The epigenetic mechanism of mechanically induced osteogenic differentiation

Abstract

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.

Figure 1

Schematic depictions of the location and designated lengths of amplicon segments and representative electrophoresis gels for MSCs exposed to biochemical and biomechanical stimulation. (A) The Osteocalcin amplicon was 467 bp in length and had one potential target for methylation. Methylation was not affected with biochemical or biomechanical stimulation. (B) The PCR product for Osteopontin was 227 bp long and had one potential target for methylation. MSCs did have this site methylated and its methylation state altered by both biochemical and biomechanical stimulation. (A) For Collagen 1, the PCR product was a 353 bp amplicon located 2 kbp upstream of the site of transcription. Our analysis indicates that this target was also methylated, but was not altered with either stimulation.

Figure 2

Quantification of gene expression and methylation in response to biochemical stimulation. (A) The gene expression levels of MSCs cultured in osteo-inductive media for 2 weeks varied in only late stage osteogenic genes. Osteocalcin and Osteopontin, were upregulated 1770.9-fold (p<0.01) and 32.5-fold (p<0.01), respectively. (B) Collagen 1 promoter methylation was not significantly altered by exposure to differentiation media. Furthermore, the methylation of osteopontin decreased by 43.9% after biochemically-induced osteogenic differentiation (p<0.01). Osteocalcin promoter methylation was not altered. [Error bars: SEM (n≥4)]

Figure 3

Mechanically induced alterations in gene expression (A) and promoter methylation (B). Neither gene expression nor promoter methylation of Collagen 1 and Osteocalcin was altered by oscillatory fluid flow. Osteopontin gene expression increased by 2.3-fold (p<0.01), corresponding to a promoter methylation decrease of 1.5-fold (p<0.05). [Error bars: SEM (n≥8)]