CYB5A promotes osteogenic differentiation of MC3T3-E1 cells through autophagy mediated by the AKT/mTOR/ULK1 signaling pathway

Bone metabolism involves complex genetic and cellular processes. While many advances have been made in understanding the molecular mechanisms of osteogenic differentiation, many aspects remain to be fully elucidated. This study investigated the role of CYB5A in promoting osteogenic differentiation of MC3T3-E1 cells and explored the influence of autophagy via the AKT/mTOR/ULK1 signaling pathway. CYB5A expression during osteogenesis was analyzed through bioinformatics, quantitative reverse transcription polymerase chain reaction, and Western blotting. CYB5A was overexpressed or knocked down via plasmid or small interfering RNA transfection, and its effects on cell proliferation, migration, and differentiation were evaluated. Results showed that CYB5A expression increased during differentiation without affecting proliferation. However, CYB5A significantly enhanced cell differentiation by stimulating autophagy, as indicated by an increased ratio of the autophagic marker LC3-II/LC3-I and reduced levels of P62. Mechanistically, CYB5A modulates autophagy by activating ULK1 and reducing active mTOR phosphorylation. Autophagy inhibitors and activators confirmed that the AKT/mTOR/ULK1 pathway mediates CYB5A's regulatory effects on osteogenesis. This study reveals that CYB5A positively regulates osteogenic differentiation through autophagy, offering insights into bone metabolism mechanisms. These findings suggest that CYB5A is a promising therapeutic target for managing bone metabolic disorders.