(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) ()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) .
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-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) .
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) ()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.
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,
600.
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 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.
Figure 6:
In situ analysis of r-SmLIM
expression in rat vascular tissue. r-SmLIM mRNA was assayed with 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).
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 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.
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 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.
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 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.
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].