Abstract
BackgroundThe potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs.ResultsMicroarray analysis and validation experiments showed that the expression of IGF2 and IGFBP2 was increased together with integrin alpha5 (ITGA5) during dexamethasone-induced osteoblast differentiation in human MSCs. This effect was functional since we found that IGF2 and IGFBP2 enhanced the expression of osteoblast phenotypic markers and in vitro osteogenic capacity of hMSCs. Interestingly, we showed that downregulation of endogenous ITGA5 using specific shRNA decreased IGF2 and IGFBP2 expression in hMSCs. Conversely, ITGA5 overexpression upregulated IGF2 and IGFBP2 expression in hMSCs, which indicates tight crosstalks between these molecules. Consistent with this concept, activation of endogenous ITGA5 using a specific antibody that primes the integrin, or a peptide that specifically activates ITGA5 increased IGF2 and IGFBP2 expression in hMSCs. Finally, we showed that pharmacological inhibition of FAK/ERK1/2-MAPKs or PI3K signalling pathways that are enhanced by ITGA5 activation, blunted IGF2 and IGFBP2 expression in hMSCs.ConclusionThe results show that ITGA5 is a key mediator of IGF2 and IGFBP2 expression that promotes osteoblast differentiation in human MSCs, and reveal that crosstalks between ITGA5 and IGF2/IGFBP2 signalling are important mechanisms that trigger osteogenic differentiation in human bone marrow derived mesenchymal stromal cells.
Highlights
The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration
Total RNA from dexamethasone-treated and untreated MSCs were collected in three different human MSCs (hMSCs) cultures and used for microarray hybridizations using HG-U133 Plus 2.0 arrays consisting of 54,675 probesets, as described in our original study [11]
Validation experiments using TaqMan Low-Density Array with two separate experiments using mRNA from two different donors confirmed that IGF2, IGFBP2 and integrin alpha5 (ITGA5) mRNA levels were increased by more than 2-fold in dexamethasonetreated MSCs compared to control cells, validating the microarray analysis (Fig. 2A-C)
Summary
The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. Short term treatment with dexamethasone was found to induce the expression of osteoblast phenotypic genes in MSCs [9,10]. Glucocorticoids cannot be use therapeutically to promote MSC differentiation because of their long term negative impact on osteoblast recruitment and function [11]. Identifying genes that are induced by dexamethasone and are involved in the osteogenic differentiation of human MSCs may help to promote their ability to differentiate into cells of the osteoblast lineage
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