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

Abstract

RationaleTumor necrosis factor-alpha (TNF-α) 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-α expression; however, whether TTP is involved in cigarette smoke-induced TNF-α 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-α mRNA stability, and the decay of TNF-α mRNA, were determined by real-time quantitative RT-PCR. TNF-α 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-α 3’-untranslated region was generated to characterize the TTP targeted site within TNF-α mRNA.ResultsCSE induced TTP down-regulation in alveolar macrophages and alveolar epithelial cells. Reduced TTP expression resulted in significantly increased TNF-α mRNA stability. Importantly, increased TNF-α mRNA stability due to impaired TTP function resulted in significantly increased TNF-α levels in these cells. Forced TTP expression abrogated the increased TNF-α mRNA stability and expression induced by CSE. By using the globin reporter construct containing TNF-α mRNA 3’-untranslated region, the data indicate that TTP directly targets the adenine- and uridine-rich region (ARE) of TNF-α mRNA and negatively regulates TNF-α 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-α mRNA stability and excessive TNF-α expression in alveolar macrophages and epithelial cells. The data suggest that TTP is a novel post-transcriptional regulator and limits excessive TNF-α expression and inflammatory response induced by cigarette smoke.

Enhancing tristetraprolin activity reduces the severity of cigarette smoke‐induced experimental chronic obstructive pulmonary disease

“…Wild-type C57BL/6J mice were treated daily (i.p.) 35 This led to increased Tnfa mRNA stability and higher subsequent levels of TNF-a in these cells. Controls were vehicle-treated mice exposed to normal air.…”

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression, sleep and behaviour

“…Expression of prepro‐hypocretin mRNA was determined by real‐time quantitative RT‐PCR as described by us previously . Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Inc), and 1 µg of total RNA was reverse‐transcribed to cDNA for real‐time quantitative RT‐PCR as described previously .…”

“…Prepro‐hypocretin mRNA stability and half‐life were determined by culturing primary murine hypothalamic neuron cells in medium with actinomycin D (10 μg/mL) to block transcription as described by us previously . Briefly, cells were treated with actinomycin D, then followed by TNF‐α or vehicle treatments for the indicated time, and harvested.…”

“…Expression of prepro-hypocretin mRNA was determined by real-time quantitative RT-PCR as described by us previously. 25 Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Inc), and 1 µg of total RNA was reverse-transcribed to cDNA for real-time quantitative RT-PCR as described previously. 26 Quantitative RT-PCR analysis was carried out with the SYBR ® Green PCR Master Mix (Applied Biosystems) using the Bio-Rad iQ5 Real-Time PCR Detection System, according to the manufacturer's instructions.…”

“…Prepro-hypocretin mRNA stability and half-life were determined by culturing primary murine hypothalamic neuron cells in medium with actinomycin D (10 μg/mL) to block transcription as described by us previously. 25 Briefly, cells were treated with actinomycin D, then followed by TNF-α or vehicle treatments for the indicated time, and harvested. Total RNA was collected, and the amounts of preprohypocretin mRNA at each time-point were quantified by real-time RT-PCR and normalized to the amounts of β-actin mRNA at the same time-point.…”