Department of Pediatrics, Stanford University, Palo Alto, California 94305
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ABSTRACT |
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Immature organisms (neonates; <12 h old) have vastly differing responses to hyperoxic injury than adults. A common feature of hyperoxic gene regulation is involvement of activator protein (AP)-1. We evaluated lung AP-1 binding as well as that of the AP-1 subunit proteins c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D after exposure to >95% O2 for 3 days. Unlike adults, neonates showed no increased AP-1 binding in hyperoxia despite a high affinity of the AP-1 binding complexes for phosphorylated c-Jun and Jun D as demonstrated by supershift of these antibodies with the AP-1 complexes. Moreover, neonatal lungs exhibited two distinguishable AP-1 binding complexes, whereas adult lungs had one. In neonates, sequential immunoprecipitation revealed that the lower AP-1 complex was composed of proteins from both the Fos and Jun families, whereas the upper complex consisted of Jun family proteins, with predominance of Jun D. In adults, the single AP-1 complex appeared to involve other Fos or non-Fos or non-Jun family proteins as well. Neonatal lungs showed a higher level of Jun B and Jun D immunoreactive proteins in both air and hyperoxia compared with those in adult lungs. These results suggest that significant maturational differences in lung AP-1 complexes exist and that these may explain transcriptional differences in hyperoxic gene regulation.
antioxidant; oxidant stress; sequential immunoprecipitation heme oxygenase
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INTRODUCTION |
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IMMATURE ORGANISMS have vastly differing responses to injury than their mature counterparts. For example, in response to oxygen toxicity and reactive oxygen species, transcriptional regulation of critical antioxidant enzymes (AOEs) such as manganese superoxide dismutase (15) and of the stress response protein heme oxygenase-1 (HO-1) (21) are noted in the mature lung. In contrast, in hyperoxia, AOE regulation is not necessarily transcriptionally mediated in the immature lung (9). Additionally, despite evidence of increased oxidative stress, hyperoxia-exposed neonatal rats (<12 h old) showed increased lung HO-1 activity that was not associated with increased HO-1 mRNA, suggesting posttranscriptional regulation of HO-1 (12). Although posttranscriptional regulation of other AOEs in immature organisms is related to increased mRNA stability (9), it is not clear whether this occurs with HO-1, and it is also not understood what dictates the refractoriness of lung HO-1 and other AOE to transcriptional regulation in neonates exposed to hyperoxia. Because adult lung HO-1 gene regulation in hyperoxia is associated with increased binding activity of activator protein (AP)-1, a known early stress response factor (21), and because the HO-1 gene has several stress response elements with the AP-1 consensus binding site (1-3), we wanted to evaluate whether differences in AP-1 binding observed in hyperoxia could explain the differences in HO-1 gene regulation between adults and neonates. To this effect, neonatal and adult rats were exposed to >95% O2 for 3 days, and lung nuclear proteins were analyzed for AP-1 binding. We also determined levels of c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D immunoreactive proteins because the components of the AP-1 complex may modulate the degree of AP-1 binding and activity (4). Observations reported here help explain maturational differences in AP-1-mediated gene regulation.
An understanding of the unique mechanisms that modulate gene regulation in neonates is critical in the development of therapeutic interventions that are specifically suited to immature organisms.
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MATERIALS AND METHODS |
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Animal models. Litters of Wistar rat pups <12 h old (neonates) with their mothers and 2-mo-old adult male rats were obtained from Simonsen (Gilroy, CA). The animals were kept in a 12:12-h light-dark cycle and allowed to feed ad libitum until the time of experimentation.
Experimental design. Neonatal or adult rats were placed in a flow-through, airtight Plexiglas cylindrical exposure chamber (Vreman Scientifics, Los Altos, CA) that had ports for gas entry and exit and access to nesting materials, food, and water. Either air or hyperoxic atmospheres (>95% O2) were provided by commercial cylinders (Liquid Carbonic, Chicago, IL). Rats were kept in a 12:12-h light-dark cycle throughout the exposure and taken out daily for 10-15 min to allow for bedding changes and chamber cleaning. At this time, mothers of neonates that had been in one environment (i.e., air or hyperoxia) were changed to the other (air to hyperoxia and vice versa) every 24 h to nurture the new litter and to obviate the effects of hyperoxia on the mothers. After 3 days of exposure, the neonates were killed by decapitation and the adults by CO2 narcosis. The lungs were excised and rinsed in cold PBS on ice and further perfused with cold 0.15 M KCl to remove any blood.
Some neonates were exposed to hyperoxia for 6 days to evaluate the effects of prolonged hyperoxic exposure.
Reagents. Buffer reagents and chemical reagents were purchased from Sigma (St. Louis, MO) except where indicated. Radiolabeled 32P isotope was obtained from Amersham (Arlington Heights, IL).
Preparation of lung nuclear proteins. Rat lung nuclear proteins
were collected according to published methods (21) with modifications.
Briefly, lung tissue was homogenized on ice with a Dounce homogenizer
in a buffer containing 0.5 M sucrose, 10 mM HEPES (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 10% glycerol, 1 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 4,000 g for 20 min at 4°C, and the pellet was resuspended in a
high-salt buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 0.5 M
KCl, 1.5 mM MgCl2, 0.4 mM EDTA, and 0.5 mM
phenylmethylsulfonyl fluoride. After incubation on ice for 10 min, the
supernatant was centrifuged at 14,000 g for 15 min at 4°C.
The supernatant was divided into aliquots and stored at
80°C. Before assays, nuclear protein content was determined
by the method of Bradford (6) (Bio-Rad Laboratories, Richmond, CA).
Determination of AP-1 binding ability of lung nuclear proteins in hyperoxia by electrophoretic mobility shift assay. A 32P-labeled oligonucleotide with the AP-1 consensus sequence (5'-CTAGTGATGAGTCAGCCGGATC-3'; Operon Technologies, Alameda, CA) containing the core AP-1 binding site TGAGTCA seen in HO-1 (1-3) was used as a probe to evaluate the AP-1 binding ability of lung nuclear protein. Nuclear proteins (20 µg) were mixed with the radiolabeled AP-1 probe in a buffer containing 10 mM HEPES (pH 7.9), 1 mM EDTA, 80 mM KCl, 1 µg of poly(dI-dC) · poly(dI-dC), and 4% Ficoll. The reaction mixture was incubated at room temperature for 30 min and electrophoresed on 6% polyacrylamide gels. To distinguish nonspecific binding of the nuclear proteins, competition reactions were performed by adding 100-fold excess nonradiolabeled AP-1, mutated AP-1 (5'-CTAGTGAACTCAGTGCCCGATC-3'; Operon Technologies), or an unrelated factor AP-3 (5'-CTAGTGGGACCTTCCACAGATC-3'; Operon Technologies) to the binding reaction mixture before electrophoresis.
In separate experiments, 2 µl of phosphorylated c-Jun, Jun B, or Jun D antibodies were added to 20 µl of reaction buffer containing nuclear extracts. The mixture was then incubated with the 32P-labeled oligonucleotide probe at room temperature for 1 h. Thereafter, the samples were electrophoresed as described above. This allowed for visualization of supershift-retarded bands in the AP-1 complex.
Antibodies. The following primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): polyclonal rabbit anti-rat c-Jun/AP-1, c-Fos, Jun B, and Jun D and mouse anti-rat monoclonal IgG1 phosphorylated c-Jun (KM-1). Horseradish peroxidase (HRP)-labeled secondary antibodies against the correspondent primary antibodies were purchased from Santa Cruz Biotechnology for c-Jun, c-Fos, Jun B, and Jun D and from Caltag (Burlingame, CA) for phosphorylated c-Jun.
Determination of lung protein carbonyl content. To detect oxidative injury with hyperoxic exposure, protein oxidation was measured by detecting oxidatively generated carbonyl groups with the OxyBlot Kit (Oncor, Gaithersburg, MD). Briefly, 20-µg protein aliquots mixed with 1 volume of 12% SDS were incubated with 2 volumes of 1× 2,4-dinitrophenylhydrazine for 15 min at room temperature. The samples were then neutralized by addition of 1.5 volumes of neutralization solution (Oncor) and electrophoresed on a 12% polyacrylamide gel. After transfer to polyvinylidene difluoride membrane, blots were briefly washed in blocking buffer [1% BSA in PBS-0.05% Tween 20 (PBS-T)] for 1 h then incubated for 1 h at 25°C with rabbit anti-2,4-binitrophenylhydrazone IgG diluted 1:150 in blocking buffer (1% BSA in PBS-T). Blots were washed in PBS-0.05% Tween and incubated for 1 h at 25°C with a 1:300 dilution of HRP-conjugated goat anti-rabbit IgG. The antigen-antibody signal was visualized by chemiluminescence with the HRP system according to the manufacturer's instructions (Bio-Rad). To compare the extent of protein oxidation, densitometric quantitation of all bands was performed (PDI, Sunnyvale, CA).
Determination of c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D immunoreactive protein levels by Western analysis. For detection of c-Fos and c-Jun, the subunits of the AP-1 complex, as well as of phosphorylated c-Jun, Jun B, and Jun D immunoreactive proteins, 80-µg aliquots of lung nuclear proteins were electrophoresed on a 12% polyacrylamide gel according to the method of Laemmli (20). Proteins were transferred overnight to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) with a Bio-Rad transblot apparatus according to the method of Towbin et al. (24). Blots were briefly washed in 1× PBS and then incubated with a 1:150 dilution of either rabbit anti-rat c-Fos, c-Jun, Jun B, or Jun D polyclonal IgG or mouse anti-rat monoclonal phosphorylated c-Jun IgG1 for 45 min at room temperature in blocking solution (3% nonfat milk in PBS-T). Blots were washed in PBS-T and incubated for 1 h at room temperature with a 1:5,000 dilution of HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for c-Fos, c-Jun, Jun B, Jun D, or goat anti-mouse IgG1 (Caltag) for phosphorylated c-Jun. The antigen-antibody signal was visualized as described in Determination of lung protein carbonyl content. Equal loading was verified by Coomassie blue staining.
Sequential immunoprecipitation of lung AP-1 subunit proteins. Immunoprecipitation was done with polyclonal c-Fos, Jun B, Jun D, and monoclonal c-Jun (KM-1) antibodies (13). Briefly, 500 µg of lung nuclear protein extract were reacted for 40 min with the first antibody [Jun D, Jun B, c-Jun (KM-1), or c-Fos] at a 1:100 dilution before addition of Staphylococcus aureus protein A-Sepharose CL-4B beads (final concentration of 10%) for an additional 20 min. After centrifugation at 10,000 g for 3 min to pellet the beads, the supernatant was reimmunoprecipitated with the same primary antibody in an identical manner to be certain that the extract was immunodepleted of all protein reacting with this primary antibody. This precleared extract was then divided into aliquots for immunoprecipitation with one of the three remaining antibodies. The immunodepleted extracts (20 µg) were then reacted with 32P-labeled AP-1 probe for electrophoretic mobility shift assay experiments as described in Determination of the AP-1 binding ability of lung nuclear proteins in hyperoxia by electrophoretic mobility shift assay.
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RESULTS |
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AP-1 binding differed in neonates versus adults. After 3 days
of hyperoxic exposure, lung AP-1 binding remained unchanged in
neonates, whereas lung AP-1 binding increased dramatically in adults
compared with air control rats. Of note, in air, neonates showed more
AP-1 binding activity than the similarly exposed adults. Furthermore,
the predominance of binding in adults occurred at a single band,
whereas neonates demonstrated binding at two bands (Fig.
1A). The lowest (third) band in
neonates was completely competed away with either cold mutated AP-1 or
cold AP-3, a nonrelated factor; therefore, it was nonspecific (Fig.
1A). A similar lack of AP-1 binding was noted in neonates
exposed to hyperoxia for 6 days despite a significant increase in
protein oxidation (2.3-fold) compared with neonates exposed to
hyperoxia for 3 days (Fig. 1B), indicating that the lack of
AP-1 binding was independent of oxidative signaling, at least in the
neonates.
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Specificity of the AP-1 binding. To test for specificity of the
AP-1 binding in both neonates and adults, AP-1 complexes were successfully competed away with 100-fold excess cold AP-1 but not with
100-fold excess cold mutated AP-1 (Fig. 2).
Furthermore, a new supershift-retarded band was noted when both adult
and neonatal nuclear proteins were incubated with antibodies to
phosphorylated c-Jun and Jun D (Fig. 2) but not with a peptide antibody
against Jun B. Supershift with c-Fos was clearly observed in adults but was barely visible in neonates (Fig. 2).
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c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D protein levels
in the lung nuclear extracts. Because AP-1 consists of Jun/Jun
homodimers or Fos/Jun heterodimers, we wanted to understand whether
these nuclear proteins were developmentally regulated, therefore
contributing to the differences in AP-1 binding seen between neonates
and adults exposed to hyperoxia for 3 days. Protein levels of c-Fos did
not increase with 3 days of hyperoxia in either adults or neonates
(Fig. 3). Additionally, c-Jun protein
levels did not change significantly in air and hyperoxia in both adults and neonates (Fig. 3). However, phosphorylated c-Jun levels were increased in adults exposed to hyperoxia but were nearly undetectable in neonates (Fig. 3). Because the presence of Jun B or Jun D can result
in less efficient AP-1-mediated transcription (7, 14, 22, 23), we
assessed the level of these proteins in the lung nuclear extracts. In
neonates, lung Jun B and Jun D protein levels were higher compared with
those in adults after air exposure and further increased in hyperoxia
(Fig. 3). In contrast, in adult lungs, Jun B decreased and Jun D did
not change after 3 days of hyperoxia (Fig. 3).
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AP-1 complexes differed in neonates versus adults. The
composition of the AP-1 complex dictates its migration on
electrophoretic mobility shift assay. Because neonates and adults had
different AP-1 binding patterns after 3 days of hyperoxia, differences
in AP-1 subunit composition were evaluated with sequential
immunoprecipitation. In neonates, after immunodepletion of Jun D, the
top band of the AP-1 complex was barely visible, and the intensity of
the lower band was substantially diminished (Fig.
4A1). Immunoprecipitation with
other antibodies was less efficient in reducing the AP-1 complexes
(Fig. 4A, 2-4). However, in combination with Jun D, loss
of the upper band was accomplished by further immunoprecipitation with
c-Jun, Jun B, and c-Fos and loss of the lower band with c-Fos only
(Fig. 4A1). In adults, immunodepletion of any of the AP-1 subunits evaluated did not result in complete loss of the single band
(Fig. 4B), suggesting that other Fos or non-Fos or non-Jun family proteins made up the AP-1 complex. Nonetheless, the band diminished by immunoprecipitation with both Jun and Fos antibodies, indicating that this band was likely composed of heterodimers (Fig.
4B).
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DISCUSSION |
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The unique nature of regulatory mechanisms observed in immature organisms in response to oxidative stress dictate that it is critical to understand maturational differences in gene regulation so as not to erroneously apply knowledge derived in adult models to the neonatal circumstance. The latter approach could lead to unsatisfactory results if similar therapeutic strategies to obviate oxidant stress were employed regardless of maturational considerations.
It is known that the hyperoxic regulation of AOE and the stress response protein HO-1 differ in maturation. Others (8-10) have shown that immature animals show posttranscriptional regulation of several AOEs in oxidative stress, but it is unclear why there is no increased transcription of these AOEs. Similarly, a lack of increased lung HO-1 mRNA was shown in hyperoxia-exposed neonatal rats (12), but it is not clear what factors dictate this refractoriness of HO-1 to transcriptional regulation. Many AOE genes possess AP-1 or AP-1-like binding motifs. For example, the rat manganese superoxide dismutase gene has one copy of the consensus sequence for the binding of AP-1 in the proximal 5'-flanking region upstream of the transcription initiation site (16), and the human glutathione peroxidase gene has several AP-1 and AP-1-like binding motifs (18). As to the mouse HO-1 gene, there are multiple stress response elements containing the AP-1 consensus binding site TGAGTCA in the two distal enhancer regions (1-3), and increased AP-1 binding activity has been associated with enhanced HO-1 gene transcription in hyperoxia, at least in adult rats (21). Here we demonstrated a lack of increase in AP-1 binding in hyperoxia-exposed neonatal rat lungs and show evidence to explain this observation. Furthermore, we demonstrated that the differences in AP-1 binding observed in neonates versus adults exposed to hyperoxia for 3 days were not merely correlated to the level of oxidative injury because AP-1 binding could not be demonstrated in neonates despite a longer exposure (6 days) and clear evidence of oxidative injury.
Although no changes in c-Fos or c-Jun immunoreactive protein were observed in either neonates or adults, increased phosphorylated c-Jun was seen in adults after 3 days of hyperoxia. In oxidative stress, phosphorylation of c-Jun occurs via signal transduction pathways (19), and this enhances AP-1-mediated transcriptional activation independent of changes in AP-1 binding (5). The increased phosphorylation of c-Jun could help explain the increased AP-1-mediated HO-1 transcription in hyperoxia in adult lungs but not in neonatal lungs as shown previously (12, 21). To further corroborate the importance of c-Jun phosphorylation in the lung AP-1 complex in hyperoxia, specificity was demonstrated by supershift gel retardation with phosphorylated c-Jun antibodies. Interestingly, hyperoxia-exposed neonates showed equal or greater specificity of the AP-1 complex to phosphorylated c-Jun than did similarly exposed adults; nonetheless, the relative absence of phosphorylated c-Jun protein in the neonatal lungs may have precluded increased AP-1-mediated transcription in hyperoxia, independent of AP-1 binding. These data also suggest that neonates may not be able to phosphorylate c-Jun as effectively as adults. Because there are many factors that modulate transcriptional activation and because the phosphorylation of c-Jun may have many effects (25), these aspects need to be further evaluated.
We also noted differences in the AP-1 complex of neonatal and adult lungs in that a single predominant band appeared in adult lungs, whereas two bands were seen in neonatal lungs. It has been shown that migration of the AP-1 complex results from the differential DNA binding affinities of the Fos/Jun heterodimer and Jun/Jun homodimer where the Fos/Jun has stronger DNA binding affinity than Jun/Jun (4). Because the phosphorylated c-Jun, Jun B, and Jun D protein levels differed between neonates and adults, the differences in the AP-1 binding pattern could have been explained by the different composition of the AP-1 subunits in the two groups. With immunoprecipitation, the upper neonatal band appeared to be composed of the Jun family proteins with a predominance of Jun D. The lower band appeared to be composed of c-Fos and Jun D heterodimers. In contrast, the single adult band appeared to be composed of c-Fos and Jun family proteins but likely consisted of other Fos or non-Fos or non-Jun family proteins as well. Taken together, the predominance of Jun D protein and the high Jun D content of AP-1 bands in neonates suggest that neonatal lungs could have less efficient AP-1-mediated transcriptional activity in hyperoxia. Unlike c-Jun, Jun B and Jun D can result in reduced AP-1 transcriptional activity either due to competition of Jun B with the more active form c-Jun (4) or through the less efficient transcriptional activation resulting from the Jun B/c-Jun or Jun D/c-Jun heterodimer (14, 22, 23). Although we did not observe specificity of the AP-1 complex for Jun B (lack of supershift-retarded band) in adult or neonatal lungs, immunodepletion with Jun B altered the AP-1 complex, suggesting a contribution of Jun B to AP-1-mediated regulation in this model. We suspect that the lack of supershift with Jun B could have resulted from inefficient binding of the Jun B peptide antibody to the AP-1 complexes. In adults, incomplete loss of the AP-1 binding by immunoprecipitation indicated that other Fos or non-Fos or non-Jun family proteins may be involved. In corroboration with this result, others (11) have shown that other Fos family proteins such as Fra can participate in the AP-1 binding complexes. Additionally, non-Jun and non-Fos proteins such as MAF and NF-E2 can form the AP-1 complexes (17).
In conclusion, maturational differences in AP-1-mediated transcription in hyperoxia appear to be regulated in the neonates by a combined relative deficiency of lung phosphorylated c-Jun protein and an abundance of Jun D and perhaps of Jun B. An understanding of the mechanisms that modulate transcriptional regulation in neonates may allow for therapeutic intervention to regulate gene expression in a manner unique to the immature organism.
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ACKNOWLEDGEMENTS |
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We express our gratitude to Arthur Tatarov and Dr. Yi-Hao Weng for expert technical assistance.
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FOOTNOTES |
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This work was funded by National Heart, Lung, and Blood Institute Grants HL-52701 and HL-58752 (to P. A. Dennery); an American Lung Association Career Investigator Award (to P. A. Dennery); and the Mary L. Johnson and Hess Funds of Stanford University.
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: P. A. Dennery, Dept. of Pediatrics, Stanford Univ., 750 Welch Rd. #315, Palo Alto, CA 94304 (E-mail: dennery{at}leland.stanford.edu).
Received 5 February 1999; accepted in final form 27 September 1999.
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REFERENCES |
---|
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---|
1.
Alam, J.
Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals.
J. Biol. Chem.
269:
25049-25056,
1994
2.
Alam, J.,
S. Camhi,
and
A. M. Choi.
Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and inducer-dependent transcription enhancer.
J. Biol. Chem.
270:
11977-11984,
1995
3.
Alam, J.,
and
Z. Den.
Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene.
J. Biol. Chem.
267:
21894-21900,
1992
4.
Angel, P.,
and
M. Karin.
The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.
Biochim. Biophys. Acta
1072:
129-157,
1991[ISI][Medline].
5.
Binetruy, B.,
T. Smeal,
and
M. Karin.
Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain.
Nature
351:
122-127,
1991[ISI][Medline].
6.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[ISI][Medline].
7.
Chiu, R.,
P. Angel,
and
M. Karin.
Jun-B differs in its biological properties from, and is a negative regulator of, c-Jun.
Cell
59:
979-986,
1989[ISI][Medline].
8.
Clerch, L. B.,
J. Iqbal,
and
D. Massaro.
Perinatal rat lung catalase gene expression: influence of corticosteroid and hyperoxia.
Am. J. Physiol. Lung Cell. Mol. Physiol.
260:
L428-L433,
1991
9.
Clerch, L. B.,
and
D. Massaro.
Rat lung antioxidant enzymes: differences in perinatal gene expression and regulation.
Am. J. Physiol. Lung Cell. Mol. Physiol.
263:
L466-L470,
1992
10.
Clerch, L. B.,
A. E. Wright,
and
J. J. Coalson.
Lung manganese superoxide dismutase protein expression increases in the baboon model of bronchopulmonary dysplasia and is regulated at a posttranscriptional level.
Pediatr. Res.
39:
253-258,
1996[Abstract].
11.
Cohen, D. R.,
P. C. Ferreira,
R. Gentz,
B. R. Franza, Jr.,
and
T. Curran.
The product of a fos-related gene, fra-1, binds cooperatively to the AP-1 site with Jun: transcription factor AP-1 is comprised of multiple protein complexes.
Genes Dev.
3:
173-184,
1989[Abstract].
12.
Dennery, P. A.,
P. A. Rodgers,
M. A. Lum,
B. C. Jennings,
and
V. Shokoohi.
Hyperoxic regulation of lung heme oxygenase in neonatal rats.
Pediatr. Res.
40:
815-821,
1996[Abstract].
13.
Harlow, E.,
and
D. Lane.
Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988.
14.
Hirai, S. I.,
R. P. Ryseck,
F. Mechta,
R. Bravo,
and
M. Yaniv.
Characterization of junD: a new member of the jun proto-oncogene family.
EMBO J.
8:
1433-1439,
1989[Abstract].
15.
Ho, Y. S.,
M. S. Dey,
and
J. D. Crapo.
Antioxidant enzyme expression in rat lungs during hyperoxia.
Am. J. Physiol. Lung Cell. Mol. Physiol.
270:
L810-L818,
1996
16.
Ho, Y. S.,
A. J. Howard,
and
J. D. Crapo.
Molecular structure of a functional rat gene for manganese-containing superoxide dismutase.
Am. J. Respir. Cell Mol. Biol.
4:
278-286,
1991[ISI][Medline].
17.
Inamdar, N. M.,
Y. I. Ahn,
and
J. Alam.
The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors.
Biochem. Biophys. Res. Commun.
221:
570-576,
1996[ISI][Medline].
18.
Jornot, L.,
and
A. F. Junod.
Hyperoxia, unlike phorbol ester, induces glutathione peroxidase through a protein kinase C-independent mechanism.
Biochem. J.
326:
117-123,
1997[ISI][Medline].
19.
Kyriakis, J. M.,
P. Banerjee,
E. Nikolakaki,
T. Dai,
E. A. Rubie,
M. F. Ahmad,
J. Avruch,
and
J. R. Woodgett.
The stress-activated protein kinase subfamily of c-Jun kinases.
Nature
369:
156-160,
1994[ISI][Medline].
20.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
21.
Lee, P. J.,
J. Alam,
S. L. Sylvester,
N. Inamdar,
L. Otterbein,
and
A. M. Choi.
Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury.
Am. J. Respir. Cell Mol. Biol.
14:
556-568,
1996[Abstract].
22.
Nakabeppu, Y.,
K. Ryder,
and
D. Nathans.
DNA binding activities of three murine Jun proteins: stimulation by Fos.
Cell
55:
907-915,
1988[ISI][Medline].
23.
Schutte, J.,
J. Viallet,
M. Nau,
S. Segal,
J. Fedorko,
and
J. Minna.
jun-B inhibits and c-Fos stimulates the transforming and trans-activating activities of c-jun.
Cell
59:
987-997,
1989[ISI][Medline].
24.
Towbin, H.,
T. Staehelin,
and
J. Gordon.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:
4350-4354,
1979[Abstract].
25.
Wisdom, R.,
R. S. Johnson,
and
C. Moore.
c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms.
EMBO J.
18:
188-197,
1999