RESUMEN
Myostatin, or GDF-8 (growth and differentiation factor-8), was first identified through sequence identity with members of the BMP (bone morphogenetic protein)/TGF-beta (transforming growth factor-beta) superfamily. The skeletal-muscle-specific expression pattern of myostatin suggested a role in muscle development. Mice with a targeted deletion of the myostatin gene exhibit a hypermuscular phenotype. In addition, inactivating mutations in the myostatin gene have been identified in 'double muscled' cattle breeds, such as the Belgian Blue and Piedmontese, as well as in a hypermuscular child. These findings define myostatin as a negative regulator of skeletal-muscle development. Myostatin binds with high affinity to the receptor serine threonine kinase ActRIIB (activin type IIB receptor), which initiates signalling through a smad2/3-dependent pathway. In an effort to validate myostatin as a therapeutic target in a post-embryonic setting, a neutralizing antibody was developed by screening for inhibition of myostatin binding to ActRIIB. Administration of this antimyostatin antibody to adult mice resulted in a significant increase in both muscle mass and functional strength. Importantly, similar results were obtained in a murine model of muscular dystrophy, the mdx mouse. Unlike the myostatin-deficient animals, which exhibit both muscle hypertrophy and hyperplasia, the antibody-treated mice demonstrate increased musculature through a hypertrophic mechanism. These results validate myostatin inhibition as a therapeutic approach to muscle wasting diseases such as muscular dystrophy, sarcopenic frailty of the elderly and amylotrophic lateral sclerosis.
Asunto(s)
Músculo Esquelético/citología , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Humanos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/fisiología , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/metabolismo , Miostatina , Transducción de Señal/fisiología , Células Madre/citología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Myostatin, a transforming growth factor beta (TGF-beta) family member, is a potent negative regulator of skeletal muscle growth. In this study we characterized the myostatin signal transduction pathway and examined its effect on bone morphogenetic protein (BMP)-induced adipogenesis. While both BMP7 and BMP2 activated transcription from the BMP-responsive I-BRE-Lux reporter and induced adipogenic differentiation, myostatin inhibited BMP7- but not BMP2-mediated responses. To dissect the molecular mechanism of this antagonism, we characterized the myostatin signal transduction pathway. We showed that myostatin binds the type II Ser/Thr kinase receptor. ActRIIB, and then partners with a type I receptor, either activin receptor-like kinase 4 (ALK4 or ActRIB) or ALK5 (TbetaRI), to induce phosphorylation of Smad2/Smad3 and activate a TGF-beta-like signaling pathway. We demonstrated that myostatin prevents BMP7 but not BMP2 binding to its receptors and that BMP7-induced heteromeric receptor complex formation is blocked by competition for the common type II receptor, ActRIIB. Thus, our results reveal a strikingly specific antagonism of BMP7-mediated processes by myostatin and suggest that myostatin is an important regulator of adipogenesis.
Asunto(s)
Adipocitos/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/metabolismo , Células COS , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Homeostasis , Humanos , Ratones , Ratones Endogámicos C3H , Modelos Biológicos , Miostatina , Células 3T3 NIH , Fosforilación , Reacción en Cadena de la Polimerasa , Unión Proteica , ARN/metabolismo , Interferencia de ARN , Proteína Smad2 , Factores de Tiempo , Transactivadores/metabolismo , Transcripción Genética , Activación Transcripcional , TransfecciónRESUMEN
Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Oocitos/fisiología , Ovario/fisiología , Animales , Proteína Morfogenética Ósea 15 , Proteínas Morfogenéticas Óseas/genética , Femenino , Marcación de Gen , Factor 9 de Diferenciación de Crecimiento , Sustancias de Crecimiento/genética , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Oocitos/citología , Ovario/anatomía & histología , Zona Pelúcida/química , Zona Pelúcida/metabolismoRESUMEN
Nodal, a member of the transforming growth factor beta (TGF-beta) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Here we define the intracellular signaling pathway induced by recombinant nodal protein treatment of P19 embryonal carcinoma cells. Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-beta. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrate that nodal induces p(CAGA)(12), a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Finally, we show that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin-TGF-beta pathway Smads, Smad2 and Smad3. We also show here that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-beta signaling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Inhibinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Glicoproteínas de Membrana , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Factores de Transcripción , Factor de Crecimiento Transformador beta/farmacología , Proteínas de Xenopus , Activinas , Animales , Proteínas Morfogenéticas Óseas/fisiología , Proteínas de Unión al ADN/genética , Células Madre de Carcinoma Embrionario , Factor de Crecimiento Epidérmico/fisiología , Proteínas Ligadas a GPI , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Sustancias de Crecimiento/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Proteína Nodal , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteína Smad2 , Proteína smad3 , Transactivadores/genética , Transfección , Células Tumorales CultivadasRESUMEN
The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Regulación del Desarrollo de la Expresión Génica/genética , Mesodermo/metabolismo , Proteínas de Xenopus , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras , Clonación Molecular , Extremidades/embriología , Extremidades/crecimiento & desarrollo , Folistatina , Glicoproteínas/farmacología , Histocitoquímica , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Proteínas/farmacología , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , Cola (estructura animal)/embriología , Cola (estructura animal)/crecimiento & desarrollo , Trasplante de TejidosRESUMEN
The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.
Asunto(s)
Proteínas Morfogenéticas Óseas , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/aislamiento & purificación , Familia de Multigenes/genética , Factor de Crecimiento Transformador beta/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN/aislamiento & purificación , Regulación de la Expresión Génica , Factor 5 de Diferenciación de Crecimiento , Factor 6 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Proteínas de Pez CebraRESUMEN
We have taken advantage of the sequence relationships among the bone morphogenetic proteins (BMPs) to identify the mouse Bmp15 and human BMP15 genes. The 392-amino acid prepropeptides encoded by these BMP genes exhibit significant homology to each other, although the 70% identity observed between the 125-amino acid mature peptides is considerably lower than that seen in comparisons of other mouse and human orthologs. Both genes share a common structural organization and encode mature peptides that lack the cysteine residue normally involved in the formation of a covalent dimer. In addition, mouse Bmp15 and human BMP15 map to conserved syntenic regions on the X chromosome. We demonstrate, through a combination of Northern blot and in situ hybridization analyses, that mouse Bmp15 is expressed specifically in the oocyte beginning at the one-layer primary follicle stage and continuing through ovulation. Interestingly, BMP-15 is most closely related to and shares a coincident expression pattern with the mouse growth/differentiation factor 9 (GDF-9) gene that is essential for female fertility. Our findings will be important for defining the role of BMP-15 in follicular development.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Expresión Génica , Ligamiento Genético , Oocitos/metabolismo , Cromosoma X , Secuencia de Aminoácidos , Animales , Northern Blotting , Proteínas Morfogenéticas Óseas/química , Mapeo Cromosómico , Femenino , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Ovario/química , ARN Mensajero/análisis , Mapeo RestrictivoRESUMEN
Cumulative evidence indicates that osteoblasts and adipocytes share a common mesenchymal precursor and that bone morphogenetic proteins (BMPs) can induce both osteoblast and adipocyte differentiation of this precursor. In the present study, we investigated the roles of BMP receptors in differentiation along these separate lineages using a well-characterized clonal cell line, 2T3, derived from the mouse calvariae. BMP-2 induced 2T3 cells to differentiate into mature osteoblasts or adipocytes depending upon culture conditions. To test the specific roles of the type IA and IB BMP receptor components, truncated and constitutively active type IA and IB BMP receptor cDNAs were stably expressed in these cells. Overexpression of truncated type IB BMP receptor (trBMPR-IB) in 2T3 cells completely blocked BMP-2-induced osteoblast differentiation and mineralized bone matrix formation. Expression of trBMPR-IB also blocked mRNA expression of the osteoblast specific transcription factor, Osf2/ Cbfa1, and the osteoblast differentiation-related genes, alkaline phosphatase (ALP) and osteocalcin (OC). BMP-2-induced ALP activity could be rescued by transfection of wild-type (wt) BMPR-IB into 2T3 clones containing trBMPR-IB. Expression of a constitutively active BMPR-IB (caBMPR-IB) induced formation of mineralized bone matrix by 2T3 cells without addition of BMP-2. In contrast, overexpression of trBMPR-IA blocked adipocyte differentiation and expression of caBMPR-IA induced adipocyte formation in 2T3 cells. Expression of the adipocyte differentiation-related genes, adipsin and PPARgamma, correlated with the distinct phenotypic changes found after overexpression of the appropriate mutant receptors. These results demonstrate that type IB and IA BMP receptors transmit different signals to bone-derived mesenchymal progenitors and play critical roles in both the specification and differentiation of osteoblasts and adipocytes.
Asunto(s)
Adipocitos/citología , Mesodermo/fisiología , Proteínas de Neoplasias , Osteoblastos/citología , Receptores de Superficie Celular/fisiología , Receptores de Factores de Crecimiento , Factor de Crecimiento Transformador beta , Adipocitos/metabolismo , Fosfatasa Alcalina/genética , Animales , Matriz Ósea/fisiología , Proteína Morfogenética Ósea 2 , Receptores de Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Calcificación Fisiológica , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Factor D del Complemento , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Expresión Génica , Indometacina/farmacología , Ratones , Osteoblastos/metabolismo , Osteocalcina/genética , Fenotipo , Receptores de Superficie Celular/genética , Receptores Citoplasmáticos y Nucleares/genética , Serina Endopeptidasas/genética , Células Madre/citología , Factores de Transcripción/genéticaRESUMEN
Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.
Asunto(s)
Sustancias de Crecimiento/farmacología , Ligamentos/crecimiento & desarrollo , Tendones/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/fisiología , Clonación Molecular , Decorina , Elastina/análisis , Proteínas de la Matriz Extracelular , Regulación del Desarrollo de la Expresión Génica/genética , Factor 5 de Diferenciación de Crecimiento , Factor 6 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento , Sustancias de Crecimiento/genética , Histocitoquímica , Humanos , Hibridación in Situ , Articulaciones/crecimiento & desarrollo , Ligamentos/citología , Ligamentos/trasplante , Ratones , Datos de Secuencia Molecular , Proteoglicanos/análisis , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tendones/citología , Tendones/trasplante , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
A new member of the transforming growth factor (TGF)-beta superfamily, BMP-9, has recently been identified and shown to be expressed in the developing mouse liver. This report demonstrates that human HepG2 liver tumor cells bind recombinant human BMP-9 (rhBMP-9) with high affinity. Cross-linking analysis indicates that HepG2 cells express two BMP-9 receptors of approximately 54 and 80 kilodaltons, similar in size to the Type I and Type II receptors reported by others for TGF-beta and BMP-4. However, cross-competition experiments demonstrate that the BMP-9 receptors on HepG2 cells do not bind other BMPs or TGF-beta s, indicating that these are novel receptors with binding specificity for BMP-9. In functional studies, rhBMP-9 stimulates HepG2 cell proliferation as indicated by [3H]thymidine incorporation and cell counting assays. A proliferative effect of rh-BMP-9 was also observed on primary rat hepatocytes. In contrast, TGF-beta had no effect on HepG2 cell proliferation and inhibited proliferation in primary hepatocytes. These results suggest that BMP-9, acting through a novel set of receptors, may play a regulatory role in hepatic growth and function.
Asunto(s)
Sustancias de Crecimiento/farmacología , Hígado/efectos de los fármacos , Proteínas/farmacología , Animales , Proteínas Morfogenéticas Óseas , División Celular/efectos de los fármacos , Hígado/citología , Hígado/metabolismo , Masculino , Proteínas/metabolismo , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
Bone morphogenetic proteins (BMPs) are a class of related growth and differentiation factors within the TGF-beta superfamily of proteins which are known to induce cartilage and bone formation in adult animals and to be involved in many inductive events throughout embryonic development. Here we describe the molecular cloning and characterization of a mammalian receptor, CFK-43a, which specifically binds BMP-2 and BMP-4. This molecule is a member of the serine/threonine kinase receptor family which includes receptors for other TGF-beta superfamily members. CFK-43a binds other BMP family members with lower affinity, but does not bind TGF-beta. During embryogenesis, in situ hybridization analysis indicates that CFK-43a mRNA is localized in developing skeletal tissues in a complementary fashion to the transcripts for its ligands.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Proteínas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Morfogenéticas Óseas , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Cinética , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Factores de Crecimiento/genéticaRESUMEN
We report the isolation of a Xenopus BMP receptor that is expressed maternally in the appropriate location to play a role in mesoderm induction. This receptor binds both BMP-2 and BMP-4 with high affinity. A truncated form of this BMP receptor specifically blocks BMP-4 signaling. Expression of this truncated BMP receptor during embryogenesis converts ventral mesoderm to dorsal mesoderm. Contrary to the popularly held view that ventral is the ground state for all mesoderm, our results suggest that formation of ventral mesoderm requires an active signal and that, in the absence of this ventral signal, dorsal mesoderm is formed.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Mesodermo/fisiología , Proteínas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Factores de Crecimiento , Xenopus/embriología , Activinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Receptores de Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas , Línea Celular , Clonación Molecular , Inducción Embrionaria , Sustancias de Crecimiento/genética , Inhibinas/genética , Datos de Secuencia Molecular , Morfogénesis , ARN , Receptores de Superficie Celular/metabolismo , Análisis de Secuencia de ADNRESUMEN
Characterization of the polypeptides present in bone-inductive protein extracts from bovine bone has led to the cloning of seven regulatory molecules, six of which are distantly related to transforming growth factor beta. The three human bone morphogenetic proteins (BMPs) we describe herein, BMP-5, BMP-6, and BMP-7, show extensive sequence similarity to BMP-2, a molecule that by itself is sufficient to induce de novo bone formation in vivo. The additive or synergistic contribution of these BMP-2-related molecules to the osteogenic activity associated with demineralized bone is strongly implicated by the presence of these growth factors in the most active fractions of highly purified bone extract.
Asunto(s)
Huesos/metabolismo , Proteínas/genética , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN de Neoplasias/genética , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Osteosarcoma , Proteínas/aislamiento & purificación , ARN Mensajero/genética , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Factor de Crecimiento Transformador beta/aislamiento & purificaciónRESUMEN
We have approached the study of growth factors affecting cartilage and bone development by investigating those factors present in bone which are able to initiate new cartilage and bone formation in vivo. This has led to the identification and molecular cloning of seven novel human factors which we have named BMP-1 through BMP-7. Six of these molecules are related to each other, and are also distantly related to TGF-beta. The presence of one of these molecules, recombinant human BMP-2 (rhBMP-2) is sufficient to produce the complex developmental system of cartilage and bone formation when implanted subcutaneously in a rat assay system. In this model, administration of rhBMP-2 ultimately results in the formation of a piece of trabecular bone, which is filled with mature bone marrow. While our studies demonstrate that rhBMP-2 by itself has the ability to induce cartilage and bone formation in vivo, we find other BMP molecules present along with BMP-2 in our highly purified nonrecombinant bone-inductive material. These results suggest that the bone inductive capacity of bone-derived proteins may reside in the combinatorial or synergistic activities of this set of BMP-2 related molecules.
Asunto(s)
Desarrollo Óseo , Sustancias de Crecimiento/fisiología , Animales , Desarrollo Óseo/efectos de los fármacos , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/farmacología , Humanos , Proteínas Recombinantes/farmacología , Homología de Secuencia de Ácido Nucleico , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.
Asunto(s)
Sustancias de Crecimiento/genética , Osteogénesis , Proteínas/genética , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas , Cartílago/citología , Cartílago/efectos de los fármacos , Línea Celular , ADN/genética , Humanos , Datos de Secuencia Molecular , Proteínas/farmacología , Proteínas Recombinantes/farmacología , Homología de Secuencia de Ácido Nucleico , Factores de Crecimiento Transformadores/genéticaRESUMEN
cDNAs which encode bone gla protein (BGP), an abundant gamma-carboxylated protein of bone, have been cloned from rat and mouse osteosarcoma cell lines. DNA sequence analysis indicates that the cDNAs code for both the 50 (rat) or 46 (mouse) amino acids of the mature proteins and a 49 amino acid leader peptide. The leader peptide of each BGP includes the expected hydrophobic signal sequence and an apparent pro sequence. Although there is no homology between the mature forms of BGP and the gamma-carboxylated clotting factors, we note that there is some homology between their leader peptides. These cDNAs have been used to examine the modulation of BGP mRNA levels by osteoblastic cells in response to hormones. The cDNAs have also allowed isolation of the human BGP gene; analysis of this gene indicates the presence of four exons. Comparison of the exon structure of the BGP gene and the Factor IX (a gamma-carboxylated clotting factor) gene suggests that the exons encoding the part of the leader peptides presumably directing gamma-carboxylation arose from a common ancestral sequence.