1 The Cardiovascular Institute and 2 Division of Neonatology, Michael Reese Hospital and Medical Center, Chicago, Illinois 60616
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ABSTRACT |
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Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 µg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.
programmed cell death; renin-angiotensin system; type II pneumocyte; pulmonary fibrosis; angiotensin-converting enzyme
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INTRODUCTION |
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PULMONARY ALVEOLAR EPITHELIAL CELLS (AECs) have varied and important roles in lung homeostasis, one of which is the replacement of cells lost to lung injury (11). The rate of AEC death is a critical factor determining the capacity of the epithelium to repair damage (18), and the large fraction of that rate composed of apoptotic cell death is only recently being realized (2, 7). A growing body of evidence suggests that Fas-induced apoptosis of AECs is an important factor in the pathogenesis of fibrotic lung diseases (4, 5).
The onset of experimental lung fibrosis has long been known to be inhibited by antagonists of the renin-angiotensin system (12, 24, 25). Earlier work from this laboratory (20) showed potent inhibition of Fas-induced apoptosis of a human lung epithelial cell line by captopril, an angiotensin (ANG)-converting enzyme (ACE) inhibitor already known to block lung fibrogenesis (12). This finding suggested that the conversion of ANG I to ANG II might be involved in Fas-induced apoptosis by AECs. That theory was made plausible by the recent demonstration by Wang et al. (23) that purified ANG II induces dose-dependent apoptosis of cultured AECs that is inhibitable by ANG II receptor antagonists.
The same study found that AECs in vitro constitutively express the mRNAs for both subtypes of ANG II receptor as well as for ACE. Furthermore, primary AECs exposed to purified angiotensinogen (ANGEN) underwent apoptosis that was both ANG II receptor dependent and inhibitable by ACE inhibitors. Those data indicated that cultured AECs express all renin-ANG system components necessary for the enzymatic conversion of ANGEN to ANG II and the subsequent induction of apoptosis. This study examines the possibility that the synthesis of ANG II de novo and its subsequent binding to the ANG II receptor might be necessary for apoptosis in response to Fas. We report here that the induction of ANGEN expression, its proteolytic processing, and the subsequent binding of ANG II to its receptor are required events in the signaling of AEC apoptosis by Fas.
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METHODS |
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Reagents and materials. Purified ANG II, ANGEN, lisinopril, saralasin, and antibodies to ANG II and ANGEN were obtained from Sigma (St. Louis, MO). Primers for RT-PCR were synthesized by Genemed Synthesis (San Francisco, CA). Lipofectin reagent (Oligofectin G) was obtained from Sequitur (Natick, MA). All other materials were from sources described earlier (20, 23) or were of reagent grade.
Cell culture and detection of apoptosis. 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 AECs were isolated from adult male Wistar rats as described earlier (21, 23). The primary cells were studied on day 2 of culture, a time at which they are type II cell-like by accepted morphological and biochemical criteria (18), and all preparations were of >90% purity as assessed by acridine orange staining as previously described (19). All cells were seeded in 24-well or 6-well chambers, and all experiments were conducted at subconfluent densities of 80-90% in serum-free Ham's F-12 medium. Test reagents were diluted with serum-free Ham's F-12 medium and were applied under serum-free conditions for 20 h at 37°C in a 5% CO2 incubator. Detection of apoptotic cells with propidium iodide was conducted as described earlier (20, 21, 23) after digestion of ethanol-fixed cells with DNase-free RNase in PBS containing 5 µg/ml of propidium iodide. In all assays, detached cells were retained by centrifugation of the culture vessels during fixation with 70% ethanol or by retention of the culture medium and recovery by centrifugation before the assay. As in earlier publications (22, 23), the induction of apoptosis was verified by annexin V binding and generation of DNA strand breaks.
RT-PCR. RT-PCR assay of ANGEN gene products was performed essentially as described earlier (23). Briefly, total RNA was isolated with the RNeasy Mini protocol (Qiagen, Santa Clarita, CA). To synthesize cDNA by RT-PCR, 3 µg of purified RNA were reverse transcribed with 2 µM oligo(dT), 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.01 mM dithiothreitol, 0.2 mM each deoxynucleotide triphosphate, 1 U/µl of RNase inhibitor (RNasin), and 2 U of avian myoblastosis virus reverse transcriptase (Promega, Madison, WI) in a total volume of 30 µl. The RT reaction was performed for 1 h at 42°C followed by 20 cycles of PCR amplification, which was at or near the center of the linear range for each gene product analyzed (data not shown).
PCR amplification was performed with 10-µl aliquots of cDNA obtained as described above, equivalent to 1 µg of the total starting RNA extract. The amplification reaction was performed in 50 µl of PCR buffer containing 5 mM MgCl2, 5 µg/ml of each 5' and 3' primer (see below), 2 µl of 10 mM deoxynucleotide triphosphates, and 1 U of Taq polymerase (Promega) with a Perkin-Elmer PCR amplifier. The PCRs included denaturation at 95°C for 30 s, annealing of primers for 30 s, and elongation of the chain at 72°C for 1 min with Taq DNA polymerase. The annealing temperatures for RT-PCRs were optimized for each primer by preliminary trials. For any given primer, the control and treated samples were multiplexed within the same RT-PCR. Samples were stored at 4°C; negative controls lacked DNA. Identity of the expressed genes was determined by the expected size of the PCR product on 1.6% agarose gels.
For RT-PCR from the human A549 cells, the primers used for ANGEN were
an "outer" primer of a two-step nested assay: coding, 5'-GCTTTCAACACCTACGTCCA-3', and uncoding,
5'-AGCTGTTGGGTAGACTCTGT-3', and an "inner" primer of
the nested assay: coding, 5'-TTCTCCCTGCTGGCCGAG-3', and
uncoding, 5'-GGGCTCTCTCTCATCCGC-3'. These primers yielded a
final PCR product of 447 bp (8). For -actin, single-step RT-PCR was
used with the primers coding,
5'-AGGCCAACCGCGAGAAGATGACC-3', and uncoding,
5'-GAAGTCCAGGGCGACGTAGC-3', which produced a PCR product of
332 bp (17).
For RT-PCR of rat-specific gene products, the primers used for ANGEN
were coding, 5'-CCTCGCTCTCTGGACTTATC-3', and uncoding, 5'-CAGACACTGAGGTGCTGTTG-3', which yielded a PCR product of
226 bp by single-step RT-PCR (16). For -microglobulin, the primers used were coding, 5'-CTCCCCAAATTCAAGTGTACTCTCG-3', and
uncoding, 5'-GAGTGACGTGTTTAACTCTGCAAGC-3', which yielded a
product of 249 bp (6).
Antisense transfection and in situ hybridization. Phosphorothioated control and antisense oligonucleotides against ANGEN (18 mers) were synthesized and transfected into A549 cells or primary rat AECs with the lipofectin reagent Oligofectin G (Sequitur) as the vehicle diluted in cell culture medium. The control nucleotides were of the same length and base composition as the antisense but with a scrambled sequence. The oligonucleotide-to-lipofectin ratio was optimized (over a 4-h transfection) to yield transfection efficiencies of 50-75%, with no apparent cell loss or detachment. Transfection efficiency was monitored with a FITC-labeled 25-mer oligonucleotide for luciferase (data not shown). Transfections were conducted for 4 h followed by five washes with serum-free cell culture medium; immediately thereafter, Fas activator or vehicle was applied as described above for 20 h. The transfection protocol itself had no significant effect on basal or Fas-induced apoptosis (data not shown). Phosphorothioated oligonucleotides used for transfection were ANGEN antisense, 5'-CCGTGGGAGTCATCACGG-3', and ANGEN scramble, 5'-CAGGGATCTCTGGCGGAC-3', as described by Phillips et al. (15).
In situ hybridization was performed essentially as described by Panoskaltsis-Mortari and Bucy (14) except that no 80°C denaturation was employed because single-stranded probes were used. Fixed A549 cells were hybridized with digoxigenin-labeled antisense and control oligonucleotide DNA probes specific for ANGEN, which were detected with an amplified biotin-avidin system linked to a nitro blue tetrazolium chromogen (purple is positive). As in the transfection experiments, the control probes were of the same length and base composition as the antisense probe but with a scrambled sequence. The digoxigenin-labeled probes used were antisense, 5'-AGGGTGGGGGAGGTGCTGAACAGC-3', and scramble, 5'-GATGGGGGTGGGGGACCGTAGCAA-3', as described by Lai et al. (8).
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RESULTS |
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Fas-induced apoptosis of A549 cells was significantly inhibited by the
nonthiol ACE inhibitor lisinopril (Fig.
1A)
and was essentially abrogated by the nonselective ANG II receptor
antagonist saralasin (maximal inhibition of 93% at 50 µg/ml; Fig.
1B). In agreement with the data from
A549 cells, apoptosis within primary cultures of rat AECs (induced by
purified recombinant Fas ligand) was also blocked by saralasin (Fig.
1C) or by captopril or lisinopril, both at 500 ng/ml (Fig. 1D).
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These findings suggested that Fas-induced apoptosis of AECs might be
dependent on de novo synthesis of ANG II and its binding to one of its
receptors. Consistent with this model, quantitative RT-PCR (Fig.
2) revealed that activation of Fas in
either A549 cells (A) or primary rat
AECs (B) causes significant
increases in the abundance of mRNA for ANGEN but not in the control
transcripts -actin and
-microglobulin. In both cell types, the
presence of saralasin during Fas activation had no effect on the
accumulation of ANGEN mRNA.
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In Fig. 3, a 4-h transient transfection of
antisense oligonucleotides against ANGEN significantly inhibited
Fas-induced apoptosis of either A549 cells
(left) or primary rat AECs
(right) over the subsequent 20 h
posttransfection (70 and 87% inhibition, respectively; both
P < 0.01 vs. Fas plus lipofectin).
In contrast, transfection of control oligonucleotides of the same size
but with a scrambled sequence produced no significant inhibition in
A549 cells and only 28% inhibition in primary AECs
(P < 0.05). The LipofectAMINE transfection itself had no significant effect on either basal or
Fas-induced apoptosis (data not shown). When related antisense oligonucleotides against ANGEN (see
METHODS) were used for in situ
hybridization (Fig. 4), an intense positive
signal (purple) was found within cells that contained the fragmented
nuclei typical of apoptotic cells (arrowheads). No positive signal was
found in cells hybridized with control oligonucleotides.
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Enzyme-linked immunosorbent assay specific for ANG II (Fig.
5) revealed that activation of Fas
significantly increased the concentration of free ANG II peptide in the
culture medium bathing primary AECs
(left) or A549 cells
(right). Figure
6 shows that Fas-induced apoptosis of
either rat AECs (top) or A549 cells
(bottom) could be completely
abrogated by neutralizing antibodies specific for ANG II. The
specificity of this experimental approach is supported by the finding
that the same antibody completely blocked AEC apoptosis induced by
purified ANG II, whereas isotype-matched nonimmune IgGs had no
significant effects. The reliability of the nuclear fragmentation assay
as an index of apoptosis is shown by the ability of the broad-spectrum
caspase inhibitor
Z-Val-Ala-Asp-fluoromethylketone (20) to abrogate the effect of Fas ligand (Fig. 6,
top).
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DISCUSSION |
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The ACE inhibitor captopril abrogated Fas-induced apoptosis in A549 cells at doses of the drug readily attained in vivo (20). Because captopril is a sulfhydryl-containing compound, the inhibition of apoptosis was speculated to occur through thiol-mediated blockade of cysteine protease (caspase) activities required for AEC apoptosis (20). However, a subsequent study (23) showed that purified ANG II is capable of inducing ANG II receptor-dependent apoptosis in both A549 cells and primary AECs isolated from rats. More importantly, the experiments reported herein show that the enzymatic activity of ACE and the ability of ANG II to interact with its receptor are not only involved in the signaling of Fas-induced apoptosis but are also required for its execution.
The notion that ANG II binding to its receptor is a required step in the signaling pathway activated by Fas is supported by the ability of the nonselective receptor antagonist (saralasin) and neutralizing anti-ANG II antibodies to completely abrogate (93 and 101% inhibition, respectively) AEC apoptosis in response to Fas (Figs. 1 and 6). Although the inhibitory actions of lisinopril and captopril on AEC apoptosis were slightly less complete (75-85% inhibition; Fig. 1) than that of saralasin, these data do not lessen the importance of ANG II function because ACE, the preferred target of these inhibitors (1), is only one of several dipeptidyl carboxypeptidases capable of cleaving ANG I to ANG II (6, 10). Although the large degree of inhibition exerted by these compounds suggests that ACE may be the primary ANG I cleavage activity expressed by these cells, other enzymes such as chymase or cathepsins (which are not sensitive to ACE inhibitors) might also be present and thus would preclude complete blockage of ANG II formation by these drugs.
The large increase in ANGEN mRNA in response to Fas activation (48-fold for A549 cells and 8-fold for rat AECs; Fig. 2) is consistent with a proposed mechanism in which Fas induces the transcription of ANGEN. Regardless of whether the increased ANGEN mRNA is due to transcriptional activation or changes in transcript stability, the finding that ANGEN antisense oligonucleotides can inhibit apoptosis in response to Fas (Fig. 3) suggests that the level of functional ANGEN mRNA is critical to the process by which Fas signals AEC apoptosis. The fact that the antisense inhibition of apoptosis was not complete (70 and 87% for A549 and primary AECs, respectively; Fig. 3) might be explained by the fact that the transfection efficiency was at best 75% (see METHODS), and thus a fraction of the cells undergoing apoptosis may not have been subject to antisense inhibition.
Regardless, the ANGEN promoter is known to contain an acute-phase
response element and to be rapidly responsive to cytokines such as
tumor necrosis factor- (3). To our knowledge, however, this is the
first report of the induction of ANGEN expression specifically by Fas.
In cardiac myocytes, apoptosis induced by mechanical strain
("stretch") was recently shown to be mediated by a mechanism in
which increased levels of p53 protein interact directly with the ANGEN
promoter to induce its transcription (9, 16); the newly synthesized
ANGEN protein is then proteolytically cleaved to ANG II, which induces
cardiomyocyte apoptosis through binding to its receptor in a manner
entirely analogous to that shown earlier for AECs (23). In some cell
types, apoptosis in response to Fas is believed to involve the
induction of p53 expression (13), which raises the interesting
possibility that Fas activation in AECs might also produce elevated
levels of p53 protein. The possibility that the Fas-induced increase in
the abundance of ANGEN mRNA (Fig. 2) is due to p53-mediated
transcriptional activation is currently under investigation.
In summary, Fas-induced apoptosis of A549 cells or primary cultures of rat AECs was significantly inhibited by nonthiol ACE inhibitors and was completely abrogated by an ANG II receptor antagonist. Activation of Fas significantly increased the abundance of ANGEN mRNA, and transfection of antisense oligonucleotides against ANGEN significantly inhibited Fas-induced apoptosis. Activation of Fas increased the amount of free ANG II in the extracellular space, and neutralizing antibodies specific for ANG II completely blocked the induction of apoptosis by Fas. Together, these data support the hypothesis that Fas-induced apoptosis by AECs requires the induction of ANGEN expression, its proteolytic processing, and subsequent binding of ANG II to at least one of its receptors. They also suggest that therapeutic manipulation of Fas-induced apoptosis in vivo will be feasible with a variety of well-characterized pharmacological antagonists of the renin-ANG system.
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ACKNOWLEDGEMENTS |
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We thank Drs. P. Denes and D. Vidisagar for support of this project.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-45136 (to B. D. Uhal) and the Women's Board Endowment to The Cardiovascular Institute and the Research and Education Foundation, Michael Reese Hospital (Chicago, IL).
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: B. D. Uhal, The Cardiovascular Institute, Michael Reese Hospital, 2929 S. Ellis Ave., Rm. 405KND, Chicago, IL 60612 (E-mail: bdu1{at}earthlink.net).
Received 29 July 1999; accepted in final form 8 September 1999.
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