Impact of melatonin on periodontal ligament fibroblasts during mechanical strain.

BACKGROUND: The endogenous hormone melatonin regulates the circadian rhythm and impacts on bone metabolism. As patient compliance to wear removable orthodontic appliances is generally higher at night, when melatonin release is increased, a boosting effect on tooth movement would be favourable for therapy, whereas an inhibiting effect would indicate daytime wear to be more therapy-effective. We hypothesize that melatonin has either a stimulating or impeding effect on the expression profile of periodontal ligament fibroblasts (PDLF) during simulated orthodontic compressive and tensile strain, which would suggest either an accelerating or inhibiting impact on orthodontic tooth movement in vivo. METHODS: PDLF were preincubated with melatonin for 24 h and then subjected to tensile or compressive strain to mimic tension and pressure sides in PDL. In addition, the selective melatonin MTNR1B-receptor antagonist 4P-PDOT was used. We investigated melatonin effects on collagen synthesis, expression of inflammatory and bone-remodelling genes/proteins by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and total collagen assays. PDLF-induced osteoclastogenesis was analysed in a coculture model by tartrate-resistant acid phosphatise (TRAP) staining. RESULTS: Expression of melatonin receptors in PDLF was not affected by compressive strain. Melatonin increased expression of inflammatory factors and elevated collagen synthesis during mechanical strain. Melatonin showed no effects on OPG or RANKL expression without mechanical strain, but increased RANKL gene expression during compression. CONCLUSIONS: Expression of melatonin receptors by PDLF enable them to detect fluctuating melatonin concentrations in the periodontal ligament. Melatonin increased collagen synthesis and expression of inflammatory mediators, but had no effect on genes involved in bone remodelling. Therefore, we suggest that melatonin has no accelerating effect on PDLF-induced osteoclastogenesis.

Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

Effect of melatonin on human dental papilla cells.

Melatonin regulates a variety of biological processes, which are the control of circadian rhythms, regulation of seasonal reproductive function and body temperature, free radical scavenging and so on. Our previous studies have shown that various cells exist in human and mouse tooth germs that express the melatonin 1a receptor (Mel1aR). However, little is known about the effects of melatonin on tooth development and growth. The present study was performed to examine the possibility that melatonin might exert its influence on tooth development. DP-805 cells, a human dental papilla cell line, were shown to express Mel1aR. Expression levels of mRNA for Mel1aR in DP-805 cells increased until 3 days after reaching confluence and decreased thereafter. Real-time reverse transcription-polymerase chain reaction showed that melatonin increased the expression of mRNAs for osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotin (DSPP). Melatonin also enhanced the mineralized matrix formation in DP-805 cell cultures in a dose-dependent manner. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.

Selenomethionine exposure affects chondrogenic differentiation and bone formation in Japanese medaka (Oryzias latipes).

Excess selenium entering the aquatic environment from anthropogenic activities has been associated with developmental abnormalities in fish including skeletal deformities of the head and spine. However, mechanisms of this developmental toxicity have not been well-characterized. In this study, Japanese medaka (Oryzias latipes) embryos were exposed to seleno-l-methionine (Se-Met) in a range of concentrations. Gene expression was evaluated for sex-determining region Y (SRY)-related box (Sox9a and Sox9b), runt-related transcription factor 2 (Runx2), and melatonin receptor (Mtr). Alterations in the length of Meckel's cartilage, tail curvature, and decreased calcification were observed in skeletal stains at 10- and 22-days post-fertilization (dpf). Embryonic exposure of Osterix-mCherry transgenic medaka resulted in fewer teeth. Sox9a and Sox9b were up-regulated, while Runx2 and Mtr were down-regulated by Se-Met prior to hatch. Whole mount in situ hybridization (WISH) localized gene expression to areas observed to be affected in vivo. In addition, Se-Met exposures of a Mtr morpholino (Mtr-MO) as well as Luzindole exposed embryos developed similar skeletal malformations, supporting involvement of Mtr. These findings demonstrate that Se-Met modulates expression of key genes involved in chondrogenic differentiation and bone formation during development.

Circadian Rhythm Regulates Development of Enamel in Mouse Mandibular First Molar.

Rhythmic incremental growth lines and the presence of melatonin receptors were discovered in tooth enamel, suggesting possible role of circadian rhythm. We therefore hypothesized that circadian rhythm may regulate enamel formation through melatonin receptors. To test this hypothesis, we examined expression of melatonin receptors (MTs) and amelogenin (AMELX), a maker of enamel formation, during tooth germ development in mouse. Using qRT-PCR and immunocytochemistry, we found that mRNA and protein levels of both MTs and AMELX in normal mandibular first molar tooth germs increased gradually after birth, peaked at 3 or 4 day postnatal, and then decreased. Expression of MTs and AMELX by immunocytochemistry was significantly delayed in neonatal mice raised in all-dark or all-light environment as well as the enamel development. Furthermore, development of tooth enamel was also delayed showing significant immature histology in those animals, especially for newborn mice raised in all daylight condition. Interestingly, disruption in circadian rhythm in pregnant mice also resulted in delayed enamel development in their babies. Treatment with melatonin receptor antagonist 4P-PDOT in pregnant mice caused underexpression of MTs and AMELX associated with long-lasting deficiency in baby enamel tissue. Electromicroscopic evidence demonstrated increased necrosis and poor enamel mineralization in ameloblasts. The above results suggest that circadian rhythm is important for normal enamel development at both pre- and postnatal stages. Melatonin receptors were partly responsible for the regulation.