19 results on '"Amorim BR"'
Search Results
2. Investigation of PPAR β / δ within Human Dental Pulp Cells: A Preliminary In Vitro Study.
- Author
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de Lima CL, Amorim BR, Royer C, Resende AP, Borin MF, Neves FAR, and Acevedo AC
- Abstract
Controlling the inflammatory response to restore tissue homeostasis is a crucial step to maintain tooth vitality after pathogen removal from caries-affected dental tissues. The nuclear peroxisome proliferator-activated receptor beta/delta (PPAR β / δ ) is a ligand-activated transcription factor with emerging anti-inflammatory roles in many cells and tissues. However, its expression and functions are poorly understood in human dental pulp cells (hDPCs). Thus, this study evaluated PPAR β / δ expression and assessed the anti-inflammatory effects evoked by activation of PPAR β / δ in lipopolysaccharide- (LPS-) induced hDPCs. Our results showed that hDPCs constitutively expressed PPAR β / δ mRNA/protein, and treatment with LPS increased PPARβ/δ mRNA expression. The selective PPAR β / δ agonist GW0742 significantly decreased inflammation-related mRNA expression in hDPCs ( IL6 , IL1β , TNFα , MMP1 , and MMP2 ) and RAW264.7 cells ( Il6 and Tnfα ). Further, PPAR β / δ agonist attenuated MMP2/9 gelatinolytic activity in hDPCs. Previously LPS-conditioned hDPCs increased the migration of RAW264.7 cells through the membrane of a Transwell coculture system. Conversely, pretreatment with GW0742 markedly decreased macrophage recruitment. These findings provide among the first evidence that hDPCs express PPAR β / δ . In addition, they suggest that activation of PPAR β / δ by GW0742 can attenuate some cellular and molecular in vitro aspects related to the inflammatory process, pointing out to investigate its potential target role in dental pulp inflammation., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Caroline L. de Lima et al.)
- Published
- 2021
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3. Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro.
- Author
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Martins L, Amorim BR, Salmon CR, Leme AFP, Kantovitz KR, and Nociti FH Jr
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- Amelogenin genetics, Animals, Cell Line, Enzyme Activation, Gene Expression Regulation, Gene Regulatory Networks, Mice, Protein Binding, Protein Interaction Maps, Signal Transduction, Amelogenin metabolism, Cell Differentiation, Cementogenesis, Dental Cementum metabolism, Dental Enamel Proteins metabolism, Plasminogen metabolism
- Abstract
Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies., (© 2019 Wiley Periodicals, Inc.)
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- 2020
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4. In vitro evaluation of Eugenia dysenterica in primary culture of human gingival fibroblast cells.
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Costa CRR, Amorim BR, Silva SMMD, Acevedo AC, Magalhães PO, and Guerra ENS
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- Animals, Brazil, Cells, Cultured, Chlorhexidine analogs & derivatives, Chlorhexidine pharmacology, Humans, Mice, Nitric Oxide analysis, Plant Leaves chemistry, RAW 264.7 Cells, Reference Values, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Wound Healing drug effects, Anti-Inflammatory Agents pharmacology, Antioxidants pharmacology, Eugenia chemistry, Fibroblasts drug effects, Gingiva cytology, Plant Extracts pharmacology
- Abstract
Eugenia dysenterica is a Brazilian tree investigated for its properties and bioactive compounds, which are believed to have both pharmacological and phytochemical therapeutic effects. The leaves of this tree contain tannins, flavonoids, terpenes, and saponins, with reportedly beneficial effects to the human body. Despite these therapeutic applications, its effects have never been tested on oral tissues. Therefore, the aim of the present study was to evaluate the cytotoxic and antioxidant effects and the anti-inflammatory and repair properties of the acetone fraction of E. dysenterica on primary culture of human gingival fibroblasts and on the immortalized murine macrophage cell line (RAW 264.7). For this purpose, a metabolic activity assay, a wound healing assay, a nitric oxide assay, and RT-qPCR were performed. The assays revealed a cytoprotective effect of this plant, suggested by the increase in the expression of SOD1 and NRF2. An antioxidant potential effect was observed in the DPPH• assay. However, the fraction of E. dysenterica did not show anti-inflammatory activity. In conclusion, Eugenia dysenterica may promote cytoprotection when associated with chlorhexidine digluconate because of its antioxidant effect. However, additional studies are necessary on other human dental tissues using other parts of the plant in order to develop a possible mouthwash to assist patients with oral disorders.
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- 2019
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5. Protocols for Genetic and Epigenetic Studies of Rare Diseases Affecting Dental Tissues.
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Amorim BR, Dos Santos PAC, de Lima CL, Andia DC, Mazzeu JF, and Acevedo AC
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- Amelogenesis Imperfecta genetics, Cell Culture Techniques methods, DNA genetics, DNA isolation & purification, Humans, Phenotype, Polymerase Chain Reaction methods, Tooth Abnormalities genetics, Epigenesis, Genetic, High-Throughput Nucleotide Sequencing methods, Rare Diseases genetics, Tooth Diseases genetics
- Abstract
This chapter describes methods related to the diagnosis of genetic dental diseases. Based on the present knowledge, clinical phenotyping and next-generation sequencing techniques are discussed. Methods necessary for Sanger sequencing, multiplex ligation-dependent probe amplification, and epigenetic modification methods are detailed. In addition, protocols for cell culture establishment and characterization from patients with inherited dental anomalies are described.
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- 2019
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6. Osteogenic potential of periodontal ligament stem cells are unaffected after exposure to lipopolysaccharides.
- Author
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Albiero ML, Amorim BR, Casati MZ, Sallum EA, Nociti FH Junior, and Silvério KG
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- Alkaline Phosphatase analysis, Cell Differentiation drug effects, Cell Proliferation drug effects, Cell Survival drug effects, Cells, Cultured, Core Binding Factor Alpha 1 Subunit analysis, Flow Cytometry, Gene Expression, Humans, Interleukin-1beta analysis, Mesenchymal Stem Cells metabolism, Octamer Transcription Factor-3 analysis, Osteocalcin analysis, Real-Time Polymerase Chain Reaction, Statistics, Nonparametric, Time Factors, Toll-Like Receptors analysis, Tumor Necrosis Factor-alpha analysis, Lipopolysaccharides toxicity, Mesenchymal Stem Cells drug effects, Osteogenesis drug effects, Periodontal Ligament cytology, Porphyromonas gingivalis
- Abstract
Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs) phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS) on mesenchymal phenotype and osteoblast/cementoblast (O/C) potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2) expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i) cell metabolic activity using MTS; (ii) gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR); (iii) flow cytometry for STRO-1 and CD105, and (iv) osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs.
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- 2017
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7. Neuropilin Controls Endothelial Differentiation by Mesenchymal Stem Cells From the Periodontal Ligament.
- Author
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Amorim BR, Silvério KG, Casati MZ, Sallum EA, Kantovitz KR, and Nociti FH Jr
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- Animals, Flow Cytometry, Mice, Cell Differentiation, Mesenchymal Stem Cells, Neuropilins physiology, Periodontal Ligament
- Abstract
Background: Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105(+)-enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)-like cells and to give insights into the mechanism involved., Methods: CD105(+)-enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM-2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real-time polymerase chain reaction; 2) Western blotting; 3) fluorescence-activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays., Results: Data analyses demonstrated that EGM-2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain-containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube-like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105(+)-enriched PDLSCs resulted in increased cord/tube-like structures in a matrigel assay., Conclusion: These findings demonstrated the potential of CD105(+)-enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.
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- 2016
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8. Effects of Plants on Osteogenic Differentiation and Mineralization of Periodontal Ligament Cells: A Systematic Review.
- Author
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Costa CR, Amorim BR, de Magalhães P, De Luca Canto G, Acevedo AC, and Guerra EN
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- Aloe chemistry, Cell Differentiation drug effects, Cell Proliferation drug effects, Centella chemistry, Coumarins pharmacology, Epimedium chemistry, Flavonoids pharmacology, Hesperidin pharmacology, Humans, Mannans pharmacology, Morinda chemistry, Periodontal Ligament drug effects, Triterpenes pharmacology, Zanthoxylum chemistry, Osteogenesis drug effects, Periodontal Ligament cytology, Plant Extracts pharmacology
- Abstract
This systematic review aimed to evaluate the effects of plants on osteogenic differentiation and mineralization of human periodontal ligament cells. The included studies were selected using five different electronic databases. The reference list of the included studies was crosschecked, and a partial gray literature search was undertaken using Google Scholar and ProQuest. The methodology of the selected studies was evaluated using GRADE. After a two-step selection process, eight studies were identified. Six different types of plants were reported in the selected studies, which were Morinda citrifolia, Aloe vera, Fructus cnidii, Zanthoxylum schinifolium, Centella asiatica, and Epimedium species. They included five types of isolated plant components: acemannan, osthole, hesperetin, asiaticoside, and icariin. In addition, some active substances of these components were identified as polysaccharides, coumarins, flavonoids, and triterpenes. The studies demonstrated the potential effects of plants on osteogenic differentiation, cell proliferation, mineral deposition, and gene and protein expression. Four studies showed that periodontal ligament cells induce mineral deposition after plant treatment. Although there are few studies on the subject, current evidence suggests that plants are potentially useful for the treatment of periodontal diseases. However, further investigations are required to confirm the promising effect of these plants in regenerative treatments., (Copyright © 2016 John Wiley & Sons, Ltd.)
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- 2016
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9. Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern.
- Author
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Albiero ML, Amorim BR, Martins L, Casati MZ, Sallum EA, Nociti FH Jr, and Silvério KG
- Subjects
- Blotting, Western, Cell Differentiation physiology, Cell Survival physiology, Cells, Cultured, Cytokines genetics, Endoglin, Humans, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism, Osteoblasts cytology, Osteogenesis physiology, Polymerase Chain Reaction, Statistics, Nonparametric, Time Factors, Toll-Like Receptor 4 metabolism, Antigens, CD metabolism, Cytokines analysis, Escherichia coli metabolism, Lipopolysaccharides toxicity, Mesenchymal Stem Cells drug effects, Osteoblasts drug effects, Periodontal Ligament cytology, Receptors, Cell Surface metabolism
- Abstract
Unlabelled: Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix., Objective: This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105(+) cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS)., Material and Methods: Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105(+) cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR., Results: PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group., Conclusions: These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities.
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- 2015
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10. Characterization of highly osteoblast/cementoblast cell clones from a CD105-enriched periodontal ligament progenitor cell population.
- Author
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Saito MT, Salmon CR, Amorim BR, Ambrosano GM, Casati MZ, Sallum EA, Nociti FH, and Silvério KG
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- Alkaline Phosphatase analysis, Antigens, CD analysis, Antigens, Surface analysis, Cell Adhesion physiology, Cell Adhesion Molecules, Neuronal analysis, Cell Culture Techniques, Cell Differentiation physiology, Cell Separation, Clone Cells, Core Binding Factor Alpha 1 Subunit analysis, Dental Cementum metabolism, Endoglin, Fetal Proteins analysis, Humans, Osteoblasts metabolism, Osteogenesis physiology, Phenotype, Receptors, Cell Surface analysis, Dental Cementum cytology, Mesenchymal Stem Cells physiology, Osteoblasts cytology, Periodontal Ligament cytology
- Abstract
Background: It is known that periodontal ligament (PDL) harbors a heterogeneous progenitor cell population at different stages of lineage commitment. However, characterization of PDL stem cells committed to osteoblast/cementoblast (O/C) differentiation remains to be elucidated. The present study is carried out to isolate single cell-derived, cluster of differentiation (CD)105-positive PDL clones and to characterize the clones that present high potential to differentiate toward O/C phenotype in vitro., Methods: Isolation of single cell-derived colonies (clones) from a CD105-enriched PDL progenitor cell population was performed by the ring-cloning technique. Cell clones were evaluated for their O/C differentiation potential, metabolic activity, and expression of STRO-1 protein. Additionally, the clones that showed potential to O/C differentiation were characterized by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for expression of runt-related transcriptor factor 2 (RUNX2), alkaline phosphatase, CD105, and CD166 during osteogenic induction., Results: Six PDL-CD105(+) clones were obtained, three being highly O/C clones (C-O) and three others that did not have the ability to produce mineralized matrix in vitro (C-F). The C-O group showed lower metabolic activity compared with the C-F group, and both cell groups were positively immunostained for STRO-1. qRT-PCR analysis demonstrated an increased expression of transcripts for RUNX2 and CD166 during the maturation of C-O cells toward O/C phenotype., Conclusions: These results provide evidence that PDL-CD105(+) purified progenitor cells comprise a heterogeneous cell population that presents a cell subset with high O/C potential and, further, that surface antigen CD166 is modulated during the O/C maturation of this cell subset.
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- 2014
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11. PKR-mediated degradation of STAT1 regulates osteoblast differentiation.
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Yoshida K, Okamura H, Amorim BR, Hinode D, Yoshida H, and Haneji T
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- Adaptor Proteins, Signal Transducing physiology, Alkaline Phosphatase metabolism, Animals, Antigens, Differentiation metabolism, Cell Line, Interleukin-6 biosynthesis, Interleukin-6 genetics, LIM Domain Proteins, Metalloproteases metabolism, Mice, Osteoblasts cytology, RNA, Messenger biosynthesis, Ubiquitin-Protein Ligases physiology, Ubiquitination, eIF-2 Kinase genetics, Cell Differentiation physiology, Osteoblasts physiology, STAT1 Transcription Factor metabolism, eIF-2 Kinase physiology
- Abstract
The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.
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- 2009
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12. Calcineurin regulates phosphorylation status of transcription factor osterix.
- Author
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Okamura H, Amorim BR, Wang J, Yoshida K, and Haneji T
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- Animals, Cell Line, Cell Nucleus metabolism, Humans, Immunoprecipitation, Mice, Phosphorylation, Sp7 Transcription Factor, Transcription Factors genetics, Calcineurin metabolism, Transcription Factors metabolism
- Abstract
Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.
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- 2009
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13. Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells.
- Author
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Okamura H, Yoshida K, Amorim BR, and Haneji T
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- Cell Line, Tumor, Cell Membrane Permeability drug effects, DNA Breaks, Double-Stranded drug effects, DNA Breaks, Single-Stranded drug effects, DNA Fragmentation drug effects, Humans, Laminin metabolism, Mitochondrial Membranes metabolism, Protein Transport drug effects, Antibiotics, Antineoplastic pharmacology, Apoptosis drug effects, Bleomycin pharmacology, Histones metabolism, Mitochondria metabolism, Signal Transduction drug effects, bcl-2 Homologous Antagonist-Killer Protein metabolism
- Abstract
Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin., (2007 Wiley-Liss, Inc.)
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- 2008
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14. Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest.
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Morimoto H, Ozaki A, Okamura H, Yoshida K, Amorim BR, Tanaka H, Kitamura S, and Haneji T
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- Cells, Cultured, Cyclin B biosynthesis, Cyclin B1, Cyclin D, Cyclins biosynthesis, Electrophoresis, Gel, Two-Dimensional, Humans, Hydroxyurea pharmacology, Isoenzymes biosynthesis, Nocodazole pharmacology, Protein Phosphatase 1, Protein Phosphatase 2, Nucleolin, Cell Cycle drug effects, Phosphoprotein Phosphatases biosynthesis, Phosphoproteins biosynthesis, RNA-Binding Proteins biosynthesis
- Abstract
In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression., ((c) 2005 John Wiley & Sons, Ltd.)
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- 2007
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15. Calyculin A stimulates the expression of TNF-alpha mRNA via phosphorylation of Akt in mouse osteoblastic MC3T3-E1 cells.
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Qiu L, Yoshida K, Amorim BR, Okamura H, and Haneji T
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- 3T3 Cells, Animals, Marine Toxins, Mice, Mitogen-Activated Protein Kinases metabolism, NF-kappa B metabolism, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism, RNA, Messenger genetics, Tumor Necrosis Factor-alpha metabolism, Enzyme Inhibitors metabolism, Oxazoles metabolism, RNA, Messenger metabolism, Signal Transduction physiology, Tumor Necrosis Factor-alpha genetics
- Abstract
Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-alpha mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that calyculin A increased the expression of TNF-alpha mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the calyculin A-stimulated TNF-alpha mRNA expression. Western blot result disclosed that calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and SB203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on calyculin A-stimulated expression of TNF-alpha mRNA. BAY11-7085 and CAPE, inhibitors of NF-kappaB activity, did not alter the calyculin A-stimulated TNF-alpha mRNA expression. Indirect immunofluorescent study confirmed that NF-kappaB was not translocated to the nucleus by calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-alpha mRNA.
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- 2007
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16. The transcriptional factor Osterix directly interacts with RNA helicase A.
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Amorim BR, Okamura H, Yoshida K, Qiu L, Morimoto H, and Haneji T
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- Amino Acid Sequence, Cell Line, Humans, Immunoprecipitation, Molecular Sequence Data, Protein Binding, RNA Helicases chemistry, Sp7 Transcription Factor, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, RNA Helicases metabolism, Transcription Factors metabolism
- Abstract
Osterix is an osteoblast-specific transcriptional factor, required for bone formation and osteoblast differentiation. Here, we identified new Osterix interacting factors by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the candidates, RNA helicase A was identified to interact with Osterix. To determine the interaction of Osterix with RNA helicase A, immunoprecipitation assay was performed. Western analysis confirmed the association between Osterix and RNA helicase A. Immunocytochemical analysis also showed that Osterix and RNA helicase A were co-localized in HEK 293 cells. Our data suggest that RNA helicase A might be a component of Osterix regulation.
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- 2007
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17. Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells.
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Okamura H, Yoshida K, Sasaki E, Qiu L, Amorim BR, Morimoto H, and Haneji T
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- Animals, Fibroblasts cytology, Gene Expression Regulation, Enzymologic drug effects, Insulin pharmacology, Macrophages cytology, Macrophages drug effects, Macrophages enzymology, Mice, NIH 3T3 Cells, Phosphorylation drug effects, RNA, Small Interfering metabolism, Signal Transduction drug effects, Fibroblasts drug effects, Fibroblasts enzymology, Lipopolysaccharides pharmacology, PTEN Phosphohydrolase genetics, PTEN Phosphohydrolase metabolism, Proto-Oncogene Proteins c-akt metabolism
- Abstract
PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.
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- 2007
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18. Okadaic acid induces phosphorylation of p65NF-kappaB on serine 536 and activates NF-kappaB transcriptional activity in human osteoblastic MG63 cells.
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Ozaki A, Morimoto H, Tanaka H, Okamura H, Yoshida K, Amorim BR, and Haneji T
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- Cell Line, Humans, Osteoblasts cytology, Phosphorylation, Promoter Regions, Genetic, Transcription Factor RelA genetics, Enzyme Inhibitors pharmacology, Okadaic Acid pharmacology, Osteoblasts drug effects, Osteoblasts metabolism, Serine metabolism, Transcription Factor RelA metabolism, Transcription, Genetic
- Abstract
Nuclear factor-kappa B (NF-kappaB) is an essential transcription factor in the control of expression of genes involved in cell growth, differentiation, inflammation, and neoplastic transformation. Previously, we reported that okadaic acid (OA), which is a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in cells of human osteosarcoma cell line MG63. However, to date, it is not clear whether the phosphorylation status of NF-kappaB could be affected by the treatment with OA. In this report, we demonstrate that treatment of MG63 cells with OA enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and a lambda protein phosphatase dephosphorylation assay. The phosphorylation level of NF-kappaB was enhanced in both time- and dose-dependent manners. In the cells treated with 100 nM OA for 3 h, consequential translocation of NF-kappaB from the cytosol to the nucleus occurred. Western blotting experiments with an anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536. Furthermore, OA stimulated the transcriptional activity of NF-kappaB in MG63 cells, as judged from the results of a luciferase assay. Our findings indicate that OA elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of genes., (2006 Wiley-Liss, Inc.)
- Published
- 2006
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19. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.
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Yoshida K, Okamura H, Amorim BR, Ozaki A, Tanaka H, Morimoto H, and Haneji T
- Subjects
- Alkaline Phosphatase, Animals, Cell Differentiation, Cell Nucleus metabolism, Core Binding Factor Alpha 1 Subunit metabolism, Genes, Dominant, In Vitro Techniques, Interferon-Stimulated Gene Factor 3 antagonists & inhibitors, Interferon-Stimulated Gene Factor 3 genetics, Interferon-Stimulated Gene Factor 3 metabolism, Mice, Phosphorylation, Protein Transport, RNA, Double-Stranded physiology, RNA, Small Interfering pharmacology, Signal Transduction, Trans-Activators, Transcription, Genetic, Bone and Bones physiology, Calcification, Physiologic, Osteoblasts metabolism, eIF-2 Kinase physiology
- Abstract
In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression.
- Published
- 2005
- Full Text
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