Abstract
Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Bone loss is mitigated by bone protective therapies, but there is a clinical need for new bone-anabolic agents. Previous work has demonstrated that Ezh2 (enhancer of zeste homolog 2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitors. Here, we investigated whether inhibition of Ezh2 can be leveraged for bone stimulatory applications. Pharmacologic inhibition and siRNA knockdown of Ezh2 enhanced osteogenic commitment of MC3T3 preosteoblasts. Next generation RNA sequencing of mRNAs and real time quantitative PCR profiling established that Ezh2 inactivation promotes expression of bone-related gene regulators and extracellular matrix proteins. Mechanistically, enhanced gene expression was linked to decreased H3K27 trimethylation (H3K27me3) near transcriptional start sites in genome-wide sequencing of chromatin immunoprecipitations assays. Administration of an Ezh2 inhibitor modestly increases bone density parameters of adult mice. Furthermore, Ezh2 inhibition also alleviated bone loss in an estrogen-deficient mammalian model for osteoporosis. Ezh2 inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. Thus, these data suggest that inhibition of Ezh2 promotes paracrine signaling in osteoblasts and has bone-anabolic and osteoprotective potential in adults.
Highlights
Decreased bone mineral density (BMD)3 and matrix material properties are associated with increased fracture risk and an imbalance in the biological activities of bone-forming osteoblasts and bone-resorbing osteoclasts [1,2,3]
We show that Ezh2 inhibition enhances osteogenic differentiation of preosteoblasts by reducing H3K27me3 near transcriptional start sites and enhances the expression of osteogenic genes
The addition of GSK126 (2 M) inhibits H3K27me3 6 h after drug administration, and this effect perseveres for at least 72 h (Fig. 1c). These results indicate that Ezh2 activity is rate-limiting for global H3K27me3 in MC3T3 cells, which parallels the established molecular function of this epigenetic regulator [18, 21]
Summary
Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Ezh inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. The commitment of MSCs into osteogenic differentiation is controlled by transcriptional and epigenetic events [9, 10]. Several signaling pathways (e.g. Bmp, Pth, and Wnt pathways) result in the activation and expression of key osteo- Bone-anabolic Effects of Ezh Inhibition genic transcription factors (e.g. Runx and Sp7) that facilitate the commitment of MSC into the osteogenic lineage [11]. Some epigenetic events suppress whereas others enhance osteogenic differentiation of MSCs [12]. We show that Ezh inhibition enhances osteogenic differentiation of preosteoblasts by reducing H3K27me near transcriptional start sites and enhances the expression of osteogenic genes. Administration of an Ezh inhibitor enhances bone formation and prevents bone loss associated with estrogen depletion in vivo
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