RESUMO
Toll-like receptors (TLRs) make a crucial contribution to the innate immune response. TLR5 was expressed in embryoid body derived from mouse embryonic stem cells (mESCs) and ßIII-tubulin-positive cells under all-trans retinoic acid-treated condition. TLR5 was upregulated during neural differentiation from mESCs and augmented the neural differentiation of mESCs via nuclear factor-κB and interleukin 6/CREB pathways. Besides, TLR5 was expressed in SOX2- or doublecortin-positive cells in the subgranular zone of the hippocampal dentate gyrus where adult neurogenesis occurs. TLR5 inhibited the proliferation of adult hippocampal neural stem cells (NSCs) by regulating the cell cycle and facilitated the neural differentiation from the adult hippocampal NSCs via JNK pathway. Also, TLR5 deficiency impaired fear memory performance in mice. Our data suggest that TLR5 is a crucial modulator of neurogenesis from mESCs and adult hippocampal NSCs in mice and represents a new therapeutic target in neurological disorders related to cognitive function.
Assuntos
Células-Tronco Neurais , Receptor 5 Toll-Like , Animais , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Hipocampo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Receptor 5 Toll-Like/metabolismoRESUMO
There has been increasing interest in adjunctive use of anti-inflammatory drugs to control periodontitis. This study was performed to examine the effects of pirfenidone (PFD) on alveolar bone loss in ligature-induced periodontitis in mice and identify the relevant mechanisms. Experimental periodontitis was established by ligating the unilateral maxillary second molar for 7 days in mice (n = 8 per group), and PFD was administered daily via intraperitoneal injection. The micro-computed tomography and histology analyses were performed to determine changes in the alveolar bone following the PFD administration. For in vitro analysis, bone marrow macrophages (BMMs) were isolated from mice and cultured with PFD in the presence of RANKL or LPS. The effectiveness of PFD on osteoclastogenesis, inflammatory cytokine expression, and NF-κB activation was determined with RT-PCR, Western blot, and immunofluorescence analyses. PFD treatment significantly inhibited the ligature-induced alveolar bone loss, with decreases in TRAP-positive osteoclasts and expression of inflammatory cytokines in mice. In cultured BMM cells, PFD also inhibited RANKL-induced osteoclast differentiation and LPS-induced proinflammatory cytokine (IL-1ß, IL-6, TNF-a) expression via suppressing the NF-κB signal pathway. These results suggest that PFD can suppress periodontitis progression by inhibiting osteoclastogenesis and inflammatory cytokine production via inhibiting the NF-κB signal pathway, and it may be a promising candidate for controlling periodontitis.
Assuntos
Perda do Osso Alveolar , Periodontite , Camundongos , Animais , NF-kappa B/metabolismo , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/metabolismo , Microtomografia por Raio-X , Lipopolissacarídeos/farmacologia , Transdução de Sinais , Osteoclastos/metabolismo , Periodontite/tratamento farmacológico , Periodontite/etiologia , Periodontite/metabolismo , Citocinas/metabolismo , Ligante RANK/metabolismoRESUMO
AIM: To study the role of sclerostin in periodontal ligament (PDL) as a homeostatic regulator in biophysical-force-induced tooth movement (BFTM). MATERIALS AND METHODS: BFTM was performed in rats, followed by microarray, immunofluorescence, in situ hybridization, and real-time polymerase chain reaction for the detection and identification of the molecules. The periodontal space was analysed via micro-computed tomography. Effects on osteoclastogenesis and bone resorption were evaluated in the bone-marrow-derived cells in mice. In vitro human PDL cells were subjected to biophysical forces. RESULTS: In the absence of BFTM, sclerostin was hardly detected in the periodontium except in the PDL and alveolar bone in the furcation region and apex of the molar roots. However, sclerostin was up-regulated in the PDL in vivo by adaptable force, which induced typical transfiguration without changes in periodontal space as well as in vitro PDL cells under compression and tension. In contrast, the sclerostin level was unaffected by heavy force, which caused severe degeneration of the PDL and narrowed periodontal space. Sclerostin inhibited osteoclastogenesis and bone resorption, which corroborates the accelerated tooth movement by the heavy force. CONCLUSIONS: Sclerostin in PDL may be a key homeostatic molecule in the periodontium and a biological target for the therapeutic modulation of BFTM.
Assuntos
Reabsorção Óssea , Ligamento Periodontal , Animais , Humanos , Camundongos , Ligante RANK , Ratos , Técnicas de Movimentação Dentária , Microtomografia por Raio-XRESUMO
AIM: We aimed to identify a key molecule that maintains periodontal tissue homeostasis during biophysical force-induced tooth movement (BTM) by orchestrating alveolar bone (AB) remodelling. MATERIALS AND METHODS: Differential display-PCR was performed to identify key molecules for BTM in rats. To investigate the localization and expression of the identified molecules, immunofluorescence, real-time RT-PCR and Western blotting were performed in rats and human periodontal ligament (PDL) cells. Functional test and micro-CT analysis were performed to examine the in vivo effects of the identified molecules on BTM. RESULTS: Secretory leucocyte peptidase inhibitor (SLPI) in the PDL was revealed as a key molecule for BTM-induced AB remodelling. SLPI was enhanced in the PDL under both compression and tension, and downregulated by an adenyl cyclases inhibitor. SLPI induced osteoblastogenic genes including runt-related transcription factor 2 (Runx2) and synergistically augmented tension-induced Runx2 expression. SLPI augmented mineralization in PDL cells. SLPI induced osteoclastogenic genes including receptor activator of nuclear factor kappa-Β ligand (RANKL) and synergistically augmented the compression-induced RANKL and macrophage colony-stimulating factor (MCSF) expression. Finally, the in vivo SLPI application into the AB significantly augmented BTM. CONCLUSIONS: SLPI or its inhibitors might serve as a biological target molecule for therapeutic interventions to modulate BTM.
Assuntos
Ligamento Periodontal , Ligante RANK , Animais , Células Cultivadas , Ratos , Inibidor Secretado de Peptidases Leucocitárias , Técnicas de Movimentação DentáriaRESUMO
Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/ western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.
RESUMO
Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation.
Assuntos
Ameloblastos/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dente Molar/metabolismo , Proteínas Nucleares/metabolismo , Fosfatase Alcalina/metabolismo , Amelogenina/genética , Amelogenina/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA , Transdução de Sinais , Fatores de Tempo , Calcificação de Dente , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Human mesenchymal stem cells (hMSCs), characterized by rapid in vitro expandability and multi-differentiation potential, have been widely used in the clinical field of tissue engineering. Recent studies have shown that various signaling networks are involved in the growth and differentiation of hMSCs. Although Wnts and their downstream signaling components have been implicated in the regulation of hMSCs, the role of Wnt signaling in hMSC self-renewal is still controversial. Here, it was observed that activation of endogenous canonical Wnt signaling with LiCl, which decreased ß-catenin phosphorylation, leads to a decrease in hMSC proliferation. The fact that this growth arrest is not linked to apoptosis was verified by annexin V-FITC/propidium iodide assay. It was associated with sealing off of the cells in the G1 phase of the cell cycle accompanied by changes in expression of cell cycle-associated genes such as cyclin A and D. In addition, activation of Wnt signaling during hMSC proliferation seemed to reduce their clonogenic potential. On the contrary, Wnt signaling activation during hMSC proliferation had little effect on the osteogenic differentiation capability of cells. These findings show that canonical Wnt signaling is a critical regulator of hMSC proliferation and clonogenicity.
Assuntos
Cloreto de Lítio/farmacologia , Células-Tronco Mesenquimais/citologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/ß activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Ácido Ascórbico/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , NecroseRESUMO
Amelogenin, an enamel matrix protein has been considered to be exclusively expressed by ameloblasts during odontogenesis. However, burgeoning evidence indicates that amelogenin is also expressed in non-mineralizing tissues. Under the hypothesis that amelogenin may be a functional molecule in developing hair follicles which share developmental features with odontogenesis, this study for the first time elucidated the presence and functional changes of amelogenin and its receptors during rat hair follicle development. Amelogenin was specifically localized in the outer epithelial root sheath of hair follicles. Its expression appeared in the deeper portion of hair follicles, i.e. the bulbar and suprabulbar regions rather than the superficial region. Lamp-1, an amelogenin receptor, was localized in either follicular cells or outer epithelial sheath cells, reflecting functional changes during development. The expression of amelogenin splicing variants increased in a time-dependent manner during postnatal development of hair follicles. Amelogenin expression was increased by treatment with cyclosporin A, which is an inducer of anagen in the hair follicle, whereas the level of Lamp-1 and -2 was decreased by cyclosporin A treatment. These results suggest that amelogenin may be a functional molecule involved in the development of the hair follicle rather than an inert hair shaft matrix protein.
Assuntos
Amelogenina/metabolismo , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Folículo Piloso/metabolismo , Amelogenina/fisiologia , Animais , Células Epiteliais/metabolismo , Folículo Piloso/efeitos dos fármacos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Modelos Animais , Organogênese/fisiologia , Isoformas de Proteínas/metabolismo , RatosRESUMO
The present study is aimed at investigating the effects of the exogenous estrogen 17ß-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Estrogênios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Biomarcadores/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentina/citologia , Dentina/efeitos dos fármacos , Dentina/metabolismo , Estradiol/química , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/químicaRESUMO
Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.
RESUMO
The dental pulp is a highly innervated tissue transmitting pain-related sensations in the tooth. Consequently, understanding the intricacies of its innervation mechanism in odontogenesis is crucial for gaining insights into dental pain and developing dental pain-modulating agents. This study examined neuroregulatory molecules such as neurotrophic factors (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], neurotrophin-4 [NTF-4], and neurturin [NRTN]) and neuroinhibitory factors (slit2, ephrin isoforms and netrin-1) in developing rat teeth with follicles. NGF, BDNF and NRTN transcriptions showed time-dependent upregulation, particularly during the root formation stage. In contrast, NTF-4 mRNA was highly expressed at the cap stage, but became downregulated over time. Slit2 and ephrin-B2 expression was distinct at the cap stage and then downregulated in a time-dependent manner. Ephrin-A5 and netrin-1 expression did not significantly change. Immunofluorescence analysis revealed a robust expression of both ephrin-B2 and slit2 in the outer and inner dental epithelia of the enamel organ, a non-neurogenic tissue, during the cap stage of 3rd molar germs. In contrast, BDNF was predominantly localized in dental papilla cells and odontoblasts during the root formation stage. These results suggest that neuroregulatory molecules, such as BDNF, slit2 and ephrin-B2, may be important in identifying therapeutic targets for modulating dental pulp pain.
Assuntos
Polpa Dentária , Animais , Polpa Dentária/inervação , Ratos , Odontogênese/fisiologia , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Ratos Sprague-Dawley , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , MasculinoRESUMO
Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.
Assuntos
Amelogênese , Odontogênese , Animais , Ratos , Ameloblastos/metabolismo , Amelogênese/genética , Dente Molar , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/genética , Odontogênese/genética , Proteínas/metabolismoRESUMO
Prolonged endoplasmic reticulum (ER) stress contributes to cell apoptosis and interferes with bone homeostasis. Although photobiomodulation (PBM) might be used for ER stress-induced diseases, the role of PBM in relieving cell apoptosis remains unknown. During ER stress, glycogen synthase kinase-3ß (GSK-3ß) is critical; however, its functions in PBM remain uncertain. Thus, this study aimed to investigate the role of GSK-3ß in 625 nm light-emitting diode irradiation (LEDI) relieving tunicamycin (TM)-induced apoptosis. Based on the results, pre-625 nm LEDI (Pre-IR) phosphorylated GSK-3ß via ROS production. Compared with the TM group, Pre-IR + TM group reduced the phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF-2α) and B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax)/Bcl-2 ratio through regulating GSK-3ß. Furthermore, a similar tendency was observed between Pre-IR + TM and Pre-LiCl+TM groups in preventing TM-induced early and late apoptosis. In summary, this study suggests that the Pre-IR treatment in TM-induced ER stress is beneficial for preventing cell apoptosis via GSK-3ß phosphorylation.
RESUMO
Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/fisiologia , Osteoblastos/metabolismo , Osteocalcina/biossíntese , Transcrição Gênica/fisiologia , Fator 6 Ativador da Transcrição/genética , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Osteoblastos/citologia , Osteocalcina/genética , Elementos de Resposta/fisiologiaRESUMO
Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.
Assuntos
Fator II de Transcrição COUP/metabolismo , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Osteoblastos/metabolismo , Animais , Fator II de Transcrição COUP/genética , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Metaloproteinase 2 da Matriz/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ligação ProteicaRESUMO
Embryonic hypoxia/ischemia is a major cause of a poor fetal outcome and future neonatal and adult handicaps. However, biochemical cellular events in mouse embryonic stem (mES) cells during hypoxia remains unclear. This study investigated the underlying mechanism of apoptosis in mES cells under CoCl2-induced hypoxic/ischemic conditions. CoCl2 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the accumulation of reactive oxygen species in mES cells. The CoCl2-treated mES cells showed a decrease in cell viability as well as typical apoptotic changes, cell shrinkage, chromatin condensation, and nuclear fragmentation and an extended G2/M phase of the cell cycle. CoCl2 augmented the release of cytochrome c into the cytosol from the mitochondria with a concomitant loss of the mitochondrial transmembrane potential (ΔΨm) and upregulated the voltage-dependent anion channel. In addition, CoCl2-induced caspase-3, -8, and -9 activation and upregulation of p53 level, whereas downregulated Bcl-2 and Bcl-xL, a member of the anti-apoptotic Bcl-2 family in mES cells. Furthermore, CoCl2 led to the upregulation of Fas and Fas-ligand, which are the death receptor assemblies, as well as the cleavage of Bid in mES cells. These results suggest that CoCl2 induces apoptosis through both mitochondria- and death receptor-mediated pathways that are regulated by the Bcl-2 family in mES cells.
Assuntos
Apoptose , Cobalto/farmacologia , Células-Tronco Embrionárias/fisiologia , Mitocôndrias/fisiologia , Receptores de Morte Celular/metabolismo , Animais , Caspases/metabolismo , Hipóxia Celular , Citocromos c/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Proteína Ligante Fas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Receptor fas/metabolismoRESUMO
The purpose of this study is to investigate the antinociceptive effects of ginsenosides on toothache. c-Fos immunoreactive (IR) neurons were examined after noxious intrapulpal stimulation (NS) by intrapulpal injection of 2 M KCl into upper and lower incisor pulps exposed by bone cutter in Sprague Dawley rats. The number of Fos-IR neurons was increased in the trigeminal subnucleus caudalis (Vc) and the transitional region between Vc and subnucleus interpolaris (Vi) by NS to tooth. The intradental NS raised arterial blood pressure (BP) and heart rate (HR). The number of Fos-IR neurons was also enhanced in thalamic ventral posteromedial nucleus (VPMN) and centrolateral nucleus (CLN) by NS to tooth. The intradental NS increased the number of Fos-IR neurons in the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN), central cardiovascular regulation centers. Ginsenosides reduced the number of c-Fos-IR increased by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Naloxone, an opioid antagonist, did not block the effect of ginsenoside on the number of Fos-IR neurons enhanced by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Ginsenosides ameliorated arterial BP and HR raised by NS to tooth and reduced the number of Fos-IR neurons increased by NS to tooth in the NTS, RVLM, hypothalamic SON, and PVN. These results suggest that ginsenosides have an antinociceptive effect on toothache through non-opioid system and attenuates BP and HR increased by NS to tooth.
RESUMO
The formation of dentin and enamel is initiated by the differentiation of odontogenic precursor cells into odontoblasts and ameloblasts, respectively. This study was performed to identify new molecules involved in the differentiation of odontogenic cells. The genes expressed differentially between the root stage (after the differentiation of odontogenic cells and dental hard-tissue formation) and the cap stage (before the differentiation of odontogenic cells and dental hard-tissue formation) were searched using differential display PCR. For the first time, synaptic vesicle protein (SV) 2b, an important transmembrane transporter of Ca(2+) -stimulated vesicle exocytosis, was identified as a differentially expressed molecule. Real-time PCR and western blotting revealed an increase in the transcriptional and translational levels of SV2b during or after the differentiation of odontogenic cells. Immunofluorescence revealed this molecule to be localized in not only fully differentiated odontoblasts but also in pre-odontoblasts before dentin matrix secretion. The expression pattern of the SV2a isoform was similar to that of the SV2b isoform, whereas the SV2c isoform showed a contrasting pattern of expression. After treatment with alendronate, an inhibitor of protein isoprenylation for the transport of secretory vesicles, the expression of SV2a and SV2b decreased, whereas that of SV2c increased. These results suggest that the SV2 isoforms are functional molecules of (pre)odontoblasts which may be involved in vesicle transport.
Assuntos
Diferenciação Celular/genética , Exocitose/genética , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Odontoblastos/metabolismo , Odontogênese/genética , Vesículas Sinápticas/metabolismo , Germe de Dente/citologia , Alendronato/metabolismo , Animais , Regulação da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Odontoblastos/citologia , Odontogênese/fisiologia , Isoformas de Proteínas/genética , Isoformas de RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Germe de Dente/metabolismoRESUMO
BACKGROUND/PURPOSE: Autophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. MATERIALS AND METHODS: Human dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. RESULTS: HDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. CONCLUSION: These findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.