Introduction: Bone homeostasis depends on the regulation of β-catenin in osteoblasts. Glucocorticoids (GCs) are known to diminish β-catenin activity via Wnt pathway signaling, leading to osteoporosis. Conversely, activating β-catenin in osteoblasts through mitogen-activated protein kinase kinase kinase 2 (Mekk2) offers an innovative approach to combat GC-induced osteoporosis (GIOP). Fufang Zhenshu Tiaozhi (FTZ) capsules have shown effectiveness in treating GIOP, but the mechanisms behind this are still unclear.
Materials and methods: In this study, Mekk2 knockout mice (Mekk2-/-) was generated by CRISPR/Cas9. These mice were then subjected to Alcian Blue-Alizarin Red staining and immunofluorescence to assess their bone and cartilage development. To establish models of GIOP, both Mekk2-/- and wild-type (WT) mice were treated with dexamethasone (DXMS) and subsequently given FTZ capsules. We analyzed the resulting phenotypic changes in these mice using Micro-CT scans and histomorphological studies. Primary osteoblasts, isolated from both Mekk2-/- and WT mice, underwent qRT-PCR to measure key osteogenesis markers, including Runx2, Sp7, Bgalp, Col1a1 and Alp. Cells were then exposed to treatments with either FTZ or Wnt3a and the phosphorylation levels of β-catenin and Mekk2, along with the protein expression of Runx2, were evaluated using Western blotting and immunoprecipitation. Additionally, C3H10T1/2 cells transfected with TOPflash-luciferase and Renilla luciferase reporters were treated with FTZ and Wnt3a to measure β-catenin activity.
Results: In our study, administering FTZ in vivo effectively prevented bone loss typically induced by GCs. However, it's important to note that this protective effect was substantially reduced in mice lacking Mekk2. Additionally, FTZ showed a significant ability to enhance osteogenic differentiation in primary osteoblasts, doing so by altering the expression of Mekk2. Intriguingly, the impact of FTZ on Mekk2 appears to function through a pathway separate from the traditional Wnt signaling route. Furthermore, our findings indicate that FTZ also promotes the deubiquitination of β-catenin, contributing further to its positive effects on bone health.
Conclusions: This study suggests that FTZ plays a significant role in protecting bone mass in cases of GIOP. The mechanism through which FTZ confers this benefit involves the activation of Mekk2/β-catenin signaling pathways, which represents a promising alternative strategy to counteract the deleterious effects of GIOP by augmenting osteoblastogenesis.
Keywords: Mekk2; Osteoporosis; Wnt signaling pathway; β-catenin.
© 2024. The Japanese Society Bone and Mineral Research.