From the Max-Planck-Institut für Biochemie, Am
Klopferspitz 18a, D-82152 Martinsried, Germany, ¶ Cellular
Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021, and
Genzentrum der
Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, D-81377
München, Germany and Friedrich-Baur-Institut der
Ludwig-Maximilians-Universität, Ziemssenstr. 1a, D-80336
München, Germany
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ABSTRACT |
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To gain insight into the molecular mechanisms
underlying the wound repair process, we searched for genes that are
regulated by skin injury. Using the differential display reverse
transcription-polymerase chain reaction technique, we identified a gene
that was strongly induced as early as 12 h after wounding.
Sequence analysis revealed the identity of the corresponding protein
with skeletal muscle nascent polypeptide-associated complex (skNAC), a
recently identified muscle-specific transcription factor. By in
situ hybridization and immunohistochemistry, we demonstrated the
specific expression of skNAC in skeletal muscle cells of the panniculus
carnosus at the wound edge. Furthermore, in vitro studies
with cultured myoblasts revealed expression of skNAC in differentiating
and differentiated, but not in proliferating, nondifferentiated cells.
Differentiation of cultured myoblasts was accompanied by simultaneous
expression of skNAC and the muscle-specific transcription factor
myogenin. Our results provide the first evidence for a role of skNAC in muscle repair processes. Furthermore, they demonstrate the usefulness of our approach in identifying new players in wound repair.
After cutaneous injury, a series of biological events take place
that aim at the reconstitution of the damaged skin. Among them are the
migration, proliferation, and differentiation of various cell types,
the production of extracellular matrix, as well as the removal of
irreversibly destructed tissue (1). These processes are well described
at the histological level, but little is known about the molecular basis.
To gain insight into the molecular mechanisms that underlie the repair
process, we used the differential display
RT-PCR1 (DDRT-PCR) technique
(2) to systematically identify genes that are differentially expressed
in wounded compared with normal skin. Because of the migration and
proliferation of various cell types after injury, differences in total
expression levels of a certain gene could be due to variation of the
cellular composition rather than actually reflecting transcriptional or
translational regulation. To minimize this risk, we exclusively
compared normal skin with very early (24 h) wounds, because only minor
changes in the cellular composition occur during this initial repair period.
We obtained several partial cDNA clones, whereby expression of the
corresponding mRNAs was either up- or down-regulated in response to
cutaneous injury. We demonstrate that the levels of skNAC mRNA
increase dramatically as early as 12 h after wounding. "NAC"
is an abbreviation for nascent polypeptide-associated complex, a
protein that crosslinks to nascent chains just emerging from the
ribosome (3) and thereby regulates protein translocation to the
endoplasmic reticulum membrane (4, 5). NAC is an ubiquitously expressed
heterodimeric protein consisting of a 33-kDa We demonstrate a strong expression of skNAC in skeletal muscle cells of
the injured panniculus carnosus at the wound edge. In vitro,
skNAC expression was confined to differentiating and/or differentiated
muscle cells. These data suggest that up-regulation of a
differentiation-associated transcription factor in injured muscle cells
could modulate muscular repair processes after wounding.
Animal Care--
BALB/c mice were obtained from the animal care
facility of the Max Planck Institute of Biochemistry, Martinsried. They
were housed and fed according to federal guidelines, and all procedures were approved by the local government of Bavaria.
Wounding, Preparation of Wound Tissue, and RNA
Isolation--
Three independent wound healing experiments were
performed. For each experiment, 20 female BALB/c mice (8-12 weeks of
age) were anesthetized with a single intraperitoneal injection of
avertin. The hairs on the animals' backs were cut with fine scissors,
and the skin was wiped with 70% ethanol. Six full-thickness excisional wounds (6-mm in diameter, 3-4 mm apart) were generated on the back of
each animal by excising skin and panniculus carnosus. The wounds were
allowed to dry to form a scab. 12 h, 1, 3, 5, and 13 days after
wounding, four animals were sacrificed, and wounds were harvested by
excising an area 7-8-mm in diameter that included the scab and 2 mm of
the epithelial margins. A similar amount of skin from three nonwounded
animals served as a control. The tissue was immediately frozen in
liquid nitrogen and stored at RT-PCR--
5 µg of total cellular RNA was reverse transcribed
using 100 units of Superscript reverse transcriptase (Life
Technologies, Inc., Eggenstein, Germany) and
oligo(dT)12-17 as a primer. Human DDRT-PCR--
DDRT-PCR was carried out essentially as described
(8). Briefly, 300 ng of total RNA from normal murine back skin or from 1-day-old wounds was reverse transcribed using
5'-(dT12CG)-3' (C1) as a 3'-primer in a total reaction
volume of 30 µl. PCR was performed in a total volume of 20 µl using
0.5 µM upstream primer (N1: 5'-d(TACAACCAGG)-3'), 2.5 µM downstream primer (C1), 5 µCi [ RNase Protection Assay--
RNase protection assays were
performed as described (9). 453-bp fragments corresponding to nt
5765-6217 of the mouse skNAC cDNA (6) or the corresponding rat
cDNA served as templates for the generation of
32P-labeled antisense riboprobes. Furthermore, cDNA
fragments from the 5' end of the coding region of murine myogenin (280 nt; primers derived from the rat sequence) (10), murine In Situ Hybridization--
A 187-bp fragment (nt 5371-5557)
specific for the skNAC cDNA was subcloned into pBluescript KSII(+)
(Stratagene) and linearized. Sense and antisense riboprobes were
generated using T3 or T7 RNA polymerases and
[ Immunohistochemistry--
6-µm frozen sections from the middle
of 5-day-old murine excisional wounds were fixed with acetone and
treated for 3 min at room temperature with 1%
H2O2 in PBS to block endogenous peroxidase activity. They were subsequently incubated overnight at 4 °C with affinity-purified polyclonal antisera recognizing human and murine Immunofluorescence--
6-µm frozen sections from the middle
of 5-day-old murine excisional wounds were fixed for 10 min with cold
acetone. Cultured cells were fixed for 10 min with a 1:1 mixture of
cold acetone and methanol. Sections or cells were washed with PBS. They
were incubated overnight at 4 °C with the primary antibody, diluted in PBS containing 12% bovine serum albumin, 0.1% Nonidet P-40, and
0.02% NaN3 (antibody dilution buffer). After washing with PBS at room temperature, sections or cells were incubated with the
secondary antibody and diluted in antibody dilution buffer for 3 h
at room temperature. After washing with PBS, rinsing with water, and
mounting, fluorescence was analyzed with a Zeiss Axioskop microscope.
Tissue Culture--
Mouse C2C12 and rat L6 myoblasts (American
Tissue Culture Collection, 4th to 10th passage) were cultured in
Dulbecco's modified Eagle's medium containing 15% fetal bovine serum
(growth medium). At 90% confluency, they were shifted from growth to
differentiation medium (Dulbecco's modified Eagle's medium containing
2% horse serum). Differentiation medium of C2C12 cells was changed
every 48 h to avoid acidification. The differentiation process was
monitored via analysis of myogenin and Identification of skNAC as a Novel Gene Strongly Induced after Skin
Injury--
To identify genes that are differentially expressed during
the process of cutaneous wound repair, we used the differential display
RT-PCR technique to compare gene expression in normal and wounded
murine skin. A 244-bp PCR product was obtained, which was significantly
more abundant after amplification of cDNA derived from 1-day-old
wounds compared with normal skin. Sequence comparison analysis revealed
that this cDNA fragment corresponded to the Upon Wounding, skNAC Expression Is Induced in Myofibers of the
Panniculus Carnosus Immediately Adjacent to the Wound--
To
determine the site of expression of skNAC in the wound, we performed
in situ hybridization studies using a 187-nt skNAC-specific antisense RNA as a probe. Furthermore, immunohistochemical staining with an affinity-purified,
The skNAC positive cells might represent myotubes or activated
satellite cells. The latter are undifferentiated myogenic precursor cells involved in muscle regeneration. Recently, c-met, the receptor for hepatocyte growth factor, was shown to be a useful marker for
satellite cells (14-16). Therefore, we analyzed expression of this
protein in our murine wounds (Fig. 2C). Indeed, both
Although skNAC Is Dramatically Up-regulated after Injury,
To determine a possible role of skNAC in muscle repair, we compared its
expression with that of the well characterized skeletal muscle-specific
transcription factor myogenin (10) in our wound healing model. Myogenin
controls early differentiation events during development (10) and has
been shown to be one of the first myogenic transcription factors to be
induced in most murine models of muscle repair (17, 18). In injured
skin, myogenin mRNA was first detected 24 h after injury (Fig.
3B), indicating that at least at the mRNA level, skNAC
induction even precedes that of myogenin. Furthermore, myogenin
expression remained high at later stages of the repair process,
reaching maximal levels around day 5 after injury. By contrast, skNAC
mRNA expression rapidly declined and was approximately half-maximal
already 24 h after wounding (Fig. 3A). In addition, as
described in several human models of skeletal muscle injury (19), we
could detect transient expression of
As assessed by double-immunofluorescence staining,
To exclude the possibility that skNAC induction might be caused by an
enrichment of striated muscle cells in early wounds, we analyzed
expression of
The dramatic overexpression of skNAC upon injury suggests a role of the
protein in muscle regeneration. This view is supported by preliminary
data, which indicate that abnormal expression and/or splicing of the
skNAC Is Exclusively Expressed by Differentiating and
Differentiated Muscle Cells in Vitro--
To further study the
regulation of skNAC expression in skeletal muscle cells, we compared
its expression during myoblast differentiation in vitro with
that of myogenin and
The strong induction of skNAC mRNA expression upon differentiation
correlates with data from Yotov and St-Arnaud (6), who demonstrated
exclusive expression of skNAC protein by differentiated mouse C2C12
myotubes but not by undifferentiated myoblasts. Similar results were
obtained with rat L6 cells (Fig. 4) as well as with primary human
myoblasts and myotubes (Fig. 6), suggesting that skNAC induction is a
general phenomenon associated with myoblast differentiation.
In contrast to the differential regulation of skNAC and myogenin
expression during muscle regeneration in vivo, these two factors displayed very similar expression kinetics after induction of
myoblast differentiation in vitro. Therefore, we wondered if the two proteins might be simultaneously expressed in one single cell
under the in vitro conditions. For this purpose we performed double-immunofluorescence labeling with antibodies directed against
In conclusion, our results not only demonstrate the usefulness of our
approach to identify new players in wound repair but also provide the
first evidence for an involvement of skNAC in skeletal muscle
regeneration after mechanical injury. The exclusive expression of skNAC
in differentiating and differentiated muscle cells in vitro
(Ref. 6 and this study) as well as the promotion of myoblast fusion
upon overexpression of skNAC in C2C12 cells (6) suggests a role of this
protein in muscle differentiation in vitro. However, the
early increase in skNAC expression after injury in vivo that
occurs before overt muscle differentiation argues against a role of
skNAC in the in vivo differentiation process itself. By
contrast, skNAC might regulate the expression of genes required for the
onset of the in vivo differentiation program. In addition,
skNAC could be involved in the activation of satellite cells or could
initiate various other processes associated with muscle repair that
remain to be elucidated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
subunit and a 21-kDa
subunit. Furthermore, a skeletal and heart muscle-specific splice
variant of the
subunit, skNAC (skeletal muscle NAC), has recently
been identified. Most interestingly, this form seems to act as a
muscle-specific transcription factor that stimulates myoglobin
expression (6). Overexpression of skNAC in C2C12 myoblasts induced
early fusion of these cells into myosacs, indicating a role of this
protein in muscle differentiation and in the regulation of myoblast
fusion (6).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
70 °C until used for RNA isolation.
Total cellular RNA was isolated as described (7). For
immunohistochemistry/immunofluorescence and in situ
hybridization, the complete wounds were isolated and bisected. One half
of the wound was frozen in tissue freezing medium (Jung, Nussloch,
Germany), sectioned, and used for
immunohistochemistry/immunofluorescence studies. The other half was
fixed overnight in 4% paraformaldehyde in phosphate-buffered saline
(PBS) at 4 °C, transferred for 4 h to 15% sucrose in PBS at
4 °C, and embedded in tissue freezing medium, sectioned, and used
for in situ hybridization studies.
NAC and skNAC
fragments were amplified by PCR. Thirty cycles, consisting of
denaturation at 94 °C for 1 min, annealing at 52 °C for 2 min,
and extension at 72 °C for 1 min, were performed. The following
primers corresponding to the murine sequence were used: 59SPL5'
(5'-d(CCCAGCAGCTTCCCTGACTCTTG)-3'), 59SPL3' (5'-d(CCCAGTTTG GACATAGCCTTCC)-3'), and 59tag5' (5'-d(TTTCCAGCCGATCCTCGTCAA)-3'). A
453-bp fragment specific for skNAC was amplified with primers 59SPL5'
and 59SPL3'; a 279-bp fragment specific for
NAC was amplified with
primers 59tag5' and 59SPL3'. The amplified fragments were separated on
a 1.5% agarose gel and visualized by ethidium bromide staining.
-35S]dATP and 1unit of Taq polymerase
(Perkin Elmer). 40 cycles were performed (30 s at 94 °C, 30 s
at 42 °C, 30 s at 72 °C). Each experiment was repeated at
least twice. PCR products were separated on denaturing 6%
polyacrylamide gels and visualized by autoradiography. Selected bands
were eluted from the gel, reamplified by PCR, cloned into pBluescript
KSII(+) (Stratagene, La Jolla, CA), and sequenced.
cardiac/fetal muscle actin (180 nt) (11), and of murine
skeletal
muscle actin (218 nt) (11) were used.
-35S]UTP. 6-µm frozen sections from the middle of
the wound were hybridized as described (12). After hybridization,
sections were coated with NTB2 nuclear emulsion (Eastman Kodak) and
exposed at 4 °C in the dark for 4 weeks. After development, sections
were counterstained with hematoxylin/eosin.
and skNAC (1:2,000 diluted in PBS, 0.1% bovine serum albumin) (3).
Slides were stained with the avidin-biotin-peroxidase system (Vector
Laboratories, Burlingame, CA) using 3-amino-9-ethyl-carbazole as a
chromogenic substrate. After development, they were rinsed with water,
counterstained with hematoxylin, and mounted.
cardiac/fetal muscle actin
expression. Furthermore, cells were immunocytochemically stained for
expression of skeletal muscle myosin using a polyclonal antiserum
(Sigma). Fluorescence-activated cell sorted primary human fetal
myoblasts were a kind gift of Dr. E.A. Shoubridge (Montreal
Neurological Institute, Canada). They were grown in a supplemented
growth medium as described previously (13).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/skNAC mRNA. As
shown schematically in Fig.
1A, skNAC is characterized by
a large 6.0-kilobase second exon, which is spliced-in between the two
short
NAC exons (exon 1 and exon 3, Fig. 1A).
Surprisingly, our PCR-fragment was not located at the ultimate 3'-end
of the
/skNAC cDNA but approximately 0.5-0.7-kilobase upstream
(Fig. 1A), where a poly(dT) cluster allows annealing of
primer C1 with only four mismatches. Furthermore, although only the
first 10 bp of our fragment (the nucleotides corresponding to primer
N1) were located immediately upstream of the splice site between exon 2 and exon 3 (Fig. 1A), sequence comparison revealed that it
unequivocally corresponded to the skNAC and not to the
NAC
sequence (Fig. 1B).
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Fig. 1.
Alignment of differential display primer N1
with the NAC and skNAC sequences.
A, the position of the 244 bp differential display RT-PCR
fragment within the skNAC cDNA is shown schematically. Exon 2, which is spliced out in the
NAC mRNA, is shown as an open
box. The differential display fragment is shown schematically.
Differential display primers N1 and C1 are shown as small
arrows. The probe used for RNase protection assay overlaps the
second splice site and therefore allows simultaneous detection of
and skNAC transcripts in one batch of RNA. B, nucleotide
sequence of differential display primer N1 compared with the sequence
of murine
NAC and skNAC cDNAs. Primer N1 can be aligned with
only two mismatches to the skNAC but not to the
NAC sequence.
/skNAC-specific polyclonal antiserum was
performed. We obtained a strong in situ hybridization signal as well as a prominent immunohistochemical staining in a certain population of cells below the granulation tissue. The morphology of
these cells suggested them to be muscle cells of the panniculus carnosus, a striated muscle that is located below the dermis of rodents. This hypothesis was supported by the expression of skeletal muscle myosin in these cells, as determined by immunofluorescence staining of serial sections with a polyclonal antiserum against this
protein (data not shown). skNAC transcripts were exclusively detected
in the myofibers of the panniculus carnosus. As shown in Fig.
2A, mRNA levels were very
low in normal striated muscle cells at a distance from the wound.
However, there was a dramatic increase in skNAC mRNA levels in
muscle cells adjacent to the site of injury. Because our antiserum is
directed against a carboxylterminal peptide, we cannot discriminate
between
and skNAC at the protein level. However, the almost
exclusive expression of the skNAC variant in muscle
tissue2 indicates that the
signal seen in these cells results from skNAC expression. With this
antiserum, we obtained a particularly strong staining of the injured
ends of the panniculus carnosus (Fig. 2B). In addition,
apart from the prominent staining of the panniculus carnosus, we found
a weak and diffuse staining of almost every cell present in the wound
(Fig. 2B and data not shown), indicating ubiquitous
expression of the
/
NAC complex.
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Fig. 2.
Localization of skNAC-expressing cells in
healing wounds. Panel A, 6-µm frozen
sections from murine wounds harvested 5 days after injury were
hybridized with a 187-bp skNAC-specific fragment. The signals generated
by the radioactive probe appear as black dots. Note the
strong expression of skNAC mRNA in the injured myofibers at the
wound edge (magnification, × 200). Panel B,
immunohistochemical staining of a 6-µm section from a 5-day-old
murine wound. Positive cells are stained red. A polyclonal,
affinity-purified antiserum directed against /skNAC was used
(magnification, × 400). Panels C and
D, double-immunofluorescence staining of serial sections to
that used for the immunohistochemical staining shown in panel
B. Sections were simultaneously incubated with the polyclonal
affinity-purified
/skNAC antiserum and a polyclonal c-met antiserum.
A fluorescein isothiocyanate-coupled anti-rabbit (
/skNAC positive
cells; green fluorescence) and a Texas red-coupled anti-goat
(c-met positive cells; red fluorescence) secondary antibody
were used. Because there were no areas exclusively with red
fluorescence in the double exposure (D), the Texas red
fluorescence is shown alone (C) (magnification, × 200).
/skNAC and c-met were coexpressed at the injured tips of the
panniculus carnosus below the wound and at the wound edge (Fig.
2D), indicating that skNAC up-regulation might indeed be
characteristic for activated satellite cells. However, because of
limitations in the resolution, it is not possible to unequivocally
determine whether these proteins are indeed expressed within the same cell.
NAC Is
Only Slightly Induced--
Because
and skNAC proteins seem to be
very different in function, we first analyzed if both of them, or only
the sk variant, were induced after injury. Therefore, we performed
RNase protection analysis with total RNA from normal and wounded skin.
A 453-bp fragment, which overlaps the 3'-splice site (Fig.
1A) was amplified by PCR and used for the generation of a
32P-labeled antisense riboprobe. This probe allows the
simultaneous detection of skNAC and
NAC in one sample of RNA. Fig.
3A shows that
NAC mRNA
was far more abundant than skNAC mRNA in both normal and wounded
skin, probably because of its ubiquitous expression. Nevertheless, this
form was only slightly induced after injury. Furthermore, expression of
the
NAC binding partner
NAC was not induced after skin injury
(data not shown). The skNAC transcript was hardly detectable in normal
skin. However, expression of this variant was dramatically induced as
early as 12 h after wounding. This result was reproduced with RNAs
from three independent wound healing experiments. As assessed by
Northern blotting,
NAC and skNAC transcripts had the expected sizes
of 0.9- and 7.0-kilobases (data not shown).
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Fig. 3.
Strong induction of skNAC mRNA expression
during wound healing. Panel A, RNase
protection assay with samples of 50 µg total RNA derived from murine
skin wounds. A 453-bp antisense riboprobe overlapping the splicing site
was used for hybridization. The film was exposed for 4 h
(upper panel) or for 3 days (lower panel). 1,000 cpm of the radioactive probe served as a size marker and were loaded in
the lane labeled Probe. 50 µg of tRNA were used as a
negative control. Panels B-D, RNase protection assays
showing expression of myogenin (panel B), cardiac/fetal
muscle actin (panel C), and
skeletal muscle actin
(panel D) in the same RNA batches. 20 µg of total RNA were
used; the films were exposed for 7 days (myogenin,
cardiac/fetal
muscle actin) or 1 h (
skeletal muscle actin).
cardiac/fetal muscle actin
(Fig. 3C) at later stages of the repair process. The good
correlation of myogenin and
cardiac/fetal muscle actin expression
with most models of muscle regeneration indicates that the murine
panniculus carnosus is a useful model to study skeletal muscle regeneration.
/skNAC and
myogenin seem to be expressed within different regions of the panniculus carnosus 5 days after wounding, although, due to the nuclear
myogenin and the cytoplasmic
/skNAC staining and the low resolution,
we could not analyze this question at the single cell level (data not
shown). This is in agreement with the RNA data, which show that
induction of skNAC expression occurs earlier than that of myogenin.
Thus, as suggested by the expression kinetics, skNAC induction seems to
precede myogenin induction in the course of the myogenic repair
process. However, we cannot exclude the possibility that in
vivo, skNAC-overexpressing cells represent a different subtype of
myogenic (precursor) cells than myogenin-expressing cells. In any case,
our data suggest the use of skNAC expression as an early marker for
muscle repair processes.
skeletal muscle actin, a marker for striated muscle
cells (11), in the same RNA batches. As shown in Fig. 3D,
expression of this gene remained equally high during the early phase of
wound repair and subsequently even declined. Therefore, skNAC
overexpression is indeed due to an increase in synthesis and/or
stability of its mRNA.
/skNAC transcript is associated with several types of inflammatory
muscle diseases in humans.3
Furthermore, preliminary results from our laboratory revealed a strong
up-regulation of skNAC expression in the skin as well as in several
individual muscles of the dystrophic (mdx) mouse, a murine model for
muscular dystrophy (data not shown). Most importantly, increased levels
of skNAC mRNA correlated with the presence of degenerated myofibers
at the histological level. On the other hand, the basal expression of
skNAC in the intact panniculus carnosus indicates that skNAC, in
contrast to several myogenic transcription factors, might also play a
role in the maintenance of functional skeletal muscle cells under
physiological circumstances.
cardiac/fetal muscle actin, the molecules that
we already used as "markers" for repair processes of the panniculus
carnosus. As shown in Fig. 4, skNAC was
induced approximately 30 h after a shift from growth to
differentiation medium, in parallel with myogenin, and expression of
both genes declined to basal levels within 4 days. Induction of
cardiac/fetal muscle actin occurred only slightly later than that of
the transcription factors.
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Fig. 4.
Induction of skNAC expression during myoblast
differentiation in vitro. Mouse C2C12
(left) and rat L6 (right) myoblasts were shifted
from growth to differentiation medium and harvested 12 h, 26 h, 56 h, and 4 days later. 20 µg of total RNA were analyzed for
/skNAC (top panels), and, with respect to C2C12 cells,
myogenin (middle panel), and
cardiac/fetal muscle actin
(bottom panel) mRNA expression. 20 µg of tRNA were
used as a negative control, 1,000 cpm of the radioactive probe served
as a size marker. The films were exposed for 7 days (NAC/C2C12 cells),
7 h (NAC, L6 cells), or 2 days (myogenin and
cardiac/fetal
muscle actin).
/skNAC and myogenin.
Only a very
weak
/skNAC fluorescence, which was homogeneously distributed over
the cytoplasm of every cell, was obtained before the induction of
differentiation (data not shown). No nuclear staining was seen in any
cell. 68 h after the induction of differentiation, some cells
stained strongly positive for
/skNAC, whereby most of them had a
fiber-shaped, differentiated morphology (Fig. 5). Although the antibody
recognizes both the
- and the skNAC variant, the exclusive induction
of skNAC mRNA expression during the differentiation process
indicates that the strong signal seen in these cells is due to
increased skNAC expression. Most interestingly, all nuclei of the
strongly stained cells were also positive for myogenin, indicating that
at least in vitro skNAC and myogenin might indeed be
coexpressed during the myogenic differentiation program. Because
myogenin is a marker for differentiating rather than for differentiated
myoblasts, coexpression of this factor and skNAC suggests a role of
skNAC in the early differentiation process in vitro.
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Fig. 5.
Coexpression of
/skNAC and myogenin during myoblast differentiation
in vitro. Murine C2C12 myoblasts were induced to
differentiate. Cells were stained with a polyclonal, affinity-purified
/skNAC antiserum and a fluorescein isothiocyanate-coupled secondary
antibody (green fluorescence) and with a monoclonal
anti-myogenin antibody and a Texas red-coupled secondary antibody
(red fluorescence) 68 h after the addition of
differentiation medium. Note the red nuclei of the cells
that are strongly positive for
/skNAC 68 h after the addition
of differentiation medium (magnification, ×400).
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Fig. 6.
skNAC is exclusively expressed in primary
human myotubes but not in undifferentiated myoblasts. Total
cellular RNA was extracted from exponentially growing and
differentiated primary human myoblasts. Subsequently, RT-PCR was
performed to analyze and skNAC expression.
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ACKNOWLEDGEMENTS |
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We thank Nancy Larochelle, Helga Riesemann, and Frédérique Wanninger for excellent technical assistance and Sibylle Blumenthal for help with the tissue culture experiments. Furthermore, we thank Peter Hans Hofschneider for support.
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FOOTNOTES |
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* This work was supported by Grants from the Deutsche Forschungsgemeinschaft (WE 1983/2-1 and WE 1983/3-1) and from the Human Frontier Science Program (to S. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Kekulé predoctoral fellowship from the Verband der Chemischen Industrie. Present address: Dept. of Molecular Pharmacology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305.
** Supported by grants from the Deutsche Forschungsgemeinschaft and the Sander-Stiftung, Germany.
A Hermann and Lilly Schilling Professor of Medical Research. To
whom correspondence should be addressed: Institute of Cell Biology,
ETH-Hönggerberg, CH-8093 Zürich, Switzerland. Tel.: 41-1-633-3941; Fax: 41-1-633-1174; E-mail:
Sabine.werner{at}cell.biol.ethz.ch.
2 B. Munz, unpublished data.
3 B. Munz, H. Lochmüller, and S. Werner, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: RT-PCR, reverse transcription-polymerase chain reaction; DDRT-PCR, differential display RT-PCR; skNAC, skeletal muscle nascent polypeptide-associated complex; PBS, phosphate-buffered saline; bp, base pair(s); nt, nucleotide(s).
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