Am J Physiol Cell Physiol 292: C70 –C81, 2007.
First published August 23, 2006; doi:10.1152/ajpcell.00386.2006.
Invited Review
Serum response factor: master regulator of the actin cytoskeleton
and contractile apparatus
Joseph M. Miano, Xiaochun Long, and Keigi Fujiwara
The Cardiovascular Research Institute, University of Rochester School of Medicine, Rochester, New York
myocardin; smooth muscle; CArG box; knockout
intricate and dynamic cytoskeleton
which, in addition to conferring cell shape and structural
support, serves to regulate such diverse processes as apoptosis,
cell adhesion, cell division, cell migration, cell polarity, chromosomal segregation, gene expression, intracellular trafficking, muscle contraction, phagocytosis, and signaling. The existence of a cytoskeleton has even been documented in the
bacterial kingdom where homologs of actin, tubulin, and an
intermediate filament function in cell shape and chromosomal
segregation (73). Among simple eukaryotes (e.g., yeast), a
larger repertoire of cytoskeleton-related proteins exists with
actin-myosin complexes, an array of actin-associated proteins,
and Rho-mediated signaling pathways (107). The emergence
of multicellular organisms and relatively simple animals heralded a commensurate expansion of the cytoskeleton and the
earliest forms of muscle. The explosive rise in vertebrate
species necessitated an increasingly complex cytoskeleton and
multiple contractile tissues (skeletal, cardiac, and smooth muscle) to carry out cell/tissue-specific functions. Collectively,
mammalian cytoskeleton-contractile systems encompass
⬎1,000 unique genes (list available upon request) whose
encoded proteins hard wire the cell’s membrane-bound, cytoplasmic, and nuclear cytoskeleton complexes that are central to
carrying out essentially all biological processes in a cell.
Although the cell biology and physiology of many cytoskeleton proteins have been studied in great detail, much less is
known about their transcriptional regulation. Here, we review
genetic and genomic data supporting a vital role for one
ALL LIVING CELLS HARBOR AN
Address for reprint requests and other correspondence: J. M. Miano, Cardiovascular Research Institute, Univ. of Rochester School of Medicine, 601 Elmwood
Ave., Box 679, Rochester, NY 14642 (e-mail: [email protected]).
C70
transcription factor, serum response factor (SRF), in the regulation of an expanding number of genes encoding actin-cytoskeleton and contractile proteins. Before we consider the
newest SRF target genes and their connection to SRF-mediated
cell motility, we provide a brief up-to-date summary of SRF’s
control of growth and differentiation programs of gene expression as well as the nature of the CArG box.
SERUM RESPONSE FACTOR: TOGGLE SWITCH FOR
DISPARATE PROGRAMS OF GENE EXPRESSION
SRF is a founding member of the MADS (Mcm1 and Arg80
in yeast, Agamous and Deficiens in plants, and SRF in animals)
domain-containing family of transcription factors and is
present in most, if not all, species from the animal, plant, and
fungus kingdoms (67, 68, 99). At least one species of protist
(Dictytostelium discoideum) harbors an SRF ortholog, but no
SRF orthologs have yet been defined in the bacterial kingdom.
SRF was so named because of its binding activity to a serum
response element located in the promoter of the immediate
early transcription factor, FOS (79, 108). Similar serum response elements were subsequently found in the promoter
regions of a number of other immediate early transcription
factors. Signaling to SRF occurs principally through mitogenactivated protein kinase (MAPK) or RhoA pathways that
converge on the nucleus to stimulate gene expression (38).
SRF-induced growth-related genes encode for proteins that
directly activate growth factor and cell cycle genes (51). Thus
SRF is largely an indirect regulator of cell growth, and the in
vitro evidence for this stems primarily from gain- and loss-offunction studies (32, 33, 86, 103). Despite the evidence supporting SRF’s role as a growth-associated transcription factor,
there are some reports demonstrating an inverse relationship
0363-6143/07 $8.00 Copyright © 2007 the American Physiological Society
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Miano JM, Long X, Fujiwara K. Serum response factor: master regulator of the
actin cytoskeleton and contractile apparatus. Am J Physiol Cell Physiol 292: C70 –C81,
2007. First published August 23, 2006; doi:10.1152/ajpcell.00386.2006.—Serum response factor (SRF) is a highly conserved and widely expressed, single copy transcription factor that theoretically binds up to 1,216 permutations of a 10-base pair cis
element known as the CArG box. SRF-binding sites were defined initially in growthrelated genes. Gene inactivation or knockdown studies in species ranging from
unicellular eukaryotes to mice have consistently shown loss of SRF to be incompatible
with life. However, rather than being critical for proliferation and growth, these genetic
studies point to a crucial role for SRF in cellular migration and normal actin cytoskeleton and contractile biology. In fact, recent genomic studies reveal nearly half of the
⬎200 SRF target genes encoding proteins with functions related to actin dynamics,
lamellipodial/filopodial formation, integrin-cytoskeletal coupling, myofibrillogenesis,
and muscle contraction. SRF has therefore emerged as a dispensable transcription factor
for cellular growth but an absolutely essential orchestrator of actin cytoskeleton and
contractile homeostasis. This review summarizes the recent genomic and genetic
analyses of CArG-SRF that support its role as an ancient, master regulator of the actin
cytoskeleton and contractile machinery.
Invited Review
C71
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that restrict SRF-mediated transcriptional activities to cellspecific gene sets.
Among SRF cofactors, the myocardin (MYOCD) family of
transcription factors has been of special interest in the context
of vascular smooth muscle cell (SMC) differentiation (65, 84).
First cloned in a bioinformatics screen for cardiac-restricted
transcription factors (110), MYOCD is among the most powerful eukaryotic transactivators known. Through a close partnership with SRF, MYOCD can execute a near-complete
program of SMC differentiation in vitro (19, 111, 121). Indeed,
MYOCD knockout mice arrest shortly after establishment of a
functional cardiovascular system because of impaired vascular
SMC differentiation (63). Two other myocardin-related transcription factors (MRTF-A and MRTF-B) have been identified
that are more widely expressed than MYOCD and appear to
direct distinct programs of gene expression (95, 112). Interestingly, levels of MYOCD mRNA are as high in vascular SMC
as they are in cardiac muscle, but the levels of MYOCD are
sharply attenuated when SMC are cultured in vitro or injured in
vivo (19, 41). Thus the phenomenon of an attenuated SMC
differentiated phenotype, first recognized 36 years ago (31),
may in part relate to altered MYOCD expression. In this
context, it will be of great interest to understand the transcriptional nature of MYOCD gene expression.
The binding of MYOCD family members to SRF has been
delimited within a region of MYOCD proteins bearing homology to the B-box of the ELK family of SRF cofactors (113,
122). In an elegant study from the Olson laboratory, the ability
of SRF to bind either ELK1 or MYOCD was shown to depend
on the phenotype of SMC. When SMC are stimulated with a
growth factor, the MAPK-ELK1-SRF axis is engaged and
SRF-ELK1 complexes are favored over SRF-MYOCD complexes. As a result, growth-related genes are more likely to be
activated than SMC contractile genes. Conversely, when serum
is withdrawn and the MAPK-ELK1-SRF axis is not engaged,
SRF-MYOCD complexes are favored and SMC contractile
gene expression ensues (113). Similar molecular events are
likely to exist with other disparate programs of SRF-dependent
gene expression. The SRF-MYOCD relationship highlights
one of several known mechanisms wherein ubiquitously expressed SRF can direct specific programs of gene expression in
a cofactor-mediated manner (Fig. 1). Another critical determinant of SRF-dependent gene expression is SRF’s binding to a
diverse assortment of DNA cis element sequences, a subject
we take up next.
FUNCTIONAL SRF BINDING SITES: THE CArGome
As indicated earlier, SRF binds serum response elements in
the promoter of various immediate early transcription factors.
The core sequence of the serum response element is a CC A/T
rich GG sequence or CArG box (74), which is common to all
SRF target genes. Serum response elements, by definition,
contain an adjacent cis element (called an ETS site) that binds
members of the ELK family of SRF cofactors (11). A misconception regarding serum response elements and CArG boxes
continues to exist in the SRF world and is in need of clarification. Specifically, serum response elements and CArG boxes
are often referred to as one in the same; however, they should
not be considered synonymous since CArG boxes are not
always associated with adjacent ETS sites. Therefore, in this
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between SRF expression and cell growth. For example, SRF
reverses the ras-transformed phenotype (54), and RNA interference (RNAi) of SRF phenocopies growth factor-stimulated
smooth muscle cell proliferation (49). Perhaps the most direct
in vitro evidence blurring the concept of SRF being essential
for growth are the studies done in embryonic stem cells null for
SRF. Here, although defective expression of immediate early
SRF target genes is evident, cells proliferate normally (94).
The latter result clearly indicates that compensatory pathways
must exist in cell systems to ensure that such a critical process
as growth occurs, even in the absence of SRF. As will be
discussed later, cytoskeleton-contractile activity cannot be
compensated for in cells where SRF expression is reduced or
absent.
What in vivo evidence exists to support SRF’s role in
promoting a growth phenotype? Balloon injury to the vessel
wall evokes rapid increases in c-fos as well as other SRF target
genes involved in growth of vascular cells (52, 71, 72). SRF
can mediate regenerating epithelium in an in vivo model of
gastric ulceration (14), and anti-sense SRF impedes angiogenesis in the same model (15). In yeast, mutation of the minichromosome maintenance 1 (Mcm1) gene results in defective
DNA replication, and there is evidence for Mcm1 binding to
bonafide origins of replication in the yeast genome (18). In
contrast to the above instances, much in vivo genetic evidence
exists for SRF playing a critical role in processes unrelated to
cell replication. For example, cardiac-restricted overexpression
of either wild-type or dominant-negative SRF results in various
forms of heart failure because of severe alterations in contractile gene expression (125, 126). Moreover, SRF inactivation or
knockdown in D. discoideum (26), Drosophila melanogaster
(91), Caenorhabditis elegans (30), and Mus musculus (1, 28,
55, 61, 70, 78, 81, 82) does not result in impaired cell
proliferation. Instead, these SRF null models reveal an essential and ancient role for SRF in the organization of normal
cytoskeleton and contractile systems (see below). Although the
in vivo data indicate, overwhelmingly, that SRF is dispensable
for cell proliferation and growth of tissues, when SRF is
present it likely plays a redundant role in growth-related
processes.
At the same time SRF was being characterized as a transcription factor activating growth-promoting genes, independent work from the muscle field showed SRF binding within
the promoter region of several contractile genes that mark the
terminally differentiated state of sarcomeric muscle (8, 59, 74).
We now appreciate that SRF can activate a number of contractile genes, the expression of which spans all three differentiated
muscle types. Indeed, developmental studies reveal the highest
levels of SRF mRNA expression in differentiating cardiac,
skeletal, and smooth muscle cell lineages (6). Smooth muscle
cell-restricted SRF target genes are of particular interest, since
their compromised expression is a hallmark of vascular and
visceral smooth muscle cell diseases as well as metastatic
cancer (80, 88). The realization that SRF controls mutually
exclusive programs of gene expression (growth vs. muscle
differentiation) led to an important question in the SRF world:
how could a widely expressed transcription factor direct such
disparate programs of gene expression? SRF, per se, effects
only subtle changes in gene expression. However, nearly 50
SRF cofactors (list available upon request) have been defined
Invited Review
C72
review, we will only refer to SRF-binding sites as CArG boxes
or CArG elements.
Binding site selection and scores of promoter studies have
helped elucidate the base sequence rules for SRF binding (60,
68, 119). Accordingly, we may classify CArG elements in two
broad categories, consensus CArG and CArG like. Consensus
CArG boxes are 10-base pair sequences of DNA conforming to
the general rule, CCWWWWWWGG (where W may be either
an A or T nucleotide). There are 64 possible consensus CArG
elements to which SRF may bind (either A or T across 6
nucleotides ⫽ 26 or 64). SRF binds with high affinity to
consensus CArG boxes. CArG-like elements, to which SRF
binds with weaker affinity, are essentially a consensus CArG
with 1 base pair deviation from the consensus CCW6GG (e.g.,
ACW6GG, CCW6TG, CCGW5GG, etc.). If we consider each
central W substituted with either C or G (e.g., CCSW5GG,
CCWSW4GG, CCWWSW3GG, etc), then there are 6 ⫻ 26 ⫽
384 deviations from the consensus CArG. Furthermore, there
are 12 potential single nucleotide changes across the terminal
CC (either C may be substituted with A, G, or T) or GG (either
G may be substituted with A, C, or T) of an otherwise
consensus CArG yielding 12 ⫻ 26 ⫽ 768 additional deviations
from consensus CArG. Thus there are 384 ⫹ 768 ⫽ 1,152
theoretical CArG-like elements. Combined with consensus
CArGs, SRF can theoretically bind 1,152 ⫹ 64 ⫽ 1,216
permutations of CArG. These theoretical data have been of
enormous utility in developing a bioinformatic approach to
define, genome-wide, functional CArG elements, which is our
next topic of discussion.
The acquisition of the genetic code of life in a growing
number of organisms heralds an unprecedented opportunity to
scan different species of genomes for similarities in sequence
composition (i.e., comparative genomics). This approach has
clearly assisted investigators in defining coding exons that
average ⬃150 base pairs. Defining regulatory sequences (e.g.,
CArG boxes), averaging between 8 and 12 base pairs, is much
more challenging, particularly if the binding rules of the
transcription factor are poorly resolved. With much of the rules
governing SRF binding to CArG understood, we set out to
perform a genome-wide scan for functional CArG elements in
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SRF-REGULATED ACTIN CYTOSKELETON GENES
An impressive list of SRF target genes with clear physiological functions in the actin cytoskeleton and contractile
systems exists with the number of actin cytoskeleton genes
now surpassing those of contractile genes (Table 1). It is
interesting to point out that, although the transcriptional regulation of most cytoskeleton genes in Table 1 has only recently
been revealed, much information exists regarding the role of
the cytoskeleton in gene transcription. In this context, SRF
target genes are known to be governed by dynamic changes in
the actin cytoskeleton. For example, the small GTPase, RhoA,
which induces the formation of stress fibers, can activate
SRF-mediated gene expression (43). Subsequent studies
showed that the increase in SRF activity via RhoA was through
the depletion of globular (G) actin during filamentous (F) actin
polymerization (102). At least two RhoA-dependent pathways
mediate a reduction in G-actin levels. One pathway involves
members of the formin family of cytoskeleton proteins (Diaphenous) and the other is through the ROCK-LIMK-cofilin
axis. Both RhoA-dependent pathways act to stabilize F-actin
(35, 102). It is evident that one consequence of decreasing
G-actin levels is the liberation of MRTF-A (normally bound to
G-actin in the cytosol), its translocation to the nucleus, and
subsequent association with SRF to direct context-dependent
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Fig. 1. Serum response factor (SRF) controls disparate programs of gene
expression through cofactor associations. Schematic illustrates some of the
SRF-dependent gene programs that are specified by the recruitment of cellrestricted cofactors. A growing number of actin cytoskeleton genes are direct
targets of SRF although the cofactors involved in such gene activation have yet
to be fully disclosed. See text for more details.
an attempt to further define the mammalian CArGome (106).
Our approach involved two important filters to reduce
“genomic noise.” First, we confined our search to CArG
elements that were 100% conserved in sequence and space
between human and mouse. It is thought that nucleotide sequence constraint signifies important functions such as transcriptional regulation (22). Second, we only scanned an 8-kb
window of sequence around protein-coding genes with annotated transcription start sites (4 kb upstream and 4 kb downstream of the annotated start site). The rationale for this filter
was that virtually all functional CArG elements are within the
boundaries of this window (68). Applying these filters to the
mouse and human genomes resulted in the recovery of nearly
200 putative CArG-containing target genes, 10 of which had
previously been validated (106). The distribution of these
CArG sequences across the 8-kb window approximated a bell
curve with an equal likelihood of finding a conserved CArG
box in the proximal promoter or downstream intronic/exonic
sequence. Interestingly, nearly 10% of the novel CArG sequences were found within coding exons, which generally do
not contain regulatory sequences for transcription (45). After
manually validating the computer’s prediction of conserved
CArG elements, a total of 89 targets were cloned for experimental validation using luciferase reporter, gel shift, chromatin
immunoprecipitation (ChIP), and RNAi knockdown of SRF.
Most (60) of the targets evaluated were found to be functional
in two or more of the assays employed. Remarkably, the Gene
Ontology categorization of each CArG element’s associated
gene revealed nearly half to be associated with the actin
cytoskeleton or contractile systems (106). Several independent
groups using different methodologies have also reported a
number of SRF target genes with known functional roles in
cytoskeleton and contractile systems (3, 83, 96, 124). A comprehensive list of all functionally validated SRF target genes is
available upon request.
Invited Review
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Table 1. Cytoskeleton-contractile SRF target genes
Cytoskeleton
Cytoplasmic
a
AKAP12A
AOC3
DMD
DTNA
ENAHc
ITGA1a
ITGA5c
ITGA9c
ITGB1c
ITGB1BP2c
SDC2c
SDC4c
SPTBc
TGFB111
TJP1
TLNc
VCLc
VIL1
Contractile
Physiological Association
e
ACTA1
ACTA2d
ACTCf
ACTG2d
ACTN1d
CALD1d
CRYAB
MYH11d
MYH4e
MYH6f
MYH7f
MYL3
MYL4
MYLKv6d
MYLKv7d
SMTNAd
TNNC1e,f
TNNT2f
TPM2d
ARHE
ATP2A2g
BARX2
BIN1
CA3
CALB1
CASQ1g
CKM
CPT1B
DMPKg
FHL1
FHL2
MYOD1e
MYOGe
NKX2-5f
NPPA
OPHN1
PLN7
SLC2A1
SLC8A1g
SRF
Hypothetical
ANKRD1
ANLN
ANXA2
ANXA3
ARPC4b
BVES
CDV1
CMYA1
CNN2
DSP
EPLIN
FLNBb
FNBP1a
FNBP3a
JPH2
KRT14
KRT18
KRT19
KRT7
KRT8
LDB3
LPP
LRRC10
MYL9
MYO1B
MYOM1
MSN
NEBL
NES
PFN2a
RHOQ
RHOU
TCAP
TMOD3
TNS1
TPM4a
VASPc
ZYXc
HUGO gene nomenclature symbols (human) are indicated for each category of serum response factor (SRF) target genes. The designations are not meant to
be absolute, and we acknowledge that some targets could be categorized in more than one column (e.g., CNN1 is known to be involved in both cytoskeleton
and contractile processes). a Actin filament-associated genes; b filament reorganization genes; c integrin coupling-associated genes; d smooth muscle cell-restricted
genes; e skeletal muscle cell-restricted genes; f cardiac muscle cell-restricted genes; g calcium homeostasis-related genes. This table was constructed from data
obtained in Refs. 3, 68, 83, 96, 106, and 124. See text for further details.
gene expression (58, 75). SRF itself is subject to cytoplasmicnuclear shuttling in some cellular contexts (48, 64). In addition
to RhoA-induced signaling to SRF, there are LIM proteins
(CRP1 and CRP2) that can associate with SRF in the nucleus
to direct SRF-dependent gene expression (16). Moreover,
␤-actin is found in the nucleus and has been implicated in the
assembly of a preinitiation complex for gene transcription (44).
These studies represent a sampling of data supporting an
increasingly important role for cytoskeleton-associated proteins in the control of gene transcription. It is noteworthy that
a growing number of actin cytoskeleton genes are themselves
direct targets of SRF, implying the existence of a feedback
loop for SRF-dependent control of actin cytoskeleton-related
processes (84, 106). The subject of actin cytoskeleton biology
is a daunting one to address, so we confine our discussion in
this review to the role of the actin cytoskeleton in one biological process where SRF is clearly of importance– cell migration.
Cell migration is central to myriad developmental (e.g.,
gastrulation) and pathological (e.g., metastasis) processes. We
can consider cell migration occurring in a series of at least four
steps (62, 100). First, a portion of the cell membrane must
buckle outwardly in the direction of migration to form a
lamellipodium in a process known as cell protrusion. Second,
focal adhesions must be made at the leading edge of the
migrating cell (i.e., at the lamellipodium) to create the necessary matrix-cell-cytoskeleton connections for migration to proceed. Third, the interior actin cytoskeleton must engage myosin
motors to elicit active contraction. Fourth, as contraction occurs, adhesions at the “rear” or trailing edge of the cell must
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dissipate to allow cell propulsion in the direction of the
lamellipodium. Each of these steps involves a complex interplay between the cell’s extracellular matrix and the interior
actin cytoskeleton, mediated by cell adhesion proteins such as
integrins (see below). The actin cytoskeleton can therefore be
thought of as a critical link between the exterior milieu surrounding a cell and the interior cell signaling pathways that
ultimately converge in the nucleus to effectuate gene expression control. For purposes of brevity, we shall focus on three
aspects of actin cytoskeleton-associated cell migration to highlight the intimate role of SRF in the control of this important
biological process.
Actin Filament Dynamics
The actin cytoskeleton, long thought to be a static scaffold
for the maintenance of cell shape, polarity, and mechanical
support, undergoes dynamic remodeling involving scores of
proteins that regulate the cytoskeleton (21, 85). One of the
central features of actin filament dynamics and the ability of a
cell to migrate is a process known as actin treadmilling, which,
as alluded to earlier, plays a key role in RhoA-mediated SRF
activation (102). Fundamentally, actin treadmilling involves
the polymerization of ATP-G-actin into an existing actin filament at the plus or barbed end and depolymerization of
ADP-actin at the minus or pointed end. Several SRF target
genes encode for proteins that play a key role in actin treadmilling (Table 1). For example, profilin 1 (PFN1) is known to
bind and sequester G-actin but can readily facilitate the polymerization of G-actin into F-actin by virtue of its ability to
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ACTB
ACTG1a
ACTR3b
CAPZA3b
CFL1a
CFL2a
CNN1a
CORO1Ab
CSRP1
CSRP2
DESa
DSTNa
FLNAb
FLNCb
GSNa
HSPB7
KRT17
PDLIM5
PFN1a
SNX2
TAGLN1d
TPM1a
TRIP6
TTN
TUFT1
Membrane
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Formation of a Leading Edge
The protrusive force at the leading edge of a migrating cell
represents the epicenter of cytoskeleton activity requisite for
cell migration to proceed. Although an explosive actin polymerization is ongoing at the leading edge, there are several
additional actin-binding proteins that reorganize actin filaments
to facilitate the formation of the lamellipodium. At this point,
it may be instructive to define lamellipodia vs. related membrane alterations called filopodia, both of which are involved in
cell migration. Lamellipodia are broad outward bucklings of
the cell membrane, whereas filopodia are “spikes” at the cell
periphery. Whether a cell membrane is deformed to create a
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lamellipodium or a filopodium relates largely to the presence or
absence of key actin-binding proteins (66). Chief among the
proteins involved in these two important membrane alterations
are actin-associated nucleating, branching, cross-linking, and
capping factors. Each of these classes of actin-binding proteins
has at least one known SRF target gene (Table 1). The
actin-related protein 3 (ACTR3) gene encodes for one component of a seven-member multiprotein composite known as
Arp2/3 complex. The Arp2/3 complex binds the side of an
existing actin filament and nucleates actin polymerization such
that the uncapped barbed end is able to elongate rapidly,
generating protrusive force for the lamellipodium (42). The
Arp2/3 complex requires signal-induced activation by the
WASp/Scar/WAVE family of proteins, which bring the Arp2/3
complex in close apposition to the existing F-actin (42). Interestingly, the Arp2/3 complex moves away from the membrane
under conditions favoring filopodial formation, suggesting this
membrane alteration does not require Arp2/3 complexes (66).
At present, ACTR3 has not been genetically inactivated, so its
direct role in cell motility is not known. The process of
nucleating an actin filament is coupled closely to filament
branching, which has the effect of creating a lattice of actin
filaments (so called dendritic nucleation; see Ref. 42). It is the
collective forces associated with branching actin filament
polymerization (absent myosin motors) that overcome the
forces of the cell membrane, thereby facilitating the formation
of lamellipodia in the direction the cell will eventually migrate
toward (42, 62). Coronin 1a (CORO1A) and two filamins
(FLNA and FLNC) are SRF-dependent target genes (Table 1)
that are also located at the leading edge of a cell to assist in
actin filament cross-linking (29, 42, 92). Several mutations in
FLNA have been defined in humans, and mice deficient in
FLNA show evidence of defects in cell migration, particularly
in the central nervous system (29). It is important to note that
the process of actin filament nucleation/branching/cross-linking is carefully regulated by capping proteins that bind barbed
ends of F-actin, thereby stabilizing these filaments. RNAi
knockdown of capping protein (CAPG) blocks lamellipodia
and accentuates the formation of filopodia that have far less
F-actin branching but instead exhibit elongating actin filaments
(66). It is unclear whether cells deficient in CAPG have any
defects in cell migration (66). One capping protein that has
been shown to be SRF dependent is CAPZA3 (Table 1). This
capping protein is particularly enriched in male germ cells
where it is thought to play a critical role in their motility (114).
This brief summary of a very complex process of leading edge
formation points out several F-actin reorganization steps that
are subject to direct control through SRF-mediated target gene
activation.
Up to this point, our discussion has focused mainly on
cytoplasmic cytoskeleton SRF target genes involved in actin
filament modifications necessary for cellular migration. Such
changes in the actin cytoskeleton largely involve signals emanating from the cell’s exterior (e.g., chemotactic factors).
Certain signaling events for cell migration, therefore, begin at
the membrane-matrix interface before the formation of lamellipodia and filopodia (100). Two classes of membrane receptors comprising several SRF target genes serve to bridge the
exterior milieu of a cell to its internal actin cytoskeleton:
integrins and syndecans. Integrins are noncovalently bound
heterodimers of ␣- and ␤-subunits that have diverse functions
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accelerate nucleotide exchange of ADP-actin for the more
polymerization-competent ATP-actin (117). Recently, PFN1mediated polymerization of actin filaments was shown to be
accelerated in the presence of various members of the formin
family (e.g., Diaphenous; see Ref. 56). Although no formin
genes have yet to be described as direct targets of SRF, there
are two formin-binding proteins (FNBP) that are hypothesized
to be SRF dependent (Table 1). PFN1 knockout mice arrest
early during embryogenesis, presumably because of a defect in
cytokinesis (117). A hypomorphic allele of the PFN1 ortholog
in Drosophila (chickadee) elicits clear defects in border cell
migration during development (109). A second, neural-restricted profilin gene (PFN2) is presently a hypothetical SRF
target (Table 1). Four of the most widely cited actin depolymerization or severing proteins [destrin (DSTN), cofilin (CFL)
1, CFL2, and gelsolin (GSN)] are now known to be direct
transcriptional targets of SRF (Table 1). GSN, which has
several functions in actin cytoskeleton remodeling, severs actin
filaments in a calcium-dependent manner (105). Fibroblasts
null for GSN exhibit elevated stress fibers and a defect in cell
migration (105). DSTN and CFL depolymerize F-actin by
increasing the dissociation constant of ADP-actin at the
pointed end of actin filaments, thereby increasing pools of
ADP-actin for sequestration by PFN1 (4). DSTN- and CFLmediated depolymerization is balanced, in part, through competitive binding of F-actin by tropomyosin (TPM), which acts
to stabilize F-actin filaments. Several isoforms of TPM are
known or hypothetical SRF targets (Table 1). Another stabilizer of F-actin (and SRF target) is the SMC-restricted calponin
(CNN1) gene, which is found in association with stress fibers
along the long axis of SMC in culture (37, 69). Importantly,
DSTN-CFL activity is negatively regulated by LIMK-mediated
phosphorylation of these proteins at an NH2-terminal serine
residue (120). Such phosphorylation can occur through RhoA
signaling, which ultimately activates SRF (see above). The fate
of phosphorylated DSTN-CFL is unclear, but it is intriguing to
consider that such proteins could be destined for degradation.
In this setting, the elevation of SRF activity could provide for
a mechanism of restoring DSTN-CFL levels. The phenotype of
DSTN-CFL null mice is unknown; however, worms deficient
in CFL exhibit a severe impairment in myofibrillogenesis (4).
Given the critical role of actin filament dynamics in a variety
of cell functions, particularly cell migration, and the fact that
several key genes in actin filament regulation are direct transcriptional targets of SRF, it is entirely predictive that cells in
which SRF is deleted or reduced will show concomitant defects
in actin dynamics and cell migration (see below).
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the cytoplasmic tail of ITGB1, is hypothesized to be a membrane sensor of mechanical stress in the heart (10). These few
examples further demonstrate the critical role SRF plays in
mediating both mechanosensory and migratory responses
through membrane-associated cytoskeleton target gene activation.
Cellular Contraction
The role of SRF in controlling muscle contractile gene
expression and differentiation has been reviewed previously
(47, 57, 68). New SRF target genes involved in sarcomeric
muscle assembly and differentiation include TGFB1I1 and
BIN1 (98, 115). In addition, many of the genes listed in Table
1 (e.g., DMD, ACTN1) associate with specialized structures in
striated muscle (Z- and intercalated disks) to stabilize sarcomeres. In SMC, the contractile apparatus is physically and
functionally linked to the actin cytoskeleton, since agents that
disrupt the actin cytoskeleton (e.g., cytochalasins) interfere
with SMC contraction; sarcomeric muscle is refractory to such
treatment apparently because their contractile filaments are
stabilized through actin-capping proteins (36). The close relationship between SMC actin cytoskeleton and contractile apparatus is thought to be necessary for the distribution of
contractile force across the cell. When SMC lose their native
phenotype in various diseases (68, 80), the actin cytoskeleton
adopts a more promigratory phenotype as contractile elements
dissipate, probably because of a decrease in SRF- and
MYOCD-mediated gene expression.
As for nonmuscle cells, the net effect of changes at focal
adhesions and lamelliopodia-filopodia after exterior stimuli for
migration is the generation of actin-myosin cross-bridging
within the cytosol. The sliding filaments create tractional
forces that drag the rear part of the cell in the direction of the
leading edge (62). Clearly, SRF target genes involved in actin
dynamics (see above) are engaged as are SRF target genes that
function to stabilize F-actin (CNN1 and TPM1), thereby extending the half-life of mature actin filaments. The principal
actin genes involved with nonmuscle contraction are the cytoplasmic ␤- and ␥-actin genes (ACTB and ACTG1), both of
which are SRF dependent (Table 1). Until recently, the underlying mechanism guiding spatial distribution of the proteins
encoded by ACTB and ACTG1 was unknown. We now understand that the ␤-actin protein undergoes arginylation, which
allows for ␤-actin redistribution to the leading edge of a cell
and the formation of a lamellipodium necessary for cell motility (50). A hypomorphic allele of ACTB has been reported in
mice that results in embryonic lethality, although insight into
the underlying mechanism for this phenotype is lacking (97).
Interestingly, none of the nonmuscle myosin genes appear to
be SRF dependent, which contrasts with many muscle myosin
genes known to require SRF for optimal expression (Table 1).
However, for actin-myosin force generation to proceed in
nonmuscle cells and muscle cells, specific myosin light chains
must be phosphorylated in a calcium-dependent manner. Several SRF target genes are involved both in the handling of
calcium (ATP2A2, CASQ1, PLN, and SLC8A1) and the phosphorylation of myosin light chains (MYLKv6 and MYLKv7).
Recently, the CArG element in MYLKv7 (also known as
telokin) was deleted in mice, and the phenotype revealed
abolished telokin expression and impaired relaxation of SMC
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in cell biology, including cell migration (46). Integrin ␤-1
(ITGB1), a known SRF target gene, dimerizes with the majority of ␣-integrin subunits, including three that are SRF dependent (Table 1). Hence, SRF indirectly regulates many distinct
integrin-dependent pathways through ITGB1 gene regulation.
Interestingly, ITGB1 is involved in a RhoA-dependent pathway of SRF stimulation beginning at focal adhesions with the
activation of focal adhesion kinase (116). Focal adhesions are
present at the leading and trailing edge of migrating cells and
function to create an attachment point for the cell to interface
its actin cytoskeleton with extracellular ligands bound to clustered integrin receptors. Gene inactivation of ITGB1 results in
peri-implantation lethality, but several conditional ITGB1
knockouts suggest important functions related to adhesion and
migration (9). Two syndecans (SDC2 and SDC4) are also
known SRF targets and function at focal adhesions to stabilize
integrin receptor-ligand interactions, thereby assisting in the
transmittance of mechanical information to the cell interior
(118). SDC4 interacts with a PDZ domain-containing protein
called synectin, which functions to increase cell spreading and
reduce cell migration (118). Moreover, fibroblasts from SDC4
null mice show impaired migration in a scrape wound assay
(118). Characterization of SDC2 null mice has yet to be
reported.
The clustering of integrins and syndecans at focal adhesions
link the matrix world to the actin cytoskeleton through an array
of SRF-regulated actin-binding proteins, including vinculin
(VCL), dystrophin (DMD), dystrobrevin-␣ (DTNA), and talin
(TLN) (Table 1). VCL is a classic actin-binding protein that
serves to stabilize focal adhesions and regulate cell motility
(20). Homozygous VCL knockout mice are embryonic lethal
because of severe dysmorphogenesis of the heart and brain
(presumably, in part, because of faulty migration), and heterozygous mice are susceptible to stress-induced cardiomyopathy stemming from cytocontractile defects in the myocyte
(123). DMD and DTNA are closely associated proteins at the
membrane cytoskeleton that have important roles in muscle
membrane structural integrity and hence normal muscle physiology. Mutations in these genes, particularly DMD, are not
well tolerated and result in muscle atrophy and death (90).
TLN activates the COOH-terminal tails of ␤-integrin receptors
and stabilizes their interaction with underlying F-actin (12, 46).
TLN gene inactivation results in a gastrulation defect resulting
from abnormal cell migration and not altered programs related
to growth or apoptosis (76). It is expected that cells without
TLN will also show defective integrin signaling, a subject that
has yet to be addressed.
Several membrane-associated cytoskeleton SRF target genes
play demonstrable or hypothetical roles in the regulation of
cytoskeleton processes. For example, AKAP12 is a tumor
suppressor gene that functions, in part, to establish a normal
cytoarchitecture conducive for a cell’s stationary state. When
AKAP12 expression is compromised, notable defects in the
actin cytoskeleton are observed, and the cells display heightened migratory behavior (34). Interestingly, the AKAP12 locus
comprises three distinct transcription units with only the
AKAP12 ␣ gene under direct control of SRF (104). Another
recently described SRF target gene is the mammalian homolog
of Enabled (ENAH), which appears to promote actin filament
extension along filopodia, enabling cell migration (89). Melusin (ITGB1BP2), a muscle-restricted gene that interacts with
Invited Review
C76
Fig. 2. SRF directs expression of genes involved in several aspects of cytoskeleton biology. Schematic illustrates concept of SRF controlling cell
migration through several aspects of actin cytoskeleton biology. ITG, integrin.
See text for more details.
GENETIC EVIDENCE SUPPORTING SRF AS A MASTER
REGULATOR OF THE ACTIN CYTOSKELETON AND
CONTRACTILE APPARATUS
Multiple species of SRF have either been genetically inactivated or knocked down with RNAi or cell-restricted, Cremediated excision techniques. In this section of the review, we
provide an up-to-date summary of the experimental data supporting SRF’s role as a master regulator of the actin cytoskeleton and contractile apparatus. A central theme that runs
parallel to the preceding section is the concept that SRF
controls many aspects of actin cytoskeleton biology related to
cell migration (Fig. 2). We therefore emphasize migratory
phenotypes in the context of SRF disruption.
In yeast, several mutant alleles of the SRF ortholog Mcm1
exist, displaying a range of phenotypes from loss in viability to
defective cell polarization and mating (23). Although no published data exist regarding the state of the cytoskeleton in
Mcm1 mutants or the presence of CArG elements in the
promoter regions of genes involved in cytoskeleton homeostasis, it is plausible that there are defects in expression of some
cytoskeleton-associated genes given the critical role of the
cytoskeleton in yeast mating (17). The power of yeast genetics
and genomics should be exploited to interrogate its genome for
functional CArG elements within cytoskeleton promoters and
to examine the basic cytoarchitecture of Mcm1 mutants.
The protist, D. discoideum, carries one SRF ortholog (Srfa),
which was originally inactivated by insertional mutagenesis
(25). Mutants show normal spore differentiation markers but
display defective spore morphology and loss of viability under
stress-induced conditions (25). Ultrastructural analysis of mutant spores reveals faulty development of intracellular actin
rods, which are required for proper spore maturation (27). At
least one known cytoskeleton gene (cofilin B) is reduced in
Srfa mutants. Cofilin B likely functions in the proper deployment of actin rods within spores (24, 27). In a separate study
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(53). The latter result provides the first (and only) genetic proof
of an “endogenous” CArG element’s essential role in controlling gene expression and contractile activity. Although each of
the above SRF target genes relates largely to muscle contractility, a role in nonmuscle contraction cannot be ruled out
entirely. In summary, scores of SRF target genes play direct or
indirect roles in several aspects of actin cytoskeleton biology
relating to migration (Fig. 2). It is particularly striking that the
bare minimum number of actin-binding proteins sufficient for
actin dynamics (i.e., Arp2/3 complex, ACTB, DSTN/CFL, and
CAPZ; see Ref. 85) are all SRF dependent. Clearly, knocking
down or inactivating SRF completely would be expected to
have an overt phenotype related to defects in actin-cytoskeleton and contractile activity. The data in support of this concept
are considered next.
with a different mutant Srfa allele, multicellular slugs showed
reduced migration compared with normal slugs, suggesting
widespread perturbations in cytoskeletal processes relating to
locomotion (26). Another invertebrate, C. elegans, also displays a defect in locomotion upon RNAi knockdown of SRF
(30). It will be of major interest to characterize functional
CArG elements in the promoter of cofilin B as well as additional SRF-dependent cytoskeleton genes in D. discoideum and
C. elegans.
D. melanogaster SRF (DSRF) plays a vital role in the
terminal branching of tracheal cells (that form oxygen-carrying
lumens) of the respiratory system (39) and the correct specification of intervein cells (carrying hemolymph) in the wing
(77). The respiratory phenotype arises because of the inability
of terminal cells of the tracheal system to migrate and extend
long cytoplasmic extensions in the correct spatial manner, and
the wing phenotype is the result of a flaw in terminal differentiation. Overexpressing DSRF causes exuberant tracheal cell
extensions in wild-type flies and can partially rescue the
tracheal phenotype in mutants (39). These results suggest that
DSRF controls cytoskeletal events surrounding adhesion, migration, and cytoplasmic outgrowth of tracheal cells. Mutants
of the fly ortholog of MRTF-A (SRF cofactor) show similar
deficiencies in the migration of tracheal cells as well as altered
cardiac and ovarian border cell migration (40, 101). Consistent
with mouse studies (see below), DSRF/DMRTF-A mutant
phenotypes do not manifest perturbations in cell proliferation
(39, 91, 101). Instead, altered cytoarchitecture appears to
underlie the migratory phenotypes in these mutant strains of
fly. In this context, the Drosophila cytoplasmic actin 5C gene
harbors two CArG boxes in as many promoters that may be
important targets of DSRF/DMRTF-A for correct migratory
responses (7). It is unknown whether these CArG elements
bind SRF and regulate directly the promoter activities of
Drosophila actin 5C.
Most of the data generated on phenotypes arising from loss
in SRF expression have been obtained in mice. The first
genetic knockout of SRF was shown to arrest mouse embryos
at gastrulation (e7.0 days), with impaired folding of the ectoderm and endoderm and the complete absence of a mesoderm
(2). In addition, no primitive streak could be found in mutant
embryos. As embryogenesis was normal through e6.5 days, a
proliferation defect was considered unlikely even though expression of several SRF-dependent growth genes was reduced
(2). It appears then that SRF is essential for mesoderm formation, possibly as a mediator of complex cell migratory events at
this stage of development. Embryonic stem (ES) cells have
provided further insight into this possibility. SRF null ES cells
show clear evidence of attenuated cortical actin networks and
cell protrusions, both of which are essential for the cell’s
ability to mount a migratory response (94). Subsequent work
showed that SRF null ES cells exhibit impaired expression of
ITGB1, TLN, VCL, and ZYX (all SRF targets; Table 1) and
faulty formation of focal adhesions (93). Moreover, levels of
F-actin were severely reduced probably because of the profound loss in SRF target genes involved with actin polymerization (see above). Consequently, cells in which SRF expression is compromised would be expected to fail to migrate, a
concept that has been demonstrated in a boyden chamber assay
using ES cells null for SRF (93). Conversely, overexpression
of SRF has been reported to increase the migration of cultured
Invited Review
C77
SMC (14). Collectively, there is mounting in vitro evidence
supporting a role for SRF in the migration of various cell types.
As discussed next, there is growing support for a link between
SRF and cell migration in vivo as well.
Over the last two years, several papers have been published
in which SRF inactivation was restricted to specific cell types
using Cre-LoxP technology. A common finding across virtually all of these studies is the severe disruption of the actin
cytoskeleton and/or the contractile apparatus, resulting in defective cellular migration and/or force generation. The first
report of a conditional knockout (cKO) of SRF used a cardiacrestricted promoter (beta myosin heavy chain) controlling Cre
expression to circumvent the early gastrulation phenotype
associated with the standard knockout (82). Although the
targeting strategy resulted in a truncated SRF protein product
of unknown function, mutant mice arrested in midgestation
with thinning of the myocardial compact zone, attenuated
trabeculations throughout the ventricular chamber, and cardiac
dilation (82). Such perturbations in the heart suggest that
embryonic development arrested because of heart failure. Significantly, no alterations in myocardial cell proliferation or
apoptosis were evident at e10.5 days of development. Higher
apoptotic indexes were noted 1 day later, but this was likely a
consequence of hypoxia. The attenuated expression of several
cardiac transcription factors led the authors to conclude that
SRF was critical in the determination and differentiation of
myocardial cells (82). A second cKO of SRF used a targeting
strategy in which LoxP sequences flanked the proximal promoter and first exon, resulting in complete loss of any SRF
product upon Cre-mediated excision with a cardiac/vascular
SMC-restricted promoter (SM22-␣; see Ref. 70). Here, ultrastructure analysis suggests that loss in SRF expression throughout the cardiovascular system results in defective myofibrillogenesis (in the heart) and cytoskeletal assembly (in the dorsal
aorta). Furthermore, an apparent decrease in SMC migration to
the dorsal aorta was noted, consistent with the absence of
lamellipodial extensions in mutant SMC. No evidence of
impaired myocardial or SMC proliferation was observed in
AJP-Cell Physiol • VOL
mutants. However, several genes involved in cytoskeleton and
contractile physiology were severely reduced in expression
(70). Similar data were obtained in a separate report where
SRF was deleted in the heart using a different cardiac-restricted
Cre driver (␣-myosin heavy chain; see Ref. 78). In addition,
cultured cardiomyocytes carrying deleted SRF alleles show
drastically reduced levels of numerous genes involved with
sarcomeric assembly and contractile function with virtually no
discernable contractile apparatus (3). Moreover, deletion of
SRF in the adult heart using tamoxifen-induced, Cre-mediated
excision results in cardiac failure and death resulting from
dilated cardiomyopathy. Consistent with the embryonic phenotypes, adult hearts absent SRF show highly disorganized
sarcomeres (81). Finally, a skeletal muscle cKO of SRF (using
a myogenin Cre driver) results in perinatal lethality because of
dysmyofibrillogenesis in skeletal muscle (61). The fact that
mice were born with skeletal muscle indicates that loss of SRF
during embryogenesis was not linked to skeletal muscle hypoplasia, further supporting the notion of SRF being dispensable
for proliferation. Thus all three muscle types in which SRF is
deleted show, consistently, the critical need for this transcription factor in directly regulating expression of target genes
necessary for proper myofilament assembly and the generation
of contractile force. Figure 3 shows how RNAi knockdown of
SRF alters the actin cytoskeleton in human coronary artery
SMC, an effect that would be expected to prohibit SMC
migration. The migratory defect seen in vascular SMC in vivo
(70) is consonant with defective migratory responses in more
ancient species of animal where SRF is deleted (see above). As
detailed next, a series of cKO in the central nervous system
provide further support for SRF’s salient role in mammalian
cell migration.
Perhaps no other organ requires as exquisitely timed and
controlled cellular migration as the brain. At least some of this
complex neuronal migration is under direct control of SRF and
its effects on the actin cytoskeleton. Alberti and colleagues (1)
made the first cKO of SRF in the forebrain using a Ca2⫹/
calmodulin-dependent protein kinase II-Cre driver and showed
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Fig. 3. Knockdown of SRF disrupts vascular
SMC actin cytoskeleton and contractile apparatus. Adenovirus (300 plaque-forming
units) carrying a short hairpin RNA to SRF
(104) or control (shEGFP) was used to transduce human coronary artery SMC. Cells
were stained 5 days later with antisera to
SRF (FITC) and CNN1 (Texas red; A and B)
or phalloidin and DAPI (blue; C and D).
Knockdown of SRF (arrows in B) results in
disruption of CNN1 expression (B) and the
actin cytoskeleton (D).
Invited Review
C78
since the number of validated SRF target genes in invertebrates
currently registers only one (13). Identifying all SRF target
genes in multiple species will allow for powerful comparative
genomic analyses to discover other common regulatory elements and their trans-acting factors that may cooperate with
SRF to effect gene transcription. We also need to understand
how CArG-SRF is controlling actin cytoskeleton (and other)
target genes (Fig. 1). What SRF cofactors (among nearly 50)
are utilized and what are the essential SRF protein interfaces
necessary for such gene transcription? In this context, an
ambitious plan would be to generate an allelic series of SRF
mutants to test in transcription-based assays and in vivo using,
for example, zebrafish as a high-throughput model system.
Finally, inspection of the human SRF locus at the UCSC
Browser (http://genome.ucsc.edu/, Human May 2004 Assembly) reveals a single synonymous coding single nucleotide
polymorphism (SNP) (G327G); however, there exists a cluster
of 3⬘-untranslated region SNPs that could have some influence
on SRF gene expression (5) in human populations. It is possible that hypomorphic alleles of SRF exist (resulting in its
attenuated expression) that are linked to human diseases involving the actin-cytoskeleton and/or contractile apparatus.
ACKNOWLEDGMENTS
J. M. Miano wants to acknowledge the memory of Brian K. Piotter, friend
and fellow Sabres’ enthusiast.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-62572 and HL-70077 to J. M. Miano and HL-69041 to K. Fujiwara
and by American Heart Association Grant 0340075N to J. M. Miano.
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Invited Review
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