Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice

Abstract

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of early in the mesenchymal lineage ( through excision via promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether controls osteoblastogenesis at later developmental stages beyond patterning. We show that loss in committed pre-osteoblasts by Cre expression via the osterix/ promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, affects bone formation stage-dependently. We further show that loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of cKO calvaria revealed that the cyclin-dependent kinase inhibitor is the most prominent cell cycle target of Hence, genetic loss of in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.

Epigenetics as a New Frontier in Orthopedic Regenerative Medicine and Oncology

“…Complete knockout of EZH2 in mice produced nonviable embryos with abnormal accumulation of mesodermal cells . EZH2 also has key roles in skeletal development as conditional loss of EZH2 , affects skeletal patterning as well as skeletal growth and bone formation . For example, conditional loss of EZH2 in neural crest–derived tissues (using the Wnt1‐Cre driver) causes craniofacial defects .…”

“…These mice also have distinct skeletal phenotypes during postnatal endochondral bone formation . Loss of EZH2 in osteoblasts (using the SP7/Osx1‐Cre driver) causes a transient low bone mass phenotype that recedes by the time the mice reach skeletal maturity . Studies on the EZH2 null mutation in chondrocytes (using the COL2a1‐Cre driver) revealed that EZH2 deficiency reduces H3K27me3 levels, yet remarkably had minimal impact on skeletal development or maturation of articular cartilage .…”

“…EZH1 appears to be expressed at different times of skeletal development. EZH2 is most abundant in proliferating uncommitted mesenchymal stem cells, while EZH1 is expressed most prominently in postproliferative lineage‐committed cells . Hence, EZH1 and EZH2 are expressed in different spatio‐temporal patterns and may have distinct mechanistic activities during mesenchymal differentiation and skeletal development.…”

β-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2

“…Consistent with these studies, previous work from our laboratories revealed that EZH2 suppresses osteogenesis and promotes adipogenesis in BMSCs . Furthermore, we found that EZH2 is required for normal cell cycle progression in BMSCs and that inhibition of EZH2 blocks cell proliferation by de‐repressing cell cycle inhibitors . Thus, self‐renewal and lineage commitment of BMSCs is tightly linked to EZH2 function.…”

Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

“…However, conflicting results on Ezh2 expression and function in pre-osteoblasts were also reported recently, Dudakovic et al published data showing that Ezh2 cko expressing the Osx-Cre driver have no changes on bone volume phenotype in young adult mice. 43 Possible explanation for that may be as follows: (i) age, animal age presented in our study was younger, deletion of Ezh2 in pre-osteoblast leads to aberrant skeletal growth in different age of mice; (ii) sample size, we only presented four animals per group as a pilot study, our original purpose was to check if SATB2 gene expression changes in bone in pre-osteoblast specific Ezh2 deletion; (iii) different methods, we used pQCT to measure bone density, although we used micro-CT method, we had some failed and did not present in the current report; and (iv) different Cre mouse model used and different control mouse groups were compared. Nonetheless, from results of our current study, it was indicated once again that bone development or growth is epigenetically regulated.…”