Background: Osteoporosis is a systemic skeletal disease with a multifactorial pathogenesis, and microgravity contributes significantly to its progression. Despite the critical role of epigenetic modifications, particularly DNA methylation, in cellular function, their involvement in microgravity-induced osteoporosis remains unclear. This study investigates the impact of DNA methylation on osteoblast phenotypes under microgravity, offering new insights into the epigenetic regulation of bone homeostasis.

Methods: Reduced representation bisulfite sequencing (RRBS), methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), and quantitative PCR (qPCR) were used to analyze the DNA methylation and transcriptional profiles of MC3T3-E1 cells under microgravity. Differentially methylated regions (DMRs) in the Ddit3 promoter and its expression were assessed. Functional assays, including Western blotting (WB), flow cytometry, and CCK-8, were performed to evaluate the impact of Dnmt3a overexpression or Ddit3 inhibition on osteoblast function. In vivo, a tail suspension (TS) mouse model was used to investigate bone phenotypic changes after Ddit3 inhibition. The role of the endoplasmic reticulum (ER) stress pathway in mediating Ddit3's effects was examined using the ER stress inhibitor 4-phenylbutyric acid (4PBA).

Results: Microgravity exposure markedly induced apoptosis in osteoblasts, with the apoptotic rate increasing from 3.83% in the control group to 13.44% in the microgravity group (p < 0.05). Pro-apoptotic proteins Bax and cleaved caspase-3 increased by 30% (p < 0.05) and 66% (p < 0.05), respectively, whereas the anti-apoptotic protein Bcl-2 decreased by 33% (p < 0.05). Reduced representation bisulfite sequencing (RRBS) revealed a significant global decrease in DNA methylation under microgravity. RT-qPCR analysis demonstrated a 48% reduction in Dnmt3a expression (p < 0.05), accompanied by hypomethylation of the Ddit3 promoter and a 2.36-fold elevation of Ddit3 mRNA (p < 0.05). BSP further confirmed a 62% (p < 0.05) decrease in Ddit3 promoter methylation, while MSP showed a 45% reduction (p < 0.05). Functional assays indicated that Dnmt3a overexpression elevated cell viability by 31% (p < 0.05) and suppressed apoptosis, reducing Bax expression by 35% (p < 0.05) and increasing Bcl-2 expression by 103% (p < 0.05) compared with the microgravity group. Moreover, the regulatory effects of Dnmt3a were Ddit3-dependent; inhibition of Ddit3 under microgravity reduced the apoptotic rate by 58% (p < 0.05) relative to Dnmt3a-inhibited cells. Microgravity also strongly activated the ER stress pathway, upregulating p-PERK, p-IRE1α, and XBP1s by 3.2-, 2.1-, and 3.8-fold (p < 0.05), respectively. Under microgravity, Ddit3 inhibition alleviated ER stress, decreasing p-PERK, p-IRE1α, and XBP1s by 57% (p < 0.05), 48% (p < 0.05), and 56% (p < 0.05), respectively. Furthermore, treatment with the ER stress inhibitor 4PBA under microgravity reduced the ER stress markers ATF4 and XBP1s by 48% (p < 0.05) and 49% (p < 0.05), respectively. In vivo studies using a TS mouse model confirmed that Ddit3 inhibition attenuated the osteoporosis-like phenotype, improving bone mass and microarchitectural integrity.

Conclusion: Under microgravity, Dnmt3a downregulation induces Ddit3 promoter hypomethylation, triggering ER stress and osteoblast apoptosis, thereby promoting osteoporosis. These findings identify the Dnmt3a–Ddit3 axis as a key epigenetic regulator of osteoblast dysfunction in microgravity and a promising therapeutic target for microgravity-induced osteoporosis.