Targeting mRNA stability as a potential therapeutic for LPS-induced inflammatory bone loss
Patil, Chetan Shyam
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The manipulation of mRNA stability has become a potent and novel method of inhibiting pro-inflammatory cytokines. Targeting mRNA stability via signaling pathways and their downstream components has been evaluated in vitro utilizing over expression assays and reporter studies. Very few studies have translated the concept to an in vivo model to mitigate disease progression. The goal of this study was to evaluate mRNA stability in vitro and in vivo as a therapeutic target for chronic inflammatory diseases such as periodontal disease. This study utilized cells associated with the periodontium such as MC3T3-E1 osteoblastic cells, mPDL fibroblasts, and RAW264.7 murine macrophages as well as an in vivo experimental periodontitis model. To determine the role of mRNA stability in the production of IL-6 in MC3T3-E1 osteoblasts, we utilized a specific inhibitor of p38 MAPK (SB203580). Cells that were pre-treated with the p38 MAPK inhibitor were capable of destabilizing IL-1β -induced IL-6 mRNA through the IL-6 3'UTR. This destabilization resulted in the immediate inhibition of IL-6 protein in a dose dependent manner. These data were expanded into other periodontal cell types including periodontal ligament fibroblast. Using pharmacological inhibitors in mPDL cells, we demonstrated the importance of ERK, JNK and p38 MAPK in the production of LPS-induced IL-6 mRNA and protein. This inhibition by pharmacological inhibitors was observed in the presence of IL-1β and LPS from E. coli and the periodontal pathogen A. actinomycetemcomitans. Utilizing mPDL cells expressing dominant negative upstream kinases of p38 MAPK, we further demonstrated a preferential pathway utilizing MKK3-p38 MAPK. It has been well established that p38-dependent signaling cascades can regulate a wide variety of cellular responses including, proliferation, apoptosis, growth, and inflammation. To gain more knowledge relative to post-transcriptional pro-inflammatory gene expression, we evaluated a common downstream substrate of p38 MAPK phosphorylation that regulates mRNA decay pathway- tristetraprolin (TTP). Macrophage cell lines stimulated with A. actinomycetemcomitans LPS demonstrated marked inhibition of IL-6 and TNFα protein and PGE 2 levels when TTP was overexpressed. This inhibition was observed for their mRNA steady state equivalents: IL-6, TNFα, and COX-2. In reporter assays utilizing HeLa cells, TTP overexpression targeted luciferase reporters containing class II AU-Rich Elements (ARE) within the 3' untranslated region (UTR). In an experimental model of periodontitis, TTP overexpression markedly reduced LPS-induced bone loss around M1 and M2 maxillary molars. Additionally, TTP overexpression reduced the presence of inflammatory cells and cells associated with osteoclast activity. These data indicate that targeting mRNA stability via the over expression of TTP inhibits the production of pro-inflammatory cytokines and protects animals from LPS-induced bone loss. Collectively, these data support the role of mRNA stability as a therapeutic target for management of inflammation-driven bone loss as found in periodontitis.