NEDD4 regulates VEGF signaling and mTOR to promote angiogenesis and the cell cycle in steroid‑induced osteonecrosis of the femoral head

Steroid‑induced osteonecrosis of the femoral head (SONFH) is a progressive hip condition marked by osteocyte apoptosis from poor blood supply, leading to femoral head collapse and hip joint dysfunction. Examination of the GSE123568 dataset revealed the important role of ubiquitination in the development of SONFH, contributing to processes such as 'apoptosis', 'protein processing in the endoplasmic reticulum', 'lysosome function', 'cell cycle regulation and autophagy'. The present research revealed that the E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) is involved in SONFH, showing positive correlations with key genes in the p53 signaling pathway, DNA damage response and cell cycle regulation. This highlights the role of NEDD4 in DNA repair and cell cycle control. Additionally, NEDD4 exhibited varying regulatory effects on integrin, TGF‑β/SMAD, Hippo/yes‑associated protein and Notch signaling pathways, underscoring its multifaceted role in cellular signaling. A NEDD4 overexpression vector was created and found to significantly boost the viability, migration and angiogenesis of bone microvascular endothelial cells (BMECs). Reverse transcription‑quantitative PCR results revealed higher mRNA levels of mTOR, VEGF and VEGFR2 in NEDD4‑overexpressing cells, suggesting that the VEGF signaling pathway was activated. Immunoprecipitation assays showed decreased mTOR ubiquitination levels following NEDD4 overexpression, suggesting NEDD4 may indirectly modulate mTOR ubiquitination rather than directly catalyzing it., Small interfering RNA experiments found that NEDD4 and mTOR cooperated to boost BMEC proliferation and migration, as confirmed by MTT, EdU and wound healing assays. Furthermore, the present research showed that glucocorticoids could suppress NEDD4 expression by increasing promoter methylation levels. These findings highlight the key roles of NEDD4 in angiogenesis, maintaining cell balance, regulating the cell cycle and repairing DNA damage in SONFH. By demonstrating the numerous functions of NEDD4 in steroid‑induced osteonecrosis and angiogenesis, the present study suggested that it may impact vascular growth and bone tissue repair through multiple pathways and mechanisms.