RESUMO
TGF-ß1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-ß1, although single low dose TGF-ß1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-ß1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-ß1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-ß1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-ß1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-ß1 administration. This study showed that persistence of TGF-ß1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Fator de Crescimento Transformador beta1/metabolismo , Biomarcadores/metabolismo , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Doenças Ósseas/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Humanos , Proteínas Substratos do Receptor de Insulina/antagonistas & inibidores , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Ligamento Periodontal/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Amelogenesis imperfecta (AI) is associated with mutations in a number of genes, including AMELX and ENAM. However, the precise mechanism leading to enamel malformation in different AI types remains to be elucidated. In the present study, we investigated morphological change in tooth germ obtained from ENAM-mutant mice (Enam(Rgsc521) homozygotes) as a model for human AI using histological and immunohistochemical methodologies. The results showed that ameloblasts detached from developing dentin and lost cell polarity in mutant mice at post-natal day 3. Cyst-like structures, including amelogenin-immunopositive materials, were observed between these detached cells and the dentin. No enamel-like structure, however, was observed in the cusp of the crown. These results suggest that enamelin acts as an adhesion molecule and is involved in ameloblast cell differentiation during the early stages of tooth development.