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

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. 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. The endothelium regulates local vascular tone and integrity through the coordinated release of vasoactive molecules. Secretion of endothelin-1 (ET-1) 1The abbreviations used are: ET-1, endothelin-1; nt, nucleotide; ORF, open-reading frame; RT, reverse transcription; UTR, untranslated region; cDNA, complementary DNA; eNOS, endothelial nitricoxide synthase; RNAi, RNA interference; HUVEC, human umbilical vein endothelial cells; WT, wild type; DE, destabilizing element; ARE, AU-rich element; BAEC, bovine aortic endothelial cells; Act D, actinomycin D; HepG2, human hepatocellular carcinoma.1The abbreviations used are: ET-1, endothelin-1; nt, nucleotide; ORF, open-reading frame; RT, reverse transcription; UTR, untranslated region; cDNA, complementary DNA; eNOS, endothelial nitricoxide synthase; RNAi, RNA interference; HUVEC, human umbilical vein endothelial cells; WT, wild type; DE, destabilizing element; ARE, AU-rich element; BAEC, bovine aortic endothelial cells; Act D, actinomycin D; HepG2, human hepatocellular carcinoma. from the endothelium signals vasoconstriction and influences local cellular growth and survival (1Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar, 2Nelson J. Bagnato A. Battistini B. Nisen P. Nat. Rev. Cancer. 2003; 3: 110-116Crossref PubMed Scopus (500) Google Scholar, 3Miyauchi T. Masaki T. Annu. Rev. Physiol. 1999; 61: 391-415Crossref PubMed Scopus (496) Google Scholar, 4Shichiri M. Kato H. Marumo F. Hirata Y. Hypertension. 1997; 30: 1198-1203Crossref PubMed Scopus (157) Google Scholar, 5Wu-Wong J.R. Chiou W.J. Dickinson R. Opgenorth T.J. Biochem. J. 1997; 328: 733-737Crossref PubMed Scopus (105) Google Scholar, 6Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10220) Google Scholar). ET-1 has been implicated in the development and progression of vascular disorders such as atherosclerosis and hypertension (1Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar, 2Nelson J. Bagnato A. Battistini B. Nisen P. Nat. Rev. Cancer. 2003; 3: 110-116Crossref PubMed Scopus (500) Google Scholar, 3Miyauchi T. Masaki T. Annu. Rev. Physiol. 1999; 61: 391-415Crossref PubMed Scopus (496) Google Scholar). Endothelial cells upregulate ET-1 in response to hypoxia, oxidized LDL, pro-inflammatory cytokines, and bacterial toxins (7Boulanger C.M. Tanner F.C. Bea M.L. Hahn A.W. Werner A. Luscher T.F. Circ. Res. 1992; 70: 1191-1197Crossref PubMed Google Scholar, 8Yoshizumi M. Kurihara H. Morita T. Yamashita T. Oh-hashi Y. Sugiyama T. Takaku F. Yanagisawa M. Masaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1990; 166: 324-329Crossref PubMed Scopus (254) Google Scholar, 9Marsden P.A. Brenner B.M. Am. J. Physiol. 1992; 262: C854-C861Crossref PubMed Google Scholar, 10Kourembanas S. Marsden P.A. McQuillan L.P. Faller D.V. J. Clin. Invest. 1991; 88: 1054-1057Crossref PubMed Scopus (632) Google Scholar). Initial studies on the ET-1 promoter provided some of the earliest mechanistic insight into endothelial-specific gene regulation (11Wilson D.B. Dorfman D.M. Orkin S.H. Mol. Cell. Biol. 1990; 10: 4854-4862Crossref PubMed Scopus (131) Google Scholar, 12Lee M.E. Temizer D.H. Clifford J.A. Quertermous T. J. Biol. Chem. 1991; 266: 16188-16192Abstract Full Text PDF PubMed Google Scholar). Numerous studies have since provided valuable insight into ET-1 promoter regulation under basal and activated cellular states (13Mawji I.A. Marsden P.A. Microsc. Res. Tech. 2003; 60: 46-58Crossref PubMed Scopus (39) Google Scholar). The ET-1 mRNA is labile with a half-life of less than an hour (14Inoue A. Yanagisawa M. Takuwa Y. Mitsui Y. Kobayashi M. Masaki T. J. Biol. Chem. 1989; 264: 14954-14959Abstract Full Text PDF PubMed Google Scholar, 15Bloch K.D. Friedrich S.P. Lee M.E. Eddy R.L. Shows T.B. Quertermous T. J. Biol. Chem. 1989; 264: 10851-10857Abstract Full Text PDF PubMed Google Scholar). Together, the combined actions of ET-1 transcription and rapid mRNA turnover allow for stringent control over its expression. We have previously that ET-1 mRNA is stabilized in response to cellular by suggesting ET-1 is regulated by post-transcriptional mechanisms Y. J. Marsden P.A. J. Clin. PubMed Scopus Google Scholar). elements mRNA half-life are found 3′-untranslated (3′-UTR) J. Rev. PubMed Google Scholar, Biochem. Full Text PDF PubMed Scopus Google Scholar, G.M. Res. Mol. Biol. 1999; PubMed Scopus Google Scholar, R. Cell. 46: Full Text PDF PubMed Scopus Google Scholar). The 3′-UTR of human ET-1 for over of the and of highly an AU-rich region (14Inoue A. Yanagisawa M. Takuwa Y. Mitsui Y. Kobayashi M. Masaki T. J. Biol. Chem. 1989; 264: 14954-14959Abstract Full Text PDF PubMed Google Scholar, 15Bloch K.D. Friedrich S.P. Lee M.E. Eddy R.L. Shows T.B. Quertermous T. J. Biol. Chem. 1989; 264: 10851-10857Abstract Full Text PDF PubMed Google Scholar). 3′-UTR AU-rich elements in and expression by half-life under basal and in response to cellular Biochem. Full Text PDF PubMed Scopus Google Scholar, R. Cell. 46: Full Text PDF PubMed Scopus Google Scholar). with for have been characterized AUF1 the and G.M. Res. Mol. Biol. 1999; PubMed Scopus Google Scholar, K. A.W. K. Mol. Cell. Biol. PubMed Scopus Google Scholar, C.M. J.A. Mol. PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar, J. J. A. M. J. H. PubMed Scopus Google Scholar, Biochem. 30: PubMed Scopus Google Scholar, P. Biochem. 30: PubMed Scopus Google Scholar, T. P. A. J.R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). mechanisms directing ARE activity have been suggest to cellular pathways that and J. Rev. PubMed Google Scholar, Biochem. Full Text PDF PubMed Scopus Google Scholar, G.M. Res. Mol. Biol. 1999; PubMed Scopus Google Scholar). studies have revealed a functional heat shock and the ubiquitin-proteasome R. 1999; PubMed Scopus Google Scholar). inhibition by inhibition or heat shock was to a model mRNA whereas of cellular pathways was to ARE mRNA turnover R. 1999; PubMed Scopus Google Scholar, Res. 30: PubMed Scopus Google Scholar, B. S. A. PubMed Scopus Google Scholar). with in proteasome suggest that the proteasome RNA destabilizing activity F. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The AUF1 has been to the ubiquitin-proteasome AUF1 mRNA destabilizing activity has been with its of and has been to with a of the R. 1999; PubMed Scopus Google Scholar, Res. 30: PubMed Scopus Google Scholar, B. S. A. PubMed Scopus Google Scholar, B. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, K. J. Biol. Chem. 2003; PubMed Scopus Google Scholar, G.M. K. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, under of cellular heat shock AUF1 with heat shock ARE activity T. P. A. J.R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R. 1999; PubMed Scopus Google Scholar, G.M. K. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). little is about pathways labile in endothelial the of ET-1 in the vascular we to ET-1 as a model labile mRNA. We evidence that the 3′-UTR the ET-1 through two destabilizing DE1 and and that DE1 functions through a ARE by the We also evidence for a pathway of ET-1 mRNA stabilization by the heat shock and were from and were using were using a were using an were using The luciferase was from with and and into to type and 3′-UTRs were from by or and into the of of the luciferase ET-1 3′-UTR are to the of the 3′-UTR (14Inoue A. Yanagisawa M. Takuwa Y. Mitsui Y. Kobayashi M. Masaki T. J. Biol. Chem. 1989; 264: 14954-14959Abstract Full Text PDF PubMed Google The 3′-UTR was by using human as a and and into to into the of of the luciferase Deletion mutants were by of in and were by or from and into the of and were using and to 3′-UTR by and into the of mutants were as previously F. H. Y. J. Y. Marsden P.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). using as a two were to and of the 3′-UTR to the The of and of were to on the of the 3′-UTR. The for and for of and an a region and B. and were with or and into the 3′-UTR with a at the of This from 3′-UTRs were by and into to and Deletion was from of the of and the of in the of and into the of was by the of a with and the in of wild type The was into the of to a 3′-UTR. The 3′-UTR was from human using into and into to The at position of the was using a as with ET-1 ET-1 are to the from human ET-1 (14Inoue A. Yanagisawa M. Takuwa Y. Mitsui Y. Kobayashi M. Masaki T. J. Biol. Chem. 1989; 264: 14954-14959Abstract Full Text PDF PubMed Google to and of the ET-1 were from human umbilical vein endothelial cells cellular RNA using and of into and and were into the of to a human ET-1 to ET-1 were from by or using the and into to and were from bovine aortic endothelial cells were from on and in with bovine and as previously P.A. Brenner B.M. Am. J. Physiol. 1992; 262: C854-C861Crossref PubMed Google Scholar). were at were from umbilical in with bovine endothelial and as previously P.A. M. S. T. 1992; PubMed Scopus Google Scholar). were at endothelial cells were from and in with bovine and and hepatocellular cells were from and in with bovine and cells were at and and actinomycin were as in in were by two of were by and by were and to for to cells were with of of of luciferase and to a of with at a to on cellular RNA was cells in were with of were for and in with 1 for than were and in with studies were RNA interference of were into of with the The for were of were and luciferase were using the was using the luciferase activity was to luciferase activity to for luciferase activity was to cellular RNA was using the P. Biochem. PubMed Scopus Google Scholar). were as Y. J. Marsden P.A. J. Clin. PubMed Scopus Google using were with to a activity of by analysis of RNA from luciferase or human ET-1 signals were to signals from the gene or to for is expressed from and on were as from the luciferase human ET-1, from the and of to the The ET-1 human ET-1 mRNA and endogenous bovine ET-1 in signals were with a and with 2 for was from cellular RNA using and reverse using were with of RNA in on an and with was under the of at and 1 at or were to were in the of of for of and for ET-1 and endothelial analysis ET-1 were with and with R. S. Res. PubMed Scopus Google Scholar). were with and with were with were with were with and were for at for cellular and by the were in and by of of AUF1 were with AUF1 by were with by were using the and using a were a of the of for data half-life and half-life and using the or analysis of or were using The ET-1 3′-UTR a of ET-1 mRNA ET-1 mRNA in the vascular endothelium are a of promoter activity and mRNA turnover. little is known about the post-transcriptional mechanisms these we expressed human ET-1 transcripts in bovine aortic endothelial cells with or without the 3′-UTR or analysis of RNA from cells revealed that of the 3′-UTR in a in ET-1 mRNA expression studies with actinomycin that of the 3′-UTR mRNA half-life We also the of the 3′-UTR to mRNA destabilizing activity to a Chimeric luciferase transcripts with control or ET-1 3′-UTRs were expressed in endothelial cells and mRNA and mRNA half-life were in the of the ET-1 3′-UTR. Taken together, these findings that the ET-1 3′-UTR potent mRNA destabilizing activity and is for the of ET-1 ET-1 3′-UTR a mRNA. were with luciferase expression as under of luciferase transcripts bearing a control or wild type ET-1 3′-UTR. and ET-1 and region. cellular were from and Chimeric activity was to a to control for cellular RNA was from and luciferase expression by with a were and for expression to control for and luciferase mRNA D, were to with Act D, cellular RNA at expression as and mRNA using in were and WT, endothelial and were and luciferase expression as in B. human endothelial cells; human the of and or The ET-1 3′-UTR mRNA in pathways mRNA turnover are Mol. Cell. Biol. 1994; PubMed Google Scholar, A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, A.W. Biochem. J. 1997; PubMed Scopus Google Scholar). the of ET-1 3′-UTR we to endothelial cells that ET-1 human endothelial as as two human that of ET-1 and The of luciferase in the was to This that the ET-1 3′-UTR functions in a variety of of the cells ET-1 The 3′-UTR DE1 and for directing 3′-UTR destabilizing we a in endothelial Chimeric transcripts bearing wild type and 3′-UTRs were expressed and for luciferase activity and mRNA expression Deletion of the or of the 3′-UTR or destabilizing as by an in luciferase activity and mRNA expression and of of the 3′-UTR to a complex expression and and we that luciferase expression to wild type 3′-UTR was with the of and whereas of of these two and in expression to control destabilizing these functional domains of the 3′-UTR that we to as DE1 and elements 1 and 2 to and of the is to that of DE1 in the in the of DE1 destabilizing activity. findings that DE1 is is for destabilizing activity. Together, findings suggest that DE1 and to ET-1 mRNA of 3′-UTR in a DE1 through a AU-rich of DE1 and and bovine and revealed domains are highly DE1 highly and with the and by AU-rich element implicated in the regulation of mRNA half-life Biochem. Full Text PDF PubMed Scopus Google Scholar, G.M. Res. Mol. Biol. 1999; PubMed Scopus Google Scholar). further we two mutants to the of Deletion of a region DE1 or of in a in luciferase activity and mRNA expression over the wild type 3′-UTR. This data DE1 as a functional AU-rich element also or DE1 as a of mRNA of DE1 were to luciferase transcripts bearing wild type and DE1 of are wild type with the and are to position the 3′-UTR. of the wild type ET-1 3′-UTR DE1 and and an of The of DE1 are by The 3′-UTR a that DE1 The 3′-UTR of DE1 with the and is by cellular were from and the of further functional we expressed mutants and of and we also two a and a We the of by the functional of a on the a model ARE R. 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The destabilizing activity of a 3′-UTR as by luciferase activity and mRNA half-life studies with ET-1 mutants revealed that luciferase activity and mRNA expression wild type ET-1 mutants failed to expression studies using DE1 mutants and that in expression were by in mRNA half-life of DE1 were with luciferase bearing wild type and or ET-1 and as a luciferase transcripts bearing wild type or were expressed in The of the human 3′-UTR is with and The region wild type with the at position of the 3′-UTR. were and cells were with Act D, cellular RNA at expression and mRNA using in were D, of the wild type ET-1 3′-UTR DE1 and and of control and DE1 the of DE1 and a and a mutants have of wild type with by luciferase bearing wild type or ET-1 3′-UTRs were into BAEC, cellular and cellular RNA was from and luciferase and expression by cells were with Act D, cellular RNA at expression as and mRNA using in were WT, the of and or Since of DE1 and or ET-1 3′-UTR destabilizing we that with functions as an AU-rich ET-1 mRNA in and in to or studies have a functional mRNA and pathways the the and of this pathway by heat shock R. 1999; PubMed Scopus Google Scholar). Furthermore, a ARE mRNA and of AUF1 and ARE activity Res. 30: PubMed Scopus Google Scholar, B. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Since the ET-1 3′-UTR functions through an AU-rich we the that its expression is regulated by this we RNA interference to AUF1 expression in human endothelial cells AUF1 of of and by mRNA of the AUF1 gene Y. G.M. PubMed Scopus Google Scholar). in and domains, functional and for Res. 30: PubMed Scopus Google Scholar, G.M. J. K. Y. S. B. S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, G.M. J. K. Y. Y. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that under control AUF1 were in human endothelial cells is of to that the and were in than and This the that and have been to and to pathways R. 1999; PubMed Scopus Google Scholar, Res. 30: PubMed Scopus Google Scholar). AUF1 or by in a than in expression for the We ET-1 and mRNA in these cells using ET-1 mRNA were in cells to cells as a or a control mRNA failed to to AUF1 further the of this pathway, we bovine and human endothelial cells to heat shock or proteasome inhibition with the inhibitor and ET-1 mRNA expression using analysis and ET-1 mRNA were observed of heat shock and ET-1 also to proteasome inhibition by in a and and mRNA failed to to heat shock or in the of ET-1 by these or endothelial cells were to with actinomycin and mRNA ET-1 mRNA was stabilized by heat shock or proteasome inhibition This data provides evidence that ET-1 mRNA turnover is regulated in by the also a novel and of ET-1 by the heat shock of ET-1 mRNA expression by AUF1 heat and proteasome inhibition ET-1 is regulated by the The ET-1 3′-UTR through the the the ET-1 3′-UTR and mRNA were that expressed luciferase transcripts with WT, or ET-1 expression in were by luciferase and and were to observed in of endothelial cells of to an of luciferase activity and mRNA in cells ET-1 3′-UTR transcripts and and mutants failed to to studies under control or that in expression were by changes in mRNA half-life Taken together, these studies suggest that DE1 regulates ET-1 mRNA half-life through a The highly of the that the endothelium to changes in local cellular and a potent vasoconstrictor and cellular the ET-1 gene has of regulation from the and of its mRNA to the and of its peptide (1Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar, 2Nelson J. Bagnato A. Battistini B. Nisen P. Nat. Rev. Cancer. 2003; 3: 110-116Crossref PubMed Scopus (500) Google Scholar, 3Miyauchi T. Masaki T. Annu. Rev. Physiol. 1999; 61: 391-415Crossref PubMed Scopus (496) Google I.A. Marsden P.A. Microsc. Res. Tech. 2003; 60: 46-58Crossref PubMed Scopus (39) Google Scholar). of its mRNA changes in the of ET-1 transcription are by changes in mRNA Together, this functions as an and in ET-1 studies have implicated mRNA stabilization as an of ET-1 expression in response to and cellular Y. J. Marsden P.A. J. Clin. PubMed Scopus Google Scholar, M. S. Yanagisawa M. Y. K. J. Pharmacol. 1991; PubMed Scopus Google Scholar, Lee S.H. Pharmacol. PubMed Scopus Google Scholar, J.A. D.M. R. Biochem. Pharmacol. 2003; PubMed Scopus Google Scholar, S. T. Y. J. Biol. Chem. 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