Center for Cellular and Molecular Biology, Hyderabad 500 007 India
* Address for correspondence (e-mail: jdhawan{at}gene.ccmbindia.org )
Accepted 11 April 2002
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Summary |
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Key words: Synchronized C2C12 myoblasts, Satellite cell, LIX, TTP
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Introduction |
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Quiescent MPC, known as satellite cells (SC), persist within adult skeletal
muscle tissue (Mauro, 1961)
and facilitate its regeneration after damage (reviewed in
Grounds, 1991
;
Bischoff, 1994
;
Seale and Rudnicki, 2000
). SC
lie sequestered between the basal lamina and plasma membrane of myofibers and
are induced to proliferate when the muscle is injured. Quiescent SC do not
express MRFs, but activated SC express MyoD, Myf5 and Myogenin
(Grounds et al., 1992
;
Cornelison and Wold, 1997
;
Cooper et al., 1999
). Until
recently, molecular studies on SC were impeded by the lack of unambiguous
markers. Owing to their proximity to the myofiber, SC are difficult to
distinguish from peripheral myonuclei and from interstitial mononucleated
cells. Recently, genes such as M-cadherin [M-cad;
(Irintchev et al., 1994
)],
c-met (Cornelison and Wold,
1997
), myocyte nuclear factor
(Garry et al., 1997
),
Pax7 (Seale et al.,
2000
) and CD34
(Beauchamp et al., 2000
), have
been demonstrated to be specifically expressed in SC. Of these, Pax7
is required for the specification of SC
(Seale et al., 2000
),
c-met has been implicated in SC activation
(Tatsumi et al., 1998
) and
CD34 has been suggested to contribute to maintenance of arrest
(Beauchamp et al., 2000
).
Activation of SC occurs when muscle experiences increased workload,
mechanical or toxic injury or genetic defects such as dystrophin deficiency
that lead to myofiber damage (reviewed by
Grounds, 1991;
Seale and Rudnicki, 2000
).
Although the proximal activating factor of SC in vivo has yet to be
identified, hepatocyte growth factor/scatter factor (HGF/SF) is a strong
candidate (Tatsumi et al.,
1998
; Sheehan and Allen,
1999
). However, little is known of early activation events in
SC.
Direct identification of molecular correlates of SC activation in vivo is
complicated by problems of asynchrony and of dilution by the non-SC components
of muscle. SC associated with isolated single myofibers
(Bischoff, 1986) are more
amenable to a molecular analysis of activation
(Cornelison and Wold, 1997
;
Cornelison et al., 2000
;
Beauchamp et al., 2000
). The
first documented molecular transformation in activated SC is the appearance of
a splice variant of CD34 mRNA (Beauchamp et
al., 2000
), the defining marker of hematopoeitic progenitors
(reviewed by Krause et al.,
1996
). Altered splicing of CD34 is detected 3 hours after the
isolation of single fibers from muscle, a process that leads to SC activation.
Induction of MyoD mRNA in activated SC is detected within 6 hours of crush
injury (Grounds et al., 1992
)
or single fiber isolation (Cornelison and
Wold, 1997
; Beauchamp et al.,
2000
).
Although single fiber cultures retain the association between myofiber and
SC, an important feature of the muscle environment, isolated myoblasts provide
cellular homogeneity and large numbers that are advantageous for molecular
analysis. Despite the loss of extrinsic interactions characteristic of the
tissue milieu, cultured cells retain some important intrinsic attributes of
SC. For example, representational difference analysis of proliferating versus
differentiated primary muscle cultures led to the identification of
Pax7 as a gene involved in SC specification
(Seale et al., 2000). However,
to date few genes other than the MRFs have been identified that are
induced during the activation of resting SC.
In this study, we report the identification of two genes that are acutely
induced in response to skeletal muscle injury and are expressed in a spatial
pattern consistent with activated SC. Our strategy employed culture conditions
(Milasincic et al., 1996) that
enabled cell cycle synchrony. Synchronized C2C12 myoblasts share with SC their
core property of reversible arrest without differentiation. Using differential
display PCR (DD PCR), we isolated cDNAs that could be detected during arrest
and activation of synchronized cultures but not during asynchronous growth or
differentiation.
We found that two of the cDNAs, LIX and TTP, are rapidly expressed in
response to muscle injury in vivo and encode molecules implicated in cell-cell
signaling. LIX, a chemokine implicated in damage-induced neutrophil
chemo-attraction (Wuyts et al.,
1996; Wuyts et al.,
1999
; Chandrasekar et al.,
2001
) is expressed in skeletal muscle within 6 hours of injury.
TTP, an RNA-binding protein that regulates cytokine mRNA stability
(Carballo et al., 1998
;
Carballo et al., 2000
), is
dramatically and transiently induced within 30 minutes of injury, before any
other recorded molecular event in this tissue. In injured muscle, LIX and TTP
transcripts appear to be located in mononuclear cells that abut myofibers and
lie beneath the basal lamina sheath, a location reminiscent of SC. Taken
together, our observations support the notion that SC could themselves be an
early source of signaling molecules that play a role in the regeneration of
damaged muscle.
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Materials and Methods |
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DNA synthesis assay
Cells were seeded on cover slips for staining. To cumulatively label S
phase cells, 10 µM BrdU was added to the culture medium for 2-48 hours,
cultures were fixed in cold 70% ethanol, DNA denatured in 2 N HCl, 0.5% Triton
X-100, 0.5% Tween-20 and neutralized with NaBH4 (1 mg/ml). Staining
with anti-BrdU monoclonal antibody (Sigma, 1:500) was detected using a
biotinylated goat anti-mouse secondary antibody (1:200) and the Vectastain ABC
reaction (Vector Labs). Antibodies were diluted in blocking buffer (10% horse
serum, 0.5% Tween 20). Controls excluding primary antibody or BrdU were
negative. The frequency of cells in S was determined after counting three
fields (250 nuclei) per sample using a Zeiss Axioskop equipped with DIC
optics.
Flow cytometry
Cells were fixed in cold 80% ethanol, washed in PBS and incubated in PBS
+1% Triton X100, 50 µg/ml propidium iodide and RNAse (100 µg/ml final)
for 30 minutes at 37°C. 104 cells/sample were analyzed on a
FACStar Plus flow cytometer (Becton Dickenson) using the CellQuest
software.
Western blot analysis
Cell pellets were solubilized in 2xSDS-PAGE sample buffer, total
protein estimated (Biorad protein assay), 100 µg samples separated by 12.5%
SDS-PAGE and transferred to Hybond C (Amersham-Pharmacia). Blots were blocked
in 25 mM Tris-Cl, pH 8.0, 125 mM NaCl, 0.05% Tween 20 (TBST) +5% nonfat dry
milk. Antibodies were diluted in blocking buffer: MyoD polyclonal (Santa Cruz)
1:400; Myf5 polyclonal (Santa Cruz) 1:1000; actin monoclonal (Developmental
Studies Hybridoma Bank) 1:500; desmin polyclonal (Sigma Chemical Co.) 1:500.
Alkaline-phosphatase-conjugated secondary antibodies (anti-rabbit or
anti-mouse, Bangalore Genei) were used at 1:2000. Washes were for 3x15
minutes in TBST. Antibody binding was detected using chemiluminescence
(CDP-Star, Amersham-Pharmacia).
Northern blot analysis
RNA was isolated from cells and tissue using Trizol (Life Technologies,
Inc). 10-20 µg samples were separated in 1% agarose gels containing 2%
HCHO, transferred to Hybond N and immobilized by UV crosslinking. Probes used
were histone H2B (DeLisle et al.,
1983), MyoD (Davis et al.,
1987
), Myf5 (Braun et al.,
1989
), HGF (Bladt et al.,
1995
), c-met (Takayama et al.,
1996
), PEA3 (Taylor et al.,
1997
), muscle creatine kinase (MCK) and ribosomal protein L7 mRNA
[loading control, (Cornelison and Wold,
1997
)]. Probes labeled with [
32P]-dCTP (>3000
Ci/mmol, BRIT, India) by PCR or by random priming of purified inserts were
used at >106 cpm/ml of hybridization solution (7% SDS, 0.5 M
sodium phosphate pH 7.0, 1 mM EDTA). Blots were washed with 1xSSC, 0.1%
SDS and 0.1xSSC, 0.1% SDS at 65°C; for initial screening of DD-PCR
cDNAs (see below) washes were at 60°C. Hybridization was detected either
by autoradiography or on a phosphor imager (Fuji); L-Process and Image Gauge
programs (Fuji) were used to quantify background-subtracted signals.
Differential display PCR analysis
RNA was isolated from growing, arrested and differentiated cultures.
Residual proliferating cells in day 3 myotube cultures were eliminated by
exposure to cytosine arabinoside (10-5 M) for a further 2 days. 0.2
µg of RNA (DNase-treated using MessageClean, GenHunter Corp.) was used for
DD RT-PCR (Liang and Pardee,
1992), with the RNAimage kit (GenHunter Corp.) and
[
33P]-dATP according to manufacturer's instructions.
Purified fragments were cloned into pBS (KS) (Stratagene). The differentially
expressed fragments described in the Results are as follows. CF1 (333 bp) is
the 3'UTR of Matrilin2 (Accession # U69262). CF2 (253 bp) spans the
junction of the coding and 3'UTR regions of Znf216 (Accession #
AF062071). 740 bp of the coding region of Znf216 was amplified from muscle RNA
using primers FZCOD, 5'-AAAATATGGCTCAGGAGAC-3' and RZCOD,
5'-CAAAGGAAAATGGCCATGC-3'. CF3 (333 bp) is the 3'UTR of TTP
(Accession # M57422). A near full-length cDNA of TTP (1.7 kb) was obtained by
RT-PCR from adult skeletal muscle RNA with primers TTP5,
5'-AATACCGCGGTCTCTTCACCAAGGCCATTC-3' and TTP3,
5'-CCCCGCGGTAGCAATATATTAATATATTATAGC-3'. CF4 (419 bp) is the
3'UTR of LIX (Accession # U27267). A near full-length cDNA encoding LIX
(1.4 kb) was amplified from G0 myoblast RNA using primers LIX5,
5'-CACACCTCCTCCAGCATATC-3' and LIX3,
5'-AGACACTATAAGATGTACAGGC-3'.
RT-PCR analysis
Relative levels of CD34 mRNA were determined using RT-PCR. A 442 bp region
common to both CD34 transcripts (exons 4-7) was amplified using primers
described by Beauchamp et al. (Beauchamp et
al., 2000). DNAse-treated RNA samples (2.5 µg) were reverse
transcribed using the Advantage RT-for PCR kit (Clontech). Volumes of RT
product were normalized to generate relatively equal amounts of PCR product
for a control mRNA (L7). Each sample was then assayed in duplicate by RT-PCR
for both CD34 (29 cycles) and L7 (24 cycles), separated on agarose gels, and
bands quantified by Southern hybridization using CD34 and L7-specific probes
and phosphorimager analysis (Fuji).
Freeze injury of muscle in vivo and isolation of tissue
Animals were handled according to the guidelines of the CCMB Institutional
Animal Ethics Committee. Balb/c and C57B1/6 mice, 3 months old, were
anaesthetized by i.p. injection of 2.5% Avertin at a dose of 375 µg/g.
Freeze injury was performed as described previously
(Dhawan et al., 1996
;
Pavlath et al., 1998
).
Briefly, the tibialis anterior (TA) muscle was exposed by a 2 mm incision in
the overlying skin, and a small piece of dry ice was directly applied to the
belly of the muscle for 15 seconds. The skin was sutured and mice were allowed
to recover for varying periods of time (30 minutes to 14 days). Mice were
sacrificed by cervical dislocation, the TA dissected free and frozen
immediately in liquid nitrogen for RNA isolation or in embedding media
(HistoPrep, Fisher Scientific) for histology. 20 µm transverse cryosections
were used for hematoxylin and eosin (HE) staining or in situ
hybridization.
RNA in situ hybridization
Detection of RNA in fresh cryosections of TA muscles was performed as
described previously (Smerdu et al.,
1994), but antisense and sense probes were labeled with
digoxigenin-11-UTP (Roche) with a transcription kit (Stratagene) and detected
with alkaline-phosphatase-coupled anti-digoxigenin antibody (Roche).
Combined RNA in situ hybridization and immunodetection of the basal
lamina
For co-detection of laminin protein and either Pax7 or TTP RNA, the ISH
protocol was modified as follows: protease treatment was reduced to 2 minutes,
the anti-laminin polyclonal (Santa Cruz, 1:500) was included during
anti-digoxigenin antibody incubation and detected with a goat anti-rabbit
secondary antibody conjugated to AlexaFluor488 (1:500, Molecular Probes).
Hybridized antisense RNA was then detected as described and nuclei were
counterstained with Hoechst 33342. Secondary antibody controls showed no
laminar staining.
Image analysis
Autoradiographs and photomicrographs were scanned using a UMAX 3200 scanner
and composites assembled using Adobe Photoshop 5.0.
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Results |
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|
To ascertain the phase of the cell cycle in which C2C12 myoblasts had arrested, we performed a flow cytometric analysis of DNA content (Fig. 1C). By 48 hours in suspension >90% of arrested myoblasts possessed G1 DNA content, and replating for 24 hours in GM (20% FBS) activated >30% of cells into S phase with a proportionate decrease in G1. Since suspended cells possess a G1 DNA content, do not synthesize DNA and re-enter S phase with kinetics consistent with a G0-G1 transition (Fig. 1B), we conclude that anchorage deprivation arrests cloned C2C12 myoblasts in G0.
Arrested C2C12 myoblasts downregulate myogenic regulators and do not
differentiate
During the arrest that accompanies myogenic differentiation, MyoD is
induced and maintained at high levels, but Myf5 is downregulated
(Yoshida et al., 1998;
Kitzmann et al., 1998
).
To determine the status of these MRFs during reversible arrest we used western blot analysis (Fig. 2A). Both MyoD and Myf5 proteins were rapidly suppressed during arrest. During reactivation of growth, both MRFs were induced, although MyoD appeared within 6 hours (mid-G1) and Myf5 by 12-18 hours (late G1/early S). Consistent with their differing roles in myogenesis, MyoD was highly expressed in myotubes, but Myf5 was not. Actin levels varied with alterations in the growth/adhesive state, whereas levels of desmin, a muscle-specific intermediate filament protein did not.
|
To assess the differentiation status of reversibly arrested C2C12 cells we
used northern blot analysis (Fig.
2B). Expression of the cell cycle dependent histone H2B confirmed
efficient arrest and re-activation. Despite a high fusion index (60%),
differentiated cultures expressed the S phase marker as
10% of nuclei
(unfused cells, see Fig. 1A)
incorporate BrdU. Muscle creatine kinase (MCK), a marker of differentiation,
was abundantly expressed in myotubes but not at any time during arrest and
reactivation. MyoD and Myf5 mRNAs were suppressed in arrested cells and
re-activated 6 hours after replating, well before the onset of DNA synthesis
correlating well with the protein expression patterns
(Fig. 2A). Thus, C2C12
myoblasts can be reversibly arrested in G0 in culture without activating the
differentiation program, as observed in quiescent SCs in vivo (reviewed by
Grounds, 1991
;
Seale and Rudnicki, 2000
). The
induction of MRFs in G1 also recapitulates their induction in activated SC
(Grounds et al., 1992
;
Cornelison and Wold,
1997
).
Expression of candidate SC regulators during arrest and
re-activation
To examine the expression of other genes implicated in SC function we used
northern blot analysis (Fig.
3). The c-met receptor, a marker of SC
(Cornelison and Wold, 1997;
Tatsumi et al., 1998
), plays a
role in their activation. As reported previously
(Anastasi et al., 1997
),
transcripts encoding c-met were detected in C2C12 myoblasts and reduced in
myotubes (Fig. 3A). However,
c-met mRNA levels in asynchronous, arrested and activated myoblasts did not
vary. Down-modulation of EGF and FGF receptors has been proposed as a
mechanism for irreversible arrest of differentiated cells
(Clegg et al., 1987
;
Olwin and Hauschka, 1988
).
Retention of c-met expression during reversible arrest may provide an option
for activation when its ligand, HGF, is available.
|
HGF is a mitogen for SC (Allen et al.,
1995; Tatsumi et al.,
1998
) that is also expressed by activated SC
(Jennische et al., 1993
;
Cornelison et al., 2000
). As
with c-met, HGF mRNA has been reported to be expressed by C2C12 myoblasts and
absent in myotubes (Anastasi et al.,
1997
). However, we did not detect HGF transcripts in either
asynchronous or differentiated cultures
(Fig. 3). Surprisingly, we
found that HGF mRNAs were strongly expressed during G0 and early G1 but
suppressed in late G1. Expression of both HGF and c-met transcripts by
synchronized C2C12 myoblasts implies that autocrine mechanisms may contribute
to growth control as suggested previously
(Anastasi et al., 1997
).
However, as suspended C2C12 myoblasts are quiescent despite expression of
transcripts encoding both receptor and ligand, additional controls may be
involved.
SC in uninjured muscle also express M-cadherin (M-cad), a muscle-specific
adhesion molecule (Irintchev et al.,
1994), but not all SC are positive for this marker
(Cornelison et al., 1997
;
Beauchamp et al., 2000
). We
found that M-cad mRNA showed a marked cell cycle dependence, as it was
abrogated in G0, reactivated during G1 and its peak was coincident with the
onset of S phase (Fig. 3A).
We also examined the expression of PEA3, an ets-domain
transcription factor reported in activated SC
(Taylor et al., 1997).
Consistent with its expression in vivo, PEA3 mRNA was not expressed in G0
myoblasts but was induced in G1 (Fig.
3A). Moreover, PEA3 induction was dependent on growth activation
since cells replated in DM did not express this transcript at the levels seen
in GM.
Recently, a marker of hematopoietic stem cells, CD34, has been localized to
SC and by its expression during arrest and early activation, suggested to
regulate the G0-G1 transition (Beauchamp et
al., 2000). Using semi-quantitative RT-PCR, we estimated the
relative levels of CD34 mRNA during reversible arrest
(Fig. 3B). Synchronization in
G0 led to a
five-fold induction of CD34 transcripts relative to the
levels seen in asynchronous C2C12 cells, declining rapidly during the G0-G1
transition but staying above basal levels at S phase. Thus, CD34 also appears
to show cell cycle regulation in culture.
Taken together, these results show that synchronization of C2C12 myoblasts in culture reveals cell cycle dependent regulation of the MRFs and other genes implicated in SC function in vivo.
Isolation of genes induced in synchronized C2C12 myoblasts using
differential display PCR
To isolate other genes induced in synchronized C2C12 cells, DD-PCR was used
to simultaneously compare RNA from asynchronous, arrested and differentiated
cultures. RNA samples were first tested for appropriate expression of markers
of proliferation and differentiation (histone H2B and MCK, respectively) (data
not shown). Fig. 4A shows a
representative gel used for identification and isolation of differentially
expressed cDNA fragments. Of 36 fragments isolated, four cDNAs (CF1-4)
hybridized to single transcripts specifically detected in G0 synchronized
myoblasts but not in either asynchronously growing or differentiated cells
(Fig. 4B-E).
|
Sequence analysis of these cDNA fragments revealed that all four are
100% identical to sequences in GenBank. CF1 is identical to a region of
the 3'UTR of the mRNA coding for Matrilin-2, an extracellular matrix
protein (Deak et al., 1997
).
CF2 spans the junction of the coding and 3'UTR region of the mRNA for
Znf216, a zinc finger protein of unknown function
(Scott et al., 1998
). CF3
aligns completely with a portion of the 3'UTR of the mRNA coding for
TTP, a zinc-finger protein that binds RNA and is implicated in mRNA decay
(Carballo et al., 1998
;
Carballo et al., 2000
). CF4 is
identical to part of the 3'UTR of LIX, a chemokine first isolated from
LPS-stimulated fibroblasts (Smith and
Herschman, 1995
). Interestingly, both LIX and TTP mRNAs contain
multiple AU-rich elements (AREs) characteristic of labile transcripts
(Shaw and Kamen, 1986
).
Dynamic expression of transcripts detected by differential display
PCR products during synchronous activation of G0 C2C12 myoblasts
To examine the expression patterns detected by the cDNAs isolated from
synchronized myoblasts, a time course of cell cycle activation was analyzed by
northern blotting (Fig. 5). DNA
synthesis monitored in parallel confirmed efficient arrest and activation (not
shown). LIX mRNA was not detected in either asynchronous or differentiating
cells. Although upregulated during G0, activation into G1 led to further
induction of LIX transcripts after 2 hours followed by a rapid extinction,
consistent with the behavior of labile ARE-containing mRNAs of immediate early
genes (Chen and Shyu,
1994).
|
As with LIX, TTP mRNA was only detected in synchronized cultures. Although induced during G0, maximum levels of TTP transcripts were detected at 1 hour after activation, followed by suppression later in G1. The rapid extinction of both LIX and TTP transcripts in activated C2C12 myoblasts coupled with the presence of AREs led us to examine the effect of translation inhibition on their expression. Cycloheximide treatment of replated cells led to a marked stabilization of both LIX and TTP transcripts (data not shown), suggesting that rapid turnover may account for their suppression in G1.
Matrilin-2 mRNA showed induction and decay kinetics similar to TTP in arrested and activated myoblasts. Differences in expression of Znf216 mRNA among asynchronous, arrested and differentiated cultures were less than that of LIX, TTP and Matrilin-2, and though induced after replating, Znf216 transcript levels declined with slower kinetics. Thus, all four transcripts identified by synchronization of C2C12 myoblasts were induced during cell cycle activation in vitro.
LIX and TTP are rapidly and transiently induced in response to muscle
injury in vivo
To determine whether the cDNAs we identified are expressed during
regeneration, the tibialis anterior (TA) muscle of adult mice was subjected to
focal injury and analyzed for molecular and histological changes. Freeze
injury reproducibly leads to the degeneration and regeneration of 20-30% of
the cross sectional area of the TA. We monitored histological changes by HE
staining of muscle sections (Fig.
6). Damage is rapidly followed by inflammatory cell infiltration
within hours, macrophage activity and MPC proliferation peaking at 2-3 days,
appearance of new myotubes by 3 days and their maturation over the next 2
weeks into centrally nucleated regenerated myofibres
(Pavlath et al., 1998).
|
RNA isolated from time courses of induced regeneration in adult C57B1/6 and
Balb/c mice was analyzed by northern blotting
(Fig. 7). Matrilin-2 mRNA was
not detected in uninjured muscle or during regeneration. Znf216 mRNA was
expressed at high levels in uninjured muscle but did not fluctuate markedly
during regeneration. By contrast, LIX and TTP mRNAs were not detected in
uninjured muscle but were rapidly and transiently induced in response to
injury. Expression of LIX was activated at 6 hours and remained high until 3
days post injury (PI), a time when proliferation in the recovering tissue is
maximal, as shown by histone H2B expression. Expression of TTP was seen within
30 minutes of injury followed by an acute downregulation between 3 and 6 hours
PI. The timing of induction of TTP RNA in damaged muscle precedes that of
MyoD, the first MRF expressed in activated SC
(Grounds, 1992;
Cooper et al., 1999
). Although
their expression in G0 myoblasts was not mirrored by uninjured muscle, the
rapid and transient induction of both LIX and TTP during growth stimulation
occurred similarly in vitro and in vivo. Thus, two of the four cDNAs isolated
from synchronized C2C12 myoblasts in culture are part of the acute response to
muscle damage in vivo. Interestingly, both are labile mRNAs encoding molecules
implicated in inflammatory processes.
|
LIX and TTP transcripts show a spatial distribution similar to MyoD
in injured muscle
To identify the cellular source of the mRNAs detected in regenerating
muscle we used RNA in situ hybridization. LIX transcripts localized to a few
mononucleated cells at 6 hours PI, with no signal in the myofiber cytoplasm
(Fig. 8A). As expected from
northern analysis, LIX transcripts were not detected in uninjured muscle
(Fig. 8a). The timing and
distribution of LIX mRNA are similar to that of MyoD mRNA
(Fig. 8B), which is expressed
only in activated SC of damaged muscle at this time
(Grounds et al., 1992)
(reviewed in Seale and Rudnicki,
2000
). As with LIX, TTP mRNA was not detected in uninjured muscle
(Fig. 8c). However, at 2 hours
after injury, approximately one third of the cross-sectional area of the TA
muscle was dotted with TTP-positive mononucleated cells
(Fig. 8C,D).
|
TTP transcripts are associated with presumptive satellite cells in
injured muscle
Prior to their division following activation, SC contain sparse cytoplasm
surrounding a condensed nucleus (Bischoff,
1994). To determine if TTP transcripts co-localized with nuclei,
sections were counter-stained with Hoechst 33342 following RNA in situ
hybridization. As seen in Fig.
9, the hybridization signals localize to a subset of nuclei at the
myofiber periphery. The absence of signal away from these nuclei suggests that
the transcripts are physically constrained from diffusing into the myofiber
interior, consistent with their location in mononucleated cells and
distinguishing them from myonuclei.
|
To determine if TTP-positive mononucleated cells are found in a sublaminar rather than an interstitial location, we combined RNA in situ hybridization with immunodetection of the basal lamina (Fig. 10). TTP-positive cells were found beneath a laminin sheath at the myofiber periphery, as were cells expressing the SC marker Pax7. Taken together, these results suggest that TTP-positive cells are mononucleated and located below the basal lamina, features typical of SC.
|
Since neither LIX nor TTP is muscle specific, we cannot rule out their expression by mononucleated cells in addition to presumptive SC. However, along with their expression in cultured myoblasts, the timing and location of LIX and TTP mRNAs in vivo suggests that activated SC are among the mononucleated cells that express these transcripts in response to muscle injury.
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Discussion |
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Stringent growth control in synchronized C2C12 myoblasts
In non-tumorigenic cells, loss of adhesion triggers arrest despite the
presence of mitogens (Benecke et al.,
1978; Dike and Farmer,
1988
; Milasincic et al.,
1996
). Anchorage-dependence of proliferation is the best in vitro
correlate of strict growth control
(Freedman and Shin, 1974
).
Therefore, we used adhesion-dependent subclones of C2C12 myoblasts to analyze
synchronous cell cycle activation. Two lines of evidence suggest that cells
arrest in G0. Firstly, FACS analysis shows that >90% of cells arrest with a
2 N DNA content. Secondly, the kinetics of return to S phase are consistent
with the correspondence of the lag period to the G0-G1 transition.
MRF expression is suppressed in G0
Differentiation is accompanied by MRF expression and irreversible cell
cycle exit (Andres and Walsh,
1996). By contrast, in quiescent SC, neither MyoD nor Myf5 are
expressed (Grounds et al.,
1992
; Cornelison and Wold,
1997
; Cooper et al.,
1999
), suggesting that MRF expression is incompatible with
reversible arrest. Similarly, we find that expression of MyoD and Myf5
proteins is extinguished in suspension-arrested C2C12 myoblasts as in
`reserve' cells of differentiating cultures
(Yoshida et al., 1998
). MyoD
inhibits the cell cycle independently of its myogenic activity
(Crescenzi et al., 1990
), but
as it is not detected in G0 myoblasts, suspension-arrest may be independent of
this MRF. Indeed, absence of MyoD in G0 may be necessary for arrest to be
reversible (Yoshida et al.,
1998
), and absence of the cyclindependent kinase inhibitor p21, a
target of MyoD during irreversible arrest
(Halevy et al., 1995
;
Guo et al., 1995
), in G0
arrested cells supports this hypothesis (J.D., unpublished). Unlike resting SC
in vivo, C2C12 myoblasts induced to enter G0 continue to express desmin,
perhaps reflecting the greater stability of this cytoskeletal protein relative
to the labile transcription factors MyoD and Myf5.
Cell cycle dependent expression of candidate SC regulators in
synchronized C2C12 myoblasts
Several genes detected in SC have been implicated in muscle regeneration
(reviewed by Seale and Rudnicki,
2000). Conceivably, genes involved in SC activation may be cell
cycle regulated, but genes specifying SC identity may not. Consistent with
this idea, MyoD is induced during SC activation in vivo
(Grounds et al., 1992
;
Cornelison and Wold, 1997
) and
is cell cycle responsive in culture
(Kitzmann et al., 1998
). By
contrast, both resting and activated SC express Pax7
(Seale et al., 2000
),
suggesting that this specification factor may not be cell cycle regulated.
Myf5 expression during G0 in C2C12 myoblasts
(Kitzmann et al., 1998
;
Yoshida et al., 1998
), in SC
on single fibers (Beauchamp et al.,
2000
; Cornelison and Wold,
1997
; Cornelison et al.,
2000
) and quiescent SC in vivo
(Cooper et al., 1999
;
Beauchamp et al., 2000
) remains
controversial. In adhesion-dependent arrest, we find Myf5 to be absent in G0,
but activated during G1, consistent with previous observations
(Yoshida et al., 1998
;
Cornelison and Wold, 1997
;
Cooper et al., 1999
).
The regulation of other genes implicated in SC function has not been previously analyzed in synchronized myoblasts. Our data show that HGF, PEA-3, M-Cad and CD34, are all cell cycle dependent in culture.
The c-met receptor and its ligand HGF play a key role in SC activation. In
uninjured muscle, whereas c-met has been detected on SC, HGF is found at the
myofiber periphery (Tatsumi et al.,
1998). Immunodepletion of HGF from crushed muscle extracts results
in a loss of SC mitogenic activity
(Tatsumi et al., 1998
). Thus,
it is thought that HGF sequestered in the ECM is released by damage and
stimulates resting SC in a paracrine fashion. HGF mRNA is not detected in SC
until after their activation (Jennische et
al., 1993
; Cornelison et al.,
2000
) and may serve to amplify the activating signal. Both c-met
and HGF transcripts were detected in asynchronous C2 myoblasts, suggesting
that autocrine activation may also play a role
(Anastasi et al., 1997
). In
synchronized cultures, we find that c-met transcripts are maintained in G0 as
well, raising the possibility that quiescent cells retain responsiveness to a
ligand whose expression/activity is regulated. Strikingly, HGF transcripts are
only detected during G0 and early G1. Although we have not assessed the levels
of HGF protein, it must be absent or inactive in arrested myoblasts, as
autocrine/paracrine effects would be readily detected as BrdU-positive
cells.
The transcription factor PEA3 is expressed by activated SC but not by
resting SC (Taylor et al.,
1997). The rapid induction of PEA3 during cell cycle re-entry of
G0 C2C12 myoblasts suggests that the activation process in vivo and in culture
show some similarities in gene expression.
M-Cad expression by SC in vivo is heterogeneous: 20% of SC in uninjured
muscle do not express either M-cad or CD34 and are proposed to comprise a
minor stem-cell-like compartment that gives rise to the lineage-restricted
marker-positive majority (Beauchamp et al.,
2000). Further, whereas <20% of SC on freshly isolated single
fibers are M-cad positive, 100% are positive after 96 hours, consistent with
an induction of M-cad in activated SC
(Cornelison and Wold, 1997
).
In this context, it is interesting that we find suppression of M-cad mRNA in
G0 myoblasts and its reactivation during G1 in culture. Heterogeneity of M-Cad
expression in vivo may also reflect cell cycle position.
CD34, a marker of hematopoeitic progenitors is routinely used for their
clinical isolation for transplantation (reviewed by
Krause et al., 1996). Although
the function of this cell surface glycoprotein is obscure, its presence on
dermal (Nickoloff, 1991
),
liver (Omori et al., 1997
) and
muscle precursors (Beauchamp et al.,
2000
) in the adult strongly suggests a role in regeneration. Our
data demonstrate that expression of CD34 mRNA is high in G0 and downregulated
in activated C2C12 myoblasts, supporting the suggestion
(Beauchamp et al., 2000
) that
it is regulated at the G0/G1 transition.
Differential display PCR reveals genes expressed specifically in
synchronized C2C12 myoblasts
Since reversible arrest is at the core of SC function, we used synchronized
cultures to search for cDNAs expressed during arrest and activation but not in
differentiated cells. Interestingly, all four transcripts identified (LIX,
TTP, Matrilin-2 and Znf216) are further induced during activation of arrested
myoblasts, but as with other early response genes, the induction is short
lived. The lack of expression of these genes at early times in suspension
suggests that they are induced as a consequence of cell cycle synchrony caused
by prolonged suspension culture and not as a stress response to non-adherent
conditions. Our screen was not designed to isolate muscle-specific factors but
cell cycle dependent genes, and those we identified show a wide tissue
distribution, but are rapidly induced in response to a number of
growth-activating stimuli (Varnum et al.,
1989; Smith and Herschman,
1995
). Such genes might also be expected to display cell cycle
dependent expression during SC activation in vivo.
Both LIX and TTP transcripts contain multiple instability elements (AREs),
hallmarks of labile cytokine and oncogene mRNAs (reviewed by
Chen and Shyu, 1995). Such
transcripts are stabilized by the inhibition of translation, and indeed both
LIX and TTP mRNAs are stable in cycloheximide-treated cells (C.S.,
unpublished). The pronounced suppression of protein synthesis accompanying
quiescence (Benecke et al.,
1980
) may account for their induction in suspension. Since stable
ARE-positive mRNAs are often not translated
(Chen and Shyu, 1995
), LIX and
TTP proteins may not be synthesized in arrested cells despite the presence of
their mRNAs. As translation is dramatically stimulated during the G0-G1
transition (J. Dhawan, PhD thesis, Boston University, 1991), a burst of
synthesis of LIX and TTP proteins may precede the decay of both transcripts
after activation into G1.
LIX and TTP are induced in response to muscle damage in vivo
Regeneration of damaged muscle is a complex process involving many cell
types and the interplay of a number of growth factors, cytokines, chemokines,
extracellular matrix components and signaling molecules (reviewed by
Grounds, 1991;
Seale and Rudnicki, 2000
).
Uninjured muscle containing quiescent SC is devoid of LIX transcripts, in
contrast to quiescent C2C12 myoblasts in culture. 6 hours after injury, LIX
mRNA is strongly induced and peaks at 48 hours, prior to the peak of
proliferation in SC (and infiltrating cells). LIX was first isolated in a
screen for LPS-inducible glucocorticoid-attenuated genes in fibroblasts
(Smith and Herschman, 1995
).
This small, secreted protein contains a CXC motif preceded by an ELR sequence
characteristic of chemokines with neutrophil-attracting activity
(Wuyts et al., 1998
). LIX is
also induced during injury of the spinal cord
(McTigue et al., 1998
) and
injury of cardiac myocytes in vivo and is a potent neutrophil chemoattractant
(Chandrasekhar et al., 2001
).
In muscle, neutrophils enter a lesion 1 to 3 hours after damage
(Orimo et al., 1991
), before
LIX is detected, but this chemokine could contribute to the massive
inflammatory influx seen 6 to 24 hours PI. The presence of LIX mRNA at 3 days,
after neutrophils have withdrawn from the site of injury
(Orimo et al., 1991
), may
suggest that LIX plays additional roles. CXC R2, a receptor of ELR-positive
chemokines has been found on endothelial cells
(Addison et al., 2000
),
implicating these proteins in angiogenesis and raising the possibility that
LIX promotes revascularization during tissue repair. Cytokines released by
inflammatory leukocytes have been proposed to activate SC
(Jesse et al., 1998
).
Conversely, regulators of inflammation such as LIX and TTP expressed by
activated SC may affect the function of infiltrating and resident cells in
regenerating muscle.
As with LIX, TTP RNA is not detected in uninjured muscle. Although uninjured muscle may contain rare activated SC, the transient expression of these early response genes may preclude their detection. Injury induces a dramatic increase of this transcript within 30 minutes, but the induction is transient and TTP is undetectable by 6 hours after damage. In situ hybridization shows that TTP transcripts are closely associated with a few nuclei at the myofiber margin and not with the myofibre interior. This sequestration is consistent with a barrier to diffusion such as the presence of a membrane and suggests that TTP transcripts are harbored in mononucleated cells at the myofiber periphery, thereby distinguishing them from myonuclei. Further, the visualization of TTP-positive cells beneath the basal lamina in a location similar to Pax7-positive cells distinguishes these cells from interstitial cells. In addition, the frequency of TTP positive cells at 3 hours is similar to that of MyoD positive cells at 6 hours. Taken together, these data support the identification of TTP positive cells as SC.
The acute expression kinetics of TTP suggest that this zinc finger protein
may perform a critical function in muscle immediately after injury. The lack
of expression by myofibers suggests a specific early response of SC to muscle
trauma. TTP is known to bind mRNAs encoding tumor necrosis factor (TNF)
and granulocyte-macrophage colony stimulating factor (GM-CSF), triggering
their decay. TTP-/- mice develop severe inflammation and cachexia
(muscle wasting) (Taylor et al.,
1996
), symptoms that are largely attenuated by antibody
neutralization of TNF
. The increased TNF
levels detected 24
hours after muscle injury (Collins and
Grounds, 2001
; Zador et al.,
2001
) have been attributed to infiltrating phagocytes. TTP's early
induction in SC may involve regulation of TNF
expression, but it is
also conceivable that as TTP binds to ARE instability motifs in other
transcripts (Carballo et al.,
2000
), its targets may vary in different cell types. Thus, TTP
could play a novel role in SC. Taken together with its localization, the
induction of TTP expression by injury suggests that this transcript may serve
as a marker for activated SC and could allow quantitative analyses of the
extent of damage and/or repair.
From our observations, myoblast activation in vitro and in vivo appear to involve similar early events such as the induction of TTP and LIX. Identification of TTP in muscle within 30 minutes of damage suggests that SC respond to injury very rapidly. Thus, identification of the factors that control expression of TTP may lead to an understanding of acute signals released in muscle in response to injury. Further, as TTP is known to regulate the half-life of cytokine mRNAs, SC may be involved not only in the generation of new myoblasts but also in cell-cell signaling during injury and repair.
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