Enhancer of Zeste Homolog 2 Inhibition Stimulates Bone Formation and Mitigates Bone Loss Caused by Ovariectomy in Skeletally Mature Mice

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.

Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing

“…Beyond studies from our own group [Dudakovic et al, 2016; Dudakovic et al, 2015b; Dudakovic et al, 2013; Farzaneh et al, 2016], other studies have used various screening methods to assess expression and function of epigenetic regulators. These studies utilized technologies such RNA interference [Bajpe et al, 2015; Cellot et al, 2013; Fazzio et al, 2008], high-content cell-spot microarrays [Bjorkman et al, 2012], as well as large-scale reverse genetic screening [Huang et al, 2013] to screen for epigenetic regulators in various tissues, organism, and disease states.…”

“…Using the method we described here in detail, we identified the H3K27 methyltransferase EZH2 as the most prominently down-regulated epigenetic regulator during osteogenic commitment of MSCs [Dudakovic et al, 2015b]. Our group and others collectively showed that EZH2 is a principal epigenetic regulator of osteogenic lineage commitment of MSCs and a suppressor of osteoblast maturation [Dudakovic et al, 2016; Dudakovic et al, 2015b; Hemming et al, 2014; Hui et al, 2014; Wei et al, 2011]. …”

“…The advent of next-generation genomic and proteomic technologies has allowed a greater understanding of the epigenetic states of different cell types [de Wit and de Laat, 2012; Laird, 2010; Rando and Chang, 2009]. Epigenetic mechanisms respond to environmental cues and external biological factors, and can be effectively applied in stem cell-based engineering strategies for musculoskeletal tissue regeneration [Dudakovic et al, 2015a; Dudakovic et al, 2016; Dudakovic et al, 2015b; Dudakovic et al, 2013]. …”

“…For example, modulating the activity of histone deacetylases (HDACs), the WD-repeat domain protein WDR5, which is associated with a histone 3 lysine 4 (H3K4) methyltransferase complex, and the H3K9 methyl transferase SUV420H2 can stimulate osteogenic differentiation of MSCs and/or pre-osteoblasts [Di Bernardo et al, 2009; Dudakovic et al, 2013; Farzaneh et al, 2016; Gordon et al, 2015; Gori et al, 2001; Gori et al, 2006; Lee et al, 2009; Schroeder and Westendorf, 2005]. Other studies have demonstrated that suppression of histone 3 lysine 27 trimethylation (H3K27me3) or altering CpG methylation can significantly enhance osteogenic differentiation and inhibit adipogenic differentiation of MSCs [Dudakovic et al, 2016; Dudakovic et al, 2015b; Hemming et al, 2014; Jing et al, 2015; Thaler et al, 2016; Wei et al, 2011]. These studies demonstrate that the epigenetic landscape is deformable to favor selective commitment of mesenchymal stromal/stem cells to the osteogenic lineage.…”

“…We have utilized ChIP-seq analysis in osteoblasts to demonstrate that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) modifies the epigenomic fingerprint of histone H4 acetylation in differentially regulated genes [Dudakovic et al, 2013]. Other genomic studies have successfully mapped multiple histone modifications and chromatin conformations in pre-osteoblasts, differentiated osteoblasts, and/or osteocytes [Barutcu et al, 2014; Dudakovic et al, 2016; Meyer et al, 2014; Pike et al, 2015; St John et al, 2014; Tai et al, 2014; Wu et al, 2014]. To improve our understanding of the epigenome in supporting differentiation of mesenchymal cell types, it is necessary to characterize which EpiRegs are expressed at specific biological stages.…”