RESUMO
Members of the TGF-beta/BMP family of growth factors induce odontoblast differentiation and reparative dentin synthesis, and their use has been proposed to stimulate pulp healing during dental therapeutics in human. However, factors that modulate TGF-beta and/or BMP signalling during odontoblast differentiation and physiology remain largely unknown. To identify them, we compared expression profiles of TGF-beta/BMP-related genes in pulp fibroblast- and odontoblast-like cells cultured from human dental pulp explants using cDNA gene arrays. We evidenced that the gene encoding ecotropic viral integration site-1 (EVI1), a transcription factor that inhibits TGF-beta/BMP signalling, was under-expressed in odontoblast-like cells. This result was verified by real-time PCR and, at the protein level, by immunohistochemistry. In vivo, real-time PCR analysis revealed that EVI1 was expressed in the dental pulp, at a level similar to brain, but lower than in lung, kidney or trachea. The protein was localized in dental pulp samples in pulp core and subodontoblast cells. Staining intensity progressively decreased from the radicular to the coronal pulp where EVI1 staining was almost undetectable in odontoblasts. Our data suggest that fine regulation of the EVI1 level in the human dental pulp might be important in the TGF-beta/BMP-induced modulation of dental pulp cell kinetics and/or odontoblast differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Ligação a DNA/análise , Polpa Dentária/metabolismo , Fatores de Transcrição/análise , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adolescente , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Polpa Dentária/citologia , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Proteína do Locus do Complexo MDS1 e EVI1 , Odontoblastos/citologia , Odontoblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
The orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays a major role in adrenal and gonadal development, as well as in sexual differentiation. It has been demonstrated that the expression of a number of genes regulated by SF-1 is inhibited by the transforming growth factor, (TGF-beta). To date, however, the influence of TGF-beta on the expression of SF-1 gene has not been reported. A Northern blot analysis with the use of a radiolabeled cDNA probe, and immunodetection with antibodies directed against SF-1, demonstrated that the Sf-1 transcript and the SF-1 protein levels were lowered by TGF-beta in Y-1 adrenocortical cells, both in untreated and adenylyl cyclase activator, forskolin-treated cells. An examination of the Sf-1 transcript stability in the presence of actinomycin D revealed no influence of TGF-beta on the rate of Sf-1 mRNA decay. Inhibition of Sf-1 expression by TGF-beta was abolished by cycloheximide, suggesting that the growth factor inhibitory effect requires ongoing protein synthesis. We conclude that in Y-1 cells TGF-beta inhibits the expression of SF-1 gene at a transcriptional level, and we postulate that the inhibitory effect of TGF-beta on steroid hormone synthesis in the adrenal cortex could be due to an attenuated transcription of Sf-1.
Assuntos
Córtex Suprarrenal/metabolismo , Proteínas de Homeodomínio/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia , Neoplasias do Córtex Suprarrenal , Animais , Northern Blotting , Linhagem Celular Tumoral , Colforsina/farmacologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Proteínas de Homeodomínio/biossíntese , Camundongos , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/biossíntese , Fator Esteroidogênico 1 , Fatores de Tempo , Fatores de Transcrição/biossíntese , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The objective of our study was to investigate the effect of stimulation of the cAMP-dependent pathway on the expression of an orphan nuclear receptor, SF-1/Ad4BP in mouse adrenal tumour, Y-1 cells in culture. We evaluated the temporal pattern of the effects of corticotropin (ACTH) and the adenylyl cyclase activator forskolin on the level of SF-1 mRNA, and compared the time course of induction of SF-1 with that of CYP11A1. Forskolin, corticotropin and 8-Br-cAMP significantly elevated the level of the SF-1 transcript, after 1.5 h of incubation, with a concomitant increase of SF-1 protein level, observed after 6 h. The CYP11A1 transcript increased gradually over the incubation period, and reached the maximal level after 12 to 24 h. The steady-state level of the SF-1 transcript was unaffected by forskolin when the cells were incubated with actinomycin D, indicating that stimulation of the cAMP pathway results in enhanced transcription of the gene. The effect of forskolin was augmented by cycloheximide, suggesting that an inhibitory protein, whose synthesis was inhibited by cycloheximide, could be involved in negative regulation of SF-1 expression. It is concluded that SF-1 expression is positively regulated by the cAMP pathway at the transcriptional level, and can represent the primary event in cAMP-mediated induction of steroid hormone synthesis in Y-1 cells.
