RESUMO
The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190bp enhancer ~2.5 kilobases 5' of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFß effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFß signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.
Assuntos
Proteína Morfogenética Óssea 6/genética , Elementos Facilitadores Genéticos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Smegmamorpha/embriologia , Peixe-Zebra/embriologia , Nadadeiras de Animais/embriologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Benzamidas/farmacologia , Sítios de Ligação/genética , Proteína Morfogenética Óssea 6/biossíntese , Cromossomos Artificiais Bacterianos/genética , Sequência Conservada/genética , Dioxóis/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Dados de Sequência Molecular , Odontogênese/genética , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Alinhamento de Sequência , Análise de Sequência de DNA , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/genética , Proteína Smad3/metabolismo , Smegmamorpha/genética , Dente/embriologia , Peixe-Zebra/genéticaRESUMO
The dental follicle (DF) plays an essential role in tooth eruption via regulation of bone resorption and bone formation. Bone morphogenetic protein-6 (BMP6) expression in the DF is coincident with bone growth in the tooth crypt. DF stem cells (DFSCs) have been shown to possess strong osteogenic capability. This study aims to determine the expression of BMP6 in DFSCs and to elucidate the role of BMP6 in the osteogenesis of DFSCs. DFSCs and their non-stem cell counterpart, DF cells (DFCs), were obtained from the DFs of rat pups. We showed that expression of BMP6 was significantly higher in the DFSCs than in the DFCs. DFSCs lost osteogenic capability during in vitro expansion, and DFSCs in late passages had reduced BMP6 expression as compared to early passages of DFSCs when they were subjected to osteogenic induction. Addition of exogenous human recombinant BMP6 (hrBMP6) to the osteogenic medium dramatically enhanced the osteogenesis of the late-passage DFSCs. Knockdown of BMP6 by short interfering RNA in the DFSCs in early passages resulted in a decrease in osteogenesis, which could be restored by addition of hrBMP6. We concluded that DFSCs need to express high levels of BMP6 to maintain their osteogenesis capability. Increased BMP6 expression seen in vivo in the DF may reflect the activation of DFSCs for osteogenic differentiation for bone growth during tooth eruption.
Assuntos
Proteína Morfogenética Óssea 6/biossíntese , Diferenciação Celular/efeitos dos fármacos , Saco Dentário/metabolismo , Osteogênese/efeitos dos fármacos , Células-Tronco/metabolismo , Animais , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/farmacologia , Diferenciação Celular/genética , Células Cultivadas , Saco Dentário/citologia , Humanos , Osteogênese/genética , Interferência de RNA , RNA Interferente Pequeno , Ratos , Células-Tronco/citologiaRESUMO
AIM: We sought to determine whether triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether), an extensively used anti-plaque agent with broad-spectrum anti-microbial activity, with reported anti-inflammatory effects via inhibition of prostaglandin E2 and interleukin 1 (IL-1)beta, could also more broadly suppress multiple inflammatory gene pathways responsible for the pathogenesis of gingivitis and periodontitis. MATERIALS AND METHODS: As an exploratory study, the effects of triclosan on the inflammatory gene expression profile were assessed ex vivo using peripheral whole blood samples from eight periodontally healthy donors. Ten-millilitres whole blood aliquots were incubated 2 h with 0.3 microg/ml Escherichia coli lipopolysaccharide (LPS) with or without 0.5 microg/ml triclosan. Affymetrix microarray gene expression profiles from isolated leucocytes and pathway-specific quantitative polymerase chain reaction arrays were used to investigate changes in expression of target cytokines and cell signalling molecules. RESULTS: Ex vivo human whole blood assays indicated that triclosan significantly down-regulated the LPS-stimulated expression of Toll-like receptor signalling molecules and other multiple inflammatory molecules including IL-1 and IL-6 and the dampening of signals that activate the T-helper type 1 acquired immune response via suppression of CD70 with concomitant up-regulation of growth factors related to bone morphogenetic protein (BMP)2 and BMP6 synthesis. CONCLUSIONS: Anti-inflammatory effects were found in this exploratory survey, including suppression of microbial-pathogen recognition pathway molecules and the suppression of acute and chronic mediators of inflammation.