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First published online 22 August 2006
doi: 10.1242/jcs.03158

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

¹ Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
² Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8675, Japan

View larger version (37K): [in a new window]   Fig. 1. Myotube formation of C2C12 myoblasts through three distinct stages. (A) Differentiation of C2C12 cells was induced in a serum-free DMEM containing 10 ng/ml IGF-I. Along with typical morphological changes, the expression levels of PCNA and MHC were examined by immunostaining, using specific antibodies. There was no overlapping of the staining for proliferation and differentiation markers, indicating that there were three distinct stages, as shown on the right. These stages were designated as a proliferation stage, an elongation stage and a fusion stage, based on their cell morphologies. Bars, 100 µm. (B) Cell and myotube nuclei were counted. A fusion ratio was calculated as nuclei in multinucleated cells/total nuclei. An increase in the number of nuclei in the early phase (24 hours), and the fusion index increase in the late phase (72, 96, 120 and 144 hours) clearly established the boundaries between the proliferation and elongation stages at 24 hours, and between the elongation and fusion stages at 72 hours.

View larger version (42K): [in a new window]   Fig. 2. Inhibition of MMP activity in the elongation stage blocks myotube formation without affecting the genetic program. (A) Differentiating C2C12 cells were treated with BB94 (10 µM) during the indicated time period, and 0.1% of DMSO was added throughout the cell culture duration. (B) The total cell number, the numbers of myogenin-positive cells and the fusion rates were measured at 144 hours. The results are presented as the mean ± s.e.m. (n = 6). (C) Expression levels of differentiation marker genes were measured by semi-quantitative RT-PCR at 144 hours, including muscle creatine kinase (MCK) and embryonic myosin heavy chain (eMyHC). The results are normalized with G3PDH and presented as the mean ± s.e.m. (n = 3). (D) Anti-MHC immonostaining (green signal) with Hoechst counterstaining (blue signal) at 144 hours. Bar, 100 µm. (E) Luciferase reporter assay for representative myogenic transcriptional factors. Reporter constructs have tandem repeats of the DNA binding sites for the indicated myogenic transcription factors. 4RE houses four tandem binding sites for MRFs, 3MEF2 contains three tandem binding sites for MEF2 and 3SRE contains three tandem binding sites for SRF. The results are presented as the mean ± s.e.m. (n = 6).

View larger version (33K): [in a new window]   Fig. 3. Induction of MMP expression in the elongation stage. (A) Transcripts for MMP-2 and TIMP-2 in the differentiating C2C12 cells were measured by semi-quantitative RT-PCR. The results are normalized with G3PDH and presented as the mean ± s.e.m. (n = 3). (B) MMP-2 activity was monitored by gelatin zymography (upper panel). pro(gly); glycosylated form of proMMP-2, pro; proMMP-2, int; intermediate form, act; activated form, NCDM; none-conditioned differentiation medium. Expression patterns of MT1-MMP (middle panel) and tubulin (lower panel) were evaluated by western blot analysis. Open arrowhead indicates active MT1-MMP and closed one shows a major autolytically degraded fragment (Osenkowski et al., 2004). These results are representative one of three independent experiments.

View larger version (35K): [in a new window]   Fig. 4. Role of MT1-MMP in myotube formation and pericellular fibronectin. (A) Differentiating cells were pulse-treated with the indicated inhibitors during the elongation stage, as indicated in the upper part of the figure. TIMP-1 and TIMP-2 were used at concentrations of 1 µg/ml. The cell fusion ratio was monitored at 144 hours and shown in the relative ratio dealing NT as 100%. The results are presented as the mean ± s.e.m. (n = 6). NT, no treatment; DMSO, solvent for BB94 alone. (B) Endogenous MT1-MMP expression in the C2C12 cells was knocked down by the stable expression of shRNA, using the U6 promoter. A western blot in the upper panel shows a significant reduction of MT1-MMP by shRNA for MT1-MMP (U6MT1), but not for the control LacZ (U6LacZ). In the lower panel, the fusion ratio was monitored at 144 hours and shown in the relative ratio dealing U6LacZ as 100%. The results are presented as the mean ± s.e.m. (n = 6). (C) Cells treated with the indicated inhibitors or with the shRNAs were collected and analyzed by western blotting using antibodies against fibronectin or tubulin.

View larger version (66K): [in a new window]   Fig. 5. Expression of MT1-MMP and MMP-2 in regenerating skeletal muscle. (A) The tibialis anterior (TA) muscles of 2-3 month old mice were injured by injecting cardiotoxin, and regeneration was monitored by histology with HE staining. Arrowheads indicate that regenerating myofibers having centrally located nuclei at day 4. Bar, 20,000 µm. (B) MMP expression levels in the regenerating muscle tissues. MMP-2 expression was monitored by gelatin zymography (upper panel). Closed and open arrowheads on the right indicate active- and pro-form of MMP-2, respectively. Western blot in the lower panel shows the expression profile of MT1-MMP. Open arrowhead indicates active MT1-MMP and closed arrowhead shows an autolytically degraded fragment.

View larger version (76K): [in a new window]   Fig. 6. Small myofibers and tissue heterogeneity in MT1-MMP-deficient skeletal muscle tissue. Transverse sections of quadricep muscles in 4 week-old wild-type (left column), and MT1-MMP-deficient mice (right column) were analyzed as follows: HE-staining (a,b), immunostaining using anti-ß-DG to visualize legible peripheral myofibers (c,d), elastica-van gieson staining (EVG; e,f), and reticulin silver impregnation (silver staining; g,h). Note that the MT1-MMP-deficiency caused myofiber heterogeneity in size and shape, and peripheral ECM accumulation. Some faintly hypertrophied fibers containing centrally located nuclei also exist (arrow heads). Bars, 200 µm.

View larger version (58K): [in a new window]   Fig. 7. Processing of laminin 2 subunit by MT1-MMP. (A) Schematic representation of the heterotrimeric laminin-2, the localization of two epitopes of the antiserum and the corresponding regions of three processed fragments `a', `b' and `c' appeared in (B) and (C). Open arrow indicates a putative cutting site by MT1-MMP that generates the band `b'. Closed arrow indicates additional cleavage site that is available only in biochemical assay (discussed in Results). (B) Western blot analysis of muscle tissues obtained from 4-week-old wild type (WT) and MT1-MMP-deficient mice (MT1-MMP-/-) using anti-300K antiserum. Three arrowheads in the right indicate the major fragments detected. (C) The purified laminin-2 from human placenta was digested with the recombinant catalytic fragment of MT1-MMP (1:100, 1:50 molecular ratio in w/w) at 37°C for 18 hours. The resulting products were analyzed by western blotting, using either anti-300K or anti-IVa. Three arrowheads in the right indicate the corresponding fragments of those detected in (B).

View larger version (32K): [in a new window]   Fig. 8. Requirement of MT1-MMP in multiple phases of skeletal muscle tissue development. Myoblast differentiation proceeds through three distinct stages. During elongation, the MMP-dependent ECM remodeling that is critical for the successive fusion step occurs. Time course of MMP-2, MT1-MMP and TIMP-2 expression supports the idea that MMPs are active in the elongation stage. Blocking of MMP activities by inhibitors or specific down-regulation of MT1-MMP by shRNA caused an accumulation of cell-associated fibronectin. Thus, fibronectin may represent ECM components having inhibitory effect for myoblast fusion and have to be cleared by MT1-MMP at this stage. A MT1-MMP deficiency also causes self-destruction of the muscle tissue, which resembles muscular dystrophy. MT1-MMP is responsible for laminin 2 processing in vivo and in vitro, and appears to regulate laminin-based cell adhesion.