1 Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224; 2 Alton Ochsner Medical Foundation, Department of Molecular Genetics, and Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70121; and 3 Section of Pulmonary and Critical Care Medicine, Yale University, New Haven, Connecticut 06516
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ABSTRACT |
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Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in cellular protection against oxidant injury. Increasing evidence also suggests that HO-1 is markedly modulated by hypoxia in vitro and in vivo. Our group has previously demonstrated that the transcription factor hypoxia-inducible factor (HIF)-1 mediates hypoxia-induced HO-1 gene transcription and expression in systemic (aortic) vascular smooth muscle (AoVSM) cells (P. J. Lee, B.-H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. K. Choi. J. Biol. Chem. 272: 5375-5381, 1997). Because the pulmonary circulation is an important target of hypoxia, this study investigated whether HO-1 gene expression in pulmonary arterial vascular smooth muscle was differentially regulated by hypoxia in comparison to AoVSM cells. Interestingly, hypoxia neither induced HO-1 gene expression nor increased HIF-1 DNA binding activity in pulmonary arterial vascular smooth muscle cells. Conversely, pulmonary arterial endothelial cells (PAECs) demonstrated a marked induction of HO-1 gene expression after hypoxia. Electrophoretic mobility shift assays detected an increase in activator protein-1 rather than in HIF-1 DNA binding activity in nuclear extracts of hypoxic PAECs. Analyses of the promoter and 5'-flanking regions of the HO-1 gene were performed by transiently transfecting PAECs with either the hypoxia response element (HIF-1 binding site) or the HO-1 gene distal enhancer element (AB1) linked to a chloramphenicol acetyltransferase reporter gene. Increased chloramphenicol acetyltransferase activity was observed only in transfectants containing the AB1 distal enhancer, and mutational analysis of this enhancer suggested that the activator protein-1 regulatory element was critical for hypoxia-induced HO-1 gene transcription. Collectively, our data demonstrate that the molecular regulation of HO-1 gene transcription during hypoxia differs between the systemic and pulmonary circulations and also provide evidence that hypoxia-induced HO-1 gene expression in PAECs and AoVSM cells is regulated through two discrete signaling pathways.
heme oxygenase-1; vascular smooth muscle cell; endothelial cell; hypoxia-inducible factor-1; activator protein-1
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INTRODUCTION |
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HYPOXIA IS AN IMPORTANT pathological stimulus that targets the vasculature affecting vascular tone, endothelial permeability, and coagulant function contingent on the vascular bed involved. For example, hypoxia elicits systemic vasodilation yet causes acute pulmonary vasoconstriction, which, if sustained, leads to profound remodeling of the pulmonary vasculature, culminating in structural-based increases in pulmonary vascular resistance leading to the development of pulmonary hypertension (13, 16, 20). Although the focus of intense research for nearly a century, the mechanism(s) underlying these differential vascular responses to hypoxia remains unclear (37). It has become increasingly appreciated that hypoxia regulates the transcription and expression of several hypoxia-inducible genes including vascular endothelial growth factor, erythropoietin, platelet-derived growth factor, endothelin-1, constitutive nitric oxide (NO) synthase (NOS), and tyrosine hydroxylase (9, 10, 16, 19, 21, 29). These genes mediate important physiological and cellular adaptive responses to hypoxic stress, including vascular remodeling during angiogenesis (vascular endothelial growth factor, platelet-derived growth factor), increased ventilation (tyrosine hydroxylase), increased erythrocyte production (erythropoietin), and regulation of vasomotor tone (constitutive NOS, endothelin-1). More recently, the expression of several hypoxia-inducible genes has been reported to be regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1), suggestive of a common signaling pathway (7).
Interestingly, the stress response gene heme oxygenase (HO)-1 (HO-1) is also induced during hypoxia (15). HO catalyzes the rate-limiting step in the oxidative degradation of heme to biliverdin, releasing equimolar amounts of biliverdin IXa, iron, and carbon monoxide (CO). Three isoforms, HO-1 (32 kDa), HO-2 (36 kDa), and HO-3 (30 kDa), exist and have been identified as products of distinct genes (17, 18). Although HO-2 and HO-3 are constitutively expressed isoforms, HO-1 is inducible (17). In addition to its main substrate heme, it is well documented that HO-1 is upregulated by a variety of nonheme inducers including heavy metals, cytokines, hormones, endotoxin, heat shock, and agents causing oxidative stress (4). Furthermore, accumulating evidence both in vivo and in vitro strongly suggests that HO-1 induction plays an important role in providing cellular and tissue protection against oxidative stress (4, 14, 24-26).
Lee et al. (15) recently demonstrated that HIF-1 mediates HO-1 gene transcription in aortic vascular smooth muscle (AoVSM) cells in response to hypoxia. Against this background, our laboratory has focused on the regulation and function of hypoxia-induced HO-1 gene expression. Interestingly CO, one of the major catalytic by-products of HO activity, has previously been shown to modulate gene expression during hypoxia (11). In addition, CO, similar to NO, can activate guanylate cyclase and stimulate cGMP and has been demonstrated to regulate vascular tone under both physiological and pathophysiological conditions. Given the disparate consequences on vascular tone between the systemic and pulmonary vasculatures during hypoxia, we hypothesized that HO-1 gene expression in response to hypoxia may be differentially regulated between these two vascular beds. We therefore designed this study to test the specific hypothesis that the regulation of HO-1 gene expression in response to hypoxia would differ between AoVSM and pulmonary arterial vascular smooth muscle (PAVSM) cells and pulmonary arterial endothelial cells (PAECs).
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EXPERIMENTAL PROCEDURES |
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Cell culture. PAVSM cells were digested from freshly isolated rat pulmonary arteries with a modified technique described by Smirnov et al. (30). In brief, main pulmonary arteries were isolated, dissected free of connective tissue, opened via a longitudinal incision, and allowed to recover for 30 min in cold HEPES-buffered physiological saline solution (HPSS) containing 1.5 mM Ca2+. The tissue was then placed at room temperature in low-Ca2+ HPSS (20 µM Ca2+) for 20 min before enzymatic digestion at 37°C for 20 min in low-Ca2+ HPSS containing 1 mg/ml of collagenase (type I, 1,750 U/mg), 1 mg/ml of papain (9.5 U/mg), 2 mg/ml of bovine serum albumin (BSA), and 1 mM dithiothreitol (all from Sigma, St. Louis, MO). The tissue was then transferred to enzyme-free Ca2+-free HPSS and triturated with a flame-polished Pasteur pipette to disperse smooth muscle cells. All vascular smooth muscle cells were maintained in Dulbecco's modified Eagle's medium (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT) and gentamicin (50 µg/ml). Primary cultures of rat AoVSM cells were generously provided by Dr. Michael Crow (National Institute of Aging, National Institutes of Health, Baltimore, MD) and isolated by similar collagenase treatment as described above for PAVSM cells. Primary cultures of rat main PAECs were generously provided by Dr. Mark N. Gillespie (University of South Alabama, Mobile, AL). The endothelial cells were maintained in one-half DMEM and one-half Ham's F-12 medium (Mediatech, Herndon, VA) supplemented with 10% FBS and gentamicin (50 µg/ml). All cultures were maintained at 37°C in a humidified atmosphere of 5% CO2-95% air.
The cells were exposed to hypoxia (1% O2-5% CO2-94% N2) in a tightly sealed modular incubator chamber (Billups-Rothberg, Del Mar, CA) at 37°C. All experiments were performed with confluent cultures of these cells (PAVSM and AoVSM cells and PAECs) between passages 5 and 18. No significant proliferative or morphological changes were detected in these cells within these passages.
RNA isolation and Northern blot analysis. Total RNA was isolated with the STAT-60 RNAzol method with direct lysis of cells in RNAzol lysis buffer followed by chloroform extraction (Tel-Test "B," Friendswood, TX). Northern blot analyses were performed as previously described (3). In brief, 10 µg of total RNA were electrophoresed on a 1% agarose gel and then transferred to Gene Screen Plus nylon membrane (DuPont, Boston, MA) by capillary action and cross-linked with a UV Stratalinker (Stratagene, La Jolla, CA). Ethidium bromide staining of the gel was used to confirm RNA integrity. The membranes were then prehybridized in hybridization buffer (1% BSA, 7% SDS, 0.5 M phosphate buffer, pH 7.0, and 1 mM EDTA) at 65°C for 2 h followed by incubation in hybridization buffer containing 32P-labeled rat HO-1 cDNA at 65°C for 24 h. The nylon membranes were then washed in wash buffer A (0.5% BSA, 5% SDS, 40 mM phosphate buffer, pH 7.0, and 1 mM EDTA) for 25 min two times at 55°C followed by washes in buffer B (1% SDS, 40 mM phosphate buffer, pH 7.0, and 1.0 mM EDTA) for 15 min three times at 55°C. Autoradiogram signals were quantified by densitometric scanning (Molecular Dynamics, Sunnyvale, CA). To control for variation in either the amount of RNA among samples or loading errors, the membranes were hybridized with an oligonucleotide probe corresponding to 18S rRNA. All densitometric values obtained with HO-1 mRNA were normalized to values for 18S rRNA obtained on the same blot. Quantification of the steady-state HO-1 mRNA level of hypoxic cells or tissues is expressed in densitometric absorbance units and normalized to normoxic control samples and expressed as multiple of induction compared with control values.
cDNA and oligonucleotide probes. A
full-length rat HO-1 cDNA, generously provided by Dr. S. Shibahara
(Tohoku University, Sendai, Japan) (28) was subcloned
into pBluescript vector, and Hind
III-EcoR I digestion was performed to
isolate a 0.9-kb HO-1 cDNA insert. A 24-bp oligonucleotide
(5'-ACGGTATCTGATCGTCTTCGAACC-3') complementary to 18S rRNA
was synthesized with a DNA synthesizer (Applied Biosystems, Foster
City, CA). HO-1 cDNA was labeled with [-32P]CTP with a
random-primer kit (Boehringer Mannheim, Mannheim, Germany). The 18S
rRNA oligonucleotide was labeled with
[
-32P]ATP at the
3'-end with terminal deoxynucleotidyltransferase (Bethesda
Research Laboratories, Gaithersburg, MD).
Western blot analyses. For HO-1 immunoblots, the cells were homogenized in lysis buffer [1% Nonidet P-40, 20 mM Tris (pH 8.0), 137.5 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml of aprotinin]. Protein concentrations of the lysates were determined by Coomassie blue dye binding assay (Bio-Rad Laboratories, Hercules, CA). An equal volume of 2× SDS sample buffer [0.125 M Tris.HCl (pH 7.4), 4% SDS, and 20% glycerol] was added, and the samples were boiled for 5 min. The samples (100 µg) were subjected to electrophoresis on a 12% SDS-polyacrylamide gel (Novex, San Diego, CA) for 2 h at 20 mA. The proteins were then transferred electrophoretically (Bio-Rad Laboratories) onto a polyvinylidene fluoride membrane (Immobilon, Bedford, MA) and were incubated for 2 h in Tris-buffered saline (TBS) and 1% Tween 20 (TBS-T) containing 5% nonfat powdered milk. The membranes were then incubated for 2 h with mouse monoclonal antibody against HO-1 (1:1,000) dilution. After three washes in TBS-T for 5 min each, the membranes were incubated with goat anti-mouse immunoglobulin G antibody (Amersham, Arlington Heights, IL) for 2 h. The membranes were then washed three times in TBS-T for 5 min each, followed by detection of the signal with an enhanced chemiluminescence detection kit (Amersham).
Cellular nuclear protein extraction.
The cells were scraped into cold phosphate-buffered saline (PBS) and
centrifuged at 14,000 g at 4°C for
10 min. After the supernatant was discarded, the cell pellet was lysed
in lysis buffer containing 10 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol (DTT), 0.5% Nonidet P-40, and 1 mM PMSF. The lysate
was chilled in ice for 5 min and then centrifuged at 1,500 g for 5 min. The supernatant was
removed, and the pellet was resuspended in nuclear resuspension buffer
containing 25 mM Tris, pH 7.8, 60 mM KCl, 1 mM DTT, and 1 mM PMSF. The
nuclei were then frozen and thawed three times to obtain nuclear
protein. The protein was kept in nuclear resuspension buffer and stored
at 80°C.
Electrophoretic mobility shift assay. Mobility shift assays were performed as described by Barberis et al. (2) with minor modifications. DNA binding activity was determined after incubation of 7 µg of nuclear protein extract with 10 fmol (25,000-60,000 counts/min) of either a 32P-labeled 22-mer oligonucleotide encompassing the activator protein (AP)-1 site (5'-CTAGTGATGAGTCAGCCGGATC-3'; Stratagene) or a 32P-labeled 35-mer oligonucleotide encompassing the HIF-1 site (5'-GATCGAGCGGACGTGCTGGCGTGGCACGTCC- TCTC-3') in reaction buffer containing 10 mM HEPES (pH 7.9), 1 mM DTT, 1 mM EDTA, 80 mM potassium chloride, 1 µg of poly(dI-dC) · poly(dI-dC) for AP-1 or 10 µg of calf thymus for HIF-1, and 4% Ficoll. After a 20-min incubation, the reaction mixture was electrophoresed on a 6% polyacrylamide gel. The gel was transferred to DE81 ion-exchange chromatography paper (Whatman, Maidstone, UK) and dried down before exposure to autoradiographic film.
Plasmid constructs and mutations.
Construction of the chloramphenicol acetyltransferase (CAT) reporter
plasmids has been previously described (1, 15). Subfragments of EH (see
Fig. 7A) were isolated after
digestion with the appropriate restriction endonucleases, blunt ended,
and cloned into the Spe I site
upstream from the minimal promoter and
CAT gene in pMHO1cat-33.
Oligonucleotide-directed mutagenesis of the enhancer AB1 was carried
out according to the method of Deng and Nickoloff (6) with slight
modifications (see Fig. 7B).
Mutations were confirmed by DNA sequence analysis (1). Plasmids (10 µg) containing the various constructs were transiently transfected
into PAECs with Lipofectin reagent (GIBCO BRL) according to
manufacturer's protocol. In brief, the cells were transfected
overnight, after which time the plates were washed two times with
serum-free medium and then incubated in DMEM containing 10% FBS and
gentamicin at 50 µg/ml until they reached confluency. The cells were
then exposed to hypoxia (1% O2)
for 24 h.
CAT assay. Cellular protein extracts were prepared immediately after termination of hypoxia, and CAT assays were performed as previously described (25, 26). In brief, cells from 10-cm plates were washed with ice-cold PBS, resuspended in 1.0 ml of 0.25 M Tris · HCl (pH 7.5), and then lysed with three cycles of freezing and thawing. Cell debris was then removed by centrifugation for 10 min at 14,000 rpm in a microcentrifuge. Protein concentrations of the supernatant fluids were determined by Coomassie blue dye binding assay. Reaction mixtures containing, in a final volume of 150 µl, 20 mM acetyl-CoA, 0.3 µCi of [14C]chloramphenicol (50 µCi/µmol; Amersham, Arlington Heights, IL), and 100 µg of protein were incubated for 4 h at 37°C. The acetylated and nonacetylated forms of chloramphenicol were then separated by ascending thin-layer chromatography.
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RESULTS |
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Hypoxia does not induce HO-1 gene expression in PAVSM
cells. Lee et al. (15) previously reported that AoVSM
cells exposed to hypoxic conditions expressed significantly higher
levels of HO-1 than cells cultured in normoxia. To ascertain whether
HO-1 gene expression differed between
vascular smooth muscle cells isolated from the systemic and pulmonary
circulations, we subjected both PAVSM and AoVSM cells to hypoxic
conditions (1% O2) for 0, 4, 8, or 24 h and examined HO-1 gene
expression by Northern blot analysis. In contrast to our
observations in AoVSM cells, no alterations in HO-1 mRNA steady-state
levels were detected in PAVSM cells (Fig.
1A).
To confirm that the lack of HO-1 mRNA induction in PAVSM cells was not
a function of cell specificity, we did observe marked HO-1 mRNA
induction in PAVSM cells after treatment with other stimuli such as the
NO donor spermine NONOate (Fig. 1B).
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Hypoxia differentially increases HO-1 protein in PAVSM
cells. To determine whether hypoxia increased HO-1
protein levels in PAVSM cells, Western blot analyses were performed in
cellular lysates from both PAVSM and AoVSM cells exposed to hypoxia
(1% O2). As seen in
Fig. 2A,
hypoxia caused a marked induction in HO-1 protein levels in AoVSM
cells. In contrast, only a modest increase in protein levels,
comparable to basal levels in AoVSM cells, was detected in PAVSM cells
(Fig. 2B).
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Hypoxia does not induce HIF-1 DNA binding activity in
PAVSM cells. Because the transcription factor HIF-1
mediates induction of HO-1 gene
expression during hypoxia in AoVSM cells and because HIF-1 has been
shown to be critical in regulating expression of other
hypoxia-inducible genes such as inducible NOS, vascular endothelial
growth factor, and erythropoietin, we hypothesized that PAVSM cells did
not exhibit increased HO-1 mRNA expression after hypoxia due to lack of
HIF-1 activation. We performed electrophoretic mobility shift assays on
both AoVSM and PAVSM cells after exposure to hypoxia (1%
O2) with oligonucleotides
corresponding to the HIF-1 DNA binding site located in the hypoxia
response element (HRE) of the HO-1
gene (15). As expected, we observed increased HIF-1 DNA binding
activity in the AoVSM cells at 3 and 6 h of hypoxic exposure compared
with normoxic control cells (Fig. 3). In
contrast to the AoVSM cells, however, we did not detect increased HIF-1
DNA binding activity in the PAVSM cells after hypoxia (Fig. 3). HIF-1
DNA binding activity was inhibited by an excess amount of unlabeled
HIF-1 oligonucleotide and was not affected by labeled SP1
oligonucleotide, confirming the specificity of HIF-1 DNA binding activity (Fig. 3).
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Hypoxia-induced HO-1 gene expression in
PAECs. We extended our studies to examine whether
pulmonary vascular endothelial cells exhibit high levels of
HO-1 gene expression after hypoxia.
The rationale for this study was based on our observation that PAVSM cells do not exhibit increased HO-1
gene expression after hypoxia but that pulmonary arteries taken from
hypoxic rats do exhibit increased HO-1 mRNA expression (data not
shown). Given that PAECs and PAVSM cells are the two predominant cell
types comprising the main pulmonary artery, we exposed primary cultures
of PAECs to hypoxic conditions as described in Cell
culture for either 0, 4, 8, or 24 h
before isolation of total RNA for Northern blot analysis.
Interestingly, we observed marked induction of HO-1 mRNA expression in
a time-dependent manner in PAECs similar to that previously observed in
AoVSM cells (Fig. 4).
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Hypoxia induces AP-1 but not HIF-1 DNA binding
activity in PAECs. Based on our observations that
hypoxia induces HO-1 mRNA expression in PAECs, we hypothesized that
hypoxia also activates the transcription factor HIF-1, the global
regulator of hypoxia-inducible genes, in PAECs. We performed
electrophoretic mobility shift assays on nuclear extracts obtained from
PAECs exposed to hypoxia (1% O2) for either 3 or 6 h and
incubated with radiolabeled probe corresponding to the HIF-1 DNA
binding sites of the HO-1 gene. Surprisingly, no increase in HIF-1 DNA binding activity was detected in
PAECs (Fig. 5); however, increased HIF-1
DNA binding activity was observed in nuclear extracts from AoVSM cells
subjected to identical hypoxic conditions (Fig. 5). We had
previously identified that the transcription factor AP-1
mediated HO-1 expression to a variety of stimuli (1, 3, 4) and
therefore postulated that AP-1 may play a role in the induction of HO-1
expression during hypoxia. Electrophoretic mobility shift assays were
repeated with a synthetic double-stranded DNA probe specific for the
consensus AP-1 DNA binding site in the
HO-1 gene. As seen in Fig.
6A,
although no detectable change in AP-1 DNA binding activity was observed in AoVSM cells exposed to hypoxia, increased AP-1 DNA binding activity
was evident after hypoxic exposure in PAECs. In addition, this
hypoxia-induced AP-1 DNA binding activity in PAECs was inhibited by an
excess amount of unlabeled AP-1 oligonucleotide but was not affected by
labeled SP1 oligonucleotide, confirming the specificity of AP-1 DNA
binding activity (Fig. 6B). These
data suggest distinct signaling pathways exist between pulmonary
vascular endothelial cells and systemic vascular smooth muscle cells in
response to hypoxia.
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HO-1 mRNA induction requires AP-1 sites specific to
the second distal enhancer. The mouse
HO-1 gene has been extensively
characterized with respect to transcription regulation, demonstrating
the requirement of distal 5' sequences for HO-1 induction in
response to most known stimuli. Therefore, to further confirm that the
increased transcription of the HO-1
gene in PAECs was not mediated through HIF-1 activation, two 5'
distal fragments of the HO-1 gene,
either the HRE (HIF-1 binding site) or the distal enhancer AB1, were linked to a CAT reporter gene and
transiently transfected in PAECs and assayed for CAT activity in
response to hypoxia (Fig.
7A). These analyses demonstrated that the transcriptional activation of the
HO-1 gene by hypoxia is mediated not
through the HIF-1 binding site located in the HRE (Fig.
8A) but
rather by the distal enhancer AB1 (Fig.
8B). This distal enhancer region
contains putative DNA binding sites for the transcriptional factors
AP-1 and CCAAT enhancer binding protein. By mutational
analysis (Fig. 7B), we demonstrated
that the AP-1 DNA binding elements were necessary for hypoxia-induced
HO-1 gene transcription (Fig.
8C).
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DISCUSSION |
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The role of HO in heme metabolism has been well established since its characterization in the late 1960s, although it took nearly a decade before it became appreciated that HO exists as two (and more recently three) distinct isoforms, separable by their unique molecular masses, tissue distribution, and ability to be induced. Studies (1, 3, 4) have since described the induction of HO-1 by a variety of cellular stresses including heat shock, reactive oxygen species, electrophiles, endotoxin, NO, and hyperoxia. The chemical diversity of these HO-1 inducers in conjunction with the properties of the various catalytic by-products of HO activity leads to the notion that HO-1 may have a broader physiological role in cellular and systemic functions beyond heme degradation. Consistent with this hypothesis, HO-1 has been shown to provide cellular protection against oxidative damage both heme and nonheme mediated (14, 24-26) and to modulate both inflammation and cell growth (14, 34).
Lee et al. (15) previously reported the induction of HO-1 mRNA steady-state levels under acute hypoxic conditions both in vivo and in vitro. In addition, they demonstrated that the upregulation of HO-1 gene expression in AoVSM cells during hypoxia was mediated through the transcription factor HIF-1. These data, in conjunction with observations of elevated HO-1 gene expression during chronic hypoxic (8), suggest that HO-1 may play important physiological roles in response to both acute and chronic hypoxic stress. In addition, emerging evidence suggests that HO-1 might also play an important role in the cellular responses to various models of ischemia and reperfusion (31, 35).
Although the role of HO-1 during hypoxia remains unclear, it is well known that hypoxia has profound effects on vascular tone dependent on the vascular bed that is involved. For example, although hypoxia causes systemic dilatation, it mediates constriction in the pulmonary circulation (33, 37). Hypoxic pulmonary vasoconstriction (HPV) initially functions as a protective response of the lung to divert blood to well-ventilated regions of the lungs in an effort to optimize arterial blood gas oxygenation (13). However, when alveolar hypoxia is generalized, such as in patients with chronic lung disease, and/or becomes prolonged, HPV becomes pathological, leading to remodeling of the vasculature and contributing to the development of pulmonary hypertension. Despite the fact that HPV has been the focus of intense research since it was first characterized nearly half a century ago (32), the underlying mechanism(s) by which hypoxia mediates pulmonary vasoconstriction remains uncertain.
Many researchers agree that the initial pulmonary vasoconstriction is dependent on the inhibition of K+ channels, allowing Ca2+ influx to trigger contraction (12, 33, 37). However, this does not explain the sustained hypoxia-induced vasoconstriction, and more recently, studies have focused on the balance between vasoconstrictors and vasodilators as mediators and/or modulators of HPV. As a major catalytic by-product of HO activity, CO has been demonstrated to modulate gene expression and signal transduction and activate guanylate cyclase in a manner analogous to NO. Specifically, CO has been shown to suppress the induction of both endothelin-1 and platelet-derived growth factor under hypoxic conditions (22) as well as of hypoxia-induced HO-1 gene expression in cultured vascular cells (23). In addition, it has been demonstrated that CO may play a role in the regulation of vascular tone under certain physiological and pathophysiological conditions via its vasodilatory effects (5, 23, 38).
The present study demonstrates marked differences in hypoxia-induced HO-1 gene expression between pulmonary and systemic vascular cells. Cultured PAVSM cells contrast sharply from systemic AoVSM cells in their lack of hypoxia-induced HO-1 mRNA steady-state levels. This apparent inability of hypoxia to induce HO-1 gene expression in PAVSM cells appears to be coupled to an inability to increase HIF-1 DNA binding activity during hypoxia (Figs. 1A and 3). Although it is plausible that the levels of HO-1 mRNA induced by hypoxia in PAVSM cells are below the detectability of our assay, this has not been problematic when using other inducers of HO-1 such as the NO donor spermine NONOate (Fig. 1B). Surprisingly, the level of HO-1 protein induced by hypoxia in PAVSM cells, as determined by Western blot analysis, was comparable to basal levels of HO-1 protein in AoVSM cells (Fig. 2). We hypothesize that this modest augmentation of HO-1 protein in hypoxic PAVSM cells may be related to posttranslational regulation; however, further experiments will be necessary to delineate the mechanism underlying the hypoxia-associated increase in protein.
Similar to our findings, Yu et al. (36) recently showed that hypoxia
induced a marked increase in HIF-1 expression and HIF-1 DNA binding
activity in AoVSM cells, although few to no changes were detected in
pulmonary smooth muscle cells. Additionally, in contrast to their
cultured results, in vivo immunohistochemical analyses did not reveal
detectable basal HIF-1
expression in pulmonary arterial smooth
muscle cells and only low levels during anoxia (0%
O2). Interestingly, in their
culture system, PAVSM cells exhibited a high basal level of HIF-1
protein and HIF-1 binding activity. It is becoming increasingly clear
that pulmonary arteries are composed of heterogenous subpopulations of
PAVSM cells; therefore, we speculate that the observed differences in HIF-1
expression and HIF-1 binding activity may be related to different subtypes of PAVSM cells and/or culture conditions.
Interestingly, PAECs exposed to acute hypoxia exhibit HO-1 induction
comparable to AoVSM cells, which correlated to our in vivo
observations, because intact but not denuded pulmonary arteries taken
from acutely hypoxic rats expressed a marked induction of HO-1 gene expression (data not shown).
The transcriptional regulation of the
HO-1 gene in PAECs, however, appears
to be mediated not through the transcription factor HIF-1 but rather
through the transcription factor AP-1 (Fig. 6). Palmer et al. (27)
recently demonstrated that hypoxic induction of type II
NOS gene expression in bovine PAECs
was mediated through HIF-1. In addition, Yu et al. observed increased
HIF-1 expression and HIF-1 DNA binding activity in sheep PAECs (36).
We postulate that these apparent differences in HIF-1 activation may,
in part, be species dependent because our experiments were conducted in
rat pulmonary endothelial cells. To further examine whether AP-1
mediated HO-1 transcription during hypoxia, analysis of the mouse
HO-1 gene was performed that confirmed
that the distal enhancer AB1 and not the HRE was necessary for
hypoxia-induced HO-1 gene expression
(Fig. 8, A and
B). More specifically,
oligonucleotide-directed mutagenesis of the AB1 enhancer demonstrated
that the AP-1 sites were critical for HO-1 transcription during hypoxia
(Fig. 8C).
Millhorn et al. (21) recently reported that tyrosine hydroxylase was regulated by both AP-1 and HIF-1 under hypoxic conditions. However, to our knowledge, this is the first report to show that a hypoxia-inducible gene such as HO-1 can involve two distinct signaling pathways depending on cell specificity. Although it is well established that hypoxia can regulate gene expression in both endothelial cells and vascular smooth muscle cells, we believe that our data are the first to suggest that hypoxia may differentially mediate gene expression in vascular cells contingent on the vascular bed in which they reside. In light of the apparent disparities in gene expression between these two important vascular beds, we caution that observations made in systemic vessels and cell cultures should not be extrapolated to the pulmonary circulation without reservation.
Our data demonstrate that HO-1 is differentially regulated between the systemic and pulmonary vasculatures in a manner dependent on both cell type and signaling pathway. We speculate that this differential signaling pathway may be important given the fact that HO-1 is the major source of cellular CO production, and it is becoming increasing clear that CO, similar to NO, can regulate vascular tone and mediate gene expression. Additional studies are necessary to determine whether CO is playing a critical role in modulating the unique responses to hypoxia between these two vascular beds through the regulation of either vascular tone or gene expression of vasoactive substances such as endothelin-1 or platelet-derived growth factor. It is interesting to speculate that the modulation of HO-1, and therefore of CO, might be a novel therapeutic strategy to modulate HPV and vascular remodeling.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Multidisciplinary Training Grant 1-F32-HL-09807-01 (to C. L. Hartsfield); NHLBI Grants RO1-HL-55330 and RO1-HL-60234; National Institute of Allergy and Infectious Diseases Grant R01-AI-42365; and an American Heart Association Established Investigator Award (all to A. M. K. Choi).
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. M. K. Choi, Section of Pulmonary and Critical Care Medicine, Yale Univ. School of Medicine, 333 Cedar St., LCI 105, New Haven, CT 06520 (E-mail: augustine.choi{at}yale.edu).
Received 29 March 1999; accepted in final form 20 July 1999.
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