From the Cardiovascular Biology Laboratory,
Harvard School of Public Health, the § Department of
Medicine, Harvard Medical School, and the ¶ Cardiovascular
Division, Brigham and Women's Hospital, Boston, Massachusetts 02115 and the
Division of Developmental Biology, National Institute
for Medical Research, London NW7 1AA, United Kingdom
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ABSTRACT |
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The change in vascular smooth muscle cells (SMC)
from a differentiated to a dedifferentiated state is the critical
phenotypic response that promotes occlusive arteriosclerotic disease.
Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an
in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent
induction of the SMC markers smooth muscle -actin, smooth muscle
myosin heavy chain, calponin, SM22
, and APEG-1. The activity of the SM22
promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the
wild-type SM22
promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we
identified new DNA-protein complexes in nuclear extracts prepared from
differentiated Monc-1 cells. One of the new complexes contained serum
response factor. This Monc-1 to SMC model should facilitate the
identification of nodal regulators of smooth muscle development and
differentiation.
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INTRODUCTION |
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Arteriosclerosis and its complications (heart attack and stroke) are the major causes of death in developed and developing countries (1), and the change in vascular smooth muscle cells (SMC)1 from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease (1-3). Despite its importance, we know little about the genes that regulate SMC differentiation (2). This is in contrast with our more thorough understanding of transcription factors involved in the differentiation of skeletal muscle cells (4-7). A major reason for the rapid progress in our understanding of skeletal as opposed to smooth muscle biology is the availability of an in vitro system for studying differentiation of skeletal muscle cells. The ability to induce myogenic differentiation by the use of demethylating agents and serum starvation in 10T1/2 cells and C2C12 cells, respectively, has been critical to the study of skeletal muscle development and differentiation (8, 9). Indeed, the establishment of immortalized myogenic cell lines may have been the single most important development in the field of skeletal myogenesis (10). Until the neural crest cell to SMC differentiation system described here, a way of rapidly and uniformly inducing precursor cells to differentiate into SMC had been lacking.
Pluripotent neural crest cells can differentiate into neurons, glia,
chondrocytes, melanocytes, and SMC (11-13). Arterial SMC of the chick
ascending and thoracic aorta are of a neural crest origin (14-16), and
various members of the transforming growth factor- superfamily can
instructively promote differentiation of primary cultured neural crest
cells into neuronal cells or SMC (12). Unfortunately, our ability to
work with neural crest cells in primary culture has been limited by the
difficulty of obtaining quantities sufficient for biochemical and
genetic analysis. This problem was solved recently by the generation of
an immortalized neural crest cell line, Monc-1, by retroviral
transfection of mouse neural crest cells with the v-myc gene
(17, 18).
We hypothesized that Monc-1 cells could be used to develop an in
vitro SMC differentiation system. We describe in this report the
culture conditions under which Monc-1 cells can be differentiated efficiently into SMC. Exclusive Monc-1 to SMC differentiation was
indicated by cellular appearance and induction of the SMC markers
smooth muscle -actin, smooth muscle myosin heavy chain, calponin,
SM22
, and APEG-1. Also, a 20-30-fold increase in the activity of
the SMC-specific promoter SM22
coincided with the formation of new
DNA-protein complexes during differentiation.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Reagents-- The Monc-1 cell line was kindly provided by David Anderson (Pasadena, CA). Monc-1 cells were cultured in the undifferentiated state on fibronectin-coated plates in an L-15 CO2-based medium supplemented with chick embryo extract, hereafter referred to as complete medium, as described by Stemple and Anderson (11). Differentiation down the neuronal and glial pathways was performed on plates coated sequentially with poly-D-lysine (0.5 mg/ml) and fibronectin (0.25 mg/ml) in complete medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) plus 5 mM forskolin (Sigma) as described (17). SMC differentiation was induced by application of M199 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone), penicillin (100 units/ml), streptomycin (100 µg/ml), and 25 mM HEPES (pH 7.4), hereafter referred to as SMC differentiation medium (SMDM).
RNA Extraction, Northern Analysis, and Reverse Transcription
Polymerase Chain Reaction (PCR)--
Total RNA from cultured cells was
prepared by guanidinium isothiocyanate extraction and centrifugation
through cesium chloride (19). RNA from mouse aorta was prepared by the
RNAzol B method (Tel-Test, Friendswood, TX) according to the
manufacturer's instructions. Total RNA was fractionated on a 1.3%
formaldehyde-agarose gel and transferred to nitrocellulose filters,
which were hybridized with the appropriate, randomly primed,
32P-labeled probe (19, 20). The smooth muscle -actin was
provided by J. Lessard (Cincinnati, OH); the angiotensin type II
receptor (AT2) cDNA was provided by A. D. Strosberg (Paris,
France). The calponin cDNA was isolated from a mouse aortic
cDNA library.
Immunocytochemistry and Western Analysis--
Monc-1 cells were
grown on glass slides coated with fibronectin or fibronectin plus
poly-D-lysine in the appropriate medium (see "Cell
Culture and Reagents"). Immunostaining for smooth muscle -actin,
calponin, glial fibrillary acidic protein, and peripherin was performed
as described (12, 21). Proteins from undifferentiated and
differentiated Monc-1 cells and mouse aortas were prepared according to
standard procedures (19) with minor modifications. Proteins were
resolved on 5% sodium dodecyl sulfate-polyacrylamide gels (22),
transferred electrophoretically to nitrocellulose membranes (Schleicher
and Schuell), and incubated with a rabbit anti-smooth muscle myosin
heavy chain antibody (23) (kindly provided by Ute Groschel-Stewart of
London, UK) diluted 1:5000, followed by incubation with a horseradish
peroxidase-conjugated goat anti-rabbit antibody diluted 1:4000.
Membranes were processed with an enhanced chemiluminescence reagent
(Pierce) and exposed to film.
Transfection and Luciferase Assays--
A 1.4-kilobase fragment
of the SM22 promoter was obtained by PCR with mouse genomic DNA and
the following primers: forward 5' CAGTGGCTGGAAAGCAAGAGC 3' and reverse
5' GGGCTGGGGCAGACGGGC 3'. The promoter fragment was subcloned into the
pGL2-Basic vector (Promega, Madison, WI). Generation of the
multimerized CArG and CArG mutant constructs in the pGL2-Basic vector
will be described elsewhere.2
Monc-1 cells were transfected transiently by electroporation as
described (24). Cell extracts were prepared 48-72 h after transfection, and luciferase and
-galactosidase assays were
performed as described (25, 26). Each construct was transfected at
least six times. Data for each construct are presented as the mean ± S.E.
Electrophoretic Mobility Shift Analysis--
Nuclear extracts
were prepared according to the method of Ritzenthaler et al.
(27) with minor modifications. Electrophoretic mobility shift analysis
was performed as described (28, 29). In brief, double-stranded
oligonucleotide probes synthesized according to the sequence of the
SM22 CArG element 5' TCGAGACTTGGTGTCTTTCCCCAAATATGGAGCCTGTGTGGAGTG 3' were radiolabeled as described (21). The reaction mixture was
incubated at room temperature for 20 min and analyzed by 5% native
polyacrylamide gel electrophoresis in 0.25 × TBE buffer (22 mM Tris base, 22 mM boric acid, and 0.5 mM EDTA). A 250-fold excess of specific or nonspecific
oligonucleotide was used for competition experiments. For supershift
experiments, 1 µl of antibody to serum response factor (sc-335x,
Santa Cruz Biotechnology, Santa Cruz, CA) or anti-YY1 antibody
(sc-281x, Santa Cruz) was incubated with nuclear extracts and
probes.
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RESULTS AND DISCUSSION |
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Identification of Culture Conditions That Promote Monc-1 to SMC Differentiation-- Monc-1 cells on fibronectin-coated plates can be maintained in the undifferentiated state in an L-15 CO2-based medium supplemented with chick embryo extract (11, 17). After confirming that Monc-1 cells incubated in this complete medium expressed the low-affinity nerve growth factor receptor (data not shown), a marker for undifferentiated neural crest cells (11, 12, 17), we looked for culture conditions that would allow us to differentiate the Monc-1 cells down the smooth muscle lineage.
Although several media had little effect on the Monc-1 phenotype, culturing the cells in M199 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone), penicillin (100 units/ml), streptomycin (100 µg/ml), and 25 mM HEPES (pH 7.4) induced a dramatic morphologic change. We studied two different lots of fetal bovine serum and observed no difference in their ability to induce Monc-1 cell differentiation. Within 24 h of placement in this SMDM, the cells began to assume a flat, fusiform appearance and the size of the cytoplasm increased. By 4 days of culture in SMDM nearly 100% of the cells had assumed this form (Fig. 1A, bottom). In comparison with undifferentiated Monc-1 cells (Fig. 1A, top), cells cultured in SMDM grew much more slowly. At confluence the differentiated cells took on the "hill and valley" appearance characteristic of differentiated SMC (not shown). Immunostaining with antibodies specific to smooth muscle
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Time-dependent Induction of SMC Markers in Response to
Monc-1 Cell Differentiation--
We also measured expression of the
mRNAs for these and other smooth muscle markers over the course of
Monc-1 cell differentiation in SMDM (at 2, 5, and 8 days). RNA from
mouse aorta was used as a positive control for differentiated SMC. As a
negative control, Monc-1 cells were induced to differentiate down the
neuronal and glial pathways (17, 18) in a parallel experiment. Smooth
muscle -actin and calponin mRNA expression increased as early as
2 days after placement in SMDM (Fig. 2).
Neither message was detected after differentiation down the neuronal
pathway.
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Induction of Smooth Muscle Myosin Heavy Chain Expression in Response to Monc-1 Cell Differentiation-- To determine whether smooth muscle myosin heavy chain, a specific marker of differentiated SMC, is expressed in Monc-1 cells, we performed reverse transcription PCR with a pair of primers that amplify 324- and 363-base pair fragments of the SM1 and SM2 isoforms, respectively. The SM1 and SM2 DNA fragments were both amplified from reverse transcribed mouse aorta RNA (Fig. 3A). SM1 was amplified from differentiated but not undifferentiated Monc-1 cell RNA. We then used primers for glyceraldehyde-3-phosphate dehydrogenase to amplify a specific band from all RNA samples. SM1 and SM2 were both detected in samples prepared from mouse aortas by high resolution Western analysis with an antibody to smooth muscle myosin heavy chain (23) (Fig. 3B). Although undifferentiated Monc-1 cells expressed only non-muscle myosin heavy chain (Fig. 3B, asterisk), differentiated Monc-1 cells expressed SM1. Taken together, these data indicate the presence of SM1 in SMC differentiated from Monc-1 cells.
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Induction of SM22 Promoter Activity by Monc-1 to SMC
Differentiation--
The molecular mechanisms regulating expression of
the SM22
gene are very well characterized. For example, we know that
a cis-acting element, the CArG box (CC A/T6 GG),
is critical for expression of SM22
in vascular SMC, in
vitro and in vivo (28, 30, 32). To see whether the same
cis-acting element is critical to induction of SM22
in
Monc-1 cells differentiated down the smooth muscle lineage, we
performed transient transfection assays with the SM22
promoter.
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Induction of Serum Response Factor DNA Binding Activity by Monc-1
to SMC Differentiation--
We hypothesized that specific
trans-acting factors may be induced after Monc-1
differentiation that would bind to the CArG element and thereby
regulate SM22 expression. We tested this hypothesis in
electrophoretic mobility shift assays with the CArG (3X)
oligonucleotide used as a probe. Five specific DNA-protein complexes
(Fig. 4B) were revealed. Complexes 1 and 4 appeared in
nuclear extracts from differentiated Monc-1 cells but not in those from
undifferentiated cells. Complex 2 was present under both conditions but
appeared to intensify after differentiation. Antibody supershift
experiments showed that complex 4 (shifted to 5) contains a protein
antigenically identical or related to serum response factor, whereas
complex 2 (shifted to 3) contains a protein related to YY1. Complex 1 was the only one visible solely in nuclear extracts from Monc-1 cells
after differentiation. The protein contained in this novel complex is
currently under investigation.
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ACKNOWLEDGEMENTS |
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The Monc-1 cell line was kindly provided by D. J. Anderson (Pasadena, CA), the antibody to smooth muscle myosin heavy chain by U. Groschel-Stewart (London), and the glyceraldehyde-3-phosphate dehydrogenase primers by A. Räisänen-Sokolowski (Boston). We thank G. K. Owens, L. Wang, N. M. Shah, and M. S. Rao for helpful suggestions, M. A. Perrella for reviewing the manuscript, B. Ith for technical assistance, and T. McVarish for editorial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL03745 (to M. T. C.), HL03274 (to N. E. S. S.), and GM53249 (to M.-E. L.).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.
This work is dedicated to the memory of Edgar Haber, M.D.
** To whom correspondence should be addressed: Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4994; Fax: 617-432-0031. E-mail: lee{at}cvlab.harvard.edu.
1 The abbreviations used are: SMC, smooth muscle cells; SMDM, SMC differentiation medium; AT2, angiotensin II receptor; PCR, polymerase chain reaction.
2 M. T. Chin and M. E. Lee, manuscript in preparation.
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REFERENCES |
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