Estrogen increases Setdb1 cytoplasmic localization to stabilize Serpinh1 and improve protein homeostasis in osteoblasts

Estrogen regulates osteoblast activity at the epigenetic level. Setdb1 is an epigenetic regulator that functions in skeleton homeostasis maintenance. Setdb1 shows nuclear and cytoplasm localization in cells; however, the subcellular distribution of Setdb1 and the role of cytoplasmic Setdb1 in osteoblasts are largely unknown. Here, immunofluorescence staining and immunoblotting analysis showed that the distribution of Setdb1 in the cytoplasm increased upon β-estradiol treatment by increasing nuclear Setdb1 stability in osteoblasts. In β-estradiol-treated MC3T3-E1 cells, knocking-down Atf7ip expression enhanced Setdb1 cytoplasmic localization, but the cytoplasmic distribution of Setdb1 decreased in cells treated with the Setdb1 inhibitor (R,R)-59. Moreover, ovariectomized (OVX) mice lacking Atf7ip in mature osteoblasts showed better bone microstructure than the OVX controls. The proteomic analysis of the cytoplasmic binding of Setdb1 showed that cytoplasmic Setdb1 in osteoblasts mainly functioned to regulate protein homeostasis. Setdb1 binds to Serpinh1, a regulator of pro-collagen folding and maturation, and enhances Serpinh1 stability. Interrupting Setdb1 cytoplasmic localization by treating the cells with Leptomycin B (LMB) or (R,R)-59 led to an accumulation of unfolded protein and the elicitation of endoplasmic reticulum (ER) stress. The findings revealed a previously unrecognized role of cytoplasmic Setdb1 in the regulation of β-estradiol-mediated osteoblast homeostasis, which could enhance the understanding of estrogen's mechanism of action in regulating osteoblasts.