(Received for publication, March 10, 1997, and in revised form, May 8, 1997)
From the Departamento de Bioquímica y
Biología Molecular, Facultad Ciencias Químicas y
Farmacéuticas, Universidad de Chile, Olivos 1007, Santiago,
Chile, the ¶ Division of Cardiac Medicine, Imperial College School
of Medicine, National Heart and Lung Institute, London SW3 6LY, United
Kingdom, and the ** Diabetes Branch, NIDDK, National Institutes of
Health, Bethesda, Maryland 20892-1770
In response to insulin-like growth factor-I
(IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response.
The elucidation of the IGF-I signal transduction system in these cells
remains unknown. We show here that cardiac myocytes present a single
class of high affinity receptors (12,446 ± 3,669 binding
sites/cell) with a dissociation constant of 0.36 ± 0.10 nM. Two different -subunits of IGF-I receptor were
detected, and their autophosphorylation was followed by increases in
the phosphotyrosine content of extracellular signal-regulated kinases
(ERKs), insulin receptor substrate 1, phospholipase C-
1, and
phosphatidylinositol 3-kinase. IGF-I transiently activates c-Raf in
cultured neonatal cardiac myocytes, whereas A-raf is activated much
less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were
resolved in cardiac myocytes treated with IGF-I by fast protein liquid
chromatography, both being stimulated by IGF-I (with EC50
values for the stimulation of ERK1 and ERK2 by IGF-I of 0.10 and 0.12 nM, respectively). Maximal activation of ERK2 (12-fold) and
ERK1 (8.3-fold) activities was attained after a 5-min exposure to
IGF-I. Maximal activation of p90 S6 kinase by IGF-I was achieved after
10 min, and then the activity decreased slowly. Interestingly, IGF-I
stimulates incorporation of [3H]phenylalanine (1.6-fold)
without any effect on [3H]thymidine incorporation. These
data suggest that IGF-I activates multiple signal transduction pathways
in cardiac myocytes some of which may be relevant to the hypertrophic
response of the heart.
Left ventricular hypertrophy is both an important cardiovascular risk factor epidemiologically associated with cardiac failure and a frequent pathology of major significance in cardiovascular medicine. The myocardium is composed of many different cell types with cardiac myocytes contributing the greatest protein mass. In response to hormonal (polypeptide growth factors, endothelin, angiotensin II) and mechanical stimuli (1-6), the myocardium adapts by hypertrophy of individual myocytes. Because these cells are terminally differentiated cells that have lost the ability to proliferate, cardiac growth during hypertrophy results primarily from an increase in cellular protein content, with little or no change in cardiac myocyte number (7). The central features of the myocardial hypertrophic response are increases in the mass, volume, and contractile protein content of the myocytes, the induction of contractile protein gene expression, and the expression of embryonic genes and proto-oncogenes (7, 8).
Among growth factors, insulin-like growth factor-I (IGF-I)1 is significant because it promotes hypertrophy by growth and differentiation in many types of cells (9-11); it also mediates the anabolic and cardiovascular actions of growth hormone in vivo (12). IGF-I and its receptor (IGF-IR) are present on the neonatal rat myocardium, consistent with IGF-I regulating growth and hypertrophy of cardiac myocytes in the developing heart in an autocrine or paracrine manner (13). IGF-I acts directly on cultured neonatal cardiomyocytes to induce myocyte hypertrophy, which leads to increases in mRNA expression and synthesis of contractile proteins (14). The administration of recombinant IGF-I enhances ventricular hypertrophy and function during the onset of experimental heart failure in the rat (15, 16). There is also clear clinical evidence for a role of IGF-I in the initiation and development of left ventricular hypertrophy. Patients with dilated cardiomyopathy and heart failure treated with recombinant growth hormone showed increased myocardial mass and contractile performance accompanied by a doubled serum IGF-I concentration (17, 18). Moreover, cardiac hypertrophy develops in acromegalic patients with concomitantly increased plasma concentrations of both growth hormone and IGF-I (19). Increased plasma IGF-I concentrations have been reported in patients with hypertension and cardiac hypertrophy (20). The hypertension induces significant increases in cardiac IGF-I mRNA and protein in parallel with the onset and early development of experimental cardiac hypertrophy; this is followed by a normalization of the IGF-I mRNA once the hypertrophic response is established (21). A very recent report has established that the overexpression of IGF-I in cardiac myocytes leads to cardiomegaly mediated by an increased number of cells in the heart (22).
The mechanism by which IGF-I exerts its growth effect on cardiac
myocytes is still unknown. The IGF-IR is an
2
2 heterotetrameric protein with
ligand-stimulated tyrosine kinase activity (23). Binding of IGF-I to
its receptor induces receptor autophosphorylation in the intracellular
kinase domain of the
-subunit and results in activation of the
intrinsic tyrosine kinase activity of the IGF-IR (24, 25). The
predominant substrate of the IGF-IR is IRS-1, a docking protein that
has multiple tyrosines in YMXM or related motifs known to
associate with proteins containing SH2 domains. Phosphorylated IRS-1
regulates the activity of certain SH2 domain-containing proteins such
as phosphatidylinositol 3-kinase (PI 3-kinase) (26, 27). IRS-1 also
associates with other SH2 domain-containing proteins involved in growth
factor signaling pathways, including Grb-2, Nck, and Syp (23). Other
phosphotyrosine substrates of the IGF-I signaling pathway are PLC-
and Shc (23); and direct association of Shc with activated IGF-IR has
also been detected (28). Phosphorylated Shc associates with Grb-2 and subsequently, through a Grb-2·SOS (Son of Sevenless) complex, activates Ras and Raf-1, an intermediate in the Ras-ERK signaling pathway (29). In the ensuing phosphorylation cascade, activation of
ERKs occurs after phosphorylation by the mixed function
threonine-tyrosine kinase MEK. Some of these kinases, as well as
protein kinase C and RSK, can regulate the activities of a diverse
array of cellular and nuclear proteins, including transcription
factors.
The signaling pathways of other hypertrophic agonists (endothelin and acidic fibroblast growth factor) may converge at or above the level of the ERK cascade in cardiac myocytes (30). The elucidation of the IGF-I signal transduction system, and especially the degree of involvement of the IGF-IR and the ERK cascade during the cardiac hypertrophic process, is of considerable interest and constitutes the aim of this work.
Harlan Sprague Dawley rats were bred in the Animal Breeding Facilities from the National Heart and Lung Institute (London, U. K.), NIDDK (Bethesda, MD), or the Faculty of Chemical and Pharmaceutical Sciences, University of Chile (Santiago).
Materials[-32P]ATP,
[3H]phenylalanine, and
[methyl-3H]thymidine were from Amersham
International (Bucks, U. K.), and 125I-IGF-I was from NEN
Life Science Products. 12-O-Tetradecanoylphorbol-13-acetate (TPA), Dulbecco's modified Eagle's medium (DMEM), medium 199 (M199), n-octyl
-D-glucopyranoside, protease
inhibitors, protein A-Sepharose, bovine myelin basic protein (MBP), and
other biochemicals were purchased from Sigma unless stated otherwise.
Culture dishes (Primaria) were from Falcon, and heat-inactivated fetal
calf serum and horse serum were from Sera-Lab. Other tissue culture
products were from Life Technologies, Inc. Glutathione-Sepharose was
from Pharmacia Biotech Inc. Affinity-purified antibodies raised against
COOH-terminal peptide sequences of c-Raf (CTLTTSPRLPVF) or A-Raf
(CLLSAARLVP), and the corresponding peptides were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA) as were the antibodies raised
against IGF-IR
-subunit, tyrosine-phosphorylated proteins
(anti-phosphotyrosine antibody clone 4G10), PLC-
1, and RSK. The
polyclonal anti-IGF-IR
-subunit antibody was raised against the
COOH-terminal peptide sequence of the
-subunit of IGF-IR
(AHMNGGRKNERALPLPQSST). We have also assessed that this antibody does
not cross-react with insulin receptor
-subunit using different cell
lines that express different numbers of insulin receptor and IGF-IR
(31, 32). Enhanced chemiluminescence (ECL) immunoblotting detection
reagents, autoradiographic film, and prestained molecular mass standard proteins were from Amersham International. Protein assay reagents were
from Bio-Rad. Human recombinant IGF-I was donated by Drs. P. Valenzuela
and C. George-Nascimento (Chiron Corp.) or was purchased from
Boehringer-Mannheim GmbH (Mannheim, Germany). IRS-1 antibody was a gift
from Dr. J. Pierce (NCI, NIH). Clones for Glutathione S-transferase (GST) fusion proteins and anti-murine ERK
antibody were donated by Prof. C. J. Marshall (Chester Beatty
Laboratories, Institute of Cancer Research, London, U. K.).
Recombinant murine ERK2 and MEK1 were expressed as GST fusion proteins
in Escherichia coli and were purified by
glutathione-Sepharose affinity chromatography as described previously
(29). For the assay of MEK phosphorylation by the immunoprecipitated
protein kinases, recombinant MEK1 was modified by site-directed
mutagenesis to produce GST-MEK1 (R97/A291/A385), a catalytically
inactive form of MEK1 in which a Lys residue essential for kinase
activity is mutated (K97R) and which also lacks ERK consensus
phosphorylation sequences (T291A and T385A). Recombinant GST-ERK2 was
stored at
20 °C in 50% (v/v) glycerol (final protein concentration 25 mg/ml), whereas recombinant MEK1 species were stored
at
80 °C in 5%(v/v) glycerol (final protein concentration 10 mg/ml).
Neonatal ventricular myocytes were prepared from hearts of 1-3-day-old Harlan Sprague Dawley rats as described previously (33). Briefly, ventricles were trisected, pooled, and myocytes dissociated in a solution of collagenase and pancreatin. After enzymatic dissociation, the cells were selectively enriched for cardiac myocytes by being preplated in DMEM/M199 (4:1) containing 10% (v/v) horse serum, 5% (v/v) heated-inactivated fetal calf serum, penicillin, and streptomycin (100 units/ml). The myocytes, plated at a final density of 1.0-1.4 × 103/mm2 on gelatin-precoated 35-mm or 60-mm dishes, respectively, were confluent and spontaneously beating after 18 h. Serum was withdrawn for 24 h before the cells were treated further with agonists IGF-I (0-100 nM for 0-60 min) or TPA (1 µM for 3-5 min) in serum-free medium (DMEM/M199) at 37 °C.
IGF-I Binding AssayIGF-I binding to ventricular cardiac
myocytes was quantified using monoiodinated 125I-IGF-I.
Confluent cardiac myocytes in 12-well culture plates were washed with 1 ml of binding buffer (100 mM Hepes, pH 7.4, 120 mM NaCl, 1 mM EDTA, 5 mg/ml bovine serum
albumin) and incubated in binding buffer containing 25,000 cpm
(approximately 20 pM) of 125I-IGF-I and
increasing amounts of unlabeled IGF-I (0-1,000 ng/ml) for 5 h at
4 °C. The cells were washed with cold PBS and solubilized in 0.2 M NaOH before being counted with a -counter (GammaTrac 1290, Tm Analytic, Brandon, FL).
Tyrosine phosphorylation of cellular proteins was analyzed by immunoblotting with an anti-phosphotyrosine antibody. Confluent cardiac myocytes in 60-mm dishes were exposed to IGF-I (0-100 nM) for 0-60 min in serum-free medium at 37 °C. Upon completion of the exposure protocol, the medium was removed by aspiration and the cells washed twice with cold Ca2+/Mg2+-free Dulbecco's PBS. Myocytes were scraped into 150 µl of cold lysis buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1% (v/v) Triton X-100, 2 µg/ml leupeptin, and 2 µg/ml aprotinin). The protein content of the lysate was determined by Bradford's method (34), and equal amounts of protein were separated by SDS-PAGE on 9% gels. Proteins were then transferred electrophoretically to a nitrocellulose membrane (0.45 µm). The membranes were blocked with 3% (w/v) bovine serum albumin in PBST (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.05% (v/v) Tween 20, pH 7.4) for 1 h at room temperature. Tyrosine-phosphorylated proteins were probed with a monoclonal anti-phosphotyrosine antibody and detected with horseradish peroxidase-conjugated anti-mouse immunoglobulin using an ECL system. The dilutions of the first and second antibodies were 1:1,000 and 1:4,000, respectively. Immunoblots were quantified by densitometry.
Immunoblot Analysis forThe
amounts of IGF-IR or IRS-1 present were determined by immunoblotting
with monoclonal anti-IGF-IR -subunit antibody or with polyclonal
anti-IRS-1 antibody as described previously (35).
Cleared cell lysates were prepared as described above.
For immunoprecipitation, 400 µg of protein was incubated with 1 µg of an anti-PLC-1 antibody at 4 °C overnight. Then, 50 µl of
10% protein A-Sepharose in 50 mM Tris-HCl buffer, pH 7.5, was added and incubated at 4 °C for 3 h. The precipitates were
washed three times with immunoprecipitation buffer (20 mM
Tris, 300 mM NaCl, 2 mM EDTA, 2 mM
EGTA, 0.4 mM Na3VO4, 0.4 mM PMSF, pH 7.4) and separated on 9% SDS-PAGE. Resolved
proteins were transferred electrophoretically to nitrocellulose
membranes. Blots were incubated with anti-phosphotyrosine antibody
(1:1,000 dilution) or anti-PLC-
1 (1:1,000 dilution). Both antibodies
were detected by horseradish peroxidase-conjugated anti-mouse or
anti-rabbit immunoglobulin, respectively (1:4,000 dilution). The blots
were then developed by ECL.
Confluent cardiac myocytes grown in
60-mm dishes were incubated in serum-free DMEM/M199. After 24 h,
IGF-I (0-100 nM) was added in fresh serum-free medium, and
dishes were reincubated for 5 min at 37 °C. The cells were quickly
washed once with ice-cold PBS and twice with wash buffer (20 mM Tris, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 100 µM Na3VO4, pH 7.5). Cells were
solubilized in 600 µl of wash buffer containing 1% (v/v) Nonidet
P-40, 10% (v/v) glycerol, and 0.35 mg/ml PMSF. The lysate was
centrifuged at 10,000 × g for 10 min at 4 °C, and
tyrosyl-phosphoproteins were immunoprecipitated with a monoclonal
anti-phosphotyrosine antibody overnight at 4 °C. Then, 50 µl of
10% protein A-Sepharose in 50 mM Tris-HCl, pH 7.4, was
added and incubated for 4 h at 4 °C. The immunoprecipitates
were washed once with PBS containing 1% (v/v) Nonidet P-40 and 100 µM Na3VO4, twice with 100 mM Tris, pH 7.5, containing 500 mM LiCl and 100 µM Na3VO4, and once with 10 mM Tris, pH 7.5, containing 100 mM NaCl, 1 mM EDTA, and 100 µM
Na3VO4. The pellets were resuspended in 40 µl
of 10 mM Tris, pH 7.5, containing 100 mM NaCl
and 1 mM EDTA. To each tube was added 10 µl of 100 mM MnCl2 and 20 µg of phosphatidylinositol. The phosphorylation reaction was started by the addition of 10 µl of
440 µM ATP containing 30 µCi of
[-32P]ATP. After 10 min the reaction was stopped by
the addition of 20 µl of 8 M HCl and 160 µl of
CHCl3/methanol (1:1). The organic phase was extracted and
applied to a silica gel TLC plate. The plates were developed in
CHCl3/CH3OH/H2O/NH4OH
(60:47:11.3:2), dried, and visualized by autoradiography. The spots
were quantified with a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA).
Serum-starved ventricular myocytes were exposed to
agonists in serum-free medium (DMEM/M199). After the medium had been
removed by aspiration, cells were washed twice with cold
Ca2+/Mg2+-free Dulbecco's PBS and scraped into
200 µl of cold buffer A (20 mM Hepes, 75 mM
NaCl, 2.5 mM MgCl2, 20 mM
-glycerophosphate, 0.5 mM DTT, 0.1 mM EDTA,
pH 7.5, containing 0.2 mM Na3VO4, 2 µg/ml leupeptin, 100 µg/ml PMSF, 0.05% (v/v) Triton X-100).
Extracts were incubated at 4 °C for 10 min, then centrifuged
(10,000 × g, 10 min, 4 °C); supernatant fractions
were retained for assay. In control experiments, we determined that the
detergent-insoluble pellets did not contain Raf isoforms as assessed by
immunoblotting, and the pellets were not analyzed further. The
detergent-soluble supernatants (approximately 1.5-2.0 mg protein/ml)
were retained. When activation of A-Raf was directly compared with
activation of c-Raf, each supernatant was divided equally. Antibodies
that recognize c-Raf or A-Raf (3 µl) were added to each portion (90 (µl), and these were incubated with mixing at 4 °C for 2 h.
Protein A-Sepharose was added, and the incubation was continued for a further hour. The immunoprecipitates were washed three times with buffer A and twice with buffer B (20 mM Hepes, 75 mM NaCl, 2.5 mM MgCl2, 20 mM
-glycerophosphate, 0.5 mM DTT, 0.1 mM EDTA, pH 7.5).
Protein A-Sepharose immunoprecipitates of c-Raf and A-Raf
were assayed for their activation of recombinant GST-MEK1 (36) using a
coupled assay in which GST-MEK1 activates GST-ERK2. The activity of
GST-ERK2 is subsequently measured by incorporation of 32P
from [-32P]ATP into MBP. Immunoprecipitates were
resuspended in 30 µl of buffer C (30 mM Tris, 0.1 mM EGTA, pH 7.5, containing 0.1% (v/v) mercaptoethanol,
0.03% (w/v) Brij 35, 10 mM magnesium acetate, and 20 mM n-octyl
-D-glucopyranoside]
that had been supplemented with 80 µM (unlabeled) ATP, 2 µg/ml GST-MEK1, and 20 µg/ml GST-ERK2. The reactions were incubated
at 30 °C for 20 min with intermittent mixing, then terminated by
mixing 15 µl of the supernatant with 15 µl of ice-cold buffer B
containing 1 mg/ml bovine serum albumin. To assay the activity of
GST-ERK2, 10 µl of the diluted supernatants was mixed with 40 µl of
MBP phosphorylation buffer (50 mM Tris, 0.1 mM
EGTA, pH 8.0, containing 0.4 mg/ml MBP, 50 µM ATP, 12.5 mM magnesium acetate, and 1 µCi of
[
-32P]ATP). Mixtures were incubated for 15 min at
30 °C; the reaction was terminated by spotting onto P-81 paper,
which was then washed three times in 75 mM
H3PO4. Controls consisted of incubations of
antibodies alone with buffer A and protein A-Sepharose; these results
were subtracted from each data point. One unit of Raf activity is that
amount that catalyzed the activation of sufficient GST-ERK2 to transfer
1 fmol of phosphate from [
-32P]ATP to MBP/min in the
second stage of the assay. Preliminary experiments showed that no
activity from IGF-I-stimulated cells was associated with the protein
A-Sepharose after incubation of these detergent-soluble lysates with
protein A-Sepharose in the absence of antibodies to c-Raf or A-Raf.
Protein A-Sepharose immunoprecipitates of c-Raf and A-Raf
were resuspended in 30 µl of buffer C containing 50 µM
ATP, 1 mg/ml GST-MEK1 (R97/A291/A385), and 2 µCi
[-32P]ATP. The reaction was incubated at 30 °C for
20 min with intermittent mixing. The reaction was stopped by
centrifugation of the immunoprecipitated kinases; 15-µl portions of
supernatants were then pipetted into SDS-PAGE sample buffer. Samples
were boiled for 5 min, and proteins were separated by electrophoresis
on 10% SDS-polyacrylamide gels. Gels were stained, dried, and
subjected to autoradiography. Preliminary experiments showed that in
the absence of antibodies to c-Raf or A-Raf, no MEK phosphorylation
activity from detergent-soluble lysates of IGF-I-stimulated cells was
associated with the protein A-Sepharose.
Ventricular myocytes were lysed in buffer A. Extracts were incubated on ice for 10 min, then centrifuged (10,000 × g, 2 × 10 min, 4 °C). Supernatants were applied to Mono Q HR5/5 column (Pharmacia) equilibrated with 50 mM Tris, 2 mM EDTA, 0.5 mM DTT, 5% (v/v) glycerol, 0.1% (v/v) Triton X-100, containing 0.3 mM Na3VO4, 50 µg/ml PMSF, pH 7.5, at a flow rate of 1 ml/min. After washing with 5 ml of equilibration buffer, proteins were eluted with a linear gradient of 0-0.5 M NaCl. Fractions (1 ml) were collected and assayed immediately for MEK and ERK activities.
Assays of ERK and MEKERK was assayed by the "direct"
assay; supernatant samples were incubated with MBP and
[-32P]ATP as described earlier (37). Reactions were
terminated by spotting 40 µl of the reaction mixture onto P-81 paper
and washing in 75 mM H3PO4 (1 × 5 min, 3 × 30 min). Phosphate incorporation was measured by
Cerenkov counting. In these assays, 1 unit of ERK catalyzes the
incorporation of 1 pmol of phosphate into MBP/min.
MEK activity was assayed by a two-step assay using the activation of
exogenous recombinant GST-ERK2 (30). Supernatant samples were diluted
20-fold in buffer B. After samples (10 µl) had been mixed with 5 µl
of GST-ERK2 in Buffer A containing 0.04% (v/v) Brij 35, 0.1% (v/v)
2-mercaptoethanol, and 0.5 µM okadaic acid, the
activation of GST-ERK2 was initiated by the addition of 5 µl of 80 mM magnesium acetate and 0.8 µM unlabeled
ATP. After 15 min at 30 °C, the activity of GST-ERK2 was assayed by
the addition of 25 µl of 0.8 mg/ml MBP in buffer A containing 2.25 µM protein kinase A inhibitor, 2.25 µM
okadaic acid, and 5 µl of 40 mM magnesium acetate, 0.4 µM [-32P]ATP (0.5 µCi/assay). After a
further 15 min at 30 °C, the reaction was terminated by spotting 40 µl of the reaction mixture onto P-81 paper that was washed with 75 mM H3PO4 as described for the ERK
assay above. To correct for endogenous ERK activity in samples, rates
in control assays carried out in the absence of GST-ERK2 were
subtracted. The autophosphorylation and autoactivation of GST-ERK2
occurring in the incubation of GST-ERK2 with ATP in these assays in the
absence of cell lysates were also subtracted. One unit of MEK is the
amount that, in 1 min of the preincubation in the presence of unlabeled
ATP, specifically catalyzes the activation of exogenous GST-ERK2,
sufficient to transfer 1 pmol of phosphate into MBP/min in the
subsequent ERK assay.
Activities of MBP kinases were examined using an in-gel
kinase assay (38). Briefly, cells were treated with agonists, lysates were prepared, and proteins resolved in 10% SDS-polyacrylamide gels
containing 0.5 mg/ml MBP. ERK1, ERK2 and additional MBP kinase activities were detected by incubation of these gels with
[-32P]ATP and autoradiography as described earlier
(38). This assay was used to confirm that ERK1 and ERK2 activities
changed in parallel with activities measured by the direct assay.
Agonist-stimulated phosphorylation of endogenous ERK2 was also
determined from the shift in electrophoretic mobility as described
(38).
RSK activity was measured by an immune
complex kinase assay using an S6 peptide (RRRLSSLRA) as a substrate as
described previously (39). The lysates were prepared from
cardiomyocytes grown in 60-mm dishes. After stimulation with IGF-I (10 nM), the cells were removed by scraping and lysed with
lysis buffer (10 mM potassium phosphate, pH 7.4, 1 mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 50 mM -glycerophosphate, 1 mM Na3VO4, 2 mM DTT, 40 µg/ml PMSF, 10 mM okadaic acid, 0.8 µg/ml leupeptin, 10 mM p-nitrophenyl phosphate, and 10 µg/ml
aprotinin). Equal amounts of lysate proteins (300 µg) were diluted
with 10 volumes of RIPA buffer (10 mM Tris, 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) deoxycholic
acid, and 0.1% (w/v) SDS, pH 7.2) and were incubated with 2 µl of
anti-S6 kinase antibody for 2 h at 4 °C. Then protein
A-Sepharose was added, and the immunoprecipitates were washed with
lysis buffer without p-nitrophenyl phosphate. Ten µl of
immunoprecipitate was incubated with 12.5 µl of 2 × kinase
buffer (50 mM MOPS, 120 mM
-glycerophosphate, 10 mM EDTA, 30 mM
MgCl2, 60 mM p-nitrophenyl
phosphate, 2 mM DTT, pH 7.2) containing 2 mM
Na3VO4, 2 µM protein kinase A
inhibitor, 12.5 µCi of [
-32P]ATP, and 12.5 µl of 5 µM S6 peptide (RRRLSSLRA) at 30 °C for 15 min. To stop
the reaction, samples were spotted onto Whatman P-81 paper and washed
five times (5 min each) with 180 mM
H3PO4 and once with 95% ethanol. Radioactivity
was determined in a
-counter.
Confluent cardiac myocytes in 12-well plates were grown to quiescence in serum-free DMEM/M199 for 24 h. IGF-I (0-100 nM) in fresh serum-free DMEM/M199 was added, and the cells were incubated for an additional 18 h. [methyl-3H]Thymidine (1 µCi/well) was then added and incubation continued for a further 6 h. Cells were rinsed twice with ice-cold PBS, twice with ice-cold 5% trichloroacetic acid, and twice with ethanol. The cells were dissolved in 0.3 ml of 1 M NaOH; the resulting solutions were neutralized with 0.3 ml of 1 M HCl and counted in a liquid scintillation counter.
Phenylalanine IncorporationConfluent cells were grown to quiescence in serum-free medium for 24 h. Cardiac myocytes were stimulated with IGF-I (0-100 nM) in fresh serum-free medium for 24 h in the presence of [3H]phenylalanine (5 µCi/well). The cells were washed with ice-cold PBS, and 10% trichloroacetic acid was added at 4 °C for 60 min to precipitate the proteins. The precipitates were washed twice with ethanol and dissolved in 1 M NaOH; the resulting solutions were neutralized with 1 M HCl and counted in a liquid scintillation counter.
Expression of ResultsResults are expressed either as means ± S.E. for the number of independent experiments indicated (n) or as examples of representative experiments performed on at least two or three separate occasions. Time course and dose-dependent analysis were performed using analysis of variance, and comparisons between groups were performed using a protected Tukey's t test.
Binding of IGF-I to neonatal cardiac myocytes was
saturable and specific (Fig. 1). The Scatchard plot was
curvilinear, and the analysis (40) suggested the presence of two
classes of binding sites (Fig. 1, inset). Cultured cardiac
myocytes had high affinity binding sites of 12,446 ± 3,669 binding sites/cell with a dissociation constant for IGF-I of 0.36 ± 0.10 nM (n = 4). Low affinity binding sites (Kd = 27 ± 11 nM), whose
number was estimated to be 77,911 ± 7,683 binding sites/cell,
were also detected in neonatal cardiac myocytes. Binding studies with
125I-IGF-I(N-3), an IGF-I analog lacking the three
NH2-terminal amino acids which binds only to high affinity
IGF-IR (41), suggest that low affinity IGF-I binding sites may be
correspond to nonreceptor IGF-I-binding proteins (data not shown).
Although there are no comparative studies between receptors for insulin
and IGF-I in neonatal cardiac myocytes, our results show that insulin
receptors appear to be in lesser amount than IGF-IRs (data not
shown).
IGF-IR Autophosphorylation and Phosphorylation of Endogenous Substrates
As shown in Fig. 2 (A and
B upper), incubation of cardiac myocytes with IGF-I resulted
in increases in the phosphotyrosine content of several proteins with
apparent molecular masses of 42, 44, 96, 100, and 185 kDa,
corresponding to the two ERK isoenzymes, the two -subunits of IGF-IR
and IRS-1. IGF-IR autophosphorylation was much greater in neonatal
cardiac myocytes exposed to IGF-I concentrations above 5 nM; two separate bands were observed at low levels of
receptor autophosphorylation. In contrast, receptor autophosphorylation
was undetectable in cells incubated without IGF-I. Membranes shown in
Fig. 2 B, upper, were stripped and reblotted with
a polyclonal antibody directed against the
-subunit of IGF-IR to
confirm both the presence of two isoforms of the
-subunit of IGF-IR
and that similar amounts of IGF-IR protein were present in all samples
(Fig. 2B, lower). Fig. 2C shows the
time course of stimulation of cultured cardiac myocytes by 10 nM IGF-I; phosphorylation of the IGF-IR
-subunit was
maximal within 1 min.
The IRS-1 pathway is activated following IGF-IR phosphorylation (42). We evaluated IRS-1 activation by incubating cells with IGF-I (0-100 nM) for 1 min at 37 °C. Exposure to concentrations of IGF-I above 10 nM was accompanied by phosphorylation of the expected 185-kDa band (Fig. 2A); phosphorylation was apparent 1 min after stimulation with 10 nM IGF-I (Fig. 2C). When membranes were stripped and reblotted with a polyclonal antibody directed against IRS-1, we confirmed the presence of IRS-1 and that similar amounts of IRS-1 protein were present in all samples (results not shown).
Tyrosine Phosphorylation of PLC-Fig. 3A shows the enhancement
of tyrosine phosphorylation of PLC-1 in cells stimulated with IGF-I.
Maximum stimulation (5-fold) occurred 2 min after the addition of IGF-I
(Fig. 3A). PLC-
1 coprecipitated with a 185-kDa
phosphotyrosine protein. When membranes were stripped and reblotted
with a polyclonal antibody directed against PLC-
1, we confirmed the
identity of PLC-
1 and that similar amounts of PLC-
1 protein were
present in all samples (Fig. 3B). Since the 185-kDa
phosphorylated protein that coprecipitated with PLC-
1 may correspond
to IRS-1, the membrane was reblotted with different anti-IRS-1
polyclonal antibodies (catalog numbers 06-248 and 06-524 from Upstate
Biotechnology Inc., Lake Placid, NY, and sc-559 from Santa Cruz
Biotechnology, Santa Cruz, CA). However, the presence of IRS-1 in the
immunoprecipitates was not detected. A similar result was obtained when
IRS-1 was first immunoprecipitated, and the membrane was blotted with
an anti-PLC-
. PI 3-kinase activity was increased in cardiac myocytes
incubated with 10 nM IGF-I for 5 min (Fig.
4A); this increase was maximal (2.5-fold)
after 5 min of incubation with 30 nM IGF-I (Fig.
4B).
IGF-I Activates c-Raf Much More Than A-Raf in Cultured Neonatal Cardiac Myocytes
c-Raf is activated after exposure of neonatal
cardiomyocytes to agonists acting through protein tyrosine kinase and
G-protein receptors (36). Both c-Raf and A-Raf from neonatal cardiac
myocytes form a stable complex with kinase inactive GST-MEK1
(R97/A291/A385) (36). Fig. 5A shows that
IGF-I (10 nM) activated c-Raf through its ability to
activate GST-MEK1 in a coupled assay in vitro. Maximal
activation occurred within 3-10 min and returned to basal levels after
30 min. We confirmed that the amounts of immunoprecipitated c-Raf did
not vary greatly (data not shown). There was no detectable activity in
c-Raf immunoprecipitated from serum-starved ventricular myocytes
exposed to serum-free medium. Although A-Raf was present in cultured
ventricular myocytes, its activation by IGF-I was much less than c-Raf
(closed circles, Fig. 5A). The amounts of A-Raf
immunoprecipitates from the cells did not vary (data not shown).
Activity of the immunoprecipitated c-Raf was also studied by the phosphorylation of GST-MEK1 (R97/A291/A385), a recombinant mutant MEK1 unable to undergo autophosphorylation and retrophosphorylation by endogenous ERK (43). This assay confirmed the rapid and transient activation of immunoprecipitated c-Raf (Fig. 5B). The maximum increase in c-Raf activity was 400% of the control value and the EC50 for IGF-I was approximately 0.1 nM (Fig. 5C). In cultured cardiac myocytes, TPA activates different isoenzymes of protein kinase C and causes sustained activation of MEK and ERK (33). Activation of c-Raf (15-fold) and A-Raf (3.3-fold) was attained after a 3-min exposure to TPA (1 µM). c-Raf was 29-fold more activated by TPA than by IGF-I (Fig. 5C).
Activation of MEK by IGF-I in Cultured Neonatal Cardiac MyocytesCrude extracts of unstimulated cultured cardiac myocytes
poorly activate exogenous inactive recombinant GST-ERK2. In contrast, IGF-I-treated cultured cardiac myocytes substantially stimulated MEK
activity (results not shown). Supernatant fractions of cultured neonatal ventricular myocytes were subjected to FPLC on a Mono Q
column, and fractions were assayed for their ability to phosphorylate and activate GST-ERK2. Extracts of cells exposed to 10 nM
IGF-I for 5 min showed two peaks of MEK activity (KK1 and KK2) eluted at 70 and 130 mM NaCl (Fig. 6A),
although the second peak was smaller than the first. The combined
stimulation of MEK activities approached 10-fold. A similar MEK
activity profile was obtained after exposure to 1 µM TPA
for 5 min (results not shown). We have demonstrated previously that
endogenous ERK activity does not interfere with the assay of GST-ERK2
activity (30).
Activation of ERK by IGF-I in Crude Extracts of Cultured Neonatal Cardiac Myocytes
To examine whether ERKs are activated by IGF-I,
we next measured ERK activity by an in-gel ERK assay. There were only
small changes in the intensity of the ERK2 band after the exposure of myocytes to IGF-I between 1 and 3 min, without any change in ERK1 band
(Fig. 7A). The maximal intensities of ERK1
(12-fold) and ERK2 (8.3-fold) were attained after a 5-min exposure to
IGF-I. Thereafter, activities declined rapidly returning to control
values within 30 min (Fig. 7A). The time course for the
effects of IGF-I (10 nM) on ERK activity is shown in Fig.
7B. The effect of IGF-I on the time course of
ligand-stimulated phosphorylation of ERK2 in cultured cardiac myocytes
was determined from the electrophoretic mobility shift of endogenous
ERK2. IGF-I provoked a transient increase in ERK2 phosphorylation which
followed a time course similar to that determined by the in-gel assay
technique (data not shown). The dependence of ERK activation on the
concentration of IGF-I was also characterized by in-gel assays (Fig. 7,
panels C and D). The EC50 values for
the stimulation of ERK1 and ERK2 in cultured cardiac myocytes by IGF-I
were 0.10 and 0.12 nM, respectively. When cardiac myocytes
were exposed for 5 min to a saturating concentration of IGF-I (10 nM), ERK1 and ERK2 activities were stimulated to 35 and
58%, respectively, of the TPA-induced activity (Fig. 7D). Exposure of cardiac myocytes to increasing concentrations of IGF-I for
5 min also caused phosphorylation of ERK2, as demonstrated by delayed
mobility on SDS-PAGE (data not shown).
Separation by FPLC of IGF-I-activated Isoforms of ERK in Cultured Neonatal Cardiac Myocytes
IGF-I stimulates ERK activity in cultured ventricular myocytes; this can be assayed directly in supernatants with MBP as substrate. FPLC of supernatant fractions from IGF-I-treated cells on the Mono Q (Fig. 6A) revealed two major peaks of MBP kinase activity eluting at 160 (peak K1) and 220 (peak K2) mM NaCl, respectively. The mean stimulation of each peak fraction was 3-fold.
Identification of ERK Isoenzymes Activated by IGF-IFractions from peaks K1 and K2 from Mono Q chromatography of extracts from cardiac myocytes incubated with 10 nM IGF-I for 5 min were pooled, concentrated, and analyzed by in situ phosphorylation of MBP gels. Two proteins of 42 and 44 kDa were identified in fractions corresponding to peak K1 and K2, respectively (Fig. 6B).
Stimulation of p90 RSK Activity by IGF-I in Cultured Cardiac Myocytesp90 RSK is activated by ERK1 and ERK2 both in
vivo and in vitro (44). The time course of activation
of the p90 RSK by IGF-I (10 nM) in cultured cardiac
myocytes is depicted in Fig. 8. p90 RSK activity reached
a peak at 10 min and then decreased slowly.
Stimulation of Phenylalanine and Thymidine Incorporation by IGF-I in Cultured Cardiac Myocytes
After a 24-h incubation of myocytes
with 10 nM IGF-I, incorporation of
[3H]phenylalanine was increased 1.6-fold over control
levels (Fig. 9A). No
IGF-I-dependent stimulation of [3H]thymidine
incorporation was observed in cultured cardiac myocytes (Fig.
9B).
The development of cardiac hypertrophy constitutes a compensatory adaptation of cardiac myocytes to increased hemodynamic stress or to loss of contractile myocytes. In addition to an overall increase in protein and RNA content, there are alterations at the level of gene expression which distinguish the hypertrophic process from normal maturational growth (7). The mechanisms by which the mechanical stress is sensed by cardiac myocytes and growth-related signals are activated and integrated to regulate gene expression programs selectively during the hypertrophic process are largely unknown. The hypothesis is that growth factors (such as IGF-I) are produced by cardiac non-muscle cells or by the myocytes themselves in response to mechanical stress and that these factors, through specific cell surface receptors and intracellular signaling cascades, regulate protein synthesis and transcription of genes of the contractile apparatus, as well as others genes involved in cell growth. In cultured neonatal cardiac myocytes, IGF-I induces an early and sustained expression of the muscle-specific genes for troponin I and myosin light chain-2 with myocyte size almost doubling after 48 h of treatment with IGF-I (14). An increase in left ventricular IGF-I mRNA and its protein has been described in pressure overload cardiac hypertrophy in various models of hypertension; this suggests that IGF-I may be an important mediator of an adaptive hypertrophic response (21, 45, 46). However, the results of overexpression of IGF-I in transgenic mice have been contradictory (22, 47). For example, when overexpression of IGF-I was restricted to the heart, IGF-I stimulated an increase in ventricular muscle cell number in vivo. Surprisingly, cardiac myocyte cellular hypertrophy was not enhanced (22). Studies with IGF-I knockout mice have shown that lack of IGF-I does not prevent the development of pressure overload hypertrophy, suggesting that IGF-I alone is not the sole precondition for the hypertrophic phenotype (48).
Our results showed that IGF-I binding was specific with a single class of high affinity sites (Kd = 0.36 nM) in neonatal cardiac myocytes (Fig. 1) in agreement with an earlier work (14). Although there are no comparative studies between receptors for insulin and IGF-I in neonatal cardiac myocytes, our results show that there appear to be fewer insulin receptors than IGF-IRs (data not shown). A previous study showed that specific binding for insulin and IGF-I/100 mg of rat cardiac tissue heart reached 52 and 20%, respectively, with Kd values of 0.25 nM for insulin receptors and of 0.40 nM for IGF-IR for its own ligand (49), indicating that insulin receptors are more abundant in non-myocyte cell types.
It is well known that IGF-I stimulates tyrosine phosphorylation of
several proteins in various cell types (23, 50, 51). We detected
several tyrosine-phosphorylated proteins with apparent molecular mass
of 42, 44, 95, 105, and 180 kDa, whose tyrosine phosphorylation in
response to IGF-I increased rapidly (Fig. 2). By immunoblotting, these
proteins were identified as p42 Erk2, p44 Erk1, IGF-IR -subunit, and
IRS-1 (Figs. 2 and 7, and data not shown) (52, 53). The results also
showed that the autophosphorylation of IGF-IR
-subunit as well as
phosphorylation of IRS-1, both on tyrosines, was IGF-I concentration
dependent, although with a different sensitivity. IGF-IR
-subunit
autophosphorylation became evident with 5 nM IGF-I, whereas
for IRS-1 phosphorylation was observed after 10 nM (Fig.
2A). The presence of two isoforms for IGF-IR
-subunits
with molecular masses of 95 and 105 kDa (Fig. 2B) was also
detected in neonatal cardiac myocytes; similar isoforms have been
reported earlier (23). Although it remains unknown whether if each
subunit isoform may have a particular signal transduction system and if
their expression changes during the myocyte development of cardiac
myocytes, the higher and lower molecular masses may correspond to a
fetal and adult forms, respectively (54, 55). Other hypertrophic
stimuli (stretch, angiotensin II) also stimulate protein tyrosine
phosphorylation in cardiac myocytes (56-58), and it has been
hypothesized that cardiac hypertrophy might be associated with altered
tyrosine phosphorylation of certain proteins (i.e. a 120-kDa
cytosolic protein) in the heart (59). Although in cardiac myocytes
there is no evidence of cross-talk between angiotensin II and IGF-I,
this interaction has been demonstrated at multiple levels in vascular
smooth muscle cells (60). Both angiotensin II and thrombin caused rapid
tyrosine phosphorylation of the IGF-IR
-subunit and of IRS-1 in this
cellular type (61). IRS and Shc are considered the two major IGF-I
signaling pathways. IRS-1 is an adapter protein phosphorylated on
multiple tyrosine residues upon receptor stimulation, providing
multiple sites of interactions for proteins with SH2 domains such as
p85 PI 3-kinase, Nck, Grb-2, and PTP1P (23, 62). PLC-
1 also contains
SH2 domains that recognize and bind to the phosphorylated tyrosine
residues of the receptor tyrosine kinases (63). In contrast with other RTKs, it is not well established whether PLC-
1 associates with IGF-IR and becomes activated by tyrosine phosphorylation upon receptor
stimulation. We demonstrated that IGF-I induces a rapid tyrosine
phosphorylation of PLC-
1 reaching 3.5-fold (Fig. 3). Although we
have detected coprecipitation of a 185-kDa phosphorylated protein with
PLC-
1 which might correspond to IRS-1, our results suggest that both
proteins are not associated after IGF-IR activation, and further
studies will be required to identify the phosphorylated protein that
coprecipitates with PLC-
. PI 3-kinase activation in IGF-I signal
transduction may play a important role because this enzyme mediates the
activation of other protein kinases such as protein kinase C, S6
kinase, and serine-threonine kinase Akt as well as the regulation of
cytoskeletal function (63-65). Our results also show that its
activation was IGF-I concentration-dependent and that it
was activated by tyrosine phosphorylation. IGF-I also causes rapid
translocation of two Shc isoforms (46 and 52 kDa) to the membrane
fraction with a maximum increase in membrane (6-11-fold) after 30 s with 10 nM IGF-I.2 It has
been shown that Shc, like IRS-1, associates with Grb-2, upon tyrosine
phosphorylation, and subsequently activates the p21 Ras-ERK pathway via
a Grb-2·SOS complex (23). Our results stress that IGF-I has multiple
signal transduction pathways through different adapter proteins in
cardiac myocytes.
We and others have previously proposed that activation of the p21 Ras-ERK pathway may be involved in the growth response of cardiac myocytes to hypertrophic agonists (66-69). In contrast, others have reported that ERK activation is not sufficient for G-protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the atrial natriuretic factor gene (70).
Diverse hypertrophic agonists converge to produce cardiac hypertrophy
following a sequential activation of Raf MEK
ERK. The upstream
event in the ERK cascade involves the activation of Raf kinases. We
have presented evidence that exposure of cardiac myocytes to IGF-I
rapidly activated c-Raf. A-Raf was activated much less than c-Raf in
neonatal cardiac myocytes. This finding is similar to that reported
previously for acidic fibroblast growth factor in cardiac myocytes but
differs from that produced by TPA (36). The reason for this
differential activation of Raf isoforms by IGF-I is unclear; and its
functional effectors, in addition to Ras and Src, are yet need to be
identified (71, 72). The differential activation of both Raf isoforms
may also reflect different requirements for effectors or the
interaction with other proteins like 14-3-3 proteins (73). We have also
demonstrated that IGF-I activated two peaks of MEK (Fig. 6) which were
capable of phosphorylating exogenously added recombinant GST-ERK2. Both peaks may correspond to the MEK1 and MEK2 isoforms activated by acidic
fibroblast growth factor and endothelin-1 (30). The two isoforms of ERK
(ERK1 and ERK2) were coordinately activated by IGF-I (Fig. 7). The
maximum extent of phosphorylation of ERK2 elicited by IGF-I
corresponded to 44% of the total ERK2 pool (results not shown). This
response is similar to that seen with other hypertrophic agonists
(endothelin-1, phenylephrine, acidic fibroblast growth factor) (33,
36-38). Nonhypertrophic agents such as bradykinin, carbachol, and ATP
do, however, also activate ERK in cardiac myocytes (70, 74). In PC-12
cells the duration of the activation of ERK1 and ERK2 is critical in
determining whether the cells differentiate (prolonged activation) or
proliferate (transient activation) (75, 76). In neonatal cardiac
myocytes in culture activation of ERKs induced by mechanical stretch
and angiotensin II is accompanied by sustained activation of p90 RSK
(37, 58). We have shown here that IGF-I causes activation of p90 RSK in
cardiac myocytes. Last, we have shown that IGF-I stimulates
[3H]phenylalanine incorporation into cardiac myocyte
proteins without changes in the incorporation of
[3H]thymidine into DNA in neonatal cardiac myocytes. This
agrees with previous reports showing the hypertrophic action of IGF-I in vitro (14). Our preliminary results using different
inhibitors of the IGF-I signal transduction pathway (i.e. PD
098059, an inhibitor of MEK) suggest that IGF-I-induced cardiac
hypertrophy may be mediated by the ERK
cascade.3
We concluded that IGF-I activates multiple signal transduction pathways
that involve tyrosine phosphorylation of IRS-1, PI 3-kinase, and
PLC-1 and the activation of the ERK cascade and p90 RSK in neonatal
cardiac myocytes in culture. The use of both selective inhibitors of
signal transduction pathways as well as the transient transfection of
cardiac myocytes with dominant-negative forms and antisense
oligodeoxynucleotide approach for the different components of signal
transduction mechanism of IGF-I will allow us to establish their
contribution to cardiac myocyte hypertrophy.
We thank Professor C. I. Pogson for helpful discussion and a critical review of the manuscript.