Abstract
Endothelin-1 (ET-1) is a potent endothelial-derived vasocon-strictor and cellular mitogen. Perturbations in ET-1 levels have been observed in a number of cardiovascular and renal disorders. Steady-state ET-1 mRNA expression is regulated in the vascular endothelium by an inducible promoter and a constitutively short mRNA half-life. Recent studies have identified mRNA stabilization as a pathophysiologically relevant mechanism of ET-1 induction in vascular endothelial cells. However, mechanistic studies on posttranscriptional pathways in physiologically relevant postconfluent primary endothelial cell monolayers have remained challenging because of endothelial resistance to DNA-based gene transfer and expression. To overcome these challenges, we developed an RNA transfection method to study ET-1 posttranscriptional regulation. Reporter transcripts transfected into either preconfluent or postconfluent primary endothelial cells were rapidly and robustly expressed. RNA transfection reconstituted poly(A)-tail–dependent protein expression and ET-1 3′-UTR–dependent mRNA destabilization, suggesting that the transfected RNA accessed endothelial cell posttranscriptional pathways. Because RNA transfection uncouples transcription from expression, the influence of the ET-1 3′-UTR on posttranscriptional expression kinetics could also be monitored. Taken together, our results suggest that RNA transfection is a versatile tool to investigate ET-1 posttranscriptional regulation in endothelial cell culture models.
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