Tristetraprolin reduces alveolar bone loss in experimental periodontitis

Destruction of periodontal tissues has been associated with an imbalance in the production of pro-inflammatory cytokines and enzymes such as TNF-α, IL-6, and COX-2. Many of these inflammatory mediators contain AU-rich elements within the 3'untranstlated region (UTR) that affect the stability and translation of the mRNA through interactions with RNA binding proteins. Binding with destabilizing RNA binding proteins, such as tristetraprolin (TTP), results in the degradation of mRNA and consequently reduction in inflammatory mediators.

Objectives: To evaluate over expression of TTP as a novel ant-inflammatory therapeutic agent in vitro and in vivo.

Methods: RAW264.7 cells were transduced with adenovirus serotype-5 (Ad5)-TTP, Ad5-GFP, or no adenovirus. Cells were then stimulated with Aggregatibacter actinomycetemcomitans LPS at 1μg/mL, RNA was harvested at 18 hours for Realtime RT-PCR, while supernatants were harvested 24 hours post stimulation for ELISA. Realtime RT-PCR analysis of steady state was performed for IL-6, TNF-α, and COX-2 mRNA which was normalized to GAPDH. ELISA measured murine IL-6, TNF-α, and PGE2 levels (pg/mL). Using an established 4-week LPS-induced inflammatory bone loss model, we measured linear, area, and volumetric bone via micro computed tomography in LPS-treated rats expressing Ad5-hTTP, Ad5-GFP, or no adenovirus (n=8/group).

Results: Realtime RT-PCR analysis of endogenous steady mRNA levels demonstrated that Ad5-hTTP overexpression reduces IL-6 [p<0.01], TNF-α[p<0.001], and COX-2 [p<0.01] mRNA to baseline levels. ELISA revealed Ad5-hTTP overexpression significantly reduces protein secretion of IL-6 (~85%), TNF-α (~50%), and PGE2 (~50%) [p<0.001]. μCT analysis demonstrates that Ad-5 hTTP prevents linear, area, and volumetric bone loss induced by Aggregatibacter actinomycetemcomitans LPS [p<0.01].

Conclusion:TTP over expression can reduce inflammatory cytokine and prostanoid production in vitro and is capable of protecting bone loss in an LPS-induced inflammatory bone loss model in rats.

This work was supported by the NIH grants DE07034,DE017966, and Department of Defense grant W81XWH-05-0075.