(Received for publication, June 12, 1995; and in revised form, August 23, 1995)
From the
Adult mammalian ventricular cardiomyocytes are terminally
differentiated cells that enlarge adaptively by hypertrophy. In this
situation, genes normally expressed in the fetal ventricular
cardiomyocyte (e.g. atrial natriuretic factor (ANF),
-myosin heavy chain (
-MHC), and skeletal muscle (SkM)
-actin) are re-expressed, and there is transient expression of
immediate early genes (e.g. c-fos). Using appropriate
reporter plasmids, we studied the effects of transfection of the
constitutively active or dominant negative mitogen-activated protein
kinase kinase MEK1 on ANF,
-MHC, and SkM
-actin promoter
activities in cultured ventricular cardiomyocytes. ANF expression was
stimulated (maximally 75-fold) by the hypertrophic agonist
phenylephrine in a dose-dependent manner (EC
, 10
µM), and this stimulation was inhibited by dominant
negative MEK1. Cotransfection of dominant negative MEK1 with a dominant
negative mitogen-activated protein kinase (extracellular
signal-regulated protein kinase (ERK2)) increased this inhibition.
Transfection with constitutively active MEK1 constructs doubled ANF
promoter activity. The additional cotransfection of wild-type ERK2
stimulated ANF promoter activity by about 5-fold. Expression of
-MHC and SkM
-actin was also stimulated. Promoter activity
regulated by activator protein-1 or c-fos serum response
element consensus sequences was also increased. We conclude that the
MEK1/ERK2 cascade may play a role in regulating gene expression during
hypertrophy.
Extracellular signal-regulated protein kinases (ERKs) ()are members of the mitogen-activated protein kinase (MAPK)
family and play an important role in intracellular signaling pathways
that lead to the division or differentiation of a number of cell types
(reviewed in (1, 2, 3, 4, 5, 6, 7) ).
This is probably attributable to their ability to phosphorylate a
variety of transcription factors and other signaling and structural
proteins (reviewed in (5) and (8) ). Three closely
related mammalian ERKs have been identified by molecular cloning (9) with ERK1 and ERK2 being the most widely
distributed(10) . ERKs are in turn activated by highly specific (11) MAPK (or ERK) kinases (MEK1 or
MEK2) by phosphorylation of a Tyr and a Thr residue in a conserved TEY
motif (reviewed in (1) and (4) ). MEKs and ERKs are
activated through protein-tyrosine kinase- and G protein
(G
- or G
-)-coupled receptors (reviewed in (3) ). The tyrosine kinase-mediated activation involves Ras and
the MEK kinase c-Raf (reviewed in (12) and (13) ),
which phosphorylates Ser
and Ser
(14) in rabbit MEK1 (or corresponding Ser residues in
other MEKs(15, 16, 17) ). Mutation of these
residues to Glu produces a rabbit MEK1 expressing increased
constitutive activity(14) . Activity can be further increased
by deleting additionally an inhibitory domain in the N-terminal
region(18) . Transfection of such constructs into cultured
cells results in differentiation and
transformation(18, 19, 20) .
In adult
mammals, the ventricular cardiomyocyte is a terminally differentiated
cell that loses its ability to mitose soon after birth. However, in
response to the imposition of an increased workload in vivo,
it adapts hypertrophically to accommodate the increased contractile
load (reviewed in (21) ). This process contributes
substantially to the clinical entity of ``cardiac
hypertrophy.'' In both the in vivo setting and primary
cultures of ventricular myocytes from neonatal rat hearts, a number of
characteristic transcriptional modifications distinguish the
hypertrophy from normal maturational growth (reviewed in (21) ). Following a hypertrophic stimulus, immediate early gene (e.g. c-fos, c-jun, egr-1)
expression is rapidly and transiently up-regulated. Following this,
genes that are only normally expressed in the fetal ventricle are
re-expressed (e.g. atrial natriuretic factor (ANF),
-myosin heavy chain (
-MHC), and skeletal muscle
-actin
(SkM
-actin)). In the slightly longer term, expression of
constitutive contractile protein genes (e.g. ventricular
myosin light chain-2, cardiac muscle
-actin) is increased.
Stimulation of promoter activity for these genes has frequently been
used as a marker of the hypertrophic response (reviewed in (21) ).
The precise physiological stimuli that induce
cardiac hypertrophy in vivo have not been identified, and the
cause may be multifactorial. In cultured myocytes, a number of
interventions lead to the acquisition of the hypertrophic phenotype.
These include sympathoadrenal agonists (especially
-adrenergic
agonists(22, 23, 24, 25) ), direct
activation of protein kinase
C(26, 27, 28, 29) , vasoactive
peptides (e.g. endothelin-1 (30, 31, 32, 33) and angiotensin
II(34, 35) ), growth factors (e.g. fibroblast
growth factors(36) , insulin-like growth factor-1(37) ,
and insulin-like growth factor-2 (38) ), and mechanical
stretch(39, 40, 41) . In the heart, the
-adrenergic agonist phenylephrine (42) and
many of other hypertrophic agonists activate ERKs(42, 43, 44, 45, 46, 47) and,
where studied, MEKs(44, 47) . On the basis of this
correlation, we suggested (42, 44) that activation of
the ERK cascade is important in the development of the hypertrophic
phenotype. Here, we show directly that specific activation of the ERK
cascade using transfected expression plasmids encoding constitutively
active MEK mutants leads to stimulation of a variety of promoters of
genes known to be up-regulated during the hypertrophic response.
Myocytes were
transfected 24 h after the initial plating by a calcium phosphate
precipitation method. Plasmids were diluted in 0.25 M CaCl, and an equal volume of 50 mM Bes (pH
6.9), 280 mM NaCl, 1.5 mM Na
HPO
was added. After 20 min, cells were transfected with this
suspension (1 ml/plate). Myocytes were standardly transfected with 15
µg of LUX reporter plasmid, 4 µg of pON249, and a total of 10
µg of test plasmid(s). In control experiments, test plasmids were
replaced by empty vectors. Controls were carried out concurrently for
each transfection. In experiments where the dependence of ANF-LUX
expression on phenylephrine concentration was studied, test plasmids
were omitted.
After transfection for 16-20 h, cells were
washed in maintenance medium containing 10% horse serum and then twice
with maintenance medium. Cells were incubated for 48 h in maintenance
medium, washed twice with ice-cold phosphate-buffered saline, and
extracted on ice with 0.1 M potassium phosphate (pH 7.9), 0.5%
(v/v) Triton X-100, 1 mM dithiothreitol (0.4 ml) for 15 min.
LUX activity was assayed in 0.5 ml of 100 mM Tricine (pH 7.8),
10 mM MgSO, 2 mM EDTA, 75 µM luciferin, and 5.5 mM ATP. Light emitted was measured
using an LKB 1219 RackBeta liquid scintillation counter with the
photomultipliers set out of coincidence.
For assessment of
transfection efficiency and cell area, cells were washed twice with
ice-cold phosphate-buffered saline, fixed with 4% formaldehyde for 10
min, and stained with 0.2 mg/ml
5-bromo-4-chloro-3-indolyl--D-galactopyranoside, 5
mM K
Fe(CN)
, 5 mM
K
Fe(CN)
, 2 mM MgCl
in
phosphate-buffered saline. The number of blue cells in 100 fields was
counted for each treatment. Cell area of transfected (blue) cells was
estimated using an image grabber and planimetry.
Figure 1: Dependence of ANF-LUX expression on phenylephrine concentration. Following transfection of cardiomyocytes with the ANF reporter plasmid (15 µg/plate) and pON249 (4 µg/plate) for 16-20 h, the cells were exposed to phenylephrine for 48 h, and expression of LUX activity was measured as described under ``Experimental Procedures.'' For each of the six separate experiments (minimum of three at any given phenylephrine concentration), data were fitted to sigmoid curves using the GraphPad (San Diego) Inplot 4 program. Data were normalized taking the derived maximum luciferase activity as 100%, averaged, and replotted. Data are means ± S.E.
Figure 2: Stimulation of ANF-LUX expression by MEK1 and ERK2 expression plasmids. Cardiomyocytes were transfected with the ANF reporter plasmid (15 µg/plate), pON249 (4 µg/plate), and MEK1 and ERK2 expression plasmids (total of 10 µg/plate, 5 µg of each or 5 µg of empty vector when appropriate) for 16-20 h as described under ``Experimental Procedures.'' Cells were extracted after a further 48 h in maintenance medium. Results (mean ± S.E., n = five separate experiments) are expressed relative to control transfections with the pEXV3 vector (10 µg). Statistical significance by a paired two-tailed t test as follows: a, p < 0.05; b, p < 0.02; c, p < 0.01 versus transfection with pEXV3 vector; d, p < 0.05 versus transfection with MEK(wt) + ERK(wt) expression plasmids.
Activation of other promoters (see Table 2) up-regulated during the hypertrophic response was also
examined (Fig. 3). Under optimal conditions for ANF promoter
stimulation (i.e. transfection with ERK2(wt) +
MEK1(E/E
)), the
-MHC promoter activity
was stimulated 15-fold, c-fos SRE activity was stimulated
10-fold, and SkM
-actin activity was stimulated 5-fold. Despite
the higher constitutive specific activity of human
MEK1(
N3.E
/D
) over rabbit
MEK1(E
/E
), transfection of cardiomyocytes
with ERK2(wt) + MEK1(
N3.E
/D
) did
not significantly increase the activities of the ANF,
-MHC, and
SkM
-actin promoters or of the c-fos SRE over ERK2(wt)
+ MEK1(E
/E
) (results not shown).
However, the reasons for this could have been trivial (e.g. from differences in levels of expression of MEK1). Levels of
expression of the constructs (even epitope-tagged constructs) cannot be
easily assessed in cultured cardiomyocytes because of the low
transfection efficiency.
Figure 3:
Stimulation of AP-1-regulated, -MHC,
c-fos SRE, and SkM
-actin promoters by MEK1 and ERK2
expression plasmids. Cardiomyocytes were transfected with reporter
plasmids for TRE/AP-1,
-MHC, c-fos SRE, or SkM
-actin (15 µg/plate) and additionally with pON249 (4
µg/plate), and MEK1 and ERK2 expression plasmids (5 µg of
each/plate) for 16-20 h as described under ``Experimental
Procedures.'' Cells were extracted after a further 48 h in
maintenance medium. Results (mean ± S.E., n =
five separate experiments) are expressed relative to control
transfections with the pEXV3 vector (10 µg). The open bar shows the vector control (100%), the solid bars represent
transfections with MEK1(wt) + ERK2(wt), whereas the cross-hatched bars represent transfections with
MEK1(E
/E
) + ERK2(wt). Statistical
significance: a, p < 0.05; b, p < 0.05; c, p < 0.001 versus transfections with pEXV3 vector by a paired two-tailed t test.
The sensitivity of TRE/AP-1 sites in the
TRE2PRL(-36) construct to activation by MEK1 was also examined.
Cotransfection of MEK1(E/E
) with ERK2(wt)
stimulated LUX activity significantly by 24.0 ± 7.3-fold (mean
± S.E.), whereas cotransfection of rabbit MEK1(wt) and ERK2(wt)
resulted in a statistically insignificant 3.0 ± 1.0-fold (mean
± S.E.) increase (Fig. 3). Human MEK1(wt) + ERK2(wt)
also increased LUX activity 5.0 ± 1.4-fold (mean ± S.E., n = five separate preparations of cardiomyocytes).
MEK1(
N3.E
/D
) + ERK2(wt) increased
LUX activity by 682 ± 205-fold (mean ± S.E., n = five separate preparations of cardiomyocytes, p < 0.02 by an unpaired two-tailed t test versus MEK1(E
/E
) + ERK2(wt)). Activation
by the wild-type MEK1 constructs alone in pEXV3 or pCEP4L did not
differ. The AP-1-regulated promoter was the only one that showed
significantly greater activation with the human
MEK1(
N3.E
/D
) expression plasmid than
with the rabbit MEK1(E
/E
) construct. No
stimulation of LUX activity by MEK1(E
/E
)
+ ERK2(wt) was detected with the promoterless vectors for ANF or
TRE/AP-1 (results not shown).
One pathway that stimulates promoter activities of genes
induced during hypertrophy involves the binding of agonists to
G-coupled receptors, thereby stimulating phospholipase
C
-mediated hydrolysis of membrane
phosphatidylinositols(30, 49, 51, 52, 53, 54, 55) .
The ensuing increase in sn-1,2-diacylglycerol concentrations
activates the appropriately sensitive isoforms of protein kinase
C(56, 57, 58, 59, 60) , and
this leads indirectly to a increase in promoter activities (reviewed in (21) ). Equally, direct activation of protein kinase C induces
the hypertrophic phenotype(26, 27, 28, 29, 57, 61, 62) as
does transfection of myocytes with constitutively active
G
(63) . Hypertrophic agonism and protein kinase C
activation also stimulate the MEK/ERK
cascade(42, 43, 44, 45, 46) ,
which is strongly implicated in the regulation of cell growth and
differentiation (reviewed in (2, 3, 4, 5) and 7). We have proposed that activation of MEK and ERK is
an important aspect of the hypertrophic
response(42, 44) .
Activation of rabbit MEK1
involves phosphorylation of Ser and/or Ser
in a LIDS
MANS
sequence (14) .
Mutation of Ser
Glu or double mutation of
Ser
and Ser
Glu produces MEK1
species, which are 30-40-fold more active than the
unphosphorylated enzyme(14) . However, in vitro, these
mutated species express only about 0.5% of the activity of recombinant
wild-type MEK1 that had been phosphorylated by c-Raf(14) .
Despite this relatively low level of activity, recombinant
MEK1(E
/E
) is able to phosphorylate and
activate recombinant ERK2 fully in vitro. (
)By
combining mutation of the analogous Ser residues in human MEK1
(Ser
Glu and Ser
Asp) with
deletion of an N-terminal 20-amino acid predicted
-helix, Mansour et al.(18) produced a MEK1 that was 400 times more
active than the unphosphorylated wild-type MEK1. As discussed in (19) , the apparent discrepancy between the low level of
constitutive activity of recombinant
MEK1(E
/E
) and the ability of transfected
MEK1(E
/E
) to activate ERK can be
rationalized as follows. First, a small activation of MEK may be
sufficient to activate ERK fully(64) . Second, following
exposure of cells to suitable agonists, endogenous MEK activity is
stimulated but rapidly returns to basal values(47) , presumably
because of protein phosphatase activity.
MEK1(E
/E
) is not subject to such
regulation.
To examine whether MEK (and hence its sole substrate,
ERK(11) ) plays any role in the regulation of expression of
hypertrophic marker genes, we transfected cardiomyocytes with plasmids
encoding constitutively active MEK1. Transfection with
MEK1(E) stimulated ANF promoter activity by about 2-fold,
and MEK1(E
/E
) showed the same trend (Fig. 2). Stimulation was increased to approximately 5-fold when
ERK2(wt) was additionally cotransfected (Fig. 2). Thus, ERK may
be limiting in the cardiomyocyte. Under optimal conditions (ERK2(wt)
and MEK1(E
/E
) cotransfected), activities of
promoters for
-MHC and SkM
-actin and the c-fos SRE
were stimulated by up to 15-fold (Fig. 3). Cotransfection of
MEK1(
N3.E
/D
) (18) with
ERK2(wt) did not increase promoter activation further (results not
shown).
The stimulation of ANF promoter activity by
MEK1(E) or MEK1(E
/E
), even
with cotransfection of ERK2(wt) (Fig. 2), was much less than the
75-fold stimulation seen with maximally effective concentrations of
phenylephrine (Fig. 1). In contrast, the agonist-stimulated and
the MEK1(E
/E
)-stimulated responses were
similar in magnitude in the PC12 cell line(19) . The relatively
low constitutive activities of MEK1(E
) and
MEK1(E
/E
) may be more important in the
primary cultures of cardiomyocytes than in established cell
lines(19) . The time courses of protein expression from the
constructs may differ between cell types. Alternatively, the MEK/ERK
cascade may not be of prime importance in the regulation of ANF
promoter activity and, by implication, development of the hypertrophic
phenotype. However, the stimulation of ANF promoter activity by
phenylephrine is decreased by transfection with a plasmid encoding
dominant negative MEK1(A
) and inhibition is increased by
cotransfecting additionally with dominant negative
ERK2(A
/A
/F
). Thorburn et
al. have also recently shown that transfection of dominant
negative ERK1 (as well as chemical inhibition of ERK) blocked
activation of the ANF, MLC-2 and c-fos promoters by
phenylephrine(45) .
The detailed mechanisms involved in the
activation of promoters for hypertrophic marker genes are unclear. For
the ANF gene, the region principally responsible for
phenylephrine-inducible expression (base pairs -323 to -638) contains
TRE/AP-1, AP-2, CRE, Egr-1 and SRE/CArG consensus
sequences(25) . Cotransfection of
MEK1(E/E
) or
MEK1(
N3.E
/D
) with ERK2(wt) stimulates
at TRE/AP-1-regulated expression by 24- or 680-fold, respectively. This
is the only example we found of MEK1(E
/E
)
and MEK1(
N3.E
/D
) differing
significantly. Activation at TRE/AP-1 sites involves the binding of
heterodimers of members of the Fos and Jun families (AP-1 complexes),
transactivation being stimulated by the phosphorylation of Jun
(reviewed in (65) ). Although ERK can phosphorylate the
N-terminal transactivation domain of c-Jun(66) , a separate Jun
N-terminal kinase (also known as stress-activated protein kinase)
family has been identified (reviewed in (67) and (68) ). However, c-Jun N-terminal kinase/stress-activated
protein kinases are not activated by MEK1(69) . Furthermore, Fig. 3shows that MEK1 + ERK2 is capable of activation at
AP-1 sites, presumably by phosphorylation of the AP-1 complex. An
alternative pathway for activation of the ANF and SkM
-actin
promoters is through their SRE/CArG
sequences(25, 70) . This sequence is present in the
native c-fos promoter (reviewed in (71) ) and in the
c-fos construct used here. Phosphorylation of the
transcription factor Elk-1 (p62-TCF) by ERK increases its
transactivating activity at SRE/CArG(72) . Alternatively, other
transcription factors (e.g. Egr-1, expression of which is
coregulated with c-fos(73) ) may be involved in the
mediation of the hypertrophic
response(24, 25, 74) .
Our overall
conclusions are that activation of the MEK/ERK cascade can stimulate
ANF-, -MHC-, c-fos SRE-, SkM
-actin-, and
TRE/AP-1-regulated promoter activity. This may be of relevance to the
development of the hypertrophic phenotype in the ventricular
cardiomyocyte.