Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182
Three hallmark features of the cardiac hypertrophic growth program are increases in cell size,
sarcomeric organization, and the induction of certain
cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by 1- adrenergic receptor agonists, such as phenylephrine
(PE) and other growth factors that activate mitogen-
activated protein kinases (MAPKs). In this study the
MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and
p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases
possessing gain-of-function mutations. Transfected
cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the
A- and B-type natriuretic peptides (NPs), as well as the
-skeletal actin (
-SkA) gene. While activation of JNK
and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and
-SkA promoter activities,
neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which
specifically activated p38, augmented cell size, induced
NP and
-Ska promoter activities by up to 130-fold, and
elicited sarcomeric organization in a manner similar to
PE. Moreover, all three growth features induced by
MKK6 (Glu) or PE were blocked with the p38-specific
inhibitor, SB 203580. These results demonstrate novel
and potentially central roles for MKK6 and p38 in the
regulation of myocardial cell hypertrophy.
CARDIAC myocytes, which are postmitotic, increase
in size during postnatal development through a
well-studied hypertrophic growth program. Myocardial cell hypertrophic growth is characterized by a number of phenotypic changes, including the activation of several immediate early genes (e.g., c-fos, c-jun, and egr-1), increased expression of genes encoding certain sarcomeric
proteins (e.g., Cultured neonatal rat ventricular cardiac myocytes have
served as a model system for studies aimed at gaining a
better understanding of this interesting mechanism of cell
growth. Primary myocardial cells respond to a variety of
stimuli by undergoing a hypertrophic growth program virtually identical to that observed in the developing neonate
and the pathologic adult myocardium (Van Bilsen and
Chien, 1993 Studies of the intracellular signaling mechanisms responsible for myocardial cell growth have focused on the mitogen-activated protein kinases (MAPK), in part because at
least one of the MAPKs, extracellular signal-regulated kinase (ERK), has been implicated in mitogenic growth in a
variety of cell types (Cobb et al., 1991 In cultured cardiac myocytes, PE activates Ras, Raf, MEK,
and ERK (Thorburn and Thorburn, 1994 Another member of the MAPK family, the stress-kinase
c-Jun NH2-terminal kinase (JNK) (Davis, 1994 JNK is activated in cultured myocardial cells by the
growth promoters PE and ET-1 (Bogoyevitch et al.,
1995a The third and most recently characterized member of
the MAPK family is the stress-kinase, p38, also known as
protein kinase HOG1 (Rouse et al., 1994 In previous studies, it was shown that a p38-like kinase is
induced in isolated perfused rat heart after preconditioning, which is a brief ischemic treatment known to protect
cardiac tissue from damage due to subsequent ischemic
episodes taking place soon thereafter (Bogoyevitch et al.,
1996 The goal of the present study was to compare the effects
of activating the p38 pathway with activation of ERK and
JNK on the three major features of the myocardial cell hypertrophic growth program- cell size, sarcomeric organization, and activation of cardiac gene expression. It was
found that only p38 activation conferred all three features
of myocardial cell growth in a manner similar to that observed using PE. These results suggest that p38 plays a
central role in mediating the cardiac hypertrophic growth
pathway, a concept that could also be applicable to other terminally differentiated cell types.
Cell Culture
Primary ventricular myocytes were prepared from 1-4-d-old neonatal rats
as previously described (Sprenkle et al., 1995 Transfections
After preplating (see above), myocardial cells were resuspended at a density of 30 million cells/ml minimal medium (DMEM:F12 [GIBCO BRL,
Gaithersburg, MD] containing 1 µg/ml BSA) and transfections were carried out as described previously (Sprenkle et al., 1995 Reporter Enzyme Assays
Transfected cells were maintained in DMEM:F12 supplemented with 10%
FBS for ~16 h after electroporation. The cells were then washed thoroughly and the medium was replaced with minimal medium. Unless otherwise
stated, 24 h later, the medium was again replaced with minimal medium ± 50 µM phenylephrine with 1 µM propranolol added to block Test Expression Constructs
To assess the effects of various signaling proteins, the following constructs
were used: pDCR H-RasV12 (codes for activated Ha-Ras; from D. Bar-Sagi, State University of New York at Stony Brook, NY), pDCR RacV12
(codes for activated Rac; from M. Cobb and J. Frost, University of Texas
Southwestern Medical Center, Dallas, TX), RSV-Raf-1 BXB (codes for
activated Raf-1 kinase; from U. Rapp, University of Wurzburg, Wurzburg,
Germany), pCMV5 MEKKCOOH (codes for activated MEKK-1; from G. Johnson, University of Colorado, Denver, CO), pcDNA3 MKK6 (Glu)
(codes for activated MKK6, or p38/MAPKK; from R. Davis, University of
Massachusetts, Worcester MA), ATF2/Gal4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA-binding domain;
from R. Davis), MEF2C/Gal4 (codes for MEF2C fused to the Gal4 DNA-
binding domain; from J. Han, The Scripps Research Institute, La Jolla,
CA), MEF2C-S/Gal4 (codes for mutant [Ser to Ala 387] MEF2C fused to
the Gal4 DNA-binding domain; from J. Han), pG5E1bLuc (codes for 5X
Gal4 sites cloned upstream of a prolactin promoter driving luciferase expression; from R. Davis). Preliminary experiments using different concentrations of each construct verified that optimal doses were chosen.
Immunocytofluorescence: Morphometric Analyses
To study the effects of activating the MAPK pathways on cell size and sarcomeric organization, myocardial cells were cotransfected with a test expression construct or an empty vector control and CMV- To study the effects of the test expression constructs on endogenous
cardiac gene expression, myocardial cells were cotransfected with a test
expression construct or an empty vector control and ANP-3003GL. After
fixation, the cells were immunostained for luciferase using a rabbit anti-
luciferase antiserum, and they were immunostained for ANP using a
mouse monoclonal antibody to rat ANP (Glembotski et al., 1987 Morphometric Analyses
Cell Size.
Transfected myocytes that immunostained positively for Sarcomeric Organization.
Transfected myocytes were identified by positive MAPK Assays
To test the effects of treatments on MAPK activity levels, hemagglutinin
(HA)-tagged forms of ERK1 (pCEP4 HA-wt-ERK1 from M. Cobb), JNK
(SRa-HA-JNK from G. Johnson) or p38 (pCEP4 HA-wt-p38Hog1 from
M. Cobb) were cotransfected with the test constructs. After the appropriate times, cultures were extracted in a buffer containing 10 mM Tris, pH
7.6, 1% Triton X-100, 0.05 M NaCl, 5 mM EDTA, 2 mM o-vanadate, and
20 µg/ml aprotinin. After brief centrifugation, extracts were incubated for
2 h at 4°C with HA monoclonal antibody (12CA5; Boehringer-Mannheim
Corp., Indiannapolis, IN), bound to protein A-Sepharose (Pharmacia
Biotech, Inc.) and immune-complex kinase assays were carried out using
the appropriate substrates, as described (Derijard et al., 1994 In each experiment, two identically treated cultures (1.5 × 106 cells/35-mm dish) were used for each treatment, and after densitometric analyses of
the exposed phosphorimage plates, values for each treatment were averaged.
MKK6 (Glu) Selectively Activates p38
in Myocardial Cells and Strongly Stimulates
Cardiac Gene Expression
To characterize the effects of overexpressing various signaling proteins on each of the three MAPK family members in the cardiac context, myocardial cells were cotransfected with constructs encoding gain-of-function forms of
Ras, Rac, Raf-1 kinase, JNKK kinase, or p38 kinase, and
constructs encoding HA-tagged p38, JNK, or ERK. In the
cardiac myocytes, Ras V12 served as a poor activator of either p38 or JNK, but as expected, it was a strong activator of ERK (Fig. 1). Rac V12 had no effect on p38 or ERK in
the cardiac cells; however, it strongly activated JNK, consistent with its hypothesized ability to serve as an upstream activator of MEKK1 (Lange-Carter et al., 1993
The abilities of the various expression constructs to activate three cardiac genes (ANP, BNP, and
MKK6 (Glu) Increases Cardiac Myocyte Size and
Sarcomeric Organization
Further studies were undertaken to compare the effects of
Raf BXB, MEKKCOOH, and MKK6 (Glu) with the gold
standard, PE, on other features of the program, such as
cell size and sarcomeric organization. Compared to cells
maintained in control media (Fig. 3, A and A
When myocardial cells were transfected with MKK6
(Glu), they were on average four times larger than control
cells (Figs. 3, E and 4 A), and notably, they displayed sarcomeric organization that was visually similar to that observed upon PE treatment (Figs. 3 E The Effects of PE and MKK6 Are Sensitive
to a p38 Inhibitor
To demonstrate that the effects of MKK6 (Glu) and PE on
myocardial cell growth and gene expression involved p38,
cultures were treated with the highly specific p38 inhibitor,
SB 203580 (Young et al., 1993
The stress-activated MAPK pathways, especially p38,
are well-known stimulators of ATF2 (Gupta et al., 1995 The SB 203580 compound also had potent inhibitory effects on myocardial cell size and sarcomeric organization
induced by PE or MKK6 (Glu) (Fig. 7). PE- or MKK6
(Glu)-mediated sarcomeric organization and increases in
cell size were reduced by >90% by SB 203580 (Fig. 8).
These results are consistent with a central role for a p38-like pathway in myocardial cell growth conferred by PE or
by MKK6 (Glu).
Recent studies have established that the transcription
factor, MEF2C, serves as a specific substrate for p38, such
that phosphorylation on serine 387 confers MEF2C-mediated transcriptional activation in RAW 264.1 cells (Han et
al., 1997
Under certain conditions, cardiac myocytes undergo nonmitotic, hypertrophic growth that is typified by dramatic
increases in cell size, high degrees of sarcomeric organization, and enhanced expression of certain cardiac-specific
genes. Several results from this study indicate that MKK6-activated p38 is sufficient to confer the three main features
of this unique hypertrophic growth program. First, MKK6
(Glu), which amongst the MAPKs induces only p38 (Fig. 1
and Raingeaud et al., 1996 To our knowledge, this is the first report to document
that the activation of the p38/MAPK pathway can mimic
the morphological changes and gene inductive effects of
growth factor treatment in any cell type. Thus, while p38/
MAPK is known as being a stress-activated kinase, it can
apparently contribute to cell growth in a manner that may
represent a compensatory response to stress. Although such a role for p38 contrasts with earlier findings that p38
induces apoptosis (Xia et al., 1995 The mechanism by which MKK6-mediated p38 activation could lead to myocardial cell hypertrophic growth remains to be elucidated; however, recent work has revealed
several downstream p38 targets that could be involved.
For example, p38 phosphorylates and activates several
transcription factors, such as ATF2 and Elk-1 (Gupta et al., 1995 Alternatively, p38 may activate other downstream kinases that serve as the final steps in the signaling program.
For example, it is well known that p38 can phosphorylate
and activate MAP kinase-activated protein kinases (MAPKAPs)-1, -2, and -3 (MAPKAP-3 a.k.a. 3pK) (Stokoe et al.,
1992a In addition to altering the function of transcription factors, p38-mediated MAPKAP/3pK activation could modulate other pathways that might favor cell survival, and in
cardiac myocytes, these pathways could contribute to the
development of myocardial cell-specific features, such as
myofilament organization. For example, MAPKAP-2 has
been shown to be activated during ischemic preconditioning of isolated rat hearts (Bogoyevitch et al., 1996 In summary, our results demonstrate a role for MKK6
and p38 in myocardial cell hypertrophic growth and gene
expression. This is consistent with the view that the hypertrophic growth program represents a compensatory response of the myocardium to stress. In a physiological context, the mediators of the hypertrophic response are often
hemodynamic stresses, such as increases in blood pressure
or volume. Accordingly, increases in myocardial cell size
and contractile function afforded by such growth could be
viewed as cellular adaptations designed to counteract a
physiological stress. Indeed, induction of the cardiac natriuretic peptide genes, which encode hormones that decrease blood pressure and volume, represent an endocrine
compensatory response by the myocardium. The findings
that the p38 pathway can mediate all three primary features of the myocardial cell growth program represent a
major advancement in our understanding of the signals
that regulate this important induction process. Future
studies aimed at determining how other signaling pathways, perhaps even the other MAPK pathways, complement the p38 pathway and how p38 itself contributes to
myocyte growth, sarcomeric organization, and cardiac
gene induction, should reveal new roles for this interesting
stress-kinase pathway in the heart.
-skeletal actin,
-myosin heavy chain, and
myosin light chain-2), and the induction of the genes for
the A- and B-type cardiac natriuretic peptides (ANP and
BNP)1 (Schneider et al., 1992
; Van Bilsen and Chien, 1993
;
Lembo et al., 1995
). Although myocardial mass in the fully
developed adult does not generally undergo significant increases in size, in some pathological conditions, such as
overload-induced hypertrophy, adult cardiac myocytes do
reenter a hypertrophic growth program very similar to
that observed in the developing neonatal heart (Schneider
et al., 1992
; Van Bilsen and Chien, 1993
; Lembo et al.,
1995
; Van Heugten et al., 1995
; Yamazaki et al., 1995
).
). For example, cultured myocardial cells treated
with the
1-adrenergic receptor agonist, phenylephrine
(PE), various other growth factors, or mechanical loading
or electrical pacing of contractions display marked increases in size, enhanced sarcomeric organization, and induction of the cardiac genes associated with the hypertrophic growth program (Simpson, 1983
; Komuro et al., 1990
; McDonough and Glembotski, 1992
; LaMorte et al.,
1994
; Sadoshima et al., 1995
; Bogoyevitch et al., 1995b
;
Karns et al., 1995
; Sprenkle et al., 1995
; LaPointe et al.,
1996
; Thuerauf and Glembotski, 1997
). The convergence
of these diverse stimuli on the features that define myocardial cell growth suggests that a select group of intracellular
signaling pathways are coordinately activated by these
treatments. Accordingly, a better understanding of myocardial cell signaling pathways that contribute to hypertrophic growth is required to fully grasp normal cardiac
development as well as counterproductive pathological
growth.
). Typically, ERK is
activated via the well-known sequential pathway sometimes referred to as a MAPK module. MAPK modules
consist of three members, a MAPKKK, followed by a
MAPKK and then the terminal MAPK, itself. In the case
of the ERK pathway, Ras stimulates the MAPKKK, Raf-1,
which activates the MAPKK known as MEK (Kyriakis et
al., 1992
; Davis, 1993
).
; Bogoyevitch et
al., 1993a
, b
Clerk et al., 1994
; Thorburn et al., 1994a
), and
transfection of constructs encoding active Ras, Raf, or
MEK can induce ANP,
-MHC,
-skeletal actin (
-SkA)
and/or MLC-2 promoter activities (Thorburn et al., 1993
,
1994b
; Abdellatif et al., 1994
; Thuerauf and Glembotski,
1997
) . Moreover, dominant interfering forms of Ras or Raf
can inhibit PE-induced ERK and cardiac gene promoter activity (Thorburn et al., 1993
, 1994b
; Thorburn, 1994
),
consistent with a role for ERK in the cardiac growth program. However, the overexpression of active forms of either Ras or ERK does not lead to the sarcomeric organization typical of hypertrophic growth (Thorburn et al.,
1994a
,b), and inhibiting MEK with PD 098059 does not
block PE-induced sarcomeric organization or ANP gene expression (Post et al., 1996
). Additionally, while myocardial cell ERK can be activated by some agonists, such as
PE, ET, or FGF, each of which can cause hypertrophic
growth, it can also be activated by agonists, such as ATP or
carbachol, neither of which support myocardial cell growth
(Post et al., 1996
). These findings suggest that the activation of ERK alone is not sufficient to confer hypertrophic
growth and the related gene expression.
; Cano and
Mahadevan, 1995
; Karin, 1995
) has received some attention as a potential mediator of growth in cardiac myocytes.
The activation cascade for JNK is somewhat less understood than that for ERK. However, it is believed that in
most cells the sequential stimulation of the Ras-like monomeric GTP-binding protein, Rac, perhaps by Ras itself, leads to the activation of MEKK1, a MAPKKK that then
activates the MAPKK, MEK4, also called SEK or JNK kinase, culminating in the activation of c-jun kinase (JNK)/
MAPK (Lange-Carter et al., 1993
; Derijard et al., 1994
).
), suggesting that it may contribute to the hypertrophic phenotype. Transfection of cultured myocardial
cells with a construct encoding active MEKK1 leads to an
approximately twofold increase in myocardial cell size and
5-50-fold enhancement of ANP,
-MHC, and
-SkA promoter activities (Bogoyevitch et al., 1995a
, 1996
). However,
there have been no reports that JNK activation fosters the
sarcomeric organization that is an obligate feature of the
hypertrophic phenotype. Thus, it appears that like the
ERK pathway, the JNK pathway alone may not be sufficient to support all of the main features associated with
the hypertrophic growth program.
; Raingeaud et
al., 1995
). In comparison to the JNK pathway but in contrast to ERK, the p38 pathway is not commonly activated
by mitogens but is induced by cell stresses such as endotoxins, osmotic shock, or metabolic inhibitors. The upstream activators of p38 are poorly understood; however,
recent studies have resulted in the cloning and characterization of members of the p38 MAPKK module, most notably MKK4, which can activate both JNK and p38 (Lin et
al., 1995
), and MKK3 (Derijard et al., 1995
) and MKK6
(Raingeaud et al., 1996
; Han et al., 1996
), either of which
specifically activate p38. MKK6 is also known as SAPKK3 (Cuenda et al., 1996
).
; Maulik et al., 1996
). The possibility that p38 activation could contribute to the protective effects of preconditioning is consistent with findings that p38 can lead to the
phosphorylation of heat-shock proteins (Stokoe et al.,
1992b
), a modification thought to enhance their cell-protective effects. However, the putative role of the p38 pathway in the hypertrophic growth of cardiac myocytes has
not been assessed.
MATERIALS AND METHODS
; Thuerauf and Glembotski,
1997
). After the enzymatic dissociation of ventricular tissue, the cells were
plated onto uncoated plastic dishes in DME/F-12 (1:1)/10% FBS for 1 h
during which time most of the fibroblasts adhered to the dish. The recovered cells were then transfected (see below), plated on fibronectin-coated
plastic dishes or glass slides (3 × 105 cells/mm2), and then maintained for
about 18 h in DME/F-12 (1:1)/10% FBS. The cultures were then washed
briefly with medium, refed with serum-free DME/F-12 (1:1), maintained
for 48 h, and then either extracted for reporter enzyme assay (see below)
or fixed and then analyzed by immunocytofluorescence or staining with
fluorescent phallicidin.
; Thuerauf and
Glembotski, 1997
). Briefly, for each transfection, 300 µl, or 9 million cells,
were mixed with 15-30 µg of either ANP-3003GL (Sprenkle et al., 1995
),
BNP-2501GL (Thuerauf and Glembotski, 1997
),
-SkA-394GL (MacLellan et al., 1994
), or pG5E1bLuc (Raingeaud et al., 1996
); 3-9 µg of cytomegalovirus-
-galactosidase (used as a normalization reporter except
where the test construct is known to activate CMV promoter activity
[Gillespie-Brown et al., 1995
; Paradis et al., 1996
; Post et al., 1996
]); and in
some experiments, 15-45 µg of an activated Ras, Rac, Raf-1, JNKKK,
MKK6, activating transcription factor-2 (ATF2)/Gal4, or MEF2C/Gal4
expression construct (see below). The levels of plasmid used in each culture within an experiment were equalized using empty vector DNA, such
as pCEP. Each 300-µl aliquot was then electroporated in a Bio-Rad (Hercules, CA) Gene Pulser at 700 V, 25 µF, 100
in a 0.2-cm-gap cuvette, a
protocol that allows for the selective transfection of only cardiac myocytes
(Sprenkle et al., 1995
). This procedure results in an ~30% viability
(Sprenkle et al., 1995
); Accordingly, the 3 million viable cells were plated
into fibronectin-coated 35-mm wells, at 1 × 106 cells/well, into 24-mm
wells at 0.5 × 106 cells/well, or into four-chamber Lab Tek chamber slides
at 0.3 × 106 cells/2 cm2 well.
-adrenergic receptors. Luciferase and
-galactosidase assays were performed as
described (Sprenkle et al., 1995
). Luciferase activity was measured for 30 s
on a Bio Orbit 1251 Luminometer (Pharmacia Biotech. Inc., Piscataway,
NJ). Data are expressed as "Relative Luciferase (Rel Luc)" = arbitrary
integrated luciferase units/
-galactosidase units, representative of at least
three independent experiments performed with two different plasmid preparations, and represent the mean and SEM of triplicate 35- or 24-mm wells.
-galactosidase.
Double-staining experiments demonstrated a cotransfection efficiency of
~85%. After treatment for 48 h with or without PE, cultures were fixed,
as described (McDonough and Glembotski, 1992
). Transfected cardiac
myocytes were identified by immunostaining for
-galactosidase using a
Texas red-conjugated anti-mouse IgG. Since the
-galactosidase is cytosolic, staining was uniform throughout the myocardial cells, facilitating the determination of cell area (see below). The same samples were also
stained with BODIPY-conjugated phallicidin, an actin-specific stain, to
evaluate sarcomeric organization, as described (McDonough and Glembotski, 1992
).
) and visualized using differential fluorescence. Generally, positive ANP staining
was visualized using a Texas red-conjugated anti-mouse IgG, and positive
luciferase staining was visualized using an FITC-conjugated anti-rabbit IgG.
-galactosidase were microscopically visualized under fluorescent illumination and photographed. The photographic images were then digitally acquired
using a scanner (model ES-1200C; Epson America, Inc., Torrance, CA)
attached to a Apple Power Mac 8500 (Cupertino, CA). The area in pixels
of each digitized image was determined using NIH Image software and
compared to a standard image possessing an area of 1 µm2. This enabled
the designation of area, in square micrometers, to each cell image; between 20 and 50 images of different cells derived from each treatment
were analyzed. The values reported are the mean areas, in square micrometers ± standard error.
-galactosidase immunostaining and observed using a Texas red-
compatible filter. The cells were then viewed after phalloidin staining
using an FITC-compatible filter and scored positively for sarcomeric organization if the myofilament alignment resembled that in cells treated with
PE. Approximately 50-100 cells from each of three cultures per treatment
were assessed. Generally, treating CMV-
-galactosidase/pCEP-transfected cells with PE (positive control) or without (negative control) resulted in ~25 and 2% of the
-galactosidase-positive cells scoring for sarcomeric organization, respectively. In experimental cultures transfected
with CMV-
-galactosidase and a test construct, the number of cells scoring positive for sarcomeres was normalized to (divided by) the number of
cells scoring positive for sarcomeres obtained with PE, which gave maximal values, and the results are displayed as percentages of maximal values.
; Post et al.,
1996
). Briefly, reactions were initiated by the addition of 1 µg of the appropriate substrate, MBP for ERK, GST-c-Jun for JNK, Phas-I for p38,
and 6 µM [
-32P]ATP (5,000 Ci/mmol) in a final volume of 30 µl of kinase
buffer (20 mM Hepes, pH 7.4, 20 mM MgCl2, 20 mM
-glycerophosphate,
2 mM DTT, 20 µM ATP). After 30 min at 25°C, the reactions were terminated by the addition of Laemmli sample buffer, and the phosphorylation level of substrate proteins was evaluated by SDS-PAGE followed by autoradiography and phosphorimage analyses.
RESULTS
;
Derijard et al., 1994
). Raf BXB, which encodes an active
form of Raf-1 kinase (Bruder et al., 1992
; Kolch et al., 1993
),
served primarily as an ERK activator, while MEKKCOOH, a truncated active form of MEKK1 (Lange-Carter et al.,
1993
), mildly activated p38 by about fourfold, moderately
activated JNK by 8-10-fold, as expected, but more strongly
stimulated ERK by about 25-fold (Fig. 1). The ability of
MEKKCOOH to activate both JNK and ERK is consistent
with results in other cell types (Minden et al., 1994
). Importantly, however, MKK6 (Glu), an activated form of the
p38 kinase, MKK6 (Raingeaud et al., 1996
), potently activated p38 in the cardiac myocytes by about 16-fold, with
no effect on either ERK or JNK (Fig. 1). A kinase-dead
form of MKK6, MKK6 (K82A) (Raingeaud et al., 1996
),
did not activate p38 in the cardiac cells (not shown). These
results verify the utility of constructs encoding activated
forms of Raf, MEKK1, and MKK6 as stimulators of the
MAPK pathways, and in particular, they clearly show that
MKK6 serves as a very selective p38 activator in cardiac myocytes.
Fig. 1.
Activation of p38, JNK, and ERK MAP kinases in myocardial cells. Myocardial cells were cotransfected with an expression construct encoding activated Ras (Ras V12), Rac (Rac
V12), Raf (Raf-1 BXB), JNK kinase (MEKKCOOH), p38 kinase
(MKK6 [Glu]), or an empty vector control (pCEP) and either
HA-p38, HA-JNK, or HA-ERK. After a 48-h incubation in serum-free media, the cultures (~3 × 106 cells each) were extracted
and incubated with an HA monoclonal antibody, and the appropriate kinase assay was carried out on the resulting immune complex, as described in the Materials and Methods. After exposing
the resulting SDS gel to a phosphorimager plate, each phosphorylated substrate band was digitized and printed (see the inset of
each panel). The relative density of each band was determined
using Molecular Dynamics Image Quant software (Sunnyvale, CA). Each treatment was carried out on two identical cultures, and the average of the band density for each treatment was then normalized to the maximal value obtained in each experiment.
Shown is the percentage of the maximum; the average variation
between duplicate samples was 10% or less. This is representative of three identical experiments that produced similar results.
[View Larger Version of this Image (26K GIF file)]
-SkA) that
serve as hallmarks of the hypertrophic growth program
were tested using ANP-3003GL, BNP-2501GL, or
-SkA-
394GL. These reporter constructs possess 3,003, 2,501, or
394 bp of the ANP, BNP, or
-SkA 5
-flanking sequences,
respectively. As expected from previous studies (Thorburn et al., 1993
; MacLellan et al., 1994
; Thuerauf and
Glembotski, 1997
), Ras V12 served as a strong activator of
both natriuretic peptide (NP) promoters, fostering up to
50-fold activation of luciferase expression (Fig. 2). The
Rac V12 construct also activated these promoters, but less
strongly than Ras, ~10-fold; this may reflect the differential efficacies of ERK and JNK as inducers of the cardiac
genes studied. Although Raf BXB and MEKKCOOH stimulated NP and
-SkA promoter activities by up to 20-fold,
most notable were the effects of the p38-activating construct, MKK6 (Glu), which stimulated up to 130-fold (Fig.
2). These findings suggest that while each of the MAPK
pathways can stimulate cardiac natriuretic peptide and
-SkA gene expression, the p38 pathway as stimulated with MKK6 (Glu) confers the strongest induction of the
three genes studied.
Fig. 2.
Effects of Ras, Rac, and MAP kinase pathway expression constructs on cardiac-specific promoter activities in myocardial cells. Myocardial cells were cotransfected with an expression
construct encoding activated Ras (Ras V12), Rac (Rac V12), Raf
(Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6
[Glu]), or an empty vector control (pCEP) and either an ANP
(ANP-3003GL), BNP (BNP-2501GL), or -SkA (
-SkA-394GL)
promoter/luciferase reporter construct. These reporter constructs
contain either the full-length, 3,003 bp of rat ANP 5
-flanking sequence, the full-length, 2,501 bp of rat BNP 5
-flanking sequence,
or
394 bp of the rat
-SkA 5
-flanking sequence driving the expression of a luciferase reporter. All cultures were also transfected with CMV-
-galactosidase. After a 48-h incubation in serum-free media, the cultures were extracted, and luciferase and
-galactosidase enzyme activities were assessed, as described in
the Materials and Methods. Values for luciferase enzyme units
obtained with each treatment were normalized to the maxima.
Values are means ± SE, n = 3 cultures. In this experiment, luciferase values were not normalized to
-galactosidase since one
of the constructs, MEKKCOOH, is a strong inducer of CMV-driven reporter expression, and it is believed that such normalization can be misleading, as previously reported (Gillespie-Brown et al.,
1995
; Paradis et al., 1996
; Post et al., 1996
).
[View Larger Version of this Image (25K GIF file)]
), the PE-treated cells were much larger (Fig. 3 B), displaying an approximately two- to three-fold increase in area (Fig. 4 A),
and they possessed a high degree of sarcomeric organization (Fig. 3 B
). In general, the PE-treated cultures possessed about 10-fold more myocytes displaying organized
sarcomeres than the control cultures (Fig. 4 B). PE-treated
cultures also displayed significantly increased levels of endogenous ANP expression, observed as the prototypical
perinuclear staining found often in hypertrophic cardiac
myocytes (Fig. 3, F [control] vs. G [PE-treated]). Interestingly, cultures transfected with Raf BXB or MEKKCOOH
displayed increases in size (Figs. 3, C [BXB] and D
[MEKKCOOH], and 4 A), and while the usual shape of the
Raf BXB-treated cells was similar to PE-treated cells, the
MEKKCOOH-treated cells were almost always very long and thin. Moreover, while either Raf BXB (Fig. 3 H
[BXB]) or MEKKCOOH (Fig. 3 I [MEKK-1]) fostered the
induction of endogenous ANP expression, neither construct supported sarcomeric organization (Fig. 3 C
[BXB]
and 3 D
[MEKK-1]; also see Fig. 4 B). These results suggested that neither ERK (Raf BXB) alone nor ERK and
JNK (MEKKCOOH) were sufficient to confer all the features of the hypertrophic phenotype.
Fig. 3.
Fluorescent microscopic analyses of the effects of Raf-1 BXB, MEKKCOOH, or MKK6 (Glu) expression constructs on size, sarcomeric organization, and endogenous cardiac-specific gene expression in myocardial cells. Myocardial cells were cotransfected with Raf
(Raf-1 BXB), JNKK kinase (MEKKCOOH), p38 kinase (MKK6 [Glu]), or an empty vector control (pCEP) and CMV--galactosidase
(A-E
) or ANP-3003GL (F-J), as described in the legend for Fig. 1. After 48 h of incubation in either serum-free control media or in the same media containing 10 µM of the
1-adrenergic receptor agonist, phenylephrine (PE) + 1 µM propranolol (the latter to block potential binding to
-adrenergic receptors), cultures were fixed in paraformaldehyde. (A-E)
-galactosidase expression (Gal), used to identify transfected cells, was visualized with a Texas red-conjugated second antibody and photographed using a rhodamine-compatible filter.
(A
-E
) Actin organization in the same
-galactosidase-positive cells shown in A-E was assessed by staining them with BODIPY-conjugated phalloidin (Phalloidin) and photographing them using an FITC-compatible filter. (F-J) In a separate experiment, luciferase expression (Luc), used to identify transfected cells, was visualized with an FITC-conjugated second antibody and photographed using an
FITC-compatible filter. The same cells were also assessed for endogenous ANP expression (ANP), viewed with a Texas red-conjugated
second antibody, and photographed with a rhodamine-compatible filter. The digitized photographic images of luciferase- and ANP-positive
cells were overlaid using Adobe Photoshop (San Jose, CA), and the resulting montage was prepared in Claris MacDraw Pro. Bar, 50 µM.
[View Larger Version of this Image (43K GIF file)]
Fig. 4.
Morphometric analyses of the effects of Raf-1 BXB,
MEKKCOOH, or MKK6 (Glu) expression constructs on size and
sarcomeric organization in myocardial cells. (A) Photographic
images of myocardial cells transfected and treated as described in
the legend to Fig. 3, A-E, were digitized and the areas (µm2) of
~20-50 cells from each treatment were determined using NIH Image software, as described in the Materials and Methods.
Shown are the mean area values for each treatment ± SE (n = 3 cultures. (B) The myofilament structure in myocardial cells transfected and treated as described in the legend to Fig. 3, A-E
, was
evaluated using BODIPY-phalloidin to stain actin. Upon visually
inspecting 50-100 transfected (i.e.,
-galactosidase-positive) cells
per treatment using a fluorescence microscope, cells were scored
for possessing organized sarcomeres (i.e., appearing similar to
cells shown in Fig. 3, B
or E
). The number of cells transfected
with test construct that scored positive for sarcomeric organization was normalized to (divided by) the maximal values for sarcomeric organization, which were obtained by PE treating cells that
had been transfected with the empty vector control; the results
are shown as a percentage of these maximal values. Generally,
~25% of the cells transfected with the empty vector control and
then treated with PE displayed highly organized sarcomeres.
Shown are the mean area values for each treatment ± SE (n = 3 cultures).
[View Larger Version of this Image (38K GIF file)]
and 4 B). Consistent
with the high degree of sarcomeric ordering was the finding that cells transfected with MKK6 (Glu) displayed spontaneous contractile activity. Moreover, like PE-treated cells, MKK6 (Glu)-transfected myocardial cells
possessed significantly elevated levels of endogenous ANP
(Fig. 3 J), consistent with the ability of MKK6 (Glu) to
strongly activate NP promoter activities (Fig. 2). Thus, it
was apparent that MKK6 (Glu), a selective p38 activator
in the cardiac myocytes, was able to mimic the three hallmark features of the hypertrophic growth program.
). At 20 µM, SB 203580 has
been shown to block p38/MAPK, while concentrations as
high as 100 µM have been shown to have no effect on 20 other protein kinases tested, including ERK and JNK
(Cuenda et al., 1995
). In the present study, SB 203580 (20 µM) blocked PE and MKK6 (Glu)-inducible NP promoter activity by between 40 and 70% (Fig. 5, A and B)
and decreased MKK6 (Glu)-activated
-SkA by >90%
(not shown). Additionally, myocardial cell p38 was shown
to be activated by PE, as was ERK; however, JNK was not stimulated under these conditions (Fig. 6). Taken together, these results confirmed a central role for p38 in
MKK6 (Glu) induction of cardiac gene expression and
strongly suggest that the ability of PE to induce NP expression is at least partly due to the MKK6/p38 pathway.
Fig. 5.
Effects of SB 203580 on ANP-, BNP-, or ATF2-dependent luciferase production in myocardial cells. Myocardial cells
were cotransfected with MKK6 (Glu) or an empty vector control
(pCEP and pCEP + PE) and either ANP-3003GL, BNP-2501GL,
or pG5E1bLuc reporter constructs. In C, cells were also transfected with ATF2/GAL4 (codes for the ATF2 transcriptional activation domain fused to the Gal4 DNA-binding domain). All
cells were also transfected with CMV--galactosidase for normalization purposes. Cultures were then maintained for 48 h with
or without SB 203580 (20 µM) or with or without DMSO (vehicle
control) and with or without PE (10 µM) + propranolol (1 µM),
as shown and then extracted and assayed for luciferase and
-galactosidase reporter activities, as described in Materials and Methods. Luciferase enzyme units were normalized to
-galactosidase, and the values obtained with each treatment were normalized to pCEP + PE. Values are means ± SE, n = 3 cultures.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Effects of PE on the activities of p38, JNK, and ERK in
myocardial cells. Myocardial cells were treated for 30 min with or without PE (10 µM) + propranolol (1 µM) and then extracted
and subjected to SDS-PAGE followed by Western analyses using
antibodies (New England Biolabs, Inc., Beverly, MA) that detect
p38, JNK, or ERK only when activated by dual phosphorylation
on Thr180 and Tyr182 (following the manufacturer's protocols).
Developed blots were then analyzed using a Molecular Dynamics
PhosphorImager. Each bar represents the mean blot intensity of
three identically treated cultures ± SE.
[View Larger Version of this Image (48K GIF file)]
;
Raingeaud et al., 1996
). ATF2 can dimerize with other
ATF family members (e.g., cAMP response element-binding protein [CREB] or ATF-1), Rb, NF-
B, or c-jun and
enhance transcription through cAMP response elements, AP-1 sites, or NF-
B sites. Accordingly, the abilities of
MKK6 (Glu) or PE to activate ATF2-dependent transcription in cardiac myocytes were tested. Myocardial cells
were cotransfected with a reporter plasmid possessing
GAL4 DNA-binding sites cloned upstream of luciferase, a
construct encoding a fusion protein composed of the
ATF2 transactivation domain and the GAL4 DNA-binding domain, and either MKK6 (Glu) or pCEP. Transfection with MKK6 (Glu) or treatment pCEP-transfected
cells with PE enhanced ATF2-mediated luciferase production (Fig. 5 C). Moreover, SB 203580 served as a potent inhibitor of MKK6 (Glu)-activated ATF2, consistent with a
major requirement for p38. Interestingly, PE-activated
ATF2 was only partially blocked by SB 203580 (Fig. 5 C),
as was PE-activated BNP transcription (Fig. 5 B), suggesting that the ability of this agonist to activate cardiac gene
expression is partly dependent on p38, or a very similar kinase.
Fig. 7.
Fluorescent microscopic analyses of the effects
of SB 203580 on size and sarcomeric organization in myocardial cells. Myocardial cells
were cotransfected with MKK6
(Glu) or an empty vector
control (pCEP and pCEP + PE) and CMV--galactosidase and then plated on glass
slides, as described in the legend for Fig. 3. After maintenance for 48 h in serum-free
control medium with or without SB 203580 (20 µM) or
DMSO (vehicle control) and
with or without PE (10 µM) + propranolol (1 µM), as
shown, cultures were fixed in
paraformaldehyde and immunostained for
-galactosidase expression (Gal) (A-
F), and the same cultures
were stained for actin with
BODIPY-phalloidin (Phall)
(A
-F
). Bar, 50 µm.
[View Larger Version of this Image (21K GIF file)]
Fig. 8.
Morphometric analyses of the effects of SB 203580 on
size and sarcomeric organization in myocardial cells. (A) Photographic images of -galactosidase-positive myocardial cells transfected and treated as described in the legend to Fig. 3 were digitized, and the areas were determined as described in the legend
to Fig. 4 A. Shown are the mean area values for each treatment ± SE (n = 3 cultures). The SB 203580 vehicle, DMSO, was included
in all controls and had a slight inhibitory effect itself on the ability of PE and the test constructs to increase cell area. (B) The myofilament structure was evaluated using BODIPY-phalloidin to
stain actin, as described in the legend to Fig. 4 B. Shown are the
mean area values for each treatment ± SE (n = 3 cultures).
[View Larger Version of this Image (26K GIF file)]
). Accordingly, the abilities of PE or MKK6 (Glu)
to activate MEF2C-dependent transcription in cardiac myocytes were tested. Myocardial cells were cotransfected with a reporter plasmid possessing GAL4 DNA-binding
sites cloned upstream of luciferase and a construct encoding a fusion protein comprised of the MEF2C transactivation domain and the GAL4 DNA-binding domain. Using
this system, the activation of MEF2C after phosphorylation on serine 387 can be studied in the cardiac myocytes
without interference from any endogenous MEF2 family
members. Treatment with PE or transfection with MKK6
(Glu)-enhanced MEF2C-mediated luciferase production
(Fig. 9, MEF2C/Gal4). When cells were transfected with an altered MEF2C/Gal4 chimera, in which serine 387 was
mutated to alanine, neither PE nor MKK6 (Glu) conferred luciferase induction (Fig. 9, MEF2C-S/Gal4). These
results indicate that like MKK6 (Glu), PE can activate
MEF2C in cardiac myocytes, an event that requires phosphorylation at serine 387 by p38. This result further supports the notion that in part, PE enhances myocardial cell growth, sarcomeric organization, and the related gene expression through a pathway involving p38 or a very similar
kinase.
Fig. 9.
Effects of PE or MKK6 (Glu) on MEF2C-dependent
luciferase production in myocardial cells. Myocardial cells were
transfected with pG5E1bLuc and either Gal4 (codes for Gal4
DNA-binding domain only), MEF2C/Gal4 (codes for MEF2C
fused to the Gal4 DNA-binding domain), or MEF2C-S/Gal4
(codes for mutant [Ser to Ala 387] MEF2C fused to the Gal4
DNA-binding domain). All cells were also transfected with
CMV--galactosidase for normalization purposes. (A) Cultures were maintained with or without PE (10 µM) + propranolol (1 µM), as shown, and then extracted and assayed for luciferase and
-galactosidase reporter activities, as described in Materials and
Methods. (B) Cultures were also transfected with the control vector, pCEP, or with the test construct, MKK6 (Glul), maintained
in control media for 24 h, and then extracted and assayed for luciferase and
-galactosidase reporter activities, as described in
Materials and Methods. Luciferase enzyme units were normalized to
-galactosidase, and the values obtained with each treatment were normalized to MEF2C/Gal 4 + PE (A) or MEF2C/
Gal 4 + MKK6 (Glu) (B). Values are means ± SE, n = 3 cultures.
[View Larger Version of this Image (22K GIF file)]
DISCUSSION
), conferred sarcomeric organization, increased cell size, and increased cardiac gene expression in a manner similar to the well-characterized
1-adrenergic receptor agonist, PE. Second, when induced by
MKK6, all three features of the growth program could be
blocked by the p38-specific inhibitor, SB 203580. Interestingly, PE-enhanced sarcomeric organization, cell size, and,
to some extent, cardiac gene induction were also blocked
by SB 203580, indicating that the p38 pathway probably plays an important role in
1-adrenergic receptor signaling
in myocardial cells. However, since the effects of PE on
BNP transcription and ATF2-enhanced transcription were
only partially blocked by SB 203580, it appears that while
p38 may be central to some features of the hypertrophic
response, it may play only a partial role in mediating other
aspects of the growth program.
), it is consistent with recent studies indicating that this stress kinase can promote
survival in certain cell types (Juo et al., 1997
); in this respect, p38 appears to function in a cell-specific manner.
; Livingstone et al., 1995
; Raingeaud et al., 1995
,
1996
), which could augment the expression of cardiac-specific genes induced during hypertrophy. Many of these inducible genes are known to possess relevant cis-acting sequences, including serum response elements, cAMP response
elements, AP-1 sites, and NF-
B sites. Most recently, it
has been demonstrated that the muscle cell-enriched transcription factor, MEF2C, a MADS box protein known to
bind to A/T-rich regions of muscle-specific genes and
known to be required for proper growth and development
of cardiac muscle (Edmondson et al., 1994
; Olson and
Srivastava, 1996
), serves as a substrate for p38 but not
ERK or JNK (Han et al., 1997
). In that report, the p38-specific phosphorylation was shown to lead to the activation of MEF2C as a transcription factor. Both the rat ANP
and BNP 5
-flanking regions, as well as regulatory regions
of other genes induced during the hypertrophic growth
program (e.g.,
-skeletal actin and
-myosin heavy chain
genes), contain A/T-rich regions that are required for transcriptional induction and could bind MEF2C or related
proteins (MacLellan et al., 1994
; Thuerauf et al., 1994
;
Karns et al., 1995
; Sprenkle et al., 1995
). Thus, it is possible
that p38 could phosphorylate and activate transcription factors that augment the expression of genes that participate in the cardiac growth program.
; Young et al., 1993
; Rouse et al., 1994
; English et al.,
1995
; Ludwig et al., 1996
; McLaughlin et al., 1996
; Sithanandam et al., 1996
; Tan et al., 1996
). In response to p38
activation by growth factors, MAPKAPs have been shown
to phosphorylate and activate selected transcription factors, such as CREB and ATF-1, usually at protein kinase
A/CaMK consensus sequences (Tan et al., 1996
). Thus, it
is possible that via the MAPKAPs, myocardial cell p38
stimulation could culminate with the activation of transcription factors often thought of as being downstream of
non-MAPKs, e.g., protein kinase A or CaMK.
; Maulik
et al., 1996
). Such preconditioning is known to serve as a
myocardial stress adaptation, resulting in enhanced protection from ischemia-induced myocardial cell death (Murry
et al., 1986
; Parratt, 1994
; Cumming et al., 1996
; Gottlieb
et al., 1996
). In part, it is believed that this cardioprotection is derived from the induction and activation of heat-shock proteins (hsp's) 27 and 70 (Marber et al., 1993
; Mestril et al., 1994
; Parratt, 1994
), both of which are known to protect cells from apoptosis (Mehlen et al., 1996
; Samali
and Cotter, 1996
; Sharma et al., 1996
). Interestingly,
MAPKAP-2 and -3 have been shown to phosphorylate hsp
27, a modification known to enhance its protective properties (Ahlers et al., 1994
; Huot et al., 1995
). Moreover, after
phosphorylation induced by either heat-shock or mitogen
stimulation, hsp 27 has been shown to bind to and stabilize
actin filaments in mouse fibroblasts (Lavoie et al., 1993
).
Such hsp 27-mediated filament stabilization in cardiac myocytes could be a major contributor to the striking sarcomeric organization observed upon MKK6-mediated p38
activation. Intracellular signaling pathways leading to hsp
activation and/or phosphorylation in cardiac myocytes
could conceivably extend back to
1-adrenergic receptors.
Indeed, adrenergic receptor stimulation has been shown to
activate hsp's in a variety of cell types; most notable is the
finding that
1-adrenergic agonists activate hsp 70 in rat
aortic cells (Chin et al., 1996
) and in rat cardiac cells
(Meng et al., 1996
).
Received for publication 9 May 1997 and in revised form 9 July 1997.
Address all correspondence to Christopher C. Glembotski, Department of Biology, San Diego State University, San Diego, CA 92182. Tel.: (619) 594-2959. Fax: (619) 594-6200. e-mail: cglembotski{at}sunstroke.sdsu.eduThis work was supported in part by National Institutes of Health Grants NS/HL-25073 (C.C. Glembotski), HL-46345 (C.C. Glembotski), HL-56861 (C.C. Glembotski) and HL-54030 (P.M. McDonough). This work was done during the tenure of a predoctoral research fellowship from the American Heart Association, California Affiliate, awarded to D.S. Hanford.
-SkA,
-skeletal actin;
ANP and
BNP, A- and B-type cardiac natriuretic peptides;
ATF, activating transcription factor;
CMV, cytomegalovirus;
ERK, extracellular signal-regulated kinase;
HA, hemagglutinin;
hsp, heat-shock protein;
JNK, NH2-terminal kinase;
MAPK, mitogen-activated protein kinase;
MAPKAP, MAPK-activated protein kinase;
PE, phenylephrine.
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