Role of phosphorylated P38 in steroid-induced osteonecrosis of the femoral head: From comprehensive phosphoproteomics to mechanistic insights

Background: Our study aims to comprehensively understand the phosphorylation status of proteins in femoral head specimens from patients with steroid-induced osteonecrosis of the femoral head (SONFH) and further investigate the role of phosphorylated proteins in SONFH. Methods: We conducted a Tandem Mass Tag (TMT) Labeling mass spectrometry (MS)-based quantitative phosphoproteomic analysis on femoral head samples from patients with SONFH and femoral neck fractures (FNF). We performed protein annotation, functional classification, and functional enrichment analysis for all proteins containing phosphorylation sites. Furthermore, we investigated the level and effects of phosphorylated P38 in SONFH rats and MC3T3-E1 cells. Finally, we examined the function and molecular mechanism of P38 phosphorylation in the context of SONFH using in vivo and in vitro approaches. Results: Our study identified over 4000 phosphorylation sites corresponding to 2289 proteins, with 134 up-regulated and 69 down-regulated phosphorylation sites in the SONFH group. Notably, many of these differentially expressed phosphorylated proteins were associated with biological processes critical to the pathogenesis of SONFH. Additionally, our findings demonstrated that phosphorylated P38 promotes apoptosis and inhibits osteogenic differentiation in vitro. Inhibition of P38 phosphorylation protected glucocorticoid-induced femoral head destruction in rat models. Conclusions: These results provide vital insights into the pathophysiology of SONFH and suggest potential therapeutic targets for SONFH.