A novel junctional epithelial cell line, mHAT-JE01, derived from incisor epithelial cells.

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.

Semaphorin-RhoA signaling regulates HERS maintenance by acting against TGF-ß-induced EMT.

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.

Energy metabolic shift contributes to the phenotype modulation of maturation stage ameloblasts.

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.

LPA6-RhoA signals regulate junctional complexes for polarity and morphology establishment of maturation stage ameloblasts.

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.

Elongation of the side chain by linear alkyl groups increases the potency of salacinol, a potent α-glucosidase inhibitor from the Ayurvedic traditional medicine "Salacia," against human intestinal maltase.

Four chain-extended analogs (12a-12d) and two related de-O-sulfonated analogs (13a and 13c) by introducing alkyl groups (a: R = C3H7, b R = C6H13, c: R = C8H17, d: R = C10H21) to the side chains of salacinol (1), a natural α-glucosidase inhibitor from Ayurvedic traditional medicine "Salacia", were synthesized. The α-glucosidase inhibitory activities of all the synthesized analogs were evaluated in vitro. Against human intestinal maltase, the inhibitory activities of 12a and 13a with seven-carbon side chain were equal to that of 1. In contrast, analogs (12b-12d, and 13c) exhibited higher level of inhibitory activity against the same enzyme than 1 and had equal or higher potency than those of the clinically used anti-diabetics, voglibose, acarbose, and miglitol. Thus, elongation of the side chains of 1 was effective for specifically increasing the inhibitory activity against human intestinal maltase.

Harmine promotes molar root development via SMAD1/5/8 phosphorylation.

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.

Regulatory mechanisms of Hertwig׳s epithelial root sheath formation and anomaly correlated with root length.

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.

Stem cell sources for tooth regeneration: current status and future prospects.

Stem cells are capable of renewing themselves through cell division and have the remarkable ability to differentiate into many different types of cells. They therefore have the potential to become a central tool in regenerative medicine. During the last decade, advances in tissue engineering and stem cell-based tooth regeneration have provided realistic and attractive means of replacing lost or damaged teeth. Investigation of embryonic and adult (tissue) stem cells as potential cell sources for tooth regeneration has led to many promising results. However, technical and ethical issues have hindered the availability of these cells for clinical application. The recent discovery of induced pluripotent stem (iPS) cells has provided the possibility to revolutionize the field of regenerative medicine (dentistry) by offering the option of autologous transplantation. In this article, we review the current progress in the field of stem cell-based tooth regeneration and discuss the possibility of using iPS cells for this purpose.

[Clinical evaluation of calculating carboplatin dosage using Japanese equation for estimating GFR for gynecologic cancer].

Glomerular filtration rate (GFR) is an important factor when considering carboplatin dosage adjustment. The Japanese equation for estimating GFR (eGFR) was recommended as a guideline for evaluating GFR in 2009 by The Japanese Society of Nephrology. However, benefits in the field of cancer chemotherapy with the use of eGFR have not yet been shown. To clarify the clinical benefits of eGFR, we investigated the renal function of 100 patients with gynecologic cancer who were treated with carboplatin from 2003 through 2009, and the carboplatin dosage was calculated by the Calvert formula in which eGFR was substituted for GFR. To predict the clinical benefit on the basis of carboplatin dosage using eGFR, we retrospectively divided the patients into two groups so that carboplatin dosage was within dosage in using eGFR and one was not. We compared response rates and adverse effects of the two groups. Renal function using eGFR was lower than renal function calculated by using the other formulae. Carboplatin dosage using eGFR was significantly lower than the dosage calculated with the other formulae (p<0.01). Moreover, the patients group actually, administered the dosage calculated by eGFR showed less side effects than the group of patients not treated this way, but the efficacy did not change. Thus, using eGFR in planning carboplatin dosage suggested clinical application to patients with Japanese gynecologic cancer.

Docking and SAR studies of salacinol derivatives as alpha-glucosidase inhibitors.

Salacinol is a potent alpha-glucosidase inhibitor isolated from Salacia reticulata, and a good lead compound for an antidiabetic drug. It is essential to clarify the binding state of salacinol to alpha-glucosidase for efficient optimization study using structure-based drug design. Redocking simulations of two inhibitors, acarbose and casuarine whose complex structures are known, were performed to assure the appropriate docking pose prediction. The simulation reproduced both experimental binding states with accuracy. Then, using the same simulation protocol, the binding mode of salacinol and its derivatives has been predicted. Salacinol bound to the protein with a similar binding mode as casuarine, and the predicted structures could explain most of the structure-activity relationships of salacinol derivatives.