Investigating the role and mechanism of methionine in different types of skeletal fluorosis based on Siglec-15 methylation.

Introduction

Primary sources of human fluoride intake encompass daily water consumption, dietary intake, inhalation of air, and tea preparation (Kabir et al., 2020). Extended exposure to higher fluoride levels may lead to a range of health impairments, with the most severe one being damage to bone tissues, including skeletal and dental fluorosis (Phipps, 1995). Pathological changes in skeletal fluorosis are characterized by a complex and diverse spectrum of phenotypes, including osteosclerosis, osteoporosis, and osteomalacia (osteoporotic manifestations are usually accompanied by osteomalacia rather than occur alone) (Boivin et al., 1990; Guan, 2021; Wei et al., 2019). Scholars have agreed that differences in nutritional levels among the fluoride-exposed populations are essential for occurrence of different phenotypes of skeletal fluorosis. According to population surveys, fluoride exposure under the low nutritional condition probably contributes to skeletal fluorosis characterized by the osteoporotic or osteomalacic phenotype, as opposed to fluoride exposure under normal nutritional condition facilitating skeletal fluorosis characterized by the osteosclerotic phenotype (Guan, 2021; Krishnamachari and Krishnaswamy, 1973; Mithal et al., 1993).

Skeletal fluorosis usually exhibits the typical feature of abnormal bone turnover, which arises from an imbalance between bone formation mediated by bone marrow mesenchymal stem cells-originating osteoblasts and bone resorption mediated by bone marrow monocytes-derived osteoclasts (Qiao et al., 2021). Therefore, under different nutritional conditions, fluoride exposure causes distinct bone turnover statuses, probably leading to different phenotypes of skeletal fluorosis. Studies have demonstrated that nutrients including calcium, protein, vitamin D, choline, and methionine combined with vitamin E can counteract the bone damage induced by fluoride (B?aszczyk et al., 2012; Hu et al., 2024; Reddy and Srikantia, 1971; Zhao et al., 2017). However, the specific nutrients involved in different types of skeletal fluorosis and logical relationships of nutritional factors with abnormalities in fluoride-induced bone turnover remain unknown. Therefore, the responses to these questions are crucial for developing relevant strategies to prevent and treat fluoride-induced bone impairment and warrant further deliberation.

According to research results of our group and other research teams through epidemiological investigation, animal experiments, and in vitro studies, DNA methylation has an important effect on skeletal fluorosis development and progression (Chen et al., 2022; Daiwile et al., 2019; Gao et al., 2020; Ma et al., 2020; Pan et al., 2020; Wu et al., 2019). Simultaneously, with the deepening of studies regarding effects of nutrients on DNA methylation, it presents a possibility to investigate the underlying logical relationship between nutritional factors and fluoride-induced bone turnover abnormalities from the DNA methylation perspective. DNA methylation, as the chemical modification process, transfers one methyl group in S-adenosylmethionine (SAM) to fifth carbon atom in cytosine under the catalysis of DNA methyltransferase, causing the formation of 5-methylcytosine (5 mC) (Martin and Fry, 2018). The deposition of methylation marks in this process depends on the catalytic activities of DNA methyltransferases (DNMTs), like DNMT3A, DNMT3B, and DNMT1, whereas active removal of methylation marks depends on the activities of ten-eleven translocation methylcytosine dioxygenases (TETs), such as TET1, TET2, and TET3 (Lyko, 2018; Wu and Zhang, 2017). It is commonly believed that gene promoter methylation is related to gene silencing (Greenberg and Bourc’his, 2019). As genome-wide DNA methylation profiles are increasingly explored, recent investigations have not only concentrated on the relation of promoter methylation with transcriptional expression but also emphasized the significant attention paid to the association between gene body methylation and transcriptional expression (Wang et al., 2022).

Methionine, as an essential amino acid, serves as the sole direct precursor of SAM. Fluctuations in dietary methionine are directly associated with changes in DNA methylation. These fluctuations impact SAM levels and exert regulatory effects on key enzymes related to DNA methylation modification process, thereby regulating DNA methylation (Gou et al., 2021; Ponnaluri et al., 2018; Poomipark et al., 2016; Soda, 2018; Takumi et al., 2015). Dietary methionine levels are also essential for maintaining bone health. Research has indicated that methionine restriction decreases bone mass by inhibiting osteoblast differentiation, thus affecting bone health (Navik et al., 2021). Conversely, supplementation with methionine has been found to improve bone density in ovariectomized rats by decreasing osteoclast activity (Vijayan et al., 2014). Based on population studies, diets rich in one-carbon metabolization-associated nutrients, like methionine, betaine, choline, vitamin B6, and folate are related to the reduced fluorosis prevalence (Chen et al., 2021). Therefore, whether methionine is associated with skeletal fluorosis development by modulating methylation of critical genes during bone turnover is a scientific question to be addressed.

In the previous study, we successfully established rat models with osteosclerotic and osteoporotic/osteomalacic skeletal fluorosis. Moreover, we conducted mRNA sequencing plus targeted bisulfite sequencing (TBS) for identifying the 8 genes (Ostn, Siglec-15, Cthrc1, Slc9b2, Mill1, Serpinh1, Dhh, and Atp6v0a4) with significant differential methylation levels in rats with different types of skeletal fluorosis (Ding et al., 2023). Among them, Siglec-15, with the second highest differential methylation level, has been identified as a key regulator in bone metabolism and a potential therapeutic target for its associated diseases (Huang et al., 2023; Kang et al., 2020). At present, SIGLEC-15 is widely suggested to mediate bone loss caused by osteoarthritis (Shimizu et al., 2015), estrogen deficiency (Kameda et al., 2015) and glucocorticoid-induced osteoporosis (Sato et al., 2020), primarily through influencing osteoclast differentiation. However, limited studies have been conducted to explore its influence on osteoblasts. Therefore, for reasons outlined above, we chose to investigate the role and mechanism of methionine in different types of skeletal fluorosis based on the Siglec-15 gene.

To sum up, this study addressed the following scientific inquiries: 1) whether methionine played a part in the development of different types of skeletal fluorosis; and 2) whether methionine affected the methylation levels of key genes during bone turnover in different skeletal fluorosis types. Hence, attempting to answer these questions, the following investigations were conducted: 1) the effect of methionine on abnormal bone turnover in rats with different types of skeletal fluorosis; 2) the methylation levels of Siglec-15 in fluoride-exposed osteoblasts and osteoclasts under different nutritional conditions and the regulatory role of methionine in Siglec-15 methylation; 3) the regulatory mechanism of methionine in Siglec-15 methylation. The present work focused on addressing logical relationships among nutrients, DNA methylation, and different skeletal fluorosis types, thereby providing the scientific basis for developing nutrition intervention strategies for skeletal fluorosis.