Regulatory effects of interleukin-1β and prostaglandin E2 on expression of receptor activator of nuclear Factor-κB ligand in human periodontal ligament cells

Background: Receptor activator of nuclear factor-kappaB ligand (RANKL), which is expressed on the cell membrane of osteoblasts/ stromal cells, stimulates osteoclastogenesis. We investigated the regulatory effects of interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE(2)) on expression of RANKL in human periodontal ligament (HPDL) cells and the mechanisms involved in the PGE(2) effect. Methods: The HPDL cells were treated with IL-1beta, alone or in combination with indomethacin (INDO) or NS398, a cyclooxygenase-2 (COX-2) inhibitor. The HPDL cells were also pretreated with H89, a protein kinase A (PKA) inhibitor or GF109203X, a protein kinase C (PKC) inhibitor and subsequently treated with PGE(2), PGE receptor (EP)2 agonist, EP4 agonist, forskolin, dibutyryl CAMP (db-cAMP), or 3-(isobutyl)-1-methylxantine (IBMX). After each treatment, expression of EP2, EP4, or RANKL mRNA was analyzed by reverse transcription-polymerase chain reaction and Southern hybridization. Expression of RANKL protein was detected by Western blotting, and CAMP accumulation was determined using a CAMP enzyme immunoassay kit. Results: ILAP stimulated the expression of RANKL at messenger RNA (mRNA) and protein levels in HPDL cells. Endogenous PGE2 partially mediated the IL-1beta-induced RANKL mRNA expression. Exogenously added PGE(2) also stimulated RANKL expression at mRNA and protein levels in the cells. The PGE(2)-stimulated RANKL expression was mediated by EP2/4 and cAMP-dependent PKA, while PKC was possibly involved in the PGE2 action. Conclusion: Human periodontal ligament cells activated with inflammatory factors such as IL-1beta and PGE(2) may directly stimulate osteoclastogenesis through RANKL, which is stimulated to express by these factors,.