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(Circulation Research. 1995;76:742-749.)
© 1995 American Heart Association, Inc.

Articles

A Retinoic Acid–Induced Clonal Cell Line Derived From Multipotential P19 Embryonal Carcinoma Cells Expresses Smooth Muscle Characteristics

Randal S. Blank, Ellen A. Swartz, Maria M. Thompson, Eric N. Olson, Gary K. Owens

From the University of Virginia School of Medicine, Department of Molecular Physiology and Biological Physics, Charlottesville, and the Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center (E.N.O.), Houston, Tex.

Correspondence to Gary K. Owens, PhD, Department of Molecular Physiology and Biological Physics, Box 449 Jordan Hall, University of Virginia, Charlottesville, VA.

	Abstract

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Abstract Despite intense interest in understanding the differentiation of vascular smooth muscle, very little is known about the cellular and molecular mechanisms that control differentiation of this cell type. Progress in this field has been hampered by the lack of an inducible in vitro system for study of the early steps of smooth muscle differentiation. In this study, we describe a model system in which multipotential mouse P19 embryonal carcinoma cells (P19s) can be induced to express multiple characteristics of differentiated smooth muscle. Treatment of P19s with retinoic acid was associated with profound changes in cell morphology and with the appearance at high frequency of smooth muscle -actin–positive cells that were absent or present at extremely low frequency in parental P19s. A clonal line derived from retinoic acid–treated P19s (9E11G) stably expressed multiple characteristics of differentiated smooth muscle, including smooth muscle–specific isoforms of -actin and myosin heavy chain, as well as functional responses to the contractile agonists phenylephrine, angiotensin II, ATP, bradykinin, histamine, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Additionally, 9E11G cells expressed transcripts for MHox, a muscle homeobox gene expressed in smooth, cardiac, and skeletal muscles, but not the skeletal muscle–specific regulatory factors, MyoD and myogenin. Results demonstrate that retinoic acid treatment of multipotential P19 cells is associated with formation of cell lines that stably express multiple properties of differentiated smooth muscle. It remains to be determined whether retinoic acid has induced commitment to a smooth muscle cell lineage as opposed to directly (or indirectly) activating genes characteristic of differentiated smooth muscle cells. However, results suggest that this cell system may be of use in attempting to identify genes involved in controlling smooth muscle differentiation and/or lineage determination.

Key Words: smooth muscle differentiation • embryonal carcinoma cells • muscle-specific gene expression • homeobox genes • transcription factors

	Introduction

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Cellular differentiation involves initial commitment of multipotential embryonic cells to a specific cellular lineage and subsequently differentiation of committed cells. Cellular differentiation is characterized by coordinate induction of a repertoire of cell-specific proteins necessary for specialized functions. In smooth muscle, differentiation involves the coordinated expression of a number of smooth muscle isoforms of myosin heavy chain (SM-MHC),^{1 2} actin,^{3 4 5} caldesmon,^{5 6 7} calponin,⁵ and tropomyosin,^{8 9} as well as a number of receptors and ion channels required for the contractile and metabolic functions of smooth muscle cells (SMCs). The identification of regulatory genes that control the coordinate and tissue-specific expression of such genes is a subject of considerable interest to both developmental biologists studying vasculogenesis and pathologists because alterations in SMC differentiation are believed to play an important role in atherogenesis.^{10 11}

Studies of skeletal myogenesis have led to the identification of a family of regulatory genes that control skeletal muscle differentiation. The first of these to be identified was MyoD, which was isolated by subtractive hybridization approaches in an inducible differentiation system in which multipotential 10T1/2 cells were converted to skeletal muscle through treatment with the DNA hypomethylating agent 5-azacytidine.¹² MyoD and the related factors myogenin,^{13 14} myf-5¹⁵ and MRF4/herculin/myf-6^{16 17 18} encode transcriptional regulatory factors that can convert a variety of cell lines to skeletal myoblasts. The actions of these factors are mediated, at least in part, by the direct activation of a number of muscle-specific genes, including muscle creatine kinase, cardiac -actin, myosin light chains 1/3, and troponin I.^{19 20} Importantly, the differentiation program in skeletal muscle depends on the continuous expression of myogenic regulatory factors such as MyoD.²¹ Positive autoregulation and cross-regulation of myogenic regulatory factor expression²² may provide a mechanism for stabilizing the skeletal muscle phenotype. The activity of MyoD and related family members is subject to regulation by changes in phosphorylation and expression of an inhibitor of DNA binding, Id.²³ Although differentiation control genes, analogous to MyoD, may be expressed in smooth muscle, none have been reported yet.

The lack of an inducible differentiation system for smooth muscle has been a major impediment in the search for lineage and differentiation control genes. The aim of the present study was to identify an inducible smooth muscle lineage system that might be useful in studies of this kind. The multipotential P19 mouse embryonal carcinoma can give rise to a number of distinct lineages, including cardiac and skeletal muscle, after treatment with retinoic acid or dimethyl sulfoxide.^{24 25} McBurney and colleagues^{26 27} described the appearance of a "fibroblast-like" cell in retinoic acid–treated P19s and showed that an isolate of these cultures (P19R1s) expresses some characteristics of smooth muscles, including smooth muscle -actin (SM -actin). While expression of this gene may signify conversion to a smooth muscle lineage, SM -actin is also expressed by developing sarcomeric muscles^{25 26 28 29} and by a variety of cultured cell types and tumor cell lines.^{30 31 32 33} The SM-MHC (designated SM-1 and SM-2 MHC)^{34 35} appear to be the most stringent markers of the smooth muscle lineage identified thus far in that their expression appears to be completely restricted to smooth muscle tissues in both adult and developing animals in vivo.^{36 37} The presence in P19R1 cells of transcripts that hybridize to a SM-MHC cDNA probe is equivocal,²⁷ however, because the probe used in these studies is known to cross-hybridize with skeletal and cardiac muscle MHC transcripts.^{35 38} Thus, while P19R1 cells appear to express some properties of differentiated smooth muscle, additional examination of smooth muscle differentiation markers is needed. Moreover, neither the stability nor the homogeneity of these cultures has been reported.

The present studies have explored the possibility that smooth muscle lineages are induced by retinoic acid treatment of P19s by determination of whether multiple characteristics of smooth muscle are expressed in a clonal line derived from retinoic acid–treated P19s. We show that retinoic acid treatment of P19 cells results in induction of a cell lineage that stably expresses multiple characteristics of smooth muscle, including SM -actin and SM-MHC, responsiveness to a variety of contractile agonists, and expression of a mesodermal homeobox gene, MHox.

	Materials and Methods

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Cell Culture
P19s were obtained from the American Type Culture Collection (No. CRL1825). Cells were cultured in -minimum essential medium (-MEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Hyclone) as described by Rudnicki and McBurney.²⁶ Cells were passaged at 2- to 3-day intervals at a ratio of 1:10. Cells were maintained in -MEM containing 7.5% FBS, 200 µg/mL L-glutamine, 100 U/mL penicillin (GIBCO), and 100 µg/mL streptomycin (GIBCO).

For induction experiments, P19s were plated at a density of 10 000 cells/cm² and treated for 48 hours with 1 µmol/L retinoic acid. Cells were then washed and maintained in -MEM plus 7.5% FBS for 5 to 7 days, at which time cultures were either fixed for immunostaining or harvested for gel electrophoretic analysis.

P19R1 cells (P19R1s) were a generous gift from Dr Michael McBurney (University of Ottawa, Canada). P19R1s were derived from P19s treated with retinoic acid and have been reported to express SM -actin and low levels of an MHC isoform recognized by the SM-MHC cDNA.³⁸ Surprisingly, immunostaining of P19R1 cultures with an SM -actin antibody demonstrated a low fraction of positive cells. In an effort to derive a clonal line that could subsequently be used in studies of the smooth muscle lineage, dilutional cloning techniques were used to isolate the 9E11G cell line described here.

Immunofluorescence Staining
Indirect immunofluorescence was performed as previously described.³⁹ Briefly, cells were grown in multiwell plates, fixed in cold methanol, and stored desiccated at 4°C. Cells were then rehydrated through a series of ethanol washes, washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), and subsequently incubated for 60 minutes with primary antibodies. Cultures were then washed three times, stained with a rhodamine-labeled secondary antibody, washed, and mounted for viewing with a Zeiss fluorescent microscope equipped with epifluorescence optics.

Western Blot Analysis
Western blot analysis was performed as previously described.⁴⁰ Actin isoforms were resolved by two-dimensional isoelectric focusing PAGE, while myosins and caldesmons were resolved on 4% and 8% polyacrylamide gels, respectively.⁴⁰ Proteins were transferred to nitrocellulose and reacted with primary antibodies to either SM -actin (Sigma Chemical Co) or SM-MHC. SM-MHC antibodies include a monoclonal antibody developed in our laboratory, designated 5F5,⁴¹ and a polyclonal antibody kindly provided by Dr Robert Adelstein (National Institutes of Health, Bethesda, Md). This antibody is specific for SM-MHC (and Fig 2).^{40 42} Western analysis of caldesmon expression was done with an antibody specific for h-caldesmon (obtained from M. Walsh, University of Calgary, Alberta, Canada).⁴³

View larger version (46K): [in this window] [in a new window]   Figure 2. Western blot analysis of smooth muscle myosin heavy chain (SM-MHC) expression in 9E11G cells. MHC isoforms were resolved on polyacrylamide gels and analyzed after Western blotting with a specific SM-MHC primary antibody (kindly provided by Dr R. Adelstein) and a horseradish peroxidase–conjugated anti-rabbit secondary antibody. This antibody did not recognize myosin in 10T1/2 cells or in skeletal muscle homogenates but showed staining of myosin isoforms in homogenates of aorta and 9E11G cells. Identical blots were reacted with rabbit preimmune serum and secondary antibody. SFM indicates serum-free medium.

Measurement of Cytosolic Calcium Concentrations
Confluent P19s or 9E11Gs were harvested by trypsinization and loaded with indo 1 by the method of Dostal et al.⁴⁴ Fluorescence measurements were made before and after addition of agonist to a stirred cell suspension on a fluorimeter (SLM 8000C, SLM Instruments Inc, SLM-Aminco; excitation, 332 nm; emission, 400 and 485 nm), and calcium concentration was estimated as described by Grynkiewicz et al.⁴⁵

RNA Isolation and Northern Blot Analysis
Generally, 9E11G cultures were grown in serum-containing medium and harvested while subconfluent or postconfluent. For growth factor stimulation experiments, 9E11G cultures were grown until confluent, maintained in serum-free medium for 3 days, and then treated with serum-free medium alone, 10% FBS, platelet-derived growth factor (PDGF)-BB (20 ng/mL, Upstate Biotechnology), or PDGF vehicle (2 mg/mL BSA; 10 mmol/L acetic acid) for 24 hours. Cultures were rinsed with PBS and removed by trypsinization, and RNA was isolated by the guanidinium isothiocyanate–CsCl method.⁴⁶ Extracted RNA was dissolved in water and stored at -70°C. Poly A⁺ RNA was selected by oligo dT chromatography (Invitrogen reagents). For gel electrophoresis of RNA, 10 µg total RNA was diluted in a solution of 4.4 mol/L formaldehyde and 5% formamide, denatured by heating for 10 minutes at 65°C, and subsequently resolved on a 1% agarose gel containing 2.2 mol/L formaldehyde. Capillary transfer of RNA to a nylon membrane (Micron Separations, Inc) was carried out overnight in 10x saline/sodium phosphate/EDTA. Blots were air dried, fixed under UV illumination for 90 seconds, and baked for 2 hours under vacuum at 80°C.

MHox,⁴⁷ MyoD,⁴⁸ and myogenin¹³ transcripts were identified after hybridization with full-length cDNAs isolated from plasmid DNA after digestion with an appropriate restriction endonuclease and glass bead purification. All probes were labeled with -³²P-dCTP (NEN) by random priming (Stratagene). Hybridizations and washes were carried out at 65°C as previously described by Church and Gilbert.⁴⁹ Probed blots were dried and exposed to Kodak X-OMAT K film at -70°C in the presence of intensifying screens to generate autoradiographic images.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Retinoic Acid Treatment Induces SM -Actin and SM-MHC Expression in P19 Cells
McBurney and colleagues^{26 27} reported the appearance of SM -actin–expressing cells after retinoic acid treatment of P19s and isolated a cell line from such cells that expresses SM -actin (P19R1). Our initial aim was to further assess SMC differentiation markers in P19R1 cells. Preliminary immunostaining experiments demonstrated heterogeneity with respect to -actin expression in the P19R1 line. Therefore, in an effort to obtain a homogeneous population of SMC, we conducted cell cloning experiments. Dilutional cloning was used to derive a homogeneous cell line expressing a variety of smooth muscle characteristics. Fig 1 shows immuno- staining of one such line, designated 9E11G, by use of antibodies to SM -actin and SM-MHC. Whereas parental P19s and retinoic acid–treated P19s showed frequencies of SM -actin staining of <5x10^-5 and 40% to 50% (data not shown), respectively, 100% of 9E11G cells stained for SM -actin and SM-MHC (Fig 1). These cells also were stained with an additional SM-MHC monoclonal antibody developed in this laboratory (designated 9A9) that recognizes SM-1 and SM-2 isoforms of SM-MHC but not MHC from skeletal, cardiac, or nonmuscle cells and tissues.⁴¹ Expression of SM-MHC was confirmed by Western blot analysis with the polyclonal SM-MHC antibody obtained from Dr Robert Adelstein⁴² (Fig 2). These studies did not resolve which of the SM-MHC isoforms (ie, SM-1 or SM-2) is expressed by 9E11G cells because we have been unable to definitely resolve these isoforms on porous SDS gels (in either mouse aortic samples [Fig 2] or in 9E11G cells [Fig 2]), and to the best of our knowledge, no antibodies are available that distinguish SM-1 MHC and SM-2 MHC isoforms that are reactive in the mouse. In addition, full-length cDNA sequences have not yet been reported, thus precluding the distinction of these isoforms at the mRNA level. Nevertheless, given the very high specificity of both SM-MHC isoforms for SMCs,^{36 50 51} these results provide very strong evidence that the 9E11G cell line either represents an SMC lineage derived by retinoic acid treatment of multipotential P19 embryonal carcinoma cells or that retinoic acid has directly or indirectly led to activation of SM-MHC expression. To further ascertain the developmental or maturational stage of 9E11G cells, we also assessed expression of h-caldesmon, which is purported to be a marker of a late stage of SMC differentiation or maturation, at least in humans in which its expression is first observed in the last trimester.^{6 7} Results of Western blot analysis showed no detectable expression of h-caldesmon, although 9E11G cells did express the nonmuscle low-molecular-weight isoform l-caldesmon (data not shown).

View larger version (137K): [in this window] [in a new window]   Figure 1. Immunostaining of the 9E11G clonal line with antibodies to smooth muscle (SM) -actin and smooth muscle myosin heavy chain (SM-MHC). P19 and 9E11G cells were grown in multiwell plates, fixed, and stained with monoclonal antibodies to either SM-MHC (5F5)41 or SM -actin (Sigma Chemical Co) followed by a rhodamine-labeled secondary antibody as described in the text. Cells were then washed, mounted, and viewed on a Zeiss fluorescent microscope equipped with epifluorescence optics. Controls lacked primary antibody.

Retinoic Acid–Induced Smooth Muscle Line Exhibits Transient Increases in Cytosolic Calcium in Response to Contractile Agonists
The 9E11G line was further analyzed for developmental-dependent changes in contractile agonist responsiveness by assessment of changes in cytosolic calcium levels after treatment with agonists known to stimulate contraction of smooth muscle in vivo. Whereas parental P19s showed little or no response to any of the agonists tested, the 9E11G line exhibited marked transient increases in cytosolic calcium in response to phenylephrine, angiotensin II, bradykinin, ATP, histamine, methacholine, PDGF-AA, PDGF-BB (Fig 3), and endothelin (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 3. Graphs showing the characterization of agonist-induced alterations of cytosolic calcium in parental P19 cells and 9E11G smooth muscle. Confluent P19 or 9E11G cells were harvested and loaded with indo 1. Cytosolic calcium determinations were performed as described in "Methods" by use of parental P19 cells (top) and 9E11G cells (bottom) before and after addition of phenylephrine (PE, 10 µmol/L), prostaglandin F2 (PGF2A, 1 µmol/L), angiotensin II (AII, 1 µmol/L), truncated angiotensin II peptide (3-8, 1 µmol/L), bradykinin (BK, 1 µmol/L), arginine vasopressin (AVP, 0.1 µmol/L), atrial natriuretic factor (ANF, 1 µmol/L), ATP (10 µmol/L), isoproterenol (INE; 10 µmol/L), histamine (HIS, 10 µmol/L), methacholine (MCH, 10 µmol/L), platelet-derived growth factor (PDGF) vehicle (10 mmol/L acetic acid, 2 mg/mL bovine serum albumin), PDGF AA (20 ng/mL), and PDGF BB (20 ng/mL).

These results demonstrate the acquisition of contractile agonist responsiveness in a clonal line derived from retinoic acid–treated P19s. It is unclear whether induction of agonist responsiveness reflects nascent receptor expression and/or activation of previously dormant signaling pathways. However, [¹²⁵I]PDGF binding studies provided evidence for appreciable levels of PDGF-AA and PDGF-BB binding in 9E11G cells but not in P19s (data not shown), supporting the idea that differentiation of P19s into smooth muscle is accompanied by induction of new receptor expression, at least for PDGF.

Mesodermally Restricted Homeobox Gene MHox Is Expressed in the Induced Smooth Muscle Lineage
Observations that 9E11G cells express SM -actin, contractile agonist responsiveness, and SM-MHC strongly support the identification of this line as smooth muscle. Of these, SM-MHC expression is most discriminating because it appears to be completely restricted to smooth muscle during development and in mature animals.^{36 37} Nevertheless, to rule out the possibility that the 9E11G line might represent a nonfusing skeletal muscle lineage that expresses SM-MHC in culture, expression of MyoD and myogenin transcripts, which are specific to skeletal muscles,^{12 13} was assayed by Northern blot analysis of total RNA from parental P19s and 9E11Gs. Both transcripts were detected in total RNA from rat thigh skeletal muscle but not in RNA from either parental P19s or 9E11G cells (Fig 4).

View larger version (70K): [in this window] [in a new window]   Figure 4. Blots showing expression of MHox but not MyoD or myogenin in 9E11G smooth muscle cells. Total RNA was isolated from confluent P19 cells, 9E11G cells, and adult rat thigh skeletal muscle. Northern blots of total RNA from these sources (10 µg per lane) were hybridized with 32P-labeled probes for MHox, MyoD, or myogenin as described in "Methods." X-OMAT K film was exposed to probed blots for 1 to 3 days.

We also examined expression of MHox, a mesodermally restricted homeobox gene expressed in smooth, cardiac, and skeletal muscle cells.⁴⁷ Northern blot analysis of RNA from these cells revealed expression of an abundant MHox transcript in 9E11G cells that comigrated with the MHox transcript from skeletal muscle (Fig 4). In addition to the primary transcript of approximately 3.6 kb, one larger transcript (4.0 kb) and several smaller transcripts were also detectable on longer exposure and on blots of poly A⁺ RNA (see Figs 5 and 6). MHox transcripts were undetectable in parental P19 cells. These results are consistent with 9E11G cells representing cells that are of a mesodermal origin.

View larger version (31K): [in this window] [in a new window]   Figure 5. Blots showing the effect of culture density on MHox and smooth muscle (SM) -actin mRNA expression in 9E11G cultures. RNA was isolated from 9E11G cultures grown in serum-containing medium and harvested while subconfluent or postconfluent. Northern blots of poly A+-selected RNA (1 µg per lane) from these sources were hybridized with either MHox or SM -actin cDNA probes.

View larger version (55K): [in this window] [in a new window]   Figure 6. Blots showing the effect of platelet-derived growth factor (PDGF)-BB and fetal bovine serum (FBS) on MHox and smooth muscle (SM) -actin mRNA expression in 9E11G cultures. RNA was isolated from postconfluent 9E11G cultures (in serum-free medium) that had been stimulated subsequently with either 10% FBS or 20 ng/mL PDGF-BB for 24 hours. Northern blots of poly A+-selected RNA (1 µg per lane) were hybridized with cDNA probes for MHox or SM -actin. SFM indicates serum-free medium.

To ascertain whether expression of MHox and SM -actin transcripts might be regulatable in 9E11G, we assessed the effect of growth state and cell density on expression of the mRNAs for these proteins by Northern analysis. RNA was isolated from 9E11G cultures grown in serum-containing medium and harvested while subconfluent or postconfluent. Northern blots of poly A⁺ RNA (1 µg per lane) from these sources were hybridized with either MHox or SM -actin cDNA probes. Our choice of the full-length rat SM -actin cDNA for these studies was based on considerations of probe availability and proven efficacy in the mouse. Moreover, we wanted to be able to monitor both muscle and nonmuscle actin isoform expression on the same blot. Finally, it was extremely unlikely that other muscle actin isoforms might be comigrating with the 1.7-kb SM -actin mRNA in that Western blotting experiments using an affinity-purified polyclonal antibody specific for the striated muscle -actins⁵² (kindly provided by Dr Jeannette Chloe Bulinski) demonstrated that 9E11G cells did not express either skeletal or cardiac muscle actin isoforms (data not shown). The expression of MHox and SM -actin was not tightly regulated as a function of cell density. In several experiments, both transcripts were either equally abundant in subconfluent and postconfluent cultures or slightly less abundant in subconfluent cells (Fig 5). RNA was also isolated from postconfluent 9E11G cells in serum-free medium stimulated with either 10% FBS or 20 ng/mL PDGF-BB for 24 hours. While both treatments were mitogenic for 9E11G cells (data not shown), neither exerted an appreciable effect on the expression of SM -actin or MHox transcripts (Fig 6).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The lack of an inducible differentiation or lineage system for smooth muscle has been a major limitation in studies of smooth muscle development and has impeded efforts to identify genetic elements involved in the regulation of smooth muscle lineage determination and/or differentiation. The positive identification of smooth muscle lineages has also been problematic for several reasons. First, SMCs do not undergo terminal differentiation or cell fusion and therefore are not readily identifiable by morphological criteria. Second, many markers of differentiated smooth muscle such as SM -actin, h-caldesmon, (, ß)-metavinculin, and -vinculin are or can be expressed by non-SMCs, including developing skeletal and cardiac muscle^{28 29} and other contractile cells in vivo^{53 54 55} and in a variety of cultured cell lines.^{30 31 32 33} Additionally, it is well established that the differentiated state of SMCs is extremely plastic^{40 56 57 58 59 60 61 62 63 64 65} and appears to be dependent on environmental cues to a greater extent than that in skeletal muscle.^{10 11} Unfortunately, the environmental cues that play a key role in regulating SMC differentiation are poorly understood, and many markers of differentiated SMCs may or may not be expressed in vitro, depending on the methods of cell culture used. As such, the positive identification of an induced smooth muscle lineage depends on assessment of multiple markers that are characteristic of normal differentiated smooth muscle, with the caveat that many properties of differentiated SMCs are unlikely to be expressed in the absence of appropriate environmental cues for complete differentiation or maturation.

The present studies describe a retinoic acid–induced P19 cell line that expresses a number of features of differentiated smooth muscle, including SM -actin, SM-MHC, functional responses to contractile agonists, and transcripts for MHox, but not the skeletal muscle–specific regulatory factors MyoD and myogenin. Importantly, expression of SM-MHC provides the most compelling evidence for the SMC lineage of 9E11G cells because it appears to be completely restricted to SMCs in both developing and mature animals.^{34 35 36 37 38 50 51} One report based on immunostaining suggests that SM-MHC is expressed in subconfluent endothelial cells in culture, although not by endothelial cells in vivo.⁶³ However, we have found that the antibody used in these studies shows some cross-reactivity with a 200-kD nonmuscle MHC B that comigrates very closely to SM-2 MHC (M.M.T. and G.K.O, unpublished observations, 1994). Interestingly, we found that 9E11G cells failed to express h-caldesmon. Koteliansky and coworkers^{6 7} presented evidence suggesting that h-caldesmon is a marker of a late stage of differentiation or maturation in vascular SMCs based on observations that its expression was first detectable in aorta late in gestation in human fetuses and then increased postnatally. Expression of SM-2 MHC appears to be induced at approximately the same time or later, whereas SM-1 MHC is detectable earlier.^{50 51} On this basis, it seems likely that the SM-MHC isoform expressed in 9E11G cells is SM-1 MHC rather than SM-2 MHC. Similarly, Miano et al³⁶ suggested that the SM-MHC isoform detected by in situ analysis in 10.5 p.c. mouse embryonic aorta was SM-1 MHC, not SM-2 MHC. Taken together, the preceding results suggest that induction of the smooth muscle differentiation program after retinoic acid treatment is arrested at an intermediate stage of SMC differentiation or maturation equivalent to day 10.5 p.c. in the mouse embryo. This culture system is stable as it expresses smooth muscle properties over a range of culture passages (up to at least passage 19).

The P19-derived smooth muscle–like cell line described here may be useful for the isolation of genes involved in commitment of a multipotential cell to the smooth muscle lineage (determination) and/or induction of a pattern of gene expression characteristic of smooth muscle (differentiation), assuming that maintenance of the differentiated state in smooth muscle is dependent on the continued expression of such regulatory genes. It is important to note that our studies have not determined whether retinoic acid induced conversion to a smooth muscle lineage or merely activated the particular smooth muscle differentiation genes that we examined. The fact that we observed expression of a number of genes characteristic of differentiated SMC would argue that activation involved an upstream "master regulator." However, we cannot rule out the possibility that each of the genes involved is directly responsive to retinoic acid. While there is no direct evidence showing that maintenance of the differentiated phenotype in smooth muscle is dependent on production of one or more transcriptional regulatory factors that regulate multiple SMC differentiation genes, studies in other cell types strongly suggest that this may be a universal mechanism for controlling and maintaining cellular differentiation.⁶⁴ The greatest value of the P19 system described here is probably not the retinoic acid–derived 9E11G clonal line per se, which appears to exhibit many characteristics similar to smooth muscle primary lines, but rather the demonstration that multipotential P19 cells can be stimulated to express multiple genes characteristic of differentiated SMCs by retinoic acid treatment. This implies first that a regulatory cDNA might be isolated by subtractive cloning techniques and second that a putative regulatory cDNA, once identified (by this or any other method), could be forcibly overexpressed in P19 cells to assess its ability to induce SMC determination or differentiation.

The mechanisms whereby retinoic acid induces different cell lineages are not known. However, studies in other systems have identified a number of retinoic acid–inducible genes that could possibly account for activation of SMC genes in the P19 system. Among these retinoic acid–inducible genes are the homeobox genes that encode transcriptional regulators and are thought to regulate various aspects of embryonic development and tissue differentiation and organization. Retinoic acid treatment of embryonal carcinoma cells is known to differentially regulate expression of 38 homeobox genes in a manner related to their position within the four HOX clusters.⁶⁵ MHox, a mesodermally restricted homeobox factor, is expressed in 9E11Gs but not in parental P19s, raising the possibility of its involvement in early smooth muscle differentiation. MHox expression in the induced lineage appears to be independent of cell density and growth state and thus may serve as an early constitutive marker of smooth muscle or mesodermal lineages. Moreover, the coexpression of MHox and SM -actin mRNAs under a variety of growth conditions may be suggestive of a regulatory relation. This possibility is particularly intriguing in light of studies showing that the human homologue of MHox (Phox) enhances binding of serum response factor to the serum response element⁶⁶ and thus may regulate the transcription of selected genes. Interestingly, the rat SM -actin promoter contains CArG box sequences that bind serum response factor and that are required for smooth muscle–specific transcriptional activation.⁶⁷ Studies to address the possible role of MHox in the induction and/or maintenance of the 9E11G lineage are in progress.

Taken together, results of the present study suggest that the 9E11G cell line represents an induced SMC or SMC-like cell lineage and that the retinoic acid–P19 system may have utility in studies to identify genes involved in regulation of smooth muscle differentiation or determination. Identification of such genes and their mechanisms of action is likely to be critical to understanding changes in SMC phenotype during vasculogenesis and in the pathogenesis of vascular diseases.

	Acknowledgments

 
This work was supported by National Institutes of Health grants RO1 HL-38854 and PO1 HL-19242 and by training grant T32 HL-O7355, the University of Virginia Cancer Center, the Muscular Dystrophy Association, the Council for Tobacco Research, and the Robert A. Welch Foundation. We would like to thank Dr Michael McBurney for his generous gift of the P19R1 cell line, Dr Robert Adelstein for the polyclonal SM-MHC antibody, Dr Jeannette Chloe Bulinski for the skeletal muscle -actin antibody, Michael Walsh for the h-caldesmon antibody, Diane P. Raines and Sallie E. Adams for technical assistance, and William D. Woolfolk and Jennifer Clatterbuck for manuscript preparation. We are also indebted to Dr Ryan E. Lesh for his assistance in analysis of caldesmon expression.

Received July 8, 1994; accepted February 13, 1995.

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