1 Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center; and 2 Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111
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ABSTRACT |
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The chemotherapeutic agent bleomycin
induces pulmonary fibrosis through the generation of reactive oxygen
species (ROS), which are thought to contribute to cellular damage and
pulmonary injury. We hypothesized that bleomycin activates oxidative
stress response pathways and regulates cellular glutathione (GSH).
Bovine pulmonary artery endothelial cells exposed to bleomycin exhibit
growth arrest and increased cellular GSH content. -Glutamylcysteine
synthetase (
-GCS) controls the key regulatory step in GSH synthesis,
and Northern blots indicate that the
-GCS catalytic subunit
[
-GCS heavy chain (
-GCSh)] is upregulated by
bleomycin within 3 h. The promoter for human
-GCSh
contains consensus sites for nuclear factor-
B (NF-
B) and the
antioxidant response element (ARE), both of which are activated in
response to oxidative stress. Electrophoretic mobility shift assays
show that bleomycin activates the transcription factor NF-
B as well
as the ARE-binding factors Nrf-1 and -2. Nrf-1 and -2 activation by
bleomycin is inhibited by the ROS quenching agent
N-acetylcysteine (NAC), but not by U-0126, a MEK1/2
inhibitor that blocks bleomycin-induced MAPK activation. In contrast,
NF-
B activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of
-GCS
expression. These data suggest that bleomycin can activate oxidative
stress response pathways and upregulate cellular GSH.
reactive oxygen species; Nrf-1 and -2; nuclear factor-B; antioxidant response element; mitogen-activated protein kinase
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INTRODUCTION |
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BLEOMYCIN IS PRIMARILY
USED for the treatment of testicular carcinoma, lymphoma, and
squamous cell carcinoma, but its use as a chemotherapeutic agent is
limited by adverse side effects, especially pulmonary injury and
fibrosis. Because of this, bleomycin has been used to develop a rodent
experimental model of pulmonary fibrosis that has allowed the study of
changes in the cellular composition of the lung and the expression of
specific proteins leading to fibrosis. Many changes identified in the
bleomycin model system have also been observed in pulmonary fibrosis
induced by other causes (2, 20, 21, 30). Treatment of
rodents with bleomycin endotracheal instillation or subcutaneous
injection results in initial pulmonary inflammation and a spike of
epithelial/endothelial apoptosis (13, 30, 38, 46).
This is followed by the proliferation of myofibroblasts (fibroblasts
expressing -smooth muscle actin) and sustained
epithelial/endothelial apoptosis. The bleomycin model has
allowed the identification of potentially critical changes in protein
expression, including the induction of transforming growth factor-
1
(18, 26, 27, 29, 30). However, the signaling mechanism(s)
inducing these changes is not well understood.
Bleomycin triggers apoptosis in growing cells by causing single- and double-stranded DNA breaks through the direct binding of bleomycin to DNA. This activity is dependent on oxygen and a bound ferrous ion. Bleomycin is also believed to produce reactive oxidative species (ROS), including superoxide, H2O2, and/or organoperoxides, which may also play a role in the toxicity of bleomycin (34). Several studies have shown that cells are partially protected from the cytotoxic effects of bleomycin by acute hypoxia (thus eliminating the oxygen source for ROS) or by the addition of superoxide dismutase enzyme or transfection with an expression vector for superoxide dismutase (thus reducing the level of ROS generated by bleomycin) (10, 34). In contrast, the detrimental effects of bleomycin can be intensified via reduction of the intrinsic cellular defenses against oxidative stress. Either buthionine sulfoximine (BSO)-induced depletion of cellular glutathione (GSH) or the lack of GSH S-transferase (a phase II detoxifying enzyme) results in hypersensitivity of cells to bleomycin-induced apoptosis (12, 31).
On the basis of studies indicating that bleomycin cytotoxicity may be
modulated by oxidants and antioxidants, we hypothesized that bleomycin
may regulate cellular defenses against oxidative stress. GSH is a
ubiquitous sulfhydryl-containing tripeptide that serves as a primary
biological defense against oxidative damage (6, 33, 37,
44). GSH, normally present in cells at millimolar levels,
directly interacts with ROS and toxins during cellular detoxification
(6, 37, 44). Adaptation of cells to oxidative stress can
occur via the induction of antioxidant enzymes and increased cellular
levels of GSH. The enzyme -glutamylcytsteine synthetase (
-GCS)
controls the rate-limiting step in GSH synthesis (16, 17,
24). This enzyme is composed of two subunits, a catalytic heavy
chain (
-GCSh) and a regulatory light chain. Both
-GCS
subunits have been shown to be upregulated in response to oxidative
stress, including ionizing radiation, ROS, heavy metals, phenolic
antioxidants, and the GSH-depleting compound BSO (16, 17, 32, 33,
36, 41, 44). The regulation of the
-GCS subunits by oxidative
stress has been demonstrated in variety of species, including human,
rat, and bovine (32, 33, 36, 44).
In the present study, we examined the effect of bleomycin on the
regulation of GSH in bovine pulmonary artery endothelial cells (BPAEC).
Our results indicate that bleomycin treatment increases total cellular
levels of GSH and upregulates the level of -GCSh mRNA.
We also show that bleomycin activates the DNA binding activity of
nuclear factor (NF)-
B and Nrf-1 and -2 transcription factors. These
factors are known to be activated by oxidative stress and to regulate
the expression of human
-GCSh (14-17, 24, 40, 41, 44, 45).
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EXPERIMENTAL METHODS |
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Reagents. Bleomycin (Blenoxane) was from Mead Johnson (Princeton, NJ). The mitogen-activated protein kinase/extracellular signal-regulated kinase [MAPK/ERK (MEK)] 1/2 inhibitor U-0126 was purchased from New England Biolabs (Beverly, MA). Other reagents are described below.
Cell culture and bleomycin treatment. BPAEC were obtained from freshly slaughtered calves as previously described (4). Passage 3-8 cells were used for all experiments. Rat pulmonary microvascular endothelial cells (RPMEC) were a gift of Dr. Una Ryan (Avant Immunotherapeutics, Needham, MA) (3); these cells were used as a control in Fig. 2. Both BPAEC and RPMEC were cultured in RPMI 1640 (GIBCO-BRL, Rockville, MD) with antibiotics (penicillin and streptomycin), fungisone, and 10% fetal bovine serum in 5% CO2 at 37°C in a humidified atmosphere. For cell culture treatment, bleomycin was dissolved in 0.9% NaCl and added to the culture medium.
Propidium iodide staining and fluorescence activated cell sorting. Propidium iodide and fluorescence-activated cell sorting (FACS) were used to determine cellular apoptosis. Cells (~3-7.5 × 105) were washed twice with phosphate-buffered saline (PBS) at 25°C and trypsinized. Cells were then pelleted by centrifugation and washed twice on ice with cold PBS. After the last wash, we resuspended cell pellets in 100 µl of cold PBS, and added 1 ml of cold 80% ethanol dropwise while vortexing on a low setting. Ethanol-treated cells were stored at 4°C for at least 4 h and up to 1 wk. Before FACS analysis, cells were pelleted at 4°C, resuspended in 0.5 ml of propidium iodide solution [0.05 mg/ml RNase A in PBS containing 5% (wt/vol) propidium iodide], and incubated for 30 min on ice in the dark. Cells were then analyzed on a Becton-Dickinson FACS Calibur (Franklin Lakes, NJ) cell sorter.
Cell counts and preparation for GSH assays.
Cell counts and GSH assays were performed as described
(43). Culture dishes were rinsed twice with PBS at 25°C
and incubated for 3-5 min with 1.0 ml of trypsin-EDTA. The cells
were rapidly suspended and placed on ice. The cellular suspension (0.1 ml) was removed, diluted, and counted on a ZM Coulter Counter (Coulter Electronics, Hialeah, FL). Of the remaining suspension, 0.8 ml was
treated with 0.1 ml of 1% perchloric acid. Lysates were sonicated on
ice for 10 s and centrifuged at 14,000 g at 4°C for
20 min. Aliquots were stored at 20°C for GSH assays.
GSH assay.
GSH assays were performed as specified (40). Frozen
perchloric acid-treated supernatants were thawed on ice and sonicated for 10 s on ice. The pH was adjusted to 7.0 with 0.3 M potassium hydroxide-3-(N-morpholino)propanesulfonic acid (MOPS). The
sonicate was then centrifuged at 14,000 g at 4°C for 20 min. The supernatant was assayed for total cellular GSH by the
spectrophotometric Tietze method (1). Briefly, the sum of
the oxidized and reduced forms of GSH was determined using a kinetic
assay in which GSH or GSH disulfide and GSH reductase reduce
5,5'-dithiobis(2-nitrobenzoic acid) to form 5-thio-2-nitrobenzoate
(TNB). The formation of TNB was followed spectrophotometrically at 412 nm. Each assay was individually calibrated with standard GSH, and the
concentration of each sample was adjusted by dilution to ensure that
the reaction rate was on the linear portion of the standard curve.
Cellular GSH levels were expressed as nanomoles per 106
cells. For the assay, brewer's yeast GSH reductase, -NADPH, and GSH
disulfide were obtained from Sigma (St. Louis, MO).
RNA purification and Northern blot.
RNA was purified using Trizol (GIBCO-BRL) according to the
manufacturer's instructions; RNA concentrations were determined by
absorbance at 260 nm. RNA (10 µg) was denatured in a loading buffer
[0.4 M MOPS, pH 7.0, 2.5% formaldehyde, 67% formamide, 0.2 µg
ethidium bromide, 1.2 mM EDTA, 6.7 mM Na acetate, and 13% dye (50%
glycerol, 1 mM EDTA, 0.25% bromophenol blue, and 0.25% xylene cyanol
FF)] for 15 min at 65°C. Denatured RNA was run in a 0.9% (wt/vol)
denaturing agarose gel (0.2 M MOPS, pH 7.0, 5 mM sodium acetate, 1 mM
EDTA, and 1.8% formaldehyde) with running buffer (0.2 M MOPS, pH 7.0, 5 mM Na acetate, 1 mM EDTA, and 1.8% formaldehyde). RNA was
transferred to a Zeta Probe blotting membrane (Bio-Rad, Hercules, CA)
using capillary blotting in 1× SSC (20× SSC = 3 M NaCl, 3 mM
sodium citrate, pH 7.0). Blots were baked at 80°C for 2 h under
vacuum and prehybridized in ExpressHyb (Clontech, Palo Alto, CA) for 30 min at 68°C under shaking. The 2 × 106
counts · min1 · ml
1 of
labeled, denatured probe were then added with denatured salmon sperm
DNA (100 ng/ml) to the prehybridized blot. Hybridization was performed
at 68°C overnight with agitation. Blots were washed at 37°C; the
first wash was performed in triplicate (2× SSC and 0.05% SDS) for 15 min each; the second wash was done twice (1× SSC and 0.1% SDS) for 15 min each at 50°C. The blot was then exposed to Kodak X-OMAT AR film
(NEN, Boston, MA) at
80°C using intensifying screens.
Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed as described by Garner and Revzin (11). To prepare nuclear extracts, cells were washed in PBS and incubated in 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM NaF, 0.1 mM sodium orthovanadate, and 1 mM tetrasodium pyrophosphate for 15 min at 4°C. (Octylphenoxy)polyethoxyethanol (Igepal CA-630, Sigma) was then added at a final concentration of 0.6%(vol/vol). Samples were vortexed and centrifuged. Pelleted nuclei were resuspended in extraction buffer [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.1 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, and 1% (vol/vol) glycerol], then mixed vigorously for 20 min, and centrifuged for 5 min. The supernatants were harvested, and protein concentrations were determined (22).
Oligonucleotides containing the sense and antisense antioxidant response element (ARE) consensus sequence were 5'-TCACAGTGACTCAGCAGAATC-3' and 5'-GATTCTGCTGAGTCA-CTGTGA-3', respectively. [The ARE consensus sequence is underlined; the ARE sequence used is identical to ARE4 of the humanWestern blot analysis.
To prepare lysates, cells were washed in PBS and solubilized with 50 mM
HEPES solution (pH 7.4) containing 1% (vol/vol) Triton X-100, 4 mM
EDTA, 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM tetrasodium
pyrophosphate, 2 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin. The lysate was cleared by centrifugation at 4°C for 15 min. Protein concentrations in the supernatant were determined as above
(22). Cell lysates (10 µg protein) were electrophoresed
through reducing (5% -mercaptoethanol) SDS polyacrylamide-bis gels
(10%) and electroblotted onto nitrocellulose membranes. After the
transfer, membranes were blocked in 5% milk in Tween- and Tris-buffered saline (20 mM Tris · HCl, pH 7.5, 150 mM NaCl,
and 0.05% Tween 20) and then blotted with the antibody for the NF-
B p65 subunit and I
B
(Santa Cruz Biotech). Levels of
proteins and phosphoproteins were detected with horseradish
peroxidase-linked secondary antibodies and ECL System (Amersham Life
Science, Arlington Heights, IL). Western blots were repeated at least
three times.
Statistics. Statistical comparisons were performed using a Student's t-test for unpaired samples and a two-way analysis of variance for multiple comparisons. Statistical significance was determined at P < 0.05.
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RESULTS |
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Bleomycin upregulates cellular GSH and the -GCS gene.
Bleomycin has been shown to induce growth arrest, apoptosis,
and necrosis in a number of cells, including immortalized cells and
primary cancer cells (31, 34). We first determined doses of bleomycin that induce growth arrest with limited cell death in
BPAEC. Cells were placed in RPMI medium containing 0.1% FBS for
24 h and then exposed to increasing doses of bleomycin for 24 h. Cell numbers indicated that 10 µg/ml of bleomycin induced high
levels of growth arrest (Fig.
1A), which was associated with visible cell death (data not shown). Levels of 1.0 and 0.1 µg/ml of
bleomycin induced lower levels of growth arrest. Propidium iodide
staining and FACS analysis showed that 1.0 µg/ml of bleomycin induced
only 8.8% (± 1.6 SD) apoptosis, compared with 5.8% (± 1.7 SD) apoptosis in control cells (n = 3).
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Bleomycin activates NF-B and the ARE-binding transcription
factors Nrf-1 and -2.
Studies of human
-GCSh gene expression have identified
two promoter cis elements that are believed to primarily
regulate its transcription: the
B element and the ARE (6, 16,
17, 37, 44). Increased expression of
-GCSh in
response to ionizing radiation, which produces physical and chemical
damage in addition to inducing the formation of ROS, is dependent on
NF-
B activation (16). However, regulation of
-GCSh by xenobiotics and ROS-generating compounds is
believed to occur through the activation of proteins that bind to the
ARE (17, 37, 44). Because bleomycin induces physical
damage to cellular proteins and DNA while at the same time producing
ROS, we investigated the activation of DNA binding to both
B and ARE.
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Bleomycin activates ARE-binding via ROS and NF-B via MAPK.
Both ARE and NF-
B site binding factors are known to be activated
through a variety of signaling pathways (7, 8, 14, 28, 40, 41,
44, 45). Both the overexpression of the ERK1-MAPK and the
induction of ROS activate NF-
B (16, 24, 28). Studies
have shown that Nrf-1 and -2 are activated in response to ROS, and the
inhibition of ERK1/2-MAPK blocks activation of ARE-binding factors by
xenobiotic compounds (17, 40, 44, 45). We previously
reported that bleomycin activates the ERK1/2 family of MAPKs
(5), and it is widely believed that bleomycin produces ROS
through its bound ferrous ion (10, 13, 34, 39). Thus we
investigated whether one or both of these mechanisms downstream of
bleomycin lead to the activation of factors that bind ARE and/or the
NF-
B site.
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DISCUSSION |
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The chemotherapeutic agent bleomycin can induce pulmonary fibrosis, thus limiting its application in cancer treatment in humans. This side effect has been utilized in an animal model system to identify cellular and molecular changes that occur during the development and progression of pulmonary fibrosis (10, 14, 18, 20, 21, 26, 27, 29, 30, 34, 38). Despite extensive work demonstrating the changes in mRNA and protein expression that occur during bleomycin-induced fibrosis, comparatively little is known about the mechanism(s) of these events. The cytotoxic effects of bleomycin are modulated by antioxidants, and we hypothesized that bleomycin regulates cellular defenses against oxidative stress.
The present study indicates that in endothelial cell culture, treatment
with sublethal levels of bleomycin (0.01-1.0 µg/ml) for 18 h causes increased total cellular GSH. We also demonstrated that in 30 min, bleomycin increases the level of -GCSh mRNA, the
catalytic subunit of
-GCS that controls the key regulatory step in
GSH synthesis. Karam et al. (19) previously reported a
~50% decrease in the level of total GSH in alveolar T2 cells isolated from Wistar rats treated for 7 or 14 days with bleomycin. This
was accompanied by a slight decrease in
-GCS enzyme levels at 7 days. Further experiments are required to determine whether the
increase in cellular GSH as observed in the present study would also
occur in vivo after acute exposure to bleomycin.
In response to oxidative or toxic stress, expression of the human
-GCSh is positively regulated at the level of
transcription by factors binding to both ARE and NF-
B promoter
elements (16, 17, 40, 41, 44). Our EMSA results indicate
that bleomycin activates both ARE-binding factors and NF-
B in BPAEC
within 30 min. Although the sequence of the bovine
-GCSh
promoter has not been reported, Shi et al. (32, 33) found
that endogenous bovine
GCSh mRNA is increased in BPAEC
in response to quinone-induced oxidative stress, similar to findings in
other species, including human (44). Therefore, in BPAEC,
the induced increase of
-GCSh mRNA by bleomycin is
consistent with a mechanism involving the induction of ARE-binding
factors and NF-
B.
A number of proteins have been shown to bind the ARE element, including Nrf-1 and -2 (members of the cap 'n' collar subfamily of basic region-leucine zipper transcription factors), members of the Jun family (c-Jun, JunB, and JunD), and several small Maf family proteins (c-Maf, hMaf, MafG, and MafK) (7, 8, 14, 17, 23, 40, 41, 44). Supershift experiments using antibodies directed against Nrf-1 or Nrf-2 caused a reduction in the level of the ARE-containing band. However, neither the Nrf-1 nor -2 antibodies caused the appearance of a supershifted species, suggesting that these antibodies dissociate Nrf proteins from a complex with ARE. Inclusion of the antibody against Nrf-2 caused both the reduction of the original ARE-containing band and the appearance of a band with increased electrophoretic mobility. The induction of a band with faster mobility suggests that complexes with Nrf-2 contain at least one additional protein (X) and that the new, faster complex contains the ARE probe bound to X. The identity of X is currently unknown. Other laboratories have shown that Nrf-2 forms heterodimers with small Maf proteins, including MafG (7, 23, 44, 45) and Jun proteins (44). Supershift experiments were performed using antibodies against c-Jun and c-Fos, but these antibodies did not alter the mobility of the ARE complex, indicating that these proteins are not activated by bleomycin.
Nrf proteins have been shown to be activated downstream of protein kinase C and the MAPKs ERK1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) (14, 44, 45). In BPAEC, bleomycin activates p38/HOG1 (unpublished results) and ERK1/2 (5), but not SAPK/JNK (unpublished results). Our current data suggest that bleomycin activates Nrf-1 and -2 through a mechanism independent of ERK1/2-MAPK, as the MEK inhibitor U-0126 failed to block bleomycin induction. Our data showing the inhibition Nrf activation by NAC suggest that ROS act as second messengers downstream of bleomycin.
In contrast to Nrf-1 and -2 activation by bleomycin, NF-B activation
is blocked by U-0126 but not by the ROS-quenching agent NAC, suggesting
that the ERK1/2-MAPK pathway is involved. NF-
B activation has been
shown to occur in response to the overexpression of ERK1-MAPK (9,
28).
In summary, our results indicate that bleomycin upregulates GSH
and -GCS through the activation of both ROS- and MAPK-dependent pathways in a mechanism that likely involves the induction of ARE-binding factors and NF-
B. The use of bleomycin in animals remains the most useful model system for the study of pulmonary fibrosis, and the understanding of mechanisms of bleomycin signal transduction pathways should help in developing therapeutic strategies.
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ACKNOWLEDGEMENTS |
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We thank Drs. Amy Simon and Daniel Rotiz for technical help.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-42376 and the United States Department of Agriculture (58-1950-9-001). R. Day is a recipient of an American Heart Association National Center Scientist Development Award and an American Lung Association Research Grant.
Address for reprint requests and other correspondence: R. M. Day, New England Medical Center, Pulmonary and Critical Care Division, NEMC #257, 750 Washington St., Boston, MA 02111 (E-mail: rday{at}lifespan.org).
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. Section 1734 solely to indicate this fact.
First published January 18, 2002;10.1152/ajplung.00338.2001
Received 27 August 2001; accepted in final form 9 January 2002.
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