Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Transient and recurrent expression of the Egr-1 gene in epithelial and mesenchymal cells during tooth morphogenesis suggests involvement in tissue interactions and in determination of cell fate.

We have analyzed the expression of early growth response gene (Egr-1) by mRNA in situ hybridization during mouse embryonic tooth development and in experimental recombinations of dental epithelium and mesenchyme. Egr-1 was transiently and recurrently expressed both in epithelial and mesenchymal cells starting from day 13 of gestation and up to 4 days after birth. The expression correlated with developmental transition points of dental mesenchymal and epithelial cells suggesting a role for Egr-1 in sequential determination and differentiation of cells. In recombination cultures of early dental epithelium and mesenchyme Egr-1 RNA was localized at the epithelial-mesenchymal interface in mesenchymal cells, and in two cases also in epithelial cells. These data indicate that Egr-1 expression may be regulated by epithelial-mesenchymal interactions when they are specific enough to initiate differentiation. We have also analyzed by in situ hybridization whether Wilms' tumour-1 gene (wt-1) is expressed in the developing tooth as it was proposed on the bases of in vitro studies that it may inhibit Egr-1 expression. No wt-1 expression was detected at any stage of tooth development showing that wt-1 is not obligatory for regulation of Egr-1 expression.     

Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.     

Diurnal expressions of four subtypes of melatonin receptor genes in the optic tectum and retina of goldfish

Four subtypes of melatonin receptor genes (Mel(1a) 1.4, Mel(1a) 1.7, Mel(1b), and Mel(1c)) are considered to be expressed to mediate various physiological functions of melatonin in goldfish (Carassius auratus). To examine their tissue distribution and diurnal changes in expression levels, we cloned partial gene fragments for these melatonin receptor subtypes, and established specific RT-PCR and quantitative real-time PCR systems. Mel(1a) 1.4 and Mel(1b) were predominantly expressed in various neuronal and peripheral tissues, while Mel(1a) 1.7 and Mel(1c) were expressed in the restricted tissues. All subtype genes were expressed in the optic tectum, diencephalon, mesencephalon, vagal lobe, retina and spleen. The real-time PCR analyses showed that significant differences among time were observed for Mel(1a) 1.4 in the optic tectum and for Mel(1a) 1.7 and Mel(1b) in the retina. In the retina, the levels of Mel(1a) 1.7 and Mel(1b) mRNAs showed diurnal changes with one peak at ZT24. The present results show differential distribution of four subtypes of melatonin receptor mRNAs in the neuronal and peripheral tissues. However, the expressions of all subtype genes in the retinorecipient brain regions and retina reinforce the role of the melatonin receptor in processing visual information. Furthermore, the present study demonstrates diurnal expressions of the major subtype genes, i.e. Mel(1a) 1.4 in the optic tectum and Mel(1a) 1.7 in the retina.     

Physiological and metabolic functions of melatonin

Melatonin is a lipophilic hormone, mainly produced and secreted at night by the pineal gland. Melatonin synthesis is under the control of postganglionic sympathetic fibers that innervates the pineal gland. Melatonin acts via high affinity G protein-coupled membrane receptors. To date, three different receptor subtypes have been identified in mammals: MT1 (Mel 1a) and MT2 (Mel 1b) and a putative binding site called MT3. The chronobiotic properties of the hormone for resynchronization of sleep and circadian rhythms disturbances has been demonstrated both in animal models or in clinical trials. Several other physiological effects of melatonin in different peripheral tissues have been described in the past years. In this way, it has been demonstrated that the hormone is involved in the regulation of seasonal reproduction, body weight and energy balance. This contribution has been focused to review some of the physiological functions of melatonin as well as the role of the hormone in the regulation of energy balance and its possible involvement in the development of obesity.     

Polymorphism and signalling of melatonin receptors.

Melatonin receptors belong to the superfamily of G protein-coupled receptors. Cloning of Mel1c receptors expressed in Xenopus skin revealed the existence of a polymorphism for these receptors. Heterologous expression of the two allelic isoforms, called Mel1c(alpha) and Mel1c(beta), indicated functional differences in their signalling properties. Both isoforms are coupled to the cAMP and cGMP pathways. However, the alpha isoform is preferentially coupled to the cAMP pathway, whereas the beta isoform couples preferentially to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between the two isoforms are localized within intracellular receptor regions potentially involved in G protein coupling. Allelic isoforms were also observed for Mel1a receptors expressed in ovine pars tuberalis, suggesting that polymorphism is a general feature of the melatonin receptor family. We also evaluated the potential of the two human melatonin receptor subtypes, Mel1a and Mel1b, to modulate the cGMP pathway. Melatonin inhibited intracellular cGMP levels in a dose-dependent manner in HEK293 cells transfected with the human Mel1b receptor. This was not the case for HEK293 cells transfected with the human Mel1a receptor. In conclusion, our results indicate that the expression of receptor subtypes and isoforms may permit differential signalling between melatonin receptors.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

Cell surface proteoglycan expression correlates with epithelial-mesenchymal interaction during tooth morphogenesis

Tooth morphogenesis and differentiation of the dental cells are guided by interactions between epithelial and mesenchymal tissues. Because the extracellular matrix is involved in these interactions, the expression of matrix receptors located at the cell surface may change during this developmental sequence. We have examined the distribution of an epithelial cell surface proteoglycan antigen, known to behave as a receptor for interstitial matrix, during tooth morphogenesis. Intense staining was seen around the cells of the embryonic oral epithelium as well as the dental epithelium at the early bud stage. With development, expression was greatly reduced in the enamel organ. Differentiation of these cells into ameloblasts was associated with the loss of expression, while the epithelial cells remaining in the stratum intermedium and stellate reticulum regained intense staining. The PG antigen was weakly expressed in the loose neural crest-derived jaw mesenchyme but it became strongly reactive in the condensed dental papilla mesenchyme when extensive morphogenetic movements took place. With development, the PG antigen disappeared from the advanced dental papilla mesenchyme but persisted in the dental sac mesenchyme, which gives rise to periodontal tissues. The PG antigen was not expressed by odontoblasts. Hence, the expression of the PG antigen changes during the epithelial-mesenchymal interactions of tooth development and is lost during terminal cell differentiation. The expression follows morphogenetic rather than histologic boundaries. The acquisition and loss of expression in epithelial and mesenchymal tissues during tooth development suggest that this proteoglycan has specific functions in the epithelial-mesenchymal interactions that guide morphogenesis.     

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.     

Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.     

Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants

The Msx2-interacting nuclear target protein (MINT) is a nuclear matrix protein that regulates the development of many tissues. However, little is known regarding the role of MINT in tooth development. In this study, we prepared polyclonal antibodies against MINT, and found that that MINT was expressed in different cells at each stage of tooth germ development by immunohistochemistry. The role of MINT in tooth development was further illustrated by the misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINT-deficient mandibular explants, the tooth germ was reduced in the overall size and lacked enamel knot, with abnormal dental lamina and collapsed stellate reticulum. Furthermore, the BrdU incorporation experiment showed that the proliferation activity was significantly reduced in MINT-deficient dental epithelium. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis.     

Epidermal growth factor regulates gene expression of both epithelial and mesenchymal cells in mouse molar tooth organs in culture

Epidermal growth factor and cis-hydroxyproline specifically inhibited synthesis of type 1 collagen, a major gene product of the differentiated dental mesenchymal cells (odontoblasts). In tandem, synthesis of enamel proteins, specific gene products of differentiated dental epithelial cells (ameloblasts), was also inhibited. Under these culture conditions, total protein synthesis in tooth organs was not inhibited but rather increased. Inhibition curves of the gene products specific for epithelial and mesenchymal phenotypes were quite similar, indicating coordinate and intimately associated regulation of gene expression under conditions that perturb cytodifferentiation.     

Dpysl4 Is Involved in Tooth Germ Morphogenesis through Growth Regulation,Polarization and Differentiation of Dental Epithelial Cells

Dihydropyrimidinase-related protein 4 (Dpysl4) is a known regulator of hippocampal neuron development. Here, we report that Dpysl4 is involved in growth regulation, polarization and differentiation of dental epithelial cells during tooth germ morphogenesis. A reduction in Dpysl4 gene expression in the tooth germ produced a loss of ameloblasts, resulting in the decrease of synthesis and secretion of enamel. The inhibition of Dpysl4 gene expression led to promotion of cell proliferation of inner enamel epithelial cells and inhibition of the differentiation of these cells into pre-ameloblasts, which was confirmed by analyzing cell polarization, columnar cell structure formation and the expression of ameloblast marker genes. By contrast, overexpression of Dpysl4 in dental epithelial cells induces inhibition of growth and increases the expression of the inner enamel epithelial cell marker gene, Msx2. These findings suggest that Dpysl4 plays essential roles in tooth germ morphogenesis through the regulation of dental epithelial cell proliferation, cell polarization and differentiation.     

Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis

Wnt/beta-catenin signaling plays key roles in tooth development, but how this pathway intersects with the complex interplay of signaling factors regulating dental morphogenesis has been unclear. We demonstrate that Wnt/beta-catenin signaling is active at multiple stages of tooth development. Mutation of beta-catenin to a constitutively active form in oral epithelium causes formation of large, misshapen tooth buds and ectopic teeth, and expanded expression of signaling molecules important for tooth development. Conversely, expression of key morphogenetic regulators including Bmp4, Msx1, and Msx2 is downregulated in embryos expressing the secreted Wnt inhibitor Dkk1 which blocks signaling in epithelial and underlying mesenchymal cells. Similar phenotypes are observed in embryos lacking epithelial beta-catenin, demonstrating a requirement for Wnt signaling within the epithelium. Inducible Dkk1 expression after the bud stage causes formation of blunted molar cusps, downregulation of the enamel knot marker p21, and loss of restricted ectodin expression, revealing requirements for Wnt activity in maintaining secondary enamel knots. These data place Wnt/beta-catenin signaling upstream of key morphogenetic signaling pathways at multiple stages of tooth development and indicate that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth.     

Reproductive phase dependent daily variation in melatonin receptors (Mel(1a) and Mel(1b)), androgen receptor (AR) and lung associated immunity of Perdicula asiatica

Our knowledge about the involvement of melatonin in the regulation of lung associated immune system (LAIS) is still poor though the melatonin receptor types (Mel(1a) and Mel(1b)) have been localized in lungs of some wild birds. We thought to explore the correlation between daily variation (within a 24h time scale) in peripheral melatonin and testosterone along with expression of melatonin receptors (Mel(1a) and Mel(1b)) and androgen receptor (AR) in lungs during reproductively active and inactive phases. Receptor expression of Mel(1b) was more prominent than Mel(1a) at all the time points during both the reproductive phases. The expression of AR was inversely related to both the melatonin and its receptor expression at the 24h time scale during both the reproductive phases. Results also reflected a parallel relationship of melatonin, melatonin receptors and all the immune parameters (total leukocyte count, lymphocyte count, % stimulation ratio) suggesting that peripheral melatonin might be responsible for daily periodicity of LAIS. The presence of androgen receptors in lung led us to propose that gonadal steroid does influence the LAIS. Therefore melatonin along with testosterone might be acting as a temporal synchronizer for daily rhythms in lung associated immunity in Perdicula asiatica during different reproductive phases.     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

Tooth regeneration from newly established cell lines from a molar tooth germ epithelium

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.