(Received for publication, October 2, 1996, and in revised form, April 14, 1997)
From the Department of Neurochemistry and Neuropharmacology, Isoform diversity of tropomyosin is generated
from the limited genes by a combination of differential transcription
and alternative splicing. In the case of the It is important to elucidate the molecular mechanism of phenotypic
modulation of smooth muscle cells (SMCs)1
such as vasculogenesis, enterogenesis, atherosclerosis, hypertension, and leiomyogenic tumorigenesis. The SMCs are derived from mesodermal precursors, but the intracellular and extracellular factors determining the SMC lineage and its phenotype remain unclear. The search for molecular parameters indicating SMC phenotype is a first step in
analyzing phenotypic modulation of SMCs. Several cytoskeletal and
contractile proteins are such candidates. Among them, changes of actin
(1, 2), caldesmon (CaD) (1, 3, 4), myosin heavy chain (5, 6), and
vinculin/meta-vinculin (1, 7) isoforms are closely
associated with phenotypic modulation of SMCs. Recent studies have
focused on the gene regulation of such parameters (8-14). In addition
to these isoform changes, expression of Tropomyosin (TM) is a predominant helical protein that binds to actin
groves. Recent evidence suggests that
Ca2+-dependent actin-myosin interaction in
smooth and nonmuscle cells is controlled by myosin- and actin-linked
dual regulation. In this regulation, the TM and CaD are involved as
actin-linked regulators (23-25). Also, it has been proposed that both
proteins are essential for the reorganization of the actin cytoskeleton
mediated by stabilization of microfilaments (26-28). Therefore, TM, in
addition to CaD, plays a vital role in motile events. One- and
two-dimensional gel electrophoreses revealed multiple TM isoforms
associated with morphological changes and tumorigenic transformation
(29-31). cDNA and genomic DNA analyses have also shown a diversity
of TM mRNAs generating from the limited TM genes by a combination
of differential promoter usage and alternative splicing (reviewed in
Ref. 32). Of these, some of the TM isoforms accumulate in a
tissue-specific manner. It has been demonstrated that the chicken and
rat Here we have been the first to demonstrate expressional change of TM
isoforms depending upon SMC phenotype. During dedifferentiation of SMCs
in primary culture, the SMC-type We prepared SMCs from 15-day-old chick embryo
gizzards. Briefly, gizzard muscles were carefully separated from serosa
and tunica mucosa, minced with scissors, and incubated at 30 °C for 60 min in 1 mg/ml collagenase (type V, Sigma) solution containing 137 mM NaCl, 5 mM KCl, 4 mM
NaHCO3, 2 mM MgCl2, 5.5 mM D-glucose, 10 mM PIPES, pH 6.5, and 2 mg/ml bovine serum albumin (Sigma) with gently shaking. Finally,
the dispersed cells were plated onto culture dishes (see below) in a
density of 1.5 × 105 cells/cm2 and were
incubated at 37 °C with 5% CO2 atmosphere. To promote dedifferentiation of SMCs, isolated SMCs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS)
on plastic culture dishes. We used SMCs cultured under these conditions
for more than 1 week as dedifferentiated SMCs. To maintain the
differentiated phenotype, SMCs were cultured in Dulbecco's modified
Eagle's medium containing 0.2% bovine serum albumin and 5 µg/ml
insulin on laminin-coated dishes.
We prepared vascular SMCs from chicken aortae by explant methods
(30). To isolate explant-derived SMCs, the aortic media was
minced into 1-2-mm segments and incubated in Dulbecco's modified Eagle's medium supplemented with 10% FCS. The cells were routinely passaged at a 1:3 ratio. We used three-passaged cells as
dedifferentiated vascular SMCs.
Two-dimensional gel
electrophoresis was performed according to the previously described
method (39) with some modifications. Cells were washed with
phosphate-buffered saline and lysed in 9.5 M urea, 2%
Triton X-100, and 5% 2-mercaptoethanol by sonication. The
concentration of ampholyte (pH 4.0-6.0) was adjusted to 2%, and then
the samples were separated by isoelectric focusing. First dimensional
gel was composed of 9.2 M urea, 4%
acrylamide/bisacrylamide, 2% Triton X-100 and 2% ampholyte (pH
4.0-6.0). Isoelectric focusing was first conducted at 500 V for 10 min, followed by at 750 V for 3.5 h and then at 1000 V for 2 h. The second dimension was SDS-PAGE with 17.5% acrylamide. Separated
proteins were then transferred to nitrocellulose membranes and were
detected by the ECL Western blotting detection kit (Amersham Corp.)
using specific antibodies. TM311, anti-chicken gizzard TM monoclonal
antibody (mAb), was purchased from Sigma. Anti-CaD polyclonal
antibodies were prepared as described elsewhere (3). We also used
polyclonal TM antibodies against chicken gizzard TMs (40) in this
study.
The SMCs
were washed with phosphate-buffered saline and were harvested in
phosphate-buffered saline containing 1 µg/ml leupeptin and 0.1 mM diisopropyl fluorophosphate. The cells were pelleted by
brief centrifugation and then sonicated in 20 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM dithiothreitol (DTT),
10 mM EGTA, 0.5 mM diisopropyl fluorophosphate,
and 5 µg/ml leupeptin. The lysates were boiled for 5 min, followed by
centrifugation at 100,000 × g for 30 min. The
supernatants thus obtained were subjected to ammonium sulfate
precipitation between 28 and 36%. The pellets were suspended in 10 mM potassium phosphate, pH 7.0, 1 M NaCl, 1 mM DTT and dialyzed against the same buffer whose pH was
lowered to 4.7 by the addition of HCl. After centrifugation at
15,000 × g for 20 min, the pellets were resuspended in
and dialyzed against 20 mM imidazole-HCl, pH 7.2, 100 mM NaCl, 0.1 mM DTT, and 0.1 mM
EGTA. The dialysates were centrifuged at 100,000 × g
for 30 min. All manipulations described above were carried out at
4 °C. The clarified dialysates (crude TM fractions) were used for
actin-binding assays. The crude TM fractions were incubated with rabbit
skeletal muscle actin at 37 °C for 30 min. The buffer conditions
were as follows: 20 mM imidazole-HCl, pH 7.2, 100 mM NaCl, 0.1 mM DTT, 0.1 mM EGTA,
and 5 mM MgCl2. After centrifugation at
100,000 × g for 30 min, the sedimented actin and its
associated proteins were dissolved in two-dimensional gel
electrophoresis buffer and subjected to two-dimensional gel
electrophoresis.
cDNA
libraries of SMCs and of dedifferentiated SMCs were constructed in
Glutathione S-transferase (GST) and truncated TM fusion
proteins were produced in Escherichia coli and purified by
glutathione-Sepharose using the GST Gene Fusion System (Pharmacia
Biotech Inc.). Each of the truncated cDNAs carrying exon 1a of
We constructed expression vectors carrying each of the cDNAs for TM
isoforms downstream of the chicken Expression of Total cellular RNAs were isolated from
SMCs using the ISOGEN (Nippongene). Ten µg of total RNAs at the
indicated culture days were separated on 1.0% agarose-formaldehyde
gels and then transferred to nylon membranes (Hybond-N+,
Amersham). The membranes were hybridized with respective TM and CaD
isoform-specific DNA probes under the conditions listed in Table
I. After removal of the hybridized probes, the membranes were reused. To visualize 28 S rRNAs, membranes were stained with 0.02% methylene blue.
Table I.
Conditions of Northern blotting
Department of Anatomy
and Neuroscience, and the § Department of Molecular
Neurobiology (TANABE), Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
-tropomyosin (
-TM)
gene, exon 2a rather than exon 2b is specifically spliced in
-TM-SM
mRNA, which is one of the major tropomyosin isoforms in smooth
muscle cells. Here we demonstrate that expressions of
-tropomyosin
and caldesmon isoforms are coordinately regulated in association with
phenotypic modulation of smooth muscle cells. Molecular cloning and
Western and Northern blottings have revealed that in addition to the
down-regulation of
-TM-SM,
-TM-SM converted to
-TM-F1 and
-TM-F2 by a selectional change from exon 2a to exon 2b during
dedifferentiation of smooth muscle cells in culture. Simultaneously, a
change of caldesmon isoforms from high Mr type
to low Mr type was also observed by alternative
selection between exons 3b and 4 in the caldesmon gene during this
process. In contrast, cultured smooth muscle cells maintaining a
differentiated phenotype continued to express
-TM-SM,
-TM-SM, and
high Mr caldesmon. In situ
hybridization revealed specific coexpression of
-TM-SM and high
Mr caldesmon in smooth muscle in
developing embryos. These results suggest a common splicing mechanism
for phenotype-dependent expression of tropomyosin and caldesmon
isoforms in both visceral and vascular smooth muscle cells.
-smooth muscle actin (
-SM
actin) (15, 16), CaD (1, 3, 4), myosin heavy and light chains (5, 6,
17), meta-vinculin (1, 7), SM22, and calponin (18-20) have
been reported to be up-regulated during differentiation of SMCs, but down-regulated during dedifferentiation, suggesting the involvement of
SMC phenotype-dependent transcriptional regulation in the
SMC-specific parameter genes. In fact, the transcriptional machineries
of
-SM actin (8, 9), CaD (12), myosin heavy chain (13), SM22 (21),
and calponin (22) have been partially characterized.
-TM genes generate multiple transcripts. A pair of internal
exons (exons 6a and 6b) are spliced in a mutually exclusive manner, and
their utilization is regulated during differentiation of skeletal
muscle cells; exons 6a and 6b are used in myoblasts and myotubes,
respectively (33, 34). In the case of the
-TM genes, exon 2b rather
than exon 2a (termed exons 3 and 2, respectively, in Ref. 35) is
spliced in the high Mr
-TM mRNAs in all
cell types except for SMCs (35, 36); exon 2a is specifically selected in SMCs. The selection of exon 2a is therefore considered to be an
SMC-specific event. The splicing mechanism of the
-TM genes has been
also studied (37, 38); however, there are no reports regarding isoform
interconversion of TM in association with phenotypic modulation of
SMCs.
-TM (
-TM-SM) converted to
fibroblast-type 1 and 2
-TM isoforms (
-TM-F1 and
-TM-F2) by a
selectional change from exon 2a to exon 2b, while expression of
SMC-type
-TM (
-TM-SM) was down-regulated. Under culture
conditions in which differentiated SMCs were caused to dedifferentiate
by serum or platelet-derived growth factor-BB (PDGF-BB), expressional change of CaD from high Mr (h-CaD) to low
Mr form (l-CaD) was coincident with that of TM.
In contrast, expression of
-TM-SM and h-CaD was maintained in
differentiated SMCs cultured on laminin-coated dishes without serum.
In situ hybridization revealed coexpression of
-TM-SM and
h-CaD mRNAs in smooth muscle in developing embryos. The present
results, therefore, suggest that the
-TM and CaD genes are
coordinately regulated in a SMC phenotype-dependent manner.
We further discuss the molecular mechanism of such gene expressions
including transcription and splicing during phenotypic modulation of
SMCs.
Cell Culture
gt11. The libraries were screened using TM311 mAb. Alternatively, we
amplified partial cDNA fragments carrying
-TM exons 3, 4, and 5 or
-TM exons 3, 4, and 5 by the polymerase chain reaction (PCR)
method. These cDNA fragments were also used as probes for screening
of the cDNA libraries. Cloned cDNAs were sequenced.
-TM, exons 2b to 9d (containing the 3
-noncoding region) of
-TM,
exon 1a of
-TM, exons 2 to 9d (containing the 3
-noncoding region)
of
-TM, or exons 1b to 6b of
-TM, was fused to the GST gene in
the pGEX-3X vector. Immunological reactivities of TM311 mAb against
fusion proteins were examined by Western blotting.
-actin promoter (pAct-vector). Expression vectors were transfected into dedifferentiated SMCs as
described elsewhere (12), and the cell lysates were analyzed by
two-dimensional gel electrophoresis and Western blotting with TM311 mAb
as described above.
-SM actin in
differentiated and dedifferentiated SMCs and chick embryo fibroblasts
(CEFs) was examined by indirect immunofluorescence microscopy as
described previously (40). In this study, fixed and permeabilized cells
were incubated with anti-
-SM actin monoclonal antibody (Sigma) and
then were double-stained with both fluorescein isothiocyanate-labeled
anti-mouse IgG antibody and rhodamine-phalloidin.
Probes
Nucleotide positions (GenBankTM accession
No.)
Labeling
Hybridization buffer/temperature
Washing
buffer/temperature
-TM/E2a
128-147 of
-TM-SM
(D87893[GenBank])
[
-32P]ATPa
HB-Ab/48 °C
WB-Ac/52 °C
-TM/E2b
264-283 of
-TM-F1
(D87891[GenBank])
[
-32P]ATP
HB-A/52 °C
WB-A/52 °C
-TM/E1a-E2
28-263 of
-TM-SM
(K02446[GenBank])
[
-32P]dCTPd
HB-Be/42 °C
WB-Bf/42 °C
CaD/E3b
811-932 of h-CaD
(M28417[GenBank])
[
-32P]dCTP
HB-B/42 °C
WB-B/42 °C
CaD/E3a-E5
757-776 of l-CaD
(M59762[GenBank])
[
-32P]ATP
HB-A/52 °C
WB-A/52 °C
a
Antisense oligo-DNA was phosphorylated by
[ -32P]ATP using T4 polynucleotide kinase (Takara).
b
cDNA fragment was amplified by PCR, and antisense strand
DNA was labeled by [ -32P]dCTP using BcaBEST DNA polymerase
(Takara).
c
HB-A consists of 6 × SSC (20 × SSC consists of
100 mM NaCl and 100 mM trisodium citrate
dihydrate), 0.5% SDS, 1 mg/ml polyvinylpyrrolidone, 1 mg/ml bovine
serum albumin, and 100 µg/ml herring sperm DNA.
d
HB-B consists of HB-A plus 50% formamide.
e
WB-A consists of 6 × SSC and 0.1% SDS.
f
WB-B consists of 2 × SSC and 0.1% SDS.
Oligo(dT)15-primed single-stranded cDNAs
were synthesized from 3 µg of total RNAs using RAV-2 reverse
transcriptase (Takara). ,
, and
volume of heat-treated single-stranded cDNA mixtures were subjected
to PCR using specific sets of primers (Table II) and Ex
Taq DNA polymerase (Takara) under the following conditions:
96 °C for 0.5 min, 55 °C for 1 min, and 72 °C for 3 min. Cycle
numbers are given in Table II. PCR products were separated on 1.5%
agarose gels and were stained with SYBR Green I (FMC). Intensity of
stained bands were relatively quantified by a FluorImager (Molecular
Dynamics). To eliminate changes of transcriptional activities of
-TM
and CaD promoters and differences in efficiency of amplification of
each PCR product, the relative contents of each mRNA were estimated
by multiregression analysis based on the equation, [common] = cA·[exon A] + cB·[exon B] + error. [common], [exon A],
and [exon B] represent the intensity of each PCR product of
-TM-common (or CaD-common),
-TM-exon2a (or h-CaD), and
-TM-exon2b (or l-CaD) that was amplified using total RNAs from the
indicated culture of SMCs. cA and
cB represent regression coefficients for each TM
(or CaD) isoform. Regression coefficients were calculated using the
computer program StatView 4.0 (Abacus Concepts). Finally, the relative
content of each mRNA, [exon A]
or [exon B]
, was calculated as
follows: [exon A]
= cA·[exon
A]/(cA·[exon A] + cB·[exon B]); [exon
B]
= cB·[exon B]/(cA·[exon A] + cB·[exon B]).
|
Chick embryos at
different stages were frozen on dry ice powder and sectioned 8-12 µm
thick by cryostat (Bright). The sections were mounted on
polylysine-coated glass slides and stored at 20 °C. Slides were
incubated in 4% formaldehyde in 0.1 M phosphate buffer for
30 min followed by three washes in 1 × SSC and dehydration in
ethanol. Oligonucleotide probes (Table III) were
radiolabeled with [
-35S]dATP (DuPont NEN) using
terminal deoxynucleotidyl transferase (Takara). Hybridization was
performed at 45 °C in 0.1 M phosphate buffer containing
50% formamide, 4 × SSC, 1 × Denhardt's solution, 0.4%
sarcosyl, 250 µg/ml yeast tRNA, 50 mM DTT, 100 µg/ml
poly(A), and labeled oligonucleotide probe (100,000-200,000 dpm).
After hybridization, slides were washed twice in 1 × SSC for 5 min at room temperature. A high stringency wash was carried out twice at 65 °C for 15 min in 1 × SSC followed by an additional two
washings at 65 °C for 15 min in 0.4 × SSC. Finally, the slides
were sequentially dehydrated in ethanol and exposed to a Fuji phosphor
imaging plate. Data were collected and analyzed by a BAS-5000 phosphor
imager (Fujifilm).
|
It is well known that primary cultured SMCs
under serum-stimulated conditions convert from a differentiated to
dedifferentiated state. We examined the isoform changes of TM in
primary cultured gizzard SMCs. Western blotting of precultured SMCs on
two-dimensional gels with an anti-chicken gizzard TM monoclonal
antibody, TM311 mAb, revealed two major spots (spots a and
c in Fig. 1I, A). In 3-day cultured cells under serum-stimulated conditions, new spots (spots 1 and 2) were detected with a more acidic
isoelectoric point and faster migration than spots a and
c, whereas the latter became faint (Fig. 1I,
B). The relative staining intensities of spots 1 and 2 gradually increased in 7-day cultured cells in
accordance with the decreases in spots a and c
(Fig. 1I, C). During this process, the intensity
of spot a reduced faster than that of spot c. In
two passaged cells, spots 1 and 2 were the only
TM isoforms detected by TM311 mAb (Fig. 1I,
D).
In this study, we used TM311 mAb as one of anti-TM antibodies. Although
the epitope of TM311 mAb has not been characterized, several reports
have suggested it to be the antibody cross-reacting with
high-Mr TM isoforms (40, 41). To identify the
epitope, we constructed bacterial expression plasmids carrying fusion
proteins between GST and truncated - or
-TM isoforms; exon 1a of
-TM; exons 2b, 3, 4, 5, 6b, 7, 8, and 9d of
-TM; exon 1a of
-TM; exons 2, 3, 4, 5, 6a, 7, 8, and 9d of
-TM; and exons 1b, 3, 4, 5, and 6b of
-TM. The TM311 mAb only cross-reacted with truncated proteins including exons 1a of both
- and
-TMs, but not with the
other truncated proteins (Fig. 2). Since amino acid
sequences encoded by exons 1a of the
- and
-TM genes are 87%
identical, the TM311 mAb is believed to recognize the limited regions
of the high Mr
- and
-TM isoforms encoded
by the respective exons 1a.
To confirm the expressional profile of TM isoforms at protein levels, TM isoforms in both differentiated and dedifferentiated gizzard SMCs were purified using heat treatment, ammonium sulfate fractionation, and isoelectric precipitation and were finally identified by an actin-binding assay. These actin-binding proteins separated by two-dimensional gel electrophoresis were detected by Coomassie Brilliant Blue staining (Fig. 1I, F and G); spots a and c and spots 1 and 2 were major TM isoforms in both phenotypes of SMC. In dedifferentiated SMCs, faint spots (spots 3a and 3b) corresponding to low Mr TM isoforms were observed by protein staining (Fig. 1I, G) and also by polyclonal TM antibodies against chicken gizzard TMs, which cross-reacted with both the high and low Mr TM isoforms (data not shown). Since a similar two-dimensional gel pattern was obtained using the TM fractions without the actin-binding assay (data not shown), the low amounts of spots 3a and 3b in the total TM fractions were not due to differences in their affinities against actin. Thus, expression of low Mr TM isoforms in SMCs was extremely low, suggesting that TM311 mAb can detect major TM isoforms expressed in SMCs.
There was a remarkable difference in expression of -SM actin between
dedifferentiated SMCs under serum-stimulated culture conditions and
CEFs;
-SM actin was strongly expressed in dedifferentiated visceral
SMCs (Fig. 3, B and E), whereas
this isoform in CEFs was a trace level (Fig. 3, C and
F).
-SM actin was also undetectable in differentiated
visceral SMCs (Fig. 3, A and D). The major actin isoform expressed in the cells is
-actin as identified by
immunoblotting and two-dimensional gel electrophoresis (data not
shown). Based on immunostaining, we assessed the contamination of CEFs
in cultured gizzard SMCs; 91 ± 6% of total 7-day cultured SMCs
under serum-stimulated conditions were
-SM actin-positive (data not
shown). This result suggests that the contamination of CEFs in cultured
SMCs was less significant. Therefore, a change of TM isoform as
presented here depends on phenotypic modulation of gizzard SMCs in
culture but does not arise from replacement of contaminated fibroblasts
derived from connective tissue and serosa.
We further analyzed isoform change of TM in vascular SMCs as associated with their phenotypic modulation (Fig. 1I, H and I). In precultured vascular SMCs (differentiated SMCs), spots a and c were seen as major spots (Fig. 1I, H). In contrast, in dedifferentiated vascular SMCs under serum-stimulated conditions, spots 1 and 2 were newly detected in place of spots a and c (Fig. 1I, I). This isoform change of TM is consistent with that of TM during dedifferentiation of cultured gizzard SMCs (Fig. 1I, A-D). Thus, isoform interconversion of TM from spots a and c to spots 1 and 2 occurs in common with phenotypic modulation of both visceral and vascular SMCs.
Identification of TM Isoforms Whose Expression Is Dependent on SMC PhenotypeTo identify the major TM isoforms detected by TM311
mAb, we screened cDNA libraries of differentiated and
dedifferentiated gizzard SMCs with TM311 mAb or specific - or
-TM
cDNA probes and obtained four different clones. Sequencing of two
clones from differentiated SMCs revealed
-TM-SM (36) and
-TM-SM
(42), and two other clones from dedifferentiated SMCs were
-TM-F1
and
-TM-F2 (36). These four clones belong to high
Mr TM isoforms including exon 1a. cDNAs
encoding other high and low Mr TM isoforms were
not detected. Although the cDNA sequences of
-TM-F1,
-TM-F2, and
-TM-SM have been speculated upon from the genomic structure of
the
-TM gene (36), the present results reveal the first sequence
data of their TM isoforms (DDBJ/EMBL/GenBankTM data bank
with accession numbers D87891-D87893[GenBank][GenBank][GenBank]).
To identify the four spots summarized in Fig. 1I,
E, the TM cDNAs obtained were transfected in
dedifferentiated gizzard SMCs. The cells transfected with -TM-SM or
-TM-SM cDNAs showed newly intensive spots corresponding to
spots a or c (Fig. 1II, B
and C). Transfection of
-TM-F1 and
-TM-F2 cDNAs
increased in spots 1 and 2, respectively (Fig.
1II, D and E). These results indicate that
-TM-SM and
-TM-SM (spots a and c)
substitute for
-TM-F1 and
-TM-F2 (spots 1 and
2) during dedifferentiation of SMCs. Based on the present
results, exon structures of the
- and
-TM genes and their
alternative splicings for
-TM-F1 and
-TM-F2 are summarized in
Fig. 4, A and B. A significant
difference among
-TM-SM,
-TM-F1, and
-TM-F2 is the selection
between exons 2a and 2b; exon 2a is specifically spliced in the
mRNA for
-TM-SM in differentiated SMCs, whereas the mRNAs
for
-TM-F1 and
-TM-F2 in dedifferentiated SMCs contain exon 2b,
indicating that this exon selection is dependent on the SMC phenotype.
In contrast, expression of
-TM-SM was down-regulated during SMC
dedifferentiation.
Coordinate Expressional Changes of TM and CaD Isoforms in Association with Phenotypic Modulation of SMCs
It has been demonstrated that isoform interconversion of CaD is associated with phenotypic modulation of SMCs (1, 3, 4). Fig. 4C represents an alternative splicing of CaD isoforms depending upon selection of exon 3a or 3ab and exon 4 (10, 11). To compare changes of TM and CaD isoforms during dedifferentiation of SMCs, we carried out Northern blotting using the respective exon-specific probes. Synthesized oligonucleotides or cDNA fragments amplified by PCR were used as probes (Table I).
cDNA probe expanding from exon 3 to exon 5 of the -TM gene,
which is spliced in every mRNA for
-TM isoforms, hybridized with
2.0-kilobase mRNAs, which were slightly decreased during serum-induced dedifferentiation of SMCs (data not shown). The mRNA
containing exon 2a, which is specifically spliced in
-TM-SM mRNA, was intensively expressed in precultured cells and was
interchanged with the mRNAs containing exon 2b within 24 h
after serum-stimulation (Fig. 5I). Exon 2b is
commonly spliced in the mRNAs for skeletal muscle or fibroblast
type high Mr
-TM isoforms. Therefore,
selection between exons 2a and 2b in the
-TM gene is highly
sensitive to serum-induced dedifferentiation. In contrast to such exon
selection, selection between exons 6a and 6b was not specific for the
SMC phenotype (data not shown). The mRNA for
-TM-F1 and the
mRNAs for
-TM-SM and
-TM-F2 contained exon 6a and exon 6b,
respectively.
cDNA probe expanding from exon 3 to exon 5 of the -TM gene was
able to detect a 1.2-kilobase mRNA for
-TM-SM and a 1.1-kilobase mRNA for fibroblast type
-TM isoform, but the latter mRNA
was expressed at an extremely low level (data not shown). During
dedifferentiation, expression of
-TM-SM mRNA probed by exons 1a
and 2 was down-regulated (Fig. 5I). The present results are
consistent with expressional change of TM isoforms at protein levels
(Figs. 1 and 5I). In regard to the expressional change of
CaD isoforms, the mRNA for h-CaD, in which exons 3b and 4 were
spliced, converted to the mRNA for l-CaD within 24 h after
serum stimulation (Fig. 5I). The switching of CaD isoforms
by alternative splicing was completely coincident with that of
-TM
isoforms.
To further compare phenotype-dependent isoform
interconversion of TM and CaD, we introduced a primary culture system
maintaining a differentiated phenotype of SMCs cultured on
laminin-coated dishes in the presence of insulin. The differentiated
phenotype-specific expression of TM and CaD was maintained even in
10-day cultured SMCs under conditions whereby -TM-SM,
-TM-SM, and
h-CaD were continuously expressed (Fig. 5II). In contrast,
serum and PDGF-BB induced the dedifferentiated phenotype-specific
expression of TM and CaD isoforms;
-TM-SM converted to
-TM-F1 and
-TM-F2,
-TM-SM was down-regulated, and h-CaD was synchronously
changed to l-CaD (Fig. 5, III and IV). Thus, exon
selection in the
-TM and CaD genes (exons 2a and 2b in the
-TM
gene and exons 3a, 3ab, and 4 in the CaD gene) would be coordinately
regulated in a SMC phenotype-dependent manner. We also
characterized such exon selection in the TM and CaD genes by
semiquantitative RT-PCR methods during dedifferentiation of SMCs by
serum stimulation. Coordinate changes from exon 2a to exon 2b in the
-TM gene and from exons 3ab and 4 to exon 3a in the CaD gene were
observed during dedifferentiation, and these exon conversions were
completed in 8-day cultured SMCs (Fig. 6). These results
are in good accord with the Northern blotting data (Fig.
5III). Laminin retarded such isoform interconversions induced by serum (Fig. 5, III and I). PDGF-BB was
induced to down-regulate the mRNAs for
-TM-SM,
-TM-SM, and
h-CaD, and this effect was more potent than that of serum (Fig. 5,
III and IV). We analyzed cell numbers of SMC
cultured on laminin under nonstimulated and serum- or
PDGF-BB-stimulated conditions. Culture conditions were identical with
those of Northern blotting analyses described above: gizzard SMCs were
cultured on laminin-coated dishes under nonstimulated conditions for 2 days, and then the cells were stimulated by 10% FCS or 20 ng/ml
PDGF-BB. A dramatic increase in cell number was found under
serum-stimulated conditions, while a significant increase in cell
number was not observed under nonstimulated and PDGF-BB-stimulated conditions, suggesting that proliferation and dedifferentiation of SMCs are not always cooperative (Fig. 5V). In the
postconfluent culture of dedifferentiated SMCs, SMC-specific
transcripts of
-TM and CaD did not reappear (Fig.
5III).
Coexpression of
To analyze the expressional profiles of -TM-SM and
h-CaD in vivo, we carried out in situ
hybridization in developing chick embryos. For this experiment, we used
specific oligoprobes described under "Materials and Methods," which
specifically hybridized with the mRNAs for
-TM exon 2a,
-TM
exon 2b, h-CaD, and l-CaD by Northern blotting (data not shown).
Expressional profiles of the mRNAs for
-TM exon 2a,
-TM exon
2b, h-CaD, and l-CaD in 8-21-day-old chick embryos are shown in Fig.
7. The h-CaD transcripts were localized in tissues
containing SMCs such as esophagus, crop, proventriculus, gizzard,
intestine, lung, and aorta in developing embryos (Fig. 7C
and 8A). As shown in Fig. 7, A and C,
the localization of
-TM-SM transcripts coincided with that of h-CaD
transcripts, and
-TM-SM transcripts were detectable in 8-day-old
embryo. In contrast to h-CaD, l-CaD transcripts were weak and were
ubiquitously distributed (Fig. 7D). The distribution of
l-CaD mRNA in gizzard was opposite to that of h-CaD mRNA; h-CaD
mRNA was highly expressed in the muscle layer, whereas l-CaD
mRNA was restricted mainly to the inner layer of the the lumen
(Fig. 8B). The localization of
-TM-SM
mRNA probed by an exon 2a-specific oligoprobe was shown to be
identical to that of h-CaD mRNA (Fig. 7, A and
C). Exon 2b in the
-TM gene is selected in almost all
cell types except for differentiated SMCs. In 8-day-old embryo,
-TM
isoforms containing exon 2b were evenly expressed in both smooth and
skeletal muscles. During development, such expressions gradually
increased in skeletal muscles but decreased in smooth muscles (Fig.
7B).
The SMCs display phenotypic modulation from a differentiated to
dedifferentiated state under conventional culture conditions. During
dedifferentiation, the morphology of SMC shows a dramatic change, from
a long spindle-like to a proliferative fibroblast-like shape. In
accordance with this process, h-CaD, which is a highly favorable
parameter for the differentiated phenotype of SMCs, converts to l-CaD
(43-45). On the other hand, a change of CaD isoforms from the low to
high Mr form links to SMC differentiation (1, 3,
4). Isoform interconversion associated with phenotypic modulation of
SMCs is also observed in other cytoskeletal and contractile proteins,
actin (1, 2), myosin heavy chain (5, 6), and
vinculin/meta-vinculin (1, 7). Here we have identified the
epitope of TM311 mAb in the amino acid sequences encoded by exons 1a of
- and
-TM genes (Fig. 2) and used this antibody to investigate
isoform interconversion of TMs in association with phenotypic
modulation of SMCs. cDNA cloning of major TM isoforms from
differentiated and dedifferentiated SMCs and identification by
overexpression of isolated cDNAs revealed the presence of four TM
isoforms:
-TM-SM changed to
-TM-F1 and
-TM-F2, and
-TM-SM was down-regulated during dedifferentiation (Fig. 1). Since an identical change of TM isoforms was found in visceral and vascular SMCs, these molecular events were common to both phenotypes of SMC.
Based on the difference of
-SM actin expression in dedifferentiated SMCs and CEFs (Fig. 3), the expressional change of TM isoforms is
caused by phenotypic modulation of SMCs themselves, and not be due to
the replacement of differentiated SMCs by contaminated fibroblasts.
It has been reported that isoform diversity of TM is caused by
alternative splicing and differential promoter usage in the TM genes
(32). Regarding the -TM gene, exon 2a is specifically spliced in the
-TM-SM mRNA, and exon 2b is spliced in other
-TM isoform
mRNAs. Selection between exons 2a and 2b is a critical event during
phenotypic modulation of SMCs. As shown in Figs. 5 and 6, exon
selection of the
-TM gene and that of the CaD gene might be
coordinately regulated during dedifferentiation of SMCs induced by
serum or PDGF-BB stimulation. Inversely, both the mRNAs for h-CaD
and
-TM-SM were developmentally coexpressed in visceral and vascular
smooth muscles at each stage of chick embryos (Figs. 7 and 8). Since
selection of exon 2a in the
-TM gene and exons 3b and 4 in the CaD
gene is specific for differentiated SMCs, such splicings seem to be
controlled under a common regulatory mechanism.
Mutually exclusive splicing between exons 2a and 2b in the -TM gene
has been intensively studied. Exon 2b is selected in almost all cell
types except for SMCs. The possible explanation of this event is that
predominant selection of exon 2b may be based on a competition between
exons 2a and 2b; the branch point/pyrimidine tract elements in the
downstream intron of exon 2b are stronger than those of exon 2a (37).
The functional strength of exon selection depends on an affinity of
splicing factor U2AF for the pyrimidine tract for either exon.
Alternatively, the SMC-specific selection of exon 2a may be due to
suppression of exon 2b selection; two conserved elements in each of the
introns flanking exon 2b are essential for such SMC-specific
suppression (38). The third possibility is that the splicing
machineries themselves may be regulated in a SMC
phenotype-dependent fashion. The SMC-specific splicing in
the CaD gene has been only scarcely investigated. Recently, it has been
reported that repeating purine-rich motifs in exon 3b act as an exon
enhancer element, causing predominant selection of distal 5
-splice
site in myofibroblasts and nonmuscle cells (46). However, a different
result was obtained in our separate experiments, suggesting that the
intron sequence between exons 3b and 4 might be involved in alternative
selection of distal and proximal 5
-splice sites within exon 3. At
present, it remains unclear whether common factor(s) are involved in
such exon selection in the
-TM and CaD genes. Further study will be
necessary to reveal the phenotype-dependent coordinate
splicing mechanism of these genes.
Compared with immediate changes in exon selection in the -TM and CaD
genes during the dedifferentiation process, the down-regulation of
-TM is moderate. These molecular events are characteristic of SMC
phenotype-dependent changes. While transcriptional
machineries of the TM genes have not been well characterized, the
upstream promoter of the
-TM gene has been partially analyzed in a
skeletal muscle cell line; a CArG box-like motif was identified as one of the essential cis-elements necessary for skeletal
muscle-specific transcription (47). A CArG box-like motif was found in
the 5
-upstream regions of muscle-specific genes such as the
-skeletal (48) and
-SM actin genes (8, 9), the CaD gene (12), the
SM22 gene (21), and the myosin heavy chain gene (9). In the case of the
-skeletal actin gene, a CArG box-like motif functions as either a
positive or negative regulatory element; trans-acting factors (serum response factor and YY1) bound to the CArG box-like motif may be involved in positive and negative regulations,
respectively (49). Detailed promoter analysis of the
-TM gene
including transcription factor(s) is of future interest.
As shown in Figs. 1 and 5I, primary cultured SMCs by
serum-stimulation convert their phenotype from a differentiated to
dedifferentiated state using molecular markers such as TM and CaD
isoforms. Serum-derived growth factors and extracellular matrices such
as fibronectin are known to promote dedifferentiation of SMCs (50). In
a separate experiment, we found that laminin and insulin show a potent
ability to maintain a differentiated phenotype of primary cultured
SMCs.2 In fact, laminin is abundant in
extracellular matrices of both aortic media and visceral muscle layers
(51), and its expression is developmentally regulated (52). It has also
been reported that laminin is involved in functional differentiation of
mammary gland epithelial cells to activate the -lactoglobulin gene
(53). In regard to gene expression, such as the
- and
-TM and CaD genes, these gene expressions were found in differentiated SMCs only in
the presence of laminin and insulin (Fig. 5II). Despite the
presence of laminin, PDGF-BB induced isoform changes of
-TM-SM and
h-CaD and down-regulation of
-TM-SM with much more potency than
serum (Fig. 5, III and IV). However, the PDGF-BB
did not promote cell proliferation (Fig. 5V). These results
suggest that dedifferentiation of SMCs is independent of cell
growth.
There is no report investigating the expressional change of TM isoforms
associated with phenotypic modulation of SMCs. Most recently, a
developmental change of TM isoforms has been demonstrated using mouse
embryonic stem cells and embryos; -TM-SM mRNA is expressed in
undifferentiated embryonic stem cells and in all stages of developing
embryoid bodies, whereas the mRNA is detectable in postcoitum
embryo (4.5 days) and increases during development, suggesting that
expression of
-TM-SM occurs at very early developmental stages (54).
However, these results are unclear with respect to localization of TM
isoform mRNAs, because such study has been performed by RT-PCR
using RNAs from whole embryoid bodies and embryos. Here we have been
the first to demonstrate the localization and developmental changes of
TM and CaD mRNAs by in situ hybridization. The mRNAs
for
-TM-SM and h-CaD were confined to tissues containing SMCs and
were clearly coexpressed in each embryonic stage. Thus, expressions of
-TM-SM and h-CaD are coordinately regulated in vivo as
well as in vitro. In conclusion, alternative splicing in
exons 2a and 2b in the
-TM gene and exons 3 and 4 in the CaD gene
might be controlled by a common mechanism that is closely associated
with SMC phenotype.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D87891[GenBank]- D87893[GenBank].