Degenerative orthopedic diseases such as osteoporosis (OP) are common and frequent in the elderly, especially the postmenopausal women, affecting 250 million people worldwide (Zheng et al., 2022). An increasing number of osteoporotic vertebral compression fractures are occurring every year due to the aging process of the world's population (Anam and Insogna, 2021). Recent studies focused on the role of OP in bone homeostasis, and how dysfunction of osteoclasts leads to osteopetrosis (Song et al., 2022; Wang et al., 2022a). Thus, it is particularly crucial to detect the early diagnosis and treatment for diagnosing osteoporosis.

A gene called HAMP (Hepcidin antimicrobial peptide) encodes Heparin, which is secreted by the liver as a polypeptide containing 25-amino acid hormone that maintains systemic iron homoeostasis in mammals (Liu et al., 2019). In recent studies, Hepcidin and iron metabolism have been linked to systemic osteoporosis (Sato et al., 2020). In addition, Hepcidin may prevent iron overload, which may increase intracellular calcium, contributing to osteoporosis prevention (Lu et al., 2015). Furthermore, Hepcidin can prevent the osteoporosis development by lowering iron levels in bone tissue, inhibiting osteoclast differentiation and bone resorption (Zhang et al., 2019). However, deletion of Hepcidin (Hepcidin−/− mice) results in the apoptosis of osteocytes, and reducing bone formation and enhancing bone resorption (Ma et al., 2022). The mechanism by which Hepcidin induces bone loss causing unbalanced bone turnover, inhibiting osteoclast differentiation, and bone resorption.

As a member of the ETS-transformation specificity (ETS) family, Spi-1 proto-oncogene (PU.1) (SPI1) plays a critical role in myeloid and B‐cell development and differentiation (Gregoricchio et al., 2022; Le Coz et al., 2021; Oikawa et al., 1999). A transcriptional activator, SPI1 binds to or near closed nucleosomes (Ghisletti et al., 2010). For instance, SPI1 binds to the closed chromatin and recruits epigenetic modifiers, such as histone acetyltransferases (HATs), to activate the transcription of its target genes (Barozzi et al., 2014; McAndrew et al., 2016). However, recent studies have reported that SPI1 controls neutrophil immune responses by repressing transcription in osteoclasts (de la Rica et al., 2013). Abnormally high SPI1 expression was involved in osteoporosis (Skinkyte-Juskiene et al., 2018), indicating that a reduction in SPI1 levels inhibits osteoclast differentiation, making it a novel osteoporosis therapeutic target. Investigating the mechanisms by which SPI1 inhibits osteoclast differentiation by repressing gene expression.

DMT1 is a major membrane transport protein, which can transport metal ions including iron, manganese, and cadmium (Gunshin et al., 1997). In mammals, DMT1 is the best characterized Fe2+ transporter. For example, DMT1 exports Fe2+ from the endosome and enters the mitochondrial intermembrane space (Wolff et al., 2018; Buracco et al., 2015). In a previous study, Zhang et al. reported there is a close association between DMT1 and osteoporosis (Zhang et al., 2015), and Chew et al. found iron overload caused by overexpression of DMT1 (Chew et al., 2011). Thus, DMT1 expression and iron overload are intimately related. Recent studies have shown that reduction of DMT1 contributed to osteogenesis and prevented bone mass loss in Diabetic osteoporosis (DOP) mice (Ren et al., 2022). However, no study has reported that the Hepcidin regulates iron overload through DMT1 in osteoporosis.

This study revealed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. Furthermore, we discovered that SPI1 inhibited Hepcidin's transcription. We discovered that SPI1 and DMT1 facilitated the osteoclasts differentiation and iron accumulation both in vitro and in vivo. Moreover, SPI1 increased DMT1 expression, and overexpression of DMT1 facilitated osteoclast differentiation and iron accumulation in OVX mice.