Background: Osteogenic-angiogenic coupling is essential for skeletal homeostasis, and its dysregulation constitutes a pathological basis for osteoporosis. This study investigates whether the E3 ubiquitin ligase Mind bomb 2 (MIB2) plays a critical role in this coupling by regulating the deubiquitinase cylindromatosis (CYLD) and oxidative stress.

Methods: Key regulatory factors were identified using integrated bioinformatics analysis. Gain-of-function studies were performed on MC3T3-E1 osteoprogenitors and bEnd.3 endothelial cells through lentiviral transduction. Protein interactions and ubiquitination were assessed using co-immunoprecipitation and ubiquitination assays. Osteogenic differentiation and angiogenic capacity were evaluated by alkaline phosphatase activity assay, Alizarin Red S staining, scratch wound, and tube formation assays. Oxidative stress markers, including reactive oxygen species, malondialdehyde, and glutathione, were measured. Cellular crosstalk was examined using a Transwell co-culture system.

Results: CYLD was identified as a key node regulating both osteogenic and angiogenic processes. Functional studies demonstrated that CYLD overexpression enhanced osteogenic differentiation and angiogenesis while attenuating oxidative stress in both cell types. Mechanistically, MIB2 was identified as an E3 ubiquitin ligase for CYLD, binding CYLD and promoting its ubiquitination and degradation. Rescue experiments confirmed that MIB2 overexpression suppressed osteogenesis and induced oxidative stress (reactive oxygen species (ROS), reduced glutathione (GSH), and malondialdehyde (MDA)) by downregulating CYLD. Co-culture experiments further revealed that disruption of the MIB2–CYLD axis in osteoblasts significantly suppressed endothelial expression of key angiogenic markers (PECAM1, EMCN, and HIF-1α), elevated intracellular ROS, and altered oxidative stress markers in the conditioned medium (GSH and MDA), thereby demonstrating that osteoblast-derived oxidative stress signals impair endothelial function and compromise osteogenic-angiogenic coupling.

Conclusion: MIB2 impairs osteogenic-angiogenic coupling through the ubiquitin-mediated degradation of CYLD, exacerbating oxidative stress and simultaneously suppressing osteogenesis and angiogenesis. These findings provide new insights into the pathogenesis of skeletal disorders and highlight the MIB2-CYLD axis as a potential therapeutic target.