Tristetraprolin Down-Regulation Contributes to Persistent TNF-Alpha Expression Induced by Cigarette Smoke Extract through a Post-Transcriptional Mechanism

Rationale Tumor necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory mediator and its expression is up-regulated in chronic obstructive pulmonary disease (COPD). Tristetraprolin (TTP) is implicated in regulation of TNF-alpha expression; however, whether TTP is involved in cigarette smoke-induced TNF-alpha expression has not been determined. Methods TTP expression was examined by western blot analysis in murine alveolar macrophages and alveolar epithelial cells challenged without or with cigarette smoke extract (CSE). TNF-alpha mRNA stability, and the decay of TNF-alpha mRNA, were determined by real-time quantitative RT-PCR. TNF-alpha protein levels were examined at the same time in these cells. To identify the molecular mechanism involved, a construct expressing the human beta-globin reporter mRNA containing the TNF-alpha 3'-untranslated region was generated to characterize the TTP targeted site within TNF-alpha mRNA. Results CSE induced TTP down-regulation in alveolar macrophages and alveolar epithelial cells. Reduced TTP expression resulted in significantly increased TNF-alpha mRNA stability. Importantly, increased TNF-alpha mRNA stability due to impaired TTP function resulted in significantly increased TNF-alpha levels in these cells. Forced TTP expression abrogated the increased TNF-alpha mRNA stability and expression induced by CSE. By using the globin reporter construct containing TNF-alpha mRNA 3'-untranslated region, the data indicate that TTP directly targets the adenine-and uridine-rich region (ARE) of TNF-alpha mRNA and negatively regulates TNF-alpha expression at the post-transcriptional level. Conclusion The data demonstrate that cigarette smoke exposure reduces TTP expression and impairs TTP function, resulting in significantly increased TNF-alpha mRNA stability and excessive TNF-alpha expression in alveolar macrophages and epithelial cells. The data suggest that TTP is a novel post-transcriptional regulator and limits excessive TNF-alpha expression and inflammatory response induced by cigarette smoke.