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(Received for publication, January 3, 1996; and in revised form, February
9, 1996) From the
Differentiated, quiescent vascular smooth muscle cells assume a
dedifferentiated, proliferative phenotype in response to injury, one of
the hallmarks of arteriosclerosis. Members of the LIM family of
zinc-finger proteins are important in the differentiation of various
cells including striated muscle. We describe here the molecular cloning
and characterization of a developmentally regulated smooth muscle LIM
protein, SmLIM, that is expressed preferentially in the rat aorta. This
194-amino acid protein has two LIM domains, and comparisons of rat
SmLIM with its mouse and human homologues reveal high levels of amino
acid sequence conservation (100 and 99%, respectively). SmLIM is a
nuclear protein and maps to human chromosome 3. SmLIM mRNA expression
was high in aorta but not in striated muscle and low in other smooth
muscle tissues such as intestine and uterus. In contrast with arterial
tissue, SmLIM mRNA was barely detectable in venous tissue. The presence
of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels
decreased by 80% in response to platelet-derived growth factor-BB in
rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased
by 60% in response to vessel wall injury during periods of maximal
smooth muscle cell proliferation. The down-regulation of SmLIM by
phenotypic change in vascular smooth muscle cells suggests that it may
be involved in their growth and differentiation.
In their normal state, vascular smooth muscle cells (VSMCs) ( Unlike VSMC differentiation, skeletal muscle differentiation is well
studied. The myogenic helix-loop-helix proteins MyoD, myogenin, myf-5,
and myf-6 have been assigned important roles in the differentiation of
skeletal muscle cells(7, 8, 9, 10) .
Recently, a muscle LIM-domain protein, MLP, has also been described as
a positive regulator of myogenic cell differentiation(11) . Its
cysteine-rich LIM domain, defined by the 50-60-amino acid
consensus sequence (CX So far there are
three classes of LIM proteins. Class 1 proteins (LIM-HD) contain two
LIM domains and a homeodomain. Lin-11, Isl-1, and
Mec-3(17, 18, 19) , the first LIM proteins to
be identified, belong to this group. Class 2 proteins (LIM-only)
contain one or more LIM domains, but lack the homeodomain(14) .
Class 3 proteins (LIM-K) contain LIM domains and a protein kinase
domain(20, 21, 22) . Two members of the
LIM-only class, RBTN2 (12) and MLP(11) , have been
shown to play important roles in cellular differentiation. Originally
identified in childhood T cell acute lymphoblastic
leukemia(23, 24) , RBTN2 is essential for erythroid
cell development; a homozygous null mutation in RBTN2 leads to failure
of yolk sac erythropoiesis and embryonic death(12) . MLP is
expressed only in the heart and skeletal muscle of rats. Overexpression
of sense MLP in C2 myoblasts potentiates myogenic cell differentiation.
In contrast, expression of antisense MLP retards myoblast
differentiation and withdrawal from the cell cycle. Although these
observations suggest that MLP could be involved in regulating skeletal
and heart muscle cell-specific gene expression (11) , MLP mRNA
is not expressed in VSMCs in vitro or in vivo (data
not shown). We hypothesized that a related but heretofore unidentified
LIM protein may play an analogous role in VSMC differentiation. We
report the identification and characterization of a LIM-only protein
expressed preferentially in aortic smooth muscle cells. Smooth muscle
LIM (SmLIM) is a nuclear protein whose expression is regulated
developmentally. Stimulation of cultured VSMCs with the potent mitogen
platelet-derived growth factor (PDGF)-BB caused a down-regulation of
SmLIM mRNA. In vivo, SmLIM mRNA levels decreased as VSMCs
changed from a quiescent to a proliferative phenotype in response to
vascular injury.
Figure 1:
Nucleotide,
deduced amino acid sequence, and in vitro transcribed and
translated product of r-SmLIM. A, Complete nucleotide (upper line) and deduced (lower line) amino acid
sequences of r-SmLIM. Residues composing the two LIM domains are in
boldface, a putative nuclear localization signal is underlined, and the polyadenylation signal is underlined and in italics. B, The entire r-SmLIM open reading frame
was cloned in the sense and antisense orientations into the eukaryotic
expression vector PCRIII. After in vitro transcription and
translation with wheat germ lysate the protein was resolved on a 10%
SDS-PAGE Tricine gel. The single intense band in the sense lane (arrow) represents full-length SmLIM at 21
kDa.
To determine whether SmLIM was
conserved across species, we obtained the human (Fig. 2A) and mouse (m) (
Figure 2:
Conservation of SmLIM among species. A, Sequence alignment of r-SmLIM and h-SmLIM proteins to the
LIM proteins r-CRP, h-CRP, and r-MLP. Shaded amino acids
designate identity to r-SmLIM. Consensus sequence indicates residues
conserved in all five proteins. Cysteine and histidine residues
composing LIM domains are underlined. B, percentage nucleotide
and amino acid identity of r-SmLIM versus m- and h-SmLIM
homologues, r- and h-CRP, and r-MLP.
Figure 3:
Cellular localization of r-SmLIM. COS
cells were transiently transfected with the c-Myc-r-SmLIM hybrid
construct or vector alone (not shown). Protein expression was assayed
48 h after transfection with an anti-c-Myc monoclonal antibody (9E10)
followed by rhodamine-conjugated secondary antibody (red, right). Nuclear counterstaining was performed with Hoechst
33258 (blue, left). Magnification,
Figure 4:
Chromosomal localization of h-SmLIM.
Individual chromosomes are numbered 1-22, X, and Y. The three
control DNA samples (human, mouse, and hamster) were provided by the
manufacturer of the kit (BIOS somatic cell hybrid blot). Arrow indicates specific signal for h-SmLIM visible only in the human, mix, and chromosome 3
lanes.
Figure 5:
Tissue distribution of SmLIM. A,
r-SmLIM mRNA expression in male and female rat tissues. Northern
analysis was performed with 10 µg of total RNA per lane. After
electrophoresis, RNA was transferred to nitrocellulose filters and
hybridized with a
Figure 6:
In situ analysis of r-SmLIM
expression in rat vascular tissue. r-SmLIM mRNA was assayed with
Figure 7:
Down-regulation of SmLIM by growth factor
and vascular injury. A, decrease in r-SmLIM mRNA expression in
response to PDGF-BB treatment. Rat aortic smooth muscle cells were made
quiescent by incubation in low serum medium (DME plus 0.4% calf serum)
for 48 h. Cells were then treated for the indicated times with PDGF-BB
(20 ng/ml). Northern analysis was performed with 10 µg of total
RNA/lane. After electrophoresis, RNA was transferred to nitrocellulose
filters and hybridized with a
In response to vessel wall injury,
VSMCs undergo a phenotypic change from a differentiated, contractile
state to a dedifferentiated, proliferative state. Balloon injury of the
rat carotid artery is a well characterized model for studying this
change in phenotype in vivo. Previous work on cellular
proliferation after arterial injury showed that smooth muscle cell
proliferation reaches a maximum in the medial layer at 48 h and a
maximum in the intimal layer at 96 h and declines
thereafter(38) . We therefore studied r-SmLIM mRNA levels in
rats at 2, 5, and 8 days after balloon injury of the carotid artery (Fig. 7B). SmLIM mRNA levels decreased by more than 60%
after day 2 in comparison with control and remained at this level
through day 8. These data suggest that r-SmLIM mRNA decreases in
response to smooth muscle cell proliferation and dedifferentiation both in vitro and in vivo.
Figure 8:
Developmental regulation of SmLIM mRNA
expression. Total RNA isolated from undifferentiated embryonic stem
cells (ES) and mouse embryos days 7.5-16.5 p.c. Northern
analysis was performed with 10 µg of total RNA/lane. After
electrophoresis, RNA was transferred to nitrocellulose filters and
hybridized with a
We have isolated a developmentally regulated nuclear LIM
protein, SmLIM, from a rat smooth muscle cell library. SmLIM is
expressed preferentially in arterial smooth muscle cells, and in
response to external cues that promote smooth muscle cell proliferation
and dedifferentiation, SmLIM mRNA is down-regulated. SmLIM is a
highly conserved, two-LIM-domain nuclear protein of the LIM-only class (Fig. 1Fig. 2Fig. 3). Other members of this class
include RBTN2, MLP, and CRP. Like SmLIM, RBTN2 and MLP are nuclear
proteins with two LIM domains, and they are highly conserved across
species(11, 12, 41) . CRP proteins also have
two LIM domains and show high cross-species
conservation(37, 42) . Sequence comparisons of SmLIM
and CRP suggest that the two gene families are related yet distinct (Fig. 2). In contrast with SmLIM, which is a nuclear protein (Fig. 3), CRP has been localized to the cytoskeletal adhesion
plaques(13, 15) . Moreover, h-SmLIM localizes to
chromosome 3 (Fig. 4), whereas h-CRP localizes to chromosome
1(43) . Finally, Northern analysis of r-CRP tissue distribution
showed that the size of its mRNA and pattern of expression were
distinct from those of r-SmLIM (data not shown). Taken together, these
data indicate that SmLIM and CRP are distinct LIM proteins. While this
manuscript was in preparation, Weiskirchen et al. (42) reported the cloning of the chicken CRP2 gene. Sequence
comparisons suggest that CRP2 is the avian homologue of SmLIM. Although SmLIM is highly expressed in smooth muscle cells, it is not
expressed in striated muscle cells (Fig. 5). This pattern is in
contrast with that of MLP, which is expressed only in the heart and
skeletal muscle(11) . When a full-length MLP probe was
hybridized to total RNA from aorta and cultured VSMCs, we were unable
to detect a message (data not shown). Thus, the expression of the two
LIM proteins is distinct within the myogenic cell lineage. Arber et
al.(11) have shown that MLP may play an essential role
in striated muscle differentiation. Perhaps SmLIM plays an analogous
role in VSMCs. SmLIM mRNA is expressed preferentially in tissue
containing vascular smooth as opposed to nonvascular smooth muscle
cells (Fig. 5). As such it joins two other recently identified
genes, SM22 Smooth muscle cells differ from striated muscle cells in their
ability to reenter the cell cycle. This reentry is accompanied by a
change from a quiescent, differentiated phenotype into a proliferative,
dedifferentiated phenotype(3, 50) . Genes important
for maintaining the differentiated state may require down-regulation or
inactivation to permit this phenotypic modulation. In this study we
evaluated r-SmLIM expression in response to two different systems that
model VSMC dedifferentiation. First, we found that r-SmLIM expression
was down-regulated in response to PDGF-BB stimulation (Fig. 7).
Second, we found an analogous decrease in SmLIM mRNA expression after
balloon injury to the rat carotid artery, with a brisk down-regulation
at 2 days after injury. In both aspects SmLIM is similar to the growth
arrest-specific homeobox gene gax(44, 51) .
During this 2-8-day period after injury, smooth muscle cells
dedifferentiate and assume a highly proliferative
phenotype(3) . Thus, both in vitro and in
vivo, SmLIM expression is down-regulated as smooth muscle cells
undergo phenotypic change. SmLIM expression also appears to be
regulated developmentally. Expression is highest at day 7.5 p.c. in
mouse embryos (Fig. 8) and plateaus by day 9.5 p.c. These early
stages represent important points in the development of the mouse heart
and vascular systems. At the late primitive streak stage (day 7.5
p.c.), discrete blood islands make their first appearance and
amalgamate shortly thereafter to form the yolk sac vasculature. Within
the embryo one also sees the early formation of a vasculature at 8.0
days p.c. and amalgamation of the embryonic and extraembryonic
circulations at 8.5 days p.c.(39, 40) . Given that
SmLIM expression is highest in the adult aorta and correlates with the
level of smooth muscle cell differentiation, it is interesting that its
embryonic expression is highest during periods critical for vascular
development. The LIM domain functions as a modular protein-binding
interface(52) . For example, the LIM-only protein RBTN2 binds
to the basic helix-loop-helix protein tal-1(53) , an
interaction thought to be critical in regulating red blood cell
development. Homozygous deletion of either RBTN2 or tal-1 results in absence of red blood cell
formation(12, 54) . Similarly, it has been suggested
that the effect of the LIM-only protein MLP on myoblast differentiation
may be as a cofactor regulating muscle-specific gene expression.
Identification of the interaction partner(s) of SmLIM may yield
important information about other factors involved in smooth muscle
cell development and differentiation.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s) U44948 [GenBank]and U46006[GenBank].
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10194-10199
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)regulate vessel tone and blood pressure. VSMCs are not
terminally differentiated, in contrast with skeletal muscle and cardiac
muscle cells. In response to mechanical, chemical, or immunologic
injury (1, 2, 3, 4, 5) the
VSMC phenotype changes rapidly from that of a differentiated, quiescent
cell to that of a dedifferentiated, proliferating cell. Although VSMC
proliferation is a hallmark of arteriosclerosis, the leading cause of
death in developed countries, little is known about the molecular
mechanisms regulating this phenotypic change. Progress in this area has
been limited by the lack of VSMC-specific markers and precursor cells
that can be differentiated into VSMCs in vitro(6) . 
-CX
±
1-H-X-C)-X-(C-X-C-X ± 1-C-X-C/D/H)(12) , is found
in proteins that function in developmental regulation, cellular
differentiation, and actin-based cytoskeletal
interaction(13, 14, 15) . Because this
sequence is conserved among highly divergent species, the LIM domain
appears to be functionally important(16) .
Cell Culture and Reagents
Aortic smooth muscle
cells were harvested from the thoracic aorta of adult male
Sprague-Dawley rats (200-250 g) by enzymatic digestion according
to the method of Gunther et al.(25) . COS-7 and 10T1/2
cells were obtained from the American Type Cell Culture Collection.
Embryonic stem cells (D3) were kindly provided by R. Rosenberg
(Massachusetts Institute of Technology, Boston, MA). Rat aortic smooth
muscle cells were grown in DME (JRH Biosciences, Lenexa, KS)
supplemented with 10% fetal calf serum, penicillin (100 units/ml),
streptomycin (100 µg/ml), and 25 mM Hepes (pH 7.4) in a
humidified incubator (37 °C, 5% CO). COS-7 and 10T1/2
cells were grown similarly, with the exceptions that DME was
supplemented with Serum Plus (Hyclone, Logan, Utah) for the former and
basal medium Eagle (JRH Biosciences) was substituted for DME for the
latter. Cells were cultured and maintained in an undifferentiated state
with leukemia inhibitory factor as described by Doetschman et
al.(26) . PDGF-BB was purchased from Collaborative
Biomedical Products (Bedford, MA).Cloning and Sequencing of Rat (r)-SmLIM and Human
(h)-SmLIM
The full-length rat MLP cDNA was amplified from rat
heart RNA by the reverse transcriptase PCR(27) . Forward
(5`-GAGTCTTCACCATGCCGAAC-3`) and reverse (5`-CTCTCCCACCCCAAAAATAG-3`)
primers, designed according to the published rat MLP
sequence(11) , were used to amplify an 801-base pair fragment.
The PCR fragment was then subcloned and sequenced by the dideoxy chain
termination method (27) . The r-MLP fragment was used to screen
a rat neonatal aortic cDNA library in 
gt11(27) .
Approximately 1.6 million phage clones were plated, transferred to
nitrocellulose paper, and screened at low stringency. One out of nine
isolated clones encoded the partial sequence of a novel LIM protein,
r-SmLIM. This partial clone was then used to screen a rat smooth muscle
cDNA library in ZAP (Clontech) to obtain the full-length clone.
The same partial rat clone was also used to screen a human aortic
gt11 cDNA library to obtain the human sequence. The sequences of
several partially overlapping clones were compiled to obtain the
full-length h-SmLIM sequence. Both strands of the entire r-SmLIM and
h-SmLIM cDNAs were sequenced at least once by the dideoxy chain
termination method or on an automated DNA Sequencer (Licor, Lincoln,
NE) according to the manufacturer's instructions.Cellular Localization of r-SmLIM
To construct the
expression plasmid Myc-SmLIM/pCR3, we added in frame a c-Myc peptide
tag (EQKLISEED) to the r-SmLIM open reading frame at the N terminus by
PCR techniques. This hybrid DNA fragment was then cloned into the
eukaryotic expression vector pCR3 (Invitrogen). COS-7 and 10T1/2 cells
were transiently transfected with the Myc-SmLIM/pCR3 plasmid by the
DEAE-dextran method (27) with minor modifications(28) .
The transfected cells were grown on chamber slides and fixed with 4%
paraformaldehyde in phosphate-buffered saline. Immunostaining was
performed 48 h after transfection with an anti-c-Myc monoclonal
antibody (9E10, Oncogene) followed by a rhodamine-conjugated goat
anti-mouse IgG secondary antibody. Nuclear counterstaining was
performed with Hoechst 33258 as recommended by the manufacturer.Chromosomal Localization of h-SmLIM
We localized
h-SmLIM with the BIOSMAP somatic cell hybrid blot (BIOS Labs, New
Haven, CT), which contains DNA from 20 somatic cell hybrid cell lines
plus three control DNAs (human, hamster, and mouse). A full-length
h-SmLIM fragment was randomly primed (27) and hybridized as
recommended by the manufacturer. This blot was washed according to the
manufacturer's recommendations and then exposed to Kodak XAR film
at -80 °C.RNA Extraction and RNA Blot Analysis
We isolated
total RNA from cultured cells, rat organs, embryonic stem cells, and
mouse embryos by guanidinium isothiocyanate extraction and
centrifugation through cesium chloride(29) . The mouse embryo
samples (days 7-10) included placenta and yolk sac. Carotid
artery total RNA was obtained by the RNA-Zol method (Cinna/Biotecx
Laboratories International, Houston, TX) from adult male Sprague-Dawley
rats that had been subjected to balloon injury (Zivic-Miller Co.,
Zelienople, PA). Human poly(A)
RNA was purchased from
Clontech Laboratories (Palo Alto, CA). All RNA was fractionated on a
1.3% formaldehyde-agarose gel and transferred to nitrocellulose
filters. The filters were then hybridized with the appropriate 
P-labeled, random primed cDNA probes as described
elsewhere(27, 30, 31) . The hybridized
filters were washed in 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS at 55 °C, and autoradiographed on
Kodak XAR film at -80 °C. To correct for differences in RNA
loading, the blots were hybridized with an 18 or 28 S oligonucleotide
probe. The filters were scanned, and radioactivity was measured on a
PhosphorImager running the ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).In Vitro Transcription and Translation
The
complete r-SmLIM open reading frame was cloned into the eukaryotic
expression vector pCR3 (Invitrogen). In vitro transcription
and translation was performed in the TNT-coupled wheat germ extract
system (Promega, Madison, WI) according to the manufacturer's
instructions. The transcribed and translated products were resolved on
a 10% SDS-PAGE Tricine gel(32) , and autoradiography was
performed with Kodak BMR film at room temperature.In Situ Hybridization
r-SmLIM mRNA was hybridized in situ as described elsewhere (33) with minor
modifications. Adult male Sprague-Dawley rats were perfused with 4%
paraformaldehyde. Organs were then postfixed with 4% paraformaldehyde,
soaked in 30% sucrose until the tissue had sunk, embedded in optimum
cutting temperature compound, and stored in isopentane at -80
°C. Tissue sections were cut at a thickness of 5 µm. SmLIM mRNA
was detected by hybridization with a 
S-UTP-labeled
antisense cRNA probe synthesized with the SP6 RNA polymerase from HindIII-linearized r-SmLIM in Bluescript II SK+. For
control experiments, a S-UTP-labeled sense cRNA probe was
synthesized under the same conditions. RNA probes were degraded to a
length of approximately 100-200 nucleotides by partial hydrolysis
for 15 min at 60 °C in 80 mM NaHCO
and 120
mM NaCO. After hybridization the
tissue sections were washed under moderately stringent conditions as
previously described(33) . The dried tissue sections were then
dipped into Kodak NTB2 emulsion (Eastman Kodak Co.) and exposed for
2-4 days at 4 °C. Counterstaining was performed with
hematoxylin-eosin.
Isolation and Characterization of r-SmLIM and h-SmLIM
cDNA
The nucleotide sequence of the r-SmLIM cDNA revealed a
582-base pair open reading frame encoding a 194-amino acid protein.
Analysis of this frame identified two LIM domains separated by a
glycine-rich region and a putative nuclear localization signal (Fig. 1A). The nucleotide sequence flanking the
putative initiation methionine complied with the Kozak consensus
sequence for initiation of translation(34) , and the r-SmLIM
open reading frame predicted a 21-kDa protein. We then cloned the
entire r-SmLIM cDNA into the PCRIII eukaryotic expression vector. In vitro transcription and translation (Promega) of this
expression plasmid with wheat germ lysate revealed a protein product of
21 kDa (Fig. 1B).
)homologues. A
comparison of the h-SmLIM and r-SmLIM open reading frames revealed 93%
identity at the cDNA level and 99% identity at the amino acid level (Fig. 2, A and B). Comparison of the open
reading frames of m-SmLIM and r-SmLIM revealed 97% identity at the cDNA
level and 100% identity at the amino acid level (Fig. 2B). A GenBank
search indicated that
SmLIM shares homology with the cysteine-rich protein (CRP)
family(13, 15, 35, 36, 37) . Fig. 2A compares r-SmLIM and h-SmLIM with their rat and
human CRP counterparts and rat MLP. Although an amino acid sequence
comparison of r-SmLIM versus h-SmLIM shows 99% identity (Fig. 2B), a comparison of r-SmLIM with r-CRP shows 79%
identity. These data indicate that SmLIM and CRP are related but
different genes.
Cellular and Chromosomal Localization of SmLIM
The
r-SmLIM deduced amino acid sequence contains the putative nuclear
localization signal KKYGPK, suggesting that SmLIM may be a nuclear
protein. To determine the cellular localization of SmLIM, we first
generated a plasmid that would express a fusion protein of the c-Myc
tag and r-SmLIM. This plasmid and the control vector alone were
transfected into COS cells and immunostained with an anti-c-Myc
antibody. Detection of the immunofluorescent signal in the nuclei of
COS cells transfected with the c-Myc-r-SmLIM fusion plasmid but not the
control vector alone localized the SmLIM protein to the nucleus (Fig. 3). We performed the same experiment in 10T1/2 fibroblasts
and found that SmLIM localized to the nucleus in these cells as well
(data not shown). We also mapped the chromosomal location of h-SmLIM
with the BIOS somatic cell hybrid blot. h-SmLIM localized to chromosome
3 (Fig. 4, arrow).
600.
Tissue Distribution of r-SmLIM and h-SmLIM (Northern
Analysis)
Total RNA were isolated from 15 tissues of adult male
and female rats and analyzed for SmLIM expression by Northern blot
analysis (Fig. 5A). A single, intense, 1.0-kb band was
detected in aorta. A much weaker signal was detected in kidney, thymus,
and intestine. SmLIM expression was not detectable in heart and
skeletal muscle and was barely detectable in brain, testis, esophagus,
lung, liver, aortic adventitia, vena cava, and uterus. Thus, r-SmLIM
appears to be expressed in tissue containing smooth rather than
striated muscle. Furthermore, because expression of SmLIM was much
greater in aorta than in intestine or uterus it would appear to be
expressed preferentially in VSMCs. Even among vascular RNAs, r-SmLIM
expression was more robust in arterial tissue (aorta) than in venous
tissue (vena cava). Consistent with the r-SmLIM expression pattern,
h-SmLIM was expressed to a high degree in aorta but not in heart or
skeletal muscle (Fig. 5B).
P-labeled r-SmLIM probe. A single
r-SmLIM transcript is visible at 1.0 kb. Filters were hybridized with
18 S to verify equivalent loading. B, h-SmLIM mRNA expression.
Northern analysis was performed with 2 µg of poly(A) RNA (Clontech). A 2.1-kb transcript is
shown.
Tissue Distribution of r-SmLIM (in Situ
Hybridization)
For each antisense experiment with the r-SmLIM
riboprobe (Fig. 6, left) a corresponding sense
(control) experiment (Fig. 6, right) was performed to
localize r-SmLIM expression within the vessel wall. Fig. 6, top left, shows intense staining of r-SmLIM in both the aorta (Ao) and a small artery (Ar) nearby. Consistent with
our Northern analysis, minimal expression of r-SmLIM was visible in the
vena cava (V). A view of the aorta at higher magnification
reveals that r-SmLIM expression was limited to smooth muscle cells in
the medial layer (Fig. 6, bottom left). SmLIM signal
expression was absent in skeletal muscle cells (data not shown). These
observations agree with our Northern analyses and indicate that r-SmLIM
was expressed preferentially in arterial smooth muscle cells.
S-UTP-labeled antisense (left) and sense (right) cRNA probes. Top panels, aorta (Ao),
small artery (Ar), and vein (V) at low magnification
( 200). Bottom panels, aorta (Ao) at high
magnification ( 600).
Down-regulation of r-SmLIM Expression in VSMCs by Growth
Factors and Arterial Wall Injury
PDGF-BB is unique among the
smooth muscle cell mitogens in its ability to selectively suppress in vitro the expression of differentiation markers such as
-actin, smooth muscle myosin heavy chain, and
-tropomyosin(6) . Therefore we evaluated the effect of
PDGF-BB on SmLIM expression in cultured VSMCs. r-SmLIM mRNA levels
decreased gradually in response to PDGF-BB stimulation (Fig. 7A). A decrease in r-SmLIM expression appeared as
early as 4 h after treatment, and a maximal decrease of 80% was
obtained at 32 h after treatment.
P-labeled r-SmLIM probe. A
single r-SmLIM transcript is visible at 1.0 kb. Filters were hybridized
with 18 S to verify equivalent loading. B, decrease in r-SmLIM
mRNA expression after balloon injury in rat carotid arteries. Northern
analysis was performed with 20 µg of total RNA/lane at 2, 5, and 8
days after injury. A single r-SmLIM transcript is visible at 1.0 kb.
Filters were hybridized with 18 S to verify equivalent
loading.
Developmental Regulation of r-SmLIM mRNA
Expression
The data described so far suggest that SmLIM is
expressed preferentially in vascular tissue and that its levels are
affected by the differentiation state of VSMCs. To determine whether
SmLIM expression is regulated during development, we isolated total RNA
from undifferentiated embryonic stem cells and whole mouse embryos at
days 7.5-16.5 post coitum (p.c.). We found that SmLIM expression
was indeed regulated developmentally (Fig. 8). Expression was
highest during the late primitive streak stage (day 7.5 p.c.), the
point at which the embryonic and extraembryonic circulations begin to
develop(39, 40) . SmLIM expression decreased rapidly
at subsequent time points. By normalizing the data to the hybridization
signal value at 7.5 days p.c., we found that relative mRNA expression
decreased by 40% at 8.5 days p.c. and by approximately 80% at
9.5-16.5 days p.c.
P-labeled r-SmLIM probe. A single
r-SmLIM transcript is visible at 1.0 kb. Filters were hybridized with
28 S to verify equivalent loading.
and gax(44, 45) , expressed
highly in VSMC. However, some differences exist in their patterns of
expression in tissue. For example, in addition to aorta, SM22 is
highly expressed in uterus and intestine(45) , whereas SmLIM is
not. Gax expression is not detected in intestine but is detected to a
high degree in heart(44) . By comparison, SmLIM expression
appears to be more restricted to aorta. Furthermore, SmLIM is expressed
preferentially in arterial as opposed to venous tissue. Arteries and
veins have been shown to respond differently to injury (46) and
various pharmacological manipulations (47, 48, 49) ; these observations suggest
that smooth muscle cells may be fundamentally different in the two
tissue types. To our knowledge the pattern of preferential expression
in arterial but not venous smooth muscle cells is unique to SmLIM.
))
We thank B. Ith for technical assistance and T.
McVarish for editorial assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 I. Najwer and B. Lilly Ca2+/calmodulin-dependent protein kinase IV activates cysteine-rich protein 1 through adjacent CRE and CArG elements Am J Physiol Cell Physiol, October 1, 2005; 289(4): C785 - C793. [Abstract] [Full Text] [PDF]
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 K. Kawai-Kowase, M. S. Kumar, M. H. Hoofnagle, T. Yoshida, and G. K. Owens PIAS1 Activates the Expression of Smooth Muscle Cell Differentiation Marker Genes by Interacting with Serum Response Factor and Class I Basic Helix-Loop-Helix Proteins Mol. Cell. Biol., September 15, 2005; 25(18): 8009 - 8023. [Abstract] [Full Text] [PDF]
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 J.-r. Kim-Kaneyama, W. Suzuki, K. Ichikawa, T. Ohki, Y. Kohno, M. Sata, K. Nose, and M. Shibanuma Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo J. Cell Sci., March 1, 2005; 118(5): 937 - 949. [Abstract] [Full Text] [PDF]
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 M. I. Dorrell, E. Aguilar, C. Weber, and M. Friedlander Global Gene Expression Analysis of the Developing Postnatal Mouse Retina Invest. Ophthalmol. Vis. Sci., March 1, 2004; 45(3): 1009 - 1019. [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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 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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 R. C. Chambers, P. Leoni, N. Kaminski, G. J. Laurent, and R. A. Heller Global Expression Profiling of Fibroblast Responses to Transforming Growth Factor-{beta}1 Reveals the Induction of Inhibitor of Differentiation-1 and Provides Evidence of Smooth Muscle Cell Phenotypic Switching Am. J. Pathol., February 1, 2003; 162(2): 533 - 546. [Abstract] [Full Text] [PDF]
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 J. R. Bermingham Jr, S. Shumas, T. Whisenhunt, E. E. Sirkowski, S. O'Connell, S. S. Scherer, and M. G. Rosenfeld Identification of Genes That Are Downregulated in the Absence of the POU Domain Transcription Factor pou3f1 (Oct-6, Tst-1, SCIP) in Sciatic Nerve J. Neurosci., December 1, 2002; 22(23): 10217 - 10231. [Abstract] [Full Text] [PDF]
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 J. R. Henderson, D. Brown, J. A. Richardson, E. N. Olson, and M. C. Beckerle Expression of the Gene Encoding the LIM Protein CRP2: A Developmental Profile J. Histochem. Cytochem., January 1, 2002; 50(1): 107 - 112. [Abstract] [Full Text] [PDF]
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J. Kirchner, K. A. Forbush, and M. J. Bevan Identification and Characterization of Thymus LIM Protein: Targeted Disruption Reduces Thymus Cellularity Mol. Cell. Biol., December 15, 2001; 21(24): 8592 - 8604. [Abstract] [Full Text] [PDF]
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A. Huber, W. L. Neuhuber, N. Klugbauer, P. Ruth, and H.-D. Allescher Cysteine-rich Protein 2, a Novel Substrate for cGMP Kinase I in Enteric Neurons and Intestinal Smooth Muscle J. Biol. Chem., February 25, 2000; 275(8): 5504 - 5511. [Abstract] [Full Text] [PDF]
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M. T. Chin, K. Maemura, S. Fukumoto, M. K. Jain, M. D. Layne, M. Watanabe, C.-M. Hsieh, and M.-E. Lee Cardiovascular Basic Helix Loop Helix Factor 1, a Novel Transcriptional Repressor Expressed Preferentially in the Developing and Adult Cardiovascular System J. Biol. Chem., February 25, 2000; 275(9): 6381 - 6387. [Abstract] [Full Text] [PDF]
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S. Fang, R. V. Sharma, and R. C. Bhalla Enhanced Recovery of Injury-Caused Downregulation of Paxillin Protein by eNOS Gene Expression in Rat Carotid Artery : Mechanism of NO Inhibition of Intimal Hyperplasia? Arterioscler. Thromb. Vasc. Biol., January 1, 1999; 19(1): 147 - 152. [Abstract] [Full Text] [PDF]
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M. K. Jain, S. Kashiki, C.-M. Hsieh, M. D. Layne, S.-F. Yet, N. E. S. Sibinga, M. T. Chin, M. W. Feinberg, I. Woo, R. L. Maas, et al. Embryonic Expression Suggests an Important Role for CRP2/SmLIM in the Developing Cardiovascular System Circ. Res., November 16, 1998; 83(10): 980 - 985. [Abstract] [Full Text] [PDF]
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T. Suzuki, R. Nagai, and Y. Yazaki Mechanisms of Transcriptional Regulation of Gene Expression in Smooth Muscle Cells Circ. Res., June 29, 1998; 82(12): 1238 - 1242. [Full Text] [PDF]
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S.-F. Yet, S. C. Folta, M. K. Jain, C.-M. Hsieh, K. Maemura, M. D. Layne, D. Zhang, P. B. Marria, M. Yoshizumi, M. T. Chin, et al. Molecular Cloning, Characterization, and Promoter Analysis of the Mouse Crp2/SmLim Gene. PREFERENTIAL EXPRESSION OF ITS PROMOTER IN THE VASCULAR SMOOTH MUSCLE CELLS OF TRANSGENIC MICE J. Biol. Chem., April 24, 1998; 273(17): 10530 - 10537. [Abstract] [Full Text] [PDF]
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H. A. Louis, J. D. Pino, K. L. Schmeichel, P. Pomies, and M. C. Beckerle Comparison of Three Members of the Cysteine-rich Protein Family Reveals Functional Conservation and Divergent Patterns of Gene Expression J. Biol. Chem., October 24, 1997; 272(43): 27484 - 27491. [Abstract] [Full Text] [PDF]
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C.-J. Kho, G. S. Huggins, W. O. Endege, C. Patterson, M. K. Jain, M.-E. Lee, and E. Haber The Polymyositis-Scleroderma Autoantigen Interacts with the Helix-Loop-Helix Proteins E12 and E47 J. Biol. Chem., May 16, 1997; 272(20): 13426 - 13431. [Abstract] [Full Text] [PDF]
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R. Konrat, R. Weiskirchen, B. Krautler, and K. Bister Solution Structure of the Carboxyl-terminal LIM Domain from Quail Cysteine-rich Protein CRP2 J. Biol. Chem., May 2, 1997; 272(18): 12001 - 12007. [Abstract] [Full Text] [PDF]
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J. Sun, C. N. Kamei, M. D. Layne, M. K. Jain, J. K. Liao, M.-E. Lee, and M. T. Chin Regulation of Myogenic Terminal Differentiation by the Hairy-related Transcription Factor CHF2 J. Biol. Chem., May 18, 2001; 276(21): 18591 - 18596. [Abstract] [Full Text] [PDF]
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