Post-transcriptional regulation of human endothelin-1 by the 3'-untranslated region in health and disease.
Published: 2005
Language: eng
Number of pages: 269
Description: Endothelin-1 (ET-1) is a potent vasoconstrictor expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through an inducible promoter and rapid mRNA turnover. Aberrant expression of ET-1 is associated with endothelial dysfunction and the progression of vascular disease. For example, E. coli O157:H7 derived-verotoxin (VT) activates the endothelium leading to vascular injury and hemolytic uremic syndrome. VT also stabilizes the ET-1 mRNA. However little is known about pathways regulating ET-1 mRNA stability in health and disease. To characterize these pathways, human ET-1 and luciferase transcripts were expressed with or without the wild-type ET-1 3'-untranslated region (3'-UTR). The 3'-UTR was found to have potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR termed d&barbelow;estabilizing e&barbelow;lements 1 and 2 (DE1 and DE2). Mutational studies localized DE1 to a 100 nt AU-rich element (ARE). Detailed mapping studies on the ARE defined a 10 nt AU-motif as the primary mediator of DE1 mRNA destabilizing activity. Depletion of endothelial cell AUF1 by RNA interference induced ET-1 expression, suggesting ET-1 mRNA stability is regulated by an AUF1-dependent pathway. Since AUF1 functions through the ubiquitin-proteasome pathway, heat shock and proteasome inhibition were used to disrupt this pathway resulting in stabilization of endogenous ET-1 mRNA in endothelial cells. Chimeric transcripts bearing wild-type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Reporter transcripts were used to investigate the role of the 3'-UTR in VT-mediated ET-1 mRNA stabilization. VT induced reporter expression in a 3'-UTR-specific manner whereas induction by VT was attenuated in DE1 mutants, suggesting VT functions through a verotoxin response element (VRE) within DE1. Although VT did not change AUF1 or HSP70 protein levels, VT decreased total cellular protein-ubiquitin conjugates. Therefore VT may stabilize ET-1 mRNA through modulation of the ubiquitin proteasome pathway. Taken together, these findings describe a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the AUF1-proteasome pathway. Finally, these data provides evidence that VT regulates ET-1 by influencing post-transcriptional regulatory pathways.
Notes: Source: Dissertation Abstracts International, Volume: 66-10, Section: B, page: 5185.

Thesis (Ph.D.)--University of Toronto, 2005.

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