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Matrix Metalloproteinase-1 Promotes Muscle Cell Migration and Differentiation

2009; Elsevier BV; Volume: 174; Issue: 2 Linguagem: Inglês

10.2353/ajpath.2009.080509

1525-2191

William Yang Wang, Haiying Pan, Kiley Murray, Bahiyyah Jefferson, Yong Li,

Protease and Inhibitor Mechanisms

Injured skeletal muscle has the capacity to regenerate through a highly coordinated sequence of events that involves both myoblast migration and differentiation into myofibers. Fibrosis may impede muscle regeneration by posing as a mechanical barrier to cell migration and fusion, providing inappropriate signals for cell differentiation, and limiting vascular perfusion of the injury site, subsequently leading to incomplete functional recovery. Our previous studies demonstrated that matrix metalloproteinase-1 (MMP-1) is able to digest fibrous scar tissue and improve muscle healing after injury. The goal of this study is to investigate whether MMP-1 could further enhance muscle regeneration by improving myoblast migration and differentiation. In vitro wound healing assays, flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot analyses demonstrated that MMP-1 enhances myoblast migration but is not chemoattractive. We discovered that MMP-1 also enhances myoblast differentiation, which is a critical step in the sequence of muscle regeneration. In addition, RT-PCR and Western blot analyses demonstrated the up-regulation of myogenic factors after MMP-1 treatment. In vivo, we observed that myoblast transplantation was greatly improved after MMP-1 treatment within the dystrophic skeletal muscles of MDX mice. MMP-1 may therefore be able to improve muscle function recovery after injury or disease by increasing both the number of myofibers that are generated by activated myoblasts and the size of myoblast coverage area by promoting migration, thus fostering a greater degree of engraftment. Injured skeletal muscle has the capacity to regenerate through a highly coordinated sequence of events that involves both myoblast migration and differentiation into myofibers. Fibrosis may impede muscle regeneration by posing as a mechanical barrier to cell migration and fusion, providing inappropriate signals for cell differentiation, and limiting vascular perfusion of the injury site, subsequently leading to incomplete functional recovery. Our previous studies demonstrated that matrix metalloproteinase-1 (MMP-1) is able to digest fibrous scar tissue and improve muscle healing after injury. The goal of this study is to investigate whether MMP-1 could further enhance muscle regeneration by improving myoblast migration and differentiation. In vitro wound healing assays, flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot analyses demonstrated that MMP-1 enhances myoblast migration but is not chemoattractive. We discovered that MMP-1 also enhances myoblast differentiation, which is a critical step in the sequence of muscle regeneration. In addition, RT-PCR and Western blot analyses demonstrated the up-regulation of myogenic factors after MMP-1 treatment. In vivo, we observed that myoblast transplantation was greatly improved after MMP-1 treatment within the dystrophic skeletal muscles of MDX mice. MMP-1 may therefore be able to improve muscle function recovery after injury or disease by increasing both the number of myofibers that are generated by activated myoblasts and the size of myoblast coverage area by promoting migration, thus fostering a greater degree of engraftment. Muscle injuries are among the most common injuries seen in orthopaedic clinics and present a challenging problem in traumatology. Fibrosis is a consequence of the local overgrowth of extracellular matrix (ECM) at the site of injury. It poses a significant barrier in the prevention of complete muscle regeneration, thus leading to incomplete recovery, pain, and functional deficits. Accelerated ECM deposition may impede muscle regeneration by creating mechanical barriers against cell migration and fusion, providing inappropriate signals for cell differentiation, and limiting vascular perfusion of the injury site; this leads to incomplete functional recovery and a propensity for re-injury.1Garrett Jr, WE Muscle strain injuries: clinical and basic aspects.Med Sci Sports Exerc. 1990; 22: 436-443Crossref PubMed Google Scholar, 2Li Y Cummins J Huard J Muscle injury and repair.Curr Opin Orthop. 2001; 12: 409-415Crossref Scopus (63) Google Scholar, 3Lehto MU Jarvinen MJ Muscle injuries, their healing process and treatment.Ann Chir Gynaecol. 1991; 80: 102-108PubMed Google Scholar Experimental studies have demonstrated the efficacy of preventing fibrosis after muscle injury by blocking key factors in the fibrotic cascade, such as transforming growth factor-β.4Chan YS Li Y Foster W Horaguchi T Somogyi G Fu FH Huard J Antifibrotic effects of suramin in injured skeletal muscle after laceration.J Appl Physiol. 2003; 95: 771-780Crossref PubMed Scopus (130) Google Scholar, 5Fukushima K Badlani N Usas A Riano F Fu F Huard J The use of an antifibrosis agent to improve muscle recovery after laceration.Am J Sports Med. 2001; 29: 394-402Crossref PubMed Scopus (204) Google Scholar, 6Li Y Huard J Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.Am J Pathol. 2002; 161: 895-907Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 7Li Y Foster W Deasy BM Chan Y Prisk V Tang Y Cummins J Huard J Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis.Am J Pathol. 2004; 164: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 8Sato K Li Y Foster W Fukushima K Badlani N Adachi N Usas A Fu FH Huard J Improvement of muscle healing through enhancement of muscle regeneration and prevention of fibrosis.Muscle Nerve. 2003; 28: 365-372Crossref PubMed Scopus (168) Google Scholar However, treating patients before the onset of fibrosis is often unlikely; most persons with muscle injuries seek treatment only after the onset of fibrosis and the concomitant pain and functional deficits it produces. Additionally, pervasive skeletal muscle fibrosis can be caused by neuromuscular dysfunction and various musculoskeletal diseases such as Duchenne muscular dystrophy (DMD). DMD is an X-linked muscle disorder characterized by progressive muscle weakness caused by a lack of dystrophin expression in the sarcolemma of muscle fibers. Patients diagnosed with DMD begin to experience fibrous scar tissue formation in their muscles during their teenage years.9Hoffman EP Brown Jr, RH Kunkel LM Dystrophin: the protein product of the Duchenne muscular dystrophy locus.Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3841) Google Scholar, 10Partridge TA Invited review: myoblast transfer: a possible therapy for inherited myopathies?.Muscle Nerve. 1991; 14: 197-212Crossref PubMed Scopus (182) Google Scholar This anomalous generation of matrix protein is thought to be driven by the repeated degeneration, inflammation, and regeneration of muscle in DMD patients.11Iimuro Y Nishio T Morimoto T Nitta T Stefanovic B Choi SK Brenner DA Yamaoka Y Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.Gastroenterology. 2003; 124: 445-458Abstract Full Text PDF PubMed Scopus (215) Google Scholar, 12Karpati G Holland P Worton RG Myoblast transfer in DMD: problems in the interpretation of efficiency.Muscle Nerve. 1992; 15: 1209-1210Crossref PubMed Scopus (1) Google Scholar Myoblast transplantation has been considered as a potential therapeutic method for DMD.10Partridge TA Invited review: myoblast transfer: a possible therapy for inherited myopathies?.Muscle Nerve. 1991; 14: 197-212Crossref PubMed Scopus (182) Google Scholar, 13Partridge TA Morgan JE Coulton GR Hoffman EP Kunkel LM Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts.Nature. 1989; 337: 176-179Crossref PubMed Scopus (765) Google Scholar, 14Huard J Bouchard JP Roy R Labrecque C Dansereau G Lemieux B Tremblay JP Myoblast transplantation produced dystrophin-positive muscle fibres in a 16-year-old patient with Duchenne muscular dystrophy.Clin Sci (Lond). 1991; 81: 287-288PubMed Google Scholar However, poor cell survival and low dispersion of grafted cells outside of the injection site after transplantation have hindered the overall success of this technology.15Skuk D Myoblast transplantation for inherited myopathies: a clinical approach.Expert Opin Biol Ther. 2004; 4: 1871-1885Crossref PubMed Scopus (46) Google Scholar, 16Caron NJ Asselin I Morel G Tremblay JP Increased myogenic potential and fusion of matrilysin-expressing myoblasts transplanted in mice.Cell Transplant. 1999; 8: 465-476PubMed Google Scholar, 17Tremblay JP Malouin F Roy R Huard J Bouchard JP Satoh A Richards CL Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy.Cell Transplant. 1993; 2: 99-112PubMed Google Scholar Fibrous scar tissue continues to impede muscle cell migration, fusion, and muscle regeneration, even when myogenic cells are transplanted into the region. Significant improvements in cell survival have been obtained after immunosuppressive therapy,18Sato H Okada Y Seiki M Membrane-type matrix metalloproteinases (MT-MMPs) in cell invasion.Thromb Haemost. 1997; 78: 497-500Crossref PubMed Scopus (103) Google Scholar, 19Werb Z Chin JR Extracellular matrix remodeling during morphogenesis.Ann NY Acad Sci. 1998; 857: 110-118Crossref PubMed Scopus (177) Google Scholar but few studies have been centered on muscle cell migration, especially after myogenic cell transplantation. Our research has indicated that it is this secondary pathological process of DMD, namely fibrous scar tissue formation, which poses the most significant obstacle to myogenic cell migration, fusion, and regeneration. Digesting fibrous scar tissue could remodel the microenvironment to make it more hospitable to migration, fusion, and myogenic cell regeneration.20Bedair H Liu TT Kaar JL Badlani S Russell AJ Li Y Huard J Matrix metalloproteinase-1 therapy improves muscle healing.J Appl Physiol. 2007; 102: 2338-2345Crossref PubMed Scopus (53) Google Scholar, 21Huard J Li Y Fu FH Muscle injuries and repair: current trends in research.J Bone Joint Surg Am. 2002; 84A: 822-832Google Scholar Subsequently, it would enhance the myogenic transplantation process and improve muscle healing, not only in injured skeletal muscle, but also in patients suffering from DMD. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes with the ability to digest specific components of the ECM.22Brinckerhoff CE Matrisian LM Matrix metalloproteinases: a tail of a frog that became a prince.Nat Rev Mol Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (976) Google Scholar They present a promising approach to treat fibrosis after skeletal muscle injury or as a consequence of neuromuscular disease. MMP-1, in particular, has the ability to digest the main constitutive proteins in fibrous scar tissue, native fibrillar collagens type I and III, while sparing collagen type IV, which is a component of the basement membrane.23Sakaida I Hironaka K Kimura T Terai S Yamasaki T Okita K Herbal medicine Sho-saiko-to (TJ-9) increases expression matrix metalloproteinases (MMPs) with reduced expression of tissue inhibitor of metalloproteinases (TIMPs) in rat stellate cell.Life Sci. 2004; 74: 2251-2263Crossref PubMed Scopus (35) Google Scholar, 24Roach DM Fitridge RA Laws PE Millard SH Varelias A Cowled PA Up-regulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury; protection by the MMP inhibitor, doxycycline.Eur J Vasc Endovasc Surg. 2002; 23: 260-269Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 25Vincenti MP Brinckerhoff CE Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors.Arthritis Res. 2002; 4: 157-164Crossref PubMed Scopus (627) Google Scholar MMP-1 may also play important roles in ECM remodeling and cell signaling with its ability to act on the cell surface, matrix, and nonmatrix substrates, such as insulin growth factor binding proteins, L-selectin, and tumor necrosis factor-α.26McCawley LJ Matrisian LM Matrix metalloproteinases: they're not just for matrix anymore!.Curr Opin Cell Biol. 2001; 13: 534-540Crossref PubMed Scopus (1112) Google Scholar, 27Pardo A Selman M MMP-1: the elder of the family.Int J Biochem Cell Biol. 2005; 37: 283-288Crossref PubMed Scopus (205) Google Scholar Previous work in our laboratory has indicated that MMP-1 can help remove the fibrous blockade to enhance muscle healing.20Bedair H Liu TT Kaar JL Badlani S Russell AJ Li Y Huard J Matrix metalloproteinase-1 therapy improves muscle healing.J Appl Physiol. 2007; 102: 2338-2345Crossref PubMed Scopus (53) Google Scholar, 28Kaar JL Li Y Blair HC Asche G Koepsel RR Huard J Russell AJ Matrix metalloproteinase-1 treatment of muscle fibrosis.Acta Biomater. 2008; 4: 1411-1420Crossref PubMed Scopus (52) Google Scholar We hypothesize that MMP-1 can further enhance muscle regeneration by directly improving myoblast migration and differentiation capability. This may ultimately enhance muscle regeneration by improving the myoblasts' ability to increase the number of regenerating myofibers within an area of injury as well as increasing the effective range of transplanted myoblast-enhanced muscle regeneration can occur. We tested this hypothesis by studying the effects of MMP-1 on myoblasts in vitro and myoblast transplantation in vivo. C2C12 cells were purchased from the American Type Culture Collection (Rockville, MD). The cells were cultured in a complete medium containing Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 10% horse serum, 0.5% chicken embryo extract, and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere in 12-well plates until 70% confluent. The 12-well plates were either uncoated or coated with type I collagen or fibronectin. Cells were then placed in serum-free DMEM supplemented with 1% penicillin/streptomycin and treated with 0, 1.0, 10, or 100 ng/ml of MMP-1 (M1802; Sigma, St. Louis, MO). An artificial wound was created by disrupting the monolayer with a sterile plastic pipette tip. Cells were incubated for 1, 4, 6, and 12 hours to allow for migration back into the wound area. Cells were then fixed in cold methanol, washed with phosphate-buffered saline (PBS), and then stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) to help visualize cell migration. Northern Eclipse software (Empix Imaging Inc., Mississauga, Canada) was used to quantify the average migration distance of C2C12 cells that traveled past the original wound demarcation. C2C12 cells were cultured in a complete media (described above). Cells were then placed in serum-free DMEM supplemented with 1% penicillin/streptomycin and treated with 0, 1.0, 10, or 100 ng/ml of MMP-1 for 18 hours. Cells were incubated with either polyclonal N-cadherin (sc-31031; Santa Cruz Biotechnology, Santa Cruz, CA) or β-catenin (sc-1496, Santa Cruz Biotechnology) primary antibodies, and subsequently with a PE-conjugated secondary antibody. Marked cell samples were analyzed with a FACS Caliber flow cytometer (BD Biosciences, Sparks, MD) and CellQuest software (BD Biosciences). The assay was performed using a multiwell insert system (BD Biosciences) with an 8.0-μm pore size polyethylene terephthalate membrane. C2C12 cells (5 × 104) were placed in the top well of the system. Serum-free DMEM or serum-free DMEM supplemented with 10 and 100 ng/ml of MMP-1 were placed in the bottom well. Cells were incubated at 37°C for 3 hours to permit migration across the membrane in response to MMP-1. The top wells were then treated with 0.5% trypsin-ethylenediaminetetraacetic acid (Invitrogen) for 5 minutes to detach cells remaining on the top surface of the membrane and washed with PBS. Cells remaining on the bottom surface of the membrane were then fixed in cold methanol, washed with PBS, and then stained with DAPI (Sigma) to help visualization under fluorescent microscopy. The migration of cells was quantified by averaging the number of cells counted in five high-power fields and compared between control and MMP-1 treatment. C2C12 cells were seeded onto six-well plates and treated with MMP-1 for 3 and 12 hours. A monophasic solution of phenol and guanidine isothiocyanate (TRIzol, 10 cm2/ml; Life Technologies, Inc., Grand Island, NY) was used to extract total RNA from these cells. Reverse transcription was performed with the RETROscript kit (Ambion, Applied Biosystems, Austin, Texas) and cDNA was prepared. Primers used for gene detection were designed using Oligo software (OligoPerfect Designer, Invitrogen). The primers included: myogenin 5′-CCAGTGAATGCAACTCCCACAGC-3′ and 5′-AGACATATCCTCCACCGTGA-3′; MyoD 5′-GGCTACGACACCGCCTACTA-3′ and 5′-GTTCTGTGTCGCTTAGGGAT-3′; muscle regulatory factor-4 (MRF4) 5′-GCACCGGCTGGATCAGCAAGAG-3′ and 5′-CTGAGGCATCCACGTTTGCTCC-3′; desmin 5′-AACCTGATAGACGACCTGCAG-3′ and 5′-GCTTGGACATGTCCATCTCC-3′; insulin-like growth factor-1 receptor (IGF1R) 5′-GGTGGATGCTCTTCAGTTCG-3′ and 5′-GACTTGGCAGGCTTGAGGG-3′; and β-actin 5′-GGGTCAGAAGGACTCCTATGTGG-3′ and 5′-CCTGGATGGCTACGTACAT-3′. The conditions for IGF1R and β-actin amplification were the following: 93°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes for 31 cycles. The conditions for desmin amplification were the following: 93°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes for 34 cycles. The conditions for MyoD were the following: 93°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes for 28 cycles. The conditions for MRF4 were the following: 93°C for 1 minute, 53°C for 1 minute, and 72°C for 2 minutes for 31 cycles. The conditions for myogenin were the following: 93°C for 1 minute, 53°C for 1 minute, and 72°C for 2 minutes for 33 cycles. Products were separated by size on 1% agarose gel. C2C12 cells were harvested after 48 hours of incubation with or without treatment (0, 1.0, 10, 100 ng/ml) in serum-free DMEM. After lysing, the samples were separated by 12% sodium dodecyl-sulfate-polyacrylamide electrophoresis gel and transferred to nitrocellulose membranes. Anti-PreMMP-2 antibodies (Sigma) at a dilution of 1:1000, anti-TIMP-1 (sc-5538, Santa Cruz Biotechnology) at a dilution of 1:1000, and anti-myogenin (Sigma) at a dilution of 1:2000 were used as primary antibodies. Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma) at a dilution of 1:2000 was used for protein quantification. Anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL) was applied at a dilution of 1:5000. Blots were developed using SuperSignal West Pico chemiluminescent substrate (Amersham Pharmacia Biotech, Piscataway, NJ), and positive bands were visualized on X-ray film. Northern Eclipse software (Empix Imaging) was used to evaluate these results. C2C12 cells were cultured in 12-well plates to 75% confluency. Cells were subsequently incubated in differentiation media containing serum-free DMEM supplemented with 1% penicillin/streptomycin and treated with 0, 1.0, 10, or 100 ng/ml of MMP-1 (M1802, Sigma) for 3, 5, and 7 days. Cells were fixed in cold methanol for 1 minute and washed with PBS. The fixed cells were immunostained for myosin heavy chain (MyHC, Sigma) and DAPI to visualize mature myotubes. Cell differentiation was quantified by averaging the number of myotubes counted in five high-power fields and compared among different concentrations of MMP-1 treatment. Nine-week-old MDX/SCID mice (C57BL/10ScSn-Dmdmdx crossed with C57BL/6J-Prkdcscid/SzJ) were injected with C2C12 cells with MMP-1 (M1802, Sigma). LacZ-positive C2C12 cells (1 × 105) were mixed with 200 ng of MMP-1 in a volume of 5 μl of PBS, which was then injected into the left gastrocnemius muscles (GMs) or tibialis anterior (TAs) of MDX/SCID mice. The same number of LacZ-positive C2C12 cells was diluted with 5 μl of PBS and was injected into the right GMs or TAs of the mouse to serve as a control. Muscle tissues were harvested for histological analysis at 2 and 4 weeks after transplantation. GMs and TAs were isolated, mounted, and frozen in 2-methylbutane cooled in liquid nitrogen. Each muscle specimen was cryostat-sectioned at 10 μm for histological analysis. LacZ staining with eosin and immunohistochemistry for dystrophin (Sigma) and β-galactosidase (Abcam, Cambridge, MA) were performed.6Li Y Huard J Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.Am J Pathol. 2002; 161: 895-907Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 7Li Y Foster W Deasy BM Chan Y Prisk V Tang Y Cummins J Huard J Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis.Am J Pathol. 2004; 164: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar The dystrophin-positive myofibers were counted and their diameters were measured to evaluate the enhancement of MMP-1 on myoblast differentiation and fusion capacities in vivo. Myoblast migration distances from the initial site of injection were also quantified using Northern Eclipse software (Empix Imaging Inc.). Statistical significance was assessed by analysis of variance and two-tailed Student's t-tests; P < 0.05 was considered significant. Our previous studies have not shown that MMP-1 actually stimulated myoblast migration directly. We used an in vitro wound-healing assay, flow cytometry, and Western blot analysis to determine whether MMP-1 could alter C2C12 migration. After culturing C2C12 cells to 70% confluence, we created artificial wounds as described previously and measured migration distances of C2C12 cells back into the wounded area using various conditions. Our results indicate that MMP-1 can enhance C2C12 myoblast migration throughout time in uncoated plates (Figure 1A) as well as under conditions that more closely represent in vivo ECM content, eg, type I collagen or fibronectin-coated plates (Figure 1B). We observed a significant difference in migration distances for C2C12 cells treated with 10 ng/ml of MMP-1 at 4 and 6 hours after wounding when compared with control cells in uncoated and fibronectin-coated plates (Figure 1B). Using type I collagen-coated plates, there were significant differences in migration distances for C2C12 cells treated with MMP-1 (10 ng/ml) at 1, 4, and 6 hours after wounding when compared with control cells (Figure 1B; *P < 0.05). N-cadherin and β-catenin are two proteins expressed with myoblast migration29Brand-Saberi B Gamel AJ Krenn V Muller TS Wilting J Christ B N-cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud.Dev Biol. 1996; 178: 160-173Crossref PubMed Scopus (68) Google Scholar, 30Woodfield RJ Hodgkin MN Akhtar N Morse MA Fuller KJ Saqib K Thompson NT Wakelam MJ The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.Biochem J. 2001; 360: 335-344Crossref PubMed Scopus (45) Google Scholar and were used as markers for C2C12 cell migration analysis. Flow cytometry results demonstrated that MMP-1 promoted the up-regulation of N-cadherin and β-catenin in C2C12 cells when treated with 10 and 100 ng/ml of MMP-1 (Figure 2, C and D, G and H). Treatment with 0.1 and 1.0 ng/ml of MMP-1 did not yield significant results (Figure 2, A and B, E and F). Western blot analysis further demonstrates that MMP-1 treatment enhances the expression of migration-related proteins. It has been reported in the literature that pre-MMP-2 and TIMP are up-regulated with myoblast migration.18Sato H Okada Y Seiki M Membrane-type matrix metalloproteinases (MT-MMPs) in cell invasion.Thromb Haemost. 1997; 78: 497-500Crossref PubMed Scopus (103) Google Scholar, 31Takino T Watanabe Y Matsui M Miyamori H Kudo T Seiki M Sato H Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration.Exp Cell Res. 2006; 312: 1381-1389Crossref PubMed Scopus (89) Google Scholar, 32Ohtake Y Tojo H Seiki M Multifunctional roles of MT1-MMP in myofiber formation and morphostatic maintenance of skeletal muscle.J Cell Sci. 2006; 119: 3822-3832Crossref PubMed Scopus (104) Google Scholar, 33Mendes O Kim HT Lungu G Stoica G MMP2 role in breast cancer brain metastasis development and its regulation by TIMP2 and ERK1/2.Clin Exp Metastasis. 2007; 24: 341-351Crossref PubMed Scopus (128) Google Scholar, 34Gong YL Xu GM Huang WD Chen LB Expression of matrix metalloproteinases and the tissue inhibitors of metalloproteinases and their local invasiveness and metastasis in Chinese human pancreatic cancer.J Surg Oncol. 2000; 73: 95-99Crossref PubMed Scopus (64) Google Scholar, 35Kim HJ Park CI Park BW Lee HD Jung WH Expression of MT-1 MMP, MMP2, MMP9 and TIMP2 mRNAs in ductal carcinoma in situ and invasive ductal carcinoma of the breast.Yonsei Med J. 2006; 47: 333-342Crossref PubMed Scopus (50) Google Scholar Our Western blot results indicate that these two proteins are up-regulated with MMP-1 stimulation in myoblasts, especially with higher doses of MMP-1 (Figure 3A). Northern Eclipse software (Empix Imaging, Inc.) was used to analyze positive bands, which demonstrated that the expression of pre-MMP-2 increased mostly in a dose-dependent manner when treated with 0.1, 1.0, 10, and 100 ng/ml of MMP-1 (Figure 3B). TIMP1 expression increased mainly with 10 and 100 ng/ml of MMP-1 treatment; C2C12 cells treated with 0.1 and 1.0 ng/ml of MMP-1 demonstrated minor increases in the expression of TIMP1 when compared with nontreated control cells (Figure 3B).Figure 3Myoblasts (C2C12) were treated with 0, 1.0, 10, or 100 ng/ml of MMP-1 for 7 hours. A: Western blot analysis showed an increase in the expression of migration-related proteins, PreMMP-2 and TIMP as well as a myogenic protein, myogenin. GAPDH staining served as the control. The positive bands were evaluated and compared with standard GAPDH. B: Results showed the dose-dependent increase of protein expression with MMP-1 treatment. C: RT-PCR results indicated some myogenic genes, eg, MRF4, myogenin, and MyoD were initially increased within 3 hours of stimulation in a dose-dependent response with MMP-1 stimulation. With 12 hours of stimulation, myogenin, IGF1R, and MRF4 were promoted, thus increasing the level of mRNA. Desmin, however, was decreased in a dose-dependent response with MMP-1 treatment in 3-hour and 12-hour time periods; MyoD also decreased, but only at the 12-hour time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Results from the chemotaxis assay demonstrated that 10 and 100 ng/ml of MMP-1 treatment did not increase the number of C2C12 that migrated in the polyethylene terephthalate membrane compared with control, on visualization under fluorescent microscopy (Figure 1C). This suggests that MMP-1 may not have a chemoattractive effect on myoblast (C2C12) migration in vitro. We have tested the mRNA level of different genes within C2C12 myoblasts after treating with MMP-1. Results indicated that MMP-1 increases MRF4, myogenin, and MyoD initially within 3 hours of stimulation in a dose-dependent manner. In the following 12 hours of stimulation with MMP-1, myogenin, IGF1R, and MRF4 were promoted, thus increasing the mRNA level. Desmin, however, was decreased in a dose-dependent response with MMP-1 treatment in both 3-hour and 12-hour time periods; MyoD also decreased, but only at the 12-hour time point (Figure 3C). Results indicate MMP-1 somehow accelerates myogenic gene activation, with the exception of desmin, which mainly relates to mature myoblasts during development.36Bischoff R The Satellite Cell and Muscle Regeneration.in: Engel AG Franzini-Armstrong C McGraw-Hill, New York1994: 97-118Google Scholar C2C12 cells proliferate when maintained in growth media containing serum, but differentiate into multinucleated myotubes when grown in serum-free media. This progression of differentiation is similar to that observed for myogenesis in vivo.37Lluri G Jaworski DM Regulation of TIMP-2, MT1-MMP, and MMP-2 expression during C2C12 differentiation.Muscle Nerve. 2005; 32: 492-499Crossref PubMed Scopus (59) Google Scholar We investigated whether MMP-1 could enhance C2C12 differentiation in vitro using a differentiation assay as well as Western blot analysis. When cultured in differentiation media, C2C12 cells displayed a dose-dependent increase in differentiation capacity when treated with 10 and 100 ng/ml of MMP-1 compared with control groups at 5 days (Figure 4, A–D). Treatment with 10 and 100 ng/ml of MMP-1 produced significantly more myotubes compared with the control group at 3 and 5 days (Figure 4, E and F) incubation, but not at 7 days (Figure 4G). To further demonstrate that MMP-1 enhances cell differentiation, we used Western blot analysis to detect myogenin, which has previously been shown to be a key protein controlling myofiber formation.32Ohtake Y Tojo H Seiki M Multifunctional roles of MT1-MMP in myofiber formation and morphostatic maintenance of skeletal muscle.J Cell Sci. 2006; 119: 3822-3832Crossref PubMed Scopus (104) Google Scholar When cultured in differentiation media, C2C12 cells displayed a dose-dependent increase in the expression of myogenin when treated with 0.1, 1.0, 10, and 100 ng/ml of MMP-1 (Figure 3, A and B). We elected to test the effects of MMP-1 on myoblast migration and differentiation by transplanting C2C12 cells in MDX mice. The MDX mouse contains a nonsense mu