Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts

Maria V. Panchenko1, Harrison W. Farber2, and Joseph H. Korn1

1 Arthritis Center and Section of Rheumatology and 2 Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, (±)-alpha -tocopherol (vitamin E), N-acetyl-L-cysteine, 5,6-dichloro-1-D-ribofuranozylbenzimidazole (DRB), and cycloheximide were purchased from Sigma; bovine holo-transferrin was purchased from GIBCO BRL (Gaithersburg, MD).

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 beta -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 alpha -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, beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of hypoxia on heme oxygenase-1 (HO-1) mRNA expression by fibroblasts. Fibroblasts were conditioned in 1.2 ml of medium supplemented with 0.5% fetal bovine serum (FBS) for 24 h. Cells were then incubated in hypoxic conditions for indicated times. Total RNA was extracted, and 10 µg of RNA from each sample were analyzed by Northern blot for HO-1 mRNA and 18S ribosomal RNA expression. HO-1 mRNA signal intensities were quantitated using scanning laser densitometry, normalized against those of 18S rRNA, and plotted as multiples of control (0 h of hypoxia). Data are means ± SD for 3 pooled experiments performed with 3 primary cell lines derived from normal individuals. A representative Northern blot is shown. * Cells were incubated in 95% air-5%CO2 for 24 h.

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.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of GSH on hypoxia-, H2O2-, and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced HO-1 mRNA. Fibroblasts were conditioned in 1.2 ml of medium supplemented with 0.5% FBS for 24 h. When used, 0.3 mM GSH was added to medium 1 h before start of hypoxia (A) or addition of 0.25 mM H2O2 or 0.03 mM CCCP (B). Cells were exposed to hypoxia or to oxidants for 8 h. HO-1 mRNA and 18S rRNA expression were visualized by Northern blot, and signal intensity of each RNA sample hybridized to HO-1 probe was divided by that of each sample hybridized to 18S probe. Shown are values for normalized HO-1 mRNA in 1 of 2 experiments showing similar results. Mean values for 2 experiments were 87 and 100% inhibition by GSH of H2O2 and CCCP induction, respectively.

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.




View larger version (151517K):
[in this window]
[in a new window]
 
Fig. 3.   Time course of free radical generation by fibroblasts. A and B: fibroblasts seeded in 12-well plates were loaded with 5 µM chloromethyl-2,7-dichlorofluorescein diacetate (DCFDA) in 0.5 ml of PBS for 1 h. When used, 0.3 mM GSH was added to cells and incubated for last 15 min of DCFDA loading. Cells were then rinsed twice with PBS, and fresh PBS was added. For GSH-treated cells, a new aliquot of 0.3 mM GSH was added, and 0.25 mM H2O2 (A) or 0.03 mM CCCP (B) was added to samples at time 0. Fluorescence was measured at indicated time points. Arrow in A indicates addition of H2O2, to control sample. Values are given as arbitrary units (AU) of fluorescence relative to control measured at time 0 (before addition of oxidant). Each experiment was repeated at least 3 times with virtually identical results. C: cells were seeded and loaded with DCFDA as in A and B. Each plate containing duplicate samples was placed in an individual hypoxic chamber, simultaneously flushed with 5% CO2-95% N2 for 10 min, and incubated for indicated times. Chambers then were unsealed, and fluorescence was measured immediately. Dashed arrow indicates start of reoxygenation by unsealing of chamber after 180 min of hypoxia. Solid arrow indicates addition of 0.25 mM H2O2 into 120-min hypoxic sample immediately after unsealing. Values represent fluorescence relative to control measured at time 0 (before start of hypoxia). Duplicate readings did not vary by more than 5%; each experiment was performed 3 times with similar results; representative experiment is shown.

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.



View larger version (4552K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of desferrioxamine mesylate (DFAM) on hypoxia- and oxidant-induced HO-1 mRNA stimulation. Fibroblasts seeded on 35-mm plates were conditioned in presence or absence of 0.3 mM DFAM in DMEM supplemented with 0.5% FBS. After 15 h of incubation, cells were subjected to hypoxia (A) or incubated with 0.25 mM H2O2 or 0.03 mM CCCP (B) for indicated times. Total RNA was extracted, and HO-1 mRNA expression was visualized by Northern blot. Signal intensity of each RNA sample hybridized to HO-1 probe was normalized by that of 18S probe and then plotted as multiples of control (no treatment, no DFAM). Means ± SD of data from 3 experiments.

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.




View larger version (454341K):
[in this window]
[in a new window]
 
Fig. 5.   Induction of HO-1 mRNA by exogenous sources of iron. A: 150 or 300 µM FeSO4 [Fe(II), final concentration]. B: 4 and 16 µg/ml ferric ammonium citrate [Fe(III), final concentration]. C: 0.4 mg/ml of holo-transferrin. Fibroblasts were conditioned with or without 0.3 mM DFAM for 15 h as described for Fig. 4 except that 0.05% FBS was present in medium. Iron source was added to cells and incubated for indicated times. Total RNA was extracted, and HO-1 mRNA expression was visualized by Northern blot. Signal intensity of each RNA sample hybridized to HO-1 probe was normalized by that of each sample hybridized to 18S probe and then plotted as multiples of control. A and B: means ± SD of normalized HO-1 mRNA signal intensities obtained in 3 experiments. C: values for normalized HO-1 mRNA of a representative experiment. Experiment was performed twice with similar results.

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.




View larger version (353827K):
[in this window]
[in a new window]
 
Fig. 6.   Hypoxia increases half-life (t1/2) of HO-1 mRNA. Fibroblasts were seeded as described for Fig. 1 and grown in hypoxic conditions for 4 or 6 h. Chambers were quickly unsealed, and 20 µg/ml 5,6-dichloro-1-D-ribofuranozylbenzimidazole (DRB) was immediately added to medium. Chambers were then gassed for an additional 5 min, and incubation continued for indicated times. Samples were collected, total RNA was extracted, and expression of HO-1 mRNA, beta -actin mRNA, and 18S rRNA was visualized by Northern blot. Signals were quantitated using scanning laser densitometry and normalized to 18S ribosomal RNA signal. A and B: relative HO-1 mRNA levels remaining (after DRB addition) plotted vs. time on semilogarithmic coordinates. HO-1 mRNA half-lives were calculated after linear regression analysis. C: to ensure that manipulations of chambers (sealing and unsealing) did not affect experimental conditions, cells were treated as described in B, except no DRB was added to fibroblasts. HO-1 mRNA induction relative to control (0 h of hypoxia) was plotted vs. time. Insets: representative Northern blots. Each line shows values for normalized HO-1 mRNA signal intensities obtained in 2 separate experiments.

The general inhibitor of translation cycloheximide did not affect the basal levels of HO-1 mRNA; however, it effectively blocked the induction of HO-1 by hypoxia (data not shown). These data indicate that newly synthesized protein is required for the induction to occur.

We hypothesize that iron-dependent cellular protein, rather than free iron, may be responsible for the induction of HO-1 by hypoxia.

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.



View larger version (2822K):
[in this window]
[in a new window]
 
Fig. 7.   Hypoxia induces more HO-1 mRNA in systemic sclerosis (SSc) fibroblasts than in normal fibroblasts. A: time course of HO-1 mRNA induction by hypoxia in normal and SSc fibroblasts; 106 cells of each strain were plated in 35-mm tissue culture dishes. After 24 h, cells were rinsed with PBS and conditioned for 24 h in DMEM supplemented with 1% FBS. Cells then were incubated in hypoxic conditions for 5, 10, 15, and 24 h. For control, cells were either harvested before hypoxia treatment (0 h of hypoxia) or cells were incubated in 95% air-5% CO2 at 37°C for 24 h (24*). B: all SSc and normal control cell lines used for this experiment were in passage 3 or 4. Normal control fibroblasts (designated N1 to N10), lesional SSc fibroblasts (designated 1A to 9A), and nonlesional SSc fibroblasts (designated 1B to 9B) were subjected to hypoxia for 10 h (top) and 15 h (bottom). Total RNA was extracted, and 10 µg of RNA in every sample were analyzed for HO-1 mRNA expression by Northern blot. Quantitation of Northern blot images was done with PhosphorImager (Image Quant, Molecular Dynamics, Sunnyvale, CA). HO-1 signal intensities were normalized against those of 18S and then plotted as multiples of control (0 h). Values are means ± SE; difference in HO-1 induction between normal controls and lesional SSc fibroblasts for 10-h experiment was statistically significant (P < 0.002, Mann-Whitney 2-sample test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, delta -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).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   Regulation of HO-1 mRNA by hypoxia and free radicals. A postulated scheme for alternative regulation of HO-1 mRNA by hypoxia and free radicals. AP-1, activator protein-1; NF-kappa B, nuclear factor-kappa B; Q ·, ubisemiquinone (free radical); UTR, untranslated region.

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.


    ACKNOWLEDGEMENTS

We thank Mikhail Panchenko and Philip Trackman for review of the manuscript and suggestions. We also thank Ante Jelaska for providing characterized cell strains.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Alam, J., S. Shibahara, and A. Smith. Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells. J. Biol. Chem. 264: 6371-6375, 1989[Abstract/Free Full Text].

2.   American Rheumatism Association, Diagnostic and Therapeutic Criteria Committee, Subcommittee for Scleroderma Criteria.. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum. 23: 581-590, 1980[ISI][Medline].

3.   Applegate, L. A., P. Luscher, and R. M. Tyrrell. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 51: 974-978, 1991[Abstract].

4.   Beerepoot, L. V., D. T. Shima, M. Kuroki, K. T. Yeo, and E. E. Voest. Up-regulation of vascular endothelial growth factor production by iron chelators. Cancer Res. 56: 3747-3751, 1996[Abstract].

5.   Borger, D. R., and D. A. Essig. Induction of HSP 32 gene in hypoxic cardiomyocytes is attenuated by treatment with N-acetyl-L-cysteine. Am. J. Physiol. Heart. Circ. Physiol. 274: H965-H973, 1998[Abstract/Free Full Text].

6.   Camhi, S. L., J. Alam, G. W. Wiegand, B. Y. Chin, and A. M. Choi. Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5' distal enhancers: role of reactive oxygen intermediates and AP-1. Am. J. Resp. Cell Mol. Biol. 18: 226-234, 1998[Abstract/Free Full Text].

7.  Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15: 532-534, 536-537, 1993.

8.   Costa, L. E., S. Llesuy, and A. Boveris. Active oxygen species in the liver of rats submitted to chronic hypobaric hypoxia. Am. J. Physiol. Cell Physiol. 264: C1395-C1400, 1993[Abstract/Free Full Text].

9.   Cross, A. R., L. Henderson, O. T. Jones, M. A. Delpiano, J. Hentschel, and H. Acker. Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body. Biochem. J. 272: 743-747, 1990[ISI][Medline].

10.   Czyzyk-Krzeska, M. F., B. A. Furnari, E. E. Lawson, and D. E. Millhorn. Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells. J. Biol. Chem. 269: 760-764, 1994[Abstract/Free Full Text].

11.   Dennery, P. A., K. J. Sridhar, C. S. Lee, H. E. Wong, V. Shokoohi, P. A. Rodgers, and D. R. Spitz. Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts. J. Biol. Chem. 272: 14937-14942, 1997[Abstract/Free Full Text].

12.   Duranteau, J., N. S. Chandel, A. Kulisz, Z. Shao, and P. T. Schumacker. Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J. Biol. Chem. 273: 11619-11624, 1998[Abstract/Free Full Text].

13.   Eisenstein, R. S., and K. P. Blemings. Iron regulatory proteins, iron responsive elements and iron homeostasis. J. Nutr. 128: 2295-2298, 1998[Abstract/Free Full Text].

14.   Eyssen-Hernandez, R., A. Ladoux, and C. Frelin. Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia. FEBS Lett. 382: 229-233, 1996[ISI][Medline].

15.   Falanga, V., T. A. Martin, H. Takagi, R. S. Kirsner, T. Helfman, J. Pardes, and M. S. Ochoa. Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts. J. Cell. Physiol. 157: 408-412, 1993[ISI][Medline].

16.   Gleadle, J. M., B. L. Ebert, J. D. Firth, and P. J. Ratcliffe. Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am. J. Physiol. Cell Physiol. 268: C1362-C1368, 1995[Abstract/Free Full Text].

17.   Goldberg, M. A., S. P. Dunning, and H. F. Bunn. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242: 1412-1415, 1988[ISI][Medline].

18.   Goldberg, M. A., C. C. Gaut, and H. F. Bunn. Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events. Blood 77: 271-277, 1991[Abstract].

19.   Graven, K. K., R. J. McDonald, and H. W. Farber. Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase. Am. J. Physiol. Cell Physiol. 274: C347-C355, 1998[Abstract/Free Full Text].

20.   Graven, K. K., R. F. Troxler, H. Kornfeld, M. V. Panchenko, and H. W. Farber. Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia. J. Biol. Chem. 269: 24446-24453, 1994[Abstract/Free Full Text].

21.   Hancock, W. W., R. Buelow, M. H. Sayegh, and L. A. Turka. Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nat. Med. 4: 1392-1396, 1998[ISI][Medline].

22.   Hanson, E. S., and E. A. Leibold. Regulation of iron regulatory protein 1 during hypoxia and hypoxia/reoxygenation. J. Biol. Chem. 273: 7588-7593, 1998[Abstract/Free Full Text].

23.   Hartsfield, C. L., J. Alam, and A. M. Choi. Transcriptional regulation of the heme oxygenase 1 gene by pyrrolidine dithiocarbamate. FASEB J. 12: 1675-1682, 1998[Abstract/Free Full Text].

24.   Hasegawa, K., H. Yoshioka, T. Sawada, and H. Nishikawa. Direct measurement of free radicals in the neonatal mouse brain subjected to hypoxia: an electron spin resonance spectroscopic study. Brain Res. 607: 161-166, 1993[ISI][Medline].

25.   Jelaska, A., M. Arakawa, G. Broketa, and J. H. Korn. Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts. Increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts. Arthritis Rheum. 39: 1338-1346, 1996[ISI][Medline].

26.   Jelaska, A., and J. H. Korn. Apoptosis as a selection factor in the pathogenesis of scleroderma (Abstract). Arthritis Rheum. 40: S200, 1997.

27.   Keyse, S. M., L. A. Applegate, Y. Tromvoukis, and R. M. Tyrrell. Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts. Mol. Cell. Biol. 10: 4967-4969, 1990[ISI][Medline].

28.   Keyse, S. M., and R. M. Tyrrell. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Natl. Acad. Sci. USA 86: 99-103, 1989[Abstract].

29.   Kroll, S. L., and M. F. Czyzyk-Krzeska. Role of H2O2 and heme-containing O2 sensors in hypoxic regulation of tyrosine hydroxylase gene expression. Am. J. Physiol. Cell Physiol. 274: C167-C174, 1998[Abstract/Free Full Text].

30.   Kuriyama-Matsumura, K., H. Sato, M. Yamaguchi, and S. Bannai. Regulation of ferritin synthesis and iron regulatory protein 1 by oxygen in mouse peritoneal macrophages. Biochem. Biophys. Res. Commun. 249: 241-246, 1998[ISI][Medline].

31.   Lee, P. J., B. H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. Choi. Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J. Biol. Chem. 272: 5375-5381, 1997[Abstract/Free Full Text].

32.   Levy, A. P., N. S. Levy, and M. A. Goldberg. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J. Biol. Chem. 271: 2746-2753, 1996[Abstract/Free Full Text].

33.   Maines, M. D., N. G. Ibrahim, and A. Kappas. Solubilization and partial purification of heme oxygenase from rat liver. J. Biol. Chem. 252: 5900-5903, 1977[Abstract].

34.   Maines, M. D., and A. Kappas. Metals as regulators of heme metabolism. Science 198: 1215-1221, 1977[ISI][Medline].

35.   Mancuso, C., P. Preziosi, A. B. Grossman, and P. Navarra. The role of carbon monoxide in the regulation of neuroendocrine function. Neuroimmunomodulation 4: 225-229, 1997[ISI][Medline].

36.   Melillo, G., L. S. Taylor, A. Brooks, T. Musso, G. W. Cox, and L. Varesio. Functional requirement of the hypoxia-responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine. J. Biol. Chem. 272: 12236-12243, 1997[Abstract/Free Full Text].

37.   Morita, T., M. A. Perrella, M. E. Lee, and S. Kourembanas. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc. Natl. Acad. Sci. USA 92: 1475-1479, 1995[Abstract].

38.   Murphy, B. J., K. R. Laderoute, S. M. Short, and R. M. Sutherland. The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells. Brit. J. Cancer 64: 69-73, 1991[ISI][Medline].

39.   Neilands, J. B. Siderophores: structure and function of microbial iron transport compounds. J. Biol. Chem. 270: 26723-26726, 1995[Free Full Text].

40.   Otterbein, L. E., J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and A. M. Choi. Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103: 1047-1054, 1999[Abstract/Free Full Text].

41.   Pantopoulos, K., S. Mueller, A. Atzberger, W. Ansorge, W. Stremmel, and M. W. Hentze. Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intracellular oxidative stress. J. Biol. Chem. 272: 9802-9808, 1997[Abstract/Free Full Text].

42.   Paulding, W. R., and M. F. Czyzyk-Krzeska. Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3'-untranslated region. J. Biol. Chem. 274: 2532-2538, 1999[Abstract/Free Full Text].

43.   Poss, K. D., and S. Tonegawa. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. USA 94: 10925-10930, 1997[Abstract/Free Full Text].

44.   Qi, Y., T. M. Jamindar, and G. Dawson. Hypoxia alters iron homeostasis and induces ferritin synthesis in oligodendrocytes. J. Neurochem. 64: 2458-2464, 1995[ISI][Medline].

45.   Samaniego, F., J. Chin, K. Iwai, T. A. Rouault, and R. D. Klausner. Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function, and post-translational regulation. J. Biol. Chem. 269: 30904-30910, 1994[Abstract/Free Full Text].

46.   Silverstein, J. L., V. D. Steen, T. A. Medsger, Jr., and V. Falanga. Cutaneous hypoxia in patients with systemic sclerosis (scleroderma). Arch. Dermatol. 124: 1379-1382, 1988[Abstract].

47.   Snyder, S. H., S. R. Jaffrey, and R. Zakhary. Nitric oxide and carbon monoxide: parallel roles as neural messengers. Brain Res. Brain Res. Rev. 26: 167-175, 1998[ISI][Medline].

48.   Soares, M. P., Y. Lin, J. Anrather, E. Csizmadia, K. Takigami, K. Sato, S. T. Grey, R. B. Colvin, A. M. Choi, K. D. Poss, and F. H. Bach. Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat. Med. 4: 1073-1077, 1998[ISI][Medline].

49.   Stocker, R., A. N. Glazer, and B. N. Ames. Antioxidant activity of albumin-bound bilirubin. Proc. Natl. Acad. Sci. USA 84: 5918-5922, 1987[Abstract].

50.   Tenhunen, R., H. S. Marver, and R. Schmid. Microsomal heme oxygenase. Characterization of the enzyme. J. Biol. Chem. 244: 6388-6394, 1969[Abstract/Free Full Text].

51.   Tohyama, M., and T. Ogawa. [Stress response of cultured astrocytes to hypoxia/reoxygenation]. Nippon Yakurigaku Zasshi 109: 145-151, 1997[Medline].

52.   Wang, G. L., and G. L. Semenza. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82: 3610-3615, 1993[Abstract].

53.   Willis, D., A. R. Moore, R. Frederick, and D. A. Willoughby. Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat. Med. 2: 87-90, 1996[ISI][Medline].

54.   Wood, S. M., M. S. Wiesener, K. M. Yeates, N. Okada, C. W. Pugh, P. H. Maxwell, and P. J. Ratcliffe. Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1alpha subunit (HIF-1alpha ). Characterization of hif- 1alpha -dependent and -independent hypoxia-inducible gene expression. J. Biol. Chem. 273: 8360-8368, 1998[Abstract/Free Full Text].

55.   Zulueta, J. J., R. Sawhney, F. S. Yu, C. C. Cote, and P. M. Hassoun. Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation. Am. J. Physiol. Lung Cell. Mol. Physiol. 272: L897-L902, 1997[Abstract/Free Full Text].

56.   Zweier, J. L., R. Broderick, P. Kuppusamy, S. Thompson-Gorman, and G. A. Lutty. Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation. J. Biol. Chem. 269: 24156-24162, 1994[Abstract/Free Full Text].


Am J Physiol Cell Physiol 278(1):C92-C101
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society