Methods: HPDL cells were treated with ATP (10-40 µM). The expression of RANKL and cyclo-oxygenase-2 (COX-2) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The level of prostaglandin E2 (PGE2) was determined using enzyme-linked immunosorbent assay (ELISA). Signaling pathways were investigated by means of inhibitors and antagonist.
Results: ATP induced the expression of RANKL at both mRNA and protein levels. Indomethacin, a non-selective inhibitor of COX, could abolish the induction of RANKL expression, suggesting the COX-dependent mechanism. H89 and PDTC inhibited RANKL expression, PGE2 production and NFkB translocation. In addition, MRS2179, a specific P2Y1 antagonist and P2Y1 small interfering RNA diminished the effect of ATP.
Conclusions: Extracellular ATP stimulates RANKL expression through P2Y1, cAMP-dependent protein kinase, NFkB and COX-dependent pathway in HPDL cells. Our results suggest that, among the molecules responsible to mechanical stress, ATP could be the one that participates in bone resorption or bone homeostasis by mediating its signal through P2Y1 and NFkB/COX/RANKL axis in periodontal tissue.