Statins regulate IL-1beta-induced RANKL and OPG production by human gingival fibroblasts

Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase competitive inhibitors, also known as statins, are widely used agents for lowering cholesterol and reducing the risk for a heart attack. Recent data suggest that statins influence bone metabolic activity by stimulating new bone formation both in vivo and in vitro. Bone resorption in periodontitis, an inflammatory disease, is orchestrated by the interaction of various cytokines in the inflamed tissue, produced by immune cells and also by resident cells such as human gingival fibroblasts (HGF) and periodontal ligament (PDL) cells. Three molecules, members of the tumor necrosis factor (TNF) ligand and receptor superfamilies, regulate the process of osteoclast formation. The first one, receptor activator for NFkB ligand (RANKL) is expressed on hematopoetic stromal cells and periosteal osteoblasts as well as on HGF and PDL cells. RANKL interacts with its corresponding receptor, RANK, on mononucleated osteoclast precursors and induces their activation to multinuclear bone resorbing osteoclasts. The effects of RANKL are blocked by its soluble decoy receptor, osteoprotegerin (OPG), thus inhibiting osteoclast differentiation, activation and survival. RANKL and OPG are produced by many cells in the body including human gingival fibroblasts and PDL cells. At the moment, no data exist comparing the effect of statins on OPG/ RANKL production by resting and interleukin-1β (IL-1β)-stimulated HGF. Objective: The purpose of this project was to evaluate OPG and RANKL production in resting and IL-1β-stimulated HGFs, and to determine the effect of statins on their production. Methods: Cytotoxicity of statins was determined using an assay that measures the activity of a mitochondrial enzyme. Fibroblasts were preincubated with atorvastatin or simvastatin for 24 hours in serum-free medium, and then incubated without a stimulus or with IL-1β for 6 days. OPG or RANKL in culture supernatants was measured by specific ELISA. Data were analyzed using ANOVA and Scheffe’s F procedure for post hoc comparison. Results: Concentrations of simvastatin or atorvastatin (5 x 10-6 M to 1 x 10-11 M) had no significant effect on the viability of the fibroblasts, after 7 days of exposure. IL-1β (1 x 10-8 M) increased OPG production significantly at day 1, 3 and 6, while IL-1β (1 x 10-10 M) increased OPG production significantly only on day 6. There was a trend towards increasing RANKL production with IL-1β stimulation, but no statistical significance was reached. When they had an effect, the statins tended to increase constitutive OPG and RANKL production and to decrease it in the presence of IL-1β, but these findings were not statistically significant. Both statins significantly increased the constitutive RANKL/OPG ratio at multiple concentrations. At the highest concentration (5 x 10-6 M), atorvastatin significantly increased the IL-1β stimulated RANKL/OPG ratio. Conclusion: Under IL-1β stimulation and in the absence of statins, OPG production by HGFs was increased significantly. Simvastatin and atorvastatin differed minimally in their effects on OPG and RANKL production by resting and IL-1β-activated HGFs. Both statins increased constitutive RANKL/OPG ratios, a finding suggesting that in the absence of inflammation statins may influence the production of RANKL and OPG by HGFs to favor bone catabolism. Document Type Thesis Degree Name Master of Dental Science (MDS) Program Periodontology