Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the pathological remodeling of air sacs as a result of excessive accumulation of extracellular matrix (ECM) proteins, but the mechanism governing the robust protein expression is poorly understood. Our recent findings demonstrate that alternative polyadenylation (APA) caused by NUDT21 reduction is important for the increased expression of fibrotic mediators and ECM proteins in lung fibroblasts by shortening the 3'-untranslated regions (3'-UTRs) of mRNAs and stabilizing their transcripts, therefore activating pathological signaling pathways. Despite the importance of NUDT21 reduction in the regulation of fibrosis, the underlying mechanisms for the depletion are unknown. We demonstrate here that NUDT21 is depleted by TGFβ1. We found that miR203, which is increased in IPF, was induced by TGFβ1 to target the NUDT21 3'-UTR, thus depleting NUDT21 in human and mouse lung fibroblasts. TGFβ1-mediated NUDT21 reduction was attenuated by the miR203 inhibitor antagomiR203 in fibroblasts. TGFβ1 transgenic mice revealed that TGFβ1 down-regulates NUDT21 in fibroblasts in vivo Furthermore, TGFβ1 promoted differential APA of fibrotic genes, including FGF14, RICTOR, TMOD2, and UCP5, in association with increased protein expression. This unique differential APA signature was also observed in IPF fibroblasts. Altogether, our results identified TGFβ1 as an APA regulator through NUDT21 depletion amplifying pulmonary fibrosis.
Highlights
Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the pathological remodeling of air sacs as a result of excessive accumulation of extracellular matrix (ECM) proteins, but the mechanism governing the robust protein expression is poorly understood
To identify a profibrotic cytokine that down-regulates NUDT21 in human lung fibroblasts, primary fibroblasts (CCD8Lu) were cultured with seven cytokines that are associated with pulmonary fibrosis: Transforming growth factor 1 (TGF1), insulin-like growth factor 1, connective tissue growth factor, platelet-derived growth factor BB, interleukin (IL) 4, IL6, and IL13 for 7 days (26 –28)
To longitudinally determine the capacity of TGF1 to deplete NUDT21, fibroblasts were cultured with TGF1 and harvested each day for Western blot analysis (Fig. 1B)
Summary
Transforming growth factor 1 (TGF1) down-regulates NUDT21 in primary human lung fibroblasts. Serpine was used as a positive control because this gene is a direct target of Smad3 [34] These two promoter regions were found to be bound by phosphorylated Smad following 3 h of TGF1 treatment in human lung fibroblasts, but SIS3 decreased the Smad binding, suggesting that phosphorylated Smad acts as a transcription factor for miR203 expression (Fig. 3E). Quantitative analysis of the images showed that TGF1 did not reduce GFP intensity or the number of GFP-positive cells in the GFP 3Ј-UTR mutant–transfected fibroblasts (Fig. 3J) These data collectively suggest that endogenous miR203 up-regulation following TGF1 treatment is sufficient to affect NUDT21 gene expression.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
