Expression of Myostatin Gene in Regenerating Skeletal Muscle of the Rat and Its Localization

Yamanouchi, Keitaro; Soeta, Chie; Naito, Kunihiko; Tojo, Hideaki

doi:10.1006/bbrc.2000.2464

Cited by 38 publications

(31 citation statements)

References 32 publications

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“…The protein is not detected by Western blot, probably because of its low concentration relative to total protein, but has been detected by ELISA. Myostatin expression in fibroblasts from injured skeletal muscle was first detected by Yamanouchi et al (2000) indicating that fibroblasts could be a source of myostatin. More recently, Zhu et al (2007) have shown that myostatin stimulates fibroblast proliferation and induces α-SMA expression, as does TGF-β1, whereas Li et al (2008) have found that biologically active myostatin is expressed and secreted by muscle-derived fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-β1 treatment

2009

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To probe pro-fibrotic mechanisms in dystrophic muscle, we isolated primary fibroblasts from Duchenne muscular dystrophy (DMD) and control muscle biopsies and induced transdifferentiation in myofibroblasts by transforming growth factor beta1 (TGF-beta1) treatment. We compared proliferating activity, soluble collagen production, and transcript and protein levels of decorin, myostatin, TGF-beta1, matrix metalloproteinase-1 (MMP-1; interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the tissue inhibitors of metalloproteinases inhibitors (TIMPs) 1-4, in fibroblasts and myofibroblasts. Principal differences included a significantly greater proliferation rate and soluble collagen production, a significant upregulation of decorin, myostatin and MMP-7 transcripts and proteins, and a significant downregulation of MMP-1 and TIMP-3 transcripts (with MMP-1 protein being reduced as shown by enzyme-linked immunosorbent assay and TIMP-3 protein apparently being reduced on Western blot), in untreated DMD fibroblasts compared with controls. TGF-beta1 transdifferentiation significantly lowered decorin and myostatin and significantly increased TGF-beta1 transcript and protein, significantly increased MMP-1 and TIMP-3, and significantly lowered MMP-7 transcript and protein in DMD cells compared with pretreatment controls. The differences between DMD and control fibroblasts showed that DMD fibroblasts had a profibrotic phenotype, accentuated by TGF-beta1 treatment. Dystrophin absence itself could exert a direct influence on the homeostasis of the extracellular matrix (ECM) by allowing leakage of cellular components to the extracellular space or by abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes influencing muscle fibroblasts either directly by altering adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells. The transdifferentiation of muscle fibroblasts might serve as a simplified model of fibrosis for further elucidation of the mechanisms of muscle fibrosis and for testing possible anti-fibrotic agents.

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Section: Discussionmentioning

confidence: 99%

Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-β1 treatment

2009

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“…We do not know whether myostatin plays a role in muscle ®ber differentiation. Although myostatin has been postulated to be associated``in vivo'' in the skeletal muscle exclusively with MHCIIb (Perry and Rudnick, 2000), there are also reports of its association with both MHC-I and -II (Yamanouchi et al, 2000). Further work is needed to determine whether myostatin can activate the expression of MHC-II in C2C12 myotubes through its effect on muscle cell differentiation.…”

Section: Discussionmentioning

confidence: 99%

Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells

Artaza

Bhasin

Mallidis

et al. 2002

Journal Cellular Physiology

105

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Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant nuclear localization suggests that it may play a role in transcriptional regulation.

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“…Equal amounts of the RNA samples were reverse transcribed using random hexamer primers (Invitrogen) and Super Script II reverse transcriptase (Invitrogen). Semi-quantitative RT-PCR was performed, as described previously (Yamanouchi et al, 2000). The sequences of the intron-spanning primer pairs were as follows: MCK forward: 5Ј-AGC ACA GAC AGA CAC TCA-3Ј, reverse: 5Ј-GGC TTG TAG TTC AGC TTG-3Ј, eMyHC forward: 5Ј-TTG GGT GGG TTC ATG GCA TA-3Ј, reverse: 5Ј-GTC AAG GGG AAA ATT AAG AGT A-3Ј, MMP-2 forward: 5Ј-TTC TTC TTC AAG GAC CGG TTT ATT TGG-3Ј, reverse: 5Ј-ATT TTC TTC TTC ACC TCA TTG TAT CTC CA-3Ј, and TIMP-2 forward: 5Ј-CCG CAA CAG GCG TTT TGC AA-3Ј, reverse: 5Ј-TCA CTT CTC TTG ATG CAG GC-3Ј.…”

Section: Semi-quantitative Rt-pcrmentioning

confidence: 99%

Multifunctional roles of MT1-MMP in myofiber formation and morphostatic maintenance of skeletal muscle

Ohtake

Tojo

Seiki

2006

Journal of Cell Science

Self Cite

114

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Sequential activation of muscle-specific transcription factors is the critical basis for myogenic differentiation. However, the complexity of this process does not exclude the possibility that other molecules and systems are regulatory as well. We observed that myogenic differentiation proceeded through three distinct stages of proliferation, elongation and fusion, which are distinguishable by their cellular morphologies and gene expression patterns of proliferation- and differentiation-specific markers. Treatment of the differentiating myoblasts with inhibitors of matrix metalloproteinases (MMPs) revealed that MMP activity at the elongation stage is a critical prerequisite to complete the successive myoblast cell fusion. The MMP regulated the myogenic differentiation independently from the genetic program that governs expression of the myogenic genes. Membrane-type 1 matrix metalloproteinase (MT1-MMP) was identified as a major contributor to this checkpoint for morphological differentiation and degraded fibronectin, a possible inhibitory factor for myogenic cell fusion. A MT1-MMP deficiency caused similar myogenic impediments forming smaller myofibers in situ. Additionally, the mutant mice demonstrated some central nucleation of the myofibers typically found in muscular dystrophy and MT1-MMP was found to cleave laminin-2/4 in the basement membrane. Thus, MT1-MMP is a new multilateral regulator for muscle differentiation and maintenance through processing of stage-specific distinct ECM substrates.

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Expression of Myostatin Gene in Regenerating Skeletal Muscle of the Rat and Its Localization

Cited by 38 publications

References 32 publications

Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-β1 treatment

Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-β1 treatment

Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells

Multifunctional roles of MT1-MMP in myofiber formation and morphostatic maintenance of skeletal muscle

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