From the Section of Cardiac Medicine, National Heart and Lung Institute Division, Imperial College School of Medicine, London SW3 6LY, United Kingdom
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ABSTRACT |
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In response to hormones and growth factors,
cultured neonatal ventricular myocytes increase in profile, exhibit
myofibrillogenesis, and re-express genes whose expression is normally
restricted to the fetal stage of ventricular development. These include
atrial natriuretic factor (ANF), -myosin heavy chain (
-MHC), and
skeletal muscle (SkM)-
-actin. By using luciferase reporter plasmids,
we examined whether oncogenes that activate the extracellular
signal-regulated kinase cascade (srcF527,
Ha-rasV12, and v-raf) increased
expression of "fetal" genes. Transfection of myocytes with
srcF527 stimulated expression of ANF,
SkM-
-actin, and
-MHC by 62-, 6.7-, and 50-fold, respectively, but
did not induce DNA synthesis. Stimulation of ANF expression by
srcF527 was greater than by
Ha-rasV12, which in turn was greater than by
v-raf. General gene expression was also increased but to a
lesser extent. The response to srcF527 was
inhibited by dominant-negative Ha-rasN17.
Myocyte area was increased by srcF527,
Ha-rasV12, and v-raf, and although
it altered myocyte morphology by causing a pseudopodial appearance,
srcF527 did not detectably increase
myofibrillogenesis either alone or in combination with
Ha-rasV12. A kinase-dead src mutant
increased myocyte size to a much lesser extent than
srcF527 and also did not inhibit ANF-luciferase
expression in response to phenylephrine. We conclude that members of
the Src family of tyrosine kinases may be important in mediating the
transcriptional changes occurring during cardiac myocyte hypertrophy
and that Ras and Raf may be downstream effectors.
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INTRODUCTION |
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The cardiac myocyte is a terminally differentiated cell that
withdraws from cell division at around birth in mammals. In these cells, adaptive hypertrophic growth in response to an increased requirement for contractile power involves increases in cell size, protein content, and myofibrillar organization (reviewed in Ref. 1).
There are also changes in gene transcription that distinguish hypertrophy from maturational growth (eutrophy). In response to hypertrophic stimuli, cultured neonatal rat ventricular myocytes initiate a rapid and transient increase in expression of immediate early genes (c-jun, c-fos, and Egr-1),
followed by the re-expression of so-called "fetal" genes including
atrial natriuretic factor (ANF),1 -myosin heavy
chain (
-MHC), and skeletal muscle (SkM)
-actin. More chronic
exposure to hypertrophic agonists also elicits an increase in the
expression of constitutively expressed contractile protein genes such
as ventricular myosin light chain-2 and cardiac muscle
-actin. Many
of these changes are seen during hypertrophy of the rat heart in
vivo, providing a justification for the use of this model system
(reviewed in Ref. 1).
A wide variety of agonists induce the hypertrophic phenotype in
cultured ventricular myocytes. These include agonists acting through G
protein-coupled receptors such as endothelin-1 (2, 3), angiotensin II
(4, 5), and 1-adrenergic agonists (4, 6, 7). Other
hypertrophic stimuli include passive stretch (8-10), phorbol esters
that activate protein kinase C isoforms (11, 12), and growth factors
acting through receptor protein tyrosine kinases such as fibroblast
growth factors (13) and insulin-like growth factor 1 (14, 15). A
feature common to each of these stimuli is their ability to activate
the extracellular-regulated kinase (ERK) members of the
mitogen-activated protein kinase (MAPK) superfamily (16-22). This has
prompted the suggestion that activation of ERKs may be a central
component of the intracellular signaling mechanism through which
hypertrophic agonists exert their effects (16, 17, 23), although it is
still not clear whether ERK activation is obligatory or sufficient (19,
24-26).
Activation of ERK1 (p44 MAPK) and ERK2 (p42 MAPK) is brought about by
phosphorylation by MAPK kinases (MEKs, for MAPK (or ERK) kinases) (reviewed in Refs. 27-31). MEKs
are themselves activated by phosphorylation by MAPK kinase kinases, the
best-characterized being the Raf family (c-Raf-1, A-Raf, and B-Raf in
higher organisms) (32-35). For agonists acting through receptor
protein tyrosine kinases, the intervening steps between receptor
occupancy and activation of the Raf MEK
ERK cascade are
relatively well established and involve SH2/SH3-containing adaptor
proteins such as Grb2 and Shc, guanine-nucleotide exchange factors such
as Sos, and activation through GDP/GTP exchange of the small G proteins of the Ras family (reviewed in Refs. 36-38). Ras·GTP mediates the activation of c-Raf-1 by recruiting it to the plasma membrane (39-42),
where other reactions that possibly include the phosphorylation of
Tyr-340/Tyr-341 by Src family protein tyrosine kinases lead to its full
activation (43, 44). Recent evidence shows that A-Raf is also activated
synergistically by oncogenic ras and oncogenic src, whereas oncogenic ras alone is sufficient
for maximal activation of B-Raf (44). In terms of the hypertrophic
response, there is good evidence for a role for activated Ras. Thus,
angiotensin II activates Ras in myocytes (45); injection of oncogenic
RasV12 initiates hypertrophic changes in myocytes (46), and
PE-induced ANF expression is inhibited by a dominant-negative
ras mutant (46).
Recent work has suggested that protein tyrosine kinases of the Src family (Src, Fyn, Lyn, etc.) may participate in the coupling of both receptor protein tyrosine kinases and G protein-coupled receptors to ERK activation (reviewed in Refs. 47-51). Indeed, a role for Fyn has been proposed in angiotensin II-induced hypertrophy of the cardiac myocyte (45). Here we have examined the effect of overexpression of srcF527, an oncogenic variant of the c-src protooncogene, on the transcriptional and morphological characteristics associated with the hypertrophic phenotype in cardiac myocytes. We show that srcF527 expression potently activates genes that are up-regulated in response to hypertrophic stimuli, a response in which it may act in concert with Ras and Raf. Our results suggest that non-receptor protein tyrosine kinases such as c-Src may play a role in some of the altered growth responses of myocytes exposed to hypertrophic stimuli.
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EXPERIMENTAL PROCEDURES |
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Materials--
Sprague-Dawley rats were bred within the National
Heart and Lung Institute. Culture medium and other reagents were from
Sigma, Life Technologies, Inc., or Merck. The plasmids encoding
constitutively active v-Raf and Ha-RasV12 and
dominant-negative (DN) Ha-RasN17 and DNRaf were from
Professor C. J. Marshall, and those encoding chicken
SrcF527 and kinase-dead mutants of c-Src
(srcK) and SrcF527
(srcF527K
) were from Dr. R. M. Marais
(both at the Chester Beatty Laboratories, Institute of Cancer Research,
London, UK). In SrcF527 the tyrosine residue at position
527 is mutated to phenylalanine rendering it insensitive to
phosphorylation and inactivation by the tyrosine kinase Csk. The
ANF-luciferase (ANF-LUX) reporter construct pANF(
638)L
5' (7) and
pON249 (52) in which
-gal is expressed from a constitutive
cytomegalovirus promoter were kindly provided by Dr. K. R. Chien
(Department of Medicine, University of California, San Diego). The
AP-1-LUX construct TRE2PRL(
36) was a gift from Drs. J. H. Brown
(Department of Pharmacology, University of California, San Diego) and
M. G. Rosenfeld (Howard Hughes Medical Institute, University of
California, San Diego). The LUX reporter constructs for
-MHC (53),
SkM-
-actin (53-55), and the c-fos serum responsive
element (SRE) (56) were gifts from Dr. M. D. Schneider (Molecular
Cardiology Unit, Baylor College of Medicine, Houston, TX). The LUX
reporter plasmids have been described in detail previously (57).
Plasmid pNG1 was constructed by subcloning a
BamHI-NotI fragment of pPD46.21 (58) (a gift from
Dr. A. Fire, Carnegie Institute, Baltimore, MD) encoding a
-gal gene
containing a nuclear localization signal into the BamHI-NotI sites in pBK-cytomegalovirus
(Stratagene). Plasmids were purified by polyethylene glycol
precipitation (59).
Transient Transfection of Cultured Neonatal Ventricular
Myocytes--
Ventricular myocytes were isolated from the hearts of
1-2-day-old rats by a modification of the method of Iwaki et
al. (60) as detailed previously (57). Transfections (by the
calcium phosphate method) involved the addition of 5 or 15 µg of LUX
reporter plasmid, 2 or 4 µg of pON249, and a total of up to 10 µg
of test plasmids to 60-mm cell culture dishes containing 1 million
cells. For sarcomeric staining, transfections involved the addition of
6 µg of ANF-LUX reporter plasmid, 1.6 µg of pNG1, and 2 µg each
of test plasmids to 100,000 cells per well of a 2-well chamber slide.
Phenylephrine (PE), when present, was added to a concentration of
10-100 µM (as stated in individual experiments)
approximately 20 h after the transfection. After a further 48 h, myocytes were extracted and assayed for LUX and -gal as detailed
previously (57). Results are presented as the mean ± S.E. of
experiments on at least four separate preparations of myocytes.
Determination of Myocyte Size--
To assess the area of
transfected myocytes, cells were washed three times with ice-cold
Dulbecco's Ca2+/Mg2+-free phosphate-buffered
saline (PBS), fixed with 4% formaldehyde in PBS for 10 min, re-washed
three times with PBS, and then stained with 0.2 mg/ml
5-bromo-4-chloro-3-indolyl--D-galactopyranoside, 5 mM K4Fe(CN)6, 5 mM
K3Fe(CN)6, 2 mM MgCl2
in PBS. Transfected (blue) cells were randomly selected from all areas
of the dishes and imaged using a video hardcopier from which the cell
area was determined by planimetry.
Immunocytochemistry--
Myocytes were cultured in Permanox
2-well chamber slides pretreated with gelatin (1%) and laminin (20 µg/ml). After fixing as for cell sizing, cells were permeabilized
with 0.3% Triton X-100 in PBS (10 min, room temperature), and
nonspecific binding sites were blocked with 10% horse serum in 0.3%
Triton X-100 in PBS (10 min, room temperature). Antibodies were diluted
in 10% horse serum in 0.3% Triton X-100 in PBS, and myocytes were
washed three times in PBS between all stages of the immunofluorescence procedure. Nuclearly localized -gal was detected using a mouse polyclonal anti-
-gal primary antibody (Sigma, 1/200 dilution, 1 h at 37 °C), a biotinylated anti-mouse IgG secondary antibody (Amersham Pharmacia Biotech, 1/200 dilution, 30 min at 37 °C) and
streptavidin-7-amino-4-methylcoumarin-3-acetic acid (Boehringer Mannheim, 1/400 dilution, 15 min at 37 °C).
-MHC was subsequently detected by staining with a mouse monoclonal anti-
-MHC primary antibody (Novacastra, 1/50 dilution, 1 h at 37 °C) and a Texas Red-conjugated anti-mouse IgG secondary antibody (Amersham Pharmacia Biotech, 1/100 dilution, 30 min at 37 °C). To assess DNA synthesis, myocytes were treated with 0.1 mM bromodeoxyuridine
(BrdUrd) for 24 h prior to fixing and permeabilizing as above. All
subsequent blocking and antibody incubation steps were carried out in
1% bovine serum albumin in 0.1% Tween 20 in PBS. BrdUrd was detected with a mouse monoclonal anti-BrdUrd antibody (clone BU-33, Sigma, 1/500
dilution), followed by a biotinylated anti-mouse IgG secondary antibody
and streptavidin-7-amino-4-methylcoumarin-3-acetic acid as above.
-Gal was detected with a rabbit polyclonal antibody (Organon
Teknika, 1/200 dilution) and a Texas Red-conjugated anti-rabbit IgG
secondary antibody (Amersham Pharmacia Biotech, 1/100 dilution).
Expression of Results and Statistical Analysis-- The absolute values of chemiluminescence differs considerably between the various reporter constructs. Thus, in Figs. 1-3, results are expressed as the ratio of reporter gene expression in the presence of srcF527, v-raf, and Ha-rasV12 expression plasmids relative to empty vector controls. This facilitates comparison of the relative potencies of the expression plasmids. Results are presented as means ± S.E. For statistical analysis, absolute values of chemiluminescence were used, and significance was assessed by using an unpaired or paired Student's t test as appropriate with a significant difference taken as being established at p < 0.05.
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RESULTS |
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Stimulation of Promoter Activities by srcF527,
Ha-rasV12, and v-raf--
The proximal 638 base pairs of
the ANF promoter are sufficient to confer tissue specificity and
inducible expression on the LUX reporter (7). Transfection with
srcF527, a constitutively active oncogenic
mutant of c-src, stimulated expression of this construct by
62.3 ± 10.3-fold (Fig. 1,
panel A). This was
significantly greater (p < 0.01) than the stimulation of ANF-LUX by oncogenic Ha-rasV12 (18.0 ± 2.7-fold). Co-transfection with srcF527 and
Ha-rasV12 stimulated ANF-LUX expression by
106.1 ± 21.0-fold which was significantly greater than
transfection with Ha-rasV12 alone
(p < 0.01) but was not significantly greater than
transfection with srcF527 alone (Fig. 1,
panel A). When LUX expression was normalized for -gal
expression (Fig. 1, panel B), the effect of
srcF527 (7.2 ± 0.8-fold) was still
significantly greater (p < 0.02) than activation by
Ha-rasV12 (3.7 ± 0.7-fold). Again, the
combined effect of srcF527 and
Ha-rasV12 (8.3 ± 1.3-fold) was
significantly greater than that of Ha-rasV12
alone (p < 0.025) but was not significantly greater
than that of srcF527 alone. Since transfection
efficiency using the calcium phosphate method is routinely about 2%
and independent of the plasmids transfected (57), the reduction in the
magnitude of the responses when normalized to
-gal is a consequence
of a global stimulation of gene expression. This is manifest by the
activation of
-gal expression despite it being driven by the
constitutive cytomegalovirus promoter. Thus, the fold induction of
-gal was 4.7 ± 1.1, 7.7 ± 1.7, and 11.6 ± 3.0 for
Ha-rasV12, srcF527, and
Ha-rasV12 plus
srcF527-transfected cells, respectively.
srcF527 is therefore a more potent activator of
ANF expression than is Ha-rasV12 and, like
oncogenic ras (53), srcF527 also has
a powerful effect on gene expression in general. Similar conclusions
can be drawn from experiments using a LUX reporter responsive to the
activation of the AP-1 transcription factor complex (Fig. 1,
panels C and D). This reporter has very low basal activity in cardiac myocytes compared with the ANF-LUX transgene, accounting for the greater variability in stimulation when expressed as
fold induction by srcF527 and
Ha-rasV12.
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Oncogenic srcF527 Does Not Induce DNA Synthesis in
Cardiac Myocytes--
To determine whether the ability of
srcF527 to induce strong activation of gene
expression (Figs. 1 and 2) might reflect a capacity to re-initiate DNA
synthesis, myocytes were transfected with
srcF527 and subsequently analyzed for BrdUrd
incorporation into nuclei. Myocytes transfected with
srcF527 were identified by staining for
co-transfected -gal and did not show any nuclear staining for BrdUrd
(results not shown). Thus, the strong effects of
srcF527 on gene expression are transcriptional
effects and are not the result of changes in DNA synthesis.
Inhibition of srcF527-stimulated Gene Expression by
Dominant-negative ras--
The relative order of potency of
srcF527, Ha-rasV12, and
v-raf is in accord with observations in other cell types
that the transforming activity of Src is dependent upon Ras (61) and
that of Ras on c-Raf-1 (62). To investigate whether the increase in
cardiac gene expression in response to srcF527
requires functional Ras and/or Raf, the effects of DN
Ha-rasN17 and DNraf were determined.
In this series of experiments ANF-LUX expression was increased about
4-fold by srcF527, and this was inhibited by
co-transfection with Ha-rasN17 (Fig.
3). DNraf had a modest but
non-significant inhibitory effect (35.9 ± 8.3%) on
srcF527-induced ANF-LUX/-gal expression and
did not significantly potentiate the inhibition by
Ha-rasN17 (Fig. 3). However, the results with
the DNraf construct were complicated by its effect on
-gal expression. Transfection with DNraf inhibited
srcF527-induced ANF-LUX expression by 50.2 ± 5.2% but also inhibited
-gal expression in the presence of
srcF527 by 36.7 ± 5.6% in these same
transfections. Similarly, the DNraf construct reduced
-gal expression in the presence of Ha-rasV12
and in the presence of 0.1 mM PE by 20.2 ± 2.5 and
71.0 ± 1.3%, respectively (n = 4 separate
preparations). Thus, the DNraf construct has a predominantly
general effect to depress gene expression and which masks the effects
of DNraf on PE-, Ha-rasV12-, and
srcF527-induced ANF-LUX expression. These
results suggest that transmission of the signal from
srcF527 to ANF-LUX expression is mediated
through Ras but that Raf may not be the only downstream Ras effector
through which the signal is propagated.
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The Effect of Genistein on ANF-LUX Expression--
It has
previously been reported that activation of Ras and induction of ANF
expression in cardiac myocytes by PE can be blocked by the tyrosine
kinase inhibitor genistein (63). Surprisingly, we found that genistein
(20 µM) enhanced ANF-LUX/-gal expression in response
to srcF527 (Fig.
4, panel A). However,
genistein also increased basal ANF-LUX activity to a similar extent in
these experiments such that fold induction of ANF-LUX/
-gal by
srcF527 was unaltered by genistein when
expressed relative to the genistein control (Fig. 4, panel
B). In the light of these unexpected results, we re-examined the
effects of genistein on PE- and TPA-induced ANF-LUX expression.
Genistein had no effect on the fold activation of ANF-LUX/
-gal by PE
or TPA when expressed relative to the Me2SO control (Fig.
4, panel A). Again, because of its effect on basal ANF-LUX/
-gal expression, when the results were expressed relative to
the matched control, genistein appeared to inhibit PE- and TPA-induced
ANF-LUX/
-gal expression (Fig. 4, panel B). Thus, interpretation of these results is dependent upon the manner in which
they are expressed. A possible explanation for the altered responses to
TPA and PE in the presence of genistein is that under basal conditions
c-Src is kept in an inactive conformation through phosphorylation of
tyrosine 527 (in chicken c-Src) in the C-terminal tail by Csk (47, 64).
If the tyrosine kinase activity of Csk is more sensitive to inhibition
by genistein than is c-Src itself, the net effect of genistein could
result in an increase in c-Src activity.
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The Effect of srcF527, Ha-rasV12, and v-raf
on Myocyte Size--
As well as characteristic alterations in
transcription, hypertrophied cardiomyocytes are larger and more regular
in shape than control cells (2, 6, 7, 25, 60, 65). In separate experiments, transfection of myocytes with
srcF527, Ha-rasV12, or
v-raf significantly increased the size of transfected cells by 68, 129, and 62%, respectively, but there was no additional stimulation in the presence of both Ha-rasV12
and srcF527 (Fig.
5). The increases in cell size induced by
these oncogenes were as great as that elicited by maximally effective
concentrations of PE (57). To determine whether the increase in cell
size by srcF527 is dependent on its kinase
activity, the effect of a kinase dead srcF527
mutant (srcF527K) on myocyte size was
determined (Fig. 5, Expt. 3). Whereas srcF527 increased myocyte area by 74.8%
(p < 0.001), srcF527K
only
increased myocyte area by 22.5% (p < 0.001 versus srcF527), although this was still
significantly greater than the controls (p < 0.02). It
is unlikely that this is due to any residual kinase activity as
srcF527K
was completely ineffective in
stimulating ANF-LUX activity (0.8 ± 0.13-fold of control compared
with 11.8 ± 0.48-fold of control for
srcF527, n = 4). Thus, the
ability of srcF527 to induce an increase in cell
size is largely dependent on its kinase activity, although there is a
small kinase-independent component that may reflect an ability of
srcF527 (and srcF527K
)
to induce cell spreading.
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The Effect of srcF527 on Sarcomeric
Organization--
Another feature characteristic of myocytes exposed
to hypertrophic agonists is increased assembly and organization of
contractile proteins into sarcomeric units (2, 25, 60). To examine the
effects of srcF527 on sarcomerogenesis, myocytes
were co-transfected with pNG1, a vector that targets -gal to the
nucleus. This helps to prevent problems with cross-reactivity which can
arise when staining proteins in the same intracellular compartment with
two primary mouse antibodies. Myocytes were subsequently stained for
-gal to identify transfected cells (results not shown) and with
-MHC to highlight the contractile apparatus (Fig.
6). Transfection with empty vector had no
effect on sarcomeric organization (Fig. 6, panel A; compare
the transfected cell (see arrow) to the surrounding
non-transfected cells). Likewise, cells transfected with
srcF527 did not display increased organization
of the contractile apparatus into sarcomeric structures (Fig. 6,
panel B), even though the transfected cell is
morphologically distinct with a more clearly defined outline and
outgrowth of extended processes/pseudopodia. These probably represent
points of contact with the substratum. A combination of
srcF527 and Ha-rasV12
enhanced this definition of shape (Fig. 6, panel C), but
despite the clear change in size and appearance, there was little
evidence of increased sarcomerogenesis when compared with the
organization of the myofibrils seen in response to PE (Fig. 6,
panel D). Myocytes treated with PE also displayed more
defined points of anchorage to the dish.
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PE-induced ANF-LUX Is Not Inhibited by Kinase-dead src--
In an
attempt to determine whether c-Src may be a component of the
intracellular signaling pathway through which PE exerts its
hypertrophic transcriptional responses, myocytes were transfected with
kinase-dead wild type c-src (srcK) or
srcF527K
, and its effect on PE-induced
ANF-LUX was determined. Transfection with srcK
did not
affect ANF-LUX/
-gal expression in response to 10-100
µM PE (fold induction by PE in the absence and presence of 3-20 µg of srcK
was 14.9 ± 3.5 and 20.5 ± 7.1, respectively, n = 7). Similar results were
obtained using the srcF527K
construct; fold
induction of ANF-LUX/
-gal by 10 µM PE was 11.6 ± 3.3 and 18.8 ± 4.9 in the absence and presence of 3 µg of
srcF527K
, respectively (n = 4). In these experiments
-gal expression in the presence of
srcF527K
was reduced to 50.5 ± 11.2%
which accounted for the apparent stimulation of ANF-LUX/
-gal
expression. These results suggest that c-Src may not be an essential
component of the pathway through which PE transduces its
transcriptional responses. However, these conclusions remain to be
substantiated because the efficacy with which these kinase-dead Src
mutants can act to inhibit the function of endogenous c-Src is not
known.2
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DISCUSSION |
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The Src family of non-receptor protein tyrosine kinases is activated by a wide variety of agents (growth factors, cytokines, G-protein-coupled receptor agonists, UV light, etc.) and is intimately connected with cell growth (reviewed in Refs. 47-51 and 66). This is reflected by the ability of oncogenic src to transform cells (61), for which there is a functional dependence on Ras (61). Similarly, the transforming ability of oncogenic ras is dependent on c-Raf-1 (62), and unregulated c-Raf-1 protein kinase activity is itself oncogenic (67). The function of all three protooncogenes is closely interconnected because, although Src and Ras can independently activate c-Raf-1, both are required for full c-Raf-1 activation (43, 44, 68). The same is true for activation of A-Raf but not B-Raf which can be fully activated by oncogenic Ras alone (44). One view is that Ras is required to recruit c-Raf-1 to the plasma membrane where it can be activated by phosphorylation on tyrosine residues 340 and 341 by membrane-bound Src (42-44, 69). B-Raf lacks the tyrosine residues in c-Raf-1 and A-Raf which are phosphorylated by Src, which may explain its lack of sensitivity to activation by Src (44). However, since the activation of c-Raf-1 autokinase activity by v-src is not inhibited by expression of dominant-negative Ha-rasN17, v-src may at least partially activate c-Raf-1 via a Ras-independent pathway (68).
The importance of Ras activation in the hypertrophic response has been
demonstrated by the ability of oncogenic Ha-RasV12 protein
to induce the characteristic morphological and transcriptional changes
in myocytes and for dominant-negative Ha-rasA15 to
inhibit the hypertrophic response to PE (46). Oncogenic raf
has also been shown to induce the characteristic transcriptional changes in myocytes, although not morphological changes associated with
hypertrophy (70). Here we have compared the effects of srcF527, Ha-rasV12, and
v-raf with respect to their ability to initiate a gene
program associated with hypertrophy (Figs. 1 and 2). The order of
potency observed (srcF527 > Ha-rasV12 > v-raf) is in accord with
their functional hierarchy as described above and which is reinforced
by the observation that Ha-rasN17 inhibits
activation of ANF-LUX expression by srcF527
(Fig. 3). DNraf had a lesser specific effect on the response of ANF-LUX to srcF527, suggesting that c-Raf-1
may not be the only downstream effector that can transduce the effects
of activated Ras. Other possible Ras effectors include MEK kinase-1,
phosphatidylinositol-3-kinase, p120GAP, Ral-GDS and protein
kinase C (reviewed in Ref. 71). Of these other potential candidates,
MEK kinase-1 and protein kinase C
have been shown to induce ANF-LUX
when constitutively active forms are overexpressed in cardiac myocytes
(72-74), and Ras-GAP (a GTPase-activating protein for Ras) has also
recently been proposed to have an effector-like function in these cells
(75). Observations in fibroblasts that oncogenic src
associates with and phosphorylates Ras-GAP suggests an additional level
of complexity to the functional interaction of Src with Ras (76).
As well as these specific transcriptional effects associated with a
hypertrophic response, srcF527 and
Ha-rasV12 also stimulated general or global gene
expression, as previously reported for rasR12 (53).
In partial agreement with that study (53), we only detected a 1.6-fold
induction of SkM--actin by Ha-rasV12 when
corrected for
-gal expression (Fig. 2, panel B), although we observed a 6.5-fold activation of
-MHC under these conditions (Fig. 2, panel F). This discrepancy may be due in part to
the different oncogenic ras constructs used or to
differences in culture conditions because the stimulation of
SkM-
-actin-LUX and
-MHC-LUX reported here was much greater than
the effect reported for the Arg-12 Ras construct using the same LUX
reporters (53). The ability of both oncogenic src and
ras to stimulate general gene transcription after serum
withdrawal probably reflects their ability to substitute for growth
factors, the responses to which are dependent on the activation of the
cellular counterparts of these oncogenes.
The most striking features of myocyte morphology evoked by powerful hypertrophic agonists such as endothelin-1 and PE are an increase in cell size and the organization of the contractile apparatus into sarcomeric structures (Fig. 6, panel D, and Refs. 2, 25, 60). In common with oncogenic ras or raf, srcF527 increased myocyte size, but the effects of Ha-rasV12 and srcF527 were not additive (Fig. 5). This may reflect a limit on the size that these cells can attain under the conditions in which they are cultured. Despite its potent effects on gene transcription and cell size, srcF527, either alone or in combination with Ha-rasV12, did not detectably induce sarcomeric organization (Fig. 6, panels B and C). This result differs from the reported similarity in the pattern of ventricular myosin light chain-2 organization in myocytes microinjected with Ras oncoprotein and myocytes treated with PE (46). However, this could be due to a difference between injection of the Ras oncoprotein and transfection with a Ha-rasV12 expression plasmid because we have consistently failed to observe effects of transfected Ha-rasV12 on sarcomeric structural organization.3
One clear morphological effect of transfection of myocytes with srcF527 or with srcF527 plus Ha-rasV12 is the increased definition of the cell periphery and in particular the contact points with the substratum. A possible explanation for this phenomenon is that c-Src is known to associate with the plasma membrane and especially with focal adhesions (77). Here, it may phosphorylate cytoskeletal proteins, several of which have been described as Src substrates including the focal adhesion kinase p125FAK (78, 79), paxillin (80), and p130Cas (81). In accord with this view, the effects of srcF527 on myocyte morphology were greatly reduced in the absence of Src kinase activity. Indeed, interaction and phosphorylation of Src and p125FAK have been proposed as critical early steps in the process by which cell adhesion initiates intracellular signaling (reviewed in Ref. 49). Another recent report suggests that Src family tyrosine kinases are essential for ERK activation in myocytes in response to hydrogen peroxide (82). It is likely that Src family tyrosine kinases will also serve to transduce the effects of other stimuli in cardiac myocytes, although our initial studies do not support a role for Src in the response to PE.
The lack of effect of srcK on PE-induced transcription
might be interpreted in one of several ways. First, and most obviously, c-Src may not be involved in the hypertrophic response to PE. Second,
the hypertrophic response to PE could require c-Src but not its kinase
activity, such that transfected srcK
may be equally as
effective as c-Src in transducing the effect of PE. However, overexpression of SrcK
or SrcF527K
did not stimulate
ANF-LUX expression making this explanation unlikely (results not
shown). Third, although PE may signal through c-Src, when this pathway
is inhibited by SrcK
other Src family members may be able to
substitute for it. Alternatively, the response to PE may require
another member of the Src family that is not inhibited by SrcK
but
whose effects on activation by PE can be reproduced by
srcF527. Another point for consideration is that it
is also possible that srcF527 does not stimulate the
same pathways as activated endogenous c-Src. Finally, SrcK
and
SrcF527K
may not be effective competitors of c-Src. Thus,
whether c-Src or one of its family members is involved in the
hypertrophic response to PE remains equivocal.
In summary, srcF527 initiates changes in transcription associated with the hypertrophic response, an effect for which it is dependent on Ras. Transfection with srcF527 also increases myocyte size, although it does not increase the organization of the contractile apparatus into sarcomeric units. We conclude that members of the Src family of non-receptor protein tyrosine kinases may be important in mediating the transcriptional responses that occur during the development of cardiac hypertrophy, although the hypertrophic stimuli which may utilize Src as part of their signaling pathway have yet to be defined.
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ACKNOWLEDGEMENTS |
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We thank Chris Marshall, Richard Marais, Ken Chien, Michael Schneider, Joan Brown, Mike Rosenfeld, and Andy Fire for providing plasmids; and Nicola Haward and Richard Taylor for preparation of the myocytes.
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FOOTNOTES |
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* This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (to P. H. S. and S. J. F.) and Grant BS1 from the British Heart Foundation (to S. J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
British Heart Foundation Lecturer in Basic Science. To whom
correspondence should be addressed: Cardiac Medicine, NHLI Division, Imperial College School of Medicine, Dovehouse St., London SW3 6LY,
United Kingdom. Tel.: 44 171-352-8121 (ext. 3309/3314); Fax: 44 171-823-3392; E-mail: stephen.fuller{at}ic.ac.uk.
§ Current address: Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom.
1
The abbreviations used are: ANF, atrial
natriuretic factor; -gal,
-galactosidase;
-MHC,
-myosin
heavy chain; BrdUrd, bromodeoxyuridine; DN, dominant-negative; ERK,
extracellular signal-regulated kinase; LUX, luciferase; MAPK,
mitogen-activated protein kinase; MEK, MAPK (or ERK) kinase; PBS,
Dulbecco's Ca2+/Mg2+-free phosphate-buffered
saline; SkM-
-actin, skeletal muscle
-actin; SRE, serum responsive
element; TPA, 12-O-tetradecanoylphorbol-13-acetate; PE,
phenylephrine.
2 Richard Marais, personal communication.
3 S. J. Fuller, J. Gillespie-Brown, and P. H. Sugden, unpublished observations.
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REFERENCES |
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