Osteoporosis, marked by reductions in bone mass and the deterioration of bone microstructure, increases vulnerability to bone fragility fractures (Compston et al., 2019). Osteoporosis is closely related to skeletal homeostasis disturbances under both physiological and pathological conditions (Cappola et al., 2023). Among the contributory causal factors, estrogen deficiency is the key etiology of primary osteoporosis (Almeida et al., 2017; Walker and Shane, 2023). However, the current therapies for post-menopausal osteoporosis include antiresorptive agents and hormonal therapy, which have adverse effects and side effects (Walker and Shane, 2023). Therefore, further investigations into the precise cellular and molecular mechanisms of action of estrogen on skeletal homeostasis are crucial for developing pharmacotherapeutics for post-menopausal osteoporosis.

Estrogen maintains bone mass predominantly by inhibiting osteoclastic bone resorption (Almeida et al., 2017). Estrogen withdrawal and deficiency increase the inflammatory response and the production of osteoclastogenic factors Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL), Tumor Necrosis Factor (TNF), and Interleukin-6 (IL-6) and activate osteoclast differentiation, maturation and activity. However, extensive studies have shown that estrogen also helps in maintaining osteoblast homeostasis by increasing osteoblast activity and attenuating osteoblast apoptosis (Almeida et al., 2017). Estrogen orchestrates genetic and epigenetic programs for the elaborate harmonization of gene activation and inhibition under specific conditions, which are directed toward the terminal phenotype of osteoblasts (Liu et al., 2022). Recently, estrogen was demonstrated to function at the epigenetic level to regulate osteoblast differentiation (Liu et al., 2022; Bitirim et al., 2021; Dashti et al., 2023). However, the detailed mechanisms of estrogen's action as an epigenetic regulator remain unclear.

Setdb1 is a well-known H3K9 methyltransferase expressed in multiple cells and tissues (Luo et al., 2023). The expression of Setdb1 was reported to increase during osteoblast differentiation (Zhang et al., 2022). Knockdown of Setdb1 impaired the osteoblast function by removing the H3K9me3 on the promotor region of miR-212-3p, an inhibitor for osteoblast differentiation, which downregulated Hmgb1 (Zhang et al., 2023). Mice with conditional Setdb1 knockout (KO) in mesenchymal cells showed defective long bone growth and trabecular bone formation (Yang et al., 2013). Setdb1 has a nuclear export signal (NES) on its N-terminus and localizes in both the nucleus and the cytoplasm (Eom et al., 2022). In the nucleus, Setdb1 H3K9 methyltransferase activity helps to maintain cell integrity by inhibiting the activity of Runx2 and silencing Sp7 gene expression in uncommitted mesenchymal multipotent cells (Yang et al., 2013; Sepulveda et al., 2017; Lawson et al., 2013). The nuclear localization of Setdb1 is modulated by its binding partner, Atf7ip (Timms et al., 2016; Tsusaka et al., 2019). Atf7ip regulates Setdb1 catalytic activity, mainly by regulating the subcellular localization and stability of Setdb1 (Tsusaka et al., 2019). Genome-wide assays showed that the loss of Atf7ip phenocopies resulted in the loss of Setdb1 (Timms et al., 2016). Setdb1 also localizes in the cytoplasm (Eom et al., 2022; Cho et al., 2013; Tachibana et al., 2015; Beyer et al., 2016), and exogenously expressed Setdb1 is largely retained in the cytoplasm (Cho et al., 2013; Tachibana et al., 2015). In the terminal differentiation of skeletal myoblasts, the re-localization of Setdb1 to the cytoplasm is Wnt signaling-dependent (Beyer et al., 2016). However, whether Setdb1 can be re-localized to the cytoplasm and the role of cytoplasmic Setdb1 in estrogen-treated osteoblasts are largely unknown.

The current study found that the cytoplasmic localization of Setdb1 increased in osteoblasts upon treatment with estrogen. In the cytoplasm, Setdb1 stabilized Serpinh1 and regulated pro-collagen maturation. Enhancing cytoplasmic Setdb1 by knocking out Atf7ip in osteoblasts improved the bone integrity of estrogen-deficient mice. In contrast, inhibiting Setdb1 localization to the cytoplasm led to the accumulation of unfolded protein and elicited endoplasmic reticulum stress.