From the
SM22
The phenotypic plasticity of smooth muscle cells
(SMCs)
Previous studies have suggested that
the SM22
In the studies described in this report, we
have used the murine SM22
To isolate the 3`-untranslated region of the
SM22
The transcriptional start site of the SM22
In this report, we have isolated and structurally
characterized the murine SM22
The unique contractile properties of
SMCs and their ability to reversibly modulate their phenotype from
primarily contractile to primarily synthetic, distinguishes this
myogenic lineage from both the skeletal and cardiac muscle cell
lineages. However, in contrast to the striated muscle lineages (for
review, see Refs. 16-19), relatively little is currently
understood about the cis-acting sequences and
trans-acting factors that regulate gene expression in SMCs.
This is due, in part, to the poorly understood lineage relationships of
SMCs, which appear to develop from multiple locations throughout the
embryo, as well as to the relative paucity of SMC-specific
markers
(48, 49, 50, 51, 52, 53, 54) .
The data presented in this report demonstrate that the level of
SM22
This differential pattern of SM22
Current developmental paradigms suggest that tissue-specific gene
expression is ultimately regulated by the expression of
lineage-specific or lineage-restricted transcription
factors
(16, 17, 18, 19) . Interestingly,
sequence analyses of the minimal SM22
The GenBank
accession number for the murine SM22
We thank Jeffrey M. Leiden and Eric N. Olson for
helpful discussions and suggestions. We also thank Lisa Gottschalk for
expert preparation of illustrations and figures.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
is expressed exclusively in smooth muscle-containing
tissues of adult animals and is one of the earliest markers of
differentiated smooth muscle cells (SMCs). To examine the molecular
mechanisms that regulate SMC-specific gene expression, we have isolated
and structurally characterized the murine SM22
gene. SM22
is
a 6.2-kilobase single copy gene composed of five exons. SM22
mRNA
is expressed at high levels in the aorta, uterus, lung, and intestine,
and in primary cultures of rat aortic SMCs, and the SMC line, A7r5. In
contrast to genes encoding SMC contractile proteins, SM22
gene
expression is not decreased in proliferating SMCs. Transient
transfection experiments demonstrated that 441 base pairs of SM22
5`-flanking sequence was necessary and sufficient to program high level
transcription of a luciferase reporter gene in both primary rat aortic
SMCs and A7r5 cells. DNA sequence analyses revealed that the 441-base
pair promoter contains two CArG/SRF boxes, a CACC box, and one
potential MEF-2 binding site, cis-acting elements which are
each important regulators of striated muscle transcription. Taken
together, these studies have identified the murine SM22
promoter
as an excellent model system for studies of developmentally regulated,
lineage-specific gene expression in SMCs.
(
)
permits this muscle cell lineage to
subserve diverse functions in multiple tissues including the arterial
wall, uterus, respiratory, urinary and digestive tracts. In contrast to
fast and slow skeletal muscle cells which fuse and terminally
differentiate before expressing contractile protein isoforms, SMCs are
capable of simultaneously proliferating and expressing a set of
lineage-restricted proteins including myofibrillar isoforms, cell
surface receptors, and SMC-restricted enzymes. Moreover, in response to
specific physiological and pathophysiological stimuli, SMCs can
modulate their phenotype by down-regulating a set of contractile
protein genes, and in so doing, convert from the so called
``contractile phenotype'' to a dedifferentiated
``secretory
phenotype''
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) .
This phenotypic modulation has been implicated in the pathogenesis of a
number of disease states including atherosclerosis and restenosis
following coronary balloon angioplasty (11-14) and may also
contribute to the airway remodeling seen in asthma
(15) . One
approach to understanding the molecular mechanisms that regulate SMC
development and differentiation is to identify and characterize the
cis-acting sequences and trans-acting factors that
regulate SMC-specific, developmentally-regulated gene expression. This
approach has provided a great deal of information concerning the
molecular mechanisms that regulate skeletal muscle and cardiac muscle
development (for review, see Refs. 16-19). In contrast,
relatively little is understood about the molecular mechanisms that
regulate SMC development due in part to the fact that few SMC-specific
markers have been identified.
gene product is expressed exclusively in smooth
muscle-containing tissues of adult animals and is one of the earliest
markers of differentiated smooth muscle
cells
(10, 20, 21, 22, 23) .
SM22
is a 22-kDa protein with structural homology to the
vertebrate thin filament myofibrillar regulatory protein calponin and
the Drosophila muscle protein mp20 (24). Interestingly,
Drosophila mp20 is expressed specifically in the synchronous
muscles and is not found in the asynchronous oscillatory flight
muscles. In addition, Drosophila mp20 has two potential
calcium binding domains that are oriented in a helix-loop-helix or
EF-hand conformation
(25) . In contrast to calponin, which is
associated physically with other SMC thin filament proteins including
actin, caldesmon, and troponin T, both SM22
and Drosophila mp20 do not appear to be physically associated with the
contractile apparatus
(20) . In vitro analyses,
including differential display performed using primary cultures of rat
aortic SMCs, have revealed that SM22
is expressed at high levels
in freshly dispersed SMCs, but is dramatically down-regulated in late
passage cells
(10) . Taken together, these data are consistent
with the hypothesis that SM22
, while not physically associated
with the contractile apparatus, plays an important functional role in
smooth muscle cells.
gene as a model system to examine the
molecular mechanisms that regulate SMC-specific gene expression.
Specifically, we have isolated the murine SM22
cDNA and used it as
a molecular probe to better define the tissue and cellular pattern of
SM22
gene expression in vivo and in primary cultures of
rat aortic SMCs during progression through the cell cycle. In addition,
we have isolated and structurally characterized a murine SM22
genomic clone and performed a series of transient transfections using
SM22
/luciferase reporter plasmids in order to identify the
functionally important cis-acting transcriptional regulatory
elements that control SM22
gene expression in SMCs. These studies
demonstrated that SM22
is expressed at high levels in all smooth
muscle cell-containing tissues of the adult mouse, as well as in
primary cultures of rat aortic SMCs and the smooth muscle cell line,
A7r5. Transient transfection analyses revealed that a relatively small
fragment in the 5`-flanking region of the SM22
gene (bp -441
to +41) is necessary and sufficient to program high level
transcription of SM22
/luciferase reporter constructs in primary
rat aortic smooth muscle cells and in A7r5 cells, but is inactive in
all non-smooth muscle cell lines analyzed. Taken together, these data
suggest that the SM22
promoter may serve as an excellent model
system to examine the molecular mechanisms that regulate SMC-specific
gene expression.
Isolation of Murine SM22
The
coding region of the murine SM22 cDNA Clones
cDNA was isolated by performing
low stringency PCR using murine uterine RNA and synthetic 5` and 3`
oligonucleotide PCR primers constructed from the previously published
sequence of the rat SM22
cDNA
(23) . The 5` PCR primer was
constructed to be identical to the first 34-bp of the rat SM22
cDNA with the addition of a 5` EcoRI site
(5`-ATCGAATTCCGCTACTCTCCTTCCAGCCCACAAACGACCAAGC-3`). The 3` primer was
constructed to include the reverse complement of bp 759 to 782 of the
rat SM22
cDNA with an additional 3` HindIII restriction
site (5`-ATCAAGCTTGGTGGGAGCTGCCCATGTGCAGTC-3`). PCR reaction products
were subcloned into EcoRI/HindIII-digested pGEM7Z
(Promega, Madison, WI) as described previously
(26) . The
nucleotide sequence of the murine SM22
cDNA was confirmed by
sequencing of the full-length murine SM22
genomic clone. MacVector
DNA sequencing software (Kodak/IBI, Rochester, NY) was used for DNA
sequence analyses.
cDNA, 5
10
recombinant clones from an
oligo(dT)-primed
gt11 C2C12 myotube cDNA library were screened
with the
P-labeled murine SM22
cDNA probe (bp
29-811) as described previously
(27) . Twelve clones were
purified to homogeneity and analyzed by Southern blot analyses as
described
(27) . Two independent clones, each of which contained
a poly(A) tail, were subcloned into EcoRI-digested pGEM7Z and
their nucleotide sequences determined. The nucleotide sequence of the
5`-untranslated region was determined from the sequence of the
SM22
genomic clone. The 5`-untranslated region was localized on
the genomic clone by Southern blot hybridizations, in addition to RNase
protection and primer extension analyses as described below.
Isolation of Murine SM22
Approximately 1 Genomic
Clones
10
recombinant phage
from a murine 129SV Lambda FIX II genomic library (Stratagene, La
Jolla, CA) were screened with the 783-bp murine SM22
cDNA probe
(bp 29-811) labeled with [
-
P]dCTP,
and three positive clones were purified to homogeneity as described
previously
(27) . One clone (SM22-
3a) was found to include
the entire coding region of the SM22
gene and 9-kb of 5`-flanking
sequence and was used for all subsequent subcloning and sequencing
experiments.
Southern Blot Analyses
High molecular weight DNA
was prepared from the tails of strain 129SV mice as described
previously (26). Southern blotting and hybridization to the
radiolabeled 783-bp murine SM22 cDNA probe were performed as
described previously
(26) . Low stringency washing conditions
were 2
SSC, 0.1% SDS at 50 °C. High stringency washing
conditions were 0.1
SSC, 0.1% SDS at 68 °C.
Northern Blot Analyses
Tissues were isolated from
12-week-old 129SV mice (Jackson Laboratories) as described previously
(26). Animals were housed and cared for according to NIH guidelines in
the University of Chicago Laboratory Animal Medicine Veterinary
Facility. RNA was prepared from organ samples and from cultures of
primary rat aortic SMCs, the rat SMC line A7r5, and non-smooth muscle
cell lines including murine NIH 3T3 cells, murine C3H10T1/2 cells,
monkey COS-7 cells, murine C2C12 myoblasts and myotubes, human HepG2
cells, and murine EL-4 cells by the single step guanidinium
isothiocyanate protocol described previously
(28) . Northern
blotting was performed using 10 µg of RNA/sample as described
previously with the exception that 36 µg/ml ethidium bromide was
added to the RNA resuspension buffer in order to permit quantitation of
the 28 S and 18 S ribosomal RNA subunits in each lane. Probes included
the 783-bp (bp 29-811) murine SM22 cDNA and the 754-bp (bp
659-1404) murine calponin cDNA probe.
(
)
Quantitative image analyses were performed using a
Molecular Dynamics PhosphorImager (Sunnyvale, CA).
Primer Extension, 5`-RACE, and RNase Protection
Analyses
A 25-mer oligonucleotide probe constructed to include
the reverse complement of base pairs +80 to +104 of the
SM22 cDNA (5`-TGCCGTAGGATGGACCCTTGTTGGC-3`) was 5` end-labeled
with [
-
P]ATP and T4 polynucleotide kinase.
40 µg of mouse uterine RNA was hybridized to 2
10
dpm of labeled probe and primer extension reactions performed at
42, 50, and 56 °C as described previously
(27) . 5`-RACE was
performed using murine uterine RNA and a synthetic antisense cDNA probe
corresponding to bp 234 to 258 of the murine SM22
cDNA according
to the manufacturer's instructions (Perkin Elmer). RNase
protection analyses were performed by subcloning the -441 to
+41 murine SM22
genomic subfragment including a synthetic 3`
HindIII linker into PstI/HindIII-digested
pGEM4Z and performing in vitro transcription of the antisense
strand of the genomic subfragment with T7 polymerase of the
NcoI-linearized plasmid (NcoI cuts at bp -88 of
the genomic clone) in order to obtain an antisense cRNA probe
corresponding to bp -88 to +44. (Of note, the
HindIII linker shares sequence identity with the SM22
cDNA resulting in a cRNA probe with sequence identity initiated at bp
+44 (not +41) of the SM22
genomic clone.) The 142-bp
probe was labeled with [
-
P]UTP and RNase
protection analyses were performed using the RPAII kit (Ambion, Austin,
TX) according to the manufacturer's instructions.
Cell Culture
The rat cell line A7r5 which was
derived from embryonic thoracic aorta was grown in Dulbecco's
modified essential media (Life Technologies, Inc.) supplemented with
10% fetal bovine serum (Life Technologies, Inc.) and 1%
penicillin/streptomycin. The human hepatocellular carcinoma cell line
HepG2 was grown in modified Eagle's medium supplemented with 10%
fetal bovine serum and 0.1 mM minimal essential medium
non-essential amino acids (Life Technologies, Inc.). Murine
lymphoma-derived EL4 cells were grown in Dulbecco's modified
Eagle's media supplemented with 10% horse serum (Life
Technologies, Inc.). Murine NIH 3T3 cells, C3H10T1/2 cells, C2C12
myoblasts and myotubes were grown as described previously (29, 30).
Primary cultures of rat aortic SMCs were isolated from
12-16-week-old Sprague-Dawley rats (Charles River Laboratories)
using the method described previously
(31) . Virtually all cells
isolated using this method stain positive with anti-smooth muscle actin
monoclonal antiserum. In all experiments, only early passage (passage 2
or 3) rat aortic SMCs were utilized. For the cell cycle analyses, SMCs
from the third passage were placed in serum-free medium (50%
Dulbecco's minimal essential medium, 50% Ham's F-12,
L-glutamine (292 mg/ml), insulin (5 mg/ml), transferrin (5
mg/ml), and selenious acid (5 ng/ml)) for 72 h in order to synchronize
the cells in G/G
as described
previously
(31) . Following 72 h of serum starvation, cells were
stimulated to proliferate by incubation in medium containing 45%
Dulbecco's modified Eagle's medium, 45% Ham's F-12,
and 10% fetal bovine serum.
Plasmids
The Rous sarcoma virus (RSV) long
terminal repeat-driven luciferase reporter plasmid, pRSVL, and the
pMSVgal reference plasmid have been described
previously
(27) . The promoterless pGL2-Basic plasmid (Promega,
Madison, WI) served as the cloning backbone for all of the luciferase
reporter plasmids described below. The p-5000/I1SM22luc plasmid
contains 5-kb of SM22
5`-flanking sequence, the untranslated
SM22
first exon, the SM22
first intron, and the first 12-bp
of exon 2 of the SM22
gene subcloned 5` of the luciferase reporter
gene. It was constructed by first subcloning the 8.5-kb
BamHI/HindIII SM22
genomic subfragment
(containing 5-kb of 5`-flanking sequence, exon 1, and 3.5 kb of intron
1) into BglII/HindIII digested pGL2-Basic vector.
Next, a 488-bp PCR-generated HindIII-linked SM22
genomic
subfragment including at its 5` end the SM22
intron 1
HindIII restriction site (see Fig. 5A) and
running to bp +76 of the SM22 cDNA (which includes 12-bp of exon
2) was subcloned into the HindIII-digested vector and its
correct orientation (5` to 3` relative to the luciferase reporter gene)
confirmed by DNA sequence analysis. The p-5000SM22luc plasmid,
containing 5-kb of SM22
5`-flanking sequence subcloned 5` of the
luciferase reporter gene, was constructed by first subcloning the
2.2-kb BamHI/EcoRI SM22
genomic subfragment
(corresponding to bp -5000 to -2800) into
BamHI/EcoRI-digested pBluescript IIKS (Stratagene La
Jolla, CA). Next, the 1251-bp EcoRI/NcoI SM22
genomic subfragment corresponding to bp -1339 to -89 and
the 130-bp PCR-generated genomic subfragment containing bp -88
(including the NcoI site at its 5` end) to +41 (including
a HindIII linker at its 3` end) was ligated into the
EcoRI/HindIII-digested vector. Then, the 1.4-kb
EcoRI SM22
genomic subfragment (corresponding to bp
-2800 to -1340) was subcloned into the
EcoRI-digested plasmid and its orientation confirmed by DNA
sequence analysis. Finally, the resulting SM22
genomic subfragment
corresponding to bp -5 kB to +41 was excised from the
Bluescript phagemid with BamHI and HindIII and
subcloned into BglII/HindIII-digested pGL2-Basic. The
p-1339SM22luc plasmid containing the 1380-bp SM22
genomic
subfragment (bp -1339 to +41) subcloned 5` of the luciferase
reporter in the pGL2-Basic vector, was constructed using the 1251-bp
EcoRI/NcoI SM22
genomic subfragment (bp
-1338 to -89) and the 130-bp (bp -88 to +41)
PCR-generated genomic subfragments described above. The p-441SM22luc
plasmid contains the 482-bp (bp -441 to +41)
PstI/HindIII SM22
genomic subfragment subcloned
into BglII/HindIII-digested pGL2-Basic plasmid. The
p-300SM22luc and p-162SM22luc luciferase reporter plasmids,
respectively, contain the PCR-generated bp -300 to +41, and
-162 to +41 SM22
genomic subfragments (including
synthetic XhoI (5` end) and HindIII (3` end)
linkers), subcloned into XhoI/HindIII-digested
pGL2-Basic vector. All PCR-generated genomic subfragments were
confirmed by dideoxy DNA sequence analysis.
Figure 5:
Structure of the murine SM22 gene.
A, a schematic representation and partial restriction
endonuclease map of the murine SM22
gene. BamHI
(B), EcoRI (R), XbaI (X),
NcoI (N), and HindIII (H)
restriction enzyme sites are shown. The transcriptional start site is
indicated with an arrow. Exons are shown as shaded boxes.B, the nucleotide sequence of the murine SM22
gene.
The nucleotide sequence of the exons (upper-case letters) and
introns (lower-case letters), as well as 1340 bp of
5`-flanking sequence are shown. Nucleotides within the minimal
functional SM22
promoter with sequence identity to previously
characterized muscle regulatory cis-acting sequence elements
are boxed (see text). The consensus splice donor-acceptor
junctions (AG/GT) are underlined.
Transfections and Luciferase Assays
1
10
passage three primary rat aortic SMCs and A7r5 cells,
respectively, were split and plated 24 h prior to transfection and
transfected with 50 µg of Lipofectin reagent (Life Technologies),
15 µg of luciferase reporter plasmid, and 5 µg of the
pMSV
gal reference plasmid as described
previously
(27, 32) . 1
10
NIH 3T3 or
COS-7 were transfected with 20 µg of Lipofectin reagent, 15 µg
of the luciferase reporter plasmid, and 5 µg of the pMSV
gal
reference plasmid as described previously
(32, 35) . 1
10
HepG2 cells were transfected using 360 µg of
Lipofectamine reagent (Life Technologies), 26 µg of luciferase
reporter plasmid, and 9 µg of the pMSV
gal reference plasmid.
Following transfection, cell lysates were prepared and luciferase and
-galactosidase assays were performed as described
previously
(27) . All experiments were repeated at least three
times to assure reproducibility and permit the calculation of standard
errors. Luciferase activities (light units) were corrected for
variations in transfection efficiencies as determined by assaying cell
extracts for
-galactosidase activities. Data are expressed as
normalized light units ± S.E.
Isolation and Structural Characterization of the Murine
SM22
Murine SM22 cDNA
cDNA clones were isolated using
the polymerase chain reaction in conjunction with synthetic
oligonucleotide primers derived from the previously published sequence
of the rat SM22
cDNA
(23) . A partial restriction
endonuclease map and the nucleotide sequence of the full-length murine
SM22
cDNA are shown in Fig. 1. The murine SM22
cDNA
encodes a 201-amino acid polypeptide with a predicted molecular mass of
22.5 kDa. It is composed of a 76-bp 5`-untranslated region, a 603-bp
open reading frame, and a 403-bp 3`-untranslated region. Of note, 23-bp
5` of the poly(A) tail there is an A/T-rich sequence (AATATA) which may
function as the polyadenylation signal (Fig. 1B, boxed).
Figure 1:
The primary structure of a full-length
murine SM22 cDNA. A, schematic representation of a
partial restriction endonuclease map of the SM22
cDNA. The size of
the cDNA in bp is shown above the map. The protein coding
region of the cDNA is shown as an open box; the
5`-untranslated region is shaded, and the 3`-untranslated
region is hatched. MscI, NcoI,
HincII (H2), PstI, and BstXI restriction
enzyme sites are shown above the map. The size of the deduced
protein in amino acids (aa) is shown below the map.
B, the nucleotide and deduced amino acid sequence of the
murine SM22
cDNA. The putative polyadenylation signal is boxed (see text). The two regions of the SM22
cDNA that share high
level sequence identity with the Drosophila muscle protein
mp20 are shaded. C, a Pustell protein matrix analysis
of the mouse SM22
amino acid sequence (x axis) versus the Drosophila muscle protein mp20 amino acid sequence
(y axis). MacVector Sequence Analysis Software (Kodak, IBI)
was used to perform the Pustell protein matrix analysis, parameters
were set to a window size of 6, min % score of 60, and hash value of 2.
Regions of sequence identity are indicated by a
line.
A comparison of the coding sequences of the murine and human
SM22 cDNAs
(10) demonstrated that the two sequences are 91
and 97% identical at the nucleotide and amino acid levels,
respectively. In addition, a comparison of the coding sequences of the
murine SM22
cDNA and the murine smooth muscle thin filament
regulatory protein, calponin
(33) , demonstrated that these two
sequences are 23% identical and 32% conserved at the amino acid level.
Interestingly, the protein sequence encoded by the murine SM22
cDNA exhibits partial sequence identity with the sequence of the
Drosophila muscle protein mp20
(20) across the entire
cDNA, suggesting that these two proteins may have evolved from a common
ancestral gene (Fig. 1C). As shown in
Fig. 1C, two domains were particularly well conserved
between these proteins. One domain with 14/19 amino acid identity
(corresponding to amino acids 104-122 of the murine SM22
protein, see Fig. 1B, shaded box) may represent a
calcium binding domain oriented in an EF-hand conformation
(25) .
The second C-terminal conserved domain with 13/24 amino acid identity
(corresponding to amino acids 158-181 of the murine SM22
protein) is a domain of unknown function (Fig. 1B, shaded
box).
SM22
The
finding of a putative calcium binding domain oriented in an EF-hand
conformation suggested that SM22 Is Encoded by a Single Copy Gene
might be related to other members
of the troponin C supergene family of intracellular calcium binding
proteins including slow/cardiac troponin C, fast skeletal troponin C,
calmodulin, myosin light chain, and parvalbumin
(25) . In order
to determine whether SM22
is encoded by a single copy gene in the
murine genome and whether SM22
is related to other troponin C
supergene family members, the murine SM22
cDNA was used to probe
Southern blots containing murine genomic DNA under both high and low
stringency conditions. As shown in Fig. 2, under high stringency
conditions, the murine SM22
cDNA probe hybridized to one or two
BamHI, EcoRI, HindIII, PstI, and
XbaI bands, suggesting that SM22
is a single copy gene in
the murine genome. Interestingly, no additional bands were demonstrated
under low stringency conditions, suggesting that although the SM22
gene may have one EF-hand calcium binding domain, it is not closely
related to other members of troponin C supergene family.
Figure 2:
A Southern blot analysis of the murine
SM22 gene. High molecular weight murine SV 129 DNA was digested
with the restriction endonucleases BamHI, EcoRI,
HindIII, PstI, and XbaI and hybridized to
the radiolabeled SM22
cDNA probe under high and low (data not
shown) stringency conditions. Size markers are shown in kb to the
left of the blot.
Lineage-restricted Expression of the SM22
Previous studies have suggested that SM22
Gene
protein is
expressed solely in smooth muscle-containing tissues of the adult and
may be one of the earliest markers of the smooth muscle cell lineage
(21-23). To determine the in vivo pattern of SM22
gene expression, the SM22
cDNA was hybridized to Northern blots
containing RNAs prepared from 12-week-old murine tissues. As shown in
Fig. 3A, the murine SM22
cDNA probe hybridized to
one predominant mRNA species of approximately 1.2-kb. SM22
mRNA is
expressed at high levels in the smooth muscle-containing tissues of
aorta, small intestine, lung, spleen, and uterus. In addition,
prolonged autoradiographic exposures revealed very low, but detectable,
levels of SM22
mRNA in heart, kidney, skeletal muscle, and thymus
(data not shown).
Figure 3:
The in vivo tissue distribution
and cellular-specificity of SM22 gene expression. A, the
top panel shows a Northern blot analysis of RNA samples
isolated form adult murine tissues hybridized to the radiolabeled
SM22
cDNA probe. RNA size markers are shown in kb to the left of the blot. The SM22
cDNA probe hybridized to a single
1.2-kb species of mRNA which was present in smooth muscle-containing
tissues (arrow). The bottom panel shows the ethidium
stained formaldehyde-containing gel prior to membrane transfer of RNA.
The locations of the 28 S and 18 S ribosomal RNA bands are indicated to
the left of the gel. B, the top panel shows
a Northern blot analysis of RNA samples isolated from primary rat
aortic SMCs (VSMC), A7r5, NIH 3T3 (3T3), C3H10T1/2
(10T1/2), COS-7, C2C12 myoblast (C2 Blasts), C2C12
myotubes (C2 Tubes), HepG2, and EL-4 cells hybridized to the
radiolabeled SM22
cDNA probe. The SM22
cDNA hybridized to a
1.2-kb species of mRNA (arrow) present in primary SMCs, A7r5
cells, C2C12 myoblasts and myotubes. The bottom panel shows
the ethidium-stained formaldehyde-containing gel prior to transfer of
RNA.
In order to determine the cell-specificity of
SM22 gene expression, the SM22
cDNA probe was hybridized to
Northern blots containing RNAs prepared from rat aortic vascular SMCs,
the rat SMC line A7r5, murine NIH 3T3 and C3H10T1/2 fibroblasts, the
SV40-transformed monkey kidney cell line COS-7, murine C2C12 myoblasts
and myotubes, the human hepatocellular carcinoma cell line HepG2, and
the murine lymphoid cell line EL4. As shown in Fig. 3B,
high levels of SM22
mRNA were detected in primary rat aortic
vascular SMCs and the smooth muscle cell line A7r5. Of note, detection
of a second larger species of mRNA may represent either
cross-hybridization of the SM22
probe to the murine calponin
mRNA
or to a rare SM22
transcript with an extended 5`
untranslated region
(70) . (
)In addition, SM22
mRNA was expressed in both undifferentiated C2C12 myoblasts and
terminally-differentiated C2C12 myotubes. Finally, a faint
hybridization signal was detectable in NIH 3T3, C3H10T1/2, and HepG2
cells after a 3-day autoradiographic exposure (data not shown).
Quantitative PhosphorImager analysis of these low level hybridization
signals revealed that SM22
mRNA is expressed in these three
non-myogenic cell lines at less than 1.5% the intensity of SM22
gene expression in A7r5 and primary SMCs. Thus, in addition to primary
SMCs and SMC lines, SM22
mRNA is expressed in other embryonic
skeletal muscle cell lineages such as C2C12 myoblasts and myotubes, but
not in other non-myogenic cell lineages.
SM22
Within the tunica media of the arterial wall
the vast majority of vascular SMCs are maintained in a
non-proliferating, quiescent state and express contractile
proteins
(2, 3, 4, 5, 9, 10, 34, 35) .
However, in response to vascular injury, SMCs migrate from the tunica
media to the intimal layer, proliferate, and assume a ``synthetic
phenotype''
(11, 12, 13, 14, 35, 36, 37) .
Previous studies have demonstrated that many genes encoding vascular
SMC contractile proteins are down-regulated during this
process
(2, 3, 5, 38) . Thus, it was of
interest to determine whether SM22 Is Expressed in Both Cell Cycle-arrested and
Proliferating SMCs
gene expression was
differentially regulated during progression through the cell cycle. In
order to address this question, cultures of low passage number primary
rat aortic SMCs were synchronized in the G
/G
stage of the cell cycle by serum starvation for 72 h.
Fluorescence activated cell sorter analyses revealed that under these
conditions approximately 90% of cells are arrested in
G
/G
(Ref. 31, and data not shown). The cells
were then serum-stimulated and RNA was prepared from replicate cultures
at the time of serum stimulation (t
), and at 8,
12, 16, and 24 h post-stimulation. After serum stimulation the arrested
vascular SMCs begin to pass through the G
/S checkpoint of
the cell cycle at approximately 12 h, and by 24 h post-stimulation
greater than 50% of cells are in the S and G
/M phases of
the cell cycle
(31) . A Northern blot analyses demonstrated no
differences in SM22
gene expression in cell cycle arrested
versus proliferating SMCs as assessed by quantitative
PhosphorImager analysis of the hybridization signal (Fig. 4).
Thus, in contrast to other smooth muscle contractile proteins, such as
smooth muscle myosin heavy chain
(3) , smooth muscle
-actin
(2) , and calponin,
(
)
SM22
appears to be constitutively expressed at high levels in both quiescent
and proliferating vascular SMCs.
Figure 4:
The cell
cycle regulation of SM22 gene expression in vitro. The
top panel shows a Northern blot analysis of RNA prepared from
G
/G
synchronized cultures of primary rat aortic
SMCs at t
and 8, 12, 16, and 24 h post-serum
stimulation hybridized to the radiolabeled SM22
cDNA probe.
Quantitative image analysis of the hybridization signal
(arrow) was performed using a Molecular Dynamics
PhosphorImager. RNA size markers are shown in kb to the left of the blot. The bottom panel shows the ethidium-stained
formaldehyde-containing gel prior to membrane transfer of RNA. The
locations of the 28 S and 18 S ribosomal RNA bands are indicated to the
left of the gel.
Isolation and Structural Characterization of a SM22
A full-length murine SM22
Genomic Clone
genomic clone was
isolated by screening a murine 129SV genomic library with a SM22
cDNA probe under high stringency conditions. A partial restriction map
of this 20-kb clone is shown in Fig. 5A. Exons were
identified by hybridization with specific cDNA fragments and their
boundaries confirmed by DNA sequence analysis (Fig. 5B).
The murine SM22
gene is composed of five exons spanning 6.2 kb of
genomic DNA.
gene
was identified by RNase protection, primer extension, and 5`-RACE PCR
analyses (Fig. 6). As shown in Fig. 6A, primer
extension analyses utilizing an antisense synthetic oligonucleotide
corresponding to bp 80-104 of the SM22
cDNA resulted in a
major extended product of 104-bp (arrow) which was generated
at reaction temperatures up to 56 °C. In addition, 5`-RACE PCR was
performed utilizing an antisense oligonucleotide primer corresponding
to bp 234-258 of the SM22
cDNA. DNA sequence analyses of
eight random 5`-RACE clones revealed a transcriptional start site 76 bp
5` of the initiation codon in seven of eight clones and 72 bp 5` of the
initiation codon in one of eight clones (data not shown). RNase
protection analyses were also performed using an antisense cDNA probe
corresponding to bp -88 to +44 of the SM22
genomic
sequence as deduced by DNA sequence and Southern blot analyses
(Fig. 6B). These analyses revealed a major protected
fragment of 44 bp (arrow) corresponding to a transcriptional
start site 76 bp 5` of the initiation codon. In addition, a second,
minor (20% relative signal intensity) protected fragment of 54 bp was
also demonstrated. Taken together, these data allowed the
identification of the major transcriptional start site of the murine
SM22
gene 76 bp 5` of the initiation codon.
Figure 6:
Localization of the transcriptional start
site of the murine SM22 gene. A, primer extension
analysis of SM22
mRNA. The reaction products of primer extension
reactions performed at 42, 50, and 56 °C utilizing a 5` end-labeled
antisense oligonucleotide primer corresponding to bp 80-104 of
the SM22
cDNA and SV129 murine uterine RNA were separated on a 6%
acrylamide/urea gel which was subject to autoradiography. The upper
band (black arrow) in the autoradiogram represents the
104-bp primer extension product. The lower band (hatched
arrow) represents the nonhybridized 25-bp radiolabeled
oligonucleotide probe. DNA size markers in bp are indicated to the
left of the autoradiogram. B, RNase protection
analysis of SM22
mRNA. Murine uterine RNA was subjected to RNase
protection analysis using an antisense cRNA probe corresponding to bp
-88 to +44 of the SM22
gene. The top panel shows the nucleotide sequence of the SM22
mRNA (RNA) and the
SM22
cRNA probe (Probe). The arrows indicate the
transcriptional start sites (see text). The bottom panel shows
an autoradiogram of a 8% sequencing gel containing the RNase protection
analysis reaction products. The first lane (Probe
only) contains the radiolabeled 142-bp antisense cRNA probe only
(dotted arrow). The second lane (-RNA)
contains the products from an RNase digestion reaction performed with
tRNA and the radiolabeled 142-bp antisense probe. The third lane (+RNA) contains the digestion products of a reaction
mixture containing murine uterine RNA and the radiolabeled antisense
cRNA probe. The 44-bp product, corresponding to a transcriptional start
site 76-bp 5` of the initiation codon, is indicated by a black
arrow.
The complete coding
sequence and 1340 bp of 5`-flanking sequence of the SM22 gene is
shown in Fig. 5B. Each of the splice junctions
(Fig. 5B, underlined) conforms to the consensus splice
donor-acceptor patterns as described by Breathnach and Chambon (39). In
order to identify potential transcriptional regulatory elements, 1340
bp of 5` sequence flanking the cap site was searched for a variety of
transcriptional regulatory elements using MacVector DNA sequencing
software (Kodak/IBI). The nucleotide sequence TTTAAA, which might
function as a TATA box was present 29-bp 5` of the start site
(Fig. 5B, boxed). A consensus CAAT box was not
identified in the immediate 5`-flanking region of the SM22
gene. A
computer homology search for previously described muscle-specific
and/or skeletal or cardiac muscle lineage-restricted transcriptional
regulatory elements revealed five consensus E boxes/bHLH myogenic
transcription factor binding sites
(CANNTG
(16, 17, 40) ) located at bp -535,
-578, -866, -899, -911, and -1268, three
consensus GATA-4 binding sites (WGATAR
(41) ) located at bp
-505, -829, -977, and two AT-rich, potential
MEF-2/rSRF binding sites (YTAWAAATAR
(42) ) located at bp
-408 (TTtAAAATcG, small letters denote mismatches from the
consensus MEF-2 sequence) and -771 (TTcAAAATAG). In addition,
functionally important nuclear protein binding sites which have been
identified in previously characterized skeletal and cardiac-specific
transcriptional regulatory elements included two consensus CArG/SRF
binding sites
(43) located at bp -150 and -273 and
one CACC box
(44) located at bp -104. Finally, four AP2
(CCCMNSSS
(45) ), one Sp1 (KRGGCKRRK
(46) ), and two NF-IL6
(TKNNGNAAK
(47) ) binding sites were located in the 5`-flanking
region.
Identification of the cis-Acting Transcriptional
Regulatory Elements That Control SM22
In
order to identify the functionally important cis-acting
sequences that regulate transcription of the SM22 Gene Expression
gene in SMCs, a
series of transient transfections were performed using
SM22
-luciferase reporter constructs and primary rat aortic
vascular SMCs and the SMC line, A7r5, both of which express high levels
of SM22
mRNA (Fig. 3B). Transfection of A7r5 cells
with the plasmid p-5000/I1SM22luc, containing 5 kb of 5`-flanking
sequence and the entire 4-kb SM22
intron 1 sequence (the
initiation codon is located in exon 2), resulted in a
250-300-fold induction in luciferase activity as compared to the
promoterless control plasmid, pGL2-Basic (Fig. 7A, lanes 1 and 2). This level of transcriptional activity was
comparable to that obtained following transfection of A7r5 cells with
the RSV-containing luciferase reporter plasmid, pRSVL
(Fig. 7A, lanes 2 and 8). In order to determine
whether this transcriptional activity was due to the immediate
5`-flanking region of the SM22
gene, or alternatively, was due to
a transcriptional regulatory element located within the first intron of
the SM22
gene, the activities of the p-5000/IlSM22luc and
p-5000SM22luc plasmid were compared (Fig. 7A, lanes 2 and 3). Transfection of A7r5 cells with the p-5000SM22luc
plasmid, containing only 5 kb of 5`-flanking sequence, resulted in high
level transcription of the luciferase reporter gene comparable (on a
molar basis) to levels obtained with the p-5000/I1SM22luc plasmid.
Thus, the 5`-flanking region of the SM22
gene contains
cis-acting sequence elements required for high level
transcription in A7r5 cells.
Figure 7:
Identification and localization of
transcriptional regulatory elements that control SM22 gene
expression in A7r5 and primary rat aortic SMCs. A, transient
transfection analyses of SM22
/luciferase reporter plasmids in the
smooth muscle cell line, A7r5. 15 µg of SM22
/luciferase
reporter plasmid and 5 µg of the pMSV
gal reference plasmid
were transiently transfected into replicate cultures of A7r5 cells.
Cells were harvested 60 h after transfection, and cell extracts were
assayed for both luciferase and
-galactosidase activities.
Luciferase activities (light units) were corrected for variations in
transfection efficiencies as determined by
-galactosidase
activities. Data are expressed as normalized light units ± S.E.
B, transient transfection analyses of SM22
/luciferase
reporter plasmids in primary rat aortic SMCs. Transient transfection
analyses were performed using a series of SM22
/luciferase reporter
plasmids and primary rat aortic SMCs as described in A above.
Data are expressed as normalized light units ±
S.E.
To further localize the 5`-flanking
elements of the SM22 gene that direct high level expression in
SMCs, a series of 5` deletion mutants were transfected into both A7r5
cells (Fig. 7A) and primary cultured rat aortic vascular
smooth muscle cells (Fig. 7B). In both A7r5 cells and
primary vascular SMCs, the p-441SM22luc plasmid, containing 441 bp of
5`-flanking sequence, increased transcription of the luciferase
reporter to levels comparable to the p-5000SM22luc plasmid and the
p-1339SM22luc plasmids (Fig. 7, A lanes 3-5 and
B, lanes 2 and 3). However, transfection of both A7r5
cells and primary vascular SMCs with the luciferase reporter
plasmidsp-300SM22luc and p-162SM22luc containing 300 and 162 bp,
respectively, of 5`-flanking sequence resulted in 50 and 90% reductions
in normalized luciferase activities as compared with those obtained
with the p-441SM22luc plasmid (Fig. 7, A, lanes 5-7 and B, lanes 3-5). These data demonstrated that 441
bp of SM22
5`-flanking sequence, containing the endogenous
SM22
promoter, is sufficient to direct high level transcriptional
activity in both A7r5 cells and primary rat aortic SMCs.
Cellular Specificity of the SM22
In
order to characterize the cellular specificity of the SM22 Promoter
promoter sequence, the transcriptional activities of the 441-bp
SM22
promoter containing plasmid, p-441SM22luc, was compared to
the positive control plasmid containing the Rous sarcoma virus-long
terminal repeat, pRSVL, in primary rat vascular SMCs, the smooth muscle
cell line A7r5, NIH 3T3 fibroblasts, COS-7, and HepG2 cells. Consistent
with the lineage-restricted pattern of SM22
mRNA expression
demonstrated in these cell lines (see Fig. 3B), the
promoter-containing plasmid, p-441SM22luc, was active in primary rat
aortic SMCs and A7r5 cells, increasing transcription of the luciferase
reporter gene approximately 2500- and 540-fold, respectively, over that
induced by transfection with the promoterless pGL2-Basic plasmid
(Fig. 8). This level of promoter activity was comparable to
levels obtained following transfection of these cells with the RSV-long
terminal repeat-driven positive control plasmid (Fig. 8). In
contrast, the 441-bp SM22
promoter was inactive in NIH 3T3, COS-7,
and HepG2 cells (Fig. 8).
Figure 8:
Cellular-specificity of the 441-bp
SM22 promoter. The p-441SM22luc (black bar) and pRSVL
(hatched bar) plasmids were transiently transfected into
primary rat aortic SMCs (VSMC), A7r5, NIH 3T3 (3T3),
COS-7, and HepG2 cells and the normalized luciferase activities for
each respective plasmid was determined as described in the legend to
Fig. 7. Data are expressed as normalized luciferase light units
± S.E.
DNA sequence analyses
(Fig. 5B, boxed) revealed that this 441-bp promoter
contains two CArG/SRF boxes
(43) , a CACC box
(44) , and
one A/T-rich potential MEF-2/rSRF binding site
(42) ,
cis-acting elements which have each been demonstrated to be
involved in the transcriptional programs that regulate skeletal and
cardiac muscle-specific gene expression. However, unlike most
previously described skeletal muscle-specific transcriptional
regulatory elements, this sequence lacked a canonical E box binding
site for the myogenic bHLH transcription
factors
(17, 40) . Thus, the endogenous 441-bp SM22
promoter contains all of the cis-acting sequence elements
required to recapitulate the smooth muscle lineage-restricted pattern
of SM22
gene expression demonstrated in vivo.
cDNA and gene. Using the murine
SM22
cDNA as a molecular probe, we have defined the tissue
distribution and cell cycle-regulated pattern of SM22
gene
expression. In addition, we have demonstrated that the immediate
5`-flanking region of the SM22
gene is necessary and sufficient to
direct high level, lineage-restricted expression of the SM22
gene
in both primary vascular SMCs and the SMC line, A7r5. Finally, we have
demonstrated that the minimal SM22
promoter lacks a binding site
for the bHLH family of myogenic transcription factors. These data are
relevant to understanding the underlying transcriptional program that
regulates SMC differentiation.
protein expression is regulated at the level of gene
expression. However, in contrast to the smooth muscle myosin heavy
chain, and possibly the
-enteric actin gene, which are expressed
exclusively in
SMCs
(3, 55, 56, 57, 58) ,
SM22
is expressed in other myogenic cell lineages including the
embryonic skeletal muscle cell lineage C2C12. In this regard, it is
noteworthy that the SM22
gene is expressed in undifferentiated
skeletal myoblasts, which do not express myofibrillar protein isoforms,
and that SM22
gene expression is not down-regulated in conjunction
with other SMC contractile proteins during serum-induced SMC
proliferation. Taken together, these data suggest that the SM22
gene is not regulated in a coordinated manner with other smooth muscle
contractile proteins. Therefore, elucidation of the cis-acting
sequences that regulate SM22
gene expression in SMCs could serve
as a valuable tool for targeting gene expression to both
contractile/arrested and synthetic/proliferative SMCs in the arterial
wall in vivo.
gene
expression in several myogenic lineages suggests that distinct
transcriptional programs have evolved to permit the regulated
expression of a single gene in multiple cell lineages. However, it is
noteworthy that Olson and co-workers
(59) recently reported that
a null mutation of the MADS box transcription factor D-MEF2 gene in
Drosophila resulted in failure of somatic, cardiac, and
visceral muscles to differentiate. These data suggest that this
evolutionarily conserved family of transcription factors may play a
critical role in coordinating muscle differentiation across lineages.
Thus, it will be of interest to determine the functional role of the
A/T-rich potential MEF-2/rSRF (8/10-bp sequence identity) binding site
located within the minimal murine SM22
promoter. In this respect,
the SM22
promoter may serve as a useful target with which to
dissect the functional role of the four individual MEF-2/rSRF family
members expressed in vertebrate species (versus the single
D-MEF-2 gene in Drosophila) in the smooth muscle lineage.
Similarly, two consensus CArG box/SRF binding sites were identified in
the minimal SM22
promoter. This motif, which has been identified
in multiple skeletal and cardiac-specific transcriptional regulatory
elements
(60) , is also present in the smooth muscle
-actin
promoter
(61, 62, 63) , suggesting that it may
play a role in the coordinate regulation of genes expressed in SMCs.
Finally, a consensus CACC box was identified in the minimal SM22
promoter. This nuclear protein binding site is present in multiple
skeletal and cardiac-specific transcriptional regulatory elements,
where it has been demonstrated to function in conjunction with other
lineage-specific nuclear protein binding
sites
(29, 30, 64, 65, 66) .
promoter failed to reveal a
consensus bHLH myogenic transcription factor/E-box binding site.
Consistent with this observation, myogenic bHLH family members
including MyoD, myogenin, myf-5, and MRF-4/herculin/myf-6 are not
expressed in SMCs and null mutations of the MyoD, myogenin, and myf-5
genes, respectively, had no effect on smooth muscle cell specification
or differentiation in vivo(67, 68) . Similarly,
the minimal SM22
promoter lacked a consensus binding site for
GATA-4, a transcription factor that has been demonstrated to
transactivate multiple cardiac-specific transcriptional regulatory
elements in non-muscle cell lines
(32, 69) . Taken
together, these studies suggest that potentially novel SMC-specific
transcription factors may play a key role in regulating SMC-specific
transcription. Future studies utilizing the SM22
promoter as a
model system should provide fundamental insight into the molecular
mechanisms that regulate SMC-specific transcription and
differentiation.
cDNA is L41154. The GenBank
accession number for the murine SM22
gene is L41161.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.