Internal Translation Initiation Mediated by the Angiogenic Factor Tie2

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5′-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5′-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5′-UTR and how its production is secured under unfavorable environmental conditions. Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5′-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5′-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5′-UTR and how its production is secured under unfavorable environmental conditions. Angiogenesis is an essential step in allowing tumors to grow beyond 1–2 mm in diameter (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4846) Google Scholar). Although the vasculature of most adult tissues is quiescent, during embryogenesis or in pathological conditions such as cancer, a pro-angiogenic setting is established. At least two families of growth factors, vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor; Ang, angiopoietin; IRES, internal ribosome entry site; UTR, untranslated region; HUVEC, human umbilical vein endothelial cell; EMCV, encephalomyocarditis virus; ORF, open reading frame; uORF, upstream ORF; RT, reverse transcriptase; nt, nucleotide; ODC, ornithine decarboxylase; RACE, rapid amplification of cDNA ends; RPA, RNase protection assay. and the angiopoietins, are required for this process. VEGF mediates its effects through VEGFR-1/Flt-1 and VEGFR-2/Flk-1/KDR, two endothelial receptors implicated as central regulators of the vascular system under both normal and abnormal physiological conditions (2Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3459) Google Scholar, 3Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3048) Google Scholar, 4Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3361) Google Scholar, 5Griffioen A.W. Molema G. Pharmacol. Rev. 2000; 52: 237-268PubMed Google Scholar). VEGF expression is stimulated under hypoxic conditions by the transcription factor hypoxia-inducible factor-1 (HIF-1), which regulates a large range of physiological responses as a consequence of reduced oxygen availability (6Maxwell P.H. Pugh C.W. Ratcliffe P.J. Curr. Opin. Genet. Dev. 2001; 11: 293-299Crossref PubMed Scopus (341) Google Scholar, 7Semenza G.L. Trends Mol. Med. 2002; 8: S62-S67Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). The angiopoietins belong to a second family of angiogenic factors essential for blood vessel maturation. Angiopoietin-1 (Ang-1) is an agonist of endothelial cell tyrosine kinase receptor Tie2/tek (8Suri C. Jones P.F. Patan S. Bartunkova S. Maisonpierre P.C. Davis S. Sato T.N. Yancopoulos G.D. Cell. 1996; 87: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (2395) Google Scholar). Studies with Tie2 null mice indicate that the angiopoietin/Tie2 signaling system plays a role in the later steps of angiogenesis (9Dumont D.J. Gradwohl G. Fong G.H. Puri M.C. Gertsenstein M. Auerbach A. Breitman M.L. Genes Dev. 1994; 8: 1897-1909Crossref PubMed Scopus (818) Google Scholar). Consistent with a central role for Tie2 in angiogenesis, germ line-activating mutations in humans are associated with vascular dysmorphogenesis (10Vikkula M. Boon L.M. Carraway 3rd, K.L. Calvert J.T. Diamonti A.J. Goumnerov B. Pasyk K.A. Marchuk D.A. Warman M.L. Cantley L.C. Mulliken J.B. Olsen B.R. Cell. 1996; 87: 1181-1190Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar). Additionally, the naturally occurring Tie2 antagonist, Ang-2, disrupts angiogenesis in vivo (11Maisonpierre P.C. Suri C. Jones P.F. Bartunkova S. Wiegand S.J. Radziejewski C. Compton D. McClain J. Aldrich T.H. Papadopoulos N. Daly T.J. Davis S. Sato T.N. Yancopoulos G.D. Science. 1997; 277: 55-60Crossref PubMed Scopus (2954) Google Scholar). Blocking Tie2 activation with recombinant Ang-2 significantly inhibits tumor growth (12Lin P. Buxton J.A. Acheson A. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Channon K.M. Hale L.P. Dewhirst M.W. George S.E. Peters K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8829-8834Crossref PubMed Scopus (338) Google Scholar) and is associated with activation of apoptosis (13Jones N. Voskas D. Master Z. Sarao R. Jones J. Dumont D.J. EMBO Rep. 2001; 2: 438-445Crossref PubMed Scopus (74) Google Scholar), possibly due to disruption of Akt signaling (14Papapetropoulos A. Fulton D. Mahboubi K. Kalb R.G. O'Connor D.S. Li F. Altieri D.C. Sessa W.C. J. Biol. Chem. 2000; 275: 9102-9105Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Hence, studies aimed at better defining the regulation of Tie2 expression are important in assessing anti-angiogenic therapeutic avenues. In addition to profound transcriptional effects (6Maxwell P.H. Pugh C.W. Ratcliffe P.J. Curr. Opin. Genet. Dev. 2001; 11: 293-299Crossref PubMed Scopus (341) Google Scholar, 7Semenza G.L. Trends Mol. Med. 2002; 8: S62-S67Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar), exposure of cells to hypoxia attenuates protein synthesis by decreasing translation initiation (15Koumenis C. Naczki C. Koritzinsky M. Rastani S. Diehl A. Sonenberg N. Koromilas A. Wouters B.G. Mol. Cell. Biol. 2002; 22: 7405-7416Crossref PubMed Scopus (549) Google Scholar, 16Arsham A.M. Howell J.J. Simon M.C. J. Biol. Chem. 2003; 278: 29655-29660Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Two main steps of initiation are targets for regulation, either 43 S ribosomal complex formation (by affecting eIF2 phosphorylation status (17Clemens M.J. J. Cell. Mol. Med. 2001; 5: 221-239Crossref PubMed Scopus (93) Google Scholar)) or the mRNA/ribosome binding step (18Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1762) Google Scholar). Ribosome recruitment in eukaryotes can occur by two mechanisms, a cap-dependent process and by internal ribosome recruitment. Cap-dependent recruitment occurs through eIF4F-mediated recognition of the m7G-cap structure and involves binding of the 43 S preinitiation complex near the mRNA 5′-end, followed by scanning to the appropriate AUG start codon (18Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1762) Google Scholar). Barriers to the scanning process, such as mRNA secondary structure and uORFs, impinge in a negative fashion on the translational efficiency of a mRNA species. The cap dependence of an mRNA is thought to be a function of 5′-cap-proximal secondary structure; hence mRNAs with reduced secondary structure will have reduced dependence on eIF4F for initiation, whereas those with increased secondary structure are more dependent on eIF4F for initiation (19Gehrke L. Auron P.E. Quigley G.J. Rich A. Sonenberg N. Biochemistry. 1983; 22: 5157-5164Crossref PubMed Scopus (60) Google Scholar, 20Svitkin Y.V. Pause A. Haghighat A. Pyronnet S. Witherell G. Belsham G.J. Sonenberg N. RNA (N. Y.). 2001; 7: 382-394Crossref PubMed Scopus (336) Google Scholar). Internal ribosome recruitment allows an mRNA to bypass the cap-dependent initiation requirement for eIF4F. These different mechanisms of initiation provide a layer of gene regulation by which expression of specific mRNAs can be maintained or altered independent of others. Although hypoxia exerts inhibitory effects on cap dependent ribosome binding (16Arsham A.M. Howell J.J. Simon M.C. J. Biol. Chem. 2003; 278: 29655-29660Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), several mRNAs implicated in angiogenesis remain efficiently translated, including VEGF (21Huez I. Creancier L. Audigier S. Gensac M.C. Prats A.C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (244) Google Scholar, 22Akiri G. Nahari D. Finkelstein Y. Le S.Y. Elroy-Stein O. Levi B.Z. Oncogene. 1998; 17: 227-236Crossref PubMed Scopus (224) Google Scholar, 23Stein I. Itin A. Einat P. Skaliter R. Grossman Z. Keshet E. Mol. Cell. Biol. 1998; 18: 3112-3119Crossref PubMed Scopus (424) Google Scholar, 24Miller D.L. Dibbens J.A. Damert A. Risau W. Vadas M.A. Goodall G.J. FEBS Lett. 1998; 434: 417-420Crossref PubMed Scopus (70) Google Scholar), HIF-1α (25Lang K.J. Kappel A. Goodall G.J. Mol. Biol. Cell. 2002; 13: 1792-1801Crossref PubMed Scopus (270) Google Scholar), and Tie2 (this report). Whereas VEGF and HIF-1α achieve this by recruiting ribosomes internally to an IRES, the issue of how Tie2 is able to circumvent this translational block has not been addressed previously. Herein, we present functional studies that identify the presence of an IRES element within the Tie2 5′-UTR, highlighting the complexities of Tie2 expression at the level of translation. Cell Lines, Transfections, and Hypoxia Treatment—Primary human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (Walkersville, MD) and maintained in endothelial growth medium-2 supplemented with growth factors, 2% fetal bovine serum, 50 μg/ml gentamicin, and 50 μg/ml amphotericin to the were as by the cells were and in endothelial growth medium-2 supplemented with 2% fetal bovine were and the system RNA were the were to were with of and mRNA and were in and analysis and RNA from under hypoxia and for were by on with were two and or RNA from RNA from to the were with kinase and on of of mRNA or of RNA and of the at for The to the were on an and the by RNase for synthesis were by a containing the to the to used with the in transcription to from the The by followed by for at in mm The efficiency of by and of were used RNase protection were the system to the the to of human for at RNA with a of RNase RNase The were on an and by and were on at were with for and in mm mm mm RNase at a the on in mm mm mm were at for at and were the and in RNA or RNA from the of and of on G. P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). a to the 5′-UTR by amplification of the 5′-UTR cDNA and In the second the containing 5′-UTR and region of were by the 5′-UTR and the of containing a structure element A. C. W. Sonenberg N. EMBO J. 7: PubMed Scopus Google Scholar)) were by the element upstream of the region of the of the recombinant in this in which a of its AUG codon with the Tie2 AUG codon in The of by expression were a RNA by the and the RNA to the The level of S used as a for within to the of specific The of used for are on RNA with of RNA with of RNA a by and to a in at with a of the at with to the with were at cells and for followed by a under either or hypoxic conditions. were with and in supplemented with fetal and to were with and in mm mm mm mm mm μg/ml μg/ml μg/ml The cell of were with protein and with and The were to followed by were at least in Tie2 mRNA under the translational of Tie2 mRNA under hypoxic we its from and hypoxic hypoxic is a in by an in of ribosomal as I. Itin A. Einat P. Skaliter R. Grossman Z. Keshet E. Mol. Cell. Biol. 1998; 18: 3112-3119Crossref PubMed Scopus (424) Google Scholar, K.J. Kappel A. Goodall G.J. Mol. Biol. 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M. 2000; PubMed Scopus Google Scholar) or mRNA mRNA despite an of the We this to the of to and that is in the To Tie2 protein under hypoxic we analysis on protein from protein in from hypoxic cells activation of the hypoxic In this used as a for Consistent with the Tie2 protein present in both and hypoxic cells this a with an is used as a for Tie2 protein translated under both and hypoxic conditions as by of followed by with In synthesis of and significantly exposure of to hypoxic To the to which translation in is by we the of in under or hypoxic conditions hypoxic protein synthesis is reduced by in in with under or conditions hypoxic indicate that translation of Tie2 mRNA is to the in protein synthesis by hypoxic of The of VEGF, and mRNAs in RNA from were by VEGF mRNA increased in to whereas those of These are with effects of hypoxia on VEGF and gene D. Itin A. D. Keshet E. Nature. PubMed Scopus Google Scholar, J.A. F. G.L. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, S. M.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). hypoxic Tie2 mRNA with the by and S.J. 1998; PubMed Scopus Google Scholar). the Tie2 an to how Tie2 translation is maintained under hypoxic during a cap-dependent protein synthesis is we undertook a structure/function to mapping of the Tie2 The transcription start of the human Tie2 mRNA have been and Biochem. 1998; PubMed Scopus Google Scholar). The the presence of transcription initiation from to upstream of the AUG codon Biochem. J. 1998; PubMed Scopus Google Scholar). from this is not to which were by those by We the transcription initiation in the human Tie2 mRNA to identify transcription start analysis with which the of the Tie2 5′-UTR specific were with or that of the transcription start two were with These to and of the Tie2 5′-UTR within To the and we the start we RNase protection The used from to by and Two of and were These to and of the Tie2 5′-UTR We that the start site at have been in the due to in this range of the the RNase protection and indicate that a Tie2 transcription site at site within of the Tie2 transcription initiation site Biochem. J. 1998; PubMed Scopus Google Scholar). analysis of with the Tie2 the of a within the Tie2 and J. Hence, the Tie2 mRNA isoforms several to be inhibitory to a cap-dependent initiation a 5′-UTR five uORFs, and an AUG codon with the In this we the translational of the The Tie2 5′-UTR Internal mechanism by which Tie2 be efficiently translated under hypoxic conditions a that cap-dependent translation initiation (16Arsham A.M. Howell J.J. Simon M.C. J. Biol. Chem. 2003; 278: 29655-29660Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, G. P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), be Tie2 translation by internal ribosome recruitment to the To this, the Tie2 5′-UTR within the the and We a in which been by AUG to to to IRES activity a negative for we a of the 5′-UTR that not internal ribosome binding The of the is to the of the Tie2 5′-UTR used in In we in which the human element upstream of to expression independent of expression and not a consequence of ribosome The secondary structure of this element is to as a to initiation and expression of the protein A. C. W. Sonenberg N. EMBO J. 7: PubMed Scopus Google Scholar). with that expression containing the element upstream of the initiation codon a in activity to the this to of activity were from cells with or the 5′-UTR not for or for internal ribosome recruitment and and were in from cells with and with or with and with These indicate that the Tie2 5′-UTR mediates the production of independent of as be an IRES IRES during function of the Tie2 IRES be to translation during hypoxia To the Tie2 IRES function in a under hypoxic we with or and cells under or hypoxic conditions were in cell We that the activity cells were to hypoxia is an of the in protein on of which will be present to the start of hypoxic of the increased under hypoxic conditions in The indicates that the Tie2 IRES activity stimulated under hypoxic conditions These indicate that the Tie2 IRES is functional during hypoxic analysis indicates that in vivo from and were of that for the expression of from and to for the be the existence of upstream of the region that a or the upstream translation of a to increased the to the existence of an a for this, we on RNA from that been with or were that the 5′-UTR of and within the region of RNA from cells and in to the the and Tie2 5′-UTR were not reverse from the this of as by the of amplification be during the To that the in were not due to the presence of a within the Tie2 5′-UTR, we expression in which the Tie2 5′-UTR upstream of the gene in a containing used as a of that and of activity the activity with and These indicate the of a activity within the Tie2 To and we a of mRNA We and mRNAs from the 5′-UTR with the IRES the IRES and the Tie2 5′-UTR containing or of mRNAs and production by or by that production of the mRNA whereas the of expression from cells with with the of an IRES in this In of mRNA of from cells with and with more from the with These were by and The from with mRNA that from cells with with the presence of an IRES in the The of cells that of the negative These indicate that the Tie2 IRES activity is and not due to the presence of a that with that the within the Tie2 5′-UTR translation The signaling plays an important role in the of normal and tumor Tie2 expression is in human with expression in at the of tumors by Peters K.G. P.C. P. L. Dewhirst M.W. S. PubMed Scopus Google have been in Tie2 protein and mRNA during in Tie2 protein expression have been to in Tie2 mRNA I. 2003; PubMed Scopus Google Scholar, C. P. M. S. U. 2000; 87: PubMed Scopus Google Scholar, K. R. P. L. J. Cell. Biochem. 2002; PubMed Scopus Google Scholar). In and S.J. 1998; PubMed Scopus Google Scholar) have Tie2 mRNA during and S. K.G. H. Biochem. 2002; PubMed Scopus Google Scholar) Tie2 protein under The for are not to hypoxia the different cell In Tie2 mRNA as a consequence of hypoxia with protein production at with those during Our indicates that Tie2 protein production is secured under hypoxic conditions translation is The presence of an IRES within the Tie2 5′-UTR an for how this mRNA is able to the in translation by of cells to hypoxic conditions has been to be associated with a in cap-dependent translation as a of increased formation of the inhibitory complex (16Arsham A.M. Howell J.J. Simon M.C. J. Biol. Chem. 2003; 278: 29655-29660Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, FEBS Lett. 1999; PubMed Scopus Google Scholar). In increased phosphorylation of associated with hypoxia to protein the production of a specific of mRNAs (15Koumenis C. Naczki C. Koritzinsky M. Rastani S. Diehl A. Sonenberg N. Koromilas A. Wouters B.G. Mol. Cell. Biol. 2002; 22: 7405-7416Crossref PubMed Scopus (549) Google Scholar). The that the Tie2 IRES allows an from translation during hypoxia is by in which the Tie2 IRES activity and under hypoxic conditions The Tie2 mRNA 5′-UTR five uORFs, an unusually for a The used to the of the mRNA in five and the the initiation codon Our that the Tie2 is associated with under both and hypoxic that Tie2 mRNA this complex 5′-UTR with its five We that in both and RNA expression of is increased of the Tie2 are and of is that of the the IRES and the and are by the internally have the consequence of translation initiation, due to ribosomes during or the initiation codon of the ORF, due to an to a for the role of the Tie2 in translation initiation be to the of ribosomes that have by a cap-dependent mechanism from the Tie2 initiation as a to initiation from with IRES activity on the Tie2 mRNA the structure of the Tie2 IRES be by scanning In this ribosomes in a cap-dependent fashion Tie2 5′-UTR secondary structure and on IRES a has been implicated in the function of the IRES, disruption of the and of an IRES internal The IRES activity is of initiation I. J. H. M. Koromilas L. D. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). a occurs on the Tie2 IRES to be addressed and indicate that translation initiation on the Tie2 mRNA is more and complex Our indicate that Tie2 to a of mRNA IRES activity to circumvent the of protein synthesis during hypoxic mRNAs in this G. P. RNA (N. Y.). 1998; PubMed Scopus Google Scholar, C. Prats A.C. L. P. G. V. Oncogene. 2001; PubMed Scopus Google Scholar, M. Le Oncogene. 1998; PubMed Scopus Google Scholar, C. I. Audigier S. Gensac M.C. S. G. Prats A.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), HIF-1α (25Lang K.J. Kappel A. Goodall G.J. Mol. Biol. Cell. 2002; 13: 1792-1801Crossref PubMed Scopus (270) Google Scholar), and VEGF (21Huez I. Creancier L. Audigier S. Gensac M.C. Prats A.C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (244) Google Scholar, 22Akiri G. Nahari D. Finkelstein Y. Le S.Y. Elroy-Stein O. Levi B.Z. Oncogene. 1998; 17: 227-236Crossref PubMed Scopus (224) Google Scholar, 23Stein I. Itin A. Einat P. Skaliter R. Grossman Z. Keshet E. Mol. Cell. Biol. 1998; 18: 3112-3119Crossref PubMed Scopus (424) Google Scholar, 24Miller D.L. Dibbens J.A. Damert A. Risau W. Vadas M.A. Goodall G.J. FEBS Lett. 1998; 434: 417-420Crossref PubMed Scopus (70) Google Scholar). The presence of in the 5′-UTRs of two mRNAs angiogenic factors and that this translation initiation mechanism plays an important role in the of The of expression during this process be to specific gene expression during a translation is We that are maintained during hypoxia for Additionally, the signaling system has been implicated in of cells in the F. A. M. Sato H. S. K. K. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Tie2 expression to be maintained during a physiological associated with reduced protein synthesis Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). Hence, the Tie2 IRES to circumvent the translation block by to Tie2 protein We the for in the

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