Tristetraprolin Down-Regulation Contributes To Persistent Tnf-Alpha Expression Induced By Cigarette Smoke Extract Through A Post-Transcriptional Mechanism

RationaleTumor 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.MethodsTTP 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.ResultsCSE 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.ConclusionThe 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.