RESUMO
Fibroblast growth factor (FGF) signalling plays a crucial role in the morphogenesis of multiple tissues including teeth. While the role of the signal has been studied in tooth crown development, little is known about root development. Of several FGF ligands involved in hard tissue formation, we suggest that FGF18 regulates the development of murine tooth roots. We implanted FGF18-soaked heparin beads into the lower first molar tooth buds at postnatal day 6 (P6), followed by transplantation under the kidney capsule. After 3 weeks, FGF18 significantly facilitated root elongation and periodontal tissue formation compared to the control. In situ hybridisation showed that Fgf18 transcripts were initially localised in the dental pulp along Hertwig's epithelial root sheath at P6 and P10 and subsequently in the dental follicle cells at P14. Fgf receptors were expressed in various dental tissues during these stages. In vitro analysis using the dental pulp stem cells revealed that FGF18 inhibited cell proliferation and decreased expression levels of osteogenic markers, Runx2, Alpl and Sp7. Consistently, after 1 week of kidney capsule transplantation, FGF18 application did not induce the expression of Sp7 and Bsp, but upregulated Periostin in the apical region of dental mesenchyme in the grafted molar. These findings suggest that FGF18 facilitates molar root development by regulating the calcification of periodontal tissues.
Assuntos
Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Raiz Dentária , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Raiz Dentária/crescimento & desenvolvimento , Raiz Dentária/metabolismo , Camundongos , Transdução de Sinais/fisiologia , Dente Molar/embriologia , Odontogênese/fisiologiaRESUMO
There exists a close connection between changes occurring in the teeth and those occurring in the jaw during the evolutionary process. In mammals, the roots of teeth are supported, along with periodontal ligaments and alveolar bones by a unique structure termed the gomphosis. In the present study, we performed combined in silico analysis using the information obtained from various DNA microarrays and identified 19 putative tooth root formation-related genes. Furthermore, quantitative PCR was performed on the candidate genes, Chd3 was confirmed as having sufficient expression levels in the early stage of tooth root formation and increased gene expression toward the middle stage. A high degree of Chd3 gene expression was observed in secretory ameloblasts and Hertwig's epithelial root sheath (HERS), but low expression was observed in developing odontoblasts and stellate reticulum. The CHD3 foci were observed in the nucleus of the HERS01a cells. In addition, knockdown experiments using SiChd3 suggested the involvement of Chd3 in the suppression of DNA synthesis. These results suggested that Chd3 plays a role in DNA synthesis in HERS cells for promoting tooth root development.
Assuntos
Células Epiteliais , Raiz Dentária , Animais , DNA , Órgão do Esmalte , OdontogêneseRESUMO
Bitter taste avoidance behavior (BAB) plays a fundamental role in the avoidance of toxic substances with a bitter taste. However, the molecular basis underlying the development of BAB is unknown. To study critical developmental events by which taste buds turn into functional organs with BAB, we investigated the early phase development of BAB in postnatal mice in response to bitter-tasting compounds, such as quinine and thiamine. Postnatal mice started to exhibit BAB for thiamine and quinine at postnatal day 5 (PD5) and PD7, respectively. Histological analyses of taste buds revealed the formation of microvilli in the taste pores starting at PD5 and the localization of type 2 taste receptor 119 (TAS2R119) at the microvilli at PD6. Treatment of the tongue epithelium with cytochalasin D (CytD), which disturbs ACTIN polymerization in the microvilli, resulted in the loss of TAS2R119 localization at the microvilli and the loss of BAB for quinine and thiamine. The release of ATP from the circumvallate papillae tissue due to taste stimuli was also declined following CytD treatment. These results suggest that the localization of TAS2R119 at the microvilli of taste pores is critical for the initiation of BAB.
Assuntos
Actinas/metabolismo , Aprendizagem da Esquiva/fisiologia , Microvilosidades/metabolismo , Frações Subcelulares/metabolismo , Papilas Gustativas/fisiologia , Paladar/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição TecidualRESUMO
Labial grooves in maxillary incisors have been reported in several wild-type rodent species. Previous studies have reported age-dependent labial grooves occur in moderate prevalence in C57BL/6 mice; however, very little is known about the occurrence of such grooves. In the present study, we observed age-dependent groove formation in C57BL/6 mice up to 26 months after birth and found that not only the frequency of the appearance of incisor grooves but also the number of grooves increased in an age-dependent manner. We examined the molecular mechanisms of age-dependent groove formation by performing DNA microarray analysis of the incisors of 12-month-old (12M) and 24-month-old (24M) mice. Amelx, encoding the major enamel matrix protein AMELOGENIN, was identified as a 12M-specific gene. Comparing with wild-type mice, the maxillary incisors of Amelx-/- mutants indicated the increase of the frequency and number of labial grooves. These findings suggested that the Amelx gene impacts the age-dependent appearance of the labial incisor groove in C57BL/6 mice.
Assuntos
Envelhecimento/genética , Amelogenina/genética , Esmalte Dentário/metabolismo , Dentina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Incisivo/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Amelogenina/deficiência , Animais , Esmalte Dentário/diagnóstico por imagem , Esmalte Dentário/patologia , Dentina/diagnóstico por imagem , Dentina/patologia , Incisivo/diagnóstico por imagem , Incisivo/patologia , Maxila/diagnóstico por imagem , Maxila/metabolismo , Maxila/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Tomografia Computadorizada por Raios XRESUMO
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.
Assuntos
Harmina/farmacologia , Dente Molar/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Raiz Dentária/efeitos dos fármacos , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Dente Molar/crescimento & desenvolvimento , Dente Molar/metabolismo , Proteína Smad1/análise , Proteína Smad5/análise , Proteína Smad8/análise , Raiz Dentária/crescimento & desenvolvimento , Raiz Dentária/metabolismoRESUMO
BACKGROUND: The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon. RESULTS: The Foxc1 loss of function mutant mouse, Foxc1(ch/ch) , showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites. CONCLUSIONS: These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon.
Assuntos
Circulação Cerebrovascular/fisiologia , Embrião de Mamíferos , Fatores de Transcrição Forkhead/metabolismo , Telencéfalo , Animais , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/embriologia , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Mutantes , Telencéfalo/irrigação sanguínea , Telencéfalo/embriologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Fibroblast growth factors (FGFs) regulate the proliferation and differentiation of various cells via their respective receptors (FGFRs). During the early stages of tooth development in fetal mice, FGFs and FGFRs have been shown to be expressed in dental epithelia and mesenchymal cells at the initial stages of odontogenesis and to regulate cell proliferation and differentiation. However, little is known about the expression patterns of FGFs in the advanced stages of tooth development. In the present study, we focused on FGF18 expression in the rat mandibular first molar (M1) during the postnatal crown and root formation stages. FGF18 signals by RT-PCR using cDNAs from M1 were very weak at postnatal day 5 and were significantly up-regulated at days 7, 9 and 15. Transcripts were undetectable by in situ hybridization (ISH) but could be detected by in situ RT-PCR in the differentiated odontoblasts and cells of the sub-odontoblastic layer in both crown and root portions of M1 at day 15. The transcripts of FGFR2c and FGFR3, possible candidate receptors of FGF18, were detected by RT-PCR and ISH in differentiated odontoblasts throughout postnatal development. These results suggest the continual involvement of FGF18 signaling in the regulation of odontoblasts during root formation where it may contribute to dentin matrix formation and/or mineralization.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Odontogênese/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Hibridização In Situ , Mandíbula , Dente Molar/fisiologia , Odontoblastos/fisiologia , Ratos , Ratos Wistar , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
OBJECTIVES: This study determined the early development of taste buds by observing the changes in the three-dimensional structures of taste pores and microvilli in the circumvallate papillae (CVP) of mice, from pre- and postnatal stages to the adult stages. METHODS: Fragments of mouse CVP tissue were collected on embryonic day (E) 18 and postnatal days (P) 0, 3, 6, 7, 14, 21, 28, and 56. The surfaces of the tissue fragments located pore apertures via scanning electron microscopy, and the sizes of the CVP and maximum diameters of the pores were estimated from the recorded images. Likewise, changes in the structures of the epithelium around the pore aperture and microvilli protruding from the pores were examined. RESULTS: The size of the CVP exhibited a linear increase with age from E18 to P56. The epithelium around the pore aperture demonstrated changes to form microridges, indicating a characteristic pattern during CVP development. The size of the pore aperture also increased with age from E18 to P56. Furthermore, an increase in the number of pores with protruding microvilli was observed at the base of the epithelial trench. A significant positive correlation was observed between the maximum diameter of the pore and the size of the CVP. CONCLUSIONS: The expansion in the lateral view of the CVP was associated with the developmental stage from E18 to P56, suggesting that the growth of the CVP leads to the opening and enlargement of the taste pores with microvillus projections during these stages.
Assuntos
Papilas Gustativas , Camundongos , Animais , Papilas Gustativas/química , Paladar , Microscopia Eletrônica de Varredura , EpitélioRESUMO
Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra "cones" to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4-mediated signaling.
Assuntos
Evolução Biológica , Incisivo , Morfogênese/fisiologia , Odontogênese/fisiologia , Ameloblastos/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Esmalte Dentário/ultraestrutura , Dentina/ultraestrutura , Peixes/anatomia & histologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Incisivo/anatomia & histologia , Incisivo/fisiologia , Proteínas Relacionadas a Receptor de LDL , Camundongos , Camundongos Knockout , Coelhos , Ratos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais/fisiologia , Anormalidades Dentárias/genética , Anormalidades Dentárias/metabolismoRESUMO
The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction), the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP), we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1(-/) (-) mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Biologia Computacional , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Papilas Gustativas/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Fosfolipase C beta/genética , Transdução de Sinais , Papilas Gustativas/química , Papilas Gustativas/crescimento & desenvolvimento , Papilas Gustativas/metabolismo , Fatores de Transcrição HES-1RESUMO
Mammalian dentitions consist of different shapes/types of teeth that are positioned in different regions of the jaw (heterodont) whereas in many fish and reptiles all teeth are of similar type (homodont). The process by which heterodont dentitions have evolved in mammals is not understood. In many teleosts teeth develop in the pharynx from endoderm (endodermal teeth), whereas mammalian teeth develop from the oral ectoderm indicating that teeth can develop (and thus possibly evolve) via different mechanisms. In this article, we compare the molecular characteristics of pharyngeal/foregut endoderm with the molecular characteristics of oral ectoderm during mouse development. The expression domains of Claudin6, Hnf3beta, alpha-fetoprotein, Rbm35a, and Sox2 in the embryonic endoderm have boundaries overlapping the molar tooth-forming region, but not the incisor region in the oral ectoderm. These results suggest that molar teeth (but not incisors) develop from epithelium that shares molecular characteristics with pharyngeal endoderm. This opens the possibility that the two different theories proposed for the evolution of teeth may both be correct. Multicuspid (eg. molars) having evolved from the externalization of endodermal teeth into the oral cavity and monocuspid (eg. incisors) having evolved from internalization of ectodermal armour odontodes of ancient fishes. The two different mechanisms of tooth development may have provided the developmental and genetic diversity on which evolution has acted to produce heterodont dentitions in mammals.
Assuntos
Evolução Biológica , Ectoderma/fisiologia , Endoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Arcada Osseodentária/embriologia , Dente/embriologia , Animais , Linhagem da Célula , Galinhas , Ectoderma/citologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Hibridização In Situ , Arcada Osseodentária/metabolismo , Camundongos , Sondas RNA , Dente/metabolismoRESUMO
The low-density lipoprotein (LDL) receptor family is a large evolutionarily conserved group of transmembrane proteins. It has been shown that LDL receptor family members can also function as direct signal transducers or modulators for a broad range of cellular signaling pathways. We have identified a novel mode of signaling pathway integration/coordination that occurs outside cells during development that involves an LDL receptor family member. Physical interaction between an extracellular protein (Wise) that binds BMP ligands and an Lrp receptor (Lrp4) that modulates Wnt signaling, acts to link these two pathways. Mutations in either Wise or Lrp4 in mice produce multiple, but identical abnormalities in tooth development that are linked to alterations in BMP and Wnt signaling. Teeth, in common with many other organs, develop by a series of epithelial-mesenchymal interactions, orchestrated by multiple cell signaling pathways. In tooth development, Lrp4 is expressed exclusively in epithelial cells and Wise mainly in mesenchymal cells. Our hypothesis, based on the mutant phenotypes, cell signaling activity changes and biochemical interactions between Wise and Lrp4 proteins, is that Wise and Lrp4 together act as an extracellular mechanism of coordinating BMP and Wnt signaling activities in epithelial-mesenchymal cell communication during development.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Face/embriologia , Receptores de LDL/metabolismo , Transdução de Sinais/fisiologia , Crânio/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Embrião de Mamíferos , Células Epiteliais/fisiologia , Espaço Extracelular/metabolismo , Proteínas Relacionadas a Receptor de LDL , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Odontogênese/fisiologia , Organogênese/fisiologia , Receptores de LDL/genética , Receptores de LDL/fisiologia , Anormalidades Dentárias/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiologiaRESUMO
OBJECTIVES: The aim of this study was to explore the relationship between the consumption of a high-fat diet and aging-dependent formation of maxillary incisor grooves in C57BL/6 mice, and to identify putative maxillary incisor groove-related genes. METHODS: We fed 2-month-old and 16-month-old C57BL/6 mice on either a chow diet or a high-fat diet for three months and observed changes in maxillary incisor grooves. We examined tissue sections of the maxillary incisors with grooves and carried out transcriptome analysis of the apical tissue fragments of maxillary incisors with/without grooves. RESULTS: Consumption of a high-fat diet for three months resulted in significant increases in both body weight and the number of incisor grooves. Both the number and frequency of incisor grooves increased in an age-dependent manner from 26 to 28 months, during which time an additional groove appeared. There was abnormal differentiation and apoptosis of ameloblasts on the labial surface at the grooves of the maxillary incisors. Transcriptome analysis identified 23 genes as being specific to 24-month-old mice; these included several genes related to apoptosis and cell differentiation. CONCLUSIONS: The study findings indicate that, in C57BL/6 mice, consumption of a high-fat diet increases labial groove formation in maxillary incisors, which is related to aging of the tissue stem cells in the apical root end of the teeth.
Assuntos
Incisivo , Ápice Dentário , Envelhecimento , Animais , Dieta Hiperlipídica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structure helps to elucidate the process of organogenesis. Turing-type reaction-diffusion mechanisms have been shown to play a critical role in regulating periodic patterning in organogenesis. Palatal rugae are periodically patterned ridges situated on the hard palate of mammals. We have previously shown that the palatal rugae develop by a Turing-type reaction-diffusion mechanism, which is reliant upon Shh (as an inhibitor) and Fgf (as an activator) signaling for appropriate organization of these structures. The disturbance of Shh and Fgf signaling lead to disorganized palatal rugae. However, the mechanism itself is not fully understood. Here we found that Lrp4 (transmembrane protein) was expressed in a complementary pattern to Wise (a secreted BMP antagonist and Wnt modulator) expression in palatal rugae development, representing Lrp4 expression in developing rugae and Wise in the inter-rugal epithelium. Highly disorganized palatal rugae was observed in both Wise and Lrp4 mutant mice, and these mutants also showed the downregulation of Shh signaling, which was accompanied with upregulation of Fgf signaling. Wise and Lrp4 are thus likely to control palatal rugae development by regulating reaction-diffusion mechanisms through Shh and Fgf signaling. We also found that Bmp and Wnt signaling were partially involved in this mechanism.
Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Palato Duro/embriologia , Palato Duro/metabolismo , Receptores de LDL/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/genética , Difusão , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Relacionadas a Receptor de LDL , Camundongos , Camundongos Mutantes , Palato Duro/patologia , Fenótipo , Receptores de LDL/genética , Transdução de SinaisRESUMO
Stress is a well-known cause of numerous digestive conditions, including gastrointestinal-function disorders. The autonomic nervous system regulates intestinal movements via cholinergic and adrenergic efferent fibers; however it is not clear how stress could affect these control mechanisms and in particular whether in a site-dependent manner. In this study we tested in vitro the effects of topical application of acetylcholine (Ach) and adrenalin (Adr) on smooth-muscle contractions of intestinal segments isolated from stress-conditioned rats. Stress was loaded by hypergravity stimulation (10min/day) for periods of 1, 6 or 30days. As a result, stress-conditioning affected intestinal sensitivity to Ach and Adr differently at sections of the ileum and colon. In the ileum no significant differences were found between control and stress-conditioned rats, whereas in the colon, samples from 6- and 30-day stress-conditioned rats showed larger amplitudes of Ach-induced contraction, as well as greater antagonization by Adr application. These results suggest that stress conditioning can modify autonomic control of intestinal movements by altering smooth-muscle sensitivity to Ach and Adr.
Assuntos
Colo/fisiopatologia , Íleo/fisiopatologia , Músculo Liso/fisiopatologia , Estresse Psicológico/fisiopatologia , Acetilcolina/metabolismo , Animais , Modelos Animais de Doenças , Epinefrina/metabolismo , Hipergravidade , Masculino , Contração Muscular/fisiologia , Ratos Wistar , Fatores de TempoRESUMO
Fibroblast growth factor (FGF) signaling is involved in skeletal development. Among total 22 FGFs, it is suggested that FGF18 functions in promotion of osteoblast differentiation. In order to elucidate the mechanism of FGF18-dependent acceleration of osteogenesis, we implanted rhFGF18 soaked beads over mouse fetal coronal sutures using ex-utero surgery. The coronal suture area comprises the peripheries of the developing frontal and parietal bones, separated by the sutural mesenchyme. rhFGF18 accelerated osteogenesis by promoting connection of the frontal and parietal bone domains, resulting in elimination of the sutural mesenchyme. Expression of Fgf receptors, Fgfr1, -2 and -3 involved in skeletal development, was maintained or upregulated in the developing bone domains, consistent with enhanced osteogenesis. Bone morphogenetic protein (Bmp) 2 was specifically upregulated in the skeletogenic layer and the application of Bmp antagonist, rmNoggin, inhibited rhFGF18-dependent upregulation of osteoblast markers. These results suggest that FGF18 accelerates osteogenesis by upregulation of Bmp2 as well as maintenance or upregulation of Fgfr1, -2 and -3 expression in osteoblasts.
Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/biossíntese , Osteoblastos/citologia , Animais , Desenvolvimento Ósseo/genética , Proteína Morfogenética Óssea 2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Osteoblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Hertwig's epithelial root sheath (HERS), a bilayered epithelial cell sheath located at the cervical loop of the enamel organ in a developing tooth, is at the forefront of root formation. However, little is known about the exact mechanisms that regulate the development of HERS. The neuropeptide vasoactive intestinal peptide (VIP) is involved in the development of various tissues and cells. In this study, we investigated the roles of VIP in HERS development. VIP-immunoreactive nerve fibers were found in the dental pulp and around the root apex of the tooth, while the expression of VIP receptor 1 (VPAC1) was observed in HERS. The expression level of VPAC1 correlated with the development of HERS and was elevated at postnatal days 14 and 21. Using ex vivo cultures of neonatal tooth germs, VIP enhanced the elongation and proliferation of HERS. In vitro, VIP also promoted the proliferation of cells from the HERS-derived cell line, HERS01a cells, and upregulated the mRNA expression of cytokeratin 14 and vimentin (typical molecular markers of HERS) in these cells. These results suggest that VIP may be an essential factor for HERS development.
Assuntos
Órgão do Esmalte/citologia , Células Epiteliais/metabolismo , Peptídeo Intestinal Vasoativo/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Órgão do Esmalte/efeitos dos fármacos , Órgão do Esmalte/crescimento & desenvolvimento , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Queratina-14/genética , Queratina-14/metabolismo , Masculino , Camundongos , Camundongos SCID , RNA Mensageiro/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Raiz Dentária/citologia , Raiz Dentária/efeitos dos fármacos , Raiz Dentária/crescimento & desenvolvimento , Peptídeo Intestinal Vasoativo/farmacologia , Vimentina/genética , Vimentina/metabolismoRESUMO
To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering.
Assuntos
Acrilatos/química , Proteína Morfogenética Óssea 2/farmacologia , Colesterol/química , Fatores de Crescimento de Fibroblastos/farmacologia , Glucanos/química , Polietilenoglicóis/química , Polietilenoimina/química , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta/farmacologia , Animais , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Linhagem da Célula/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nanogéis , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Proteínas Recombinantes/farmacologia , Rodaminas/metabolismo , Tomografia Computadorizada por Raios X , Cicatrização/efeitos dos fármacosRESUMO
The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.
Assuntos
Receptores de LDL/metabolismo , Transdução de Sinais , Dente/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Embrião de Mamíferos/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de LDL/genética , Dente Supranumerário/embriologia , Proteínas Wnt/metabolismoRESUMO
Cajal bodies contain cyclin E/cdk2 and the substrate p220(NPAT) to regulate the transcription of histones, which is essential for cell proliferation, however, recent mouse knockout studies indicate that cyclin E and cdk2 are dispensable for these events. Because the CBP/p300 histone acetyltransferase are also known to be involved in cell proliferation, we examined the molecular and functional interactions of p220(NPAT) with the CBP/p300 at the G1/S boundary as cell cycle regulators. The subnuclear localization of p220(NPAT) and CBP/p300 proteins showed that their foci partially overlapped in a cell cycle dependent manner. Overexpression of p220(NPAT) and CBP/p300 cooperatively enhanced G1/S transition and DNA synthesis even without cdk2 phosphorylation site. Finally, molecular alignment analysis indicated that p220(NPAT) contains several potential substrate sites for CBP/p300. Overall, our findings demonstrate that p220(NPAT) and CBP/p300 form a transient complex at the G1/S boundary to play cooperative roles to promote the S-phase entry.