© 1999 American Heart Association, Inc.
Rapid Communication |
Regulation of Smooth Muscle 
-Actin Expression In Vivo Is Dependent on CArG Elements Within the 5' and First Intron Promoter Regions
From the Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, Va.
Correspondence to Gary K. Owens, PhD, Box 449 Health Sciences Center, University of Virginia, Charlottesville, VA 22908.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abstract—The aims of the present studies were to define sufficient promoter sequences required to drive endogenous expression of smooth muscle (SM)
-actin and to determine whether regulation of SM -actin
expression in vivo is dependent on CArG (CC(A/T)6GG)
cis elements. Promoter deletions and site directed
mutagenesis techniques were used to study gene regulation in transgenic
mice as well as in smooth muscle cell (SMC) cultures. Results
demonstrated that a Lac Z transgene that contained 547 bp of the 5' rat
SM -actin promoter was sufficient to drive embryonic expression of
SM -actin in the heart and in skeletal muscle but not in SMCs.
Transient transfections into SMC cultures demonstrated that the
conserved CArG element in the first intron had significant positive
activity, and gel shift analyses demonstrated that the intronic
CArG bound serum response factor. A transgene construct from -2600
through the first intron (p2600Int/Lac Z) was expressed in embryos and
adults in a pattern that closely mimicked endogenous SM
-actin expression. Expression in adult mice was completely
restricted to SMCs and was detected in esophagus, stomach, intestine,
lung, and nearly all blood vessels, including coronary,
mesenteric, and renal vascular beds. Mutation of CArG B completely
inhibited expression in all cell types, whereas mutation of the
intronic CArG selectively abolished expression in SMCs, which suggests
that it may act as an SMC-specific enhancer-like element. Taken
together, these results provide the first in vivo evidence for the
importance of multiple CArG cis elements in the
regulation of SM -actin expression.
Key Words: muscle, smooth • transgenic mice • transfection • gene expression
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several cis elements and trans-acting factors have recently been described that regulate muscle-specific gene expression in skeletal and cardiac muscle and are required for the terminal differentiation of these muscle cell types.1 2 In contrast, the mechanisms that regulate smooth muscle cell (SMC) differentiation are only poorly understood, and, to date, no transcription factors have been identified that direct SMC-specific gene expression. Because SMC maturation and differentiation are required for the full development of arteries and veins during angiogenesis and vasculogenesis, the identification of the molecular mechanisms that control SMC differentiation is important to our understanding of these processes that occur not only during development but also under pathological conditions. Furthermore, it may lead to a better understanding of SMC phenotypic modulation that has been shown to contribute to atherosclerosis and restenosis after balloon angioplasty.3 4
A major goal of our laboratory has been to elucidate control processes
that regulate SMC differentiation by identifying mechanisms that
control the transcription of genes such as SM -actin and SM myosin
heavy chain (MHC) that are required for the contractile function of
SMC.5 SM -actin is a contractile protein that comprises
40% of total SMC protein.6 It is required for the
contractile function of SMCs and is the first SMC differentiation
marker to appear during development.7 Although it is also
transiently expressed in the myocardium and skeletal muscle
during the development of the embryo and is expressed in
myofibroblasts during wound healing, SM -actin expression in adult
animals is highly restricted to SMCs or SM-like
cells.8 9
Our laboratory and others have shown that the regulation of SM
-actin expression involves a complex interaction of multiple
positive and negative cis elements that act in a cell-type
specific fashion.10 11 12 13 14 15 16 For example, a 547 bp SM
-actin promoter/CAT construct (p547CAT) had high activity in
cultured SMCs and in L6 skeletal myotubes, which are cell types that
express SM -actin in culture. However, the same construct was
inactive in non-SMC-types such as endothelial
cells14 and AKR2B fibroblasts.17 Of
particular interest, we have demonstrated that two completely conserved
CArG (CC(A/T)6GG) elements, CArG A at -62 and
CArG B at -112, within the first 125 bps of the SM -actin 5'
promoter are required for high level of expression in cultured SMCs in
that mutation of either CArG abolished transcriptional
activity.14 Additional positive and negative
cis elements have been described in the sequences further
upstream to -2.8 Kb, but in cell culture systems, this region acts
mainly to inhibit expression in cell types that do not normally express
SM -actin.13 14 The SM -actin first intron
contains another completely conserved CArG element,12 but
the activity of the intronic CArG and its contribution to SMC-specific
regulation have not been studied.
The CArG element was first described as the core sequence of the serum
response element (SRE) within early response genes, such as
c-fos (reviewed in Reference 1818 ) but has also been shown to
be required for the activity of many muscle-specific gene
promoters.19 20 21 22 23 Of interest, nearly all of the SMC
differentiation marker genes characterized to date, including SM MHC,
caldesmon, and telokin, contain two or more CArG elements that are
required for maximal expression in cultured SMCs.14 24 25 26 27 28
In addition, separate laboratories have reported that a conserved CArG
element in the SM-22 promoter is required for the arterial
expression of a Lac Z transgene in the mouse.29 30
Electrophoretic mobility supershift studies demonstrated that the SM
-actin CArG elements, like the SRE, bind the serum response factor
(SRF).14 Although recent evidence suggests that
muscle-derived tissues express higher levels of SRF than non–muscle
derived tissues,31 SRF is thought to be ubiquitously
expressed, and a critical yet presently unresolved question remains
as to the mechanism of CArG-dependent regulation of SMC-specific gene
expression. Evidence from this laboratory suggests that both CArGs A
and B, together with highly conserved CArG flanking sequences, act
cooperatively to coordinate the formation of an SRF-containing
SMC-specific activation complex that may contain an SMC-selective SRF
accessory protein (C.P.M., Mike M. Thompson, Susan Lawrenz-Smith,
G.K.O., unpublished results, 1998).
It is well established that SMC differentiation is dependent on a large number of local environmental cues, including extracellular matrix interactions, local production of growth factors, and mechanical stresses that cannot be accurately reproduced in culture.5 32 Moreover, recent studies have provided evidence that gene regulation in SMC culture systems often does not completely mimic regulation in vivo.30 33 Therefore, when studying SMC differentiation, it is critical that regulatory pathways initially identified in cultured SMC are tested in vivo through the use of transgenic mice. In addition, analysis of SM-22 and SM MHC gene expression in transgenic mice has demonstrated that expression of SMC marker genes is complex and may involve "regulatory cassettes" that drive expression within some but not all SM tissues.25 29 33 Therefore, transgenic studies are also critical for detecting possible heterogeneity in SMC gene regulation.
No studies to date have reported the complete characterization of the
regulatory regions required to drive in vivo expression of SM -actin
during development and maturation. Wang et al34 recently
reported that a SM -actin promoter that contains 1100 bp of 5'
promoter and the entire first intron could drive expression of an IGF-1
transgene in many SM tissues. However, these studies were restricted to
analysis in adult animals and focused on examination of the
effects of IGF-1 overexpression in SMC rather than on the
characterization of the promoter regions required for SMC-specific
expression. This is a critical issue because the SM -actin
gene is known to be expressed by all three muscle types during
development. In addition, although a large number of regulatory
elements have been identified on the basis of studies in cultured SMCs,
no studies have been reported examining whether specific cis
elements regulate SM -actin expression in vivo. As such, the goals
of the present experiments were to define the sufficient promoter
sequences required to drive expression of SM -actin in vivo and
whether this regulation is CArG dependent. Data presented
demonstrate that a promoter construct from 2600 through the first
intron is sufficient to drive high-level expression in vivo in a manner
that appears to mimic the endogenous SM -actin gene.
Moreover, we provide evidence that the CArG elements are required for
transcriptional activation of the promoter in vivo but exhibit
differential activity in SMCs versus non-SMCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of Rat SM
-Actin Lac Z ReportersThe pUC19-Lac Z plasmid used to generate reporter gene constructs was a generous gift of Dr Eric Olson (University of Texas Southwestern, Dallas, Tex). Several deletion constructs were generated for analysis in transgenic mice. The p125/Lac Z, p547/Lac Z, and p2800/Lac Z reporters were made by subcloning the corresponding promoter regions from previously described CAT reporter constructs14 into the Lac Z vector after HindII/Xba I restriction digestion. Constructs that contained the first intron, p547Int/Lac Z and p2600Int/Lac Z, were subcloned from a larger genomic fragment isolated and described previously with PmlI/XhoI and ScaI/XhoI digestion, respectively.
CArG mutations in the p2600Int/Lac Z construct were made with the polymerase chain reaction (PCR)–based Excite method (Promega) as per protocol. To avoid potential PCR-induced mutations in the Lac Z reporter, the promoter was subcloned into pBluescript, and after the mutagenesis protocol was returned to the Lac Z vector. The oligonucleotides used to make these mutations contained the following sequences that have been shown to abolish SRF binding in gel shift analyses (mutated sequences are in bold): CArG A, 5'-AATTGTTTAA; CArG B, CCCTATATCA; and intronic CArG, AATAATTAAA. Final subcloning steps and all mutations were verified by direct DNA sequencing. Before transgenic injections, all constructs were tested for Lac Z expression by transient transfection into cultured rat aortic SMC cultures to ensure functional activity of all constructs. All clones, including those that contained CArG mutations, showed at least some activity in these assays.
Generation and Analysis of Transgenic Mice
All constructs were prepared for transgenic injection by removal
of pUC19 backbone sequences by NotI/EcoRI
digestion and subsequent agarose gel purification of the linearized
promoter/Lac Z fragment. Transgenic mice were generated with standard
methods25 35 either commercially (DNX, Princeton,
NJ) or within the transgenic core facility at the University of
Virginia, Charlottesville. Mice (C57/B6) were analyzed
transiently at several embryonic stages or by establishing founder
lines that allowed more detailed analysis of transgene
expression throughout development and in adult animals. Transgene
presence was analyzed by PCR with genomic DNA purified from
placentas (transients) or tail clips (founders) according to the method
of Vemet.36 Mice were euthanized by IP injection of
pentobarbital (100 mg/kg), and transgene expression was
analyzed as previously described.25 37 All animal
procedures used in these studies were reviewed and approved by the
University of Virginia Animal Use and Care Committee.
Cell Culture, Transient Transfections, and Reporter Gene
Assays
SMCs from rat thoracic aorta were isolated and cultured as
previously described.13 SMCs were seeded into 6-well
plates and transfected 24 hours after plating at 70% to 80%
confluency. Transfections were performed with 4 µg of plasmid DNA and
the transfection reagent DOTAP (Boehringer Mannheim). Growth
conditions and preparation of cell lysates for measurement of Lac Z
activity were performed as previously described.14 The
enzyme activity of each sample was normalized to the protein
concentration of each cell lysate as measured by the DC protein assay
(BioRad). In each experiment, the promoterless Lac Z construct was also
transfected to serve as the baseline indicator of Lac Z activity, and
the activity of each promoter construct is expressed relative to
promoterless activity. All activities represent at least three
independent experiments, with each construct tested in triplicate per
experiment. Relative Lac Z activities are expressed as the mean±SD
computed from the results obtained from each set of transfection
experiments. We did not cotransfect a viral promoter/reporter construct
as a control for transfection efficiency because we have previously
shown that such constructs exhibit unknown and variable squelching
effects on the SM -actin promoter presumably because of competition
for common transcription factors.14 Moreover, we have
found that inclusion of such controls are unnecessary because
variations in transfection efficiency between independent experimental
samples are routinely small (<10%).14
Preparation of Nuclear Extracts, In Vitro Synthesis of SRF, and
Electromobility Shift Assays
Nuclear extracts were prepared from confluent rat aortic SMCs by
the methods of Dignam.38 Culture conditions matched those
used for transient transfection assays.
Oligonucleotides used in electrophoretic mobility shift
assays (EMSAs) were purchased commercially (Operon Technologies) and
include the following: CArG A, 5'-ttgctccttgtttgggaagc-3'; CArG B,
5'-gaggtccctatatggttgtg-3'; and intronic CArG,
5'-ttttacctaattaggaaatg-3'. Probes were 32P end
labeled and annealed. All probes were purified on a 6%
acrylamide gel, eluted in TE, and precipitated twice in
ethanol.
EMSAs were performed with 20 µL of binding reaction that included
30 pg of labeled probe, 5 µg of SMC nuclear extract, 0.2 to 0.6
µg of poly(dI-dC) in 1x binding buffer (10 mmol/L Tris-HCl, pH
7.5; 100 mmol/L KCl; 50 mmol/L NaCl; 1 mmol/L
dithiothreitol; 1 mmol/L EDTA; and 5% glycerol). After a
30-minute incubation at room temperature, the samples were subjected to
electrophoresis on a 5% polyacrylamide gel that had been
prerun at 170 V for 1 hour. Electrophoresis was performed at 170 V in
0.5x TBE (45 mmol/L Tris borate and 1 mmol/L EDTA). Gels
were dried and exposed to film for 24 to 72 hours at -70°C. For
supershift studies, 1 µL of SRF antibody was added after the
30-minute incubation period and the reaction was incubated for an
additional 15 minutes and loaded onto the gel for electrophoresis.
Immunohistochemical Staining of SM -Actin Expression
Embryos were fixed overnight in formalin. Tissues were
dehydrated, incubated in 100% xylene, and embedded in paraffin. Thin
sections (6 µm) were placed on uncoated slides and dried on a
slide warmer. Sections were cleared in 100% xylene and rehydrated
through a graded ethanol series to a final incubation in PBS.
Endogenous peroxidase activity was quenched by incubating
the slides for 30 minutes in methanol that contained 0.3% hydrogen
peroxide. Slides were subsequently rehydrated in PBS and blocked in a
1:50 solution of normal goat serum made in PBS. Sections were then
incubated with SM -actin primary antibody for 1 hour and washed with
three changes of PBS. Detection of primary antibody was performed with
a Vectastain ABC kit (Vector Laboratories) according to the
manufacturer's instructions, with 3,3'-diaminobenzidine as the
chromagen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SM
-Actin Promoter Region From -2600 Through the First Intron
Conferred In Vivo Expression of a Lac Z Reporter in a Manner Similar to
That of the Endogenous GenePrevious results from transient transfections into rat aortic SMC cultures demonstrated that reporter constructs that contained the first 547 bp of the SM
-actin 5' promoter were expressed at high levels in
only SMCs or other muscle cells that are known to express their
endogenous SM -actin gene.14 Therefore, we
initiated our transgenic mouse studies with a construct that contained
this promoter region (Figure 1A
). Figure 2A shows a p547/Lac Z positive embryo at
embryonic day 13.5 (E13.5), a time point when SM -actin is expressed
in skeletal, cardiac, and smooth muscle.9 Results showed
that this promoter region was sufficient to drive transgene expression
in skeletal and cardiac muscle but not in the vasculature or in any
other SMC tissue. Note that in subsequent studies, a construct that
contains a larger 5' region (up to -2800 bps) did not result in
expression in SMCs. The p2800/Lac Z construct, like p547/Lac Z, was
expressed in only embryonic cardiac and skeletal muscle (data not
shown).
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The preceding observations indicated that additional regions of the SM
-actin promoter were necessary for expression of SM -actin in
SMCs in vivo. Nakano et al12 previously reported that the
first intron of the human gene had significant enhancer activity in
cultured SMCs, an observation consistent with observations in
this laboratory for the rat first intron (see Figure 4).
Therefore, constructs were generated from a genomic clone that included
the first intron and 547 or 2600 bp of the 5' promoter (Figure 1B
and 1C). Results shown in Figure 2B demonstrate that
p547Int/Lac Z, like the p547 construct, was expressed highly in
embryonic cardiac and skeletal muscle. In addition, all independent
transgenic founder embryos (E13.5) generated with this construct (n=8)
expressed high levels of Lac Z in the umbilical arteries and one half
showed expression in the lower portion of the abdominal aorta. These
data demonstrated that the addition of the first intron to 547 bp of
the 5' promoter promoted transgene expression in only a small subset of
SMCs.
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We next tested a transgenic construct that contained sequences from
-2600 bp through the first intron (p2600Int/Lac Z). Results shown in
Figure 2C demonstrated that this construct was expressed at
E13.5 in a pattern that closely followed expression of the
endogenous SM -actin gene, with staining in heart and
skeletal muscle as well as in multiple SM tissues, including the aorta,
carotids, multiple small and large arteries, esophagus, stomach,
intestine, bladder, ureter, and airway SM. Examination of
histological sections from p2600Int/Lac Z animals at
E10.5 to E16.5 showed that Lac Z staining was highly restricted to the
vasculature or the SMC layers of SM-containing organs as well as to
cardiac and skeletal muscle. Figure 3
shows representative sections at E16.5 with panel 3D
showing immunohistochemical detection of SM -actin expression for
comparison. Figure 4 shows p2600Int/Lac Z
expression in various organs taken from adult mice 4 to 6 weeks of age.
Lac Z staining was seen in nearly all adult SM tissues examined,
including the esophagus, stomach, intestine, bladder, trachea, bronchi,
and most blood vessels, including the coronary, mesenteric, and
renal vascular beds. Histological sections taken from
adult tissues are shown in Figure 5. Note
that expression was completely restricted to SMCs and that the
p2600Int/Lac Z transgene, which was highly expressed in skeletal and
cardiac muscle during embryonic development, was no longer expressed in
adult skeletal or cardiac muscle cells. The latter observation is
consistent with the absence of SM -actin expression in these
tissues in adult animals5 and indicates that the -2600 to
+2784 bp promoter region tested is sufficient to confer appropriate
developmental regulation of this gene in multiple cell types.
Expression in most structures was found to be homogeneous
between individual cells with most, if not all, SMC being stained. This
is in contrast to previous observations with certain SM MHC and SM-22
promoter constructs25 29 33 and suggests that the
p2600Int/Lac Z transgene also contains sufficient information to drive
expression in SMC subtypes that have been shown to differentially
express SM-22 or SM MHC transgenic constructs within a given SMC
tissue.
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A total of 10 independent founder lines were established with the
p2600Int/Lac Z construct. Of these, six showed expression patterns
during embryonic development, and as adults, that virtually mimicked
expression of the endogenous SM -actin gene with two
exceptions. Only one founder exhibited expression in uterine SMC, and
most founders showed relatively low expression in small cranial
arteries during development. However, in adult animals, we
consistently detected expression in the basilar artery and
other cerebral vessels (data not shown) in each of these six
independent founders, which suggests that developmental signals may be
important for expression of the p2600Int/Lac Z transgene in some SMC
subtypes. Of the four remaining founders, two showed high expression in
all vascular SMCs but only limited expression in SM-containing organs
such as stomach, intestine, and bladder; one founder showed expression
only in cardiac and skeletal muscle during development; and one was
expressed only in a small subset of skeletal muscle in the head and
neck. Thus, although there were clearly some modest effects of
insertion site on the expression pattern of the p2600Int/Lac Z
transgene in a small number of founders, a high level of
reproducibility of transgene expression across multiple independent
founder lines existed. This provides strong evidence that the observed
expression pattern was the result of sequences contained within the
p2600Int/Lac Z construct and not insertional locus.
CArG Mutations Attenuated the Activity of p2600Int/Lac Z Activity
in Cultured SMCs
Previous studies demonstrated that CArGs A and B (when contained
within a construct that contained either 125 or 547 bp of the 5'
promoter region) were absolutely required for expression in SMC
cultures.14 However, the transgenic results shown above
demonstrate that additional sequences, including the CArG-containing
first intron, are required for expression in vivo. Therefore, to
measure the transcriptional activity of the first intron and to test
the effects of mutations to CArGs A and B and the intronic CArG in the
context of the promoter region shown to be sufficient for in vivo
expression, we transfected cultured rat SMCs with equimolar amounts of
the deletion or site-directed mutant constructs shown in Figure 6. Results demonstrated that the first
intron had significant transcriptional activity in the -547 and -2600
context and that mutation of either CArG A or B or the intronic CArG
decreased p2600Int/Lac Z activity in cultured SMCs.
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SRF Bound the Intronic CArG
EMSA supershift analysis was performed to determine
whether the intronic CArG, like CArGs A and B, binds SRF (Figure 7). Results demonstrated that SRF bound
to the intronic CArG (lanes 3 and 6). The intronic CArG appeared to
bind SRF more avidly than CArGs A and B (compare lane 3 with lanes 1
and 2), a result that is consistent with the fact that these
CArGs contain a conserved G or C substitution in their internal A/T
rich nucleotide region14 and that such
substitutions lower SRF binding affinity.39
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CArG B Was Required for Expression of the p2600Int/Lac Z Transgene
in Skeletal, Cardiac, and Smooth Muscle at E13.5, Whereas the Intronic
CArG Was Required Only in SMCs
Results from our transgenic analyses of the SM -actin
promoter demonstrated that the first intron was required for transgene
expression in SMCs. Combined with the cell culture studies described
above, these results suggest that the intronic CArG, and perhaps CArGs
A and B, are required for expression of SM -actin in SMCs in vivo.
Therefore, we tested whether CArG mutations affected expression of the
p2600Int/Lac Z transgene in developing embryos and in adult mice. At
least five independent founder lines were generated for each CArG
mutant construct. Results shown in Figure 8 compare the effects of CArG mutations
on Lac Z expression in mouse embryos at E13.5 when the
endogenous SM -actin gene and our p2600Int/Lac Z
transgene (wt) is expressed in all three muscle cell types. Mutation of
CArG B (B mut) completely abolished Lac Z expression in all three
muscle cell types, which indicated that it is absolutely required for
SM -actin expression. Of major significance, mutation of the
intronic CArG (Int mut) had no effect on cardiac or skeletal muscle
expression but completely abolished expression in all SM tissues, which
indicated that it is required for expression in SMCs but not in cardiac
and skeletal muscle. Mutation of CArG A had no visible effect on
staining in skeletal or heart muscle, but reduced or eliminated
staining in some SM tissues (data not shown). However, these effects
varied somewhat between founders, which suggested that the activity of
this construct was somewhat sensitive to the site of transgene
insertion.
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Mutations to CArG B and the Intronic CArG Abolished Expression of
the p2600Int/Lac Z Transgene in SMCs in Adult Mice
To determine whether CArG elements are also required for
expression in adult mice, we compared the expression of the wild-type
p2600Int/Lac Z transgene construct and respective CArG mutants in 4- to
6-week-old mice (Figure 8). Results demonstrated that mutation
of CArG B or the intronic CArG abolished expression in SMCs from all
tissues, including trachea, lung, bladder, stomach, and intestine, and
from all blood vessels, including the aorta, carotids, and
coronary mesenteric, renal, and skeletal muscle arteries.
Interestingly, in many founder lines, mutation of CArG A eliminated
expression in SM organs and large vessels such as the aorta and
carotids but only partially inhibited expression in smaller vessels
such as those found in the mesenteric and skeletal muscle vascular
beds.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because it is well established that many of the factors that affect SMC differentiation and the transcription of SMC-specific genes cannot be reproduced accurately in cultured SMCs, the goal of the present study was to identify mechanisms that regulate SM
-actin
expression in vivo in transgenic mice. Results demonstrated that the SM
-actin first intron was required for expression of a Lac Z transgene
in SMC and that the promoter regions from -2600 bp through the first
intron were sufficient to drive transgene expression in a pattern
virtually identical to that of the endogenous gene. We also
provided clear evidence that SM -actin expression is CArG-dependent
and that SMC-specific regulation requires unique cooperative
interactions between the intronic CArG and CArGs A and B.
Results of our transgenic analyses illustrated a number of
interesting features of SM -actin gene regulation that both confirm
and extend previous observations in cultured SMCs but also point out
some key differences. First, these results provide clear evidence that
shows that SM -actin expression is differentially regulated
depending on cell type. Previous studies from our laboratory
demonstrated that 2800 bp of the SM -actin 5' promoter were
sufficient to drive high-level expression of SM -actin only in
cultured SMCs or other cell types such as L6 myotubes that are known to
express their endogenous gene.13 14 In
contrast, this same construct was completely inactive in a variety of
cell types, such as endothelial cells and AKR2B
fibroblasts that do not express SM -actin.14 The
results presented in this study demonstrated that neither the
p2800/Lac Z nor the p547/Lac Z transgenes were expressed in SMCs in
vivo. However, these same constructs were expressed highly in embryonic
skeletal and heart muscle, which are known to express SM -actin
during embryonic development.9 More extensive promoter
analyses provided conclusive evidence that both the first
intron and sequences from -547 to -2600 bp contain promoter elements
required for transgenic expression in SMCs. When combined with the
results of Wang et al, who used a mouse SM -actin promoter fragment
from -1100 bp through the first intron to overexpress IGF-1 in SMCs in
vivo, our data suggest that the promoter region from -547 through
-1100 bp contains cis elements necessary for expression of
SM -actin in vivo. However, because of poorly defined differences
between the sensitivities of the in situ methods used by Wang and the
Lac Z detection methods used in our studies, it is difficult to make
direct comparisons between these two model systems. Second, the
differences in activity of promoter constructs in cultured SMCs versus
in transgenic mice further emphasize the critical importance of
studying SMC gene regulation in transgenic animals in order to
reproduce complex local environmental cues that are necessary for SMC
differentiation (ie, matrix interactions, neuronal and hormonal input,
and mechanical stresses). Third, our transgenic studies extend results
of previous studies in cell culture10 14 16 17 40 41
by providing evidence that specific cis elements exhibit
cell-type–specific activity in vivo. For example, the intronic CArG
was absolutely required for expression in SMCs but not in skeletal and
cardiac muscle during development. A key unresolved issue will be to
determine the precise combinatorial interactions of cis and
trans acting factors that are required for expression in the
various cell-types that express SM -actin under different
physiological and
pathophysiological conditions.
Because of the qualitative nature of Lac Z analysis in
transgenic animals, the possibility of insertional variegation, and
known SMC heterogeneity, considerable caution must be
used when analyzing expression patterns among different transgenic
promoters and even among independent founder lines that contain the
same transgene. Nevertheless, it is interesting that expression of the
p2600Int/Lac Z transgene was readily detected in nearly all SM tissues
in 6 of 10 independent founder lines, and expression in those lines was
remarkably homogeneous both between and within SMC
populations. Recently published transgenic studies with other SMC
marker gene promoters resulted in considerably different patterns of
SMC expression and provided evidence for significant SMC
heterogeneity. For example, a transgene driven by 441
or 1110 bp of the SM-22 5' promoter, although expressed in
arterial SMCs, was not expressed in any other SM
tissues.29 30 In addition, a Lac Z transgene construct
under the control of the SM MHC promoter region from -4299 through
+11 600 bp was expressed in most SMC tissues but lacked any detectable
activity in the renal and pulmonary vasculature and also showed
significant cell-to-cell heterogeneity between SMCs
within the same tissue.33 Although the apparent
homogeneity of expression observed could simply be a function of the
relative strength of the SM -actin promoter, they also indicate that
the p2600Int/Lac Z transgene contains sufficient information to drive
expression in nearly all SMC types and that some transcriptional
regulatory pathways such as those involving the CArGs may be common to
all SMC subtypes. Finally, it is worth noting that the high level of
SMC-specific expression observed with the p2600Int/Lac Z transgene in
adult animals may make it an attractive vector for gene therapies
targeted to SM-containing tissues.
These studies are the first to report the activity of the SM -actin
CArG elements in vivo and provide several interesting findings
concerning CArG-dependent regulation of SM -actin expression. First,
CArG B was absolutely required for in vivo expression in all three
muscle cell types and may be the only CArG element required for
transcriptional activity in skeletal and cardiac muscle during
embryonic development. Second, CArG A, which is a much weaker CArG
because it binds SRF poorly, was required for expression in nearly all
SMC tissues except for the smaller resistance vessels (Figure 9). Although SMCs within large and small
vessels are believed to be derived from a common mesenchymal
source,42 unknown differences in SMC lineage could
contribute to these differences in transgene expression. Alternatively,
differences may be ascribed to known differences in
hemodynamic and/or other environmental stresses that
could possibly regulate SM -actin expression independent of CArG A.
The effects of the CArG A and B mutations on in vivo expression of the
SM -actin transgene are somewhat analogous to the effects of
mutations to the "near" (-141) and "far" (-264) CArGs
described in the SM-22 promoter.29 30 In those studies,
mutation of the "strong" near CArG abolished expression in all cell
types, whereas mutation of the much "weaker" far CArG had only
limited effects on expression. Third, we showed that the intronic CArG
functioned as an SMC-specific enhancer-like element that was required
for expression in SMCs but not in embryonic skeletal and cardiac
muscle. SRF was shown to bind intronic CArG more avidly than CArGs A
and B (Figure 6), suggesting that SRF binding to the SM
-actin promoter may be rate limiting in SMCs in vivo, thus making
the presence of the strong intronic CArG required for expression. It is
also possible that the intronic CArG or other elements within the first
intron that interact with the intronic CArG recruit SMC-specific
factors that are required for SM -actin expression in vivo. Although
we did not detect such a factor in our gel shift analyses, this
was not surprising because we only used 20 bp intronic CArG oligos as
shift probes.
|
The requirement of multiple CArGs for p2600Int/Lac Z expression in SMCs
and the fact that the CArGs have differential effects in SMC versus
non-SMC indicates that these elements act interdependently in vivo to
regulate SM -actin expression. Recent evidence from studies in
cultured SMCs from our laboratory demonstrated that CArG phasing and
spacing are important determinants of the activity of a reporter
construct that contains the first 125 bp of the 5' promoter. This
suggests that CArGs A and B coordinate the formation of a transcription
activation complex sufficient to drive expression at least in SMC
cultures (C.P.M., Mike M. Thompson, Susan Lawrenz-Smith, G.K.O.,
unpublished results, 1998). The in vivo requirement for the intronic
CArG, which is 1,000 bp 3' to CArGs A and B, suggests that this
model is probably more complex. Moreover, we have shown that the highly
conserved intronic region functions in only one orientation, which
argues that it also has specific structural requirements important for
transcription complex assembly or activation (C.P.M., unpublished
results, 1998). Our results are somewhat analogous to those of Lee et
al,23 which demonstrated that skeletal -actin
expression in cultured cells was regulated by two "strong" CArGs
that facilitate binding of SRF to a third relatively weak CArG element.
Given the high prevalence of multiple conserved CArG elements in many
skeletal, cardiac, and SMC genes,19 20 21 22 23 this model that
involves interdependence of multiple CArG elements may be an important
general mechanism for regulating muscle-specific gene expression.
In summary, these studies are the first to provide evidence that CArG
elements play a critical role in transcriptional regulation of the SM
-actin gene in vivo and that they exhibit differential activity in
SMCs versus non-SMCs. This study and others have shown that the
activity of each of these elements is regulated by binding to SRF.
Therefore, a key goal for further studies is to determine the
mechanisms that confer SMC-specific regulation by a ubiquitously
expressed transcription factor such as SRF.
|
Acknowledgments |
|---|
This work was supported by grants RO1 HL-38854, PO1 19242, and F32 HL-10038 from the National Institutes of Health, Fellowship Grant VA-96-F-20 from the Virginia Affiliate of the American Heart Association, and by the Cardiovascular Research Center at the University of Virginia. We gratefully acknowledge the excellent technical assistance of Margaret Ober, Diane Raines, and Andrea Tanner. We would also like to thank Jill Hungerford for her assistance in immunohistochemical analysis of SM
-actin
expression. Received November 3, 1998; accepted February 4, 1999.
|
References |
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T. Yoshida, M. H. Hoofnagle, and G. K. Owens Myocardin and Prx1 Contribute to Angiotensin II-Induced Expression of Smooth Muscle {alpha}-Actin Circ. Res., April 30, 2004; 94(8): 1075 - 1082. [Abstract] [Full Text] [PDF]
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K. L. Du, M. Chen, J. Li, J. J. Lepore, P. Mericko, and M. S. Parmacek Megakaryoblastic Leukemia Factor-1 Transduces Cytoskeletal Signals and Induces Smooth Muscle Cell Differentiation from Undifferentiated Embryonic Stem Cells J. Biol. Chem., April 23, 2004; 279(17): 17578 - 17586. [Abstract] [Full Text] [PDF]
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J. Zhou, A. M. Hoggatt, and B. P. Herring Activation of the Smooth Muscle-specific Telokin Gene by Thyrotroph Embryonic Factor (TEF) J. Biol. Chem., April 16, 2004; 279(16): 15929 - 15937. [Abstract] [Full Text] [PDF]
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Y. Liu, S. Sinha, and G. Owens A Transforming Growth Factor-{beta} Control Element Required for SM {alpha}-Actin Expression in Vivo Also Partially Mediates GKLF-dependent Transcriptional Repression J. Biol. Chem., November 28, 2003; 278(48): 48004 - 48011. [Abstract] [Full Text] [PDF]
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N. Kaplan-Albuquerque, C. Garat, C. Desseva, P. L. Jones, and R. A. Nemenoff Platelet-derived Growth Factor-BB-mediated Activation of Akt Suppresses Smooth Muscle-specific Gene Expression through Inhibition of Mitogen-activated Protein Kinase and Redistribution of Serum Response Factor J. Biol. Chem., October 10, 2003; 278(41): 39830 - 39838. [Abstract] [Full Text] [PDF]
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R. J. Kelm Jr., S.-X. Wang, J. A. Polikandriotis, and A. R. Strauch Structure/Function Analysis of Mouse Pur{beta}, a Single-stranded DNA-binding Repressor of Vascular Smooth Muscle {alpha}-Actin Gene Transcription J. Biol. Chem., October 3, 2003; 278(40): 38749 - 38757. [Abstract] [Full Text] [PDF]
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Y.-F. Chang, J. Wei, X. Liu, Y.-H. Chen, M. D. Layne, and S.-F. Yet Identification of a CArG-independent region of the cysteine-rich protein 2 promoter that directs expression in the developing vasculature Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1675 - H1683. [Abstract] [Full Text] [PDF]
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 B. Hu, Z. Wu, and S. H. Phan Smad3 Mediates Transforming Growth Factor-{beta}-Induced {alpha}-Smooth Muscle Actin Expression Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 397 - 404. [Abstract] [Full Text] [PDF]
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S. Li, D.-Z. Wang, Z. Wang, J. A. Richardson, and E. N. Olson The serum response factor coactivator myocardin is required for vascular smooth muscle development PNAS, August 5, 2003; 100(16): 9366 - 9370. [Abstract] [Full Text] [PDF]
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Z. Wang, D.-Z. Wang, G. C. T. Pipes, and E. N. Olson Myocardin is a master regulator of smooth muscle gene expression PNAS, June 10, 2003; 100(12): 7129 - 7134. [Abstract] [Full Text] [PDF]
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T. Yoshida, S. Sinha, F. Dandre, B. R. Wamhoff, M. H. Hoofnagle, B. E. Kremer, D.-Z. Wang, E. N. Olson, and G. K. Owens Myocardin Is a Key Regulator of CArG-Dependent Transcription of Multiple Smooth Muscle Marker Genes Circ. Res., May 2, 2003; 92(8): 856 - 864. [Abstract] [Full Text] [PDF]
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M. S. Kumar, J. A. Hendrix, A. D. Johnson, and G. K. Owens Smooth Muscle {alpha}-Actin Gene Requires Two E-Boxes for Proper Expression In Vivo and Is a Target of Class I Basic Helix-Loop-Helix Proteins Circ. Res., May 2, 2003; 92(8): 840 - 847. [Abstract] [Full Text] [PDF]
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M. S. Kumar and G. K. Owens Combinatorial Control of Smooth Muscle-Specific Gene Expression Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 737 - 747. [Abstract] [Full Text] [PDF]
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K. L. Du, H. S. Ip, J. Li, M. Chen, F. Dandre, W. Yu, M. M. Lu, G. K. Owens, and M. S. Parmacek Myocardin Is a Critical Serum Response Factor Cofactor in the Transcriptional Program Regulating Smooth Muscle Cell Differentiation Mol. Cell. Biol., April 1, 2003; 23(7): 2425 - 2437. [Abstract] [Full Text] [PDF]
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R. Xu, Y.-S. Ho, R. P. Ritchie, and L. Li Human SM22alpha BAC encompasses regulatory sequences for expression in vascular and visceral smooth muscles at fetal and adult stages Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1398 - H1407. [Abstract] [Full Text] [PDF]
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D. Schauwienold, C. Plum, T. Helbing, P. Voigt, T. Bobbert, D. Hoffmann, M. Paul, and H. P. Reusch ERK1/2-Dependent Contractile Protein Expression in Vascular Smooth Muscle Cells Hypertension, March 1, 2003; 41(3): 546 - 552. [Abstract] [Full Text] [PDF]
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I. Manabe, T. Shindo, and R. Nagai Gene Expression in Fibroblasts and Fibrosis: Involvement in Cardiac Hypertrophy Circ. Res., December 13, 2002; 91(12): 1103 - 1113. [Abstract] [Full Text] [PDF]
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A. M. Hoggatt, G. M. Simon, and B. P. Herring Cell-Specific Regulatory Modules Control Expression of Genes in Vascular and Visceral Smooth Muscle Tissues Circ. Res., December 13, 2002; 91(12): 1151 - 1159. [Abstract] [Full Text] [PDF]
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 B. Chaqour, C. Whitbeck, J.-S. Han, E. Macarak, P. Horan, P. Chichester, and R. Levin Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E765 - E774. [Abstract] [Full Text] [PDF]
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M. Watanabe, M. D. Layne, C.-M. Hsieh, K. Maemura, S. Gray, M.-E. Lee, and M. K. Jain Regulation of Smooth Muscle Cell Differentiation by AT-Rich Interaction Domain Transcription Factors Mrf2{alpha} and Mrf2{beta} Circ. Res., September 6, 2002; 91(5): 382 - 389. [Abstract] [Full Text] [PDF]
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 H.-B. Xin, K.-Y. Deng, M. Rishniw, G. Ji, and M. I. Kotlikoff Smooth muscle expression of Cre recombinase and eGFP in transgenic mice Physiol Genomics, September 3, 2002; 10(3): 211 - 215. [Abstract] [Full Text] [PDF]
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A. Arai, J. A. Spencer, and E. N. Olson STARS, a Striated Muscle Activator of Rho Signaling and Serum Response Factor-dependent Transcription J. Biol. Chem., June 28, 2002; 277(27): 24453 - 24459. [Abstract] [Full Text] [PDF]
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J. Wang, M. Su, J. Fan, A. Seth, and C. A. McCulloch Transcriptional Regulation of a Contractile Gene by Mechanical Forces Applied through Integrins in Osteoblasts J. Biol. Chem., June 14, 2002; 277(25): 22889 - 22895. [Abstract] [Full Text] [PDF]
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P. Qiu and L. Li Histone Acetylation and Recruitment of Serum Responsive Factor and CREB-Binding Protein Onto SM22 Promoter During SM22 Gene Expression Circ. Res., May 3, 2002; 90(8): 858 - 865. [Abstract] [Full Text] [PDF]
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M. W. Majesky Smooth Muscle-Specific Transcription Without a CArG Box Element Circ. Res., April 5, 2002; 90(6): 628 - 630. [Full Text] [PDF]
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M. D. Layne, S.-F. Yet, K. Maemura, C.-M. Hsieh, X. Liu, B. Ith, M.-E. Lee, and M. A. Perrella Characterization of the Mouse Aortic Carboxypeptidase-Like Protein Promoter Reveals Activity in Differentiated and Dedifferentiated Vascular Smooth Muscle Cells Circ. Res., April 5, 2002; 90(6): 728 - 736. [Abstract] [Full Text] [PDF]
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 G. Schratt, U. Philippar, J. Berger, H. Schwarz, O. Heidenreich, and A. Nordheim Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells J. Cell Biol., February 18, 2002; 156(4): 737 - 750. [Abstract] [Full Text] [PDF]
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I. Manabe and G. K. Owens The Smooth Muscle Myosin Heavy Chain Gene Exhibits Smooth Muscle Subtype-selective Modular Regulation in Vivo J. Biol. Chem., October 12, 2001; 276(42): 39076 - 39087. [Abstract] [Full Text] [PDF]
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J. C. L. Zhang, S. Kim, B. P. Helmke, W. W. Yu, K. L. Du, M. M. Lu, M. Strobeck, Q.-C. Yu, and M. S. Parmacek Analysis of SM22{alpha}-Deficient Mice Reveals Unanticipated Insights into Smooth Muscle Cell Differentiation and Function Mol. Cell. Biol., February 15, 2001; 21(4): 1336 - 1344. [Abstract] [Full Text]
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A. M. Mikheev, S. A. Mikheev, Y. Zhang, R. Aebersold, and H. Zarbl CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter Nucleic Acids Res., October 1, 2000; 28(19): 3762 - 3770. [Abstract] [Full Text] [PDF]
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J. M. Miano and B. C. Berk Retinoids : Versatile Biological Response Modifiers of Vascular Smooth Muscle Phenotype Circ. Res., September 1, 2000; 87(5): 355 - 362. [Full Text] [PDF]
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C. P. Regan, I. Manabe, and G. K. Owens Development of a Smooth Muscle-Targeted Cre Recombinase Mouse Reveals Novel Insights Regarding Smooth Muscle Myosin Heavy Chain Promoter Regulation Circ. Res., September 1, 2000; 87(5): 363 - 369. [Abstract] [Full Text] [PDF]
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J. M. Miano, M. J. Carlson, J. A. Spencer, and R. P. Misra Serum Response Factor-dependent Regulation of the Smooth Muscle Calponin Gene J. Biol. Chem., March 24, 2000; 275(13): 9814 - 9822. [Abstract] [Full Text] [PDF]
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C. P. Mack, M. M. Thompson, S. Lawrenz-Smith, and G. K. Owens Smooth Muscle {alpha}-Actin CArG Elements Coordinate Formation of a Smooth Muscle Cell-Selective, Serum Response Factor-Containing Activation Complex Circ. Res., February 4, 2000; 86(2): 221 - 232. [Abstract] [Full Text] [PDF]
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F. Jung, A. D. Johnson, M. S. Kumar, B. Wei, M. Hautmann, G. K. Owens, and C. McNamara Characterization of an E-box-Dependent cis Element in the Smooth Muscle {alpha}-Actin Promoter Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2591 - 2599. [Abstract] [Full Text] [PDF]
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A. D. Johnson and G. K. Owens Differential activation of the SMalpha A promoter in smooth vs. skeletal muscle cells by bHLH factors Am J Physiol Cell Physiol, June 1, 1999; 276(6): C1420 - C1431. [Abstract] [Full Text] [PDF]
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B. Camoretti-Mercado, H.-W. Liu, A. J. Halayko, S. M. Forsythe, J. W. Kyle, B. Li, Y. Fu, J. McConville, P. Kogut, J. E. Vieira, et al. Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor J. Biol. Chem., September 22, 2000; 275(39): 30387 - 30393. [Abstract] [Full Text] [PDF]
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C. P. Mack, A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization J. Biol. Chem., January 5, 2001; 276(1): 341 - 347. [Abstract] [Full Text] [PDF]
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A. M. Hoggatt, A. M. Kriegel, A. F. Smith, and B. P. Herring Hepatocyte Nuclear Factor-3 Homologue 1 (HFH-1) Represses Transcription of Smooth Muscle-specific Genes J. Biol. Chem., September 29, 2000; 275(40): 31162 - 31170. [Abstract] [Full Text] [PDF]
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P. J. Adam, C. P. Regan, M. B. Hautmann, and G. K. Owens Positive- and Negative-acting Kruppel-like Transcription Factors Bind a Transforming Growth Factor beta Control Element Required for Expression of the Smooth Muscle Cell Differentiation Marker SM22alpha in Vivo J. Biol. Chem., November 22, 2000; 275(48): 37798 - 37806. [Abstract] [Full Text] [PDF]
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P. S. Chang, L. Li, J. McAnally, and E. N. Olson Muscle Specificity Encoded by Specific Serum Response Factor-binding Sites J. Biol. Chem., May 11, 2001; 276(20): 17206 - 17212. [Abstract] [Full Text] [PDF]
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B. P. Herring, A. M. Kriegel, and A. M. Hoggatt Identification of Barx2B, a Serum Response Factor-associated Homeodomain Protein J. Biol. Chem., April 20, 2001; 276(17): 14482 - 14489. [Abstract] [Full Text] [PDF]
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M. Strobeck, S. Kim, J. C. L. Zhang, C. Clendenin, K. L. Du, and M. S. Parmacek Binding of Serum Response Factor to CArG Box Sequences Is Necessary but Not Sufficient to Restrict Gene Expression to Arterial Smooth Muscle Cells J. Biol. Chem., May 4, 2001; 276(19): 16418 - 16424. [Abstract] [Full Text] [PDF]
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M. D'Addario, P. D. Arora, J. Fan, B. Ganss, R. P. Ellen, and C. A. G. McCulloch Cytoprotection against Mechanical Forces Delivered through beta 1 Integrins Requires Induction of Filamin A J. Biol. Chem., August 17, 2001; 276(34): 31969 - 31977. [Abstract] [Full Text] [PDF]
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C. J. Phiel, V. Gabbeta, L. M. Parsons, D. Rothblat, R. P. Harvey, and K. M. McHugh Differential Binding of an SRF/NK-2/MEF2 Transcription Factor Complex in Normal Versus Neoplastic Smooth Muscle Tissues J. Biol. Chem., September 7, 2001; 276(37): 34637 - 34650. [Abstract] [Full Text] [PDF]
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G. Schratt, U. Philippar, J. Berger, H. Schwarz, O. Heidenreich, and A. Nordheim Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells J. Cell Biol., February 18, 2002; 156(4): 737 - 750. [Abstract] [Full Text] [PDF]
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J. M. Miano, C. M. Kitchen, J. Chen, K. M. Maltby, L. A. Kelly, H. Weiler, R. Krahe, L. K. Ashworth, and E. Garcia Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1793 - H1803. [Abstract] [Full Text] [PDF]
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I. Manabe and G. K. Owens Recruitment of Serum Response Factor and Hyperacetylation of Histones at Smooth Muscle-Specific Regulatory Regions During Differentiation of a Novel P19-Derived In Vitro Smooth Muscle Differentiation System Circ. Res., June 8, 2001; 88(11): 1127 - 1134. [Abstract] [Full Text] [PDF]
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M. D. Layne, S.-F. Yet, K. Maemura, C.-M. Hsieh, X. Liu, B. Ith, M.-E. Lee, and M. A. Perrella Characterization of the Mouse Aortic Carboxypeptidase-Like Protein Promoter Reveals Activity in Differentiated and Dedifferentiated Vascular Smooth Muscle Cells Circ. Res., April 5, 2002; 90(6): 728 - 736. [Abstract] [Full Text] [PDF]
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P. Qiu and L. Li Histone Acetylation and Recruitment of Serum Responsive Factor and CREB-Binding Protein Onto SM22 Promoter During SM22 Gene Expression Circ. Res., May 3, 2002; 90(8): 858 - 865. [Abstract] [Full Text] [PDF]
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