RESUMO
Growth/differentiation factor-5 (GDF-5) is a new member of the BMP family supposed to be involved in chondrogenesis. We cloned the human GDF-5 gene from lambda phage library and sequenced its 3.5-kb 5'-flanking region. The transcription start site was mapped by 5'-RACE to the sequence that coincides with the initiator element. Electrophoresis mobility shift assays (EMSA) demonstrated a zinc finger transcription factor, YY1, to bind to the sequence surrounding the transcription start site. To localize positive and negative regulatory elements in the GDF-5 5'-upstream region, we constructed a series of progressively deleted promoter-reporter plasmids. The transient transfection assay with human osteoblastic Hos cells indicated that a minimal enhancer element resides within -117 to -27 relative to the transcription initiation site. Since the GDF-5 promoter was active even in fibroblastic L cells, a mechanism governing its chondrocyte-restrictive expression needs to be explored.
Assuntos
Proteínas Morfogenéticas Ósseas , Substâncias de Crescimento/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Regiões 5' não Traduzidas/genética , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Biblioteca Gênica , Genes Reporter , Fator 5 de Diferenciação de Crescimento , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/genética , Fator de Transcrição YY1 , Dedos de ZincoRESUMO
In order to generate dimeric recombinant transforming growth factor-beta (TGF-beta) proteins, expensive eucaryotic cell systems, such as CHO cells, are usually used. An alternative represents the expression of such proteins in insects using a baculovirus expression system. In this study, recombinant human activin C protein was expressed in Noctuidae larvae. On SDS-PAGE, the expressed protein has a size of about 15 kD under reducing conditions and of about 20 kD under non-reducing conditions. This suggests that activin C is expressed as a dimer and disulfide bridges can be formed. Compared with expression in eucaryotic cell culture systems, expression in insect larvae presents a rapid and low cost method, without the need for expensive tissue culture scale-ups or special equipment.
Assuntos
Baculoviridae , Vetores Genéticos , Inibinas/biossíntese , Células 3T3 , Animais , Bombyx , Linhagem Celular , Expressão Gênica , Humanos , Inibinas/genética , Larva , Manduca , Camundongos , Mariposas , SpodopteraRESUMO
Growth/differentiation factor 5 (GDF5), a novel member of the transforming growth factor beta superfamily, promotes the survival of dopaminergic neurones in vitro. We present here the first evidence for a neuroprotective action of GDF5 in vivo. We investigated the effects of intracerebral administration of GDF5 on a rat model of Parkinson's disease. GDF5 was administered just above the substantia nigra and into the lateral ventricle immediately before ipsilateral injection of 6-hydroxydopamine into the medial forebrain bundle. GDF5 prevented the development of amphetamine-induced rotations and preserved the integrity of striatal dopaminergic nerve terminals, as measured by positron emission tomography. Post-mortem studies showed that GDF5 spared dopamine levels in the striatum and tyrosine hydroxylase positive neurones in the midbrain. This study suggests that GDF5 has potential for the treatment of Parkinson's disease.
Assuntos
Proteínas Morfogenéticas Ósseas , Corpo Estriado/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson Secundária/tratamento farmacológico , Substância Negra/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Corpo Estriado/patologia , Dopamina/metabolismo , Fator 5 de Diferenciação de Crescimento , Ácido Homovanílico/metabolismo , Injeções Intraventriculares , Oxidopamina , Doença de Parkinson Secundária/patologia , Ratos , Proteínas Recombinantes/farmacologia , Substância Negra/patologiaRESUMO
The mouse inhibin/activin beta C gene (Inhbc), a member of the transforming growth factor-beta (TGF-beta) superfamily, was cloned, mapped, and characterized. The gene spans approximately 14 kb, is composed of two exons, and maps to the distal region of mouse chromosome 10, which is syntenic to chromosome 12q13.1, where the human inhibin/activin beta C gene (INHBC) maps. The primary translation product is a preproprotein of 352 amino acids. The mature C-terminal domain of 116 amino acids shares 94% identity with its human homolog. Primer extension analysis shows that transcription starts approximately 130 bp upstream of the translation initiation site, and no TATA box was found in the promoter. Ribonuclease protection analyses reveal that mouse Inhbc is predominantly expressed in adult liver. Embryonic expression is detected beginning from Day 14.5 of gestation.
Assuntos
Mapeamento Cromossômico , Regulação da Expressão Gênica no Desenvolvimento , Inibinas/genética , Ativinas , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Genes/genética , Humanos , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Especificidade de Órgãos , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genéticaRESUMO
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
Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. We show here that GDF5 is expresed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum-free conditions, to almost the same extent as TGF-beta 3, and is as effective as glial cell line-derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF-beta and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF-beta and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N-methylpyridinium ion (MPP+), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD.
Assuntos
Proteínas Morfogenéticas Ósseas , Dopamina/metabolismo , Substâncias de Crescimento/farmacologia , Mesencéfalo/metabolismo , Neurônios/metabolismo , Fator de Crescimento Transformador beta/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Dopaminérgicos/farmacologia , Fator 5 de Diferenciação de Crescimento , Humanos , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RatosRESUMO
The complete amino acid sequence of human Growth/differentiation factor 5 (huGdf5), a new member of the TGF-beta superfamily, has been determined through initial degenerate PCR and subsequent cloning and nucleotide sequencing of genomic DNA and cDNA encoding the precursor and flanking regions. The huGdf5 gene consists of only two coding exons. The protein is highly homologous to its murine equivalent, particularly the mature part which differs only by one amino acid. Expression in HuTK- cells using recombinant vaccinia virus revealed the expected processed dimeric mature protein. Antibodies against huGdf5 were raised in chicken.
