Studies have demonstrated that many epigenetic mechanisms regulate the differentiation of osteoblasts, which can have a downstream effect on bone development and homeostasis. Huang et al have demonstrated that miR-22 in human adipose derived MSCs can affect the balance between adipogenesis and osteogenesis by leading to an increase in osteogenic differentiation. When this miRNA is overexpressed in these cells, it causes an increase in ALP activity, matrix mineralisation and transcription of osteogenic specific genes whilst simultaneously causing repression of adipogenic transcription factors (206). Further experiments showed that this promotion of osteogenesis was due to direct repression of HDAC6 via miR-22. miR-17-5p and miR-106a have been shown to have to opposite effects by upregulating adipogenesis and suppressing osteogenesis. In this study, these miRNAs were shown to inhibit BMP2, which caused downregulation of genes such as TAZ2, MSX2 and RUNX2, which are essential for osteoblast differentiation (207).
Histone modifications have also been shown to have a role in regulating osteoblast differentiation. A study from Hassan and colleagues has demonstrated that HOXA10 promotes osteoblastogenesis by directly regulating the activation of RUNX2. It was also shown to upregulate other genes such as ALP, OCN and BGLAP, which are also essential for the progression of osteogenesis (208). HOXA10 mediates chromosome hyperacetylation and H3K4 trimethylation of these genes, which allows for binding of transcription factor complexes and the activation of transcription. Hemming et al have shown that EZH2 and KDMA6, a lysine demethylase, act as an epigenetic switch regulating H3K27 trimethylation to control lineage determination of MSCs. Overexpression of EZH2 caused an increase in adipogenesis and suppressed osteogenesis, whereas overexpression of KDMA6 had the opposite effect (209), demonstrating an important epigenetic switch centred around histone methylation that determines MSC differentiation.
In rats, reduced CpG methylation has been associated with the transcriptional activation of bone-specific osteocalcin. Villagra et al, using primary rat osteoblasts, demonstrated that during the differentiation process methylation levels of CpGs within the osteocalcin promoter was significantly reduced, which coincided with an increase in expression of bone-specific osteocalcin (210). Delgado-Calle et al have demonstrated that DNA methylation has a role in regulating the expression of ALP and the RANKL-OPG pathway in human bone. They found that across a variety of bone related cells the methylation of CpGs within the ALP promoter was inversely related to
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the expression of alkaline phosphatase and when cells were treated with a demethylating agent, there was a 30-fold increase in the expression of this gene (211). This group have also demonstrated that the demethylation of CpGs within the promoter of RANKL and OPG genes, two osteoblast-derived factors that are important in osteoclastogenesis, caused an upregulation in the expression of these two genes. Treatment of HEK-293 cells with a demethylating agent led to a 170-fold increase in RANKL expression and a 20-fold increase in OPG expression (212). However, when looking at methylation across patients with osteoporotic fracture and those with osteoarthritis, there was no difference in methylation levels even though RANKL:OPG ratio was significantly higher in those patients with osteoporotic fracture. This suggests that other mechanisms other than methylation could be at play to cause the increased ratios seen in patients with osteoporotic fractures.
Research from independent groups has now demonstrated roles for specific lncRNAs in the osteogenic differentiation process. A recent expression profile by Zuo et al has shown that there is differential expression of lncRNAs during the early differentiation of C3H10T1/2 mesenchymal stem cells (murine mesenchymal stem cell line obtained from embryos) into osteoblasts (213). Zuo and colleagues used an Arraystar lncRNA array to look at the expression of lncRNAs between control MSCs and those that had been treated with BMP2 to initiate osteoblast differentiation. This expression array identified 116 lncRNAs that were differentially expressed between the two treatment groups – 59 upregulated and 57 downregulated in the BMP2 treated group. Through bioinformatics analysis, this group also found potential regulatory mechanisms that allow lncRNAs to control osteoblast differentiation and identified 24 lncRNAs with nearby protein-coding gene pairs. Zhu and colleagues have demonstrated that the lncRNA ANCR interacts with enhancer of zeste homolog 2 (EZH2), a subunit of PCR2, to repress RUNX2 expression (214). This was done through quantitative PCR and ChIP analyses, which demonstrated that it was the association of ANCR with EZH2 and their translocation to the promoter of the RUNX2 gene that caused this suppression. When ANCR expression is knocked down via a siRNA in hFOB1.19 cells, a human fetal osteoblastic cell line, there is an increase in alkaline phosphatase and osteocalcin expression, differentiation and mineralisation markers respectively. van de Peppel et al have shown that lncRNA H19 expression increases during osteogenic differentiation (215). When expression of this lncRNA is knocked down by short hairpin RNAs (shRNAs) in human mesenchymal stromal cells, results showed a 70-90% decrease in ALP expression and an 80-95% decrease in matrix mineralisation. This coincided with a decrease in the expression of RUNX2 and collagen type I, an extracellular matrix protein, which suggests that H19 may have a direct role in regulating osteoblastic differentiation. This research does
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suggest that there may be a role for lncRNAs in regulating the differentiation process of mesenchymal stem cells into osteoblasts.
1.5 Identification of potential predictive markers of offspring bone outcomes