(Received for publication, October 25, 1995; and in revised form, January 2, 1996)
From the
Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5` sequence. In contrast to the strict cell type-specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression.
The discovery of cell-specific transcription factors that
trigger differentiation in skeletal and cardiac muscle has led to a
search for similar regulatory factors in smooth muscle cells (SMCs), ()whose differentiation program is impaired during the
course of intimal disease(1) . Although several transcription
factors have been documented in
SMCs(2, 3, 4, 5, 6, 7, 8) ,
none display the specificity commonly associated with factors that
control cell identity by activating batteries of cell-specific
genes(9) . Given the similarities between skeletal, cardiac,
and smooth muscle cells, it is reasonable to anticipate that these
different muscle cell types may share certain aspects of a myogenic
gene regulatory program.
In contrast to skeletal and cardiac muscle,
which are derived from distinct populations of mesodermal precursor
cells, SMCs arise throughout the embryo from diverse precursor cell
types. The mechanisms that specify the SMC phenotype and the embryonic
origins of the many different types of SMCs remain unclear. There have
been relatively few studies that have examined the temporospatial
patterns of expression of SMC-specific genes during embryogenesis.
However, the few SMC genes that have been examined have been found to
exhibit different expression patterns. Smooth muscle myosin heavy chain
(SMMHC), for example, is expressed only in the SMC lineage, appearing
initially in the dorsal aorta at 10.5 days postcoitum(10) . In
contrast, smooth muscle -actin (SM
-actin) is expressed in
the cardiac, skeletal, and smooth muscle cell lineages during
embryogenesis and in the adult (11, 12, 13, 14) . SM22
is also
expressed in cardiac, skeletal, and smooth muscle cells in the embryo
before becoming restricted to SMCs late in
embryogenesis(14, 15) . Dissection of the cis-acting
regulatory elements associated with these and other SMC genes will be
an important step toward understanding the similarities and differences
in the myogenic regulatory programs in the three major muscle cell
types.
One approach to identify SMC-specific regulatory factors is
to analyze promoters of genes that are unique to SMC lineages. The best
studied SMC promoter is that of SM
-actin(16, 17) . Defining SMC-specific
transcription factors that activate the SM
-actin promoter,
however, is complicated by its expression in multiple cell types during
embryogenesis and in the adult(11, 12, 13) .
In addition to SM
-actin, other SMC gene promoters have been
cloned and partially characterized including elastin(18) ,
SMMHC(19) , and SM22
(15, 20) . As yet, no cis elements have been shown to confer SMC-specific expression
of these promoters.
Calponin is a thin filament-associated protein that apparently functions as a negative regulatory element for SMC contraction(21) , but may also have more broad cellular activities independent of contractility(22) . Three distinct mammalian calponin genes have been described based on their expression and nucleotide sequence differences (23, 24, 25) . Whereas much effort has been directed toward understanding the function of different calponin proteins, relatively little is known about their specificity of mRNA expression. Based on studies conducted with antisera and cDNA probes, calponin was shown to predominate in SMCs(23, 26, 27) , but was also present in other cell types(28, 29) . These studies, however, could not adequately distinguish between the three calponin genes. A similar problem was recently approached with respect to SMMHC mRNA expression using stringent assays for the unambiguous assignment of this marker to only SMC lineages(10) .
In this study, we examined the temporospatial expression pattern of the basic or h1 calponin (hereafter referred as SMC calponin) during mouse embryogenesis and in adult mouse tissues. Our results demonstrate that SMC calponin is strictly specific for adult SMCs, but that during embryogenesis it is expressed throughout the early cardiac tube. While the SMC calponin gene is expressed exclusively in SMC lineages and embryonic heart, its promoter, which lacks core sequence elements typical of other muscle genes, displays activity in cell lines that do not express the endogenous transcript. These results reveal similarities between the cardiac and smooth muscle gene regulatory programs during early embryogenesis and suggest that complex mechanisms govern the cell type-specific expression of SMC calponin.
Figure 4: Partial map of two SMC calponin genomic clones and structure of the SMC calponin gene. From 16 plaque pure clones, two (CALP-8 and CALP-5) were extensively analyzed by PCR and restriction digestion. The SMC calponin gene, shown schematically within the CALP-5 clone, is about 9.5 kilobase pairs in length and is comprised of seven exons. The arrow in exon I denotes the initiation of translation. Abbreviations are: H, HindIII; S, SacI; and X, XbaI.
The CALP-5 clone was cut with SacI and all fragments subcloned into Bluescript SK+ (Stratagene) for further restriction mapping and sequencing. These analyses coupled with PCR indicated that the CALP-5 clone harbored all of the SMC calponin coding sequence. Therefore, this clone was sequenced in its entirety on both strands with an ABI 373A automated DNA sequencer (Foster City, CA). The sequence has been deposited in GenBank (accession number U28932).
RNase protection analysis and 5` RACE were used in conjunction with primer extension for mapping the start sites. For RNase protection, total RNA was hybridized to one of two independent riboprobes (see Fig. 5A) and the protected fragments resolved in a 5% polyacrylamide, 7 M urea gel. Sequencing reactions of a calponin cDNA were carried out using primers to the 3` most end of each riboprobe. Total RNA from uterus or liver was also subjected to 5` RACE according to the manufacturer's instructions (BRL). A final nested PCR was performed using the 5` anchor primer provided in the kit (BRL) and a 3` SMC calponin-specific primer, 5`-cagacaagccgtaggcaggacc-3`. A total of 10 RACE products were sequenced, two of which are presented in Fig. 5D.
Figure 5:
Transcription initiation site mapping of
the SMC calponin gene. Schematic (A) of SMC calponin 5`
sequence including the initiating ATG of exon I, intron I (denoted by
the broken line), and a portion of exon II. The arrows on the gene indicate the position of the primers used for primer
extension (B; PXT) or 5` RACE (D). The
probes used for RNase protection (C) are shown below the gene.
P1 is a 258-nt riboprobe that protects a 142- and 139-nt fragment of
the SMC calponin gene (C). Note that the 3` end of P1 was used
as a primer for PXT which yielded two bands (B) of the same
size as that with the P1 riboprobe. The P2 probe, which represents the
original PCR clone used for tissue/cell line RNase protections and in situ hybridizations, is a 195-nt riboprobe that protects
fragments of 195 and 192 nt (C). The 5` RACE products, whose
sequences are shown in D, were obtained by nested PCR using a
primer (arrow in schematic) described under ``Materials
and Methods.'' The poly(G) tail represents the 5` end of the two
SMC calponin cDNAs. E, sequence around the two transcription
initiation sites, designated S and S
. Note the
absence of a consensus TATAA element 20-30 nt upstream of
S
. The initiating ATG for translation is boxed .
Transfections were
typically carried out for 12-16 h followed by 48 h of recovery
and growth. Cells were harvested in cold phosphate-buffered saline,
spun down, and resuspended in 200 µl of 0.25 M Tris-HCl,
pH 7.8. Cell lysates were then briefly sonicated, spun down and stored
at -80 °C before use. Neither mild sonication nor multiple
freeze thawing influenced luciferase activity. Total
protein was measured by the Bradford assay (Bio-Rad). Luciferase
activity was assayed according to the manufacturer's
specifications (Promega). A Turner Model 20 luminometer was used to
measure the light reactivity of firefly luciferase. The relative light
units were then normalized to total protein and expressed as a percent
of the normalized luciferase activity obtained with the pGL3 control
vector, which contains the SV40 promoter/enhancer (Promega). The data
reflect the means (±S.E.) of at least four independent
experiments done in duplicate.
Figure 1:
Specificity of SMC
calponin mRNA and protein. A, 15 µg of total RNA from each
adult mouse tissue was hybridized to both a SMC calponin and an 18 S
rRNA riboprobe as described under ``Materials and Methods,''
digested with RNase A/T, resolved through a denaturing 5%
polyacrylamide gel, and dried for autoradiography. The size of each
protected fragment is 195 and 80 nt for SMC calponin and 18 S rRNA,
respectively. Exposure time was for 24 h. B, 15 µg of
total RNA from the indicated mouse cell lines and stomach were
processed for RNase protection as described in A. A third
riboprobe corresponding to the 3` 157 nt of SM -actin was also
used. Note the more widespread expression of SM
-actin as compared
to SMC calponin. Exposure time was for 24 h. U,
undifferentiated and D, differentiated cells as described in (10) . C, a rat SMC calponin riboprobe was hybridized
to 15 µg of total RNA from the indicated rat cell lines and tissues
and processed for RNase protection as described in A. Note
that SMC calponin transcripts were present both in low passaged (P4) and high passaged (P55) RASMC and were not
modulated if cells were made quiescent by serum deprivation for 3 days (Q) or serum stimulated (10%) for 24 h following quiescence.
Exposure time was for 8 h. The PC12 cell line was induced to
differentiate by treating subconfluent cells with 10 ng/ml nerve growth
factor. D, protein extracts (
50 µg) from the
indicated cell lines were processed for Western blotting and incubated
with a monoclonal antibody to human SMC calponin as described under
``Materials and Methods.'' Cells were either serum-deprived
for 3 days (Q) or serum-deprived and then stimulated with 10%
fetal bovine serum for 24 h. The growth state of each SMC line did not
have any discernable effect on SMC calponin protein levels. The absence
of any immunoreactive signal in 10T1/2 cells verifies the specificity
of the antisera for only SMC calponin.
No SMC calponin transcripts were detected in the rat L6 skeletal myoblast line, PC12 cells, or the HepG2 liver cell line (Fig. 1C). On the other hand, a prominent signal was observed in the A7r5 fetal rat aortic SMC line as well as primary RASMC (Fig. 1C). These latter cells also expressed SMC calponin protein as determined by Western blotting (Fig. 1D). Importantly, SMC calponin mRNA was expressed at high levels in rat SMC irrespective of passage number or growth state (Fig. 1C). Together, these results show SMC calponin to be a highly restricted marker for SMC lineages. They also demonstrate the utility of both the A7r5 fetal rat aortic SMC line and multiply passaged primary RASMC for analyzing SMC calponin promoter activity (see below).
Figure 2: Localization of SMC calponin transcripts in adult mouse tissues. Sections of adult mouse heart (A), skeletal muscle (B), small intestine (C), and uterus (D) were processed for in situ hybridization as described under ``Materials and Methods'' and photographed under darkfield microscopy. SMC calponin was detected in the SMC-containing tissues of small intestine (C) and uterus (D), but was only detected in blood vessels (arrows) of the heart (A) and skeletal muscle (B). Abbreviations are: en, endometrium; ve, villous epithelium.
To
ascertain the spatiotemporal pattern of SMC calponin mRNA expression
during embryogenesis, staged mouse embryos were processed for in
situ hybridization. Surprisingly, SMC calponin mRNA first appeared
at low levels in the heart at 8.5 days postcoitum. As shown
in Fig. 3B, 9.5-day postcoitum embryos displayed a
strong hybridization signal throughout all chambers of the heart. This
embryonic cardiac expression of SMC calponin persisted up to 13.5 days
postcoitum at which time the mRNA could be detected in several
SMC-containing tissues including lung, gut, and blood vessels (Fig. 3D). This expression pattern differs from that of
SMMHC mRNA which, as shown previously (10) , is only expressed
in SMC lineages during development (Fig. 3C). The
expression of SMC calponin mRNA in the heart subsided by 15.5 days
postcoitum.
At no time during development did we observe
SMC calponin mRNA in skeletal muscle or its precursors. Sense probes
showed no specific hybridization signal in the embryonic heart or
elsewhere.
Together our findings show clearly that with the
exception of early heart expression, SMC calponin is restricted to SMC
lineages.
Figure 3: mRNA expression of SMC markers in staged mouse embryos. Adjacent saggital sections of 9.5 days postcoitum (A and B) or 13.5 days postcoitum (C and D) mouse embryos were hybridized to a mouse riboprobe corresponding either to SMMHC (A and C) or SMC calponin (B and D). Note the intense SMC calponin hybridization signal in the heart of both 9.5- and 13.5-day postcoitum embryos. Consistent with a previous report(10) , no SMMHC signal was ever observed in tissues without a SMC component. Abbreviations are: br, brain; fb, forebrain; gu, gut; he, heart; and li, liver.
Interestingly, the initial cloning
of SMC calponin in chickens revealed two transcripts (designated
and
) that apparently arose due to alternative
splicing(23) . The putative splice site in the chicken SMC
calponin cDNA maps precisely to the boundary of the sixth intron and
seventh exon of the mouse SMC calponin gene. RT-PCR analysis of several
mouse tissues with primers flanking this region, however, failed to
reveal any splice variants of the mouse SMC calponin gene.
Thus, the splicing phenomenon of SMC calponin described by
Takahashi and Nadal-Ginard (23) appears to be restricted to
chickens.
Most SMC structural or cytosolic genes
contain a TATA box in their 5`
promoter(16, 17, 19, 20, 39) .
SMC calponin, however, has no consensus TATAA box (Fig. 5E). Moreover, no consensus initiator sequence (42) is present around its transcription start sites. There is,
however, a sequence (TTCAAAAA) that may serve as a weak binding site
for TATA binding protein (Fig. 5E and Fig. 6).
Immediately 5` of S is a stretch of 14 purines (underlined sequence in Fig. 6). Further upstream, a
consensus CCAAT box is present as is a GC box (Fig. 6). Sequence
analysis of the 5` 3000 nt of SMC calponin promoter revealed several
consensus binding sites for regulatory factors involved with muscle
transcription including E-boxes (43) and GATA binding sites (44) (Fig. 6; accession number U37071). No consensus
MEF-2 binding sites (45) are present in the 5` 3000 nt of SMC
calponin. Finally, several stretches of alternating purine-pyrimidine
dinucleotides are present (double underlined sequences in Fig. 6).
Figure 6: Nucleotide sequence of mouse SMC calponin 5`-flanking region. Known muscle regulatory elements are boxed in black. The single underlined sequences are polypurine tracts and the double underlined sequences represent alternating purine/pyrimidine dinucleotide tracts. Note that the 3` most purine/pyrimidine repeat is contiguous with a polypurine tract. Triangles designate the 5` most boundary of each SMC calponin promoter construct (see Fig. 7A).
Figure 7:
SMC calponin promoter activity in cultured
RASMC. A, schematic of progressive 5` SMC calponin promoter
deletions. Shown is a partial restriction map of the sites used to
construct each deletion construct into the pGL3 basic luciferase
vector. Note that the two BamHI sites were artificially
engineered by PCR (see ``Materials and Methods''). The two
transcription start sites are indicated by arrows. The black box corresponds to the 5` 60 nt of untranslated SMC
calponin cDNA sequence, the 3` end of which is 19 nt upstream of the
initiating methionine. The numbers represent the distance of
the 5` end of each promoter construct from the S start
site. Abbreviations are: B, BamHI; H, HindIII; and N, NcoI. B, five independent
transfections of RASMC (passage number 15-30) with each indicated
SMC calponin promoter construct were performed as described under
``Materials and Methods.'' The relative light units of
luciferase were normalized to total protein and then expressed as a
percent of the pGL3 control vector (containing the SV40
promoter/enhancer). Note that the -115 CALPLuc construct was less
than the promoterless pGL3 basic vector. Values represent the mean
percent of pGL3 control ±S.E. of
mean.
To ascertain the specificity of SMC calponin promoter activity in vitro, several cell lines were transfected with either the promoterless pGL3 basic vector, the -3000 CALPLuc promoter, or the -549 CALPLuc promoter. Although these promoters showed higher relative activity in two SMC lines, several cell lines that do not express the endogenous SMC calponin transcript displayed some SMC calponin promoter activity (Fig. 8). Only the F9 teratocarcinoma cell line exhibited relatively low luciferase activity with both SMC calponin promoter constructs (Fig. 8). In general, the -549 CALPLuc promoter construct displayed higher activity than the -3000 CALPLuc construct, particularly in the 10T1/2 cell line (Fig. 8). This suggests the presence of negative regulatory elements between -3000 and -549. These results demonstrate functional SMC calponin promoter activity in vascular SMC and, to a lesser extent, several cell lines that do not express the SMC calponin transcript. This activity is largely imparted by sequences between -549 and -115 of the SMC calponin promoter.
Figure 8: SMC calponin promoter activity in different cell lines. Four independent transfections were carried out in the indicated cell lines with the promoterless pGL3 basic vector, -549 CALPLuc and -3000 CALPLuc as described under ``Materials and Methods.'' Activity was computed as described in the legend to Fig. 7B. Values represent the mean percent of pGL3 control ± S.E. of mean.
The mRNA expression of SM -actin, SM22
,
and SMC calponin in the embryonic mouse heart should be contrasted with
the notable absence of SMMHC transcripts in developing cardiac tissue ( Fig. 3and (10) ). These unique patterns of SMC gene
expression during mammalian development suggest that distinct
regulatory factors control each gene. Identifying these regulatory
factors should provide insight into the mechanisms for SMC
transcription and may contribute to an understanding of the complexity
of SMC phenotypes that characterize many vascular lesions.
The
functional significance of a shared genetic program between developing
cardiac and smooth muscle has yet to be elucidated. One intriguing
possibility is that cardiac muscle traverses a SMC-like phenotype
during its ontogeny, a concept that is supported by the distinct
differences in embryonic versus postnatal cardiac
contractility(47) . In this regard, it will be of interest to
determine whether the decompensated adult heart, which expresses a
fetal cardiac phenotype(48) , expresses SMC calponin. Support
for such expression is provided by studies in hypertrophied rat hearts,
which express the SM -actin gene(13) . If SMC proteins
participate in some aspect of cardiac contractility, then they would do
so in the absence of their native thick filament, SMMHC.
In addition
to embryonic heart expression, SMC calponin mRNA was noted in
proliferating BCH1 cells. This cell line, originally
thought to be of SMC origin(49) , is defective for terminal
differentiation into sarcomeric muscle(50) . When stimulated to
exit the cell cycle and differentiate, BC
H1 cells acquire a
number of sarcomeric markers and, at the same time, lose several SMC
markers(50) . The reversible expression of SMC calponin mRNA in
BC
H1 cells is consistent with these findings and suggests
that proliferating BC
H1 cells more closely resemble a
SMC-like phenotype. Moreover, as with other SMC markers(50) ,
SMC calponin mRNA can be re-induced by serum-stimulating differentiated
BC
H1, which leads to their re-entry into the cell
cycle.
Thus, this cell line may be useful as a tool to
uncover regulatory factors that activate SMC calponin gene expression in vitro.
With the exception of embryonic heart and
proliferating BCH1 cells, SMC calponin mRNA was only
expressed in SMC lineages. No evidence of expression was noted in
skeletal muscle or cell lines derived from skeletal muscle.
Furthermore, no transcripts were detected in embryonic cell lines (ES,
F9), fibroblasts (3T3 or 10T1/2), or endothelial (human umbilical vein)
cells.
Thus, early reports of calponin protein expression
in such tissues as the adrenal gland (51) and such cells as
platelets, fibroblasts, and endothelial cells (28.29), probably
reflected the presence of a recently cloned non-muscle (acidic)
calponin(25) .
The proximal promoter of SMC calponin shares many features with another TATAA/initiator-less promoter, thymidylate synthase(54) . Both genes have a stretch of purines immediately 5` of an initiation site. In addition, the SMC calponin promoter has an upstream polypurine tract that immediately follows a purine/pyrimidine dinucleotide repeat (see Fig. 6). These unique DNA sequences have been shown to play a role in transcription control. For example, polypurine tracts form DNA triple helices that serve as directional attenuators of transcription(55) . Purine/pyrimidine dinucleotide repeats promote the formation of Z DNA (56) , which has recently been shown to regulate the transcription of the c-myc proto-oncogene(57) . Such mechanisms of control could exclude transcription of SMC calponin in non-SMC-containing tissues.
Another shared feature between SMC calponin and thymidylate synthase is the presence of GC-rich sequences in their 5`-flanking region. Both genes, for example, have a GC box whose binding factors may interact with the basal transcriptional machinery to facilitate preinitiation complex formation and transcription(54) . Despite the presence of a GC box, as well as a CCAAT box and sequences around both initiation sites, the -115 CALPLuc promoter construct was totally inactive. This result indicates that upstream activators between -115 and -549 play a crucial role in the initiation of SMC calponin transcription. Sequence analysis of this region reveals two E-boxes and one GATA site.
Based on its highly restricted pattern of mRNA expression, we predicted that the SMC calponin promoter would be similarly restricted in activity. Although promoter activity was very high in SMC, we found it to be active in several cell lines, including L6, C2 myoblasts, and 10T1/2 fibroblasts, none of which express the endogenous gene. Only the F9 teratocarcinoma cell line showed low promoter activity. These findings suggest either a distal inhibition element involved in long range repression of SMC calponin expression in non-SMCs is missing from our promoter constructs or some other mode of transcriptional regulation confers SMC calponin's specificity for SMC lineages.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37071 [GenBank]and U28932[GenBank].