Division of Neonatology, Department of Pediatrics, and Department of Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642
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ABSTRACT |
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Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.
apoptosis; deoxyribonucleic acid damage; lung injury
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INTRODUCTION |
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SUPPLEMENTAL OXYGEN is commonly used to treat patients with pulmonary failure in an effort to increase arterial oxygen tension and minimize hypoxia. Unfortunately, high concentrations of oxygen result in the generation of toxic reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radical. ROS cause cellular damage through the oxidation of lipids, proteins, and nucleic acids (19). The nucleus is susceptible to oxidative damage because it contains metals that maintain chromatin structure and act as cofactors for binding transcription factors to DNA. Metals also catalyze the formation of ROS from molecular oxygen through the Haber-Weiss and Fenton reactions (18, 19). In addition, a recent study (4) revealed that lipid peroxidation products can promote the formation of DNA adducts. DNA strand breaks, nucleotide depletion, and dimer formation are all associated with oxidative DNA damage (18). Although the specific ROS generated by hyperoxia that causes DNA damage remains to be identified, one study (5) found that hyperoxia alone causes the same degree of DNA damage as low levels of hydrogen peroxide. Because supplemental oxygen is clearly necessary and beneficial to treat respiratory failure, it is important to understand how cells respond to injury caused by hyperoxia.
Numerous studies (1, 10) in rodents have demonstrated that morphological signs of injury can be detected in alveolar endothelial and type I epithelial cells within 48-72 h of exposure to lethal levels of oxygen. Continued exposure for >72 h results in acute respiratory distress associated with cell death, loss of alveolar integrity, and, ultimately, mortality. Recent studies (3, 21, 24, 33) have used terminal deoxyribonucleotidyltransferase dUTP nick end-label (TUNEL) staining to show that hyperoxia causes DNA fragmentation. TUNEL staining was observed in the terminal bronchiolar epithelium and throughout the parenchyma of mouse lungs exposed to hyperoxia. TUNEL staining was associated with increased expression of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21Cip1/WAF1 (p21) (23-25). p53 accumulates in cells with DNA damage and inhibits proliferation by transcriptionally increasing the expression of p21 (11). p21 inhibits growth of proliferating cells by binding and inhibiting G1 cyclin-dependent kinases (16) and has been suggested to facilitate repair of DNA damage caused by exposure to ultraviolet (UV) light (27). p53 also induces apoptosis by increasing the expression of Bax, a proapoptotic member of the Bcl-2 family (14). Although it remains unclear how p53 distinguishes between regulating growth arrest and inducing apoptosis, one elegant study (8) using an inducible p53-expressing cell line found that low levels of p53 inhibited proliferation, whereas higher levels stimulated apoptosis. Collectively, these observations are consistent with the hypothesis that hyperoxia damages DNA in pulmonary cells, which respond by expressing genes that participate in repair and/or apoptosis.
Like p53, the growth arrest and DNA damage (GADD) genes are induced in cells exposed to genotoxic stress. GADD genes were originally identified by subtraction hybridization from a cDNA library constructed from UV-irradiated Chinese hamster ovary cells (13). The regulation of these genes by stress is complex and appears to be mediated by multiple pathways. For example, ionizing radiation induces the transcription of GADD45, which inhibits proliferation and stimulates DNA excision repair, through a p53-dependent mechanism (17, 20). In contrast, UV irradiation or the alkylating agent methylmethane sulfonate (MMS) increases GADD45 expression in the absence of p53 binding directly to the GADD45 promoter (35). GADD153, a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, is not induced by ionizing radiation (26). However, UV irradiation, MMS, and hydrogen peroxide induce GADD153 expression through an activator protein-1 element in its promoter (15). In addition to their ability to inhibit proliferation and stimulate DNA repair, GADD45 and GADD153 can also induce apoptosis when overexpressed in cells in vitro (12, 30).
Because marked TUNEL staining is detected in bronchiolar epithelial and parenchymal cells exposed to hyperoxia, the present study investigates whether hyperoxia induces the expression of GADD genes. Here we show that TUNEL staining is associated with increased expression of GADD45 and GADD153. Furthermore, changes in GADD expression were independent of p53. These findings further support the hypothesis that hyperoxia damages DNA, resulting in the expression of genes that promote repair and/or death of the cell.
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MATERIALS AND METHODS |
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Mouse exposures. Normal adult (8-12 wk) pathogen-free male
C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and 129Sv/C57BL hybrid p53 wild-type (+/+) and
p53-null (/
) mice were obtained from Taconic
(Germantown, NY). Mice were housed for 1 wk in standard barrier
conditions before exposure to oxygen. Mice were kept in room air
(control) or exposed to >95% oxygen by placing the cages inside a
Plexiglas chamber through which prewarmed, humidified, and filtered
oxygen was delivered through a 0.22-µm filter as described (24, 25).
Oxygen concentrations were monitored each day with a MiniOX I analyzer
(Catalyst Research, Owings Mills, MD). Food and water were provided ad
libitum. Mice were killed with intraperitoneal pentobarbital sodium (65 mg/kg) and bled through the abdominal aorta. The right and left lung lobes from each mouse were used for analysis of DNA integrity or
changes in gene expression. A minimum of three mice were used for each
analysis. The University of Rochester's University Committee on Animal
Resources approved all exposures and handling of the mice.
Analysis of gene expression. GADD45 and GADD153 cDNAs were obtained by RT-PCR with RNA from lungs of mice exposed to hyperoxia as templates. Primers containing the translation initiation and termination codons of the target genes with restriction enzyme sites were used to amplify products that were cloned into the Bluescript vector (Stratagene). Primer sequences for GADD45 were 5'-AATATGACTTTGGAGGAATTC-3' and 5'-ATTCGGATGCCATCACCGTTC-3'. Primer sequences for GADD153 were 5'-ATGGCAGCTGAGTCCCTGC-3' and 5'-TCATGCTTGGTGCAGGCTG-3'. cDNA templates were synthesized at 42°C for 30 min with RT and 0.5 µg of RNA with an oligo(dT) primer (Perkin-Elmer, Foster City, CA). Synthesis was terminated by heating to 99°C for 5 min and then cooling on ice. Products were amplified with 15 µM primers and cycling 35 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The DNA was extracted with phenol and chloroform and precipitated with ethanol. GADD45 and GADD153 cDNAs were cloned into Bluescript vectors with unique restriction enzyme sites added to the 5'-end of the primers (not shown in primers). Cloned products were sequenced and aligned to their respective genes with MacVector sequence analysis software to confirm their identity (Eastman Kodak, Rochester, NY).
The lungs were exposed, and the right lobes of some mice were ligated
and removed for isolation of total RNA with acid phenol and phase lock
gel columns (5 Prime 3 Prime, Boulder, CO). RNA was separated
electrophoretically on a 1.0% agarose-formaldehyde gel and transferred
to Nytran. Blots were prehybridized and hybridized at 65°C in 1%
BSA, 7% SDS, 0.5 M sodium phosphate, and 1 mM EDTA with
32P-labeled cDNAs for mouse GADD45 or
GADD153. Changes in gene expression were normalized to
expression of mRNA for the ribosomal subunit L32 as previously
described (24, 25).
The left lobe of these mice was inflation fixed through the trachea with 100 mM cacodylic acid, pH 7.4, with 2% glutaraldehyde at 10 cmH2O pressure for 10 min, dehydrated in ethanol, and embedded in paraffin for in situ and immunohistochemical analysis. All images were digitally color balanced with Adobe Photoshop (Adobe Systems, San Jose, CA). Sections were hybridized with RNA probes derived by transcription from the same GADD cDNA templates used in the Northern blot analyses. 33P-radiolabeled sense and antisense probes were synthesized using T3 and T7 RNA polymerases to a specific activity of 3.33 × 109 dpm/µg and digested to a length of ~200 bp by alkaline hydrolysis. Sections were hybridized for 16 h at 55°C, washed, and digested with RNase A as described (25). The slides were washed stringently for 30 min in 0.1× saline-sodium citrate at 65°C, dipped in a 1:1 dilution of NTB-2 emulsion (Eastman Kodak), and exposed at 4°C for 2 wk before being developed and counterstained with hematoxylin and eosin.
Immunohistochemistry was performed on sections that were deparaffinized and hydrated before endogenous peroxidase was blocked with hydrogen peroxide-methanol as described (24, 25). Antibodies specific for GADD45 and GADD153 were obtained (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were first incubated with primary antibody that was then detected with biotinylated secondary antibody, avidin-enzyme complexes (Vector Laboratories, Burlingame, CA), and 3,3'-diaminobenzidine (Sigma, St. Louis, MO) before being counterstained with methyl green. Primary antibody controls used an equivalent amount of nonimmune IgG that resulted in sections without staining (data not shown).
Antibody specificity was confirmed by Western blot analysis of Escherichia coli cell lysates containing polyhistidine-tagged GADD45 or glutathione S-transferase-GADD153 fusion proteins (Santa Cruz). Proteins were electrophoretically separated on polyacrylamide-SDS gels and transferred to polyvinyl pyrolidine membrane. Membranes were blocked in PBS containing 5% nonfat dry milk before being incubated in primary antibody (1 µg/ml) overnight at 4°C. Nonspecific interactions were removed by washing in PBS containing 0.05% Tween 20 before incubation in peroxidase-conjugated secondary antibody at a 1:5,000 dilution (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were extensively washed, and conjugates were visualized by chemiluminescence (Amersham, Arlington Heights, IL) and exposure to Kodak Bio-Max film.
Analysis of DNA integrity. TUNEL was performed with an ApopTag kit (Oncor, Gaithersburg, MD) as previously described (24). Nuclei of TUNEL-positive cells stained brown due to reaction with 3,3'-diaminobenzidine. Adjacent tissue sections were reacted in the absence of terminal deoxyribonucleotidyltransferase to ensure specificity of the reaction. Four random fields of parenchyma per lung were counted under ×40 magnification to determine the percentage of TUNEL-positive cells. Each field contained 154 ± 4.4 cells, and a total of four mice were examined for each condition.
Statistical analysis. Values are expressed as means ± SE. Group means were compared by ANOVA with Fisher's procedure post hoc analysis with StatView software for Macintosh, with P < 0.05 considered significant.
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RESULTS |
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Hyperoxia induces GADD mRNA expression. Northern blot analysis
was used to determine whether hyperoxia altered the expression of
GADD45 and GADD153 mRNAs. GADD45 mRNA was not readily detected in lungs
exposed to room air or 24 h of hyperoxia (Fig.
1A). A 1.4-kb GADD45 mRNA
transcript was readily detected after exposure to oxygen for 48 h and
did not continue to increase after 72 h. Although the GADD45 cDNA does
not have any significant homology to 18S rRNA, it weakly
cross-hybridized in all lanes to 18S on the blot. Similarly, GADD153
mRNA was not readily detected in lungs exposed to room air or hyperoxia
for 24 h (Fig. 1B). However, two mRNA transcripts of ~0.9 kb
were detected after 48 h and continued to increase at 72 h. Although
multiple GADD153 mRNAs have not been described before, a recent
unpublished submission to the GenBank database (accession no. AB029497)
reports that human GADD153 may undergo alternative splicing to yield a
novel protein. It remains to be determined whether the larger mRNA
transcript represents the mouse homolog of the novel human transcript.
Because GADD mRNAs were not readily detected in room air-exposed
samples, the blots were reprobed for expression of the ribosomal mRNA
L32 to confirm that they were equally loaded. In contrast to the marked induction of GADD mRNAs by hyperoxia, the expression of L32 remained unchanged throughout the exposures (Fig. 1C).
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In situ hybridization was used to identify the GADD-expressing cells.
Room air-exposed lungs had few silver grains uniformly distributed
throughout the tissue, which indicates minimal or no GADD45 expression
(Fig. 2A). Exposure to hyperoxia
for 48 h increased the expression of GADD45 in bronchiolar epithelial
cells and in distinct cell populations throughout the parenchyma (Fig. 2B). GADD45 expression did not increase with continued exposure through 72 h, consistent with the Northern analysis (Fig 2C). Higher magnification of the parenchyma revealed that GADD45 expression increased predominantly in cuboidal epithelial cells,
characteristic of alveolar type II cells (Fig. 2D). It is
likely that GADD45 was expressed to a lesser extent by interstitial
fibroblasts, type I epithelial, and/or endothelial cells because
alveolar septae had some grains. Sections of lung hybridized with a
sense GADD45 probe had few grains, similar to the room air tissues
(data not shown).
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Similarly, room air-exposed lungs hybridized for GADD153 mRNA had
minimal silver grains, indicating minimal or no expression (Fig.
3A). In contrast, GADD153
expression increased predominantly in bronchiolar epithelial cells and
to a lesser extent throughout the parenchyma of lungs exposed to
hyperoxia for 48 h (Fig. 3B). GADD153 mRNA remained elevated in
the bronchiolar epithelium and increased modestly throughout the
parenchyma after 72 h of exposure, consistent with the Northern
analysis (Fig. 3C). Higher magnification revealed that GADD153
was diffusely expressed throughout the parenchyma, with an occasional
cell with robust expression. Sections hybridized with a sense GADD153
probe had minimal grains, similar to the room air-exposed lungs (Fig.
3D). In general, parenchymal expression of GADD153 was
modest and uniform, whereas GADD45 expression was intense and appeared
to be restricted predominantly to alveolar type II cells.
GADD45 and GADD153 were not detected in fibroblasts underlying
the bronchiolar epithelium, smooth muscle cells, or endothelium of
large blood vessels.
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Hyperoxia increases GADD protein expression.
Immunohistochemistry was used to determine whether hyperoxia increased
GADD protein expression. Minimal GADD45 staining was detected in
terminal bronchioles or parenchyma of lungs exposed to room air (Fig.
4, A and B). Exposure to hyperoxia for 48 h resulted in intense staining throughout the bronchiolar epithelium (Fig. 4C) and in cuboidal alveolar cells characteristic of type II cells (Fig. 4D). Faint staining was detected in alveolar septae, suggesting that GADD45 may also be
expressed in interstitial fibroblasts, type I epithelial, and/or endothelial cells. Western blot analysis using cell lysates prepared from E. coli expressing a polyhistidine-tagged GADD45 fusion
protein was used to demonstrate antibody specificity because the
competing immunogen was not available. Western blotting of serial
dilutions of lysates identified the expected fusion protein of ~25
kDa (Fig. 4E).
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The expression of GADD153 protein was not detected in room air-exposed
lungs (Fig. 5, A and B). In
contrast, modest nuclear staining was observed in most bronchiolar
epithelial cells exposed to hyperoxia for 48 h, consistent with GADD153
being a transcription factor (Fig. 5C). In contrast, GADD153
protein was not readily detected in parenchymal cells exposed to
hyperoxia for 48 (Fig. 5D) or 72 h (data not shown). Based on
the modest induction of GADD153 mRNA in the parenchyma after 72 h of
exposure, it is likely that GADD153 protein was simply too low to be
detected with this technique. Western blot analysis of cell lysates
expressing glutathione S-transferase-coupled GADD153 identified
a 53-kDa fusion protein that confirmed the specificity of this
antiserum (Fig. 5E). GADD45 and GADD153 were not detected in
larger airways, fibroblasts underlying the airway epithelium, or
endothelial cells of larger blood vessels.
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Hyperoxia increases GADD expression independent of
p53. Genotoxic stresses induce GADD expression through
p53-dependent and -independent pathways (35). p53(+/+) and
p53(/
) mice were exposed to room air or >95%
oxygen for 48 h to determine whether p53 regulated GADD expression
during hyperoxia. GADD45 (Fig. 6A) and GADD153 (Fig. 6B) mRNAs were assessed by Northern blot
analysis and compared with the expression of L32 (Fig. 6C).
Room air-exposed mice had minimal expression of GADD45 and
GADD153. Hyperoxia increased GADD45 and GADD153 expression
in the presence and absence of p53. In situ hybridization confirmed
that hyperoxia increased GADD expression in the same cellular locations
independent of p53 (data not shown).
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DNA fragmentation. A previous study by our laboratory (24) has
shown increased TUNEL staining in the terminal bronchiolar epithelium
and throughout the parenchyma of mouse lungs exposed to hyperoxia for
72 h. Because GADD induction was observed after 48 h of exposure, we performed TUNEL staining to determine whether DNA
fragmentation could be detected at this time. Quantitative analysis
revealed that 64.5 ± 2.1% of parenchymal cells in p53(+/+) mice exposed to hyperoxia were TUNEL positive (Fig.
7). Similarly, 61.8 ± 2.4% of
parenchymal cells in p53(/
) mice exposed to
hyperoxia were TUNEL positive. These findings demonstrate that DNA
fragmentation is detected within 48 h of exposure to hyperoxia and that
the degree of fragmentation is not modified by p53 deficiency.
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DISCUSSION |
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All cellular compartments are at risk for oxidative damage because molecular oxygen is converted to cytotoxic free radicals that can oxidize nucleic acids, lipids, and proteins. The present study demonstrates that lungs of adult mice exposed to lethal levels of oxygen had increased expression of two members of the GADD gene family. GADD45 and GADD153 increase in cells damaged by environmental stress such as DNA damage, which also induces the expression of the tumor suppressor p53. Although p53 induces GADD45 expression in cells damaged by ionizing radiation, it was not required for GADD45 or GADD153 expression during exposure to hyperoxia. GADD gene induction was associated with marked TUNEL staining that also was not quantitatively different in the absence of p53. This finding is consistent with another study (3) in which p53 deficiency did not modify lung injury as assessed by lung wet-to-dry weight ratios. Nevertheless, these observations along with previous studies (23-25) suggest that hyperoxia causes DNA fragmentation, resulting in the expression of p53, p21, and now GADD genes.
Detailed morphometric and ultrastructural analyses over the past 30 years have attempted to clarify how hyperoxia injures and kills cells (1, 10). Adult rats and mice exposed to lethal levels of oxygen (>90%) for 48 h have focal cytoplasmic swelling of microvascular endothelial cells that leads to trapping of erythrocytes. Endothelial cell death rapidly follows and is due to rupture of the plasma and nuclear membranes, with swelling of intracellular organelles such as the mitochondria and endoplasmic reticulum. Similar swelling and destruction of the alveolar type I epithelial cell are observed after 72 h of exposure and result in mortality. In contrast, alveolar type II cells and the bronchiolar epithelium remain morphologically intact except for modest interstitial edema near the bronchiolar-alveolar junction. Based on these findings, it was concluded that hyperoxia selectively kills alveolar endothelial and type I epithelial cells by necrosis, with bronchiolar and alveolar type II epithelial cells being somewhat resistant.
Recently, TUNEL staining and DNA laddering have been used to determine whether hyperoxia kills cells through an apoptotic pathway (3, 21, 24, 33). Cells undergoing apoptosis express endonucleases that cleave genomic DNA into nucleosomal fragments, which can be identified by gel electrophoresis and TUNEL staining (2). Although TUNEL staining has often been used to detect nucleosomal strand breaks associated with apoptosis, it also recognizes randomly nicked DNA (2). We and others (3, 21, 24, 33) have shown DNA laddering or increased TUNEL staining in the bronchiolar epithelium and throughout the parenchyma of mice exposed to lethal levels of hyperoxia. Although >60% of pulmonary cells become TUNEL positive after exposure to hyperoxia (Fig. 7), the amount of laddered DNA is very low and can be detected only after radiolabeling with 32P or size-selected enrichment (3, 33). It remains to be determined why mice remain alive when so many pulmonary cells become TUNEL positive. In contrast, genomic DNA isolated from mouse lungs exposed to hyperoxia for 90 h migrated predominantly as a smear, which was interpreted as necrotic cell death due to random nicking of DNA (3). Collectively, these observations suggest that TUNEL staining may also represent random DNA fragmentation.
The cellular response to DNA damage is complex and frequently involves
the posttranscriptional stabilization of the tumor suppressor protein
p53, which regulates genes required for growth arrest, DNA repair, and
apoptosis. GADDs were originally identified in Chinese hamster
ovary cells as genes in which expression was increased by alkylating
agents and UV light (13). Each gene encodes a distinct gene product
that participates in the cellular response to stress. GADD45 binds
proliferating cell nuclear antigen and translocates it from sites of
DNA replication to sites of DNA repair (28). This is consistent with
the finding that decreased GADD45 expression sensitizes cells to DNA
damage caused by UV irradiation or cisplatin (29). GADD45 also binds
and synergizes with p21 to elicit growth arrest (31). Recently, two
additional proteins have been identified that are structurally
homologous to GADD45, suggesting that there is a family of GADD45-like
proteins involved in the stress response (30). Hyperoxia also increases mRNA levels of one member, GADD45-, in terminal bronchioles and parenchymal cells of mice (O'Reilly, Staversky, and Watkins,
unpublished observations). GADD153 is a member of the
C/EBP family of transcription factors (26). Because GADD153 lacks a DNA
binding domain, it inhibits cell proliferation by forming heterodimers
with other members of the C/EBP family, leading to their
inactivation. Overexpression of GADD45 or GADD153 in vitro can
also lead to apoptosis (12). The finding that hyperoxia induces TUNEL
staining and the expression of GADD45 and GADD153 in bronchiolar
epithelial and parenchymal cells is consistent with the hypothesis that
pulmonary cells are responding to DNA fragmentation caused by
hyperoxia. Based on the known activities of GADD genes, it is
likely that they coordinate whether pulmonary cells injured by
hyperoxia either repair damaged DNA or undergo apoptosis.
Numerous studies have demonstrated that cellular stress increases the
expression of GADD genes through distinct molecular pathways.
Deletion analysis of the human and hamster GADD45 promoters identified
a conserved p53 binding site within the third intron that functions in
p53-dependent induction after ionizing radiation (20). Although this
element is not necessary for GADD45 induction by UV irradiation, MMS,
or medium depletion, abrogation of p53 activity in cell lines results
in modest GADD45 expression (34). p53 contributes to this GADD45
induction through protein-protein interactions with a Egr1/WT1 binding
site in the GADD45 promoter. In addition to p53, GADD45 is
transcriptionally increased by C/EBP- (9). GADD153, another member
of the C/EBP family, is induced by UV irradiation, MMS, hydrogen
peroxide, glucose deprivation, and drugs such as tunicamycin that
affect the function of the endoplasmic reticulum (6, 13, 32). Moreover,
GADD153 is not regulated by p53 or induced by ionizing radiation.
Analysis of the GADD153 promoter identified an activator protein-1
element that participates in induction by UV irradiation or hydrogen
peroxide (15). This finding was consistent with another study (22), which demonstrated that hyperosmotic stress induced GADD45 and GADD153
expression through stress-activated protein kinase (c-Jun NH2-terminal kinase) pathways. Collectively, these studies
reveal that DNA damage as well as other forms of cellular stress induce GADD genes.
The present study demonstrates that hyperoxia increased GADD expression independent of p53 in bronchiolar and parenchymal cells. Although the bronchiolar epithelium expressed both GADD genes, parenchymal expression of GADD45 was restricted to individual cell types, and GADD153 was induced to a lesser extent throughout the parenchyma. Thus it is likely that there are overlapping and distinct pathways by which GADD genes are regulated by hyperoxia in these different cell types. This concept is supported by a recent study (7), which demonstrated that cellular stress differentially induces GADD genes through distinct kinase families. It remains unknown how molecular oxygen injures cells because it is converted to cytotoxic-reduced oxygen species such as superoxide anion, hydrogen peroxide, and hydroxyl radicals. Because hydrogen peroxide can induce a p53-independent GADD response, future studies comparing GADD expression in cells exposed to hyperoxia or hydrogen peroxide should provide insight into how molecular oxygen induces GADD genes.
In conclusion, the current study provides further molecular evidence that hyperoxia injures pulmonary cells and that DNA fragmentation may play an important role in the cellular response to hyperoxic injury. Although it remains to be determined whether TUNEL staining reflects DNA damage and/or apoptosis, it is clear that hyperoxia stimulates the expression of a DNA damage response involving p53, p21, and now GADD45 and GADD153. All of these proteins participate in the cellular response to genotoxic stress by inhibiting proliferation, promoting repair, and/or apoptosis. It is worth noting that these same genes are most abundantly expressed in bronchiolar and alveolar type II epithelial cells that are thought to be resistant to hyperoxia. Perhaps resistance is conferred by their ability to express p53, p21, and GADDs and other related genes. Alternatively, these proteins may promote apoptosis of severely injured cells to diminish inflammation and further tissue injury. Future studies are necessary to clarify the role of these proteins in hyperoxic injury and may lead to therapeutic agents designed to ameliorate the toxic effects of oxygen.
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ACKNOWLEDGEMENTS |
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We thank Jacob Finkelstein for thoughtful and stimulating advice.
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FOOTNOTES |
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This work was supported by The American Lung Association, American Heart Association Beginning Grant-in-Aid 9860004T, National Institute of Environmental Health Sciences Pilot Project Grant P30-ES-01247, and National Heart, Lung, and Blood Institute Grant HL-58774 (to M. A. O'Reilly). Additional support was provided by National Heart, Lung, and Blood Institute Grant HL-36543 (to W. M. Maniscalco) and National Cancer Institute Grants CA-73725 and CA-11198 (to P. C. Keng). The animal exposures were performed at the core facilities supplied through the Environmental Health Sciences Center (University of Rochester, Rochester, NY; supported by National Institute of Environmental Health Sciences Grant P30-ES-01247).
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. A. O'Reilly,
Dept. of Pediatrics (Neonatology), Box 777, Children's Hospital at
Strong, Univ. of Rochester, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: michaeloreilly{at}urmc.rochester.edu).
Received 20 May 1999; accepted in final form 4 October 1999.
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