Retinoic acid is a potential negative regulator for differentiation of human periodontal ligament cells

Background and objectives: Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells. Methods and results: Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and β-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 μM RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-α agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-α was expressed on the cells, suggesting that RAR-α was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-α, PDGF receptor-β, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses. Conclusions: These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.