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1.
Development ; 148(4)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33472844

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.


Asunto(s)
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 YAP
2.
J Periodontal Res ; 58(1): 184-194, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36517910

RESUMEN

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.


Asunto(s)
Transición Epitelial-Mesenquimal , Factor de Crecimiento Transformador beta , Femenino , Ratones , Animales , Factor de Crecimiento Transformador beta/farmacología , Células Epiteliales , Raíz del Diente/fisiología , Cadherinas/metabolismo
3.
J Cell Physiol ; 236(7): 5387-5398, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33377198

RESUMEN

ß-catenin, a key mediator of Wnt signaling, plays multiple roles in tooth development. However, the role of ß-catenin in Hertwig's epithelial root sheath (HERS) during root formation remains unclear. In this study, we generated inducible tissue-specific ß-catenin conditional knockout mice (Ctnnb1i∆shh ) to investigate how ß-catenin in HERS affects tooth root development. The inactivation of ß-catenin in HERS led to interrupted root elongation due to premature disruption of HERS. This phenotype was accompanied by reduced cell-cell adhesion and decreased expression of junctional proteins, as well as increased epithelial-to-mesenchymal transition of HERS cells upon ß-catenin depletion. Accordingly, stabilization of ß-catenin in HERS (Catnbi∆shh ) led to the formation of unfragmented HERS and resulted in the failure of HERS dissociation, with increased expression of junctional proteins. Our results suggest that fine control of ß-catenin is important for HERS to guide root formation through regulating its structural integrity.


Asunto(s)
Células Epiteliales/metabolismo , Odontogénesis/fisiología , Raíz del Diente/crecimiento & desarrollo , Raíz del Diente/metabolismo , beta Catenina/metabolismo , Animales , Ratones , Ratones Noqueados
4.
Dev Dyn ; 248(12): 1264-1272, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31464047

RESUMEN

BACKGROUND: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process. RESULTS: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression. CONCLUSION: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development.


Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Huesos Faciales/embriología , Proteínas de Homeodominio/genética , Cresta Neural/embriología , Cráneo/embriología , Animales , Animales Modificados Genéticamente , Embrión de Pollo , Anomalías Craneofaciales/genética , Desarrollo Embrionario/genética , Huesos Faciales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mandíbula/embriología , Mandíbula/metabolismo , Maxilar/embriología , Maxilar/metabolismo , Cresta Neural/metabolismo , Cráneo/metabolismo
5.
Dev Dyn ; 248(1): 129-139, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30106495

RESUMEN

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.


Asunto(s)
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 & desarrollo
6.
Cell Tissue Res ; 365(1): 77-84, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26846112

RESUMEN

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.


Asunto(s)
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 Tiempo
7.
Cell Tissue Res ; 362(3): 633-42, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26246398

RESUMEN

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.


Asunto(s)
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/metabolismo
8.
Exp Cell Res ; 325(2): 78-82, 2014 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-24560742

RESUMEN

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.


Asunto(s)
Fenómenos Fisiológicos Celulares , Epitelio/crecimiento & desarrollo , Raíz del Diente/crecimiento & desarrollo , Animales , Epitelio/metabolismo , Humanos , Raíz del Diente/metabolismo
9.
Am J Pathol ; 183(1): 108-18, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23764278

RESUMEN

Endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA), are environmental ubiquitous pollutants and associated with a growing health concern. Anecdotally, molar incisor hypomineralization (MIH) is increasing concurrently with EDC-related conditions, which has led us to investigate the effect of BPA on amelogenesis. Rats were exposed daily to BPA from conception until day 30 or 100. At day 30, BPA-affected enamel exhibited hypomineralization similar to human MIH. Scanning electron microscopy and elemental analysis revealed an abnormal accumulation of organic material in erupted enamel. BPA-affected enamel had an abnormal accumulation of exogenous albumin in the maturation stage. Quantitative real-time PCR, Western blotting, and luciferase reporter assays revealed increased expression of enamelin but decreased expression of kallikrein 4 (protease essential for removing enamel proteins) via transcriptional regulation. Data suggest that BPA exerts its effects on amelogenesis by disrupting normal protein removal from the enamel matrix. Interestingly, in 100-day-old rats, erupting incisor enamel was normal, suggesting amelogenesis is only sensitive to MIH-causing agents during a specific time window during development (as reported for human MIH). The present work documents the first experimental model that replicates MIH and presents BPA as a potential causative agent of MIH. Because human enamel defects are irreversible, MIH may provide an easily accessible marker for reporting early EDC exposure in humans.


Asunto(s)
Compuestos de Bencidrilo/toxicidad , Hipoplasia del Esmalte Dental/inducido químicamente , Disruptores Endocrinos/toxicidad , Fenoles/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Amelogénesis/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Western Blotting , Hipoplasia del Esmalte Dental/metabolismo , Proteínas del Esmalte Dental/metabolismo , Femenino , Humanos , Calicreínas/metabolismo , Masculino , Microscopía Electrónica de Rastreo , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa
10.
J Oral Biosci ; 2024 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-39159676

RESUMEN

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.

11.
Dev Biol ; 363(1): 52-61, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22226978

RESUMEN

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.


Asunto(s)
Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/farmacocinética , Diente Molar/metabolismo , Odontogénesis , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Órgano del Esmalte/embriología , Órgano del Esmalte/crecimiento & desarrollo , Órgano del Esmalte/metabolismo , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos ICR , Diente Molar/embriología , Diente Molar/crecimiento & desarrollo , Floretina/farmacología , Embarazo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Germen Dentario/embriología , Germen Dentario/crecimiento & desarrollo , Germen Dentario/metabolismo
12.
J Biol Chem ; 287(13): 10590-10601, 2012 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-22298769

RESUMEN

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.


Asunto(s)
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/citología
13.
J Oral Biosci ; 65(1): 47-54, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36693475

RESUMEN

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.


Asunto(s)
Células Epiteliales , Incisivo , Incisivo/química , Incisivo/metabolismo , Células Epiteliales/química , Células Epiteliales/metabolismo , Epitelio/química , Epitelio/metabolismo , Proteínas/análisis , Proteínas/metabolismo , Línea Celular
14.
Sci Rep ; 13(1): 18829, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37914726

RESUMEN

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.


Asunto(s)
Ameloblastos , Metaloproteinasa 20 de la Matriz , Ratones , Animales , Ameloblastos/metabolismo , Metaloproteinasa 20 de la Matriz/metabolismo , Ratones Transgénicos , Cadherinas/metabolismo , Expresión Génica
15.
Stem Cells ; 29(11): 1792-803, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21898689

RESUMEN

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor ß (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.


Asunto(s)
Diferenciación Celular/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Incisivo/citología , Células Madre/citología , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Proliferación Celular , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos/genética , Factor 3 de Crecimiento de Fibroblastos/metabolismo , Factor 9 de Crecimiento de Fibroblastos/genética , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microtomografía por Rayos X
16.
Int J Stem Cells ; 15(4): 415-421, 2022 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-36310025

RESUMEN

Cancer initiation and progression are profoundly along with the crosstalk between cancer cells and the surrounding stroma. Accumulating evidence has shown that the therapy targeting the extracellular matrix (ECM) would regress tumor growth and invasion in the most common carcinomas. However, it remains largely unexplored in several rare tumors like odontogenic tumors. Ameloblastoma (AM) is the representative odontogenic epithelial tumor in the jawbone, and it usually infiltrates into adjacent bone marrow and has unlimited growth capacity and a high potential for recurrence. This study aims to investigate the role of collagen-rich ECM during the invasion of AM. Transcriptomic analysis revealed that ECM- and epithelial-to-mesenchymal transition (EMT)-related genes were up-regulated in AM compared to ameloblastoma cell line, AM-1. Tumoroid forming analysis showed that Collagen-rich ECM is indispensable for AM progression, especially for aggressive growth patterns and collective invasion.

17.
J Oral Biosci ; 64(1): 85-92, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35074512

RESUMEN

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.


Asunto(s)
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/metabolismo
18.
Front Physiol ; 13: 1062042, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36523561

RESUMEN

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.

19.
Cell Prolif ; 55(11): e13305, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35794842

RESUMEN

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.


Asunto(s)
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 , Animales
20.
J Cell Physiol ; 226(10): 2527-34, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21792909

RESUMEN

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.


Asunto(s)
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/genética
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