SUMMARY In vitro, topographical and biophysical cues arising from the extracellular matrix (ECM) direct skeletal stem cell (SSC) commitment and differentiation. However, the mechanisms by which the SSC-ECM interface is regulated and the outcome of such interactions on stem cell fate in vivo remain unknown. Here we demonstrate that conditional deletion of the membrane-anchored metalloproteinase MT1-MMP (Mmp14) in mesenchymal progenitors, but not in committed osteoblasts, redirects SSC fate decisions from osteogenesis to adipo- and chondrogenesis. By effecting ECM remodeling, MT1-MMP regulates stem cell shape, thereby activating a β1-integrin/RhoGTPase signaling cascade and triggering the nuclear localization of the transcriptional coactivators YAP and TAZ, which serve to control SSC lineage commitment. These data identify a critical MT1-MMP/integrin/YAP/TAZ axis operative in the stem cell niche that oversees SSC fate determination.

Bone is a complex endocrine organ that facilitates structural support, protection to vital organs, sites for hematopoiesis, and calcium homeostasis. The bone marrow microenvironment is a heterogeneous niche consisting of multipotent musculoskeletal and hematopoietic progenitors and their derivative terminal cell types. Amongst these progenitors, bone marrow mesenchymal stem/stromal cells (BMSCs) may differentiate into osteogenic, adipogenic, myogenic, and chondrogenic lineages to support musculoskeletal development as well as tissue homeostasis, regeneration and repair during adulthood. With age, the commitment of BMSCs to osteogenesis slows, bone formation decreases, fracture risk rises, and marrow adiposity increases. An unresolved question is whether osteogenesis and adipogenesis are co-regulated in the bone marrow. Osteogenesis and adipogenesis are controlled by specific signaling mechanisms, circulating cytokines, and transcription factors such as Runx2 and Pparγ, respectively. One hypothesis is that adipogenesis is the default pathway if osteogenic stimuli are absent. However, recent work revealed that Runx2 and Osx1-expressing preosteoblasts form lipid droplets under pathological and aging conditions. Histone deacetylase 3 (Hdac3) and other epigenetic regulators suppress lipid storage in preosteoblasts and/or control marrow adiposity. Establishing a better understanding of fat storage in bone marrow cells, as well as the osteoblast-adipocyte relationship within the bone marrow niche is necessary to understand the mechanisms underlying disease- and aging-related marrow fat storage and may lead to the development of new therapeutic targets for "fatty bone" and osteoporosis.

Bone can adapt its structure in response to mechanical stimuli. At the cellular level, this involves changes in chromatin organization, gene expression, and differentiation, but the underlying mechanisms are poorly understood. Here we report on the involvement of RUNX2, a bone-related transcription factor, in this process. Fluid flow shear stress loading of preosteoblasts stimulated translocation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) to the nucleus where it phosphorylated RUNX2 on the chromatin of target genes, and increased histone acetylation and gene expression. MAPK signaling and two RUNX2 phosphoacceptor sites, S301 and S319, were critical for this response. Similarly, in vivo loading of mouse ulnae dramatically increased ERK and RUNX2 phosphorylation as well as expression of osteoblast-related genes. These findings establish ERK/MAPK-mediated phosphorylation of RUNX2 as a critical step in the response of preosteoblasts to dynamic loading and define a novel mechanism to explain how mechanical signals induce gene expression in bone.

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.