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Coordinate expression of matrix-degrading proteinases and their activators and inhibitors in bovine skeletal muscle D. Balcerzak, L. Querengesser, W. T. Dixon, and V. E. Baracos2 Department of Agriculture, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 ments. Statistical analysis (n = 35) revealed a strong positive correlation among the mRNA levels of several elements of the MMP system, including MMP-2, MMP14, TIMP-1, -2, and -3 (r = 0.614 to 0.930, P < 0.0001). Our results provide an extensive profile of an extracellular proteolytic cascade involving MMP in skeletal muscle and suggest that 1) the activation cascades of muscle MMP may be initiated by both plasmin and membrane-type MMP; 2) a group of genes involved in the same “arm” of zymogen activation are coexpressed in this tissue; and 3) skeletal muscle cells, in addition to the intramuscular fibroblasts, express an extensive complement of MMP and related proteins. ABSTRACT: Matrix metalloproteinases (MMP) responsible for degradation of connective tissue are found in most tissues. The MMP are regulated at the levels of transcription, zymogen activation by plasmin or membrane-type- (MT) MMP, and control of enzyme activity by tissue inhibitors of metalloproteinases (TIMP). Whole bovine skeletal muscle showed multiple MMP activities on gelatin zymography and also expressed mRNA encoding MMP-1, -2, -9, -14, and -16, tissue inhibitors of metalloproteinase (TIMP)-1, -2, and -3 and plasminogen activator and its receptor. Purified intramuscular fibroblasts and myogenic cell culture derived from satellite cells expressed most or all of these ele- Key Words: Connective Tissue, Fibroblasts, Metalloproteinases, Satellite Cells, Skeletal Muscle 2001 American Society of Animal Science. All rights reserved. Introduction J. Anim. Sci. 2001. 79:94–107 sons et al., 1997). The MMP gene expression also is regulated and is influenced by multiple cytokines and growth factors (Emonard and Grimaud, 1990; Ries and Petrides, 1995). The role of gene expression in regulation of the alternative zymogen activation cascades is less clear, but a few published results suggest coordinate expression within the MT-MMP-dependent cascade (Sakamato et al., 1999). A third level of regulation involves local production of polypeptide tissue inhibitors of metalloproteinases (TIMP) (Murphy and Willembrock, 1995). Studies of extracellular matrix catabolism in other organs provide biochemical tools for determination of MMP activity and gene expression. We used currently available approaches to undertake a comprehensive evaluation of the MMP system of bovine skeletal muscle, including the participating proteinases, activators, and inhibitors of MMP. Our work is the first systematic study of this system in muscle to include all of these potential elements and was done with a long-term view to determining its role in dynamic physiologic, pathologic, and postmortem changes in the intramuscular connective tissue as it relates to meat tenderization. The intracellular proteolytic systems of muscle that degrade the myofibrillar proteins are now well characterized. These processes have been demonstrated to participate in muscle growth, atrophy, and tissue remodeling in living animals and in postmortem proteolysis. By contrast, the proteolytic processes responsible for catabolism of the intramuscular connective tissue remain undefined. In tissues other than muscle, the matrix metalloproteinases (MMP) represent a family of enzymes (Parsons et al., 1997) responsible for connective tissue catabolism. Regulation of MMP activities is complex. The MMP are secreted in a latent form and activated sequentially in a cascade initiated by plasmin or membrane-type MMP (MT-MMP) (Figure 1) (Par- 1 This work was supported by the Canadian Beef Industry Development Fund and grants from the Natural Sciences and Engineering Research Council of Canada to V. E. Baracos and W. T. Dixon. The authors would like to thank Renate Meuser, Richard Chai, and Linghuo Jiang for their valuable technical assistance and the staff of the Edmonton Research Station for animal care. 2 Correspondence: phone: (780) 492-7664; fax: (780) 492-4265; E-mail: [email protected]. Received March 20, 2000. Accepted August 23, 2000. Materials and Methods We adapted currently available approaches for the study of MMP to bovine skeletal muscle. Because mus94 Skeletal muscle extracellular proteolysis 95 the larger sheets of perimysium (referred to as the “dissectable connective tissue”). Dissectable connective tissues were immediately frozen in liquid nitrogen for enzyme detection or placed in sterile Eagle’s Minimum Essential Medium (MEM, Life Technologies, Burlington, ON, Canada) for isolation of fibroblasts. Muscles were frozen in liquid nitrogen for later use in detection of MMP activities and RNA isolation or transferred to MEM for isolation of intramuscular fibroblasts and myogenic cells. Intramuscular Fibroblast Isolation and Culture Figure 1. Proposed cascade for activation of the matrix metalloproteinases. One “arm” of zymogen activation is thought to involve plasmin in a cascade of events where the urokinase-type plasminogen activator (uPa) is activated through binding to its cell surface receptor (uPaR) (Werb et al., 1977; He et al., 1989). Pro-MMP-1, pro-MMP3, and pro-MMP-9 are activated via this way. Another “arm” of activation may also stem from membrane-typeMMP (MT-MMP), which are expressed at the cell surface (Sato et al., 1994; Takino et al., 1995; Knäuper et al., 1996). Pro-MMP-2 and pro-MMP-13 can be activated by these MT-MMP involving a multimeric complex between MT1MMP, pro-MMP-2, and TIMP-2 (Yu et al., 1998). Black arrows represent the activation and light grey arrows represent active MMP degrading the extracellular matrix. MMP, matrix metalloproteinase; MT-MMP, membrane type-matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase. Adapted from Parson et al. (1997). cle is < 5% connective tissue on a protein basis and the turnover of these proteins is known to be slow, we anticipated a challenge in terms of the sensitivity of available methods. Whole muscle and potentially concentrated sources of MMP, including dissectible connective tissue, cultured intramuscular fibroblasts, and satellite cells, were used. Samples were obtained from animals from a beef research herd maintained at the University of Alberta. All animals were the same age and sex and on a similar plane of nutrition at the time of slaughter. Masseter and semimembranosus muscles were collected at a commercial abattoir immediately after animals were killed. Muscles were dissected to remove the epimysium and Single cell suspensions were obtained by enzymatic digestion (Yablonka-Reuveni et al., 1988) with some modifications. Briefly, muscle was finely minced and incubated in trypsin (Life Technologies) adjusted to 0.1% with MEM at 37°C for 50 min with shaking. Samples were centrifuged at 400 × g for 10 min and the supernate was discarded. The pellets were washed once with MEM and resuspended in medium 1 (MEM, 10% horse serum [Life Technologies] 1% antibiotic/antimycotic [Life Technologies]). Cells were dissociated by vigorously mixing the pellet and trituration. The resulting cell suspension was centrifuged at 400 × g for 10 min. Pellets were resuspended in medium 1 and immediately subjected to Percoll density gradient centrifugation, with the following modifications. The gradients (Percoll-medium 1) were prepared in 15-mL polypropylene tubes and centrifuged in a fixed angle rotor (Beckman J-21A, Mississauga, ON, Canada) at 4°C for 10 min at 10,000 × g. Fibroblasts were recovered from the 20% Percoll region of the gradient by centrifugation (400 × g, 10 min) following dilution with two volumes of medium 1. Cell pellets were resuspended in the same medium and cultured in 75-cm2 culture flasks in a humidified atmosphere at 37°C, under 5% CO2 until they reached confluence. Subsequently, cells were cultured in serum-free medium for 24 h or as indicated. Media were collected and cells washed with sterile phosphatebuffered saline and lysed for RNA extraction or analysis of MMP activity. Flow densitometry using 90° light scattering as the parameter for sorting has been used to estimate the homogeneity of cell suspensions (Yablonka-Reuveni et al., 1988). The majority of cells (93%) presented the same size and cytoplasm complexity. When placed in culture under conditions that would be expected to promote fusion of myogenic cells (Dulbecco’s Modified Eagle Medium (DMEM) [Life Technologies] containing 5% horse serum), these cells never fused. Myogenic Cell Isolation and Culture This protocol was adapted from Dodson et al. (1987). Muscle was diced and ground in a sterile meat grinder in DMEM. The ground tissue was incubated with pronase (1 mg/mL, Sigma Chemical Co., Oakville, ON, Canada) in DMEM at 37°C for 45 min. The suspension 96 Balcerzak et al. was centrifuged at 1,500 × g for 10 min. The supernate was discarded and the pellet was resuspended in prewarmed DMEM. Cells were separated from muscle debris by filtration (70-m cell strainer, Falcon, Franklin Lakes, NJ) and submitted to a preplating (2 h in a humidified environment and 5% CO2) to eliminate adherent contaminating fibroblasts. A unique feature of myoblasts and satellite cells is that when they are placed under specific conditions, proliferation ceases and cells migrate, align, and fuse (Dodson et al., 1987). Cells were thus grown in DMEM containing 15% horse serum during the 1st 4 d, then transferred to a 5% horse serum-containing medium for d 5 and 6 to initiate cell migration, alignment, and fusion. This fusion is never 100%, and a few myogenic cells become quiescent (i.e., cease proliferation but do not fuse). Under culture conditions that promote fusion of myogenic cells, the only proliferating cells in the culture are the fibroblasts, and these are sensitive to treatment with cytosine arabinoside (Ara C). Cytosine arabinoside (10−5 M, Sigma Chemical Co.) was used to treat cultures for 24 h on d 6 of culture to further eliminate fibroblast contamination (Kufe et al., 1980). Cells were grown from d 7 to d 14 in 1% horse serum-containing medium (to limit proliferation of any surviving fibroblasts) and placed into serum-free medium on d 14 for 12 h. Media were collected and cells washed with sterile PBS and lysed for RNA extraction or measurement of MMP activity. As a measure of fibroblast contamination, cells were grown, after 24 h of Ara C treatment, in DMEM containing 15% horse serum to stimulate fibroblast proliferation. No fibroblast proliferation was observed under these conditions. On d 14 of culture, fusion was finished and the myotubes began their spontaneous contractions. The fusion rate was between 55 and 65% at d 14, and the culture comprised a mixture of myotubes and latent satellite cells. For this reason, we refer to this mixed cell population as “myogenic cell culture.” We also carried out co-cultures of myogenic cells and fibroblasts. After the preplating, cells were grown in DMEM containing 15% horse serum for 1 d, followed by further culturing in DMEM containing 5% horse serum to initiate both satellite cell fusion and fibroblast proliferation. HT-1080 Culture Human fibrosarcoma HT-1080 cells (CCL-121, American Type Culture Collection, Rockville MD), which are known to synthesize MMP-1, MMP-2, MMP-3, MMP7, and MMP-9, were used as a positive control (Giambernardi et al., 1998). The HT-1080 cells were cultivated in MEM containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 1.0 mM sodium pyruvate, heat-inactivated 10% fetal calf serum, and 1% antibiotic/antimycotic. The cultures were incubated at 37°C in a humidified 5% CO2 atmosphere until confluency. Cells were then placed in serum-free medium for 24 h or in serum-free medium containing phorbol myristate acetate (25 ng/ mL; Sigma Chemical Co.), which stimulates the expression of MMP-1 and -3 in this cell line. Media were collected and cells were washed with sterile PBS and lysed for RNA extraction or MMP activity characterization. Dissectable Connective Tissue The pieces of dissectable connective tissue were used for the collagen degradation assay in explant culture, for zymography, and for the isolation of fibroblasts. Connective tissue pieces were incubated intact in explant culture using [3H]-type I collagen for detection of collagenase (Zucker et al., 1985). For casein and gelatin zymographies, frozen pieces of dissected connective tissue were homogenized in extraction buffer (0.01 M cacodylic acid, pH 5.2, 0.15 M CaCl2, 0.1 mM ZnCl2, 2 mM NaCl; 1 mL buffer/100 mg tissue) (Tyagi et al., 1993). For isolation of cellular material, 10 g of connective tissue was cut into small pieces and incubated in DMEM containing 0.25 mg/mL of bacterial collagenase (type IA) (Sigma Chemical Co.) at 37°C for 45 min to isolate the cellular component. After filtration (70-m cell strainer; Falcon) to eliminate the undigested debris and centrifugation (1,500 × g for 10 min) of this filtrate, we kept the pellet containing connective tissue cells. This was used to isolate the RNA. Isolation of RNA, Northern Blot Analysis, and Reverse Transcription-PCR RNA Isolation. Total RNA was isolated using the TRIzol extraction kit (Life Technologies). For muscle, 500 mg of frozen tissue was ground in liquid nitrogen and homogenized in 5 mL of TRIzol. For the connective tissue, 5 mL of TRIzol was added to the cell pellet. For fibroblasts and myogenic cell cultures, 2.5 mL of TRIzol was added to PBS-washed cells attached to a 175-cm2 flask. Following isolation, RNA was dissolved in distilled water and quantified by UV-spectrophotometry. Total RNA was used directly for Northern blotting or reverse-transcription polymerase chain reaction (RTPCR). The quality of RNA samples was confirmed by observation of ribosomal RNA integrity following electrophoresis and ethidium bromide staining (Chomczynski and Sacchi, 1987). Northern Blot Analysis. Samples were applied to a 1% agarose gel containing 2% formaldehyde and transferred to a nitrocellulose membrane (Nitropure, MSI, Westborough, MA) by an 18-h capillary transfer in 10× saline-sodium citrate (SSC) (1.5 M NaCl, 0.15 M trisodium citrate, pH 7.0). The RNA was fixed to the membrane by baking in a vacuum oven for 2 h at 80°C. Northern blots were conducted essentially as described by Kherif et al. (1999). Plasmids containing cDNA inserts for porcine MMP-1, MMP-2, MMP-3, and MMP9 that hybridize with bovine transcripts were developed in our laboratory. RT-PCR. For reverse transcription, 2 g of RNA was incubated 15 min at 37°C with 5× first-strand buffer, Skeletal muscle extracellular proteolysis dithiothreitol, and RNase inhibitor (Boehringer Mannheim, Laval, QC, Canada). Oligo dT primer, dideoxynucleotide triphosphate (dNTP) (10 mM each) and 250 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies) were added and the mixture was incubated 60 min at 37°C. An aliquot (5 L) of the reverse transcription reaction solution was amplified by PCR using 1 U Taq DNA Polymerase (Life Technologies) in a 50-L reaction volume that contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 M of the primers for the template of interest. Reaction mixtures were amplified in a DNA thermal cycler (PCR system 2400, Perkin Elmer, Mississauga, ON, Canada) under the following conditions: 1) an initial denaturation step for 3 min at 95°C; 2) variable number of cycles with denaturation at 95°C for 1 min, annealing for 1 min, and elongation at 72°C for 2 min; and 3) incubation at 72°C for 8 min. The number of cycles of amplification required to obtain a detectable signal is dependent on the abundance of the mRNA in the sample material; as few as 18 or as many as 30 cycles were required for the different gene products. The cycle number was determined empirically and is reported in the figure legends. The annealing temperature depends on the sequence of the primers and is specific for each gene product and is also reported in the figure legends. The PCR products were separated on 2% agarose gels. After migration, gels were stained with 0.1 mg/mL ethidium bromide in distilled water. The amplification products of bovine MMP mRNA were cloned with the pGEM-T Easy Vector System I (Promega, Madison, WI) and sequenced (ABI PRISM Dye terminator cycle sequencing core kit, Perkin Elmer). These sequences have been submitted to GenBank, where they are listed under the accession numbers presented in Table 1. A semiquantitative RT-PCR was validated for each gene product. The quantity of amplified product was determined as a function of the quantity of RNA used in the RT step. The linear portion of this relationship was identified, and an RNA quantity from the middle of this linear range was selected for comparative analysis of RNA samples from different animals. To control the homogeneity of the RNA concentrations in the different samples and variations of the RT-PCR reactions, we used the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), known as a “house-keeping” gene (Yelich et al., 1997). The GAPDH product was amplified using the following primers: antisense: 5′TCCACCACCCTGTTGCTGTA; sense: 5′ACCACAGT CCATGCCATCAC. Custom primer synthesis was done for the specified sequences by the DNA Synthesis Laboratory in the Department of Biochemistry at the University of Alberta. 97 buffer (0.01 M cacodylic acid, pH 5.2, 0.15 M CaCl2, 0.1 mM ZnCl2, 2 mM NaCl) (1 mL buffer/100 mg tissue). Following this incubation, the sample was homogenized with a Polytron (Brinkman, Mississauga, ON, Canada), and centrifuged for 10 min at 350 × g at 4°C. The pellet was then resuspended in extraction buffer (1 mL buffer/ 100 mg tissue), re-homogenized, and centrifuged, and the supernates pooled with the first one. This step was repeated twice. Cells were scraped from culture dishes with 1 mL of extraction buffer. The homogenate was sonicated and centrifuged for 10 min at 350 × g at 4°C. Soluble proteins contained in the supernate were quantified by the BCA protein assay (Pierce, Rockford, IL) using BSA as a protein standard. Electrophoresis and MMP Zymography. Samples prepared from cells or tissues containing 15 g of protein were mixed with 0.25 volumes of nonreducing sample buffer consisting of 0.3 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 0.03% bromophenol blue. Electrophoresis was run on 15% SDS-PAGE gels containing gelatin (Type I, 1 mg/mL [Sigma Chemical Co.]) for the gelatin zymography (Lantz and Ciborowski (1994) or α-casein (1 mg/mL [Sigma Chemical Co.]) for casein zymography (Rawdanowicz et al., 1994). After electrophoresis, the gel was removed and incubated for 30 min at 25°C in Triton X-100 (2.5% in distilled water). After two 15-min washes in Tris-HCl (50 mM, pH 7.5), gels were incubated 20 h at 37°C, with gentle shaking, in 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 0.05% Brij35. Gels were stained with 0.1% Naphthol blue black solution in acetic acid/methanol/distilled water (1:4.5:4.5 by volume) and destained with distilled water. Electrophoresis and Western Blot Samples prepared from cells or tissues containing 15 g of protein were mixed with 0.25 volumes of reducing sample buffer (0.05 M Tris-HCl, pH 6.80, 10% SDS, 0.01% bromophenol blue, 30% glycerol, 1% dithiothreitol), boiled 5 min, and run on a 15% acrylamide SDSPAGE gel (Laemmli, 1970). Fractionated proteins were then electrotransferred onto nitrocellulose membranes (Nitropure, MSI, Westborough, MA). After an overnight incubation at 4°C in blocking solution (Tris-buffered saline [TBS] containing 5% BSA), the membranes were placed in TBS containing 1% BSA and specific antibody (anti-MMP-2 at a dilution of 1/200, Calbiochem, San Diego, CA). The identification of the antigen-antibody complex was carried out (via a second antibody coupled to horseradish peroxidase) using the ECL Western blotting detection kit (Amersham, Bais d’Urfé, QC, Canada) according to the manufacturer’s instructions. Collagen Degradation Assay Zymography Sample Preparation. After grinding in liquid N, tissue samples were incubated (24 h at 4°C) in extraction Thin pieces (∼1 mm) of dissectable connective tissue (1 cm2) were placed into a Krebs Ringer bicarbonate buffer, 119 mM NaCl, 25 mM NaHCO3, 4.82 mM KCl, 98 Balcerzak et al. Table 1. Characteristics of PCR primers and amplified products of a bovine matrix metalloproteinase systema Primer sequences PCR fragment size, bp Sense: GCA GCC CAG ATG TGG GGT GCC CG Antisense: ACA CTT CTG GGG TTT GGG GGC CG 571 Sense: CTT CCC CCG CCA GCC CAA GTG GG Antisense: GGT GAA CAG GGC TTC ATG GGG GC 510 Sense: GAC TCC ACT CAC ATT CTC CAG G Antisense: CCT GAA GGA AGA GAT GGC 586 Sense: CCC AAA GAA TGG CCA AGT TC Antisense: TGC AGA AGC CCA GAT GTG GA 418 Sense: ACG TGG ACA TCT TCG ACG C Antisense: CGA ACC TCC AGA AGC TCT GC 359 Sense: TCT GGT CTG CTG GCT CAC GC Antisense: TAG GCA GCA TCA ATA CGG TTG G 472 Sense: ACC ATG AAG GCT ATG AGG CGC CC Antisense: GTT GAT GGA TGC AGG CAG GCC CC 831 Sense: CTG GTT CTG GCG AGT CAG GC Antisense: CCT TGT AGA AGT AGG TGT AGG C 424 Sense: CTG GAA GAA GGT TGG ATT TCG TGC Antisense: ACA TTC TGC CAC ACA TCA AAG G 461 Sense: GTC TGG TGA ATC GAA CTG TGG C Antisense: GGC TGC AAA CCA AGG CTG 511 Sense: TCC AGG CTC TTG GGG CCT GC Antisense: CGG CAG TCA ATG AGG AAA GT 665 Sense: GTA CCT GCG TCC CAC CCC ACC Antisense: GGC AGG CAG GCC AGG TGG CGG 504 Sense: CCT CCT GCT GCT GGG GAC GCT GC Antisense: AGT CCT GGT GGC CTG CTT ACG GG 643 Sense: GAC CCC TTG GCT CGG GCT CAT CG Antisense: GCT GGT CCC ACC TCT CCA CGA AG 377 Target mRNA MMP-1 MMP-2 MMP-3 MMP-7 MMP-9 MMP-13 MMP-14 MMP-15 MMP-16 uPa uPaR TIMP-1 TIMP-2 TIMP-3 GenBank accession no. Homology with human sequence AF134714 83.58% AF135231 87.04% AF135232 80.40% AF135233 80.68% AF135234 82.39% AF135235 92.29% AF144758 86.75% AF144759 97.10% AF144760 89.88% AF144761 79.83% AF144762 72.15% AF144763 86.65% AF144764 90.64% AF144765 93.95% a The amplified product of the expected size for each couple of primers, obtained by reverse transcription-polymerase chain reaction, was cloned with pGEM-T easy Vector System I (Promega, Madison, WI). Sequence information from these DNA permitted confirmation of their identity (by homology with human sequences). Sequences have been submitted to GenBank and are saved under the accession numbers listed. MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; uPa, urokinase-type plasminogen activator; uPaR, urokinasetype plasminogen activator receptor. 1 mM CaCl2, 1.25 mM MgSO4, 1.24 mM NaH2PO4, 2 mM HEPES, and 5 mM glucose (pH 7.4). Samples were then transferred to fresh medium containing 0.3 mCi/ mL of N-[propionate-2-3-3H] propionylate collagen (rat type I, specific activity: 0.2 mCi/mg; Dupont NEN, Boston, MA). Explants were incubated at 30°C, 5% CO2, in a humidified atmosphere. Samples were dried on a SpeedVac (Savant Inc., Holbrook, NY) and run on a 12.5% SDS-PAGE gel under reducing conditions (Laemmli, 1970). Gels were processed for fluorography prior to drying and exposure to x-ray film (Kodak X-OMAT, Rochester, NY) (Laskey and Mills, 1975). These films were scanned and analyzed using the Molecular Analyst software (BioRad, Mississauga, ON, Canada). Statistical Analysis To observe possible correlation of expression between the different elements of the extracellular proteolytic system in bovine semimembranosus muscle (n = 35), simple regression analyses were performed using the General Linear Models procedures of SAS (SAS Inst. Inc., Cary, NC). Results Characterization of an Extracellular Proteolytic System in Skeletal Muscle Degradation of [3H]-Type I Collagen. Dissectable con- nective tissue was incubated with [3H]-type I collagen for up to 4 h. We observed two characteristic fragments of degradation that corresponded to the 3/4-length fragments of the α1 and α2 chains of the collagen (data not shown). The presence of these 3/4-length fragments demonstrated the presence of a collagenase activity able to degrade type I collagen in the extracellular matrix of skeletal muscle. The MMP-1, MMP-8, and MMP13 are known to exhibit this proteolytic specificity and were suspected to be responsible for the observed degradation. No detectable activity was seen in incubated explants of skeletal muscle using this assay. There may Skeletal muscle extracellular proteolysis be no net collagenase activity in muscle (i.e., due to the presence of TIMP); an alternative possibility is that the assay was not sensitive enough to detect the activity present. Zymography. Two major gelatinase activities were observed (Figure 2) in the homogenate of masseter muscle (lane 6), in connective tissue derived from this muscle (lane 7), and in the positive control cell line HT1080 (lane 5) using zymography. These activities had apparent molecular weights of 100 kDa and 66 kDa (the major signal) characteristic of pro-MMP-9 and proMMP-2, respectively, which are known to be expressed by HT-1080 cells (Huhtala et al., 1991). Activities with apparent molecular weights of 80 kDa and 62 kDa corresponded to the activated forms of MMP-9 and MMP2, respectively. It seems that the majority of MMP2 exists in its zymogen form in muscle (lane 6) and connective tissue (lane 7) homogenates. For MMP-9, no active form was detected in the same samples. A strong pro-MMP-2 activity was detected in the conditioned medium obtained from cultured fibroblasts (lane 3), whereas this activity was less intense in the satellite cell-conditioned culture medium (lane 1). In the conditioned medium obtained from co-cultures of satellite cells and fibroblasts, we observed an intermediate level of pro-MMP-2 activity (lane 2) as compared to individual cultures, showing the influence of fibroblasts in the satellite cell culture on the quantity of this pro-enzyme in the conditioned culture medium. Surprisingly, satellite cell culture and co-culture conditioned media exhibited the active form of MMP-2, whereas this was not seen in the fibroblast-derived medium. This activated form was observed, however, in the fibroblast homogenate (lane 4). In this sample, an unidentified activity 99 was also observed that had an apparent molecular weight of about 50 kDa. This could be the zymogen form of MMP-1 or MMP-3. These results suggest that fibroblasts may not be the exclusive source of gelatinases within the muscle tissue, because myogenic cells and whole muscle also expressed these activities. Western Blot. Western blot analysis was limited by the lack of commercially available antibodies known to cross-react with bovine MMP. Additionally, the sensitivity of this technique was problematic. Using HT1080 culture medium as a positive control, we were able to detect a signal with an anti-MMP-2 antibody, but only when this conditioned medium was concentrated 30-fold (data not shown). We were also able to detect MMP-2 with this antibody in conditioned culture media from intramuscular fibroblasts, also concentrated 30fold (data not shown). No signal was detectable in skeletal muscle homogenates or cultured fibroblast homogenates. Moreover, unconcentrated culture media gave no signal. It seems that this approach was at least 30fold less sensitive than zymography, which could reveal activity in unconcentrated culture media and tissue extracts. Northern Blot. Northern hybridization analysis was attempted using MMP-1, MMP-3, and MMP-9 probes on RNA extracted from cultured intramuscular fibroblasts and whole skeletal muscle, but no signal was detected. Using this technique, we were able to detect a signal corresponding to MMP-2 mRNA in cultured intramuscular fibroblasts, but not from whole skeletal muscle total RNA (data not shown). Because we had observed proMMP-2 in both samples as well as proMMP-9 by zymography, it seemed likely that the sensitivity of the Northern technique was limiting, and Figure 2. Detection of gelatinase activities by zymography. Proteins (15 g) were separated on a 15% SDS-PAGE gel containing gelatin (1 mg/mL). After migration the gel was washed in a triton X-100 solution (2.5% in distilled water), incubated 20 h at 37°C in enzyme buffer, and stained with Naphthol blue black solution. Lanes from left to right contained the following samples: M, Molecular Weight Markers; 1) bovine myogenic cell culture medium; 2) coculture bovine myogenic cell-fibroblast culture medium; 3) bovine fibroblast culture medium; 4) bovine fibroblast culture homogenate; 5) human fibrosarcoma HT-1080 culture medium; 6) bovine skeletal muscle homogenate; and 7) bovine connective tissue homogenate. 100 Balcerzak et al. Table 2. Summary of gene expression in bovine whole muscle, connective tissue, isolated fibroblasts, myogenic cells, and human fibrosarcoma HT-1080 cells Cultured cells Controls Whole muscle Connective tissue Isolated fibroblasts Myogenic cells HT-1080 Collagenases MMP-1 MMP-13 + − − + + − + − + − 3a 3b Gelatinases MMP-2 MMP-9 + + + + + − + − + + 3c 3d Stromelysins MMP-3 MMP-7 − − − − + − + − + + MT-MMP MMP-14 MMP-15 MMP-16 − − + − + + + − + − − + + + + 4a 4b 4c TIMP TIMP-1 TIMP-2 TIMP-3 + + + + + + + + + + + + + + + 4d 4e 4f uPa cascade uPa uPaR + + + + + + + + + + 4g 4h Enzyme Bovine tissue + Duodenum Figurea 3e 3f a Representative PCR results are shown in the figures indicated. “+” denotes the expression and “−” the absence of expression. MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; uPa, urokinase-type plasminogen activator; uPaR, urokinase-type plasminogen activator receptor. we thus proceeded to the highly sensitive RT-PCR approach. RT-PCR. The technique is based on amplification of cDNA corresponding to RNA of interest and requires knowledge of the individual cDNA sequences in order to design primers for the amplification. For the bovine, only sequences for MMP-1 (Tamura et al., 1994), MMP9 (Baylis et al., 1995), TIMP-1 (Satoh et al., 1994), TIMP-2 (Boone et al., 1990), and urokinase-type plasminogen activator (uPa) (Kratzschmar et al., 1993) have been published, and primers were designed to amplify bovine MMP-9 and uPa (Menino et al., 1997). Primers to amplify MMP-7 and uPaR were kindly provided by Linghuo Jiang (University of Alberta). For all the other MMP, TIMP, MT-MMP, and factors involved in regulation of MMP activity, it was necessary to design our own primer sets and validate the amplified products. We designed consensus primers, validated the amplified products, and submitted to the GenBank database partial sequences for bovine MMP-1, -2, -3, 7, -13, -14 (MT1-MMP), -15 (MT2-MMP), -16 (MT3MMP), TIMP-1, -2, -3, uPa, and urokinase-type plasminogen activator receptor (uPaR). A summary of the RT-PCR analysis of the expression of these elements is shown in Table 2. The identity of all amplified products was confirmed by DNA sequencing and through the use of positive controls, including a human cell line known to express multiple MMP (Giambernardi et al., 1998) and bovine duodenum (a source of MMP-7, unpublished data). Whole Muscle Tissue. Bovine masseter muscle expressed mRNA encoding MMP-1, -2, -9, -16, TIMP-1, 2, and -3, uPa, and its receptor, uPaR (Figures 3 and 4, lane 2). This indicates the expression of all the elements potentially involved in the degradation of the protein components of the extracellular matrix: one collagenase, two gelatinases, and one MT-MMP. The MMP-16 (MT3-MMP) is thought to be responsible for activation of pro-MMP-2, and plasmin, which is activated by uPa, is thought to activate pro-MMP-1 and pro-MMP-9. Of the MMP studied, MMP-2 and MMP-16 gave the strongest signals in the PCR reaction at 30 cycles of amplification; these results concur with results of zymography and Western blotting, suggesting high levels of MMP2. Skeletal muscle also expressed all three TIMP tested. Several tissues are known to express more than one TIMP (Tanney et al., 1998), although the role of these in regulation of the specific MMP is not known. When a tissue expresses more than one MMP or TIMP with overlapping specificity, it may be that these are present at different sites within the tissue. Freshly Isolated Connective Tissue Cells. The expression of elements of the MMP system was also studied in RNA extracted from skeletal muscle connective tissue cells. The overall pattern of expression was similar to that of whole muscle in that it included MMP-2, -9, and -16, TIMP-1, -2, and -3, and uPa and uPaR (Figures 3 and 4, lane 3). At the same time, two MMP not seen in whole muscle were expressed, a collagenase (MMP13) and MMP-15 (MT2-MMP). Assuming that these Skeletal muscle extracellular proteolysis latter two enzymes are expressed only by fibroblasts, their expression would be more apparent in this relatively enriched source of fibroblast mRNA, compared with whole muscle in which the vast majority of RNA is derived from muscle cells. Cultured Fibroblasts. The pattern of expression of the intramuscular fibroblast population was not identical to that in the connective tissue cell fraction, possibly because the cells had been cultured for several passages prior to harvest of the RNA. However, the overall pattern of expression was similar to that of whole muscle in that it included MMP-1, -2, and -16, TIMP-1, -2, and 3, uPa, and uPaR (Figures 3 and 4, lane 4). There were again some unique aspects of expression in this cell population: lack of MMP-9 and expression of MMP-3 and MMP-14 (MT1-MMP). Myogenic Cells. To test the hypothesis that myogenic cells could synthesize elements of the MMP system, muscle cells free of contaminating fibroblasts was prepared. This myogenic cell fraction from bovine muscle, however, represents a relatively undifferentiated cell type compared to mature muscle cells. This cell popula- 101 tion showed an expression pattern highly similar to that of whole muscle: MMP-1, -2, and -16, TIMP-1, -2, and -3, uPa, and uPaR (Figures 3 and 4, lane 5). No expression of MMP-9 or MMP-3 was observed in these cells. This pattern was also similar to that seen in isolated fibroblast expression, except for the absence of MMP-14 (highly expressed in cultured fibroblasts) and MMP-15 (highly expressed in freshly isolated connective tissue cells) (Figure 4). The absence of expression of these connective tissue-specific elements further suggests the absence of major fibroblast contamination in the satellite cell culture. Coordinate Expression of Elements of the Extracellular Proteolytic System in Muscle Tissue Skeletal muscle expressed MMP-1, -2, -9, and -16 (MT3-MMP), TIMP-1, -2, and -3, uPa, and uPaR. In an attempt to understand how expression of this system is regulated, we studied semimembranosus muscle from 35 individual animals. We observed a few differences in the pattern of MMP expression in this muscle Figure 3. Determination of matrix metalloproteinase expression by reverse transcription-polymerase chain reaction. Reverse transcription was done with total RNA isolated from cultured human fibrosarcoma HT-1080 cells (lane 1) and bovine skeletal muscle (lane 2), dissectable connective tissue (lane 3), cultured fibroblasts (lane 4), cultured myogenic cells (lane 5), and duodenum (lane 6). The 100-bp DNA ladder is presented in the lane denoted M in each panel. The PCR reactions were conducted with the primers for a) MMP-1 (30 cycles; annealing at 65°C), b) MMP-13 (30 cycles, annealing at 57°C), c) MMP-2 (25 cycles, annealing at 65°C), d) MMP-9 (30 cycles, annealing at 55°C), e) MMP-3 (30 cycles, annealing at 65°C), and f) MMP-7 (30 cycles, annealing at 60°C). MMP, matrix metalloproteinase. 102 Balcerzak et al. Figure 4. Expression of modulators of matrix metalloproteinase activity determined by reverse transcription-polymerase chain reaction. Reverse transcription was done with total RNA isolated from cultured human fibrosarcoma HT-1080 cells (lane 1) and bovine skeletal muscle (lane 2), dissectable connective tissue (lane 3), cultured fibroblasts (lane 4), and cultured myogenic cells (lane 5). The 100-bp DNA ladder is presented in the lane denoted M in each panel. The PCR reactions were conducted with the primers for a) MMP-14 (30 cycles, annealing at 65°C), b) MMP15 (30 cycles, annealing at 57°C), c) MMP-16 (25 cycles, annealing at 65°C), d) TIMP-1 (30 cycles, annealing at 65°C), e) TIMP-2 (30 cycles, annealing at 65°C), f) TIMP-3 (30 cycles, annealing at 65°C), g) uPa (30 cycles, annealing at 65°C), and h) uPaR (30 cycles, annealing at 60°C). MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; uPa, urokinase-type plasminogen activator; uPaR, urokinase-type plasminogen activator receptor. compared to masseter, with the absence of MMP-1 and MMP-9 and the presence of MT1-MMP. There may be fiber type specificities in the MMP system of different muscles, but these remain to be determined. To study variations in gene expression within the MMP system of skeletal muscle, a semiquantitative RTPCR approach was developed. The number of PCR cycles and the quantity of total RNA necessary in the RT step were optimized to observe the quantity of amplified product obtained in the linear part of the process of cDNA amplification. First, using 2 g of total RNA in the RT step, the quantity of amplified product formed as a function of cycle number was determined. Second, the quantity of amplified product formed as a function of the level of total RNA in the RT step was determined. For example, for MMP-2 at 24 cycles, the linear range of amplification was 0.7 to 2.2 g of total RNA in the assay (Figure 5). Thus, in all assays in which levels of 103 Skeletal muscle extracellular proteolysis MMP-2 expression were compared among animals, 1.5 g of total RNA was used. The quantification of amplified product was similarly validated with other elements of the MMP system, as well as GAPDH (Figure 5). Because the optimal RNA quantity was different depending on the primer set used, we prepared different RNA solutions (0.25, 0.5, 1, and 1.5 g/L) from a 2g/L stock by serial dilutions. We tested the expression of GAPDH in the 35 samples and observed a very similar quantity of amplified product showing the uniformity in the level of expression in these samples (Figure 6C). This also suggests the homogeneity of the RNA concentration in the samples and the absence of major variations in the RT-PCR reactions. Using this semiquantitative RT-PCR technique, the levels of MMP-2, -14 (MT1-MMP), and -16 (MT3-MMP), TIMP-1, -2, and -3, uPa, and uPaR expression were determined. A regression analysis was done for all these genes to test for coordinated expression (the r-values [coefficient of correlation] and the corresponding P-values are summarized in Figure 6A). For example, the relationship between TIMP-1 and TIMP-2 expression is illustrated in Figure 6B. We observed a significant correlation (r > 0.614, P < 0.0003) of expression among TIMP-1, -2, and -3, MMP-2, and MMP-14 (MT1-MMP). We observed also a strong relationship among uPa, MMP-2 and MMP-14 expression (r > 0.621, P < 0.0004). Concerning MMP-16 (MT3-MMP), we observed a strong correlation of its expression with TIMP-3 and MMP-2 expression (r > 0.615, P < 0.0005). Discussion Figure 5. Determination of the optimal quantity of bovine RNA for reverse transcription. (A) Graph and picture representing the evolution of MMP-2 amplified product as a function of the quantity of total RNA in the reverse transcription. The PCR reaction was running at 24 cycles. (B) Graph and picture representing the evolution of GAPDH amplified product as a function of the quantity of total RNA in the reverse transcription. The PCR reaction was running at 17 cycles. (C) Summary of the PCR conditions used for each couple of primers, concentration range for which the increase of amplified product quantity was linear, and quantity of total RNA chosen for the reverse transcription step. MMP, matrix metalloproteinase; GAPDH, glyceraldehyde phosphate dehydrogenase. Little is known of the MMP system of skeletal muscle. Expression of MMP-1, but not MMP-2, -3, or -10 was reported in mouse muscle (Gack et al., 1994). Another study revealed the expression of MMP-2 and TIMP-1 in unfused human satellite cells; MMP-1 and MMP9 were also expressed after stimulation with phorbol myristate acetate (Guerin and Holland, 1995). These findings suggest that myogenic precursors synthesize and secrete selected members of the MMP family. However, the relationship of these observations to differentiated skeletal muscle is unclear. The activity, expression, and localization of the gelatinases MMP-2 and MMP-9 have been recently reported in muscle after injury and denervation (Kherif et al., 1998, 1999). However, in addition to gelatinases, the full spectrum of MMP participating in matrix catabolism must clearly also include collagenases, stromelysins, activators, and inhibitors of MMP. Our work is the first systematic study of the MMP system of skeletal muscle to include all of these potential elements. The predominant cell type in skeletal muscle tissue is the myofiber, but this tissue also contains fibroblasts, vascular endothelial and smooth muscle cells, nerves, adipocytes, and mast cells. Whereas fibroblasts are considered to be the classic source of connective tissue 104 Balcerzak et al. Figure 6. Regression analysis of the expression of elements of a bovine matrix metalloproteinase system. (A) Regression analysis between the expression of different genes of the extracellular proteolytic system in bovine semimembranosus muscle (n = 35). The r-value (coefficient of correlation) is on the top and the corresponding P-value in parentheses on the bottom. (B) Illustration of the regression analysis between TIMP-1 and TIMP-2 expression. The rvalue (coefficient of correlation) and the corresponding P-value are presented with the graph. (C) GAPDH expression in the 35 samples tested. The mean and its standard deviation are presented with the graph. TIMP, tissue inhibitor of metalloproteinase; GAPDH, glyceraldehyde phosphate dehydrogenase. components, including MMP, it seems that the muscle fibers themselves also express elements of the MMP cascade, including MMP-1, -2, -9, and -16, uPa, uPaR, and TIMP-1, -2, and -3. The MMP-13 and MMP-15 are examples of enzymes expressed specifically in connective tissue cells, and at 30 cycles of amplification in PCR, there was no detectable signal in RNA from whole skeletal muscle for either. This is in contrast with MMP-2, -9, and -16, uPa, uPaR, and TIMP-1, -2, and -3, which all showed a similar signal, using an equivalent number of cycles of amplification, in both whole muscle tissue and connective tissue cells. If connective tissue cells were the sole source of these mRNA in muscle, given the abundance of these cells (< 1% of total nuclei), one would expect a much lower signal in the RT-PCR for whole muscle than for connective tissue, as seen for MMP-13 and MMP-15. Although the myogenic cell cultures used here are not differentiated adult myofibers, results from study of these cells support the idea that cells of a myogenic Skeletal muscle extracellular proteolysis lineage express MMP-1, -2, -3, and -16, uPa, uPaR, and TIMP-1, -2, and -3. These results concur with observations by Guerin and Holland (1995), who showed expression of MMP-2 and TIMP-1 in human satellite cells. The expression of multiple elements of an MMP cascade in muscle cells would point to a high degree of interrelationship between muscle cells and the dynamic modulation of the connective tissue. Muscle cell-derived MMP and fibroblast MMP may act coordinately and(or) independently to control connective tissue catabolism. The skeletal muscle cells and structurally related connective tissue function cooperatively in their mutual task of force transduction, so the myofibers could participate in some manner in the dynamic remodeling of connective tissue as the muscle hypertrophies, atrophies, stretches, or changes aspects of its force generation. Further work will reveal the relative roles of muscle cells and connective tissue cells in the expression and regulation of the intramuscular MMP system. Based on what we have observed here and the related literature, both cell-cell and cell-matrix interactions would be anticipated to have a major influence. When the promoter sequences of some of the elements of the MMP system are examined, consensus sequences such as E-box, CCAC box, SRE 3, MAPF1/2, M1, and other muscle-specific motifs are observed. For example, the promoter of human MMP-2 (Bian and Sun, 1997) (GenBank accession no. U96098) contains at least six E-boxes, two MAPF1/2, and one M1, all of which are potential targets for muscle-specific transcription factors. This is also true of the promoters for human MMP1 (Rutter et al., 1997) (GenBank accession no. AF023338), human MMP-3 (Kirstein et al., 1996) (GenBank accession no. U43511), and rat TIMP-1 (Bugno et al., 1995) (GenBank accession no. X90486). The presence of these motifs suggests the possibility that these genes could be expressed specifically under certain physiological conditions in the muscle, independently of the other tissues. The extracellular matrix of skeletal muscle is structurally and functionally complex. This matrix, which contains collagen (Types I, III, IV, and V), glycoproteins (laminin and fibronectin), and proteoglycans (Kuhn, 1987; Bailey and Light, 1989; Pearson and Young, 1989), contributes to the mechanical characteristics of muscle (Partridge and Benton, 1981; Moore, 1983) and the transmission of force from muscle to tendon. We attempted to address the complexity of the MMP system of skeletal muscle. The expression of as many as two collagenases (MMP-1 and MMP-13), one gelatinase (MMP-2 or MMP-9), elements of proMMP activation (uPa and uPaR, MT1-MMP, MT2-MMP, and MT3MMP), and metalloproteinase inhibitors (TIMP-1, -2, and -3) were demonstrated in the different samples. The individual cellular components (whole muscle, dissectible connective tissue, muscular fibroblasts in culture, and myogenic cells in culture) correspond to various potential sources of elements of the extracellular matrix proteolytic system in muscle. With respect to 105 the identity and potential substrates of these enzymes, this proteolytic system in its entirety would be able to degrade all of the components of muscle extracellular matrix. The collagenases (MMP-1 or MMP-13) denature the fibrillar collagen (Miller et al., 1976a,b; Velgus et al., 1981). This denatured collagen can be degraded to small peptides by gelatinase activity (MMP-2 or MMP9) (Seltzer et al., 1981, 1990). These gelatinases are also known to degrade the fibrillar type V and the nonfibrillar type IV collagen. In parallel with these enzymes, all of the elements for their activation are also expressed. Urokinase-type plasminogen activator and uPaR are expressed by the whole muscle and fibroblasts in the muscular connective tissue, allowing for plasminogen activation. This plasminogen, synthesized by the liver and secreted into the blood circulation, diffuses from blood vessels to the connective tissue and will encounter all of the elements for its activation on muscle and fibroblast cell surfaces. The resulting plasmin will be able to activate the zymogens of MMP-1 and MMP-9. The MT-MMP, the second major route for proMMP activation, are also expressed by muscle cells and fibroblasts in the connective tissue. In masseter muscle, MMP-16 (MT3-MMP) is expressed by both cell types. This enzyme can participate in the activation of pro-MMP-2 and also, perhaps, pro-MMP13. The presence of MT-MMP and(or) plasmin activity could also complete the proteolytic process by acting on glycoproteins (fibronectin, vitronectin, and laminin) and on proteoglycans. Production of TIMP is one of the mechanisms by which MMP activity is controlled. In all of the samples tested, we detected expression of TIMP-1, TIMP-2, and TIMP-3. The presence of these three proteinase inhibitors shows the important negative regulatory arm of this proteolytic system, which is necessary to protect muscle connective tissue from uncontrolled metalloproteinase activities. Variations in the expression of elements of this proteolytic system were analyzed in semimembranosus muscle from 35 animals. A strong correlation among expression of TIMP-1, TIMP-2, TIMP-3, MMP-2, and MMP-14 (MT1-MMP) was observed. Another correlation was observed among MMP-16 (MT3-MMP), MMP2, and TIMP-3. All these genes are involved in the activation of pro-MMP-2 (Figure 1). Tissue inhibitor of metalloproteinase-2 and MMP-14 are known to be involved in the multimeric complex during the process of proMMP-2 activation, which could explain their coordinate expression. Sakamoto et al. (1999) observed a similar relationship among these three genes in human cartilagenous tumors. Our results indicate also a strong correlation among the expression of these three genes and expression of TIMP-1 and TIMP-3. Tissue inhibitor of metalloproteinase -3, which inhibits both MMP-2 and MMP-14 activity, is a likely regulator of this activation system, whereas TIMP-1, which inhibits MMP-2 activity (and not MMP-14), could be expressed to control the active MMP-2. The MMP-16 is also known to be 106 Balcerzak et al. a physiological activator of pro-MMP-2, which could explain the strong correlation of expression observed between them. This type of pro-MMP-2 activation may be the major pathway in the masseter muscle, which expressed only MMP-16, in comparison with semimembranosus muscle, which expressed both MMP-14 and MMP-16. Interestingly, uPa expression was also found to be strongly correlated with MMP-2 and MMP-14 expression. The most likely role of uPa in this system is the activation of plasminogen to plasmin, which then activates pro-MMP-1, pro-MMP-3, and pro-MMP-9 (Figure 1), rather than its involvement in pro-MMP-2 activation. Recently, Kazes et al. (1998) mentioned the presence in vitro of a soluble form of MT1-MMP (which could be the extracellular domain of MT1-MMP cleaved from its transmembrane domain) and its activation by uPa in human mesangial cells. In light of these observations, uPa may be in some way involved in both activation pathways (MT-MMP and plasmin). Conventional approaches to studying MMP seem to have some limitations in skeletal muscle tissue. It seems that gelatin zymography and RT-PCR were sensitive enough to detect gelatinase activities and MMP expression, respectively. Other techniques ([3H]-type I collagen degradation, α-casein zymography, Western, and Northern blot analysis) lacked sensitivity to detect low quantities of RNA and enzymes. Similar problems were observed in previous studies of MMP in muscle. Guerin and Holland (1995) did not observe the expression of MMP-1 in satellite cells using the Northern blot technique unless these cells had been activated with phorbol ester. Our results show the possibility of detecting these mRNA with the RT-PCR technique in unstimulated satellite cells. Kherif et al. (1999) attempted in situ hybridization using an MMP-2 riboprobe, but this did not allow for the identification of the cellular source of MMP-2 mRNA, which they attributed to a low number of MMP-2 transcripts per cell. Low levels of enzyme and gene expression may pose significant problems for localization of MMP in situ in the different cell types and structural divisions of the connective tissue. Implications To date, a number of results implicate intracellular proteolytic systems in the physiologic modulation of muscle protein deposition and in the meat tenderization processes through continued activity postmortem. There has been a lack of information concerning the proteolytic system degrading the components of connective tissue in muscle. This work reveals a highly complex system for extracellular proteolysis in muscle and opens new possibilities to study the proteolytic processes touching upon muscle growth and remodeling in vivo and connective tissue postmortem. Literature Cited Bailey, A. J., and N. D. Light. 1989. Structure and localisation of connective tissue components in muscle. In: Connective Tissue in Meat and Meat Products. p 120. Elsevier Science Publishing Co., New York. Baylis H. A., A. Megson, and R. Hall. 1995. Infection with theileria annulata induces expression of matrix metalloproteinase 9 and transcription factor AP-1 in bovine leucocytes. Mol. Biochem. Parasitol. 69:211–222. Bian, J., and Y. Sun. 1997. Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase 2) promoter. Mol. Cel. Biol. 17:6330–6338. Boone, T. C., M. J. Johnson, Y. A. DeClerck, and K. E. Langley. 1990. cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases. Proc. Natl. Acad. Sci. USA 87:2800–2804. Bugno, M., L. Graeve, P. Gatsios, A. Koj, P. C. Heinrich, J. Travis, and T. Kordula. 1995. Identification of the interleukin-6/oncostatin M response element in the rat tissue inhibitor of metalloproteinases-1 (TIMP-1) promotor. Nucleic Acids Res. 23:5041– 5047. Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159. Dodson, M. V., E. L. Martin, M. A. Brannon, B. A. Mathison, and D. C. McFarland. 1987. Optimization of bovine satellite cell-derived myotube formation in vitro. Tissue Cell 19:159–166. Emonard, H., and J. A. Grimaud. 1990. Matrix metalloproteinases. A review. Cell. Mol. Biol. 36:131–153. Gack, S., R. Vallon, J. Schaper, U. Ruther, and P. Angel. 1994. Phenotypic alteration in Fos transgenic mice correlates with changes in Fos/Jun-dependent collagenase type I expression. J. Biol. Chem. 269:10363–10369. Giambernardi, T. A., G. M. Grant, G. P. Taylor, R. J. Hay, V. M. Maher, J. J. McCormick, and R. J. Klebe. 1998. Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol. 16:483–496. Guerin, C. W., and P. C. Holland. 1995. Synthesis and secretion of matrix-degrading metalloproteinases by human skeletal muscle satellite cells. Dev. Dyn. 202:91–99. He, C., S. M. Wilhelm, A. P. Pentland, B. L. Marner, G. A. Grant, A. Z. Eisen, and G. I. Goldberg. 1989. Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc. Natl. Acad. Sci. USA 86:2632–2636. Huhtala, P., A. Tuuttila, L. T. Chow, J. Lohi, J. Keski-Oja, and K. Tryggvason. 1991. Complete structure of the human gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells. J. Biol. Chem. 266:16485–16490. Kazes, I., F. Delarue, J. Hagege, L. Bouzhir-Sima, E. Rondeau, J. D. Sraer, and G. Nguyen. 1998. Soluble latent membrane-type 1 matrix metalloproteinase secreted by human mesangial cells is activated by urokinase. Kidney Int. 54:1976–1984. Kherif, S., M. Dehaupas, C. Lafuma, M. Fardeau, and H. S. Alameddine. 1998. Matrix metalloproteinases MMP-2 and MMP-9 in denervated muscle and injured nerve. Neuropathol. Appl. Neurobiol. 24:309–319. Kherif, S., C. Lafuma, M. Dehaupas, S. Lachkar, J. G. Fournier, M. Verdière-Sahuqué, M. Fardeau, and H. S. Alameddine. 1999. Expression of matrix metalloproteinases 2 and 9 in regenerating skeletal muscle: A study in experimentally injured and mdx muscles. Dev. Biol. 205:158–170. Kirstein, M., L. Sanz, S. Quinones, J. Moscat, M. T. Diaz-Meco, and J. Saus. 1996. Cross-talk between different enhancer elements during mitogenic induction of the human stromelysin-1 gene. J. Biol. Chem. 271:18231–18236. Knäuper, V., C. Lopez-Otin, B. Smith, G. Knight, and G. Murphy. 1996. Biochemical characterization of human collagenase 3. J. Biol. Chem. 271:1544–1550. Skeletal muscle extracellular proteolysis Kratzschmar, J., B. Haendler, S. Kojima, D. B. Rifkin, and W. D. Schleuning. 1993. Bovine urokinase-type plasminogen activator and its receptor: cloning and induction by retinoic acid. Gene (Amsterdam) 125:177–183. Kufe, D. W., P. P. Major, E. M. Egan, and G. P. Beardsley. 1980. Correlation of cytotoxicity with incorporation of ara-C into DNA. J. Biol. Chem. 255:8997–9000. Kuhn, K. 1987. The classical collagens: Types I, II and III. In: R. Mayne and R. E. Burgeon (ed.) Biology of Extracellular Matrix: A Series, Structure and Function of Collagen Types. pp 1–42. Academic Press, Orlando, FL. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227:680–685. Lantz, M. S., and P. Ciborowski. 1994. Zymographic techniques for detection and characterization of microbial proteinases. Methods Enzymol. 235:563–594. Laskey, R. A., and A. D. Mills. 1975. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335–341. Menino, A. R. Jr., A. Hogan, G. A. Schultz, S. Novak, W. T. Dixon, and G. H. Foxcroft. 1997. Expression of proteinases and proteinases inhibitors during embryo-uterine contact in the pig. Dev. Genet. 21:68–74. Miller, E. J., J. E. Finch, E. Chung, W. T. Butler, and P. B. Robertson. 1976a. Specific cleavage of the native type III collagen molecule with trypsin. Similarity of the cleavage products to collagenaseproduced fragments and primary structure at the cleavage site. Arch. Biochem. Biophys. 173:631–637. Miller, E. J., E. D. Harris, E. Chung, J. E. Finch, J. E. McCroskery, and W. T. Butler. 1976b. Cleavage of type I and II collagens with the mammalian collagenase: Site of cleavage and primary structure at the NH2 terminal portion of the smaller fragment released from both collagens. Biochemistry 15:787–792. Moore, M. J. 1983. The dual connective tissue system of rat soleus muscle. Muscle Nerve 6:416–422. Murphy, G., and F. Willembrock. 1995. Tissue inhibitors of matrix metalloproteinases. Methods Enzymol. 248:496–510. Parsons, S. L., S. A. Watson, P. D. Brown, H. M. Collins, and R. J. C. Steele. 1997. Matrix metalloproteinases. Br. J. Surg. 84:160–166. Partridge, L. D., and L. A. Benton. 1981. Muscle: The motor. In: V. B. Brooks (ed.) Handbook of Physiology. American Physiology Society, Bethesda, MD. Pearson, A. M., and R. B. Young. 1989. The connective tissues: Collagen, elastin, and ground substance. In: Muscle and Meat Biochemistry. p 338. Academic Press, San Diego, CA. Rawdanowicz, T. J., A. L. Hampton, H. Nagase, D. E. Woolley, and L. A. Salamonsen. 1994. Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha. J. Clin. Endocrinol. Metab. 79:530–536. Ries, C., and P. E. Petrides. 1995. Cytokine regulation of matrix metalloproteinase activity and its regulatory dysfunction in disease. Biol. Chem. 376:345–355. Rutter, J. L., U. Benbow, C. I. Coon, and C. E. Brinckerhoff. 1997. Cell-type specific regulation of human interstitial collagenase- 107 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells. J. Cell. Biochem. 66:322–336. Sakamoto, A., Y. Oda, Y. Iwamoto, and M. Tsuneyoshi. 1999. Expression of membrane type 1 matrix metalloproteinase, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 in human cartilaginous tumors with special emphasis on mesenchymal and dedifferentiated chondrosarcoma. J. Cancer Res. Clin. Oncol. 125:541–548. Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yama-moto, and M. Seiki. 1994. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature (Lond.) 370:61–65. Satoh, T., K. Kobayashi, S. Yamashita, M. Kibuchi, Y. Sendai, and H. Hoshi. 1994. Tissue inhibitor of metalloproteinases (TIMP1) produced by granulosa and oviduct cells enhances in vitro development of bovine embryo. Biol. Reprod. 50:835–844. Seltzer, J. L., S. A. Adams, G. A. Grant, and A. Z. Eisen. 1981. Purification and properties of a gelatin specificity neutral proteinase from human skin. J. Biol. Chem. 256:4662–4668. Seltzer, J. L., K. T. Akers, H. Weingarten, G. A. Grant, D. W. Mc Court, and A. Z. Eisen. 1990. Cleavage specificity of human skin type IV collagenase (gelatinase). J. Biol. Chem. 265:20409– 20413. Takino, T., H. Sato, A. Shinagawa, and M. Seiki. 1995. Identification of the second membrane-type matrix metalloproteinase (MTMMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. J. Biol. Chem. 270:23013–23020. Tamura, M., H. Shimokawa, and S. Sasaki. 1994. Primary structure of bovine interstitial collagenase deduced from cDNA sequence. DNA Seq. 5:63–66. Tanney, D. C., L. Feng, A. S. Pollock, and D. H. Lovett. 1998. Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis. Dev. Dyn. 213:121–129. Tyagi, S. C., L. Matsubara, and K. T. Weber. 1993. Direct extraction and estimation of collagenase(s) activity by zymography in microquantities of rat myocardium and uterus. Clin. Biochem. 26:191–198. Velgus, H. G., J. J. Jeffrey, and A. Z. Eisen. 1981. The collagen substrate specificity of human skin fibroblast collagenase. J. Biol. Chem. 256:9511–9515. Werb, Z., C. L. Mainardi, C. A. Vater, and E. D. Jr Harris. 1977. Endogenous activation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator. N. Engl. J. Med. 296:1017–1023. Yablonka-Reuveni, Z., S. K. Anderson, D. F. Bowen-Pope, and M. Nameroff. 1988. Biochemical and morphological differences between fibroblasts and myoblasts from embryonic chicken skeletal muscle. Cell Tissue Res. 252:339–348. Yelich, J. V., D. Pomp, and R. D. Geisert. 1997. Ontogeny of elongation and gene expression in the early developing porcine conceptus. Biol. Reprod. 57:1256–1265. Yu, A. E., A. N. Murphy and W. G. Stetler-Stevenson. 1998. 72-kDa gelatinase (gelatinase A): structure, activation, regulation and substrate specificity. In: Matrix Metalloproteinases. p 85. Academic Press, San Diego, CA. Zucker, S., R. M. Lysik, J. Wieman, D. P. Wilkie, and B. Lane. 1985. Diversity of human pancreatic cancer cell proteinases: role of cell membrane metalloproteinases in collagenolysis and cytolysis. Cancer Res. 45:6168–6178.