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© The Rockefeller University Press,
0021-9525/1997//1323 $5.00
The Journal of Cell Biology, Volume 138, Number 6,
, 1997 1323-1331
Article |
Cadherins Promote Skeletal Muscle Differentiation in Three-dimensional Cultures
Department of Biology, University of Toledo, Toledo, Ohio 43606
The cell–cell adhesion molecule N-cadherin, with its associated catenins, is expressed by differentiating skeletal muscle and its precursors. Although N-cadherin's role in later events of skeletal myogenesis such as adhesion during myoblast fusion is well established, less is known about its role in earlier events such as commitment and differentiation. Using an in vitro model system, we have determined that N-cadherin– mediated adhesion enhances skeletal muscle differentiation in three-dimensional cell aggregates. We transfected the cadherin-negative BHK fibroblastlike cell line with N-cadherin. Expression of exogenous N-cadherin upregulated endogenous β-catenin and induced strong cell–cell adhesion. When BHK cells were cultured as three-dimensional aggregates, N-cadherin enhanced withdrawal from the cell cycle and stimulated differentiation into skeletal muscle as measured by increased expression of sarcomeric myosin and the 12/101 antigen. In contrast, N-cadherin did not stimulate differentiation of BHK cells in monolayer cultures. The effect of N-cadherin was not unique since E-cadherin also increased the level of sarcomeric myosin in BHK aggregates. However, a nonfunctional mutant N-cadherin that increased the level of β-catenin failed to promote skeletal muscle differentiation suggesting an adhesion-competent cadherin is required. Our results suggest that cadherin-mediated cell–cell interactions during embryogenesis can dramatically influence skeletal myogenesis.
THE association of similarly fated cells is a critical aspect of embryonic development. The cadherin family of proteins mediates cell–cell adhesion through homophilic interactions and thus promotes homogeneity within tissues (Takeichi, 1995). Their spatiotemporal pattern of expression during development suggests cadherins play an important role in the formation and maintenance of tissues (Takeichi, 1988). The best-characterized cadherins are classical cadherins including E-cadherin (uvomorulin), N-cadherin, and P-cadherin. Cadherins are calcium-dependent transmembrane proteins that interact intracellularly with a group of proteins known as catenins (Wheelock and Knudsen, 1991; Kemler, 1993; Gumbiner, 1996). The catenins link the cadherins to the actin cytoskeleton and are required for full adhesive activity of most cadherins (Nagafuchi and Takeichi, 1988, 1989; Ozawa et al., 1990; Knudsen et al., 1995; Rimm et al., 1995). β-catenin, which interacts with cadherins directly, has a role in signal transduction and the specification of cell fate (McCrea and Gumbiner, 1991; Ozawa and Kemler, 1992; Miller and Moon, 1996). Cadherin-mediated junctions serve as signaling centers and disruption of these junctions can lead to growth and developmental defects (Huber et al., 1996; Larue et al., 1996).
The formation of skeletal muscle is a developmental process that requires the commitment of mesodermal precursors, withdrawal from the cell cycle, and differentiation of myoblasts into terminally differentiated multinucleate myofibers (Olson, 1992). This process is controlled through a number of developmental checkpoints that regulate cell cycle arrest and the expression of skeletal muscle–specific genes (Ludolph and Konieczny, 1995). Skeletal muscle– specific transcription factors, including MyoD, control the program of tissue-specific transcription within the developing myoblasts and serve as early markers of skeletal myogenesis (Olson and Klein, 1994).
In one myogenic model system, the pluripotent P19 embryonal carcinoma cells can be induced to form skeletal muscle through the addition of DMSO or exogenous MyoD (Edwards et al., 1983). The induction of differentiation by either of these methods requires aggregation of cells in suspension (Edwards et al., 1983; Skerjanc et al., 1994). This requirement for close contact of similar cells during skeletal muscle myogenesis is known as the community effect. The community effect is important for the differentiation of somites, cell lines, and embryonic stem cells into skeletal muscle (Gurdon et al., 1993; Kato and Gurdon, 1993; Slager et al., 1993; Skerjanc et al., 1994; Cossu et al., 1995). Cadherin-mediated adhesion has been implicated in the community effect and skeletal muscle differentiation (Gurdon et al., 1993; George-Weinstein et al., 1997).
Injection of mRNA encoding a dominant-negative cadherin into early Xenopus laevis embryos blocks the expression of MyoD in the early stages of skeletal muscle myogenesis (Holt et al., 1994). N-cadherin has been identified as a predominant cadherin in developing skeletal muscle and thus most studies have focused on its role in myogenesis. For example, function perturbing antibodies to N-cadherin inhibit skeletal muscle differentiation by primitive streak stage chick epiblast cells in vitro (George-Weinstein et al., 1997). Perturbation studies also demonstrated N-cadherin plays a role in myoblast interaction, the formation of myotubes, and in myofibrillogenesis (Knudsen et al., 1990; Mege et al., 1992; Peralta Soler and Knudsen, 1994). Recently, N-cadherin has been shown to play a role in the migration of skeletal muscle precursors to the limb bud (Brand-Saberi et al., 1996).
Our laboratory is interested in the role of cadherins in myogenesis and the potential role cadherins play in the specification of cell fate. In these studies we used the BHK cell line 2254-62.2. BHK cells exhibit myogenic potential through the expression of MyoD and low levels of sarcomeric myosin (Schaart et al., 1991; this study). However, they do not contain detectable cadherin. We introduced exogenous cadherin into this cell line to investigate the role of cadherins in myogenesis. Our results demonstrate that cadherins dramatically increase the expression of sarcomeric myosin and thus promote the differentiation of BHK cells into skeletal muscle. Differentiation in these cells is dependent on the formation of aggregates, or a community effect, with concomitant withdrawal from the cell cycle.
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Antibodies
Myosin was detected with the mouse monoclonal anti–chicken pectoralis myosin antibody MF20 (Developmental Studies Hybridoma Bank; Bader et al., 1982). The 12/101 antibody is a skeletal muscle–specific mouse monoclonal antibody to newt skeletal muscle homogenate (Developmental Studies Hybridoma Bank, Johns Hopkins University, Baltimore, MD; Kintner and Brockes, 1984). N-cadherin was detected with 6B3 (George-Weinstein et al., 1997) or 13A9 (Knudsen et al., 1995), β-catenin was detected with 15B8 (Johnson et al., 1993), E-cadherin was detected with the E9 rat monoclonal anti–E-cadherin antibody (Wheelock et al., 1987). Mouse monoclonal antibodies to E- and P-cadherin were purchased from Transduction Laboratories (Lexington, KY). A polyclonal pan–cadherin antibody was purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal anti–bromo-deoxyuridine (BrdU)1 was purchased from Amersham Corp. (Arlington Heights, IL). Polyclonal anti-MEF2 recognizing all isoforms was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal anti-MyoD was a gift from A.J. Harris (University of Otago Medical School, Otago, New Zealand).
Transfection of BHK Cells
Transcription of the chicken N-cadherin cDNA was controlled by the human β-actin promoter in the expression vector pHβApr-1neo (pHβ-Ncad) (Murphy-Erdosh et al., 1995) (gift of G. Grunwald, Thomas Jefferson University, Philadelphia, PA). A HindIII fragment containing the entire human E-cadherin cDNA was cloned into the HindIII site of pHβApr-1neo to create pHβ-Ecad (Lewis et al., 1997). A mutant cDNA encoding an N-cadherin missing 390 bp from the extracellular domain (Fujimori and Takeichi, 1992) was cloned into pHβApr-1neo to create pHβ-Ncad
. BHK cells were cotransfected with 1.0 µg of cadherin DNA and 0.1 µg of pLKpac, which served as a more efficient selectable marker than the neomycin resistance gene within pHβApr-1neo. pLKpac was constructed by placing an AvaII/SmaI fragment containing the puromycin resistance gene from pBSpacp in pLK-neo-1 (de la Luna et al., 1988; Hirt et al., 1992). Control cells were transfected with pLKpac alone. Transfections were carried out using Lipofectamine (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions.
Formation of Cell Aggregates
Aggregates were formed by harvesting cells with trypsin/EDTA and resuspending the cells at 1.5 x 105 cells per ml in DME with FBS. 20-µl drops of media containing 3,000 cells per drop were pipetted onto the inner surface of the lid of a Petri plate. The lid was then placed on the Petri plate so that the drops were hanging from the lid with the cells suspended within them. To eliminate evaporation within the hanging drops, 5 ml of PBS was placed in the bottom of the Petri plate. For analysis, aggregates were harvested by pipette and dispersed for counting, extracted for Western blot analysis, or replated on slides for immunofluorescence microscopy.
Cell Growth Assay
A total of 1.5 x 105 cells were plated in 20-µl drops containing 3,000 cells per drop and either suspended from Petri plate lids or placed as individual drops on tissue culture dishes. Cells were grown for 72 h and harvested with trypsin for cells on culture dishes or dispersed into single cells with trypsin for cells in hanging aggregates. Cells were counted microscopically using a hemocytometer and viability determined using Trypan blue exclusion.
BrdU Labeling of Cells
BHK cells were labeled with a 1:1,000 dilution of cell proliferation labeling reagent (Amersham Corp.) containing BrdU and fluoro-deoxyuridine for 2.5 h and fixed using paraformaldehyde. BrdU was detected with a monoclonal anti-BrdU antibody.
Western Blot Analysis
Cell monolayers were harvested by scraping, or suspended cell aggregates were collected by pipet. The cells were washed in PBS, and extracted in a 10-fold excess of 1x SDS sample buffer (50 mM Tris, pH 6.8, 20 mM DTT, 2% SDS, 0.1% bromphenol blue, 10% glycerol). Insoluble material was removed by centrifugation. An equal amount of protein from each sample was separated on SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose as described (Knudsen et al., 1995). Blots were blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20. Proteins of interest were detected with various primary monoclonal and polyclonal antibodies and the appropriate species-specific, alkaline phosphatase–conjugated secondary antibodies (Fisher Scientific). Blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Prestained molecular weight markers were purchased from Bio Rad Laboratories (Richmond, CA). High range molecular weight markers include myosin (205 kD), β-galactosidase (116.5 kD), bovine serum albumin (77 kD), and ovalbumin (46.5 kD). Low range molecular weight markers include phosphorylase B (110 kD), bovine serum albumin (84 kD), ovalbumin (47 kD), carbonic anhydrase (33 kD), soybean trypsin inhibitor (24 kD), and lysozyme (16 kD). The presence of dye in the prestained standards causes some proteins to migrate at the indicated molecular weights rather than their true molecular weights.
Immunofluorescence
Cells were grown on glass slides for 2 d and fixed in methanol for 10 min at –20°C. Staining was performed as described (Knudsen et al., 1995). For double labeling, cells were labeled sequentially for BrdU incorporation and myosin expression using anti-BrdU, indocarbocyanate conjugated to anti–mouse IgG (Jackson Immuno Research, West Grove, PA), MF20, and FITC conjugated to anti–mouse IgG2b (ICN Biomedicals, Irvine, CA).
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Given the significant role of cadherins in many aspects of skeletal muscle development, and the myogenic potential of the BHK cells that lack detectable cadherins, these cells presented a unique model system to examine the role of cadherins in differentiation. Thus, we transfected the BHK cells with exogenous cadherin and examined the effect of its expression on a biochemical marker of differentiation, i.e., sarcomeric myosin. We initiated our studies with N-cadherin due to its established role in myogenesis.
We transfected chicken N-cadherin into BHK cells and established several cell lines that express stable levels of N-cadherin (Fig. 1). The exogenous N-cadherin localized to sites of cell–cell adhesion but had little effect on the morphology of cells in monolayer cultures (Fig. 1 A), although it increased their ability to aggregate in suspension in the presence of calcium (not shown). Introduction of the exogenous cadherin resulted in a dramatic increase in the levels of β-catenin detected at cell–cell junctions by immunofluorescence light microscopy (Fig. 1 A) and in cell extracts by Western blot analysis (Fig. 1 B). This increase in catenin levels upon introduction of exogenous cadherin in cells lacking endogenous cadherin has been shown previously (Nagafuchi et al., 1991; Tanihara et al., 1994).
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N-cadherin Alters the Growth of BHK Cells in Aggregates
Differentiation into skeletal muscle requires coordinated withdrawal from the cell cycle and activation of myogenic transcription factors (Ludolph and Konieczny, 1995). Since N-cadherin was capable of promoting differentiation of BHKs in aggregates but not the monolayer cultures, we were interested in its potential to affect the growth rate of these cells in monolayer and suspension cultures.
Exogenous N-cadherin had no effect on the proliferation of BHK cells grown as monolayer cultures in the presence of serum (Fig. 4 A). However, when BHK cells were cultured as aggregates in the presence of serum, their growth decreased significantly in comparison to the attached cells. Moreover, this inhibition was enhanced in cells expressing N-cadherin (Fig. 4 A). There was no difference in the percentage of nonviable cells in any of the cultures. These results suggest that suspension culture induces withdrawal from the cell cycle and that this is further enhanced by N-cadherin. The inhibition of cell proliferation in control aggregates did not stimulate differentiation. However, the enhanced inhibition of growth induced by N-cadherin in the aggregates may play a role in promoting their differentiation into skeletal muscle.
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Two of the transcription factors that cooperate to activate skeletal muscle myogenesis, MyoD and MEF2, both respond to growth control signals during differentiation (Olson et al., 1991; Ludolph and Konieczny, 1995; Molkentin et al., 1996). Given our finding that N-cadherin inhibits proliferation and enhances sarcomeric myosin levels in aggregated BHK cells, we were interested in the possible effect N-cadherin–mediated aggregation may have on levels of MyoD and MEF2. Therefore, we examined the levels of MEF2 and MyoD in suspended aggregates using Western blot analysis. We found that suspension culture with concomitant withdrawal from the cell cycle slightly increased the levels of MEF2 and MyoD above those found in cells grown as a monolayer (Fig. 5). However, the presence of N-cadherin did not have any detectable effect on the levels of MyoD or MEF2 in either monolayer or suspension cultures (Fig. 5).
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Discussion |
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The increase in sarcomeric myosin in cadherin-expressing BHK cells required their aggregation in suspension, which disrupts the extracellular matrix interactions found in cells grown as monolayers. In this regard, the BHK cells are similar to P19 teratocarcinoma cells that differentiate to skeletal muscle only after being aggregated in suspension (Edwards et al., 1983). In other systems, perturbation of cell–matrix adhesion inhibits myogenesis. Inhibition of matrix synthesis and perturbation of integrin function with peptides or antibodies inhibit the differentiation of cultured embryonic chick pectoral muscle myoblasts (Menko and Boettiger, 1987; Nandan et al., 1990; Saitoh et al., 1992). Moreover, myogenic cells grown in suspension as single cells growth arrest but fail to differentiate without substrate adhesion (Milasincic et al., 1996). These apparently conflicting results are likely due to differences in the model systems and how the experiments were conducted. It is possible that cadherin-mediated adhesion alters the production of matrix or the interaction of cells with extracellular matrix in a way that favors differentiation. N-cadherin–mediated adhesion also could bypass the signals required for the integrin-mediated promotion of myogenesis.
Many growth factors inhibit myogenesis by negatively regulating myogenic transcription factors, including MyoD and MEF2, that are upregulated during skeletal muscle myogenesis (Vaidya et al., 1989; Olson et al., 1991; Yu et al., 1992; Breitbart et al., 1993; Buchberger et al., 1994). In addition, MyoD positively autoregulates its own expression and in some systems can induce cells to withdraw from the cell cycle through induction of the cell cycle inhibitor p21 (Thayer et al., 1989; Halevy et al., 1995). Therefore, it is not surprising that the aggregated BHK cells that withdrew from the cell cycle showed increased levels of these myogenic transcription factors. However, only the cadherin-expressing BHK cells expressed increased sarcomeric myosin. This may be due to the enhanced growth suppression in the cadherin-expressing cells. Alternatively, cadherin-mediated adhesion may alter the activity of the myogenic transcription factors. The activities of both MyoD and MEF2 are regulated through changes in phosphorylation state and interaction with binding partners (Benezra et al., 1990; Jen et al., 1992; Li et al., 1992; Ornatasky and McDermott, 1996). However, we did not observe a shift in molecular weight of MyoD or MEF2, nor did we see a change in serine phosphorylation of MEF2, suggesting the cadherin did not alter phosphorylation of these transcription factors (not shown).
Cadherins are responsible for the close association of groups of cells with a similar developmental fate. The associated cells then initiate and respond to further developmental signals. One possible result of this cadherin-mediated association is intracellular signaling via the cadherin–catenin complex. Several lines of evidence suggest β-catenin has an important role in development and signal transduction. β-Catenin shares homology with the Drosophila segment polarity protein Armadillo, which also is found in cell junctions (Gumbiner, 1995; Peifer, 1995). Changes in β-catenin levels result in alterations of mesoderm formation in Xenopus and mouse (Heasman et al., 1994; Funayama et al., 1995; Haegel et al., 1995). In addition, signaling by Wnt leads to the accumulation of β-catenin in the cytoplasm and β-catenin can be translocated to the nucleus (Orsulic and Peifer, 1996; Papkoff et al., 1996; Schneider et al., 1996; Yost et al., 1996). Although the role of β-catenin in signal transduction makes it an attractive candidate for an enhancer of myogenesis, our studies suggest that the primary stimulatory effect of exogenous cadherins on myogenesis comes from the cadherin–catenin complex itself and cadherin-mediated adhesion. It is possible, however, that β-catenin stimulates myogenesis through a pathway that is dependent on functional cadherins.
The requirement of a close community of cells for differentiation has been demonstrated in several developmental systems, and cell–cell adhesion plays an important role in many of these systems (Gurdon et al., 1993). The formation of aggregates by primary thyroid cells alters cell–cell contacts and promotes their differentiation (Yap and Manley, 1993). The differentiation of trophoblast cells in culture also requires their aggregation and is associated with an increase in E-cadherin expression (Rebut-Bonneton et al., 1993). The importance of tissue morphology and the balance between cell–cell and cell–matrix adhesion in development was demonstrated recently in a tumorigenic breast cell line (Weaver et al., 1997). In this model system, nontumorigenic breast cancer cells cultured as three-dimensional aggregates differentiated and formed a basement membrane. In contrast, a spontaneous tumorigenic subline with known oncogenic mutations failed to undergo morphogenesis when grown as aggregates, and instead produced invasive colonies with poor cell–cell adhesion. The addition of integrin function perturbing antibodies to these tumorigenic aggregates promoted the formation of cell–cell junctions, withdrawal from the cell cycle, and differentiation. However, the same antibodies caused decreased cell–cell adhesion in the nontumorigenic cells and inhibited their differentiation. This study, like ours, illustrates the impact that tissue morphology and cellular interactions play in the regulation of cell growth and differentiation. Cellular condensation involving changes in cell shape and cell adhesion is a frequent occurrence in embryogenesis, i.e. somitogenesis. These changes are likely to alter many aspects of cell behavior, including proliferation and expression of transcription factors, all of which contribute to the control of cell and tissue differentiation both in vivo and in vitro.
In summary, our studies show that cadherin-mediated cell–cell adhesion, along with the presence of myogenic transcription factors (i.e., MyoD and MEF2) and withdrawal from the cell cycle, is necessary for skeletal muscle differentiation. Muscle differentiation is not stimulated by increased MyoD/MEF2 and decreased cell proliferation in the absence of cadherin-mediated cell–cell adhesion, even if the cells are brought into close contact. The signal(s) generated by the cadherin-mediated adhesion and the mechanism by which sarcomeric myosin levels are increased are subjects for future studies.
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Acknowledgments |
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Submitted: 5 May 1997
Revised: 16 July 1997
Address all correspondence to Karen A. Knudsen, The Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA 19096. Tel.: (610) 645-3581. Fax: (610) 645-2205.
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