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1.
Cell Mol Biol (Noisy-le-grand) ; 70(3): 22-28, 2024 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-38650160

RESUMO

This study aimed to evaluate the physiological role of NAMPT associated with MDPC-23 odontoblast cell proliferation. Cell viability was measured using the (DAPI) staining, caspase activation analysis and immunoblotting were performed. Visfatin promoted MDPC-23 odontoblast cell growth in a dose-dependent manner. Furthermore, the up-regulation of Visfatin promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers in MDPC-23 cells. However, FK-866 cell growth in a dose-dependent manner induced nuclear condensation and fragmentation. FK-866-treated cells showed H&E staining and increased apoptosis compared to control cells. The expression of anti-apoptotic factors components of the mitochondria-dependent intrinsic apoptotic pathway significantly decreased following FK-866 treatment. The expression of pro-apoptotic increased upon FK-866 treatment. In addition, FK-866 activated caspase-3 and PARP to induce cell death. In addition, after treating FK-866 for 72 h, the 3/7 activity of MDPC-23 cells increased in a concentration-dependent manner, and the IHC results also confirmed that Caspase-3 increased in a concentration-dependent. Therefore, the presence or absence of NAMPT expression in dentin cells was closely related to cell proliferation and formation of extracellular substrates.


Assuntos
Apoptose , Proliferação de Células , Nicotinamida Fosforribosiltransferase , Odontoblastos , Nicotinamida Fosforribosiltransferase/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Odontoblastos/citologia , Odontoblastos/metabolismo , Animais , Camundongos , Linhagem Celular , Citocinas/metabolismo , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Acrilamidas/farmacologia , Odontogênese/efeitos dos fármacos
2.
Oral Dis ; 29(4): 1644-1656, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-35199415

RESUMO

OBJECTIVES: This study aimed to identify formation of tubular dentin induced by transforming growth factor-ß (TGF-ß) and bone morphogenic protein (BMP) signaling pathway in dental epithelial cells. METHODS: We collected conditioned medium (CM) of rTGF-ß1/rBMP-2-treated HAT-7 and treated to MDPC-23 cells. The expression levels of odontoblast differentiation markers, KLF4, DMP1, and DSP were evaluated by real-time PCR and Western blot analysis. To evaluate whether CM of rTGF-ß1/rBMP-2 induces tubular dentin formation, we made a beagle dog tooth defect model. RESULTS: Here, we show that Cpne7 is regulated by Smad4-dependent TGF-ß1/BMP2 signaling pathway in dental epithelial cells. CM of rTGF-ß1/rBMP-2 treated HAT-7 or rCPNE7 raises the expression levels of KLF4, DMP1, and DSP in MDPC-23 cells. When rTGF-ß1 or rBMP-2 is directly treated to MDPC-23 cells, however, expression levels of Cpne7-regulated genes remain unchanged. In a beagle dog defect model, application of rTGF-ß1/BMP2-treated CM resulted in tubular tertiary dentin mixed with osteodentin at cavity-prepared sites, while rTGF-ß1 group exhibited homogenous osteodentin. CONCLUSIONS: Taken together, Smad4-dependent TGF-ß1/BMP2 signaling regulates Cpne7 in dental epithelial cells, and CPNE7 protein secreted from pre-ameloblasts mediates odontoblast differentiation via epithelial-mesenchymal interaction.


Assuntos
Proteínas da Matriz Extracelular , Fator de Crescimento Transformador beta1 , Cães , Animais , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Sialoglicoproteínas/genética , Fosfoproteínas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Odontoblastos , Transdução de Sinais , Células Epiteliais/metabolismo , Diferenciação Celular , Dentina/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(9): 4910-4920, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071240

RESUMO

Growth and differentiation factor 11 (GDF11) and myostatin (MSTN) are closely related transforming growth factor ß (TGF-ß) family members, but their biological functions are quite distinct. While MSTN has been widely shown to inhibit muscle growth, GDF11 regulates skeletal patterning and organ development during embryogenesis. Postnatal functions of GDF11, however, remain less clear and controversial. Due to the perinatal lethality of Gdf11 null mice, previous studies used recombinant GDF11 protein to prove its postnatal function. However, recombinant GDF11 and MSTN proteins share nearly identical biochemical properties, and most GDF11-binding molecules have also been shown to bind MSTN, generating the possibility that the effects mediated by recombinant GDF11 protein actually reproduce the endogenous functions of MSTN. To clarify the endogenous functions of GDF11, here, we focus on genetic studies and show that Gdf11 null mice, despite significantly down-regulating Mstn expression, exhibit reduced bone mass through impaired osteoblast (OB) and chondrocyte (CH) maturations and increased osteoclastogenesis, while the opposite is observed in Mstn null mice that display enhanced bone mass. Mechanistically, Mstn deletion up-regulates Gdf11 expression, which activates bone morphogenetic protein (BMP) signaling pathway to enhance osteogenesis. Also, mice overexpressing follistatin (FST), a MSTN/GDF11 inhibitor, exhibit increased muscle mass accompanied by bone fractures, unlike Mstn null mice that display increased muscle mass without fractures, indicating that inhibition of GDF11 impairs bone strength. Together, our findings suggest that GDF11 promotes osteogenesis in contrast to MSTN, and these opposing roles of GDF11 and MSTN must be considered to avoid the detrimental effect of GDF11 inhibition when developing MSTN/GDF11 inhibitors for therapeutic purposes.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Desenvolvimento Muscular/fisiologia , Miostatina/metabolismo , Osteogênese/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Osso e Ossos/patologia , Condrócitos/metabolismo , Regulação para Baixo , Folistatina , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Diferenciação de Crescimento/genética , Camundongos , Camundongos Knockout , Músculos/patologia , Osteoblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
4.
Int Endod J ; 56(4): 432-446, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36462163

RESUMO

AIM: The physiological effects and cellular mechanism of 25-hydroxycholesterol (25-HC), which is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase (CH25H) expressed under inflammatory conditions, are still largely unknown during odontoclastogenesis. This study aimed to evaluate 25-HC-induced odontoclastogenesis and its cellular mechanisms in odontoblast-like MDPC-23 cells. METHODOLOGY: To investigate 25-HC-induced odontoclastogenesis of MDPC-23 cells and its cellular mechanism, haemotoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, dentine resorption assay, zymography, reactive oxygen species (ROS) detection, immunocytochemistry, and nuclear translocation were performed. The experimental values are presented as mean ± standard deviation and were compared using analysis of variance, followed by post hoc multiple comparisons (Tukey's test) using SPSS software version 22 (IBM Corp.). A p-value <.05 was considered statistically significant. RESULTS: Lipopolysaccharide or receptor activator of nuclear factor-κB ligand (RANKL) induced the synthesis of 25-HC via the expression of CH25H in MDPC-23 cells (p < .01). Multinucleated giant cells with morphological characteristics and TRAP activity of the odontoclast were increased by 25-HC in MDPC-23 cells (p < .01). Moreover, 25-HC increased dentine resorption through the expression and activity of matrix metalloproteinases in MDPC-23 cells. It not only increased the expression of odontoclastogenic biomarkers but also translocated cytosolic nuclear factor-κB (NF-κB) to the nucleus in MDPC-23 cells. Additionally, 25-HC not only increased the production of ROS (p < .01), expression of inflammatory mediators (p < .01), pro-inflammatory cytokines, receptor activator of NF-κB (RANK), and RANKL but also suppressed the expression of osteoprotegerin (OPG) in MDPC-23 cells. In contrast, CDDO-Me, a chemical NF-κB inhibitor, decreased TRAP activity (p < .01) and downregulated the expression of the odontoclastogenic biomarkers, including RANK and RANKL, in MDPC-23 cells. CONCLUSION: 25-HC induced odontoclastogenesis by modulating the RANK-RANKL-OPG axis via NF-κB activation in MDPC-23 cells. Therefore, these findings provide that 25-HC derived from cholesterol metabolism may be involved in the pathophysiological etiological factors of internal tooth resorption.


Assuntos
NF-kappa B , Odontoblastos , Diferenciação Celular , NF-kappa B/metabolismo , Odontoblastos/metabolismo , Osteoclastos , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima , Animais , Camundongos
5.
J Clin Periodontol ; 49(6): 609-620, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35373365

RESUMO

AIM: Once the periodontal ligament (PDL) is damaged, it is difficult to regenerate its characteristic structure. Copine7 (CPNE7) reportedly plays a functional role in supporting periodontal attachment and PDL alignment. Here we demonstrate the regulatory mechanism of CPNE7 coordination with cytoskeleton reorganization and cementum attachment protein (CAP)-mediated attachment in PDL regeneration. MATERIALS AND METHODS: The expression and localization of CPNE7, α-TUBULIN, ACTIN, and microtubule associated protein tau (TAU) were investigated in vitro. The effects of recombinant CPNE7 (rCPNE7) and CPNE7-derived peptides (CPNE7-DP) on the regulation of CAP were analysed in vitro, and PDL repair capacity was analysed in vivo. RESULTS: CPNE7 co-localized with F-ACTIN and induced α-TUBULIN expansion to the edge of human PDL cells (hPDLCs). ACTIN and α-TUBULIN protein expressions were not elevated in rCPNE7-treated hPDLCs. rCPNE7 elevated the protein expression of TAU, which co-localized with F-ACTIN and α-TUBULIN. Replantation studies on mice revealed that well-attached and well-aligned PDLs were repaired in the rCPNE7 group. CPNE7-DP directly up-regulate the expression of CAP in vitro and promote PDL regeneration in three-wall defect canine models in vivo. CONCLUSIONS: Our findings suggest that CPNE7 helps in PDL repair by supporting PDL alignment through TAU-mediated cytoskeleton reorganization and direct regulation of CAP-mediated PDL attachments of PDLCs.


Assuntos
Cemento Dentário , Ligamento Periodontal , Actinas , Animais , Células Cultivadas , Camundongos , Regeneração , Tubulina (Proteína)/farmacologia
6.
BMC Oral Health ; 22(1): 483, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36368979

RESUMO

BACKGROUND: Dentin hypersensitivity is a painful response to external stimuli applied to exposed dentinal tubules. Various toothpastes with active desensitizing ingredients for the relief of dentin hypersensitivity are commercially available. However, data from several studies suggest that the effects of desensitizing toothpastes are unstable and brief. This study aimed to investigate the effect of toothpastes containing CPNE7-derived oligopeptide (CPNE7-DP) and other active desensitizing ingredients in the dentin microleakage, tubule occlusion and tertiary dentin formation. METHODS: Using scanning electron microscopy (SEM), we evaluated the patency of dentinal tubules on the surface of human dentin disks after brushing experiments with the various toothpastes. Dentin was histologically evaluated in a hypersensitivity model of canine teeth, after the exposed dentin area was brushed for 6 weeks. The toothpaste used in group 1 (control) did not contain any desensitizing ingredients; that used in group 2 contained CPNE7-DP; Colgate Sensitive was used in group 3; and Sensodyne Rapid Relief was used in group 4. Finally, we conducted microleakage analysis to investigate the dentin sealing effect. The microleakage analysis data were subjected to one-way ANOVA and post-hoc Tukey tests (alpha = 0.05). RESULTS: In the SEM images, all four groups of teeth exhibited partial occlusion of the dentinal tubules on the tooth surface. In the in vivo hypersensitivity model, group 2 exhibited a newly formed tertiary dentin, whereas no new hard tissue formation was observed in groups 1, 3, and 4. Microleakage analysis revealed that the volume of dentinal fluid flow was significantly smaller in group 2 than in group 1. CONCLUSIONS: These results indicate that CPNE7-DP is a promising active ingredient with long-term dentin sealing effects.


Assuntos
Sensibilidade da Dentina , Cremes Dentais , Humanos , Cremes Dentais/farmacologia , Cremes Dentais/uso terapêutico , Sensibilidade da Dentina/tratamento farmacológico , Dentina , Escovação Dentária/métodos , Fluoreto de Sódio , Microscopia Eletrônica de Varredura
7.
Korean J Physiol Pharmacol ; 26(1): 37-45, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34965994

RESUMO

The aim of the present study was to investigate the physiological role of nicotinamide phosphoribosyltransferase (NAMPT) associated with odontogenic differentiation during tooth development in mice. Mouse dental papilla cell-23 (MDPC- 23) cells cultured in differentiation media were stimulated with the specific NAMPT inhibitor, FK866, and Visfatin (NAMPT) for up to 10 days. The cells were evaluated after 0, 4, 7, and 10 days. Cell viability was measured using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay. The mineralization assay was performed by staining MDPC-23 cells with Alizarin Red S solution. After cultivation, MDPC-23 cells were harvested for quantitative PCR or Western blotting. Analysis of variance was performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05. The expression of NAMPT increased during the differentiation of murine odontoblast-like MDPC-23 cells. Furthermore, the up-regulation of NAMPT promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers, such as dentin sialophosphoprotein, dentin matrix protein-1, and alkaline phosphatase in MDPC-23 cells. However, treatment of the cells with the NAMPT inhibitor, FK866, attenuated odontogenic differentiation, as evidenced by the suppression of odontoblastic biomarkers. These data indicate that NAMPT regulated odontoblastic differentiation through the regulation of odontoblastic biomarkers. The increase in NAMPT expression in odontoblasts was closely related to the formation of the extracellular matrix and dentin via the Runx signaling pathway. Therefore, these data suggest that NAMPT is a critical regulator of odontoblast differentiation during tooth development.

8.
Int J Mol Sci ; 22(23)2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34884488

RESUMO

In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray's clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.


Assuntos
Antígeno CD11c/metabolismo , Células Dendríticas/imunologia , Polpa Dentária/imunologia , Imageamento Tridimensional/métodos , Pulpite/imunologia , Dente/imunologia , Animais , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pulpite/metabolismo , Pulpite/patologia , Dente/metabolismo , Dente/patologia
9.
Connect Tissue Res ; 60(5): 419-430, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30734591

RESUMO

Introduction: Preameloblast-conditioned medium (PA-CM), as a mixture of dental epithelium-derived factors, has been reported to regenerate dentin and periodontal tissues in vitro and in vivo. The aim of this study was to investigate the biological effect of Cpne7 on the proliferation, migration, and cementoblast differentiation of periodontal cells in vitro, and on the regeneration of periodontal tissue using periodontal defect model with canine in vivo. Materials and methods: The effect of Cpne7 on cell proliferation, migration, and cementoblast differentiation of periodontal cells were evaluated in vitro. A periodontal defect canine model was designed and the defects were divided into five groups: Group 1: No treatment (negative control), Group 2: Collagen carrier only, Group 3: PA-CM with collagen carrier (positive control), Group 4: PA-CM + CPNE7 Antibody (Ab) with collagen carrier, and Group 5: recombinant CPNE7 (rCPNE7) protein with collagen carrier. Results: Cpne7 was expressed in HERS cells and periodontal ligament (PDL) fibers. By real-time PCR, Cpne7 increased expression of Cap compared to the control. In the periodontal defect canine model, rCPNE7 or PA-CM regenerated periodontal complex, and the arrangement of the newly formed PDL-like fibers were perpendicular to the newly formed cementum and alveolar bone like Sharpey's fibers in natural teeth, while PA-CM + CPNE7 Ab showed irregular arrangement of the newly formed PDL-like fibers compared to the rCPNE7 or PA-CM group. Conclusion: These findings suggest that Cpne7 may have a functional role in periodontal regeneration by supporting periodontal cell attachment to cementum and facilitating physiological arrangement of PDL fibers.


Assuntos
Proteínas de Membrana/metabolismo , Periodonto/fisiologia , Regeneração , Adolescente , Ameloblastos/citologia , Ameloblastos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cementogênese/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Cães , Humanos , Camundongos , Periodonto/citologia , Proteínas Recombinantes/farmacologia , Regeneração/efeitos dos fármacos , Dente/crescimento & desenvolvimento , Dente/metabolismo , Adulto Jovem
10.
Cell Tissue Res ; 374(3): 531-540, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30091046

RESUMO

Enamel makes up the outermost layer of the crown and its hardness protects other dental tissues from various stimuli. Enamel cannot be regenerated once damaged because ameloblasts are lost during the tooth eruption. Since the ameloblast differentiation mechanism is still unknown, further research is essential for developing treatments for defective or damaged enamel. Previously, we have reported that osteoblast differentiation and bone formation were regulated through the runt-related transcription factor 2 (Runx2)-nuclear factor 1-C (Nfic)-osterix (Osx) pathway where Nfic directly controls Osx expression. This pathway regulates odontoblast differentiation and dentin formation as well. The aim of this study was to investigate if the same pathway is applicable for ameloblast differentiation. Structural enamel defects with disorganized ameloblasts and decreased proliferation activity of the cervical loop were observed in Nfic-/- mice incisors. Expression of the ameloblast differentiation markers was also downregulated significantly in Nfic-/- mice. Real-time PCR analyses suggested that Runx2, Nfic, and Osx regulate the expression of ameloblast differentiation markers, where Runx2 is upstream of Nfic, and Nfic controls Osx expression. Therefore, we suggest the Runx2-Nfic-Osx pathway as one of the key factors that regulate ameloblast differentiation.


Assuntos
Ameloblastos/citologia , Ameloblastos/metabolismo , Diferenciação Celular , Esmalte Dentário/metabolismo , Fatores de Transcrição NFI/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Esmalte Dentário/ultraestrutura , Camundongos , Fatores de Transcrição NFI/deficiência , Dente/metabolismo , Dente/ultraestrutura , Microtomografia por Raio-X
11.
BMC Oral Health ; 18(1): 148, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143043

RESUMO

BACKGROUND: Odontogenic Ameloblast-Associated Protein (ODAM) in gingival crevicular fluid (GCF) can provide evidence of the detachment of junctional epithelium from the tooth surface by periodontitis. This study sought to investigate the ability of ODAM to reflect the severity of periodontitis at a site-specific level; thus whether there was a relationship between clinical diagnostic parameters and the value of ODAM in GCF was analyzed. METHODS: Eight periodontitis patients with various severities were enrolled, and the clinical parameters and samples of GCF were obtained from 44 to 60 sites of each subject. The ODAM concentration was quantified by enzyme-linked immunosorbent assay. Correlation analyses between clinical parameters and ODAM values and unadjusted and adjusted (linear) mixed model analyses were performed. The accuracy of ODAM to reflect sites having a probing depth (PD) ≥ 5 mm and a positive bleeding on probing (BOP) was evaluated by receiver-operating characteristic analysis. RESULTS: A total of 424 GCF samples were collected. The mean ODAM concentration from each patient varied from 0.2 to 1.52 ng/ml. Correlations between PD or clinical attachment level (CAL) and ODAM values were found (p <  0.0001). An adjusted linear mixed model showed that PD or CAL were associated with ODAM values (p <  0.05). The area under the curve of ODAM, which reflected sites with PD ≥ 5 mm and positive BOP, was 0.661 (p <  0.0001). CONCLUSION: This result shows the possibility of GCF ODAM as a site-specific biomarker for periodontal tissue destruction.


Assuntos
Proteínas de Transporte/metabolismo , Líquido do Sulco Gengival/química , Periodontite/diagnóstico , Adulto , Idoso , Amiloide , Biomarcadores/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias , Índice Periodontal , Periodontite/metabolismo , Projetos Piloto
12.
J Biol Chem ; 290(23): 14740-53, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25911094

RESUMO

Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin ß3- and ß6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.


Assuntos
Proteínas de Transporte/metabolismo , Inserção Epitelial/metabolismo , Periodontite/metabolismo , Proteínas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Dente/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amiloide , Animais , Proteínas de Transporte/análise , Linhagem Celular , Inserção Epitelial/patologia , Fibronectinas/análise , Fibronectinas/metabolismo , Humanos , Integrinas/análise , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Laminina/análise , Laminina/metabolismo , Camundongos , Proteínas de Neoplasias , Periodontite/patologia , Proteínas/análise , Fatores de Troca de Nucleotídeo Guanina Rho/análise , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/análise
13.
J Cell Physiol ; 231(7): 1484-94, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26335354

RESUMO

The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans-differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5'-aza-dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3-L1 adipocytes with 5'-aza-dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5'-aza-dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue-engineering applications.


Assuntos
Transdiferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética/genética , Osteogênese/genética , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Transdiferenciação Celular/efeitos dos fármacos , Monofosfato de Citidina/administração & dosagem , Monofosfato de Citidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Proteína Wnt3A/genética
14.
Bioelectromagnetics ; 37(3): 163-174, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26991921

RESUMO

The purpose of this study was to investigate the effect of Schwann-like cells combined with pulsed electromagnetic field (PEMF) on peripheral nerve regeneration. Schwann-like cells were derived from human dental pulp stem cells (hDPSCs) and verified with CD104, S100, glial fibrillary acidic protein (GFAP), laminin, and P75NTR immunocytochemistry. Gene expression of P75NTR and S100 were analyzed. Male Sprague-Dawley rats (200-250g, 6-week-old) were divided into seven groups (n = 10 each): control, sham, PEMF, hDPSCs, hDPSCs + PEMF, Schwann-like cells, Schwann-like cells + PEMF. Cells were transplanted (1 × 106 /10µl/rat) at crush-injury site or combined with PEMF (50 Hz, 1 h/day, 1 mT). Nerve regeneration was evaluated with functional test, histomorphometry and retrograde labelled neurons. Schwann-like cells expressed CD104, S100, GFAP, laminin, and p75 neurotrophin receptor (P75NTR ). P75NTR and S100 mRNA expression was highest in Schwann-like cells + PEMF group, which also showed increased Difference and Gap scores. Axons and retrograde labeled neurons increased in all treatment groups. Schwann-like cells, hDPSCs with or without PEMF, and PEMF only improved peripheral nerve regeneration. Schwann-like cells + PEMF showed highest regeneration ability; PEMF has additive effect on hDPSCs, Schwann-like cell in vitro and nerve regeneration ability after transplantation in vivo. Bioelectromagnetics. 37:163-174, 2016. © 2016 Wiley Periodicals, Inc.

15.
Int J Neurosci ; 126(8): 739-48, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26010211

RESUMO

PURPOSE: The purpose was to clarify the influence of frequency and exposure time of pulsed electromagnetic fields (PEMF) on the peripheral nerve regeneration. MATERIALS AND METHODS: Immortalized rat Schwann cells (iSCs) (1 × 10(2)/well) were exposed at four different conditions in 1 mT (50 Hz 1 h/d, 50 Hz 12 h/d, 150 Hz 1 h/d and 150 Hz 12h/d). Cell proliferation, mRNA expression of S100 and brain-derived neurotrophic factor (BDNF) were analyzed. Sprague-Dawley rats (200-250 g) were divided into six groups (n = 10 each): control, sham, 50 Hz 1 h/d, 50 Hz 12 h/d, 150 Hz 1 h/d and 150 Hz 12 Hr/d. Mental nerve was crush-injured and exposed at four different conditions in 1 mT (50 Hz 1 Hr/d, 50 Hz 12 Hr/d, 150 Hz 1 h/d and 150 Hz 12 h/d). Nerve regeneration was evaluated with functional test, histomorphometry and retrograde labeling of trigeminal ganglion. RESULTS: iSCs proliferation with 50 Hz, 1 h/d was increased from fourth to seventh day; mRNA expression of S100 and BDNF was significantly increased at the same condition from first week to third week (p < .05 vs. control); difference score was increased at the second and third week, and gap score was increased at the third under 50 Hz 1 h PEMF compared with control while other conditions showed no statistical meaning. Axon counts and retrograde labeled neurons were significantly increased under PEMF of four different conditions compared with control. Although there was no statistical difference, 50 Hz, 1 h PEMF showed highest regeneration ability than other conditions. CONCLUSION: PEMF enhanced peripheral nerve regeneration, and that it may be due to cell proliferation and increase in BDNF and S100 gene expression.


Assuntos
Magnetoterapia/métodos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/terapia , Células de Schwann/fisiologia , Animais , Técnicas de Cultura de Células , Masculino , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , Ratos , Ratos Sprague-Dawley
16.
J Biol Chem ; 289(41): 28225-36, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25138274

RESUMO

Odontoblasts are a type of terminally differentiated matrix-secreting cells. A number of molecular mechanisms are involved in the differentiation of odontoblasts. Several studies demonstrated that Krüppel-like factor 4 (KLF4) promotes odontoblast differentiation via control of dentin sialophosphoprotein (DSPP). Because nuclear factor I-C (NFIC) is also known to control DSPP, we investigated the relationship between NFIC and KLF4 during odontoblast differentiation. Klf4 mRNA expression was significantly decreased in Nfic(-/-) pulp cells compared with wild type cells. In immunohistochemistry assays, dentin matrix protein 1 (Dmp1), and DSP protein expression was barely observed in Nfic(-/-) odontoblasts and dentin matrix. Nfic bound directly to the Klf4 promoter and stimulated Klf4 transcriptional activity, thereby regulating Dmp1 and DSPP expression during odontoblast differentiation. Nfic or Klf4 overexpression promoted mineralized nodule formation in MDPC-23 cells. In addition, Nfic overexpression also decreased Slug luciferase activity but augmented E-cadherin promoter activity via up-regulation of Klf4 in odontoblasts. Our study reveals important signaling pathways during dentinogenesis: the Nfic-Klf4-Dmp1-Dspp and the Nfic-Klf4-E-cadherin pathways in odontoblasts. Our results indicate the important role of NFIC in regulating KLF4 during dentinogenesis.


Assuntos
Caderinas/genética , Dentinogênese/genética , Proteínas da Matriz Extracelular/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição NFI/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Ameloblastos/citologia , Ameloblastos/metabolismo , Animais , Caderinas/metabolismo , Diferenciação Celular , Dentina/citologia , Dentina/crescimento & desenvolvimento , Dentina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Fatores de Transcrição NFI/metabolismo , Odontoblastos/citologia , Odontoblastos/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Transcrição Gênica
17.
Stem Cells ; 32(9): 2467-79, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24801901

RESUMO

In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age-related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I-C (NFI-C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI-C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI-C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic-overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator-activated receptor gamma expression in Nfic(-/-) mice showing an age-related osteoporosis-like phenotype. Finally, NFI-C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein-2-Runx2 pathway. These results suggest that NFI-C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI-C expression in BMSCs could be a novel therapeutic approach for treating age-related osteoporosis.


Assuntos
Fatores de Transcrição NFI/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Transcrição/biossíntese , Idoso , Animais , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição NFI/genética , Osteogênese/fisiologia , Fator de Transcrição Sp7 , Transfecção
18.
BMC Cancer ; 15: 113, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25879941

RESUMO

BACKGROUND: Progression to metastasis is the leading cause of most cancer-related mortality; however, much remains to be understood about what facilitates the spread of tumor cells. In the present study, we describe a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. METHODS: We examined nuclear factor I-C (NFI-C) expression in MCF10A human breast epithelial cells, MCF7 non-invasive breast cancer cells, and MDA-MB231 invasive breast cancer cells by real-time PCR and western blotting. To investigate the loss- and gain-function of NFI-C, we determined whether NFI-C regulated KLF4 expression by real-time PCR, western blotting, and promoter assay. To understand the biological functions of NFI-C, we observed cell invasion, migration, adhesion in human tumor cells by transwell assay, wound healing assay, quantitative RT-PCR, cell adhesion assay, western blotting, and immunohistochemistry. RESULTS: We identified the downstream factors of NFI-C, such as KLF4 and E-cadherin, which play roles in EMT. NFI-C is expressed in normal mammary gland or noninvasive breast cancer cells with epithelial characteristics. NFI-C overexpression induced expression of KLF4 and E-cadherin, but not Slug, in breast cancer cells. NFI-C bound directly to the KLF4 promoter and stimulated KLF4 transcriptional activity, thereby regulating E-cadherin expression during tumorigenesis. Cells overexpressing NFI-C maintained their epithelial differentiation status, which could drive mesenchymal-epithelial transition (MET) via the NFI-C-KLF4-E-cadherin axis in breast cancer cells. Consequently, NFI-C suppressed EMT, migration, and invasion in breast cancer cells. CONCLUSIONS: Our study reveals a novel signaling pathway that is important during breast cancer tumorigenesis: the NFI-C-KLF4-E-cadherin pathway. The results indicate the important role of NFI-C in regulating KLF4 during tumorigenesis.


Assuntos
Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição NFI/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição NFI/genética , Transdução de Sinais , Transcrição Gênica
19.
Cell Biochem Funct ; 33(7): 451-61, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26358398

RESUMO

Odontogenic ameloblast-associated protein (ODAM) contributes to cell adhesion. In human cancer, ODAM is down-regulated, and the overexpression of ODAM results in a favourable prognosis; however, the molecular mechanisms underlying ODAM-mediated inhibition of cancer invasion and metastasis remain unclear. Here, we identify a critical role for ODAM in inducing cancer cell adhesion. ODAM induced RhoA activity and the expression of downstream factors, including Rho-associated kinase (ROCK). ODAM-mediated RhoA signalling resulted in actin filament rearrangement by activating PTEN and inhibiting the phosphorylation of AKT. When ODAM is overexpressed in MCF7 breast cancer cells and AGS gastric cancer cells that activate RhoA at high levels, it decreases motility, increases adhesion and inhibits the metastasis of MCF7 cells. Conversely, depletion of ODAM in cancer cells inhibits Rho GTPase activation, resulting in increased cancer migration and invasion. These results suggest that ODAM expression in cells maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells. SIGNIFICANCE Breast cancer represents the first most frequent cancer, and the ratio of mortality is high in women. Of utmost importance for reducing risk by breast cancer are their anti-invasion mechanisms, particularly in the non-invasive cancer cells because metastasis is the principal cause of death among cancer patients. ODAM induced RhoA activity. ODAM-mediated RhoA signalling resulted in actin filament rearrangement, increased cell adhesion and inhibited the migration/invasion of MCF7 cells. These results suggest that ODAM expression maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells.


Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Adesão Celular , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Adenocarcinoma/metabolismo , Amiloide , Animais , Neoplasias da Mama/metabolismo , Carcinogênese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Proteínas de Neoplasias , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
20.
Biochem Biophys Res Commun ; 446(4): 894-900, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24642258

RESUMO

MicroRNAs (miRNAs) regulate cell differentiation by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontogenic differentiation is largely unknown. In this present study, we observed that the expression of miR-663 increased significantly during differentiation of MDPC-23 cells to odontoblasts. Furthermore, up-regulation of miR-663 expression promoted odontogenic differentiation and accelerated mineralization without proliferation in MDPC-23 cells. In addition, target gene prediction for miR-663 revealed that the mRNA of the adenomatous polyposis coli (APC) gene, which is associated with the Wnt/ß-catenin signaling pathway, has a miR-663 binding site in its 3'-untranslated region (3'UTR). Furthermore, APC expressional was suppressed significantly by miR-663, and this down-regulation of APC expression triggered activation of Wnt/ß-catenin signaling through accumulation of ß-catenin in the nucleus. Taken together, these findings suggest that miR-663 promotes differentiation of MDPC-23 cells to odontoblasts by targeting APC-mediated activation of Wnt/ß-catenin signaling. Therefore, miR-663 can be considered a critical regulator of odontoblast differentiation and can be utilized for developing miRNA-based therapeutic agents.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Regulação para Baixo , Genes APC , MicroRNAs/metabolismo , Odontogênese , Via de Sinalização Wnt , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
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