From the Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México, Apartado Postal
70-246, México City, D.F., 04510, México and the
¶ Centro de Regulación Celular y Patología, Facultad
de Ciencias Biológicas, Millenium Institute for Fundamental and
Applied Biology, P. Universidad Católica de Chile,
Casilla 114-D, Santiago, Chile
Received for publication, August 20, 2002, and in revised form, October 8, 2002
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ABSTRACT |
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Betaglycan is a
membrane-anchored proteoglycan co-receptor that binds transforming
growth factor Skeletal muscle myoblasts are the precursors to skeletal muscle
fibers. During development these cells are maintained in a proliferative and undifferentiated state until appropriate signals cause them to undergo conversion into multinucleated myotubes.
A network of muscle regulatory factors
(MRFs),1 some of which have
been identified and characterized, governs this process. When
myogenesis begins, myogenic regulatory genes encoding factors of the
MyoD family are activated (myogenin, MRF4, and MRF5). These factors
bind to specific DNA consensus sites called E boxes, which function as
transcriptional enhancers of muscle differentiation genes (for review
see Ref. 1). Factors of the MEF2 family are also required to specify
muscle fate or to direct muscle differentiation (2). The expression and
activity of these master regulatory genes are regulated by polypeptide
growth factors, such as basic fibroblast growth factor (FGF-2) (3),
transforming growth factor type The onset and progression of the differentiation process are controlled
by a complex set of interactions between myoblasts and their
environment. The presence of the extracellular matrix (ECM) is
essential for normal myogenesis (8-11). It has been demonstrated that
the activities of FGF-2, HGF, and TGF- Cell interaction with TGF- Cell Culture--
The mouse skeletal muscle cell line
C2C12 (ATCC, Manassas, VA) (23) was grown and
induced to differentiate as described (15). Briefly, 3 days after
plating (80% confluence), the medium was changed to Dulbecco's
modified Eagle's medium supplemented with 5% horse serum to trigger
myoblast differentiation. Two days later 0.1 mM
cytosine- RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from cell cultures at the indicated times using Trizol
(Invitrogen). The RNA samples were electrophoresed in 1.2%
agarose/formaldehyde gels, transferred to Nytran membranes, hybridized
with probes for mouse betaglycan (24), and exposed to Kodak x-ray film.
Generation of the Adenoviral Vector for the Expression of Wild
Type Betaglycan (Adv-BG)--
The DNA vectors and hosts employed were
generous gifts of Bert Vogelstein (Johns Hopkins University Oncology
Center), and the method employed has been described by He et
al. (25). Briefly, to generate the appropriate transfer vector, a
DNA fragment containing the 186 upstream bp of the 5'-untranslated
region, the complete open reading frame, and the first 513 bp of
the 3'-untranslated region of the wild type rat betaglycan cDNA
(19) was subcloned at the NotI-HindIII sites of
pAdenoTrack. The created transfer vector was recombined with
pAdenoEasy-1, and the recombinant adenoviral genome was transfected in
293 cells to recover the recombinant viral particle (Adv-BG).
Enrichment and purification of an Adv-BG viral stock was carried out
according to standard methods.
Cloning of Murine Betaglycan Gene Promoter and Construction of
Betaglycan Promoter Reporter Vector--
A bacterial artificial
chromosome mouse genomic library (Research Genetics, Huntsville, AL)
was screened with the 190 nucleotides (bases 15-205) at the 5' end of
the reported mouse betaglycan cDNA (24), and eight positive clones
were analyzed by restriction mapping. A 6.5-kilobase pair
HindIII fragment that hybridized to bases 88-105 of the
cDNA was recovered from bacterial artificial chromosome clone B70.
A smaller HindIII-BamHI fragment containing the
2542 bp located at the 5' end was further isolated and fully sequenced
at Instituto de Fisiologia Celular Molecular Biology Core Facility
(deposited in the GenBankTM with accession number
AF537325). For the construction of betaglycan reporter plasmid,
pBG-Lux, the 2307-bp HindIII-NotI
Klenow-filled-in fragment was subcloned in the SmaI of
pGL3-basic (Promega, Madison, WI), and the proper orientation was
verified by sequencing (see Fig. 2 for more details). The pMyo-Luc
reporter construction was reported previously (14); pRLSV40 was from
Promega; and pEMSV, pEMSV-MyoD, and pEMSV-myogenin were gifts from Dr.
Stephen F. Konieczny (Purdue University).
Transient Transfection--
The cells were plated at a density
of 8,000 cells/cm2 in 6-well plates for 48 h and
transfected for 5 h using with 4 µg of pBG-Lux or 4 µg of
pMyoLuc and 0.01 µg of pRLSV40 in each well mixed in Opti-MEM I with
4 µl of PLUS reagent and 6 µl of LipofectAMINE (Promega). Following
transfection, fetal bovine serum and chicken embryo extract were added
to restore their complete growth medium concentrations, and the cells
were cultured for a further 14 h. The cells were then trypsinized
and plated in 24-well plates in normal growth medium. Under these
conditions, FGF-2 (3 ng/ml) (Invitrogen), BMP-2 (1 nM)
(generous gift from Genetic Institute, Cambridge, MA), HGF (3 ng/ml)
(R & D Systems, Minneapolis, MN), TGF- Immunoblot Analysis--
For immunoblot analysis aliquots were
subjected to SDS gel electrophoresis in 4-12% polyacrylamide gels,
electrophoretically transferred to Immobilon membranes (Millipore,
Bedford, MA), probed with a rabbit antiserum raised against rat
betaglycan ectodomain (1:2000) (22), rabbit anti-myogenin (1:500), or
rabbit anti-MyoD (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA),
and visualized by enhanced chemiluminescence (Pierce). For betaglycan
core protein analysis, the samples were previously treated with
heparitinase and chondroitinase ABC (Seikagaku, Tokyo, Japan) (9,
26).
TGF- Immunofluorescence Microscopy--
Cells to be analyzed by
immunofluorescence microscopy were grown on glass coverslips, as
described previously (27). For cell surface staining the live unfixed
cells were incubated in the first antibody solution 1:200
anti-betaglycan, 1:1000 anti-perlecan (27) in 50 mM
Tris-HCl, pH 7.7, 0.1 M NaCl, 2% bovine serum albumin for
1 h on ice. After removal of this solution, the cells were fixed
in 3% paraformaldehyde and permeabilized with 0.05% Triton X-100 and
incubated with 1:100 monoclonal anti-myosin antibody (Developmental
Studies Hybridoma Bank, University of Iowa, Iowa City, IA). The bound
antibodies were detected by incubating the cells with 1:100
affinity-purified fluorescein isothiocyanate-conjugated anti-rabbit IgG and 1:100 TRITC anti-mouse IgG (Pierce), respectively. After rinsing, the slides were viewed with a Nikon upright microscope equipped for epifluorescence.
Analysis of Creatine Kinase Activity--
Creatine kinase
activity was determined as previously described (9).
DNA and Protein Determination--
DNA (28) and protein (29)
were determined in aliquots of cell extracts as described.
Skeletal Muscle Myoblasts Up-regulate Betaglycan Expression during
Differentiation--
To evaluate the expression of betaglycan during
myoblast differentiation, the levels of its mRNA were determined in
total RNA isolated from myoblasts and myotubes induced to differentiate for 2, 4, and 6 days. As shown in Fig.
1A, betaglycan mRNA was detected after 2 days of induced differentiation with an increase by
day 4 and small decrease by day 6. At the same time, the level of
cellular betaglycan during skeletal muscle differentiation was
determined by Western blot analysis of detergent extracts treated
previously with heparitinase and chondroitinase ABC, a procedure that
reveals the total content of betaglycan as its core protein. Fig.
1B shows that cells allowed to differentiate for 4 days
increased betaglycan expression significantly compared with the amount
synthesized by myoblasts. Betaglycan has the ability to bind the three
isoforms of TGF- Cloning of Mouse Betaglycan Gene Promoter and Construction of Mouse
Betaglycan Gene Reporter--
To characterize the mechanism underlying
the control of up-regulation of betaglycan mRNA, the murine
betaglycan gene promoter was cloned. For that purpose, a bacterial
artificial chromosome library was screened with the most 5' end
sequences of the murine betaglycan cDNA, and the
HindIII-BamHI 2542-nucleotide-long fragment (Fig.
2A) containing the most
upstream cDNA region was isolated and sequenced (Fig.
2B). This genomic DNA fragment had 86% sequence identity
with the corresponding region of its rat ortholog (1). In addition, it
contained a stretch of 127 nucleotides (2265-2391) that corresponded
to bases 20-141 of the published murine betaglycan cDNA and that
was followed by a GT, presumably the splicing donor site of the first
intron. Other relevant functional cis-regulatory elements,
SP1 and GC boxes, including the putative BMP-2-responsive element, that
have been experimentally determined in the rat gene by the Centrella
and co-workers (1) were also conserved in the mouse promoter. These
similarities strongly suggest that the cloned sequences contain the
promoter and first exon of the mouse betaglycan gene.
Importantly, the mouse sequences contained several MRFs binding sites:
two MyoD- and five MEF2-binding sites, elements that have been shown to
be essential for skeletal muscle differentiation (30). In addition,
they also showed the presence of one RA receptor and four Smad4- and
one Smad3-binding elements, factors that mediate responses to RA and
TGF-
To determine whether or not the isolated
HindIII-BamHI 2542-bp fragment had promoter
activity, it was used to construct a luciferase reporter plasmid, which
was transiently transfected in cell lines capable of expressing the
endogenous betaglycan. Fig. 2C shows that, as compared with
the empty vector, the cloned HindIII-NotI
fragment drove the expression of luciferase in Rat1 fibroblasts.
Importantly, transfected L6E9 rat myoblasts, which do not express
betaglycan (22), failed to express the reporter gene. Together, these
findings confirmed that the cloned region contains the bona
fide mouse betaglycan gene promoter.
Activity of Betaglycan Reporter Increases during
Myogenesis--
To characterize the mechanisms controlling the
up-regulation of betaglycan during the myogenic differentiation,
C2C12 myoblasts were transiently transfected
using the betaglycan reporter vector and induced to differentiate for 6 days. As expected from the results described above, Fig.
3 shows that the activity of the pBG-Lux
increased 8.4-fold during skeletal muscle differentiation. In parallel,
as a control of the differentiation process, the increase in expression
of myogenin (40-fold) evaluated with pMyo-Luc reporter is shown.
Betaglycan Expression Increases during Myogenesis Are
MyoD-dependent--
As indicated before, betaglycan
promoter contain several DNA-binding sites for MyoD and MEF2 proteins.
Upon induction of muscle differentiation, these proteins bind to the
regulatory regions and activate the transcription of skeletal muscle
genes, such as creatine kinase. Thus, we asked whether or not by
forcing the expression of myogenin or MyoD in myoblasts the
transcriptional activity of the betaglycan reporter could be enhanced.
For that purpose, myoblasts cells were transiently co-transfected with an expression vector containing the full-length myogenin cDNA (pEMSV-Myog) or with an expression vector containing the full-length MyoD cDNA (pEMSV-MyoD), together with pBG-Lux and pRLSV40. The transfected cells were maintained under proliferating conditions, and
dual luciferase activity was measured after 24 h. Fig.
4 (open bars) shows that MyoD
overexpression produced an important increase of the betaglycan
reporter activity. However, no effect on the betaglycan promoter
activity was observed by myogenin overexpression. The same results were
observed when the cells were triggered to differentiate for 24 h
(Fig. 4, dark bars). The inset of Fig. 4 shows
the protein levels of MyoD or myogenin in the corresponding transfected
cells, evaluated by Western blot analyses, and is compared with the
induction of those MRFs in myoblasts induced to differentiate. The
levels of expression of MyoD or myogenin in the transfected cells were
comparable with or even higher than the levels observed during normal
differentiation. These results demonstrate that betaglycan expression
is under the control of MyoD but not of myogenin activity.
Betaglycan Expression Is Inhibited by TGF- Betaglycan Enhances TGF- Betaglycan Increases on the Extracellular Matrix and on the Surface
of Myotubes during Skeletal Muscle Differentiation--
To further
evaluate the expression and localization of betaglycan during skeletal
muscle differentiation, nonpermeabilized cell cultures were stained
with anti-betaglycan antibodies. Fig. 7D shows that myoblasts
express betaglycan that can be detected on the cell surface by
immunofluorescent staining. However, cells stained 6 days after
initiation of differentiation (Fig. 7E) showed a much higher
betaglycan immunostaining on the cell surface and on fibrillar
structures characteristic of ECM components (34, 35). Myotubes were
revealed by staining using anti-myosin antibodies after
permeabilization (Fig. 7, H and I). The ECM
localization of perlecan, a heparan sulfate proteoglycan, is shown in
Fig. 7F. Panels K and L of Fig. 7
correspond to the merge of the immunostaining for betaglycan or
perlecan, respectively, with the immunostaining for myosin. These
results indicate that up-regulated betaglycan is mainly localized to
the myotubes cell surface and associated to the ECM.
The results presented in this paper demonstrate that the
expression of betaglycan, a transmembrane heparan/chondroitin sulfate proteoglycan (19), increases during the differentiation of skeletal muscle cells. This conclusion is based on analysis of betaglycan mRNA and protein levels, activity of a reporter construct
containing a portion of the mouse betaglycan promoter, analysis of
functional betaglycan through binding, presentation of TGF- These observations are of interest in the context of the regulation of
the skeletal muscle formation by proteoglycans. Skeletal muscle cell
lines provide a very informative model to study the involvement of
proteoglycans and growth factors in this process. Heparin-binding
growth factors such as FGFs, HGF, and TGF- In the present work we provide evidence that betaglycan, another
important proteoglycan, is modulated during the myogenic differentiation. Betaglycan is a versatile co-receptor capable of
regulating the effects of several members of the TGF- In the present work we show that the expression of betaglycan is
regulated during the skeletal muscle differentiation and that this
up-regulation is mainly accounted for at the level of transcription.
Cloning of a genomic DNA containing the first exon and the first
2140-bp upstream region of mouse betaglycan gene allowed us to
test its activity during differentiation. Here we show unequivocally
that the up-regulation of betaglycan is controlled at least by the
expression of the MRF MyoD. In cells transiently transfected with MyoD,
the activity of the betaglycan gene promoter, as measured by our
reporter pBG-lux, was up-regulated, even in the presence of high serum
condition where muscle differentiation is inhibited (4). Interestingly,
transient expression of myogenin, another MRF, had no effect on the
activation of betaglycan transcription. We also found that treatment of
myoblasts with any of the TGF- The function of betaglycan in skeletal muscle cells and the
consequences of betaglycan up-regulation during differentiation are not
known and can only be determined experimentally. However, it is
reasonable to guess that they will depend on the versatile ability of
betaglycan as a co-receptor of diverse growth factors. The literature
on the role of TGF- On the other hand, although it has not been demonstrated yet, the fact
that betaglycan binds FGF-2 via its heparan sulfate chains makes it a
candidate to regulate the association of this growth factor with its
kinase receptor and therefore its actions (64). In this regard, the
effects of betaglycan on muscle cell differentiation may resemble those
of the other heparan sulfate proteoglycans that we have studied (5, 9,
11, 27). Another fact to consider is the localization of the
up-regulated betaglycan. It is possible to speculate that betaglycan is
shed from the surface and incorporated into the ECM serving there as a
TGF- In summary, we have found that the expression of betaglycan is
up-regulated during skeletal muscle differentiation. This phenomenon is
modulated by growth factors and RA and is MyoD-dependent.
The regulatory sequences responsible for this modulation are contained within a 2100-bp segment of the gene that contains consensus binding sites for several transcriptions factor such as SP1, Smad3, Smad4, MyoD, MEF2, and RA binding proteins. These results would help to
understand the complex regulation during development and
differentiation of this key integral membrane heparan/chondroitin
sulfate proteoglycan.
(TGF-
) via its core protein and basic
fibroblast growth factor through its glycosaminoglycan chains. In this
study we evaluated the expression of betaglycan during the
C2C12 skeletal muscle differentiation.
Betaglycan expression, as determined by Northern and Western blot, was
up-regulated during the conversion of myoblasts to myotubes. The mouse
betaglycan gene promoter was cloned, and its sequence showed putative
binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also
up-regulated in differentiating C2C12 cells. We
found that MyoD, but not myogenin, stimulated this transcriptional
activity even in the presence of high serum. Betaglycan promoter
activity was increased by RA and inhibited by the three isoforms of
TGF-
. On the other hand, basic fibroblast growth factor, BMP-2, and
hepatocyte growth factor/scatter factor, which are inhibitors of
myogenesis, had little effect. In myotubes, up-regulated betaglycan was
also detectable by TGF-
affinity labeling and immunofluorescence
microscopy studies. The latter indicated that betaglycan was localized
both on the cell surface and in the ECM. Forced expression of
betaglycan in C2C12 myoblasts increases their
responsiveness to TGF-
2, suggesting that it performs a TGF-
presentation function in this cell lineage. These results indicate that
betaglycan expression is up-regulated during myogenesis and that MyoD
and RA modulate its expression by a mechanism that is independent of myogenin.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
) (4), hepatocyte growth
factor/scatter factor (HGF) (5), and insulin-like growth factor (6), as
well as by retinoic acid (RA) (7).
can be regulated by binding
to proteoglycans (2-6). Cell surface heparan sulfate proteoglycans, in
particular, have been suggested to play a role in modulating the
activities of heparin-binding growth factors (7, 8). They modulate
terminal myogenesis probably by acting as low affinity receptors for
some growth factors such as FGF-2 (5, 9) and HGF (10). We have shown
that constitutive expression of syndecan-1, a heparan sulfate
proteoglycan whose expression is down-regulated during skeletal muscle
terminal differentiation, inhibits the differentiation of myoblasts in
culture (5, 11). In contrast, abolishment of the expression of
syndecan-3, another heparan sulfate proteoglycan synthesized by
myoblasts, results in acceleration of skeletal muscle differentiation
by a mechanism being dependent on FGF-2 (9). Decorin is a dermatan
sulfate proteoglycan that has the ability to bind TGF-
by its core
protein (12, 13). Decorin seems to be critical in
C2C12 cells to repress skeletal muscle
differentiation by a TGF-
-dependent mechanism (14), and
its expression is up-regulated during the process (15).
is mediated by a complex group of
proteins that includes the type I and II TGF-
receptors, which have
signal transducing activity, and the membrane-bound proteoglycan betaglycan (16-18). Betaglycan, a heparan/chondroitin sulfate
proteoglycan also known as type III TGF-
receptor (T
RIII), is
present on the cell surface of several cell types (19, 20) and has the ability to bind TGF-
through the core protein and FGF-2 through the
heparan sulfate chains (21). Although betaglycan is not directly
involved in the intracellular TGF-
signaling, it controls its access
to the T
RII receptor, thereby modulating its actions (6, 22). In
this study we show that expression of betaglycan is up-regulated during
skeletal muscle differentiation of C2C12 myoblasts, increasing its levels on the cell surface of the myotubes and in the ECM. Our data also indicate that the proximal region of the
betaglycan gene promoter controls this process in a manner that is
dependent of MyoD expression. Although it is not clear at this moment
what is the function of betaglycan during the skeletal muscle
differentiation process, the fact that its ectopic forced expression in
myoblasts enhances their responsiveness to TGF-
2 suggests a
potential regulatory role.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-arabinofuranoside was added to the culture medium. Thereafter the incubation medium was changed daily. Rat1 and
L6E9 cells were routinely cultured in Dulbecco's modified Eagle's
medium containing 10% or 20% fetal bovine serum, respectively, as
described (19).
(1 ng/ml) (R & D
Systems), and RA (1 µM) (Sigma) were added at the
indicated concentrations. After 24 h, the cells were harvested and
assayed for dual luciferase activity (Promega). All of the transfections were performed at least three times.
Affinity Labeling and Immunoprecipitation--
TGF-
was radiolabeled with 125I using the chloramine T, and
affinity labeling and immunoprecipitations were done as described before (22).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
through its core protein. To evaluate this property
of the betaglycan induced during muscle differentiation, affinity
labeling experiments using 125I-labeled TGF-
2 were
performed. Fig. 1C shows that the up-regulated betaglycan,
displaying its characteristic pattern of migration in this kind of
experiments, bound TGF-
2 and that this binding increased during
differentiation when the total extracts were analyzed. The specificity
of this result was confirmed when immunoprecipitates from the total
cell extract were analyzed using a specific anti-betaglycan antibody
(Fig. 1D). Fig. 1E shows the densitometric
analysis for the quantitative analyses of the data. As a test of
concomitant differentiation, the level of creatine kinase activity, a
specific skeletal muscle marker, was evaluated (Fig. 1F).
Together, these results demonstrate that the synthesis of betaglycan
increased during skeletal muscle differentiation.
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Fig. 1.
Betaglycan expression is up-regulated during
myoblasts differentiation. A, 15 µg of total RNA isolated
from myoblasts (lane 0) and myoblasts induced to
differentiate for the indicated days were analyzed by Northern blot
with 32P-labeled betaglycan cDNA probe. The
arrows indicate ribosomal RNA size, and the
arrowhead indicates betaglycan mRNA (BG).
B, cell extracts were prepared from myoblasts (lane
0), and cells were induced to differentiate for 4 days (lane
4) and then treated with heparitinase and chondroitinase
ABC. The extracts were analyzed by SDS-PAGE. Betaglycan was visualized
by immunoblotting with an antibody specific for betaglycan. The
arrow indicates the core protein of betaglycan.
C2C12 myoblasts were culture under growth
conditions for 48 h and switched to differentiate for several
days. At the indicated times of differentiation the cells were affinity
labeled with 100 pM of 125I-labeled TGF- 2.
The total extracts were analyzed by SDS-PAGE (C) or by
anti-betaglycan immunoprecipitation (D). Phosphorimages of
the gels are shown indicating the migration of the molecular
mass standards (kDa) and of the betaglycan-TGF-
2 complex
(bracket). E, the graphical representation of the
phosphorimaging quantitation of the betaglycan-TGF-
2 complex.
Open bars correspond to total extracts, and striped
bars correspond to the immunoprecipitates. F, cell
extracts were prepared from myoblasts (0), cells were
induced to differentiate for the indicated days, and creatine kinase
activity was determined. For graphical representation, the enzymatic
activity values were normalized by DNA content. When indicated the
results are the means ± S.D. of two independent experiments
performed in triplicate.
, regulators of skeletal muscle differentiation.
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Fig. 2.
Cloning of murine betaglycan gene promoter.
A, schematic representation of mouse betaglycan promoter and
first exon. The 2542-bp HindIII-BamHI fragment
containing the betaglycan promoter and first exon are shown. Putative
boundaries of first exon (box) were established by
comparison with rat betaglycan initial exon as experimentally
determined by Ji et al. (1) and by alignment to mouse
betaglycan cDNA (24). B, nucleotide sequence of mouse
betaglycan promoter and first exon. The positive numbers in
the right margin correspond to the 2542-bp
HindIII-BamHI sequence submitted to the
GenBankTM (accession number AF537325). For description
purposes, the nucleotide sequence of was also numbered taking the A
that opens the reading frame in betaglycan cDNA as base +1
(negative numbers in the left margin). The
predicted first exon in the murine betaglycan gene spans from position
341 to
91 (underlined). Thus, the G at position 2391, which corresponds to position
91 and belongs to the 5'-untranslated
region, is the last base in the putative first exon. The C at position
2195 (asterisk) is the first base that aligns with the
published cDNA. The partial sequence of the putative first intron
is shown in lowercase letters. Sequence analysis of the
2542-bp HindIII-BamHI fragment using Professional
MatInspector software identified several potential
cis-regulatory elements (consensus sequences higher than
0.9). The subset of these elements as well as restriction endonuclease
sites that are relevant to the present work are shown in bold
type. For clarity, if there is an overlap between two
cis-elements, one of them is underlined, and the
other is shaded. C, transactivating activity of
murine betaglycan promoter. The 2307-bp
HindIII-NotI fragment was used for the
construction of betaglycan reporter plasmid, pBG-Lux, as described
under "Experimental Procedures." Luciferase activity was measured
in total cell extracts obtained from L6E9 or Rat1 cells after 48 h
of transient transfection with control empty vector (pGL3-b) or
pBG-Lux.
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Fig. 3.
Activity of betaglycan reporter plasmid
increases in differentiating C2C12
myoblasts. Myoblasts C2C12 were
transiently co-transfected with the reporter plasmid for betaglycan
expression (pBG-Lux, columns) or myogenin expression
(pMyoLuc, solid line) and a plasmid to normalize the
transfection (pRLSV40). After transfection the cells were incubated in
growth medium for 24 h and then switched to differentiation
medium. The cells were harvested at 0, 2, 4, and 6 days after
triggering differentiation, and dual luciferase activity was
determined. The results show the means ± S.D. of two experiments
performed in triplicate.
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Fig. 4.
Betaglycan reporter activity is under the
control of MyoD expression. Myoblasts
C2C12 were transiently co-transfected with the
reporter plasmid pBG-Lux and expression vectors containing no insert
(pEMSV empty vector), the cDNA sequence for myogenin (pEMSV-myog),
or MyoD (pEMSV-MyoD). Plasmid pRLSV40 was included in all the cases to
normalize the transfection. Myoblasts were cultured in growth medium
for 24 h (open bars) and extracted to measure dual
luciferase activity. The other set of myoblasts was cultured for
24 h in differentiation media (dark bars) and processed
in the same way. Luciferase activity was expressed as arbitrary units
of pBG-Lux/pRLSV40, and the results show the means ± S.D. of two
independent experiments in triplicate. The inset shows as a
control the MRFs expression after transfection. MyoD and myogenin
expression was determined by Western blot after 0, 1, and 2 days of
differentiation of untransfected myoblasts and the levels of both MRFs
after 24 h of transfection (lane T) with pEMSV-MyoD or
pEMSV-myogenin.
and Stimulated by
Retinoic Acid--
The promoter sequence for betaglycan contains
putative binding sites for Smads, the major transcriptional mediators
of the TGF-
responses, as well as for RA receptor. Because the
induction of myoblast differentiation is modulated by removal of
diverse growth factors (26) or by the addition of RA to the medium
(11), we decided to evaluate their role on the activity of betaglycan promoter. For that purpose, myoblasts were transiently transfected with
pBG-Lux and cultured in the presence and absence of defined growth
factors FGF-2, BMP-2, HGF, and TGF-
and growth medium (10% fetal
bovine serum). Fig. 5 shows that
treatment of the cells for 24 h with FGF-2, BMP-2, or HGF did not
modify luciferase activity compared with growth medium (10% fetal
bovine serum), whereas treatment with any of the three TGF-
mammalian isoforms resulted in a 50% inhibition. These results suggest
that growth factors like FGF-2, BMP-2, and HGF are not necessary to
maintain the expression of betaglycan, whereas TGF-
is inhibitory.
On the other hand, RA, a vitamin A metabolite that plays a major role
in skeletal muscle development (31) and differentiation, strongly
stimulated the transcriptional activity of pBG-Lux (Fig. 5). This
stimulation is observed under growth or differentiation conditions
(data not shown), indicating that RA, an inducer of skeletal muscle
differentiation, stimulates the expression of betaglycan.
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Fig. 5.
Retinoic acid enhances betaglycan reporter
activity, whereas TGF- inhibits it.
Myoblasts C2C12 were transiently co-transfected
with the pBG-Lux and a plasmid to normalize the transfection (pRLSV40).
After transfection, the cells were cultured in growth medium for
24 h followed by the addition of FGF-2 (3 ng/ml), TGF-
1 (1 ng/ml), TGF-
2 (1 ng/ml), TGF-
3 (1 ng/ml), BMP2 (1 nM), HGF (3 ng/ml), or RA (1 µM). Control
cells were maintained in growth medium (SFB) without
additives. Myoblasts were incubated for 24 h with growth factors
and extracted to measure dual luciferase activity. The luciferase
activity was expressed as arbitrary units of pBG-Lux/pRLSV40. The
results show the means ± S.D. of two independent experiments
performed in triplicate.
2 Potency in
C2C12 Myoblasts--
Since the recent paper by
Eickelberg et al. (32) reporting that depending on the type
and quantity of the glycosaminoglycan attached to betaglycan, it may
prevent the access of TGF-
to the signaling receptors, thereby
acting as an inhibitor of the factor, the membrane-bound form of this
co-receptor can not be assumed to be an enhancer of TGF-
. Therefore,
to determine whether or not betaglycan in the
C2C12 myoblasts was a TGF-
enhancer or
inhibitor, we analyzed its effect on a prototypical TGF-
assay. For
this purpose we forced the expression of betaglycan in
C2C12 myoblasts using the adenoviral vector
Adv-BG, which drives the expression of the wild type receptor. Cells
infected with Adv-BG and a control empty vector were then transiently
transfected with p3TP-lux, a well known and widely used TGF-
responsive luciferase reporter (33). After another 24 h of
incubation in growth medium, TGF-
2 was added to the cells at the
concentrations indicated in Fig. 6. The
luciferase activity of these cells indicated that both the magnitude
and the sensitivity of the response greatly increased in the Adv-BG
infected cells as compared with the control infected cells.
Importantly, the presence of betaglycan in the Adv-BG-infected cells
was verified by Western blot (Fig. 6B). These results
clearly show that betaglycan forced expression in the
C2C12 cells enhances TGF-
function at least
in nondifferentiated myoblasts.
View larger version (21K):
[in a new window]
Fig. 6.
Betaglycan enhances
TGF- 2 potency in C2C12
myoblasts. A, C2C12 myoblasts
(control, closed circle) were infected with 100 plaque-forming units/cell of Adv-BG (closed triangle). After
24 h the myoblasts were transiently transfected with p3TP-Lux and
pRL-SV40, an expression vector for Renilla reniformis
luciferase, whose activity was used to normalize the transfection
(pRL). After transfection the cells were incubated in growth medium for
24 h, and then increasing concentrations of TGF-
2 were added.
The cells were harvested after 24 h, and dual luciferase activity
was determined. The results show the means of two independent
experiments in triplicate. B, C2C12
myoblasts were infected with the indicated plaque-forming units/cell
(pfu) of Adv-BG. After 48 h, the cells were harvested,
and betaglycan BG was determined by Western blot analyses as
done for the experiment in Fig. 1B. The arrow
indicates the migration position of betaglycan core protein.
View larger version (69K):
[in a new window]
Fig. 7.
Betaglycan is located on the surface of
differentiated myotubes and ECM. Betaglycan localization was
studied in C2C12 cells under proliferating
conditions (A, D, and G) or after
induced to differentiate for 6 days (B, C,
E, F, H, and I).
Nonpermeabilized cells were processed for indirect immunofluorescent
staining with anti-betaglycan (D and E) or
anti-perlecan (F) antibodies and revealed with fluorescein
isothiocyanate-conjugated secondary antibodies
(green). The same cells were permeabilized and
processed for indirect immunofluorescent staining with anti-myosin
antibodies (G-I) using TRITC-conjugated secondary
antibodies (red). J-L correspond to D
plus G, E plus H, and F
plus I (merged images), respectively. Betaglycan localizes
on the surface of myotubes and in the ECM. The fields were photographed
and printed under identical conditions. Phase contrast microscopy is
shown (A-C). The bar corresponds to 50 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 cells,
and immunofluorescent staining during differentiation.
are among the principal
known regulators of skeletal muscle regeneration and may be normally
present in muscle tissue and released upon injury or secreted auto- or
paracrinally by the cells during the regeneration process (36-38).
Quiescent and activated myoblasts express the c-Met receptor as well as
FGF receptors (specially FGFR-1 and FGFR-4) (39, 40). Growth factor
responsiveness and consequently skeletal myogenesis are regulated by
ligands and receptor availability (41), as well as by the presence of proteoglycans at the cell surface or at the ECM (either presenting or
sequestering possible ligands). Thus, the expression, processing, and
cellular localization of proteoglycans is critical to their modulatory
growth factor function. As a matter of fact, we have shown before that
perlecan, syndecan-1, and syndecan-3, all of them heparan sulfate
proteoglycans, are regulated during in vitro skeletal muscle
differentiation and play a role in modulating this process (5, 9, 11,
27).
superfamily (42-44). In several cell lines, including rat L6E9 myoblasts lacking endogenous betaglycan, it significantly increases the affinity of
T
RII for TGF-
, thereby enhancing the responses to TGF-
(22, 45). It is postulated that this effect eliminates differences in
efficacy among the three TGF-
isoforms. TGF-
2, which binds to
T
RI and T
RII with low affinity and has minimal consequence on
growth inhibitory effects in the absence of betaglycan, behaves similarly to TGF-
1 in the presence of betaglycan (22, 45). Importantly, it has been shown that betaglycan, in the presence of the
type II activin receptor, binds inhibin and thereby mediates its
well known activin antagonism (42, 43, 46). Another proposed function
of betaglycan derives from the existence of a soluble form of the
receptor that is produced by the proteolytic cleavage of its ectodomain
(47-49). Soluble betaglycan binds TGF-
with the same affinities as
its membrane precursor, but contrary to it, this binding sequesters and
neutralizes the factor (33, 49). This capacity has been exploited
therapeutically to prevent the deleterious effects of TGF-
in the
progression of some kind of malignancies (50, 51). Interestingly, a
novel and surprising function of betaglycan has been shown recently.
Eickelberg et al. (32) demonstrated that membrane betaglycan
may inhibit TGF-
signaling. This inhibition is not caused by
enhanced secretion of soluble betaglycan. It is proposed that in some
cell lines, the type and amount of the glycosaminoglycan chains
attached to betaglycan core protein, may sterically prevent the access
of TGF-
to the T
RI and T
RII, thereby preventing its downstream signaling (32).
isoforms was inhibitory. On the other
hand, treatment of the cells with RA, an inducer of skeletal muscle
differentiation in vivo (52) and in vitro (11),
increased the activity of the betaglycan reporter. Furthermore,
the increase in activity mediated by RA was observed independently of
the presence of serum. This effect of RA is consistent with a report by
Nakayama et al. (53), who found that RA increases the levels
of betaglycan mRNA in osteoblast-like cells. It is particularly
interesting that these growth factors and RA, which are known to be
expressed in the vicinity of condensing mesenchymal cells and limb buds
during early developmental stages (54-57), modulate betaglycan
expression. Interestingly, it has been described that RA induces
differentiation only in MyoD-expressing muscle cells by the formation
of the RA receptor-MyoD complex (58). Other growth factors such as
FGF-2, BMP-2, or HGF did not significantly affect the promoter
activity. This lack of BMP-2 effects contrasts with the findings of Ji
et al. (1), who, using osteoblasts, demonstrated that BMP-2
inhibited the rat betaglycan gene promoter and identified a 180-bp
region responsible of this silencing. The fact that we did not observe
any BMP-2 effect may be explained simply by a difference in the cell
line used in our experiments. Additionally, it may be possible that to
detect the activity of the BMP cis-acting element it has to
be removed form the basal promoter to reveal its silencing activity
(1). Finally, although we have emphasized on the transcriptional
control of betaglycan gene expression, we are aware that other levels
of regulation may exist. This is particularly possible in view of the
fact that our betaglycan reporter remains fully active at day 6, even
when the levels of its mRNA and cell surface protein have decreased
from their levels at day 4. Additionally, the fact that some of the
up-regulated betaglycan is being localized to the ECM (Fig. 7) makes it
very difficult to fully account for all of the betaglycan induced
during differentiation.
in muscle differentiation is abundant but
controversial. TGF-
has been shown to be a strong inhibitor of
skeletal muscle differentiation (14, 59, 60) but also to be a promoter
of terminal differentiation (61). However, the fact that the expression
of a truncated dominant negative T
RII receptor inhibits the
C2C12 differentiation suggests the need for an
active TGF-
pathway for its differentiation (62). One clue to the
role of betaglycan in C2C12 differentiation can be anticipated from our finding that betaglycan performs a TGF-
presentation role in nondifferentiated C2C12
myoblasts (Fig. 6). Reasoning based on these data would lead us to
conclude that betaglycan induction may be acting to promote the effects
of TGF-
, and therefore it may act to promote the effects of this
growth factor on the differentiation process. The regulation of TGF-
activity can be more intricate considering that TGF-
also binds to
other proteoglycans. In myoblasts that do not express decorin, a
proteoglycan with high affinity for TGF-
(63), we have observed an
inhibition of TGF-
signaling (14) together with a significant
increase in the binding of TGF-
to betaglycan, suggesting a
competition between both proteoglycans for
TGF-
.2
and/or FGF-2 reservoir. Thus, betaglycan roles during skeletal
muscle differentiation are to be determined. Experiments to test the effects of constitutive betaglycan expression and inhibition of endogenous betaglycan expression in C2C12
myoblasts are in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Kornelia Polyak (Dana-Farber Cancer Institute, Boston, MA) for generous advice and assistance in the generation of recombinant adenoviruses. Also, we thank Dr. Laura Ongay-Larios for technical assistance with the nucleotide sequencing.
![]() |
FOOTNOTES |
---|
* This work was supported in part by FONDAP-Biomedicine Grant 13980001 (to E. B.). The Millenium Institute for Fundamental and Applied Biology 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF537325.
§ Supported in part by International Research Scholars grants from the Howard Hughes Medical Institute.
Recipient of a Presidential Chair in Science from the Chilean
Government. To whom correspondence should be addressed: Dept. de
Biología Celular y Molecular, Facultad de Ciencias
Biológicas, P. Universidad Católica de Chile, Casilla
114-D, Santiago, Chile. Fax: 56-2-635-5395; E-mail:
ebrandan@genes.bio.puc.cl.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208520200
2 C. Riquelme, C. Cabello-Verrugio, and E. Brandan, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MRF, muscle
regulatory factor;
FGF-2, basic fibroblast growth factor;
HGF, hepatocyte growth factor/scatter factor;
ECM, extracellular matrix;
TGF-, transforming growth factor
;
T
R, TGF-
receptor;
RA, retinoic acid;
TRITC, tetramethylrhodamine
isothiocyanate.
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