1 Centro de Regulación Celular y Patología, Departamento de
Biología Celular y Molecular, Facultad de Ciencias Biológicas,
MIFAB, P. Universidad Católica de Chile, Santiago, Chile
2 Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, PA
17822-2613 USA
* Author for correspondence (e-mail: ebrandan{at}genes.bio.puc.cl )
Accepted 25 February 2002
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Summary |
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[35S]heparin ligand blot assays identified a 33/30 kDa doublet (p33/30) in detergent/high ionic strength extracts and heparin soluble fractions obtained from intact C2C12 myoblasts. p33/30 is localized on the plasma membrane or in the extracellular matrix where its level increases during muscle differentiation. Heparin-agarose-purified p33/30 was identified as histone H1. In vitro binding assays showed that histone H1 binds specifically to perlecan. Immunofluorescence microscopy showed that an extracellular pool of histone H1 colocalizes with perlecan in the extracellular matrix of myotube cultures and in regenerating skeletal muscle. Furthermore, histone H1 incorporated into the extracellular matrix strongly stimulated myoblast proliferation via a heparan-sulfate-dependent mechanism.
These results indicate that histone H1 is present in the extracellular matrix of skeletal muscle cells, where it interacts specifically with perlecan and exerts a strong proliferative effect on myoblasts, suggesting a role for histone H1 during skeletal muscle regeneration.
Key words: Histone H1, Proteoglycans, Myogenesis, Extracellular matrix, Skeletal muscle regeneration
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Introduction |
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We are interested in the role that heparan sulfate proteoglycans play
during skeletal muscle formation. In this process, committed myogenic
progenitor cells, or myoblasts, are maintained in a proliferative,
undifferentiated state until appropriate signals trigger their conversion to
multinucleated myotubes. The differentiation of muscle cells is controlled in
a negative manner by specific heparan-sulfate-binding mitogens, such as basic
fibroblast growth factor (FGF-2), hepatocyte growth factor and transforming
growth factor-ß (Brunetti and
Goldfine, 1990; Heino and
Massague, 1990
; Takayama et
al., 1996
).
Sodium chlorate, a specific inhibitor of proteoglycan sulfation, has
previously been shown to decrease the deposition and assembly of ECM
components in cultured C2C12 myoblasts that are induced
to differentiate. This results in the inhibition of cell fusion
(Melo et al., 1996;
Olwin and Rapraeger, 1992
;
Osses and Brandan, 2001
) and
FGF-2-dependent suppression of myoblast differentiation
(Larraín et al., 1998
).
We have also shown that the expression of heparan sulfate proteoglycans during
the terminal skeletal muscle differentiation of C2C12
cells is highly regulated. The expression of glypican, a
glycosylphosphatidylinositolanchored heparan sulfate proteoglycan that is also
released into the ECM, increases (Brandan
et al., 1996
; Campos et al.,
1993
), whereas expression of perlecan, an ECM proteoglycan, and
the transmembrane proteoglycans syndecan-1 and -3 decreases during
differentiation (Fuentealba et al.,
1999
; Larraín et al.,
1997a
; Larraín et al.,
1997b
). Furthermore, heparan sulfate proteoglycans may regulate
muscle physiology. We have demonstrated that expression of syndecan-1
(Larraín et al., 1998
)
and syndecan-3 (Fuentealba et al.,
1999
) inhibit the differentiation of cultured myoblasts in an
FGF-2-dependent manner. On the other hand, the presence of perlecan in muscle
basement membrane has been suggested to participate in cell-ECM interactions
(Villar et al., 1999
),
neuromuscular junction formation (Jacobson
et al., 2001
) and muscle regeneration
(Gulati et al., 1983
).
In the present study we searched for endogenous skeletal muscle cell ligand(s) for heparan sulfate proteoglycans that had not previously been described. We did this on the premise that knowledge of such proteins might aid in elucidating the role(s) that these macromolecules exert in muscle physiology. Here, we demonstrate that an extracellularly occurring histone H1 colocalizes with the heparan sulfate proteoglycan perlecan in the ECM of myotube cultures and in regenerating skeletal muscle. In vitro assays show that histone H1 stimulates myoblast proliferation via a heparan-sulfate-dependent mechanism. We propose that this extracellular heparan sulfate proteoglycan-binding protein may play a functional role during muscle regeneration.
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Materials and Methods |
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Cell culture
The mouse skeletal muscle cell line C2C12 was grown
and induced to differentiate as previously described
(Larraín et al.,
1997b). In some experiments, cells were cultured and maintained
during differentiation in the presence of sodium chlorate at a final
concentration of 30 mM (Melo et al.,
1996
). MST cells were grown as previously described
(Montgomery et al., 1992
).
Protein extractions and [35S]heparin ligand blot
assay
For sequential extractions, C2C12 myotubes were
rinsed twice with phosphate buffered saline (PBS) and harvested by scraping in
the same buffer. The cells were homogenized and a PBS-soluble extract was
obtained by centrifuging for 10 minutes at 13,000 g and 4°C. The
resulting pellet was homogenized in TX-100 buffer (0.05 M Tris-HCl, pH 7.4,
0.5% Triton X-100, 0.15 M NaCl) and centrifuged under the same conditions. The
final pellet was homogenized in TX-100/KCl solution (0.05 M Tris-HCl, pH 7.4,
0.5% Triton X-100, 0.5 M KCl). For heparin displacement of extracellular
proteins from intact muscle cells, myoblasts or differentiating myoblasts were
washed three times for 10 minutes with cold PBS containing 0.1 mM
CaCl2 and 1 mM MgCl2 under gentle agitation at 4°C.
The cells were then incubated in 2 mg/ml heparin in the same buffer for 20
minutes. The PBS-heparin extract was clarified by centrifugation at 13,000
g for 2 minutes at 4°C. Acid extraction of nuclear proteins was
carried out essentially as described previously
(Schmiedeke et al., 1989).
For ligand blot assays, [35S]heparin was biosynthetically
labeled and isolated from MST cells as previously described
(Montogomery et al., 1992).
Proteins in the various muscle cell extracts were separated by SDS-PAGE and
electrotransferred onto nitrocellulose membranes at 100 V for 4 hours.
Membranes were then blocked with 5% bovine serum albumin (BSA) in PBS for 1
hour and incubated overnight with 2x106 cpm/ml
[35S]heparin at 4°C under gentle agitation. After washing the
membranes, heparin-binding proteins were then visualized by
autoradiography.
Heparin-agarose chromatography
Myotubes were subjected to the sequential extraction described above.
TX-100/KCl extracts were dialyzed against 0.05 M NaCl, 0.05 M Tris-HCl, pH 7.8
and loaded onto a 0.5 ml heparin-agarose column pre-equilibrated in 0.3 M
NaCl, 0.05 M Tris-HCl, pH 7.8, and 1 mM PMSF
(Brandan and Inestrosa, 1984).
After rinsing the column extensively with equilibration buffer, bound material
was eluted using a linear gradient of 0.3 to 1 M NaCl in 0.05 M Tris-HCl, pH
7.8. Total, unbound and eluted fractions were analyzed by SDS-PAGE followed by
Coomassie blue staining.
Identification of p33/30
Identification of p33/30 was carried out at HHMI Biopolymer and W. M. Keck
Biotechnology Resource Laboratory, Yale University, CT, USA. Briefly,
molecular masses of in-gel digested p33 peptides were obtained by
matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)
carried out on a Research Grade, VG Tofspec SE instrument. Resulting peptides
were further purified on a C-18 reverse-phase HPLC column and a single peptide
subjected to conventional Edman degradation for amino-acid sequencing using an
Applied Biosystems sequencer. Immunoblots were developed as previously
described (Brix et al., 1998)
using anti-histone H1 antibody and visualized by enhanced chemiluminescence
(ECL).
Binding of heparan sulfate proteoglycans to histone H1
Histone H1 (0.5 mg) was separated by SDS-PAGE and electrotransferred onto a
nitrocellulose sheet. After brief staining with 0.5% Ponceau S red, 5% acetic
acid, histone H1-containing nitrocellulose pieces were excised and then
blocked with 5% dry nonfat milk, 0.05 M Tris-HCl, pH 7.4, 0.15 M NaCl. A
proteoglycanenriched fraction obtained from myotube-conditioned media was
partially purified on a 1 ml DEAE column pre-equilibrated in 0.15 M NaCl, 0.05
M Tris-HCl, pH 7.4 and eluted with 1 M NaCl prepared in the same buffer. The
nitrocellulose immobilized-histone H1 was incubated overnight with the
dialyzed proteoglycan-containing fraction in 2% BSA at 4°C and washed with
the same buffer. Heparan sulfate proteoglycans bound to immobilized histone-H1
were then eluted with 8 M urea, 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4. Total,
unbound and bound fractions were dialyzed and treated with heparitinase as
described (Brandan et al.,
1996). The core proteins of heparan sulfate proteoglycans were
visualized by western blot using the anti-
-heparan sulfate monoclonal
antibody and polyclonal antiperlecan specific antisera, as previously
described (Fuentealba et al.,
1999
; Larraín et al.,
1997a
).
Induction of rabbit skeletal muscle regeneration
The tibialis anterior muscle of adult hamsters were injected
intramuscularly, under ketamine/xylazine anesthesia, with 0.25 ml of an
aqueous solution of BaCl2 (1.2% w/v)
(Caldwell et al., 1990). Four
and five days later the injected muscle area was dissected out of the
anesthetized animal and the tissue quickly frozen in isopentane previously
cooled in liquid nitrogen. Frozen tissue was embedded in Tissue Freezing
Medium gel and a series of 6 µm cryostat sections was obtained.
Immunofluorescence
Immunofluorescence microscopy of C2C12 cultures and
skeletal muscle cryosections was carried out essentially as described
previously (Riquelme et al.,
2001). In some experiments, 95% ethanol/5% acetic acid was used
for fixation and permeabilization of cells.
Proliferation assays
Myoblast proliferation was assayed by measuring [3H]thymidine
incorporation, as described previously
(Currie et al., 1997). For
some assays, skeletal muscle ECM was obtained after detaching myotubes
differentiated during six days by incubating with 10 mM EDTA in PBS at
37°C for 10 minutes, as described previously
(Andress, 1995
). Anti-histone
H1 (C17) or anti-ß galactosidase antibodies were diluted 1:100 for an
overnight preincubation of the ECM-containing dishes and 1:200 through the
proliferation assay.
Others
Adhesion assays were performed as described previously
(Chernousov et al., 1996). DNA
and protein content determinations were performed as described previously
(Riquelme et al., 2001
). Cell
death was determined by measuring histone-complexed DNA fragments (mono and
oligonucleosomes) in conditioned media (necrosis) and cell extracts
(apoptosis) using specific ELISA assays as described by the supplier.
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Results |
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To determine whether p33/30 was present in the extracellular space, intact C2C12 cell monolayers on various days of differentiation were incubated with a heparin solution. The solubilized material was analyzed by [35S]heparin ligand blot assay. As shown in Fig. 1B heparin was able to solubilize p33/30 from intact cells. In control experiments CK, LDH and DNA were poorly detected in the heparin extracts under these conditions (Table 1), demonstrating that the permeability of the cell monolayers was unaffected by heparin treatment. Furthermore, as shown in Fig. 1B, the binding of [35S]heparin to heparin-solubilized p33/30 increased during skeletal muscle differentiation. These data strongly suggest that p33/30 binds to heparan sulfate, is present on the cell surface or in the extracellular space of muscle cell cultures, and increases during differentiation.
|
p33/30 is histone H1
In order to purify p33/30, TX-100/KCl-soluble proteins obtained from
differentiated myotubes were subjected to heparin-agarose chromatography.
Proteins were eluted by a linear NaCl gradient, analyzed by SDS-PAGE and
stained with Coomassie blue. Fig.
2A shows a 30 and 33 kDa doublet that was almost completely
retained by the column and was eluted between 0.55 and 0.75 M NaCl. The
identity of this p33/30 doublet was determined by MALDI-MS analysis of heparin
affinity-purified fractions. The highest probability scores were obtained with
histone H1 from different species (data not shown). Moreover, N-terminal
sequencing of an HPLC-purified tryptic peptide from p33 showed highest
identity with different subclasses of histone H1 from at least four species
(Fig. 2B). This identification
was confirmed by western blot analysis using an anti-histone H1 monoclonal
antibody. As shown in Fig. 2C,
an immunoreactive 33/30 kDa doublet was detected in the fractions shown above
to contain p33/30 by the [35S]heparin ligand blot assay. The high
specificity of the antibody for histone H1 was confirmed by specific staining
of a nuclear-acid-soluble protein fraction containing nuclear skeletal muscle
histones (Fig. 2C).
Furthermore, like p33/30, histone H1 was specifically solubilized from
myoblasts by heparin, unlike other GAGs
(Fig. 2D). Collectively, these
data allow the identification of p33/30 as a subclass of histone H1.
|
Extracellular histone H1 binds specifically to perlecan in skeletal
muscle ECM
To study a possible interaction between histone H1 and heparan sulfate
proteoglycan(s), a solid-phase assay was performed by incubating a partially
purified total proteoglycan fraction obtained from C2C12
myotube-conditioned media with immobilized histone H1. Total, unbound and
histone-H1-bound proteoglycans were analyzed by western blot using
anti--heparan sulfate monoclonal antibody, which recognizes heparan
sulfate proteoglycan core proteins after heparitinase treatment
(Fig. 3A), and a polyclonal
anti-perlecan-specific antibody (Fig.
3B). Although several core proteins of heparan sulfate
proteoglycans, including glypican, were detected with the anti-
-heparan
sulfate antibody in the total sample, only perlecan was observed to bind to
the immobilized histone H1. In a negative control experiment, heparan sulfate
proteoglycans did not bind to a blank nitrocellulose sheet (data not show).
These in vitro data strongly suggest that histone H1 binds specifically to
perlecan.
|
To evaluate the extracellular localization of histone H1 in skeletal muscle cells cultured in vitro, dual immunofluorescent staining was performed on non-permeabilized C2C12 myotubes. As shown in Fig. 4 (left and middle panels), anti-histone H1 antibody (H1) produced an extracellular filamentous staining pattern that colocalized extensively with the heparan sulfate proteoglycans perlecan (per) and glypican (gly). Interestingly, as is the case with perlecan, the extracellular histone H1 distribution was restricted to the ECM in myotubes, whereas glypican was also present on the plasma membrane. Control experiments in permeabilized myotubes showed, as expected, anti-histone H1 antibody staining primarily in cell nuclei (pH1).
|
To investigate the relation between heparan sulfate proteoglycans present
in the ECM and the extracellular localization of histone H1,
C2C12 cultures were treated with sodium chlorate, a
metabolic inhibitor of heparan sulfate synthesis
(Fig. 4, right panel). Chlorate
treatment resulted in fewer and shorter myotubes and a strong disorganization
of the ECM (Melo et al.,
1996), as revealed by phase-contrast microscopy as well as
anti-perlecan (per) and anti-glypican (data not shown) staining. Under these
conditions, the immunoreactivity of extracellular histone H1 was strongly
diminished, suggesting that heparan sulfate proteoglycans are essential for
its extracellular localization in the ECM.
The extracellular localization of histone H1 was evaluated in hamster skeletal muscle that had been induced to regenerate after barium chloride injection. As observed by hematoxilyn/eosin staining (Fig. 5A), four days after the injection, abundant mononucleated cells probably both inflammatory and muscle precursor cells were encountered together with degenerating myofibers and remnants of the original basement membranes. Newly formed centrally nucleated myotubes could be observed at this stage, but they were more evident and abundant five days after the injection. Fig. 5B shows that in normal adult skeletal muscle (ctrl) only nuclear histone H1 staining was observed. However, in regenerating muscle at day four after barium chloride injection, a pool of extracellular histone H1 was detected in a filamentous pattern between myofibers and mononucleated cells (H1, insert) that does not colocalize with nuclei stained with Hoechst 33258 (Ho, insert). Histone H1 extracellular staining colocalized with anti-perlecan antibody staining in the basement membrane of degenerated fibers (per, insert). At the fifth day of regeneration, extracellular histone H1 staining was weaker and was found mostly associated with the basal lamina surrounding the newly formed regenerating myotubes, where again it colocalized with perlecan. Similar myotubes can be detected by indirect immunofluorescence anti-embryonic myosin (em), a transient early skeletal muscle differentiation marker in similar regenerating foci of adjacent cryosections. No staining was observed with this antibody in control skeletal muscle (data not shown).
|
Taken together, these results demonstrate that a pool of extracellular histone H1 binds specifically to perlecan in skeletal muscle ECM.
Cell death may be a source of histone H1 in the ECM
To determine whether cell lysis could account for the presence of histone
H1 in the ECM, LDH activity was assayed in conditioned media collected over 2
day intervals following induction of cell differentiation.
Fig. 6A shows that LDH was
released into the medium during the 6 days after induction of differentiation,
with the highest level of LDH activity detected within the first two days. In
these experiments, necrotic and apoptotic cell death were also evaluated using
ELISA assays. As shown in Fig.
6B, necrosis was the main cause of cell death and showed a similar
temporal pattern to LDH release during differentiation, whereas very little
apoptotic cell death was detected.
|
The cumulative LDH release, calculated using the data in Fig. 6A (Fig. 6A, insert), closely paralleled the accumulation of extracellular heparin-displaced histone H1 detected during the differentiation of C2C12 cells (Fig. 6C, Fig. 1B). These findings support the possibility of cell death as the source of the pool of histone H1 in the ECM of skeletal muscle cells.
Histone H1 stimulates the proliferation of myoblasts
To study the possible functions of extracellular histone H1, myoblasts were
plated on dishes coated with histone H1 purified from myoblasts by
heparin-affinity chromatography. Myoblast attachment and spreading was
monitored by phase contrast microscopy.
Fig. 7A (upper panel) shows
that 2 hours after plating myoblasts on histone H1-coated dishes assumed a
flattened polygonal morphology characteristic of C2C12
myoblasts. For comparison, laminin, fibronectin and BSA were also used as
attachment substrates. Myoblasts were well spread on laminin but not BSA.
Interestingly, 21 hours after plating (Fig.
7A, lower panel), there was a large increase in the number of
cells on histone-H1-containing dishes. Laminin showed a similar apparent
increase in cell number, whereas fibronectin and BSA failed to produce such an
effect. This result led us to examine a possible role for extracellular
histone H1 in myoblast proliferation.
|
Cell proliferation, assayed by measuring [3H]thymidine incorporation, was seen to increase upon addition of exogenous histone H1 in a time- and concentration-dependent manner, reaching a maximal value at 10 µg/ml (Fig. 7B,C). To evaluate the involvement of heparan-sulfate-like molecules in the proliferative effect observed for histone H1, myoblasts were incubated with increasing concentrations of histone H1 in the absence or presence of heparin. Fig. 7D shows that histone H1-dependent [3H]thymidine incorporation was increased in cells that were also treated with heparin. Moreover, the incorporation of [3H]thymidine induced by histone H1 was almost completely abolished in cells treated with sodium chlorate (data not shown). To directly evaluate the function of histone H1 externalized by C2C12 cells, [3H]thymidine incorporation was determined on myoblasts seeded on myotube ECM, which were prepared by detaching the myotube monolayer. This histone H1-containing ECM (data not shown) was previously incubated with anti-histone H1 (C17) and the antibody was maintained during the assay. As a control, an unrelated anti-ß galactosidase antibody was also used. Anti-histone H1 antibody caused a 40% reduction of the proliferative effect induced by the myotube ECM (Fig. 7E). Collectively, these results strongly suggest that endogenously released histone H1, which is bound to the ECM, stimulates myoblast proliferation via a mechanism dependent on heparan sulfate chains.
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Discussion |
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Previous studies have shown that nucleosome-complexed core histones, namely
H2A, H2B, H3 and H4 (Schmiedeke et al.,
1989) as well as histone-like proteins
(Kohnke-Godt and Gabius,
1991
), may bind to basement membranes and particularly to cell
surface heparan sulfate proteoglycans
(Watson et al., 1999
).
Extracellular nucleosomes have been linked either to the formation of
auto-antibodies in the pathogenesis of systemic lupus erythematosus
(Schmiedeke et al., 1989
) or
the stimulation of the synthesis of immunoglobulins and other immunomodulatory
factors (Emlen et al.,
1992
).
Our data unequivocally demonstrate that the extracellular 33/30 kDa doublet, which was displaced from intact cell monolayers by heparin, is a subclass of histone H1, as determined by MALDI-MS, N-terminal sequencing and immunological analyses. During this investigation, extracellular histone H1 was never found complexed to DNA or other core histones.
Histone H1 is expressed in a wide variety of isoforms
(Schulze et al., 1994) and
shows different degrees of post-translational modification
(Spencer and Davie, 1999
).
Nuclear histone H1 functions not only as a structural protein but also acts as
a positive and negative regulator of gene transcription
(Wolffe et al., 1997
). Indeed,
genes controlling muscle differentiation are known to be selectively repressed
by histone H1 (Steinbach et al.,
1997
). Cytoplasmic pools of histone H1 have also been described in
mammalian cells (Zlatanova et al.,
1990
), and an extracellular histone H1 was identified at the cell
surface of a macrophage cell line, where it acts as a thyroglobulin-binding
protein that mediates its endocytosis via a mechanism that is inhibited by
heparin (Brix et al., 1998
).
Moreover, addition of histone H1 to different cell types has been correlated
with cell division (Kundahl et al.,
1981
), differentiation
(Okabe-Kado et al., 1981
) and
cytotoxicity (Class et al.,
1996
). These studies have led to the notion that histone H1 may
act as a multifunctional protein, which is consistent with the findings
described in this study.
The present data suggest that among several GAGs, histone H1 binds specifically to heparan sulfate chains. This interaction seems to be dependent on GAG sulfation, given that N-desulfated heparin failed to solubilize histone H1 from myoblasts. More specifically, a biochemical approach showed that, among the whole population of heparan sulfate proteoglycans externalized by myotubes, histone H1 exclusively binds to perlecan. Immunohistological studies in non-permeabilized myotubes showed that histone H1 colocalizes extensively with perlecan in the ECM, even though glypican is also present on the cell surface. Similarly, a filamentous pattern of extracellular histone H1 was observed in skeletal muscle regeneration foci. This pattern was also observed for the immunostaining of heparan sulfate proteoglycan core proteins (data not shown), and the specific colocalization of histone H1 with perlecan suggests that it is deposited in the ECM of regenerating skeletal muscle.
The specific interaction of histone H1 with perlecan is an intriguing
finding given that many extracellular ligands bind to the heparan sulfate
chains of different proteoglycans with similar affinities
(Bernfield et al., 1999;
Tumova et al., 2000
).
Moreover, we have demonstrated the presence of at least perlecan and glypican
in the ECM of differentiating myotubes
(Brandan et al., 1996
;
Larraín et al., 1997a
).
However, there is an increasing body of evidence that suggests a role for
specific heparan sulfate sequence motifs in ligand specificity (for a review,
see Turnbull et al., 2001
).
Alternatively, the expression of multiple isoforms of perlecan
(Iozzo, 1998
) might influence
its specific binding to histone H1.
The results of this study point to a functional role for extracellular
histone H1 in cell proliferation. We observed a five- to seven-fold increase
in [3H]thymidine incorporation in histone-H1-treated muscle cells,
which was correlated with an increase in cell number. Moreover, an
anti-histone H1 antibody caused a 40% reduction of the proliferation induced
by the ECM obtained from differentiated skeletal muscle cells. Heparan sulfate
proteoglycans have been extensively characterized as co-receptors of mitogenic
factors, with the FGF-heparan sulfate interaction constituting an archetype
example (Bernfield et al.,
1999). We have previously demonstrated that the myogenic
inhibitory activity of FGF-2 is potentiated by the transmembrane heparan
sulfate proteoglycans syndecan-1 and -3
(Fuentealba et al., 1999
;
Larraín et al., 1998
).
Extracellularly occurring heparan sulfate proteoglycans such as perlecan or
shed syndecan-1 may have the ability to inhibit signaling by sequestering the
growth factor (Aviezer et al.,
1994b
; Kato et al.,
1998
). Nevertheless, perlecan has also been described as a
positive regulator of FGF-2 activity in some conditions
(Aviezer et al., 1994a
),
suggesting that cellular context may be important in determining functional
roles.
As a potential mechanism for the observed effect of histone H1 on myoblast
proliferation, the entry of extracellular histone H1 to the nucleus, which can
be increased by the addition of heparin
(Villeponteau, 1992), has been
shown previously to generate new initiation sites for DNA replication
(Kundahl et al., 1981
).
Although our results do not provide direct evidence, it is tempting to suggest
that heparan sulfate may physiologically mediate the internalization of
histone H1, as heparin addition was found to increase histone-H1-induced
myoblast proliferation. In further support of this hypothesis, perlecan has
been reported to mediate the internalization of different ligands
(Fuki et al., 2000
), in
addition to its localization on the surface of proliferative myoblasts
(Larraín et al.,
1997a
).
On the basis of the present novel data, we suggest that the presence of
histone H1 in the ECM may play a role in muscle physiology. The regenerative
capacity of skeletal muscle arises mainly from the activation of a small
population of quiescent mononucleated cells, called satellite cells, located
between the basal lamina and the sarcolemma of mature muscle fibers
(Mauro, 1961). After injury,
satellite cells, from which C2C12 cells are derived
(Andres and Walsh, 1996
),
proliferate and fuse in response to extracellular signals in order to
regenerate the damaged muscle fibers. Several well-known heparin-binding
mitogenic factors, such as FGF-2, are released from muscle cells following
injury (Clarke et al., 1993
),
and here we provide evidence suggesting that cell death may be involved in
histone H1 externalization. Infiltrating leukocytes accumulate early at the
site of damage and phagocytose the necrotic tissue. However, empty muscle
fiber basement membrane structures and specific molecular components,
including type IV collagen, laminin and heparan sulfate proteoglycan, are
initially preserved (Gulati et al.,
1983
) and are repopulated by proliferating myoblasts. The
persistence of basement membranes has been shown to accelerate muscle
regeneration (Caldwell et al.,
1990
) and favor the reinnervation at original synaptic sites
(Sanes et al., 1978
). In this
context, our results support the idea that after damage histone H1 may act as
an externalized growth signal in regenerating muscle that can be stored by
heparan sulfate proteoglycan ECM components, such as perlecan, to subsequently
induce the proliferation of satellite cells. Indeed, during inflammatory
processes, active growth-factorheparan-sulfate complexes have been
shown to be released from ECM via controlled proteolytic cleavage
(Benezra et al., 1993
;
Whitelock et al., 1996
).
In summary, our results demonstrate that histone H1 localizes to the ECM of cultured muscle cells and in regenerating skeletal muscle, where it binds specifically to perlecan. Our results also suggest that extracellular histone H1 may stimulate the proliferation of satellite myoblasts during skeletal muscle regeneration.
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Acknowledgments |
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