From the Centro de Regulación Celular y
Patología, Departamento de Biología Celular y
Molecular, Facultad de Ciencias Biológicas, MIFAB,P.
Universidad Católica de Chile, P. O. Box 114-D,
Santiago, Chile and the ¶ Institute of Physiological
Chemistry and Pathobiochemistry, Waldeyerstrasse 15, D-48149
Münster, Germany
Received for publication, May 28, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Decorin is a member of the family of the small
leucine-rich proteoglycans. In addition to its function as an
extracellular matrix organizer, it has the ability to activate
the epidermal growth factor receptor, and it forms complexes with
various isoforms of transforming growth factor The process of myogenic development involves an ordered sequence
of molecular events, which includes commitment of muscle precursor
cells, cessation of cell division, myoblast terminal differentiation,
and formation of myotubes that express muscle-specific genes involved
in the specialized functions of the myofiber (1). A network of muscle
regulatory factors (2) governs these processes. One of the factors
responsible for the induction of terminal differentiation is myogenin,
a transcription factor of the basic helix-loop-helix family that
activates skeletal muscle-specific products such as creatine kinase,
myosin heavy chain, acetylcholine receptor, etc. (3, 4). Terminal
differentiation is critical as well during skeletal muscle formation as
during muscle regeneration after injury (5).
The onset and progression of this process are controlled by a complex
set of interactions between myoblasts and their environment. The
presence of the extracellular matrix
(ECM)1 is essential for
normal myogenesis (6-9). The ability of myoblasts to differentiate is
controlled in a negative manner by the extracellular concentration of
specific mitogens, such as FGF-2, HGF/SF, and TGF- It has been demonstrated that the activities of FGF-2, HGF/SF, and
TGF- Decorin is a member of the small leucine-rich proteoglycans gene
family, which consists of a core protein and a single covalently linked glycosaminoglycan chain (23). Decorin binds several types of
collagen in vivo, among them being types I, II, and VI, and promotes fibril stability. By interacting with ECM molecules like fibronectin and thrombospondin it influences cell adhesion.
Additionally, at least in some types of cells, decorin activates the
epidermal growth factor receptor, thereby triggering a signaling
cascade that leads to phosphorylation of mitogen-activated
protein kinase, to the induction of p21, and to growth
suppression (24, 25). Great attention has been given to the observation
that its core protein interacts with TGF- In skeletal muscle decorin is found mainly in the perimysium (34). The
synthesis and expression of decorin is up-regulated during skeletal
muscle differentiation (35). Interestingly, the synthesis of decorin is
also augmented in dystrophic mdx mouse skeletal muscle (36).
In the present study we investigated the function of decorin during
muscle differentiation. C2C12 myoblasts were
transfected with an expression plasmid containing antisense decorin
cDNA. Suppression of decorin production was accompanied with
accelerated terminal differentiation and with a significant decrease in
the sensitivity to TGF- Materials--
The C2C12 cell line was
purchased from ATCC, Manassas, VA. Trizol LS, LipofectAMINE,
Dulbecco's modification of Eagle's minimal essential medium, CEE,
horse serum, FCS, Opti-MEM I, Hanks' balanced salt solution, G418, and
human FGF-2 were obtained from Life Technologies, Inc. Wizard plus
maxipreps and prime-a-gene labeling system dual-luciferase reporter
assay system, pGL3 basic vector, and pRL were from Promega, Madison, WI. F-12 medium, creatine kinase assay kit, bovine cartilage decorin, and fluorescein isothiocyanate- and
tetramethylrhodamine B isothiocyanate-conjugated goat
anti-rabbit IgG were from Sigma. TGF-
The pMyoLuc reporter construct was synthesized as described for pMyoCAT
(20) with some modifications. Briefly, a 670-base pair DNA
fragment corresponding to the myogenin promoter region, from +55 to
The construction of a replication-deficient adenoviral vector
containing the full-length human decorin cDNA under the control of
the EF-1 promoter (38), the purification of decorin from mouse skeletal
muscle (34), and the production of species-specific decorin antisera
(38) have been described previously.
Cell Culture--
The mouse skeletal cell line
C2C12 was grown and induced to differentiate as
described previously (20, 39). Collagen gels were prepared using
collagen type I from rat tail, and the lattice was performed as
described previously (38), with some modifications: cell-free
collagen lattice was obtained by mixing 450 µl of collagen type I (5 µg/ml), 675 µl of minimum medium, and 75 µl of 0.1 N NaOH in hydrophobic dishes and incubated at 37° for
1 h. The formed lattice was cultured on myoblasts monolayers for
different period of time.
Stable Transfection and Isolation of
Clones--
C2C12 cells were plated at a
density of 6000 cells/cm2 in 100-mm dishes in growth
medium. 24 h later the cells were transfected with the mammalian
expression plasmid pCMVneo containing a 1372-bp cDNA insert
containing the full-length mouse decorin sequence, in the antisense
orientation, kindly donated by Dr. Renato Iozzo, Thomas Jefferson
University, Philadelphia. The complete insert and a pCMVneo plasmid
were digested with EcoRI to generate the pCMVneo-decorin
recombinant plasmid; positive clones were selected by ampicillin
resistance, and the orientation of the insert was determined by
polymerase chain reaction; the antisense orientation was confirmed by
DNA sequencing. Control cells were transfected with pCMVneo containing
no insert. For transfections, 40 µg of plasmid DNA and 30 µg of
LipofectAMINE in a total volume of 600 µl of Opti-MEM I were used
according to the instructions of the manufacturer. After 6 h at
37 °C, FCS and CEE were added to final concentrations of 10 and
0.5%, respectively, and the cells were incubated overnight. The next
day the cells were rinsed twice with Hanks' balanced salt solution and
cultured in normal growth medium. Medium was supplemented with G418
(400 µg/ml) after 3 days. After 2-3 weeks colonies were selected
using cloning rings.
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from cell cultures using Trizol. RNA samples were
electrophoresed in 1.2% agarose/formaldehyde gels, transferred to
Nytran membranes, and hybridized with probes for creatine kinase,
myogenin, MyoD, and tubulin as described previously (20, 21, 40). Blots
were hybridized with random primed labeled probes in a hybridization
buffer at 65 °C. Hybridized membranes were washed twice at 65 °C
and exposed to Kodak x-ray film. For quantitative determination of the
mRNA levels the intensity of the hybridization signals were
measured by densitometric scanning (epson scanning densitometer).
Gel Filtration Chromatography and SDS-PAGE--
Conditioned
media from wild type and antisense decorin transfected cells, obtained
after metabolic labeling during 18 h with 100 µCi/ml
[35S]sulfate, were fractionated on a DEAE-Sephacel column
(0.5 ml of resin) pre-equilibrated in 10 mM Tris-HCl, pH
7,5, 0.2 M NaCl, 0.1% Triton X-100 and eluted with a
linear NaCl gradient (0.2-1.0 M) at a flow rate of 5 ml/h.
Fraction of 1.0 ml were collected, and radioactivity and conductivity
were determined. Pooled fractions containing radioactive proteoglycans
were then chromatographed on an analytical Sepharose CL-4B column (100 x 1 cm) equilibrated and eluted with 1% SDS, 0.1 M NaCl,
50 mM Tris-HCl buffer, pH 8.0. Samples (0.5 ml) were
applied to the column together with prefractionated dextran blue (2000)
and phenol red to mark void and total volumes, respectively.
Columns were eluted at a flow rate of 5.0 ml/h, and effluent
fractions of 0.8 ml were collected and aliquots counted for
radioactivity. Selected fractions obtained from the Sepharose CL-4B
column were analyzed by SDS-PAGE as described previously (41).
Decorin Immunoprecipitation and Enzymatic
Degradation--
Immunoprecipitation of secreted labeled decorin
followed by SDS-PAGE and fluorography were carried out as described
previously (42). Treatment of purified decorin with
chondroitinase ABC was performed as described previously (41).
Immunofluorescence Microscopy--
For immunostaining cells were
grown on coverslips for 72 h. For staining of decorin the cells
were rinsed with phosphate-buffered saline and fixed with 3%
paraformaldehyde for 30 min at room temperature. They were then
permeabilized with 0.05% Triton X-100 and incubated with the primary
antibodies for 1 h at room temperature. After blocking unspecific
binding sites with Blotto they were incubated for 1 h at
room temperature with affinity-purified tetramethylrhodamine B
isothiocyanate-labeled secondary antibodies diluted in blotto. The coverslips were viewed with a Nikon Diaphot inverted microscope equipped for epifluorescence. Immunostaining of fibronectin, biglycan, fibromodulin, lumican, and perlecan was carried out as described previously (7, 39).
Analysis of Creatine Kinase Activity--
Myoblasts and
myoblasts induced to differentiate for the indicated days were washed
twice with phosphate-buffered saline, lysed by incubation with
phosphate-buffered saline containing 0.1% Triton X-100 for 10 min at
25 °C, and harvested by scraping. Creatine kinase activity was
determined using the creatine phosphokinase assay kit. All data
points represent the means of duplicate determinations from two
independent experiments.
Transient Transfection--
Transfections with decorin
adenoviral constructs or insert-free adenoviral preparations were
performed as described previously (38).
C2C12 cells, which had been plated the day
before (6000 cells/cm2 in 25 cm2 flasks), were
treated with 7.5 × 107 plaque-forming units of
adenovirus containing the decorin sequence or with control virus in 1.3 ml/25 cm2 flask of Dulbecco's modification of Eagle's
minimal essential medium/F-12 containing 2% heat-inactivated FCS.
After 90 min of incubation 4 ml of standard medium/25-cm2
flask was added and incubation continued for an additional period of
23 h. Subsequently, the medium was changed for fresh medium, and
the cells were grown for RNA analysis for 2 days.
For transfection with pMYOLuc and pRL, cells were plated at a density
of 8000 cells/cm2 in six-well plates for 48 h and
transfected for 6 h using Opti-MEM I containing 5 µg of pMyoLuc
and 0.01 µg of pRL DNA and 12 µg of LipofectaMINE. Following
transfection, 10% FCS and 0.5% CEE were added to the medium, and the
cells were cultured for a further 14 h. Cells were then
trypsinized and plated in 24-well plates in normal growth medium. Under
these conditions, decorin or TGF-
For transfection with TGF DNA and Protein Determination--
DNA (43) and protein (44)
were determined in aliquots of cell extracts as described.
Stable Transfection of C2C12 Myoblasts with
Antisense Decorin cDNA--
Skeletal muscle myoblasts up-regulate
decorin during differentiation (35). Because decorin binds TGF
The specificity of the effect on decorin synthesis is shown in Fig.
1B. SDS-PAGE followed by fluorography of three selected fractions obtained after the Sepharose CL-4B chromatography showed that
in contrast to wild type cells, the antisense clones A4 and A6 did not
synthesize detectable amounts of decorin. Decorin migrates like an
Mr 100,000~110,000 protein during SDS-PAGE
(34) indicated by a bracket in Fig. 1B; this
material can completely be degraded by chondroitinase ABC (data not
shown). On the contrary, the synthesis of cell surface proteoglycans,
composed mainly of heparan sulfate-containing species, was unchanged
compared with wild type cells (data not shown). Fig. 1C
shows that there is no change in the synthesis of proteins by antisense
clone compared with wild type cells. To further confirm the decreased
synthesis of decorin in the antisense-transfected cells, antibodies
against decorin were used to stain myoblast cultures. Fig.
2 shows that decorin was readily
detectable in control transfected cells (35), whereas significantly
lower levels were seen in the antisense clones. As a control measure, the localization of biglycan, fibromodulin, lumican, fibronectin, and
perlecan was also assessed in control and transfected myoblasts and was
found to indicate no changes in localization. Taken together, these
results show unequivocally that stable clones with strongly reduced
decorin expression were obtained by transfection with antisense decorin
cDNA.
Skeletal Muscle Differentiation Is Accelerated in Antisense
Decorin-transfected Myoblasts--
To evaluate whether the diminished
synthesis of decorin in the antisense clones had any effect on skeletal
muscle differentiation, the expression of myogenin, a master regulatory
gene of skeletal muscle differentiation, was determined. Total RNA was
isolated from antisense clones and wild type cells and analyzed by
Northern blotting, during growth and differentiation conditions. Fig.
3A shows that in contrast to
wild-type cells, myogenin was expressed in both antisense clones
already under growth conditions. However, MyoD, a transcription factor
involved in skeletal muscle commitment, was not up-regulated by decorin
suppression (Fig. 3B), and the expression of myogenin in the
transfected clones was further augmented upon triggering muscle
differentiation for 30 h.
To further determine the relationship between decorin repression and
myogenin induction under growth conditions, the induction of decorin
expression was achieved episomally by infection using adenovirus
containing the full-length sequence for human decorin or by adding
purified decorin to the myoblast cultures. Fig.
4A, top left panel,
shows that after viral infection, antisense cells did not express
myogenin when maintained under growth conditions (compare
A6i and A6), and wild type cells did not express myogenin in
growth conditions (WT) or after infection with the
adenovirus containing the decorin sequence (WTi), as
expected. Wild type and antisense cells expressed myogenin after
triggering differentiation in the above conditions (data not shown).
Use of species-specific antibodies demonstrated the virus-mediated
expression of decorin in antisense and wild-type cells after infection
with the adenovirus containing decorin sequence (Fig. 4A,
right panel). The viral infection did not affect the overall
synthesis of proteins as shown in Fig. 1C. Fig.
4B shows that the application of exogenous decorin reverted
myogenin expression. The effect of bovine cartilage decorin is shown in
the left panel, whereas the effect of skeletal muscle-purified decorin is shown in the right panel.
Treatment of purified skeletal muscle decorin with chondroitinase ABC
had no effect on the reversion of myogenin expression (Fig.
4C). For both types of decorin, the IC50 was
around 50~100 nM. To evaluate whether this decorin
concentration was relevant to the conditions encountered by the cells,
the amount of decorin in myoblast-conditioned medium was measured by
Western blot assay, using skeletal muscle decorin as the standard.
Conditioned medium, obtained after 24 h of culture under growth
conditions, contained decorin at a concentration of ~70
nM (data not shown). These results strongly suggest that inhibition of decorin synthesis triggers the expression of myogenin under growth conditions and that this effect can be reversed by the
endogenous production of decorin or by its exogenous application.
It is well established that skeletal muscle differentiation, including
the expression of creatine kinase, is dependent on the expression of
myogenin. In accordance with this fact was the finding that the enzyme
became induced more rapidly in antisense-transfected clones than in
wild type cells when the cells were transferred to differentiation
medium (Fig. 5). The expression of
muscle-specific creatine kinase was measured in antisense-transfected
clones and compared with wild type cells. As shown in Fig.
5A, creatine kinase mRNA levels were detected after 2 days of induced differentiation in antisense cells. In contrast
creatine kinase mRNA was not detected until 4 days of
differentiation. Creatine kinase activity was also detected earlier in
the antisense-transfected cells than in wild type myoblasts (Fig.
5B). These results demonstrate that the expression of
creatine kinase a skeletal muscle specific marker, controlled by the
expression of myogenin, is accelerated in antisense decorin-transfected
cells compared with wild type cells.
Inhibition of Decorin Expression Decreases the Sensitivity of
Myoblasts to TGF- Modulation of TGF-
The levels of TGF- The expression of decorin is up-regulated during skeletal muscle
differentiation (35). The physiological relevance of this process is
not yet known. Mice with deleted decorin genes have not been reported
to exhibit clinical signs of abnormal muscle development (45), although
detailed studies on muscle differentiation in these mice are not
available in the literature. The absence of a striking muscular
phenotype in decorin-deficient mice, however, may simply be an
indication of the redundancy of the small leucine-rich proteoglycans
and of the possibility that other members of this family may fulfil the
function of decorin in skeletal muscle during development. We have,
therefore, studied muscle differentiation in a less complex model,
i.e. in cultured C2C12 myoblasts.
Decorin expression was inhibited by stable transfection with
full-length decorin antisense cDNA. As a result of abolishing
decorin expression, skeletal muscle differentiation was triggered in
myoblasts under growth conditions. This was evidenced by the
accelerated expression of the transcription factor myogenin, a master
regulator of expression of muscle-specific genes, which is silent in
wild type cells under growth conditions (46, 47). Expression of
myogenin in the nondecorin-expressing cells was reverted by infecting
the cells with an adenovirus containing the full-length sequence for
endogenously produced decorin and by adding exogenous decorin from two
different sources. The data obtained support the hypothesis that
decorin is able to suppress myoblast differentiation. This effect could be either the consequence of a direct signaling function of the proteoglycan or an indirect one mediated by the ability of decorin to
interact with ECM components and growth factors.
Recent studies indicated that, at least in some tumor cells, decorin is
influencing gene expression by direct interaction with a cell membrane
receptor. Clear evidence was obtained that decorin binds to the
epidermal growth factor receptor with a relatively high apparent
Kd value of about 90 nM (25). Binding was followed by receptor phosphorylation and an induction of p21, an
inhibitor of cyclin-dependent kinases (24). Another
candidate molecule for signaling is the 51-kDa endocytosis receptor
protein, although evidence has not yet been obtained that decorin
endocytosis is accompanied by altered gene expression (48-50).
Myoblasts have been shown to respond to epidermal growth factor (51)
and C2C12 myoblasts endocytose decorin very
efficiently.2 In the present
study, however, we have been unable to show unequivocally that decorin
itself is a signaling molecule for C2C12
myoblasts. When decorin, purified under nondenaturing conditions, was
added to the culture medium of wild type myoblasts undergoing
differentiation, no significant change in myogenin expression was
detected. As mentioned above, however, decorin is able to interact with
a variety of serum components like vitronectin, thrombospondin, and
fibronectin, and it is not known whether these interactions have an
influence on the signaling properties of decorin. The observation that
decorin undergoes a secretion-recapture pathway (52) is also compatible with the idea that it is the endogenously produced and not the exogeneously added decorin that directly influences cell behavior.
The data obtained in the present investigation can best be explained by
the assumption that decorin forms complexes as well with endogenously
produced as with exogeneously added TGF- Interestingly, proteoglycans seem to be modulators of the biological
activity of several growth factors, as we observed in this paper for
TGF- Decorin posses multiple functions besides modulation of TGF- In summary our results support the idea that decorin plays an important
role during skeletal muscle differentiation by promoting the ability of
the differentiation inhibitory growth factor TGF- (TGF-
). Decorin
is expressed during skeletal muscle differentiation and is up-regulated
in dystrophic muscle. In this study we investigated the role of decorin in TGF-
dependent inhibition of myogenesis. To probe the
function of decorin during myogenesis,
C2C12 myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a
master transcription factor for muscle differentiation, under growth
conditions and accelerated skeletal muscle differentiation as
determined by the expression of creatine kinase. In contrast myogenin
expression was inhibited by adenovirally induced decorin expression or
by adding exogenous decorin. Reduced synthesis of decorin resulted in a
7-fold decreased sensitivity to TGF-
mediated inhibition of
myogenin expression. In contrast, adenovirally induced decorin
expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-
mediated inhibition of myogenin expression. Transfection studies with the TGF-
-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that
the transducing receptors for TGF-
1 and TGF-
2 were involved in
the different responses of wild type and antisense decorin myoblasts.
These results demonstrate that a reduction of decorin expression or of
decorin availability results in a decreased responsiveness to TGF-
.
These findings strongly suggest a new role for decorin during skeletal
muscle terminal differentiation by activating TGF-
-dependent signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(10-13). In the
presence of these inhibitory growth factors, myoblasts continue to
proliferate and fail to fuse or to express muscle-specific gene
products. Conversely, the reduction in the concentration of these
growth factors below a critical level results in cell culture
experiments in an irreversible arrest in the G0 phase and
in terminal differentiation.
can be regulated by binding to proteoglycans (14, 15). Cell
surface heparan sulfate proteoglycans, in particular, have been
suggested to play a role in modulating the activities of
heparin-binding growth factors (16, 17). They modulate terminal
myogenesis probably by acting as low affinity receptors for some growth
factors such as FGF-2 (18) and HGF/SF (19). We have shown that the
constitutive expression of syndecan-1, a heparan sulfate proteoglycan
whose expression is down-regulated during terminal skeletal muscle
differentiation, inhibits the differentiation of myoblasts in culture
(20, 21). In contrast, abolishing the expression of syndecan-3, which
is another heparan sulfate proteoglycan synthesized by myoblasts,
results in an acceleration of skeletal muscle differentiation by a
mechanism being dependent on FGF-2, too (22).
(26-29). There are
independent binding sites of decorin core protein for TGF-
and type
I collagen (30). Affinity measurements of the interaction between
decorin and TGF-
indicate the presence of at least two sites with
Kd values of 1-20 and 20-200 nM for
the high and low affinity binding sites, respectively (31). The
biological consequences of the interaction between decorin and TGF-
are still a matter of debate. In analogy to the curative effect of
blocking antibodies against TGF-
on the course of the
anti-Thy-1-induced glomerulonephritis (32), the beneficial effect of
decorin in the same disease model has been interpreted as the
consequence of the inactivation of the cytokine by the proteoglycan.
The reversal of TGF-
-mediated effects in this and other disease
models by decorin gene transfer (27-29) supported this conclusion. On
the other hand, the addition of decorin to osteoblasts resulted in an
increased biological activity of TGF-
(33). Therefore, the precise
mechanism whereby decorin modulates TGF-
activity remains to be elucidated.
-dependent inhibition of
myogenesis. It will be shown that this inhibition is mediated directly
by interfering with the signaling cascade triggered upon binding of
TGF-
to its transducing receptors. These findings demonstrate that
the responsiveness of myoblasts to TGF-
, an inhibitor of skeletal
muscle differentiation, is directly modulated by decorin expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and TGF-
2 were from
R & D Systems, Minneapolis, MN.
H2[35S]SO4 carrier-free
(1050-1600 Ci/mmol) and [
-32P]dCTP (3000 mCi/mmol)
and L-[35S]methionine (1175 Ci/mmol) were
obtained from PerkinElmer Life Sciences. S & S Nytran Plus
membranes were from Schleicher & Schuell GmbH, Dassel, Germany.
p3TP-Lux is a TGF
-inducible luciferase reporter construct kindly
donated by Dr. Fernando Lopez-Casilla, National University of Mexico,
Mexico City, Mexico. Briefly, a modified plasmid containing
adenovirus E4 promoter sequences was used to construct the 3TP
promoter; a TGF-
-responsive element from positions
636 to
740 in
the human plasminogen activator inhibitor-1 promoter was synthesized
and inserted upstream of the adenovirus E4 sequences; a fragment
(XhoI-EcoRI) containing the 3TP promoter was
isolated and cloned into XhoI-HindIII digested luciferase expression vector generating p3TP-Lux (37).
615 nucleotides, was subcloned into the pGL3 basic vector, which
contains the cDNA encoding the firefly luciferase enzyme.
1 was added at the indicated
concentrations. After 24 h, the cells were harvested and assayed
for dual luciferase activity.
-inducible luciferase reporter construct
(3TP-Lux), the cells were plated in growth medium 1 day before
transfection at a density of 8000 cells/cm2 in 60-mm
plates. Subsequently, the cells were incubated for 6 h in Opti-MEM
I containing 2 µg of reporter plasmid (3TP-Lux), 2 µg of
-galactosidase plasmid and 15 µg of LipofectAMINE. After transfection the cells were incubated for 14 h in Opti-MEM I
containing 10% FCS and 0.5% CEE. The cells were then washed twice
with Hanks' balanced salt solution and incubated for 2 days in growth
medium followed by 1 day in differentiation medium or 30 h in
differentiation medium containing TGF-
1, TGF-
2 (0-1.0 ng/ml), or
FGF-2 (0-30 ng/ml). The cells were harvested and assayed for
luciferase (37) and
-galactosidase activities (39), as a control of
the transfection among different plates. All transfections were
performed at least three times.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a
strong inhibitor of skeletal muscle differentiation, we reasoned that
the down-regulation of decorin expression would affect skeletal muscle
differentiation by modulating TGF-
activity. To test this hypothesis
C2C12 myoblasts were stably transfected with a
plasmid containing full-length decorin cDNA in the antisense
orientation or with the control vector. Of the nine clones showing
reduced [35S]sulfate incorporation into proteoglycans,
two were chosen for more detailed studies. Myoblasts were labeled with
[35S]sulfate followed by chromatographic analysis of the
conditioned media on a Sepharose CL-4B column. Fig.
1A shows that both antisense clones, A4 and A6, synthesized significantly lower levels of sulfated proteoglycan compared with wild type cells.
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Fig. 1.
Decreased expression of decorin in myoblasts
stably transfected with antisense decorin cDNA. A,
incorporation of [35S]sulfate (100 µCi/ml) into
secreted proteoglycans of stably transfected
C2C12 myoblasts: C1, vector control
(closed circles); A4 (open squares), and A6
(open triangles), clones with decorin cDNA in antisense
orientation. Medium was concentrated on DEAE-Sephacel after 18 h
of incubation and chromatographed on a Sepharose CL-4B column; 800 µl
were collected and analyzed in a scintillation counter. The counts/min
are referred to the total volume of the fraction. B,
fractions indicated by I, II, and III
in A were concentrated and subjected to 10% SDS-PAGE
followed by fluorography. Decorin migrates like an
Mr 95,000-110,000 globular protein indicated by
the bracket. C, synthesis of proteins was
evaluated in wild type (WT) and stably transfected myoblasts
(A6) and in transfected cells infected with an adenovirus
containing the full-length sequence for human decorin (WTi
and A6i). The cultures were metabolically labeled with
[35S]methionine (50 µCi/ml for 18 h), and cell
extract (left panel) and medium (right panel)
were concentrated and subjected to 8% SDS-PAGE followed by
fluorography.
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Fig. 2.
Antisense decorin-expressing clones
synthesized normal levels of other proteoglycans. Control
transfected cells (C1) and antisense decorin cells
(A4 and A6) were stained with anti-rat decorin
(DCN), anti-chicken biglycan (BGN), anti-rat
fibromodulin (FMD), anti-rat lumican (LUM),
anti-human fibronectin (FN) or anti-mouse perlecan
(PER). Fluorescein isothiocyanate-conjugated anti-rabbit IgG
and tetramethylrhodamine B isothiocyanate-conjugated
anti-chicken IgG were used as secondary antibodies. The bar
corresponds to 25 µm.
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Fig. 3.
Myogenin expression is augmented in antisense
decorin-expressing clones under growth conditions. A,
myogenin expression in control transfected cells (C1) and
antisense decorin-transfected cells (A4 and A6)
was evaluated by Northern blot analysis. 10 µg of total RNA isolated
from myoblasts incubated under growth conditions (1) or in
differentiation medium for 30 h (2) was separated by
electrophoresis, blotted onto nylon membranes, and hybridized with
32P-labeled myogenin cDNA probe. The ethidium
bromide-stained gel is shown in the lower part of the panel,
and the ribosomal RNAs are indicated. B, the Northern blot
was hybridized with 32P-labeled myogenin, MyoD, and tubulin
cDNA probes. The transcript sizes for myogenin (Myo),
MyoD, and tubulin (Tub) are indicated in each panel.
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Fig. 4.
The expression of myogenin in antisense
decorin clones is reverted by specific decorin re-expression and by the
addition of exogenous decorin. A: left panel, myogenin
expression was evaluated in wild type cells and antisense
decorin-transfected cells (WT and A6) and in
transfected cells infected with an adenovirus containing the
full-length sequence for human decorin (WTi and
A6i). Northern blot analysis for myogenin under growth
conditions was performed. 10 µg of total RNA isolated from myoblasts
was separated by electrophoresis, blotted onto nylon membranes, and
hybridized with a 32P-labeled myogenin probe
(Myo). The methylene blue-stained nylon membrane is shown in
the lower part of the panel, and the ribosomal RNAs are
indicated. The transcript size is indicated. Right panel,
the extent of decorin synthesized by the adenovirus-infected cells
described in A was evaluated by specific immunoprecipitation
with antibodies against human decorin as described under
"Experimental Procedures." B, A6 cells were incubated
for 48 h with decorin purified from bovine cartilage (left
panel) or from mouse skeletal muscle (right panel).
Northern blot analysis for the effect of bovine decorin is shown in the
left panel as described above. The reversion effect of
skeletal muscle decorin on myogenin expression was evaluated using A6
myoblasts transiently cotransfected with pMyoLuc and pRL plasmids. The
cells were incubated for 24 h in growth medium, harvested, and
dual luciferase activity was determined as described under
"Experimental Procedures." C, A6 cells were incubated
with skeletal muscle decorin pretreated with chondroitinase ABC
(Cabc) and assayed as explained in the legend of
B. Decorin concentrations are as follows: D1 = 0.1 µM and D2 = 0.5 µM. The results shown
are the means from two different experiments performed in
duplicate.
View larger version (32K):
[in a new window]
Fig. 5.
Creatine kinase expression is augmented in
antisense decorin-transfected clones. A, creatine
kinase expression in wild type (WT) and antisense
decorin-transfected cells (A6) was evaluated by Northern
blot analysis. 10 µg of total RNA, isolated from myoblasts incubated
in differentiation medium for 0 h, 2, or 4 days, was separated by
electrophoresis, blotted onto nylon membranes, and hybridized with a
32P-labeled creatine kinase cDNA probe (CK).
The transcript size is indicated. The ethidium bromide (EtBr)-stained
gel is shown in the lower part of the panel, and the
ribosomal RNAs are indicated. B, creatine kinase activity
was quantitated as a function of time of differentiation as described
under "Experimental Procedures."
-dependent Inhibition of Myogenin
Expression--
To determine whether the accelerated expression of
myogenin and other skeletal muscle markers observed in the antisense
decorin-transfected cells resulted from a change in TGF-
signaling
activity, we measured the TGF
1-dependent inhibition of
myogenin mRNA expression as a function of growth factor
concentration. Wild type- and antisense-transfected myoblasts were
triggered to differentiate for 30 h in the presence of different
concentrations of TGF-
1. As shown in the Fig.
6A (top left
panel), exposure of wild type cells to TGF-
1 resulted, as
expected, in a significant inhibition of myogenin mRNA expression. However, the antisense-transfected cells were considerably less sensitive toward the cytokine (bottom left panel). Whereas
in wild-type cells half-maximal inhibition required 0.1 ng/ml, 0.7 ng/ml was needed to achieve the same effect in the cells with suppressed decorin synthesis (Fig. 6A, right
panel). This change in responsiveness to TGF
1 was specific,
since no significant difference in the inhibitory activity of FGF-2 on
myogenin expression was observed in both cell types (Fig.
6B, left and right panels). To confirm
this modulatory effect of decorin on myogenin expression, wild type and
wild type cells infected with adenovirus containing the full-length
sequence for human decorin were triggered to differentiate for 30 h in the presence of different concentrations of TGF-
1. As shown in
Fig. 6C, decorin-transfected cells exposed to TGF-
1 were
more susceptible to the cytokine-induced inhibition of myogenin mRNA expression than wild type cells. Whereas in decorin-infected cells half-maximal inhibition required 0.018 ng/ml, the same effect in
noninfected wild type cells required 0.09 ng/ml.
View larger version (30K):
[in a new window]
Fig. 6.
Concentration dependence of
TGF- -mediated inhibition of myogenin
expression in control and antisense decorin cells. Wild type
(WT) and antisense decorin-transfected cells (A6)
were incubated for 30 h in differentiation medium containing the
indicated concentrations of TGF-
1 (A, left
panel) and FGF-2 (B, left panel). RNA was
isolated from the cells, and 10 µg of total RNA was analyzed by
Northern blot with a 32P-labeled myogenin (Myo)
cDNA probe. Right panels show the graphical
representations of TGF-
1 (A)- and FGF-2
(B)-dependent inhibition of myogenin expression
in antisense decorin cells (open circles) and wild type
cells (closed circles). Values correspond to the means of
three independent experiments. C shows
TGF-
1-dependent inhibition of myogenin expression in
wild type cells (closed circles) and wild type cells
infected with adenovirus containing the full-length sequence for human
decorin (open circles). Myogenin expression was evaluated as
described in the legend of Fig. 4B.
Signaling by Decorin Suppression--
To
determine whether decorin suppression had an influence on TGF
signaling, a reporter construct was used where the promoter of
plasminogen activation inhibitor-1 was coupled with luciferase cDNA. This reporter, p3TPLux, is activated upon binding of TGF-
to its transducing receptors (37). The antisense clone responded less
to TGF-
1 and TGF-
2 when compared with wild type cells. The
promoter activity was also diminished in the absence of exogeneously added TGF-
, suggesting that the autocrine response to the cytokine was reduced (data not shown). To further examine this point, we titrated the response of the antisense clone, control transfected, and
wild type cells to TGF-
1 and TGF-
2. Fig.
7 shows that TGF-
1 and -
2 increased
reporter activity in all three cell lines. The antisense cells,
however, showed a significant decrease in response mediated by TGF-
transducing receptors compared with control transfected and wild type
cells (57 and 68%, respectively). Interestingly, the half-maximal
effect was measured at similar cytokine concentration.
View larger version (15K):
[in a new window]
Fig. 7.
Concentration dependence of
TGF- 1 and -
2 on the
plasminogen activator inhibitor-1 promoter activity in myoblasts of
different capacity for decorin production. Wild type (closed
circles), control transfected (open triangles), and
antisense decorin cells (open circles) were incubated for
30 h in growth medium containing the indicated concentrations of
TGF-
1 (A) or TGF-
2 (B). TGF-
-mediated
activities were evaluated as described under "Experimental
Procedures." The results shown are the means from three different
experiments performed in duplicate.
-RII, determined by the immunoprecipitation of
metabolic labeled extract of myoblasts and the levels of betaglycan or
(TGF-
-RIII), determined by Western blot analyses, were the same in
antisense-transfected and wild type cells (data not shown). These
results demonstrate, therefore, that the lowered sensitivity toward
TGF-
in the myoblasts with suppressed decorin expression was
mediated by the transducing TGF-
receptors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and that these complexes
allow a more efficient presentation of the cytokine to its signaling
receptors than the free effector molecule. Myoblasts, being unable to
synthesize relevant amounts of decorin or being deprived of the
proteoglycan due to its sequestering to collagen lattices, are,
therefore, less prone to the inhibitory effect of TGF-
on myoblast
differentiation. What molecular mechanisms are involved in the
activation of TGF-
by decorin remains to be investigated. This
effect is specific to TGF-
, because antisense-transfected cells
treated with FGF-2, another strong inhibitor of myogenin (10, 21, 22),
exhibited the same FGF-2-dependent inhibition of myogenin
expression than wild type cells. These observations suggest that
decorin expression can modulate the biological activity of TGF-
on
skeletal muscle cells. However, as stated above, this activation is not
a unique property of the experimental system used in this study, since
osteoblasts, too, have been reported to become activated by
TGF-
-decorin complexes (33). In light of the data presented
in this study, one wonders how the up-regulation of decorin as an
indirect, and even perhaps direct, inhibitor of muscle differentiation
is controlled during muscle development. However, skeletal muscle cells
possess several signaling pathways that modulate the expression of
myogenin. As mentioned FGF-2 and HGF/SF are strong inhibitors of
skeletal muscle differentiation via activation of specific receptors to
transduce their signals.
. We have shown that constitutive expression of syndecan-1, a
heparan sulfate proteoglycan that is down-regulated during skeletal
muscle differentiation (20), produces a 7-fold decrease in the
IC50 for FGF-2 dependent inhibition of myogenin expression
(21). Inhibition of syndecan-3 expression produces a 13-fold increase
in the IC50 for FGF-2 inhibitory activity (22). The
presence in the plasma membrane of syndecan-1 and -3 is critical for
presentation of FGF-2 to the transducing receptors. Although decorin
has been normally considered as an ECM proteoglycan, it has been shown
to be associated to the plasma membrane through a specific decorin
endocytosis receptor in a variety of eukaryotic cells (48-50, 53). The
ability of decorin to interact with the plasma membrane via its
specific receptor raises the possibility that decorin might modulate
the TGF-
activity in a similar fashion to that described for FGF-2
by heparan sulfate proteoglycans (16, 17).
activity. Decorin has been implicated in the control of collagen fibrillogenesis (54), cell proliferation (55), and corneal transparency
(56). Some of these functions resides in the core protein, whereas
other are attributed to the glycosaminoglycan chain (26). Some of the
binding sites for molecules, such as TGF-
and collagen type
I, have been mapped and reside in different parts of the decorin core
protein (30). The versatility of the decorin binding activity suggests
that decorin may function as a modulator of fundamental biological
processes during skeletal muscle differentiation, where its expression
is up-regulated (35). In addition decorin is an important
structural constituent of the skeletal muscle ECM in normal and
dystrophic mice (34, 36). It remains a challenge for further work to
study the relative contributions of these growth factors and ECM
components for muscle development.
to interact with
its signaling receptors.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. D. J. Carey for critical reading of the manuscript and Carolina Achondo for technical support.
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FOOTNOTES |
---|
* This work was supported in part by Fondo de Investigación Avanzade en Areas Prioritarias Grant 13980001, by Fondo Nacional de Investigación Centifica y Tecnológica Grants 1960634 and 1990151 (to E. B.) and 2980053 (to C. R.), and by Volkswagen-Stiftung Grant I/71 498. The Millenium Institute for Fundamental and Applied Biology (MIFAB) is financed in part by the Ministerio de Planificación y Cooperación (Chile).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662.
Both authors supported by Deutsche Forschungsgemeinschaft
Grant SFB 492, Project A6.
** Supported in part by an International Research Scholars grant from the Howard Hughes Medical Institute, Presidential Chair in Science from the Chilean Government, and Fondo de Investigación Avanzade en Areas Prioritarias in Biomedicine. To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Faculty of Biological Sciences, Pontifical Catholic University of Chile, P. O. Box 114-D, Santiago, Chile. Fax: 56-2-635-5395; E-mail: ebrandan@genes.bio.puc.cl.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M004602200
2 C. Riquelme, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular matrix;
CEE, chicken embryo extract;
FCS, fetal calf
serum;
FGF-2, basic fibroblast growth factor;
HGF/SF, hepatocyte growth
factor/scatter factor;
TGF-, transforming growth factor
;
TGF-
-R, transforming growth factor-
receptor;
PAGE, polyacrylamide gel electrophoresis.
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