RESUMEN
Stem cells are maintained in specific niches that strictly regulate their proliferation and differentiation for proper tissue regeneration and renewal. Molecular oxygen (O2) is an important component of the niche microenvironment, but little is known about how O2 governs epithelial stem cell (ESC) behavior. Here, we demonstrate that O2 plays a crucial role in regulating the proliferation of ESCs using the continuously growing mouse incisors. We have revealed that slow-cycling cells in the niche are maintained under relatively hypoxic conditions compared with actively proliferating cells, based on the blood vessel distribution and metabolic status. Mechanistically, we have demonstrated that, during hypoxia, HIF1α upregulation activates the RhoA signal, thereby promoting cortical actomyosin and stabilizing the adherens junction complex, including merlin. This leads to the cytoplasmic retention of YAP/TAZ to attenuate cell proliferation. These results shed light on the biological significance of blood-vessel geometry and the signaling mechanism through microenvironmental O2 to orchestrate ESC behavior, providing a novel molecular basis for the microenvironmental O2-mediated stem cell regulation during tissue development and renewal.
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Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Epitelio/metabolismo , Incisivo/metabolismo , Oxígeno/metabolismo , Células Madre/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Hipoxia , Inmunohistoquímica , Transducción de Señal , Células Madre/citología , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND AND OBJECTIVES: Hertwig's epithelial root sheath (HERS) plays a role in root dentin formation. It produces the epithelial rests of Malassez (ERM) for the induction of periodontal tissue development during root formation. Although ERM is thought to be caused by epithelial-mesenchymal transition (EMT), the mechanism by which HERS is maintained as epithelium is unknown. Here, we aimed to elucidate the molecular mechanisms regulating the relationship between HERS maintenance and ERM development. METHODS: To understand the relationship between HERS and ERM development during root formation, we observed the developing molar root using cytokeratin14 (CK14) Cre/tdTomato mice via stereomicroscopy. The relationship between semaphorin and transforming growth factor (TGF) signaling in the maintenance of HERS and ERM development was examined using CK14cre/R26-tdTomato mice and a HERS cell line. RESULTS: tdTomato-positive cells were observed on HERS and the migrating cells from HERS. The migrating cells showed reduced E-cadherin expression. In contrast, HERS cells expressed semaphorin receptors and active RhoA. Semaphorin signaling was associated with RhoA activation and cell-cell adhesion, while TGF-ß induced decreased E-cadherin and active RhoA expression, and consequently enhanced cell migration. CONCLUSION: HERS induces root formation by controlling epithelial maintenance and EMT through the opposing effects of semaphorin and TGF-ß signaling.
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Transición Epitelial-Mesenquimal , Factor de Crecimiento Transformador beta , Femenino , Ratones , Animales , Factor de Crecimiento Transformador beta/farmacología , Células Epiteliales , Raíz del Diente/fisiología , Cadherinas/metabolismoRESUMEN
The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.
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Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales/métodos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fluoroacetatos , Expresión Génica , Compuestos Heterocíclicos de 4 o más Anillos/química , Xenoinjertos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Moluscos/química , Mutagénesis Sitio-Dirigida , Mutación , Inhibidores de Proteínas Quinasas/químicaRESUMEN
BACKGROUND: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. RESULTS: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. CONCLUSIONS: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129-139, 2019. © 2018 Wiley Periodicals, Inc.
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Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Diente/citología , Animales , Biomimética/métodos , Biomimética/tendencias , Diferenciación Celular , Células Epiteliales/fisiología , Humanos , Mesodermo/citología , Mesodermo/fisiología , Diente/crecimiento & desarrolloRESUMEN
Tooth and bone are major tissues involved in physiological calcification in the body, and they use similar molecular pathways for development, homeostasis, and regeneration. Harmine (HMN) is a natural small compound that stimulates osteoblast differentiation in vitro and in vivo. Here we examined the biological effect of HMN on the postnatal development of molar tooth roots and periodontal tissues. HMN supported the formation of tooth roots and periodontal tissues in developing tooth germs. In tooth germ organ culture, HMN promoted the elongation of Hertwig's epithelial root sheath (HERS) and stimulated cell proliferation in HERS and dental follicle-derived tissues, including dental papillae and dental follicles. HMN stimulated cell proliferation and cell movement of HERS-derived cells without mesenchymal cells in vitro and directly induced the phosphorylation of SMAD1/5/8 protein in HERS-derived cells. Our results indicated that HMN was the first natural small compound to stimulate postnatal development of tooth germs.
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Harmina/farmacología , Diente Molar/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Raíz del Diente/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Proteína Smad1/análisis , Proteína Smad5/análisis , Proteína Smad8/análisis , Raíz del Diente/crecimiento & desarrollo , Raíz del Diente/metabolismoRESUMEN
The transcription factor Sox2 is a stem cell marker that dictates cell lineage. It has been shown to mark the epithelial stem cells of the continuously growing mouse incisors. Sox2 also interferes with Wnt signaling by binding to ß-catenin, a central mediator of the Wnt pathway. We show that these functions of Sox2 are essential for mouse molar development. Sox2 has previously been shown to play a role in the formation of new teeth from the existing dental epithelium. To assess Sox2 function related to cell migration within a tooth, we monitored cell movement by using a DiI system and observed that DiI moves from molar 1 to molar 2 during tooth development. However, upon temporal knockdown of Sox2, DiI remains in the molar 1 region. This study also provides novel insights into the role of Sox2 and the important validation of Sox2 as a potent target in Wnt signaling during tooth development. Our data reveal that the degradation of Wnt signaling caused by the knockdown of Sox2 results in a lack of cell migration during tooth development.
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Factores de Transcripción SOXB1/metabolismo , Diente/embriología , Diente/metabolismo , Vía de Señalización Wnt , Animales , Movimiento Celular , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , ARN Interferente Pequeño/metabolismo , Factores de TiempoRESUMEN
Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.
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Movimiento Celular , Quimiocina CXCL12/metabolismo , Esmalte Dental/citología , Receptores CXCR4/metabolismo , Transducción de Señal , Nicho de Células Madre , Células Madre/citología , Animales , Agregación Celular , Línea Celular , Proliferación Celular , Forma de la Célula , Quimiocina CXCL12/deficiencia , Quimiocina CXCL12/genética , Células Epiteliales , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Incisivo/citología , Incisivo/embriología , Ratones Noqueados , Mutación , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/deficiencia , Receptores CXCR4/genética , Células Madre/metabolismoRESUMEN
Teeth are composed of two domains, the enamel-covered crown and cementum-covered root. The mechanism for determining the transition from crown to root is important for understanding root anomaly diseases. Hertwig׳s epithelial root sheath (HERS) is derived from the dental epithelium and is known to drive the growth of root dentin and periodontal tissue. Some clinical cases of hypoplastic tooth root are caused by the cessation of HERS development. Understanding the mechanisms of HERS development will contribute to the study of the disease and dental regenerative medicine. However, the developmental biology of tooth root formation has not been fully studied, particularly regarding HERS formation. Here, we describe the mechanisms of HERS formation on the basis of analysis of cell dynamics using imaging and summarize how the growth factor and its receptor regulate cell behavior of the dental epithelium.
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Fenómenos Fisiológicos Celulares , Epitelio/crecimiento & desarrollo , Raíz del Diente/crecimiento & desarrollo , Animales , Epitelio/metabolismo , Humanos , Raíz del Diente/metabolismoRESUMEN
OBJECTIVES: Details about salivary gland tumor histogenesis remain unknown. Here, we established a newly generated murine salivary gland tumor model that could overexpress pleomorphic adenoma gene 1 (PLAG1) and attempted to clarify the events that occur during the early phase of salivary gland tumor histogenesis. METHODS: Salivary gland tumors were generated using murine models (Sox9IRES-CreERT2; ROSA26-PLAG1). Lineage tracing of Sox9-expressing cells was performed using Sox9IRES-CreERT2; ROSA26-tdTomato mice, which were generated by crossing Sox9CreERT2/- and ROSA26-tdTomato mice (expressing the tdTomato fluorescent protein). Organ-cultured embryonic salivary glands from the murine model were morphologically analyzed, and mRNA sequencing was conducted two days after tumor induction for gene enrichment and functional annotation analysis. RESULTS: Salivary gland tumors exhibited epithelial features with acinar-like structures because of gene rearrangements in the luminal cells. Structural disturbances in the duct-acinar unit of the salivary gland were observed and cancer-related pathways were enriched among the differentially upregulated genes in the early phase of tumor induction in an organ-cultured embryonic salivary gland tumor model. CONCLUSIONS: The newly generated murine salivary gland tumor model may show that the tumorization of luminal stem/progenitor cells can result in the development of salivary gland tumors comprising only luminal cells.
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Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.
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Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Pulpa Dental/fisiología , Células Epiteliales/fisiología , Células Madre Multipotentes/fisiología , Odontoblastos/fisiología , Células Madre/fisiología , Animales , Animales Recién Nacidos , Antígenos de Diferenciación/biosíntesis , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Técnicas de Cocultivo , Pulpa Dental/citología , Células Epiteliales/citología , Transición Epitelial-Mesenquimal/fisiología , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos ICR , Células Madre Multipotentes/citología , Odontoblastos/citología , Ratas , Células Madre/citologíaRESUMEN
OBJECTIVES: Junctional epithelium (JE) connects the tooth surface and gingival epithelium and adheres directly to the tooth enamel. JE plays an important role as a barrier preventing the invasion of exogenous bacteria and substances. However, the cellular characteristics of this epithelium have not been adequately described, because no useful in vitro experimental model exists for JE. METHODS: We generated a novel JE cell line, mHAT-JE01, using naturally immortalized dental epithelium derived from incisor labial cervical cells and by selecting cells that adhered to apatite. mHAT-JE01 was characterized by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction and compared with the gingival epithelial cell line, mOE-PE01. RESULTS: The mHAT-JE01 cells had a higher capacity for producing JE-specific markers than oral mucous epithelial cells. In addition, the presence of lipopolysaccharides from Porphyromonas gingivalis downregulated the expression of JE protein markers in mHAT-JE01 cells. CONCLUSIONS: This cell line is stable and presents the opportunity to characterize JE efficiently, which is essential for the prevention and treatment of periodontal disease.
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Células Epiteliales , Incisivo , Incisivo/química , Incisivo/metabolismo , Células Epiteliales/química , Células Epiteliales/metabolismo , Epitelio/química , Epitelio/metabolismo , Proteínas/análisis , Proteínas/metabolismo , Línea CelularRESUMEN
Tooth development is governed largely by epithelial-mesenchymal interactions and is mediated by numerous signaling pathways. This type of morphogenetic processes has been explained by reaction-diffusion systems, especially in the framework of a Turing model. Here we focus on morphological and developmental differences between upper and lower molars in mice by modeling 2D pattern formation in a Turing system. Stripe vs. spot patterns are the primary types of variation in a Turing model. We show that the complexity of the cusp cross-sections can distinguish between stripe vs. spot patterns, and mice have stripe-like upper and spot-like lower molar morphologies. Additionally, our computational modeling that incorporates empirical data on tooth germ growth traces the order of cusp formation and relative position of the cusps in upper and lower molars in mice. We further propose a hypothetical framework of developmental mechanism that could help us understand the evolution of the highly variable nature of mammalian molars associated with the acquisition of the hypocone and the increase of lophedness.
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Diente Molar , Odontogénesis , Animales , Mamíferos , Ratones , Diente Molar/anatomía & histología , Morfogénesis , Transducción de Señal , Germen DentarioRESUMEN
OBJECTIVES: Lysophosphatidic acid (LPA) is a potent bioactive phospholipid that exerts various functions upon binding to six known G protein-coupled receptors (LPA1-6); however; its role in a tooth remains unclear. This study aimed to explore the impact of the LPA/LPA receptor 6 (LPA6)/RhoA signaling axis on maturation stage ameloblasts (M-ABs), which are responsible for enamel mineralization. METHODS: The expression of LPA6 and LPA-producing synthetic enzymes during ameloblast differentiation was explored through immunobiological analysis of mouse incisors and molars. To elucidate the role of LPA6 in ameloblasts, incisors of LPA6 KO mice were analyzed. In vitro experiments using ameloblast cell lines were performed to validate the function of LPA-LPA6-RhoA signaling in ameloblasts. RESULTS: LPA6 and LPA-producing enzymes were strongly expressed in M-ABs. In LPA6 knockout mice, M-ABs exhibited abnormal morphology with the loss of cell polarity, and an abnormal enamel epithelium containing cyst-like structures was formed. Moreover, the expression of E-cadherin and zonula occludens-1 (ZO-1) significantly decreased in M-ABs. In vitro experiments demonstrated that LPA upregulated the expression of E-cadherin, ZO-1, and filamentous actin (F-actin) at the cellular membrane, whereas LPA6 knockdown decreased their expression and changed cell morphology. Furthermore, we showed that RhoA signaling mediates LPA-LPA6-induced junctional complexes. CONCLUSIONS: This study demonstrated that LPA-LPA6-RhoA signaling is essential for establishing proper cell morphology and polarity, via cell-cell junction and actin cytoskeleton expression and stability, of M-ABs. These results highlight the biological significance of bioactive lipids in a tooth, providing a novel molecular regulatory mechanism of ameloblasts.
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Ameloblastos , Lisofosfolípidos , Receptores del Ácido Lisofosfatídico , Proteína de Unión al GTP rhoA , Ameloblastos/metabolismo , Amelogénesis , Animales , Cadherinas/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Receptores del Ácido Lisofosfatídico/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Maturation stage ameloblasts (M-ABs) are responsible for terminal enamel mineralization in teeth and undergo characteristic cyclic changes in both morphology and function between ruffle-ended ameloblasts (RA) and smooth-ended ameloblasts (SA). Energy metabolism has recently emerged as a potential regulator of cell differentiation and fate decisions; however, its implication in M-ABs remains unclear. To elucidate the relationship between M-ABs and energy metabolism, we examined the expression pattern of energy metabolic enzymes in M-ABs of mouse incisors. Further, using the HAT7 cell line with M-AB characteristics, we designed experiments to induce an energy metabolic shift by changes in oxygen concentration. We revealed that RA preferentially utilizes oxidative phosphorylation, whereas SA depends on glycolysis-dominant energy metabolism in mouse incisors. In HAT7 cells, hypoxia induced an energy metabolic shift toward a more glycolytic-dominant state, and the energy metabolic shift reduced alkaline phosphatase (ALP) activity and calcium transport and deposition with a change in calcium-related gene expression, implying a phenotype shift from RA to SA. Taken together, these results indicate that the energy metabolic state is an important determinant of the RA/SA phenotype in M-ABs. This study sheds light on the biological significance of energy metabolism in governing M-ABs, providing a novel molecular basis for understanding enamel mineralization and elucidating the pathogenesis of enamel hypomineralization.
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OBJECTIVES: Ameloblastoma (AM) has been known as a benign but locally invasive tumour with high recurrence rates. Invasive behaviour of the AM results in destruction of the adjacent jawbone and the non-detectable remnants during surgery, interrupting the complete elimination of cancer cells. METHODS: To explore novel targets for the tumour cell invasion, a transcriptomic analysis between AM and odontogenic keratocyst were performed through next-generation sequencing in detail. RESULTS: Enrichment of CACNA1C gene (encoding Cav1.2) in AM, a subunit of the L-type voltage-gated calcium channel (VGCC) was observed for the first time. The expression and channel activity of Cav1.2 was confirmed by immunostaining and calcium imaging in the patient samples or primary cells. Verapamil, L-type VGCC blocker revealed suppression of the Ca2+ -induced cell aggregation and collective invasion of AM cells in vitro. Furthermore, the effect of verapamil in suppressing AM invasion into the adjacent bone was confirmed through orthotopic xenograft model specifically. CONCLUSION: Taken together, Cav1.2 maybe considered to be a therapeutic candidate to decrease the collective migration and invasion of AM.
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Ameloblastoma , Bloqueadores de los Canales de Calcio , Canales de Calcio Tipo L , Humanos , Ameloblastoma/tratamiento farmacológico , Ameloblastoma/genética , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/fisiología , Verapamilo/farmacología , AnimalesRESUMEN
During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and ß-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.
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Ameloblastos/enzimología , Diferenciación Celular/fisiología , Quinasas Asociadas a rho/fisiología , Ameloblastos/citología , Amelogénesis/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Incisivo/citología , Incisivo/enzimología , Incisivo/fisiología , Ratones , Técnicas de Cultivo de Órganos , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
The epithelial-mesenchymal transition (EMT) is an important event in the developmental process of various organs. In periodontal development during root formation of a tooth, this EMT has been a subject of controversy. Hertwig's epithelial root sheath (HERS), consisting of two epithelial layers, plays a role of inducing odontogenesis during root development and thereafter becomes fragmented. Some researchers have maintained that in the process of this fragmentation, some HERS cells change from epithelial to mesenchymal cells. Here, we established a HERS cell line (HERS01a) and examined its gene and protein expression. Immunohistochemical staining and real-time PCR analysis showed that HERS01a cells expressed vimentin and N-cadherin as mesenchymal markers as well as cytokeratin14, E-cadherin, and p63 as epithelial stem cell markers. In the presence of TGF-ß, HERS01a cells also expressed many more mesenchymal markers, as well as snail1 and 2 as EMT markers. Taken together, our data show that HERS01a displayed unique features associated with EMT in the root formation process, and will thus be useful for analyzing the biological characteristics of HERS and the molecular mechanism underlying the EMT.
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Línea Celular , Células Epiteliales/citología , Transición Epitelial-Mesenquimal , Raíz del Diente/citología , Animales , Separación Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Expresión Génica , Inmunohistoquímica , Ratones , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Raíz del Diente/crecimiento & desarrollo , Raíz del Diente/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Mesenchymal stem cells (MSCs), which potentially transdifferentiate into multiple cell types, are increasingly reported to be beneficial in models of organ system injury. However, the molecular mechanisms underlying interactions between MSCs and host cells, in particular endothelial cells (ECs), remain unclear. We show here in a matrigel angiogenesis assay that MSCs are capable of inhibiting capillary growth. After addition of MSCs to EC-derived capillaries in matrigel at EC:MSC ratio of 1:1, MSCs migrated toward the capillaries, intercalated between ECs, established Cx43-based intercellular gap junctional communication (GJC) with ECs, and increased production of reactive oxygen species (ROS). These events led to EC apoptosis and capillary degeneration. In an in vivo tumor model, direct MSC inoculation into subcutaneous melanomas induced apoptosis and abrogated tumor growth. Thus, our findings show for the first time that at high numbers, MSCs are potentially cytotoxic and that when injected locally in tumor tissue they might be effective antiangiogenesis agents suitable for cancer therapy.
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Inhibidores de la Angiogénesis/uso terapéutico , Endotelio Vascular/citología , Melanoma Experimental/irrigación sanguínea , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Neovascularización Patológica/terapia , Animales , Apoptosis/fisiología , Comunicación Celular , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Uniones Comunicantes/fisiología , Immunoblotting , Técnicas para Inmunoenzimas , Inmunoprecipitación , Laminina/metabolismo , Pulmón/citología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Ingeniería de TejidosRESUMEN
The use of agents that target both glia and neurons may represent a new strategy for the treatment of ageing disorders. Here, we confirmed the presence of the novel cyclic peptide Naturido that originates from a medicinal fungus (Isaria japonica) grown on domestic silkworm (Bombyx mori). We found that Naturido significantly enhanced astrocyte proliferation and activated the single copy gene encoding the neuropeptide VGF and the neuron-derived NGF gene. The addition of the peptide to the culture medium of primary hippocampal neurons increased dendrite length, dendrite number and axon length. Furthermore, the addition of the peptide to primary microglial cultures shifted CGA-activated microglia towards anti-inflammatory and neuroprotective phenotypes. These findings of in vitro glia-neuron interactions led us to evaluate the effects of oral administration of the peptide on brain function and hair ageing in senescence-accelerated mice (SAMP8). In vivo analyses revealed that spatial learning ability and hair quality were improved in Naturido-treated mice compared with untreated mice, to the same level observed in the normal ageing control (SAMR1). These data suggest that Naturido may be a promising glia-neuron modulator for the treatment of not only senescence, but also Alzheimer's disease and other neurodegenerative diseases.
Asunto(s)
Envejecimiento/efectos de los fármacos , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Axones/efectos de los fármacos , Axones/fisiología , Proliferación Celular/efectos de los fármacos , Dendritas/efectos de los fármacos , Dendritas/fisiología , Femenino , Humanos , Hypocreales/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Microglía/citología , Microglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Péptidos Cíclicos/administración & dosificación , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Spermatogenesis is an important cellular differentiation process that produces the male gametes and remains active throughout the individual's lifespan. Sertoli cell-only syndrome (SCO) refers to the dysfunction of the male reproductive system, including infertility. Accurate self-renewal of spermatogonial stem cells (SSCs) is essential to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice and MAST4 expression shifted to Leydig cells and spermatids throughout puberty. Mast4 knockout (KO) testes were reduced in size compared to wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. In addition, MAST4 phosphorylated the Ets-related molecule (ERM), specifically the serine 367 residue. The phosphorylation of ERM ultimately controls the transcription of ERM target genes related to SSC self-renewal. The expression of spermatogenesis-associated proteins was significantly decreased whereas Sertoli cell markers were increased in Mast4 KO testes, which was well-founded by RNA-sequencing analysis. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway and this association helps us explore the capacity of SSCs to maintain a vertebrate stem cell niche.