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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2004 as doi:10.1096/fj.04-2215fje. |
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* Departments of Pharmacology and
Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
1 Correspondence: Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. E-mail: feg5{at}pitt.edu
SPECIFIC AIMS
The aim of the present study is to investigate whether caveolin-1, the structural protein component of caveolar membranes, contributes to signaling events that lead to activation of satellite cells during muscle regeneration.
PRINCIPAL FINDINGS
1. Caveolin-1 is expressed in skeletal muscle satellite cells in vivo
Cross sections of skeletal muscle tissue derived from normal control C57Bl6 mice were stained with an antibody probe specific for caveolin-1. Skeletal muscle sections were costained with anti-caveolin-3 IgGs to detect myofibers. Caveolin-3 was expressed only at the sarcolemma whereas caveolin-1 was selectively expressed in cells surrounding myofibers in vivo (Fig. 1
). We performed double staining with caveolin-1 and each of these two markers of satellite cells, CD34 and M-cadherin. Caveolin-1 was expressed in CD34+ and M-cadherin+ cells (Fig. 1)
, indicating that caveolin-1 is expressed in skeletal muscle satellite cells in vivo.
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2. Caveolin-1 is expressed in myogenic precursor cells, but not myotubes, in vitro
We investigated the expression of caveolin-1 in skeletal muscle cells in vitro. Undifferentiated myogenic precursor cells (MPCs) were derived from mouse limb muscles and subjected to immunofluorescence analysis using pairs of antibodies (caveolin-1/caveolin-3, caveolin-1/CD34, and caveolin-1/M-cadherin). We found that caveolin-1 was expressed in CD34+, M-cadherin+ cells before differentiation. Caveolin-3 was not detected before differentiation. More than 95% of myogenic precursor cells were positive for caveolin-1, CD34, and M-cadherin. More than 95% of undifferentiated MPCs expressed caveolin-1, M-cadherin, and desmin, but not CD34, after 3 and 10 days of culture. Expression of desmin in >95% of cells indicates that fibroblast contamination was not a concern.
Caveolin-1 was not expressed in multinucleated myotubes after differentiation, which expressed caveolin-3. Caveolin-1 expression was restricted to round cells that surrounded multinucleated myotubes and resembled undifferentiated MPCs. We also observed caveolin-1 expression in cells that did not differentiate to myotubes, but lost their original "round" cell morphology. After 3 days of differentiation, >95% of cells in differentiated cultures were either caveolin-1+/desmin+/caveolin-3–/CD34–/troponinT– or caveolin-3+/troponin-T+/caveolin-1–/desmin–/CD34–.
3. Caveolin-1 is down-regulated during in vitro muscle regeneration
Myogenic precursor cells were differentiated for 3 days. Muscle wounding was performed and cells were observed at 0, 1, and 3 days. Cells began to migrate toward the wounded area after 1 day. After 3 days, the damaged area was covered with migrated cells, which fused to form new myotubes. Newly formed myotubes were troponin-T+, MHC+ and possessed multiple nuclei, as verified by DAPI staining. We demonstrated that caveolin-1 was transiently down-regulated 1 day after muscle wounding, which temporarily matched migration of myogenic precursor cells toward the wounded area. Caveolin-1 expression returned to basal levels 3 days after muscle injury, which coincided with the differentiation of migrated cells to multinucleated myotubes and formation of caveolin-1+ cells surrounding newly developed myotubes. Thus, down-regulation of caveolin-1 expression occurs in myotube-surrounding myogenic precursor cells during muscle regeneration.
4. Down-regulation of caveolin-1 expression during muscle regeneration is promoted by a hepatocyte growth factor-mediated mechanism
Hepatocyte growth factor (HGF) is believed to contribute to activation of satellite cells during muscle regeneration. We asked whether HGF may mediate down-regulation of caveolin-1 expression in satellite cells during muscle regeneration.
Cells were collected at 0, 2, 6, 12 h, and 1 day after muscle damage and RT-PCR was performed using a pair of primers specific for mouse HGF. We show that HGF mRNA was transiently up-regulated 6–12 h after in vitro muscle wounding.
We examined whether activation of HGF signaling mediates down-regulation of caveolin-1 expression during muscle regeneration. Caveolin-1 protein expression was down-regulated in differentiated myogenic precursor cells after treatment with recombinant HGF (100 nM for 16 h). Significant down-regulation of caveolin-1 protein expression was also obtained after stimulation of undifferentiated MPCs with 100 nM HGF for 16 h. Recombinant HGF significantly inhibited the activity of the caveolin-1 promoter. These data indicate that HGF down-regulates caveolin-1 expression by inhibiting caveolin-1 gene transcription.
5. Overexpressing caveolin-1 cells fail to repair muscle damage after in vitro muscle wounding
We asked whether overexpression of caveolin-1 affects muscle repair mechanisms in vitro. We took advantage of caveolin-1 transgenic mice, where caveolin-1 is overexpressed in a variety of tissues, including skeletal muscle. MPCs were derived from caveolin-1 transgenic mice, differentiated for 3 days; in vitro muscle wounding was performed. Myogenic precursor cells derived from normal C57Bl6 mice were used as controls. Cells were collected 0, 1, and 3 days after muscle damage and caveolin-1 expression was determined by immunoblot analysis. Unlike normal MPCs, caveolin-1 was not down-regulated in caveolin-1 transgenic cells 1 day after muscle injury. Caveolin-1-overexpressing cells, in contrast to control cells, failed to migrate toward the wounded area and form new myotubes after 3 days.
6. Overexpression of caveolin-1 delays muscle regeneration in vivo
Our results suggest that overexpression of caveolin-1 inhibits muscle regeneration in vitro. We investigated this possibility in vivo as well. Muscle damage was induced by injecting cardiotoxin (100 µL of 5 µM cardiotoxin) into hind limb muscles of caveolin-1 transgenic mice. Normal mice were used as controls. Skeletal muscle tissue was collected after 1, 3, and 10 days and immunoblot analysis was performed using an antibody probe specific for caveolin-1. Figure 2
A illustrates that caveolin-1 was overexpressed before cardiotoxin injection in skeletal muscle tissue of caveolin-1 transgenic mice compared with control mice. In contrast, caveolin-1 transgenic mice failed to down-regulate caveolin-1 expression 1 day after muscle injury (Fig. 2A
). We performed a histological examination on skeletal muscle tissue extracted 0, 3, 10, and 28 days after cardiotoxin injection. Before cardiotoxin injection (–Cdtx), structural integrity of the muscle appeared essentially indistinguishable in control and caveolin-1 transgenic mice (Fig. 2B
); 12 h after cardiotoxin injection, necrosis and degeneration of muscle fibers was identical in control and caveolin-1 transgenic mice. In control mice, an extensive cellular proliferative response was observed by day 3 as indicated by accumulation of mononucleated cells at the wounded site. A combination of regenerating myofibers with central nuclei and mature fibers with peripheral nuclei was detected by day 10. Restoration of hind limb architecture occurred by day 28. In contrast, caveolin-1 transgenic mice showed a delay in the regeneration process: cellular proliferative response was significantly reduced by day 3 (Fig. 2B
). Ten days after cardiotoxin injection, the great majority of muscle fibers in caveolin-1 transgenic mice were represented by regenerating myofibers with central nuclei, with few mature fibers with peripheral nuclei. Full restoration of muscle architecture was observed after 28 days (Fig. 2B
). These data indicate that down-regulation of caveolin-1 expression plays a major role in activation of satellite cells during muscle regeneration in vivo.
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CONCLUSIONS AND SIGNIFICANCE
We investigated the role of caveolin-1 as a mediator of satellite cell activation and demonstrated that caveolin-1 is expressed in satellite cells in vivo and myogenic precursor cells in vitro. We showed that caveolin-1 is down-regulated in MPCs and satellite cells during muscle regeneration and that overexpression of caveolin-1 inhibited muscle repair mechanisms both in vitro and in vivo.
Satellite cells are quiescent cells that exit the cell cycle. Reentry into the cell cycle is necessary for their activation and expansion. A possible molecular explanation for the phenotype of caveolin-1 transgenic satellite cells/MPCs is that expression of caveolin-1 is important for the exit of satellite cells from the cell cycle while down-regulation of caveolin-1 expression contributes to their reentry during muscle regeneration. Satellite cells that constitutively overexpress caveolin-1 fail to reenter the cell cycle and expand to repair muscle damage. In support, we previously demonstrated that overexpression of caveolin-1 arrests NIH 3T3 cells in the G0/G1 phase of the cell cycle and have shown that caveolin-1 overexpression in mouse embryonic fibroblasts is sufficient to induce a G0/G1 block, inhibit cellular proliferation, reduce the proliferative life span, and promote a premature senescent phenotype.
We demonstrated that HGF is up-regulated during muscle regeneration. Expression of HGF was increased early during the regeneration process (6–12 h after muscle injury). In contrast, down-regulation of caveolin-1 occurred 24 h after muscle injury. It is evident that activation of HGF signaling preceded down-regulation of caveolin-1. Up-regulation of HGF as well as down-regulation of caveolin-1 was transient. HGF levels returned to basal values 24 h after cardiotoxin injection and muscle wounding whereas caveolin-1 expression returned to preinjury levels after 3 days. Thus, it appears that a reciprocal inverse correlation exists between HGF signaling and caveolin-1 expression. We conclude that activation of the signaling cascade initiated by HGF may down-regulate caveolin-1 in satellite cells. In support, recombinant HGF down-regulated caveolin-1 protein expression and caveolin-1 promoter activity in normal myogenic precursor cells.
Data presented here indicate that caveolin-1 is a novel marker of satellite cells and demonstrate that the HGF-mediated down-regulation of caveolin-1 expression is a necessary step for satellite cell activation during muscle regeneration, providing new insight into molecular mechanisms that regulate adult muscle repair (Fig. 3
).
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2215fje;
This article has been cited by other articles:
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 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]
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