RESUMO
This study concerns the use of low-energy pulsed ultrasound as nondestructive photodynamic antimicrobial therapy for controlling dental plaque. We examined the antibacterial and bactericidal effects of low-energy pulsed ultrasound on mutans streptococci and its inhibitory effects on bacterial cell adhesion of Streptococcus mutans. The results indicated weak antibacterial and bactericidal effects. However, ultrasonic stimulation for less than 20 min markedly decreased bacterial cell adhesion. To analyze the mechanism underlying the inhibitory effect, we examined cell surface protein antigen C (PAc) and glucosyltransferase I (GTF-I) expression in S. mutans. The levels of PAc gene and protein expression were markedly decreased by ultrasonic stimulation for 20 min. However, no change in GTF-I expression was observed. The expression of stress response heat shock proteins GroEL and DnaK was also examined. GroEL and DnaK levels were significantly decreased by ultrasonic stimulation, and the expression of the PAc protein was also diminished upon the addition of GroEL or DnaK inhibitors without ultrasonic stimulation. These observations suggest that the expression of the PAc protein in S. mutans may be dependent on heat shock proteins. Thus, low-energy pulsed ultrasound decreases bacterial adhesion by the inhibitory effect on the PAc protein and heat shock protein expression and may be useful as photodynamic antimicrobial chemotherapy in controlling dental plaque.
Assuntos
Antígenos de Bactérias/metabolismo , Chaperonina 60/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Streptococcus mutans/metabolismo , Ultrassom , Adenosina Trifosfatases/metabolismo , Antígenos de Bactérias/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Chaperonina 60/genética , Cárie Dentária/prevenção & controle , Placa Dentária/terapia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Glucosiltransferases/metabolismo , Proteínas de Choque Térmico HSP70/genética , Resposta ao Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , RNA Bacteriano/metabolismo , Streptococcus mutans/genética , Streptococcus mutans/imunologiaRESUMO
Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-alpha stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-alpha-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-alpha and BMP signaling pathways.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osteoblastos/metabolismo , Proteína Smad4/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Núcleo Celular/patologia , Inativação Gênica , Camundongos , Fosforilação , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Previously, we showed that recombinant human bone morphogenetic protein-2 (rhBMP-2) increased bone augmentation beyond the skeletal envelope within a titanium cap in a rabbit calvarium; many cuboidal osteoblastic cells were observed histologically. These results suggested that the new osteoblastic cells might have differentiated and matured via stimulation by rhBMP-2. To date, however, no studies have reported the characteristics of osteoblastic cells derived from adult rabbit calvarium, after addition of rhBMP-2. To determine the effects of rhBMP-2 on osteoblastic cells, we observed morphological characteristics and alkaline phosphatase activity of osteoblastic cells from an adult rabbit calvarium. The expression of proteins in the BMP signaling pathway and extracellular matrix were analyzed, and mineralized nodule formation was assessed. The alkaline phosphatase activity increased significantly after rhBMP-2 stimulation. The protein levels of phosphorylated-Smad1, Runx2, osteocalcin, osteopontin, and type I collagen were augmented by rhBMP-2 stimulation using Western blotting or ELISA; rhBMP-2 also stimulated mineralized nodule formation with alizarin red staining. The results suggest that primary osteoblastic cells derived from a rabbit calvarium have osteogenetic characteristics in vitro, underscoring the potential use of these cells as a model for studying bone formation. These cells may play an important role in in vivo bone augmentation in a rabbit experimental model.
Assuntos
Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Crânio/citologia , Crânio/efeitos dos fármacos , Fosfatase Alcalina/análise , Animais , Antraquinonas , Western Blotting , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno Tipo I/análise , Corantes , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/análise , Humanos , Masculino , Modelos Animais , Osteoblastos/fisiologia , Osteocalcina/análise , Osteogênese/fisiologia , Osteopontina/análise , Fosforilação , Coelhos , Proteínas Recombinantes/farmacologia , Proteína Smad1/análise , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Peróxido de Hidrogênio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cisteína/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Oxirredução , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate within the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from human bone marrow to identify the proteins interacting with Smad4. Two full-length cDNA clones for Ubc9 were identified, and the potential functions of Ubc9 were investigated. To determine the role of Ubc9 in the BMP signaling pathway, the endogenous transcription of Ubc9 in the human osteoblast cell line Saos-2 was silenced using siRNA. The expression of BMP-induced transcription factors, including Runx2, Dlx5, Msx2, and Osterix, was examined using real-time reverse transcription polymerase chain reaction (qRT-PCR), and the protein expression of Smad4, Smad1, phosphorylated Smad1, and BMP type I receptors was determined by Western blotting. The subcellular localization of Smad1 and Smad4 was observed using immunofluorescence staining after Ubc9 silencing. To determine whether Smad4 is sumoylated in vitro, recombinant Smad4 was purified and sumoylated Smad4 was visualized using Western blotting. The mRNA expression of various transcription factors was markedly inhibited after Ubc9 silencing. The protein levels of Smad4 and phosphorylated Smad1 decreased in a dose-dependent manner according to the amount of siRNA applied. Gene silencing also decreased the nuclear accumulation of Smad1 and Smad4. The sumoylation assay showed that sumoylated Smad4 is present and dependent on Ubc9 in vitro, which was confirmed by pretreatment with Senp2, a SUMO-protease. These results suggest that Ubc9 promotes the stability of sumoylated Smad4. Furthermore, the expression of key transcription factors, phosphorylated Smad1 protein, and the nuclear accumulation of Smad1 and Smad4 are inhibited by Ubc9 silencing. Thus, Ubc9 plays an important role in the up-regulation of the BMP signaling pathway.
Assuntos
Núcleo Celular/metabolismo , Osteoblastos/metabolismo , Proteína Smad1/metabolismo , Proteína Smad4/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologia , Transporte Ativo do Núcleo Celular , Western Blotting , Proteína Morfogenética Óssea 2 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína SUMO-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/genética , Proteína Smad4/genética , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Low-intensity pulsed ultrasound (LIPUS) is known to accelerate bone regeneration, but the precise cellular mechanism is still unclear. The purpose of this study was to determine the effect of LIPUS on the differentiation of pluripotent mesenchymal cell line C2C12. The cells were cultured in differentiation medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm2 for 20 min for all cultures. To verify the cell lineage after LIPUS stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARgamma) was studied using real-time polymerase chain reaction analysis. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were performed using Western blotting. The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the LIPUS stimulation, whereas the expression of MyoD, C/EBP, and PPARgamma was drastically decreased. In the Western blot analysis, LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. Our study demonstrated that LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK.