(Received for publication, March 2, 1995; and in revised form, May 22, 1995)
From the
Caldesmon, which plays a vital role in the actomyosin system, is
distributed in smooth muscle and non-muscle cells, and its isoformal
interconversion between a high M form and low M
form is a favorable molecular event for studying
phenotypic modulation of smooth muscle cells. Genomic analysis reveals
two promoters, of which the gizzard-type promoter displays much higher
activity than the brain-type promoter. Here, we have characterized
transcriptional regulation of the gizzard-type promoter. Transient
transfection assays in chick gizzard smooth muscle cells, chick embryo
fibroblasts, mouse skeletal muscle cell line (C2C12), and HeLa cells
revealed that the promoter activity was high in smooth muscle cells and
fibroblasts, but was extremely low in other cells. Cell type-specific
promoter activity depended on an element, CArG1, containing a unique
CArG box-like motif (CCAAAAAAGG) at -315, while multiple E boxes
were not directly involved in this event. Gel shift assays showed the
specific interaction between the CArG1 and nuclear protein factors in
smooth muscle cells and fibroblasts. These results suggest that the
CArG1 is an essential cis-element for cell type-specific
expression of caldesmon and that the function of CArG1 might be
controlled under phenotypic modulation of smooth muscle cells.
Smooth muscle cells (SMCs) ()undergo remarkable
phenotypic modulation during embryogenesis. A converse transition of
arterial SMCs from a differentiated to dedifferentiated phenotype is
one of major events in the onset of
atherosclerosis(1, 2) . Although molecular approaches
of such phenotypic modulation are important for understanding vascular
pathogenesis, only limited information is available. Of these,
-smooth muscle actin (
-SM actin) was considered to be a
suitable molecular marker for differentiation of SMCs(3) .
Recent studies have led to suspect the significance of this protein
because its expression has been found in skeletal muscle cell line (4) and certain stromal cells(5) .
Caldesmon (CaD)
plays a vital role in the Ca-dependent regulation of
smooth muscle and non-muscle contraction(6, 7) . The
two CaD isoforms have been identified. h-CaD (high M
form) is dominantly expressed in differentiated SMCs, while l-CaD
(low M
form) is widely distributed in non-muscle
tissues and cells(8, 9, 10) . In particular,
the isoformal interconversion of CaD is tightly associated with
phenotypic modulation of SMCs, in which the CaD isoform converts from
the l- to h-form during differentiation and vice
versa(11, 12, 13) . CaD is therefore a
favorable molecular marker for studying phenotypic modulation of SMCs.
Genomic analysis has revealed that the expression of h- or l-CaDs
depends on a unique selection of two 5`-splice sites within the exon
3(14, 15) . Another important molecular event is the
up-regulation of CaD expression during SMC
differentiation(11) . Several cytoskeletal proteins such as
myosin heavy and light chains(16, 17) ,
-SM
actin(3) , tropomyosin(18) , vinculin(12) ,
metavinculin(12) , calponin(13, 19) , and SM22 (20) are also up-regulated in association with SMC
differentiation. Contrarily, their expressions are down-regulated
during dedifferentiation. The expressional changes of these
cytoskeletal proteins in their amounts might be controlled at a
transcriptional level. However, their transcriptional regulations have
been scarcely investigated. The
-SM actin and vinculin genes have
been partially
characterized(4, 21, 22, 23) . In
our previous report(24) , we have identified two CaD promoters,
gizzard-type and brain-type promoters, in which the gizzard-type
promoter shows much higher activity than the brain-type promoter. Here,
we have characterized the transcriptional regulation of the
gizzard-type promoter, which actively functions in SMCs and chick
embryo fibroblasts (CEFs) but is unable to promote high levels of
transcriptional activity in other cell types such as C2C12 and HeLa
cells. The promoter activity in differentiated SMCs was higher than
that in dedifferentiated SMCs. This result coincided with the high
expression of h-CaD in differentiated SMCs compared with the low
expression of l-CaD in dedifferentiated cells. We have also
demonstrated that the cell type-specific expression of the CaD gene is
regulated by a single cis-element, CArG box-like motif
(CCAAAAAAGG), located between -309 to -300, whereas
multiple E boxes located in the 5`-upstream region are not directly
involved in this event.
Figure 1: CaD expression analysis in SMCs, CEFs, C2C12, and HeLa cells by immunoblot. Cell homogenates from a 2-day culture of SMCs under serum-free conditions (differentiated phenotype) (lane 1) and SMCs cultured in the medium containing 10% fetal calf serum for 9 days (dedifferentiated phenotype) (lane 2), CEFs (lane 3), C2C12 cells cultured in growth medium (lane 4), and HeLa cells (lane 5) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contents of cell homogenates were based on the actin contents. Respective cell homogenates from C2C12 cells cultured in growth medium and in low serum medium for 96 h were loaded in lanes 6 and 7 based on DNA contents. Immunoblot of CaD was carried out using the COOH-terminal domain-specific 35-kDa CaD antibody(42) . Arrowheads indicate h-CaD and l-CaD, respectively.
Figure 2: Relative CAT activity of the gizzard-type CaD promoter deleted constructs in SMCs, CEFs, C2C12, and HeLa cells. A, restriction sites, locations of canonical and putative cis-elements, and the transcriptional starting site are shown in the alignment map of the 5`-upstream region of exon 1a-1 of chicken CaD gene (-3041 to +60)(24) . Schematic structures of deleted or chimeric CAT constructs are shown under the map. Thick and thin lines indicate the sequences of the gizzard- and brain-type CaD promoters, respectively. Open boxes indicate the GE100 and the CArG1. B, relative CAT activities of respective CAT constructs in differentiated SMCs, dedifferentiated SMCs, CEFs, C2C12 myoblasts, C2C12 myotubes, and HeLa cells are shown. They were normalized to the activity of pUC2CAT in respective cells as 100%. BP1CAT and GE100/BP1CAT were transfected into only both types of SMCs and CEFs. To account for differences in transfection efficiencies, the level of luciferase activities from control plasmid carrying RSV promoter and luciferase cDNA were assayed.
Figure 3:
In vivo competition assay and
specific DNA-protein interaction using GE100. A, In vivo competition assay was carried out by cotransfection with
GP1Db-21CAT (4 µg) and competitor (20 µg), pUCGE100, or
control, pUC18, into CEFs. Relative CAT activity was based on the
activity of the cells cotransfected with GP1Db-21CAT and pUC18. B and C, specific DNA-protein interaction was analyzed by
gel shift assay using P-labeled GE100 and CEF nuclear
extracts (B, lanes 1-4, and C, lane 2, 4 µg, and C, lane 1, 8 µg)
or respective SMC nuclear extracts (C, lanes
3-8, 4 µg) as described under ``Experimental
Procedures.'' The arrowheads indicate a specific
DNA-protein complex and free fragment (F). Competitors used
are indicated at the top of respective lanes. -, without
competitor. (D) and (DD) indicate the phenotype of
SMCs, differentiated and dedifferentiated SMCs,
respectively.
Figure 4:
Structures of probes and nucleotide
sequences of synthetic DNA duplexes used for the promoter analysis. The
CArG box-like motif is deleted in GE80(CArG). Mutated nucleotides
in CArG1 are indicated by negative scripts.
Figure 5:
Effect of the CArG1 on the basal
promoter. A, schematic structures of deleted/mutated CAT
constructs derived from GP1Db-21CAT are shown. Respective CAT
constructs are numbered on the right. Open boxes indicate
GE100 and CArG1, and mutated CArG1 is presented by a closed
box, respectively. Deleted 3`-regions of GE100 are shown by dashed lines (3`GE100/GP1(SphI)CAT c14). The
CArG1 inserted in the antisense orientation is indicated by shaded box. B, relative CAT activities in differentiated and
dedifferentiated SMCs and CEFs are shown. CAT activity was quantified
as described in legend of Fig. 2. Numbers under the
graph indicate the respective CAT constructs shown in A.
Gel
shift assay using CEF and differentiated SMC nuclear extracts revealed
the specific CArG1-protein complex formation with identical migration
in gels, because such complexes were suppressed by unlabeled CArG1, but
not CArG2, CArG3, and CArG1M ( Fig. 4and Fig. 6, A and B). Conversely, radiolabeled deletion and/or mutation
probes did not form the DNA-protein complex (data not shown). The
amounts of such complex were also high in the differentiated SMC
extracts (Fig. 6B). The results of transient
transfection assay (Fig. 5) and gel shift assay (Fig. 6)
indicate that the interaction between the CArG1 and nuclear protein
factors is essential for enhancement of the basal promoter activity.
Since the promoter activities in differentiated SMCs and CEFs were
nearly equal in spite of the difference in the amounts of the
CArG1-protein complex, the quantities of the complex were not directly
correlated to the enhancement. The CArG1-protein complex was resistant
to high salt (120 mM NaCl) and did not require
Mg, but was sensitive to orthophenanthroline, a
Zn
chelator; 5 mM orthophenanthroline
suppressed the complex formation (data not shown).
Figure 6:
Characterization of the CArG1-protein
interaction by gel-shift assay. Binding assays were carried out using 4
µg of CEF or differentiated SMC nuclear extracts and P-labeled CArG1 as described under ``Experimental
Procedures.'' Competitors used are indicated at the top of
respective lanes. -, without competitor. The arrowheads indicate a specific DNA-protein complex and free fragment (F). (D) indicates phenotype of SMCs, differentiated
SMCs.
Figure 7:
Serum
effect on CAT activity (A), the endogenous CaD expression (B), and positive control of serum inducibility in CEFs (C). A, GP1CAT and GP1Db-21CAT were independently
transfected into CEFs. Cells were cultured either serum-starved for 50
h (minus fetal calf serum) or serum-starved for 42 h and then
restimulated in the growth medium for 8 h (plus fetal calf serum).
Relative CAT activity was based on the activity of each CAT construct
under conditions of serum starved. B, total RNAs (5 µg)
prepared from CEFs under serum-starved(-) or serum-stimulated
after starvation (+) were analyzed by Northern blotting using a
oligonucleotide probe specific to the gizzard-type CaD. Ethidium
bromide staining of the gels (at the bottom) is also shown. C,
relative luciferase activities from the -actin promoter under
respective conditions of serum starved and stimulated are
shown.
The expressional changes of CaD isoforms and in their
contents are closely associated with phenotypic modulation of
SMCs(11, 12, 13) . CaD is therefore
considered to be a favorable molecular marker for studying such
phenotypic modulation. The CaD expression is regulated by two means,
splicing and transcription, within a single
gene(14, 15, 24) . Maturation of mRNAs for
CaD isoforms is determined by a unique splicing; the expression of h-
or l-CaDs depends on a selection of two 5`-splice sites within exon
3(14, 15) . The change in CaD content is determined by
the regulation of promoter activities. Characterization of the factors
involved in the expressional regulation of the key genes is important
for phenotypic modulation of SMCs. Although little is known about the
transcriptional regulation of the -SM actin gene in differentiated
SMCs, the CArG boxes are reported to be key cis-elements in
the regulation of
-SM actin promoter in dedifferentiated SMCs and
skeletal muscle cell lines(4, 21, 22) .
Recent studies have expressed doubt that the
-SM actin is suitable
for a molecular marker of SMC phenotypic modulation because its
expression is not restricted within SMCs(4, 5) .
Recently, the promoter region of the human vinculin gene has been
partially analyzed (23) . Although it contains a CArG box and
shows serum inducibility, the involvement of cis-elements in
the activation of the vinculin promoter is unknown. At present, the
expressional regulations of SMC-specific molecular markers have not
been well characterized, and cis-elements and trans-acting factors involving in SMC-specific transcription
also remain unclear.
We have previously demonstrated the cloning of
the gizzard- and brain-type CaD promoters, in which the gizzard-type
promoter displays much higher activity than the brain-type
promoter(24) . In the present study, we have characterized the
transcriptional regulation of the gizzard-type promoter in SMCs, CEFs,
C2C12, and HeLa cells. The gizzard-type promoter displays SMC-specific
high expression except for CEFs (Fig. 2). In addition, both the
promoter activity and the protein level of CaD in differentiated SMCs
were higher than in dedifferentiated SMCs ( Fig. 1and Fig. 2). At present, it is unknown why the promoter activity is
high in CEFs and the h-CaD is expressed in these cells. At any rate,
CEFs are one of suitable subjects for the present purpose. It has been
further clarified that only a limited region expanding from -315
to -218, GE100, enhances the basal promoter (-217 to
+1) activity in SMCs and CEFs, while the upstream region from
-3041 to -316 containing multiple E boxes is not directly
involved in this event (Fig. 2). In vivo competition
and gel shift assays suggest the presence of trans-acting
factors bound to cis-element in GE100 (Fig. 3A). Detailed analyses indicate that the target
element is a unique CArG box-like motif, located at -309 to
-300 and that the CArG1 composition of this motif in addition to
its 5`- and 3`-flanking sequences is essential for binding of trans-acting factors ( Fig. 5and Fig. 6). CArG
boxes have been found in several actin genes as well as the c-fos gene(36, 37) . They interact with multiple
nuclear protein factors and are required for skeletal or cardiac
muscle-specific expression of the actin genes, for basal constitutive
expression of the non-muscle actin genes, and for rapid and transient
activation of the c-fos gene in response to serum growth
factors. The sequence of the CArG1 is specific because both the binding
and transcriptional activities were decreased by deletion or mutation
in CArG1 ( Fig. 5and Fig. 6). Therefore, the inner core
of the CArG1 in the CaD gene, CCAAAAAAGG, is unique compared with other
CArG boxes. The CArG1 was also able to activate the promoter activity
in spite of its position and orientation. Based on these findings, we
conclude that the CArG1 plays a role as a cell type-specific enhancer.
The interaction between the CArG1 and nuclear protein factors was
essential for activation of the gizzard-type promoter, while the
amounts of the CArG1-protein complex were variable in differentiated
and dedifferentiated SMCs and CEFs (Fig. 3, A and B, and 6). Therefore, the quantities of CArG1-binding protein
factors would not be directly related to the promoter activity. These
variations suggest the multiple interactions between the CArG1-protein
complex and basal promoter units including CCAAT box, Sp1 site, and
TATA box in respective cell types. Compared with the factors
interacting with CArG boxes in the skeletal -actin
gene(38, 39) , the CArG1-binding factors were
resistant against high salt concentrations. Our preliminary studies by
UV cross-linking using the CArG1 or the c-fos serum response
element as probes suggest that distinct proteins with different M
values bind to the each probe. (
)Based on our results, we speculate that the CArG1-binding
factors would be distinct from such CArG box-binding factors which have
already been characterized. Further studies will be necessary to
establish the CArG1-binding protein factors in the cell type-specific
expression of the CaD gene.
The CArG1 fails to function as a
serum-responsive element because the gizzard-type promoter was not
affected by serum (Fig. 7A). This result coincided with
the expression of endogenous CaD gene in CEFs (Fig. 7B)
and high levels of promoter activity in differentiated SMCs cultured
under serum-free condition ( Fig. 2and 5). Considering the serum
responsiveness of vinculin and - and
-actin
genes(23, 35, 38, 40, 41) ,
serum inducibility might depend on cell type and might require another
factor to mediate between the CArG box-binding factor and basal
transcription initiation factors.
In summary, the present studies demonstrated that the gizzard-type CaD promoter exhibits high levels of transcriptional activity in SMCs and CEFs, but extremely low levels in other cell types such as C2C12 and HeLa cells, and that the promoter activity in differentiated SMCs is higher than that in dedifferentiated SMCs. The protein levels of CaD in differentiated and dedifferentiated SMCs were in good agreement with the promoter activities in the respective cells. These results suggest that the gizzard-type CaD promoter activity might be controlled under phenotypic modulation of SMCs. In addition, we have identified that the CArG1, located at -309 to -300 upstream of the transcriptional starting site of the gizzard-type CaD promoter is an essential cis-element for the SMC-specific expression, and that specific DNA-protein complex formation is found between the CArG1 and nuclear extracts from SMCs and CEFs. Further studies regarding SMC-specific gene expression are required for understanding the molecular events of phenotypic modulation of SMCs.
Addendum-During submission of this paper, promoter elements of the smooth muscle myosin heavy chain gene have been identified(43) . E boxes, myocyte enhancer binding factor 2 (MEF2)-like motifs, and CArG box-like motifs are found in the myosin heavy chain gene and are involved in the SMC-specific expression. Based on their study, the protein binding to the MEF2-like motifs is revealed to be different from a MEF2 protein, while the CArG box-like motif does not show protein binding. In our present studies, a MEF2-site is absent in the gizzard-type CaD promoter, and E boxes are not important in the CaD expression, whereas only CArG1 is the essential cis-element for activation of the promoter.