Effect on MyoD Conversion of Cultured Aortic Smooth ... - NCBI - NIH

American Journal of Pathology, Vol. 143, No. 1, July 1993 Copyright X) American Society for Investigative Pathology

Basic Fibroblast Growth Factor Has a Differential Effect on MyoD Conversion of Cultured Aortic Smooth Muscle Cells from Newborn and Adult Rats

Johan W. van Neck,* John J. Medina,t Carla Onnekink,* Peter F. M. van der Vent, Henri R J. Bloemers,* and Stephen M. Schwartzt Departments of Biochemistry* and of Cell Biology and Histology,t University of Nijmegen, the Netherlands, and Department of Pathology,t University of Washington, Seattle,

Washington

MyoD is a master regulatory genefor myogenesis that also converts many mesoderm-derived ceUs into the skeletal muscle phenotype. Rat aortic smooth muscle ceUs do not contain MyoD homologous mRNA. However, expression of an exogenously supplied MyoDgene in aortic smooth muscle ceUs culturedfrom newborn and adult animals converts these ceUs to elongated myoblasts and myotubes expressing the skeletal muscle genes for titin, nebulin, myosin, and skeletal a-actin. The presence of basic flbroblast growth factor during growth and serum starvation completely inhibits MyoD-mediated conversion in cultures of newborn smooth muscle ceUs. However, in smooth muscle ceUl cultures derived from adult rats the presence ofjfbroblast growth factor increases the conversion frequency. The dfferential response of exogenous MyoD suggests that the two morphological types ofaortic smooth muscle ceUs, one typicalfor the newborn rat, the otherfor the adult rat, represent two distinctive states of differentiation. (Am J

Pathol 1993, 143:269-282)

Cultured smooth muscle cells derived from the aortas of newborn rats differ from cultured smooth muscle cells from the aortas of adult rats in morphology,1 growth characteristics,2 and gene expression. 4 Moreover, differences between newborn and adult aortic smooth muscle also can be seen in vivo, sug-

gesting that these distinctions are inherent to the cell rather than influenced by other extrinsic controls of cell phenotype. The differences in vivo include the smaller numbers of newborn smooth muscle cells expressing some of the proteins seen in adult smooth muscle cells.3'5-7 The most impressive difference for the same cells in vitro is in growth control. Smooth muscle cells derived from the newborn aorta, in contrast to smooth muscle cells derived from the adult aorta, do not require platelet-derived mitogens and, in fact, constitutively secrete mitogens.2 Smooth muscle cells from the adult aorta forming the neointima after balloon angioplasty show a phenotype very similar to that of the cells cultured from the newborn wall.89 Again, these cultured cells grow in medium prepared without platelet release and secrete growth factors. Moreover, in vivo, the neointima has an extensive proliferative response that persists for months after injury.10 Finally, smooth muscle cells derived from the newborn aorta or from the neointima express a number of genes not seen, or seen at much lower levels, in cultured cells from adult, uninjured vessels.3'410 Some of these genes are also differentially expressed at high levels in the neointima in vivo, suggesting that intimal cells represent a recrudescence of an immature phenotype. Taken together, these studies suggest that the arterial media contains two subsets of smooth muscle cells, one of which may be an immature precursor of the other. Establishing such a relationship between immature and adult arterial smooth muscle cells in vitro has been difficult since cells cultured from the arterial media tend to lose expression of many of the proteins believed to identify the smooth muscle cell type in Supported by NIH grant HL03174,

a grant from the Netherlands Heart Foundation (NHS 87033), and a NATO collaborative research grant (CRG 900213). Accepted for publication January 26, 1993. Address reprint requests to Dr. Johan W. van Neck, Department of Pathology/Pediatrics, Erasmus University, P. 0. Box 1738, 3000 DR Rotterdam, the Netherlands.

269

270

van Neck et al

AJPJuly 1993, Vol. 143, No. 1

vivo.11 13 In contrast, a great deal is known about skeletal muscle differentiation at the level of control of transcription, in large part because of experimental systems that permit the observation of cells being converted from undifferentiated fibroblasts to striated muscle myoblasts and myocytes. Davis et al identified a gene product, MyoD, that was absent in fibroblasts but characteristic of myoblasts even when these cells were not differentiated into myocytes or myotubes.14 MyoD-containing myoblasts will differentiate into skeletal muscle if appropriate growth factors are removed from the medium. MyoD thus marks and determines the skeletal muscle lineage. This protein belongs to the family of homologous helix-loophelix proteins.15 MyoD expression occurs late in the myoblast lineage, suggesting that skeletal muscle cells express other, as yet unidentified, cis- or transacting factors that determine the muscle phenotype. 1 Since many of the cytoskeletal proteins seen in the smooth muscle cell are also expressed in precursors of skeletal muscle,16,17 it is reasonable to think that skeletal muscle cell precursors and smooth muscle cells may share elements that control the expression of lineage-specific markers. The experiments described here were designed to explore the interactions of MyoD with other controls of gene expression found in newborn and adult smooth muscle. We report that neither smooth muscle type expresses MyoD. However, there is a marked difference in the percentage of cells converted to skeletal muscle when exogenous MyoD is introduced into the two cell types. Under appropriate conditions, MyoD causes massive conversion of the adult cell to skeletal muscle. The converted cells express much of the striated muscle phenotype. In contrast, under these same conditions, newborn cells do not convert. Our data suggest that the differences between newborn and adult smooth muscle cells may be related to differences seen in the developmental precursors of striated muscle.

Materials and Methods Cell Culture Thoracic aortas from 12-day-old (newborn) or 3-month-old (adult) male Wistar rats were removed. The tunica media was isolated, and smooth muscle cells were placed into culture as described.1 Smooth muscle cells were grown in Waymouth's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and were used between the 15th and 20th passages.

Retroviral Infection MDSN, a MyoD complementary DNA (cDNA) containing retrovirus, and LNL-6, the parental retrovirus, lacking the MyoD insert, were generously provided by Dr. A. D. Miller (Fred Hutchinson Cancer Research Center, Seattle, WA). Both MDSN and LNL-6 are constructed from an amphotropic retrovirus in which a neomycin phosphotransferase gene is transcribed from an internal simian virus 40 early promoter enhancer18 enabling the selection of infected cells from the neomycin analogue G418 (Gibco, Paisley, Scotland). MyoD expression is driven con-

stitutively by the viral long terminal repeat.19 Smooth muscle cells were plated in 60-mm Petri dishes (Costar, Cambridge, England) at a density of 30,000 cells/cm2 and allowed to settle for 24 hours. Then the medium was removed and replaced with medium containing 4 pi/ml polybrene (Sigma Chemical Company, St. Louis, MO) and 100 pl viral stock (titer: 107 neomycin-resistant colony-forming units/ ml). After 16 hours the medium was changed, G418 (300 pg/ml) and, in the cases indicated, 10 ng/ml bFGF (Promega, Madison, WI) were added. The cells were passaged and grown to 80% confluency, i.e., 80% of the surface of the tissue culture dish was covered with cells. Then the medium was changed to serum-free Waymouth's medium for 7 days, which initiates skeletal muscle terminal differentiation.

Immunostaining Smooth muscle cells were grown on glass microscopic slides until they reached 80% confluency. Then the medium was changed and the cells were serum-starved as described above. In order to immunostain for skeletal muscle-specific proteins, the cells were rinsed three times in phosphate-buffered saline (PBS) and incubated for 2 minutes in 70% ethanol, 3.7% formaldehyde, and 5% glacial acetic acid (for assays using peroxidase conjugated secondary antibodies), or the glass microscopic slides were dipped in cold methanol for 3 seconds, followed by acetone fixation and air-dried at room temperature (for immunofluorescence experiments). Fixed smooth muscle cells were incubated with the following antibodies: Against myosin: a mouse anti-chicken myosin heavy chain monoclonal antibody, MF-20,20 which was a kind gift of Dr. Stephen D. Hauschka (Department of Biochemistry, University of Washington, Seattle, WA). This antibody reacts with all sarcomeric myosins. Against titin: a polyclonal antiserum to titin isolated from chicken

MyoD Conversion of Smooth Muscle Cells 271 AJPJuly 1993, Vol. 143, No. 1

gizzard21 which was a kind gift of Dr. D. Gassner (University of Bonn, Bonn, Germany). Against titin: a mouse monoclonal antiserum (9D10),22 and against sarcomeric tropomyosin: a mouse monoclonal antiserum (CH-1 ),23 both obtained from the Developmental Studies Hybridoma Bank, maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD), and the Department of Biology, University of Iowa (Iowa City, IA), under contract N01-HD-62915 from the National Institute of Child Health and Human Development. Against nebulin: a mouse monoclonal antibody NB2 (Sigma). Against desmin: a mouse monoclonal antibody D3,24 a kind gift of Dr. D. A. Fishman (Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY). Against von Willebrand factor: a mouse monoclonal antibody, A082 (Dakopatts, Glostrup, Denmark).25 Against smooth muscle a-actin: a mouse monoclonal antibody 1A4 (Sigma). The cells were incubated with the primary antibody for 30 minutes at room temperature. They were rinsed in PBS and, when double labeling was performed, incubated with a second antiserum. After washing with PBS, they were incubated with the appropriate anti-mouse or anti-rabbit immunoglobulins and conjugated with peroxidase, fluorescein isothiocyanate (both from Nordic, Tilburg, the Netherlands), or Texas red (Southern Biotechnology Associates, Birmingham, AL). After rinsing with PBS the cells were mounted in GELVATOL (Monsanto, St. Louis, MO), or we proceeded with the detection of peroxidase by rinsing in 50 mmol/L Tris (pH 7.5) and 10 mmol/L imidazole and incubating in 50 mmol/L Tris (pH 7.5), 10 mmol/L imidazole, 0.05% diamino benzidine (DAKO, Glostrup, Denmark), and 0.025% hydrogen peroxide for 30-45 minutes. The reaction was stopped by rinsing in 50 mmol/L Tris (pH 7.5) and 10 mmol/L imidazole.

DNA Autoradiography Smooth muscle cells were grown on glass microscopic slides until they reached 80% confluency. Then the medium was changed and the cells were serum-starved as described above. Waymouth's medium supplemented with 10% fetal bovine serum and 1 pCi/ml [3H]methylthymidine (Amersham, Hertogenbosch, the Netherlands) was added for 12 hours. Then the medium was removed, and the cells were washed with PBS and immunostained for sarcomeric myosin as described above. The slides

were then dipped in Kodak NTB2 emulsion. Autoradiography took place for 4 weeks. Subsequently the slides were developed in Kodak D19 developer and fixed in 24% sodium thiosulfate. The cells were counterstained in hematoxylin, dehydrated in graded ethanol concentrations and xylene, and mounted in synthetic resins.

RNA Isolation Total cellular RNA was isolated by the lithium-urea procedure as described by Auffray and Rougeon.26

Northern Blot Analysis Glyoxylated RNA samples were electrophoresed through 1% agarose gels submerged in 10 mmol/L sodium phosphate (pH 7.2),27 transferred to a nylon membrane (HYBOND N, Amersham, Amersham, England), and hybridized with 1-2 x 106 cpm/ml of 32P-labeled DNA probes according to the method described by Church and Gilbert.28 Membranes were washed twice at 65 C in 0.1 mol/L sodium phosphate (pH 7.2), 1% sodium dodecyl sulfate (SDS), and 1 mmol/L EDTA (pH 8.0) for 30 minutes. RNA gels were normalized by direct visualization of total RNA in each lane of the gel and on the blots. Blot hybridization with 32P 5'-terminal labeled oligonucleotides was carried out in 5x Denhardt's mix, 0.5% SDS, 6x standard saline citrate, 100 pg/ml denatured salmon sperm DNA, and 0.05% sodium pyrophosphate at 45 C. Membranes were washed twice at 42 C in 0.2x standard saline citrate and 0.1% SDS for 20 minutes. Hybridizations with the inosine oligonucleotide DL27 (see below) were executed using the hybridization mix described by Church and Gilbert at temperatures between 42 C and 32 C.28 The filters were washed in 40 mmol/L sodium phosphate (pH 7.2), 1% SDS, 0.05% bovine serum albumin, and 1 mmol/L EDTA, at a temperature 8 C below the hybridization temperature.

DNA Probes DNA probes used for Northern blot hybridization analysis were as follows: For MyoD: a 1.8-kb mouse cDNA probe, kindly provided by Dr. A. B. Lassar (Fred Hutchinson Cancer Research Center).14 For MyoD low-stringency hybridizations, a 38-bp degenerate MyoD inosine-oligonucleotide DL27 (5' ATG TA(G/C) l(G/C)G ITG GCI ITI ll(G/C) AGG ATC ICI ACC TTI GG 3'), kindly donated by Dr. Harold

272

van Neck et al


Weintraub (Fred Hutchinson Cancer Research Center). For desmin: a 0.8-kb hamster desmin cDNA kindly provided by Dr. H. Bloemendal of our laboratory.29 For myosin: a 0.5-kb human adult myosin light chain 2 cDNA, kindly provided by Dr. B. Nadal Ginard (Department of Pediatrics, Harvard Medical School, Boston, MA).30 For troponin, a 0.5-kb fragment of the mouse fast troponin cDNA clone cMl 13a, kindly provided by Dr. K. Hastings (Neurological Institute, University of Montreal, Qu6bec, Canada).31 For cardiac muscle a-actin: a 40-bp oligonucleotide (5' CTG ATG GGA GAT GGG AGA GGG CCT CAG AGG ATT CCA AGC A 3') representing part of the rat cardiac a-actin 3' untranslated region, and for skeletal muscle a-actin: a 40-bp oligonucleotide (5'AAT CTA TGT ACA CGT CAA AAA CAG GCG CCG GCT CGA GTG G 3') representing part of the rat skeletal a-actin 3' untranslated region, both kindly provided by Dr. Robert Schwartz (Department of Cell Biology, Baylor College of Medicine, Houston, TX). For smooth muscle a-actin: a 25-bp oligonucleotide (5' AGT GCT GTC CTC TTC TTC ACA CAT A 3'), nucleotides -8 to 18 relative to the coding region of the human smooth muscle a-actin gene, was prepared by Dr. Rick Meek of our laboratory following an established procedure.32

MyoD consensus oligonucleotide under lowstringency conditions.33 Rat skeletal muscle control RNA showed a single hybridization signal (Figure 1a). No specific hybridization signal was detectable in RNA samples isolated from adult rat aortae (Figure 1b) and cultured adult and newborn smooth muscle cells (Figures lc and ld, respectively).

MyoD Converts Newborn and Adult Smooth Muscle Cells to Myosin-Positive Cells at an Equal Percentage Cultures of newborn and adult smooth muscle cells were infected with a MyoD-containing amphotropic retrovirus (MDSN) or the MyoD-lacking parental retrovirus (LNL-6), both containing the neomycin phosphotransferase gene. After G418 selection, the cells

kb

23.1 6.:6 9.4 Results Throughout this paper, to avoid the repeated use of long descriptions, we will use the terms "adult smooth muscle cells" and "newborn smooth muscle cells" for cultures of smooth muscle cells derived from the aortas of, respectively, 3-month-old or 12day-old rats.

..

:21-.O3, -*.to

_:.

No MyoD Homologue Is Detectable in Smooth Muscle Cells Cultured adult smooth muscle cells have a spindleshaped morphology and grow in overlapping bundles organized in multilayers at confluence.1 In contrast, cultured newborn smooth muscle cells grow in a monolayer fashion and have an endothelial-like morphology. Both by Northern blot and immunocytochemistry, newborn cells, as well as adult cells, express the smooth muscle marker a-actin1 but do not express von Willebrand factor,25 an established endothelial cell marker (results not shown). To search for MyoD homologous transcripts, we screened Northern blots containing equal amounts of total RNA from cultures of newborn and adult smooth muscle cells with an inosine-containing

0$-

Figure 1. Northern analysis of MyoD expression in rat skeletal and smooth muscle. Total RNA from rat tissues and cell cultures was isolated and analyzed by Northern blotting (30 4g total RNA/lane) by hybridization to 32P 5-terminal labeled MyoD inosine oligonucleotide: lane A, leg muscle; lane B, adult aortic; lane C, cultured adult smooth muscle cell; lane D, cultured newborn smooth muscle cell.

MyoD Conversion of Smooth Muscle Cells 273 AJP July 1993, Vol. 143, No. 1

were grown to 80% confluency, placed in serumfree medium, and immunostained for sarcomeric myosin. About 2% of the MDSN-infected newborn (Figure 2a) and adult smooth muscle cells (Figure 2b) stained positively for skeletal myosin. Morphologically, these converted cells were elongated and resembled postmitotic myoblasts. In the LNL-6 controls no sarcomeric myosin-positive cells were detected (Figures 2c and 2d).

bFGF Enhances MyoD Conversion of Adult Smooth Muscle Cells to Skeletal Muscle We explored the effects of bFGF on this system because of evidence that this mitogen plays a role in myoblast differentiation. bFGF is known to inhibit myotube formation and to repress MyoD expression in myoblasts,34 although in Xenopus embryos, bFGF plays an important role in the induction of mesoderm formation.35 The continuous presence of bFGF, beginning at the start of the G418 selection, completely inhibited conversion in newborn smooth muscle cells even when serum was withdrawn (Figure 3a). The addition of bFGF 1 week after the be-

ginning of the G418 selection did not affect the conversion (not shown). In adult smooth muscle cells, on the other hand, the presence of bFGF raised the percentage of converted cells seen upon withdrawal of serum. The adult cultures showed cells positive for sarcomeric myosin. These cells were grouped in bundles in which complete fusion was seen. Depending on the experiment, 10-30% of the nuclei present in the culture were found in these myotubes (Figure 3b). Other cells expressed sarcomeric myosin but did not fuse (Figure 3c, 3d). If we count these nuclei as well as those in myotubes in converted adult cells, grown in the presence of bFGF, the total percentage of nuclei seen in myosin-positive cells was 20-50%. In control cultures infected with LNL-6, no converted cells were detectable (results not shown). bFGF had to be given immediately after infection in order to see the enhancement of conversion. The presence of bFGF during serum starvation only does not increase the frequency of converted cells compared to cells not treated with bFGF (results not shown). Although all cells in these cultures were resistent to G418 and, consequently, were infected by

M-

9a9SSw:4s-ha£

Figure 2. Conversion of cultured newborn and adult aortic smooth muscle cell by MyoD. Newborn (a and C) and adult (b and d) smooth muscle cells were infected with a MyoD containing retrovirus MDSN (a and b) or with the parental retrovirus LNL-6 (c and d). Neomycin-resistant cells were selected with G418 and serum-starved for 7 days followed by immunostaining for sarcomeric myosin.

274

van Neck et al


Figure 3. Conversion by MyoD of cultured newborn and adult aortic smooth muscle cells grown in the presence of bFGF. MDSN-infected newborn (a) and adult (b, C, d) smooth muscle cells were grown and serum-starved for 7 days continuously in the presence of 10 ng/ml bFGF. The cultures were immunostained for sarcomeric myosin. Different morphologies seen in the adult smooth muscle cell are shown (b, c, d).

the virus (see Materials and Methods), many of the cells did not convert. Since true skeletal muscle cells become postreplicative when they differentiate, we examined the ability of converted smooth muscle cells to replicate. We converted an adult smooth muscle cell culture, previously grown and serum-starved in the presence of bFGF. Serum was re-added, and after 24 hours the converted culture was grown for 12 hours in the presence of [3H]thymidine. The culture then was stained for sarcomeric myosin and analyzed by autoradiography. About 10% of the cells were stained positive for sarcomeric myosin, and none of these cells incorporated [3H]thymidine into their nuclei. Almost all cells that stained negative for sarcomeric myosin did incorporate [3H]thymidine (Figure 4). These observations suggest that cultured adult smooth muscle cells contain a subpopulation of cells that are convertible under the direction of MyoD and once converted by removal of serum undergo terminal differentiation and are no longer capable of replication. The majority of the cells, on the other hand, do not convert and retain their capability to grow in culture.

Adult Smooth Muscle Cells, Converted in the Presence of bFGF; Transcribe Many Skeletal Muscle Genes To determine the status of the myogenic differentiation program of converted smooth muscle cells, MDSN-infected cultures of newborn and adult smooth muscle cells grown with and without bFGF were serum-starved, and total RNA was isolated. Transcription of the viral encoded MyoD was, as expected, exclusively linked with MDSN infection. Activation of the endogenous MyoD gene was not seen in these cells (Figure 5), since expression of the endogenous gene should lead to a hybridization signal of 2.5 kb (Figure 1a). Expression of a number of skeletal muscle genes was assayed on Northern blots containing equal amounts of total RNA from MDSN-infected converted newborn and MDSN-infected converted adult smooth muscle cell cultures, serum-starved MDSN-infected newborn smooth muscle cells grown in the presence of bFGF (without detectable conversion), and MDSN-infected adult smooth


*kb 23.1 :9.4 6.6

4.3 *n.. x

*. ,,l.w

2:.0

:.

0.6 ::::: .. :.

..

::

::

*: .:

*::

:: .. *: ....

:.

.....

*. .:...

Figure 4. Replication of converted MDSN-infected adult smooth muscle cells. MDSN-infected adult smooth muscle cells were grown on coverslips and serum-starved for 7 days. The culture was refed with serum containing medium in the presence of [PHlthymidine for 12 hours. The culture then was immunostained for sarcomeric myosin and analyzed by autoradiography (as described in Materials and Methods).

muscle cells grown in the continuous presence of bFGF (with a high conversion percentage). Northern blot analysis of the cells using myosin light chain 2 cDNA as a probe showed a hybridization signal only in the bFGF-treated MDSN-infected adult smooth muscle cells (Figure 6a), corresponding to the expected mRNA size of 0.9 kb.30 Expression of skeletal muscle genes (myosin light chain, skeletal muscle a-actin, and troponin 1) in bFGFtreated MDSN-infected adult smooth muscle cells, but not in any of the other culture types or FGF conditions, was observed consistently (Figure 6a, b, and c). In contrast, expression of cardiac muscle a-actin was not observed (results not shown). To examine the possibility that MyoD also influences the level of expression of genes normally expressed in smooth muscle cells we assayed the expression of smooth muscle a-actin and desmin. Desmin is of particular interest because this gene is expressed in both skeletal muscle cells and in smooth muscle cells. The level of expression of the smooth muscle cell a-actin gene is unaffected by

..

:.

.::

.:

.........

..............

Figure 5. Northern blot analysis of MyoD transcripts in cultures of uninfected and MDSN-infected newborn and adult aortic smooth muscle cells. Newborn (lane N), MDSN-infected newborn (lane IN), adult (lane A), and MDSN-infected adult (lane IA) smooth muscle cells were treated as descnbed in Figure 1. Total RNA was isolated, and a Northern blot was prepared and screened with 32P-labeled MyoD cDNA. The positions and sizes of the phage A HindIII marker fragments are indicated.

the presence of MyoD (Figure 7). The expression pattern of desmin is not clear. In some cultures the amount of desmin mRNA was increased when MyoD was present, as shown by the increased intensity of the 2.1-kb hybridization signal (Figure 8). In other cultures desmin transcription is very low and seems to be unaffected by MyoD or cannot be detected, irrespective of the presence of MyoD. The reason for this variability remains unclear. It is of interest to note, however, that these results at the RNA level contradict the common experience that cultured smooth muscle cells lose desmin, as seen by immunocytochemistry or by Western blotting.36 We confirmed the absence of desmin protein detectable by immunostaining (results not shown). The detection of RNA for various skeletal muscle genes in MDSN-infected adult smooth muscle cells treated with bFGF suggests a conversion into the

276

van Neck et al


.i

Figure 6. Northern blot analysis of expression of genes coding for skeletal muscle contractile proteins in cultures of MDSN-infected newborn and adult aortic smooth muscle cells maintained and converted in the presence or absence of bFGF. MDSN-infected smooth muscle cells were maintained and serum-starved in the absence (marked IN and IA for MDSN-infected newborn and adult smooth mtuscle cells, respectively) or presence of 10 ng/ml bFGF (marked INF and IAFfor MDSN-infected newborn and adult smooth muscle cells, respectively). Total RNA uwas isolated, and a Northern blot was made and hybridized with a 32P-labeled myosin light chain 2 cDNA (a), a skeletal a-actin oligonucleotide (b), and a skeletal muscle-specific fast troponin I cDNA (c). 7Te positions and sizes of the phage A HindIII marker fragments are indicated.

skeletal muscle phenotype. We were unable to detect the transcription of skeletal muscle genes in MDSN-infected smooth muscle cells grown in the absence of bFGF, probably because of the low number of converted cells in these cultures. This possibility can be assessed by immunostaining using antibodies to the corresponding proteins (see below).

Converted Smooth Muscle Cells Express Many Myofibrillar Proteins Cultures of newborn and adult smooth muscle cells, infected with MDSN and converted, were stained for various skeletal muscle myofibrillar or intermediate filament proteins to determine whether the myogenic conversion included a spectrum of skeletal muscle proteins and, as noted, to characterize the small frequency of cells that convert when bFGF is

absent (Figure 9). As shown before, the presence of bFGF during growth and conversion influences the percentage of cells converting to the skeletal muscle phenotype. However, irrespective of the presence of bFGF in the culture medium, all cells that appear converted express the full set of skeletal muscle genes assayed (results not shown). Immunostaining for sarcomeric tropomyosin (Figure 9a), myosin (Figure 9b), titin (Figure 9c), nebulin (Figure 9d), and desmin (Figure 9e) showed the characteristic banding pattern for these proteins. These data indicate that MyoD activates the complete myogenic differentiation program in these smooth muscle cell cultures whether the frequency of conversion without FGF is low or whether the frequency is high, eg, in adult cells with FGF. No expression of these skeletal muscle proteins, however, could be found in uninfected smooth muscle cells or in newborn smooth muscle cells that have been


lineage. This is consistent with the earlier report by Choi et al of striated muscle conversion after transfection of cultured gizzard smooth muscle cells.37 The observed absence of endogenous MyoD expression in cultured arterial smooth muscle is in agreement with the results reported in human uterus38 and in Xenopus embryos.39 While smooth muscle cells do not contain a MyoD-like protein, our results indicate that some but not all smooth muscle cells may contain other factors that regulate the ability of MyoD to elicit expression of the skeletal muscle phenotype. Benezra et al. isolated the so-called Id protein, another member of the helix-loop-helix protein family that antagonizes other helix-loop-helix proteins, including MyoD.40 Id is expressed in many tissue culture cell lines,41 including primary cultures of rat vascular smooth muscle cells.42 In the latter cell, Id gene expression is inhibited when serum is taken away. This

Figure 7. Northern blot analysis of smooth muscle a-actin transcripts in uninfected and MDSN-infected cultures of newborn and adult aortic smooth muscle cells. Cultures of newborn (lane N), adult (lane A), MDSN-infected newborn (lane IN), and MDSN-infected adult (lane IA) smooth muscle cells were serum-starved for 7 days. Total RNA was isolated, and a Northern blot was hybridized with a 32p_ labeled smooth muscle a-actin oligonucleotide. The positions and sizes of the phage A HindIII markerfragments are indicated.

infected with the virus, grown in the presence of bFGF, and serum-starved (results not shown). To investigate the expression of smooth muscle cell-specific proteins in converted cells, converted smooth muscle cells were double-stained for titin and for the smooth muscle-specific form of a-actin. Single converted smooth muscle cells express smooth muscle a-actin (Figure 10a) and titin (Figure 10b). These data show that MyoD expression is sufficient to activate the myogenic program in smooth muscle cells without repressing expression of smooth muscle cell a-actin.

Discussion Unlike skeletal muscle cells, smooth muscle cells lack MyoD or any closely related mRNA detectable by degenerate oligonucleotide probes. Our observations presented here, however, show that retrovirally mediated MyoD expression is able to induce differentiation steps in cultured aortic smooth muscle cells that normally occur in the skeletal muscle

Figure 8. Northern analysis of desmin transcripts in uninfected and MDSN-infected cultures of newborn and adult aortic smooth muscle cells. Cultures of newborn (lane N), adult (lane A), MDSN-infected newborn (lane IN), and MDSN-infected adult (lane IA) smooth muscle cells were serum-starvedfor 7 days. Total RNA was isolated, and a Northern blot, containing 50 ug total RNVA/lane, was hybridized with 32P-labeled desmin cDNA. The positions and sizes of the phage A HindIII markerfragments are indicated.

278

van Neck et al


Figure 9. Representative elongated myoblasts from different converted smooth muscle cell cultures immunostained for various sarcomeric proteins and desmin. MDSN-infected newborn and adult smooth muscle cells were seeded on coverslips. The cells uere converted, fixed on the glass, atnd immunostainedfor striated muscle tropomyosin (a), myosin (b), titin (c), nebulin (d), and desmin (e) with the use ofafluorescein-labeled second-

ary antibody.

finding correlates well with our observation that conversion to the skeletal muscle phenotype only occurs when MyoD is present and only in the absence of serum.

The most dramatic evidence is the differentiation response to MyoD of smooth muscle cells obtained from newborn rats versus the response of smooth muscle cells obtained from the adult animal. MyoD


Figure 10. Double-stained elongated myoblasts from converted cultures of MDSN-infected newborn and adult aortic smooth muscle cells. MDSNinfected smooth muscle cells were seeded on coverslips. 7Te cells were converted, fixed on the glass, and double-stained for smooth muscle a-actin (a) and titin (b), with the use offluorescein-labeled secondaty antibodies.

converts 2% of newborn smooth muscle cells, but the conversion is inhibited by bFGF. Adult smooth muscle cells also show 2% myogenic conversion in the absence of bFGF but show 20-50% conversion in the presence of bFGF. Thus, at least in the presence of FGF, the frequency of myogenic conversion is much higher in adult than in pup cells. This difference is consistent with other evidence distinguishing the properties of smooth muscle cells obtained from the aortas of newborn and adult animals. Smooth muscle cells obtained from the arterial media of newborn rats or from the neointima formed after balloon injury of adult rats, when placed into culture, grow independently of platelet-derived factors and display a characteristic epithelioid monolayer growth. In contrast, smooth muscle cells cultured from the arterial media of adult rats require platelet factors and display the typical spindle-shaped "hill and valley" growth pattern.9 10 Furthermore, both newborn smooth muscle cells and neointimal smooth muscle cells express a number of genes that are not expressed in adult smooth muscle cells.4 10 Because these expression patterns are stable in passaged cells, they presumably represent changes in differentiation rather than reversible changes in function such as the changes in expression of contractile proteins described as "modulation" during the adaptation of smooth muscle cells to cell culture.43 Along with the effects of MyoD reported here, it seems likely that the pup and adult smooth muscle phenotypes represent the result of constitutive expression of factors that differentially control gene expression. We do not have a satisfactory explanation for the ability of bFGF to influence differentially the myogenic conversion of adult and pup smooth muscle cells. One possibility is that the cell types differ in the expression of FGF receptors. However, we have

not been able to detect differences in the expression of fig or bek, and binding of FGF does not appear to be affected by the expression of MyoD (data not shown). The mechanism by which bFGF influences conversion into myoblasts and myotubes is unknown. Spizz et al showed that bFGF completely blocks conversion of myoblasts at concentrations at least tenfold lower than that required for mitogenic activity.44 Prior to the terminal differentiation of skeletal muscle FGF receptors are present, but these receptors are lost as the cells differentiate.45 Vaidya et al reported that bFGF inhibits expression of MyoD in myoblast cultures.34 Such a mechanism is clearly not operative after infection of cells with the viral construct MDSN in which the MyoD gene is not directed by authentic regulatory elements. Perhaps, under the direction of MyoD, a subset of highly convertible adult smooth muscle cells become bFGF dependent. Perhaps similar changes occur during the early phases of infection in our system in some way not detected by our measurements of the FGF receptor. The addition of bFGF right after infection might enable these cells to survive, giving rise to a high percentage of conversion to the skeletal muscle phenotype. Intriguingly, a similar subset of FGF-dependent chick embryo skeletal myoblasts was described by Seed and Hauschka.46 They suggested that FGF might be required at one stage of differentiation but prevent differentiation at a later stage. It should be noted that, independently of the age of the rats or the presence of bFGF, conversion of MyoD-infected smooth muscle cells always includes the formation of elongated muscle cells and myotubes and the expression of many skeletal muscle genes in all converted cells (Figures 6 and 9). However, even with the adult cell population, not all cells respond to MyoD. Moreover, the failure to con-

280

van Neck et al


vert may represent a property of a subset of adult cells, since nonconverted cells, after propagation in the presence of the neomycin analogue G418, do not convert at a later stage (Figure 4). This diversity of response is consistent with other evidence that the vessel wall contains a mixture of smooth muscle cell phenotypes47'48 and with evidence for stable morphological differences between clones of smooth muscle cells derived from the adult wall.49'50 Finally, our data suggest that smooth muscle cells, pup as well as adult, contain some factor which prevents expression of the endogenous MyoD. All infected smooth muscle cells in these experiments, converted as well as nonconverted and adult as well as newborn, with or without FGF, express the exogenous MyoD gene but not the endogenous MyoD gene (Figure 5). Thayer et al described the induction of expression of endogenous MyoD mRNA by transfection with a MyoD cDNA in 1OT1/2 and Swiss 3T6 cells but not in several other fibroblast cell lines.51 They suggested that other factors need to be present in order to activate the endogenous MyoD gene. In support of this hypothesis, Goldhamer et al have identified a distal 5' element that is required for skeletal muscle-specific expression of MyoD in transgenic animals.52 Our data suggest that, at least in the presence of FGF, expression of the endogenous MyoD gene is prevented in arterial smooth muscle cells by. either the absence of necessary cofactors or the presence of inhibitors.

Acknowledgments We thank DeeAnn Gregory and Hein van der Lee for immunostainings; Marylene Denyn for DNA autoradiography; Marius Coelen for photography; and Trudy Bartosek, Linda Steijns, and Isa Werny for excellent technical assistance. We also want to express our appreciation to Dr. David Hajjar for determining the binding of FGF to our cells. Finally, we express our appreciation to Steve Hauschka for his advice and comment.

References 1. Gordon D, Mohai LG, Schwartz SM: Induction of polyploidy in cultures of neonatal rat aortic smooth muscle cells. Circ Res 1986, 59:633-643 2. Seifert RA, Schwartz SM, Bowen-Pope DF: Developmental regulated production of platelet-derived growth

factor-like molecules. Nature 1984, 311:669-671 3. Majesky MW, Benditt EP, Schwartz SM: Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sis genes in rat aortic smooth muscle cells. Proc Natl Acad Sci USA 1988, 85:1524-1528 4. Giachelli CM, Majesky MW, Schwartz SM: Developmentally regulated cytochrome P-4501A1 expression in cultured rat vascular smooth muscle cells. J Biol Chem 1991, 266:3981-3986 5. Kocher 0, Skalli 0, Cerutti D, Gabbiani F, Gabbiani G: Cytoskeletal features of rat aortic cells during development. Circ Res 1985, 56:829-838 6. Glukhova MA, Frid MG, Koteliansky VE: Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle. J Biol Chem 1990, 265:13042-13046 7. Kocher 0, Gabbiani G: Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental intimal thickening and culture Differentiation 1986, 32:245-251 8. Walker LN, Bowen-Pope DF, Ross R, Reidy MA: Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Proc Natl Acad Sci USA 1986, 83:7311-7315 9. Schwartz SM, Foy L, Bowen-Pope DF, Ross R: Derivation and properties of platelet-derived growth factorindependent rat smooth muscle cells. Am J Pathol 1990, 136:1417-1428 10. Majesky MW, Giachelli CM, Reidy MA, Schwartz SM: Rat carotid neointimal smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury. Circ Res 1992, 71:759-768 11. Hammerle H, Fingerle J, Rupp J, Grunwald J, Betz E, Haudenschild CC: Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells. Exp Cell Res 1988, 178:390-400 12. Glukhova MA, Kabakov AE, Belkin AM, Frid MG, Ornatsky 01, Zhidkova NI, Koteliansky VE: Meta-vinculin distribution in adult human tissues and cultured cells. FEBS Lett 1986, 207:139-141 13. Sobue K, Sellers JR: Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J Biol Chem 1991, 266:12113-12118 14. Davis RL, Weintraub H, Lassar AB: Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987, 51:987-1000 15. Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Blackwell TK, Turner D, Rupp R, Hollenberg S, Zhuang Y, Lassar A: The MyoD gene family: Nodal point during specification of the muscle cell lineage. Science 1991, 251:761-766 16. Emerson CP, Bernstein SI: Molecular genetics of myosin. Annu Rev Biochem 1987, 56:695-726 17. Otey CA, Kalnoski MH, Bulinski JC: Identification and quantitation of actin isoforms in vertebrate cells and tissues. J Cell Biochem 1987, 34:113-124

MyoD Conversion of Smooth Muscle Cells

281


18. Bender MA, Palmer TD, Gelinas RE, Miller AD: Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol 1987, 61:1639-1646 19. Weintraub H, Tapscott SJ, Davis RL, Thayer MJ, Adam MA, Lassar AB, Miller AD: Activation of musclespecific genes in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci USA 1989, 86:5434-5438 20. Bader D, Masaki T, Fishman DA: Immunochemical analysis of myosin-heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol 1982, 95:763-770 21. Gassner D: Myofibrillar interaction of blot immunoaffinity-purified antibodies against native titin as studied by indirect immunofluorescence and immunogold stainings. Eur J Cell Biol 1986, 40:176-184 22. Wang SM, Greaser ML: Immunochemical studies using a monoclonal antibody to bovine cardiac titin on intact and extracted myofibrils. J Muscle Res Cell Motil 1985, 6:293-312 23. Linn JJC, Chou CS, Linn JLC: Monoclonal antibodies against tropomyosin isoforms: Production, characterization and application. Hybridoma 1985, 4:223-242 24. Danto Si, Fishman DA: Immunochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin. J Cell Biol 1984, 98: 2179-2191 25. Sehested M, Hou-Jensen K: Factor Vill-related antigen as an endothelial cell marker in benign and malignant diseases. Virchows Arch 1981, 391:217-252 26. Auffray C, Rougeon F: Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem 1980, 107:303-314 27. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Edited by C Nolan. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1982 28. Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad Sci USA 1984, 81:1991-1995 29. Quax W, van den Broek L, Vree Egberts W, Ramaekers F, Bloemendal H: Characterization of the hamster desmin gene: Expression and formation of desmin filament in non-muscle cells after gene transfer. Cell 1985, 43:327-338 30. Garfinkel LI, Periasamy M, Nadal-Ginard B: Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2 and 3, troponin-c, troponin-t and a-tropomyosin, and a-actin. J Biol Chem 1982, 18:11078-11086 31. Koppe RI, Hallauer PL, Karpati G, Hastings KEM: cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin mRNAs. J Biol Chem 1989, 264:14327-14333 32. Ueyama H, Hamada H, Battula N, Kakunaga T: Structure of a human smooth muscle actin gene (aortic type) with a unique intron site. Mol Cell Biol 1984, 4:1073-1078 33. Martin FH, Castro MM: Base pairing involving deoxy-

inosine: Implications for probe design. Nucleic Acids Res 1985, 13:8927-8938 34. Vaidya TB, Rhodes SJ, Taparowsky E, Koniecnky SF: Fibroblast growth factor and transforming growth factor f repress transcription of the myogenic regulatory gene MyoDl. Mol Cell Biol 1989, 9:3576-3579 35. Amaya E, Musci TJ, Kirschner MW: Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 1991, 66:257-270 36. Campbell JH, Kocher 0, Skalli 0, Gabbiani G, Campbell GR: Cytodifferentiation and expression of a-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Arteriosclerosis 1989, 9:633-643 37. Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer H: MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc Natl Acad Sci USA 1990, 87:7988-7992 38. Braun T, Bober E, Busschhausen-Denker G, Kotz S, Grzeschik K-H, Arnold H: Differential expression of myogenic determination genes in muscle cells: Possible autoactivation by the Myf gene products. EMBO J 1989, 8:3617-3625 39. Hopwood ND, Pluck A, Gurdon JB: MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J 1989, 8:3409-3417 40. Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H: The protein Id: a negative regulator of helixloop-helix DNA binding protein. Cell 1990, 61:49-59 41. Benezra R, Davis RL, Lassar A, Tapscott S, Thayer M, Lockshon D, Weintraub H: Id: a negative regulator of helix-loop-helix DNA binding protein. Control of terminal myogenic differentiation. Ann NY Acad Sci 1990, 599:1-11 42. Kemp PR, Grainger DJ, Shanahan M, Weissberg PL, Metcalfe JC: The Id gene is activated by serum but is not required for de-differentiation in rat vascular smooth muscle cells. Biochem J 1991, 277:285-288 43. Campbell GR, Campbell JH, Manderson JA: Arterial smooth muscle: A multifunctional mesenchymal cell. Arch Pathol Lab Med 1988, 112:977-986 44. Spizz G, Roman D, Strauss A, Olson EN: Serum and fibroblast growth factor inhibit myogenic differentiation through a mechanism dependent on protein synthesis and independent of cell proliferation. J Biol Chem 1986, 261:9483-9488 45. Olwin B, Hauschka SD: Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture. J Cell Biol 1988, 107:761-769 46. Seed J, Hauschka SD: Clonal analysis of vertebrate myogenesis. VIII. Fibroblast growth factor (FGF)dependent and FGF-independent muscle colony

282 van Neck et al AJPJuly 1993, Vol. 143, No. 1

types during chick wing development. Dev Biol 1988, 128:40-49 47. Babaev VR, Bobryshev YV, Stenina OV, Tararak EM, Gabbiani G: Heterogeneity of smooth muscle cells in atheromatous plaque of human aorta. Am J Pathol 1990, 136:1031-1042 48. Zanellato AMC, Borrione AC, Tonello M, Scannapieco G, Pauletto P, Sartone S: Myosin isoform expression and smooth muscle cell heterogeneity in normal and atherosclerotic rabbit aorta. Arteriosclerosis 1990, 10:

996-1009 49. Dreher KL, Cowen K: Expression of antisense transcripts encoding an extracellular matrix protein by sta-

bly transfected vascular smooth muscle cells. Eur J Cell Biol 1991, 54:1-9 50. Hall KL, Harding JW, Hosick HL: Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas. In Vitro Cell Differ Biol 1991, 27A:791-798 51. Thayer MJ, Tapscott SJ, Davis RL, Wright WE, Lassar AB, Weintraub H; Positive autoregulation of the myogenic determination gene MyoD. Cell 1989, 58:241248 52. Goldhamer DJ, Faeman A, Shani M, Emerson CP Jr: Regulatory elements that control the lineage-specific expression of MyoD. Science 1992, 256:538-542