Cloning of Novel Injury-regulated Genes
IMPLICATIONS FOR AN IMPORTANT ROLE OF THE MUSCLE-SPECIFIC PROTEIN skNAC IN MUSCLE REPAIR*

Barbara MunzDagger §, Martin Wiedmann, Hanns Lochmüllerparallel **, and Sabine WernerDagger Dagger Dagger

From the Dagger  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 parallel  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 alpha  subunit and a 21-kDa beta  subunit. Furthermore, a skeletal and heart muscle-specific splice variant of the alpha  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).

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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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 -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.

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 alpha 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 alpha 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.

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 [alpha -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.

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 alpha  cardiac/fetal muscle actin (180 nt) (11), and of murine alpha  skeletal muscle actin (218 nt) (11) were used.

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 [alpha -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.

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 alpha  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.

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 alpha  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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 alpha /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 alpha NAC exons (exon 1 and exon 3, Fig. 1A). Surprisingly, our PCR-fragment was not located at the ultimate 3'-end of the alpha /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 alpha NAC sequence (Fig. 1B).


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Fig. 1.   Alignment of differential display primer N1 with the alpha 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 alpha 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 alpha  and skNAC transcripts in one batch of RNA. B, nucleotide sequence of differential display primer N1 compared with the sequence of murine alpha NAC and skNAC cDNAs. Primer N1 can be aligned with only two mismatches to the skNAC but not to the alpha NAC sequence.

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, alpha /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 alpha  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 alpha /beta 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 alpha /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 alpha /skNAC antiserum and a polyclonal c-met antiserum. A fluorescein isothiocyanate-coupled anti-rabbit (alpha /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).

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 alpha /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.

Although skNAC Is Dramatically Up-regulated after Injury, alpha NAC Is Only Slightly Induced-- Because alpha  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 alpha NAC in one sample of RNA. Fig. 3A shows that alpha 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 alpha NAC binding partner beta 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, alpha 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), alpha  cardiac/fetal muscle actin (panel C), and alpha  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, alpha  cardiac/fetal muscle actin) or 1 h (alpha  skeletal muscle actin).

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 alpha  cardiac/fetal muscle actin (Fig. 3C) at later stages of the repair process. The good correlation of myogenin and alpha  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.

As assessed by double-immunofluorescence staining, alpha /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 alpha /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.

To exclude the possibility that skNAC induction might be caused by an enrichment of striated muscle cells in early wounds, we analyzed expression of alpha  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.

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 alpha /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.

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 alpha  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 alpha  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 alpha /skNAC (top panels), and, with respect to C2C12 cells, myogenin (middle panel), and alpha  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 alpha  cardiac/fetal muscle actin).

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 alpha /skNAC and myogenin. Only a very weak alpha /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 alpha /skNAC, whereby most of them had a fiber-shaped, differentiated morphology (Fig. 5). Although the antibody recognizes both the alpha - 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 alpha /skNAC and myogenin during myoblast differentiation in vitro. Murine C2C12 myoblasts were induced to differentiate. Cells were stained with a polyclonal, affinity-purified alpha /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 alpha /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 alpha  and skNAC expression.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger Dagger 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.

    ABBREVIATIONS

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).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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