Abstract
Osteoclasts are absorptive cells that play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role of epigenetic regulation in osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation (H3K79me), during osteoclast formation. Using RANKL-induced RAW264.7 macrophage cells as an osteoclast differentiation model, we found that DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. In addition, DOT1L inhibition increased osteoclast surface area and accelerated bone-mass reduction in a mouse ovariectomy (OVX) model of osteoporosis without alter osteoblast differentiation. DOT1L inhibition increase reactive oxygen species (ROS) generation and autophagy activity, and cell migration in pre-osteoclasts. Moreover, it strengthened expression of osteoclast fusion and resorption-related protein CD9 and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-regulated, H3K79me2-mediated, epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.
Highlights
Osteoclasts (OCs) are the primary effectors of bone resorption and are indispensable for bone remodeling and repair and maintaining mineral homeostasis
To determine the influence of DOT1L on OC differentiation, we knocked down DOT1L in RAW264.7 cells and induced OC differentiation by RANKL stimulation
We found that the EPZ5676induced differentially expressed proteins (DEPs) of RANKL-induced RAW264.7 cells were remarkably different from those of untreated RAW264.7 cells (Fig. 4b, c), which indicated that DOT1L regulates a group of OC differentiationassociated proteins
Summary
Osteoclasts (OCs) are the primary effectors of bone resorption and are indispensable for bone remodeling and repair and maintaining mineral homeostasis. Dysregulation of these large, multinucleate cells is the main cause of most bone disorders (nearly 90%) such as osteoporosis (OP) (systemic) or osteolysis (local), which are usually accompanied by spontaneous fractures and hypercalcemia[1]. OC formation is triggered by a series of RANKLinduced signaling events that lead to activation of transcription factors such as NFATc1 and NF-κB2,3. These transcription factors induce the expression of OC-specific genes such as TRAP, CTSK, and MMP9. After RANKL stimulation, the H3K27 methyltransferase EZH2 downregulates the Official journal of the Cell Death Differentiation Association
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