<scp>MECP2</scp> Insufficiency Attenuates <scp>RUNX2</scp> ‐Dependent Osteoblast Differentiation via <scp>miR</scp> ‐126‐3p/ <scp>DKK1</scp> ‐Mediated Canonical Wnt Signaling Inhibition in Rett Syndrome

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2) that is located on the X chromosome. Affected individuals also exhibit a variety of non-neurological symptoms such as kyphoscoliosis and osteoporosis. Thus, MECP2 may play a functional role in bone remodeling and osteoblast differentiation. This study aimed to clarify the molecular mechanisms underlying the deregulation of bone remodeling in RTT. Human deciduous tooth-derived mesenchymal stem cells that exhibit osteoblast plasticity were used as a cellular model of RTT. Using a small interfering RNA-mediated MECP2 (MECP2-siR) knockdown system, we quantitatively analyzed the RUNX2-dependent and canonical Wnt signaling pathways during osteoblast differentiation. Expression of active β-catenin, RUNX2, and their downstream targets (osteocalcin and alkaline phosphatase) and mineralization were decreased in MECP2-siR-treated osteoblasts compared to that in control osteoblasts. In contrast, the MECP2-siR-treated osteoblasts exhibited an increase in the endogenous Wnt antagonist DKK1. Notably, MECP2/DKK1 double-knockdown osteoblasts possessed greater β-catenin and RUNX2 levels than MECP2 single-knockdown osteoblasts. Furthermore, microRNA126-3p was upregulated in MECP2-siR-treated osteoblasts, and an antagomir of microRNA126-3p prevented DKK1 upregulation, thereby improving the levels of active β-catenin and other osteoblastic phenotypes. These results suggest that MECP2 insufficiency enhances DKK1 expression via the upregulation of microRNA126-3p, suppressing the canonical Wnt signaling and subsequent RUNX2-dependent osteoblast differentiation. The present study provides insights into the molecular mechanisms involved in impaired osteoblast differentiation that contribute to the development of osteoporosis in RTT.