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INTRODUCTION |
Vascular smooth muscle cell
(SMC)1 differentiation is an
important process during vasculogenesis and angiogenesis, and it is recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis (1-3). It is
well established that SMC do not terminally differentiate and that SMC
phenotype is regulated by a complex array of local environmental cues
including humoral factors, cell-cell and cell-matrix interactions,
inflammatory stimuli, and mechanical stresses (reviewed in Refs. 4 and
5). However, the mechanisms by which these diverse signals regulate SMC
phenotype and the transcription mechanisms that ultimately regulate SMC
differentiation are largely unknown. It is clear though that the
identification of the signaling and transcription pathways that control
SMC-specific gene expression will be an important step toward our
understanding of blood vessel development and the role played by SMC
during the development of cardiovascular disease.
To date, no transcription factors have been identified that specify SMC
lineage or by themselves can explain SMC-specific gene expression.
However, serum response factor (SRF), a MADS box transcription
factor, has been shown to contribute to the regulation of most SMC
differentiation marker genes and may be a key transcriptional regulator
of SMC differentiation (6-9). Interestingly, CArG
cis-elements (CC(A/T)6GG) that bind SRF are present in the promoters of virtually all of the SMC-specific marker
genes including SM
-actin, SM myosin heavy chain, SM22, h-caldesmon,
calponin, and telokin. The CArGs within any given SMC-specific promoter
are highly conserved among species and have been shown to be required
for the high level of activity that these promoters exhibit in SMC
cultures (7, 8, 10-14). Results from transgenic studies demonstrate
that CArG elements within the SM22 and SM
-actin promoters were also
required for the expression of these genes in vivo (8-10).
Indeed, we have shown that interactions among multiple CArG elements
are important for the in vivo expression of SM
-actin and
that a CArG element within the SM
-actin first intron functions as a
SMC-specific enhancer-like element (10). Because SRF is ubiquitously
expressed, the mechanisms whereby it contributes to SMC differentiation
are somewhat unclear. However, evidence suggests that other factors
including additional cis- and trans-regulatory
elements may mediate cell-type-specific regulation by SRF (6,
15-22).
The CArG motif was first identified as the core sequence of the serum
response element (SRE) within early response genes such as
c-fos, and much of what is known regarding the signaling
pathways that regulate SRF-dependent transcription resulted
from studies on the c-fos promoter (reviewed in Ref. 23).
During serum- or growth factor-mediated stimulation, SRF forms an
initiation complex with a protein from the ternary complex factor
family whose members (Elk-1, SAP-1, and Net/Erp) are regulated by MAP
kinase phosphorylation (24-26). The ternary complex is
stabilized by protein interactions between SRF and the ternary complex
factor and by interactions between the ternary complex factor
and the Ets cis-element that flanks the SRE CArG (27).
Recent evidence suggests that SRF-dependent transcription
is also regulated by the small GTP-binding protein RhoA, which is
required for serum-induced c-fos expression, and may be
important for regulating CArG-dependent genes in the
skeletal muscle as well (28-31). The effects of RhoA on
SRF-dependent transcription are not dependent upon the
presence of an Ets domain or on the known SRF accessory proteins
suggesting that RhoA activates SRF-dependent transcription
in a ternary complex-independent manner. Importantly, the CArG elements
within the SMC-specific promoters are not flanked by a consensus Ets
domain and do not appear to be regulated by an
Ets-dependent mechanism.
Recently, Sotiropoulos et al. (32) have shown that in NIH
3T3 cells, the effects of RhoA on SRF-mediated transcription may be
secondary to the actions of RhoA on the actin cytoskeleton. These
results are of particular importance because they are the first to
suggest that SRF-dependent transcription is positively regulated through increases in actin polymerization. The exact mechanisms for SRF activation remain to be identified, and the significance of these findings in regard to the expression of SRF-dependent gene targets is unclear because an artificial
c-fos promoter (lacking a consensus Ets domain) was used as
the primary measure of Rho-dependent SRF activation.
In the present studies, we extend the observations of
Sotiropoulos et al. (32) by demonstrating in SMC that
multiple SMC-specific promoters are regulated via
RhoA-dependent changes in actin dynamics. In addition, we
demonstrate that these RhoA-induced changes are at least partially
mediated by Rho kinase. Because virtually all of the SMC-specific
promoters identified to date are regulated by SRF, these results
suggest that RhoA signaling regulates SMC phenotype. Moreover, because
many of the environmental factors that regulate SMC phenotype have also
been shown to activate RhoA, these results also suggest that RhoA may
help to integrate multiple environmental signals that regulate SMC differentiation.
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MATERIALS AND METHODS |
Cell Culture Transient Transfections and Reporter
Assays--
SMC from rat thoracic aorta were isolated, cultured, and
transfected as described previously (33, 34). In short, cells were
maintained in 10% serum and were transfected 24 h after plating at 70-80% confluency with 2 µg of reporter plasmid DNA, using the
transfection reagent Superfect (Qiagen) as per protocol. The SM22
promoter (from
450 to +88), the SM
-actin promoters (p2600Int, from
2560 to +2784, and p125, from
125 to +44), and the
c-fos promoter (from
356 to +109) that were used in this
study have been described previously (8-10, 35). Coexpression
experiments were carried out by including 0.25 µg of the following
expression constructs: empty Prk5 vector; Prk5 containing
constitutively active RhoA (L63), Cdc42 (L61), or Rac (L61); or
Prk5 containing C3 transferase (expression plasmids were a
generous gift from Alan Hall, University College, London, United
Kingdom). Cells expressing C3 were maintained in 10% serum,
whereas cells expressing the small G proteins were maintained in
defined serum-free medium. After 48 h, cell lysates were
prepared for the measurement of CAT activity using standard methods
(34). The CAT activity of each sample was normalized to the protein
concentration of each cell lysate as measured by the Bradford assay. A
promoterless CAT construct was also transfected to serve as a base-line
indicator of CAT activity, and the activity of each promoter construct
was expressed relative to promoterless activity. Additionally, an SV40
promoter-CAT construct with enhancer (Promega) served as a positive
control of transfection and CAT activity. All CAT activities represent
at least three independent experiments with each construct tested in
triplicate per experiment. Relative CAT activity data are expressed as
the means ± S.D. computed from the results obtained from each set
of transfection experiments. We did not cotransfect a viral
promoter/LacZ construct as a control for transfection efficiency
because we have previously shown that such constructs exhibit unknown
and variable squelching effects on the SM-specific promoters,
presumably due to competition for common transcription factors (34).
Moreover, we have found that the inclusion of such controls is
unnecessary in that variations in transfection efficiency among
independent experimental samples are routinely very small (<10%)
(34).
Drug Treatment and F-actin Visualization--
Latrunculin B and
cytochalasin D were purchased from Calbiochem (San Diego, CA).
Jasplakinolide was purchased from Molecular Probes (Eugene, OR).
Y-27632 was a generous gift from Akiko Yoshimura (Welfide Corporation,
Osaka, Japan). Before drug treatment, SMC were transfected with
SMC-specific and c-fos reporter constructs as described
above. Immediately after the removal of the Superfect transfection
reagent, cells were treated with either latrunculin B (0.5 µM), jasplakinolide (0.5 µM), cytochalasin
D (2.0 µM), Y-27632 (10 µM), or
vehicle. SMC treated with latrunculin B or Y-27632 were
maintained in 10% serum, and those treated with jasplakinolide or
cytochalasin D were maintained in defined serum-free medium. After 12 h, cells were harvested, and reporter assays were
performed as described above. Drug-induced effects on actin
polymerization were measured in parallel experiments for all groups. At
0, 5, 10, and 24 h after drug treatment, cells were rinsed three
times with ice-cold phosphate-buffered saline, fixed with 4%
paraformaldehyde, stained with FITC-phalloidin, and visualized with a
fluorescent microscope. SMC transfected with a constitutively
active Myc-tagged L63 RhoA were immunostained with an anti-c-Myc
antibody and counterstained with FITC-phalloidin.
Measurement of Endogenous Actin Expression--
Control- or
Y-27632-treated SMC were maintained in minimal methionine medium
containing 10% fetal calf serum for 12 h. Cells were
then switched to fresh medium containing 50 µCi/ml
[35S]methionine and were incubated for an additional
12 h. Cells were then rinsed three times with ice-cold
phosphate-buffered saline and lysed in loading buffer. Equal amounts of
DNA were loaded onto tube gels, and actin isoforms were visualized by
standard two-dimensional gel electrophoresis as described
previously (36). Multiple labeling experiments were performed, and
three separate gels were run for each treatment group per experiment.
The ratio of radiolabeled SM
-actin to nonmuscle
-actin was
determined by densitometry using ImageQuant software (Molecular
Dynamics, Sunnydale, CA).
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RESULTS |
SMC-specific Promoter Activity Was Regulated by RhoA
Signaling--
Current evidence suggests that signaling through RhoA
is required for SRE-dependent c-fos expression
and regulates several CArG-containing genes in the skeletal muscle
(28-31). To determine whether RhoA was important for
CArG-dependent SMC-specific gene regulation, we
cotransfected CAT reporter constructs driven by the SM22 and SM
-actin promoters into rat aortic SMC along with an expression vector
containing C3 transferase, which specifically ADP-ribosylates and
irreversibly inhibits RhoA. The SM22 promoter (from
450 to +88) and
the SM
-actin promoter (from
2600 to +2754) used in these studies
were shown to direct SMC-specific regulation of these genes in
transgenic models (8-10). C3 transferase expression completely
abolished the activity of both promoters, indicating that RhoA activity
is required for the expression of these constructs in SMC (Fig.
1A). To test the involvement
of RhoA more directly, we also cotransfected a constitutively active RhoA (L63) into serum-starved SMC. L63 RhoA trans-activated
the SM22 and the full-length SM
-actin promoters by 10-fold and
6-fold, respectively (Fig. 1B). Constitutively active RhoA
also dramatically increased the activity of a minimal SM
-actin
promoter (
125 to +44) that is regulated by two CArG elements within
this short promoter region but had no effect on the same construct that
contained mutations to the CArG elements (data not shown). These
results indicate that the effects of RhoA on SMC-specific gene
expression were mediated by a CArG/SRF-dependent mechanism.
Significantly, L63 RhoA had relatively modest effects on the activity
of an Ets-containing SRE-dependent c-fos
promoter in SMC.

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Fig. 1.
Regulation of SMC-specific transcription by
RhoA signaling. A, cultured rat aortic SMC were
transfected with 2 µg of the indicated SMC-specific promoter/CAT
reporter construct along with either 0.25 µg of the empty Prk5
expression vector or the same vector that contained C3 transferase, an
irreversible inhibitor of RhoA. CAT activity (±S.D.) measured after
48 h in 10% serum was expressed relative to the base-line CAT
activity of a promoterless CAT construct set to 1. B, SMC
were transfected with 2 µg of the indicated CAT reporter along with
the Prk5 expression vector containing constitutively active L63
RhoA. CAT activity was measured after 48 h in serum-free
media and expressed relative to the CAT activity measured in
cells containing empty vector. Note that constitutively active RhoA
only modestly increased the activity of a CArG-containing
c-fos promoter. Promoter sequences are SM22 ( 450 to +88),
SM -actin ( 2560 to +2784), and c-fos ( 356 to +109).
The 125 construct is the SM -actin promoter region from 125
to +44 that contains two CArG elements important for expression
in vivo. C, a comparison of the effects of
constitutively active RhoA, Cdc42, and Rac on SM -actin and
c-fos promoter activities.
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Because the Rho family members Cdc42 and Rac have also been shown to
up-regulate the SRF activity of c-fos, we also tested whether the constitutively active forms of these proteins would regulate SMC differentiation marker gene expression. Although all three
small GTPases slightly but significantly increased c-fos promoter activity (p < 0.01), SM
-actin promoter
activity was not affected by constitutively active Rac and was slightly
inhibited by constitutively active Cdc42 (Fig. 1C).
RhoA regulated Stress Fiber Formation in SMC--
Sotiropoulos
et al. (32) have suggested that RhoA activation
regulates gene expression through its effects on the actin cytoskeleton. Therefore, to test the effects of RhoA activation on
actin polymerization, we transfected a Myc-tagged L63 RhoA expression
plasmid into SMC that were then stained for F-actin stress fibers with
FITC-phalloidin. Fig. 2B shows
that untransfected SMC maintained in 10% serum had fairly well
developed stress fibers (small arrows). However, SMC
expressing L63 RhoA had dramatically increased actin stress fiber
networks (large arrows), demonstrating that RhoA activation
regulates stress fiber formation in SMC.

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Fig. 2.
Effects of constitutively active RhoA on
actin stress fiber formation in SMC. SMC maintained in 10% serum
were transfected with 1.0 µg of the Prk5 expression plasmid
containing constitutively active c-Myc-RhoA. After 48 h,
cells were fixed in 4% paraformaldehyde and immunostained with an
anti-c-Myc antibody to visualize Rho expression (A) and
immunostained with FITC-phalloidin to visualize actin stress fibers
(B).
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SMC Differentiation Marker Gene Transcription Was Regulated by
Actin Polymerization--
Our results suggested that RhoA activation
regulates both SMC-specific gene transcription and stress fiber
formation in SMC. To test whether the effects of RhoA on SMC-specific
promoter activity were mediated by its effects on actin dynamics, we
treated SMC with latrunculin B, a toxin that disrupts actin
polymerization. Latrunculin B treatment (0.5 µM) of SMC
in 10% serum disrupted stress fiber formation for up to 12 h
(Fig. 3A). After this time, actin polymerization gradually returned, and by 24 h SMC treated with latrunculin B were virtually indistinguishable from untreated cells. In addition, latrunculin B also had little effect on total cellular protein, which indicated that this drug was not overtly cytotoxic under these conditions. Latrunculin B did not affect c-fos promoter activity but dramatically attenuated the
activities of the SM22 and SM
-actin promoters (Fig. 3B).
Importantly, latrunculin B also inhibited trans-activation
of the SM
-actin promoter by L63 RhoA as well as L63 RhoA-induced
stress fiber formation.

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Fig. 3.
Effects of latrunculin B on actin
polymerization and promoter activity in SMC. A, SMC
were treated with 0.5 µM latrunculin B (LB), a
toxin that inhibits actin polymerization, for the indicated time
period. Cells were then fixed and immunostained for F-actin with
FITC-phalloidin. B, SMC were transfected with the indicated
reporter constructs and were immediately treated with 0.5 µM latrunculin B. CAT activity was measured after 12 h and is expressed as a percentage of CAT activity in vehicle-treated
cells. Note that trans-activation by L63 RhoA was also
inhibited by latrunculin B.
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The results presented in Fig. 3 suggest that RhoA activation induces
SMC-specific gene expression by increasing actin polymerization. Therefore, to further test whether SM
-actin transcription was sensitive to actin dynamics, we treated SMC with jasplakinolide, a
toxin known to increase actin polymerization (37). SMC treated with 0.5 µM jasplakinolide and labeled with FITC-phalloidin had very disorganized actin structures and contained large amorphous actin
aggregates (data not shown). These effects are consistent with the
known actions of this toxin, which severs F-actin, creating new
nucleation sites that support actin polymerization (37). Jasplakinolide
increased SM22 and SM
-actin promoter activities by 22-fold and
14-fold, respectively, but increased c-fos promoter activity
by only 3-fold. Cytochalasin D had similar effects increasing the
activities of the SM22 and SM
-actin promoters but having no effect
on the c-fos promoter. The fact that cytochalasin D (which
disrupts actin polymerization) increased SMC-specific promoter activity
suggests that cytochalasin-induced SRF activity is not directly
regulated by increased actin polymerization per se and that
additional mechanisms that sense actin dynamics in SMC may be involved
in regulating SMC-specific transcription. Interestingly, Sotiropoulos
et al. (32) made similar observations in their studies on the c-fos SRE and suggested that
SRF-dependent transcription may be negatively regulated by
monomeric G-actin and that cytochalasin D may sequester monomeric
G-actin in a manner that effectively reduces the free pool. To
determine whether the effects of cytochalasin D were independent of
those mediated by RhoA, we treated SMC transfected with L63 RhoA with
cytochalasin D. The treatment of SMC with cytochalasin D prevented both
the L63 RhoA-induced increase in differentiation marker gene expression
(Fig. 4B) and L63 RhoA-induced
stress fiber formation (data not shown).

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Fig. 4.
Effects of jasplakinolide and cytochalasin D
on SMC-specific promoter activity. A, SMC were
transfected with the indicated reporter plasmids and were immediately
treated with 0.5 µM jasplakinolide, a toxin that
stimulates actin polymerization, or 2.0 µM cytochalasin
D, a drug that disrupts actin polymerization by binding to G-actin. CAT
activity was measured after 12 h and is expressed relative to the
CAT activity of vehicle-treated cells set to 1. Results showing
trans-activation of the SM -actin promoter by L63 RhoA
are included for comparison. B, SMC cotransfected with
constitutively active L63 RhoA were then treated with 2.0 µM cytochalasin D. The effects of cytochalasin D alone
are also included.
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Transcriptional Regulation by RhoA in SMC Was Partially Mediated by
Rho Kinase--
It is well known that RhoA-dependent
activation of Rho kinase induces actin polymerization and stress fiber
formation in many cell types (reviewed in Ref. 38). However, it is
unclear whether the activity of Rho kinase correlates with the effects
of RhoA on the transcription of c-fos (39-42). Our results
indicated that in SMC, the SMC-specific promoters were significantly
more sensitive than the c-fos promoter was to activation by
RhoA signaling. Therefore, to test whether SMC-specific
transcription was regulated through the activation of Rho kinase, we
treated SMC with the highly specific Rho kinase inhibitor Y-27632 (43).
The micrograph in Fig. 5A shows that 10 µM Y-27632 significantly inhibited stress
fiber formation in SMC and also led to the appearance of long thin
cellular extensions. Y-27632 also reduced the activity of transiently
transfected SMC-specific promoters by 50-60% (Fig. 5B),
although it had little effect on the c-fos promoter or on
the minimal thymidine kinase (TK) promoter that does not contain CArG
elements. Fig. 5C is an autoradiograph from a representative
two-dimensional gel that illustrates the effects of Y-27632 on the
expression of endogenous actin isoforms. Interestingly, the results
showed that all of these CArG-containing actin genes were
down-regulated to some extent. However, the synthesis of the
SM-specific
-actin isoform was inhibited to a greater extent than
the ubiquitously expressed
-actin isoform (
:
synthesis ratios
were reduced by greater than 40% in Y-27632-treated SMC,
p < 0.01). Taken together, these results suggest that
Rho kinase plays an important role in the regulation of SMC
differentiation marker gene expression and that ubiquitously expressed
CArG-dependent genes, such as c-fos and nonmuscle
-actin, may be less sensitive to this regulatory
pathway.

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Fig. 5.
Effects of the Rho kinase inhibitor Y-27632
on actin stress fiber formation, SMC-specific transcription, and
endogenous actin expression. A, SMC maintained in 10%
serum were treated with Y-27632 (10 µM). After 12 h,
cells were fixed in 4% paraformaldehyde and stained with
FITC-phalloidin to visualize actin stress fibers. B, SMC
were transfected with the indicated reporter constructs and immediately
treated with Y-27632. After 24 h, CAT activity was measured and
expressed relative to a promoterless CAT construct (pless)
set to 1. Note that Y-27632 had little effect on the c-fos
promoter or the minimal thymidine kinase promoter (TK).
C, rat aortic SMC maintained in 10% serum were treated with
Y-27632 for 12 h. Cells were then switched to
[35S]methionine containing medium and
restimulated with Y-27632 for an additional 12 h. Equal amounts of
control- and drug-treated SMC lysates were run on two-dimensional gels
using standard protocols for visualizing actin isoforms. A
representative autoradiograph is shown.
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|
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DISCUSSION |
A major goal of our laboratory has been to elucidate control
processes that regulate SMC differentiation by identifying the mechanisms that coordinately control the transcription of SMC differentiation marker genes. The focus of the present study was to
investigate the role of RhoA signaling in the regulation of SMC-specific gene transcription. Because RhoA activates SRF, a transcription factor that has been shown to regulate virtually all of
the SMC-specific marker genes identified to date (7, 8, 10-14), we
hypothesized that RhoA signaling contributes to the coordinate
regulation of SMC-specific gene expression. The data presented indicate
that: 1) RhoA activation is important for the expression of several
SMC-specific marker genes; 2) regulation of actin polymerization by
RhoA (but not by Cdc42 or Rac) may be an important determinant of
SMC-specific gene transcription; 3) RhoA-dependent
regulation of SMC-specific gene transcription is mediated at least in
part by Rho kinase; and 4) in SMC, the CArG-dependent
SMC-specific genes (i.e. SM22, SM
-actin) and the
CArG-dependent genes (i.e. c-fos,
-actin) that are ubiquitously expressed are differentially regulated
by RhoA.
Results presented in Fig. 1 provide strong evidence that RhoA activity
is required for SMC-specific promoter activity because C3 transferase,
which ADP-ribosylates and irreversibly inactivates RhoA, completely
inhibited the activities of the SM22 and SM
-actin promoters.
Constitutively active RhoA up-regulated several SMC-specific CArG-dependent genes, further indicating that RhoA
activation is important for this response. RhoA activation has been
shown to have similar effects on the SRF-dependent activity
of several CArG-dependent skeletal muscle-specific genes
(30, 31), suggesting that RhoA activation may be a common signaling
pathway that regulates muscle-specific gene expression. In fact, RhoA
also regulates SRF expression (probably through CArG elements within
the SRF promoter) and may be part of an important positive feedback
mechanism for maintaining high levels of SRF expression in all three
muscle cell types (44, 45). Although our results indicate that
RhoA-dependent activation of SRF plays a major role in
SMC-specific transcription, other mechanisms must be involved that
distinguish SRF-dependent transcriptional regulation in SMC
from SRF-dependent transcriptional regulation in the other
muscle cell types. For example, it is well known that in cardiac and
skeletal muscles, SRF interacts with other cardiac and skeletal
muscle-specific factors, such as myoD, GATA-4, and Nkx2.5, to regulate
cell-type-specific expression (22, 46-48). However, factors that
confer SMC-specific gene regulation have not been identified in SMC. We
have recently provided evidence that an SRF-associated protein that is
selectively expressed in SMC may contribute to SMC-specific gene
regulation by SRF (6). Several splice variants of SRF have also been
described recently, but a detailed description of the expression
patterns and activities of these variants has not been reported, which
makes it difficult to determine whether alternative splicing of SRF
contributes to cell-type-specific gene regulation (49, 50).
Our results also indicate that multiple SMC-specific differentiation
marker genes are regulated by RhoA-induced changes in the actin
cytoskeleton. Constitutively active RhoA trans-activated the
SM22 and SM
-actin promoters but had little effect on the CArG-containing c-fos promoter. Inhibiting actin
polymerization with latrunculin B blocked trans-activation
by constitutively active RhoA, which supports a model in which
RhoA-mediated increases in F-actin promote SMC differentiation marker
gene transcription. Our observation that jasplakinolide, an actin
polymerizing agent, dramatically increased SMC-specific promoter
activity is also consistent with such a model. The constitutively
active forms of Cdc42 and Rac did not stimulate SMC-specific promoter
activity, suggesting that actin polymerization at the cell periphery
does not regulate SMC-specific transcription and suggests that separate pools of actin may differentially regulate SMC function. Our data also
indicate that signaling through Rho kinase is significant, but because
Y-27632 only inhibited promoter activity by 50-60%, we cannot rule
out the involvement of other Rho effectors, such as PKN, citron
kinase, rhotekin, and the diaphanous family of proteins.
The precise mechanisms by which actin dynamics affect
SRF-dependent transcription are currently unclear. Based in
part on the observed effects of cytochalasin D, Sotiropoulos
(32) has suggested a model whereby G-actin inhibits SRF directly
or sequesters cofactors required for SRF activation. Our data indicate
that cytochalasin D-induced activation of SRF-dependent
SMC-specific transcription does not require an intact actin stress
fiber network. However, we did show that the expression of
constitutively active RhoA in cytochalasin D-treated SMC did not result
in stress fiber formation or in an increase in SM
-actin promoter
activity above that seen with cytochalasin D alone. These data indicate
that in SMC these interventions probably converge at some level and that RhoA must affect actin polymerization to increase SMC marker gene
transcription. There is some evidence to suggest that nuclear actin may
regulate transcription by helping to modify chromatin structure (51),
and it has been shown that G-actin shuttles through the nucleus (52)
and binds specifically to important nuclear proteins including the
SWI·SNF complex (53, 54). It is also important to consider
that the observed effects of RhoA activation (and of the cytoskeletal
inhibitors) may be partially due to secondary changes in cell
morphology that occur under these conditions. In addition, the
pharmacological mechanisms of the cytoskeletal inhibitors used in this
study are only partially understood, and undescribed side effects of
these drugs may have affected SMC differentiation marker gene expression.
The signal transduction pathways that regulate SMC differentiation are
only partially understood, but results from the present studies suggest
that RhoA activation may serve as a convergence point for multiple
environmental factors known to regulate SMC-specific gene expression
and differentiation. For example, integrin-matrix interactions,
mechanical stretch, and contractile agonists, such as endothelin-1 and
angiotensin II, are known to regulate SMC-specific gene expression as
well as the activity of RhoA (40, 55-57). Whether these environmental
factors regulate SMC differentiation through RhoA-dependent
signaling mechanisms and/or changes in actin dynamics will be an
important area for future studies.
RhoA activation is also required for growth factor-induced
expression of c-fos, and recent evidence suggests that RhoA
activation may mediate the growth response of SMC to angiotensin II,
thrombin, and mechanical stretch (40, 55, 58). The data presented in
this manuscript, however, demonstrate that c-fos expression in SMC is not dramatically up-regulated by constitutively active RhoA
or by drug-induced modulation of the actin cytoskeleton, indicating
that the RhoA signaling pathways that regulate SMC differentiation
marker gene expression diverge from those that regulate cell growth and
proliferation. SMC proliferation and differentiation are not
necessarily mutually exclusive (4), and it will be very important to
further clarify the specific signals that differentiate these two
SRF-dependent pathways. Indeed, Seasholtz et al.
(58) found that constitutively active RhoA only potentiated SMC
proliferation in the presence of activated Ras, and results from
Alberts et al. (28) indicate that additional signaling
through the SAPK/JNK pathway may be important for the Rho-mediated
regulation of c-fos. Integrin signaling also requires Rho
activation (59-61), and it interesting to speculate that Rho and
integrin pathways may converge (perhaps at the level of the actin
cytoskeleton) to regulate SMC growth and/or differentiation.
In summary, the data presented herein indicate that RhoA signaling
plays a major role in the SRF-dependent regulation of SMC differentiation marker gene expression. Although the exact mechanisms involved in controlling the transcriptional activation of SRF are not
completely clear, the activation of Rho kinase and RhoA-specific changes in actin polymerization seem to be involved. Interestingly, most SMC-specific differentiation marker genes identified to date code
for contractile or contractile-associated proteins (4). Therefore, RhoA
signaling may also help to determine SMC phenotype (and possibly the
phenotype of other muscle cell types as well) by increasing contractile
protein gene expression. Moreover, when coupled with extensive evidence
that RhoA increases SMC contractility (43, 62), our data suggest that
short term regulation of SMC contractile force may be coupled to long
term regulation of SMC contractile-associated gene expression by a
RhoA-dependent mechanism. Finally, given the importance of
SMC growth and differentiation during blood vessel development and
cardiovascular pathophysiology, further studies to identify the
mechanisms that regulate RhoA-dependent changes in SMC
differentiation marker gene expression may provide novel insights
regarding the mechanisms that contribute to the abnormal regulation of
SMC differentiation characteristic of vascular diseases such as atherosclerosis.