|
|
|
|
 |
|||
|
|||
doi:10.1369/jhc.5A6675.2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Volume 54 (4): 375-384, 2006
Copyright ©The Histochemical Society, Inc.
Sphingomyelin Levels in the Plasma Membrane Correlate with the Activation State of Muscle Satellite Cells
Department of Life Sciences, The University of Tokyo, Tokyo, Japan (YN,RM); Muscle Cell Biology Group, Medical Research Council Clinical Sciences Centre, Imperial College, London, United Kingdom (YN,PSZ); Randall Division of Cell and Molecular Biophysics, King's College London School of Biomedical and Health Sciences, Guy's Campus, London, United Kingdom (PSZ); Department of Molecular Biodynamics, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (HK,MU); Research Laboratory, Zenyaku Kogyo Co. Ltd., Tokyo, Japan (NO,SK)
Correspondence to: Dr. Ryoichi Matsuda, Department of Life Sciences, The University of Tokyo, Rm. 15-309A, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. E-mail: cmatsuda{at}mail.ecc.u-tokyo.ac.jp
 |
Summary |
|---|
 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Satellite cells are responsible for postnatal growth, hypertrophy, and regeneration of skeletal muscle. They are normally quiescent, and must be activated to fulfill these functions, yet little is known of how this is regulated. As a first step in determining the role of lipids in this process, we examined the dynamics of sphingomyelin in the plasma membrane. Sphingomyelin contributes to caveolae/lipid rafts, which act to concentrate signaling molecules, and is also a precursor of several bioactive lipids. Proliferating or differentiated C2C12 muscle cells did not bind lysenin, a sphingomyelin-specific binding protein, but noncycling reserve cells did. Quiescent satellite cells also bound lysenin, revealing high levels of sphingomyelin in their plasma membranes. On activation, however, the levels of sphingomyelin drop, so that lysenin did not label proliferating satellite cells. Although most satellite cell progeny differentiate, others stop cycling, maintain Pax7, downregulate MyoD, and escape immediate differentiation. Importantly, many of these Pax7-positive/MyoD-negative cells also regained lysenin binding on their surface, showing that the levels of sphingomyelin had again increased. Our observations show that quiescent satellite cells are characterized by high levels of sphingomyelin in their plasma membranes and that lysenin provides a novel marker of myogenic quiescence. (J Histochem Cytochem 54:375–384, 2006)
Key Words: satellite cell • stem cell • skeletal muscle • activation • regeneration • sphingolipid • sphingomyelin • lysenin
|
Introduction |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
SKELETAL MUSCLE CONTAINS both differentiated myofibers and stem cells, termed satellite cells. Myofibers are long, multinucleated cells specialized for rapid force generation. Muscle satellite cells are mononucleated and closely associated with myofibers, being located within the basal lamina that surrounds each myofiber (Mauro 1961
). Satellite cells are responsible for postnatal growth, hypertrophy, and repair of skeletal muscle [reviewed in Zammit and Beauchamp (2001)]. In adult muscle, satellite cells are mitotically quiescent (Schultz et al. 1978), but can be activated to enter the cell cycle and produce myogenic precursor cells that then differentiate and fuse into multinucleated myotubes or existing myofibers (Snow 1977,1978). The numbers and replicative capacity of satellite cells are severely reduced in myopathic conditions such as Duchenne muscular dystrophy (Schultz and Jaryszak 1985; Webster and Blau 1990) showing the importance of regulated satellite cell activation for maintaining functional skeletal muscle.
Signals released from crushed myofibers, invading macrophages, and connective tissue have been implicated in the initiation of satellite cell activation [reviewed in Charge and Rudnicki (2004)]. However, the molecular mechanisms responsible for the transduction of such extracellular signals in satellite cells remain poorly defined and the potential role of lipid-mediated signaling has not previously been considered in this context.
Lipids have been recognized to play vital roles in various cellular functions. For example, phosphatidylserine is involved in myoblast fusion (van den Eijnde et al. 2001), and phosphatidylethanolamine is essential in cytokinesis (Emoto and Umeda 2000). Although differences in phospholipid composition during myoblast differentiation and fusion have been reported (Kent et al. 1974; Sessions and Horwitz 1983; Anderson 1991; Pediconi et al. 1992), the dynamics of phospholipid composition during satellite cell activation remains unknown.
Sphingomyelin is an integral lipid component of cell membranes in animals. Although sphingomyelin is located in intracellular membranes such as those of the endosomes, lysosomes, and Golgi apparatus and nucleus, significant amounts are also found in the plasma membrane [reviewed in van Meer and Holthuis (2000)]. Sphingomyelin in the plasma membrane forms lipid microdomains with other sphingolipids, cholesterols, and proteins. It has been suggested that these rafts or caveolae lipid microdomains facilitate cytoplasmic signaling by acting to concentrate signaling molecules [reviewed in Simons and Toomre (2000)]. Moreover, sphingomyelin in the plasma membrane also acts as a reservoir of bioactive sphingolipids. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate, are emerging as important regulators of a variety of cellular events, including cell proliferation, differentiation, and apoptosis [reviewed in Ohanian and Ohanian (2001); Spiegel and Milstien (2003)]. Despite the importance of these sphingomyelin metabolites, few studies have attempted to examine the dynamics of cellular sphingomyelin, and those that have, have relied on indirect biochemical analysis of extracted lipids lacking single-cell resolution [e.g., Kent et al. (1974)].
In this study, we focus on sphingomyelin levels in the plasma membrane of myogenic cells using lysenin, a protein isolated from the earthworm Eisenia foetida [Sekizawa et al. (1997); reviewed in Kobayashi et al. (2004)]. Because lysenin binds specifically to sphingomyelin (Yamaji et al. 1998), we used it to directly assay sphingomyelin levels in individual myogenic cells as they activate from quiescence. We initially used C2 reserve cells, which model myogenic cell quiescence (Yoshida et al. 1998). Lysenin bound selectively to quiescent reserve cells, but not cycling and differentiated cells, indicating sphingomyelin levels fell with activation. Quiescent satellite cells also had robust lysenin immunostaining, showing that sphingomyelin levels were also high on myogenic cells in vivo. During satellite cell activation and subsequent proliferation, however, sphingomyelin levels became practically undetectable. Some satellite cells maintained in culture adopt characteristics consistent with a return to a quiescent-like state (Zammit et al. 2004). Importantly, many of these cells also regained lysenin binding, showing that sphingomyelin levels in their plasma membranes had increased to levels seen in quiescent satellite cells.
|
Materials and Methods |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Cell Culture
C2C12 myogenic cells (Yaffe and Saxel 1977
; Blau et al. 1983) were maintained in growth medium comprising DMEM (Gibco; Grand Island, NY) containing 20% FBS (Equitec-Bio; Kerrville, TX), 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma; St. Louis, MO) at 37C in 5% CO2. To induce differentiation and produce reserve cells, 5 x 104 cells were seeded on a 35-mm tissue culture dish in growth medium, and, 24 hr later, the medium was replaced with serum-free differentiation medium [DMEM supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, and 1 mg/ml BSA (Sigma)] . To isolate reserve cells from myotubes, C2C12 cells were cultured in differentiation medium for 4–5 days and then all cells were detached using with 0.05% trypsin-0.53 mM EDTA (Gibco; Grand Island, NY) for 5 min at 37C. Cells were then plated in fresh 20% FBS/DMEM in new Petri dishes. After 30 min at 37C, cells were gently rinsed with PBS to remove floating cells (mostly myotubes) and further cultured in 20% FBS/DMEM.
Phospholipid Analysis
To determine the phospholipid content, 8 x 105 C2C12 cells were plated per 300-mm tissue culture dish in growth medium, and 24 hr later, the medium was switched to serum-free differentiation medium. Four days later, reserve cells and myotubes were separated using a modified version of the method of Kitzmann et al. (1998). Briefly, cells were rinsed with PBS containing 0.1 g/liter MgCl2/6H2O and 0.13 g/liter CaCl2/2H2O before incubation with 0.05% trypsin (without EDTA) at room temperature for 5–10 min. Myotubes detach under these conditions and were collected. Residual cells remained attached and were then rinsed with PBS and collected using standard trypsin-EDTA, as detailed previously. Lipids were then extracted from each fraction according to the method of Bligh and Dyer (1959) and were separated on a silica gel 60 TLC plate (Merck; Darmstadt, Germany) using chloroform/methanol/acetic acid/water (100:75:7:4, v/v). Lipids were then visualized with iodine vapor and compared with those of standards before being assayed for phosphorous content with malachite green reagent (Sigma) (Zhou and Arthur 1992).
Isolation and Culture of Mouse Myofibers
Single myofibers with associated satellite cells were prepared as previously described (Rosenblatt et al. 1995). Briefly, adult (
8 weeks old) C57BL/6 mice were killed by cervical dislocation and the extensor digitorum longus muscle was carefully removed. After washing with PBS, the muscles were immersed in 0.6% Collagenase Type I (Worthington Biochemical; Lakewood, NJ) in DMEM and incubated at 38C for 100 min with agitation. Single myofibers were then liberated from the muscle by repeatedly triturating with a Pasteur pipette and then washed in several changes of DMEM. For the analysis of quiescent satellite cells, myofibers were immediately fixed by the addition of 4% paraformaldehyde/PBS for 10 min before washing in PBS. To activate the associated satellite cells, myofibers were cultured in suspension in DMEM containing 10% horse serum/0.5% chick embryo extract at 37C and 5% CO2 for up to 72 hr, and then fixed as described previously.
Lysenin Probing and Immunostaining
To label sphingomyelin with lysenin (Sekizawa et al. 1997; Yamaji et al. 1998), cultured cells or myofibers were fixed with 4% paraformaldehyde/PBS for 10 min, blocked with 2% BSA/PBS, and then incubated with 0.5 µg/ml lysenin (Peptide Institute Inc; Osaka, Japan, or Sigma) in 2% BSA/PBS for 60 min. To remove sphingomyelin from the plasma membrane, cells were pretreated with 10 mU/ml bacterial sphingomyelinase from Bacillus cereus (Sigma) at 37C for 1 hr before incubation with lysenin. Where used, BrdU (10 µM) was added to the cultures for 3 hr before fixation.
For immunostaining, fixed cells were permeabilized with 0.5% TritonX-100. For BrdU detection, cells were then treated with 3 N hydrochloric acid for 10 min at room temperature. Cells were then incubated with primary antibodies (mouse monoclonal anti-MyoD clone 5.8A [Dakocytomation; Carpinteria, CA]), anti-myogenin clone F5D (a gift from Dr. W. Wright at University of Texas), anti-sarcomeric myosin heavy chain (sMyHC) clone MF20, anti-BrdU clone G3G4 and anti-PAX7 (Developmental Studies Hybridoma Bank; Iowa City, IA), hamster monoclonal anti-Bcl-2 clone 3F11 (BD Pharmingen; San Diego, CA), and rabbit polyclonal anti-lysenin (Sekizawa et al. 1996) (Peptide Institute Inc; Osaka, Japan). After washes in PBS, primary antibody binding was visualized with Alexa Fluor dye-conjugated secondary antibodies (Molecular Probes; Eugene, OR) for 30 min before washing and mounting in Fluoromount fluorescent mounting medium (DakoCytomation) containing 100 ng/ml 4',6-diamidino-2-phenylindole (DAPI) or Hoechst 33258. Myofibers were immunostained as above and mounted in Fluoromount fluorescent mounting medium (DakoCytomation) containing 100 ng/ml DAPI.
Western Blotting
Cells were lysed in SDS-sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1 mM phenylmethansulfonylfluoride, bromophenol blue), followed by boiling for 3 min. Fifty µg of protein were analyzed by 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with Odyssey Blocking Buffer (LI-COR; Lincoln, NE) and incubated for 1 hr with primary antibodies. Membranes were washed and incubated with Alexa-Fluor 680-conjugated secondary antibodies and analyzed with an Odyssey Infrared Imaging System (LI-COR).
|
Results |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
To detect cell surface sphingomyelin, we used lysenin, a peptide isolated from the earthworm Eisenia foetida [Sekizawa et al. (1997)
; reviewed in Kobayashi et al. (2004)], which binds specifically to sphingomyelin (Yamaji et al. 1998). We first examined the levels of sphingomyelin on the surface of proliferating C2C12 cells maintained in high-serum medium by probing fixed cells with 0.5 µg/ml lysenin. Subsequent immunostaining using a specific anti-lysenin antibody (Sekizawa et al. 1996) showed that sphingomyelin on the surface of proliferating C2C12 cells was barely detectable (Figures 1A
and 1B and quantified in Figure 2A
).
|
|
When C2C12 cells were switched to differentiation medium, lysenin-positive (lysenin+ve) cells appeared within 24 hr (Figures 1C and 1D and quantified in Figure 2A). At this time, the lysenin+ve cell population contained both MyoD+ve and MyoD-negative (MyoD-ve) cells (Figures 1C and 1D and quantified in Figure 2A). After 4 days however, lysenin+ve mononucleated cells invariably became MyoD-ve (Figures 1E and 1F and quantified in Figure 2A). On serum deprivation, most C2C12 cells undergo terminal differentiation (Figure 2B), but differentiated mononucleated cells identified with myogenin (Figures 1G and 1H) and multinucleated myotubes (Figures 1E, 1F, and 1K) did not label with lysenin. Differentiation, however, is not the only response to serum withdrawal. Other myogenic cells stop dividing, downregulate MyoD, and escape immediate differentiation to form mononucleated reserve cells (Yoshida et al. 1998
). Because the mononucleated cells that bound lysenin after 4 days in differentiation medium were also MyoD-ve (Figures 1E and 1F and quantified in Figure 2A) and did not incorporate BrdU and so were no longer cycling (Figures 1I and 1J), these cells were identified as reserve cells. Because only 8.1 ± 1.2% (mean ± SEM, n=3) lysenin+ve cells incorporated BrdU on Day 1 and virtually none did on Day 2 (0.3 ± 0.3%, mean ± SEM, n=3), lysenin+ve cells rapidly exit the cell cycle after serum withdrawal to become reserve cells. This is also supported by the observation that the percentage of lysenin+ve cells did not increase after Day 1 (Figure 2B). To ensure that lysenin was faithfully reporting sphingomyelin levels, reserve cells were treated with 10 mU/ml bacterial sphingomyelinase for 60 min at 37C to digest sphingomyelin, and then incubated with 0.5 µg/ml lysenin. As expected, lysenin+ve immunosignals disappeared when reserve cells were pretreated with bacterial sphingomyelinase, confirming that the lysenin specifically labels sphingomyelin (Figures 1K and 1L). Thus sphingomyelin is highly expressed in reserve cells, but significantly less so in proliferating and differentiating myogenic cells.
When stimulated with serum, reserve cells reinduce MyoD expression, proliferate, and subsequently differentiate (Yoshida et al. 1998). Reserve cells were separated from differentiated cells and reseeded in fresh growth medium. Lysenin binding was still present on the surface of stimulated reserve cells after 60 min, when most of the cells remained MyoD-ve (Figures 3A
and 3B and quantified in Figure 3I), and also after 3 hr, when MyoD expression was beginning to be induced (Figures 3C and 3D and quantified in Figure 3I). By 24 hr, however, significant number of cells did not bind lysenin (Figures 3E and 3F and quantified in Figure 3I), and by 48 hr practically no cells did (Figures 3G and 3H and quantified in Figure 3I). Therefore, sphingomyelin levels in the plasma membrane of reserve cells decrease as the cells are stimulated to proliferate.
|
Proportion of Sphingomyelin is Higher in Reserve Cells than in Myotubes
Changes in the other components of the plasma membrane, such as glycolipids and cholesterols, can interfere with the efficient binding of lysenin to sphingomyelin (Yamaji et al. 1998
, Ishitsuka et al. 2004). To ensure therefore that the differences in lysenin binding observed during reserve cell activation accurately reflects a drop in sphingomyelin levels, we directly quantified their phospholipid composition. After 4 days in serum-free differentiation medium, many myotubes were present, as identified by sMyHC content (Figure 4A
) and morphology (Figure 4C), whereas reserve cells expressed Bcl-2 (Figure 4B). Cultures were partially trypsinized to separate myotubes from reserve cells (Kitzmann et al. 1998). Western blot analysis confirmed that this separation was effective with sMyHC, myogenin, and MyoD detectable in both isolated myotubes and total C2C12 cultures, whereas Bcl-2 was enriched in the reserve cell fraction as expected (Dominov et al. 1998) (Figure 4D). Analysis of separated myotube and reserve cell fractions for total phospholipid composition using thin layer chromatography showed that sphingomyelin made up 7.4 mol% of the total phospholipid content in reserve cells, but only 3.8 mol% in myotubes (Table 1
). This increase in sphingomyelin content of noncycling reserve cells is consistent with the increase of sphingomyelin in the plasma membrane, as revealed by the binding of lysenin to their surface (Figures 1E and 1F).
|
-tubulin.
|
High Levels of Sphingomyelin in the Plasma Membrane Are Characteristic of Quiescent Satellite Cells
Our observations on reserve cells show that sphingomyelin levels in the plasma membrane of noncycling myogenic reserve cells are high, but then fall as they activate. To see if quiescent myogenic cells in vivo also have high levels of sphingomyelin in their plasma membranes, adult extensor digitorum longus myofibers and their associated quiescent satellite cells were isolated, immediately fixed, and incubated in 0.5 µg/ml lysenin. Immunostaining showed that quiescent satellite cells on the edge of the myofiber, identified by Pax7 expression (Seale et al. 2000
), had strong cell surface lysenin labeling (Figures 5A
–5C), showing that they too have high levels of sphingomyelin in their plasma membranes. Notably, almost all (98.2 ± 0.8%, mean ± SEM, n=60 myofibers) Pax7+ve quiescent satellite cells were lysenin+ve, whereas 96.9 ± 1.0% (mean ± SEM, n=60) of lysenin+ve satellite cells were Pax7+ve.
|
Sphingomyelin Levels Drop during Satellite Cell Activation
Next, we investigated the levels of sphingomyelin during satellite cell activation. Incubation of isolated myofibers in serum stimulates the associated satellite cells to activate, as shown by the induction of MyoD (Yablonka-Reuveni and Rivera 1994
; Beauchamp et al. 2000). Coimmunostaining of quiescent satellite cells showed that the majority were lysenin+ve and MyoD-ve (Figures 5D–5F and quantified in Figures 5M and 5N), but, on stimulation, MyoD+ve satellite cells rapidly appeared, with a concomitant drop in the number of cells binding lysenin (Figures 5G–5I and quantified in Figures 5M and 5N). After 48 hr, lysenin staining was absent from virtually all activated MyoD+ve satellite cells (Figures 5J–5L and quantified in Figures 5M and 5N). Satellite cells immunostaining for lysenin and myogenin were mutually exclusive, showing that sphingomyelin levels drop significantly before the cells enter terminal differentiation (data not shown).
Some Satellite Cells Again Increase the Level of Sphingomyelin in Their Plasma Membranes
We have recently shown that when satellite cells are activated on an isolated myofiber, most are destined for differentiation. Some, however, maintain Pax7, downregulate MyoD, and stop cycling (Zammit et al. 2004), thus reacquiring characteristics of quiescence. Although lysenin immunostaining was universally lost in activated/proliferating satellite cells (Figures 5J–5L and quantified in Figures 5M and 5N), some satellite cell progeny later reacquired lysenin binding (Figure 6
). Lysenin+ve cells tended to express Pax7 (Figures 6A and 6B), but not MyoD (Figures 6C and 6D). At this time in culture, most MyoD+ve satellite cells also express myogenin (data not shown; Zammit et al. 2004) and are thus committed to differentiation. Therefore the increased levels of sphingomyelin in the plasma membrane of Pax7+ve/MyoD-ve satellite cell progeny to levels characteristic of quiescent satellite cells is further evidence that some satellite cells have returned to a quiescent-like state.
|
|
Discussion |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
In this study, we have examined the levels of sphingomyelin at the cell surface of myogenic cells using the sphingomyelin-specific binding protein lysenin (Yamaji et al. 1998
). Cycling C2C12 cells are either MyoD+ve or MyoD-ve (Yablonka-Reuveni and Rivera 1997), but do not bind lysenin, showing that proliferating myogenic cells have little sphingomyelin in their plasma membranes. This remains the case in differentiated myocytes and myotubes. However, other myogenic cells respond to serum withdrawal by downregulating MyoD and escaping immediate differentiation to become reserve cells (Yoshida et al. 1998). In the course of this process, the levels of sphingomyelin in their plasma membranes of reserve cells significantly increased. Indeed, direct measurement showed that reserve cells contained almost twice as much sphingomyelin as myotubes. The differences in the level of sphingomyelin may appear smaller than expected from lysenin staining, but only 50% of cellular sphingomyelin is located in the plasma membrane [reviewed in van Meer and Holthuis (2000)], with the rest in intracellular membranes. In addition, the low levels of sphingomyelin on the surface of myotubes may be below the level of detection with lysenin at the concentration used in this study. Taken together, our data suggest that quiescence is accompanied by a specific increase in sphingomyelin levels in the plasma membrane. We are examining the changes in sphingomyelin metabolism during the activation of satellite cells to determine how much breakdown or synthesis of sphingomyelin occurs during this event.
Muscle satellite cells contribute myogenic cells to growing muscle to provide new myonuclei, but then become quiescent in normal adult muscle (Schultz et al. 1978). The rapid isolation of intact myofibers allows their associated satellite cells to be examined while still quiescent (Yablonka-Reuveni and Rivera 1994; Beauchamp et al. 2000). Using this preparation, we found that virtually all quiescent satellite cells bound lysenin, demonstrating that they too have high levels of sphingomyelin in their plasma membranes. The culture of myofibers in mitogen-rich medium stimulates their associated satellite cells to activate, proliferate, and subsequently differentiate, and this process is accompanied by a significant decrease of sphingomyelin on their cell surface. However, although lysenin is useful for identifying quiescent satellite cells on isolated myofibers, it should be noted that sphingomyelin is present to varying degrees in the membranes of all cells. Significantly, sphingomyelin levels fall rapidly on activation, whereas most other commonly used molecular markers for satellite cells, including M-cadherin, Pax7, and the Myf-5/ß-galactosidase fusion protein, persist in proliferating cells (Irintchev et al. 1994; Beauchamp et al. 2000; Seale et al. 2000).
Why is the transition from quiescence to activation in myogenic cells associated with a change in the levels of sphingomyelin in the plasma membrane? Because activation itself is rapid, the change in sphingomyelin observed in this study might be secondary to the activation. It would be important to examine the relationship between sphingomyelin and the certain stimuli known to involved in satellite cell activation such as hepatocyte growth factor (Allen et al. 1995), mechanical stretch (Tatsumi et al. 2001), and nitric oxide (Anderson and Pilipowicz 2002). One possible role for sphingomyelin in the plasma membrane is to act to concentrate signaling molecules. It is well established that sphingolipids, together with cholesterol and signaling molecules, are organized into lateral assemblies within cell membranes. These assemblies, including lipid rafts and caveolae, are emerging as important centers for cell signaling [reviewed in Simons and Toomre (2000)] and could act as platforms to coordinate molecules needed to initiate myogenic cell activation. Alternatively, the loss of sphingomyelin may reflect its metabolism to produce bioactive lipids. It has been proposed that sphingomyelin in the plasma membrane acts as a store that can then be cleaved to generate ceramide, sphingosine, or sphingosine-1-phosphate, which have been shown to act as second messengers in a variety of cell types (Ohanian and Ohanian 2001). Ceramide, generated by agonist-induced sphingomyelin hydrolysis, has been implicated in cell differentiation, growth arrest, and apoptosis (Hannun 1996). Ceramide is further metabolized into sphingosine-1-phosphate, which is mitogenic in diverse cell types and has been shown to oppose ceramide-mediated apoptosis (Spiegel and Milstien 2003). Recent studies have revealed that sphingolipids are also active in skeletal muscle. Sphingosine controls muscle contraction by regulating calcium concentration in myofibers (Sabbadini et al. 1999), whereas ceramide has an inhibitory effect on insulin-like growth factor-I–induced protein synthesis in mouse myogenic C2C12 cells (Strle et al. 2004). Neutral sphingomyelinase catalyses the cleavage of sphingomyelin to produce bioactive lipid metabolites and is present in skeletal muscle (Ghosh et al. 1998); therefore, the sphingomyelin in the plasma membrane could be accessed for signaling purposes. It is then intriguing to speculate that bioactive lipids may also play a role in satellite cell activation.
When both reserve cells and satellite cells were stimulated with serum, sphingomyelin levels in their plasma membranes dropped significantly. However, at later times in culture, a limited number of satellite cell progeny reacquired lysenin binding, showing that the level of sphingomyelin in their plasma membranes had again increased. The effective response to repeated injury shows that the satellite cell pool is maintained (Sadeh et al. 1985; Luz et al. 2002), but there is debate at present about how this is achieved. It has been proposed that satellite cells may be part of a hierarchical system and merely represent a committed myogenic precursor that is restricted to providing myonuclei. In this system, the replacement of satellite cells occurs from a stem cell located within the muscle interstitium (Gussoni et al. 1999; Asakura et al. 2002) or outside muscle tissue (Fukada et al. 2002; LaBarge and Blau 2002), but there is no evidence that this accounts for more than a very minor contribution. More likely is that satellite cells self-renew, as originally proposed by Moss and Leblond (1971). Indeed we have recently shown that satellite cell progeny maintained on isolated myofibers adopt divergent fates in culture. When they activate, satellite cells coexpress Pax7 with MyoD and most then proliferate, downregulate Pax7, and differentiate. In contrast, other proliferating cells maintain Pax7 but lose MyoD and withdraw from both the cell cycle and immediate myogenic differentiation, characteristics consistent with a quiescent state (Zammit et al. 2004). Similar observations have been made in chicken during muscle growth, showing that this may be a widespread mechanism in vertebrates (Halevy et al. 2004). The demonstration that some of these Pax7+ve/MyoD-ve cells also reacquire lysenin binding on their surface, a characteristic of quiescence in myogenic cells, is further evidence that these cells have reentered a quiescent state.
In conclusion, sphingomyelin levels are high in the plasma membrane of quiescent satellite cells and then fall as they activate and proliferate. These results implicate lipid rafts/caveolae as platforms to coordinate activation signals or the involvement of bioactive lipid metabolism in the process of activation and the subsequent return to quiescence. This increase in sphingomyelin levels on some satellite cells that appear to be reentering quiescence supports the conclusion that they are able to self-renew. Assaying the sphingomyelin level in the plasma membrane using lysenin therefore provides a positive marker of quiescence that actually drops during activation, in contrast to most other molecular markers of satellite cells.
|
Acknowledgments |
|---|
This work was supported by a research grant (11B-1) for Nervous and Mental Disorders and by a grant for research in Brain Science from the Ministry of Health, Labor and Welfare, Japan, and by a grant from the Fugaku Trust for Medical Research. The Japan Scholarship Foundation funded Y.N., whereas P.S.Z. was supported by The Muscular Dystrophy Association and the Medical Research Council.
We thank Jon Beauchamp and Terry Partridge for invaluable discussions and their comments on the manuscript and Drs. Y. Miyagoe-Suzuki and S. Takeda (National Institute of Neuroscience, National Center of Neurology and Psychiatry) for advice on single myofiber isolation. P.S.Z. acknowledges the invaluable assistance of the late, great John Peel. The monoclonal antibodies, MF20, G3G4, and PAX7 were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa.
|
Footnotes |
|---|
Received for publication March 2, 2005; accepted September 14, 2005
|
Literature Cited |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Allen RE, Sheehan SM, Taylor RG, Kendall TL, Rice GM (1995) Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro. J Cell Physiol 165:307–312[CrossRef][Medline]
Anderson J, Pilipowicz O (2002) Activation of muscle satellite cells in single-fiber cultures. Nitric Oxide 7:36–41[CrossRef][Medline]
Anderson JE (1991) Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle. Biochem Cell Biol 69:835–841[Medline]
Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA (2002) Myogenic specification of side population cells in skeletal muscle. J Cell Biol 159:123–134
Beauchamp JR, Heslop L, Yu DS, Tajbakhsh S, Kelly RG, Wernig A, Buckingham ME, et al. (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151:1221–1234
Blau HM, Chiu CP, Webster C (1983) Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell 32:1171–1180[CrossRef][Medline]
Bligh EG, Dyer WJ (1959) A rapid method of lipid extraction and purification. Can J Biochem Physiol 37:911–917
Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84:209–238
Dominov JA, Dunn JJ, Miller JB (1998) Bcl-2 expression identifies an early stage of myogenesis and promotes clonal expansion of muscle cells. J Cell Biol 142:537–544
Emoto K, Umeda M (2000) An essential role for a membrane lipid in cytokinesis: regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine. J Cell Biol 149:1215–1224
Fukada S, Miyagoe-Suzuki Y, Tsukihara H, Yuasa K, Higuchi S, Ono S, Tsujikawa K, et al. (2002) Muscle regeneration by reconstitution with bone marrow or fetal liver cells from green fluorescent protein-gene transgenic mice. J Cell Sci 115:1285–1293
Ghosh N, Sabbadini R, Chatterjee S (1998) Identification, partial purification, and localization of a neutral sphingomyelinase in rabbit skeletal muscle: neutral sphingomyelinase in skeletal muscle. Mol Cell Biochem 189:161–168[CrossRef][Medline]
Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, et al. (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401:390–394[CrossRef][Medline]
Halevy O, Piestun Y, Allouh MZ, Rosser BW, Rinkevich Y, Reshef R, Rozenboim I, et al. (2004) Pattern of Pax7 expression during myogenesis in the posthatch chicken establishes a model for satellite cell differentiation and renewal. Dev Dyn 231:489–502[CrossRef][Medline]
Hannun YA (1996) Functions of ceramide in coordinating cellular responses to stress. Science 274:1855–1859
Irintchev A, Zeschnigk M, Starzinski-Powitz A, Wernig A (1994) Expression pattern of M-cadherin in normal, denervated, and regenerating mouse muscles. Dev Dyn 199:326–337[Medline]
Ishitsuka R, Yamaji-Hasegawa A, Makino A, Hirabayashi Y, Kobayashi T (2004) A lipid-specific toxin reveals heterogeneity of sphingomyelin-containing membranes. Biophys J 86:296–307
Kent C, Schimmel SD, Vagelos PR (1974) Lipid composition of plasma membranes from developing chick muscle cells in culture. Biochim Biophys Acta 360:312–321[Medline]
Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJ, Fernandez A (1998) The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142:1447–1459
Kobayashi H, Ohta N, Umeda M (2004) Biology of lysenin, a protein in the coelomic fluid of the earthworm Eisenia foetida. Int Rev Cytol 236:45–99[Medline]
LaBarge MA, Blau HM (2002) Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 111:589–601[CrossRef][Medline]
Luz MA, Marques MJ, Santo Neto H (2002) Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells. Braz J Med Biol Res 35:691–695[Medline]
Mauro A (1961) Satellite cells of skeletal muscle fibres. J Biophys Biochem Cytol 9:493–495
Moss FP, Leblond CP (1971) Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec 170:421–435[CrossRef][Medline]
Ohanian J, Ohanian V (2001) Sphingolipids in mammalian cell signalling. Cell Mol Life Sci 58:2053–2068[CrossRef][Medline]
Pediconi MF, Politi LE, Bouzat CB, De Los Santos EB, Barrantes FJ (1992) Myogenic differentiation of the muscle clonal cell line BC3H-1 is accompanied by changes in its lipid composition. Lipids 27:669–675[Medline]
Rosenblatt JD, Lunt AI, Parry DJ, Partridge TA (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31:773–779[Medline]
Sabbadini RA, Danieli-Betto D, Betto R (1999) The role of sphingolipids in the control of skeletal muscle function: a review. Ital J Neurol Sci 20:423–430[Medline]
Sadeh M, Czyewski K, Stern LZ (1985) Chronic myopathy induced by repeated bupivacaine injections. J Neurol Sci 67:229–238[CrossRef][Medline]
Schultz E, Gibson MC, Champion T (1978) Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. J Exp Zool 206:451–456[CrossRef][Medline]
Schultz E, Jaryszak DL (1985) Effects of skeletal muscle regeneration on the proliferation potential of satellite cells. Mech Ageing Dev 30:63–72[CrossRef][Medline]
Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102:777–786[CrossRef][Medline]
Sekizawa Y, Kubo T, Kobayashi H, Nakajima T, Natori S (1997) Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle. Gene 191:97–102[CrossRef][Medline]
Sekizawa Y, Ohta N, Natori S, Kobayashi H (1996) Immunocytochemical localization of lysenin, a novel protein isolated from the coelomic fluid of the earthworm Eisenia foetida. Biomed Res 17:327–330
Sessions A, Horwitz AF (1983) Differentiation-related differences in the plasma membrane phospholipid asymmetry of myogenic and fibrogenic cells. Biochim Biophys Acta 728:103–111[Medline]
Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39[CrossRef][Medline]
Snow MH (1977) Myogenic cell formation in regenerating rat skeletal muscle injured by mincing: II: an autoradiographic study. Anat Rec 188:201–217[CrossRef][Medline]
Snow MH (1978) An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res 186:535–540[Medline]
Spiegel S, Milstien S (2003) Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 4:397–407[CrossRef][Medline]
Strle K, Broussard SR, McCusker RH, Shen WH, Johnson RW, Freund GG, Dantzer R, et al. (2004) Proinflammatory cytokine impairment of insulin-like growth factor I-induced protein synthesis in skeletal muscle myoblasts requires ceramide. Endocrinology 145:4592–4602
Tatsumi R, Sheehan SM, Iwasaki H, Hattori A, Allen RE (2001) Mechanical stretch induces activation of skeletal muscle satellite cells in vitro. Exp Cell Res 267:107–114[CrossRef][Medline]
van den Eijnde SM, van den Hoff MJ, Reutelingsperger CP, van Heerde WL, Henfling ME, Vermeij-Keers C, Schutte B, et al. (2001) Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation. J Cell Sci 114:3631–3642
van Meer G, Holthuis JC (2000) Sphingolipid transport in eukaryotic cells. Biochim Biophys Acta 1486:145–170[Medline]
Webster C, Blau HM (1990) Accelerated age-related decline in replicative life-span of Duchenne muscular dystrophy myoblasts: implications for cell and gene therapy. Somat Cell Mol Genet 16:557–565[CrossRef][Medline]
Yablonka-Reuveni Z, Rivera AJ (1997) Influence of PDGF-BB on proliferation and transition through the MyoD-myogenin-MEF2A expression program during myogenesis in mouse C2 myoblasts. Growth Factors 15:1–27[Medline]
Yablonka-Reuveni Z, Rivera AJ (1994) Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat fibers. Dev Biol 164:588–603[CrossRef][Medline]
Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727[CrossRef][Medline]
Yamaji A, Sekizawa Y, Emoto K, Sakuraba H, Inoue K, Kobayashi H, Umeda M (1998) Lysenin, a novel sphingomyelin-specific binding protein. J Biol Chem 273:5300–5306
Yoshida N, Yoshida S, Koishi K, Masuda K, Nabeshima Y (1998) Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’. J Cell Sci 111:769–779[Abstract]
Zammit P, Beauchamp J (2001) The skeletal muscle satellite cell: stem cell or son of stem cell? Differentiation 68:193–204[CrossRef][Medline]
Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166:347–357
Zhou X, Arthur G (1992) Improved procedures for the determination of lipid phosphorus by malachite green. J Lipid Res 33:1233–1236[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. S. Zammit All muscle satellite cells are equal, but are some more equal than others? J. Cell Sci., September 15, 2008; 121(18): 2975 - 2982. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Zammit, T. A. Partridge, and Z. Yablonka-Reuveni The Skeletal Muscle Satellite Cell: The Stem Cell That Came in From the Cold J. Histochem. Cytochem., November 1, 2006; 54(11): 1177 - 1191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nagata, T. A. Partridge, R. Matsuda, and P. S. Zammit Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling J. Cell Biol., July 17, 2006; 174(2): 245 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Zammit, F. Relaix, Y. Nagata, A. P. Ruiz, C. A. Collins, T. A. Partridge, and J. R. Beauchamp Pax7 and myogenic progression in skeletal muscle satellite cells J. Cell Sci., May 1, 2006; 119(9): 1824 - 1832. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||