© 1996 American Heart Association, Inc.
Articles |
Preferential Differentiation of P19 Mouse Embryonal Carcinoma Cells Into Smooth Muscle Cells
Use of Retinoic Acid and Antisense Against the Central Nervous System–Specific POU Transcription Factor Brn-2
From the Third Department of Internal Medicine (T.S., H.-S.K., M.K., M.A., M.W., N.W., Y.S., Y.Y., R.N.), University of Tokyo (Japan) and the Department of Developmental Biology and Cancer Prevention (H.H., H.F.), Tokyo Metropolitan Institute for Medical Science.
Correspondence to Ryozo Nagai, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Showa-machi, Maebashi, Gunma 371, Japan. E-mail nagai@sb.gunma-u.ac.jp.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 µmol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage–specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and
-smooth
muscle actin. Our results show that the pathway of SMC differentiation
may provide an in vitro system useful in the investigation of SMC
regulatory mechanisms (eg, transcriptional regulation) and in the
further understanding of SMC development and differentiation.
Key Words: P19 cells • smooth muscle cells • retinoic acid • transcription factors
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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The development and differentiation of SMCs play important roles in angiogenesis and the pathogenesis of vascular diseases, such as restenosis after coronary angioplasty, and in arteriosclerosis.1 2 The elucidation of the regulatory mechanisms involved in the development and differentiation of SMCs is therefore critical in further understanding the physiological and pathological vascular processes. However, little is known of the regulatory mechanisms of SMC development and differentiation. The investigation of the involved mechanisms has remained elusive because of a lack of an established in vitro model of SMC differentiation and a lack of markers of SMC differentiation and development. Recent advancements in vascular biology have addressed the latter, with notable achievements, including characterization of a number of SMC-specific genes by the authors2 3 4 5 6 and other groups.7 8 9 However, an in vitro system for SMC differentiation remained to be established. The present study was conducted to establish an in vitro model of SMC differentiation. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm.10 11 12 13 14 15 16 17 18 19 20 21 In the present study, we have shown a unique strategy to specifically enhance SMC differentiation in P19 cells. First, optimum conditions for chemical induction were determined to be high concentrations of RA (1 µmol/L). Because P19 cells with induction by a high concentration of RA also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation in order to enhance SMC differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage–specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, SMC differentiation by P19 cells was greatly enhanced. Differentiation into SMCs was documented by immunohistochemical studies, RNA expression analysis (eg, RNase protection assay), and protein analysis (eg, Western blot) using SMC specific markers (eg, SM1 and calponin) and
-SM actin. Our results show that the
presented pathway of SMC differentiation may provide an in
vitro system useful in the investigation of SMC regulatory mechanisms
(eg, transcriptional regulation) and in the further understanding of
SMC development and differentiation. |
Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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P19 Cell Culture and Induction of Differentiation
P19 cells were cultured in modified
-minimal
essential medium (Sigma Chemical Co) supplemented with 10% fetal calf
serum (GIBCO Laboratories) as previously
described.17 18 20 Cells in the
exponential growth phase
were trypsinized and transferred to bacterial grade Petri dishes to
form aggregates with a cell density of 
105 cells per
milliliter. Fresh medium was added every other day. After 4 days of
culture in bacterial grade dishes, 200 cell aggregates were plated
on 100-mm tissue culture dishes. To induce P19 cells to differentiate
into SMCs, various regimens were used, including low concentrations of
RA (low RA, 10 nmol/L) and high concentrations of RA (high RA, 1
µmol/L) for 2 days12 (RA for both low and high
concentrations was from Sigma), DMSO (1%, Nacalai Tesque) for 2
days,13 and HMBA (2 mmol/L, Sigma) for 4
days.21
Stable Transformant With Brn-2 Antisense Expression
Vector
A stable transformant with an antisense RNA expression vector
of
the POU-domain transcription factor Brn-2 was prepared as described
previously.22 In the present study, P19 cells were
used with either a Brn-2 antisense RNA expression vector (cell line
227) or pGKneo alone (cell line 215), with the latter used as a
control.
cDNA Cloning of Mouse SM1 and Sequencing
To clone mouse SM1
cDNA, a mouse uterine 
gt 11 cDNA library
(CloneTech) was screened with a 32P-labeled DNA fragment
that was derived from the EcoRI-HindIII fragment
(-43 nt to +389 nt) of the 1H61 rabbit SM1 clone, which had
been isolated by screening a rabbit aorta ZapII cDNA library with
SMHC40.4 Clones (1x106) were
transferred to plaque/colony hybridization transfer membranes (New
England Nuclear) and hybridized with 32P-labeled DNA
fragments in 50% formamide, 0.1% SDS, 5x Denhardt's solution,
denatured salmon sperm DNA, and 5x SSPE (0.75 mol/L NaCl, 50 mmol/L
NaH2PO4, and 5 mmol/L EDTA) at 42°C
for 18 hours. Membranes were washed in 2x SSC and 0.1% SDS at 42°C.
Positive clones were subcloned into pBluescript II SK(-)
(Stratagene Inc), and enzymatic extension reactions were performed
using the Taq dye terminator cycle sequencing kit (Applied Biosystems
Inc) with DNA thermal cycler 480 (The Perkin-Elmer Corp and Cetus
Corp). Nucleotide sequences were analyzed by DNA
sequencing systems (model 373A, Applied Biosystems).
RNA Preparation and RNase Protection Assay
Total RNA was
prepared from cultured P19 cells using RNAzol
(Biotecx Laboratory Inc).
The SM1 probe was prepared by subcloning the
EcoRI-BstXI fragment (Fig 1a
) of the mouse
SM1
cDNA clone into pBluescript II SK(-). This plasmid was linearized
with EcoRI, and a riboprobe of 235 nt was synthesized using
T3 polymerase (Promega Corp). The protected fragment of SM1 RNA was 175
nt (-76 to +99 nt; translation initiation site, +1 nt)
(Fig 1b).
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The calponin probe was prepared by subcloning the EcoRI–Pst I fragment of the mouse calponin cDNA clone (MMH1CALA,23 a kind gift from Dr Shunichiro Taniguchi, Kyushu University, Japan) into pBluescript II SK(-). The plasmid was linearized with EcoRI, and a riboprobe of 377 nt was synthesized using T3 polymerase (Promega Corp). The protected fragment of calponin RNA was 310 nt.
As for the -SM
actin probe, two oligonucleotides
were synthesized on the basis of the mouse -SM actin cDNA
sequence24 : a sense primer,
5'-CCTGAGAAGCTTCTCCAGCTATGTG-3' (-20 to +5 nt), and
an
antisense primer, 5'- AGCCCTGGTACCATCATCA-3' (+123 to
+142 nt),
which contain HindIII and Kpn I sites,
respectively. A 162-bp fragment, which was not homologous with other
actin isoforms, was amplified by polymerase chain reaction using DNA
reverse-transcribed from poly(A)+ RNA of mouse aorta
with the cDNA synthesis system plus (Amersham International) and then
subcloned into the HindIII–Kpn I sites of
pBluescript II SK(-). This plasmid was linearized with
EcoRI, and a riboprobe was synthesized using T7 polymerase.
This riboprobe was 125 nt in length and contained a protected fragment
of 95 nt with -SM actin RNA.
As an internal control, a probe for
GAPDH was prepared with two
oligomers synthesized on the basis of the mouse GAPDH cDNA
sequence25 : sense primer,
5'-GCCAAGGATATCCATGACAACT-3'
(+474 to +495 nt), and antisense primer,
5'-CATCCACAGAATTCTGGGTGGCAGTGAT-3' (+534 to 561 nt),
which contain
EcoRV and EcoRI sites, respectively. An 87-bp
fragment was amplified by polymerase chain reaction using cDNA
reverse-transcribed from poly(A)+ RNA of mouse liver
and subcloned into EcoRI-EcoRV sites of
pBluescript II SK(-). The plasmid was linearized with
Xho I, and a riboprobe of 163 nt was synthesized using T3
polymerase. The protected fragment with GAPDH RNA was 68 nt.
RNA
samples were hybridized with a mixture of the two probes (either
GAPDH and -SM actin probes, GAPDH and SM1 probes, or GAPDH and
calponin probes), which was then digested with RNase using the
ribonuclease protection assay RPA II kit (Ambion Inc) and
analyzed on polyacrylamide gels as previously
described.6
SDS-PAGE and Immunoblotting
Myosin was extracted from
cultured cells according to methods
described elsewhere.26 The protein concentration was
measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories).
Protein (120 µg) was loaded in each lane and separated on 5%
SDS-PAGE. The gels were stained with Coomassie blue, or the proteins
were electrotransferred onto nitrocellulose membranes. The membranes
were stained immunologically as previously described.5
Indirect Immunofluorescence
Cultured cells were fixed in 2%
paraformaldehyde for 10 minutes and then
permeabilized with 0.2% Triton X-100 for 10 minutes,
as previously described.18 After blocking with 5% skim
milk in PBS, the samples were incubated for 100 minutes with
anti–-SM actin antibody (1A4, Dako, A/S) diluted at 1:25, anti-SM1
antibody diluted at 1:10, or anti-SM2 antibody diluted at
1:155 6 27 as the primary antibodies.
Rhodamine-conjugated goat anti-mouse IgG antibody was used as
the second antibody and was incubated for 60 minutes. Between each
step, samples were rinsed four times with PBS for 10 minutes each. All
procedures were performed at room temperature.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Analysis of Nucleotide and Amino Acid Sequences of Mouse SM1 cDNA
Two positive clones were isolated by screening a mouse uterine cDNA library with a 32P-labeled DNA fragment that encodes rabbit SM1. These clones showed identical lengths and restriction maps (Fig 1a
). An EcoRI-HincII fragment
at the 5' end including the translation-initiation site was
sequenced. Nucleotide sequence homology between mouse and
rabbit SM1 genes was 89% in the sequenced translated region and 63%
in the 5' untranslated region. The amino acid sequence homology between
mouse and rabbit SM1 was 97% (Fig 1b and 1c).
Optimum Conditions for Inducing SMC Expression in P19
Cells
Previous reports have documented a number of chemical regimens
that induce P19 cells to differentiate into cells of different
embryological
origin.10 11 12 13 14 15 16 17 18 19 20
To establish the optimum
chemical regimen to induce differentiation of P19 cells into SMCs,
various regimens that induce specific embryological differentiation
were investigated. Because vascular SMCS are derived from the
neuroectoderm as well as from the mesodermal and endodermal layers, we
first used DMSO, HMBA, and low concentrations of RA to see whether P19
cells could be induced to differentiate into SMCs, because DMSO, HMBA,
and low concentrations of RA have been previously reported to induce
P19 cell aggregates to differentiate into cells of mesodermal and
endodermal layers.10 12 13 21
Although P19 cells under
these conditions could be induced to differentiate into
fibroblast-like cells after day 8 of treatment (Fig 2a),
expression of the SMC-specific gene, SM1, which is
expressed ubiquitously in SMCs, could not be documented by RNase
protection assay (Fig 2b). A majority of the P19 cells
differentiated
into multinucleated myotubes as well as into beating
cardiomyocytes. Next, because high concentrations of RA had
been previously reported to induce P19 cells to differentiate into
cells derived from the neuroectoderm,11 12 we tried
high
concentrations of RA to see whether P19 cells could be induced to
differentiate into SMCs, since SMCs are also derived from the neural
crest.28 Compared with P19 cells treated with low
concentrations of RA (10 nmol/L), those treated with high
concentrations of RA (1 µmol/L) showed expression of SMC-specific
genes, as documented by RNase protection assay of SM1, from day 8 after
induction along with -SM actin expression (Fig 3a and
3b). Although P19 cells could be induced to
differentiate into SMCs in the presence of a high concentration of RA,
a great number of P19 cells also differentiated into neurons and glial
cells, as revealed by microscopy.
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To increase the population of SMCs in high-concentration RA–treated P19 cells, a strategy to block differentiation to the neuronal lineage was used by inhibiting the expression of Brn-2, a central nervous system–specific POU-domain transcription factor required for differentiation of P19 cells to the neuronal lineage, with an antisense expression vector construct. In the presence of high concentrations of RA, P19 cells with Brn-2 blocked (cell line 227) could differentiate into SM1-positive SMCs more efficiently than cells without Brn-2 blocked (cell line 215). As shown by the immunohistochemical staining and RNA analysis, the cells with Brn-2 blocked showed a marked increase in the SMC population.
Quantitative RNA Analysis of SMC Expression
To quantify the
level of SMC expression in the Brn-2–blocked P19
cells, RNase protection assay using SMC-specific markers was conducted.
By using probes for the SMC-specific markers (SM1 and calponin) and
-SM actin, with GAPDH as the internal control, SMC RNA expression
was investigated. The results show that -SM actin showed expression
from as early as day 12 in both Brn-2–blocked and –nonblocked
groups,
with increased expression in Brn-2–blocked cells at day 16 (Fig
4a). Analysis of the SMC-specific marker SM1,
which is found constitutively in all types of SMCs regardless of the
phenotype (eg, differentiated and dedifferentiated), showed
clearly increased expression at day 16 in Brn-2–blocked cells compared
with nonblocked cells, with a 1.8-fold increase in RNA as assessed by
densitometry (Fig 4b). Calponin, a marker of SMCs as well, also
showed
tendencies similar to those for SM1, with increased expression in cells
with Brn-2–blocked compared with –nonblocked cells, with a
1.7-fold
increase in RNA as assessed by densitometry (data not shown). The
results clearly showed that SMC expression is increased in
Brn-2–blocked compared with –nonblocked P19 cells.
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Confirmation of SMC-Specific Protein Expression in
P19-Induced Cells
We performed immunoblot analysis of the
protein extracts from high-concentration RA–treated P19 cells with
Brn-2 blocked to confirm and quantify SM1 expression at the protein
level as well. SM1 protein was detected at 204 kD by use of anti-SM1
monoclonal antibody (Fig 5). The amount of SM1 protein
expression was more abundant in high-concentration RA–treated P19
cells with Brn-2 blocked than those with Brn-2 not blocked. This
finding was consistent with the analysis of RNA
levels.
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Immunohistochemical Examination of SMC
Differentiation
To further investigate the SMC population in culture,
indirect
immunofluorescence studies using -SM actin, SM1,
and SM2 were performed sequentially for the high-concentration
RA–treated P19 cells with Brn-2 blocked. In the undifferentiated P19
cells, the cells were not positive for -SM actin, SM1, or SM2 (data
not shown). When high-concentration RA–treated P19 cells with
Brn-2 blocked were immunostained with monoclonal
anti–-SM actin antibody, cells resembling fibroblasts and
myofibroblasts positive for -SM actin were frequently observed at
earlier stages (Fig 6a and 6e). With continuing
culture,
these positive cells proliferated and increased in number (Fig
6b, 6c, 6f, and
6g). A majority of the positive cells could be maintained until
day 20 after induction (Fig 6d and 6h). As for
smooth muscle myosin
heavy chain, expression of SM1, which is found specifically yet
constitutively in all developmental stages of SMCs, was delayed
compared with that of -SM actin. SM1-positive cells could be seen at
days 12 and 16 (Fig 7a , 7b, 7e, and 7f). SM1-positive
cells could be frequently observed at day 18 (Fig 7c and
7g) and
maintained until day 20 after induction (Fig 7d and
7h). Additionally,
the expression of SM2, the product of alternative splicing of the
same smooth muscle myosin heavy chain gene as SM1 and which is
expressed specifically in differentiated SMCs, was also investigated. A
few SM2-positive cells could be seen as clusters at day 12 (Fig
8a and 8e), with differentiating cells growing
out of the
clusters at day 16 (Fig 8b and 8f). SM2-positive
cells increased at day
18 (Fig 8c and 8g) and could be maintained until
day 20 after induction
(Fig 8d and 8h).
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The low-power
magnification photographs at day 18 clearly
demonstrate that P19 cells with Brn-2 blocked compared with cells with
normal expression of Brn-2 showed marked increases in staining of
SMC-positive regions (Fig 9). Whereas at earlier stages
(eg, day 12), areas staining positive for -SM actin were in great
abundance compared with SM1; by day 18, areas staining positive for
-SM actin and SM1 were similar, showing increased expression of
SMCs, especially in the cells with Brn-2 blocked. It is also important
to emphasize that a greater number of cells in the surrounding regions
of the aggregates were positive for SM1 and displayed an increase in
the positive-staining population within the aggregates and bands
for the Brn-2–blocked P19 cells. Approximately 40% of the total cell
population was positive for SM1, as determined by immunohistochemical
examination. The staining properties at day 20 were similar.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Little is known of the mechanisms that regulate SMC differentiation compared with skeletal and cardiac muscle differentiation.29 30 31 32 Characterization of a number of SMC-specific genes (eg, smooth muscle myosin heavy chain gene and isoforms and calponin) by the authors2 3 4 5 6 and others7 8 9 23 have provided useful markers for the documentation of SMC development and differentiation. The major drawback in investigation of SMC-specific gene expression, however, has been the lack of an established SMC system suitable for investigation of gene expression. The present study demonstrates that the P19 embryonal carcinoma cell system provides a potential in vitro model of a pathway of SMC differentiation.
P19 Cells Can Efficiently Differentiate Into SMCs Under
Appropriate Conditions
SMCs are generally considered to be of
neuroectodermal and
mesodermal
origin.10 11 12 13 14 15 16 17 18 19 20 21
In order to induce P19 cells to
differentiate into SMCs, regimens that have been previously reported to
induce P19 cells into cells of mesodermal origin, including DMSO, HMBA,
and a low concentration of RA, were
tried.10 12 13 21
However, these regimens could not induce P19 cells to express
SMC-specific genes. After the induction of P19 cells with a high
concentration of RA, which has been reported to induce P19 cells into
cells of neuroectoderm origin,11 12 SM1-positive SMCs
could be found abundantly and in markedly greater numbers by inhibiting
differentiation to the neural cell lineage by blocking the POU-domain
transcription factor Brn-2. The optimum chemical regimen of high
concentrations of RA was consistent with the results of a
recent report.33
During the course of development of SMCs
in the P19 cells, in the early
stages (days 8 to 12), a number of cells stained positive for -SM
actin, although markers specific to the SMC lineage (eg, SM1) were
negative. When the characteristics of the markers used are taken into
consideration, whereas the smooth muscle myosin heavy chain gene is
specific to the SMC lineage34 and -SM actin is
expressed in nonmuscle cells (notably fibroblasts) and in
SMCs,35 36 37 38 39
the fibroblast-like cells in the early
stages of differentiation may have been of fibroblastic (eg,
fibroblast, myofibroblast) nature rather than SMCs. Confirmation and
quantification of SMC differentiation by RNase protection assay by use
of the SMC-specific markers has clearly demonstrated the cells found in
later stages after day 12 to be of the SMC lineage.
Immunoblot analysis
by use of SM1-specific antibody
revealed two bands at 204 kD. The faster migrating protein was
confirmed to be a smooth muscle myosin heavy chain isoform observed in
cultured vascular SMCs but not in smooth muscles in
vivo.40 SMCs differentiated from P19 cells may therefore
closely resemble primary cultured SMCs. The discrepancy in marked
increases in expression of SM1 protein compared with the mild increases
of SM1 RNA during differentiation of high-concentration RA–treated
P19 cells compared with Brn-2–blocked P19 cells may suggest the
presence of posttranscriptional regulation, which has been observed in
cytoplasmic and muscle-specific isoactins.35
Furthermore, numerous cells positive for SM2, a marker of differentiated SMCs usually found in nonpathological media and downregulated in dedifferentiated SMCs of intimal hyperplasia,2 6 were frequently observed in later stages, paralleling the expression of SM1 in immunofluorescence studies. The induction regimen of the present study may have led to differentiation of SMCs to a well-differentiated SMC phenotype. Investigations are under way to determine the characteristics of the SMCs obtained by the method used in the present study.
What Are the Roles of a High Concentration of RA and
Antisense Brn-2?
The investigation of the role of RA as a morphogen
has been under
extensive research in recent years. The previous data suggest RA to be
a critical and decisive factor in cell differentiation. The
concentration gradient of diffusible morphogens such as RA has been
reported to be an important factor of pattern formation in limb
morphogenesis41 42 and to play an essential role in
the
formation of the heart and vascular system.43 In the past,
the authors have shown the embryonic octamer binding transcription
factor Oct 3, whose expression is linked with regulation of early
embryogenesis, to be rapidly repressed by RA through an RA-repressible
enhancer.18 In the present study, optimum SM1-positive
SMC differentiation of P19 cells could be induced by high
concentrations of RA (1 µmol/L), which was consistent with
the results of a recent report.33 Because lower
concentrations of RA can induce fibroblast or myofibroblast formation
but not SMC differentiation,19 perhaps a continuous
spectrum of differentiation consisting of fibroblasts, myofibroblasts,
and SMCs may exist, with the degree of differentiation regulated
according to RA concentration. In conclusion, the results of the
present study suggest RA-sensitive differentiation of P19 cells,
with the concentration of RA being the most likely determinant of the
fate of SMC differentiation.
When inhibited in P19 cells treated with high concentrations of RA, Brn-2, a POU-domain transcription factor that plays a critical role in neural development,22 44 45 induced large amounts of cells to differentiate into SMCs. When Brn-2 was not blocked, a majority of cells differentiated into neural and glial cells; when the expression of Brn-2 was inhibited, the cells did not differentiate into the neuronal lineage but into cells including SMCs, fibroblasts, myofibroblasts, and myocardiocytes. Under high-concentration RA induction, the Brn-2–blocked P19 cells showed efficient differentiation into SMCs.
Our previous studies suggest that loss of Brn-2 expression at early stages most probably affects early essential stages of neural cell differentiation.22 Blocking Brn-2 expression during the first 2 days in the antisense Brn-2 cell line alone is sufficient to inhibit differentiation to the neuronal lineage.22 Impaired coordination of a Brn-2–dependent pathway of neuronal cell differentiation may lead to disrupted neural cell differentiation.
The cell system used presents a model for a pathway of SMC differentiation. By using the presented construct, elucidation of regulatory mechanisms involved in SMC differentiation may become possible. Comparison of differential screening of Brn-2–expressed and Brn-2–blocked high-concentration RA–treated P-19 cells should allow determination of factors responsible for regulation of SMC differentiation. Studies are presently under way to determine the involved SMC factors (eg, transcription factors). The P19 cell system is a promising tool for the investigation of SMC gene expression and regulation and will be useful in further understanding the regulatory mechanisms of SMCs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Suzuki is a fellow of the Japanese Academy for the Promotion of Sciences. The authors thank Dr Shunichiro Taniguchi (Kyushu University, Japan) for kindly providing mouse calponin cDNA.
Received July 3, 1995; accepted December 5, 1995.
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