Mice and cattle with genetic deficiencies in myostatin exhibit dramatic increases in skeletal muscle mass, suggesting that myostatin normally suppresses muscle growth. Whether this increased muscling results from prenatal or postnatal lack of myostatin activity is unknown. Here we show that myostatin circulates in the blood of adult mice in a latent form that can be activated by acid treatment. Systemic overexpression of myostatin in adult mice was found to induce profound muscle and fat loss analogous to that seen in human cachexia syndromes. These data indicate that myostatin acts systemically in adult animals and may be a useful pharmacologic target in clinical settings such as cachexia, where muscle growth is desired.
Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn ؊/؊ mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo. Mice carrying a targeted mutation in the myostatin gene have muscles that are about twice the normal size as a result of a combination of muscle fiber hyperplasia and hypertrophy (2). Myostatin appears to play a similar role in other species as well; naturally occurring mutations in the myostatin gene have been shown to be responsible for the double-muscling phenotype in cattle (3-6), and recent studies have demonstrated that a human baby with approximately twice the normal muscle mass is also homozygous for a loss-of-function mutation in the MSTN gene (7). These findings have raised the possibility that agents capable of targeting the myostatin signaling pathway may be useful for increasing muscle mass for both agricultural and human therapeutic applications. In this regard, loss of myostatin signaling has been shown to have beneficial effects in mouse models of muscle degenerative (8, 9) and metabolic (10) diseases.Various myostatin-binding proteins have been identified that are capable of inhibiting myostatin activity in vitro (8,(11)(12)(13)(14)(15)(16). Two of these proteins, the JA16 neutralizing monoclonal antibody (Ab) directed against myostatin (8, 15) and a mutant form of the myostatin propeptide resistant to members of the BMP-1͞tolloid family of metalloproteases (16), have been shown to be capable of increasing muscle mass by Ϸ25% when administered to wild-type (WT) mice. To determine whether these increases in muscle growth are the maximal achievable by targeting this signaling pathway, we sought additional myostatin inhibitors that might have a broader specificity in their ability to target additional members of the TGF- superfamily. Previous studies have demonstrated that myostatin is capable of binding the two activin type II receptors, ACVR2B and, to a lesser extent, ACVR2, in transfected COS cells (11,17). Moreover, transgenic mice in which a myosin light chain promoter͞ enhancer was used to express a truncated form of ACVR2B in skeletal muscle were found to have dramatic increases in muscle mass (11). Because the activin type II receptors have been shown to be capable of binding a number of other TGF- family members in addition to ...
GDF-8 is a new member of the TGF-beta superfamily which appears to be a negative regulator of skeletal muscle mass. Factors which regulate the biological activity of GDF-8 have not yet been identified. However, the biological activities of TGF-beta superfamily members, TGF-beta1, -beta2 and -beta3, can be inhibited by noncovalent association with TGF-beta1, -beta2 and beta3 propeptides cleaved from the amino-termini of the TGF-beta precursor proteins. In contrast, the propeptides of other TGF-beta superfamily members do not appear to be inhibitory. In this investigation, we demonstrate that purified recombinant GDF-8 propeptide associates with purified recombinant GDF-8 to form a noncovalent complex, as evidenced by size exclusion chromatography and chemical crosslinking analysis. Furthermore, we show that GDF-8 propeptide inhibits the biological activity of GDF-8 assayed on A204 rhabdomyosarcoma cells transfected with a (CAGA)12 reporter construct. Finally, we demonstrate that GDF-8 propeptide inhibits specific GDF-8 binding to L6 myoblast cells. Collectively, these data identify the GDF-8 propeptide as an inhibitor of GDF-8 biological activity.
ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M r ϳ68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M r ϳ53,000 and M r ϳ40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAGbinding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the Cterminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/ spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.Extracellular metalloproteases play a pivotal role in the proteolytic processing and turnover of the component molecules of a variety of tissues. Although a number of matrix metalloproteinases (MMPs) 1 may participate in such events, evidence for the involvement of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases in these processes is increasing. For example, ADAMTS-2, ADAMTS-3, and ADAMTS-14 can function as procollagen N-proteinases (1-3), and ADAMTS-13 has been identified as a von Willebrand factor-cleaving protease (4 -6).Within the extracellular matrix of cartilage, ADAMTS-4 (7), ADAMTS-5 (8), and ADAMTS-1 (9 -11) may all potentially function as aggrecanases, glutamyl endopeptidases that cleave specific Glu-Xaa bonds of the aggrecan core protein (reviewed in (21)), indicating that regulation of the proteolytic activities of ADAMTS family members is likely to be important for maintenance of homeostasis in a variety of extracellular matrices. Unlike most of the MMPs, which are secreted in a state of latency conferred by the cysteine switch region of the retained propeptide (22), ADAMTS proteases can be cleaved N-terminally by furin or related pro-protein convertase(s) within the trans-Golgi, resulting in secretion of mature, potentially active enzymes lacking the propeptide region. Interestingly, however, ADAMTS family members such as ADAMTS-1 and AD-AMTS-12 have been shown to undergo proteolytic processing within their C-terminal regions, resulting in removal of domains that can bin...
Aggrecan is the major cartilage hyalectan (1), which, together with the collagen network, provides this tissue with its unique mechanical properties of compressibility and stiffness (2-4). Extraction of aggrecan in its native form (5) and subsequent structural analysis (6) have revealed that the molecular organization of aggrecan is perfectly suited to its central functional role in articular cartilage. The N-terminal region of aggrecan is composed of two globular domains (G1 1 and G2) separated by the interglobular domain (IGD). G1 interacts with hyaluronan and link protein, thereby keeping the aggrecan molecule anchored within the cartilage tissue. Further interactions with other matrix components such as tenascin-R and fibulin-1 and fibulin-2 (7, 8) may occur through a third globular domain (G3) at the extreme C terminus of the core protein. The extended core protein between G2 and G3 is composed of a short keratan sulfate-rich region followed by a longer chondroitin sulfate-substituted domain. The charge repulsion and hydration of the long negatively charged glycosaminoglycan (GAG) chains are thought to maintain the C-terminal portions of aggrecan in an extended conformation (9). The swelling pressure of the aggrecan-link protein complex with hyaluronan is restrained by the tension in the collagen network; and together, these components form a fiber-reinforced concentrated gel within the cartilage, which transmits forces across the articular joint.In diseases characterized by cartilage degradation such as rheumatoid arthritis and osteoarthritis, increased aggrecan release from the cartilage occurs early (10, 11) and before the bulk of the collagen network is degraded (12). Proteolytic cleavage of aggrecan within the IGD separates the GAG-rich region from the hyaluronan-anchored G1 domain, resulting in GAG release from the cartilage matrix to the synovial fluid. Biomechanical tests on cartilage discs have shown that proteolysis within the IGD of aggrecan, and not cleavages near the C terminus, is primarily responsible for the loss of compressive resistance that accompanies interleukin-1-mediated degradation of the tissue (13). Identification of the proteinases responsible for this "destructive" cleavage of aggrecan has therefore been a major focus of experimentation in arthritis-related research.In this regard, two major cleavage sites that occur in vivo have been identified in the IGD of human aggrecan. One is a matrix metalloproteinase (MMP)-sensitive site at VDIPEN 341 2F 342 FGVGG, which can be cleaved at neutral pH by any one of a range of MMPs, including MMP-1-3, -7-9, -13,
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