Melatonin Contributes to The Hypertrophic Differentiation of Mesenchymal Stem Cell-Derived Chondrocytes Via Activation of The WNT/β-Catenin Signaling Pathway

Abstract BackgroundHypertrophy is a critical process for chondrocyte differentiation and maturation during endochondral ossification, which is responsible for the formation of long bone and its postnatal longitudinal growth. Increasing evidence suggests that melatonin, an indole hormone, plays a pivotal role in chondrogenesis; however, little is known about its effects on the terminal differentiation of chondrocytes. MethodsMesenchymal stem cells (MSCs) derived chondrocytes generated by a high-density micromass culture system were further induced to hypertrophic differentiation. The melatonin-mediated hypertrophic differentiation were examined by reverse transcription polymerase chain reaction analysis (RT-PCR) analysis and histological staining and immunohistochemistry. The expression of downstream factors of WNT signaling pathway was evaluated by RT-PCR, western blotting and immunofluorescence. The WNT signaling pathway antagonist XAV-939 was used to further demonstrate melatonin-induced hypertrophic chondrocytes and WNT signaling pathway activation.ResultsHistological staining showed melatonin increased the chondrocytes cell volume and the expression of type X collagen, but decreased the expression of type II collagen compare to the control group. RT-PCR showed that melatonin significantly up-regulated the expression of biomarkers of hypertrophic chondrocytes, including type X collagen (COL10A1), alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), Indian hedgehog (IHH), and parathyroid hormone-related protein receptor (PTHrP-R) and down-regulated the hallmarks of chondrocytes, including parathyroid hormone-related protein (PTHrP). The WNT signaling pathway PCR array showed that the effect of melatonin was accompanied by the up-regulation of multiple target genes of the canonical WNT signaling pathway and the melatonin-mediated effect can be blocked by XAV-939. ConclusionsThese findings demonstrate that melatonin can enhance the hypertrophic differentiation of MSCs-derived chondrocytes through the WNT signaling pathway. It adds evidence to the role of melatonin in promoting bone development and highlights its positive effects on chondrocytes terminal differentiation.