The epigenetic mechanism of mechanically induced osteogenic differentiation

Epigenetic regulation of gene expression occurs due to alterations in chromatin proteins that do not change DNA sequence, but alter the chromatin architecture and the accessibility of genes, resulting in changes to gene expression that are preserved during cell division. Through this process genes are switched on or off in a more durable fashion than other transient mechanisms of gene regulation, such as transcription factors. Thus, epigenetics is central to cellular differentiation and stem cell linage commitment. One such mechanism is DNA methylation, which is associated with gene silencing and is involved in a cell’s progression towards a specific fate. Mechanical signals are a crucial regulator of stem cell behavior and important in tissue differentiation; however, there has been no demonstration of a mechanism whereby mechanics can affect gene regulation at the epigenetic level. In this study, we identified candidate DNA methylation sites in the promoter regions of three osteogenic genes from bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that mechanical stimulation alters their epigenetic state by reducing DNA methylation and show an associated increase in expression. We contrast these results with biochemically induced differentiation and distinguish expression changes associated with durable epigenetic regulation from those likely to be due to transient changes in regulation. This is an important advance in stem cell mechanobiology as it is the first demonstration of a mechanism by which the mechanical micro-environment is able to induce epigenetic changes that control osteogenic cell fate, and that can be passed to daughter cells. This is a first step to understanding that will be vital to successful bone tissue engineering and regenerative medicine, where continued expression of a desired long-term phenotype is crucial

Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate.

Understanding how the mechanical microenvironment influences cell fate, and more importantly, by what molecular mechanisms, will enhance not only the knowledge of mesenchymal stem cell biology but also the field of regenerative medicine. Mechanical stimuli, specifically loading induced oscillatory fluid flow, plays a vital role in promoting healthy bone development, homeostasis and morphology. Recent studies suggest that such loading induced fluid flow has the potential to regulate osteogenic differentiation via the upregulation of multiple osteogenic genes; however, the molecular mechanisms involved in the transduction of a physical signal into altered cell fate have yet to be determined.Using immuno-staining, western blot analysis and luciferase assays, we demonstrate the oscillatory fluid flow regulates beta-catenin nuclear translocation and gene transcription. Additionally, real time RT-PCR analysis suggests that flow induces Wnt5a and Ror2 upregulation, both of which are essential for activating the small GTPase, RhoA, upon flow exposure. Furthermore, although beta-catenin phosphorylation is not altered by flow, its association with N-cadherin is, indicating that flow-induced beta-catenin signaling is initiated by adherens junction signaling.We propose that the mechanical microenvironment of bone has the potential to regulate osteogenic differentiation by initiating multiple key molecular pathways that are essential for such lineage commitment. Specifically, non-canonical Wnt5a signaling involving Ror2 and RhoA as well as N-cadherin mediated beta-catenin signaling are necessary for mechanically induced osteogenic differentiation

A schematic diagram of potential signaling mechanisms involved in mechanically stimulated osteogenic differentiation.

<p>Oscillatory fluid flow, a potent mechanical signal within the microenvironment of bone has the potential to regulate non-canonical Wnt5a and β-catenin signaling pathways in MSCs, both of which are essential for fluid flow induced osteogenic lineage commitment via Runx2 upregulation. Furthermore, Wnt5a signals through Ror2 to activate RhoA, a small GTPase that is necessary and sufficient for osteogenic differentiation. Finally, flow induced β-catenin signaling appears to be mediated by alterations in N-cadherin/β-catenin association indicating that adherens junctions may be involved in the transduction of a mechanical signaling into a cell fate decision.</p

Wnt5a and Ror2 are necessary for flow-induced RhoA activation, but do not effect β-catenin translocation.

<p>(A) Western blots of nuclear β-catenin indicate that both scrambled and Wnt5a siRNA treated cells maintained their potential to initiate β-catenin signaling with flow. (B) Analysis of the western blots demonstrates that scrambled and Wnt5a siRNA treated cells had a 1.9±0.2-fold and 1.8±0.3-fold increase in nuclear β-catenin with flow, respectively. (C) β-catenin/TCF/LEF transcription of downstream genes was also maintained in both scramble and Wnt5a treated cells with a 1.8±0.4-fold and 2.2±0.3-fold increase in luciferase activity, respectively, indicating that Wnt5a is not necessary for mechanically induced β-catenin signaling. (D) Western blots were used to assay Rho activation in response to flow in scrambled, Wnt5a and Ror2 siRNA treated cells. (E) Analysis of the blots indicates that the 1.7±0.1-fold increase observed in scrambled treated cells is significantly greater than Wnt5a (p<0.01) and Ror2 (p<0.05) siRNA treated cells, both of which lost flow-induced RhoA activation. (Error bars: SEM (n≥4)).</p

Wnt5a and β-catenin signaling are both necessary for flow induced Runx2 upregulation.

<p>(A) Flow induced Runx2 expression was upregulated 2.2±0.2-fold in scrambled siRNA treated cells exposed to flow verses scrambled siRNA control cells. This fold increase was significantly different (p<0.05) than Wnt5a siRNA treated cells, in which the flow induced Runx2 expression was abrogated. (B) The fold change in Runx2 expression with flow was significantly different between untreated cells and cells with inhibited β-catenin signaling via endostatin treatment (p<0.01). Untreated cells exposed to oscillatory fluid flow had a 2.8±0.5-fold increase in Runx2 expression over control cells; while endostatin treated cells resulted in no difference between flowed and control cells. (C) Western blot analysis demonstrates that there is a significant decrease in β-catenin levels after a 24 hour incubation with endostatin. (Error bars: SEM (n≥6)).</p

Mechanically induced osteogenic differentiation – the role of RhoA, ROCKII and cytoskeletal dynamics

Many biochemical factors regulating progenitor cell differentiation have been examined in detail; however, the role of the local mechanical environment on stem cell fate has only recently been investigated. In this study, we examined whether oscillatory fluid flow, an exogenous mechanical signal within bone, regulates osteogenic, adipogenic or chondrogenic differentiation of C3H10T1/2 murine mesenchymal stem cells by measuring Runx2, PPARγ and SOX9 gene expression, respectively. Furthermore, we hypothesized that the small GTPase RhoA and isometric tension within the actin cytoskeleton are essential in flow-induced differentiation. We found that oscillatory fluid flow induces the upregulation of Runx2, Sox9 and PPARγ, indicating that it has the potential to regulate transcription factors involved in multiple unique lineage pathways. Furthermore, we demonstrate that the small GTPase RhoA and its effector protein ROCKII regulate fluid-flow-induced osteogenic differentiation. Additionally, activated RhoA and fluid flow have an additive effect on Runx2 expression. Finally, we show RhoA activation and actin tension are negative regulators of both adipogenic and chondrogenic differentiation. However, an intact, dynamic actin cytoskeleton under tension is necessary for flow-induced gene expression

Flow-induced β-catenin signaling may be mediated by cadherin signaling rather than canonical Wnt signaling.

<p>(A) Western blots were used to determine the level of phosphorylated β-catenin in cells exposed to oscillatory fluid flow versus controls. (B) Analysis of the western blot indicates that there is no significant difference between control and experimental cells in the level of phosphorylated β-catenin. (C) A western blot was also used to assay N-cadherin association with β-catenin as a function of mechanical stimulation. (D) Analysis of the western blot demonstrates that there is a significant 30% decrease in β-catenin/N-cadherin association with exposure to 35 minutes of oscillatory fluid flow (p<0.01). (Error bars: SEM (n≥4)).</p

The Periosteum as a Cellular Source for Functional Tissue Engineering

The periosteum, a specialized fibrous tissue composed of fibroblast, osteoblast, and progenitor cells, may be an optimal cell source for tissue engineering based on its accessibility, the ability of periosteal cells to proliferate rapidly both in vivo and in vitro, and the observed differentiation potential of these cells. However, the functional use of periosteum-derived cells as a source for tissue engineering requires an understanding of the ability of such cells to elaborate matrix of different tissues. In this study, we subjected a population of adherent primary periosteum-derived cells to both adipogenic and osteogenic culture conditions. The commitment propensity of periosteal cells was contrasted with that of well-characterized phenotypically pure populations of NIH3T3 fibroblast and MC3T3-E1 osteoblast cell lines. Our results demonstrate that the heterogeneous populations of periosteal cells and NIH3T3 fibroblasts have the ability to express both osteoblast-like and adipocyte-like markers with similar potential. This raises the question of whether fibroblasts within the periosteum may, in fact, have the potential to behave like progenitor cells and play a role in the tissue's multilineage potential or whether there are true stem cells within the periosteum. Further, this study suggests that expanded periosteal cultures may be a source for tissue engineering applications without extensive enrichment or sorting by molecular markers. Thus, this study lays the groundwork for future investigations that will more deeply enumerate the cellular sources and molecular events governing periosteal cell differentiation