ATP and UTP stimulate bone morphogenetic protein-2,-4 and -5 gene expression and mineralization by rat primary osteoblasts involving PI3K/AKT pathway

The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast. On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-β superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation. In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100 μM ATP, ATPγS and UTP, but not ADP, ADPβS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100 μM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.