Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL-induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway

The overactivation of the osteoclasts is a crucial pathological factor in the development of osteoporosis. MZF1, belonging to the scan-zinc finger family, plays a significant role in various processes associated with tumor malignant progression and acts as an essential transcription factor regulating osteoblast expression. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis. First, we established MZF1-deficient female mice and evaluated the femur bone phenotype by micro-computed tomography and histological staining. Our findings indicate that MZF1-/- mice exhibited a low bone mass osteoporosis phenotype. RANKL could independently induce the differentiation of RAW264.7 cells into osteoclasts, and we found that the expression level of MZF1 protein decreased gradually. Then, the CRISPR/Cas 9 gene-editing technique was used to build a RAW264.7 cell model with MZF1 knockout, and RANKL was used to independently induce MZF1-/- and wild-type cells to differentiate into mature osteoclasts. Tartrate-resistant acid phosphatase staining and F-actin fluorescence results showed that the MZF1-/- group produced more tartrate-resistant acid phosphatase-positive mature osteoclasts and larger actin rings. The expression of osteoclast-associated genes (including tartrate-resistant acid phosphatase, CTSK, c-Fos, and NFATc1) was evaluated by reverse transcription quantitative polymerase chain reaction and Western blot. The expression of key genes of osteoclast differentiation in the MZF1-/- group was significantly increased. Furthermore, we found that cell viability was increased in the early stages of RANKL-induced cell differentiation in the MZF1-/- group cells. We examined some prevalent ferroptosis markers, including malondialdehyde, glutathione, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis. The results suggest that MZF1 may be involved in osteoclast differentiation by regulating RANKL-induced ferroptosis of osteoclasts. Collectively, our findings shed light on the essential involvement of MZF1 in the regulation of osteoclastogenesis in osteoporosis and provide insights into its potential underlying mechanism. MZF1 knockdown promotes multinucleated osteoclast formation and differentiation.