Mechanical Loading remodels transcriptional regulation in Osteocytes through Nucleus translocation of NRF2 [ChIP-seq]

The mechano-responsiveness of osteocytes is critical for maintaining bone health, yet the molecular mechanisms underlying this process remain poorly understood. Here, we investigate the impact of mechanical loading on transcriptional and epigenetic remodeling in osteocytes. Using MLO-Y4 cells, we subjected osteocytes to mechanical loading and profiled their transcriptome and histone modifications. Our findings revealed significant transcriptional and epigenetic changes, with NRF2 emerging as a key regulator. We demonstrated that mechanical loading stabilizes NRF2 protein levels and promotes its nucleus translocation, leading to enhanced genomic binding of NRF2 and activation of its target genes. Further analysis confirmed cell type-specific functions of NRF2, with distinct binding profiles in osteocytes compared to astrocytes. These results highlight the pivotal role of NRF2 in the mechano-responsiveness of osteocytes. Unlike pharmacologic agents that stabilize NRF2, mechanical loading uniquely promoted NRF2 nucleus translocation and transcriptional activity. This underscores the irreplaceable role of mechanical stimuli, such as exercise, in promoting bone health. This study advances our understanding of osteocyte mechanotransduction and identifies potential therapeutic interventions for bone diseases. For the loaded group, MLO-Y4 cells were plated onto UniFlex™ Culture Plates coated with type I collagen (Flexcell International Corporation), which are 6-well plates with a soft silicone rubber membrane at the bottom of each well. When the cells reached 60% confluency, the plates were subjected to the Flexcell FX5000 Tension System and treated with equiaxial dynamic stretching at 0.5 Hz and 20% strain for 12 hours. For the unloaded group, , MLO-Y4 cells were plated onto 6-well cell culture plate (Corning). When the cell density reached 60% confluency, the plates were transferred to the cell incubator of the Flexcell FX5000 Tension System for 12 hours, serving as the control group. MLO-Y4 cells of each group were fixed by 1% formaldehyde solution (Sigma, F8775-25ML) and quenched with 1.25M glycine (Amresco, 200-272-2). Chromatin was collected and sheared using the Bioruptor Plus with 30 seconds on and 30 seconds off for 18 cycles. Immunoprecipitation was performed using 3–5 μg of H3K27ac (Abcam, ab4729); H3K4me3 (Abcam, ab8580) or NRF2 (Proteintech, 80593-1-RR) antibodies. DNA was purified using the DNA Clean & Concentrator-5 kit (Zymo, D4014). Libraries were then constructed from ChIP samples using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, E7370) and sequenced on a NovaSeq by Annoroad Gene Tech (Beijing) Co., Ltd. Two biological replicates were performed for each group.