Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.
Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.
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Abstract Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.
The breakup length of ligaments and the diameter of drops formed from ligaments were measured by feeding of aqueous glycerol solution and liquid paraffin on disks rotating at 10002500 rpm. The breakup length increases with the flow rate and with the rotating velocity. The diameter of drops formed from ligaments increases with the flow rate and decreases with rotating velocity.The stability theory of liquid jets is applied to predict the breakup length of ligaments and the drop diameter by modeling of ligaments as liquid jets ejected from imaginary rotating nozzles. Theoretical predictions show good agreements with both experimental data of breakup length and drop diameter. For practical use, furthermore, the diameter of drops formed from ligaments is correlated with dimensionless groups.
Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.
