All-trans Retinoic Acid Modulates Wnt3A-induced Osteogenic Differentiation of Mesenchymal Stem Cells Via Activating the PI3K/AKT/GSK3β Signalling Pathway

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a vital process for the maintenance of healthy bone tissue and is mediated by numerous factors. Canonical Wnt signalling is essential for MSC osteogenic differentiation, and it interacts with several nuclear receptors, including the retinoic acid receptor, vitamin D receptor, and glucocorticoid receptor. Here, we explored whether Wnt3A and all-trans-retinoic acid (ATRA) play synergistic roles in MSC osteogenic differentiation. We found that ATRA potentiated the Wnt3A-induced expression of early and late osteogenic markers as well as matrix mineralization and further confirmed the phenomena using foetal limb explant culture and MSC implantation experiments. Mechanistically, ATRA cooperated with Wnt3A to induce β-catenin translocation from cell-cell contacts into the cytosol and nucleus, thereby activating Wnt/β-catenin signalling. Additionally, Wnt3A attenuated ATRA-induced Cyp26a1 expression, inhibiting the degradation of ATRA into its oxidative forms. β-catenin silencing abolished the stimulatory effect of ATRA on Wnt3A-induced alkaline phosphatase (ALP) activity and reversed its inhibitory effect on Cyp26a1 expression. Furthermore, ATRA and Wnt3A synergistically promoted AKT phosphorylation, enhancing β-catenin-dependent transcription through GSK3β inhibition or direct β-catenin phosphorylation at Ser552. This event was largely abolished by LY294002 pre-treatment, suggesting that ATRA and Wnt3A at least partially promote osteogenic differentiation via activating the PI3K/AKT/GSK3β signalling pathway. Thus, crosstalk between the Wnt/β-catenin and retinoic acid signalling pathways may be an effective therapeutic target for bone diseases, such as osteoporosis.