1 Chicago College of Osteopathic Medicine, Downers Grove, Illinois 60551; 2 Department of Physiology, Michigan State University, East Lansing, Michigan 48824; and 3 Abbott Laboratories, Abbott Park, Waukegan, Illinois 60085
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ABSTRACT |
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Previous work from this laboratory
demonstrated induction of apoptosis in lung alveolar epithelial
cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs
for both ANG II receptor subtypes AT1 and AT2
(Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S,
Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J
Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The
present study was designed to determine the ANG II receptor subtype
mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT1-selective
receptor antagonists L-158809 or losartan inhibited ANG II-induced
apoptosis by 90% at concentrations of 108 M and
10
7 M, respectively. The inhibition was concentration
dependent with IC50 of 10
12 M and
10
11 M on the primary rat AEC. In contrast, the
AT2-selective antagonists PD-123319 or PD-126055
could not block ANG II-induced apoptosis in either cell type.
In primary rat AEC, apoptosis in response to ANG II was blunted
in a dose-dependent manner by the protein kinase C inhibitor
chelerythrine but not by the tyrosine phosphatase inhibitor sodium
orthovanadate. Together, these data indicate that AEC apoptosis
in response to ANG II is mediated by receptor subtype AT1,
despite the expression of mRNAs for both AT1 and AT2.
type II pneumocyte; programmed cell death; angiotensin-converting enzyme inhibitor; pulmonary fibrosis; lung injury; angiotensin II
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INTRODUCTION |
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PULMONARY ALVEOLAR
EPITHELIAL CELLS (AEC) have critical roles in surfactant
synthesis and secretion, in the maintenance of lung barrier function,
and in the repair of epithelial damage after lung injury
(14). Apoptosis of AEC is now believed to be an
important mechanism in normal alveolar epithelial turnover, in the
removal of excess epithelial stem cells after hyperplastic repair
processes, and in the pathogenesis of a variety of lung diseases
(6). Recent work in this laboratory demonstrated
dose-dependent induction of apoptosis by purified angiotensin
II (ANG II) in the human AEC-derived carcinoma cell line A549 and in
primary cultures of well-differentiated AEC isolated from rats
(22). Shortly thereafter, the autocrine synthesis and
binding of ANG II to its receptor(s) on AEC was shown to be required
for the induction of apoptosis in response to Fas ligand
(21) or tumor necrosis factor (TNF)- (19).
Those results also indicated that the alveolar epithelium, in addition to the capillary endothelium, can be a producer of ANG II under conditions that promote the presence of these cytokines. The lung myofibroblast, which accumulates in fibrotic human lung and in animal models of lung fibrosis (17), also synthesizes the precursor of ANG II, the 58-kDa proprotein angiotensinogen (20). Although isolated human lung myofibroblasts have little capacity to proteolytically process the parent protein, related studies have shown that AEC can convert the parent molecule to ANG II and respond to it by undergoing apoptosis (22). For all these reasons, the mechanisms by which ANG II interacts with AEC are critical to understanding the regulation of cell death in the alveolar epithelium.
Apoptosis of AEC induced by purified ANG II could be completely abrogated by ANG II-neutralizing antibodies or by the nonselective ANG receptor antagonist saralasin (22). Analyses of total RNA by RT-PCR indicated that both the A549 cell line and primary AEC from rats express the mRNAs for the two major subtypes of the ANG receptor, AT1 and AT2. Evidence from other cell types indicate that either or both of these subtypes may be involved in signaling apoptosis, depending on the cell type in question and the experimental conditions. Moreover, the relative importance of the AT1 vs. the AT2 receptors in signaling apoptosis can change in a manner dependent on other stimuli (5).
The purpose of the present study, therefore, was to determine the relative importance of the ANG receptor subtypes AT1 and AT2 in the induction of AEC apoptosis with well-differentiated primary cultures of normal AEC in the absence of additional stimuli. We report here the use of functional assays and subtype-selective receptor antagonists to determine that the ANG receptor subtype AT1 mediates AEC apoptosis, under basal conditions, in a manner apparently independent of receptor subtype AT2.
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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 antagonists PD-126055 and PD-123319 were obtained from Parke Davis Research Division (Ann Arbor, MI). The caspase inhibitor ZVAD-fmk (N-benzylcarboxy-Val-Ala-Asp-fluoromethylketone) was obtained from Kamiya Biomedical (Seattle, WA). DEVD-fmk (Asp-Glu-Val-Asp-fluoromethylketone) and YVAD-cmk (Tyr-Val-Ala-Asp-chloromethylketone) were 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). Anti-phosphotyrosine antibodies were obtained from Upstate Biotechnology (Saranac Lake, NY). Reagents for detection of alkaline phosphatase and other secondary reagents for in situ end labeling (ISEL) of DNA or Western blotting were from sources described earlier (16). All other materials were of reagent grade and were obtained from Sigma Chemical (St. Louis, MO).
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 AEC were isolated from adult male Wistar rats as described earlier (17). The primary cells were studied at day 2 of culture, a time at which they are type II cell-like by accepted morphological and biochemical criteria (14). All primary cell preparations were of better than 90% purity assessed by acridine orange staining as described previously (15). All cells were seeded in 24-well chambers at subconfluent densities of 80-90% in serum-free Ham's F-12 medium. Test reagents were diluted with Ham's F-12 medium. The cells were exposed to caspase inhibitors, chelerythrine, sodium orthovanadate, or antagonists of angiotensin receptors 30 min before exposure to ANG II for 20 h.
Quantitations of apoptosis and cell loss.
Detection of apoptotic cells with propidium iodide (PI) was
conducted as described earlier (19, 21) after digestion of ethanol-fixed cells with DNase-free RNase in PBS containing 5 µg/ml
of 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 annexin V binding (19, 22) and by ISEL and/or TdT-mediated dUTP nick end
labeling (TUNEL) of fragmented DNA (16, 17) (see Fig.
1).
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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Western blotting. Total protein lysates were obtained from AEC scraped from the culture vessels in Nonidet P-40 lysis buffer supplemented with a commercially available protease inhibitor cocktail. Proteins were resolved on 12% ReadyGels (Bio-Rad), transferred to polyvinylidene difluoride blotting membranes, and incubated with anti-phosphotyrosine polyclonal antibodies. Bound primary antibody was detected with biotin-conjugated secondary antibodies and a streptavidin-chromogen amplification system (20).
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RESULTS |
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Previous studies of the ability of ANG II to induce apoptosis in human and rat AEC (22) revealed a dose-dependent induction that became maximal at ANG II concentrations of 5 µM and higher. For this reason, the single concentration of 5 µM ANG II was used to test the ability of various inhibitors to block ANG II-induced apoptosis. The assay of nuclear fragmentation revealed by PI (Fig. 1A, arrows) was shown to detect the same nuclear fragments that label by ISEL (Fig. 1A, top inset) or by TUNEL (bottom inset), each applied after exposure of AEC to ANG II. In Fig. 1B, the caspase inhibitors ZVAD-fmk, DEVD-fmk, and YVAD-cmk blocked apoptosis in response to ANG II, although to varying degrees that are expressed as percent inhibition relative to ANG II alone (Fig. 1B, inset). The apoptotic indexes detected under these conditions were 1.8 ± 0.2% vs. 8.8 ± 1.1% (control vs. ANG II, respectively).
Over 20 h of incubation, this level of apoptosis induction
was sufficient to reduce the total cell number (attached + detached) in a given culture vessel by >50% (Fig. 1C).
These results are consistent with an estimated duration of AEC
apoptosis of ~4-5 h, which is in agreement with that
reported by Bursch et al. (2) in their kinetic study of
hepatocyte apoptosis. The net decrease in cell number induced
by ANG II was blocked by either ZVAD-fmk (60 µM), by the nonselective
ANG II receptor antagonist saralasin (50 µg/ml), or by the receptor
AT1-selective antagonist L-158809 (109 M).
Without additives, cell number was constant during the 20-h incubation
(compare time 0 with control at 20 h), confirming the absence of cell proliferation (15).
Under the same experimental conditions, application of
subtype-selective ANG receptor antagonists revealed complete abrogation of ANG-induced apoptosis by the AT1-selective
antagonist L-158809 in either primary cultures of rat AEC (Fig.
2) or in the human A549 carcinoma cell
line (Fig. 3). In contrast, the
AT2-selective antagonist PD-126055 (Figs. 2B and
3B) did not achieve statistically significant
inhibition, at any concentration, compared with treatment with ANG II
alone (as in Fig. 1B). The inhibition by L-158809 was
maximal at 108 M in both cell types but exhibited a
slightly lower IC50 in primary rat AEC (10
12
M) than in A549 cells (10
11 M).
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In primary cultures of rat AEC, qualitatively similar results were
obtained with additional antagonists. The AT1-selective receptor blocker losartan (Fig.
4A) completely inhibited ANG
II-induced apoptosis at 108 M, but the
AT2-selective antagonist PD-123319 had no statistically significant inhibitory effect. Losartan exhibited an
IC50 for ANG II-induced apoptosis of
~10
11 M.
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In other cell types, the action of the ANG II receptor subtype
AT1 is mediated, at least in part, by protein kinase C
(PKC), whereas the action of AT2 is dependent on protein
tyrosine phosphatases. Consistent with those observations, the PKC
inhibitor chelerythrine (Fig. 5) showed
dose-dependent inhibition of ANG-induced apoptosis in primary
rat AEC with an IC50 of ~0.5 µM. In contrast, the
tyrosine phosphatase inhibitor sodium orthovanadate (SOV; Fig.
6A) had no inhibitory capacity
toward AEC apoptosis at any concentration up to 1 mM, despite
the ability of 100 µM SOV to significantly increase the abundance of
phosphotyrosine residues on AEC proteins observed by Western blotting
(Fig. 6B).
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DISCUSSION |
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The induction of apoptosis by ANG II has been studied most extensively in cardiac myocytes (3, 7, 10, 11), in capillary endothelial cells (4, 12), and in some transformed cell lines (9, 23). To date, the issue of which ANG receptor subtype mediates the induction of apoptosis is controversial and highly dependent on the type of experimental system under study. Investigations of transformed R3T3 and phaeochromocytoma PC12W cells (9, 23), which express primarily the AT2 receptor subtype, suggested that AT2 is the mediator of apoptosis in response to ANG II. In the cardiac myocyte system, however, neonatal ventricular myocytes undergo ANG II-induced apoptosis that was abrogated by AT1-selective blockers but not by AT2-selective antagonists (3, 11). The same blockade of ANG II-mediated apoptosis by AT1-selective antagonists, but not by AT2-selective antagonists, was also observed in adult ventricular myocytes in the studies of Kajstura et al. (10). These data are in contrast with those of other investigators, who found essentially the opposite result using the neonatal myocyte culture system (6).
In cultured human coronary artery endothelial cells, the
AT1-selective antagonist losartan blocked apoptosis
in response to ANG II, TNF-, and anoxia reoxygenation applied
simultaneously (12). In contrast, the
AT2-selective agonist CGP-42112 induced apoptosis
of cultured coronary endothelial cells (13), but neither AT1- nor AT2-selective antagonists could block
ANG II-induced apoptosis in that study. Moreover, Dimmeler et
al. (4) found that AT1- and
AT2-selective antagonists could block apoptosis in
human umbilical vein endothelial cells when the agents were applied together.
The interpretation of these studies is complicated by the fact that an unusually high dose of the AT2 agonist CGP-42112 (2 mM) was required for the induction of apoptosis in coronary artery cells (13); the normal serum concentration of ANG II is ~1-10 pM (5). Thus it is possible that the partial AT1 agonist activity of this otherwise AT2-selective compound was acting through the AT1 receptor. Moreover, studies in which the AT2 receptor has been implicated as the primary mediator of apoptosis have generally been conducted with cell lines that express AT2, but little or no AT1, receptor (9, 23). Together, the available data imply that the ANG receptor subtype most important in mediating apoptosis in a given cell type is likely dependent on the relative expression of AT1 vs. AT2.
Regardless, the ability of AT1-selective antagonists to
block ANG-induced apoptosis in the present study clearly
suggests that AT1 is the functional receptor for
apoptosis in normal AEC, despite the expression by these cells
of both mRNAs for AT1 and AT2
(22). This interpretation is supported by the finding that the benzophenanthridine alkaloid chelerythrine, a specific inhibitor of
PKC (8), could block AEC apoptosis, but an
inhibitor of tyrosine phosphatases (SOV) could not. The presumption
that the SOV treatment actually blocked tyrosine phosphatase activity
but not apoptosis is supported by the large increases in
phosphotyrosines detected by Western blotting of AEC proteins (Fig.
6B). In other cell types, AT1-mediated effects
have been demonstrated to involve PKC activation (10, 12),
whereas AT2 signaling is believed to be mediated by a
protein tyrosine phosphatase(s) inhibitable by SOV (1,
23). Together, the failure of SOV and the success of
chelerythrine to inhibit ANG II-induced apoptosis support the conclusion that receptor subtype AT1 is active in mediating
AEC apoptosis, at least under challenge by purified ANG II.
Evaluation of ANG receptor specificity after exposure of AEC to Fas
ligand or TNF-, both of which require ANG II receptor interaction
for the induction of apoptosis (19, 21), will be
an interesting topic for future inquiry.
In summary, apoptosis of AEC in response to purified ANG II was inhibited by the AT1-selective antagonists L-158809 or losartan in a concentration-dependent manner, but not by the AT2-selective antagonists PD-123319 or PD-126055. A PKC inhibitor (chelerythrine) known to block AT1 receptor signaling in other cell types inhibited ANG II-stimulated apoptosis of AEC. However, an inhibitor of protein tyrosine phosphatases (SOV) known to block AT2 signaling had no effect on ANG II-induced apoptosis of AEC. These data indicate that receptor subtype AT1 mediates apoptosis of AEC in response to ANG II in the absence of additional stimuli.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-45136 (to B. D. Uhal).
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. D. Uhal, Dept. of Physiology, Michigan State Univ., 310 Giltner Hall, East 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.
10.1152/ajplung.00103.2001
Received 21 March 2001; accepted in final form 31 October 2001.
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