Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator

¹ The Burnham Institute, La Jolla, California 92037; ² Institut National de la Santé et de la Recherche Médicale-Institut National de la Recherche Agronomique U418, Communications Cellulaires et Différenciation, Hôpital Debrousse, 69322 Lyon, France; ³ Research Administration, Immunex Corporation, Seattle, Washington 98101; and ⁴ Centre National de la Recherche Scientifique UPR 2163, Physiologie Moléculaire et Cellulaire, Centre Hospitalier Universitaire Purpan, 31059 Toulouse Cedex 03, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C₂C₁₂ myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C₂C₁₂ cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C₂C₁₂ cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.

metalloendopeptidases; protease inhibitor; growth and differentiation factor-8

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DIFFERENTIATION AND REGENERATION of skeletal muscle are controlled by many factors, including insulin-like growth factors (IGFs), hepatocyte growth factor, fibroblast growth factor-2, platelet-derived growth factor, and some members of the transforming growth factor- (TGF-) superfamily (reviewed in Refs. 13, 17, and 25). The biological activity of many such growth and differentiation factors is regulated by proteolysis, such as shedding of membrane-bound growth factors and their receptors (42, 45), activation of secreted latent growth factors (46), and degradation of growth factor binding proteins (12). Metalloproteases are known to regulate many developmental events (for review see Refs. 49, 66, and 75). In particular, members of the ADAM (a disintegrin and metalloprotease) family of membrane-bound metalloproteases are believed to activate growth factors and cytokines (for review see Refs. 6, 7, and 62), including tumor necrosis factor- (TNF-) (5, 44, 54, 59), TNF- receptor II, L-selectin, TGF-, heparin-binding epidermal growth factor (26), Notch receptor (8, 52) and its ligand delta (56), amyloid protein precursor (9, 30, 31, 70), and IGF binding protein-3 (68).

The C₂C₁₂ cell line of mouse myoblasts derived from satellite cells is frequently used to study skeletal muscle differentiation in vitro and is considered to be a good model for myogenesis and muscle regeneration. High fetal calf serum concentration is used to keep C₂C₁₂ cells in an undifferentiated, proliferating myoblast stage, but in low concentration of horse serum, C₂C₁₂ cells stop proliferating and differentiate into multinucleated myotubes. This indicates that high concentrations of factors present in the bovine serum, probably including TGF-, exert an inhibitory effect on skeletal muscle cell differentiation and that muscle differentiation requires finely tuned regulation of growth factor activation, possibly involving proteases. In the present study, we investigated the role of metalloproteases in skeletal muscle cell differentiation. Little is known about the possible role of metalloproteases in modulating differentiation factors in skeletal muscle cells. An exception is ADAM 12, also called meltrin-, which has been shown to affect skeletal muscle differentiation (78). ADAM 12 appears to play a dual role in myogenesis; its disintegrin and cysteine-rich domains are promyogenic (20, 78), whereas an antimyogenic activity is observed when its metalloprotease domain is present (78). However, other ADAMs and metalloproteases are expressed in C₂C₁₂ cells (19), and ADAM 12 may not be the only metalloproteases involved in differentiation of C₂C₁₂ cells.

We show here that hydroxamate-based inhibitors of metalloproteases (HIMPs), added at an early stage of differentiation, stimulate muscle cell fusion and myotube growth and ultimately provoke myotube hypertrophy. HIMP treatment was accompanied by a reduction in the proteolytic maturation of myostatin, a member of the TGF-/bone morphogenetic protein (BMP) superfamily and known as a negative regulator of myogenesis. Thus our results indicate that the inhibition of metalloproteases by HIMPs triggers skeletal muscle hypertrophy by a mechanism yet to be determined but that seems to involve a lack of myostatin processing.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents. HIMPs, IC-3 [also called TAPI (tumor necrosis factor- processing inhibitor)], and BB-3103 were obtained from Immunex (Seattle, WA) and British Biotech (Oxford, UK), respectively. Stock solutions of TAPI and BB-3103 were prepared at 5 mM in water and DMSO, respectively. GM-6001 (Ilomastat) was purchased from Chemicon (Temecula, CA) and stored at 2.5 mM in DMSO. 1-10-Phenanthroline was purchased from Sigma and stored at 200 mM in DMSO. Cyclosporin was purchased from Calbiochem (La Jolla, CA) and stored at 42 mM in ethanol. TNF- was obtained from Calbiochem and stored at 10 µg/ml in PBS containing 0.1% BSA.

Culture of C₂C₁₂ cells. C₂C₁₂ cells (American Type Culture Collection, Manassas, VA) were cultured on gelatin-coated dishes in proliferation medium composed of DMEM, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 U/ml penicillin and containing 10% fetal calf serum. When cells reached confluence (day 0), differentiation was induced by culture in differentiation medium composed of DMEM, glutamine, sodium pyruvate, streptomycin, and penicillin and containing 2% horse serum. The medium was changed every 1 or 2 days depending on the type of experiment.

Quantification. For morphological analysis and measurement of diameter of myotubes, cells were fixed with 0.5% glutaraldehyde, permeabilized with 0.1% Triton X-100, and stained with hematoxylin and eosin. For quantification of nuclei, cells were fixed with 4% paraformaldehyde and stained with eosin and Hoechst 33258. 1) To quantify the effect of HIMPs on myotube size throughout differentiation, we attempted to measure the maximum myotube diameter reached in control and HIMP-treated conditions at various stages of culture. We randomly selected 10 microscopic fields from 3 independent culture wells, and the diameter of the 4 largest myotubes in each field (40 myotubes in total) was measured (Fig. 1B). 2) To define myotubes according to their number of nuclei, 5 microscopic fields were randomly selected from 3 independent culture wells, and the number of nuclei per myotube was measured in 20 randomly selected myotubes per field (100 myotubes in total). Myotubes were then defined on the basis of the number of nuclei (2-9, 10-19, 20-29, 30-49, 55-99, and >100). Data (Fig. 4A) are shown as the percentage distribution of myotubes in control and HIMP-treated cultures. 3) The cultures also were characterized by how many nuclei could be maximally found in myotubes after various treatment protocols. The number of nuclei in the 10 apparently largest myotubes found in each of 3 culture wells (30 myotubes in total) was measured (Fig. 4B). Statistical analyses of the results of quantification were performed by one-way ANOVA followed by the Newman-Keuls multiple comparison test. Results were considered significantly different when P < 0.05. 

View larger version (95K): [in this window] [in a new window]   Fig. 1.   Effect of hydroxamate-based inhibitors of metalloproteases (HIMPs) on C2C12 cell differentiation. A: tumor necrosis factor- processing inhibitor (TAPI) induces C2C12 myotube hypertrophy. Parental C2C12 cells were cultured as described in MATERIALS AND METHODS. After differentiation was induced at day 0, cells were treated with various concentrations of TAPI from day 2. Cells were fixed at day 7 and stained with hematoxylin and eosin. Scale bar, 100 µm. B: BB-3103 increases the diameter of myotubes. C2C12 clone 18 cells were cultured as described in MATERIALS AND METHODS. After differentiation was induced at day 0, cells were treated with DMSO or various concentrations of BB-3103 from day 1. Cells were fixed and stained at different stages of differentiation, and the diameter of the 4 largest myotubes present within 10 fields was measured. Data represent means ± SD (n = 40). *P < 0.05; **P < 0.001, significant differences between DMSO- and BB-3103-treated cells at the same stage.

Generation of C₂C₁₂ cell clones. Parental C₂C₁₂ cells were grown at very low density to generate colonies. Cells from individual colonies were collected with a pipette tip and cultured in separate microplate wells. Eighteen individual clones were expanded and used up to four passages.

Gelatin and casein substrate zymography. Ten microliters of culture medium from differentiating C₂C₁₂ cells were subjected to SDS-PAGE in 10% gelatin zymogram or 12% casein zymogram gels (Novex; Invitrogen, Carlsbad, CA) under nonreducing conditions at 4°C. After electrophoresis, gels were incubated in 2.5% Triton X-100 in distilled water for 1 h at room temperature to renature proteins. Gels were then incubated overnight at 37°C in 50 mM Tris · HCl (pH 7.7) containing 5 mM CaCl₂ to allow proteinases to digest the substrate in the gel. Gels were then stained with Coomassie blue (G-250). Finally, gels were rinsed in 30% methanol-5% glycerol and dried. Areas of proteolysis appeared as clear zones against a blue background.

Ligand blotting with biotinylated IGF-I. Recombinant human IGF-I (rhIGF-I; kindly provided by Dr. P. Monget, Nouzilly, France) was biotinylated as previously described (18, 51). Briefly, 0.1 mg of rhIGF-I was incubated for 1 h at room temperature with 0.32 mg of EZ-Link sulfo-NHS-LC-biotin (Pierce, Rockford, IL) in a total volume of 100 µl of 0.23 M NaHCO₃ (pH 9.2). The reaction was stopped by adding 200 µl of 1M Tris (pH 7.4). Biotinylated IGF-I was dialyzed against PBS containing 0.05% sodium azide for 24 h in Slide Lyser CO 3,500 (Pierce). Biotinylated IGF-I was then stored at 4°C at a final concentration of 60 µg/ml in PBS containing 1% BSA.

Ten microliters of medium, conditioned by C₂C₁₂ cells for 48 h, were subjected to SDS-PAGE in 16% polyacrylamide gels under nonreducing conditions. Proteins were transferred onto a nitrocellulose filter. Filters were washed with Tris-buffered saline (TBS), and proteins were renatured for 30 min in TBS-3% Nonidet P-40 (NP-40) at room temperature. Filters were then treated with TBS-0.1% Tween 20 (TBS-T) containing 1% BSA for 1 h at room temperature, washed with TBS-T, and incubated with biotinylated rhIGF-I (0.6 µg/ml) in TBS-T for 1 h at room temperature. After being washed with TBS-T, filters were incubated with horseradish peroxidase (HRP)-coupled Immunopure NeutrAvidin (Pierce) diluted 1:5,000 in TBS-T for 45min at room temperature. Filters were washed extensively, and the signal was detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) on XAR Kodak autoradiography film.

Immunoblotting. Skeletal muscle tissues (tibialis anterior, soleus, and plantaris) were collected from an adult IRC male mouse and homogenized with a Tissue-Tearor (Biospec Products, Bartlesville, OK) in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). C₂C₁₂ cells were lysed on ice in lysis buffer and collected with a cell scraper. Samples were briefly centrifuged, and the supernatant was stored at 20°C until use. Protein concentration in each lysate was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). Samples were subjected to 4-20% gradient SDS-PAGE (Novex; Invitrogen) under reducing conditions. After electrophoresis, proteins were transferred onto nitrocellulose membranes. Membranes were treated for 1 h at room temperature with TBS containing 10% nonfat dry milk, incubated for 1 h at room temperature with a rabbit antiserum directed against myostatin NH₂-terminal portion (kindly provided by Dr. James G. Tidball) or a goat antiserum directed against myostatin COOH-terminal portion (C-20; Santa-Cruz Biotechnology, Santa Cruz, CA) diluted 1:500 in TBS containing 1% BSA, and finally incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG or rabbit-anti-goat IgG antibody (Calbiochem) diluted 1:2,000 in TBS containing 10% nonfat dry milk. The signal from ECL (Amersham Pharmacia Biotech) was detected on XAR Kodak autoradiography film.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

HIMPs induce hypertrophy of C₂C₁₂ myotubes. To test the possible involvement of metalloproteases in skeletal muscle cell proliferation and differentiation, we cultured C₂C₁₂ cells in the presence of various concentrations of HIMPs. Treatment of differentiating C₂C₁₂ cells with TAPI or BB-3103 had a dramatic effect on myoblast fusion and myotube morphology. Myotubes resulting from C₂C₁₂ cells differentiating in the presence of TAPI or BB-3103 (added from day 2 to day 7) were much larger (hypertrophic) and exhibited frequent branching (Fig. 1A). The effects of TAPI and BB-3103 were indistinguishable from one another and were dose dependent, with a maximum effect at 5-15 µM and no effect at 0.15 µM or lower concentrations. Treatment with DMSO (0.1% vol/vol) had no effect. Quantification showed that BB-3103 dramatically increased the diameter of C₂C₁₂ myotubes during differentiation (Fig. 1B). GM-6001, another HIMP, also induced C₂C₁₂ myotube hypertrophy at 5 µM (not shown). The zinc chelator 1-10-phenanthroline, an inhibitor of metalloproteases with broad specificity, was also tested, but the results obtained were inconclusive because of its lower specific activity and higher toxicity to C₂C₁₂ cells.

Treatment of subconfluent C₂C₁₂ myoblasts with TAPI (not shown) or BB-3103 up to 5 µM in proliferation medium had no significant effect on C₂C₁₂ number or on C₂C₁₂ cell survival (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of BB-3103 on proliferation and survival of C2C12 cells. C2C12 clone 18 cells were cultured in 96-well dishes at low density in proliferation medium containing DMSO or various concentrations of BB-3103 for 2 days. After trypsinization, the number of live cells (open bars) and dead cells (solid bars) per well was determined in 6 independent culture wells. Cell viability was assessed with trypan blue dye. Data represent means ± SD (n = 6).

HIMPs enhance myoblast fusion. As previously described by us and others, C₂C₁₂ cells are genetically unstable and usually consist of a heterogeneous population of cells. Cell lines with different fusion abilities and myotube shapes can be obtained by cloning (74). We took advantage of this variation in C₂C₁₂ cells to investigate the response to TAPI and BB-3103 of 18 clonal cell lines derived from the parental cells. As expected, the 18 cell lines showed large variation in their ability to fuse in the differentiation medium and also variation in the shape of the resulting myotubes. We divided the 18 clones into 5 groups, each characterized by a distinct fusion phenotype: fusion incompetent (n = 1), fusion inefficient (n = 6), fusion competent with normal myotube shape (n = 5), fusion competent with myosacs (n = 2), fusion competent with thinner myotubes (n = 4). When treated with HIMPs, cells from the clones within each group responded in a characteristic and reproducible manner, as described below. Clonal cells with fusion capacity similar to the parental C₂C₁₂ cells (fusion competent with normal myotube shape) responded to HIMPs in a way similar to the parental C₂C₁₂ cells. There was a significant acceleration in fusion during the first few days of differentiation and hypertrophy of the resulting myotubes at a later stage. Cells from this group differentiated even faster and fused even more efficiently than the parental cells, probably because of the homogeneity of the cloned cells. In this group, clone 18 was chosen for later experiments (see below). Clonal cells with inefficient fusion also responded to HIMPs by an increase in fusion efficiency, characterized by the appearance of a larger number of multinucleated cells and an increase in the size of the myotubes (Fig. 3, clone 6). Clonal cells showing a defect in myotube elongation (fusion competent with myosacs) responded to HIMPs by an increase in both fusion and elongation, as shown by an increase in size and length of the multinucleated cells (Fig. 3, clone 17). Cells from one clone with a complete inability to fuse (fusion incompetent) showed no response to HIMPs (Fig. 3, clone 8). Finally, some clonal cells with abnormally thin myotubes (fusion competent with thinner myotubes) did not respond to treatment with HIMPs (Fig. 3, clone 2).

View larger version (133K): [in this window] [in a new window]   Fig. 3.   Effect of BB-3103 on the differentiation of C2C12 clonal cell lines. C2C12 clonal cell lines were obtained and cultured as described in MATERIALS AND METHODS. After differentiation was induced at day 0, cells were treated with DMSO or 5 µM BB-3103 from day 1. Images show cells after 6 days of differentiation. Bar, 100 µm.

Noting that HIMPs increased the fusion efficiency of C₂C₁₂ clonal lines with inefficient fusion, we subsequently investigated whether myotube hypertrophy was associated not only with an increased diameter of myotubes but also with an increase in the number of nuclei per myotube. In these experiments, we used the C₂C₁₂ clone 18 cells, which are more homogeneous but differentiate in a manner similar to parental C₂C₁₂ cells. Figure 4A shows the range of nuclei within myotubes in control and HIMP-treated conditions at day 5 of differentiation. The results clearly show that HIMP treatment is accompanied by an increase in the number of nuclei per myotube.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of BB-3103 on cell fusion. A: BB-3103 increased the number of nuclei per myotube. C2C12 clone 18 cells were cultured as described in MATERIALS AND METHODS. After differentiation was induced at day 0, cells were treated with DMSO (open bars) or 5 µM BB-3103 (filled bars) from day 1. Cells were fixed and stained at day 5. The number of nuclei per myotube was measured on 20 myotubes randomly selected in 5 fields (n = 100). B: the effect of BB-3103 on C2C12 cell fusion is stage and time dependent. C2C12 clone 18 cells were cultured as described in MATERIALS AND METHODS. After differentiation was induced at day 0, cells were treated with DMSO or 5 µM BB-3103 at different stages of differentiation and for various periods of time. Cells were fixed and stained at day 5. The number of nuclei per myotube was measured in the 10 largest myotubes of 3 independent culture wells. Data represent means ± SD (n = 30). *P < 0.05; **P < 0.001, significant difference between DMSO- and BB-3103-treated cells. Data in A and B correspond to separate culture experiments.

We then investigated whether the hypertrophy observed at late stages of differentiation resulted from an early increase in fusion at the onset of differentiation. Cells were treated with 5 µM BB-3103 at different stages of differentiation and for variable periods of time, and the resulting hypertrophy was assessed on day 5 by measuring the number of nuclei within the largest myotubes (Fig. 4B). Continuous presence of BB-3103 (from day 1 to day 5) provoked a dramatic hypertrophy of the myotubes on day 5 (see Fig. 7, control). The average number of nuclei within the hypertrophic myotubes at day 5 was more than threefold higher than in the control myotubes at the same stage (Fig. 4B). When the cells were treated with BB-3103 from day 2, 3, or 4 and up to day 5, myotubes showed progressively less hypertrophy on day 5 (not shown), and the number of nuclei per myotube on day 5 decreased (Fig. 4B). A short and early treatment with BB-3103, started on day 1 and terminated on day 2, 3 or 4, appeared to be sufficient to produce hypertrophic myotubes on day 5. These results indicate that the hypertrophy induced by BB-3103, and most obvious at day 5 of differentiation, may result from events triggered by BB-3103 at early stages of differentiation.

Possible targets of HIMPs. HIMPs are efficient inhibitors of metalloproteases of the metzincin family including matrix metalloproteases (MMPs) and ADAMs. They act by chelating the zinc within the catalytic site of these enzymes. We previously showed that C₂C₁₂ cells express several ADAMs, including ADAM 9, 10, 12, 15, 17, and 19 (19). We now tested for expression of MMPs in C₂C₁₂ cells. Differentiated C₂C₁₂ clone 18 cells expressed two gelatinases of 96 and 75 kDa, likely corresponding to MMP-9 and MMP-2. These two gelatinases are mainly present as precursors, as shown in gelatin gel zymography (Fig. 5). No changes in the level of expression or in the activation state of MMP-2 and MMP-9 were observed in the culture medium of C₂C₁₂ cells regardless of treatment with HIMPs (Fig. 5). Casein gel zymography did not reveal any band (not shown), suggesting that differentiated C₂C₁₂ clone 18 cells do not express MMP-1, MMP-3, MMP-7, MMP-10, or plasminogen activators in their medium.

View larger version (60K): [in this window] [in a new window]   Fig. 5.   Detection of gelatinases in the medium of C2C12 cells. Parental C2C12 cells were cultured and either treated or not with 5 µM TAPI from day 1 to day 5. Gelatinases secreted into the culture medium between day 3 and day 5 were analyzed by gelatin-substrate zymography. Two major bands were detected at 96 and 75 kDa, representing the latent forms of matrix metalloprotease (MMP)-9 and MMP-2, respectively.

Mechanism of action of HIMPs in C₂C₁₂ cell differentiation. To elucidate the mechanisms by which HIMPs increase C₂C₁₂ cell fusion and provoke myotube hypertrophy, we investigated whether some of the factors known to be involved in muscle hypertrophy or atrophy could be mediating the effect of HIMPs.

IGFs play a critical role in skeletal muscle growth and differentiation (17) and have been implicated in skeletal muscle hypertrophy both in vivo (15, 34) and in vitro (48, 64, 65). Recent reports have shown that IGF-I-transfected myoblasts differentiate into hypertrophic myotubes in a calcineurin-dependent manner (47, 48, 65) reminiscent of the pathway involved in cardiac muscle hypertrophy (43, 71). ADAM 12 (68) and other metalloproteases (32) have been shown recently to have the capacity to cleave the IGF binding proteins (IGFBPs) and may thus regulate the bioavailability of IGF-I. Therefore, we investigated whether treatment with HIMPs would be associated with changes in levels of IGFBPs in the culture medium of differentiating C₂C₁₂ cells. As shown in Fig. 6, C₂C₁₂ cells expressed high amounts of IGFBP-5 and small amounts of IGFBP-4 at late stage of differentiation. Fetal calf serum also contained IGFBPs, mainly IGFBP-2 but also some IGFBP-3 and IGFBP-4. Unidentified high-molecular-weight binding proteins were found in both fetal calf serum and horse serum. No significant changes in the levels of any of these IGFBPs were detected in the medium of C₂C₁₂ cells, treated or untreated with TAPI.

View larger version (59K): [in this window] [in a new window]   Fig. 6.   Detection of insulin-like growth factor-I (IGF-I) binding proteins (IGFBP) in the medium of C2C12 cells. Media were subjected to 16% SDS-PAGE, and IGFBPs were detected by ligand blotting with biotinylated recombinant human IGF-I prepared as described in MATERIALS AND METHODS. Left: nonconditioned differentiation and proliferation media. HS, horse serum; FCS, fetal calf serum. Right: media conditioned by C2C12 cells for 2 days and collected at day 0, 1, 3, and 7. Three bands at 40, 35, and 24 kDa were detected in proliferation media containing 10% FCS, likely corresponding to IGFBP-3, IGFBP-2, and IGFBP-4, respectively. Media conditioned by C2C12 cells also contained a major band at 29 kDa and a minor band at 24 kDa, likely corresponding to IGFBP-5 and IGFBP-4, respectively. Two additional high-molecular-weight bands corresponding to unidentified binding proteins (unlabeled arroweads) were detected in all differentiation media containing 2% HS.

We then tested the effects of IGF-I and the calcineurin inhibitor cyclosporin A (CsA) in our system (Fig. 7). IGF-I at 25 ng/ml did not provoke hypertrophy when added to C₂C₁₂ cells at day 1 of differentiation, in agreement with previous reports (64, 65). In addition, IGF-I did not affect the BB-3103-induced hypertrophy at this concentration. In the presence of 250 ng/ml of IGF-I, the number of myotubes increased. However, this effect was not inhibited by 1 µM CsA. In fact, CsA by itself induced a moderate hypertrophy of C₂C₁₂ myotubes when used at 1 µM (Fig. 7) and 10 µM (not shown), and BB-3103 had an additive effect on the size of hypertrophic myotubes. Together, these results suggest that HIMP-induced hypertrophy does not involve an IGF pathway and occurs in a calcineurin-independent manner.

View larger version (85K): [in this window] [in a new window]   Fig. 7.   Effect of BB-3103, cyclosporin A (CsA), IGF-I, and tumor necrosis factor- (TNF-) on C2C12 clone 18 differentiation. C2C12 clone 18 cells were cultured as described in MATERIALS AND METHODS. Cells were treated with the indicated combinations of BB-3103 (5 µM), CsA (1 µM), IGF-I (25 or 250 ng/ml), and TNF- (1 ng/ml) from day 1. Cells were fixed and stained with hematoxylin and eosin at day 5. Bar, 100 µm.

TNF- has been shown previously to reduce C₂C₁₂ cell differentiation and to induce protein loss in muscle in vitro (35, 41) and in vivo (22, 50). Because TNF- is activated by HIMP-sensitive metalloproteases such as ADAM 10 (59), ADAM 17 (5, 44), and MMP-17 (16), inhibition of these ADAMs could block the activation of endogenous TNF-, thereby obliterating its negative effect on C₂C₁₂ cell differentiation and myotube growth. Supplying soluble active TNF- would thus be expected to counteract the effect of HIMPs. In our system, TNF- induced widespread apoptosis at 10 ng/ml, and when used at subapoptotic doses such as 1 ng/ml, TNF- did not inhibit the effect of BB-3103 on myotube hypertrophy (Fig. 7). Moreover, although skeletal muscle is a source of TNF- in humans (60), the levels of TNF- produced by C₂C₁₂ cells were undetectable by ELISA (not shown). Together, our data suggest that TNF- does not play any role in HIMP-induced C₂C₁₂ myotube hypertrophy.

We next investigated whether myostatin, another known negative regulator of muscle growth that requires proteolysis for its activation, could be involved in our hypertrophy model. Absence of functional myostatin (4, 21, 33, 39, 40) or overexpression of a dominant negative form of myostatin (81) has indeed been shown to provoke skeletal muscle hypertrophy in vivo. A reduction of the amount of active myostatin in our system would be expected to provoke myotube hypertrophy. Expression analysis by cDNA array (not shown) indicated that myostatin was indeed expressed in differentiating C₂C₁₂ cells and that its level of expression was not significantly different in HIMP-treated C₂C₁₂ cells compared with untreated cells. Therefore, we investigated whether the proteolytic processing of myostatin would be affected upon treatment with HIMPs. As with other members of the TGF-/BMP superfamily, myostatin is synthesized as a 50-kDa precursor protein. During its maturation, a proteolytic process generates a COOH-terminal 15-kDa peptide corresponding to the bioactive growth factor and a 37-kDa fragment of unknown function also called latency-associated peptide (LAP) (39). Immunoblotting analysis of myostatin in cells indicated that myostatin was produced and fully processed in C₂C₁₂ cells and in various mouse muscles, as shown by the presence of the 37-kDa LAP in cell and tissue lysates (Fig. 8). Notably, the expression of LAP was high in undifferentiated C₂C₁₂ cells, decreased at the onset of differentiation, and progressively increased as differentiation progressed. This result agrees with a recent RT-PCR analysis of myostatin expression in C₂C₁₂ cells (57) and with the pattern of expression of myostatin previously described during embryonic muscle development in the chicken (29). Interestingly, whereas the 50-kDa myostatin precursor was undetectable in control C₂C₁₂ cells as an indicator of a complete constitutive processing, a result that also agrees with recent data from Rios et al. (57), the precursor was clearly present in C₂C₁₂ cells treated with BB-3103 as shown with antisera directed against both the COOH- and the NH₂-terminal portions of the protein. The processed COOH-terminal 15-kDa bioactive portion of myostatin was never detected in tissues and cell lysates with the anti-COOH-terminal antibody. The difficulty to detect the 15-kDa COOH-terminal peptide in cell and tissue lysates is possibly due to the instability of the active myostatin peptide or to its diffusion into the extracellular milieu. The 37-kDa LAP may be detected because it is presumably complexed to a latent TGF- binding protein (LTBP)-like molecule and stored in the extracellular matrix, as previously shown for other TGF- family members (72).

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of BB-3103 on myostatin expression and processing. Myostatin was detected by immunoblotting with antibodies against the NH2-terminal portion (anti-Nterm) or the COOH-terminal portion (anti-Cterm) of myostatin. Samples correspond to lysates prepared from C2C12 clone 18 cells at various stages of differentiation, treated with DMSO or 5 µM BB-3103, and lysates prepared from plantaris (pl), soleus (so), and tibialis anterior (ta) muscles, as described in MATERIALS AND METHODS. A 37-kDa band, corresponding to the latent-associated peptide of myostatin, was detected in C2C12 cells and muscles with the anti-Nterm antibody. A 50-kDa band, representing the nonprocessed precursor of myostatin, was detected with both antibodies only in cells treated with BB-3103.

Together, our results suggest that the HIMP-induced myotube hypertrophy results from a mechanism independent of IGF-I, calcineurin, and TNF- and rather implicates lack of myostatin activation as the mechanism of action.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Proteases targeted by HIMPs. HIMPs are specific and powerful inhibitors of the metzincin family of metalloproteases, including MMPs and ADAMs. HIMPs show some degree of specificity toward different members of the metzincin family, depending on the chemical nature of their lateral groups (1, 2, 11, 79). HIMPs seem to inhibit MMPs efficiently at concentrations in the nanomolar range and ADAMs in the micromolar range (11). Whether such differences in the sensitivity of MMPs and ADAMs toward HIMPs are always true or depend on the nature of the system used to assess their activity (cell-containing vs. cell-free systems) remains unclear (3). Here we show that skeletal muscle cell hypertrophy was induced by both BB-3103 and TAPI at 5 µM concentrations and higher, indicating that in our system ADAMs may be the targets, rather than MMPs. As shown here, C₂C₁₂ cells produce only a few MMPs, and their level of expression or activation state did not significantly change with cell differentiation, suggesting that MMPs may not play a critical role in this in vitro model of myogenesis. Moreover, ADAMs, as membrane-bound metalloproteases, are more likely candidates than MMPs to participate in the local activation and release of growth factors and receptors. Although unknown effects of HIMPs are possible, we thus speculate that myotube hypertrophy induced by treatment with HIMPs might result from the inhibition of one or several endogenous ADAM-type metalloproteases. This suggestion is in agreement with the effect of various processed forms of ADAM 12 on muscle cell differentiation (20, 78). In a previous study, we showed that C₂C₁₂ cells express mRNA for ADAM 9, 10, 12, 15, 17 and 19 (19), all of which have proven or predicted catalytic activities and are thus potential targets for HIMPs in this system.

Myostatin in HIMP-induced skeletal muscle hypertrophy. In a series of experiments, we ruled out the involvement of IGFs, calcineurin, and TNF-, all factors previously shown to regulate skeletal muscle growth, in HIMP-induced myotube hypertrophy. Instead, we found that the proteolytic activation of myostatin is affected in HIMP-treated myotubes.

Myostatin, also called growth and differentiation factor-8, is a negative regulator of skeletal muscle growth. Its absence or mutation in mice and bovines leads to skeletal muscle hypertrophy and hyperplasia (33, 39, 40). Myostatin is variably expressed during degeneration and regeneration of different types of muscles (10, 28, 61, 67, 76, 80, 81) and has been detected in C₂C₁₂ cells (73). Here we found that C₂C₁₂ cells expressed myostatin mRNA and protein and that hypertrophy induced by HIMPs is associated with changes in myostatin processing in differentiating C₂C₁₂ cells. Indeed, the full-length precursor form of myostatin was detected in C₂C₁₂ cells only upon treatment with HIMPs, suggesting that the proteolytic maturation of myostatin was reduced or prevented in HIMP-treated cells. Therefore, the reduced maturation of myostatin might be at least a part of the mechanism by which HIMPs induce skeletal muscle cell hypertrophy. The mechanism by which myostatin negatively regulates muscle growth is not clear. Its total absence in mice leads to both hypertrophy and hyperplasia, suggesting that myostatin controls both the number and size of the myofibers (39). Two recent publications (57, 73) described that myostatin, overexpressed or provided as a recombinant protein, reduces proliferation and enhances survival of C₂C₁₂ myoblasts. On the other hand, mice expressing a mutant, dominant negative form of myostatin in the already formed muscle, with the use of the creatine kinase promoter, displayed muscle hypertrophy without hyperplasia (81). Together, these results suggest that myostatin may be involved both in controlling the number of myoblasts before fusion and in controlling the degree of fusion during muscle development and/or regeneration. The absence of clear effect of HIMPs on C₂C₁₂ cell proliferation in our study could be explained by the presence of saturating levels of protease inhibitors in high serum conditions.

The processes of synthesis, maturation, and activation of myostatin are yet unknown. For other members of the TGF-/BMP superfamily, the polypeptide chains initially dimerize and are cleaved inside the cell by furin proteases to release a latent growth factor consisting of the bioactive growth factor noncovalently bound to its LAP. The latent growth factor, once secreted, can be stored in the matrix by interacting with other proteins such as LTBP. The growth factor has to be activated by a process that may involve various proteolytic or nonproteolytic events (for review see Refs. 38, 46, 55, and 72). A recent report (81) showed that mice expressing a mutant, processing-deficient myostatin in skeletal muscle exhibited skeletal muscle hypertrophy. The mutant myostatin protein acted in a dominant negative fashion in that it reduced the processing of the endogenous wild-type myostatin protein. Together with our results, this observation strongly suggests that the reduction in myostatin processing occurring in HIMP-treated cells could be one of the causes of the resulting hypertrophy. A study from Wells and Strickland (77) showed that aprotinin, an inhibitor of serine proteases, stimulates skeletal muscle cell differentiation by decreasing the extracellular activation of latent TGF- in the culture medium. The production of active TGF-/BMPs may therefore require the successive actions of several proteases with different substrate specificity.

The link between metalloprotease inhibition by HIMPs and the reduction in myostatin processing remains to be determined. The possibility that myostatin would be directly processed by a HIMP-sensitive ADAM is theoretically possible but would require that 1) myostatin and active ADAM proteases coexist in the same cellular compartment and that 2) HIMPs such as TAPI and BB-3103 can cross cellular membranes. Both requirements have in fact been validated by previous reports. Although nonprocessed precursor forms of myostatin have been detected extracellularly in a model of transfected Chinese hamster ovary cells (39), it is believed that myostatin is processed intracellularly, possibly by furins, similarly to other members of the TGF-/BMP superfamily. Although ADAMs are known as plasma membrane-bound proteases that shed extracellular substrates, some ADAMs may function as intracellular proteases (14, 23, 27, 37, 63). ADAMs are activated by furins or by autocatalysis in the Golgi apparatus (24, 58) and may exert their proteolytic activity in the same organelle (70). For instance, ADAM 10 and/or ADAM 17 has been identified as a protein kinase C-regulated -secretase responsible for the nonconstitutive cleavage of amyloid protein precursor in the Golgi network (70). ADAM 19 has been shown to cleave Neuregulin  along its secretory pathway (69). Finally, the ability of HIMPs to penetrate cellular membranes to reach intracellular targets has been suggested by previous studies (36, 53, 70). Although our results show that myostatin processing is influenced by the activity of one or more HIMP-sensitive metalloproteases, further experiments are necessary to provide conclusive evidence that myostatin is directly processed by one of these proteases.

	ACKNOWLEDGEMENTS

We thank Dr. James Tidball for the antiserum to myostatin, Immunex Corporation for TAPI, and British Biotech for BB-3103.

	FOOTNOTES

This work was supported in part by the National Institutes of Health and by the Muscular Dystrophy Association.

Address for reprint requests and other correspondence: E. Engvall, The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037 (E-mail: eengvall{at}burnham.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 February 2001; accepted in final form 11 July 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Amour, A, Knight CG, Webster A, Slocombe PM, Stephens PE, Knäuper V, Docherty APJ, and Murphy G. The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3. FEBS Lett 473: 275-279, 2000[Web of Science][Medline].

2.   Amour, A, Slocombe PM, Webster A, Butler M, Knight CG, Smith BJ, Stephens PE, Shelley C, Hutton M, Knäuper V, Docherty APJ, and Murphy G. TNF- converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett 435: 39-44, 1998[Web of Science][Medline].

3.   Barlaam, B, Bird TG, Lambert-van der Brempt C, Campbell D, Foster SJ, and Maciewicz R. New -substituted succinate-based hydroxamic acids as TNF convertase inhibitors. J Med Chem 42: 4890-4908, 1999[Web of Science][Medline].

4.   Bass, J, Oldham J, Sharma M, and Kambadur R. Growth factors controlling muscle development. Domest Anim Endocrinol 17: 191-197, 1999[Web of Science][Medline].

5.   Black, RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, and Cerretti DP. A metalloproteinase disintegrin that releases tumour-necrosis factor alpha from cells. Nature 385: 729-733, 1997[Medline].

6.   Black, RA, and White JM. ADAMs: focus on the protease domain. Curr Opin Cell Biol 10: 654-659, 1998[Web of Science][Medline].

7.   Blobel, CP. Remarkable roles of proteolysis on and beyond the cell surface. Curr Opin Cell Biol 12: 606-612, 2000[Web of Science][Medline].

8.   Brou, C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black RA, and Israel A. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5: 207-216, 2000[Web of Science][Medline].

9.   Buxbaum, JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ, Johnson RS, Castner BJ, Cerretti DP, and Black RA. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 273: 27765-27767, 1998[Abstract/Free Full Text].

10.   Carlson, CJ, Booth FW, and Gordon SE. skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol Regulatory Integrative Comp Physiol 277: R601-R606, 1999[Abstract/Free Full Text].

11.   Cherney, RJ, Wang L, Meyer DT, Xue CB, Arner EC, Copeland RA, Covington MB, Hardman KD, Wasserman ZR, Jaffee BD, and Decicco CP. Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF- production. Bioorg Med Chem Lett 9: 1279-1284, 1999[Medline].

12.   Clemmons, DR, Busby W, Clarke JB, Parker A, Duan C, and Nam TJ. Modifications of insulin-like growth factor binding proteins and their role in controlling IGF actions. Endocr J 45, Suppl: S1-S8, 1998.

13.   Cusella-De Angelis, MG, Molinari S, Le Donne A, Coletta M, Vivarelli E, Bouche M, Molinaro M, Ferrari S, and Cossu G. Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis. Development 120: 925-933, 1994[Abstract].

14.   Dallas, DJ, Genever PG, Patton AJ, Millichip MI, McKie N, and Skerry TM. Localization of ADAM10 and Notch receptors in bone. Bone 25: 9-15, 1999[Medline].

15.   DeVol, DL, Rotwein P, Sadow JL, Novakofski J, and Bechtel PJ. Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. Am J Physiol Endocrinol Metab 259: E89-E95, 1990.

16.   English, WR, Puente XS, Freije JM, Knauper V, Amour A, Merryweather A, Lopez-Otin C, and Murphy G. Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2. J Biol Chem 275: 14046-14055, 2000[Abstract/Free Full Text].

17.   Florini, JR, Ewton DZ, and Coolican SA. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17: 481-517, 1996[Abstract/Free Full Text].

18.   Fowlkes, JL, and Serra D. A rapid, non-radioactive method for the detection of insulin-like growth factor binding proteins by western ligand blotting. Endocrinology 137: 5751-5754, 1996[Abstract].

19.   Galliano, MF, Huet C, Frygelius J, Polgren A, Wewer UM, and Engvall E. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha-actinin-2, is required for myoblast fusion. J Biol Chem 275: 13933-13939, 2000[Abstract/Free Full Text].

20.   Gilpin, BJ, Loechel F, Mattei GM, Engvall E, Albrechtsen R, and Wewer UM. A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo. J Biol Chem 273: 157-166, 1998[Abstract/Free Full Text].

21.   Grobet, L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Menissier F, Massabanda J, Fries R, Hanset R, and Georges M. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet 17: 71-74, 1997[Web of Science][Medline].

22.   Grunfeld, C, Wilking H, Neese R, Gavin LA, Moser AH, Gilli R, Serio MK, and Feingold KR. Persistence of the hypertriglyceridemic effect of tumor necrosis factor despite development of tachyphylaxis to its anorectic/cachectic effects in rats. Cancer Res 49: 2554-2560, 1989[Abstract/Free Full Text].

23.   Hougaard, S, Loechel F, Xu X, Tajima R, Albrechtsen R, and Wewer UM. Trafficking of human ADAM 12-L: retention in the trans-Golgi network. Biochem Biophys Res Commun 275: 261-267, 2000[Web of Science][Medline].

24.   Howard, L, Maciewicz RA, and Blobel CP. Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28. Biochem J 348: 21-27, 2000.

25.   Husmann, I, Soulet L, Gautron J, Martelly I, and Barritault D. Growth factors in skeletal muscle regeneration. Cytokine Growth Factor Rev 7: 249-258, 1996[Medline].

26.   Izumi, Y, Hirata M, Hasuwa H, Iwamoto R, Umata T, Miyado K, Tamai Y, Kurisaki T, Sehara-Fujisawa A, Ohno S, and Mekada E. A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J 17: 7260-7272, 1998[Web of Science][Medline].

27.   Kadota, N, Suzuki A, Nakagami Y, Izumi T, and Endo T. Endogenous meltrin alpha is ubiquitously expressed and associated with the plasma membrane but exogenous meltrin alpha is retained in the endoplasmic reticulum. J Biochem (Tokyo) 128: 941-949, 2000[Abstract/Free Full Text].

28.   Kirk, S, Oldham J, Kambadur R, Sharma M, Dobbie P, and Bass J. Myostatin regulation during skeletal muscle regeneration. J Cell Physiol 184: 356-363, 2000[Web of Science][Medline].

29.   Kocamis, H, Kirkpatrick-Keller DC, Richter J, and Killefer J. The ontogeny of myostatin, follistatin and activin-B mRNA expression during chicken embryonic development. Growth Dev Aging 63: 143-150, 1999[Web of Science][Medline].

30.   Koike, H, Tomioka S, Sorimachi H, Saido TC, Maruyama K, Okuyama A, Fujisawa-Sehara A, Ohno S, Suzuki K, and Ishiura S. Membrane-anchored metalloprotease MDC9 has an alpha-secretase activity responsible for processing the amyloid precursor protein. Biochem J 343: 371-375, 1999.

31.   Lammich, S, Kojro E, Postina R, Gilbert S, Pfeiffer R, Jasionowski M, Haass C, and Fahrenholz F. Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci USA 96: 3922-3927, 1999[Abstract/Free Full Text].

32.   Lawrence, JB, Oxvig C, Overgaard MT, Sottrup-Jensen L, Gleich GJ, Hays LG, Yates JR, III, and Conover CA. The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblast is pregnancy-associated plasma protein-A. Proc Natl Acad Sci USA 96: 3149-3153, 1999[Abstract/Free Full Text].

33.   Lee, SJ, and McPherron AC. Myostatin and the control of skeletal muscle mass. Curr Opin Genet Dev 9: 604-607, 1999[Web of Science][Medline].

34.   Levinovitz, A, Jennische E, Oldfors A, Edwall D, and Norstedt G. Activation of insulin-like growth factor II expression during skeletal muscle regeneration in the rat: correlation with myotube formation. Mol Endocrinol 6: 1227-1234, 1992[Abstract/Free Full Text].

35.   Li, YP, Schwartz RJ, Waddel ID, Holloway BR, and Reid MB. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor necrosis factor alpha. FASEB J 12: 871-880, 1998[Abstract/Free Full Text].

36.   Lopez-Perez, E, Zhang Y, Frank SJ, Creemers J, Seidah N, and Checler F. Constitutive -secretase cleavage of the -amyloid precursor protein in the furin-deficient LoVo cell line: involvement of the pro-hormone convertase 7 and the disintegrin metalloprotase ADAM10. J Neurochem 76: 1532-1539, 2001[Web of Science][Medline].

37.   Lum, L, Reid MS, and Blobel CP. Intracellular maturation of the mouse metalloprotease disintegrin MDC15. J Biol Chem 273: 26236-26247, 1998[Abstract/Free Full Text].

38.   Massagué, J. TGF signal transduction. Annu Rev Biochem 67: 753-791, 1998[Web of Science][Medline].

39.   McPherron, AC, Lawler AM, and Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF- superfamily member. Nature 387: 83-90, 1997[Medline].

40.   McPherron, AC, and Lee SJ. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94: 12457-12461, 1997[Abstract/Free Full Text].

41.   Meadows, KA, Holly JMP, and Stewart CEH Tumor necrosis factor--induced apoptosis is associated with suppression of insulin-like growth factor binding protein-5 secretion in differentiating murine skeletal myoblasts. J Cell Physiol 183: 330-337, 2000[Web of Science][Medline].

42.   Merlos-Suarez, A, and Arribas J. Mechanisms controlling the shedding of transmembrane molecules. Biochem Soc Trans 27: 243-246, 1999[Web of Science][Medline].

43.   Molkentin, JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215-228, 1998[Web of Science][Medline].

44.   Moss, ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, and Becherer JD. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385: 733-736, 1997[Medline].

45.   Müllberg, J, Althoff K, Jostock T, and Rose-John S. The importance of shedding of membrane proteins for cytokine biology. Eur Cytokine Netw 11: 27-38, 2000[Web of Science][Medline].

46.   Munger, JS, Harpel JG, Gleizes PE, Mazzieri R, Nunes I, and Rifkin DB. Latent transforming growth factor-beta: structural features and mechanisms of activation. Kidney Int 51: 1376-1382, 1997[Web of Science][Medline].

47.   Musarò, A, McCullagh KJA, Naya FJ, Olson EN, and Rosenthal N. IGF-I induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature 400: 581-585, 1999[Medline].

48.   Musarò, A, and Rosenthal N. Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I. Mol Cell Biol 19: 3115-3124, 1999[Abstract/Free Full Text].

49.   Nagase, H, and Woessner JF. Matrix metalloproteinases. J Biol Chem 274: 21491-21494, 1999[Free Full Text].

50.   Oliff, A, Defeo-Jones D, Boyer M, D, Martinez Keifer D, Vuocolo G, Wolfe A, and Socher SH. Tumours secreting human TNF/cachectin induce cachexia in mice. Cell 50: 555-563, 1987[Web of Science][Medline].

51.   Op De Beeck, L, Verlooy JE, Van Buul-Offers SC, and Du Caju MV. Detection of serum insulin-like growth factor binding proteins on western ligand blots by biotinylated IGF and enhanced chemiluminescence. J Endocrinol 154: R1-R5, 1997[Abstract/Free Full Text].

52.   Pan, D, and Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 9: 271-280, 1997.

53.   Pei, D. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP. J Biol Chem 274: 8925-8932, 1999[Abstract/Free Full Text].

54.   Peschon, JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, and Black RA. An essential role for ectodomain shedding in mammalian development. Science 282: 1281-1284, 1998[Abstract/Free Full Text].

55.   Piek, E, Heldin CH, and Dijke PT. Specificity, diversity, and regulation in TGF- superfamily signaling. FASEB J 13: 2105-2124, 1999[Abstract/Free Full Text].

56.   Qi, H, Rand MD, Wu X, Sestan N, Wang W, Rakic P, Xu T, and Artavanis-Tsakonas S. Processing of the notch ligand delta by the metalloprotease Kuzbanian. Science 283: 91-94, 1999[Abstract/Free Full Text].

57.   Rios, R, Carneiro I, Arce VM, and Devesa J. Myostatin regulates cell survival during C2C12 myogenesis. Biochem Biophys Res Commun 280: 561-566, 2001[Web of Science][Medline].

58.   Roghani, M, Becherer JD, Moss ML, Atherton RE, Erdjument-Bromage H, Arribas J, Blackburn RK, Weskamp G, Tempst P, and Blobel CP. Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity. J Biol Chem 274: 3531-3540, 1999[Abstract/Free Full Text].

59.   Rosendahl, MS, Ko SC, Long DL, Brewer MT, Rosenzweig B, Hedl E, Anderson L, Pyle SM, Moreland J, Meyers MA, Kohno T, Lyons D, and Lichenstein HS. Identification and characterization of a pro-tumor necrosis factor-alpha-processing enzyme from the ADAM family of zinc metalloproteases. J Biol Chem 272: 24588-24593, 1997[Abstract/Free Full Text].

60.   Saghizaden, M, Ong JM, Garvey WT, Henry RR, and Kern PA. The expression of TNF by human muscle. Relationship to insulin resistance. J Clin Invest 94: 1111-1116, 1996.

61.   Sakuma, K, Watanabe K, Sano M, Uramoto I, and Totsuka T. Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles. Biochim Biophys Acta 1497: 77-88, 2000[Medline].

62.   Schlondorff, J, and Blobel CP. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by ectomain shedding. J Cell Sci 112: 3603-3617, 1999[Abstract].

63.   Schlondorff, J, and Blobel CP. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by ectomain shedding. J Cell Sci 112: 3603-3617, 1999.

64.   Semsarian, C, Sutrave P, Richmond DR, and Graham RM. Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model. Biochem J 339: 443-451, 1999.

65.   Semsarian, C, Wu MJ, Ju YK, Marciniec T, Yeoh T, Allen DG, Harvey RP, and Graham RM. Skeletal muscle hypertrophy is mediated by a Ca²⁺-dependent calcineurin signalling pathway. Nature 400: 576-581, 1999[Medline].

66.   Shapiro, SD. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr Opin Cell Biol 10: 602-608, 1998[Web of Science][Medline].

67.   Sharma, M, Kambadur R, Matthews KG, Somers WG, Devlin GP, Conaglen JV, Fowke PJ, and Bass JJ. Myostatin, a transforming growth factor- superfamily member, is expressed in heart muscle and is upregulated in cardiomyocytes after infarct. J Cell Physiol 180: 1-9, 1999[Web of Science][Medline].

68.   Shi, Z, Xu W, Loechel F, Wewer UM, and Murphy LJ. ADAM 12, A disintegrin metalloprotease, interacts with insulin-like growth factor binding protein-3. J Biol Chem 275: 18574-18580, 2000[Abstract/Free Full Text].

69.   Shirakabe, K, Wakatsuki S, Kurisaki T, and Fujisawa-Sehara A. Roles of Meltrin /ADAM19 in the processing of Neuregulin. J Biol Chem 276: 9352-9358, 2001[Abstract/Free Full Text].

70.   Skovronsky, DM, Moore DB, Milla ME, Doms RW, and Lee VM. Protein kinase C-dependent alpha-secretase competes with beta-secretase for cleavage of amyloid-beta precursor protein in the trans-golgi network. J Biol Chem 275: 2568-2575, 2000[Abstract/Free Full Text].

71.   Sussman, MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, Colbert MC, Gualberto A, Wieczorek DF, and Molkentin JD. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 281: 1690-1693, 1998[Abstract/Free Full Text].

72.   Taipale, J, Saharinen J, and Keski-Oja J. Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion. Adv Cancer Res 75: 87-134, 1998[Web of Science][Medline].

73.   Thomas, M, Langley B, Berry C, Sharma M, Kirk S, Bass J, and Kambadur R. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275: 40235-40243, 2000[Abstract/Free Full Text].

74.   Vachon, PH, Loechel F, Xu H, Wewer UM, and Engvall E. Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival. J Cell Biol 134: 1483-1497, 1996[Abstract/Free Full Text].

75.   Vu, TH, and Werb Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 14: 2123-2133, 2000[Free Full Text].

76.   Wehling, M, Cai B, and Tidball JG. Modulation of myostatin expression during modified muscle use. FASEB J 14: 103-110, 2000[Abstract/Free Full Text].

77.   Wells, JM, and Strickland S. Aprotinin, a Kunitz-type protease inhibitor, stimulates skeletal muscle differentiation. Development 120: 3639-3647, 1994[Abstract].

78.   Yagami-Hiromasa, T, Sato T, Kurisaki T, Kamijo K, Nabeshima Y, and Fujisawa-Sehara A. A metalloprotease-disintegrin participating in myoblast fusion. Nature 377: 652-656, 1995[Medline].

79.   Yamamoto, M, Tsujishita H, Hori N, Ohishi Y, Inoue S, Ikeda S, and Okada Y. Inhibition of membrane-type 1 matrix metalloproteinase by hydroxamate inhibitors: an examination of the subsite pocket. J Med Chem 41: 1209-1217, 1998[Web of Science][Medline].

80.   Yamanouchi, K, Soeta C, Naito K, and Tojo H. Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization. Biochem Biophys Res Commun 270: 510-516, 2000[Web of Science][Medline].

81.   Zhu, X, Hadhazy M, Wehling M, Tidball JG, and McNally EM. Dominant negative myostatin produces hypertrophy without hyperplasia in muscle. FEBS Lett 474: 1-5, 2000[Web of Science][Medline].