Coordinate Expression of alpha -Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells*

(Received for publication, October 2, 1996, and in revised form, April 14, 1997)

Kouji Kashiwada , Wataru Nishida , Ken'ichiro Hayashi , Kentaro Ozawa , Yuka Yamanaka , Hiroshi Saga , Toshihide Yamashita Dagger §, Masaya Tohyama Dagger , Shoichi Shimada Dagger , Kohji Sato Dagger and Kenji Sobue

From the Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, the Dagger  Department of Anatomy and Neuroscience, and the § Department of Molecular Neurobiology (TANABE), Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the alpha -tropomyosin (alpha -TM) gene, exon 2a rather than exon 2b is specifically spliced in alpha -TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of alpha -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 beta -TM-SM, alpha -TM-SM converted to alpha -TM-F1 and alpha -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 alpha -TM-SM, beta -TM-SM, and high Mr caldesmon. In situ hybridization revealed specific coexpression of alpha -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.


INTRODUCTION

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 alpha -smooth muscle actin (alpha -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 alpha -SM actin (8, 9), CaD (12), myosin heavy chain (13), SM22 (21), and calponin (22) have been partially characterized.

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 beta -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 alpha -TM genes, exon 2b rather than exon 2a (termed exons 3 and 2, respectively, in Ref. 35) is spliced in the high Mr alpha -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 alpha -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.

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 alpha -TM (alpha -TM-SM) converted to fibroblast-type 1 and 2 alpha -TM isoforms (alpha -TM-F1 and alpha -TM-F2) by a selectional change from exon 2a to exon 2b, while expression of SMC-type beta -TM (beta -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 alpha -TM-SM and h-CaD was maintained in differentiated SMCs cultured on laminin-coated dishes without serum. In situ hybridization revealed coexpression of alpha -TM-SM and h-CaD mRNAs in smooth muscle in developing embryos. The present results, therefore, suggest that the alpha -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.


MATERIALS AND METHODS

Cell Culture

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

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.

Preparation of Tropomyosin and Actin-binding Assay

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 Cloning and Expression of TM Isoforms

cDNA libraries of SMCs and of dedifferentiated SMCs were constructed in lambda gt11. The libraries were screened using TM311 mAb. Alternatively, we amplified partial cDNA fragments carrying alpha -TM exons 3, 4, and 5 or beta -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.

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 alpha -TM, exons 2b to 9d (containing the 3'-noncoding region) of alpha -TM, exon 1a of beta -TM, exons 2 to 9d (containing the 3'-noncoding region) of beta -TM, or exons 1b to 6b of alpha -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.

We constructed expression vectors carrying each of the cDNAs for TM isoforms downstream of the chicken beta -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.

Immunofluorescence Microscopy

Expression of alpha -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-alpha -SM actin monoclonal antibody (Sigma) and then were double-stained with both fluorescein isothiocyanate-labeled anti-mouse IgG antibody and rhodamine-phalloidin.

Northern Blotting

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


Probes Nucleotide positions (GenBankTM accession No.) Labeling Hybridization buffer/temperature Washing buffer/temperature

 alpha -TM/E2a 128-147 of alpha -TM-SM  (D87893[GenBank]) [gamma -32P]ATPa HB-Ab/48 °C WB-Ac/52 °C
 alpha -TM/E2b 264-283 of alpha -TM-F1  (D87891[GenBank]) [gamma -32P]ATP HB-A/52 °C WB-A/52 °C
 beta -TM/E1a-E2 28-263 of beta -TM-SM  (K02446[GenBank]) [alpha -32P]dCTPd HB-Be/42 °C WB-Bf/42 °C
CaD/E3b 811-932 of h-CaD  (M28417[GenBank]) [alpha -32P]dCTP HB-B/42 °C WB-B/42 °C
CaD/E3a-E5 757-776 of l-CaD  (M59762[GenBank]) [gamma -32P]ATP HB-A/52 °C WB-A/52 °C

a Antisense oligo-DNA was phosphorylated by [gamma -32P]ATP using T4 polynucleotide kinase (Takara).
b cDNA fragment was amplified by PCR, and antisense strand DNA was labeled by [alpha -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.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis

Oligo(dT)15-primed single-stranded cDNAs were synthesized from 3 µg of total RNAs using RAV-2 reverse transcriptase (Takara). <FR><NU>1</NU><DE>80</DE></FR>, <FR><NU>2</NU><DE>80</DE></FR>, and <FR><NU>4</NU><DE>80</DE></FR> 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 alpha -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 alpha -TM-common (or CaD-common), alpha -TM-exon2a (or h-CaD), and alpha -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]).

Table II. PCR primer sets


Primer sets 5'-Primera 3'-Primera Cycle number

 alpha -TM-exon 2a 5'-TTCCCTGCTCTGATTTCGGC-3' 5'-GAAGAGGAGGCTGTCCTCAG-3' 24
  24 -43 of alpha -TM-F1 (D87891[GenBank])   202 -221 of alpha -TM-SM (D87893[GenBank])
 alpha -TM-exon 2b 5'-TTCCCTGCTCTGATTTCGGC-3' 5'-GTGCATCTTTAAGGGACTCG-3' 24
  24 -43 of alpha -TM-F1 (D87891[GenBank])   264 -283 of alpha -TM-F1 (D87891[GenBank])
 alpha -TM-common 5'-GTAGCTTCCCTGAACAGACG-3' 5'-GATAGTTCAGCACGCTCCTC-3' 24
  334 -353 of alpha -TM-F1 (D87891[GenBank])   619 -638 of alpha -TM-F1 (D87891[GenBank])
h-CaD 5'-TTGTTGGAGAGACTGGCAAGACGG-3' 5'-CTTTAGCCTCTTTGTCTTCC-3' 24
  478 -501 of h-CaD (M28417[GenBank])   1473 -1492 of h-CaD (M28417[GenBank])
l-CaD 5'-TTGTTGGAGAGACTGGCAAGACGG-3' 5'-TGTCTTTTACCTGATTTTCC-3' 27
  434 -457 of l-CaD (M59762[GenBank])   757 -776 of l-CaD (M59762[GenBank])
CaD-common 5'-TTGTTGGAGAGACTGGCAAGACGG-3' 5'-CTGATTTTCCTCTGTGGGTA-3' 22
  478 -501 of h-CaD (M28417[GenBank])   791 -810 of h-CaD (M28417[GenBank])

a Nucleotide positions and GenBankTM accession numbers (in parentheses) are shown below each primer.

In Situ Hybridization of Chick Embryos

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 [alpha -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).

Table III. In situ hybridization oligonucleotide probes


Targets Sequencea

 alpha -TM exon 2a 5'-AAGAGGAGGCTGTCCTCAGACTTGTGCAGCTCTTCCAGCACTTG-3'
  177 -220 of alpha -TM-SM (D87893[GenBank])
 alpha -TM exon 2b 5'-AGTTCCAACTTTTCCTGTGCATCTTTAAGGGACTCGGAGTATTT-3'
  256 -299 of alpha -TM-F1 (D87891[GenBank])
h-CaD (exon 3b) 5'-GCTTGAGCTTTTTCCTCTTGGGCTTTCTTCTCTTCCATCTTTTTC-3'
  1407 -1451 of h-CaD (M28417[GenBank])
l-CaD (short bridge region of exons 3a and 5) 5'-TTCTTTATCCTTGTTGTCTTTTACCTGATTTTCCTCTGTGGGTA-3'
  747 -790 of l-CaD (M59762[GenBank])

a Nucleotide positions and GenBankTM accession numbers (in parentheses) are shown below each primer.


RESULTS

Expressional Change of TM Isoforms during SMC Dedifferentiation

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).


Fig. 1. Expressional change of TM isoforms during dedifferentiation of SMCs. I, cell lysates from precultured and cultured gizzard and vascular SMCs were analyzed by two-dimensional gel electrophoreses (left, basic; right, acidic) followed by Western blotting using TM311 mAb (A-D, H, and I). A, precultured SMCs; B, 3-day cultured SMCs; C, 7-day cultured SMCs; D, two-passaged gizzard SMCs; H, precultured SMCs; I, 7-day cultured vascular SMCs. TM isoforms detected by this antibody (spots a, c, 1, and 2) and low Mr TM isoforms (spots 3a and 3b) are illustrated (E). The identification of TM isoforms by actin-binding assay is shown (F and G). The TM fraction prepared from differentiated (F) and dedifferentiated gizzard SMCs (G) were cosedimented with rabbit skeletal muscle F-actin, and precipitated proteins were separated by two-dimensional gel electrophoresis and stained by Coomassie Brilliant Blue. II, identification of TM isoforms by forced expression of cloned TM cDNAs (A-E). Cell lysates from dedifferentiated SMCs transfected with expression vectors carrying cloned TM cDNAs were analyzed by the same procedure as described above. A, vector only; B, SM-alpha -TM; C, SM-beta -TM; D, alpha -TM-F1; E, alpha -TM-F2 cDNAs.
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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 alpha - or beta -TM isoforms; exon 1a of alpha -TM; exons 2b, 3, 4, 5, 6b, 7, 8, and 9d of alpha -TM; exon 1a of beta -TM; exons 2, 3, 4, 5, 6a, 7, 8, and 9d of beta -TM; and exons 1b, 3, 4, 5, and 6b of alpha -TM. The TM311 mAb only cross-reacted with truncated proteins including exons 1a of both alpha - and beta -TMs, but not with the other truncated proteins (Fig. 2). Since amino acid sequences encoded by exons 1a of the alpha - and beta -TM genes are 87% identical, the TM311 mAb is believed to recognize the limited regions of the high Mr alpha - and beta -TM isoforms encoded by the respective exons 1a.


Fig. 2. Epitope mapping of TM311 mAb. GST (a) and GST-truncated TM fusion proteins such as GST-alpha -TM exon 1a (b), GST-alpha -TM exons 2b, 3, 4, 5, 6b, 7, 8, and 9d (c), GST-beta -TM exon 1a (d), GST-beta -TM exons 2, 3, 4, 5, 6a, 7, 8, and 9d (e), and GST-alpha -TM exons 1b, 3, 4, 5, and 6b (f) were expressed in E. coli. Purified fusion proteins were subjected to SDS-PAGE followed by Western blotting using anti-GST antiserum (A) or TM311 mAb (B).
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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 alpha -SM actin between dedifferentiated SMCs under serum-stimulated culture conditions and CEFs; alpha -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). alpha -SM actin was also undetectable in differentiated visceral SMCs (Fig. 3, A and D). The major actin isoform expressed in the cells is gamma -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 alpha -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.


Fig. 3. Expression of alpha -SM actin in different phenotypes of gizzard SMCs and CEFs. Differentiated gizzard SMCs (A and D), dedifferentiated gizzard SMCs under serum-stimulated culture conditions (B and E), and CEFs (C and F) were stained with rhodamine-phalloidin (A, B, and C) or anti-alpha -SM actin monoclonal antibody (D, E, and F). Gizzard SMCs started to express alpha -SM actin during phenotypic modulation. alpha -SM actin is only scarcely expressed in CEFs.
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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 Phenotype

To identify the major TM isoforms detected by TM311 mAb, we screened cDNA libraries of differentiated and dedifferentiated gizzard SMCs with TM311 mAb or specific alpha - or beta -TM cDNA probes and obtained four different clones. Sequencing of two clones from differentiated SMCs revealed alpha -TM-SM (36) and beta -TM-SM (42), and two other clones from dedifferentiated SMCs were alpha -TM-F1 and alpha -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 alpha -TM-F1, alpha -TM-F2, and alpha -TM-SM have been speculated upon from the genomic structure of the alpha -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 alpha -TM-SM or beta -TM-SM cDNAs showed newly intensive spots corresponding to spots a or c (Fig. 1II, B and C). Transfection of alpha -TM-F1 and alpha -TM-F2 cDNAs increased in spots 1 and 2, respectively (Fig. 1II, D and E). These results indicate that alpha -TM-SM and beta -TM-SM (spots a and c) substitute for alpha -TM-F1 and alpha -TM-F2 (spots 1 and 2) during dedifferentiation of SMCs. Based on the present results, exon structures of the alpha - and beta -TM genes and their alternative splicings for alpha -TM-F1 and alpha -TM-F2 are summarized in Fig. 4, A and B. A significant difference among alpha -TM-SM, alpha -TM-F1, and alpha -TM-F2 is the selection between exons 2a and 2b; exon 2a is specifically spliced in the mRNA for alpha -TM-SM in differentiated SMCs, whereas the mRNAs for alpha -TM-F1 and alpha -TM-F2 in dedifferentiated SMCs contain exon 2b, indicating that this exon selection is dependent on the SMC phenotype. In contrast, expression of beta -TM-SM was down-regulated during SMC dedifferentiation.


Fig. 4. Structures of alpha -, beta -TM, and CaD genes and splicing pathways associated with the phenotype of SMCs. Exon alignment and numbering are according to previous reports. A, alpha -TM gene (36); B, beta -TM gene (55); C, CaD gene (11). mRNA sizes of TM and CaD isoforms are shown on the right.
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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 alpha -TM gene, which is spliced in every mRNA for alpha -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 alpha -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 alpha -TM isoforms. Therefore, selection between exons 2a and 2b in the alpha -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 alpha -TM-F1 and the mRNAs for alpha -TM-SM and alpha -TM-F2 contained exon 6a and exon 6b, respectively.


Fig. 5. Expression of alternatively spliced TM and CaD mRNAs during phenotypic modulation of SMCs. Gizzard SMCs were cultured on uncoated dishes under serum stimulation (I) and on laminin-coated dishes (II-IV) as described under "Materials and Methods." The SMCs in the presence of 10% FCS were cultured on uncoated dishes (I). The cells were cultured on laminin-coated dishes in the presence of bovine serum albumin and insulin (II). At 3 days of culture, SMCs on laminin-coated dishes were stimulated by 10% FCS (III) or 20 ng/ml PDGF-BB (IV), and they were further cultured for 1-7 days under the same conditions (corresponding to 4-10 days of culture). Total RNAs were isolated from precultured gizzard SMCs (P) and cultured gizzard SMCs at the indicated days (days 3-10). Ten micrograms of RNAs were separated on formalin-agarose gels, transferred to nylon membranes, and hybridized with the indicated exon-specific DNA probes. 28 S rRNAs were stained by methylene blue. Relative cell numbers of SMCs cultured on laminin under nonstimulated (circles) and serum-stimulated (squares), or PDGF-BB-stimulated (triangles) conditions are shown (V). Culture conditions were identical with those of Northern blotting analyses.
[View Larger Version of this Image (33K GIF file)]

cDNA probe expanding from exon 3 to exon 5 of the beta -TM gene was able to detect a 1.2-kilobase mRNA for beta -TM-SM and a 1.1-kilobase mRNA for fibroblast type beta -TM isoform, but the latter mRNA was expressed at an extremely low level (data not shown). During dedifferentiation, expression of beta -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 alpha -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 alpha -TM-SM, beta -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; alpha -TM-SM converted to alpha -TM-F1 and alpha -TM-F2, beta -TM-SM was down-regulated, and h-CaD was synchronously changed to l-CaD (Fig. 5, III and IV). Thus, exon selection in the alpha -TM and CaD genes (exons 2a and 2b in the alpha -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 alpha -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 alpha -TM-SM, beta -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 alpha -TM and CaD did not reappear (Fig. 5III).


Fig. 6. RT-PCR analysis of expressional changes of alpha -TM and CaD isoforms during dedifferentiation of SMCs under serum-stimulated culture conditions. Three micrograms of total RNAs prepared for Northern blotting (Fig. 5III) were subjected to RT-PCR (I). Oligo(dT)15-primed single-stranded cDNAs were used as PCR templates. PCRs were carried out under conditions summarized in Table II, and the products were stained with SYBR Green I. Relative contents of each mRNA were estimated by multiregression analysis as described under "Materials and Methods" (II). The relative ratios of alpha -TM mRNA containing exon 2a or exon 2b to total alpha -TM mRNAs and the ratios of high or low Mr CaD mRNA to total CaD mRNAs are shown. Open triangles, alpha -TM mRNA containing exon 2a/total alpha -TM mRNAs; closed triangles, alpha -TM mRNA containing exon 2b/total alpha -TM mRNAs; open circles, h-CaD mRNA/total CaD mRNAs; closed circles, l-CaD mRNA/total CaD mRNAs.
[View Larger Version of this Image (26K GIF file)]

Coexpression of alpha -TM-SM and h-CaD mRNAs in Developing Chick Embryos

To analyze the expressional profiles of alpha -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 alpha -TM exon 2a, alpha -TM exon 2b, h-CaD, and l-CaD by Northern blotting (data not shown). Expressional profiles of the mRNAs for alpha -TM exon 2a, alpha -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 alpha -TM-SM transcripts coincided with that of h-CaD transcripts, and alpha -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 alpha -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 alpha -TM gene is selected in almost all cell types except for differentiated SMCs. In 8-day-old embryo, alpha -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).


Fig. 7. Localization of CaD and TM mRNAs in developing chick embryos. In situ hybridization using alpha -TM exon 2a (A), alpha -TM exon 2b (B), h-CaD (C), and l-CaD (D) probes was performed. Sagital sections through 8- (E8), 12- (E12), 15- (E15), and 21- (E21) day-old chick embryos are represented. As a negative control, a sense probe was hybridized to 21-day-old embryo (Sense). br, brain; ey, eye; he, heart; lu, lung; pr, proventriculus; gi, gizzard; in, intestine; sk, skeletal muscle.
[View Larger Version of this Image (52K GIF file)]


Fig. 8. Localization of h-CaD (A) and l-CaD (B) mRNAs in 15-day-old chick embryo. In situ hybridization using an h-CaD-specific probe (A and B (left)) and a l-CaD probe (B, right) was performed. br, brain; ey, eye; es, esophagus; cr, crop; ao, aorta; he, heart; lu, lung; pr, proventriculus; gi, gizzard; in, intestine.
[View Larger Version of this Image (55K GIF file)]


DISCUSSION

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 alpha - and beta -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: alpha -TM-SM changed to alpha -TM-F1 and alpha -TM-F2, and beta -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 alpha -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 alpha -TM gene, exon 2a is specifically spliced in the alpha -TM-SM mRNA, and exon 2b is spliced in other alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -TM and CaD genes during the dedifferentiation process, the down-regulation of beta -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 beta -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 alpha -skeletal (48) and alpha -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 alpha -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 beta -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 beta -lactoglobulin gene (53). In regard to gene expression, such as the alpha - and beta -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 alpha -TM-SM and h-CaD and down-regulation of beta -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; alpha -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 alpha -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 alpha -TM-SM and h-CaD were confined to tissues containing SMCs and were clearly coexpressed in each embryonic stage. Thus, expressions of alpha -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 alpha -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.


FOOTNOTES

*   This research was supported by Grants-in-Aid for COE research from the Ministry of Education, Science and, Sports Culture of Japan (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].


   To whom all correspondence should be addressed. Tel.: 81-6-879-3680; Fax: 81-6-879-3689; E-mail: sobue{at}nbiochem.med.osaka-u.ac.jp.
1   The abbreviations used are: SMC, smooth muscle cell; CaD, caldesmon; h-CaD, high Mr CaD; l-CaD, low Mr CaD; TM, tropomyosin; alpha -SM actin, alpha -smooth muscle actin; PDGF-BB, platelet-derived growth factor-BB; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); FCS, fetal calf serum; mAb, monoclonal antibody; DTT, dithiothreitol; PCR, polymerase chain reaction; GST, glutathione S-transferase; CEFs, chick embryo fibroblasts; RT-PCR, reverse transcriptase-polymerase chain reaction.
2   K. Hayashi, Y. Chimori, H. Saga, and K. Sobue, manuscript in preparation.

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