5.1 Tissue distribution of melatonin receptors
Melatonin receptors have been found in many tissues, but the studies published are largely on membrane melatonin receptors, MT1 and MT2. Both MT1 and MT2 receptors are expressed in the SCN, retina, vasculature, immune system, reproductive system, pancreas, skin, gastrointestinal tract, and kidneys, while studies only reported that MT1 receptor had been detected in bone (Slominski et al., 2012). MT1 receptors have been found in osteoblasts, osteoclasts, osteosarcoma cells and also bone marrow stromal cells (Suzuki, Somei, Kitamura, Reiter, & Hattori, 2008; Toma et al., 2007). In dental tissues, the MT1 receptor has been detected in fibroblasts of the oral mucosa (Cutando et al., 2011). Kumasaka et al. reported that MT1 receptors had been detected by immunohistochemical analysis in secretory ameloblasts, odontoblasts and the cells of stratum intermedium, stellate reticulum, outer enamel epithelium in the tooth germs of human mandibular third molars. In addition, RT-PCR and Western blot analysis also revealed that HAT-7, a rat dental epithelial cell line, expressed MT1 receptors (Kumasaka et al., 2010).
In contrast to the MT1 and MT2 receptors, MT3 is a low-affinity melatonin binding site and has been characterized as the cytosolic enzyme, NQO2. It is expressed in the brain, liver, kidney, heart, lung, adipose tissue, and eye (Nosjean et al., 2000; Pintor, Peláez, Hoyle, & Peral, 2003). As for the nuclear receptors of melatonin, they belong to the ROR/RZR family. RORβ is a brain and retina-specific receptor, while RORα is also expressed in many tissues including liver, heart, and skin (Pozo, García‐Mauriño,
Guerrero, & Calvo, 2004; Smirnov, 2001). However, these receptors have not yet been detected in dental tissue.
Our study first demonstrated that apical papilla cells expressed melatonin receptors, MT1, MT2, MT3/NQO2, and RORα. Previous research has shown that both 1 mM and 2.5 mM melatonin enhanced the protein expression of MT1, MT3, and RORα in HepG2 human hepatocarcinoma cells (Carbajo-Pescador et al., 2009). However, the expression of these melatonin receptors showed no obvious change by melatonin treatment in our study.
5.2 Effect of melatonin in odontogenesis and osteogenesis
Stimulatory effects of melatonin on bone formation and its inhibitory effects on bone resorption have been reported in many studies (Maria & Witt-Enderby, 2014). It has been suggested that the osteoblast-enhancing function of melatonin is mediated by its direct action on the differentiation and proliferation of the bone-forming cells.
Nakade et al. demonstrated the effect of melatonin on osteogenic actin in normal human bone cells (HOB-M cells) and human osteoblastic cell line (SV-HFO). Melatonin dose-dependently increased the proliferation of HOB-M cells and SV-HFO with a maximal effect at a concentration of 50 µM after 24-hour incubation. The effect on bone cell differentiation was evaluated after 48-hour melatonin treatment. While the result showed that melatonin did not affect either the ALP activity or the OCN secretion, it significantly increased the type I collagen synthesis at concentrations between 50 and 100 µM in human bone cells (Nakade, Koyama, Ariji, Yajima, & Kaku, 1999). Radio et al.
reported that melatonin promoted the differentiation of human adult mesenchymal stem cells (hAMSCs) into osteogenic lineages. Melatonin at a physiological concentration (50
nM) in combination with an osteogenic medium following a 10-day incubation, significantly increased ALP activity relative to osteogenic medium alone (Radio, Doctor,
& Witt-Enderby, 2006).
Satomura et al. performed a study to evaluate the effect of melatonin on the proliferation and differentiation of primary cultured human osteoblasts and also measured newly bone formation after intraperitoneal administration of melatonin to mice. RT-PCR and Western blot analysis showed that human osteoblasts expressed MT1 receptor. Later than 5-days incubation, melatonin at pharmacological concentrations (100 µM or over) stimulated the proliferation, ALP activity, mineralized matrix formation and promoted the gene expression of type I collagen, osteopontin, BSP, and OCN. Moreover, the volume of the newly formed cortical bone of femur increased after the mice were treated with for 21 days (Satomura et al., 2007).
In addition, Park et al. demonstrated that melatonin promoted differentiation and migration in MC3T3-E1 cells (mouse osteoblastic cells) at physiological concentrations (25, 50, 75, 100 nM). After melatonin treatment for 2 and 3 days, the expression of osteogenic markers such as OCN, BMP-2 and -4 increased in a dose-dependent manner.
Melatonin treatment also activated Runx2 expression following a short period incubation (2- and 3-hour) (Park et al., 2011). Han et al. found that melatonin (1µM) promoted osteoblast differentiation of C2C12 cells (mouse pre-myoblast cells) (Han, Kim, Kim, &
Lee, 2017). After C2C12 cells cultured in BMP-4 osteogenic induction medium and treated with melatonin for 3 days, the expression of osteogenic markers, Runx2, ALP, BSP, OCN and Osterix significantly increased relative to BMP-4 osteogenic medium alone. The enhancement was especially on Osterix, which is an essential transcription factor in the late stage of osteogenesis for the differentiation of preosteoblasts into mature
osteoblasts. Furthermore, treatment with PKA inhibitor H89 and PKC inhibitor Go6976 blocked the melatonin-induced transcriptional activity of Osterix, indicating that melatonin regulated Osterix expression via the PKA and PKC signaling pathways.
Another study has also been reported that physiological concentrations of melatonin (nanomolar range) induced mRNA expression of nestin in the C17.2 neural stem cell line.
However, nestin was thought to be a neural stem cell marker in this study.
In our examination, we did not find the similar result that melatonin has the stimulatory effect on the expression of odontogenic/osteogenic differentiation markers BSP, OCN, ALP and Nestin in apical papilla cells. As for collagen, our results demonstrated that collagen I expression would be inhibited by high-dose melatonin.
However, we found the mRNA expression of Runx2 and the protein level SP7 expression were in a trend to increase in a melatonin dose-dependent manner. These different results between transcription factors and differentiation markers might be the regulation involved in different stages of cell differentiation. While the treatment periods, 24-hour and 5-day, in our experiment did not short or long enough distinguish the difference. In addition, the melatonin concentrations in our study are different from previous studies which used the physiological concentrations or less than 1µM melatonin. Our results of MTT assays showed the significant suppression of cell number in 500 µg/ml melatonin-treated groups.
This higher dose of melatonin might also be a reason to explain that our results were different to which in previous studies.
5.3 Effect of melatonin on actin filaments
Reorganization of actin filaments plays a central role in cell migration. To migration, normally a cell must become polarized and continuously protrude a
lamellipodium toward the direction of movement. The initial step of lamellipodium formation is actin assembly and forms a protrusion with polarized actin filaments near the plasma membrane. Later, actin filaments near the front of the cell are disassembled to provide actin monomers for further polymerization at the tip of the lamellipodium that pushing the membrane forward (Nishita et al., 2005). cofilin1 (a non-muscle type of cofilin) plays an essential role in actin filament dynamics and reorganization by stimulating depolymerization and severance of actin filaments. This action is negatively regulated by cofilin phosphorylation at Ser-3 and play an essential role in the lamellipodial protrusion. In the initial stage of the cell migration, phospho-cofilin leads actin assembly for lamellipodium formation and then stabilizes actin filaments at the front and rear of the cell that helps lamellipodium extension and cell polarization. In later stages, the continuous lamellipodial protrusion is mediated by cofilin which stimulates actin filament disassembly and replenishes actin monomers for further polymerization at the leading edge of the migrating cell (Mizuno, 2013). Nishita et al. demonstrated that the phosphorylation of cofilin by LIMK1 plays a critical role in cell migration by a model of stromal cell-derived factor-1α (SDF-1α) induced chemotactic response of T lymphocytes.
Besides, Chen et al. recently reported that could be chemoattracted by SDF-1α, and their result of phalloidin staining showed that F-actin stress fiber assembly in SCAP which treated with SDF-1α for 24 hours (X. Chen, Liu, Yue, Huang, & Zou, 2016).
Our study showed the effect of melatonin on stimulating p-cofilin expression in apical papilla cells. As for total cofilin1 expression, no influence was found in different concentrations of melatonin-treated groups. This result may indicate that the effect of melatonin is on the phosphorylation of cofilin1 rather than on the stimulatory or inhibitory effects of cofilin1 expression. For the 24-hour groups in our study, the increase in the
level of p-cofilin showed that melatonin might have a potential ability to promote the initial stage of cell migration. Moreover, previous studies also revealed the chemotactic effect of melatonin on leukocytes (Pena, Rincon, Pedreanez, Viera, & Mosquera, 2007).
Considering the increase of N-cadherin expression by melatonin treatment in our study, melatonin can be considered as potential chemotaxis which may help to recruit SCAP into root canal space.
The expression of p-cofilin in the 5-day groups was stimulated by melatonin in a dose-dependent manner. This finding is consistent with the actin filaments expression in phalloidin staining. However, the cofilin inactivation can also inhibit lamellipodium extension by suppressing cofilin-mediated G-actin replenishment (Ohashi et al., 2011).
Regarding the actin filament stabilization for a relatively extended period, Chen et al.
recently demonstrated that inhibiting actin depolymerization and stabilizing actin filaments can enhance osteoblast differentiation and bone formation in human stromal stem cells (hMSCs). They cultured the hMSCs with osteogenic induction medium and evaluated the expression of cofilin1, p-cofilin1, LIMK1, p-LIMK1 and actin filament on day 0, 3, 7, 10 and 13. The results showed that actin filament ratio was increased and cofilin1 was phosphorylated after induction of osteoblast differentiation (L. Chen et al., 2015).
5.4 Potential signaling pathways involved in the biological activity of melatonin on human apical papilla cells
Melatonin can bind to the membrane receptors, MT1 and MT2, and exerts the biological functions through the PKA or PKC signaling pathway (Cutando et al., 2011).
In our study, we examined the PKA and PKC mediated pathway by treating the apical
papilla cells with/without H89 or H7 before melatonin treatment. The enhancement of N-cadherin and the suppression of Collagen I expression were not influenced by H89, but the expression of p-cofilin was even more increased. However, in the experiment of H7 pretreatment, we found the stimulation effect of melatonin on cofilin1 phosphorylation was significantly suppressed by 10 µM H7. Sakuma et al. previously reported a PKCα-mediated phosphorylation event on cofilin that inhibiting its ability to depolymerize F-actin and thereby promoting F-F-actin polymerization (Sakuma et al., 2012). Taken together with the findings that we found the expression of p-CREB in apical papilla cells was regulated by melatonin. We assumed that melatonin exerts this biological functions through the PKC-CREB pathway.