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nidogen-2 in myogenic differentiation
r 2006, Copyright the Authors Differentiation (2006) 74:573–582 DOI: 10.1111/j.1432-0436.2006.00100.x Journal compilation r 2006, International Society of Differentiation
O RI G INA L AR T I C L E
Ricarda Neu . Stephanie Adams . Barbara Munz
Differential expression of entactin-1/nidogen-1 and entactin-2/ nidogen-2 in myogenic differentiation
Received April 18, 2006; accepted in revised form June 14, 2006
Abstract Here, we show that entactin-2 expression is strongly, but transiently, induced in myogenic differentiation. Treatment of C2C12 myoblasts with actinomycin D in parallel to the induction of differentiation could demonstrate that this is due to enhanced transcription of the entactin-2 gene. Furthermore, treatment with the translation inhibitor cycloheximide could show that entactin-2 is a primary response gene. As p38 MAP kinase is an important regulator of myogenic differentiation, we also analyzed the possibility that entactin-2 might be a target of this pathway. However, using various p38 MAPK inhibitors, we could not detect involvement of p38 in entactin-2 up-regulation. Most remarkably, expression of the entactin-2 homolog entactin-1 dramatically declined in myogenesis, suggesting different functions of the two entactins in this process. A similar effect was seen in primary myoblasts isolated from two different mouse strains. Expression of high levels of entactin-1 in myoblasts using a retroviral expression system led to a higher proliferation rate both in growth and in differentiation medium and to reduced expression of various myogenic differentiation markers after the induction of differentiation. Furthermore, decreased expression of the entactin-2 gene after treatment of the cells with ent-2-specific siRNA preparation led to reduced expression of the cell cycle inhibitor p21. These data suggest important and distinct functions of entactin-1 and -2 in myogenic differentiation. Key words entactin nidogen myogenic differentation . ) Ricarda Neu Stephanie Adams Barbara Munz (* Institute of Physiology Charite´—University Medicine Berlin Arnimallee 22, 14195 Berlin, Germany Tel.: 149 30 8445 1643 Fax: 149 30 8445 1634 E-mail: [email protected] U.S. Copyright Clearance Center Code Statement:
Introduction Entactin-1 and -2 are ubiquitous components of basement membranes. They bind to various other basement membrane proteins, such as collagen IV, laminin, and perlecan, and are cell-adhesive (Carlin et al., 1981; Timpl et al., 1983; Kimura et al., 1998; Kohfeldt et al., 1998). In addition to their function as structural elements of basement membranes, entactins have been implicated in the regulation of cell attachment (Chakravarti et al., 1990), neutrophil chemotaxis (Senior et al., 1992), trophoblast outgrowth (Yelian et al., 1993), angiogenesis (Nicosia et al., 1994), and osteoblast differentiation (Kimura et al., 1998). Both entactin isoforms are glycosylated proteins that consist of three globular domains (G1 to G3) connected by a flexible link and a rod (Fox et al., 1991; Mayer et al., 1995; Kimura et al., 1998; Kohfeldt et al., 1998). In addition to their structural similarities, the expression pattern of the two entactin genes in various embryonic and adult tissues and cultured cells is very similar (Kimura et al., 1998; Kohfeldt et al., 1998; Miosge et al., 2001, 2002; Salmivirta et al., 2002). Furthermore, mice genetically null for either entactin-1 or -2 showed no overt phenotype initially (Murshed et al., 2000; Schymeinsky et al., 2002), although a few years later, Dong et al. (2002) could detect subtle neurologic defects and structural abnormalities of the basement membrane in the entactin-1 knock-out mice. As entactin-2 expression is increased in some tissues of entactin-1 knock-out mice (Murshed et al., 2000; Dong et al., 2002; Miosge et al., 2002; Schymeinsky et al., 2002), these data suggest that entactin-1 and -2 might have at least partially redundant functions. Remarkably, compound genetic ablation of both entactin isoforms leads to perinatal lethality associated with defects in the heart, lung, and kidney morphogenesis, suggesting important
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functions of the entactins in these tissues (Bader et al., 2005). Differentiation of skeletal myoblasts into mature myotubes is a complex and highly organized process (reviewed by Sabourin and Rudnicki, 2000). During embryonic development, skeletal myoblasts differentiate under the influence of various extra- and intracellular factors, such as growth factors, cytokines, proteins of the extracellular matrix, and transcription factors. Although in the past few decades, a large number of proteins have been implicated as crucial modulators of myogenic differentiation, the complexity of this process strongly suggests that more remain to be discovered. Little is known about a possible role of the entactins in myogenic differentiation. In 1998, Godfrey and Gradall demonstrated that during embryonic development, basement membrane proteins such as laminin, collagen IV, and also entactin are concentrated in myogenic regions of the mouse limb bud, suggesting a role of these factors in the early stages of myogenesis. Accordingly, in vitro studies demonstrated that the same basement membrane proteins have a stimulatory effect in the early stages of myogenesis (Langen et al., 2003). Furthermore, entactin alone might promote adhesion and long-term maintenance of skeletal myotubes in culture (Funanage et al., 1992). Nothing is known about isoform-specific functions of the two entactins in myogenesis. However, most interestingly, in contrast to most other organs, the expression patterns of the two entactins in adult heart and skeletal muscle are different (Kohfeldt et al., 1998), suggesting unique functions of the two isoforms in these tissues. Here, we demonstrate that entactin-2 expression is strongly induced in myogenic differentiation, whereas entactin-1 expression decreases after the induction of differentiation, and that high levels of entactin-1 can suppress myogenesis. These data suggest that entactins might have important, but different functions in myogenesis.
Materials and methods Tissue culture Murine C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum (growth medium; GM). Primary mouse C57 and C3H myoblasts were isolated and cultured as described previously (Rando and Blau, 1994). To induce differentiation, cells were grown to 80%–90% confluency and then switched to differentiation medium (DM; Dulbecco’s modified Eagle’s medium containing 2% horse serum). Generation of myoblasts constitutively expressing high levels of ent-1 and ent-2 The full-length wild-type murine entactin-1 and entactin-2 cDNAs were kind gifts from Dr. R. Timpl (Martinsried, Germany) and
Dr. M. Shimane (Chugai Institute for Molecular Medicine, Ibaraki, Japan). They were cloned into the retroviral vector pBabeMN-Iresneo (Morgenstern and Land, 1990). The ecotrophic producer cell line FE, a gift from Dr. Gary Nolan (Stanford University), was transfected with the resulting constructs by using Fugene (Roche, Mannheim, Germany). C2C12 cells were subsequently infected with the viral supernatants at high efficiency, as described previously for primary myoblasts (Springer and Blau, 1997). siRNA technology 2 105 C2C12 myoblasts were trypsinized, resuspended in normal growth medium, and transfected in suspension with 100 nM (final concentration) of two different siRNAs specific for the murine entactin-2 gene (Ambion, Kassel, Germany) or a random (scrambled) control siRNA according to the instructions of the manufacturer. They were then seeded in six-well plates and incubated at 371C for 24 hr. The next morning, the medium was either replaced by fresh growth medium or cells were incubated in DM for another 24 hr. Subsequently, total cellular RNA or protein was isolated from the cells. RNA isolation Total cellular RNA was extracted using the RNeasy RNA isolation kit (Qiagen, Hilden, Germany), and poly(A) RNA was isolated by using oligo(dT) cellulose spin columns (Clontech, Palo Alto, CA) according to the instructions of the manufacturer. Subtractive cDNA library hybridization For subtractive cDNA hybridization, the PCR Select Kit (Clontech) was used according to the instructions of the manufacturer. Northern blot analysis One to eight micrograms of total RNA isolated as described above was separated on a 1% agarose gel containing formaldehyde (2%). Subsequently, RNA was transferred onto a nylon membrane. Filters were hybridized overnight with antisense riboprobes that had been labeled with the digoxigenin RNA labeling kit (Roche). After washing, the blots were incubated with an alkaline phosphatasecoupled antidigoxigenin antibody (Roche), washed, and developed with CDP-Star (Tropix [Bedford, MA] or Roche) as a chemiluminescent substrate for alkaline phosphatase. Signals were detected via exposure to X-ray films (ranging from 2 sec to 5 min). Preparation of protein lysates and Western blot analysis Cultured cells were lysed in lysis buffer (1% Triton X-100, 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA pH 8.0). Ten micrograms of total protein were loaded on a sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were pre-blocked in 3% powdered milk in Tris-buffered saline containing 0.5% Tween 20 (TBS-T) for 30 min; incubated with a 1:1,000 dilution of the first antibody in blocking solution for 1 hr, washed three times with TBS-T; incubated for 30 min with a 1:2,500 dilution of the second antibody, a peroxidase-conjugated anti-rabbit or anti-mouse monoclonal antibody (Amersham, Little Chalfont, UK) in blocking solution; washed three times with TBS-T; and developed with the ECL Western blot detection system (Amersham). Cell proliferation assay Cells were seeded in chamber slides, pulse-labeled with bromodeoxyuridine (BrdU) for 20 min, and stained with the 5-bromo-2 0 -deoxy Labeling and Detection Kit II (Roche).
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Results Entactin-2 expression is induced in myogenic differentiation To identify novel genes that are differentially expressed during early myoblast differentiation, cultured C2C12 myoblasts were induced to differentiate with and without addition of 1 ng of transforming growth factor b1 (TGF-b1)/ml, a potent inhibitor of the differentiation process. Rather than a simple comparison of cells in GM and DM, this approach was chosen to minimize the detection of nonspecific changes in gene expression due to numerous differences in the composition of growth and DM. After 16 hr, RNA was isolated and assayed for differential gene expression by a subtractive cDNA library hybridization approach. Increased myogenin mRNA levels after 16–24 hr and increased myosin heavy chain protein amounts after 48 hr confirmed that the cells underwent differentiation. Differential expression was confirmed by Northern blot analysis. All transcript levels were normalized to mRNA levels of rpl32, a ubiquitous ribosomal protein. One of the genes that exhibited markedly increased expression within a few hours of differentiation was identified as entactin-2/nidogen-2. As shown in Fig. 1, entactin-2 expression was enhanced within the first 16 hr after the induction of differentiation. Specifically, we could detect a two- to threefold increase in entactin-2 mRNA levels as early as 3–7 hr after the addition of DM. Entactin-2 expression levels peaked around 24 hr, and then declined again and reached baseline levels again around 62 hr after the induction of differentiation. These data suggest that expression of the entactin-2 gene is tightly controlled in myogenic differentiation.
Fig. 2 Inhibition of transcription. C2C12 myoblasts were treated with differentiation medium (DM) alone or with DM containing the transcriptional inhibitor Actinomycin D or an equivalent volume of dimethylsulfoxide (DMSO), the solvent for the Actinomycin D. After 20 hr, total cellular RNA was extracted and analyzed via Northern blot for the presence of entactin-2 mRNA. Note the strong induction of entactin-2 gene expression after treatment with DM alone or with DM containing DMSO. By contrast, addition of Actinomycin D to the DM completely abolished this induction. Hybridization with a probe directed against the rpl32 mRNA was used as a loading control as indicated.
Entactin-2 expression is regulated at the transcriptional level To study the mechanism of ent-2 induction, myoblasts were treated with the transcription inhibitor Actinomycin D in parallel to the induction of differentiation. As shown in Fig. 2, this treatment completely abolished ent-2 induction, indicating that gene expression is regulated at the transcriptional level.
Entactin-2 is a primary response gene
Fig. 1 Entactin-2 expression in myogenic differentiation. C2C12 cells were treated with differentiation medium (DM). After the indicated time periods, cells were harvested and total cellular RNA was extracted. Eight microgram of each RNA sample was analyzed via Northern blotting using a probe specific for the murine entactin2 gene (top panel). To monitor the differentiation process, hybridization with a probe recognizing the myogenic differentiation marker myogenin was also performed (bottom panel). Hybridization with a probe directed against the mRNA encoding the ribosomal protein rpl32 was used as a loading control (both panels). Note the strong induction of entactin-2 gene expression after 16 hr in DM. Expression of the myogenin gene is characterized by similar kinetics.
To further analyze the mechanism of regulation of entactin-2 gene expression in myogenic differentiation, C2C12 cells were treated with the translation inhibitor cycloheximide (Cyc) in parallel to the induction of differentiation. As shown in Fig. 3, this treatment did not inhibit up-regulation of the entactin-2 gene. By contrast, a superinduction was observed. This is a phenomenon often seen when treating with translation inhibitors and possibly reflects reduced translation of proteins that destabilize the entactin-2 protein or enhance its degradation (Vilcek et al., 1976). By contrast, as shown in Fig. 3, induction of the gene encoding the myogenic transcription factor myogenin was completely abolished upon Cyc treatment, suggesting that the drug was effective and that myogenin is not a primary response gene in myogenesis.
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Fig. 3 Inhibition of translation. C2C12 myoblasts were treated with differentiation medium (DM) alone or with DM containing the translational inhibitor cycloheximide (Cyc) or dimethylsulfoxide (DMSO), the solvent for Cyc. At the indicated time points, total cellular RNA was extracted and analyzed via Northern blot using an antisense riboprobe directed against the entactin-2 mRNA (top panel). Note the strong induction of entactin-2 gene expression after transfer of the cells to DM, which is not inhibited by Cyc. Expression of the myogenin gene was also analyzed to monitor the differentiation process and as a positive control for the Cyc treatment (bottom panel). Note that myogenin induction was completely abolished upon Cyc treatment. Hybridization with a probe specific for the murine rpl32 mRNA was used as a control for equal loading (both panels).
Entactin-2 expression is not regulated by p38 MAP kinase To identify factors that are responsible for entactin-2 induction, cells were treated with specific inhibitors of the p38 MAP kinase pathway in parallel to the induction of differentiation. Activation of this signaling pathway has been shown to be crucial in myogenesis (Cuenda and Cohen, 1999; Zetser et al., 1999). As shown in Fig. 4, two different p38 inhibitors, SB 202190 and SB203580, did not abolish entactin-2 induction, indicating that this gene is not a target of the p38 pathway, whereas the structurally similar, but non-functional compound SB 202474 had no effect. By contrast, induction of the gene encoding the myogenic transcription factor myogenin was strongly repressed (Fig. 4). These data indicate that in contrast to various genes that regulate myogenesis, the entactin-2 gene is not a target of the p38 MAP kinase pathway.
Fig. 4 Inhibition of p38 MAP kinase. C2C12 myoblasts were treated with differentiation medium (DM) alone, with DM containing specific inhibitors of the p38 MAP kinase pathway (SB 202190 and SB 203580) or a structurally similar, but non-functional control (SB 202474), or with DM containing DMSO, the solvent for SB 202190, SB 202474, and SB 203580. After 18 hr, total cellular RNA was extracted and analyzed for expression of the entactin-2 gene via Northern blot (top panel). Note that induction of entactin-2 gene expression was not inhibited by treatment with the p38 inhibitors. To monitor the differentiation process and as a positive control for treatment with the p38 inhibitors, expression of the myogenin gene was also analyzed in the same samples (bottom panel). Note that myogenin induction was almost completely abolished upon treatment with the two functional p38 inhibitors. As a control for equal loading, both blots were also hybridized with an rpl32-specific probe.
Differential expression of entactin-1 and -2 in primary myoblasts To confirm the physiological relevance of our results, expression of the two entactin genes was also analyzed in primary cells. For this purpose, myoblasts were isolated from skeletal muscle tissue of two different mouse strains: C3H and C57BL6. Similar to the results
Entactin-1 expression decreases in myogenesis As the entactin-2 gene is usually co-expressed with its homolog entactin-1, we speculated that this might also be the case in myogenic differentiation. Thus, we analyzed entactin-1 expression in this process. Surprisingly, we did not find co-expression of the two genes. By contrast, as shown in Fig. 5, entactin-1 expression strongly decreased as early as 16 hr after the induction of differentiation and was hardly detectable after 40 hr. These data suggest that expression of each entactin gene is regulated in a distinct manner in myogenesis, indicating that they might have unique and distinct functions in this process.
Fig. 5 Entactin-1 expression in myogenic differentiation. C2C12 cells were treated with differentiation medium (DM) for the time periods indicated. Total cellular RNA was extracted and analyzed for expression of the entactin-1 gene via Northern blot. Note the strongly decreased expression of entactin-1 after 16 hr in DM (top panel). Expression of the myogenin gene was analyzed to monitor the differentiation process (bottom panel); hybridization with an rpl32-specific probe was used as a control for equal loading.
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High levels of entactin-1 inhibit myogenic differentiation The strong and early regulation of the expression levels of the two entactin genes in myogenesis suggests that entactins might be important regulators of myogenic differentiation. To analyze this question in more detail, we generated cell lines that strongly express the entactin-
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obtained with the myoblast cell line C2C12, entactin-1 expression also decreased upon induction of differentiation of these cells (Fig. 6 and data not shown). By contrast, we could detect a high basal expression, but only a slight induction of entactin-2 expression in these cells upon induction of differentiation (data not shown), which might be due to the fact that these cells had been isolated from neonatal and not embryonic mice and were characterized by a higher degree of differentiation already. In general, the degree and the time course of entactin gene expression in primary myoblasts were variable, depending on the respective mouse strain, the age of the animals the cells were isolated from, and the isolation procedure. However, in all our experiments, we found a strong repression of entactin-1 expression and at least a slight induction of entactin-2 expression after the induction of myogenic differentiation, suggesting that this is a general feature seen in all types of skeletal myoblasts. These data indicate that differential expression of the two entactin genes might be a general and physiologically relevant phenomenon.
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Fig. 6 Expression of the entactin-1 gene in primary myoblasts. Primary myoblasts isolated from neonatal C3H mice were treated with differentiation medium (DM) for 22 hr. Total cellular RNA was analyzed for expression of the entactin-1 gene in comparison with C3H myoblasts that had been maintained in growth medium only. Note the strongly decreased expression of the entactin-1 gene in DM. By contrast, expression of the myogenic differentiation marker myogenin was induced. Hybridization with an rpl32-specific probe served as a loading control.
1 or the entactin-2 gene, in the sense and antisense direction, respectively. For this purpose, we used a retroviral vector system. The advantage of this system is that with one or only a few rounds of infection, more than 99% of the cells can be reached, so that whole-cell populations in contrast to single clones can be analyzed. This approach is superior to clonal analysis, as it minimizes the risk of artifacts due to the long selection procedure of single clones. As shown in Fig. 7, at the mRNA level, the recombinant entactin-1 and entactin-2 genes were expressed at high levels in our cells. By contrast, high levels of antisense entactin-1 and -2 had only a slight inhibitory effect on expression of the endogenous genes (data not shown). By Western blot analysis, we could also detect higher levels of entactin-1 and entactin-2 proteins; however, the effects were weaker than at the mRNA level (data not shown). This might be due to the fact that the entactins are difficult to immunoprecipitate from the C2C12 medium. It is likely that after secretion, they remain attached to the surface of the cells, either directly or indirectly by binding to other proteins of the extracellular matrix (Mann et al., 1989; Kohfeldt et al., 1998; Salmivirta et al., 2002). In general, expression of the recombinant genes declined during differentiation (Fig. 7). This is probably due to silencing of the viral LTR in this process and has been observed earlier (B. Munz and H.M. Blau, unpublished observations). Remarkably, we did not find cross-regulation of the two isoforms or of recombinant and
Ent-1 rpl32
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Fig. 7 Expression of high levels of the entactin-1 and entactin-2 genes in myoblasts. The full-length murine entactin-1 and entactin-2 cDNAs were cloned into retroviral vectors in sense (s) and antisense (as) direction; specific retroviruses were produced and used to infect C2C12 cells. As a control, cells were also infected with a retrovirus containing the lacZ gene. Cells were grown in growth medium only (0 hr) or in differentiation medium (DM) for the time periods indicated. Proper differentiation of control cells was monitored by repeated light microscopical analysis and by expression analysis of certain myogenic differentiation markers (not shown). Total cellular RNA was extracted and analyzed for ent-1 (top panels) and ent2 (bottom panels) expression via Northern blot. The blots were also hybridized with probes specific for the rpl32 mRNAs. Note the high expression levels of the recombinant entactin-1 and entactin-2 genes. Expression of endogenous entactin-1 and entactin-2 is not visible due to the short exposure time of the respective films.
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Fig. 8 Proliferation and differentiation in cells engineered to express high levels of entactin-1. (A) C2C12 cells (ctrl) and myoblasts transduced with the ent-1 retrovirus were pulse-labeled with bromodeoxyuridine (BrdU) both in proliferation medium and after 24 hr in differentiation medium (dm). BrdU incorporation was then detected using an FITC-coupled anti-BrdU antibody, and the percentage of labeled nuclei relative to the total number of nuclei (visualized via DAPI-staining) is shown. Note the high proliferation rate of myoblasts expressing high levels of ent-1 both in pm and in dm when compared with controls. (B) Myoblasts transduced with the ent-1 retrovirus were cultured in DM for 26 hr and then analyzed for expression of Ki67, p21, and caveolin-3 via Western blot
analysis. Note the strong expression of Ki67 in cells expressing high levels of entactin-1 even in DM and the low expression levels of p21 when compared with control cells, keeping in mind that induction of p21 expression during differentiation of wild-type C2C12 cells does often not occur before at least 48 hr in DM (Andre´s and Walsh, 1996). Furthermore, the strong expression of caveolin-3 in the cells expressing high levels of entactin-1 when compared with controls is obvious. As expected, no caveolin-3 protein could be detected in both cell types when grown in proliferation medium, i.e., before the induction of differentiation. Incubation with an antibody directed against the a-tubulin protein served as a loading control (bottom panel).
endogenous entactin-1 and/or entactin-2 (data not shown), suggesting that in contrast to various other tissues and organs, they might not be functionally redundant in myogenesis. Interestingly, in myoblasts engineered to express high levels of entactin-2, expression of the recombinant gene declined rapidly, even when cells were kept under selection pressure permanently, with a neomycin concentration of 400 mg/ml. This finding indicates that entactin-2 might indeed promote differentiation, so that productively infected cells that express high levels of the recombinant gene withdraw from the cell cycle, initiate differentiation, and are eventually overgrown by the initially small pool of myoblasts (o5% immediately after infection, as judged by lacZ staining) that do not express high levels of recombinant entactin-2. Therefore, it was not possible to further characterize these cells with respect to myoblast proliferation and differentiation. By contrast, cells engineered to express high levels of entactin-1 were characterized by stable expression levels of the recombinant gene over at least 10 passages, even when not kept in selection medium all the time. This finding indicates that entactin-1 does not suppress myogenic differentiation. When cells were pulse-labeled with BrdU, we found that cells engineered to express high levels of entactin-1 had a higher proliferation rate than wild-type myoblasts, both in growth and—to an even greater extent with respect to control myoblasts—in DM (Fig. 8). In addition, myoblasts that express high levels of recombinant entactin-1 were characterized by higher expres-
sion levels of Ki67 specifically in DM (Fig. 8), and lower expression levels of p21 (Fig. 8), indicating that high expression levels of the entactin-1 gene have a positive effect on myoblast proliferation, especially in low-serum medium. Furthermore, when cells were switched to DM, we found that cells engineered to express high levels of entactin-1 showed lower expression levels of certain myogenic differentiation markers such as caveolin-3 (Fig. 8). These results indicate that high levels of entactin-1 can cause an increased myoblast proliferation rate and can inhibit certain aspects of myogenic differentiation. Inhibition of entactin-2 expression leads to decreased expression levels of the cell cycle inhibitor p21 To further study the role of the entactin-2 gene in myogenesis, we inhibited its expression in C2C12 cells using specific siRNA preparations. As shown in Fig. 9, we were able to block expression of the endogenous gene by at least 80% in these cells. When we studied expression of various myogenic differentiation markers, we found a higher expression of myoblast proliferation markers, such as cyclin D1, in the cells that had been treated with the siRNAs (data not shown). By contrast, expression levels of anti-proliferative and differentiation-associated genes, such as p21, were reduced (Fig. 10). These data suggest that induction of entactin-2 expression might indeed play a crucial role in the regulation of myogenic differentiation.
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Fig. 9 Inhibition of entactin-2 gene expression using specific siRNAs. Exponentially growing C2C12 cells were treated with three different ent-2-specific siRNA preparations, with a random control (siRNA control), with transfection medium alone, or left untreated. After 24 hr, total cellular RNA was extracted from the cells and analyzed for expression of the entactin-2 gene by Northern blot. The bottom panel shows a methylene blue stain of the same filter as a control for equal loading.
Discussion
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Muscle basement membrane is produced in part by the myoblasts it ensheates, in part by surrounding cells. Not only does it provide mechanical stability and structural integrity of mature muscle fibers, but it is also involved in the regulation of myogenic differentiation. Interestingly, different basement membrane proteins have ago-
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Fig. 10 Inhibition of entactin-2 expression leads to decreased expression of the p21 gene. C2C12 cells treated with siRNA #1 or control cells were analyzed for expression of the p21 gene immediately after transfection. The bottom panel shows a methylene blue stain of the same blot as a control for equal loading.
nistic, but also antagonistic effects on myogenesis. For example, laminin and collagen IV stimulate differentiation of skeletal muscle cells in culture, whereas fibronectin inhibits this process (von der Mark and Ocalan, 1989; Li et al., 2001, reviewed by Sanes, 2003). From this background, one would expect that expression and secretion of each basement membrane component is tightly and individually controlled in myogenic differentiation. In fact, differential expression during myogenesis has been demonstrated for several individual genes encoding basement membrane proteins: specifically, Rao et al. could demonstrate enhanced synthesis of type IV collagen and laminin, but decreased levels of fibronectin during differentiation of murine skeletal myoblasts in culture as early as in 1985, which is consistent with antagonistic functions of laminin and collagen IV versus fibronectin in this process. Subsequently, a broad variety of studies could demonstrate differential expression of other genes encoding basement membrane proteins, such as type XIX collagen, during myogenesis in vitro and in vivo (Godfrey et al., 1988; Solursh and Jensen, 1988; Mundegar et al., 1995; Schuler and Sorokin, 1995; Godfrey and Gradall, 1998; Gullberg et al., 1999; Myers et al., 1999; Li et al., 2001; Sumiyoshi et al., 2001). We could show that expression of the entactin-2 gene is strongly induced in myogenic differentiation. Most remarkably, expression peaked as early as 24 hr after the induction of differentiation and then declined again, concomitantly to increased myogenin gene expression. This is an expression pattern that resembles that of the myogenic transcription factor myf-5. These results suggest a role of entactin-2 in early myogenesis. Interestingly, several other studies also support the assumption that basement membrane proteins, in contrast to other ECM proteins, might specifically be involved in the regulation of the first few steps of the myogenic program: Solursh and Jensen (1988) and Godfrey and Gradall (1998) could show that laminin and other basement membrane components accumulate in myogenic regions of the chick and mouse limb buds, respectively, before the onset of myogenesis. Furthermore, Langen et al. (2003) carried out in vitro experiments with cultured C2C12 myoblasts that also argue for a role of several basement membrane proteins in early myogenesis. To analyze the mechanism of entactin-2 regulation in myoblasts, we first carried out pulse-labeling studies with the transcription inhibitor Actinomycin D. Thereby, we found that expression of entactin-2 is primarily regulated at the transcriptional level in myoblasts. Interestingly, transcriptional regulation is involved in the regulation of expression of most muscle-specific genes (Shimokawa et al., 1998, and references therein). Furthermore, pulse-labeling studies with Cyc showed that entactin-2 is a primary response gene with respect to myogenic differentiation. This finding indicates that
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entactin-2 expression is not dependent on de novo synthesis of other proteins regulating myogenesis, such as the myogenic transcription factors. From this background, we aimed at analyzing the specific signaling pathways that are responsible for induction of entactin-2 gene expression in myogenesis. Activation of p38 MAP kinase has been shown to be absolutely necessary for myogenic differentiation (Cuenda and Cohen, 1999; Zetser et al., 1999) and expression of several crucial regulators of myogenesis, such as caveolin-3 (Galbiati et al., 1999), myf4 (Suelves et al., 2004), p21, p27, myoD, troponin T (Cabane et al., 2003), and myogenin (our data), is controlled by this pathway. However, using specific inhibitors, we found that entactin-2 expression is not regulated by p38 MAP kinase. Further studies will elucidate which pathways control expression of this gene. A possible candidate is the PI3 kinase/Akt pathway, which is essential for the differentiation of skeletal muscle cells and is functionally linked to the p38 pathway in myogenesis (Li et al., 2000; Gonzalez et al., 2004), especially as it has recently been shown that expression of the genes encoding two other basement membrane proteins, laminin and collagen IV, is controlled by PI3 kinase (Li et al., 2001). Surprisingly, entactin-1 expression decreased in myogenesis. This is in contrast with various other studies that suggest that expression of the two entactin genes is mostly coregulated in human and murine embryonic development as well as in most adult tissues and organs (Kimura et al., 1998; Kohfeldt et al., 1998; Miosge et al., 2001; Salmivirta et al., 2002). Interestingly, however, Kohfeldt et al. (1998) could find spatially distinct expression patterns of the two forms in skeletal and heart muscle, indicating that in contrast to most other tissues and organs, the two entactin isoforms might exert different functions here. In this context, it seems interesting that Schuler and Sorokin (1995) could demonstrate differential expression of the laminin 1 and 2 genes in myogenesis and they also postulate different functions of these two laminin variants in skeletal muscle differentiation. To analyze the function of entactin-1 and -2 in myogenesis, we generated stable myoblast cell lines engineered to express high levels of entactin-1 and -2. In these studies, we did not find cross-regulation of the two entactin isoforms in myoblasts. Specifically, expression of high levels of recombinant entactin-1 or -2 had no effect on expression of the two endogenous variants. This is in contrast with the results obtained when analyzing entactin-1-deficient mice: in these animals, elevated levels of entactin-2 could be found in most tissues and organs, suggesting partial functional redundancy of the two isoforms (Murshed et al., 2000; Miosge et al., 2002). However, a similar result, i.e., induction of entactin-1 expression in entactin-2-deficient animals, cannot be observed (Schymeinsky et al., 2002). Using siRNA technology, we are currently analyzing
the question of a possible cross-regulation of entactin-1 and entactin-2 expression in myoblasts in more detail. Under growth conditions, myoblasts engineered to express high levels of entactin-1 were characterized by a higher proliferation rate when compared with control cells. When we induced differentiation, we found that expression of high levels of the entactin-1 gene had a negative effect on certain aspects of myogenesis, such as exit from the cell cycle or induction of marker genes for myogenic differentiation such as caveolin-3. Unfortunately, due to a putative negative selection pressure for productively infected cells, we were not able to further analyze the growth and differentiation of myoblasts engineered to express high levels of entactin2. Most likely, cell lines that allow induction of expression of the recombinant gene might be the model system of choice here. However, as in contrast to the endogenous entactin-1 gene, the endogenous entactin-2 gene is induced in myogenesis, it is possible that the levels of endogenous entactin-2 are already sufficient to modulate myogenesis maximally, so that an additionally produced recombinant variant might have little effect. To study this question in more detail, we used siRNA technology to inhibit entactin-2 production in myoblasts. Most interestingly, we found that low levels of entactin-2 indeed led to higher expression of proliferation and lower expression of myogenic differentiation markers, suggesting that entactin-2 might be an important modulator of myogenesis. Taken together, these results suggest that differential expression of the two entactin genes is crucial for the regulation of myogenic differentiation, whereby each isoform is likely to have unique functions in this process. In the future, it might be interesting to analyze expression of the two entactin genes in heart and skeletal muscle in vivo under different physiological and pathological conditions. This might have important therapeutical implications. Acknowledgments We thank the late Dr. R. Timpl (Martinsried, Germany) for nidogen-1 and nidogen-2 antibodies and for the full-length murine entactin-1 cDNA, and Dr. Shimane (Ibaraki, Japan) for the full-length murine entactin-2 cDNA. This work was supported by grants from the Sonnenfeld-Stiftung, Berlin, Germany, and from the Deutsche Forschungsgemeinschaft (Mu 1556/3-1) (to B.M.).
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ment membrane protein with binding activity for laminin, collagen IV and cells. EMBO J 8:65–72. Mayer, U., Zimmermann, K., Mann, K., Reinhardt, D., Timpl, R. and Nischt, R. (1995) Binding properties and protease stability of recombinant human nidogen. Eur J Biochem 227:681–686. Miosge, N., Holzhausen, S., Zelent, C., Sprysch, P. and Herken, R. (2001) Nidogen-1 and nidogen-2 are found in basement membranes during human embryonic development. Histochem J 33:523–530. Miosge, N., Sasaki, T. and Timpl, R. (2002) Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice. Matrix Biol 21:611–621. Morgenstern, J.P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18:3587–3596. Mundegar, R.R., von Oertzen, J. and Zierz, S. (1995) Increased laminin A expression in regenerating myofibers in neuromuscular disorders. Muscle Nerve 18:992–999. Murshed, M., Smyth, N., Miosge, N., Karolat, J., Krieg, T., Paulsson, M. and Nischt, R. (2000) The absence of nidogen-1 does not affect murine basement membrane formation. Mol Cell Biol 20:7007–7012. Myers, J.C., Li, D., Rubinstein, N.A. and Clark, C.C. (1999) Upregulation of type XIX collagen in rhabdomyosarcoma cells accompanies myogenic differentiation. Exp Cell Res 253:587–598. Nicosia, R.F., Bonanno, E., Smith, M. and Yurchenko, P. (1994) Modulation of angiogenesis in vitro by laminin-entactin complex. Dev Biol 164:197–206. Rando, T.A. and Blau, H.M. (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125:1275–1287. Rao, J.S., Beach, R.L. and Festoff, B.W. (1985) Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis. Biochem Biophys Res Commun 130:440–446. Sabourin, L.A. and Rudnicki, M.A. (2000) The molecular regulation of myogenesis. Clin Genet 57:16–25. Salmivirta, K., Talts, J.F., Olsson, M., Sasaki, T., Timpl, R. and Ekblom, P. (2002) Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens. Exp Cell Res 279:188–201. Sanes, J.R. (2003) The basement membrane/basal lamina of skeletal muscle. J Biol Chem 278:12601–12604. Schuler, F. and Sorokin, L.M. (1995) Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo. J Cell Sci 108:3795–3805. Schymeinsky, J., Nedbal, S., Miosge, N., Poschl, E., Rao, C., Beier, D.R., Skarnes, W.C., Timpl, R. and Bader, B.L. (2002) Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice. Mol Cell Biol 22:6820–6830. Senior, R.M., Gresham, H.D., Griffin, G.L., Brown, E.J. and Chung, A.E. (1992) Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. J Clin Invest 90:2251–2257. Shimokawa, T., Kato, M., Ezaki, O. and Hashimoto, S. (1998) Transcriptional regulation of muscle-specific genes during myoblast differentiation. Biochem Biophys Res Commun 246: 287–292. Solursh, M. and Jensen, K.L. (1988) The accumulation of basement membrane components during the onset of chondrogenesis and myogenesis in the chick wing bud. Development 104:41–49. Springer, M.L. and Blau, H.M. (1997) High-efficiency retroviral infection of primary myoblasts. Somat Cell Mol Genet 23: 203–209. Suelves, M., Lluis, F., Ruiz, V., Nebreda, A.R. and MunozCanoves, P. (2004) Phosphorylation of MRF4 transactivation
582 domain by p38 mediated repression of specific myogenic genes. EMBO J 23:365–375. Sumiyoshi, H., Laub, F., Yoshioka, H. and Ramirez, F. (2001) Embryonic expression of type XIX collagen is transient and confined to muscle cells. Dev Dyn 220:155–162. Timpl, R., Dziadek, M., Fujiwara, S., Nowack, H. and Wick, G. (1983) Nidogen: a new, self-aggregating basement membrane protein. Eur J Biochem 137:455–465. Vilcek, J., Havell, E.A. and Kohase, M. (1976) Superinduction of interferon with metabolic inhibitors: possible mechanisms and practical applications. J Infect Dis 133(Suppl.): A22–A29.
Von der Mark, K. and Ocalan, M. (1989) Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 40:150–157. Yelian, F.D., Edgeworth, N.A., Dong, L.J., Chung, A.E. and Armant, D.R. (1993) Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the ArgGly-Asp (RGD) recognition sequence. J Cell Biol 121:923–929. Zetser, A., Gredinger, E. and Bengal, E. (1999) p38 mitogenactivated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor. J Biol Chem 174:5193–5200.
O RI G INA L AR T I C L E
Ricarda Neu . Stephanie Adams . Barbara Munz
Differential expression of entactin-1/nidogen-1 and entactin-2/ nidogen-2 in myogenic differentiation
Received April 18, 2006; accepted in revised form June 14, 2006
Abstract Here, we show that entactin-2 expression is strongly, but transiently, induced in myogenic differentiation. Treatment of C2C12 myoblasts with actinomycin D in parallel to the induction of differentiation could demonstrate that this is due to enhanced transcription of the entactin-2 gene. Furthermore, treatment with the translation inhibitor cycloheximide could show that entactin-2 is a primary response gene. As p38 MAP kinase is an important regulator of myogenic differentiation, we also analyzed the possibility that entactin-2 might be a target of this pathway. However, using various p38 MAPK inhibitors, we could not detect involvement of p38 in entactin-2 up-regulation. Most remarkably, expression of the entactin-2 homolog entactin-1 dramatically declined in myogenesis, suggesting different functions of the two entactins in this process. A similar effect was seen in primary myoblasts isolated from two different mouse strains. Expression of high levels of entactin-1 in myoblasts using a retroviral expression system led to a higher proliferation rate both in growth and in differentiation medium and to reduced expression of various myogenic differentiation markers after the induction of differentiation. Furthermore, decreased expression of the entactin-2 gene after treatment of the cells with ent-2-specific siRNA preparation led to reduced expression of the cell cycle inhibitor p21. These data suggest important and distinct functions of entactin-1 and -2 in myogenic differentiation. Key words entactin nidogen myogenic differentation . ) Ricarda Neu Stephanie Adams Barbara Munz (* Institute of Physiology Charite´—University Medicine Berlin Arnimallee 22, 14195 Berlin, Germany Tel.: 149 30 8445 1643 Fax: 149 30 8445 1634 E-mail: [email protected] U.S. Copyright Clearance Center Code Statement:
Introduction Entactin-1 and -2 are ubiquitous components of basement membranes. They bind to various other basement membrane proteins, such as collagen IV, laminin, and perlecan, and are cell-adhesive (Carlin et al., 1981; Timpl et al., 1983; Kimura et al., 1998; Kohfeldt et al., 1998). In addition to their function as structural elements of basement membranes, entactins have been implicated in the regulation of cell attachment (Chakravarti et al., 1990), neutrophil chemotaxis (Senior et al., 1992), trophoblast outgrowth (Yelian et al., 1993), angiogenesis (Nicosia et al., 1994), and osteoblast differentiation (Kimura et al., 1998). Both entactin isoforms are glycosylated proteins that consist of three globular domains (G1 to G3) connected by a flexible link and a rod (Fox et al., 1991; Mayer et al., 1995; Kimura et al., 1998; Kohfeldt et al., 1998). In addition to their structural similarities, the expression pattern of the two entactin genes in various embryonic and adult tissues and cultured cells is very similar (Kimura et al., 1998; Kohfeldt et al., 1998; Miosge et al., 2001, 2002; Salmivirta et al., 2002). Furthermore, mice genetically null for either entactin-1 or -2 showed no overt phenotype initially (Murshed et al., 2000; Schymeinsky et al., 2002), although a few years later, Dong et al. (2002) could detect subtle neurologic defects and structural abnormalities of the basement membrane in the entactin-1 knock-out mice. As entactin-2 expression is increased in some tissues of entactin-1 knock-out mice (Murshed et al., 2000; Dong et al., 2002; Miosge et al., 2002; Schymeinsky et al., 2002), these data suggest that entactin-1 and -2 might have at least partially redundant functions. Remarkably, compound genetic ablation of both entactin isoforms leads to perinatal lethality associated with defects in the heart, lung, and kidney morphogenesis, suggesting important
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functions of the entactins in these tissues (Bader et al., 2005). Differentiation of skeletal myoblasts into mature myotubes is a complex and highly organized process (reviewed by Sabourin and Rudnicki, 2000). During embryonic development, skeletal myoblasts differentiate under the influence of various extra- and intracellular factors, such as growth factors, cytokines, proteins of the extracellular matrix, and transcription factors. Although in the past few decades, a large number of proteins have been implicated as crucial modulators of myogenic differentiation, the complexity of this process strongly suggests that more remain to be discovered. Little is known about a possible role of the entactins in myogenic differentiation. In 1998, Godfrey and Gradall demonstrated that during embryonic development, basement membrane proteins such as laminin, collagen IV, and also entactin are concentrated in myogenic regions of the mouse limb bud, suggesting a role of these factors in the early stages of myogenesis. Accordingly, in vitro studies demonstrated that the same basement membrane proteins have a stimulatory effect in the early stages of myogenesis (Langen et al., 2003). Furthermore, entactin alone might promote adhesion and long-term maintenance of skeletal myotubes in culture (Funanage et al., 1992). Nothing is known about isoform-specific functions of the two entactins in myogenesis. However, most interestingly, in contrast to most other organs, the expression patterns of the two entactins in adult heart and skeletal muscle are different (Kohfeldt et al., 1998), suggesting unique functions of the two isoforms in these tissues. Here, we demonstrate that entactin-2 expression is strongly induced in myogenic differentiation, whereas entactin-1 expression decreases after the induction of differentiation, and that high levels of entactin-1 can suppress myogenesis. These data suggest that entactins might have important, but different functions in myogenesis.
Materials and methods Tissue culture Murine C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum (growth medium; GM). Primary mouse C57 and C3H myoblasts were isolated and cultured as described previously (Rando and Blau, 1994). To induce differentiation, cells were grown to 80%–90% confluency and then switched to differentiation medium (DM; Dulbecco’s modified Eagle’s medium containing 2% horse serum). Generation of myoblasts constitutively expressing high levels of ent-1 and ent-2 The full-length wild-type murine entactin-1 and entactin-2 cDNAs were kind gifts from Dr. R. Timpl (Martinsried, Germany) and
Dr. M. Shimane (Chugai Institute for Molecular Medicine, Ibaraki, Japan). They were cloned into the retroviral vector pBabeMN-Iresneo (Morgenstern and Land, 1990). The ecotrophic producer cell line FE, a gift from Dr. Gary Nolan (Stanford University), was transfected with the resulting constructs by using Fugene (Roche, Mannheim, Germany). C2C12 cells were subsequently infected with the viral supernatants at high efficiency, as described previously for primary myoblasts (Springer and Blau, 1997). siRNA technology 2 105 C2C12 myoblasts were trypsinized, resuspended in normal growth medium, and transfected in suspension with 100 nM (final concentration) of two different siRNAs specific for the murine entactin-2 gene (Ambion, Kassel, Germany) or a random (scrambled) control siRNA according to the instructions of the manufacturer. They were then seeded in six-well plates and incubated at 371C for 24 hr. The next morning, the medium was either replaced by fresh growth medium or cells were incubated in DM for another 24 hr. Subsequently, total cellular RNA or protein was isolated from the cells. RNA isolation Total cellular RNA was extracted using the RNeasy RNA isolation kit (Qiagen, Hilden, Germany), and poly(A) RNA was isolated by using oligo(dT) cellulose spin columns (Clontech, Palo Alto, CA) according to the instructions of the manufacturer. Subtractive cDNA library hybridization For subtractive cDNA hybridization, the PCR Select Kit (Clontech) was used according to the instructions of the manufacturer. Northern blot analysis One to eight micrograms of total RNA isolated as described above was separated on a 1% agarose gel containing formaldehyde (2%). Subsequently, RNA was transferred onto a nylon membrane. Filters were hybridized overnight with antisense riboprobes that had been labeled with the digoxigenin RNA labeling kit (Roche). After washing, the blots were incubated with an alkaline phosphatasecoupled antidigoxigenin antibody (Roche), washed, and developed with CDP-Star (Tropix [Bedford, MA] or Roche) as a chemiluminescent substrate for alkaline phosphatase. Signals were detected via exposure to X-ray films (ranging from 2 sec to 5 min). Preparation of protein lysates and Western blot analysis Cultured cells were lysed in lysis buffer (1% Triton X-100, 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA pH 8.0). Ten micrograms of total protein were loaded on a sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were pre-blocked in 3% powdered milk in Tris-buffered saline containing 0.5% Tween 20 (TBS-T) for 30 min; incubated with a 1:1,000 dilution of the first antibody in blocking solution for 1 hr, washed three times with TBS-T; incubated for 30 min with a 1:2,500 dilution of the second antibody, a peroxidase-conjugated anti-rabbit or anti-mouse monoclonal antibody (Amersham, Little Chalfont, UK) in blocking solution; washed three times with TBS-T; and developed with the ECL Western blot detection system (Amersham). Cell proliferation assay Cells were seeded in chamber slides, pulse-labeled with bromodeoxyuridine (BrdU) for 20 min, and stained with the 5-bromo-2 0 -deoxy Labeling and Detection Kit II (Roche).
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Results Entactin-2 expression is induced in myogenic differentiation To identify novel genes that are differentially expressed during early myoblast differentiation, cultured C2C12 myoblasts were induced to differentiate with and without addition of 1 ng of transforming growth factor b1 (TGF-b1)/ml, a potent inhibitor of the differentiation process. Rather than a simple comparison of cells in GM and DM, this approach was chosen to minimize the detection of nonspecific changes in gene expression due to numerous differences in the composition of growth and DM. After 16 hr, RNA was isolated and assayed for differential gene expression by a subtractive cDNA library hybridization approach. Increased myogenin mRNA levels after 16–24 hr and increased myosin heavy chain protein amounts after 48 hr confirmed that the cells underwent differentiation. Differential expression was confirmed by Northern blot analysis. All transcript levels were normalized to mRNA levels of rpl32, a ubiquitous ribosomal protein. One of the genes that exhibited markedly increased expression within a few hours of differentiation was identified as entactin-2/nidogen-2. As shown in Fig. 1, entactin-2 expression was enhanced within the first 16 hr after the induction of differentiation. Specifically, we could detect a two- to threefold increase in entactin-2 mRNA levels as early as 3–7 hr after the addition of DM. Entactin-2 expression levels peaked around 24 hr, and then declined again and reached baseline levels again around 62 hr after the induction of differentiation. These data suggest that expression of the entactin-2 gene is tightly controlled in myogenic differentiation.
Fig. 2 Inhibition of transcription. C2C12 myoblasts were treated with differentiation medium (DM) alone or with DM containing the transcriptional inhibitor Actinomycin D or an equivalent volume of dimethylsulfoxide (DMSO), the solvent for the Actinomycin D. After 20 hr, total cellular RNA was extracted and analyzed via Northern blot for the presence of entactin-2 mRNA. Note the strong induction of entactin-2 gene expression after treatment with DM alone or with DM containing DMSO. By contrast, addition of Actinomycin D to the DM completely abolished this induction. Hybridization with a probe directed against the rpl32 mRNA was used as a loading control as indicated.
Entactin-2 expression is regulated at the transcriptional level To study the mechanism of ent-2 induction, myoblasts were treated with the transcription inhibitor Actinomycin D in parallel to the induction of differentiation. As shown in Fig. 2, this treatment completely abolished ent-2 induction, indicating that gene expression is regulated at the transcriptional level.
Entactin-2 is a primary response gene
Fig. 1 Entactin-2 expression in myogenic differentiation. C2C12 cells were treated with differentiation medium (DM). After the indicated time periods, cells were harvested and total cellular RNA was extracted. Eight microgram of each RNA sample was analyzed via Northern blotting using a probe specific for the murine entactin2 gene (top panel). To monitor the differentiation process, hybridization with a probe recognizing the myogenic differentiation marker myogenin was also performed (bottom panel). Hybridization with a probe directed against the mRNA encoding the ribosomal protein rpl32 was used as a loading control (both panels). Note the strong induction of entactin-2 gene expression after 16 hr in DM. Expression of the myogenin gene is characterized by similar kinetics.
To further analyze the mechanism of regulation of entactin-2 gene expression in myogenic differentiation, C2C12 cells were treated with the translation inhibitor cycloheximide (Cyc) in parallel to the induction of differentiation. As shown in Fig. 3, this treatment did not inhibit up-regulation of the entactin-2 gene. By contrast, a superinduction was observed. This is a phenomenon often seen when treating with translation inhibitors and possibly reflects reduced translation of proteins that destabilize the entactin-2 protein or enhance its degradation (Vilcek et al., 1976). By contrast, as shown in Fig. 3, induction of the gene encoding the myogenic transcription factor myogenin was completely abolished upon Cyc treatment, suggesting that the drug was effective and that myogenin is not a primary response gene in myogenesis.
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Fig. 3 Inhibition of translation. C2C12 myoblasts were treated with differentiation medium (DM) alone or with DM containing the translational inhibitor cycloheximide (Cyc) or dimethylsulfoxide (DMSO), the solvent for Cyc. At the indicated time points, total cellular RNA was extracted and analyzed via Northern blot using an antisense riboprobe directed against the entactin-2 mRNA (top panel). Note the strong induction of entactin-2 gene expression after transfer of the cells to DM, which is not inhibited by Cyc. Expression of the myogenin gene was also analyzed to monitor the differentiation process and as a positive control for the Cyc treatment (bottom panel). Note that myogenin induction was completely abolished upon Cyc treatment. Hybridization with a probe specific for the murine rpl32 mRNA was used as a control for equal loading (both panels).
Entactin-2 expression is not regulated by p38 MAP kinase To identify factors that are responsible for entactin-2 induction, cells were treated with specific inhibitors of the p38 MAP kinase pathway in parallel to the induction of differentiation. Activation of this signaling pathway has been shown to be crucial in myogenesis (Cuenda and Cohen, 1999; Zetser et al., 1999). As shown in Fig. 4, two different p38 inhibitors, SB 202190 and SB203580, did not abolish entactin-2 induction, indicating that this gene is not a target of the p38 pathway, whereas the structurally similar, but non-functional compound SB 202474 had no effect. By contrast, induction of the gene encoding the myogenic transcription factor myogenin was strongly repressed (Fig. 4). These data indicate that in contrast to various genes that regulate myogenesis, the entactin-2 gene is not a target of the p38 MAP kinase pathway.
Fig. 4 Inhibition of p38 MAP kinase. C2C12 myoblasts were treated with differentiation medium (DM) alone, with DM containing specific inhibitors of the p38 MAP kinase pathway (SB 202190 and SB 203580) or a structurally similar, but non-functional control (SB 202474), or with DM containing DMSO, the solvent for SB 202190, SB 202474, and SB 203580. After 18 hr, total cellular RNA was extracted and analyzed for expression of the entactin-2 gene via Northern blot (top panel). Note that induction of entactin-2 gene expression was not inhibited by treatment with the p38 inhibitors. To monitor the differentiation process and as a positive control for treatment with the p38 inhibitors, expression of the myogenin gene was also analyzed in the same samples (bottom panel). Note that myogenin induction was almost completely abolished upon treatment with the two functional p38 inhibitors. As a control for equal loading, both blots were also hybridized with an rpl32-specific probe.
Differential expression of entactin-1 and -2 in primary myoblasts To confirm the physiological relevance of our results, expression of the two entactin genes was also analyzed in primary cells. For this purpose, myoblasts were isolated from skeletal muscle tissue of two different mouse strains: C3H and C57BL6. Similar to the results
Entactin-1 expression decreases in myogenesis As the entactin-2 gene is usually co-expressed with its homolog entactin-1, we speculated that this might also be the case in myogenic differentiation. Thus, we analyzed entactin-1 expression in this process. Surprisingly, we did not find co-expression of the two genes. By contrast, as shown in Fig. 5, entactin-1 expression strongly decreased as early as 16 hr after the induction of differentiation and was hardly detectable after 40 hr. These data suggest that expression of each entactin gene is regulated in a distinct manner in myogenesis, indicating that they might have unique and distinct functions in this process.
Fig. 5 Entactin-1 expression in myogenic differentiation. C2C12 cells were treated with differentiation medium (DM) for the time periods indicated. Total cellular RNA was extracted and analyzed for expression of the entactin-1 gene via Northern blot. Note the strongly decreased expression of entactin-1 after 16 hr in DM (top panel). Expression of the myogenin gene was analyzed to monitor the differentiation process (bottom panel); hybridization with an rpl32-specific probe was used as a control for equal loading.
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High levels of entactin-1 inhibit myogenic differentiation The strong and early regulation of the expression levels of the two entactin genes in myogenesis suggests that entactins might be important regulators of myogenic differentiation. To analyze this question in more detail, we generated cell lines that strongly express the entactin-
C1 lac 2 0h Z0 En h t-1 as En t-1 0h s0 h En t-2 as En 0h t-2 C2 s 0h C1 En 2 24 h t-1 as En t-1 24h s2 4h En t-2 as En 24 h t-2 s2 C2 4h C1 lac 2 48 Z4 h En 8h t-1 as En t-1 48h s4 8h En t-2 as En 48 h t-2 s4 8h
obtained with the myoblast cell line C2C12, entactin-1 expression also decreased upon induction of differentiation of these cells (Fig. 6 and data not shown). By contrast, we could detect a high basal expression, but only a slight induction of entactin-2 expression in these cells upon induction of differentiation (data not shown), which might be due to the fact that these cells had been isolated from neonatal and not embryonic mice and were characterized by a higher degree of differentiation already. In general, the degree and the time course of entactin gene expression in primary myoblasts were variable, depending on the respective mouse strain, the age of the animals the cells were isolated from, and the isolation procedure. However, in all our experiments, we found a strong repression of entactin-1 expression and at least a slight induction of entactin-2 expression after the induction of myogenic differentiation, suggesting that this is a general feature seen in all types of skeletal myoblasts. These data indicate that differential expression of the two entactin genes might be a general and physiologically relevant phenomenon.
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Fig. 6 Expression of the entactin-1 gene in primary myoblasts. Primary myoblasts isolated from neonatal C3H mice were treated with differentiation medium (DM) for 22 hr. Total cellular RNA was analyzed for expression of the entactin-1 gene in comparison with C3H myoblasts that had been maintained in growth medium only. Note the strongly decreased expression of the entactin-1 gene in DM. By contrast, expression of the myogenic differentiation marker myogenin was induced. Hybridization with an rpl32-specific probe served as a loading control.
1 or the entactin-2 gene, in the sense and antisense direction, respectively. For this purpose, we used a retroviral vector system. The advantage of this system is that with one or only a few rounds of infection, more than 99% of the cells can be reached, so that whole-cell populations in contrast to single clones can be analyzed. This approach is superior to clonal analysis, as it minimizes the risk of artifacts due to the long selection procedure of single clones. As shown in Fig. 7, at the mRNA level, the recombinant entactin-1 and entactin-2 genes were expressed at high levels in our cells. By contrast, high levels of antisense entactin-1 and -2 had only a slight inhibitory effect on expression of the endogenous genes (data not shown). By Western blot analysis, we could also detect higher levels of entactin-1 and entactin-2 proteins; however, the effects were weaker than at the mRNA level (data not shown). This might be due to the fact that the entactins are difficult to immunoprecipitate from the C2C12 medium. It is likely that after secretion, they remain attached to the surface of the cells, either directly or indirectly by binding to other proteins of the extracellular matrix (Mann et al., 1989; Kohfeldt et al., 1998; Salmivirta et al., 2002). In general, expression of the recombinant genes declined during differentiation (Fig. 7). This is probably due to silencing of the viral LTR in this process and has been observed earlier (B. Munz and H.M. Blau, unpublished observations). Remarkably, we did not find cross-regulation of the two isoforms or of recombinant and
Ent-1 rpl32
Ent-2 rpl32
Fig. 7 Expression of high levels of the entactin-1 and entactin-2 genes in myoblasts. The full-length murine entactin-1 and entactin-2 cDNAs were cloned into retroviral vectors in sense (s) and antisense (as) direction; specific retroviruses were produced and used to infect C2C12 cells. As a control, cells were also infected with a retrovirus containing the lacZ gene. Cells were grown in growth medium only (0 hr) or in differentiation medium (DM) for the time periods indicated. Proper differentiation of control cells was monitored by repeated light microscopical analysis and by expression analysis of certain myogenic differentiation markers (not shown). Total cellular RNA was extracted and analyzed for ent-1 (top panels) and ent2 (bottom panels) expression via Northern blot. The blots were also hybridized with probes specific for the rpl32 mRNAs. Note the high expression levels of the recombinant entactin-1 and entactin-2 genes. Expression of endogenous entactin-1 and entactin-2 is not visible due to the short exposure time of the respective films.
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Fig. 8 Proliferation and differentiation in cells engineered to express high levels of entactin-1. (A) C2C12 cells (ctrl) and myoblasts transduced with the ent-1 retrovirus were pulse-labeled with bromodeoxyuridine (BrdU) both in proliferation medium and after 24 hr in differentiation medium (dm). BrdU incorporation was then detected using an FITC-coupled anti-BrdU antibody, and the percentage of labeled nuclei relative to the total number of nuclei (visualized via DAPI-staining) is shown. Note the high proliferation rate of myoblasts expressing high levels of ent-1 both in pm and in dm when compared with controls. (B) Myoblasts transduced with the ent-1 retrovirus were cultured in DM for 26 hr and then analyzed for expression of Ki67, p21, and caveolin-3 via Western blot
analysis. Note the strong expression of Ki67 in cells expressing high levels of entactin-1 even in DM and the low expression levels of p21 when compared with control cells, keeping in mind that induction of p21 expression during differentiation of wild-type C2C12 cells does often not occur before at least 48 hr in DM (Andre´s and Walsh, 1996). Furthermore, the strong expression of caveolin-3 in the cells expressing high levels of entactin-1 when compared with controls is obvious. As expected, no caveolin-3 protein could be detected in both cell types when grown in proliferation medium, i.e., before the induction of differentiation. Incubation with an antibody directed against the a-tubulin protein served as a loading control (bottom panel).
endogenous entactin-1 and/or entactin-2 (data not shown), suggesting that in contrast to various other tissues and organs, they might not be functionally redundant in myogenesis. Interestingly, in myoblasts engineered to express high levels of entactin-2, expression of the recombinant gene declined rapidly, even when cells were kept under selection pressure permanently, with a neomycin concentration of 400 mg/ml. This finding indicates that entactin-2 might indeed promote differentiation, so that productively infected cells that express high levels of the recombinant gene withdraw from the cell cycle, initiate differentiation, and are eventually overgrown by the initially small pool of myoblasts (o5% immediately after infection, as judged by lacZ staining) that do not express high levels of recombinant entactin-2. Therefore, it was not possible to further characterize these cells with respect to myoblast proliferation and differentiation. By contrast, cells engineered to express high levels of entactin-1 were characterized by stable expression levels of the recombinant gene over at least 10 passages, even when not kept in selection medium all the time. This finding indicates that entactin-1 does not suppress myogenic differentiation. When cells were pulse-labeled with BrdU, we found that cells engineered to express high levels of entactin-1 had a higher proliferation rate than wild-type myoblasts, both in growth and—to an even greater extent with respect to control myoblasts—in DM (Fig. 8). In addition, myoblasts that express high levels of recombinant entactin-1 were characterized by higher expres-
sion levels of Ki67 specifically in DM (Fig. 8), and lower expression levels of p21 (Fig. 8), indicating that high expression levels of the entactin-1 gene have a positive effect on myoblast proliferation, especially in low-serum medium. Furthermore, when cells were switched to DM, we found that cells engineered to express high levels of entactin-1 showed lower expression levels of certain myogenic differentiation markers such as caveolin-3 (Fig. 8). These results indicate that high levels of entactin-1 can cause an increased myoblast proliferation rate and can inhibit certain aspects of myogenic differentiation. Inhibition of entactin-2 expression leads to decreased expression levels of the cell cycle inhibitor p21 To further study the role of the entactin-2 gene in myogenesis, we inhibited its expression in C2C12 cells using specific siRNA preparations. As shown in Fig. 9, we were able to block expression of the endogenous gene by at least 80% in these cells. When we studied expression of various myogenic differentiation markers, we found a higher expression of myoblast proliferation markers, such as cyclin D1, in the cells that had been treated with the siRNAs (data not shown). By contrast, expression levels of anti-proliferative and differentiation-associated genes, such as p21, were reduced (Fig. 10). These data suggest that induction of entactin-2 expression might indeed play a crucial role in the regulation of myogenic differentiation.
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Fig. 9 Inhibition of entactin-2 gene expression using specific siRNAs. Exponentially growing C2C12 cells were treated with three different ent-2-specific siRNA preparations, with a random control (siRNA control), with transfection medium alone, or left untreated. After 24 hr, total cellular RNA was extracted from the cells and analyzed for expression of the entactin-2 gene by Northern blot. The bottom panel shows a methylene blue stain of the same filter as a control for equal loading.
Discussion
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Muscle basement membrane is produced in part by the myoblasts it ensheates, in part by surrounding cells. Not only does it provide mechanical stability and structural integrity of mature muscle fibers, but it is also involved in the regulation of myogenic differentiation. Interestingly, different basement membrane proteins have ago-
p21
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Fig. 10 Inhibition of entactin-2 expression leads to decreased expression of the p21 gene. C2C12 cells treated with siRNA #1 or control cells were analyzed for expression of the p21 gene immediately after transfection. The bottom panel shows a methylene blue stain of the same blot as a control for equal loading.
nistic, but also antagonistic effects on myogenesis. For example, laminin and collagen IV stimulate differentiation of skeletal muscle cells in culture, whereas fibronectin inhibits this process (von der Mark and Ocalan, 1989; Li et al., 2001, reviewed by Sanes, 2003). From this background, one would expect that expression and secretion of each basement membrane component is tightly and individually controlled in myogenic differentiation. In fact, differential expression during myogenesis has been demonstrated for several individual genes encoding basement membrane proteins: specifically, Rao et al. could demonstrate enhanced synthesis of type IV collagen and laminin, but decreased levels of fibronectin during differentiation of murine skeletal myoblasts in culture as early as in 1985, which is consistent with antagonistic functions of laminin and collagen IV versus fibronectin in this process. Subsequently, a broad variety of studies could demonstrate differential expression of other genes encoding basement membrane proteins, such as type XIX collagen, during myogenesis in vitro and in vivo (Godfrey et al., 1988; Solursh and Jensen, 1988; Mundegar et al., 1995; Schuler and Sorokin, 1995; Godfrey and Gradall, 1998; Gullberg et al., 1999; Myers et al., 1999; Li et al., 2001; Sumiyoshi et al., 2001). We could show that expression of the entactin-2 gene is strongly induced in myogenic differentiation. Most remarkably, expression peaked as early as 24 hr after the induction of differentiation and then declined again, concomitantly to increased myogenin gene expression. This is an expression pattern that resembles that of the myogenic transcription factor myf-5. These results suggest a role of entactin-2 in early myogenesis. Interestingly, several other studies also support the assumption that basement membrane proteins, in contrast to other ECM proteins, might specifically be involved in the regulation of the first few steps of the myogenic program: Solursh and Jensen (1988) and Godfrey and Gradall (1998) could show that laminin and other basement membrane components accumulate in myogenic regions of the chick and mouse limb buds, respectively, before the onset of myogenesis. Furthermore, Langen et al. (2003) carried out in vitro experiments with cultured C2C12 myoblasts that also argue for a role of several basement membrane proteins in early myogenesis. To analyze the mechanism of entactin-2 regulation in myoblasts, we first carried out pulse-labeling studies with the transcription inhibitor Actinomycin D. Thereby, we found that expression of entactin-2 is primarily regulated at the transcriptional level in myoblasts. Interestingly, transcriptional regulation is involved in the regulation of expression of most muscle-specific genes (Shimokawa et al., 1998, and references therein). Furthermore, pulse-labeling studies with Cyc showed that entactin-2 is a primary response gene with respect to myogenic differentiation. This finding indicates that
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entactin-2 expression is not dependent on de novo synthesis of other proteins regulating myogenesis, such as the myogenic transcription factors. From this background, we aimed at analyzing the specific signaling pathways that are responsible for induction of entactin-2 gene expression in myogenesis. Activation of p38 MAP kinase has been shown to be absolutely necessary for myogenic differentiation (Cuenda and Cohen, 1999; Zetser et al., 1999) and expression of several crucial regulators of myogenesis, such as caveolin-3 (Galbiati et al., 1999), myf4 (Suelves et al., 2004), p21, p27, myoD, troponin T (Cabane et al., 2003), and myogenin (our data), is controlled by this pathway. However, using specific inhibitors, we found that entactin-2 expression is not regulated by p38 MAP kinase. Further studies will elucidate which pathways control expression of this gene. A possible candidate is the PI3 kinase/Akt pathway, which is essential for the differentiation of skeletal muscle cells and is functionally linked to the p38 pathway in myogenesis (Li et al., 2000; Gonzalez et al., 2004), especially as it has recently been shown that expression of the genes encoding two other basement membrane proteins, laminin and collagen IV, is controlled by PI3 kinase (Li et al., 2001). Surprisingly, entactin-1 expression decreased in myogenesis. This is in contrast with various other studies that suggest that expression of the two entactin genes is mostly coregulated in human and murine embryonic development as well as in most adult tissues and organs (Kimura et al., 1998; Kohfeldt et al., 1998; Miosge et al., 2001; Salmivirta et al., 2002). Interestingly, however, Kohfeldt et al. (1998) could find spatially distinct expression patterns of the two forms in skeletal and heart muscle, indicating that in contrast to most other tissues and organs, the two entactin isoforms might exert different functions here. In this context, it seems interesting that Schuler and Sorokin (1995) could demonstrate differential expression of the laminin 1 and 2 genes in myogenesis and they also postulate different functions of these two laminin variants in skeletal muscle differentiation. To analyze the function of entactin-1 and -2 in myogenesis, we generated stable myoblast cell lines engineered to express high levels of entactin-1 and -2. In these studies, we did not find cross-regulation of the two entactin isoforms in myoblasts. Specifically, expression of high levels of recombinant entactin-1 or -2 had no effect on expression of the two endogenous variants. This is in contrast with the results obtained when analyzing entactin-1-deficient mice: in these animals, elevated levels of entactin-2 could be found in most tissues and organs, suggesting partial functional redundancy of the two isoforms (Murshed et al., 2000; Miosge et al., 2002). However, a similar result, i.e., induction of entactin-1 expression in entactin-2-deficient animals, cannot be observed (Schymeinsky et al., 2002). Using siRNA technology, we are currently analyzing
the question of a possible cross-regulation of entactin-1 and entactin-2 expression in myoblasts in more detail. Under growth conditions, myoblasts engineered to express high levels of entactin-1 were characterized by a higher proliferation rate when compared with control cells. When we induced differentiation, we found that expression of high levels of the entactin-1 gene had a negative effect on certain aspects of myogenesis, such as exit from the cell cycle or induction of marker genes for myogenic differentiation such as caveolin-3. Unfortunately, due to a putative negative selection pressure for productively infected cells, we were not able to further analyze the growth and differentiation of myoblasts engineered to express high levels of entactin2. Most likely, cell lines that allow induction of expression of the recombinant gene might be the model system of choice here. However, as in contrast to the endogenous entactin-1 gene, the endogenous entactin-2 gene is induced in myogenesis, it is possible that the levels of endogenous entactin-2 are already sufficient to modulate myogenesis maximally, so that an additionally produced recombinant variant might have little effect. To study this question in more detail, we used siRNA technology to inhibit entactin-2 production in myoblasts. Most interestingly, we found that low levels of entactin-2 indeed led to higher expression of proliferation and lower expression of myogenic differentiation markers, suggesting that entactin-2 might be an important modulator of myogenesis. Taken together, these results suggest that differential expression of the two entactin genes is crucial for the regulation of myogenic differentiation, whereby each isoform is likely to have unique functions in this process. In the future, it might be interesting to analyze expression of the two entactin genes in heart and skeletal muscle in vivo under different physiological and pathological conditions. This might have important therapeutical implications. Acknowledgments We thank the late Dr. R. Timpl (Martinsried, Germany) for nidogen-1 and nidogen-2 antibodies and for the full-length murine entactin-1 cDNA, and Dr. Shimane (Ibaraki, Japan) for the full-length murine entactin-2 cDNA. This work was supported by grants from the Sonnenfeld-Stiftung, Berlin, Germany, and from the Deutsche Forschungsgemeinschaft (Mu 1556/3-1) (to B.M.).
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ment membrane protein with binding activity for laminin, collagen IV and cells. EMBO J 8:65–72. Mayer, U., Zimmermann, K., Mann, K., Reinhardt, D., Timpl, R. and Nischt, R. (1995) Binding properties and protease stability of recombinant human nidogen. Eur J Biochem 227:681–686. Miosge, N., Holzhausen, S., Zelent, C., Sprysch, P. and Herken, R. (2001) Nidogen-1 and nidogen-2 are found in basement membranes during human embryonic development. Histochem J 33:523–530. Miosge, N., Sasaki, T. and Timpl, R. (2002) Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice. Matrix Biol 21:611–621. Morgenstern, J.P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18:3587–3596. Mundegar, R.R., von Oertzen, J. and Zierz, S. (1995) Increased laminin A expression in regenerating myofibers in neuromuscular disorders. Muscle Nerve 18:992–999. Murshed, M., Smyth, N., Miosge, N., Karolat, J., Krieg, T., Paulsson, M. and Nischt, R. (2000) The absence of nidogen-1 does not affect murine basement membrane formation. Mol Cell Biol 20:7007–7012. Myers, J.C., Li, D., Rubinstein, N.A. and Clark, C.C. (1999) Upregulation of type XIX collagen in rhabdomyosarcoma cells accompanies myogenic differentiation. Exp Cell Res 253:587–598. Nicosia, R.F., Bonanno, E., Smith, M. and Yurchenko, P. (1994) Modulation of angiogenesis in vitro by laminin-entactin complex. Dev Biol 164:197–206. Rando, T.A. and Blau, H.M. (1994) Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125:1275–1287. Rao, J.S., Beach, R.L. and Festoff, B.W. (1985) Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis. Biochem Biophys Res Commun 130:440–446. Sabourin, L.A. and Rudnicki, M.A. (2000) The molecular regulation of myogenesis. Clin Genet 57:16–25. Salmivirta, K., Talts, J.F., Olsson, M., Sasaki, T., Timpl, R. and Ekblom, P. (2002) Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens. Exp Cell Res 279:188–201. Sanes, J.R. (2003) The basement membrane/basal lamina of skeletal muscle. J Biol Chem 278:12601–12604. Schuler, F. and Sorokin, L.M. (1995) Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo. J Cell Sci 108:3795–3805. Schymeinsky, J., Nedbal, S., Miosge, N., Poschl, E., Rao, C., Beier, D.R., Skarnes, W.C., Timpl, R. and Bader, B.L. (2002) Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice. Mol Cell Biol 22:6820–6830. Senior, R.M., Gresham, H.D., Griffin, G.L., Brown, E.J. and Chung, A.E. (1992) Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. J Clin Invest 90:2251–2257. Shimokawa, T., Kato, M., Ezaki, O. and Hashimoto, S. (1998) Transcriptional regulation of muscle-specific genes during myoblast differentiation. Biochem Biophys Res Commun 246: 287–292. Solursh, M. and Jensen, K.L. (1988) The accumulation of basement membrane components during the onset of chondrogenesis and myogenesis in the chick wing bud. Development 104:41–49. Springer, M.L. and Blau, H.M. (1997) High-efficiency retroviral infection of primary myoblasts. Somat Cell Mol Genet 23: 203–209. Suelves, M., Lluis, F., Ruiz, V., Nebreda, A.R. and MunozCanoves, P. (2004) Phosphorylation of MRF4 transactivation
582 domain by p38 mediated repression of specific myogenic genes. EMBO J 23:365–375. Sumiyoshi, H., Laub, F., Yoshioka, H. and Ramirez, F. (2001) Embryonic expression of type XIX collagen is transient and confined to muscle cells. Dev Dyn 220:155–162. Timpl, R., Dziadek, M., Fujiwara, S., Nowack, H. and Wick, G. (1983) Nidogen: a new, self-aggregating basement membrane protein. Eur J Biochem 137:455–465. Vilcek, J., Havell, E.A. and Kohase, M. (1976) Superinduction of interferon with metabolic inhibitors: possible mechanisms and practical applications. J Infect Dis 133(Suppl.): A22–A29.
Von der Mark, K. and Ocalan, M. (1989) Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 40:150–157. Yelian, F.D., Edgeworth, N.A., Dong, L.J., Chung, A.E. and Armant, D.R. (1993) Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the ArgGly-Asp (RGD) recognition sequence. J Cell Biol 121:923–929. Zetser, A., Gredinger, E. and Bengal, E. (1999) p38 mitogenactivated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor. J Biol Chem 174:5193–5200.