* The Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096; and 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 -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
-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 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 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 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 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 Cells
The BHK 2254-62.2 cell line was obtained from American Type Culture
Collection (Rockville, MD) and grown in DME (Fisher Scientific, Pittsburgh, PA) with 7% FBS.
Antibodies
Myosin was detected with the mouse monoclonal anti-chicken pectoralis
myosin antibody MF20 (Developmental Studies Hybridoma Bank; Bader
et al., 1982 Transfection of BHK Cells
Transcription of the chicken N-cadherin cDNA was controlled by the
human Formation of Cell Aggregates
Aggregates were formed by harvesting cells with trypsin/EDTA and resuspending the cells at 1.5 × 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 × 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 1× 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 Immunofluorescence
Cells were grown on glass slides for 2 d and fixed in methanol for 10 min at
Exogenous N-cadherin Promotes Adhesion and
Increases The BHK 2254-62.2 cells used in this study were derived
from BHK-21/C13 cells isolated from BHK. The BHK-21/
C13 cells exhibit skeletal muscle characteristics including
the presence of desmin, titin, and muscle-specific tropomyosin, and the ability to fuse into multinucleate myotubes (Frank et al., 1982 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
N-cadherin Promotes Myogenesis Only in Aggregated
BHK Cells
Introduction of N-cadherin had no detectable effect on the
overall level of sarcomeric myosin in BHK cells growing as
a monolayer (Fig. 2 B), which at first glance suggested that
N-cadherin does not promote differentiation. Differentiation of skeletal muscle cell lines can be triggered by removing FBS or other growth factors. A similar approach
with monolayer cultures of BHK cells failed to induce differentiation, even when the cells expressed exogenous N-cadherin. However, other cells with myogenic potential,
such as P19 teratocarcinoma cells or embryonic stem cells,
are induced to differentiate into skeletal muscle through
aggregation in suspension (Edwards et al., 1983
Aggregation of the N-cadherin-containing transfectants by suspension culture resulted in a dramatic increase
in sarcomeric myosin heavy chain, a marker of muscle differentiation, whereas control cell aggregates showed no
detectable change in myosin expression (Fig. 2). The increase in myosin expression in the N-cadherin transfectants was paralleled by an increase in the 12/101 antigen, a
marker of skeletal muscle differentiation (Kintner and
Brockes, 1984 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 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.
Given the inhibition of growth induced by N-cadherin-
mediated aggregation and the role of N-cadherin in the promotion of myogenesis, we wanted to determine whether
the cells expressing myosin were postmitotic. Our experiments demonstrated that cells within the N-cadherin-
expressing aggregates express myosin (Fig. 2), whereas the
majority of those in a monolayer around the aggregate did
not. We examined the mitotic state of the cells within these aggregates using BrdU labeling and found that BrdU was
incorporated into many cells on the periphery of the aggregates, whereas the vast majority of cells within the aggregates failed to incorporate BrdU (Fig. 4 B). Double
labeling using anti-myosin and anti-BrdU monoclonal
antibodies and isotype-specific secondary antibodies revealed that myosin positive cells on the periphery of the
aggregate did not incorporate BrdU. A total of 517 cells
within 10 independent fields on the periphery of an aggregate were analyzed and 45% of the cells were positive for
BrdU, 16% were positive for myosin, but none were positive for both myosin and BrdU. The lack of BrdU incorporation within the myosin positive aggregates provides further evidence that N-cadherin promotes differentiation
and withdrawal from the cell cycle.
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
E-cadherin Also Promotes the Differentiation of
BHK Cells
N-cadherin is expressed by skeletal muscle precursors and
has been implicated in the differentiation of skeletal muscle (George-Weinstein et al., 1997
Nonfunctional N-cadherin Increases Enhanced differentiation of BHK cells into skeletal muscle does not appear to be N-cadherin specific. At this
point, we wanted to distinguish between cadherin-mediated adhesion and the cadherin-mediated increase in
Skeletal muscle precursors contain multiple cadherins that
together have an important role in establishing tissue homogeneity. M-cadherin is involved in myotube formation
and the adhesion of satellite cells to mature muscle (Donalies
et al., 1991 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 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 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
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 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.
). 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
).
-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
).
). 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
).
). 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
).
). 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
).
; 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.
Materials and Methods
). 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
),
-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).
-actin promoter in the expression vector pH
Apr-1neo (pH
-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 pH
Apr-1neo to create pH
-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 pH
Apr-1neo to create pH
-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 pH
Apr-1neo. pLKpac was constructed by
placing an AvaII/SmaI fragment containing the puromycin resistance
gene from pBSpac
p 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.
). 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),
-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.
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).
Results
-catenin Levels in BHK Cells
; Quinlan and Franke, 1982
;
Schaart et al., 1991
; Van der Loop et al., 1996
). The subline BHK 2254-62.2 used in this study expresses MyoD and low
levels of sarcomeric myosin but does not fuse. These cells
exhibit fibroblastlike morphology with poor cell-cell adhesion. Immunofluorescence light microscopy revealed that
the BHK cells lack detectable N-, E-, or P-cadherin (not
shown). Moreover,
-catenin was found at low levels in
the cytoplasm but was absent at the plasma membrane, suggesting the cells lack significant levels of transmembrane cadherin. In aggregation assays, the BHK cells exhibit a small degree of calcium-dependent cell-cell adhesion, which may result from a low level of an unidentified
cadherin or from some other adhesion mechanism (Urushihara et al., 1977
; data not shown). The BHK cells do express N-CAM and thus exhibit calcium-independent aggregation (not shown).
-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
).
Fig. 1.
Immunofluorescent
light microscopic analysis of
N-cadherin and -catenin in
control and N-cadherin-
transfected BHK cells. (A)
BHK cells containing either
pH
-Ncad plus pLKpac (N-cadherin) or pLKpac alone (control) were grown on slides, fixed,
and stained for the presence of either N-cadherin with 6B3 or
-catenin with 15B8. Both N-cadherin and
-catenin localized to
sites of cell-cell contact in the transfected cells but not in control
cells. (B) Extracts from two independent clones stably expressing
exogenous N-cadherin and from two control cell lines were resolved by SDS-PAGE and immunoblotted with antibodies to
N-cadherin (6B3) or
-catenin (15B8). Two separate gels were
probed for either N-cadherin or
-catenin. Low range molecular
weight markers were used and the positions of the 110- and 84-kD bands are shown at the right side of the figure.
[View Larger Versions of these Images (72 + 28K GIF file)]
; Slager et
al., 1993
). Aggregation likely mimics conditions in which
tight cell-cell contacts form in vivo, such as within somites.
To replicate these conditions, we brought the BHK cells
into close contact by culturing them in hanging drops of
DME containing 7% FBS, and examined the effect of this aggregation on their differentiation. Aggregation of cells
containing exogenous N-cadherin resulted in the formation of large aggregates that did not disperse after replating the cells for 48 h (Fig. 3). In contrast, the suspended
control cells lacking N-cadherin failed to form stable aggregates and readily dispersed upon replating (Fig. 3).
Fig. 2.
N-cadherin increases
sarcomeric myosin and the
12/101 antigen in aggregated
BHK cells. Control cells and
cells expressing exogenous
N-cadherin were aggregated
and examined for the expression of two markers of skeletal
muscle differentiation. (A) Cells
were aggregated for 24 h, then collected, and placed on slides for 48 h. Sarcomeric myosin was detected with MF20 and the 12/101 antigen was detected with a monoclonal antibody. The apparent differences in the number and morphology between control cells and cells expressing N-cadherin are due to the different cell-cell adhesion properties of the cells (Fig. 3). (B) Two independent clones expressing N-cadherin (N-cad) and two control clones
were grown as a monolayer and as aggregates in hanging drops
(agg.) for 72 h. The cells were collected and extracted in SDS
sample buffer. Extracts were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with MF20 to sarcomeric myosin. High range molecular weight markers were used
and the positions of the 205- and 116.5-kD bands are indicated at
the right side of the figure. No increase in myosin or 12/101 was
seen in N-cadherin-expressing cells grown in a monolayer.
[View Larger Versions of these Images (73 + 40K GIF file)]
Fig. 3.
N-cadherin promotes aggregation in BHK
cells. BHK cells expressing
exogenous N-cadherin (N-cadherin) and control cells
(control) were placed in
hanging drops for 24 h to
promote aggregation. The
cells were then collected,
placed on slides, and grown
for 48 h to permit strong attachment to the slides. During this time N-cadherin-
expressing cells remained as
aggregates whereas the control cells spread out as a
monolayer. Cells were fixed
and observed by phase contrast microscopy.
[View Larger Version of this Image (121K GIF file)]
) (Fig. 2 A). To test the specificity of the N-cadherin-mediated induction of differentiation, we also
aggregated the BHK cells with the lectins wheat germ agglutinin and concanavalin A. Aggregation in the presence
of either of these lectins did increase cell-cell adhesion but
did not increase sarcomeric myosin levels, suggesting
N-cadherin has a role in promoting differentiation beyond
simple agglutination of the cells (not shown). The differentiation process observed in the N-cadherin-containing
BHKs proceeded over a 72-h-period in suspension culture
with a gradual increase in myosin expression (not shown).
For each of the following experiments we examined differentiation in suspension cultures at the 72-h-time point.
). 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.
Fig. 4.
Culturing BHK
cells in aggregates inhibits
their growth, and this inhibition is enhanced by N-cadherin. (A) An equal number of cells (1.5 × 105) expressing
N-cadherin (N-cad) or of
control cells (control) was
grown for 72 h as hanging
drops (aggregates) or as individual drops on tissue culture
dishes (monolayer). Cells
were harvested and dispersed into single cells with
trypsin and counted microscopically using a hemocytometer. Viability was determined by Trypan blue exclusion. Viability was >95% in all of the cultures. The
mean of four individual experiments is shown and the standard deviation is indicated. Student's test was used to determine there was no significant difference between the growth of N-cadherin-expressing cells and control cells cultured as monolayers (P = 0.3736). There were significant differences between the growth of control cells in aggregates vs monolayers (P < 0.001), the growth of N-cadherin- expressing cells in aggregates vs monolayers (P < 0.001), and the growth of N-cadherin-expressing cells vs control cells cultured as aggregates (P < 0.001). (B) BHK cells expressing N-cadherin were placed in hanging drops for 24 h to promote aggregation. The cells
were collected, placed on slides, and grown for 24 h to permit strong attachment to the slides. The cells were then labeled with BrdU
and probed for BrdU using a monoclonal anti-BrdU. The position of the aggregate is indicated by an arrow in each panel.
[View Larger Versions of these Images (38 + 72K GIF file)]
; 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).
Fig. 5.
N-cadherin does
not alter levels of myogenic
transcription factors in BHK
cells. BHK cells expressing
exogenous N-cadherin (N-cad) and control cells (control) were grown as a monolayer and as hanging drops
(agg.) for 72 h and prepared
for Western blot analysis as
described in Fig. 2. Blots
were probed with polyclonal anti-MEF2 and MyoD. Low range molecular weight markers
were used and the positions of the 110-, 84-, and 47-kD bands are
indicated on the figure.
[View Larger Version of this Image (59K GIF file)]
). Other cadherins, such
as E-cadherin, can induce strong cell-cell adhesion but are
not found in skeletal muscle. Therefore, we sought to determine whether stimulation of myogenesis by N-cadherin
in BHK cells was specific to N-cadherin or if another cadherin, even one not normally expressed by skeletal muscle, also could stimulate differentiation. Therefore, we transfected the BHK cells with human E-cadherin. Similar to
those with N-cadherin, cells expressing E-cadherin showed
increased cell-cell adhesion in an aggregation assay (not
shown) and also had increased levels of
-catenin relative
to control cells (Fig. 6). Moreover, cells containing E-cadherin showed differentiation properties similar to those with
N-cadherin (i.e., upon aggregation in suspension culture,
the cells expressed increased levels of sarcomeric myosin) (Fig. 6). These results demonstrate that cadherin-mediated stimulation of skeletal muscle differentiation is not
N-cadherin specific, and likely can be accomplished by any
cadherin expressed by skeletal muscle cells or their precursors.
Fig. 6.
E-cadherin promotes skeletal muscle myogenesis. Stable cell lines transfected with either pH-Ecad plus pLKpac
(E-cadherin) or pLKpac alone (control) were aggregated and analyzed by immunofluorescence light microscopy as described in
Fig. 2. Both E-cadherin and
-catenin localized to sites of cell-
cell contact. E-cadherin was detected with E9,
-catenin was detected with 15B8, and myosin was detected with MF20. As with
N-cadherin, increased myosin was observed only when the cells
were aggregated in suspension.
[View Larger Version of this Image (64K GIF file)]
-catenin but
Does Not Promote Differentiation
-catenin. Expression of any exogenous classical cadherin by these cells would be expected to increase both cell-cell
adhesion and the level of endogenous
-catenin. Since
-catenin has been implicated in cell fate determination
and intracellular signaling (Miller and Moon, 1996
), it is
possible that the stimulation of differentiation by exogenous cadherin is due to increased levels of
-catenin. To
test this possibility we transfected the BHK cells with a
cDNA encoding mutant N-cadherin defective in cell-cell adhesion. The intracellular domain of this cadherin is intact and therefore
-catenin levels increase in its presence
(Fig. 7). However, introduction of this mutant N-cadherin
into BHK cells failed to promote differentiation in either
monolayer cultures or aggregates, suggesting that cadherin-mediated adhesion and not simply elevated levels of
-catenin stimulates the differentiation of BHKs into skeletal
muscle (Fig. 7).
Fig. 7.
Nonfunctional N-cadherin increases -catenin but does
not promote skeletal muscle differentiation. Stable cell lines
transfected with either pH
-Ncad
plus pLKpac (truncated
N-cadherin) or pLKpac alone (control) were aggregated and analyzed by immunofluorescence light microscopy as described in
Fig. 2. Truncated N-cadherin was detected with 6B3,
-catenin
with 15B8, and sarcomeric myosin with MF20. Cells expressing
truncated N-cadherin and control cells spread as a monolayer after replating due to lack of strong cell-cell adhesion in either of
these cultures.
[View Larger Version of this Image (50K GIF file)]
Discussion
; Zeschnigk et al., 1995
). N-cadherin is involved
in myoblast interaction and the formation of myotubes
(Knudsen et al., 1990
; Mege et al., 1992
). Skeletal muscle
precursors also contain cadherin-11 (Kimura et al., 1995
),
R-cadherin (Inuzuka et al., 1991
), and T-cadherin (Ranscht,
1994
). Although roles for N-cadherin and M-cadherin in
later stages of myogenesis have been firmly established,
less is known about the requirement of cadherins in the
early stages of myogenesis. Holt et al. (1994)
demonstrated
that a dominant-negative cadherin mRNA, which perturbs
the function of all cadherins, can inhibit early stages of
myogenesis in Xenopus embryos. In addition, George-Weinstein et al. (1997)
recently showed that function-perturbing antibodies to N-cadherin inhibit the differentiation of cultured primitive streak stage chick epiblast cells
into skeletal muscle. Here, we show that N-cadherin-
mediated adhesion promotes skeletal myogenesis in a
myogenic cell line by increasing the level of sarcomeric
myosin but that this effect is not specific for N-cadherin. The lack of N-cadherin specificity is consistent with previous studies in the N-cadherin null mouse, which demonstrated that N-cadherin is not essential for skeletal muscle
differentiation (Radice et al., 1997
). Since skeletal muscle
expresses multiple cadherins, it is likely that upon chronic
loss of N-cadherin another cadherin(s) substitutes functionally for it.
). 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.
; 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).
-catenin has an important role in development and signal transduction.
-Catenin shares homology with the Drosophila segment polarity protein Armadillo, which also is
found in cell junctions (Gumbiner, 1995
; Peifer, 1995
).
Changes in
-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
-catenin in the cytoplasm and
-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
-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
-catenin stimulates myogenesis through
a pathway that is dependent on functional cadherins.
). 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.
Received for publication 5 May 1997 and in revised form 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.We thank L. Myers for expert technical assistance, M. Wheelock and A. Peralta Soler for critical reading of the manuscript, and M. George-Weinstein and K. Linask for helpful discussions. The MF20 and 12/101 antibodies were obtained from the Developmental Studies Hybridoma Bank maintained in the department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD), and the Department of Biological Sciences, University of Iowa (Iowa City, IA) under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development.
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