Role of NOD and MyD88 proteins on RANKL/OPG regulation

Alveolar bone resorption is a common finding related to periodontal disease. Osteoclastogenesis is determined by the ratio between levels of osteoprotegerin (OPG) and RANKL which balances bone resorption-apposition process. OPG and RANKL expression can be modulated by TLR ligands which may utilize various adapter proteins. Recently, Nucleotide-binding Oligomerization Domain (NOD) and myeloid differentiation primary response gene 88 (MyD88) proteins were implicated in apoptosis and inflammatory responses to certain pathogenic bacteria via NFkB and p38 activation. Objective: to evaluate the influence of NOD-1, NOD-2 and MyD88 on RANKL/OPG regulation. Methods: bone marrow stromal cells of wild-type (WT) animals and knockout (KO) for MyD88, NOD-1 and NOD-2 were cultivated, deinduced and treated with 1µg/mL P. gingivalis LPS (TLR-2 agonist), 1µg/mL E. coli LPS (TLR-4 agonist), 1ng/mL interleukin-1 beta (IL1r agonist) for 10 and 45 minutes and 24 hours. RANKL, OPG and GAPDH (housekeeping gene) mRNA expression were evaluated by RT-PCR in 24-hour samples. Western blot was employed to evaluate NFkB and p38 MAPKinase activation in 10 and 45-minute samples. Results: RANKL mRNA levels increased with all agonists, whereas OPG decreased in WT animals. MyD88 and NOD-1 are necessary in positive regulation of RANKL, although only the former performed a role on OPG regulation. NOD-2 is required for OPG inhibition and increase of RANKL mRNA levels only for TLR-2 and -4 ligands. NFkB activation pattern was similar for WT and NOD-1 KO cells with increasing levels after stimulation which was more evident for the latter, suggesting a role of NOD-1 on NFkB activation. NOD-2 was required for NFkB activation only after TLR-4 stimulation. Phosphorylation of p38 was evident after TLR stimulation although TLR-2 activation was faster in this process than -4. Conclusion: NOD and MyD88 proteins can play a role on RANKL/OPG balance. Support: The State of São Paulo Research Foundation (FAPESP)