Department of Stem Cell Sciences, Graduate School of Health Sciences, Hacettepe University, Ankara, Turkey.
Department of Histology and Embryology, Faculty of Medicine, TOBB Economics and Technology University, Ankara, Turkey.
Neurochem Int. 2021 Sep;148:105079. doi: 10.1016/j.neuint.2021.105079. Epub 2021 May 25.
Dental pulp stem cells (DPSCs) have a 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 suggest the possible interaction between 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 need 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-YAP) 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-YAP. 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 YAP and reduced YAP expression. In conclusion, melatonin has potential to induce neuronal differentiation and reduce the proliferation of DPSCs by increasing phosphorylation of YAP and eliminating the activity of YAP, which indicates the active state of Hippo signaling pathway.
牙髓干细胞（DPSCs）具有高度分化为神经元细胞谱系的能力。同时，Hippo 信号通路和褪黑素都是神经元祖细胞向神经元分化的关键调节因子。最近的研究证据表明，褪黑素和 Hippo 信号通路在不同细胞系之间可能存在相互作用。但是，通过这种联系，在 DPSCs 中启动或促进神经发生分化的潜在机制仍需要进一步探索。因此，本研究的目的是研究褪黑素通过其下游效应物（YAP/p-YAP）在 DPSCs 向神经元分化过程中对 Hippo 信号通路的影响。为此，将 DPSCs 分别在含有生长和多巴胺能神经元分化培养基的条件下，或添加 10 μM 褪黑素的条件下孵育 21 天。通过相差显微镜观察细胞形态变化，通过免疫荧光标记 NeuN、GFAP 和酪氨酸羟化酶来评估 DPSCs 的分化情况。此外，通过巢蛋白免疫反应性评估神经前体细胞的存在。通过检测 YAP 和 p-YAP 的免疫反应性来研究 Hippo 信号通路。Western blot 分析也支持我们的结果，还评估了 SOX2、PCNA 和 caspase-3 的表达情况。NeuN、酪氨酸羟化酶的阳性免疫反应和 GFAP 的阴性免疫反应表明 DPSCs 向神经元分化，而不是向神经胶质细胞分化。向多巴胺能培养基中添加褪黑素可诱导酪氨酸羟化酶的表达，并显著降低 nestin 的表达。在生长培养基中添加褪黑素还可显著降低 PCNA 和 caspase-3 的表达。褪黑素处理可诱导 YAP 的磷酸化并减少 YAP 的表达。综上所述，褪黑素通过增加 YAP 的磷酸化和消除 YAP 的活性，从而诱导神经元分化并减少 DPSCs 的增殖，这表明 Hippo 信号通路处于活跃状态。
