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Regulation of Myostatin in Vivo by Growth and Differentiation Factor-Associated Serum Protein-1: A Novel Protein with Protease Inhibitor and Follistatin Domains
Jennifer J. Hill, Yongchang Qiu, Rodney M. Hewick and Neil M. Wolfman
Department of Protein Chemistry and Proteomics (J.J.H., Y.Q., R.M.H.), and Department of Musculoskeletal Sciences (N.M.W.), Wyeth Research, Cambridge, Massachusetts 02140
Address all correspondence and requests for reprints to: Jennifer J. Hill, Department of Protein Chemistry and Proteomics, Wyeth Research, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140. E-mail: [email protected].
Myostatin, a member of the TGFß superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFß superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFß binding proteins.
Differential Response to Exogenous and Endogenous Myostatin in Myoblasts Suggests that Myostatin Acts as an Autocrine Factor in Vivo
Ramón Ríos1, Susana Fernández-Nocelos, Isabel Carneiro, Víctor M. Arce and Jesús Devesa
Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, A Coruña 15782, Spain
Address all correspondence and requests for reprints to: Víctor M. Arce, M.D., Ph.D., Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, San Francisco 1, 15782 Santiago de Compostela, Spain. E-mail: [email protected].
Myostatin is a member of the TGF-ß superfamily that is essential for proper regulation of skeletal muscle growth. As do other TGF-ß superfamily members, myostatin signals into the cell via a receptor complex that consists of two distinct transmembrane proteins, known as the type I and type II receptors. Vertebrates have seven distinct type I receptors, each of which can mix and match with one of five type I receptors to mediate signals for all the TGF-ß family ligands. Accumulating evidence indicates that myostatin shares its pair of receptors with activin, and therefore, the question arises about how specificity in signaling is achieved. Our hypothesis is that a mechanism has to exist to restrict myostatin actions to the muscle cells. To investigate this possibility, we compared the effect of endogenous myostatin (myostatin overexpressed by myoblasts) and exogenous myostatin (recombinant myostatin added to the culture medium) in cultured myoblasts. As opposed to exogenous myostatin, endogenous myostatin induced the transcription of a reporter vector in cultured myoblasts. Notably, the myostatin concentrations that failed to induce a response in myoblasts were effective in MCF-7 cells (human mammary carcinoma) and in HepG2 cells (human hepatic carcinoma). Based on our observations, we propose that a mechanism exists that differentially regulates the bioavailability of endogenous and exogenous myostatin to muscle cells. This is consistent with a model in which myostatin actions are exerted in vivo in an autocrine fashion
Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases
Neil M. Wolfman * , Alexandra C. McPherron , William N. Pappano ¶, Monique V. Davies ||, Kening Song **, Kathleen N. Tomkinson *, Jill F. Wright *, Liz Zhao **, Suzanne M. Sebald , Daniel S. Greenspan ¶ and Se-Jin Lee
*Department of Inflammation, ||Antibody Technology Group, and **Department of Cardiovascular and Metabolic Diseases, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140; Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and Departments of ¶Biomolecular Chemistry and Pathology and Laboratory Medicine, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX and approved October 6, 2003 (received for review August 4, 2003)
Myostatin is a transforming growth factor family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.
Jennifer J. Hill, Yongchang Qiu, Rodney M. Hewick and Neil M. Wolfman
Department of Protein Chemistry and Proteomics (J.J.H., Y.Q., R.M.H.), and Department of Musculoskeletal Sciences (N.M.W.), Wyeth Research, Cambridge, Massachusetts 02140
Address all correspondence and requests for reprints to: Jennifer J. Hill, Department of Protein Chemistry and Proteomics, Wyeth Research, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140. E-mail: [email protected].
Myostatin, a member of the TGFß superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFß superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFß binding proteins.
Differential Response to Exogenous and Endogenous Myostatin in Myoblasts Suggests that Myostatin Acts as an Autocrine Factor in Vivo
Ramón Ríos1, Susana Fernández-Nocelos, Isabel Carneiro, Víctor M. Arce and Jesús Devesa
Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, A Coruña 15782, Spain
Address all correspondence and requests for reprints to: Víctor M. Arce, M.D., Ph.D., Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, San Francisco 1, 15782 Santiago de Compostela, Spain. E-mail: [email protected].
Myostatin is a member of the TGF-ß superfamily that is essential for proper regulation of skeletal muscle growth. As do other TGF-ß superfamily members, myostatin signals into the cell via a receptor complex that consists of two distinct transmembrane proteins, known as the type I and type II receptors. Vertebrates have seven distinct type I receptors, each of which can mix and match with one of five type I receptors to mediate signals for all the TGF-ß family ligands. Accumulating evidence indicates that myostatin shares its pair of receptors with activin, and therefore, the question arises about how specificity in signaling is achieved. Our hypothesis is that a mechanism has to exist to restrict myostatin actions to the muscle cells. To investigate this possibility, we compared the effect of endogenous myostatin (myostatin overexpressed by myoblasts) and exogenous myostatin (recombinant myostatin added to the culture medium) in cultured myoblasts. As opposed to exogenous myostatin, endogenous myostatin induced the transcription of a reporter vector in cultured myoblasts. Notably, the myostatin concentrations that failed to induce a response in myoblasts were effective in MCF-7 cells (human mammary carcinoma) and in HepG2 cells (human hepatic carcinoma). Based on our observations, we propose that a mechanism exists that differentially regulates the bioavailability of endogenous and exogenous myostatin to muscle cells. This is consistent with a model in which myostatin actions are exerted in vivo in an autocrine fashion
Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases
Neil M. Wolfman * , Alexandra C. McPherron , William N. Pappano ¶, Monique V. Davies ||, Kening Song **, Kathleen N. Tomkinson *, Jill F. Wright *, Liz Zhao **, Suzanne M. Sebald , Daniel S. Greenspan ¶ and Se-Jin Lee
*Department of Inflammation, ||Antibody Technology Group, and **Department of Cardiovascular and Metabolic Diseases, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140; Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and Departments of ¶Biomolecular Chemistry and Pathology and Laboratory Medicine, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX and approved October 6, 2003 (received for review August 4, 2003)
Myostatin is a transforming growth factor family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.
