(Received for publication, September 6, 1995)
From the
Calponin-h1 is a 34-kDa myofibrillar thin filament,
actin-binding protein that is expressed exclusively in smooth muscle
cells (SMCs) in adult animals. To examine the molecular mechanisms that
regulate SMC-specific gene expression, we have examined the temporal,
spatial, and cell cycle-regulated patterns of expression of calponin-h1
gene expression and isolated and structurally characterized the murine
calponin-h1 gene. Calponin-h1 mRNA is expressed exclusively in
SMC-containing tissues in adult animals. During murine embryonic
development, calponin-h1 gene expression is (i) detectable in E9.5
embryos in the dorsal aorta, cardiac outflow tract, and tubular heart,
(ii) sequentially up-regulated in SMC-containing tissues, and (iii)
down-regulated to non-detectable levels in the heart during late fetal
development. In addition, the gene is expressed in resting rat aortic
SMCs, but its expression is rapidly down-regulated when growth-arrested
cells re-enter phase G of the cell cycle and proliferate.
Calponin-h1 is encoded by a 10.7-kilobase single copy gene composed of
seven exons, which is part of a multigene family. Transient
transfection analyses demonstrated that 1.5 kilobases of calponin-h1
5`-flanking sequence is sufficient to program high level transcription
of a luciferase reporter gene in cultured primary rat aortic SMCs and
the smooth muscle cell line, A7r5. Taken together, these data suggest
that the calponin-h1 gene will serve as an excellent model system with
which to examine the molecular mechanisms that regulate SMC lineage
specification, differentiation, and phenotypic modulation.
The vascular smooth muscle cell (SMC) ()is
responsible for maintaining both arterial tone via
contraction-relaxation and vessel integrity by proliferation and
synthesis of extracellular matrix(1, 2) . During
postnatal development, SMCs located in the arterial tunica media are
maintained in the resting, or G
/G
, stage of the
cell cycle and express high levels of contractile protein
isoforms(3) . However, in response to vascular injury and the
concomitant release of growth factors, vascular SMCs re-enter the cell
cycle, proliferate, and modulate their phenotype to subserve a
primarily synthetic function (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
This phenotypic modulation has been implicated in the pathogenesis of a
number of disease states, including atherosclerosis and restenosis
following percutaneous transluminal coronary angioplasty (3, 14, 15, 16, 17) .
Despite the central role of SMCs in vascular biology, relatively little
is currently understood about the molecular mechanisms that control SMC
lineage specification, differentiation, and phenotypic modulation. This
is due, in part, to the fact that relatively few SMC lineage-specific
markers have been identified and to the poorly understood embryological
origin(s) of the SMC lineage(s) (18, 19, 20, 21, 22, 23, 24, 25, 26, 27) .
Previous studies have suggested that the myofibrillar thin filament
protein calponin is expressed exclusively in
SMCs(11, 28, 29, 30, 31) .
Two species of calponin mRNA, designated h1 and h2, have been
identified, each of which is encoded by a separate gene(32) .
Interestingly, only the h1 protein product is detectable in
SMC-containing tissues(32) . In addition, a non-muscle acidic
isoform of calponin with distinct functional properties has recently
been described(33) . Several findings suggest that calponin may
regulate SMC contraction: (i) calponin binds to the thin filament
proteins actin, tropomyosin, calmodulin, and
caltropin(34, 35, 36, 37, 38) ,
(ii) calponin can reversibly inhibit the actin-activated myosin
MgATPase(39, 40) , (iii) calponin can inhibit the
Ca-dependent mobility of actin on immobilized
myosin(41) , (iv) calponin induces conformational changes in
F-actin(42) , and (v) calponin decreases the rate of
cross-bridge cycling and increases maximum force production by smooth
muscle myosin (43) . Despite these in vitro findings,
the molecular mechanism by which calponin regulates smooth muscle cell
contraction in vivo remains unknown.
In the studies described in this report, we have used the murine calponin-h1 cDNA as a molecular probe to define the spatial and temporal pattern of calponin-h1 gene expression during development. In addition, we have isolated and structurally characterized a murine calponin-h1 genomic clone and performed a series of transient transfection analyses using calponin/luciferase reporter plasmids. These studies demonstrated that the calponin-h1 gene is expressed in a developmentally regulated, SMC-specific pattern. The 10.7-kb calponin-h1 gene is a single copy gene that is a member of a multigene family composed of proteins with an evolutionarily conserved actin-binding domain. Finally, transient transfection analyses demonstrate that the murine calponin-h1 promoter programs high level transcription of a luciferase reporter gene in primary cultures of rat aortic SMCs and the SMC line, A7r5. Taken together, these data suggest that the murine calponin-h1 gene should serve as an excellent model system in which to examine the transcriptional mechanisms that control SMC lineage specification and differentiation.
Figure 6: Structure of the murine calponin-h1 gene. A, a schematic representation and partial restriction endonuclease map of the murine calponin-h1 gene. HindIII (H), XbaI (X), EcoRI (R), and BamHI (B) restriction sites are shown. The transcriptional start site is indicated with an arrow. Exons are shown as shaded boxes. B, a schematic representation of the deduced full-length murine calponin cDNA. The size of the cDNA in base pairs is shown above the map. The 5`-untranslated region is shaded. The protein coding region is shown as an open box. The 3`-untranslated region is hatched. The locations of the three 29-amino acid direct repeats are shown in gray boxes. The size of the deduced calponin-h1 protein in amino acids (aa) is shown below the map. C, the nucleotide sequence of the calponin-h1 gene. The nucleotide sequence of the exons (uppercase letters) and introns (lowercase letters) as well as 1216 bp of 5`-flanking sequence are shown. The consensus splice donor and acceptor junctions are underlined. The polyadenylation signal is boxed. The deduced amino acid sequence of the murine calponin-h1 protein is shown below the nucleotide sequence. The transcriptional start site as deduced by primer extension, and RNase protection analyses (see Fig. 7) is indicated by a black arrowhead above the nucleotide sequence. Alternative transcriptional start sites as deduced by primer extension (black dots) and RNase protection (open arrowheads) are also shown.
Figure 7: Localization of the transcriptional start site of the murine calponin-h1 gene. A, primer extension analysis of calponin-h1 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 to +115 of the previously published murine calponin-h1 cDNA and SV129 murine uterine RNA were separated on a 8% acrylamide/urea gel, which was subjected to autoradiography. The upper band (dashed arrow) in the autoradiogram represents the 118-bp primer extension product, and the lower two bands (arrow) represent the 93- and 95-bp primer extension products. DNA size markers in base pairs are indicated to the left of the autoradiogram. B, RNase protection analysis of calponin-h1 mRNA. Murine uterine RNA subjected to RNase protection analysis using an antisense cRNA probe corresponding to bp -160 to +97 of the murine calponin-h1 gene was performed, and the reaction products were run down an 8% sequencing gel. The first lane (Probe only) contains the radiolabeled 257-bp probe. The second lane (-RNA) contains the products from an RNase digestion reaction performed with tRNA and the radiolabeled 257-bp probe. The third lane (+RNA) contains the digestion products of a reaction mixture containing murine uterine RNA and the radiolabeled antisense cRNA probe. The 97-bp product, corresponding to a transcriptional start site 101 bp 5` of the initiation codon, is shown (arrow). In addition, the 73-, 71-, and 69-bp products that represent alternative transcriptional start sites are shown (arrows).
Figure 1: The in vivo tissue distribution and cellular specificity of calponin-h1 gene expression. A, the top panel shows a Northern blot analysis of RNA samples isolated from adult murine tissues hybridized to the radiolabeled calponin-h1 cDNA probe. RNA size markers are shown in kilobases to the left of the blot. The calponin-h1 cDNA hybridized to a single 1.5-kb species of mRNA, which was present in SMC-containing tissues (arrow). The bottom panel shows the ethidium bromide-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, Sol8 myoblasts and myotubes (Sol8 Blasts and Tubes), Hep G2, and EL-4 cells hybridized to the radiolabeled calponin-h1 cDNA probe. The calponin-h1 cDNA probe hybridized to a 1.5-kb species of mRNA (arrow) present in primary VSMCs, A7r5 cells, and C2C12 myoblasts. The bottom panel shows an ethidium bromide-stained formaldehyde-containing gel prior to transfer of RNA.
To determine the cell specificity of calponin-h1 gene expression, the calponin-h1 cDNA probe was hybridized to Northern blots containing RNAs prepared from freshly isolated primary rat aortic SMCs, the smooth muscle cell line, A7r5, murine NIH 3T3 and C3H10T1/2 fibroblasts, the monkey SV-40 transformed kidney cell line, COS-7, murine Sol8 myoblasts and myotubes, the human hepatocellular carcinoma-derived cell line, HepG2, and the murine lymphoid cell line, EL-4. High levels of calponin-h1 mRNA were detected in primary rat aortic SMCs and the smooth muscle cell line, A7r5 (Fig. 1B, lanes 1 and 2), while the gene was not expressed in other cell lines including NIH 3T3, C3H10T1/2, COS-7, Sol8, Hep G2, and EL-4 cells even after prolonged autoradiographic exposure. Thus, calponin is expressed in a lineage-restricted fashion in primary vascular SMCs and the SMC line, A7r5.
Figure 2:
The
temporal and spatial pattern of calponin-h1 gene expression during
murine embryogenesis. In situ hybridization analyses were
performed using radiolabeled calponin-h1 antisense (panels A, C, and E) and sense (panels B, D,
and F) riboprobes on adjacent sections of staged murine
embryos. Following hybridization, each slide was subjected to emulsion
autoradiography, and the sections were visualized and photographed
through a Ziess Axiophot microscope under darkfield conditions. A and B, longitudinal sections through an E9.5 mouse embryo
hybridized to the calponin-h1 antisense (panel A) and sense (panel B) probes. Specific hybridization (white) is
seen in the embryonic heart tube, the cardiac outflow tract (open
arrow), and the dorsal aorta (white arrow) at this early
stage of development. Magnification, 25. C and D, longitudinal sections through an E14.0 mouse embryo
hybridized to the calponin-h1 antisense (panel C) and sense (panel D) probes. Specific hybridization (white
staining) is seen in the heart, aorta, and smaller branch arteries
(see also Fig. 4A), the foregut, midgut, and hindgut
(see also Fig. 4B), the developing lung bud (see also Fig. 4C), and the urogenital ridge (see also Fig. 4D). Magnification, 12.5
. E and F, longitudinal sections through an E18.5 mouse embryo
hybridized to the calponin-h1 antisense (panel E) and sense (panel F) probes. Specific hybridization is seen throughout
the arterial tree, in the esophagus, stomach, small and large
intestinal mucosa, the pulmonary bronchiole tree, and the bladder. Of
note, at this late fetal stage of development, hybridization in the
heart has decreased to background levels (compare panels E and F). Magnification, 12.5
.
Figure 4:
Down-regulation of calponin-h1 gene
expression in proliferating SMCs. A, the left panel shows the FACS cell cycle analysis of propidium iodide-stained
SMCs following 72 h of serum starvation. 85% of cells were in
G/G
phase, 6% of cells in S phase, and 9% of
cells in G
+ M phase as assessed using CellFIT
software program. The right panel shows the FACS analysis of
propidium iodide-stained SMCs 24 h post-serum stimulation. 47% of cells
were in G
/G
phase, 22% of cells were in S
phase, and 30% of cells were in G
+ M phase. B, 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 both the radiolabeled
calponin-h1 cDNA (Calponin) and the human Rb cDNA probes. The bottom panel shows the ethidium bromide-stained
formaldehyde-containing gel prior to membrane transfer of RNA. The
location of the 28 S and 18 S ribosomal RNA bands are indicated to the left of the gel. C, quantitative image analysis of
the Northern blot hybridization signals. The autoradiographic
hybridization signals (dpm) for calponin-h1 mRNA (filled
circles) and the retinoblastoma gene product mRNA (open
squares) were quantitated using a Molecular Dynamics
PhosphorImager, and each signal was normalized to the quantitated dpm
obtained at time t
and is reported as normalized
gene expression.
In E14.0 embryos, calponin mRNA is detectable in the branch arteries, foregut, midgut, hindgut, developing lung bud, and urogenital ridge (Fig. 2C). Once again, the sense probe did not hybridize to the E14.0 embryo (Fig. 2D). Higher power views reveal that calponin-h1 mRNA is expressed exclusively in the lamina propria surrounding the gut epithelium (white arrows, Fig. 3B), bronchial epithelium (white arrow, Fig. 3C), the urogenital ridge surrounding the urogenital sinus (white arrow, Fig. 3D), and the medial layer surrounding large arteries such as the dorsal aorta (Fig. 2C) as well as the smaller branch arteries (white arrow, Fig. 3A).
Figure 3:
High power views of the pattern of
calponin-h1 gene expression in E14.0 mouse embryos. In situ hybridization was performed using a radiolabeled calponin-h1
antisense riboprobe to mouse E14.0 embryos as described in the legend
to Fig. 3. In this figure, the sections were visualized and
photographed through a Zeiss Axiophot microscope under epifluorescence
and darkfield conditions. Specific hybridization is indicated by white staining. A, hybridization of the calponin-h1
probe to branch arteries in the head (white arrows).
Magnification, 200. B, hybridization of the calponin-h1
probe to the embryonic gut mucosa (white arrows).
Magnification, 50
. C, hybridization of the calponin-h1
probe to the bronchus in the lung bud (white arrow) and the
esophagus (open arrow). Magnification, 100
. D,
hybridization of the calponin-h1 probe to the urogenital ridge (open arrow) surrounding the urogenital sinus (white
arrow). Magnification, 50
.
In E18.5 mouse embryos, calponin-h1 mRNA gene expression can no longer be detected above background levels in the heart, while the gene continues to be expressed abundantly in the aorta as well as smaller branch arteries, the esophagus, stomach, upper and lower intestine, pulmonary bronchi and bronchioles, and the bladder (Fig. 2E). At this late fetal stage, low level nonspecific hybridization to the sense probe was detected in the heart, liver, and some skeletal muscles (Fig. 2F). Taken together, these analyses demonstrate that calponin-h1 gene expression is (i) initially detectable at least as early as embryonic day 9.5 in the dorsal aorta, the aortic outflow tract, and the developing heart tube, (ii) sequentially up-regulated in SMC-containing tissues including the smaller branch arteries, primitive gut, lung bud, and urinary tract where it continues to be expressed throughout adult development, and (iii) down-regulated to non-detectable levels during late fetal development in the four-chambered heart.
Figure 5: Southern blot analysis of the murine calponin-h1 gene. A, high stringency Southern blot analysis of the murine calponin-h1 gene. High molecular weight murine SV129 DNA was digested with restriction endonucleases BamHI, EcoRI, HindIII, PstI, and XbaI and hybridized to the radiolabeled calponin-h1 cDNA probe under high stringency conditions. B, low stringency Southern blot analysis of the murine calponin-h1 gene. The filter described above was rehybridized to the radiolabeled calponin-h1 cDNA probe under low stringency conditions. Size markers are shown in kilobases to the left of each blot.
The transcriptional start site was identified by RNase protection and primer extension analyses (Fig. 7). As shown in Fig. 7A, primer extension analyses utilizing an antisense synthetic oligonucleotide corresponding to bp +80 to +115 of the previously published sequence of the murine calponin-h1 cDNA (GenBank Accession No. Z19542) (32) resulted in two major extended products of 93 and 95 bp, respectively (arrows), each of which was thermostable up to a temperature of 56 °C. In addition, a less abundant (10% relative signal intensity) 118-bp primer extension product was also detected upon prolonged autoradiographic exposure (dashed arrow). Thus, primer extension analyses suggests that the 5` most transcriptional start site is located 101 bp 5` of the translational initiation codon (Fig. 6C, closed arrowhead) with alternative transcriptional start sites located 78 and 76 bp 5` of the initiation codon (Fig. 6C, black dots). To complement the primer extension analyses, RNase protection analyses were also performed using an antisense cRNA probe corresponding to bp -160 to +97 of the calponin-h1 genomic sequence as deduced by DNA sequence, Southern blot, and primer extension analyses (Fig. 7B). These analyses revealed several major protected fragments of 97 (37% relative signal intensity), 73, 71, and 69 bp, respectively (Fig. 7B, arrows). Thus, RNase protection analyses suggests that the 5` most transcriptional start site is located 101 bp 5` of the initiation codon (Fig. 6C, closed arrowhead) with alternative transcriptional start sites located 78, 76, and 74 bp (Fig. 6C, closed arrowheads) 5` of the initiation codon. Taken together, these analyses served to identify the 5` most transcriptional start site 101 bp 5` of the initiation codon, which heretofore will be designated as +1 (Fig. 6C). Additional alternative transcriptional start sites are located between bp +24 and +28.
As shown in Fig. 6B, the full-length calponin-h1 cDNA is composed
of a 101-bp 5`-untranslated region, an 892-bp open reading frame, and a
494-bp 3`-untranslated region. The predicted protein contains three
29-amino acid direct repeats (amino acids 164-192, 204-232,
and 243-271) (Fig. 6B, gray boxes). A
sequence homology search of the protein sequence data bases
demonstrated that this 29-amino acid motif is conserved across species
within each of the calponin
isoforms(29, 32, 33, 55) (Fig. 8). Interestingly, five direct repeats of
this amino acid motif are present in the recently identified unc-87
body wall muscle protein of Caenorhabditis elegans(55) (Fig. 8). In addition, single copies of this
motif are present in the chicken, rat, and murine SM22
proteins(13, 18, 46, 53, 56, 57) ,
the rat neuronal protein NP25(58) , and the Drosophila muscle protein mp20 (59) (Fig. 8). Comparison of
the amino acid sequence of this motif across species and family members
revealed a consensus sequence of
(I/V)GLQMGTNKXASQAGMTXYGX(R/P/K)R (Fig. 8, Consensus). Of note, each of the threonines in the
consensus sequence are potential sites for protein kinase C-mediated
phosphorylation(60) . Structural analysis of the calponin-h1
gene revealed that the nucleotide sequences encoding the first two
29-amino acid repeats are each interrupted by an intervening intron (Fig. 6C). Taken together, these data suggest that the
calponin multigene family consists of proteins that contain a conserved
amino acid motif that has been conserved over 600 million years of
evolution.
Figure 8:
Evolutionary conservation of the 29-amino
acid calponin repeat domain. A computer-based search of the protein
data bases revealed that the calponin 29-amino acid repeat domain is
conserved in other proteins including chicken calponin , chicken
calponin
, pig calponin-h1, rat acidic calponin, C. elegans unc-87, mouse SM22
, human SM22
, and Drosophila mp20. The location of this motif within each respective protein is
indicated in the column under aa. The actual amino acid
sequence of each motif is indicated. Amino acid identity or
conservation is indicated by gray shading. A consensus amino
acid motif derived from this analysis is shown below the
protein sequences.
1216 bp of 5`-sequence flanking the cap site was searched
for potential transcriptional regulatory elements using MacVector DNA
sequence analysis software (Eastman Kodak Co., IBI). The immediate
5`-flanking sequence did not contain a consensus TATA or CAAT box. A
search for previously described cis-acting sequences present
in the transcriptional regulatory regions of skeletal, cardiac, and
smooth muscle genes identified one consensus GATA binding site (WGATAR) (61) located at bp -273, two consensus CACCC boxes (62) located at bp -322 and -214, and seven
consensus E boxes (63) located at bp -244, -441,
-660, -916, -954, -961, -1150, and
-1214. However, unlike each of the three previously characterized
smooth muscle lineage-restricted or lineage-specific genes, including
the SM22(46) , SM-myosin heavy chain(64) , and
SM-
-actin(65, 66) , the immediate 5`-flanking
sequence of the calponin-h1 gene did not contain a consensus CArG/SRF
binding site(67, 68) . In addition, four consensus
binding sites for the ubiquitously expressed transcription factor AP2
were identified at bps -39, -214, -237, and
-322 and one consensus binding site for Sp1 was identified at bp
-983.
Figure 9:
Activity of the calponin-h1 promoter in
A7r5 and primary rat aortic SMCs. A, transient transfection
analyses of calponin-h1/luciferase reporter plasmids in the SMC line,
A7r5. 15 µg of calponin-h1/luciferase reporter plasmid and 5 µg
of the pMSVgal 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 calponin-h1/luciferase reporter plasmids in confluent
cultures of primary rat aortic SMCs. Transient transfection analyses
were performed utilizing a slight modification of the Lipofectin
transfection technique described above (see ``Materials and
Methods''). Data are expressed as normalized light units ±
S.E.
To test the activity of the calponin-h1 promoter in primary SMCs, confluent cultures of low passage rat aortic SMCs (which express abundant levels of calponin mRNA) were transiently transfected with the p-1500cal/luc reporter plasmid. Under these transfection conditions, the p-1500cal/luc plasmid increased luciferase activity approximately 40-fold above levels obtained following transfection with the promoterless pGL2-Basic plasmid (Fig. 9B). Once again, this level of transcriptional activity was approximately 50% of that obtained following transfection with the positive control plasmid, pGL2-Control (Fig. 9B). Of note, transfection of subconfluent cultures of proliferating primary rat aortic SMCs with the p-1500cal/luc plasmid resulted in significantly lower levels of transcriptional activity relative to the positive control plasmid, suggesting that the down-regulation of calponin-h1 gene expression in proliferating SMCs may be transcriptionally regulated (data not shown). Taken together, these data demonstrate that the immediate 5`-flanking region of the calponin-h1 gene contains a promoter that is active in both quiescent primary cultures of rat aortic SMCs and the smooth muscle cell line, A7r5.
The myofibrillar thin filament actin-binding protein, calponin, is believed to regulate SMC contraction and has been proposed as a candidate marker of the SMC lineage(11, 13, 28, 29, 30, 31) . In this report, we have examined the spatial and temporal pattern of calponin-h1 gene expression during murine development. In addition, we have defined the pattern of calponin-h1 gene expression in quiescent and proliferating cultured rat aortic SMCs. We have isolated and structurally characterized the murine calponin-h1 gene, which has provided novel insights into the evolution and function of the calponin multigene family. Finally, we have demonstrated that the immediate 5`-flanking region of the calponin-h1 gene directs high level transcription of the calponin-h1 gene in primary cultures of rat aortic SMCs. These data are relevant to several unresolved issues in SMC biology including (i) the developmental programs that control SMC lineage specification, (ii) the molecular mechanisms that regulate SMC phenotypic modulation, and (iii) the evolution and function of the calponin multigene family.
In contrast to the skeletal muscle
lineage (for review, see (69) ), relatively little is currently
understood about the molecular mechanisms that control SMC lineage
specification and differentiation. This is due in part to the poorly
defined embryological origin of SMCs (18, 19, 20, 21, 22, 23, 24, 25, 26) ,
the lack of definitive markers of the SMC lineage(27) , and the
phenotypic plasticity of this muscle cell
lineage(4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
In this report, we have determined the pattern of expression of the
murine calponin-h1 gene during murine embryogenesis and in adult mice
to investigate its utility as a marker of the SMC lineage. As
anticipated, the calponin-h1 gene is expressed solely in SMC-containing
tissues in adult animals demonstrating that the pattern of expression
of the protein is regulated at the level of gene expression. Calponin
mRNA was detected at least as early as embryonic day 9.5 in the dorsal
aorta as well as the aortic outflow tract. Thus, the calponin-h1 gene
represents one of the earliest developmental markers of the SMC
lineage. Second, we have shown that calponin-h1 gene expression is
rapidly down-regulated when primary rat aortic SMCs begin to pass
through the G/S checkpoint of the cell cycle and
proliferate. Taken together, these data suggest that the calponin-h1
gene will serve as an excellent model system to elucidate the molecular
mechanisms that regulate the SMC specification and differentiation.
Our results demonstrated that the calponin-h1 gene is not only
expressed in SMCs but is also transiently expressed in the early
embryonic heart. Of note, several other genes that are expressed in a
SMC-specific or lineage-restricted fashion during postnatal development
including SM22 (
)and SM-
-actin (70, 71) are also expressed in embryonic cardiac
myocytes. Similarly, expression of vascular smooth muscle
-actin
in avian cardiogenesis correlates with the onset of cardiomyocyte
differentiation(70) . Given the common embryological origin of
some SMCs and cardiac progenitors from the lateral plate mesoderm, it
is tempting to speculate that a common developmental program might
direct early cardiac and SMC lineage specification. In this regard, it
will be of interest to determine whether distinct or similar sets of cis-acting sequences and trans-acting factors control
transcription of the calponin-h1 gene in each of these two early muscle
cell lineages. In this regard, it is noteworthy that the calponin-h1
promoter contains a GATA and CACCC box/Sp1 binding sites. Similar sites
have been identified previously in several cardiac-specific
transcriptional regulatory
elements(72, 73, 74) .
Finally, structural
analyses of the calponin-h1 gene revealed some new insights into the
function and evolution of the calponin multigene family. The
calponin-h1 protein contains three conserved direct repeats of 29 amino
acids (amino acids 164-192, 204-232, and 243-271).
Previous investigators have demonstrated that amino acids 145-182
encode an actin-binding domain(39, 75) . In addition,
each of these repeats contains a conserved threonine residue, which is
phosphorylatable by protein kinase C in vitro(60) .
This motif is present in other putative actin-binding proteins
including the non-muscle, acidic isoform of calponin, the human,
murine, rat, and chicken SM22
proteins(13, 18, 46, 53, 56, 57) ,
the Drosophila muscle protein mp20(59) , and the
recently described C. elegans unc-87 body wall protein (55) (Fig. 8). Interestingly, mutation of the unc-87
protein, which contains five copies of this motif, results in variable
degrees of paralysis(55) . As such, these data are consistent
with the hypothesis that the calponin multigene family evolved from a
common ancestral protein with a conserved amino acid motif that encodes
an actin-binding domain, which is regulated by phosphorylation. Future
studies of this conserved domain may provide insights into the function
of calponin and related proteins in modulating the contractile
phenotype.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38930[GenBank], U38929[GenBank], U40348[GenBank], U40349[GenBank], U40350[GenBank], U40351[GenBank].