Fufang Zhenshu Tiaozhi capsule enhances bone formation and safeguards against glucocorticoid-induced osteoporosis through innovative Mekk2-mediated β-catenin deubiquitination

• Published in: Journal of bone and mineral metabolism 2024-09, Vol.42 (5), p.516-528
• Main Authors: Hong, Guoju, Tang, Lin, Zhou, Tianyu, Xie, Youhong, Wang, Jiangyan, Ge, Dongdong, Dong, Qunwei, Sun, Ping
• Format: Article
• Language: English
• Subjects: Animal models; Animals; beta Catenin - metabolism; Bone growth; Bone loss; Bone mass; Bone turnover; Capsules; Cbfa-1 protein; Cell Differentiation - drug effects; Collagen (type I); Computed tomography; CRISPR; Dexamethasone; Dexamethasone - pharmacology; Drugs, Chinese Herbal - pharmacology; Glucocorticoids; Glucocorticoids - pharmacology; Homeostasis; Immunofluorescence; Immunoprecipitation; Kinases; MAP kinase; MAP Kinase Kinase Kinase 2 - metabolism; Medicine; Medicine & Public Health; Metabolic Diseases; Mice; Mice, Knockout; Original Article; Orthopedics; Osteoblastogenesis; Osteoblasts; Osteoblasts - drug effects; Osteoblasts - metabolism; Osteogenesis; Osteogenesis - drug effects; Osteoporosis; Osteoporosis - chemically induced; Osteoporosis - metabolism; Osteoporosis - pathology; Phosphorylation; Signal transduction; Ubiquitination - drug effects; Western blotting; Wnt protein; β-Catenin
• ISSN: 0914-8779; 1435-5604; 1435-5604
• DOI: 10.1007/s00774-024-01516-4

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 c