The Role Of Histone Methyltransferase DOT1L In The Pathogenesis Of Osteoporosis

Osteoporosis is a systemic skeletal disorder caused by an imbalance in bone metabolism.Hundreds of millions of people suffer from Osteoporosis.The balance of bone metabolism is maintained by osteoblasts that perform osteogenesis and osteoclasts that perform bone resorption.Therefore,studying the regulatory factors that affect the differentiation and function of osteoclasts and osteoblasts is the key to understanding the pathogenesis of osteoporosis,and provides potential targets for the treatment of osteoporosis.Epigenetics refers to inheritably gene expression or cellular phenotypic changes through certain mechanisms without changing the DNA sequence.Research pointed that epigenetic factors regulate the differentiation of osteoclasts and osteoblasts,but the role of post-translational modification of histones in the differentiation of osteoclasts and osteoblasts has not been thoroughly studied.In this study,we used mass spectrometry to screen the changes of post-translational modification before and after osteoclast differentiation.We found that H3K79mel/2 increased after osteoclast differentiation and the expression level of DOT1L was also upregulated.In vitro cell experiments demonstrated that small molecule inhibitors mediated DOT1L enzyme activity inhibition or shRNA mediated reduction of DOT1L expression promoted osteoclast differentiation and bone resorption.In addition,In vivo experiments showed that DOT1L inhibitor treatment increased osteoclast surface area and aggravate the loss of femur and tibial bone in ovariectomized mice without alter osteoblast differentiation.To clarify how DOT1L regulates osteoclast differentiation,RANKL-induced RAW264.7 macrophage cells was used as an osteoclast differentiation model.Quantitative detection of changes in protein expression after treatment with DOT1L inhibitor EPZ5676 was performed using quantitative proteomic techniques.The results indicate that DOT 1L mainly regulates the early stages of osteoclasts and mainly regulates mitochondrial proteins,autophagy-related proteins,and cytoskeletal regulatory proteins.These differential proteins are closely related to reactive oxygen species(ROS)production,autophagic activity,and cell migration in cells.Further experiments verified that DOT1L inhibitor EPZ5676 enhanced ROS,up-regulate autophagic activity of osteoclasts,and enhance osteoclast migration.Moreover,ROS mediated the regulation of DOT1L on autophagy and cell migration of osteoclasts.In addition,DOT1L inhibition also caused other factors that enhance osteoclast differentiation and bone resorption.For example,the expression of cell fusion protein CD9 and bone resorption protein MMP9 was enhanced after DOT1L inhibition,and the transcriptional activation of NFATcl and NF-κB,essential transcription factors in osteoclast differentiation,was also increased.The results suggest that DOT1L-regulated,H3K79me-mediated epigenetic regulation regulates osteoclast differentiation and is involved in the onset of osteoporosis,suggesting that DOT1L and DOT1L-regulated protein networks may serve as potential targets for the treatment of osteoporosis.