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J. Biol. Chem., Vol. 279, Issue 10, 8655-8667, March 5, 2004

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Role of the 3'-Untranslated Region of Human Endothelin-1 in Vascular Endothelial Cells

CONTRIBUTION TO TRANSCRIPT LABILITY AND THE CELLULAR HEAT SHOCK RESPONSE^*

Imtiaz A. Mawji¶, G. Brett Robb, Sharon C. Tai, and Philip A. Marsden||**

From the Department of Laboratory Medicine and Pathobiology, University of Toronto, the Department of Medicine, University of Toronto, and the ^||Renal Division, St. Michael's Hospital, Toronto, Ontario M5S 1A8, Canada

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978–987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest 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 proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.

Received for publication, November 7, 2003 , and in revised form, December 1, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of Canadian Institute of Health Research/Heart & Stroke Foundation of Canada Doctoral Research Award.

^** Supported by a grant from the Kidney Foundation of Canada and is a Heart and Stroke Foundation of Canada Career Investigator. To whom correspondence should be addressed: University of Toronto, Medical Sciences Building, Rm. 7358, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-2441; Fax: 416-978-8765; E-mail: p.marsden{at}utoronto.ca.

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