ARTICLE |
Correspondence to: Zipora YablonkaReuveni, Dept. of Biological Structure, Box 357420, School of Medicine, U. of Washington, Seattle, WA 98195. E-mail: reuveni@u.washington.edu
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Summary |
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Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity. (J Histochem Cytochem 48:11731193, 2000)
Key Words: smooth muscle, skeletal muscle, Myf5, MyoD, myogenin, FGFR4, IGF1, IGF2
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Introduction |
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Smooth and skeletal muscle cells are two of the cell types used by the vertebrate body to achieve mechanical contraction. Vascular smooth muscle cells provide structural support and help regulate internal pressure within the blood vessels of vertebrates. This cell type has been characterized by the expression of various smooth muscle isoforms of structural genes involved in contraction, including smooth muscle myosin heavy chains (smMHCs), smooth muscle calponin (smCalponin), smooth muscle -actin (sm-
-actin), smooth muscle myosin light chain kinase (smMLCK), and
-tropomyosin (
Although the structural gene isoforms are for the most part characteristic of each muscle cell type, some smooth muscle cell isoforms have been detected in developing or regenerating skeletal muscle cells (e.g., smMLCK, -actin,
-actin has been seen in the developing heart (
-actin by itself is not a sufficient condition to warrant a smooth muscle cell identity. In addition, the set of smooth muscle structural gene isoforms being expressed by a given smooth muscle cell can change as a function of its environment (
Skeletal muscle cells express a family of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin, and MRF4. The MRFs are involved in the specification of the skeletal myogenic lineage during early embryogenesis and in the control of the skeletal muscle differentiated phenotype. MyoD and Myf5 are expressed earlier during skeletal muscle development, followed by myogenin and MRF4 (reviewed in
Although the smooth and skeletal muscle cells share some of the molecular underpinnings of their contractile roles, they are believed to have different developmental origins. The majority of skeletal muscle arises from discrete bodies of mesoderm sequestered and determined early in development (
Although smooth muscle cells and skeletal muscle cells are largely distinct in their structure, function, and developmental origin, cases have been described in which smooth muscle cells acquire a skeletal muscle phenotype.
The experiments detailed here demonstrate a change from smooth-to-skeletal muscle character occurring in three smooth muscle cell lines: PAC1, A10, and A7r5. -actin, smMHC, myosin regulatory light chain, the smooth muscle isoform of
-tropomyosin, and the surface receptors for angiotensin II, norepinephrine, and
-thrombin. It has also been shown to direct the smooth muscle-specific splicing of transgenes and to exhibit normal morphology and smooth muscle myosin heavy chain expression at passage levels of up to 100 (
-actin, smMHC, smCalponin, SM22, and tropoelastin. We obtained this cell line to serve as a negative control for immunofluorescence experiments conducted on primary skeletal muscle satellite cells, and quite unexpectedly found that cultures of PAC1 cells exhibited positive immunofluorescence for the MRFs MyoD and myogenin and formed myotubes. The A10 and A7r5 cell lines were established from rat thoracic aorta by
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Materials and Methods |
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Source of Cell Lines and Culture Conditions
The following smooth muscle, skeletal muscle and non-muscle rat cells were used.
PAC1 and PAC1R Cells.
The PAC1 rat smooth muscle cell line was originally established from the pulmonary artery of adult SpragueDawley rats as described by
A7r5 and A10 Cells.
The smooth muscle cell lines A7r5 and A10 were originally derived from the thoracic aortas of embryonic BDIX rats as described by
WKY3m22 Cells.
The rat WKY3m22 cell isolate was derived from the thoracic aorta smooth muscle of a 3-month old WistarKyoto rat (
ClB Cells.
The ClB smooth muscle cells were isolated in the authors' laboratory from the pulmonary artery smooth muscle layer of an 8-week old SpragueDawley rat (B&K Universal; Kent, WA), using a modification of the method described by
L6 and L8 Skeletal Muscle Myoblasts.
The L6 and L8 lines were originated by Dr. David Yaffe from the thigh muscle of newborn Wistar rats (
Rat2 Embryonic Fibroblasts.
The rat embryonic fibroblast line Rat2, derived from the Rat1 cell line, also called F2408, and originally established from Fisher rat embryos (
NRK52e Rat Kidney Epithelial Cells.
The NRK52e rat kidney epithelial cell line (
All cell types were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Gaithersberg, MD) with 10% fetal bovine serum (Sigma) and antibiotics, with the exception that the A10 cells received 20% fetal bovine serum. Cells were plated for immunocytochemical studies at a density of 500010,000 cells/cm2 in 35-mm tissue culture dishes and for RT-PCR analysis at a density of 20,000 cells/cm2 in 60-mm tissue culture dishes.
When the effect of insulin was examined, the cells were cultured in DMEM with 10% fetal bovine serum in 35-mm plates as above until reaching about 9095% confluency. The medium was then replaced with DMEM containing 10% fetal bovine serum or 2% horse serum (Sigma), with or without insulin (bovine; Sigma, added at 1 µM). The four different media were replaced every 23 days.
Immunocytochemistry
Single and double immunolabeling of methanol-fixed cultures were performed using a previously described indirect immunofluorescence protocol (
The following primary antibodies were used.
Anti-MyoD.
The mouse MAb 5.8A, made against murine MyoD (
Anti-myogenin.
The hybridoma producing the mouse MAb F5D made against rodent myogenin was developed and kindly supplied by Dr. W. Wright (University of Texas Southwestern Medical Center, Dallas, TX). The use of this antibody in immunofluorescence analyses of rat and mice myoblasts has been described (
Anti-MEF2A.
The C-21 rabbit polyclonal antibody against human MEF2A was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The use of this antibody to detect MEF2A in extracts of C2 cultures via immunoblotting was previously described by
Anti-sarcomeric Myosin.
The mouse MAb MF20, raised against chicken sarcomeric myosin, was developed by
Anti-smooth Muscle Calponin (smCalponin). The hCP mouse MAb prepared against human uterus smooth muscle calponin was obtained from Sigma. This MAb recognizes smooth muscle calponin from a variety of mammals and does not crossreact with skeletal muscle, cardiac muscle, or non-muscle forms of calponin.
Anti-smooth Muscle Myosin Light Chain Kinase (smMLCK).
The K36 mouse MAb, made against chicken gizzard smooth muscle myosin light chain kinase, was obtained from Sigma. This MAb selectively recognizes the smooth muscle form of this kinase and has been used to distinguish between smooth and skeletal muscle cells of developing rodents (
Anti-smooth Muscle Myosin Heavy Chain (smMHC).
A rabbit polyclonal antibody, made against the 204-kD isoform of bovine aortic smooth muscle myosin heavy chain, was kindly provided by the originators, Drs. C. Kelley and R. Adelstein (
Anti-smooth Muscle -Actin (sm-
-actin).
The mouse MAb against sm-
-actin (Sigma; clone 1A4) was originally developed by
-actin in various species, including rodents (
Anti-desmin.
The D3 mouse MAb against chicken desmin was developed by
RT-PCR
RNA was isolated from cell cultures at the indicated day after plating using TriZOL reagent (Gibco BRL; Gaithersburg, MD) according to the manufacturer's instructions. After isolation the RNA was digested with DNase I (RQ1 DNase; Promega, Madison, WI) to eliminate trace amounts of genomic DNA. The treatment was with 1 unit DNase per 10 µg of RNA, in a buffer containing 40 mM Tris, pH 7.9, 10 mM NaCl, 6 mM MgCl2, 10 mM CaCl2, and 0.1 U/µl RNasin (Promega) for 30 min at 37C, followed by phenol/chloroform extraction to remove the enzyme.
Reverse transcription of the RNAs was done using Maloney murine leukemia virus reverse transcriptase (MMLV-RT; Promega) and Oligo dT15 obtained as a custom primer from Gibco BRL. Two µg of RNA was reverse-transcribed in a 100-µl reaction containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTPs, 19 µg/ml Oligo dT15. After a denaturation step (95C, 2 min), this reaction was brought to 42C, 400 units of MMLV-RT was added, and the reactions were carried out for 1 hr at 42C, then heated to 90C for 10 min and stored at -20C until used in a PCR.
PCRs were performed in a PerkinElmer 9700 thermocylcer (PerkinElmer Applied Biosystems; Foster City, CA). Each 20-µl reaction received 2 µl of a reverse transcription reaction and contained the following reagents in the concentrations/amounts listed (i.e., these are the final concentrations including whatever the reverse transcription reaction contributed): 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 300 µM dNTPs, 400 nM each primer, and 1 unit of enzyme (AmpliTAQ Gold; Perkin-Elmer). The standard PCR profile included a preincubation step, one cycle at 95C for 12 min, followed by 2040 cycles of 95C for 45 sec, 65C for 59 sec, 72C for 2 min, and finishing with one cycle at 72C for 8 min. For the genes pax3 and smMLCK, an annealing temperature of 62C was used rather than 65C. Sixteen µl of the PCR was run on 1.2% agarose gels containing 0.5 µg/ml ethidium bromide (Gibco BRL) and viewed and photographed on a UV transilluminator.
The primers used in this study are shown in Fig 1. Most of these were designed de novo on the basis of published sequence information using the Entrez Browser program and with the help of the programs Primer and Tm. The primers were further evaluated with the help of the program Amplify (
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Results |
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Culture Conditions That Elicit Morphological Change in PAC1 Cells
During preliminary studies we detected myotube-like structures in crowded PAC1 cultures. These myotube-like structures, although at times containing many nuclei, were not always readily apparent because of the high density of the single cells also present in the culture. When we employed immunofluorescence with an antibody against sarcomeric myosin, these myotubes were easily detected (see below). These multinucleated myotubes were not observed in sparse cultures. As indicated in Materials and Methods, our subsequent studies were conducted using plating densities of 500010,000/cm2 for immunofluorescence studies and 20,000 cells/cm2 for RT-PCR, and maintaining the cells in DMEM containing 10% fetal bovine serum. Under these conditions, myotubes appeared at 68 and 45 days after plating, respectively. Unless otherwise noted, all studies detailed below were conducted using these conditions.
Immunofluorescence Analysis of Smooth and Skeletal Muscle Protein Expression in PAC1 Cells
We first examined the PAC1 cells for their expression of a variety of smooth muscle associated proteins to verify their original characterization as a smooth muscle cell line that retains its capacity to differentiate in vitro (-actin. Fig 2E shows the sm-
-actin expression in a 10-day culture, demonstrating the appearance of myotube-like structures in the later PAC1 cultures and the expression of this protein in these myotubes.
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The bottom four panels of Fig 2 depict double immunofluorescent micrographs that show the expression of several skeletal muscle-specific proteins in PAC1 cells. Fig 2F shows the immunostaining of a Day 6 culture with mono- and polyclonal antibodies against MyoD. Most of the cells in this field are MyoD+ and co-stained with both antibodies. Fig 2G shows the co-expression of MyoD and myogenin in the same PAC1 cells in a Day 2 culture. Fig 2H shows a Day 10 culture containing multinucleated myotubes positive for both myogenin and MyoD. Many more myogenin+ cells were seen at the later time point. Fig 2I shows the expression of sarcomeric myosin in the same cells expressing the transcription factor MEF2A. The expression of myogenin, MEF2A, and sarcomeric myosin in the myotube-like structures is a strong indication that some of cells in the PAC1 cultures have adopted a differentiated skeletal muscle cell identity (
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We next examined, via immunofluorescence, cultures of PAC1 cells obtained from the originator, Dr. A. Rothman (referred to as PAC1R), to see if the tendency of PAC1 cells to adopt a skeletal muscle phenotype was a general characteristic of this cell line. A summary of the PAC1R cell analysis is shown in Table 1 and examples of immunostained cultures are provided in Fig 3. Micrographs in Fig 3 were taken at a lower magnification than that used in Fig 2. As in PAC1 cells, many of the PAC1R cells were positive for smCalponin, desmin, smMLCK, and sm--actin. In addition, the PAC1R cultures contained a significant proportion of cells expressing MyoD, although at somewhat reduced numbers compared to the PAC1 cultures. Myogenin+ cells were also detected in PAC1R, but their frequency was far lower compared to PAC1 cultures. Many fields in the PAC1R cultures displayed no myogenin expression and a typical positive field contained only one or two myogenin+ cells. In extremely rare cases, we detected in the PAC1R cultures clusters of elongated myotube-like cells that stained positive for both sarcomeric myosin and MEF2A (myosin stain is shown in Fig 3F) or for myogenin and MEF2A (data not shown). In general, far less than 0.1% of the PAC1R cells expressed sarcomeric myosin.
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Collectively, the lower-passage cultures (PAC1R) and the higher-passage cultures (PAC1) of the pulmonary artery-derived cell line express the same repertoire of smooth muscle-specific proteins. In addition, both PAC1R and PAC1 cultures contain cells exhibiting skeletal muscle-characteristic proteins. However, the frequency of cells exhibiting the skeletal muscle differentiated phenotype is far reduced in the PAC1R cells compared to the PAC1 cells.
Expression of Smooth and Skeletal Muscle Differentiation-associated Genes in Smooth Muscle Cell Cultures
We used RT-PCR to compare smooth and skeletal muscle gene expression changes that take place during the PAC1 and PAC1R differentiation to those in established skeletal muscle cell lines (L6 and L8) and in non-myogenic lines (Rat2 and NRK52e). ClB cells were included in the comparison as a low-passage pulmonary smooth muscle line originated in our laboratory. This analysis was done on RNA taken from cells at 1, 5, and 10 days after plating, and the results are shown in Fig 4. The expression of smooth and skeletal muscle proteins in the different cell lines was also compared by immunofluorescene and is summarized in Table 1. In the RT-PCR analysis shown in Fig 4 (as in all subsequent RT-PCR analyses), the cycle number was chosen such that the best range of expression was observable among the various cell lines examined. Under these conditions, the level of some PCR products was saturated for certain cell types or time points. Lower PCR cycles allowed the comparison of the level of the more saturated PCR products. The last row in Fig 4 and in all of the other RT-PCR figures shows the expression of the ribosomal protein rpS6 in the various cell cultures. We used rpS6 expression as an indication of the quality of the RNA samples being used in the RT-PCR reactions and as a measure of the total reaction-to-reaction variation. The ribosomal protein genes are generally held not to be transcriptionally regulated in vertebrate cells (
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As shown in Fig 4, PAC1R, PAC1, and ClB cells (but not the other lines examined) express the three smooth muscle genes: smMLCK, smMHC, and smCalponin. Consistently, a high number of PCR cycles was required to detect smMLCK even in cultures in which all cells were positive for smMLCK by immunofluorescence (data not shown). A trace of smMLCK was also detected in some of the non-smooth muscle lines at this PCR sensitivity.
As shown in Fig 4, PAC1R and PAC1 cultures gave robust MyoD bands with a tendency towards decreasing intensity with time. This trend was even more prominent at lower cycle numbers (data not shown). The ClB smooth muscle cells and the kidney epithelial NRK52e cells showed no expression of MyoD. Despite their skeletal muscle origin, the myogenic lines L6 and L8 expressed a far lower level of MyoD than the PAC1R and PAC1 cells. The L6 and L8 lines were previously reported not to express MyoD at all (
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Myogenin was also expressed by the PAC1R and PAC1 cultures. In agreement with the immunofluorescence studies, the PAC1 cells expressed a far higher level of myogenin compared to the PAC1R cells. This level of myogenin expression the PAC1 cells was similar to the maximal level of myogenin seen in the skeletal myoblast lines L6 and L8 cells. The Rat2 cell line, which displayed robust MyoD expression but whose cultures were never seen to develop myotubes, gave only a trace band for myogenin. Immunofluorescence experiments confirmed the RT-PCR data with the Rat2 cells, showing myogenin expression in extremely rare cases (far less than 0.1% of cells). Neither the NRK52e nor the ClB cells showed detectable myogenin expression by RT-PCR or immunofluorescence (Fig 4; Table 1). MRF4 expression was also increased in the PAC1 cultures compared to PAC1R cultures. However, the intensity of the MRF4 signal produced by PAC1 cells was far reduced compared to that displayed by the differentiating L6 and L8 myoblasts. A faint MRF4 product was also detected in Rat2 and NRK52e cultures.
The Myf5 expression pattern was striking (Fig 4). Only L6 and L8 cells displayed Myf5 expression. This robust Myf5 expression in the L6 and L8 cultures was also demonstrated with an antibody against Myf5 (
The expression of the transcription factors Id-1 and pax3 is depicted next in Fig 4. The Id-1 gene product was displayed by all cell lines analyzed, showing a similar pattern of decreasing expression with days in culture (only one time point is shown in Fig 4 for Rat 2 and NRK52e cells). Different from Id-1, the Id-3 gene was expressed at similar levels by the cells at the time points shown in Fig 4 (data not shown). Pax3 expression was displayed by PAC1R cells at all times in culture and was rapidly decreasing in cultured PAC1 cells. This expression was far reduced compared to the level of pax3 in L6, L8 and NRK52e cells. Collectively, the Id-1, Id-3, and pax3 genes did not show explicit expression patterns that might be associated with the appearance of the skeletal muscle phenotype in the PAC1 smooth muscle cells.
The structural genes slow-twitch muscle troponin I (TnIsl), fast-twitch muscle troponin I (TnIfst) and muscle lim protein (MLP), which are associated with the differentiated state of skeletal muscle and myofiber specification (
Our ongoing studies have linked the expression of fibroblast growth factor receptor 4 (FGFR4) and differentiation of skeletal muscle myoblasts. This led us to analyze FGFR4 gene expression in the present analysis. As shown in Fig 4, the expression of FGFR4 in PAC1 cells parallels the expression of IGF1 and IGF2 in a similar manner to the expression of these three genes in L6 and L8 cultures. FGFR4 is also expressed in PAC1R cultures but not in the other cell types examined. It is noteworthy that the L6 cells are widely held to lack FGF receptors based on FGF binding studies (
In summary, the study shown in Fig 4 indicates that the skeletal myogenic differentiation program seen in the rat smooth muscle cell line PAC1 shares many common features with that seen in the established rat skeletal muscle lines L6 and L8. Nevertheless, the PAC1 cells were never found to express Myf5. The PAC1R cells, the predecessors of the PAC1 cells, expressed MyoD and, to a lesser degree, myogenin, but did not display the expression of many of the genes linked to skeletal muscle differentiation.
Sublines Created from PAC1 Cultures Exhibit Variable Penetrance into the Skeletal Myogenic Phenotype
We undertook to clone out sublines from the PAC1 cultures to obtain homogeneous cell populations for further studies on the smooth-to-skeletal muscle phenotypic switch. Thirteen sublines produced by limiting dilution cloning were screened using immunofluorescence staining for myogenin, and we found that all expressed myogenin to various degrees. Some clonal cultures, such as PAC1i and PAC16, displayed many myogenin+ cells even during the initial days in culture. Other clonal cultures, such as PAC1a, contained only a small number of myogenin+ cells, which became more apparent during later days in culture. The frequency and size of the multinucleated myotubes also varied among the sublines, with the highest morphological differentiation seen in the PAC1i cells. The various degrees of expression of the skeletal muscle phenotype in the PAC1 sublines were an inherited property of the cells which was maintained even after prolonged storage in liquid nitrogen. This permitted the expansion of the subline cell stocks for the subsequent studies described below.
Fig 6 shows an RT-PCR analysis done on RNA from Day 5 cultures of the 13 PAC1 sublines along with the control lines PAC1R, L8, NRK52e, and ClB. The RT-PCR results verified the initial immunofluorescence characterization. On culture Day 5, all sublines except for PAC1a expressed MyoD and myogenin. Experiments conducted at the cycle numbers shown in Fig 6 revealed a wide range in the level of MyoD and myogenin expression among the sublines. A lower PCR cycle further revealed that the highest myogenin expression was in the PAC1i and PAC16 sublines. In all sublines, the expression level of myogenin nearly paralleled MyoD. Sublines that expressed more or less myogenin also expressed more or less MyoD. The three markers of skeletal muscle terminal differentiation, TnIsl, TnIfst, and MLP, are also shown in Fig 6. The expression level of these three genes in the individual PAC1 sublines followed a parallel pattern. Sublines that expressed more or less TnIsl also expressed more or less TnIfst and MLP. The PAC1i and PAC16 sublines (and to a slightly lesser extent the PAC1c line) expressed more of these genes than did the other sublines.
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The expression patterns of IGF1, IGF2, and FGFR4 in the clonal PAC1 sublines shown in Fig 6 are in agreement with the expression of these genes in the parental PAC1 line. The expression of IGF1 in the PAC1 sublines followed the same pattern as the skeletal muscle differentiation markers. The highest levels of IGF1 expression was seen in PAC1i and PAC16 sublines, followed by PAC1c cells. The second group of clonal cultures, PAC1f, PAC1h, and PAC12, which display a reduced expression of the differentiation markers TnIsl, TnIfst, and MLP, demonstrate a similar reduced expression of IGF1. The sublines that showed expression of TnIsl, TnIfst, and MLP (PAC1 Clones c, f, h, i, 2, and 6) showed the expression of IGF2, and FGFR4 in addition to IGF1.
The smooth muscle-specific genes smMLCK, smMHC, and smCalponin were expressed to some degree in all of the PAC1 sublines (Fig 6), supporting the idea that these clonal populations retained smooth muscle cell identity. A reciprocal trend was seen in the sublines' expression of smMHC and skeletal muscle structural genes. Sublines i and 6, which expressed the highest levels of TnIsl, TnIfst, and MLP, showed the least expression of smMHC.
Analysis of Id-I and pax3 expression, also shown in Fig 6, did not reveal any obvious correlation between the levels of these two genes and the skeletal muscle-specific genes among the PAC1 sublines. It is possible that the expression of Id-1 and pax3 was downregulated at different rates in the different PAC1 sublines and that the summation of these different patterns resulted in the overall downregulation of these genes in the parental PAC1 line (Fig 4).
PAC1i Cells Have a High Capacity to Undergo Skeletal Muscle Differentiation
The analysis shown in Fig 6, combined with observations on the degree of morphological skeletal muscle differentiation undergone by the PAC1 sublines, indicated that the PAC1i subline was exhibiting the greatest degree of skeletal muscle differentiation. Fig 7 shows phase-contrast micrographs of PAC1R and PAC1i cells at low and high densities. The low-density appearance of the PAC1i cells seen in Fig 7C is identical to that of low-density PAC1R cells shown in Fig 7A. The high-density PAC1i culture shown in Fig 7D was generated by allowing the culture to grow for 8 days beyond that shown in Fig 7C. The Day 10 PAC1i cells formed extensive arrays of multinucleated myotubes that accounted for most of the cells on the dish. This multinucleated array was positive for sarcomeric myosin, as determined by immunofluorescence with the MF20 antibody (data not shown). Fig 7B shows the high-density appearance of the PAC1R cells cultured for 6 days. This culture is devoid of multinucleated myotubes. The absence of myotubes was also confirmed in Day 10 PAC1R cultures (data not shown). Fig 7E and Fig 7F show immunofluorescence micrographs of Day 4 and Day 10 cultures of PAC1i cells stained for myogenin (a higher and a lower magnification for culture Days 4 and 10, respectively). Comparison of the pattern of myogenin expression with the corresponding pattern of DAPI nuclear staining (Fig 7E' and 7F') shows that a very high percentage of the PAC1i cells express myogenin and are fused in large myotube-like syncytia. Smaller myotubes containing circular clusters of nuclei were already seen in earlier days in culture.
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Fig 8 shows a comparison of the time course of gene expression in the PAC1i subline and in the skeletal myoblast line L6. The general trend towards differentiation was similar in the PAC1i and L6 cultures, as demonstrated by the expression pattern of the skeletal muscle structural genes. The expression patterns of all genes analyzed in the PAC1i cultures are in agreement with the results shown in Fig 4 for the parental PAC1 line, with the exception of IGF1, whose expression is increased in the clonal subline. The comparison of IGF1 and IGF2 expression patterns in PAC1i cells demonstrated a strong induction of IGF1 with time in culture, paralleling the induction pattern of the skeletal muscle differentiation markers. Differently from IGF1, IGF2 was expressed in the PAC1i cultures at all time points studied, and its expression was only moderately upregulated with time in culture. In the study shown in Fig 8 (similar to the study shown in Fig 4), MRF4 expression level is far higher in the L6 cells compared to the PAC1 cells. It is further notable that the increase in MRF4 expression precedes the increase in myogenin expression. This observation contrasts with studies suggesting that MRF4 expression is linked to differentiation and is typically displayed coincidentally with or shortly after myogenin (
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Co-expression of Smooth and Skeletal Muscle Proteins in Individual PAC1 Cells
Double immunofluorescence analyses of PAC1R, PAC1, and PAC1i cells for the expression of proteins specific to the smooth and skeletal muscle cell phenotypes were carried out to determine whether the smooth and skeletal muscle genes were co-expressed in individual cells. The results of this study are shown in Fig 9. Fig 9A shows double staining of Day 4 PAC1R cells for smCalponin and MyoD. Fig 9B similarly demonstrates the co-expression of smMLCK and MyoD in single cells in Day 5 PAC1i cultures. Fig 9C shows double immunofluorescence staining of cells in a Day 3 PAC1 culture with antibodies specific for sarcomeric and smooth muscle myosins. In each case, arrows indicate single cells that stain for both the smooth and skeletal muscle-specific proteins. An arrowhead in Fig 9C indicates a cell that is stained only for a smooth muscle protein. In all cases, the number of cells in a given culture exhibiting double staining for smooth and skeletal muscle specific proteins was only a small proportion of the total cells, suggesting that perhaps this stage was an unstable intermediate between the two differentiated phenotypes.
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Fig 9D and Fig 9E show the double immunofluorescence staining of Day 12 cultures of PAC1 cells for smMLCK along with the transcription factor MEF2A, which is included to mark the myotubes. Fig 9D shows a field of cells with distinct subpopulations exhibiting either smMLCK cytoplasmic staining or MEF2A-labeled nuclei in myotubes. The large MEF2A+ myotube marked by an arrowhead is clearly negative for smMLCK. Fig 9E contains a binucleated cell that stains positive for both smMLCK and MEF2A and other cells (an example is indicated with an arrowhead) that stain only for MEF2A. Our preliminary surveys indicated that whereas smaller myotubes in these cultures are often positive for the smooth muscle-associated proteins such as smCalponin and smMLCK, the expression of these proteins in the more extensive myotubes seen at later times was below the level of detection by this technique.
Vascular Smooth Muscle Cell Lines A7r5 and A10 Exhibit Skeletal Muscle Gene Expression
We next examined the two widely used rat aortic smooth muscle cell lines A7r5 and A10 for the possible expression of skeletal muscle differentiation genes. Fig 10 depicts the immunofluorescence analysis of the A7r5 cell line for smooth and skeletal myogenic protein expression. Fig 10A shows that many cells in Day 1 A7r5 cultures were positive for smCalponin. Fig 10B10D show the staining at Day 5 for the skeletal muscle proteins: sarcomeric myosin (Fig 10B), MyoD (Fig 10C), and myogenin (Fig 10D). No large myotubes were observed in the A7r5 cultures, but elongated binucleated cells were infrequently seen that stained brightly for sarcomeric myosin. An example of this is shown in Fig 10E for a Day 11 culture. The frequency of cells in the A7r5 cultures expressing skeletal muscle characteristic proteins was far less than in PAC1 cultures (no more than 5% of the cells). By immunofluorescence, the A10 cell line was positive for smCalponin, MyoD, myogenin, and sarcomeric myosin, similar in pattern and frequency to the A7r5 cell line (data not shown). In agreement with a previous report (
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The smooth muscle cells WKY3m22 were also analyzed by immunofluorescene and were found negative for all skeletal muscle proteins examined (Table 1). The WKY3m22 cells were derived from the thoracic aorta and have been used by several laboratories as a model for adult aortic smooth muscle cells (
The level of skeletal muscle gene expression in the different smooth muscle lines was additionally compared by an RT-PCR cycle titration analysis, as shown in Fig 11. The study was conducted using total RNA from Day 5 cultures. Except for the L6 cells, no cell line displayed Myf5 expression at any level of RT-PCR amplification. Neither the ClB nor the WKY3m22 cells showed expression of the MRFs or the skeletal muscle structural genes at any PCR cycle level, whereas the A7r5, A10, and PAC1R lines showed weak expression of these genes.
Effect of Insulin on Skeletal Muscle Differentiation in Vascular Smooth Muscle Cultures
The studies described above showed a correlation between the degree of skeletal muscle differentiation and the level of expression of the IGFs in PAC1 parental cultures and sublines. Furthermore, it is well established that endogenously produced IGF1 and IGF2 can exert a strong positive effect on skeletal muscle differentiation (
The effect of insulin on differentiation was examined in selective PAC1 sublines showing higher and lower degrees of skeletal muscle differentiation. The parental PAC1R cells, along with ClB and Rat2 cells, were examined as well. Parallel cultures were initiated in 35-mm plates at 10,000 cells/cm2 using the standard growth medium. When the cultures were almost confluent the medium was changed to DMEM-based media containing 10% fetal bovine serum (FBS) ± insulin or 2% horse serum (HS) ± insulin. Dishes from the four treatment groups were analyzed after 38 days in the test medium for the frequency of myogenin+ cells, employing immunofluorescence of DAPI-stained cultures. The results, which are summarized in Table 2, show that the frequency of myogenin+ cells was enhanced by insulin in cultures of both PAC1R cells and the PAC1a subline. The effect of insulin was seen regardless of whether the cells were maintained in 10% FBS or 2% HS. Differentiation in PAC1J cells and in PAC16 cells was dependent on the presence of insulin when cells were maintained in 2% HS but not when cells were maintained in 10% FBS. The frequency of myogenin+ cells in the PAC1i cultures was independent of insulin regardless of the serum conditions. We noted, however, that the size of the myotubes in PAC1i cultures was enlarged earlier when insulin was present (data not shown). This phenomenon is reminiscent of recent reports on the regulation of myotube hypertrophy by IGF1 in cultures of established skeletal muscle cell lines (
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Discussion |
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In this study we have shown that cultures of three smooth muscle cell lines, PAC1, A7r5, and A10, contain cells that have begun to express a skeletal muscle cell phenotype. In the case of the PAC1 cells, several lines of evidence indicate that this phenomenon does not represent a simple contamination by skeletal muscle cells from an external source. First, all PAC1 sublines underwent skeletal muscle differentiation and also displayed expression of the smooth muscle-associated genes smMLCK, smMHC, and smCalponin. Second, two stocks of PAC1 cells (PAC1 and PAC1R) that had been obtained from different laboratories showed both skeletal and smooth muscle gene expression. Third, the double immunofluorescence data demonstrated that individual cells were expressing both smooth and skeletal muscle-specific genes. Most importantly, the absence of Myf5 expression uniquely distinguishes the different smooth muscle cell lines we have analyzed from the widely used rodent skeletal muscle lines that do express Myf5.
Whereas the skeletal muscle phenotype was characterized in the different smooth muscle cell populations by the expression of a common set of genes, the various cell lines and sublines expressed the skeletal muscle phenotype to a variable degree. The converted cells in the A7r5 and A10 populations were never seen to make large multinucleated myotubes, but instead remained mononuclear or made binucleated cells. Although a higher percentage of cells in PAC1R cultures expressed MyoD than did cells in A7r5 and A10 cultures (Table 1), the PAC1R cultures exhibited far less expression of the genes associated with skeletal muscle differentiation: sarcomeric myosin (Table 1), troponin I, and MLP (Fig 11). The clonal sublines generated from the high-passage PAC1 cultures also exhibited a wide range of capacities to express skeletal muscle genes. Certain sublines showed only weak expression of many of the myogenic genes and a poor capacity to form myotubes. In contrast, the PAC1i subline exhibited a pronounced phenotypic change over just a few days in culture. PAC1i cultures reliably produced myotubes at high frequency and exhibited an expression pattern of various skeletal muscle-associated genes. The differences in the penetrance of the various subpopulations of smooth muscle cells into the full myogenic phenotype might represent discrete stages in the transition from a smooth to the skeletal muscle identity.
A dual smooth/skeletal muscle program was previously identified in the B3CH1 cell line. This line was derived from a mouse brain tumor and its cell origin is unknown. Nevertheless, on the basis of their expression of sm--actin along with several other characteristics, the B3CH1 cells have been considered to be smooth muscle cells (
-actin is expressed by various cells, skeletal myoblasts included, and therefore the expression of this actin by itself is insufficient to attribute a smooth muscle nature to the B3CH1 cells. Unlike the B3CH1 cells, which were isolated from a mouse brain tumor and whose actual origin is unknown, the PAC1, A7r5, and A10 cells were isolated from vascular smooth muscle and express multiple smooth muscle genes. Furthermore, the expression of MyoD but not Myf5 by the three vascular-derived smooth muscle lines analyzed in the present study distinguishes the cells from the B3CH1 cells, which express Myf5 but not MyoD (
MyoD Expression by Itself is Insufficient for the Promotion of the Skeletal Muscle Phenotype in Vascular Smooth Muscle Cells
MyoD was among the genes showing differential expression in the different smooth muscle lines and within the PAC1 sublines. In most but not all cases, the level of MyoD expression was generally a good predictor of the level of expression of downstream skeletal muscle genes such as myogenin and troponin I. Hence, a simple model explaining the expression of skeletal muscle genes in smooth muscle cell lines would postulate that the MyoD gene became transcriptionally active in the cultured cells and that this "leaky" expression of MyoD caused the subsequent change in phenotype. Indeed, enforced expression of MyoD is able to foster a skeletal muscle phenotype in many but not all non-muscle cell types (
Investigating the different PAC1 cultures, we showed that the degree of skeletal muscle differentiation correlates with the increased expression of the IGFs and the growth factor receptor FGFR4. We also demonstrated that expression of IGF2 precedes expression of IGF1. Hence, IGF2 might be involved in the initiation of the skeletal myogenic program, and IGF1 expression might be the result of skeletal muscle differentiation in the PAC1 cultures. It is notable that
The present study on the effect of insulin in PAC1 cultures provides further support for the possible involvement of the IGF signaling system during the smooth-to-skeletal muscle switch. We showed that different PAC1 populations possess a capacity to switch into the myogenin+ state, depending on the environment they are in. This switch in PAC1R and PAC1a was highly dependent on the addition of insulin, possibly because the cells made very little of the IGFs. The switch to the myogenin+ state in PAC1J and PAC16 cells required insulin only when the cells were maintained in low serum. Therefore, the PAC1J and PAC16 cells might produce (or receive from the medium) sufficient IGFs in the high-serum conditions but not in the low-serum conditions. The PAC1i cells probably produced sufficient IGFs under both serum conditions. Alternatively, different levels of the IGF receptors among the various sublines could also account for some of the observations, but that aspect has not been yet investigated. PAC1R and PAC1a cells might express only a low level of the receptors, and therefore a high concentration of insulin would be required for activating the IGF receptors. PAC1J and PAC16 cultures might express a lower level of the receptors when maintained in the low serum and therefore would need the addition of insulin at a high level to initiate IGF signaling and enhance skeletal muscle differentiation in the low-serum conditions only. PAC1i cells might express a sufficient level of the IGF receptors along with a sufficient level of the IGFs in both high and low serum.
The role of FGFR4 in skeletal myogenesis is under investigation in our laboratory. FGFR4 expression in adult myoblasts (satellite cells) correlates well with the progression of the cells through the MyoD and myogenin compartments (
Myf5 Is Not Expressed During the Smooth-to-skeletal Muscle Transition
One of the more notable characteristics of our data was the observation that Myf5 expression was not activated in any smooth muscle cell lines or even in the PAC1i subline, which showed the most robust phenotypic conversion. Primary cultures of skeletal muscle myoblasts typically express both MyoD and Myf5, the latter being expressed at various levels depending on the source of the cells (reviewed in
During development, it is believed that MyoD and Myf5 are initially induced in defined and separate sets of skeletal muscle cell precursors (
Possible In Vivo Role of the Smooth-to-skeletal Muscle Switch
The fact that not all of the smooth muscle cell populations analyzed in the present study possess the capacity to undergo skeletal muscle differentiation suggests that skeletal myogenic potential is acquired in culture, potentially by initiating MyoD expression and activating the IGF signaling system. Nevertheless, the expression of skeletal muscle genes or the appearance of the myogenic phenotype has been noted in several developing smooth muscle tissues, including the esophagus, the iris, and the urethral sphincter (
We have begun to explore the possible occurrence of a smooth-to-skeletal muscle phenotype within the elaborated skeletal muscle vasculature as an alternative source for skeletal muscle myoblasts in vivo. Such a source may provide a means to produce skeletal muscle myoblasts after a massive injury, when there is a demand for a large and rapid supply of myoblasts for tissue repair (reviewed in
In conclusion, the present cell culture study provides evidence for a phenotypic transition from smooth to skeletal muscle and a detailed examination of the gene expression program associated with this transition. The observations made in the present study can now be used to begin investigating mechanisms that underlie the emergence of the skeletal muscle phenotype in various tissues and organs that do not have an obvious developmental linkage to the skeletal muscle lineage.
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Acknowledgments |
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Supported in part by grants to Z.Y.-R. from the National Institutes of Health (AG13798) and from the Cooperative State Research ServiceUS Department of Agriculture (agreement no. 95-37206-2356).
We thank Priscilla Natanson for excellent technical support. We also thank Stefanie Kästner for helpful comments on the RT-PCR analysis and Dr D. Han for important input during the preliminary stage of the investigation. We are grateful to the following investigators who kindly provided valuable reagents: Dr A. Rothman (PAC1R cells); Drs D. Han and S. Schwartz (WKY3m22 and PAC1 cells); Drs R. Seifert and D. BowenPope (Rat2 and NRK52e cells); Drs R. Adelstein and C. Kelley (anti-smMHC); Dr S. Alemá (anti-MyoD, polyclonal); Drs P. Houghton and P. Dias (anti-MyoD, monoclonal); and Dr W. Wright (anti-myogenin).
Received for publication March 15, 2000; accepted May 17, 2000.
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