© 1997 American Heart Association, Inc.
Articles |
Rapamycin Inhibits 
1-Adrenergic Receptor–Stimulated Cardiac Myocyte Hypertrophy but Not Activation of Hypertrophy-Associated Genes
Evidence for Involvement of p70 S6 Kinase
From the Laboratory of Cardiovascular Science (M.O.B., J-S.Z., X.L., L.O., E.G.L., M.T.C.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md; Département de Biologie Appliquée (A.Y.), Institut Universitaire de Technologie de Clermont-Ferrand, Aubière, France; and the Division of Cardiology (H.S.), Johns Hopkins Hospital, Baltimore, Md.
Correspondence to Marvin O. Boluyt, PhD, Laboratory of Cardiovascular Science, National Institute on Aging, Gerontology Research Center, 4940 Eastern Ave, Baltimore, MD 21224. E-mail marvinb{at}vax.grc.nia.nih.gov
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Abstract The 70-kD S6 kinase (p70S6K) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II–stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70S6K plays a role in cardiomyocyte hypertrophy induced by the
1-adrenergic receptor (1-AR) agonist
phenylephrine (PE). PE stimulated the activity of
p70S6K >3-fold, and this increase was blocked by
rapamycin, an immunosuppressant macrolide that selectively inhibits
p70S6K. When administered for 3 days, PE stimulated a 30%
increase in total protein content, a 2-fold increase in the
incorporation of [14C]phenylalanine (14C-Phe)
into protein, and a 50% increase in two-dimensional myocyte area.
Rapamycin pretreatment (
500 pg/mL) significantly inhibited each of
these PE-stimulated changes. Two days of PE treatment resulted in a
1.6-fold increase in total RNA yield per dish, a 2-fold increase in
incorporation of [14C]uridine into myocyte RNA, and
increases in relative mRNA levels of the
hypertrophy-associated atrial natriuretic
factor (ANF, 2.1-fold) and skeletal -actin (SK, 2.2-fold) genes.
Although rapamycin abolished the PE-stimulated increases in total RNA
and incorporation of [14C]uridine, it had no effect on
the induction of the ANF and SK genes. LY294002, a specific
inhibitor of phosphatidylinositol 3-kinase (PI3-K)
activity, inhibited PE-stimulated increases in p70S6K
activity and the incorporation of labeled precursors into myocyte
protein and RNA. These results demonstrate that p70S6K is
activated by the hypertrophic agent PE and that a PI3-K or
PI3-K–like activity is required for p70S6K activation and
myocyte hypertrophy. The data suggest that
p70S6K activation may be required for PE-stimulated
hypertrophy of cardiac myocytes. Our results demonstrate
that intracellular signaling pathways responsible for transcriptional
and translational responses diverge early after 1-AR
stimulation in cardiac myocytes.
Key Words: ribosomal S6-kinase • 1-adrenergic receptor • immunosuppressant drug • rapamycin • phosphatidylinositol-3 kinase
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Introduction |
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Cardiac myocyte hypertrophy requires increased synthesis of cellular protein and is associated with increased capacity for and efficiency of protein synthesis.1 The mechanisms governing these events, however, are not well understood and have been the subject of considerable attention in recent years. Cell culture models of neonatal cardiac myocyte hypertrophy have proven particularly useful in elucidating intracellular signaling events involved in hypertrophic growth. Stimulation of
1-adrenergic receptors by PE results in an elevated rate
of protein synthesis, altered gene expression, and myocyte
enlargement.2 3 4 Intracellular signal transduction pathways
activated by PE, other soluble agents,2 3 4 or
stretch5 culminate in nuclear regulation of
transcriptional events and activation of the protein synthetic
machinery in the cytoplasm. Although numerous studies have investigated
mechanisms regulating the transcription of specific genes, less has
been learned regarding the regulation of protein synthesis during
cardiac hypertrophy. Recent work in other cell types has suggested that phosphorylation of the ribosomal S6 protein regulates the translation of mRNAs into proteins.6 Ribosomal protein S6 is uniquely positioned to regulate the protein synthetic machinery from its location at the tRNA-mRNA binding site of the 40S ribosome.7 It appears to play a role in the activation of protein synthesis and to regulate translation.6 Ribosomal S6 protein is phosphorylated on a number of residues by a family of 70- and 85-kD protein kinases, termed p70S6K.6 8 Phosphorylation of these residues results in a modest increase in the rate of serum-stimulated protein synthesis and a selective increase in the translation of mRNAs containing a polypyrimidine tract at the 5' terminus.6
Rapamycin is an immunosuppressive macrolide that inhibits p70S6K activity in fibroblast cells and inhibits the rate at which fibroblast cells and T lymphocytes enter S phase.8 9 The effects of rapamycin are mediated through its binding to FKBP, an intracellular immunophilin known to bind FK506.10 11 12 Inhibition of p70S6K activity by rapamycin in fibroblasts is accompanied by a modest inhibition of protein synthesis and a selective repression of the translation of "polypyrimidine tract" mRNAs that encode elongation factors and ribosomal proteins.6 13 14 Selective regulation of this class of mRNAs may be particularly relevant in cardiac myocytes, because an important early event in the hypertrophic growth of the heart is an accumulation of ribosomal subunits.15 16
Recently, it was demonstrated that rapamycin inhibits Ang II–mediated
hypertrophy of cardiac myocytes.17 Moreover,
it was demonstrated that rapamycin selectively inhibits
hypertrophy but not hypertrophy-associated gene
expression or sarcomere assembly.17 These findings
indicate that distinct pathways regulate Ang II–stimulated changes in
translation and transcription and suggest that p70S6K plays
an important role in regulating Ang II–mediated cardiac myocyte
hypertrophy. It is not known whether p70S6K
plays a role in cardiac myocyte hypertrophy mediated by
other stimuli, such as the 1-adrenergic receptor
antagonist PE. p70S6K is regulated by an
intracellular signal transduction pathway that is distinct from other
well-delineated pathways. For example, rapamycin does not inhibit the
mitogen-activated protein kinase cascade or PKC, the activation
of which has been implicated in the changes in
hypertrophy-associated gene expression.8 17
Upstream members of the p70S6K signaling pathway are not
well defined, but there is evidence that a PI3-K is an essential link,
at least in some instances.18 19 Whether PI3-Ks are
involved in the hypertrophic growth of cardiac myocytes has not been
examined.
In the present study, we investigated the role of
p70S6K in PE-stimulated hypertrophy of cultured
neonatal cardiac myocytes. Our purpose was to determine whether PE
would activate p70S6K and whether inhibition of
p70S6K activation by rapamycin would inhibit
1-AR–mediated cardiomyocyte
hypertrophy. Furthermore, we examined the effect of
rapamycin on the accumulation of total RNA and on the expression of
specific hypertrophy-associated genes. Finally, we sought
to determine whether p70S6K and hypertrophic growth were
sensitive to inhibitors of PI3-K in cardiac myocytes.
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Materials and Methods |
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Materials
Rapamycin was a gift from Dr Suren Sehgal, Wyeth-Ayerst Research, Princeton, NJ. FK506 was a gift from Fujisawa Pharmaceutical Co, Deerfield, Ill. Rapamycin and FK506 were initially dissolved in ethanol and subsequently diluted in culture medium befoere addition. PE was obtained from Sigma Chemical Co and dissolved in ascorbic acid, such that the final concentrations in the cultures were 100 µmol/L PE and 100 µmol/L ascorbic acid. The synthetic peptide RRRLSSLRA and PKA inhibitor were obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti-p70S6K antisera (C-2 and C-18) were obtained from John Blenis, Harvard Medical School and from Santa Cruz Biotechnology, respectively. Protein A conjugated to Sepharose beads was obtained from Sigma, and wortmannin was also obtained from Sigma. LY294002 was purchased from BIOMOL Research Laboratories, Inc.
Cell Culture
Neonatal ventricular myocytes were cultured as
previously described.20 Heart ventricles from 1- to
3-day-old Wistar rats were removed, separated from the atria,
trisected, and then digested with collagenase type II (0.5
mg/mL, Worthington Biochemical) and pancreatin (0.6
mg/mL, Sigma) for 20 minutes at 37°C. The cell supernatant was
collected by centrifugation, and the pellet was
resuspended in horse serum. The above steps were repeated 7 to 10 times
until the hearts were completely digested. The cells from all
digestions were combined, washed, and then subjected to
centrifugation through a discontinuous Percoll gradient
of 1.050, 1.062, and 1.082 g/mL, respectively. The band at the
1.062/1.082 interface was collected and used as the sole source of
purified myocytes. These myocytes were resuspended in culture medium
consisting of a 4:1 (vol/vol) mixture of DMEM containing high
glucose and medium 199 (GIBCO Laboratories) supplemented with 10%
preselected horse serum, 5% heat-inactivated fetal bovine
serum, penicillin (100 U/mL), and streptomycin (100 mg/mL, GIBCO
Laboratories) and then plated on gelatin-coated 100-mm plastic tissue
culture plates or six-well culture plates at a density of
1.08x105 cells/cm2. Plating efficiency was
such that cell-cell contact was minimal at the time the cells were
harvested for various biochemical analyses. Myocyte cultures
were maintained in serum-containing media at 37°C for 24 hours in
humidified air with 5% carbon dioxide. The medium was then changed to
serum-free DMEM/medium 199. All experiments were initiated 24 hours
after the change to serum-free conditions and 48 hours after plating.
The purity of the cultures was determined at this time by
immunocytochemistry with antibodies to sarcomeric myosin (monoclonal
antibody F59) or sarcomeric actin (Sigma).20 Only cultures
consisting of >90% myocytes as determined 48 hours after plating were
used for experiments reported here.
Immunoprecipitation and Assay of p70S6K
Activity
Procedures were essentially as described by
others,8 17 with minor modifications. Extracts were
obtained from four or five 100-mm dishes of sparsely plated cultures of
neonatal cardiac myocytes. Cells were stimulated with vehicle or PE for
20 minutes, followed by aspiration of the media, two ice-cold rinses
with PBS, and addition of 200 to 250 µL of ice-cold lysis buffer.
Lysis buffer consisted of (mmol/L) KPO4 10 (pH 7.4),
EDTA 1, EGTA 5, MgCl2 10, ß-glycerophosphate 50, sodium
orthovanadate 1, and dithiothreitol 2, along with 10 nmol/L
okadaic acid, 10 µg/mL leupeptin, and 10 µg/mL
aprotinin. The lysate was incubated on ice for 20 minutes, followed by
centrifugation in a microfuge for 20 minutes at 4°C.
Lysates containing 400 µg of protein were diluted with 10 vol of RIPA
buffer (10 mmol/L Tris, pH 7.2, 150 mmol/L
NaCl, 1% deoxycholic acid, 1% Triton X-100, and 0.1% SDS) and were
incubated with 4 µL of rabbit polyclonal anti-p70S6K
antibody on an oscillating platform for 2 hours at 4°C. Protein
A–Sepharose (160 µL) was added, and the incubation was continued for
an additional 30 minutes. After centrifugation,
immunoprecipitates were washed three times with lysis buffer and then
rid of any residual supernatant.
To the remaining immune complex (
80 µL) the following were added:
50 µL of 2x kinase assay buffer (mmol/L: MOPS 50, pH 7.2,
ß-glycerophosphate 120, p-nitrophenylphosphate 60, EGTA
10, MgCl2 30, dithiothreitol 2, and sodium
orthovanadate 2, along with 500 nmol/L PKA
inhibitor) and 25 µL of ATP solution (100
µmol/L ATP and 20 µCi [
-32P]ATP). The
reaction was started by the addition of 5 µL of RRRLSSLRA (5
mg/mL) with mixing and incubated at 30°C. At 10, 20, and 30
minutes, aliquots of 40 µL were removed into microtubes containing 8
µL of 12% TCA. After centrifugation, the supernatant
was spotted onto Whatman P-81 phosphocellulose paper and washed five
times (2 minutes each) with 180 mmol/L phosphoric acid and
once with 95% ethanol. Radioactivity was quantified by scintillation
counting in Bio-Safe II scintillation cocktail.
Immunoblotting
Myocyte lysates prepared as described above were diluted with
lysis buffer to achieve the desired protein concentration and then
mixed 1:1 with 2x sample buffer (125 mmol/L Tris, pH 6.8,
4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.001% bromphenol
blue). Protein (25 to 40 µg) was loaded into each lane of a 7.5%
SDS-polyacrylamide gel and size-fractionated by electrophoresis
at a constant current of 20 mA for 16 to 20 hours. Proteins were then
electrophoretically transferred to PVDF membranes at 5 V/cm for 16 to
20 hours at 4°C. After incubation in blocking solution (PBS-T and 1%
bovine serum albumin) for 2 hours, membranes were washed three
times in Western buffer (50 mmol/L NaCl, 10
mmol/L Tris, pH 7.0, 1 mmol/L EDTA, and 0.1%
Tween-20) before incubation with the primary antibody (rabbit
anti-p70S6K and C-18) for 16 to 20 hours, followed by three
more washes and incubation for 1 hour with a 1:10 000 dilution of the
secondary antibody (goat anti-rabbit IgG conjugated with horseradish
peroxidase). Antibody binding was detected using the enhanced
chemiluminescence method according to the manufacturer's instructions
(Amersham Corp).
Measurements of Protein-to-DNA Ratio, 14C-Phe
Incorporation, and Cell Size
After cell isolation, the myocytes were cultured in
serum-containing media and plated in six-well tissue culture plates at
a density of 1x104 cells/cm2 for
protein-to-DNA ratio experiments and 2.5x104
cells/cm2 for labeling experiments. To inhibit fibroblast
growth, 10-4 mol/L bromodeoxyuridine
(Sigma) was added to the cultures at the time of plating and was
maintained in the culture medium throughout the experiment. After 1 day
in serum-containing medium, the cells were cultured in serum-free
medium (DMEM/medium 199 containing antibiotics) supplemented with
1 µmol/L insulin, 5 µmol/L transferrin, and
10 nmol/L selenium. After 1 day in serum-free medium, cells were
treated with experimental agents and harvested 72 hours later.
Protein-to-DNA ratios were determined by a modification of the
procedure described by McDermott et al.21 After two
rinses with PBS, cells were detached by scraping in 100 µL of 1x
standard sodium citrate containing 0.25% SDS and frozen at -30°C.
After they were thawed, the contents were vortexed extensively. Total
cell protein and DNA content were determined by the Nano-Orange and
PicoGreen reagents (Molecular Probes, Inc), respectively. Protein and
DNA assays were conducted in 96-well plates according to the
manufacturers' instructions. Quantification of signals was acquired
using a STORM imager (Molecular Dynamics) in the
chemifluorescence/blue fluorescence mode. Bovine serum
albumin and calf thymus DNA were used as standards for protein
and DNA determinations, respectively.
The incorporation of labeled phenylalanine into myocyte protein was assessed as described previously.22 After 1 day in serum-free medium, 14C-Phe (0.1 µCi/mL) was added to the cultures along with various drug treatments. Cells were then analyzed for changes in cell size and 14C-Phe incorporation into cellular protein 3 days later. For cell size measurements, two or three fields were randomly chosen and photographed at high power (x400), and 75 individual cell areas were measured by planimetry. For 14C-Phe incorporation into cellular protein, the amount of radiolabel incorporated into TCA-insoluble material was determined as described previously.22 An aliquot of SDS solution obtained from each well was used for measurement of DNA concentration using the PicoGreen reagent as described by the manufacturer (Molecular Probes, Inc).
Measurements of [14C]Uridine Incorporation and
RNA Yield
[14C]Uridine incorporation was measured
essentially as described above for 14C-Phe, except that
cells were harvested 48 hours after the addition of 0.2 µCi/mL
[14C]uridine (NEC 598; specific activity, 521 mCi/mmol;
New England Nuclear) and the various drug treatments. Results were then
normalized to total DNA content as described above.
RNA Blotting
Total RNA was isolated from cultured ventricular
myocytes using the guanidinium isothiocyanate method23 as
described in detail previously.22 After denaturation in
formamide and formaldehyde, equal amounts of total RNA (10 µg per
lane) were size-fractionated by electrophoresis through 1% agarose
gels containing 3% formaldehyde. The fractionated RNA was
electrophoretically transferred to nylon membranes (Duralon, Stratagene
Cloning Systems) at 5 V/cm, cross-linked by ultraviolet radiation (120
mJ), and then hybridized at 63.5°C as described by Church and
Gilbert24 with 32P-labeled
oligonucleotide and cDNA probes. Probes for the
translated region of rat ANF and the 3' untranslated region of rat
skeletal -actin were prepared as described
previously.25 The probe for 18S rRNA was a synthetic
oligonucleotide previously described.26
Complementary DNA probes were radiolabeled using the random priming
method.27 28 Oligonucleotides were
radiolabeled by terminal deoxynucleotide transferase with
[-32P]dATP.29
Statistics
Data are expressed as mean±SE. For the p70S6K
activity assay, an independent-samples t test was used to
compare the slopes of the phosphorylation rate
regressions for PE versus control cultures.30 Comparisons
between treatments and control for Northern blot analyses were
made with a one-sample t test.30 Comparisons
among treatments were made with an independent-samples t
test.30 P values were corrected by the
Bonferroni method.30 For the protein-to-DNA ratio, for
cell size measurements, for 14C-Phe or
[14C]uridine incorporation, and for RNA yield values, a
two-way ANOVA and Tukey's procedure were used.30 A value
of P<.05 was considered significant.
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Results |
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1-AR Stimulation Activates
p70S6K ActivityTo determine whether PE activates p70S6K in myocytes, cultures of sparsely plated neonatal cardiac myocytes were harvested after treatment with PE (100 µmol/L) or vehicle (ascorbic acid) for 20 minutes, 24 hours, or 3 days. After immunoprecipitation of cell lysates with an antibody directed against p70S6K (C-18), incorporation of radiolabeled phosphate into a synthetic substrate (RRRLSSLRA) driven by the immune complex was measured serially at 10, 20, and 30 minutes in the assay tubes. For cells harvested after 20 minutes of stimulation, the rate of phosphate incorporation into substrate was >3-fold greater for PE-treated myocyte extracts than for those of vehicle-treated control cultures (Fig 1A
). The rate of
phosphorylation measured after incubating the antibody
with a 10-fold excess of peptide antigen before immunoprecipitation was
essentially zero (data not shown). Activation of p70S6K was
sustained for at least 72 hours and was inhibited by rapamycin. The
inhibitory action of rapamycin was reversed by FK506, a
related macrolide that competitively displaces rapamycin from its FKBP
binding site (Fig 1B).
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Immunoblotting of whole-cell lysates with an antibody
directed against p70S6K (C-2) detected a band at 70 kD
and up to four electrophoretically retarded bands that have been shown
by others to be due to sequential phosphorylation of
serine and threonine residues (Fig 1C
).18 31 The
specificity of the antibody used for immunoblotting was
verified by preincubation of the antibody with excess peptide antigen,
which eliminated the signal (Fig 1C). Stimulation of myocyte cultures
with PE resulted in increased prominence of the electrophoretically
retarded bands, consistent with phosphorylation
and activation of p70S6K. Rapamycin pretreatment of
vehicle- and PE-treated cultures eliminated the appearance of the
retarded bands, consistent with
dephosphorylation and inactivation of
p70S6K. When a 500-fold excess of FK506 was added before
rapamycin and PE, it abolished the inhibitory effect of
rapamycin, restoring the ability of PE to induce the appearance of
electrophoretically retarded bands. Another set of bands at 85 kD
exhibited a similar pattern of mobility shifts in response to PE,
rapamycin, and FK506. These bands correspond to the 85-kD S6 kinase
that is a second product of the p70S6K gene, identical
in sequence to p70S6K but containing a 23–amino acid
nuclear targeting N-terminal extension.32 33 In aggregate,
these data indicate that both 70- and 85-kD products of the
p70S6K gene are expressed in cardiac myocytes and that
p70S6K is subject to activation by 1-AR
stimulation through a rapamycin-sensitive pathway.
Rapamycin Inhibits PE-Stimulated Cardiac Myocyte
Hypertrophy
To determine whether rapamycin inhibits PE-stimulated cardiac
myocyte hypertrophy, three independent assessments of
myocyte growth were made. Myocytes were treated with vehicle or PE in
the presence or absence of rapamycin for 3 days. As
we20 22 and others2 3 4 have previously shown,
PE treatment induced myocyte hypertrophy, as evidenced by
an increase in total protein content relative to DNA (Fig 2). Rapamycin exerted a significant
independent effect on the protein-to-DNA ratio, as well as a
significant interactive effect on PE-treated myocytes, indicating that
rapamycin inhibits both basal and PE-stimulated myocyte
hypertrophy.
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DNA content was also significantly affected by rapamycin treatment (0.518±0.065 and 0.527±0.069 µg/well for rapamycin and rapamycin+PE–treated cultures, respectively, versus 0.652±0.085 and 0.617±0.082 µg/well for vehicle- and PE-treated myocytes, respectively; P=.031, two-way ANOVA). We considered three possible explanations for this effect of rapamycin, including proliferation of nonmyocytes, detachment of myocytes, and binucleation of myocytes. Myocyte purity as assessed by staining with myocyte-specific antibodies averaged 96±1% (n=13) at the initiation of treatment (2 days after plating). After the 3-day experimental period (5 days after plating), the percentage of myocytes in vehicle-treated cultures was 94±1% (n=13). Thus, if rapamycin inhibits this small degree of nonmyocyte proliferation, it may contribute to the lower DNA content in rapamycin-treated cultures. To assess the possibility of myocyte detachment, myocyte number was estimated by counting cells in a total of one to five randomly selected high-power fields per well from each of 15 separate preparations of myocytes. The average numbers of myocytes per field were as follows (mean±SE): vehicle, 24±3; rapamycin, 21±3; PE, 24±2; and rapamycin/PE, 29±3. There were no significant differences among the groups (two-way ANOVA), indicating that myocyte detachment could not account for the lower DNA values in rapamycin-treated cultures. In two other experiments, the percentage of binucleated myocytes was estimated by counting the number of mononucleated and binucleated myocytes in 10 randomly chosen high-power fields visualized by phase-contrast microsopy. At the initiation of treatment (2 days after plating), the number of binucleated myocytes was 3.7% of the total. After 72 hours of treatment, the percentages of binucleated myocytes were 8.1%, 4.1%, 10.6%, and 6.2% for vehicle, rapamycin, PE, and rapamycin/PE treatments, respectively. Thus, the most likely explanation for the slightly lower DNA content in rapamycin-treated cultures at 72 hours is a combination of small contributions from rapamycin-mediated inhibition of nonmyocyte proliferation and myocyte binucleation.
As an independent measure of the effects of rapamycin on myocyte
growth, incorporation of labeled precursor into myocyte protein was
determined. The progressive incorporation of 14C-Phe in
myocytes resulted in a 2-fold-greater accumulation of incorporated
label over the 3-day period relative to vehicle-treated controls (Fig 3). There were no significant differences
in incorporation of label among the other three groups. DNA content in
this set of labeling experiments was similar among the groups and did
not change significantly over the 3-day treatment period (Table 1). It should be noted that this labeling
approach is used as an estimate of myocyte growth and cannot be used to
accurately assess rates of protein synthesis. In a separate set of
labeling experiments in which raw counts (not normalized to DNA) were
used as a measure of growth, rapamycin completely inhibited the
PE-stimulated growth at doses of 500 pg/mL, with the
half-maximal effect observed between 5 and 50 pg/mL (not shown).
This estimated half-maximal dose is similar to that observed for the
inhibition of T-cell proliferation and the inhibition of S6
phosphorylation in 3T3 cells.8 34
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As a third independent assessment of cellular hypertrophy,
two-dimensional cell area was quantified in cultures of myocytes
treated with vehicle, PE, rapamycin, or a combination of the two drugs.
Rapamycin (500 pg/mL) had no effect on the size of control
myocytes but completely inhibited the PE-induced increase in
two-dimensional cell area (Fig 4, Table 2). To demonstrate the specificity of the
rapamycin-FKBP interaction in the inhibition of myocyte protein
accumulation, cultures were pretreated with various doses of FK506
before the addition of rapamycin and PE. Since FK506 and rapamycin both
bind FKBP and are mutually antagonistic, it would be
predicted that a high molar excess of FK506 would competitively reverse
the rapamycin effect.34 35 The inhibition of the
PE-stimulated increase in myocyte size by rapamycin was competitively
reversed by coadministration of FK506. The reversal by FK506 indicates
that the rapamycin effect was mediated by a rapamycin-FKBP complex and
not through a nonspecific effect of rapamycin.
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Rapamycin Inhibits PE-Induced Increases in RNA
Accumulation
Since an increase in ribosomal RNA content,15
reflected in elevated levels of total RNA,16 22 is a
common feature of cardiac hypertrophy in vivo and in
culture, we examined the levels of total RNA 72 hours after treatment
with PE with and without rapamycin. The p70S6K
inhibitor rapamycin significantly reduced the increase in
accumulation of total RNA by PE (Fig 5A).
To further investigate this phenomenon, myocyte cultures were treated
with vehicle, PE, rapamycin, or rapamycin+PE in the presence of
[14C]uridine for 48 hours. PE-treated cultures exhibited
a 2-fold increase in the incorporation of labeled uridine into cellular
RNA (Fig 5B). Rapamycin effectively inhibited the PE-stimulated
increase in incorporation of label into cellular RNA. Since the vast
majority of RNA in the cell is ribosomal (>95%), it seems evident
from these data that the PE-stimulated increase in accumulation of rRNA
is abrogated by rapamycin treatment.
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RAP indicates significant main effect for RAP (two-way ANOVA). B,
Incorporation of [14C]uridine into myocyte RNA. Cultures
were stimulated with 100 µmol/L PE, 500 pg/mL RAP, 250 ng/mL
FK506, or combinations thereof for 48 hours in the presence of 0.2
µCi/mL [14C]uridine. Values are mean±SE for duplicate
determination on four independent preparations of myocytes.
*P<.05 vs VEH;
Rapamycin Does Not Inhibit Expression of
Hypertrophy-Associated Genes
To determine whether inhibition of hypertrophy by
rapamycin also results in repression of PE-stimulated increases in
expression of hypertrophy-associated genes, total RNA was
harvested from myocyte cultures treated for 48 hours with 100
µmol/L PE, 5 ng/mL rapamycin, or a combination of
rapamycin and PE. Rapamycin had no significant effect on PE-stimulated
expression of either ANF or skeletal -actin (Fig 6). This was true when ANF and skeletal
-actin signals were normalized to levels of either 18S ribosomal RNA
(Fig 6) or to glyceraldehyde-3-phosphate dehydrogenase
mRNA (data not shown). The effectiveness of the rapamycin aliquots used
in Northern blotting experiments to dephosphorylate
p70S6K was verified by its effectiveness in inhibiting the
electrophoretic mobility changes of p70S6K on
immunoblots (not shown).
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Potential Role of PI3-Ks in 1-AR–Stimulated
Hypertrophy
Recent studies have implicated a PI3-K as an upstream
effector of p70S6K in some cell types.18 To
investigate the potential involvement of PI3-K in the PE-stimulated
activation of p70S6K, two inhibitors of PI3-Ks,
wortmannin and LY294002, were used. Both agents inhibited PE-mediated
retardation of the electrophoretic mobility of p70S6K when
cultures were harvested after 20 minutes of treatment (Fig 7A and 7B). Wortmannin also inhibited activity
of p70S6K in the immune complex assay when cells were
harvested at 20 minutes and 24 hours (data not shown) but required a
higher dose in our myocyte cultures than was reported for its specific
action on PI3-K. Wortmannin partially inhibited incorporation of
14C-Phe into myocyte protein (n=3 experiments), but results
were variable, probably because of the lack of stability of this
compound.36 LY294002, on the other hand, inhibited
p70S6K activity completely at 10 µmol/L (Fig 7C), in keeping with its reported IC50 of 1.4
µmol/L for inhibition of PI3-K activity in
vitro.37 We used LY294002 for longer-term experiments on
14C-Phe incorporation because of its efficacy in myocytes
and its superior stability.37 To determine whether
LY294002 would inhibit the accumulation of protein and RNA, experiments
were conducted in the presence of radiolabeled protein and RNA
precursors for 72 hours. LY294002 inhibited the incorporation of
14C-Phe into myocyte protein in a dose-dependent manner
(Fig 8A and 8B). Similarly, LY294002
inhibited the PE-stimulated increase in [14C]uridine
incorporation into cellular RNA in a dose-dependent manner (Fig 8C and 8D). Although LY294002 alone had no significant effect on the basal
incorporation of labeled precursors into cellular RNA or protein, there
appears to be a trend for it to do so, particularly for RNA (Fig 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiac hypertrophy is an adaptation to increased hemodynamic workload that involves autocrine, paracrine, and endocrine signals.1 2 3 4 5 38 39 40 At the cellular level, cardiac hypertrophy is manifested by the enlargement of cardiac myocytes and an accumulation of contractile and noncontractile proteins resulting from a positive protein synthesis-to-degradation ratio.41 42 43 44 Protein synthesis is a tightly regulated process involving translation of available mRNAs into nascent protein. In most cases, translation initiation is the rate-limiting step.45 Activation of p70S6K has been implicated in the regulation of translation in conjunction with phosphorylation of the ribosomal S6 protein6 and in the regulation of Ang II–stimulated hypertrophy of cardiac myocytes and vascular smooth muscle cells.17 46 47
The present results demonstrate that stimulation of
1-ARs by PE leads to a rapid and sustained elevation in
p70S6K activity (Fig 1). Rapamycin, an immunosuppressant
macrolide that selectively blocks activation of p70S6K (Fig 1B and 1C), markedly inhibited hypertrophic growth in cardiac myocytes
induced by PE. The inhibitory effects of rapamycin were
noted on three independent indexes of hypertrophy,
including total protein content, incorporation of 14C-Phe
into myocyte protein, and two-dimensional myocyte size (Figs 2 through 4, Table 2). The significant interaction between rapamycin and PE for
each of the three hypertrophic indexes demonstrates that rapamycin does
not simply reduce basal myocyte growth but specifically inhibits
PE-stimulated growth processes. It can be concluded that these effects
were not due to toxic or nonspecific effects of rapamycin because the
results of protein content and labeling experiments were normalized to
DNA content (Figs 2, 3, and 5) and because the effects of rapamycin
were reversed by a high molar excess of FK506 (Figs 1 and 4, Table 2),
a macrolide related to rapamycin that competitively displaces rapamycin
from its FKBP binding site.48 Thus, the present
results indicate that a rapamycin-sensitive pathway is required for
1-AR–stimulated cardiomyocyte
hypertrophy and suggest that inhibition of
p70S6K activity may account, at least in part, for the
rapamycin-mediated inhibition of PE-stimulated
hypertrophy.
The role of rapamycin-sensitive pathways has been studied most
extensively in proliferating cell lines, where the selective inhibition
of these kinases by rapamycin prevents or delays the entry of these
cells into S phase after stimulation by mitogens.6 8 9 34
Although rapamycin leads to growth arrest of these cells, its direct
effects on incorporation of label are rather modest (
20% inhibition
by 20 ng/mL rapamycin).6 We report in the
present study that rapamycin completely inhibits the increases in
total protein, 14C-Phe incorporation, and myocyte size that
are otherwise stimulated by PE. Similarly, rapamycin has been shown to
completely inhibit the increase in protein synthesis induced by Ang
II.17 These results suggest that rapamycin-sensitive
signaling plays a larger role in protein accumulation during
hypertrophic compared with hyperplastic growth and that the precise
role of p70S6K in growth processes is cell-type
dependent.
One way that rapamycin might exert its regulatory influence in
hypertrophying myocytes is to participate in the
accelerated synthesis of ribosomes, an early event in cardiac
hypertrophy.1 15 This is plausible because
many ribosomal proteins harbor polypyrimidine tracts at their extreme
5' end,13 rendering them susceptible to selective
translational repression by rapamycin.6 Terada et
al14 have proposed that the less pronounced effect of
rapamycin on protein synthesis in cycling cells is due to the fact that
cycling cells have more ribosomal "machinery" on hand and that,
therefore, de novo synthesis of translational apparatus is
not as crucial as it is in quiescent cells. Consistent with
this notion are data in the present study showing that both
accumulation of total RNA and incorporation of labeled uridine into RNA
are inhibited by rapamycin (Fig 5) in cardiac myocytes.
Whether these effects are mediated directly via regulation of S6 phosphorylation by p70S6K is not clear, but it seems likely that they may involve other pathways that regulate translational control via phosphorylation of a protein termed PHAS-I (also referred to as 4E-BP1),49 since phosphorylation of PHAS-I (which disinhibits translation by releasing the translation initiation factor eIf-4E) by insulin is rapamycin sensitive.50 51 52 53 54 This would place PHAS-I downstream from rapamycin-FKBP, but whether PHAS-I is regulated by p70S6K or whether it represents a post–rapamycin-FKBP, p70S6K-independent bifurcation of the signaling pathway remains to be determined.
Rapamycin-FKBP is believed to act via its interaction with a family of proteins first identified in yeast and termed TOR. In yeast, rapamycin-FKBP complexes mediate their effects by interaction with two intracellular TOR proteins, TOR1 and TOR2. TOR2 mutations are lethal in yeast, since TOR2 is required for G1 progression.55 Both TOR1 and TOR2 display strong sequence homology with PI3-K. The targets of rapamycin-FKBP complexes in mammalian cells (variously termed FRAP, RAFT1, and mTOR) show significant homology with TORs, particularly in the region encoding the putative PI3-K catalytic domain.56 57 58 Brown et al59 have recently provided convincing evidence that FRAP regulates p70S6K activity in Tag Jurkat and Sf9 cells by a mechanism involving a kinase activity. FRAP is widely expressed in mammalian tissues, including the heart.56 We have observed that FRAP mRNA is expressed in cultured neonatal cardiac myocytes (authors' unpublished data, 1996). The fact that FRAP is expressed in myocytes, coupled with the data demonstrating the effects of rapamycin and FK506 on p70S6K activity in myocytes, suggests that FRAP regulates p70S6K in myocytes as it does in other cell types. The molecular link(s) between FRAP and p70S6K remains unidentified.60
Data derived from both in vitro and in vivo models of
hypertrophy have demonstrated that fetal/neonatal
genes, such as ANF and skeletal -actin, are reexpressed in response
to a hypertrophic stimulus.38 39 40 Because of the strong
association between cardiac myocyte hypertrophy and
induction of fetal/neonatal genes, expression of some of these
genes has often been used as a marker of hypertrophy. It
was recently shown that rapamycin is capable of dissociating
fetal/neonatal gene expression from Ang II–stimulated growth of
myocytes.17 In the present study, inhibition of
p70S6K activation by rapamycin also failed to block the
PE-stimulated induction of the hypertrophy-associated genes
ANF and skeletal -actin, while effectively inhibiting protein
accumulation. These findings indicate that the signaling pathway(s)
leading from the 1-adrenergic receptor to the
transcriptional (changes in gene expression) and translational (changes
in protein accumulation and cell size) effectors diverges at an early
postreceptor stage. Furthermore, these data show that
p70S6K and other putative rapamycin-sensitive translational
control mechanisms are downstream from the point at which the signaling
pathways separate in myocytes. Together with several other
studies,17 20 22 61 these data demonstrate that the
induction of hypertrophy-associated genes is not sufficient
for hypertrophic growth of myocytes. Although induction of the
hypertrophy-associated genes reported in the present
study and elsewhere17 has been shown to be independent of
p70S6K activation, it is possible that some genes relevant
to hypertrophy may be dependent on p70S6K
activation. It has been shown, for example, that the transcription
factor cAMP-responsive element modulator (CREM) is
phosphorylated and activated by
p70S6K, although endogenous target genes
directly related to this transcription factor have not been
identified.62
The intracellular signaling pathway by which hypertrophic stimuli
activate p70S6K in myocytes is not well delineated.
Work in other cell types has implicated a ras-independent
pathway involving activation of a wortmannin- and LY294002-sensitive
PI3-K–like protein.18 In addition to the TOR homologues
that contain a PI3-K domain, a wortmannin-sensitive PI3-K that is
linked to G proteins via the ß subunit has also been
identified.63 This protein does not require association
with receptor tyrosine kinases for activation, a feature that is
particularly relevant to the mechanism of S6 kinase activation in
proliferating fibroblasts by PDGF, since mutation of the PI3-K binding
sites on the PDGF receptor has demonstrated that this pathway is not
required for p70S6K activation.64 Which, if
any, of these candidate PI3-Ks or PI3-kinase–like molecules is
involved in the regulation of protein accumulation in
cardiomyocytes undergoing hypertrophy is not
known.
Because S6 kinases can be activated by phorbol esters via a
pathway that is inhibited by rapamycin, but not by
wortmannin,19 and because PKC is activated by
1-AR stimulation,4 65 it was a necessary
first step to determine whether the activation of p70S6K
and the increase in protein synthesis in cardiomyocytes by
PE are, in fact, sensitive to inhibitors of PI3-K. In the
present study, we show that in cardiac myocytes both basal and
PE-stimulated p70S6K activation are inhibited by wortmannin
and LY294002. Furthermore, LY294002 inhibited the incorporation of
labeled precursors into cellular protein and RNA in a dose-dependent
manner. The fact that wortmannin and LY294002 were effective in
inhibiting PE-stimulated events suggests that a PI3-K or a PI3-K–like
molecule plays an essential role in the transduction of signals from
the 1-AR to p70S6K and other putative
mechanisms regulating protein and RNA accumulation during
hypertrophy of myocytes. Since some of the
phorbol-sensitive PKCs are activated by PE,65 the
present data point out a possible difference in signaling between
cardiac myocytes and lymphoid cells, where phorbol ester–mediated
activation of p70S6K is insensitive to
wortmannin.19 Because the dose-response curve for the
effect of LY294002 on PE-stimulated p70S6K activity (Fig 7C) differs somewhat from those for incorporation of labeled protein
and RNA precursors (Fig 8), an alternative explanation may be that this
drug inhibits multiple PI3-Ks with effects that both parallel and
converge on rapamycin-sensitive pathways. Although FRAP has not yet
been shown to exhibit PI3-K activity,66 it possesses a
striking homology to PI3-K,56 57 58 and its kinase domain is
required for autophosphorylation as well as its effects
on p70S6K activation.59 Thus, the possibility
that inhibitors of PI3-K may interact with this domain,
thereby disrupting the conveyance of upstream signals to
p70S6K, should not be overlooked.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This article is hereby dedicated to the memory of our esteemed colleague and friend, Dr Howard Silverman. His enthusiasm, intellect, resourcefulness, energy, and warmth will be sorely missed. The authors gratefully acknowledge Dr John Blenis for generously supplying the anti-p70/85S6K antibody, Fujisawa Pharmaceutical Co for the gift of FK506, and Dr Suren Sehgal of Wyeth-Ayerst for the gift of rapamycin. We also thank Jun-ichi Sadoshima for helpful discussions regarding the S6 kinase assay and Leslie Heckendorf for excellent technical support.
|
Footnotes |
|---|
1 Deceased.
Received September 18, 1996; accepted May 16, 1997.
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References |
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N. Frey, H. A. Katus, E. N. Olson, and J. A. Hill Hypertrophy of the Heart: A New Therapeutic Target? Circulation, April 6, 2004; 109(13): 1580 - 1589. [Abstract] [Full Text] [PDF]
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J. B. Morris, T. M. Pham, B. Kenney, K. E. Sheppard, and E. A. Woodcock UTP Transactivates Epidermal Growth Factor Receptors and Promotes Cardiomyocyte Hypertrophy Despite Inhibiting Transcription of the Hypertrophic Marker Gene, Atrial Natriuretic Peptide J. Biol. Chem., March 5, 2004; 279(10): 8740 - 8746. [Abstract] [Full Text] [PDF]
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M. O. Boluyt, A. M. Loyd, M. H. Roth, M. J. Randall, and E. Y. M. Song Activation of JNK in rat heart by exercise: effect of training Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2639 - H2647. [Abstract] [Full Text] [PDF]
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T. Shioi, J. R. McMullen, O. Tarnavski, K. Converso, M. C. Sherwood, W. J. Manning, and S. Izumo Rapamycin Attenuates Load-Induced Cardiac Hypertrophy in Mice Circulation, April 1, 2003; 107(12): 1664 - 1670. [Abstract] [Full Text] [PDF]
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T. Shioi, J. R. McMullen, P. M. Kang, P. S. Douglas, T. Obata, T. F. Franke, L. C. Cantley, and S. Izumo Akt/Protein Kinase B Promotes Organ Growth in Transgenic Mice Mol. Cell. Biol., April 15, 2002; 22(8): 2799 - 2809. [Abstract] [Full Text] [PDF]
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V. C. Tu, J. J. Bahl, and Q. M. Chen Signals of Oxidant-Induced Cardiomyocyte Hypertrophy: Key Activation of p70 S6 Kinase-1 and Phosphoinositide 3-Kinase J. Pharmacol. Exp. Ther., March 1, 2002; 300(3): 1101 - 1110. [Abstract] [Full Text] [PDF]
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W. Zhang Old and new tools to dissect calcineurin's role in pressure-overload cardiac hypertrophy Cardiovasc Res, February 1, 2002; 53(2): 294 - 303. [Abstract] [Full Text] [PDF]
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D. H. Korzick, D. A. Holiman, M. O. Boluyt, M. H. Laughlin, and E. G. Lakatta Diminished {alpha}1-adrenergic-mediated contraction and translocation of PKC in senescent rat heart Am J Physiol Heart Circ Physiol, August 1, 2001; 281(2): H581 - H589. [Abstract] [Full Text] [PDF]
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A. D. Everett, T. D. Stoops, A. C. Nairn, and D. Brautigan Angiotensin II regulates phosphorylation of translation elongation factor-2 in cardiac myocytes Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H161 - H167. [Abstract] [Full Text] [PDF]
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H. Kodama, K. Fukuda, J. Pan, M. Sano, T. Takahashi, T. Kato, S. Makino, T. Manabe, M. Murata, and S. Ogawa Significance of ERK cascade compared with JAK/STAT and PI3-K pathway in gp130-mediated cardiac hypertrophy Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1635 - H1644. [Abstract] [Full Text] [PDF]
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F. H. Pham, P. H. Sugden, and A. Clerk Regulation of Protein Kinase B and 4E-BP1 by Oxidative Stress in Cardiac Myocytes Circ. Res., June 23, 2000; 86(12): 1252 - 1258. [Abstract] [Full Text] [PDF]
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A. Sabri, G. Muske, H. Zhang, E. Pak, A. Darrow, P. Andrade-Gordon, and S. F. Steinberg Signaling Properties and Functions of Two Distinct Cardiomyocyte Protease-Activated Receptors Circ. Res., May 26, 2000; 86(10): 1054 - 1061. [Abstract] [Full Text] [PDF]
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M. O. Boluyt and O. H.L. Bing Matrix gene expression and decompensated heart failure: The aged SHR model Cardiovasc Res, May 1, 2000; 46(2): 239 - 249. [Abstract] [Full Text] [PDF]
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J. Liu, X. Shen, V.-A. Nguyen, G. Kunos, and B. Gao alpha 1 Adrenergic Agonist Induction of p21waf1/cip1 mRNA Stability in Transfected HepG2 Cells Correlates with the Increased Binding of an AU-rich Element Binding Factor J. Biol. Chem., April 14, 2000; 275(16): 11846 - 11851. [Abstract] [Full Text] [PDF]
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I. I. Rybkin, M. E. Cross, E. M. McReynolds, R. Z. Lin, and L. M. Ballou alpha 1A Adrenergic Receptor Induces Eukaryotic Initiation Factor 4E-binding Protein 1 Phosphorylation via a Ca2+-dependent Pathway Independent of Phosphatidylinositol 3-kinase/Akt J. Biol. Chem., February 25, 2000; 275(8): 5460 - 5465. [Abstract] [Full Text] [PDF]
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L. M. Ballou, M. E. Cross, S. Huang, E. M. McReynolds, B.-X. Zhang, and R. Z. Lin Differential Regulation of the Phosphatidylinositol 3-Kinase/Akt and p70 S6 Kinase Pathways by the alpha 1A-Adrenergic Receptor in Rat-1 Fibroblasts J. Biol. Chem., February 18, 2000; 275(7): 4803 - 4809. [Abstract] [Full Text] [PDF]
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V.-A. T. Nguyen and B. Gao Cross-talk between alpha 1B-Adrenergic Receptor (alpha 1BAR) and Interleukin-6 (IL-6) Signaling Pathways. ACTIVATION OF alpha 1BAR INHIBITS IL-6-ACTIVATED STAT3 IN HEPATIC CELLS BY A p42/44 MITOGEN-ACTIVATED PROTEIN KINASE-DEPENDENT MECHANISM J. Biol. Chem., December 10, 1999; 274(50): 35492 - 35498. [Abstract] [Full Text] [PDF]
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R. R. Baliga, D. R. Pimental, Y.-Y. Zhao, W. W. Simmons, M. A. Marchionni, D. B. Sawyer, and R. A. Kelly NRG-1-induced cardiomyocyte hypertrophy. Role of PI-3-kinase, p70S6K, and MEK-MAPK-RSK Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2026 - H2037. [Abstract] [Full Text] [PDF]
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G. Nikcevic, M. C. Heidkamp, M. Perhonen, and B. Russell Mechanical activity in heart regulates translation of alpha -myosin heavy chain mRNA but not its localization Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2013 - H2019. [Abstract] [Full Text] [PDF]
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M. Laser, V. S. Kasi, M. Hamawaki, G. Cooper IV, C. M. Kerr, and D. Kuppuswamy Differential Activation of p70 and p85 S6 Kinase Isoforms during Cardiac Hypertrophy in the Adult Mammal J. Biol. Chem., September 18, 1998; 273(38): 24610 - 24619. [Abstract] [Full Text] [PDF]
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P. Kunapuli, J. A. Lawson, J. A. Rokach, J. L. Meinkoth, and G. A. FitzGerald Prostaglandin F2alpha (PGF2alpha ) and the Isoprostane, 8,12-iso-Isoprostane F2alpha -III, Induce Cardiomyocyte Hypertrophy. DIFFERENTIAL ACTIVATION OF DOWNSTREAM SIGNALING PATHWAYS J. Biol. Chem., August 28, 1998; 273(35): 22442 - 22452. [Abstract] [Full Text] [PDF]
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H. Koyama, N. E. Olson, F. F. Dastvan, and M. A. Reidy Cell Replication in the Arterial Wall : Activation of Signaling Pathway Following In Vivo Injury Circ. Res., April 6, 1998; 82(6): 713 - 721. [Abstract] [Full Text] [PDF]
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J.-S. Zheng, L. O'Neill, X. Long, T. E Webb, E. A Barnard, E. G Lakatta, and M. O Boluyt Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts Cardiovasc Res, March 1, 1998; 37(3): 718 - 728. [Abstract] [Full Text] [PDF]
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F. Lin, W. A. Owens, S. Chen, M. E. Stevens, S. Kesteven, J. F. Arthur, E. A. Woodcock, M. P. Feneley, and R. M. Graham Targeted {alpha}1A-Adrenergic Receptor Overexpression Induces Enhanced Cardiac Contractility but not Hypertrophy Circ. Res., August 17, 2001; 89(4): 343 - 350. [Abstract] [Full Text] [PDF]
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