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Second Department of Internal Medicine (M.S., F.M., Y.H.) Tokyo
Medical and Dental University Tokyo 113, Japan
Department
of Molecular Biology, Cell Biology and Biochemistry (J.M.S.) Brown
University Providence, Rhode Island 02912
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The c-myc protooncogene belongs to the family of immediate early growth response genes, and is believed to participate in regulating the cascade of events that follow mitogenic stimulation of quiescent cells. The c-myc gene is expressed at a low constitutive level in most growing cells, at elevated levels in a variety of tumors, and is down-regulated in both quiescent and differentiated cells. Numerous lines of evidence implicate c-Myc in the regulation of proliferation, mitogenesis, differentiation, and programmed cell death (12, 13, 14). However, much of our understanding of c-Myc function derives from studies of cells overexpressing the c-myc gene. For example, fibroblasts expressing high levels of c-Myc protein are more prone to apoptosis upon serum deprivation (14), and this effect can be suppressed by two cytokines, PDGF and insulin-like growth factor I (IGF-I) (15). Ras triggers both a phosphatidylinositol-3 (PI3) kinase-dependent antiapoptotic pathway and a Raf-MAP kinase proapoptotic pathway in fibroblasts overexpressing c-Myc (16). Recent studies have suggested a role for a balance between MAP kinase and stress-activated JUN kinase-p38 pathways in determining apoptotic frequency of PC12 pheochromocytoma cells (17), and a role for PI3 kinase in the prevention of apoptosis by nerve growth factor (18). However, functional Max protein, the dimerization partner of Myc, is not expressed in PC12 cells because of the synthesis of a mutant max transcript (19). To investigate the consequences of physiological c-myc expression, we have previously disrupted one c-myc gene copy in a diploid fibroblast cell line (20). This subtle perturbation of c-myc expression resulted in slower growth rates, lengthening of the G0-to-S cell cycle transition, and modulation of cyclin E expression (21).
In the present report, we have confirmed that apoptosis observed in fibroblasts with diploid c-myc is also a c-Myc-dependent process and further show a striking role for the endothelin-1 peptide acting through the ETA receptor as a potent apoptosis survival factor via MAP kinase activation. Our data imply that both circulating as well as local endothelin-1 may act on fibroblasts to protect them from apoptotic death under physiological conditions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M5) derived from Rat-1 cell line: TGR-1 is a nontransformed,
diploid rat fibroblast cell line (22); HET15 and HET16 are two
independent TGR-1 derivatives with one endogenous c-myc copy
knocked out by gene targeting, and express 50% of normal diploid c-Myc
levels (20). The M5 cell line is HET15 infected with a retrovirus
vector expressing the c-Myc-Estrogen Receptor (Myc-ER) chimeric protein
(23, 24). Myc-ER was functionally activated by the addition of 10
nM ß-estradiol to the medium. When rendered quiescent by
serum deprivation, fibroblast cultures always contain a fraction of
floating cells, which displayed characteristic features of apoptosis,
such as condensed chromatin and subnuclear bodies, and electrophoresis
of DNA samples showed a nucleosomal ladder (Fig. 1B
). Although the fraction of cells
susceptible to apoptosis is dependent on the density of the cultures,
HET15 and HET16 cultures always displayed fewer apoptotic cells
(13.9 ± 2.3% and 16.9 ± 2.7% of TGR-1 in exponentially
growing cultures, respectively) than TGR-1 (Fig. 1A). M5 cells,
undergoing a low level of apoptosis in the absence of ß-estradiol,
showed massive apoptosis of rapid onset upon serum withdrawal when
pretreated with 100 nM ß-estradiol, and approximately
70% of cell death when pretreated with 10 nM ß-estradiol
for 48 h (
2.7-fold greater than TGR-1) (Fig. 1A).
Electrophoresis of DNA samples extracted from M5 pretreated with
ß-estradiol showed a marked enhancement of nucleosomal laddering
(Fig. 1B). Flow cytometric analysis revealed that the number of TGR-1
with subdiploid DNA content increased after serum deprivation (Figs. 1B and 5C): 0.2% before serum withdrawal, 6.0% after 4 h, and
17.0% after 24 h. Adherent TGR-1 cells in serum-starved culture
were stained with antibody against single-stranded DNA.
Immunohistochemically positive cells coincided with those showing
cellular and nuclear fragments (Fig. 2A).
Taken together, the results indicate that serum deprivation induces
c-Myc-dependent apoptosis in fibroblasts, which is manifested as
floating dead cells.
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). Apoptosis of M5 elicited by 10
nM ß-estradiol was also significantly inhibited by
addition of endothelin-1 in concentrations lower
(10-13-10-11 M) than those for
TGR-1 (Fig. 3A). We therefore conclude that endothelin-1 blocks
c-Myc-dependent apoptosis in rat fibroblasts, thus functioning as a
survival factor.
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). The
approximate half-maximum effective concentrations
([EC50]) for TGR-1 and HET15 cells were 2.1 x
10-9 M and 5.6 x 10-9
M, respectively. The maximal response of HET15 cells was
about two-thirds that of TGR-1. Endothelin-1-induced mitogenesis was
not observed at lower concentrations
(10-13-10-9 M), at which
concentrations a significant apoptosis protection was induced.
To characterize the endothelin-1 receptor subtype mediating the
protective effect, binding studies using
[125I]endothelin-1 as a radioligand were performed in
TGR-1 (Fig. 4A) and M5 cells (data not
shown). Unlabeled endothelin-1 competitively inhibited the binding of
[125I]endothelin-1 to both cell lines. Scatchard analysis
of the binding data indicated the presence of a single class of
noninteracting binding sites for endothelin-1 (Fig. 4A, inset): the apparent Kd and Bmax
were 7.9 x 10-11 M and 1.7 x
105 sites per cell (TGR-1), and 13.6 x
10-11 M and 2.1 x 105 sites
per cell (M5), respectively. The ETA receptor
antagonist, BQ123, completely inhibited the binding of
[125I]-endothelin-1, but endothelin-3 was far less potent
than endothelin-1, suggesting the predominant expression of
ETA receptor (Fig. 4A). Northern hybridizations revealed
two bands (5.2 and 4.2 kb) corresponding to the sizes of rat
ETA mRNAs (25) (Fig. 4B), whereas ETB mRNA was
not detectable in any of the cell lines.
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). Neither BQ123 nor BQ788 added alone affected the apoptotic
frequency of TGR-1. Likewise, BQ123, but not BQ788, abrogated the
survival effect provoked by endothelin-1 on M5 (Fig. 4C). The above
data are complemented by the immunohistochemical study shown in Fig. 2.
TGR-1 cells were changed into serum-free medium, and all adherent cells
after 24 h were stained simultaneously with antibody against
single-stranded DNA and with hematoxylin. Immunohistochemically
positive cells coincided with those showing cellular and nuclear
fragments. Addition of endothelin-1 markedly reduced the number of
adherent apoptotic cells (Fig. 2B). BQ123 abolished apoptosis
protection by endothelin-1 (Fig. 2C), whereas BQ788 did not affect the
endothelin-1-induced apoptosis inhibition (Fig. 2D). These results
indicate a role for endothelin-1 as a survival factor from
c-Myc-dependent apoptosis and show that this effect is mediated
by the ETA receptor.
ETA Receptor-Mediated Apoptosis Protection Acts via MAP
Kinase Activation
We searched for intracellular signal(s) essential for the
endothelin-1-induced cell survival by a pharmacological approach.
Pretreatment of TGR-1 with the following compounds neither affected
basal cell viability nor antagonized the cell survival effect of
endothelin-1 (data not shown): PI3 kinase inhibitors, wortmannin
(10-6 M) and LY294002 (2 x
10-6 M); Jak-2 inhibitor, tyrphostin B42
(10-5 M); tyrosine kinase inhibitors,
genistein (10-5 M), herbimycin A
(10-5 M), ST638 (10-4
M); Ca2+ channel blockers, nicardipine
(10-6 M) and benidipine (10-6
M); protein kinase C inhibitor, GF109203X
(10-8 M); phospholipase C inhibitor, U73122
(10-5 M); NF-
B inhibitors, MG115
(10-6 M) and MG132 (10-7
M); ras farnesyltransferase inhibitor (10-5
M manumycin); p38 MAP kinase inhibitor, SB203580
(10-5 M); interleukin-1ß converting enzyme
inhibitor, Ac-Tyr-Val-Ala-Asp-H (10-4 M);
inhibitor for apopain/CPP32/Yama, Ac-Asp-Glu-Val-Asp-H
(10-4 M). Endothelin-1 at higher
concentrations (10-8 to 10-6 M)
induced rapid and transient increases in intracellular free
Ca2+ concentration and ITP formation in all four cell
lines. Although this effect was completely blocked by BQ123, it was not
elicited at lower concentrations (10-13 to
10-9 M) of endothelin-1, suggesting that
ETA receptor-mediated activation of phospholipase C is not
involved in the endothelin-1-induced cell survival. We considered the
possibility that, at these low concentrations, endothelin-1 may
stimulate the production of certain growth factors, such as IGF-I and
PDGF, which in turn could inhibit c-Myc-dependent apoptosis by a
paracrine mechanism. However, the cell lines released IGF-I and PDGF-B
at far lower concentrations than those reported to suppress apoptosis
as determined by RIAs, and addition of the antibodies for IGF-I or
PDGF-BB did not affect the protective effect induced by endothelin-1
(data not shown). These data argue against an intermediate role of
these growth factors for the cell survival effect of endothelin-1.
We examined whether the MAP kinase pathway is involved in the
endothelin-1-induced cell survival. Pretreatment of TGR-1 with PD98059
(5 x 10-5 M), a specific inhibitor of
MAP kinase kinase (26, 27, 28), did not affect cell viability, but blocked
the apoptosis protection of endothelin-1 (Fig. 5). Treatment of the cells with a
liposome-encapsulated antisense oligonucleotide directed against the
translation initiation sites of the p42 and p44 rat MAP kinase isoform
mRNAs antagonized the cell survival effect of endothelin-1, whereas
random and sense oligonucleotides were without effect (Fig. 6A). Furthermore, transfection of TGR-1
cells with a dominant-interfering form of MAP kinase kinase 1 (MAPKK1
S222A) prevented the apoptosis survival activity of endothelin-1 (Fig. 6B). Transfection of TGR-1 cells with a constitutively activated form
of MAP kinase kinase 1 (MAPKK1 S218D/S222A) prevented apoptosis induced
by serum deprivation (data not shown). These results indicate a central
role for the MAP kinase pathway in mediating cell survival by
endothelin-1.
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). Addition of endothelin-1
(10-7 M) to quiescent TGR-1 stimulated MAP
kinase activity, which peaked at 5 min and declined after 60 min (Fig. 7B). These results were confirmed by phosphorylation of p42 and p44 as
demonstrated by protein immunoblot analysis with antibodies to
phosphotyrosine (data not shown). The endothelin-1-stimulated MAP
kinase activity was concentration-dependent
(10-11-10-7 M) and was completely
blocked by pretreatment with the MAP kinase kinase inhibitor, PD98059
(5 x 10-5 M) (Fig. 7C). The
EC50 of endothelin-1-mediated induction of MAP kinase
activities (5 x 10-10 M) coincides well
with the EC50 to inhibit apoptosis (10-11
M) (Figs. 3A and 7C). We conclude that the MAP kinase
pathway plays a central role in ETA-mediated protection
against c-Myc-dependent apoptosis in fibroblasts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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c-Myc-dependent apoptotic processes require p53 expression (31), while apoptosis induced by DNA damage is antagonized by bcl-2 expression via a p53-independent mechanism (32). In addition to Bcl-2, certain growth factors, such as IGF-I, insulin, and PDGF, have been demonstrated to rescue cells, at least partially, from c-Myc-induced apoptosis (15). Since the potency of these growth factors to induce cell division and to antagonize apoptosis do not correlate, it has been suggested that these peptides can act as distinct cell survival factors. In this communication, we present evidence that endothelin-1, a potent vasoconstrictor and mitogen originally identified in vascular endothelial cells, can also be categorized as an apoptosis survival factor. The present study shows that endothelin-1 can antagonize apoptosis and promote cell survival at concentrations (10-13-10-9 M) distinctly below those required for stimulation of DNA synthesis (10-9-10-7 M).
Since fibroblasts do not produce and secrete endothelin-1, their
survival in an organismal context could be mediated by endothelin-1
secreted by other cells. It should be emphasized that the minimum
effective concentrations to induce apoptotic protection in TGR-1 and
M5 cells by endothelin-1 are 10-11 M and
10-13 M, respectively. These concentrations
are comparable with those (10-12 M) of
circulating endothelin-1 in animals and humans (33), which are also
distinctly below those required for stimulation of DNA synthesis
(10-9-10-7 M). In addition, the
concentrations of endothelin-1 are lower than those of other growth
factors and cytokines known to block apoptosis (IGF-I, 100 ng/ml;
insulin, 5 µg/ml; PDGF, 10 ng/ml) (15).
Since endothelin-1-induced cell survival occurs in cells expressing physiological c-Myc levels, our data imply that endogenous endothelin-1 may also serve as a survival factor in vivo. In fact, in primary rat endothelial cells, endogenous endothelin-1 secretion protects the cells from apoptotic death in an autocrine/paracrine fashion via the ETB receptor (34), while in vascular smooth muscle cells, low doses of endothelin-1 suppress apoptosis induced by serum deprivation via the ETA receptor (our unpublished observation). Furthermore, endothelin-receptor antagonists have been demonstrated to prevent ventricular remodeling (35) and to suppress postangioplasty-induced neointima formation in rats (36). Taken together, these results suggest a physiological signi-ficance of endothelin-1 as an apoptotic survival factor.
The endothelin peptides family (endothelin-1, endothelin-2, and endothelin-3) mediate their diverse effects through two distinct subtypes of G protein-coupled heptahelical receptors, termed ETA and ETB (3, 4). The ETA receptor is selective for endothelin-1 and endothelin-2, whereas the ETB receptor does not distinguish between isopeptides. Vascular smooth muscle cells mainly express ETA receptors that mediate contraction, while vascular endothelium expresses ETB receptors, which are involved in vasodilation via generation of nitric oxide (3, 4, 37). Both receptor subtypes mediate the activation of MAP kinase (8, 9, 38) and are functionally coupled to phospholipase C to induce phosphoinositide breakdown (39). In the present study, binding studies and Northern hybridization revealed that rat fibroblast cell lines under investigation express the ETA receptor, but not the ETB receptor. The protective effects of endothelin-1 from c-Myc-induced apoptosis were clearly mediated via the ETA receptor, as evidenced by the fact that the ETA receptor antagonist (BQ123), but not ETB receptor antagonist (BQ788), completely blocked the survival effect.
Our present experiments have uncovered an intriguing role of ETA receptor-mediated MAP kinase pathway activation for cell survival. MAP kinase activation can inhibit induction of apoptosis in PC-12 cells deprived of nerve growth factor (17), in ceramide-treated HL60 cells (40), and in cerebellar neurons deprived of potassium (41). Raf-1 activation has been shown to protect Rat-1 fibroblasts from c-Myc-induced apoptosis (42), while a contrasting report suggests proapoptotic effects of a Raf-MAP kinase pathway (16). In our system, neither the farnesyltransferase inhibitor, manumycin, nor PI3 kinase inhibitors, wortmannin and LY294002, had any effect on endothelin-1-mediated cell survival, while the activation of MAP kinase had clear antiapoptotic effects. However, it remains to be determined whether a complete abrogation of Ras activity, for example, using a dominant-defective Ras mutant, would influence the survival effect of endothelin-1.
In summary, using a panel of isogenic rat fibroblast cell lines, we demonstrate a novel role of endothelin-1 as an apoptosis survival factor. Both physiological c-myc expression, as well as unphysiologically high levels, caused apoptosis upon serum deprivation. The effect of endothelin-1 to block c-Myc-dependent apoptosis was mediated through ETA receptor via MAP kinase activation and was clearly distinct from its proliferative effect.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M5) are derivatives of the Rat-1 cell line and cultured in DMEM
supplemented with 10% calf serum, glutamine, penicillin, and
streptomycin sulfate in a 5% CO2 atmosphere at 37 C.
Reagents
Synthetic endothelin-1, synthetic endothelin-3, MG115, MG132,
Ac-Tyr-Val-Ala-Asp-H, and Ac-Asp-Glu-Val-Asp-H were purchased from
Peptide Institute, Inc. (Osaka, Japan); tyrphostin B42, U73122,
GF109203X, genistein, herbimycin A, and ST638 were from
Calbiochem-Novabiochem Intl. (La Jolla, CA); nicardipine and wortmannin
were from Sigma-Aldrich (Tokyo, Japan). ETA receptor
antagonist (BQ123) and ETB receptor antagonist (BQ788) were
provided by Banyu Research Laboratory (Tsukuba, Japan); benizipine was
from Kyowa Hakko Pharmaceuticals (Tokyo, Japan); SB203580 from
SmithKline Beecham Pharmaceuticals (Philadelphia, PA).
Plasmids
The plasmid MAPKK1 S222A (kindly provided by Drs. Anne Brunet
and Jacques Pouyssegur) is a dominant-interfering form of MAP kinase
kinase 1 expressed from the SV40 early promoter, and the plasmid MAPKK1
S218D/S222D (kindly provided by Drs. Anne Brunet and Jacques
Pouyssegur) is a constitutively activated form of MAP kinase kinase 1
(43).
Cell Death Analysis
Exponentially growing cells in 24-well dishes were extensively
washed and replaced with serum-free medium containing endothelin-1, and
after 24 h floating cell numbers were determined with a Sysmex
CDA-500 Autoanalyzer (Toa Medical Electronics, Kobe, Japan). To
demonstrate nucleosome laddering, cellular fragmented DNAs were
extracted from total cell cultures 4 h after serum starvation
using NP-40 lysis, which eliminates intact chromatin (44), and
fractionated on 1.2% agarose gels. Flow cytometric analysis was
performed using a FACS Calibur (Becton Dickinson, San Jose, CA) on
cells stained with propidium iodide. For transfection of MAPKK1 S222A
and MAPKK1 S218D/S222D plasmids (42), TGR-1 cells grown to 60%
confluence in 10-cm plates were transfected with 5 µg plasmid DNA by
the transferrin receptor-mediated transfer method (45) for 16 h,
incubated in 10% calf serum-DMEM for 48 h, serum-starved, and
subjected to cell death analysis.
Immunohistochemical Staining for Single-Stranded DNA
Adherent TGR-1 cells, serum-deprived and incubated with or
without endothelin-1 (10-7 M) in the presence
or absence of BQ123 or BQ788 for 24 h, were fixed in 70% acetone
and stained with rabbit polyclonal antibody raised against
single-stranded DNA (46, 47) (dilution 1:500), as previously described
(48).
DNA Synthesis
Cells were serum-deprived for 24 h, incubated with or
without endothelin-1 for 16 h, and pulsed with 0.5 µCi
[3H]thymidine (Amersham, Arlington Heights, IL) for
2 h. After incubation, cells were extensively washed with ice-cold
5% trichloroacetic acid, and radioactivity incorporated into cells was
determined.
Binding Experiments
Cells were incubated at 37 C and 4 C for 2 h with 8.6
x 10-12 M [125I]endothelin-1
(specific activity 2000 Ci/mmol, Amersham) in the presence or absence
of unlabeled endothelin-1, extensively washed, and solubilized with 1
N NaOH, and the cell-bound radioactivity was measured (10).
Specific binding was calculated as the total radioactivity bound minus
nonspecific binding in the presence of excess (10-6
M) unlabeled endothelin-1. The apparent dissociation
constant (Kd) and maximal binding capacity
(Bmax) were calculated by Scatchard analysis of the binding
data.
Northern Hybridization Analysis
RNA was extracted by the guanidinium thiocyanate method, as
described (10). Total RNA (15 µg) was electrophoresed on
formaldehyde-agarose gels. Blotting was onto MagnaGraph nylon membranes
(Micron Separations, Inc., Westborough, MA). cDNA probes for rat
ETA and ETB receptor
genes and GAPDH genes were labeled with
-[32P]dCTP using the random-priming method.
Oligodeoxynucleotides
An antisense oligodeoxyribonucleotide, a
phosphorothioate-protected 17-mer directed against the initiation of
translation site of rat p42 and p44 MAP kinase mRNA
(5'-GCCGCCGCCGCCGCCAT-3'), sense (5'-ATGGCGGCGGCGGCGGC-3') and random
(5'-CGCGCGCTCGCGCACCC-3') oligonucleotides were synthesized and
introduced into TGR-1 using transferrin receptor-mediated-transfer
(45). The efficiency was determined using FITC-conjugated
oligonucleotides: at least 80% of total cells became transfected
within 5 h of transfection, and the intensity of cellular
fluorescence correlated well with the oligonucleotide concentration
used (0.2–10 µM).
MAP Kinase Activity
TGR-1 cells, incubated in the presence or absence of
endothelin-1, were lysed and sonicated. After centrifugation at
25,000 x g for 20 min, the supernatant was assayed for
MAP kinase activity with an p42/p44 MAP kinase enzyme assay system
using [
-32P]ATP (Amersham).
Statistical Analysis
Data are expressed as mean ± SEM. Statistical
analysis were performed by using ANOVAs for repeated measures.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by the Ministry of Education, Science and Culture, Japan (to M.S. and Y.H.), by the Ministry of Health and Welfare, Japan (to Y.H.), by the Uehara Memorial Foundation Grant (to M.S.), by the Tanabe Medical Frontier Conference (to M.S.), by NIH Grant GM-R01-41690 (to J.M.S.), and by a Presidential Young Investigator Award from the National Science Foundation (to J.M.S.).
Received for publication September 19, 1997. Revision received November 10, 1997. Accepted for publication November 11, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and in cells constitutively
overexpressing a conditional c-Myc transgene (
) is shown.
Each point represents mean ± SEM (n = 6); values
were calculated as the percentage of the number of floating cells in
the absence of endothelin-1, which was set to 100%. *,
P < 0.05, **, P < 0.01,
treated vs. untreated cells. B, Effect of endothelin-1
on DNA synthesis of TGR-1 and HET15. Both cell lines were treated with
various doses (10-11 to 10-7 M)
of endothelin-1 for 24 h, and [3H]thymidine
incorporation was measured. Each point represents mean ±
SEM (n = 4).
). Inset,
Scatchard plot of binding data. B, Expression of ETA
receptor mRNA in TGR-1 cells. Total cellular RNA (15 µg) was
subjected to Northern hybridization with rat ETA and
ETB cDNA probes. The positions of 28S and 18S ribosomal
RNAs are indicated with arrows. C, Effects of endothelin
receptor antagonists on apoptosis. Apoptosis assays were performed
using TGR-1 and 
) endothelin-1 (10-7
M) plus PD98059 (40 µM).