Induction of Max by Adrenomedullin and Calcitonin Gene-Related Peptide Antagonizes Endothelial Apoptosis
Masayoshi Shichiri,
Hiroki Kato,
Masaru Doi,
Fumiaki Marumo and
Yukio Hirata
Division of Endocrinology and Metabolism Second Department of
Internal Medicine Tokyo Medical and Dental University Tokyo
113-8519, Japan
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ABSTRACT
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Adrenomedullin is a novel vasodilatory peptide
originally isolated from pheochromocytoma. Recently, we found that
adrenomedullin acts as an autocrine/paracrine apoptosis survival factor
for rat endothelial cells. In the present study, we show that
adrenomedullin induces the expression of Max, a heterodimeric partner
of c-Myc, which may contribute to its ability to rescue endothelial
cells from apoptosis. Max is a basic-helix-loop-helix-leucine zipper
protein that forms heterodimers with its alternative partners, Mad and
Mxi-1, to behave as an antagonist for Myc-Max heterodimer through
competition for common DNA targets. The expression of Max is reported
to be constitutive and more stable than c-Myc, and serum induces
immediate c-Myc stimulation followed by modest Max up-regulation. In
quiescent rat endothelial cells, adrenomedullin stimulated the
expression of Max without affecting c-Myc. Quantitation with
real-time quantitative PCR detected on the ABI Prism 7700 Sequence
Detection System revealed that adrenomedullin and calcitonin
gene-related peptide (CGRP), as well as serum, up-regulated Max mRNA
levels and that down-regulation of Max mRNA after serum deprivation was
prevented by adrenomedullin. Neither adrenomedullin nor CGRP affected
c-Myc expression. Transfection of a Max-expressing plasmid into
endothelial cells rescued the apoptosis induced by serum deprivation.
Neutralization with anti-adrenomedullin antiserum or blockade with a
CGRP receptor antagonist, CGRP(837), reduced Max mRNA levels in
growing endothelial cells and enhanced apoptosis after serum
starvation. Introduction of an antisense oligodeoxynucleotide against
Max mRNA using transferrin receptor-operated transfer led to inhibition
of both adrenomedullin-induced up-regulation of Max transcripts and its
cell survival effect, whereas random, sense, or missense
oligonucleotides were without effect. The negative regulation of
E-box-driven transcription by adrenomedullin was demonstrated by using
preproendothelin-1 promoter containing c-Myc-Max binding consensus
sequence; the promoter activity of preproendothelin-1 was reduced by
cotransfecting Max- and Mad-expressing plasmids as well as addition of
adrenomedullin and CGRP. The present results demonstrate that
adrenomedullin antagonizes serum deprivation-induced endothelial
apoptosis by up-regulation of the max gene in an
autocrine/paracrine manner.
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INTRODUCTION
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Adrenomedullin is a potent vasorelaxant/hypotensive peptide with
52-amino acid residue originally isolated from human pheochromocytoma
(1). Adrenomedullin has a conserved structure among mammals (2) and
shows a partial homology with calcitonin gene-related peptide (CGRP)
(1). Adrenomedullin is widely expressed in a variety of tissues,
including vascular smooth muscle cells (VSMC) (3) and endothelial cells
(4). Adrenomedullin receptors are functionally coupled to adenylate
cyclase in VSMC and endothelial cells (5). We have recently found that
adrenomedullin is not mitogenic in endothelial cells, but protects
against apoptosis independent of cAMP activation (6), while it acts as
a potent growth-promoting factor for quiescent VSMC via protein
tyrosine kinase-mediated mitogen-activated protein kinase
activation (7). Mitogenic activity of adrenomedullin is also reported
in rat fibroblast cell lines (8), human tumor cell lines (9), and rat
VSMC (7), whereas it inhibits proliferation of mesangial cells via
cAMP-dependent mechanism (10).
Apoptosis, an intrinsic program to eliminate unwanted, aged, or damaged
cells, is involved in the regulation of cell number in certain
physiological and pathological conditions. Diverse stimuli, such as
serum deprivation, radiation, chemotherapeutic agents, and
antioxidants, induce apoptosis in many cell types (11, 12, 13), whereas
growth factors and cytokines are known to prevent apoptosis triggered
by such environmental signals (14). Forced expression of the cellular
protooncogene c-myc combined with signals for entry into
G0 has been shown to trigger apoptosis in fibroblasts (12)
and in VSMC (15). Removal of serum growth factors from exponentially
growing fibroblasts elicits a rapid loss of c-Myc at any position in
the cell cycle (16). It has been suggested that c-Myc may integrate
several signaling pathways since its induction is not solely dependent
on any specific growth factors. Identification of the Myc dimerization
partner Max has allowed a significant advance in our understanding of
c-Myc function. Unlike c-Myc, Max expression is relatively stable
throughout the cell cycle in hematopoietic cells and fibroblasts (17, 18), and the ability of c-Myc to promote proliferation, transformation,
and apoptosis is believed to require dimerization with Max (19, 20).
Max expression is reported to be growth regulated after a transient
expression of c-Myc (21). Both Myc-Max heterodimers and Max-Max
homodimers compete each other to bind to the E-box recognition site
(CACGTG) and several related noncanonical sequences (22, 23, 24, 25). Myc-Max
activates transcription to promote cell proliferation and apoptosis,
while Max-Max, which lacks a transcriptional activation domain,
represses it (26, 27). Max also forms a heterodimer with its
alternative partners, Mad and Mxi-1, to behave as an antagonist for
Myc-Max by competition for common DNA targets (28). Whether endogenous
Max expression is affected by adrenomedullin to modulate apoptosis in
endothelial cells with physiological c-Myc expression is yet unknown.
In the present study, we have identified a unique function of
adrenomedullin to up-regulate Max without inducing c-Myc expression,
which prevented endothelial apoptosis triggered by serum
deprivation.
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RESULTS
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Northern blot hybridization of total RNA from unstimulated rat
endothelial cells revealed a distinct band corresponding to the size of
Max mRNA (2.0 kb). Adrenomedullin (10-6 M)
induced 2- to 3-fold increases in steady state Max mRNA levels in
quiescent cells after 46 h (Fig. 1A
),
whereas it did not induce c-myc expression (data not shown).
Western blot analysis using polyclonal anti-p21 Max antibody revealed
that both adrenomedullin (10-6 M) and CGRP
(10-6 M) increased p21 Max protein after
6 h (Fig. 1B
), whereas neither forskolin (10-5
M) nor 8-bromo-cAMP (10-3 M)
affected Max expression. Quantitation of Max mRNA transcripts using
real-time quantitative PCR method revealed a time-dependent (26 h)
increase in Max mRNA levels after treatment with adrenomedullin
(10-7 M) (Fig. 2A
), the effect of which was dose
dependent (10-910-6 M) (Fig. 2B
). Max mRNA expression was also up-regulated by 10% serum and CGRP
(10-7 M) to the same extent as adrenomedullin
(Fig. 2C
). In contrast, c-Myc mRNA levels were unaffected by
adrenomedullin or CGRP, but rapidly induced by 10% serum;
adrenomedullin increased the ratio of Max to c-Myc in a time- (Fig. 2D
)
and dose-dependent (Fig. 2E
) fashion, whereas the ratio after serum
addition was comparable to that of control, which was in marked
contrast to those after adrenomedullin and CGRP (Fig. 2F
).

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Figure 1. Adrenomedullin Induces max
Expression
A, Serum-deprived rat endothelial cells were treated with
adrenomedullin (10-6 M) for 26 h. Total RNA
(20 µg) was subjected to Northern blot analysis with
max and GAPDH probes, respectively.
Northern blotting was repeated four times with qualitatively similar
results. B, Protein samples (15 µg) prepared from cell lysates
after treatment with adrenomedullin, CGRP, forskolin, and 8-bromo cAMP
for 6 h were subjected to Western blot analysis with anti-Max
antibody (1:1000). An arrow shows the position of p21
Max protein. Western blotting was repeated three times with
qualitatively similar results.
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Figure 2. Adrenomedullin Up-Regulates Max mRNA as Detected by
Real-Time Quantitative PCR
Rat endothelial cells were rendered quiescent by incubating with medium
containing 0.1% FBS for 24 h. Quiescent cells were stimulated
with adrenomedullin (10-6 M) for the indicated
time (panels A and D), the indicated concentrations of adrenomedullin
for 6 h (panels B and E), or 10% serum, adrenomedullin
(10-7 M), CGRP (10-7
M) for 6 h (panels C and F). Extracted total RNAs were
subjected to quantitative PCR using the ABI Prism 7700 Sequence
Detection System for the estimation of relative Max mRNA levels (AC)
and Max to c-Myc mRNA ratio (DF) as described in Materials and
Methods. Each column with bar shows mean ±
SD (n = 4). *, P < 0.05; **,
P < 0.01, treated vs. untreated or
control cells.
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Compared with quiescent cells, exponentially growing endothelial cells
express about 4-fold greater Max mRNA levels, which decreased upon
withdrawal of serum as a function of time (26 h) (Fig. 3A
). The time-dependent down-regulation
of Max mRNA expression by serum deprivation was antagonized by the
addition of adrenomedullin (10-7 M). Serum
withdrawal decreased c-Myc mRNA with or without adrenomedullin; thus
adrenomedullin increased the ratio of Max to c-Myc (Fig. 3B
). Addition
of CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) (10-6 M), a CGRP/adrenomedullin
receptor antagonist, and polyclonal antiadrenomedullin antiserum
(1:100) resulted in a decrease in the steady state levels of Max mRNA
(Fig. 4A
) as well as the Max to c-Myc
ratio (Fig. 4B
); nonimmune serum (1:100) had no effect. Pretreatment
with CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and antiadrenomedullin antiserum enhanced apoptosis
upon serum deprivation, while nonimmune serum was without effect (Fig. 4C
). The results suggest that endogenous adrenomedullin contributes to
maintain constitutive Max expression as an autocrine/paracrine factor
to protect against apoptosis by serum deprivation.

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Figure 4. Endogenous Adrenomedullin Induces Up-Regulation of
Max mRNA and Protection from Apoptosis
A and B, Exponentially growing rat endothelial cells were incubated
with or without CGRP(8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) (10-7 M) or with
either antiadrenomedullin antiserum or nonimmune serum (1:1000) for
72 h. Total RNAs were extracted and subjected to quantitative PCR
for Max mRNA (A) and Max to c-Myc mRNA (B) ratio. Each column
with bar shows mean ± SD (n = 4). C,
Floating apoptotic cell number in a total cell population was counted
6 h after serum starvation after pretreatment with or without
CGRP(8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 ) (10-6 M) or with
antiadrenomedullin antiserum or nonimmune serum (1:1,000) for 72
h. Each column with bar shows mean ±
SD (n = 6). *, P < 0.05, treated
vs. untreated or control cells.
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We next determined whether overexpression of Max affects endothelial
apoptosis. Transfection of a Max-expressing vector (pSP-max) into
endothelial cells induced a preferential expression of Max p21 protein
in a dose-dependent manner (2080 µg/ml electroporation buffer) with
a marginal p22 protein expression (Fig. 5A
). After transfection with pSP-max
plasmid, serum deprivation-induced apoptosis was inhibited as a
function of doses used (Fig. 5B
). The results suggest that rescue by
Max from serum starvation-induced endothelial apoptosis is a
Myc/Max-dependent process.

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Figure 5. Overexpression of Max Rescues Endothelial Cells
from Serum Deprivation-Induced Apoptosis
A, Plasmid pSP-max in the indicated concentrations was electroporated
into rat endothelial cells and cultured for 48 h. Protein samples
(15 µg) were subjected to Western blot analysis with anti-Max
antibody. Western blotting was repeated three times with qualitatively
similar results. B, After electroporation and incubation for 48 h,
cells were serum deprived for 6 h; all floating dead cells were
collected and counted. Each bar represents mean ±
SEM (n = 6); values were calculated to the percentage
to the number of floating dead cells in the absence of pSP-max.
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We reasoned that inhibition of Max down-regulation by adrenomedullin
added upon serum deprivation protected against endothelial apoptosis.
To block the adrenomedullin-induced Max mRNA expression, we treated the
cells with antisense oligonucleotide against Max mRNAs by a transferrin
receptor-operated transfer of liposome-encapsulated oligonucleotides.
Quantitation of Max mRNA by a quantitative PCR method revealed that the
adrenomedullin-induced up-regulation of Max mRNA was completely blocked
by antisense oligonucleotide (0.2 µM), but not by sense,
reverse, or missense oligonucleotides in the same concentrations (data
not shown). Antisense oligonucleotide (0.2 µM) similarly
antagonized cell survival effect of adrenomedullin, whereas sense,
reverse, and missense oligonucleotides in the same concentrations were
ineffective (Fig. 6
).

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Figure 6. Max Antisense Oligonucleotides Abrogate
Adreno-medullin (AM)-Induced Protection of Endothelial Apoptosis
Rat endothelial cells treated with or without 0.2 µM each
of antisense, sense, reverse, and missense oligonucleotides against Max
mRNA via transferrin receptor-mediated transfer were incubated in
serum-free medium with or without 10-7 M
adrenomedullin (AM) for 4 h as described in Materials and
Methods; all floating dead cells were collected and counted.
Data (n = 6) are plotted as in Fig. 5B . *, P
< 0.05; **, P < 0.01, treated vs.
untreated cells.
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We examined whether adrenomedullin and CGRP in effect suppress E
box-driven transcriptional activity using 1535 bp of 5'-flanking
sequence of the human preproendothelin-1 gene containing noncannonical
c-Myc-binding consensus (CACGTTG) (29) fused to the luciferase reporter
gene (plasmid pPPET-luc). Cotransfection of pPPET-luc construct into
growing rat endothelial cells with pSP-max and/or pSP-mad led to a
marked suppression of the activity of pPPET-luc compared with that of
control empty vector (Fig. 7A
),
suggesting negative regulation of preproendothelin-1 transcription by
Max and Mad. Adrenomedullin and CGRP similarly and dose-dependently
(10-1010-6 M) suppressed the
reporter gene expression, the effect of which was reversed by
CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) (Fig. 7B
).

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Figure 7. Preproendothelin-1 Promoter Activity Is Suppressed
by Cotransfection with Max/Mad and by Adrenomedullin/CGRP
A, Plasmid pGL3-PPET1 was cotransfected with Renilla
luciferase-expressing vector and with empty vector (pSP),
Max-expressing plasmid (pSP-Max), and/or Mad-expressing plasmid
(pSP-Mad) into rat endothelial cells and incubated for 48 h. B,
Plasmid pGL3-PPET1 cotransfected with pRL-TK into rat endothelial cells
was incubated for 24 h; the cells were then incubated with or
without adrenomedullin (AM) and CGRP in concentrations indicated for
24 h. The ratio of Firefly to Renilla luciferase activity relative
to that elicited by plasmid pGL3-PPET1 plus pSP are shown. Each
bar represents mean ± SD (n = 8).
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DISCUSSION
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The present study demonstrates for the first time that a novel
vasodilatory peptide, adrenomedullin, and CGRP induced Max gene
expression in cultured rat endothelial cells without affecting its
dimerization partner, c-Myc, as evidenced by the induction of Max mRNA
and protein by Northern and Western blot analyses, respectively. This
was further supported by quantitation with the real-time PCR method
showing dose- and time-dependent up-regulation of Max transcripts by
adrenomedullin. Our findings also revealed that serum deprivation
induced down-regulation of Max mRNA, the effect of which was prevented
by the addition of adrenomedullin, and that neutralization of
endogenous adrenomedullin with antiadrenomedullin antiserum or by
blockade of adrenomedullin/CGRP receptor with CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) reduced Max
mRNA levels. Since rat endothelial cells used in the present study not
only express the adrenomedullin gene and secrete mature peptide into
the media, but contain its own receptors (6), our results are
consistent with the notion that adrenomedullin produced by and released
from endothelial cells could act in an autocrine/paracrine fashion to
maintain constitutive Max expression.
The effect of adrenomedullin on Max expression is not restricted to
endothelial cells. Adrenomedullin induces an immediate induction
of c-Myc, but gradual and sustained expression of Max in rat VSMC
(our unpublished observation). Such concomitant induction of Myc
and Max by adrenomedullin may form Myc-Max heterodimers, possibly
leading to transcription activation and mitogenic response. In
contrast, adrenomedullin induces up-regulation of Max without affecting
Myc levels in endothelial cells. Abundant Max protein in the absence of
a corresponding increase in Myc protein may cause formation of Max-Mad
or Max-Mxi1 heterodimers that are known to compete with Myc-Max
heterodimer to its binding sites (Fig. 8
). Thus, it is anticipated that
up-regulation of Max protein may result in opposing physiological
consequences depending upon the availability of Myc protein: in the
presence of an abundant amount of Myc, Max may function as a
transcription activator by forming Myc-Max heterodimer, whereas in the
absence of sufficient Myc, Max may act as a transcription inhibitor for
Myc-Max.

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Figure 8. A Proposed Mechanism of Antiapoptosis Induced by
Adrenomedullin (AM) and CGRP in Rat Endothelial Cells
Up-regulation of Max protein in the absence of c-Myc induction may
result in formation of Max-Mad or Max-Mxi heterodimers, which compete
with Myc-Max to E-box as a transcription inhibitor.
Boxes indicate genes; Myc, Max, Mad, and Mxi indicate
the encoded gene products. An arrowhead with a straight lineindicates a positive effect. A bold solid arrow
indicates a promoter action. CACGTG, a cannonical binding site for
Myc-Max, Max-Mad, or Max-Mxi.
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Transcriptional inhibition by adrenomedullin and CGRP was further
confirmed by the experiments using preproendothelin-1
promoter-luciferase construct, which contains a noncannonical c-Myc-Max
binding site (29). Cotransfection of a Max- and/or Mad-expressing
plasmid suppressed the promoter activity, suggesting a transcription
inhibition by antagonizing c-Myc-Max. Addition of either adrenomedullin
or CGRP to the cells transfected with preproendothelin-1
promoter-luciferase construct similarly and dose-dependently inhibited
the promoter activity, the effect of which was reversed by a CGRP
receptor antagonist. These results suggest that both adrenomedullin and
CGRP reduced E-box-driven transcriptional activity via common
receptor.
Myc-induced apoptosis is dependent upon the level at which it is
expressed, and induction of apoptosis by c-Myc requires association
with Max, suggesting that c-Myc drives apoptosis through a
transcriptional mechanism (20, 30, 31). Myc-Max dimerization is also a
prerequisite for cell cycle progression in fibroblasts (20). The
apparently paradoxical functions have been demonstrated to be
antagonized by overexpressing Max, whose homodimers or heterodimers
with Mad compete with Myc-Max heterodimers for its specific DNA
target sites (28). Recent studies report apparently contradictory
apoptotic responses to Max overexpression experiments using different
experimental designs or cell types (32, 33), implying differential
effects on apoptotic events depending on Max expression levels and
availability of Myc protein. In a nontransformed fibroblast cell line
selected from Rat-1 cells where c-myc is diploid and
constitutive c-Myc expression level is very low without N- or L-Myc
expression (34), overexpression of Max also resulted in a marked
suppression of apoptosis (our unpublished observation). It is
presently unknown how Myc or Max is involved in endothelial apoptosis.
The present study revealed that cell death induced by serum deprivation
was rescued by overexpressing Max, suggesting that Myc protein,
possibly by dimerizing with constitutive Max, plays an important role
in the mechanism of apoptosis in endothelial cells (Fig. 8
).
The significance of Max function was further tested in vitro
by using an efficient transferrin receptor-operated transfer of
oligonucleotides that clearly showed that the adrenomedullin-induced
up-regulation of Max mRNA was completely blocked by antisense
oligonucleotide, but not by either sense, reverse, or missense
oligonucleotides. Concomitantly, suppression of Max up-regulation after
selective transfer of antisense oligonucleotide led to abrogation of
adrenomedullin-induced protection from endothelial apoptosis.
Furthermore, both neutralization of adrenomedullin with
antiadrenomedullin antibody and blockade of CGRP/adrenomedullin
receptor with CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) caused down-regulation of Max mRNA and
increased apoptotic events upon serum deprivation. Taken together, the
results are consistent with the notion that Max plays a pivotal role in
the mechanism of survival effect by adrenomedullin from endothelial
apoptosis.
In this study, adrenomedullin and CGRP similarly up-regulated Max
without affecting c-Myc expression and suppressed E-box-driven
transcriptional activity, the concentrations of which appear to be
comparable. These data suggest that adrenomedullin and CGRP share the
same and/or very similar, if not identical, receptors. Notably, it has
recently been shown that calcitonin-receptor-like receptor
functions as either CGRP receptor or adrenomedullin receptor depending
upon whether receptor activity-modifying protein (RAMP) 1 or RAMP2 is
simultaneously expressed, respectively (35). It remains to be clarified
as to which RAMP isoforms are predominantly expressed by rat
endothelial cells.
Adrenomedullin was initially identified with its ability to stimulate
cAMP formation in platelets, and cAMP has been suggested as a second
messenger for its observed vasorelaxation effects (1). However, we
reported previously that protection of endothelial apoptosis by
adrenomedullin is not mediated via cAMP, because cAMP-elevating
agonists (forskolin, PGI2) did not inhibit apoptosis, and a
cAMP antagonist (Rp-cAMP) failed to block adrenomedullin-induced
antiapoptotic effect (6). The present study revealed that both
forskolin, an activator of adenylate cyclase, and 8-bromo-cAMP failed
to induce Max protein expression, suggesting that up-regulation of Max
is independent of cAMP. We have recently shown that adrenomedullin has
a growth-promoting effect via tyrosine kinase-mediated
mitogen-activated protein kinase activation in rat VSMC (7).
Furthermore, it has been reported that adrenomedullin stimulates
phospholipase C to generate inositol-1,4,5-trisphosphate (IP3), which
mobilized intracellular Ca2+, thereby leading to nitric
oxide production in bovine endothelial cells (36), although
adrenomedullin had no effects on IP3 generation or intracellular
Ca2+ concentrations in our rat endothelial cells (6). Thus,
there appear to exist multiple signal transduction pathways of
adrenomedullin in addition to the cAMP-dependent pathway. The
exact signal transduction of adrenomedullin linked to Max induction
remains to be determined.
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MATERIALS AND METHODS
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Cell Culture
Rat endothelial cells were prepared from 15-week-old male Wistar
rat aorta (Saitama Experimental Animals Supply Co., Saitama, Japan)
by collagenase and elastase digestion, as described previously
(6, 37). The endothelial origin of the cultures was confirmed by the
cobblestone appearance and the presence of Factor VIII by
immunohistochemical method. Cells (passages 510) were cultured
in DMEM in a 5% CO2 atmosphere at 37 C, supplemented with
10% FBS.
Reagents
Synthetic rat adrenomedullin, rat CGRP, and human CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
were purchased from Peptide Institute (Osaka, Japan), DMEM
from Life Technologies, Inc. (Rockville, MD), FBS
from HyClone Laboratories, Inc. (Logan, UT), forskolin
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan),
and 8-bromo-cAMP from Sigma Chemical Co. (St. Louis, MO).
All other reagents were of analytical grade. pSP-Max and pSP-Mad
plasmids were kindly supplied by Dr. R. N. Eisenman, Fred
Hutchinson Cancer Research Center (Seattle, WA).
Detection of Apoptosis
We described previously that primary rat endothelial cells
undergoing apoptosis are detached from culture plates after serum
deprivation and that floating apoptotic cell number increases as a
function of time, reaching a significant fraction (
40%) within
24 h (6, 37). This was proven by 1) demonstration of nucleosomal
ladders of extracted fragmented DNA from total culture using a highly
efficient NP-40 lysis method that efficiently eliminates intact
chromatin; 2) the presence of hypodiploid cells almost exclusively in
the floating fraction of endothelial culture stained with propidium
iodide by flow cytometric analysis; 3) terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method;
4) immunohistochemical detection using antibody against
single-stranded DNA; and 5) morphological features characteristic of
apoptosis. Determination of floating and adherent fractions of
endothelial culture by FACS Calibur flow cytometer (Becton
Dickinson, San Jose, CA) confirmed our previous results that
collections of floating fraction by two washes with PBS and adherent
fraction by trypsinization clearly separate apoptotic and viable cells,
respectively (6, 37). Therefore, the percentages of apoptotic cells
were calculated by counting floating apoptotic and trypsinized viable
cell number using a Sysmex CDA-500 Autoanalyzer (Toa Medical
Electronics, Kobe, Japan). In brief, rat endothelial cells were plated
in 24-well dishes in serum-containing medium and incubated for 24
h. The cells were extensively washed with PBS, replaced with serum-free
DMEM, and incubated with or without the indicated reagent. After 4
h, all floating cells were collected after two washes with PBS as
described previously (6, 37, 38). All adherent cells were also
collected after trypsinization for a quantitative analysis of total
apoptotic events in a given cell population. For determination of the
involvement of endogenous adrenomedullin, cells were plated in
24-well dishes in serum-containing medium and incubated with or without
CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), nonimmune control rabbit serum, or rabbit polyclonal
antiadrenomedullin antiserum (39) for 72 h. The medium was then
changed to serum-free DMEM, and all floating and adherent cells
were counted after 4 h.
Northern Blot Analysis
RNA was extracted from rat endothelial cells by the
guanidinium thiocyanate method as described (40). Total RNA (20 µg
per lane) was electrophoresed on formaldehyde-agarose gels. cDNA probes
for rat adrenomedullin, max, c-myc, and human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were
labeled with
-[32P]dCTP using the random-priming
method. Signals were quantitated using a BAS2000 Imaging Analyzer
(Fuji Photo Film Co., Ltd.). All values were corrected
against the GAPDH internal control. To confirm
reproducibility of the Northern hybridization detection, each RNA
sample was assayed independently on four occasions.
Real-Time Quantitative PCR
Real-time quantitative PCR method was used to detect accurately
the changes of max gene copies. The cycle at which the
amplification plot crosses the threshold (CT) is known to accurately
reflect relative mRNA values (41, 42). Total RNA was harvested from
cells treated with or without adrenomedullin or indicated reagents. Rat
max and c-myc mRNAs were amplified
(max: forward primer 5'-ACGA-TGACATCGAGGTGGAGAG-3' and
reverse primer 5'-GCA-TTATGGTGAGCCCGTTT-3', c-myc:
forward primer 5'-AAAGGCCCCCAAGGTAGTTATC-3' and reverse primer
5'-GTGCTCATCTGCTTGAACGG-3'), detected, and quantitated in real time
using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) as described previously (41, 42).
The TaqMan probes for max and c-myc were
5'-ACCGAGGTTTCAATCTGCGGCTGA-3' and 5'-TCAAAAAAGCCACCGCCTACATCCTG-3',
respectively. The amplification mixture contained 5 nM of
template DNA and 50 µM of primer DNA in 50 mM
salt and 1 mM Mg2+. A three-step PCR was
performed for 35 cycles. Denaturation was done at 94 C for 20 sec,
annealing at 55 C for 20 sec, and extension at 72 C for 30 sec. The
reaction produced a 79-bp PCR product for max and 70-bp
product for c-myc. The PCR cycle at which the amplification
plot crosses a threshold of 10 SD above the baseline was
defined as a CT value. In comparison to control sample without RNA,
which did not show any increase in normalized reporter signal (Rn),
addition of RNA from rat endothelial cells resulted in PCR
amplification reaching the threshold value. Relative mRNA level was
calculated based on the assumption that CT values increase by
approximately 1 for each 2-fold dilution (41, 42). To determine
precision of the assay, mRNA from endothelial cells was reverse
transcribed and amplified on three separate days. The mean CT values
ranged from 22.34 to 22.59, and an intraassay precision from 0.17% to
0.42% coefficient of variation. The interassay precision of
amplification for the 3 days was 3.2%.
Western Blot Analysis
Confluent cells on 10-cm dishes were harvested in PBS on ice,
pelleted and resuspended in 200 µl of ice-cold 62.5 mM
Tris (pH 6.8), 10% (wt/vol) glycerol, and 1 mM
phenylmethylsulfonyl fluoride. Fifteen microliters of 20% SDS were
added, vortexed, and immediately boiled for 5 min. Protein was
quantitated using the Micro BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL). Western blotting analysis was
performed as described (43, 44). SDS polyacrylamide gels were
transferred onto nitrocellulose, which was blocked with Blotto (5%
dried nonfat milk in PBS), and incubated with affinity-purified rabbit
polyclonal antibody raised against the carboxy terminus of Max p21
(C-17, Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
followed by horseradish peroxidase-conjugated antirabbit IgG antiserum
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA). Signals were visualized using the ECL Chemiluminescence System
(Amersham Pharmacia Biotech, Arlington Heights, IL).
Membranes were stained with Pelikan Fount India Ink to verify
electrotransfer uniformity (45).
Overexpression of max Gene
For experiments overexpressing max gene,
pSP-Max plasmid (2080 µg/ml electroporation buffer) was
electroporated into subconfluent cells cultured in 10-cm dishes using a
Gene Pulser apparatus (Bio-Rad Laboratories, Inc.,
Richmond, CA), as described previously (29), and plated in 24-well
dishes. After 48 h incubation, the cells are deprived of serum and
incubated for 4 h; floating and adherent cells were
counted. pSVßGAL plasmid DNA (5 µg) was included in all
transfections to assure almost equal electroporation efficiency. Salmon
sperm DNA was used as carrier to adjust the total DNA content of each
electroporation sample to 85 µg/ml.
Transfer of Oligodeoxynucleotides for Max mRNA
An antisense oligodeoxyribonucleotide, a
phosphorothioate-protected 15-mer directed against the initiation of
translation site of rat Max mRNA (5'-ATCGTTATCGCTCAT-3'), sense
(5'-ATGAGCGATAACGAT-3'), missense (5'-ATCGTGA-TCTCTCAT-3'), and
reverse (5'-TAGCAATAGCGAGTA-3') oligonucleotides was synthesized and
introduced into endothelial cells using transferrin receptor-mediated
transfer (46). In brief, 3 µl of Lipofectin (1 mg/ml, Life Technologies, Inc.) was added to 100 µl HEPES-buffered saline
(HBS, 20 mM HEPES, pH 7.4, and 100 mM NaCl)
containing 16 µg human holo-transferrin (Sigma Chemical Co.), incubated for 20 min at room temperature, mixed with 100
µl HBS containing various amount of oligonucleotide complexes, and
further incubated for 15 min. The mixture was then overlaid on cells
that had been covered with 300 µl of serum-free DMEM and incubated
for 4 h with or without adrenomedullin. To determine the
transfection efficiency, fluorescein isothiocyanate-conjugated
oligonucleotides were introduced into rat endothelial cells in the same
manner as indicated above. Regardless of the oligonucleotide
concentration used (0.110 µM), nearly 100% of total
cells were transfected, and the intensity of cellular fluorescence
correlated well with the oligonucleotide concentration used. In a
control experiment, the antisense oligomer (0.21 µM)
markedly reduced Max mRNA levels; 0.51 µM of the
antisense reduced the Max mRNA levels below the baseline value, and 0.2
µM of antisense blocked the rise of Max mRNA by
adrenomedullin, whereas sense, missense, and reverse in the same
concentrations were without effect. Therefore, a 0.2 µM
concentration was used in oligonucleotide transfer experiment.
Therefore, cells plated in 24-well dishes and incubated for 24 h
were treated with 0.2 µM each of oligonucleotides via
transferrin receptor-operated transfer; floating and adherent cells
were counted after 4 h.
Transcriptional Activity of Preproendothelin-1 Promoter
A PCR-generated, XhoI- and
HindIII-tailed, DNA fragment (-1535 to +86 bp) of sequence
5' to the human preproendothelin-1 gene containing noncannonical
c-Myc-binding consensus was cloned into the XhoI and
HindIII site of pGL3 basic (Promega Corp.,
Madison, WI) to create pGL3-PPET1. Cells plated in 96-well plates were
transiently cotransfected with pGL3-PPET1 and pRL-TK vector (1 µg
each/well) (Promega Corp.) expressing Renilla luciferase
as an internal control using synthetic cationic lipid,
(+)-N,N [bis
(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl]
ammonium iodide (Promega Corp.) according to the
manufacturers suggestion. Firefly and Renilla luciferase activities
were measured using the Dual Luciferase Reporter Assay System
(Promega Corp.) in a single-tube assay format using
MicroLumatPlus (EG&G Berthold, Wildbad,
Germany).
 |
ACKNOWLEDGMENTS
|
---|
The authors gratefully acknowledge R. N. Eisenman, Ph.D.,
Fred Hutchinson Cancer Research Center (Seattle, WA) for pSP-Max and
pSP-Mad plasmids, Shinobu Yamaguchi for her expert technical
assistance, and Nakanobu Hayashi, M.D., Omgen Inc., for allowing us to
use ABI 7700 TaqMan Sequence Detector and for help in quantitation of
Max and c-Myc mRNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Masayoshi Shichiri, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, 15-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
This work was supported in part by the Ministry of Education, Science
and Culture, Japan, and by the Ministry of Health and Welfare,
Japan.
Received for publication December 14, 1998.
Revision received April 28, 1999.
Accepted for publication May 3, 1999.
 |
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