The methyltransferase, Enhancer of Zeste homology 2 (EZH2), trimethylates histone 3 lysine 27 (H3K27me3) on chromatin and this repressive mark is removed by lysine demethylase 6A (KDM6A). Loss of these epigenetic modifiers results in developmental defects. We demonstrate that Ezh2 and Kdm6a transcript levels change during differentiation of multipotential human bone marrow-derived mesenchymal stem cells (MSC). Enforced expression of Ezh2 in MSC promoted adipogenic in vitro and inhibited osteogenic differentiation potential in vitro and in vivo, whereas Kdm6a inhibited adipogenesis in vitro and promoted osteogenic differentiation in vitro and in vivo. Inhibition of EZH2 activity and knockdown of Ezh2 gene expression in human MSC resulted in decreased adipogenesis and increased osteogenesis. Conversely, knockdown of Kdm6a gene expression in MSC leads to increased adipogenesis and decreased osteogenesis. Both Ezh2 and Kdm6a were shown to affect expression of master regulatory genes involved in adipogenesis and osteogenesis and H3K27me3 on the promoters of master regulatory genes. These findings demonstrate an important epigenetic switch centered on H3K27me3 which dictates MSC lineage determination. STEM CELLS 2014;32:802-815

eThe main impairment to tissue maintenance during aging is the reduced capacity for stem cell self-renewal over time due to senescence, the irreversible block in proliferation. We have previously described that the basic helix-loop-helix (bHLH) transcription factor Twist-1 can greatly enhance the life span of bone marrow-derived mesenchymal stem/stromal cells (MSCs). In the present study, we show that Twist-1 potently suppresses senescence and the Ink4A/Arf locus with a dramatic decrease in the expression of p16 and to some extent a decrease in p14. Furthermore, the polycomb group protein and histone methyltransferase Ezh2, which suppresses the Ink4A/Arf locus, was found to be induced by Twist-1, resulting in an increase in H3K27me3 along the Ink4A/Arf locus, repressing transcription of both p16/p14 and senescence of human MSCs. Furthermore, Twist-1 inhibits the expression of the bHLH transcription factor E47, which is normally expressed in senescent MSCs and induces transcription of the p16 promoter. Reduced Twist-1 wild-type expression and function in bone cells derived from SaethreChotzen patients also revealed an increase in senescence. These studies for the first time link Twist-1 to histone methylation of the Ink4A/Arf locus by controlling the expression of histone methyltransferases as well as the expression of other bHLH factors. C lonogenic bone marrow (BM)-derived mesenchymal stem/ stromal cells (BMSCs) are a heterogeneous mix of stem cells and committed progenitors that vary in their morphology, proliferation, and differentiation potential (9-12, 18, 23). This is attributed to the existence of a developmental hierarchy of stromal cellular differentiation, comprised largely of committed progenitor cells and a minor population of self-renewing multipotent stem cells capable of differentiating into adipocytes, osteoblasts (OB), chondrocytes, and myocytes (6, 21).Adult somatic stem cells including BMSCs exhibit an increased propensity for cellular senescence during ex vivo expansion, which is accompanied by a reduction in self-renewal and multidifferentiation potential. Senescence is a "fail-safe" mechanism which is activated in response to various stresses such as DNA damage, oxidative damage, and oncogene activation (13, 24). During senescence, cells fail to respond to mitogenic stimuli, undergo dramatic changes in chromatin structure and gene expression, become enlarged and flattened, and remain viable yet nondividing. The Ink4A/Arf locus (the locus of p14Arf and p16) is central to cellular senescence as it signals via p53 and retinoblastoma (Rb) to inhibit proliferation. p14ARF activates p53 by sequestering Mdm2, an E3 ubiquitin ligase, to the nucleolus, thereby preventing the Mdm2-mediated targeting of p53 to proteolytic degradation (16). p53 subsequently activates p21 CIP1/WAF1, which inhibits the cell cycle. Moreover, when activated, p16 inhibits the cyclin-dependent kinases CDK4/6 and leads to the hypophosphorylation of Rb, which in turn represses E2F-mediated cell cycle progression (2,22).Transcr...

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1-EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1-EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild-type mice. The fractured bones of Col1-EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild-type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1-EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU-F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild-type control mice. Furthermore, Col1-EphB4 mice had significantly lower numbers of TRAP-positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones. ß