Regulation of RANKL/OPG by Porphyromonas gingivalis: involvement of Arg-gingipains

Porphyromonas gingivalis is highly implicated in the pathogenesis of periodontitis. By producing a number of virulence factors, including its gingipains, it impairs the function of immune and resident cells of the periodontium. Receptor Activator of NF-kB Ligand (RANKL) is a membrane-bound ligand produced by osteoblasts, fibroblasts and activated lymphocytes, which triggers osteoclast differentiation. In contrast, the soluble decoy receptor osteoprotegerin (OPG) can bind to RANKL and block its action. Thus, an elevated RANKL/OPG ratio has been demonstrated in periodontitis. Objectives: The aim of this work was a) to investigate the regulation of RANKL and OPG gene expression in human gingival fibroblasts (GF) by P. gingivalis and b) to start to identify putative virulence factors involved in this process. Methods: Human gingival fibroblast cultures were challenged with P. gingivalis culture supernatants and expression of RANKL and OPG mRNA were determined by quantitative real time PCR. In addition the effect of heat inactivation of supernatants was tested. Finally, supernatants from Lys-gingipain and Arg-gingipain mutant P. gingivalis strains were tested. Results: P. gingivalis induced RANKL and reduced OPG mRNA expression in GF in a concentration-dependent and time-dependent manner. These changes in RANKL and OPG mRNA expression resulted in an increased RANKL/OPG relative expression ratio. Heat-inactivation of supernatants resulted in a 4-fold reduction of RANKL mRNA expression, but did not affect OPG expression. Compared to the P. gingivalis wild-type strain, the Arg-gingipain mutant strain caused a 6-fold greater enhancement of RANKL expression, whereas the Lys-gingipain mutant strain had a similar effect to the wild type. There were no differences in regulation of OPG expression between these three strains. Conclusions: P. gingivalis up-regulates RANKL and down-regulates OPG mRNA expression in GF, resulting in an increased RANKL/OPG ratio. The component mainly responsible for RANKL induction is proteinaceous, and appears to be regulated by the Arg-gingipains.