Silencing P2Y(12) and P2Y(13) receptors rehabilitates the ADP-induced P2Y(1)-mediated osteogenic commitment of post-menopausal mesenchymal stromal cells.

BACKGROUND: Participation of ADP-sensitive metabotropic P2Y(1), P2Y(12) and P2Y(13) receptors in human osteogenesis is controversial. Here, we investigated the variations in the expression and bone-forming properties of the P2Y(1)R in osteogenic-differentiating bone marrow-derived mesenchymal stromal cells (BM-MSCs) isolated from post-menopausal (Pm) women. We also tested whether observed P2Y(1)-related functional deficits result from the crosstalk with co-localized P2Y(12) and P2Y(13) receptors. METHODS: Pm BM-MSCs were cultured in an osteogenic-inducing medium in either the absence or presence of the selective P2Y(1) receptor agonist, MR2365; this compound was applied alone or after cells' incubation with selective P2Y(12) and P2Y(13) receptor antagonists or short hairpin RNAs designed to silence P2Y(12) or P2Y(13) receptors gene expression. RESULTS: BM-MSCs present immunoreactivity against all ADP-sensitive P2Y receptor subtypes, but their relative density varied among different Pm women and with the time of the cells in the culture. The P2Y(1)receptor agonist increased the alkaline phosphatase activity and bone nodule formation in BM-MSCs originating from a younger female, but it failed to promote the osteogenic differentiation of BM-MSCs from Pm women unless P2Y(12) or P2Y(13) receptors are blocked with AR-C66096 and MRS211, respectively. Silencing the P2Y(13), but not the P2Y(12), receptor gene expression restored the P2Y(1)-mediated osteogenic commitment of Pm BM-MSCs. The P2Y(1) receptor agonist failed to elicit [Ca(2+)](i) transients inside Pm BM-MSCs except after acute cholesterol depletion and lipid rafts disruption with methyl-β-cyclodextrin to prevent the P2Y(1)/P2Y(13) receptors interplay. CONCLUSIONS: Thus, personalized offsetting the activity and/or expression of P2Y(13) receptor (and P2Y(12)) may be a good strategy to rehabilitate the P2Y(1)-mediated osteogenic potential of BM-MSCs and to reduce the fracture risk in Pm women.