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1.
Mol Cell ; 68(4): 645-658.e5, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29149593

RESUMO

Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.


Assuntos
Proteína 7 com Repetições F-Box-WD , Síndrome de Hajdu-Cheney , Mutação , Osteoporose , Proteólise , Receptor Notch2 , Animais , Linhagem Celular , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Síndrome de Hajdu-Cheney/genética , Síndrome de Hajdu-Cheney/metabolismo , Camundongos Knockout , Osteoporose/genética , Osteoporose/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Ubiquitinação/genética
2.
Eur J Oral Sci ; 131(2): e12920, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36794562

RESUMO

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.


Assuntos
Canais de Cátion TRPM , Camundongos , Ratos , Animais , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Camundongos Knockout , Esmalte Dentário/metabolismo , Ameloblastos/metabolismo , Epitélio , Amelogênese/genética , Proteínas de Transporte/metabolismo , Incisivo
3.
Lab Invest ; 101(11): 1475-1483, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34504305

RESUMO

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial-mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Ósseas/secundário , Melanoma/secundário , Neoplasias Bucais/patologia , Proteínas Smad Reguladas por Receptor/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Masculino , Melanoma/metabolismo , Camundongos , Neoplasias Bucais/metabolismo , Invasividade Neoplásica , Transdução de Sinais
4.
Cancer Sci ; 111(4): 1113-1123, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32012401

RESUMO

Programmed cell death ligands (PD-Ls) are expressed in tumor cells where they bind to programmed cell death-1, an immunocyte co-receptor, resulting in tumor cell evasion from the immune system. Chemotherapeutic drugs have been recently reported to induce the expression of PD-L, such as PD-L1, in some cancer cells. However, little is known regarding PD-L2 expression and its role in oral squamous cell carcinoma (OSCC). In this study, we examined the effect of cisplatin on the expression and regulation of PD-L2 in OSCC cell lines and analyzed malignant behavior in PD-L2-expressing cells using colony, transwell and transformation assays. In addition, we examined PD-L2 expression in the tumor tissues of OSCC patients using cytology and tissue microarray methods. In OSCC cell lines, cisplatin treatment upregulated PD-L2 expression, along with that of the drug efflux transporter ABCG2, via signal transducers and activator of transcription (STAT) 1/3 activation. Moreover, PD-L2-positive or PD-L2-overexpressing cells demonstrated upregulation in both invasion and transformation ability but not in proliferation compared with PD-L2-negative or PD-L2-silencing cells. PD-L2 expression was also observed in OSCC cells of cytology samples and tissue from OSCC patients. The intensity of PD-L2 expression was correlated with more malignant morphological features in the histological appearance and an invasive pattern. Our findings indicate that cisplatin-upregulated PD-L2 expression in OSCC via STAT1/3 activation and the expression of PD-L2 are likely to be associated with malignancy in OSCC. The PD-L2 expression in cisplatin-resistant OSCC cells may be a critical factor in prognosis of advanced OSCC patients.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Bucais/tratamento farmacológico , Proteína 2 Ligante de Morte Celular Programada 1/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Análise Serial de Tecidos
5.
Int J Mol Sci ; 22(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375370

RESUMO

Calcium (Ca2+) plays an important role in regulating the differentiation and function of osteoclasts. Calcium oscillations (Ca oscillations) are well-known phenomena in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption via calcineurin. Many modifiers are involved in the fine-tuning of Ca oscillations in osteoclasts. In addition to macrophage colony-stimulating factors (M-CSF; CSF-1) and RANKL, costimulatory signaling by immunoreceptor tyrosine-based activation motif-harboring adaptors is important for Ca oscillation generation and osteoclast differentiation. DNAX-activating protein of 12 kD is always necessary for osteoclastogenesis. In contrast, Fc receptor gamma (FcRγ) works as a key controller of osteoclastogenesis especially in inflammatory situation. FcRγ has a cofactor in fine-tuning of Ca oscillations. Some calcium channels and transporters are also necessary for Ca oscillations. Transient receptor potential (TRP) channels are well-known environmental sensors, and TRP vanilloid channels play an important role in osteoclastogenesis. Lysosomes, mitochondria, and endoplasmic reticulum (ER) are typical organelles for intracellular Ca2+ storage. Ryanodine receptor, inositol trisphosphate receptor, and sarco/endoplasmic reticulum Ca2+ ATPase on the ER modulate Ca oscillations. Research on Ca oscillations in osteoclasts has still many problems. Surprisingly, there is no objective definition of Ca oscillations. Causality between Ca oscillations and osteoclast differentiation and/or function remains to be examined.


Assuntos
Reabsorção Óssea/patologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Diferenciação Celular , Osteoclastos/citologia , Osteogênese , Animais , Reabsorção Óssea/metabolismo , Humanos , Osteoclastos/metabolismo
6.
Cell Mol Life Sci ; 75(1): 33-48, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28791425

RESUMO

Rab44 is an atypical Rab GTPase that contains some additional domains such as the EF-hand and coiled-coil domains as well as Rab-GTPase domain. Although Rab44 genes have been found in mammalian genomes, no studies concerning Rab44 have been reported yet. Here, we identified Rab44 as an upregulated protein during osteoclast differentiation. Knockdown of Rab44 by small interfering RNA promotes RANKL-induced osteoclast differentiation of the murine monocytic cell line, RAW-D or of bone marrow-derived macrophages (BMMs). In contrast, overexpression of Rab44 prevents osteoclast differentiation. Rab44 was localized in the Golgi complex and lysosomes, and Rab44 overexpression caused an enlargement of early endosomes. A series of deletion mutant studies of Rab44 showed that the coiled-coil domain and lipidation sites of Rab44 is important for regulation of osteoclast differentiation. Mechanistically, Rab44 affects nuclear factor of activated T-cells c1 (NFATc1) signaling in RANKL-stimulated macrophages. Moreover, Rab44 depletion caused an elevation in intracellular Ca2+ transients upon RANKL stimulation, and particularly regulated lysosomal Ca2+ influx. Taken together, these results suggest that Rab44 negatively regulates osteoclast differentiation by modulating intracellular Ca2+ levels followed by NFATc1 activation.


Assuntos
Cálcio/metabolismo , Diferenciação Celular , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Complexo de Golgi/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Osteoclastos/citologia , Ligante RANK/farmacologia , Células RAW 264.7 , Interferência de RNA , Proteínas rab de Ligação ao GTP/genética
7.
Lipids Health Dis ; 17(1): 132, 2018 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-29859535

RESUMO

BACKGROUND: Lectin-like oxidized low-density-lipoprotein receptor 1 (Lox-1) is the receptor for oxidized low-density lipoprotein (oxLDL), a mediator in dyslipidemia. Toll-like receptor (TLR)-2 and - 4 are receptors of lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major pathogen of chronic periodontitis. Although some reports have demonstrated that periodontitis has an adverse effect on dyslipidemia, little is clear that the mechanism is explained the effects of dyslipidemia on osteoclastogenesis. We have hypothesized that osteoclast oxLDL has directly effect on osteoclasts (OCs), and therefore alveolar bone loss on periodontitis may be increased by dyslipidemia. The present study aimed to elucidate the effect of Lox-1 on osteoclastogenesis associated with TLRs in vitro. METHODS: Mouse bone marrow cells (BMCs) were stimulated with macrophage colony-stimulating factor into bone marrow macrophages (BMMs). The cells were also stimulated with synthetic ligands for TLR2 (Pam3CSK4) or TLR4 (Lipid A), with or without receptor activator of nuclear factor kappa-B ligand (RANKL), and assessed for osteoclastogenesis by tartrate-resistant acid phosphatase (TRAP) staining, immunostaining, western blotting, flow activated cell sorting (FACS) analysis, real-time polymerase chain reaction (PCR), and reverse transcription PCR. RESULTS: Lox-1 expression was significantly upregulated by Pam3CSK4 and Lipid A in BMCs (p < 0.05), but not in BMMs. FACS analysis identified that Pam3CSK4 upregulated RANK and Lox-1 expression in BMCs. TRAP-positive cells were not increased by stimulation with Pam3CSK4 alone, but were increased by stimulation with combination combined Pam3CSK and oxLDL. Expression of both Lox-1 and myeloid differentiation factor 88 (MyD88), an essential adaptor protein in the TLR signaling pathway, were suppressed by inhibitors of TLR2, TLR4 and mitogen-activated protein kinase (MAPK). CONCLUSIONS: This study supports that osteoclastogenesis is promoted under the coexistence of oxLDL by TLR2-induced upregulation of Lox-1 in BMCs. This indicates that periodontitis could worsen with progression of dyslipidemia.


Assuntos
Células da Medula Óssea/metabolismo , Osteogênese , Receptores Depuradores Classe E/fisiologia , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular , Lipoproteínas LDL , Macrófagos , Masculino , Camundongos , Periodontite , Receptores Depuradores Classe E/metabolismo
8.
Biochem Biophys Res Commun ; 468(4): 622-8, 2015 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-26551467

RESUMO

We previously reported the promotion of bone regeneration in calvarial defects of both normal and ovariectomy-induced osteoporotic rats, with the use of biodegradable DNA/protamine scaffold. However, the method by which this DNA-containing scaffold promotes bone formation is still not understood. We hypothesize that the salmon DNA, from which this scaffold is derived, has an osteoinductive effect on pre-osteoblasts and osteoblasts. We examined the effects of salmon DNA on osteoblastic differentiation and calcification in MC3T3-E1 cells, mouse osteoblasts, in vitro and bone regeneration in a calvarial defect model of aged mouse in vivo. The salmon DNA fragments (300 bps) upregulated the expression of the osteogenic markers, such as alkaline phosphatase, Runx2, and osterix (Osx) in MC3T3E1 cells compared with incubation with osteogenic induction medium alone. Measurement of phosphate ion concentrations in cultures showed that the DNA scaffold degraded phosphate ions were released to the cell cultures. Interestingly, we found that the inclusion of DNA in osteoblastic cell cultures upregulated the expression of sodium-dependent phosphate (NaPi) cotransporters, SLC20A1 and SLC34A2, in MC3T3-E1 cells in a time dependent manner. Furthermore, the inclusion of DNA in cell cultures increased the transcellular permeability of phosphate. Conversely, the incubation of phosphonoformic acid, an inhibitor of NaPi cotransporters, attenuated the DNA-induced expression and activation of SLC20A1 and SLC34A2 in MC3T3-E1 cells, resulting in suppression of the osteogenic markers. The implantation of a salmon DNA scaffold disk promoted bone regeneration using calvarial defect models in 30-week-old mice. Our results indicate that the phosphate released from salmon DNA upregulated the expression and activation of NaPi cotransporters, resulting in the promotion of bone regeneration.


Assuntos
DNA/genética , Osteoblastos/citologia , Osteogênese/genética , Fraturas Cranianas/terapia , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Alicerces Teciduais , Células 3T3 , Animais , Diferenciação Celular/genética , DNA/administração & dosagem , Implantes de Medicamento/administração & dosagem , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Osteoblastos/fisiologia , Radiografia , Salmão/genética , Fraturas Cranianas/diagnóstico por imagem , Fraturas Cranianas/fisiopatologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Resultado do Tratamento
9.
Biochem Biophys Res Commun ; 452(3): 622-8, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25181340

RESUMO

Reactive oxygen species (ROS) can cause severe damage to DNA, proteins and lipids in normal cells, contributing to carcinogenesis and various pathological conditions. While cellular senescence arrests the early phase of cell cycle without any detectable telomere loss or dysfunction. ROS is reported to contribute to induction of cellular senescence, as evidence by its premature onset upon treatment with antioxidants or inhibitors of cellular oxidant scavengers. Although cellular senescence is known to be implicated in tumor suppression, it remains unknown whether ROS initially contributed to be cellular senescence in normal human epidermal keratinocytes (NHEK) and their malignant counterparts. To clarify whether ROS induce cellular senescence in NHEKs, we examined the effect of hydrogen peroxide (H2O2) on the expression of cellular senescence-associated molecules in NHEKs, compared to in squamous carcinoma cells (SCCs). Hydrogen peroxide increased the number of cells positive in senescence associated-ß-galactosidase (SA-ß-Gal) activity in NHEKs, but not SCCs. The expression of cyclin-dependent kinase (CDK) inhibitors, especially p16(INK4a) was upregulated in NHEKs treated with H2O2. Interestingly, H2O2 suppressed the methylation of p16(INK4a), promoter region in NHEKs, but not in SCCs. Hydrogen peroxide also suppressed the expression of phosphorylated Rb and CDK4, resulting in arrest in G0/G1 phase in NHEKs, but not SCCs. Our results indicate that the ROS-induced cellular senescence in NHEKs was caused by the upregulation p16(INK4a) through demethylation in its promoter region, which is not detected in SCCs, suggesting that ROS-induced cellular senescence contributes to tumor suppression of NHEKs.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Epiderme/metabolismo , Epigênese Genética , Queratinócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Senescência Celular , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Células Epidérmicas , Epiderme/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/agonistas , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
11.
Exp Cell Res ; 318(15): 1926-32, 2012 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-22664326

RESUMO

Epithelial to mesenchymal transition (EMT) plays an important role in tumor progression, and is an early step in carcinogenesis. Although reactive oxygen species (ROS) are known to be implicated in EMT in many tumor cell types, its exact role in EMT initiation in normal human cells, especially epidermal keratinocytes (NHEKs), remains unknown. To clarify whether ROS induce EMT in NHEKs, and to establish how ROS regulate EMT, we examined the effect of hydrogen peroxide (H(2)O(2)) on the expression of molecules involved in EMT and cell morphology in NHEKs. H(2)O(2) altered the expression of EMT biomarkers, including downregulation of epithelial cadherin and upregulation of α-smooth muscle actin, through a transcriptional modulator, Snail1. H(2)O(2) also induced epithelial to fibroblast-like morphological changes, together with upregulation of EMT biomarkers, and promoted phosphorylation of ERK1/2 and JNK in a time-dependent manner. Interestingly, H(2)O(2) stimulated the expression and secretion of TGF-ß1 in NHEKs. Exogenous TGF-ß1 also induced the expression of EMT biomarkers. In contrast, neutralizing antibody anti-TGF-ß1 antibody or inhibitor of TGF-ß receptor type I suppressed the expression of EMT biomarkers. Our results suggest that ROS stimulated TGF-ß1 secretion and MAPK activation, resulting in EMT initiation in NHEKs.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Peróxido de Hidrogênio/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Actinas/genética , Actinas/metabolismo , Sequência de Bases , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Células Cultivadas , Primers do DNA/genética , Transição Epitelial-Mesenquimal/genética , Humanos , Queratinócitos/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos
12.
Eur J Oral Sci ; 121(6): 538-44, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24206072

RESUMO

Transient receptor potential type A1 (TRPA1) is reported to be a Ca(2+) -permeable channel and is activated by cold temperatures and mechanical stimuli in the hair cells and in dorsal root ganglion. Using a DNA microarray, we found that TRPA1 was significantly up-regulated in human periodontal ligament (hPDL) cells 2 d after intermittent mechanical stimulation (iMS) loading compared with unloaded cells. Although hPDL cells are known to respond to mechanical stimulation induced by occlusal force, little is known about the expression and functional role of TRPA1 in these cells. Therefore, we investigated the effects of iMS on TRPA1 expression and its signaling pathway in hPDL cells. Intermittent mechanical stimulation loading up-regulated TRPA1 expression in hPDL cells in a time-dependent manner, but had no effect on other mechanoreceptors. Furthermore, iMS significantly increased the phosphorylation of mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, and the expression of C-C chemokine ligand 2 (CCL2). Transient receptor potential type A1 agonists also increased MAPK phosphorylation and the intracellular Ca(2+) concentration. By contrast, inhibition or silencing of TRPA1 partially suppressed iMS-induced MAPK phosphorylation. In summary, iMS during occlusion activates TRPA1 and MAPK signaling in periodontal ligament tissues, suggesting that TRPA1 regulates the mechanosensitivity of occlusal force via activation of MAPKs in hPDL cells.


Assuntos
Força de Mordida , Canais de Cálcio/metabolismo , Mecanorreceptores/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ligamento Periodontal/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Análise de Variância , Canais de Cálcio/genética , Células Cultivadas , Expressão Gênica , Inativação Gênica , Humanos , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ligamento Periodontal/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/genética , Regulação para Cima
13.
Sci Rep ; 13(1): 18829, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37914726

RESUMO

Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20-/- mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20-/- and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20-/- mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.


Assuntos
Ameloblastos , Metaloproteinase 20 da Matriz , Camundongos , Animais , Ameloblastos/metabolismo , Metaloproteinase 20 da Matriz/metabolismo , Camundongos Transgênicos , Caderinas/metabolismo , Expressão Gênica
14.
Bone ; 166: 116579, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36210025

RESUMO

Transient receptor potential melastatin-subfamily member 7 (TRPM7) is a bifunctional protein containing a kinase fused to an ion channel permeated with cations, including Ca2+ and Mg2+. Trpm7-null mice show embryonic lethality. Paired related homeobox 1 (Prx1) is expressed in undifferentiated mesenchymal cells such as the progenitor cells of both chondrocytes and osteoblasts involved in limb skeleton formation. Prx1-Cre-dependent Trpm7 mesenchymal-deleted mice were generated to examine the role of TRPM7 in bone development. We found that Prx1-Cre;Trpm7fl/fl mice had shortened bones and impaired trabecular bone formation. Trabecular bone parameters, such as the bone volume (BV/TV), and trabecular number (Tb.N), were decreased in Prx1-Cre;Trpm7fl/fl mice. The cortical bone parameters of cortical bone area (Ct.Ar) and cortical bone thickness (Ct.Th) were also down-regulated in these mice. The bone formation rate in Prx1-Cre;Trpm7fl/fl mice was unchanged, but the hypertrophic area and cell size of the zone were smaller, and the expression of Col2a1, Col10a1 and Mmp13 was downregulated compared with control mice. These findings suggest impaired chondrogenesis in Prx1-Cre;Trpm7fl/fl mice compared to control mice. The receptor activator of nuclear factor-kappa B ligand (RANKL) expression was increased, and RANKL-positive cells and osteoclasts were markedly accumulated in the boundary region between the growth plate and trabecular bone. In contrast, TRPM7 KR mice, which are kinase-dead mutants in which the TRPM7 ion channel function has not been altered, showed no marked differences in trabecular or cortical bone parameters compared to wild-type mice. These findings suggest that TRPM7 is critical as a cation channel rather than as a kinase in bone development via the regulation of chondrogenesis.


Assuntos
Células-Tronco Mesenquimais , Canais de Cátion TRPM , Camundongos , Animais , Osteogênese , Condrogênese , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Células-Tronco Mesenquimais/metabolismo , Lâmina de Crescimento/metabolismo
15.
Clin Calcium ; 22(1): 19-26, 2012 Jan.
Artigo em Japonês | MEDLINE | ID: mdl-22201095

RESUMO

Although it is believed that odontoclasts, which mediated root resorption of deciduous teeth, possess common properties to osteoclasts, these regulatory mechanisms differ from osteoclastic bone resorption. It is well established that calcitonin receptor is an important osteoclast marker and that calcitonin is a potent inhibitory hormone of osteoclastic bone resorption. However, the presence and function of calcitonin receptors in human odontoclasts are still controversial. We summarize the physiological properties and differentiation mechanisms of odontoclasts, and the effects of calcitonin on root resorption, including our recent results using human odontoclasts and periodontal ligament cells freshly isolated from deciduous tooth roots.


Assuntos
Calcitonina/fisiologia , Osteoclastos/fisiologia , Dente Decíduo/citologia , Animais , Diferenciação Celular , Humanos , Osteoclastos/metabolismo , Ligamento Periodontal/citologia , Ligante RANK/fisiologia , Receptores da Calcitonina/metabolismo , Receptores da Calcitonina/fisiologia , Reabsorção da Raiz , Transdução de Sinais/fisiologia
16.
Bone ; 154: 116210, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34592494

RESUMO

Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130CasΔepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130CasΔepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130CasΔepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130CasΔepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca2++K+-exchanger in the maturation stage were reduced in p130CasΔepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).


Assuntos
Amelogênese , Proteína Substrato Associada a Crk/metabolismo , Proteínas do Esmalte Dentário , Ameloblastos/metabolismo , Animais , Proteínas do Esmalte Dentário/metabolismo , Células Epiteliais/metabolismo , Camundongos , Microtomografia por Raio-X
17.
Nat Med ; 10(6): 617-24, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15156202

RESUMO

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-kappa B (NF-kappa B) has a crucial role in osteoclast differentiation, and blocking NF-kappa B is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the I kappa B-kinase complex, a crucial component of signal transduction pathways to NF-kappa B. The peptide inhibited RANKL-stimulated NF-kappa B activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-alpha and interleukin-1 beta, abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-kappa B activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption.


Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Proteínas I-kappa B/antagonistas & inibidores , Inflamação/metabolismo , NF-kappa B/antagonistas & inibidores , Osteoclastos/fisiologia , Peptídeos/metabolismo , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Reabsorção Óssea/imunologia , Osso e Ossos/citologia , Osso e Ossos/patologia , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Inflamação/imunologia , Interleucina-1/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , NF-kappa B/metabolismo , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
18.
Bone ; 150: 116010, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34020080

RESUMO

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration.


Assuntos
Dentina , Odontoblastos , Diferenciação Celular , Polpa Dentária , Células-Tronco , Cicatrização
19.
Pflugers Arch ; 458(6): 1049-59, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19543743

RESUMO

ClC7 Cl(-) channels (Clcn7) are crucial for osteoclastic bone resorption and have heterozygous mutation in autosomal osteopetrosis type II (ADO II) patients. Although extracellular acidification is known to induce ClC7 Cl(-) currents in Clcn7-transfected oocytes, other characteristics of this acid-induced Cl(-) current, as well as the effects of mutant Clcn7 in ADO II, remain to be determined. The present study showed that extracellular acidification evoked outward Cl(-) currents in mouse osteoclasts. Expression of wild-type human Clcn7 in HEK293 cells also induced a significant increase in acid-activated Cl(-) currents. These acid-activated Cl(-) currents were independent of intracellular acidification and [Ca(2+)]( i ) increase. HEK293 cells with the Clcn7 mutation associated with ADO II at G215R did not display these Cl(-) currents. These results suggest that osteoclastic ClC7 Cl(-) channels are activated under extracellar acidification and suppressed in Clcn7 mutant associated with ADO II during bone resorption.


Assuntos
Canais de Cloreto/fisiologia , Osteoclastos/metabolismo , Osteopetrose/metabolismo , Animais , Reabsorção Óssea/fisiopatologia , Linhagem Celular , Células Cultivadas , Canais de Cloreto/genética , Clonagem Molecular , Humanos , Camundongos , Osteopetrose/genética
20.
J Biomed Mater Res B Appl Biomater ; 107(1): 122-128, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29521019

RESUMO

Scaffolds implanted into bone defect sites must achieve optimal biodegradation rates while appropriately filling the void as new bone formation progresses. We recently developed a unique biomaterial consisting of salmon deoxyribose nucleic acid (DNA) and protamine, which can be used as an osteoconductive scaffold for tissue engineering. The aim of the present study was to elucidate how the degradation rate of the scaffold affects bone regeneration. We examined the relationships between the degradation rate of salmon DNA scaffolds and new bone formation using a rat skin flank subcutaneous model and rat calvarial defect model. The degradation rates of the scaffolds were proportional to the durations of pretreatment with ultraviolet (UV) light irradiation. The biodegradation rates of the scaffolds were also dependent on the duration of UV irradiation, as tested a subcutaneous tissue implantation. Scaffolds irradiated with UV light for 0.5 h maintained gradual biodegradation of phosphate compared with scaffolds irradiated for 0 or 3 h. In the calvarial defect model, we found that new bone formation was higher in rats treated with scaffolds irradiated with UV light for 0.5 h compared with those irradiated with UV light for 0 or 3.0 h. The present results suggest that bioengineering of scaffolds for biodegradation is important to regenerate bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 122-128, 2019.


Assuntos
Implantes Absorvíveis , Regeneração Óssea , DNA/química , Crânio , Alicerces Teciduais/química , Animais , Masculino , Protaminas/química , Ratos , Ratos Sprague-Dawley , Salmão , Crânio/lesões , Crânio/metabolismo , Crânio/patologia , Raios Ultravioleta
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