Department of Physiology, Michigan State University, East Lansing, Michigan 48824
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ABSTRACT |
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Primary cultures of rat
type II alveolar epithelial cells (AECs) or human AEC-derived A549
cells, when exposed to bleomycin (Bleo), exhibited
concentration-dependent apoptosis detected by altered nuclear
morphology, fragmentation of DNA, activation of caspase-3, and net cell
loss over time. In both cell culture models, exposure to Bleo caused
time-dependent increases in angiotensinogen (ANGEN) mRNA.
Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced
apoptosis of rat AEC or A549 cells by 83 and 84%, respectively
(P < 0.01 and P < 0.05), and
prevented Bleo-induced net cell loss. Apoptosis of rat AECs or
A549 cells in response to Bleo was inhibited 91% by the ANG-converting
enzyme inhibitor captopril or 82%, respectively, by neutralizing
antibodies specific for ANG II (both P < 0.01).
Antagonists of ANG receptor AT1 (losartan, L-158809, or
saralasin), but not an AT2-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%,
P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52%
(P < 0.05). These data indicate that Bleo, like
FasL or TNF-, induces transactivation of ANG synthesis
de novo that is required for AEC apoptosis. They also support
the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.
renin-angiotensin system; type II pneumocyte; lung fibrosis; programmed cell death; angiotensinogen
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INTRODUCTION |
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ALVEOLAR EPITHELIAL
CELLS (AECs) have many important roles that are critical to
normal lung function (14). The death of AECs by
apoptosis is now believed to be an important event in the
pathogenesis of lung fibrosis (9) and in more acute lung injury (15, 24). A variety of investigations have
implicated important roles for key molecules such as tumor necrosis
factor-alpha (TNF-) and Fas ligand (FasL), both known
inducers of apoptosis in a variety of cell types, in the events
that lead to fibrogenesis in the lung (6, 7, 16).
In earlier work (28, 31), we showed that exposure of
either primary cultures of rat AECs or the human AEC-derived A549 cell
line to FasL or TNF- increases angiotensinogen (ANGEN)
mRNA and protein and evokes its subsequent conversion to angiotensin II
(ANG II). Moreover, we found that transactivation of the ANGEN gene is
required for AEC apoptosis in response to TNF-
or
FasL. Thus AEC death in response to these agents could be
blocked by ANG receptor antagonists or ANG-converting enzyme (ACE)
inhibitors (ACEis), at least in vitro. Studies of another inducer of
AEC apoptosis, the antiarrhythmic agent amiodarone, also showed
that antagonists of ANG production or receptor interaction could
prevent apoptosis of AECs in response to this benzofuran
compound (1).
For these reasons we hypothesized that AEC apoptosis in response to bleomycin (Bleo) might also require ANG synthesis and might therefore be inhibitable by ANG system antagonists. We report here that Bleo, if applied to rat or human AECs in vitro, induces the expression of ANGEN mRNA and subsequent apoptosis that can be blocked by ANGEN antisense oligonucleotides, by ACEis, or by ANG receptor antagonists of the AT1-selective subtype.
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MATERIALS AND METHODS |
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Reagents and materials. The AT1-selective antagonists L-158809 and losartan were obtained from Merck (West Point, PA). The AT2-selective antagonist PD-123319 was obtained from Parke Davis Research Division (Ann Arbor, MI). The caspase inhibitor N-benzylcarboxy-Val-Ala-Asp-[O-Me]-CH2F (ZVAD-fmk) was obtained from Kamiya Biomedical (Seattle, WA). Asp-Glu-Val-Asp-[O-Me]-CH2F (DEVD-fmk) was obtained from Pharmingen (San Diego, CA). Alkaline phosphatase-conjugated streptavidin, digoxigenin-labeled deoxyuridine trisphosphate, and biotinylated deoxyuridine trisphosphate were obtained from Boehringer Mannheim (Indianapolis, IN). Bleo, anti-ANG antibodies, aurintricarboxylic acid (ATA), captopril, and saralasin were obtained from Sigma (St. Louis, MO). Reagents for detection of alkaline phosphatase and other secondary reagents for in situ end labeling of DNA or Western blotting were from sources described earlier (29). All other materials were of reagent grade and were obtained from Sigma.
Cell culture. The human lung adenocarcinoma cell line A549 was obtained from American Type Culture Collection and cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum. Primary alveolar epithelial cells were isolated from adult male Wistar rats as described earlier (31). The primary cells were studied at day two of culture, a time at which they are type II cell-like by accepted morphological and biochemical criteria (17). Primary cell preparations were of >90% purity as assessed by acridine orange staining as described previously (25, 28, 31). All cells were grown in 24- or 6-well chambers and were analyzed at subconfluent densities of 80-90%. All subsequent incubations with Bleo and/or other test agents were performed in serum-free medium. The cells were exposed to caspase inhibitors or antagonists of the ANG system 30 min before exposure to Bleo for 1-20 h as indicated.
Quantitation of apoptosis and cell loss.
Detection of apoptotic cells with propidium iodide (PI) was
conducted as described earlier (28, 31) following
digestion of ethanol-fixed cells with DNase-free RNase in PBS
containing 5 µg/ml PI. In these assays, detached cells were retained
by centrifugation of the 24-well culture vessels during fixation with
70% ethanol. Cells with discrete nuclear fragments containing
condensed chromatin were scored as apoptotic. As in earlier
publications, the induction of apoptosis was verified by in
situ end labeling of fragmented DNA (see Fig.
1 and Refs. 29 and
126). Apoptotic cells were scored over a minimum of
four separate microscopic fields from each of at least three culture
vessels per treatment group.
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Detection and quantitation of caspase-3 activity. Activation of caspase-3 was detected through immunolabeling of fixed cells adherent to plastic culture surfaces with an antibody that recognizes only the active form of the enzyme (Biovision, Mountain View, CA). The primary antibody was detected with an alkaline phosphatase-conjugated secondary antibody followed by nitro-blue tetrazolium. The enzymatic activity of caspase-3 was measured in adherent cells incubated for 20 h with the membrane-permeable substrate N-acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Upstate Biotech, Saranac Lake, NY) at 50 µM. Quantitation of the fluorescent product was achieved with a Biotek FL600 fluorescence plate reader. Fluorescence values were normalized to cell number determined on the same culture well after cell fixing and staining of DNA with PI (27).
RT-PCR and antisense experiments. Semiquantitative RT-PCR was performed as described earlier (28, 31). The annealing temperatures for PCR reactions were optimized for each primer by preliminary trials. All PCR amplifications were terminated at or near the center of the linear range for each gene product analyzed, as determined by sequential withdrawal of sample at five-cycle intervals between 20 and 40 cycles (not shown). The identity of expressed genes was determined by expected size of the PCR product in 1.6% agarose gels.
For RT-PCR of rat-specific gene products, the following primers were used: for ANGEN, coding = 5'-CCTCGCTCTCTGGACTTATC-3' and uncoding = 5'-CAGACACTGAGGTGCTGTTG-3', which yields a PCR product of 226 bp by single-step RT-PCR (20). For ![]() |
RESULTS |
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Exposure of primary cultures of rat AECs or A549 cells to Bleo for
20 h caused apoptosis detectable by nuclear fragmentation, DNA fragmentation, and immunolabeling of the active form of caspase-3 (Fig. 1). Although these markers were detected in a minor fraction of
the cells, the apoptosis induced was sufficient to reduce the total cell number significantly over time (see Fig. 1,
D-F, and quantitation in Fig. 7B). Scoring
of fragmented nuclei revealed dose-dependent apoptosis that
reached statistical significance beginning at 0.5 mU/ml in primary AECs
(Fig. 2) and at 1 mU/ml in A549 cells
(not shown). The nuclear fragmentation was blocked by the
broad-spectrum caspase inhibitor ZVAD-fmk (60 µM) or by the
endonuclease inhibitor ATA (10 µM), confirming the specificity of the
assay for apoptosis. Apoptosis of the rat AECs also was blocked by the ACE inhibitor captopril (500 ng/ml) and by the nonselective ANG receptor antagonist saralasin (50 µg/ml), in agreement with earlier studies of Fas- and TNF-induced AEC
apoptosis (28, 31).
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In a separate experiment (Fig. 3),
Bleo-induced apoptosis of primary AECs was blocked by the
caspase-3-selective blocker DEVD-fmk (60 µM) and by the ANG receptor
subtype AT1-selective blocker losartan (106
M, P < 0.01), suggesting that subtype AT1
mediates Bleo-induced apoptosis as it does AEC
apoptosis in response to ANG II (18). This
was found to be the case in human AECs as well (Fig.
4); Bleo-induced apoptosis of
A549 cells was inhibited 83% by the AT1-selective blocker
L-158809 but was not reduced by the AT2-selective antagonist PD-123319. Moreover, Bleo-induced apoptosis was also prevented by a neutralizing antibody specific for ANG II, but not by an
isotype-matched nonimmune immunoglobulin. Furthermore, the total
enzymatic activity of caspase-3 was elevated by exposure of A549 cells
to Bleo (Fig. 5), but the increase was
inhibited 52% by saralasin.
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These data suggested that Bleo induces ANG II synthesis de novo in
primary AECs and A549 cells. Consistent with this theory, semiquantitative RT-PCR for ANGEN revealed more abundant ANGEN mRNA in
primary AECs at 3 h and especially at 20 h after challenge with 25 mU/ml Bleo (Fig. 6A).
In A549 cells (Fig. 6B), 25 mU/ml Bleo stimulated a
significant increase in ANGEN mRNA that was detectable at 1 h
after addition of Bleo and increased by 7 h.
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To determine whether functional ANGEN mRNA is required for the
apoptotic response to Bleo, we transfected phosphorothioated antisense or scrambled-sequence control oligonucleotides against ANGEN mRNA into rat AECs and A549 cells immediately before challenge with Bleo for an additional 20 h. As shown in Fig.
7A, Bleo-induced apoptosis of primary AECs was inhibited by 83% by the ANGEN
antisense but not by the scrambled control oligonucleotides. In A549
cells (Fig. 7B), the antisense also reduced Bleo-induced
apoptosis by 84%, but the scrambled oligonucleotide had no
significant effect. Moreover, exposure to Bleo for 20 h reduced
the total cell number of A549 cells (Fig 7B,
bottom) by 43%, but the ANGEN antisense prevented the
Bleo-induced cell loss.
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DISCUSSION |
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The results described here agree with previous studies from this
laboratory that showed a requirement for autocrine ANG II production
and receptor interaction for AEC apoptosis in response to
FasL or TNF- (28, 31). Other authors also
have reported DNA damage and death of AECs in response to Bleo
(7, 10). Although a previous report of Bleo action on A549
cells described no influence of the drug on cell viability in vitro
(22), the levels of apoptosis described here
(apoptotic index ~10-20%, see Figs. 1 and 2) suggest that
cell death at the relatively low doses used in that study (0.1-2
mU/ml) may have gone undetected. Nonetheless, the levels of
apoptosis reported here are more than sufficient to result in
significant net cell loss in a relatively short period of time (see
Fig. 7). More importantly, both the cell loss and markers of
apoptosis could be blocked by ANG system antagonists, consistent with the theory that these agents can prevent
apoptosis, and thus cell loss, in response to Bleo.
The findings that Bleo-induced apoptosis of AECs could be blocked by the AT1-selective antagonists losartan (Fig. 3) and L-158809 (Fig. 4) but not by the AT2-selective antagonist PD-123319 are in agreement with our recent demonstration that the AT1 receptor subtype mediates AEC apoptosis in response to purified ANG II (18). They also support the contention that autocrine production of ANG II and binding to its receptor(s) are required for the apoptotic response. This contention is also supported by the ability of ANGEN antisense oligonucleotides or a neutralizing antibody that recognizes ANG II, but not ANG I or ANGEN, to essentially abrogate apoptosis and prevent net cell loss in response to bleomycin (Figs. 4 and 7).
In an earlier report we described the induction of apoptosis in AECs by exposure to purified ANG II (32), which also occurred in a concentration-dependent manner with an EC50 of 10 and 50 nM for primary AECs and A549 cells, respectively. Those data indicated that exposure of AECs to exogenous ANG II is sufficient, in the absence of other stimuli, for the induction of apoptosis. Measurements of the ANG II concentration in the cell culture media at a single sampling time (20 h) suggest that Bleo increases ANG II in the medium by at least two- to threefold, to a level close to that required for induction of apoptosis by purified ANG II (2-3 nM, data not shown). However, intracellular receptors for ANG II have been demonstrated in other cell types (4, 8), and ANG II administered intracellularly by microinjection (8) has been shown to invoke a variety of signaling pathways. Thus, in the present study, intracellular generation of ANG II by Bleo may be sufficient to stimulate apoptosis independently of extracellular ANG II. In both serum and interstitial fluid, the half-life of ANG II is short (15 s-15 min), and receptor-bound ANG II is known to be internalized in many cell types (5); for all these reasons, the interpretation of extracellular ANG II levels is difficult at best.
The RT-PCR data of Fig. 7 and measurements of ANGEN protein by Western
blotting (not shown) suggest that there is a basal level of ANGEN
expression by AECs, even in the absence of other proapoptotic
stimuli. In experiments not reported here, the ANG receptor antagonist
saralasin, applied alone, decreased the basal rate of spontaneous AEC
apoptosis (normally 1-2% of total cells at steady state);
this finding supports the notion that basal ANGEN expression is
involved in the basal rate of AEC death. On the other hand, the rate at
which ANGEN protein is proteolytically cleaved, both inside and outside
the cell, likely constitutes another point of regulation, but at
present we have little data to indicate which point of control is most
critical. Moreover, it seems reasonable to suspect that Bleo and other
inducers of AEC apoptosis alter the expression of additional
components of the local renin-angiotensin system in AECs, such as
angiotensin-converting enzymes and receptors as well as ANGEN
expression. Consistent with this theory, recent work by Day et al.
(3) demonstrated upregulation of ACE in cultured
endothelial cells by Bleo. Alveolar epithelial cells also express ACE
mRNA, and ACE inhibitors block AEC apoptosis in response to
FasL (31), TNF- (28), and Bleo (Fig. 2). Investigations to define the possible regulation of ACE and
other peptidases in AECs are currently underway.
Although the mechanism by which Bleo upregulates ANGEN mRNA was not
addressed in this study, the findings that Bleo, FasL, and
TNF- all increased ANGEN mRNA (28, 31) imply
the involvement of a pathway common to these inducers. Regulation
of ANGEN expression is best studied in the hepatocyte, in
which the stimulatory effects of TNF-
and interleukin-6 have been
shown to be mediated through the interaction of the transcription
factors NF-
B and STAT3 with the acute phase response element of the
ANGEN promoter (2, 23). In contrast, studies of the
regulation of ANGEN expression by the cardiac myocyte have shown that
p53 is a key regulator of its expression in response to a variety of
stimuli (13, 20). Whether this difference reflects the
distinct developmental lineages of these cell types or other factors is
unknown. Viewed in this context, future investigations of the
regulation of ANGEN expression by cells of the lung will be most
interesting, particularly in light of the fact that the lung contains
many different cell types that are in very close proximity but arose
from distinct embryologic origins. As an example, the results reported
here for AECs complement our earlier report of ANGEN expression by
human lung myofibroblasts (30), which reside immediately
adjacent to AECs in fibrotic lung in situ.
In summary, exposure of primary cultures of rat alveolar epithelial cells or the AEC-derived A549 cell line to Bleo caused a time-dependent increase in ANGEN mRNA and caspase-dependent apoptosis. In either cell type, the apoptosis could be prevented by agents that prevent the synthesis of ANGEN protein, its conversion to the mature peptide ANG II, or the binding of the peptide to its receptors. These findings may have important implications toward possible therapeutic strategies to prevent lung injury and/or fibrogenesis. They also raise the possibility that previous clinical trials involving ACEis or ANG receptor antagonists may hold useful information related to the potential of these agents to affect pulmonary disease.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Public Health Service Grant HL-45136, American Heart Association Grant-In-Aid 0250269N, and by the Michigan State University Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. D. Uhal, Dept. of Physiology, Michigan St. Univ., 3185 Biomedical and Physical Sciences Bldg., E. Lansing, MI 48824 (E-mail: uhal{at}msu.edu).
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 November 15, 2002;10.1152/ajplung.00273.2002
Received 8 August 2002; accepted in final form 5 November 2002.
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