Myocardin inhibits cellular proliferation by inhibiting NF- B(p65)-dependent cell cycle progression

Article (PDF Available)inProceedings of the National Academy of Sciences 105(9):3362-7 · April 2008with61 Reads
DOI: 10.1073/pnas.0705842105 · Source: PubMed
We previously reported the importance of the serum response factor (SRF) cofactor myocardin in controlling muscle gene expression as well as the fundamental role for the inflammatory transcription factor NF-κB in governing cellular fate. Inactivation of myocardin has been implicated in malignant tumor growth. However, the underlying mechanism of myocardin regulation of cellular growth remains unclear. Here we show that NF-κB(p65) represses myocardin activation of cardiac and smooth muscle genes in a CArG-box-dependent manner. Consistent with their functional interaction, p65 directly interacts with myocardin and inhibits the formation of the myocardin/SRF/CArG ternary complex in vitro and in vivo. Conversely, myocardin decreases p65-mediated target gene activation by interfering with p65 DNA binding and abrogates LPS-induced TNF-α expression. Importantly, myocardin inhibits cellular proliferation by interfering with NF-κB-dependent cell-cycle regulation. Cumulatively, these findings identify a function for myocardin as an SRF-independent transcriptional repressor and cell-cycle regulator and provide a molecular mechanism by which interaction between NF-κB and myocardin plays a central role in modulating cellular proliferation and differentiation. • cell differentiation • serum response factor


Myocardin inhibits cellular proliferation by inhibiting
B(p65)-dependent cell cycle progression
Ru-hang Tang*
, Xi-Long Zheng
, Thomas E. Callis
, William E. Stansfield*, Jiayin He
, Albert S. Baldwin
Da-Zhi Wang
, and Craig H. Selzman*
Departments of *Surgery and
Cell and Developmental Biology,
Carolina Cardiovascular Biology Center, and
Lineberger Comprehensive Cancer Center,
University of North Carolina, Chapel Hill, NC 27599; and
Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, and Department of
Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada T2N 4N1
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 7, 2008 (received for review June 28, 2007)
We previously reported the importance of the serum response
factor (SRF) cofactor myocardin in controlling muscle gene expres-
sion as well as the fundamental role for the inflammatory tran-
scription factor NF-
B in governing cellular fate. Inactivation of
myocardin has been implicated in malignant tumor growth. How-
ever, the underlying mechanism of myocardin regulation of cellular
growth remains unclear. Here we show that NF-
B(p65) represses
myocardin activation of cardiac and smooth muscle genes in a
CArG-box-dependent manner. Consistent with their functional
interaction, p65 directly interacts with myocardin and inhibits the
formation of the myocardin/SRF/CArG ternary complex in vitro and
in vivo. Conversely, myocardin decreases p65-mediated target
gene activation by interfering with p65 DNA binding and abro-
gates LPS-induced TNF-
expression. Importantly, myocardin in-
hibits cellular proliferation by interfering with NF-
cell-cycle regulation. Cumulatively, these findings identify a func-
tion for myocardin as an SRF-independent transcriptional repressor
and cell-cycle regulator and provide a molecular mechanism by
which interaction between NF-
B and myocardin plays a central
role in modulating cellular proliferation and differentiation.
cell differentiation serum response factor
eter mining mechan isms that preferentially promote cardi-
omyoc yte and vascular smooth muscle cell (VSMC) g rowth
or differentiation remains an active challenge in cardiovascular
biology (1). This process is regulated in part by serum response
factor (SRF), a transcription factor that is widely expressed but
enriched in muscle lineages during development (2). SRF binds
to the CArG box (CC[A/T]
GG), a DNA consensus sequence
located in the c ontrol regions of numerous growth factor-
regulated and muscle-specific genes (3). Emerging evidence
suggests that the regulation of SRF-driven gene transcription
depends on its relationship with a wide variety of positive and
negative cofactors (4, 5). Myocardin is ex pressed specifically in
cardiac and smooth muscle cells and potently activates their gene
ex pression by associating with SRF bound to CArG boxes (6).
We have shown that the toggling of SRF toward growth-
dependent or dif ferentiation pathways is related to interplay
bet ween myocardin and another SRF-associated factor, Elk-1, in
a manner where myocardin directly competes with Elk-1 for SRF
binding and antagonizes proliferative signaling induced by
PDGF (7). Although previous studies have shown that overex-
pression of myocardin can retard cell grow th (8) and inactivation
of myocardin promotes tumor g rowth (9), the molecular mech-
an ism related to myocardin and cellular proliferation remains
unk nown.
The NF-
B family of transcription factors play key roles in
regulating inflammatory and immune responses as well as
cellular proliferation, differentiation, and survival (10, 11).
Interactions between NF-
B dimers and other families of tran-
scription factors have been widely reported, including that of
SRF (10, 12). We have previously demonstrated that NF-
B(p65) can strongly induce cellular proliferation (13, 14). In
light of our previous work detailing the effects of myocardin
promoting cardiac and smooth muscle differentiation and p65
promoting cell growth, we hypothesized that p65 c ould inhibit
myocardin-induced differentiation by interfering with its asso-
ciation with SRF. Conversely, we also wanted to determine the
influence of myocardin as a transcriptional regulator of p65 and
its downstream ef fect on cellular growth. Herein we describe a
relationship bet ween NF-
B and myocardin in regulating
smooth muscle and cardiac gene expression and demonstrate a
un ique role for myocardin as a transcriptional repressor and
inhibitor of cell proliferation.
B Suppresses Myocardin Activation of Cardiac and Smooth Muscle
Smooth muscle 22 (SM22) and atrial natriuretic factor
(ANF) promoters att ached to luciferase reporters were trans-
fected with myocardin and p65 expression plasmids in COS7
cells. Myocardin-induced transactivation of both SM22 and ANF
was inhibited by cotransfection with p65 in a dose-dependent
manner (Fig. 1a). Similar findings were also observed by using
-MHC-luc reporter (data not shown) and c orroborated in
rat neonatal cardiomyocy tes (Fig. 1b). Cotransfection of the p50
B subunit had no ef fect on myocardin transactivation (data
not shown). Thus, p65 appears to functionally interfere with
myocardin-dependent gene transcription.
To investigate whether p65-mediated inhibition of myocardin
transactivation is CArG-dependent, CArG-box mutation anal-
ysis of the SM22 promoter was performed. Although mutations
of the distal CArG box (CArG-far) and the proximal CArG box
(CArG-near) reduced responsiveness to myocardin (6), p65
nevertheless eliminated the abilit y of these mutant promoters to
respond to myocardin (Fig. 1c). Similarly, p65 repressed myo-
cardin activation on the ANF promoter with CArG-far box
mut ation. Next, we tested whether the CArG box is sufficient to
mediate p65 repression of myocardin transactivity by using a
luciferase reporter driven by four tandem CArG boxes derived
f rom SM22 or c-fos promoters. Indeed, p65 inhibited myocardin
activity on both CArG-box reporters (Fig. 1c and data not
Author contributions: R.-h.T. and X.-L.Z. contributed equally to this work; R.-h.T., D.-Z.W.,
and C.H.S. designed research; R.-h.T., X.-L.Z., W.E.S., and J.H. performed research; X.-L.Z.,
W.E.S., J.H., A.S.B., and D.-Z.W. contributed new reagents/analytic tools; R.-h.T., T.E.C.,
A.S.B., D.-Z.W., and C.H.S. analyzed data; and R.-h.T., T.E.C., D.-Z.W., and C.H.S. wrote the
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
To whom correspondence may be addressed at: Department of Cell and Developmental
Biology, University of North Carolina, 8336 Medical Biomolecular Research Building, CB
7126, Chapel Hill, NC 27599-7126. E-mail:
**To whom correspondence may be addressed at: Division of Cardiothoracic Surgery,
University of North Carolina, 3040 Burnett Womack Building, CB 7065, Chapel Hill,
NC 27599-7065. E-mail:
This article contains supporting information online at
© 2008 by The National Academy of Sciences of the USA
March 4, 2008
vol. 105
no. 9 www.pnas.orgcgidoi10.1073pnas.0705842105
shown). Together, these data suggest that the CArG box is
required for p65 to repress myocardin transactivation.
We have previously demonstrated that TNF-
is a potent
mitogen for vascular smooth muscle and that this effect is
B-dependent (13–16). Indeed, TNF-
repressed myocardin
activation of SM22 and ANF reporters in both COS cells (Fig.
1d) and cardiomyocy tes (Fig. 1e). Next, we examined the effect
of block ing endogenous NF-
B on myocardin transactivity.
Cotransfection with an inhibitory
superrepressor (I
SR), a potent inhibitor of NF-
B activity (11), significantly
enhanced myocardin activit y for SM22 (Fig. 1 d and e) and ANF
(dat a not shown). Together, these data demonstrate that TNF-
B signaling is both necessary and sufficient to modulate
myocardin transactivity in activation of cardiac and smooth
muscle gene expression.
B Inhibits Myocardin Transactivity by Physically Interacting with
Myocardin. To characterize their mechanistic interaction, we
tested whether p65 could still repress the activit y of myocardin
when it was fused to the DNA binding domain of GAL4, in such
a way that its transcriptional activity is independent of SRF.
Indeed, we observed that p65 repressed the myocardin-activated
GAL4-dependent luciferase reporter (UAS-luciferase) in a
dose-dependent manner (Fig. 2a), suggesting that p65 acts
directly on myocardin to repress myocardin-dependent gene
ex pression, rather than indirectly through SRF (12). Next, we
used coimmunoprecipitation to demonstrate that myocardin and
p65 had a direct physical association (Fig. 2b). With a series of
myocardin deletion mutant proteins, we mapped the domains of
myocardin responsible for its protein–protein interaction with
p65. As shown (Fig. 2c), p65 interacts with GST fused to
myocardin at amino acids 1–560, amino acids 129689, and
amino acids 78669, but not with amino acids 328670, amino
acids 669–935, or GST alone. Deletion of N-terminal sequences
of myocardin up to amino acid 328 abolished this interaction.
Therefore, the N terminus of myocardin, including the basic and
Q-rich domains, was necessary and sufficient for its interaction
with p65. Interestingly, the p65-interaction region of myocardin
protein was previously shown to be used for its interaction with
SRF (7, 17), indicating that p65 and SRF might directly compete
for myocardin binding.
Having identified the region on myocardin for p65 binding, we
next sought to define the region of p65 that interacts with
myocardin using truncated p65 proteins (Fig. 2d). Indeed, the
N-ter minal regions of p65 specifically interact with myocardin
(Fig. 2e). Whereas the region including the Rel homology
domain of p65 (amino acids 1–276) is sufficient to interact with
myocardin, no interaction was detected in the C-terminal region
of p65 (amino acids 270–551) (Fig. 2f ). This finding compares
favorably to a previous report demonstrating that SRF binds p65
bet ween residues 234 and 245 (12).
B Prevents the Formation of the Myocardin/SRF/CArG Ternary
Complex. Myocardin does not directly bind to DNA; rather, it is
recr uited to target promoters by forming a stable complex with
SRF bound to CArG box (6, 7). We therefore hypothesized that
p65 could interfere with the myocardin/SRF protein c omplex
bound to its associated CArG box. Gel shift assays were per-
for med by using a radiolabeled c onsensus CArG box (derived
f rom the c-fos promoter) in which SRF binds to the CArG box
and SRF/myocardin associate with the CArG box to form a
ternary complex (Fig. 3a). When p65 protein was added to the
reaction, there was no additional complex formation and the
SRF/myocardin/CArG ternary complex was not further shifted.
However, the intensity of the ternary complex was decreased
when increasing amounts of p65 were added (Fig. 3a, lanes 5–7).
A lthough p65 is not a part of the stable c omponent of the
SRF/CArG or the myocardin/SRF/CArG complex, these results
indicate that p65 does af fect the formation of the ternary
c omplex. This observation was further confirmed by using a
series of increased amounts of p65 relative to the fixed amounts
of SRF and myocardin (Fig. 3b Left). Conversely, addition of
increased amounts of myocardin relative to fixed amounts of
SRF and p65 resulted in enhancement of the ternary complex
for mation (Fig. 3b Right). Similarly, p65 interferes with the
ternary complex formation of myocardin/SRF on the SM22
SM22-luc ANF-luc
+++ +++
SM22-luc ANF-luc
p65 protein
+ +
+ +
+ +
far CArG-luc
near CArG-luc
far CArG-luc
4X SM22
Fig. 1. Suppression of myocardin transcriptional activity by p65. Luciferase
reporters controlled by SM22 and ANF were transfected into COS7 cells (a)or
rat neonatal cardiomyocytes (b) and cotransfected with expression plasmids
for myocardin (0.1
g) and increasing amounts of p65 (COS7: 0.01, 0.05, 0.1,
and 0.2
g; cardiomyocytes: 0.02 and 0.1
g). p65 protein assayed by Western
blot. (c) COS7 cells were cotransfected with the indicated deleted CArG box
within either the SM22 or ANF promoter. (d and e) COS7 cells (d) or cardio-
myocytes (e) treated with TNF-
(10 ng/ml for 6 h) or cotransfected with an
superrepressor. Values are the fold increase of luciferase activity relative
to activation of the reporter alone of at least three experiments. Student’s t
test: P 0.05, myocardin alone vs. myocardin plus p65, TNF-
Tang et al. PNAS
March 4, 2008
vol. 105
no. 9
CArG box [supporting infor mation (SI) Fig. 7]. Together, these
dat a demonstrate that p65 and myocardin directly compete for
their SRF association.
We next sought to test whether p65 could inhibit formation of
the SRF/myocardin/CArG complex under physiologic condi-
tions in an in vivo sepsis model (18). LPS (15
g/ml) was injected
in mice, and hearts were later procured for myocardial protein.
As expected, the p65 protein level was sign ificantly induced in
the LPS-treated hearts (Fig. 3c Lower). When nuclear protein
extracts isolated from control mouse hearts were used in
EMSAs, the formation of the SRF/CArG complex and the
SRF/myocardin/CArG ternary c omplex was evident (Fig. 3c,
lanes 1–3). Antibody supershift confirmed the identities of this
c omplex (Fig. 3c, lane 2). Importantly, LPS treatment dramat-
ically attenuated the SRF/myocardin/CArG ternary complex
for mation without affecting SRF CArG-box binding (Fig. 3c,
c ompare lanes 6 and 3). These data demonstrate that p65 is a
potent nuclear factor that inhibits myocardin transactivation by
disr upting the for mation of the SRF/myocardin/CArG complex
both in vitro and in vivo.
Myocardin Inhibits NF-
B Signaling and Its Target Gene Expression.
We next tested the effect of myocardin binding to p65 on the
transcriptional activit y of p65 itself. Luciferase constructs with
three tandem
B-box promoters and its mut ant were transfected
with p65, and p65 appropriately induced luciferase activ ity of the
B promoters but not its mutant (Fig. 4a). When
c otransfected with myocardin, p65-mediated luciferase activity
was decreased in a dose-dependent manner (Fig. 4a). These dat a
demonstrate that myocardin can inhibit p65 transcriptional
The functional inhibition of p65 transactivit y by myocardin
prompted us to determine whether myocardin interferes with
DNA binding of p65. EMSAs were conducted by using a
c onsensus
B box as a probe. Addition of p65 protein resulted
in a specific shifted band (Fig. 4 b, lane 2). Concomit ant admin-
istration of anti-p65 antibody resulted in a supershift (Fig. 4b,
lane 1). However, when the reaction was incubated w ith increas-
ing amounts of myocardin protein, the p65 and
B-box com-
plexes were gradually diminished (Fig. 4b, lanes 3–6). These
results demonstrate that myocardin can indeed block p65 binding
to its specific DNA element.
We next sought to determine whether myocardin could phys-
iologically repress transcriptional activit y of p65. Aortic smooth
muscle cells were infected with Ad-myocardin (or Ad-GFP as a
c ontrol) viruses for 48 h, and then 1
g/ml LPS was added for
2 h. As expected, LPS increased TNF-
ex pression (Fig. 4c) that
was essentially abolished with increased c oncentrations of myo-
Fig. 2. Physical interaction between p65 and myocardin. (a) COS7 cells transfected with expression plasmids encoding full-length myocardin fused to GAL4 and
UAS-luciferase reporter, with or without expression plasmid for p65. Values are the fold increase of luciferase activity relative to activation of the reporter alone
in at least three experiments. Student’s t test: P 0.05, Gal4-myocardin vs. Gal4-myocardin plus p65. (b) Anti-Myc antibodies detected myocardin in
immunoprecipitates prepared with anti-Flag antibodies and COS7 cells transfected with Flag-p65 and Myc-myocardin expression plasmids. (c)(Left) Coomas-
sie-stained proteins corresponding to the amounts of GST and GST-myocardin protein in the pull-down assay are shown. (Right) Summary of p65 interaction with
myocardin domains. (d) Autoradiogram indicating 1/20 input of radiolabeled p65 proteins used in pull-down assay. (e) Interaction between full-length and
truncated p65 and GST-myocardin (Left) and GST alone (Right). (f) Summary of myocardin interaction with p65 domains.
Fig. 3. p65 inhibits the formation of myocardin/SRF/CArG ternary complex. (a) Myc-tagged myocardin, Flag-tagged p65, and SRF proteins were expressed by
in vitro transcription and translation and incubated with radiolabeled probes, and gel mobility shift assays were performed. (b) Gel mobility shift assays were
performed with increasing amounts of p65 (Left) and myocardin (Right). (c) Gel mobility shift assays were conducted from mouse hearts with or without LPS.
Anti-SRF antibodies were applied for supershift. p65 protein was analyzed by Western blot.
www.pnas.orgcgidoi10.1073pnas.0705842105 Tang et al.
cardin. Interestingly, the expression levels of total p65 and
phospho-Ser-536-p65 were not altered by myocardin (Fig. 4c).
These dat a suggest that myocardin interferes with NF-
B sig-
naling, not by its ef fect on the upstream phosphorylation cas-
cade, but rather by directly inhibiting p65 transcriptional activity
and expression of p65-dependent downstream target genes.
Myocardin Inhibits Cellular Proliferation. Because myocardin can
functionally repress p65 transcriptional activity, and in particular
decrease expression of TNF-
, we next sought to determine the
influence of myocardin on cellular proliferation. We therefore
st ably overexpressed myocardin using a tetracycline-regulated
ex pression system in CHO cells. Doxyc ycline (Dox) treatment of
transfected cells that harbor wild-type myocardin (M33) suc-
cessfully promoted a time-dependent overexpression of myocar-
din (SI Fig. 8a). When myocardin was induced, cellular g rowth
and proliferation as measured by histology, cell numbers, and
DNA synthesis was markedly reduced compared with cells that
harbor either a control vector (TR-CHO) or the dominant
negative myocardin (DN5) (Fig. 5 a and b and SI Fig. 8b).
Myocardin overexpression was not associated with apoptosis
because a sub-G
peak was not identified, nor did we detect
DNA f ragmentation or positive T UNEL stain ing (data not
shown). The antiproliferative ef fect of myocardin was related to
–M phase arrest and for mation of polyploid cells (Fig. 5 c and
d). To define transcriptional targets linking myocardin to cell-
c ycle regulation, we demonstrate that myocardin induction is
associated with down-regulation of several import ant mediators
of cell-cycle prog ression, including those previously known to be
regulated by NF-
B such as c-myc (19) and c yclin-dependent
k inase 2 (CDK2) (20), as well as ribosomal S6 kinase (S6K) and
CDK1, but not p21 and p27 (Fig. 5e).
To investigate the endogenous influence of myocardin and p65
on cell proliferation and differentiation, we perfor med overex-
pression and loss-of-function studies in VSMC. Again, overex-
pression of myocardin using the tetracycline-regulated expres-
sion system decreased VSMC numbers, c onsistent with that seen
in CHO cells (Fig. 6a). Interestingly, overex pression of the
dominant negative myocardin increased numbers of VSMCs,
whereas dominant negative myocardin in CHO cells had no such
ef fect. We attribute this difference to interference of endoge-
nous myocardin by dominant negative myocardin in VSMC,
whereas CHO cells do not express myocardin. Quantitative
analyses by direct cell counting over 3 successive days further
support these results (Fig. 6b). Next, we knocked down myocar-
din or p65 expression using siRNAs in VSMC. Inhibition of
myocardin resulted in increased expression of NF-
and mitogenic c ytok ines TNF-
and IL-6. As expected, siRNA
to p65 reduced both TNF-
and IL-6 levels (Fig. 6c). In contrast,
myocardin inhibition decreased ex pression of SM-
whereas p65-inhibition increased its ex pression, c onsistent with
previous reports (Fig. 6c). Finally, we corroborated our overex-
pression studies in both CHO and VSMC by demonstrating that
inhibition of myocardin increased VSMC numbers, whereas
Fig. 4. Myocardin repression of p65 transcriptional activity. (a) COS7 cells transfected with a luciferase reporter construct of three tandem
B sites or its mutant,
together with expression plasmids for p65 (0.1
g), myocardin (0.1 and 0.2
g), or both. Values are the fold increase in luciferase activity relative to activation
of the reporter alone. Error bars represent SD of at least three experiments. Student’s t test: P 0.05, p65 alone vs. p65 plus myocardin. (b) Gel mobility shift
assays were performed with a
B probe with stable levels of in vitro translated p65 with or without increasing amounts of in vitro translated
myocardin. (c) Aortic VSMCs infected with increasing amounts of adenoviral myocardin (multiplicity of infection: 20, 50, and 200) for 48 h and then stimulated
with LPS (1
g/ml) for 2 h. Western blots were performed on extracted proteins.
Fig. 5. Myocardin inhibits CHO cell proliferation. (a) Numbers of cells harboring the myocardin-inducible system were counted 72 h after treatment with or
without Dox (n 7).
, P 0.01. (b) Cells were treated for 3 days, pulse-labeled with BrdU for 60 min, and stained with BrdU (for both cell count and BrdU
experiments (n 7).
, P 0.01. (c) Representative DNA histograms obtained through laser scanning cytometry in M33 CHO cells treated for 24 h. (d) Cell numbers
in both G
–M phase and polyploid cell regions as detected by cytometry (n 5).
, P 0.01. (e) Western blot with corresponding antibodies for cell-cycle
regulatory proteins in cells with () and without () Dox treatment for 3 days.
Tang et al. PNAS
March 4, 2008
vol. 105
no. 9
inhibition of p65 resulted in decreased VSMC growth (Fig. 6d).
Taken together, these dat a establish that myocardin functions as
an antiproliferative factor and show that this effect is c oupled
with its repression of NF-
B transcriptional activity.
This study reveals a relationship between t wo transcription
factors, myocardin and NF-
B, in regulation of cellular prolif-
eration and muscle differentiation. We found that p65 sup-
presses myocardin activation of CArG-dependent smooth mus-
cle and cardiac genes and is mediated by physical interruption of
the myocardin/SRF/CArG ternary complex. Conversely, myo-
cardin inhibits p65 DNA binding, thereby repressing NF-
mediated target gene expression and function, including cellular
proliferation. These findings add several layers to our current
underst anding of both NF-
B and myocardin biology. First, we
of fer evidence of the expanding complexity of myocardin-
dependent signaling by demonstrating its unique regulation by
p65. In addition, we establish additional DNA binding-
independent effects of NF-
B. Finally, we demonstrate that
myocardin can behave as a negative transcriptional cofactor in a
SRF-independent manner to inhibit cellular proliferation.
Several reports have recently described mechanisms related to
myocardin regulation of smooth muscle and cardiac gene ex-
pression and point to an intricate web of c ofactors that control
the influence of myocardin on SRF (21). Whereas regulation of
myocardin activity is typically a function of its ability to physically
associate with SRF (7), NF-
B regulation of gene transcription
is classically a result of its binding to DNA. A functional
interaction bet ween p65 and SRF has been previously described
and suggested that p65 complex formation with the SRF MADS
domain may neutralize the inhibitory function of this region,
thereby promoting SRF transcription (12, 22). In this article we
of fer an alternative mechanism of p65 regulation of SRF:
disr uption of the myocardin/SRF/CArG ternary complex, thus
functionally inhibiting myocardin transactivation of SRF-
regulated cardiac and smooth muscle genes.
In addition to demonstrating that p65 physically binds with
myocardin and interferes with its transcriptional activation, we
reveal an unexpected role of myocardin as a transcriptional
repressor of p65 rather than as a SRF c ofactor. Indeed, we
demonstrate that myocardin has SRF-independent effects by
directly interacting and preventing binding of p65 to its DNA
t arget. Further more, myocardin completely abrogated TNF-
protein production after stimulation with LPS despite persis-
tently high and stable levels of p65, suggesting that the influence
of myocardin was not on upstream NF-
B activation but on its
downstream activit y. Our studies therefore assign a molecular
mechan ism for myocardin function.
We found that myocardin potently inhibits cell proliferation,
at least in part by repressing NF-
B-dependent cell proliferation
signals. This observation is consistent with a recent report in
Fig. 6. Effect of endogenous myocardin on VSMC growth. (a) Photomicrographs of VSMC harboring the tetracycline-regulated (TR) inducible system for
myocardin and its dominant negative treated with and without Dox for 24 h. (b) Cell numbers counted over 3 days in control (TR), myocardin (Myocd), and
dominant negative (Myocd-DN) expressing VSMC (n 5).
, P 0.01. (c) Mouse aortic VSMC were transfected with siRNA to either myocardin or p65 for 24 h.
Real-time PCR was performed to detect relative fold changes in mRNA expression (normalized to 18s rRNA). Values are the relative fold change in mRNA
expression relative to control of at least three experiments. P 0.05 for both siRNA vs. control and siRNA myocardin vs. siRNA p65. (d) VSMC directly counted
48 h after being transfected with either myocardin or p65 siRNA. Error bars represent SD of five experiments. P 0.05 for both siRNA vs. control and siRNA
myocardin vs. siRNA p65. (e) Working model balancing NF-
B(p65) and myocardin regulation of cellular growth and differentiation.
www.pnas.orgcgidoi10.1073pnas.0705842105 Tang et al.
which inactivation of myocardin is associated with malignant
tumor growth (9). In addition, we demonstrate the endogenous
influence of myocardin to hinder cell growth. Our study specif-
ically links myocardin to the control of G
–M transition of
cell-c ycle prog ression. Interestingly, a recent study also reported
that NF-
B participates in regulation of G
–M cell-cycle pro-
gression through ERK5 (23). ERK5 activated NF-
B not
through its classic upstream kinase but by S6K-dependent phos-
phorylation of I
. The observation that myocardin decreased
ex pression of S6K and pS6K without associated changes in p65
levels is intriguing and may provide an alternative feedback
mechan ism to modulate cell-cycle progression. Similarly, acti-
vation of the cyclin-B–CDK1 complex is required for G
transition, and myocardin did effectively down-regulate CDK1
ex pression. In contrast, the cyclin-dependent kinase inhibitors
p21 and p27, principally acting at the G
–S transition, were not
influenced by myocardin induction, further supporting the view
that myocardin specifically targets the G
–M phase. Taken
together (Fig. 6), our results define a unique relationship
bet ween NF-
B and myocardin that govern signals balancing
cardiac and smooth muscle cell growth and dif ferentiation. We
also reveal a function for myocardin in regulation of cell
proliferation and provide a molecular mechanism by which
myocardin can act as a transcriptional repressor to inhibit the
B pathway.
Detailed materials and methods are in SI Methods. In brief, COS7, rat neonatal
cardiomyocytes, and murine aortic VSMC were isolated and cultured (5).
Luciferase reporter constructs were fused with the SM22 and ANF promoters
(6). The myocardin adenoviral expression construct (Ad-MYCD) contained a
cDNA encoding amino acids 129–935 of mouse myocardin (24).
Coimmunoprecipitation Assays. COS7 cells were transiently transfected with
plasmids encoding the epitope-tagged myocardin, SRF, and p65 proteins with
FuGENE6. Epitope-tagged proteins were precipitated with antibodies and
protein A/G beads and analyzed by Western blotting with directed antibodies.
GST Protein-Binding Assays. Plasmids encoding a GST fusion with truncated
myocardin were transformed into BL21 cells and added to the culture to
induce protein expression. p65 full-length and truncated proteins translated
in vitro were radiolabeled with [
S]methionine. Glutathione beads conju-
gated with GST fusion protein were incubated with radiolabeled proteins and
analyzed by autoradiography.
EMSAs. EMSAs were performed by using c-fos CArG probe (6) or NF-
probe (AGT TGA GGG GAC TTT CCC AGGC). For mouse studies, mice were
killed, the left ventricular muscles were isolated, and cytoplasmic and nuclear
proteins were extracted and subsequently incubated with labeled probes.
Antibody supershift experiments were conducted with anti-SRF (Santa Cruz
Biotechnology), anti-FLAG, or anti-Myc (Sigma).
Myocardin Tetracycline-Regulated System and Cellular Proliferation. A tetra-
cycline-regulated system in CHO cells or human aortic VSMCs (American Type
Culture Collection) harboring wild-type myocardin (M33) and dominant neg-
ative (DN5) myocardin were analyzed by Western blot using anti-myocardin
and anti-Flag M2 monoclonal antibody, respectively. Cell counting, BrdU
staining, and laser scanning cytometry were performed and compared with
control cells (TR) with or without Dox.
siRNA Transfection and Real-Time PCR. Primary aortic VSMCs were transfected
with predesigned siRNAs of myocardin and p65 (Ambion) by applying pro-
gram D-33 and using Basic Nucleofector Kit for primary smooth muscle cells
(VPI-1004; Amaxa). TaqMan polymerase, as well as TaqMan matched probes
and primers, were applied for real-time PCR (Applied Biosystems).
ACKNOWLEDGMENTS. This work was supported by the American College of
Surgeons (C.H.S.), the National Institutes of Health (A.S.B. and D.-Z.W.), the
Canadian Institutes of Health Research (X.-L.Z.), the March of Dimes Birth
Defects Foundation (D.-Z.W.), the Muscular Dystrophy Association (D.-Z.W.),
and the American Heart Association (D.-Z.W.).
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March 4, 2008
vol. 105
no. 9
    • "Conditional cardiac ablation of Myocardin at late stages of development results in a postnatal cardiac physiology imbalance, an increase of fibrotic tissue and an increase of cell death leading to cardiac enlargement (Huang et al., 2009). Conversely, Myocardin over-expression in primary human mesenchymal stem cells or human vascular smooth muscle cells results in a reduction of cell proliferation and a forced expression of cardiac and smooth muscle molecular markers (Chen et al., 2011; Tang et al., 2008; Van Tuyn et al., 2005; Wang et al., 2003; Wystub et al., 2013). Furthermore, Myocardin over-expression induces cardiac hypertrophy in neonatal rat cardiomyocytes, as well as in transgenic mice (Wystub et al., 2013; Xing et al., 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: The molecular events that control cell fate determination in cardiac and smooth muscle lineages remain elusive. Myocardin is an important transcription co-factor that regulates cell proliferation, differentiation and development of the cardiovascular system. Here, we describe the construction and analysis of a dual Cre and Enhanced Green Fluorescent Protein (EGFP) knock-in mouse line in the Myocardin locus (MyocdKI). We report that the MyocdKI allele expresses the Cre enzyme and the EGFP in a manner that recapitulates endogenous Myocardin expression patterns. We show that Myocardin expression marks the earliest cardiac and smooth muscle lineages. Furthermore, this genetic model allows for the identification of a cardiac cell population which maintains both Myocardin and Isl-1 expression, in E7.75 - E8.0 embryos, highlighting the contributions and merge of the first and second heart fields during cardiogenesis. Therefore, the MyocdKI allele is a unique tool for studying cardiovascular development and lineage-specific gene manipulation. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Oct 2014
    • "Regarding the underlying mechanisms, Zuckerbraun et al9 showed that the antiproliferative activity of IκB was related to cell‐cycle arrest through upregulation of the cyclin‐dependent kinase inhibitors p21WAF1/Cip1 and p27Kip1 in cultured SMCs. Moreover, of interest, myocardin has been shown to suppress SMC proliferation by inhibiting NF‐κB‐dependent cell‐cycle progression in cultured SMCs.40 Although the aforementioned mechanistic studies were mostly performed in cultured SMCs, it is highly possible that the decreased proliferation rate in our transgenic mice was also caused by these mechanisms. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Vascular proliferative diseases such as atherosclerosis are inflammatory disorders involving multiple cell types including macrophages, lymphocytes, endothelial cells, and smooth muscle cells (SMCs). Although activation of the nuclear factor‐κB (NF‐κB) pathway in vessels has been shown to be critical for the progression of vascular diseases, the cell‐autonomous role of NF‐κB within SMCs has not been fully understood. Methods and Results We generated SMC‐selective truncated IκB expressing (SM22α‐Cre/IκBΔN) mice, in which NF‐κB was inhibited selectively in SMCs, and analyzed their phenotype following carotid injury. Results showed that neointima formation was markedly reduced in SM22α‐Cre/IκBΔN mice after injury. Although vascular injury induced downregulation of expression of SMC differentiation markers and myocardin, a potent activator of SMC differentiation markers, repression of these markers and myocardin was attenuated in SM22α‐Cre/IκBΔN mice. Consistent with these findings, NF‐κB activation by interleukin‐1β (IL‐1β) decreased expression of SMC differentiation markers as well as myocardin in cultured SMCs. Inhibition of NF‐κB signaling by BAY 11‐7082 attenuated repressive effects of IL‐1β. Of interest, Krüppel‐like factor 4 (Klf4), a transcription factor critical for regulating SMC differentiation and proliferation, was also involved in IL‐1β‐mediated myocardin repression. Promoter analyses and chromatin immunoprecipitation assays revealed that NF‐κB repressed myocardin by binding to the myocardin promoter region in concert with Klf4. Conclusions These results provide novel evidence that activation of the NF‐κB pathway cell‐autonomously mediates SMC phenotypic switching and contributes to neointima formation following vascular injury.
    Full-text · Article · Apr 2013
    • "Despite the popular assumption that MYOCD is expressed almost exclusively in cardiac and SM cells, MYOCD expression was also detected in human fibroblasts [65] where it is involved in functional differentiation and has a negative role in cell proliferation [66]. In fact, in model cell-based assays overexpression of Myocd resulted in inhibition of cell-cycle progression at the G2/M phase and formation of polyploidy cells [67]. MRTF-A and MRTF-B also exert antiproliferative effects on fibroblasts [68]. "
    [Show abstract] [Hide abstract] ABSTRACT: Growing evidence suggests that gene-regulatory networks, which are responsible for directing cardiovascular development, are altered under stress conditions in the adult heart. The cardiac gene regulatory network is controlled by cardioenriched transcription factors and multiple-cell-signaling inputs. Transcriptional coactivators also participate in gene-regulatory circuits as the primary targets of both physiological and pathological signals. Here, we focus on the recently discovered myocardin-(MYOCD) related family of transcriptional cofactors (MRTF-A and MRTF-B) which associate with the serum response transcription factor and activate the expression of a variety of target genes involved in cardiac growth and adaptation to stress via overlapping but distinct mechanisms. We discuss the involvement of MYOCD, MRTF-A, and MRTF-B in the development of cardiac dysfunction and to what extent modulation of the expression of these factors in vivo can correlate with cardiac disease outcomes. A close examination of the findings identifies the MYOCD-related transcriptional cofactors as putative therapeutic targets to improve cardiac function in heart failure conditions through distinct context-dependent mechanisms. Nevertheless, we are in support of further research to better understand the precise role of individual MYOCD-related factors in cardiac function and disease, before any therapeutic intervention is to be entertained in preclinical trials.
    Full-text · Article · May 2012
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