Porphyromonas gingivalis regulates RANKL and OPG expression in PDL cells

Objectives: Receptor Activator of NF-kB Ligand (RANKL) is required for induction of osteoclast formation, whereas the decoy receptor osteoprotegerin (OPG) competitively inhibits its action. Thus the RANKL/OPG expression ratio is a major determinant of bone resorption. The aims of this study were to investigate i) the effect of the periodontal pathogen Porphyromonas gingivalis on RANKL and OPG expression in periodontal ligament (PDL) cells, and ii) any putatively elicited cytotoxicity. Methods: Human PDL tissue was obtained from pre-molars extracted during the course of orthodontic treatment. The established PDL cell cultures were challenged with ascending protein concentrations of P. gingivalis W50 strain culture supernatants. Cell lysis was determined by measuring the extracellular release of lactate dehydrogenase (LDH), whereas intracellular LDH content corresponded to relative cell number. The expression of RANKL and OPG mRNA was analysed by quantitative TaqMan real-time, as well as semi-quantitative RT-PCR. Results: After 6 h of challenge, P. gingivalis protein concentrations up-to 50 µg/ml did not affect PDL cell viability, but higher concentrations, or prolongation of the cell culture resulted in cell detachment, and subsequently in cell death. The basal level of RANKL expression was very low compared to that of OPG. However, our initial data showed that after 6 h stimulation with 50 µg/ml P. gingivalis, RANKL mRNA expression was enhanced 13-fold, whereas that of OPG was down-regulated nearly 5-fold. Overall, there was an approximately 65-fold increase in RANKL/OPG expression ratio using these culture conditions. Conclusion: Sub-toxic concentrations of P. gingivalis culture supernatants positively regulate the RANKL/OPG mRNA expression ratio in PDL cells. Therefore, P. gingivalis-challenged PDL cells may confer an increased capacity for local stimulation of osteoclastogenesis and consequently bone resorption, which is a hallmark of periodontitis.