Myocardin inhibits cellular proliferation by inhibiting NF-kappaB(p65)-dependent cell cycle progression.
ABSTRACT 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-kappaB 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-kappaB(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-alpha expression. Importantly, myocardin inhibits cellular proliferation by interfering with NF-kappaB-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-kappaB and myocardin plays a central role in modulating cellular proliferation and differentiation.
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ABSTRACT: De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.PLoS ONE 11/2014; 9(11):e112120. · 3.53 Impact Factor
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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.genesis 08/2014; · 2.04 Impact Factor
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ABSTRACT: Recently, the architectural remodeling of venous vessel wall ranks as the basis of varicose veins development based on the phenotypic state of vascular smooth muscle cells (VSMCs). In this study, we firstly demonstrated an obvious up-regulation of IQ-domain GTPase-activating protein 1 (IQGAP1) in patients with varicose veins. Importantly, following stimulation with PDGF-BB for 4 h, a common inducer of phenotypic switch in VSMCs, a dramatically time-dependent increase in IQGAP1 expression was observed in human venous smooth muscle cells (HUVSMCs), concomitant with the down-regulation of SMC markers [including α-smooth muscle actin (SMA), smooth muscle calponin (CNN), SM22α (SM22)], suggesting a critical function of IQGAP1 during the switch of synthetic VSMC phenotype. Further analysis ascertained that IQGAP1 overexpression significantly inhibited the expression of SMA, SM and CNN, while its silencing dramatically promoted their expression levels. Moreover, the elevated IQGAP1 enhanced cell proliferation, migration and rearrangement. Mechanism assay confirmed that IQGAP1 overexpression notably blocked myocardin levels. Importantly, after transfection with myocardin siRNA, IQGAP1 down-regulation-induced decrease in cell proliferation, migration and cell rearrangement was remarkably attenuated. Together, these results demonstrated that IQGAP1 may regulate the phenotypic switch of VSMCs by myocardin pathway, which is critical for the pathological progression of varicose vein. Therefore, this study supports a prominent insight into how IQGAP1 possesses its benefit function in varicose veins development by regulating vascular remodeling.International journal of clinical and experimental pathology. 01/2014; 7(10):6475-85.
Myocardin inhibits cellular proliferation by inhibiting
NF-?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-
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
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-?B-dependent
cell-cycle regulation. Cumulatively, these findings identify a func-
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
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]6GG), a DNA consensus sequence
located in the control 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 expressed specifically in
cardiac and smooth muscle cells and potently activates their gene
expression by associating with SRF bound to CArG boxes (6).
We have shown that the toggling of SRF toward growth-
dependent or differentiation pathways is related to interplay
between 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 growth (8) and inactivation
of myocardin promotes tumor growth (9), the molecular mech-
anism related to myocardin and cellular proliferation remains
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
etermining mechanisms that preferentially promote cardi-
omyocyte and vascular smooth muscle cell (VSMC) growth
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 could 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 effect on cellular growth. Herein we describe a
relationship between NF-?B and myocardin in regulating
smooth muscle and cardiac gene expression and demonstrate a
unique role for myocardin as a transcriptional repressor and
inhibitor of cell proliferation.
Genes. Smooth muscle 22 (SM22) and atrial natriuretic factor
(ANF) promoters attached 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
an ?-MHC-luc reporter (data not shown) and corroborated in
rat neonatal cardiomyocytes (Fig. 1b). Cotransfection of the p50
NF-?B subunit had no effect 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 ability of these mutant promoters to
respond to myocardin (Fig. 1c). Similarly, p65 repressed myo-
cardin activation on the ANF promoter with CArG-far box
mutation. 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
from 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: email@example.com.
**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: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
March 4, 2008 ?
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no. 9 www.pnas.org?cgi?doi?10.1073?pnas.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
NF-?B-dependent (13–16). Indeed, TNF-? repressed myocardin
activation of SM22 and ANF reporters in both COS cells (Fig.
1d) and cardiomyocytes (Fig. 1e). Next, we examined the effect
of blocking endogenous NF-?B on myocardin transactivity.
Cotransfection with an inhibitory ?B? superrepressor (I?B?-
SR), a potent inhibitor of NF-?B activity (11), significantly
enhanced myocardin activity for SM22 (Fig. 1 d and e) and ANF
(data not shown). Together, these data demonstrate that TNF-
?/NF-?B signaling is both necessary and sufficient to modulate
myocardin transactivity in activation of cardiac and smooth
muscle gene expression.
NF-?B Inhibits Myocardin Transactivity by Physically Interacting with
Myocardin. To characterize their mechanistic interaction, we
tested whether p65 could still repress the activity 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
expression, 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 129–689, and
amino acids 78–669, but not with amino acids 328–670, 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-terminal 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
between residues 234 and 245 (12).
NF-?B Prevents the Formation of the Myocardin/SRF/CArG Ternary
Complex. Myocardin does not directly bind to DNA; rather, it is
recruited 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 complex
bound to its associated CArG box. Gel shift assays were per-
formed by using a radiolabeled consensus CArG box (derived
from 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).
Although p65 is not a part of the stable component of the
SRF/CArG or the myocardin/SRF/CArG complex, these results
indicate that p65 does affect the formation of the ternary
complex. 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
formation (Fig. 3b Right). Similarly, p65 interferes with the
ternary complex formation of myocardin/SRF on the SM22
+ + + + +
+ + + + +
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
I?B? 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-?, or I?B?-SR.
Suppression of myocardin transcriptional activity by p65. Luciferase
Tang et al.
March 4, 2008 ?
vol. 105 ?
no. 9 ?
CArG box [supporting information (SI) Fig. 7]. Together, these
data 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 significantly 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 complex was evident (Fig. 3c,
lanes 1–3). Antibody supershift confirmed the identities of this
complex (Fig. 3c, lane 2). Importantly, LPS treatment dramat-
ically attenuated the SRF/myocardin/CArG ternary complex
formation without affecting SRF CArG-box binding (Fig. 3c,
compare lanes 6 and 3). These data demonstrate that p65 is a
potent nuclear factor that inhibits myocardin transactivation by
disrupting the formation 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 activity of p65 itself. Luciferase constructs with
three tandem ?B-box promoters and its mutant were transfected
with p65, and p65 appropriately induced luciferase activity of the
wild-type ?B promoters but not its mutant (Fig. 4a). When
cotransfected with myocardin, p65-mediated luciferase activity
was decreased in a dose-dependent manner (Fig. 4a). These data
demonstrate that myocardin can inhibit p65 transcriptional
The functional inhibition of p65 transactivity by myocardin
prompted us to determine whether myocardin interferes with
DNA binding of p65. EMSAs were conducted by using a
consensus ?B box as a probe. Addition of p65 protein resulted
in a specific shifted band (Fig. 4b, lane 2). Concomitant admin-
istration of anti-p65 antibody resulted in a supershift (Fig. 4b,
lane 1). However, when the reaction was incubated with increas-
ing amounts of myocardin protein, the p65 and ?B-box com-
plexes were gradually diminished (Fig. 4b, lanes 3–6). These
to its specific DNA element.
We next sought to determine whether myocardin could phys-
iologically repress transcriptional activity of p65. Aortic smooth
muscle cells were infected with Ad-myocardin (or Ad-GFP as a
control) viruses for 48 h, and then 1 ?g/ml LPS was added for
2 h. As expected, LPS increased TNF-? expression (Fig. 4c) that
was essentially abolished with increased concentrations of myo-
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-
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.
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.
p65 inhibits the formation of myocardin/SRF/CArG ternary complex. (a) Myc-tagged myocardin, Flag-tagged p65, and SRF proteins were expressed by
www.pnas.org?cgi?doi?10.1073?pnas.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 data suggest that myocardin interferes with NF-?B sig-
naling, not by its effect 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
stably overexpressed myocardin using a tetracycline-regulated
expression system in CHO cells. Doxycycline (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 growth
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-G0/G1peak was not identified, nor did we detect
DNA fragmentation or positive TUNEL staining (data not
shown). The antiproliferative effect of myocardin was related to
G2–M phase arrest and formation of polyploid cells (Fig. 5 c and
d). To define transcriptional targets linking myocardin to cell-
cycle regulation, we demonstrate that myocardin induction is
associated with down-regulation of several important mediators
of cell-cycle progression, including those previously known to be
regulated by NF-?B such as c-myc (19) and cyclin-dependent
kinase 2 (CDK2) (20), as well as ribosomal S6 kinase (S6K) and
CDK1, but not p21 and p27 (Fig. 5e).
on cell proliferation and differentiation, we performed overex-
pression and loss-of-function studies in VSMC. Again, overex-
pression of myocardin using the tetracycline-regulated expres-
sion system decreased VSMC numbers, consistent with that seen
in CHO cells (Fig. 6a). Interestingly, overexpression of the
dominant negative myocardin increased numbers of VSMCs,
whereas dominant negative myocardin in CHO cells had no such
effect. 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-?B-dependent
and mitogenic cytokines TNF-? and IL-6. As expected, siRNA
to p65 reduced both TNF-? and IL-6 levels (Fig. 6c). In contrast,
myocardin inhibition decreased expression of SM-?-actin
whereas p65-inhibition increased its expression, consistent 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
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 a32P-labeled ?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.
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
in both G2–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.
Myocardin inhibits CHO cell proliferation. (a) Numbers of cells harboring the myocardin-inducible system were counted 72 h after treatment with or
Tang et al.
March 4, 2008 ?
vol. 105 ?
no. 9 ?
inhibition of p65 resulted in decreased VSMC growth (Fig. 6d).
Taken together, these data establish that myocardin functions as
an antiproliferative factor and show that this effect is coupled
with its repression of NF-?B transcriptional activity.
This study reveals a relationship between two 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-?B-
mediated target gene expression and function, including cellular
proliferation. These findings add several layers to our current
understanding of both NF-?B and myocardin biology. First, we
offer 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 cofactors that control
the influence of myocardin on SRF (21). Whereas regulation of
associate with SRF (7), NF-?B regulation of gene transcription
is classically a result of its binding to DNA. A functional
interaction between 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
offer an alternative mechanism of p65 regulation of SRF:
disruption 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 cofactor. Indeed, we
demonstrate that myocardin has SRF-independent effects by
directly interacting and preventing binding of p65 to its DNA
target. Furthermore, 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 activity. Our studies therefore assign a molecular
mechanism 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
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.
Effect of endogenous myocardin on VSMC growth. (a) Photomicrographs of VSMC harboring the tetracycline-regulated (TR) inducible system for
www.pnas.org?cgi?doi?10.1073?pnas.0705842105Tang et al.