Mécanismes modulant la stabilité de l’ARNm alphaENaC des cellules épithéliales alvéolaires dans un environnement inflammatoire
The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays an important role in lung liquid clearance at birth and in acute respiratory distress syndrome (ARDS). Preliminary work in the laboratory has shown that TNF-α downregulates expression of αENaC mRNA in part via post-transcriptional modulation of its stability. In the present study, five 3'UTR αENaC sequences that are highly conserved (CS) across different species were studied to determine the impact of each of them on the modulation of αENaC mRNA under basal conditions, as well as in the presence of TNF-α. To study the involvement of these CS on the modulation of αENaC mRNA, a Tet-Off model was used to inhibit transcript expression using doxycycline. Transcript levels over time were estimated by RT-qPCR. With this technique, the half-life of αENaC mRNA is ~100 min. Punctual deletion mutants of 3'UTR were generated by specifically removing the CS1 (31pb), CS3 (22bp), CS2&3 (82bp), CS4&5 (74bp), CS1-5 (187bp) and CS3+ (165bp) domains. Our results show that the deletion of each CS affects their respective half-lives at 169 min, 172 min, 246 min, 193 min, 208 min and 237 min. The stability of the different mutants was then evaluated in the presence of TNF-α (a cytokine found in the lungs of patients with ARDS). In the presence of the complete 3’UTR, the degradation curve of the mRNA in presence of TNF-α was different from the one observed in under basal conditions. In presence of TNF-α, the curve turns biphasic with a rapid degradation followed by a plateau at about 50% of the expression level. The same thing was observed for the deletion mutant CS1-5. We therefore conclude that in general the 3'UTR of αENaC mRNA via the conserved domains is involved in the destabilization of the transcript. CSs studied did not contribute to the decreased expression of αENaC mRNA in the presence of TNF-α. However, we cannot exclude the possibility that other 3’UTR sequences could play an important role in the rapid degradation of αENaC mRNA during inflammation or lung injury.
inflammation TNF-alpha epithelial sodium channel (ENaC) messenger RNA stability 3' untranslated region (3'UTR) half-life alveolar epithelial cells ARDS