RESUMO
We have expressed and biologically characterized recombinant human growth/differentiation factor 5 (huGDF5). This protein is composed of a mature homodimer consisting of 15 kD subunits. Using recombinant expressed protein, we have demonstrated that huGDF5 in vitro stimulated mesenchyme aggregation and chondrogenesis in rat limb bud cells. In vivo, partially purified huGDF5 induced cartilage and bone formation in muscular tissues of rodents. However, in contrast to the effects of other BMPs, as for example BMP-2, the osteoblastic MC3T3-E1 cells did not respond to huGDF5 as measured by alkaline phosphatase activity. These results suggest that the action of GDF5 may be relatively specific for chondrogenesis during the entire process of the endochondral bone formation. GDF5 may control the morphogenesis of cartilaginous tissue, including joints, in the skeletal development of limbs.
Assuntos
Proteínas Morfogenéticas Ósseas , Substâncias de Crescimento/farmacologia , Mesoderma/metabolismo , Osteogênese/genética , Proteínas Recombinantes/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Cartilagem/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosaminoglicanos/metabolismo , Fator 5 de Diferenciação de Crescimento , Histocitoquímica , Humanos , Camundongos , Morfogênese/genética , Osteogênese/efeitos dos fármacos , Ratos , Vaccinia virus/metabolismoRESUMO
The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.
