The effect of melatonin on Hippo signaling pathway in dental pulp stem cells

Abstract

Dental pulp stem cells (DPSCs) have high capacity to differentiate into the neuronal cell lineage. Meanwhile, both Hippo signaling and melatonin are key regulators in neuronal differentiation of neuronal progenitor cells. Recently emerging evidences suggests the possible interaction between the melatonin and Hippo signaling in different cell lines. But underlying mechanisms involved in the initiation or progression of neurogenic differentiation in DPSCs through this connection needs to be explored. Therefore, the scope of this study is to investigate the effect of melatonin on Hippo signaling pathway through the expression of its downstream effector (YAP/ p-YAPY357) after the neuronal differentiation of DPSCs. In regard with this, DPSCs were incubated with growth and dopaminergic neuronal differentiation medium with or without melatonin (10 μM) for 21 days. The morphological changes were followed by phase contrast microscopy and differentiation of DPSCs was evaluated by immunofluorescence labelling with NeuN, GFAP, and tyrosine hydroxylase. Furthermore, we evaluated the presence of neural progenitor cells by nestin immunoreactivity. Hippo signaling pathway was investigated by evaluating the immunoreactivity of YAP and p-YAPY357. Our results were also supported by western-blot analysis and SOX2, PCNA and caspase-3 were also evaluated. The positive immunoreactivity for NeuN, tyrosine hydroxylase and negative immunoreactivity for GFAP showed the successful differentiation of DPSCs to neurons, not glial cells. Melatonin addition to dopaminergic media induced tyrosine hydroxylase and decreased significantly nestin expression. The expressions of PCNA and caspase-3 were also decreased significantly with melatonin addition into growth media. Melatonin treatment induced phosphorylation of YAPY357 and reduced YAP expression. In conclusion, melatonin has potential to induce the neuronal differentiation and reduce proliferation of DPSCs by increasing phosphorylation of YAPY357 and eliminating the activity of YAP, which indicates the active state of Hippo signaling pathway.