogated when cells were treated
with DFAM (Fig.
4A). The inhibitory effect of DFAM was evident at both 4 and 8 h of hypoxia. In
contrast,
H2O2-
and CCCP-mediated induction of HO-1 mRNA was unaffected by DFAM (Fig.
4B). These results suggest that iron or an iron-containing factor may play a role in HO-1 mRNA induction by
hypoxia but not in HO-1 mRNA stimulation by oxidative free radical
stress.
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HO-1 mRNA is induced by externally added sources of iron: Fe(II),
Fe(III), or holo-transferrin.
To determine whether free iron is able to stimulate HO-1 mRNA, ferrous
sulfate (Fig
5A) or
ferric ammonium citrate (Fig. 5B) was added to the cells. Both compounds were capable of inducing HO-1
mRNA more than twofold in a time- and concentration-dependent fashion.
This induction was abolished by the presence of DFAM. Similar results
were obtained when holo-transferrin
was used as a source of iron (Fig.
5C). The transient stimulatory
effect of either form of iron peaked after 4 h of treatment and
decreased to near basal levels of expression by 7 h. These data suggest that levels of intracellular iron in skin fibroblasts were able to
affect the HO-1 mRNA.
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Effect of hypoxia on stability of HO-1 mRNA.
Hypoxia has been shown to increase the mRNA stability of a number of
genes (10, 18, 32). It is also known that a family of iron-binding
proteins regulates mRNA expression by binding to target mRNAs and
prolonging their half-lives (13, 45). Therefore, we examined whether
hypoxia or addition of iron could affect HO-1 mRNA half-life, and we
compared these effects with that of
H2O2
on HO-1 mRNA half-life. Figure
6A
demonstrates the rate of HO-1 mRNA decay in basal conditions, when no
stimulus was applied to the cells. The estimated half-life of HO-1 mRNA in these conditions was 1.6 h. The half-life of HO-1 mRNA increased to
3.1 h after 4 h of hypoxia (Fig.
6B). Importantly, more prolonged hypoxia (6 h of treatment) led to a stronger stabilization of HO-1
mRNA; no decay of HO-1 mRNA was detected under these conditions (Fig.
6B). The steady-state levels of HO-1
mRNA (without inhibition of transcription) in this experiment rose as
expected, indicating that hypoxic conditions were effective during the
entire experiment (Fig. 6C). In
contrast, addition of either source of iron (ferric ammonium citrate or
holo-transferrin) or
H2O2
to the cells did not affect half-life of HO-1 mRNA (data not shown).
These data suggest that induction of HO-1 mRNA by hypoxia involves a
strong posttranscriptional mRNA stabilization mechanism, whereas
induction by either free iron or
H2O2
does not affect stability of HO-1 mRNA and most likely occurs at the
transcriptional level.
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SSC fibroblasts demonstrate stronger induction of HO-1 mRNA with
hypoxia than normal fibroblasts.
As noted earlier, dermal SSc fibroblasts are exposed to hypoxic
conditions in vivo. We hypothesized that HO-1 mRNA expression or the
HO-1 response to hypoxic stress in SSc fibroblasts might be different.
Thus we examined the HO-1 hypoxic response in SSc cells and compared it
with that of normal fibroblasts. Constitutive (basal) levels of HO-1
mRNA expression showed no significant differences between SSc and
normal fibroblasts tested. However, hypoxia induced HO-1 mRNA more
strongly in SSc cells than it did in normal fibroblasts (Fig.
7, A and
B). In seven SSc fibroblast strains,
the average HO-1 mRNA increase above the basal level at 10 h was
7.5-fold for SSc and 3.0-fold for normal fibroblasts
(Fig.7B,
top). At 15 h
(Fig.7B,
bottom) the average increase was
4.6-fold for SSc and 1.9-fold for normal fibroblasts. SSc fibroblasts
derived from clinically lesional tissue responded more strongly than
nonlesional SSc fibroblasts from the same individual: the average
levels of HO-1 induction at 10 h were 7.5- and 4.1-fold for involved
and uninvolved fibroblasts, respectively. Similar differences were observed after 15 h of incubation in hypoxic conditions. In five of
seven SSc patients tested, cells from lesional skin showed a greater
HO-1 response to hypoxia than their uninvolved counterparts isolated
from the same patient. Thus SSc fibroblasts demonstrated a stronger
induction of HO-1 mRNA in response to hypoxia than did normal
fibroblasts.
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DISCUSSION |
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HO-1 is induced by hypoxia in several cell types: vascular smooth muscle cells, neuronal cells, cardiomyocytes, and CHO cells (14, 37, 38, 51). It is unclear, however, whether cells grown in hypoxic conditions generate more free radicals, as is the case for oxidative stress (3, 6). Hypoxia could inhibit and/or disrupt cellular redox enzymes and other enzyme systems (respiratory electron-transporting chain, microsomal cytochrome oxidases, and others), and this could potentially affect free radical formation. There are several reports that, in some cells, hypoxia causes a decrease in the normal steady-state levels of free radicals whereas reoxygenation increases it (8, 24, 29, 55, 56). Recently, however, generation of free radicals by hypoxia alone was shown for cardiomyocytes (5, 12).
In the current study, we demonstrated that skin fibroblasts are able to respond to hypoxia by HO-1 mRNA induction. This response is not inhibited by GSH or other antioxidants. In contrast, the HO-1 response to H2O2 is reversed by antioxidants. We also examined whether antioxidants can affect free radical-mediated HO-1 induction when free radicals are generated metabolically by the cells rather than added externally. Treatment of skin fibroblasts by CCCP, an uncoupler of mitochondrial phosphorylation, led to a strong induction of HO-1 mRNA, an effect abrogated by GSH. These data confirm the potential role of free radicals in HO-1 regulation during an oxidative stress but argue against free radical involvement in HO-1 regulation during hypoxia.
To more directly address whether low oxygen tension could affect the steady-state levels of free radical production by the cells, a free radical-sensitive fluorescent probe, DCFDA, was used. Both H2O2 and CCCP increased the rate of free radical formation in the cell; this increase was significantly diminished or completely abrogated in the presence of free radical scavenger. In contrast, hypoxia induced a moderate decrease of DCFDA fluorescence. This was not due to dye leakage or inactivation, since addition of H2O2 to hypoxic samples resulted in rapid induction of fluorescence. Thus hypoxia does not appear to increase free radicals in the cell, and they are not responsible for HO-1 mRNA induction in hypoxia.
Intracellular iron plays a role in regulating gene expression at both translational and transcriptional levels (13, 16, 22, 30, 36, 41, 44, 45, 52). Recently, it was shown that hypoxia alters cellular iron homeostasis and iron regulatory protein-1 (IRP-1) activity in oligodendrocytes and macrophages (16, 22, 30, 44). We examined whether iron depletion could affect the HO-1 mRNA hypoxic stimulation. The induction of HO-1 mRNA by hypoxia was completely prevented by DFAM, suggesting that iron or iron-containing cellular protein is involved in HO-1 mRNA upregulation during hypoxic stress. Stimulatory effects of H2O2 and CCCP were unaffected by iron depletion, suggesting that free radical-mediated HO-1 mRNA induction is not dependent on iron. Exogenous sources of iron (ferrous sulfate, ferric ammonium citrate, or holo-transferrin) were able to induce HO-1 mRNA in skin fibroblasts.
Our studies of HO-1 mRNA half-life demonstrated that exposure of cells to hypoxia resulted in remarkable stabilization of HO-1 mRNA. Importantly, longer exposure of cells to hypoxia yielded higher induction of HO-1 mRNA and demonstrated greater mRNA stabilization. This strongly suggests that posttranscriptional mechanisms are involved in HO-1 mRNA upregulation by hypoxia. In contrast, neither iron nor holo-transferrin affected the half-life of HO-1 mRNA. This argues against direct involvement of free intracellular iron in stimulation of HO-1 mRNA by hypoxia and further confirms that induction of HO-1 mRNA by hypoxia does not depend on H2O2 and free radicals.
Transcriptional activation of the HO-1 promoter by a variety of agents generating cellular oxidative stress has been extensively studied (1, 6, 27). It appears that transcriptional activation of promoter is the major mechanism of stimulation of HO-1 gene expression by oxidative stress. In one published report, the mechanism of HO-1 mRNA induction by a proposed antioxidant, pyrrolidine dithiocarbamate, involves mRNA stabilization as well as promoter activation (23). To our knowledge, ours is the first study that describes regulation of HO-1 gene expression at the level of HO-1 mRNA stability by a physiological stimulus such as hypoxia.
Hypoxia has been shown to activate the expression of a number of genes at posttranscriptional level. Thus low oxygen pressure induces expression of vascular endothelial growth factor, erythropoietin, and tyrosine hydroxylase genes in part by increasing the stability of their mRNA (10, 18, 32). The presence of a pyrimidine-rich consensus sequence in the 3' end of tyrosine hydroxylase mRNA is responsible for binding of a specific hypoxia-inducible RNA binding protein (HIP). It was proposed that this protein(s) protects target mRNAs from the degradation by ribonucleases, thus prolonging their half-lives (42).
Another recently discovered group of proteins, IRPs (e.g., IRP-1 and
IRP-2), plays a role in posttranscriptional regulation of genes
involved in iron homeostasis, such as ferritin, transferrin, transferrin receptor, -aminolevulinate synthase, and mitochondrial aconitase (see Ref. 13 for review). Depending on availability of iron,
these proteins can bind to target mRNA and either block translation or
stabilize mRNA. Hypoxia and free radicals were shown to affect the
activity of IRPs (22, 30, 41).
There are several reports in the literature of hypoxia-inducible genes
that are also stimulated by iron depletion, both at the transcriptional
and posttranscriptional levels (4, 16, 52). Moreover, in some models,
DFAM is proposed to mimic hypoxia by interacting with the heme compound
of a hypothetical oxygen sensor (9, 17). HO-1 is the first example of a
gene in which iron depletion prevents its mRNA induction by hypoxia; by
this measure, it does not follow known examples. This suggests that other as yet unknown mechanisms dependent on iron could regulate gene
expression in hypoxia. We speculate that hypoxia induces synthesis of iron-dependent regulatory proteins that are involved in
HO-1 mRNA stabilization (Fig. 8).
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Cutaneous hypoxia is thought to play a role in the pathogenesis of SSc. The effect of hypoxia on dermal fibroblast metabolism is poorly understood, but it has been shown to increase collagen synthesis (15). Previous studies have shown that in SSc there is overrepresentation of a high-collagen-producing subset of cells (25). It has also been demonstrated that SSc fibroblasts are more resistant to apoptosis than normal fibroblasts (26). In this study, we demonstrate that hypoxia induced HO-1 mRNA in SSc fibroblasts to a greater extent than in their normal counterparts. The role of this alteration in the pathogenesis of scleroderma is, as yet, unclear. We hypothesize that perhaps the protective role of HO-1 overexpression in hypoxic SSc skin accounts for prolonged survival of a subset of hypoxia-adapted cells and thus contributes to the skin fibrosis.
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ACKNOWLEDGEMENTS |
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We thank Mikhail Panchenko and Philip Trackman for review of the manuscript and suggestions. We also thank Ante Jelaska for providing characterized cell strains.
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FOOTNOTES |
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This research was supported by grants from the Scleroderma Research Fund of Boston and by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-32343 and AR-20613.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Panchenko, Arthritis Center and Section of Rheumatology, Boston University School of Medicine, Boston, MA 02118 (E-mail: mpanchenko{at}med-med1.bu.edu).
Received 15 July 1999; accepted in final form 7 September 1999.
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