(Received for publication, January 31, 1997, and in revised form, May 15, 1997)
From the Department of Cell and Molecular Biology,
Faculty of Biological Sciences, Catholic University of Chile,
Casilla 114-D, Santiago, Chile, and the ¶ Sigfried and Janet Weis
Center for Research, Danville, Pennsylvania 17822
Syndecan-1 is an integral membrane proteoglycan
involved in the interaction of cells with extracellular matrix proteins
and growth factors. It is transiently expressed in several condensing mesenchymal tissues after epithelial induction. In this study we
evaluated the expression of syndecan-1 during skeletal muscle differentiation. The expression of syndecan-1 as determined by Northern
blot analyses and immunofluorescence microscopy is down-regulated during differentiation. The transcriptional activity of a syndecan-1 promoter construct is also down-regulated in differentiating muscle cells. The decrease in syndecan-1 gene expression is not dependent on
the presence of E-boxes, binding sites for the MyoD family of
transcription factors in the promoter region, or myogenin expression. Deletion of the region containing the E-boxes or treatment of differentiating cells with sodium butyrate, an inhibitor of myogenin expression, had no effect on syndecan-1 expression. Basic fibroblast growth factor and transforming growth factor type , which are inhibitors of myogenesis, had little effect on syndecan-1 expression. When added together, however, they induced syndecan-1 expression. Retinoic acid, an inducer of myogenesis, inhibited syndecan-1 expression and abolished the effect of the growth factors. These results indicate that syndecan-1 expression is down-regulated during
myogenesis and that growth factors and retinoic acid modulate syndecan-1 expression by a mechanism that is independent of
myogenin.
Heparan sulfate is present on the cell surface of most, if not all, adherent cells in the form of heparan sulfate proteoglycans. The best characterized cell surface proteoglycans are the syndecans, a family of transmembrane heparan sulfate proteoglycans that are implicated in a variety of interactions with the pericellular microenvironment (for review, see Refs. 1 and 2). The first member of this family to be discovered, syndecan-1, is a hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans (3). This proteoglycan binds a variety of extracellular matrix constituents, such as fibronectin (4); thrombospondin (5); tenascin (6); and collagen types I, III, and V (7) as well as basic fibroblast growth factor (bFGF)1 (8). Syndecan-1 has been suggested to be a morphoregulatory molecule because its expression during embryonic development is dynamically regulated and corresponds to morphological boundaries (9). Changes in syndecan-1 expression correlate with morphologic changes during interactions between epithelial and mesenchymal cells in tooth (10), kidney (11), female reproductive tract (12), and limb bud (13) development.
The molecular mechanisms that regulate syndecan expression are only
beginning to be explored. Sequences upstream of the first exon of
murine syndecan-1 gene have promoter activity. This region contains an
array of consensus transcription factors binding sites (14). These
sites, which include Antennapedia, NF-B, SP1 (GC and GT box), TATA
and CAAT boxes, and five E-boxes (CANNTG), provide potential mechanisms
for regulation of syndecan-1 expression.
Skeletal muscle cells are a useful model for studying cell
differentiation. The fusion of mononucleated myoblasts to form multinucleated myotubes is a central event in skeletal muscle development. The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their
environment. Some of the regulatory proteins that control this process
have been identified. Thus, when myogenesis begins, myogenic regulatory genes of the MyoD family (myogenin, mrf5, mrf4)
are activated (15). These factors bind to specific DNA consensus sites
called E-boxes, which function as transcriptional enhancers of muscle differentiation genes (for review, see Ref. 16). Recent data suggest
that factors of the MEF2 family are also required to specify muscle
fate or to direct muscle differentiation (17). The expression and
activity of these master genes are regulated by polypeptide growth
factors including bFGF (18), transforming growth factor (TGF-
)
(19), and insulin-like growth factor (20), as well as by retinoic acid
(RA) (21). One or more of these growth factors, when present at high
levels, hold myoblasts in the undifferentiated state (for review, see
Ref. 22), whereas RA induces myoblast differentiation (21).
It has been demonstrated that heparan sulfate proteoglycans are necessary for the modulation of terminal myogenesis (23), probably by acting as low affinity receptor for bFGF (24, 25), a potent inhibitor of myogenesis (18). We have shown that the expression of glypican (26, 27) and perlecan (28) varies during skeletal muscle differentiation of the C2C12 myoblast cell line. Syndecan-1 is expressed transiently during limb development (13) but is absent in adult skeletal muscle (29), making this proteoglycan a good candidate for modulation of bFGF signaling during myoblast differentiation. The presence of E-boxes in the syndecan-1 promoter has led to the suggestion that MyoD or a related protein binds to these sites to cause down-regulation of syndecan-1 expression. The decrease in syndecan-1 expression would attenuate bFGF signaling and promote differentiation of myoblasts (30).
In this study we show that expression of syndecan-1 is down-regulated
during skeletal muscle differentiation of C2C12
myoblasts but by a myogenin- and E-box-independent pathway. Syndecan-1
expression is controlled by a proximal region of the promoter which
contains putative SP1, NF-B, and RA response element binding sites
and is strongly influenced by bFGF, TGF-
, and RA.
The mouse skeletal muscle cell line
C2C12 (31) was grown as described by Brandan
et al. (32). Three days after plating (80% confluence), the
medium was changed to differentiation medium (Dulbecco's modified
Eagle's medium supplemented with 5% horse serum). Two days later, 0.1 mM cytosine--D-arabinofuranoside was added
to the culture medium. Thereafter the incubation medium was changed
daily. NMuMG mouse mammary epithelial cells and C3H10T1/2 mouse
fibroblasts (ATCC) were routinely cultured in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum (FCS) as described
(33). For experiments, cells were plated at equal density and grow to
60-70% confluence.
Total RNA was isolated from cell cultures at the indicated times by guanidium thiocyanate/phenol/chloroform extraction and isopropyl alcohol precipitation using RNAzol B (Cinna/Biotecx Laboratories, Inc., Houston, TX) (34). RNA samples (15 µg/lane) were electrophoresed through 1.2% agarose/formaldehyde gels, transferred to Nylon membranes (Sigma), and hybridized with probes for myogenin, syndecan-1, and creatine kinase. The probes were prepared as follows. For myogenin, a conserved 672-bp fragment of human myogenin cDNA was amplified by PCR (35). For syndecan-1 and creatine kinase a fragment of 529 bp (from 1505 to 2033 in the cDNA) and 486 bp from 389 to 874 in the cDNA), respectively, were amplified by reverse transcriptase PCR from total RNA obtained from C2C12 cells. Blots were hybridized with random primed labeled probes in a buffer containing 1.0 M NaCl, 1% sodium dodecyl sulfate, 10% dextran sulfate, and 100 µg/ml denatured salmon testes DNA at 65 °C overnight. Hybridized membranes were washed twice at 65 °C in 0.2 × SSC, 0.1% sodium dodecyl sulfate for 5 min and exposed to Kodak x-ray film.
Immunofluorescence MicroscopyCells 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 (1:20 of affinity-purified anti-syndecan-1 (36) in 5% non-fat milk, 0.1 M sodium chloride, 0.02 M sodium phosphate, pH 7.4) for 30 min on ice. After removing this solution the cells were fixed in 3% paraformaldehyde, and the bound antibodies were detected by incubating the cells with affinity-purified Texas Red-conjugated secondary antibodies. Nuclei were stained by incubating the cells in 0.1 µg/ml DAPI, 20 mM Tris-HCl, pH 7.5, 0.1 M NaCl for 20 min. After rinsing, the slides were viewed with a Nikon upright microscope equipped for epifluorescence.
Immunoblot AnalysisConditioned medium from approximately 6 × 108 cells was concentrated in a Q-Sepharose column (Sigma) (2.5 × 15 cm) equilibrated with 50 mM Tris-HCl, pH 7.4. After application of the conditioned medium the column was washed with the equilibration buffer until the A280 of the effluent was 0. Bound proteins were eluted with the same buffer containing 1.0 M NaCl until phenol red was eluted. After dialysis against the equilibration buffer the sample was subjected to high performance liquid chromatography on a 7.5 × 75-mm column of DEAE (Beckman Spherogel TSK DEAE-5PW) equilibrated with the same buffer. The bound proteins were eluted with a linear gradient from 0 to 1 M NaCl in the equilibration buffer at a flow rate of 1 ml/min. For immunoblot analysis aliquots were subjected to sodium dodecyl sulfate-gel electrophoresis in 7.5% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (Millipore, Bedford, MA), and stained with affinity-purified rabbit anti-syndecan-1 antibodies (36) and visualized by enhanced chemiluminescence (Pierce).
Cell Transient TransfectionsPlasmids were purified by
WizardTM Plus Maxiprep kits (Promega). The cells were
plated in growth medium 1 day before transfection at a density of
8,000/cm2 in 60-mm plates (Corning). For transfection the
cells were incubated for 6 h with Opti-MEM I (Life Technologies,
Inc.) containing 5 µg of plasmid DNA and 20 µg of LipofectAMINE
(Life Technologies, Inc.). After transfection the cells were incubated
for 14 h in Opti-MEM I containing 10% FCS and 0.5% chick embryo
extract. The cells were then washed twice with Hanks' balanced salt
solution and incubated for 2 days in growth medium, followed by 1, 2, or 3 days in differentiation medium. To analyze the effects of growth factors and RA the cells were incubated for 2 days in differentiation medium containing 10 ng/ml bFGF, 2.5 ng/ml TGF-, or the indicated concentrations of RA. All transfections were performed at least three
times with at least two plasmid DNA preparations.
At various times the cells were harvested and assayed for
chloramphenicol acetyltransferase (CAT) and -gal activities. CAT activity was measured as follows. Cell extracts were incubated for
14 h at 37 °C in a reaction mixture containing
14C-labeled chloramphenicol (NEN Life Science Products) and
n-butyryl-CoA (Promega). The reaction products were
extracted with xylene, and the organic phase was counted in a
scintillation counter. For the
-gal assay the cell extracts were
incubated for 14 h at 37 °C with a buffer containing the
substrate ONPG (Promega), and the absorbance was read at 420 nm with a
spectrophotometer.
For the construction of
the syndecan-1 reporter (p-667CAT), the published mouse promoter
sequence (14) was used to design oligonucleotides that were used to
amplify by nested PCR a 667-bp upstream fragment that begins 50 bp
downstream from the TATA box and contains potential transcription
binding sites for SP1 (GC and GT boxes), MyoD (E-boxes) and NF-B.
The fragment was subcloned into the pCAT-basic vector (Promega). For
the preparation of a reporter construct without E-boxes (p-244CAT) a
244-bp fragment was amplified by PCR with sense primer (CCT AGG AGG CGT
AGA AGG) and antisense primer (CTG CGT TAG GCT CTG TCT CC) using
p-667CAT plasmid as template.
For construction of the p-myoCAT reporter plasmid, a fragment of 688 bp
corresponding to a region of the myogenin promoter which contains the
MEF1 and E-boxes sites (from +62 to 626 (37)) was amplified by nested
PCR using a mouse BALB/c genomic library (ATCC) as template. The
amplified fragment was ligated to the pCAT-basic vector as described
above. All the pCAT vectors were sequenced using the
SequenaseTM kit. A
-gal expression plasmid (RSV-
gal)
was obtained from ATCC. This plasmid was co-transfected with the CAT
constructs to monitor transfection efficiencies.
To evaluate the expression of
syndecan-1 in myoblasts, total RNA was isolated from
C2C12 myoblasts and from 10T1/2 fibroblasts and
NMuMG epithelial cells, two cell lines known to express syndecan-1. Fig. 1A shows that
C2C12 myoblasts express both the major
2.5-kilobase and minor 3.1-kilobase forms of syndecan-1 mRNA at
levels comparable to the mouse epithelial cell line (29). Fig.
1B shows Western blot analyses of conditioned medium from
myoblasts. The proteoglycans were eluted from a DEAE column at
different NaCl concentrations (lane 2, 0.85 and lane
3, 1.0 M NaCl) and stained with affinity-purified anti-syndecan-1 antibodies (38). The antibodies recognized a high
molecular weight smear in the conditioned medium of myoblasts, which
was eluted from the DEAE column at high salt concentration, consistent
with staining of a proteoglycan. For comparison, lane 1 shows an immunoblot of syndecan-1 present in conditioned medium of
Schwann cells transfected with syndecan-1 cDNA (36). Together, these results indicate that myoblasts synthesize and release
syndecan-1.
Syndecan-1 Expression Decreases during Myogenesis
To evaluate
the expression of syndecan-1 during myoblast differentiation total RNA
was isolated from myoblasts and from myoblasts induced to differentiate
for 2 and 5 days and evaluated by Northern blot analysis. Fig.
2 shows a significant decrease in the amount of
syndecan-1 mRNA during differentiation, which was evident by day 2 of differentiation and essentially complete by day 5. Fig. 2 also shows
the increase of mRNAs coding for creatine kinase and myogenin,
skeletal muscle-specific markers that increase during differentiation.
To evaluate further the expression of syndecan-1 during skeletal muscle
differentiation cell cultures were stained with anti-syndecan-1 antibodies. Fig. 3 shows that myoblasts (panel
A) express syndecan-1 that can be detected on the cell surface by
immunofluorescent staining. Cells stained 2 days after initiation of
differentiation (panel C) showed less immunoreactive
syndecan-1. Essentially no staining was observed after 5 days of
differentiation (myotubes) (panel E). Together, these
results clearly demonstrate that the synthesis of syndecan-1 during
differentiation of C2C12 skeletal muscle cells
is significantly diminished.
To characterize the mechanism underlying this down-regulation
syndecan-1 transcriptional activity was measured in transient transfection experiments using a reporter vector consisting of 667 bp
of the rat syndecan-1 promoter linked to a CAT reporter gene
(p-667CAT). This promoter region contains a putative TATA box sequence
and consensus binding sites for several transcriptions factors,
including two E-boxes (414 and 289 bp upstream of TATA box). Fig.
4 shows the CAT activity in myoblasts transiently
transfected with p-667CAT and induced to differentiate. The
transcriptional activity decreases significantly in differentiating
cells so that by 3 days the activity is essentially abolished. The
inset in Fig. 4 shows transcriptional activity obtained with
an expression construct that contains a myogenin promoter fused to a
CAT reporter gene. As expected, and in contrast to syndecan-1 promoter
activity, transcription driven by the myogenin promoter increases
significantly after differentiation is triggered. These results
demonstrate that the decrease in syndecan-1 expression which occurs
during skeletal muscle differentiation results from a decreased rate of
transcription.
The Decrease in Syndecan-1 Expression during Myogenesis Is Myogenin- and E-box-independent
The syndecan-1 promoter contains
several E-boxes, which are DNA binding sites for MyoD and related
proteins, including myogenin. Upon induction of muscle differentiation
these proteins bind to E-box regions and activate the transcription of
skeletal muscle genes, such as creatine kinase (39). To evaluate the
role of myogenin in the expression of syndecan-1, myoblasts were
incubated for 2 days in differentiation medium with or without sodium
butyrate (40), an agent known to block transcriptional activity of
myogenin. The level of syndecan-1 mRNA was evaluated by Northern
blot analyses. Fig. 5A shows that the
decrease in the syndecan-1 mRNA level was independent of the
presence of butyrate. In contrast, the expression of creatine kinase
was totally abolished by the treatment. These results suggest that
myogenin expression is not involved in the decrease of syndecan-1
transcription observed during differentiation.
To evaluate directly the role of myogenin and other MyoD proteins on
the expression of syndecan-1, myoblasts were transiently transfected
with a syndecan-1 promoter that contained the binding sequences for
transcription factors SP1 (GC and GT boxes) and NF-B, but lacked
E-boxes (p-244CAT). The expression driven by this promoter was compared
with the expression from the p-667CAT vector during differentiation.
Fig. 5B shows that the reporter expression obtained with
both constructs was identical, and both were strongly inhibited during
differentiation. Together these results suggest that the decrease in
syndecan-1 expression observed during myogenesis is not related to the
presence of E-boxes in the promoter region.
In vitro induction of
myoblast differentiation can be obtained by removal of growth factors
from the medium. To evaluate the role of growth factors on the
expression of syndecan-1, myoblasts were induced to differentiate in
the presence and absence of defined growth factors (bFGF and TGF-)
and growth medium (10% FCS), and the expression of p-244CAT was
evaluated in transient transfection experiments. Fig. 6
shows that treatment of the cells for 2 days with bFGF (10 ng/ml)
increased CAT activity slightly compared with differentiation medium
(5% horse serum), whereas treatment with TGF-
(2.5 ng/ml) had no
effect on syndecan-1 expression. Under these conditions a strong
inhibitory effect of bFGF and TGF-
on myogenin expression was
observed (data not shown) (18, 19). However, when bFGF and TGF-
were
added together, activation of syndecan-1 promoter activity was obtained
to values similar to those achieved when 10% FCS was used. These
results suggest that growth factors like bFGF and TGF-
are necessary
to maintain the expression of syndecan-1 and that the sequences
contained in the proximal region of the promoter are sufficient to
exert such regulation.
RA Inhibits Syndecan-1 Expression
RA, a vitamin A metabolite,
plays a major role in skeletal muscle development (41) and skeletal
muscle differentiation (21). As shown in Fig.
7A, the presence of RA strongly inhibited the transcriptional activity of p-244CAT. Maximal inhibitory activity was
observed at 106 M RA. Interestingly, as shown
in Fig. 7B, the synergistic stimulatory effect observed for
bFGF and TGF-
on syndecan-1 expression was totally abolished by RA.
RA treatment also blocked the increase in syndecan-1 transcriptional
activity caused by 10% FCS (Fig. 7B). These results
indicate that RA, an inducer of skeletal muscle differentiation,
inhibits the expression of syndecan-1 and abolishes the stimulatory
effect of bFGF and TGF-
or serum. The sequences responsible of this
inhibitory effect are contained in the proximal region of the promoter
gene contained in p-224CAT.
The results presented in this paper demonstrate that the expression of syndecan-1, a transmembrane heparan sulfate proteoglycan (1), decreases during differentiation of skeletal muscle cells. This conclusion is based on analysis of syndecan-1 mRNA levels, immunofluorescent staining of cells, and the activity of a reporter construct containing a portion of the rat syndecan-1 promoter. This phenomenon correlates well with the observation that in adult skeletal muscle tissue syndecan-1 is absent (29) and with the loss of syndecan-1 in later stages of limb development (13).
The function of syndecan-1 in myoblasts and the consequences of syndecan-1 down-regulation during differentiation are not known. However, several roles for syndecan-1 and its down-regulation can be postulated. It is well known that proliferation of myoblasts is strongly stimulated by bFGF. At the same time this growth factor is a strong inhibitor of skeletal muscle differentiation (18). Heparan sulfate proteoglycans act as co-receptors for bFGF (24), allowing the binding of the growth factor to its signaling receptor on the plasma membrane and stimulating its biological effects. Therefore, the presence of syndecan-1 in the membrane might be critical for bFGF activity. Previously, we demonstrated that the expression of perlecan, a basal lamina-associated heparan sulfate proteoglycan, also decreases during differentiation (28) and that the synthesis of glypican (26), a lipid-anchored membrane-associated heparan sulfate proteoglycan, increases during differentiation (27).
These observations are potentially important in the context of the specificity of action of different heparan sulfate proteoglycans. It has been shown that addition of perlecan but not soluble syndecans or glypican restores bFGF signaling and biological activity to heparan sulfate-deficient fibroblasts (25). On the other hand, recombinant membrane-associated syndecans or glypican have been shown to promote bFGF signaling in a hematopoietic cell line that expresses low levels of heparan sulfate proteoglycans (42). It can be speculated that down-regulation of syndecan-1 expression, together with the bFGF receptor down-regulation (43), makes the cells refractory to the presence of bFGF and therefore allows differentiation of skeletal muscle (44). Experiments to test the effects of constitutive syndecan-1 expression and inhibition of endogenous syndecan-1 expression in myoblasts are in progress.
Another potential role for syndecan-1 on the surface of myoblasts is the binding of extracellular matrix adhesive proteins. Syndecan-1 has been shown to bind several extracellular matrix adhesive molecules, including fibronectin (4); thrombospondin (5); tenascin (6); and collagen types I, III and V (7). Cell adhesion to these matrix proteins might not be required after differentiation when individual muscle fibers are in direct contact with basement membrane extracellular matrix. Furthermore, it has been shown that syndecan-1 can influence cell invasion (45). Myoblasts are able to migrate through basal lamina during early stages of differentiation (46). It is tempting to speculate that the presence of syndecan-1 on the surface of myoblasts may influence their migratory pathway to give rise to slow or fast primary myotubes (47).
Syndecan-1 expression in myoblasts appears to be regulated at the level
of transcription. It has been suggested that down-regulation of
syndecan-1 expression during muscle differentiation could result from
the presence of E-boxes, target sites for the action of myogenic regulators such MyoD and myogenin (39), in the syndecan-1 promoter (30). We tested this by transiently transfecting myoblasts with CAT
reporter vectors containing the rat syndecan-1 promoter that contained
or lacked E-boxes. Our results clearly demonstrate that the presence of
the E-boxes is not required for the decrease in expression of
syndecan-1 which is observed after differentiation is triggered. These
results are supported by the finding that treatment of differentiating
myoblasts with sodium butyrate, an agent known to inhibit myogenin
expression (40, 48), had no effect on the pattern of syndecan-1
expression. Furthermore, treatment of the myoblasts with bFGF or
TGF- strongly inhibited myogenin expression (18, 19, and data not
shown), without affecting syndecan-1 expression. Together, these
results suggest that syndecan-1 expression in myoblasts is not directly
regulated by myogenin.
In this study we also found that promoter activity was not
significantly affected by bFGF or TGF-. However, exposure to both growth factors resulted in a significant increase in syndecan-1 gene
activity. These results are similar to the finding of Elenius et
al. (33) on syndecan-1 expression in fibroblasts. On the other
hand, treatment of the cells with RA, an inducer of skeletal muscle
differentiation (21), inhibited the activity of the syndecan-1 reporter. Furthermore, RA was able to abolish the stimulatory effect of
bFGF and TGF-
as well as FCS. This is particularly interesting
because both growth factors and RA are known to be expressed in the
vicinity of condensing mesenchymal cells (49, 50) and limb buds during
early developmental stages (41).
These results indicate that the main regulatory elements responsible
for the transcriptional regulation of syndecan-1 expression during
myoblasts differentiation are contained in the proximal 277-bp segment
of the syndecan-1 promoter. This region contains a putative TATA box
sequence and consensus binding sites for several transcription factors
such as SP1 and NF-B. Vihinen et al. (51) have
demonstrated that SP1-like transcription factors have an essential role
in the regulation of the transcriptional activity of the syndecan-1
gene. This suggests that the down-regulation of syndecan-1 expression
during myoblast terminal differentiation could be explained by
variations in SP1 activity. There are several possible explanations for
a decrease in SP1 activity. Changes in the level of expression of Sp1
during differentiation could occur, as was shown previously for
different cells and tissues (52). An increase of SP1 phosphorylation
which decreases its DNA binding activity, as demonstrated in terminal
differentiation of liver (53), is also possible. Alterations of
co-activators that are required by SP1 to modify gene expression have
also been described (54). Finally, recent data demonstrated that SP1
and E2F, a cell cycle-regulated transcription factor, act
synergistically to activate dihydrofolate reductase gene transcription
in the absence of E2F DNA binding sites (55). E2F is sequestered by the
retinoblastoma protein during differentiation (56), making SP1
possible less active and likely explaining the observed decrease in syndecan-1 expression during myoblast terminal differentiation.
The inhibitory effect of RA is interesting not only as an explanation
for the decrease of syndecan-1 expression during myoblasts differentiation, but also because suppression of syndecan-1 expression has been shown to be associated with malignant conversion (57). Tumor
necrosis factor- is the only other factor described which restricts
the expression of syndecan-1 (58). The inhibitory effect of RA on
syndecan-1 expression could be explained by the presence of putative RA
response elements in the promoter region. This sequence is sufficient
to cause the inhibitory effect of RA on the murine Oct4 promoter (59).
The syndecan-1 promoter region contains sequences that are similar, but
not identical to RA response elements. These are located 55, 80, and
100 bp upstream of the TATA box and are included in the p-244CAT
reporter.
The expression of perlecan during myogenesis is also down-regulated (28). As indicated by Vihinen et al. (51), the promoter region of perlecan (60) resembles that of mouse syndecan-1. The upstream regions of these genes lack canonical TATA and CAAT boxes, but several SP1 transcription factor binding sites are present within the first 200 bp of the promoter. These observations suggest that similar regulatory mechanisms are involved in the regulation of expression of these cell surface macromolecules.
In summary, we have found that the expression of syndecan-1 is
down-regulated during skeletal muscle differentiation. This phenomenon
is modulated by growth factors and RA but is E-box- and
myogenin-independent. The regulatory sequences responsible for this
modulation are contained within a 277-bp segment of the gene which
contains consensus binding sites for several transcriptions factor such
as SP1, NF-B, and RA-binding proteins. These results will help
toward understanding the complex regulation during development and
differentiation of this key integral membrane heparan sulfate proteoglycan.