Articles |
From the First Department of Internal Medicine, Kobe (Japan) University School of Medicine.
Correspondence to Mitsuhiro Yokoyama, MD, The First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
| Abstract |
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Key Words: RhoA myocardial cell c-fos promoter/enhancer
| Introduction |
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The c-fos gene is the most frequently studied member of the
cellular immediate-early genes, whose transcription is
activated rapidly and transiently within minutes of growth
stimulation in a variety of cell types.10 11 These
immediate-early genes are themselves "third messengers" of
intracellular signaling cascades, encoding proteins in transcriptional
regulatory complexes. c-fos gene expression is rarely
detected in MCs of the normal adult rat or mouse. However,
hemodynamic stress and
1-adrenergic
agonists, both of which are known to produce cardiac
hypertrophy, rapidly provoke c-fos gene
expression in MCs.12 13 Thus, the c-fos gene
may play an important role in myocardial signal transduction.
Recently, it has been reported that RhoA regulates
c-fos promoter/enhancer activity through the
c-fos SRE in NIH 3T3 cells14 and that
RhoA is involved in G
q and
1-adrenergic receptor signaling in MCs.15
Although Rho and Rho GDI are known to be
present in the heart,16 17 the exact mechanism for
signal transduction of Rho has not been elucidated in MCs.
Since the c-fos promoter/enhancer region contains major
inducible cis-acting elements, ie, SRE, CRE, and TRE, we
attempted to explore the signaling pathway by which Rho
activates the c-fos promoter/enhancer, including the
c-fos SRE, CRE, and TRE in MCs. The present study
demonstrates for the first time that the c-fos
promoter/enhancer, the c-fos SRE, and the TRE are stimulated
by activated RhoA in MCs. Particularly in the
c-fos SRE, the SRF binding site rather than the TCF binding
site mediates the effect of activated RhoA.
Furthermore, the signaling pathway from activated
RhoA to the c-fos promoter/enhancer is
insensitive to PKC inhibitors or tyrosine kinase
inhibitors.
| Materials and Methods |
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Plasmids
The wild type of RhoA, RhoA Val14 (the
mutant of Gly to Val at codon 14), which is a point-mutated active form
of RhoA,6 RhoA Ala37 (the mutant of
Thr to Ala at codon 37), which is a biologically inactive effector
domain mutant,18 and Rho GDI constructed into
the pcDSR
expression plasmid were kindly provided by Prof Y. Takai
(Osaka University, Suita, Japan). The c-fos
promoter/enhancer linked to the luciferase gene (c-fos
luciferase), SRE CAT, CRE CAT, TRE CAT, pTKCAT, and
PKCß
expression plasmids were generous gifts from Prof K. Kaibuchi (Nara
Institute of Science and Technology, Ikoma, Japan).19 20 21
The 445-bp fragment of the c-fos promoter/enhancer
(positions -404 to +41 in the c-fos gene) was cloned into
pSVO-luciferase to construct c-fos luciferase. The deletion
fragment (-323 to +41) of the c-fos promoter/enhancer,
which contains the c-fos SRE and TRE, was cloned into
pSVO-luciferase to construct -323 c-fos luciferase. The
deletion fragment (-305 to +41) of the c-fos
promoter/enhancer, which contains the c-fos TRE but lacks
the c-fos SRE, was cloned into pSVO-luciferase to construct
-305 c-fos luciferase. The deletion fragment (-293 to +41)
of the c-fos promoter/enhancer, which lacks both the
c-fos SRE and TRE, was cloned into pSVO-luciferase to
construct -293 c-fos luciferase. The deletion fragment
(-323 to +41 without -300 to -294) of the c-fos
promoter/enhancer, which contains the c-fos SRE but lacks
the c-fos TRE, was cloned into pSVO-luciferase to construct
-323
-300/-294 c-fos luciferase. These deletion
fragments were made by use of polymerase chain reaction.
Nucleotide sequencing confirmed the structure of each
deletion fragment. The c-fos SRE, the CRE, and the TRE
(5'-CAGGATGTCCATATTAGGACATCTG-3', 5'-GAGC CCGTGACGTTTACAC-3', and
5'-ATGAGTCAGCGCG GATC-3', respectively) were synthesized by a DNA
synthesizer. SRE CAT, CRE CAT, and TRE CAT were synthesized by
introducing a synthetic c-fos SRE, three synthetic CREs, and
three synthetic TREs, respectively, upstream from the IL3 promoter
(from positions -50 to +10 in the mouse IL3 gene) fused to the CAT
sequence.19 20 According to the previous
reports,22 23 24 25 SRE.L
(5'-CTGTATGTCCATATTAGGACATCTG-3'), a derivative
of the c-fos SRE that contains the intact binding site for
SRF but lacks the TCF binding site, SRE.M
(5'-CAGGATGTC CCAATCGGGACATCTG-3'), a
derivative of the c-fos SRE that contains the intact binding
site for TCF but lacks the SRF binding site, SRE.LM
(5'-CTGTATGTCCCAATCGGGACATCTG-3'),
a derivative of the c-fos SRE that lacks both TCF and SRF
binding sites, and TRE.M
(5'-AGGAGTTGGCGCGGATC-3'), a derivative of the
TRE that lacks the AP-1 binding site, were synthesized by a DNA
synthesizer. SRE.L CAT, SRE.M CAT, SRE.LM CAT, and TRE.M CAT were
synthesized by introducing a synthetic SRE.L, a synthetic SRE.M, a
synthetic SRE.LM, and three synthetic TRE.Ms, respectively, upstream
from the IL3 promoter fused to the CAT sequence. The Pvu
IIPst I fragment (from positions -197 to +18) of the
promoter region of the thymidine kinase gene of herpes simplex virus
was cloned upstream from the CAT sequence to construct pTKCAT. pcDSR
PKCß, the expression plasmid for a constitutively
activated mutant of PKC-ß, was constructed by deleting the
coding region for amino acids 6 to 159 of PKCß (25% of the
V1 and 90% of the C1 region) into the pcDSR
expression plasmid.21 pTKCAT or the RSV promoter linked to
the luciferase reporter gene (RSV luciferase) was used as an internal
control to standardize the transfection efficiency.
Cell Culture and Transfection
Single-cell cultures were prepared from neonatal rat hearts as
described previously with slight modifications.13 MCs were
distributed to 60-mm dishes at a density of 6.5x105 cells
per dish. The population of nonmyocytes was <10% of the total
cell population. The culture medium was DMEM/F-12 supplemented with 5%
calf serum. The medium was changed 24 hours after seeding the cells to
serum-free medium, which is DMEM/F-12 containing 0.1% bovine serum
albumin, ITS (10 µg/mL insulin, 10 µg/mL
transferrin, and 10 ng/mL selenious acid), and 30
mmol/L HEPES at pH 7.5. MCs in duplicate dishes were transfected
with reporter plasmids by using the modified calcium phosphate
precipitation method as described previously.26 The
DNA/CaPO4 prepicitates in each dish (5.0 mL) contained the
following if not specifically identified: 5.0 µg of c-fos
luciferase, SRE CAT, SRE.L CAT, SRE.M CAT, SRE.LM CAT, CRE CAT, TRE
CAT, or TRE.M CAT reporter plasmid; 4.0 µg of pcDSR
PKCß,
pcDSR
RhoA, pcDSR
RhoA Val14, pcDSR
RhoA Ala37, and/or pcDSR
Rho GDI; 4.0 µg of
pTKCAT or 0.1 µg of RSV luciferase as an internal control for
variations in transfection efficiency; and variable amounts of
pcDSR
plasmid vector to adjust total DNA. Transfection was carried
out using 17 µg of total DNA. Precipitates were removed after 2
hours, and then the cells were maintained in serum-free medium for 48
hours. For the last 24-hour incubation, Ro 31-8220 or herbimycin A was
added to inhibit PKC or tyrosine kinase, respectively.
Luciferase and CAT Assays
The transfected MCs were harvested by scraping. Supernatants
were collected after lysis by three cycles of freeze and thaw in 100
µL of lysis buffer including the luciferase assay kit. For luciferase
assay, an aliquot of supernatant was added to a buffer containing
luciferin in accordance with the luciferase assay kit. Luciferase
expression was measured by a luminometer (Bio-Orbit Oy). For the CAT
assay, the lysates were incubated in 0.2 mL of a reaction mixture
containing 50 nmol/L [14C]chloramphenicol (0.5
µCi) and 2 mmol/L acetyl coenzyme A in 250
mmol/L Tris-HCl at pH 7.8 for 2 hours at 37°C. CAT expression
was assayed by thin-layer chromatography as
described.19 20 27 The radioactivity was analyzed
using a Fujix bioimaging analyzer (BAS2000). Under these
conditions, both assays were within the linear range. c-fos
luciferase expression in MCs transfected with each expression plasmid
and/or treated with each agent was divided by CAT expression in MCs
cotransfected with pTKCAT in the same experiment. Similarly, SRE,
SRE.L, SRE.M, SRE.LM, CRE, TRE, or TRE.M CAT expression in MCs
transfected with pcDSR
RhoA Val14 was divided by
luciferase expression in MCs cotransfected with RSV luciferase.
Luciferase and CAT expressions are expressed relative to the basal
condition without stimulation (control) value (1.0). There was no
difference in CAT expression among MCs cotransfected with pTKCAT and
other expression plasmids. CAT expressions derived from pTKCAT in MCs
cotransfected with RhoA expression plasmids or
PKCß
expression plasmid divided by CAT expressions derived from pTKCAT in
MCs transfected with the expression vector (pcDSR
) in the same
experiment were as follows: pcDSR
RhoA, 1.0±0.2;
pcDSR
RhoA Val14, 1.0±0.0; pcDSR
RhoA
Ala37, 1.1±0.0; pcDSR
Rho GDI, 1.0±0.1; pcDSR
RhoA with pcDSR
Rho GDI, 0.9±0.2; and
pcDSR
PKCß, 1.1±0.1 (mean±SEM, n=3, P=NS).
Similarly, there was no difference in CAT expression derived from
pTKCAT between control and TPA-treated MCs (treated/control ratio,
1.1±0.0 [mean±SEM]; n=3; P=NS). There was no difference
in CAT expression derived from pTKCAT between control and Ro
31-8220treated MCs (treated/control ratio, 1.0±0.1 for 100
nmol/L Ro 31-8220 and 0.9±0.1 for 1 µmol/L Ro
31-8220 (mean±SEM); n=3; P=NS). Similarly, there was no
difference in CAT expression derived from pTKCAT between control and
herbimycin Atreated MCs (treated/control ratio, 1.0±0.1 for 100
nmol/L herbimycin A and 0.8±0.1 for 1 µmol/L
herbimycin A (mean±SEM); n=3; P=NS). Furthermore, there was
no difference in RSV luciferase expression between the expression
vector (pcDSR
) and RhoA Val14 expression
plasmids/transfected MCs (RhoA Val14/expression vector
[pcDSR
] ratio, 1.1±0.1 [mean±SEM]; n=9; P=NS).
Downregulation of PKC by Treatment With TPA in MCs
MCs were transfected with the c-fos luciferase and/or
the pcDSR
RhoA Val14 by addition of DNA/CaPO4
solution. After an 8-hour incubation, MCs were then washed with 5 mL of
PBS and incubated in 5 mL of serum-free medium with 1
µmol/L TPA for 48 hours at 37°C. The cells were washed with
5 mL of PBS twice and incubated in 5 mL of serum-free medium for 9
hours with or without 1 µmol/L TPA. The luciferase and
CAT expressions were then assayed.
Statistical Procedures
All values were expressed as mean±SEM. Statistical evaluation
of the data was performed by Student's t test for unpaired
observations. When more than two groups were compared, the significance
of the difference between group means was analyzed by one-way
ANOVA and the Bonferroni test for samples. Values were considered
statistically significant at P<.05.
| Results |
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RhoA or pcDSR
RhoA
Val14 was transfected with c-fos luciferase and pTKCAT into
MCs. c-fos luciferase expression was activated by
the wild type of RhoA and RhoA Val14 in
time-dependent (data not shown) and dose-dependent (Fig 1
Rho
GDI+pcDSR
RhoA or pcDSR
RhoA Ala37 alone
into MCs (Fig 2
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Activated RhoA Stimulates the c-fos
Promoter/Enhancer Through the-c-fos SRE and TRE in
MCs
To determine the elements responsible for the c-fos
promoter/enhancer stimulated by activated RhoA, we
performed a deletion analysis of the c-fos
promoter/enhancer in MCs. RhoA Val14 activated -323
c-fos luciferase expression as well as the native
c-fos luciferase expression (Fig 3
). In -305 c-fos luciferase,
-293 c-fos luciferase, and -323
-300/-294
c-fos luciferase, the responses to RhoA Val14
were reduced. These results indicated that both the c-fos
SRE and TRE are necessary for the stimulation of the c-fos
promoter/enhancer by activated RhoA in MCs.
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Activated RhoA Stimulates the c-fos
SRE and the TRE in MCs
To confirm the stimulation of the c-fos
promoter/enhancer by activated RhoA, we investigated
the individual cis-acting elements, the c-fos
SRE, the CRE, and the consensus sequence of TRE. RhoA Val14
activated SRE CAT and TRE CAT expressions (2.7±0.2-fold
[P<.01] and 2.1±0.3-fold [P<.05] compared
with control, respectively) but not CRE CAT expression (1.1±0.1-fold)
(Fig 4A
). Furthermore, to confirm that
RhoA Val14 actually activates the c-fos
SRE and the TRE, we transfected pcDSR
RhoA Val14 with
SRE.L CAT, SRE.M CAT, SRE.LM CAT, or TRE.M CAT into MCs (Fig 4B
). By
following the methods of previous studies,22 23 24 25 we
constructed SRE.L CAT, SRE.M CAT, SRE.LM CAT, and TRE.M CAT. SRE.L CAT
and SRE.M CAT have the SRE mutant lacking the TCF binding site and the
SRF binding site, respectively, and SRE.LM CAT has the SRE mutant
lacking both TCF and SRF binding sites. RhoA Val14
activated SRE.L CAT expression (2.8±0.3-fold,
P<.01 compared with control) but not SRE.M CAT and SRE.LM
CAT expressions (1.2±0.1-fold and 1.0±0.0-fold, respectively). SRE.L
CAT and SRE CAT expressions were almost equally activated by
RhoA Val14. Furthermore, RhoA Val14 did not
activate TRE.M CAT expression (1.0±0.1-fold). These results
indicate that the signaling pathway from activated
RhoA to the c-fos SRE is linked to SRF but not
TCF and that activated RhoA stimulates the TRE in
MCs.
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Effect of PKC or Tyrosine Kinase in the Signaling Pathway From
Activated RhoA to the c-fos
Promoter/Enhancer in MCs
Since activation of the c-fos SRE and the TRE is known
to be linked to both PKC-dependent and PKC-independent
pathways,19 28 29 30 31 32 we investigated the role of PKC in the
action of RhoA in MCs. PKC was downregulated by treatment
with 1 µmol/L TPA for 48 hours after transfection of
c-fos luciferase with pcDSR
RhoA Val14.
Without pretreatment with TPA for 48 hours after transfection,
RhoA Val14 activated c-fos luciferase
expression (4.5±0.3-fold, P<.01 compared with control that
was not pretreated with TPA). Addition of TPA without downregulation of
PKC also activated c-fos luciferase expression
(6.2±0.2-fold, P<.01 compared with control that was not
pretreated with TPA) (Fig 5A
).
c-fos luciferase expression after pretreatment with TPA for
48 hours after transfection did not return completely to the basal
level (untreated control). c-fos luciferase expression after
pretreatment with TPA was about twice that without pretreatment.
Further TPA stimulation after the pretreatment period did not cause any
additional activation of c-fos luciferase expression because
of downregulated PKC. c-fos luciferase expression
activated by RhoA Val14 was not inhibited by
pretreatment with TPA (4.6±0.4-fold, P<.01 compared with
control that was pretreated with TPA). Furthermore, we investigated the
effect of selective PKC inhibitors on the action of
RhoA Val14 in MCs (Fig 5B
). Ro 31-8220 belongs to a series
of bisindolylmaleimides that selectively inhibit PKC, including PKC
subtypes
, ß,
, and
.33 Ro 31-8220 inhibited
the action of
PKCß on c-fos luciferase expression in a
dose-dependent manner. However, Ro 31-8220 did not inhibit the effect
of RhoA Val14 on c-fos luciferase expression. In
addition, GF 109203X, which inhibited the action of
PKCß on
c-fos luciferase expression, did not inhibit RhoA
Val14activated c-fos luciferase expression (data
not shown).
|
Next, we investigated the role of tyrosine kinase on the action of
RhoA in MCs. Herbimycin A, which is a tyrosine kinase
inhibitor, did not inhibit RhoA
Val14activated c-fos luciferase expression (Fig 5C
). Genistein (10 µmol/L for the last 24-hour
incubation) also did not inhibit RhoA
Val14activated c-fos luciferase expression (data
not shown). These results suggest that the tyrosine kinase
inhibitorsensitive pathway is not involved in the
signaling pathway from activated RhoA to the
c-fos promoter/enhancer in MCs.
| Discussion |
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In the present study, the deletion mutant of the c-fos promoter/enhancer, which lacks both the c-fos SRE and TRE, did not completely abolish luciferase expression by RhoA Val14. In addition, in our assay system, this deletion mutant also did not abolish luciferase expression by TPA. These results are consistent with the previous report demonstrating that the deletion mutant of the c-fos promoter/enhancer, which lacks both the c-fos SRE and TRE, did not abolish CAT expression by epidermal growth factor or TPA.34 Our results may indicate that both the c-fos SRE and TRE are necessary but not sufficient for the stimulation of the c-fos promoter/enhancer by activated RhoA in MCs. Other RhoA-mediating sites in the c-fos promoter/enhancer may exist.
The c-fos SRE is a regulatory sequence required for serum-induced and growth factorinduced c-fos transcriptional activation. This response is mediated by TCF and/or SRF, which are known to bind the SRE both in vitro and in vivo.35 We have also shown that RhoA Val14 activates SRE.L CAT expression but not SRE.M CAT and SRE.LM CAT expressions and that SRE.L CAT expression activated by RhoA Val14 is almost equal to SRE CAT expression. Our results indicate that the signaling pathway from activated RhoA to the SRE may link SRF, but not TCF. These results are consistent with the previous report demonstrating that in NIH 3T3 cells two kinds of signaling pathways converge at the SRE: one links TCF activity to MAP kinase activation, and the other links SRF activity to the Rho familyinduced signals.14
The TRE is defined as an AP-1 binding site. AP-1 activity appears to be
regulated by the following signaling pathways: the TPA-inducible PKC
pathway,36 PKC-independent pathways, including cell
surface tyrosine kinase and Ras proteins,19 31
and another pathway that is independent of PKC and PKA.32
Recently, it has been reported that in Saccharomyces cerevisiae
PKC1 is a downstream target of RHO1.37
Therefore, to investigate whether the signaling pathway from
activated RhoA to the c-fos
promoter/enhancer is mediated by PKC, we carried out the experiment of
PKC downregulation by TPA and that using selective PKC
inhibitors. We demonstrated that neither PKC downregulation
by TPA nor selective PKC inhibitors (such as Ro 31-8220 and
GF 109203X) inhibited RhoA Val14activated
c-fos luciferase expression in MCs. The conventional PKC
subfamily (PKC
and PKCß1), novel PKC subfamily (PKC
, PKC
,
and PKC
), and atypical PKC subfamily (PKC
) are present in rat
cultured neonatal MCs.38 GF 109203X has been shown to act
as a competitive inhibitor of ATP for the conventional PKC
subfamily (PKC
, PKCß, and PKC
).39 Ro 31-8220 is an
inhibitor of not only the conventional PKC subfamily
(PKC
, PKCß, and PKC
) but also PKC
.33 PKC
is
neither activated by phorbol esters nor downregulated by TPA
treatment.40 Therefore, our study has demonstrated that at
least phorbol estersensitive or bisindolylmaleimide (Ro 31-8220 and
GF 109203X)sensitive PKC is not involved in the downstream effector
of RhoA on the activation of the c-fos
promoter/enhancer in MCs.
It has been reported that Rho regulates the signal
transduction pathway of tyrosine kinase in Swiss 3T3
cells.41 Therefore, we further investigated whether the
signaling pathway from activated RhoA to the
c-fos promoter/enhancer is mediated by tyrosine kinase. Our
results have indicated that the signaling pathway from
activated RhoA to the c-fos
promoter/enhancer is insensitive to tyrosine kinase
inhibitors (herbimycin A and genistein) in MCs. In COS-7
cells, It has been recently reported that activated
RhoA forms a complex with PKN, which is a serine-threonine
protein kinase, and activates it.42 43 Although
the functional role of PKN is not clear in MCs, there is a possibility
that RhoA activates c-fos gene expression
through the PKN pathway. Furthermore, Rho family GTPases
Rac and Cdc42 are shown to regulate the activity
of the c-Jun amino-terminal kinase through p21-activated kinase
1 without affecting PKC, PKA, or "classical" MAP kinase in COS-7
cells and NIH 3T3 cells.44 45 46 We have shown here that the
PKC inhibitoror tyrosine kinase
inhibitorsensitive pathway does not work on the
downstream of activated RhoA. However, it is
suggested that Rho works on the downstream of PKC in
platelets and lymphocytes.4 5 Therefore, in MCs
RhoA may act as the downstream effector molecule of PKC, as
in platelets and lymphocytes, or operate in quite different
pathways, including PKC
and PKN as its downstream molecules. Further
experiments are required to examine the mechanisms.
In summary, activated RhoA stimulated the c-fos promoter/enhancer in MCs in the transient transfection assay. This stimulation by activated RhoA was mediated by the c-fos SRE and TRE. At the c-fos SRE, the signaling pathway of activated RhoA linked to the SRF binding site. In addition, the signaling pathway from activated RhoA to the c-fos promoter/enhancer was insensitive to PKC inhibitors and tyrosine kinase inhibitors. The potentially important role of Rho in myocardial signal transduction and myocardial hypertrophy should be explored further.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
|---|
Received June 11, 1996; accepted August 15, 1997.
| References |
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1-adrenergic and stretch-induced
c-fos mRNA expression in rat myocardial cells.
Circ Res. 1994;75:8-14.
q and
1-adrenergic receptor
signaling in cardiomyocytes: dissociation of Ras and Rho
pathways. J Biol Chem. 1996;271:31185-31190.
, -
, -
, and PKC-L (
):
comparison of properties of recombinant proteins in
vitro and in vivo. Biochem J. 1992;283:781-787.
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