Gene Therapy and Molecular Biology Vol 7, page 71 the target sequence points to an inclination towards binding a particular structure rather than a primary sequence (Kim et al, 1974).
it binds and stabilizes VEGF.
VI. Perspectives The manipulation of angiogenic events as a therapeutic tool marks the dawn of a new era in treating numerous afflictions in medicine today. Whether angiogenesis is stimulated to feed the ischemic heart or anti angiogenic agents are applied to prevent tumor growth and metastases, understanding and controlling angiogeneic factors will be critical to the outcome. As a major participant in the angiogenic process, VEGF has been the target of a worldwide research effort in the past two decades. Cancer research has focused on the importance of VEGF in tumor growth and metastases and the findings support this notion. VEGF is now recognized as a key regulator in tumor induced angiogenesis and several anti VEGF treatments that utilize antibodies against VEGF and VEGF receptors have successfully blocked tumor growth in mouse models (Kim et al, 1993; Ferrara, 1999; Gerber et al, 2000; Lee et al, 2000). Cardiovascular research has also benefited from the advances in VEGF research. Gene therapy techniques that augment VEGF expression in the diseased heart or ischemic leg are currently under way. The success of these VEGF treatments will depend to a large extent on our understanding the complex regulation of VEGF. In addition, deciphering the molecular mechanisms that govern the expression of VEGF and other angiogenic factors will give rise to new possibilities not only in the ever growing field of vascular biology but also in the vast area of embryonic development and tissue transplantation.
V. HuR binding site on VEGF mRNA 3´UTR The 3´UTR of VEGF mRNA contains long stretches of AU residues, which confer rapid mRNA degradation through the binding of ribonucleases to the AU rich elements. However, under hypoxic conditions, these AU rich elements allow the binding of RNA binding proteins such as HuR, which block binding of ribonucleases and thus increase the stability of the VEGF mRNA and VEGF expression under hypoxia (Stein et al, 1995;; Damert et al, 1997; Claffey et al, 1998). Attempts to characterize the minimal binding site of HuR on the 3´UTR of VEGF mRNA that is still able to confer increased stability with hypoxia were carried out in our lab and resulted in the identification of a 40 base pair element at position 1285 of the 3´UTR of VEGF mRNA (nucleotides 1285-1325 of the VEGF 3´UTR, GenBank accession number U22372)(Goldberg-Cohen et al, 2002). Transient cotransfection of a vector carrying the 40 base pair element positioned 3´ to the luciferase reporter gene and a plasmid overexpressing HuR showed an increase in reporter activity that correlated with an increase in cotransfected HuR. Furthermore, when incubated overnight under hypoxic conditions, cells transfected with the reporter vector containing the 40 base pair element had greater reporter activity than cells transfected with reporter vector alone. These observations were confirmed in an in vitro model where the stability of an RNA containing the 40 base pair element was shown to be increased in an RNA degradation assay in the presence of HuR. RNase T1 and lead protection assays mapped the HuR binding site to nucleotides 23-39 of the 40 base pair element. Deletion of the HuR specific binding site dramatically reduced reporter activity in the transient transfection assay (Goldberg-Cohen et al, 2002). In view of the ability of HuR to bind and stabilize VEGF mRNA with hypoxia, a model was constructed. In this model, under normoxic conditions VEGF mRNA is extremely unstable by virtue of its recognition by ribonucleases that bind the VEGF mRNA 3´UTR and cause its rapid degradation. This labile character of VEGF mRNA can be overcome under hypoxic conditions through the binding of HuR to its recognition site on the 3´UTR of VEGF mRNA rendering it less vulnerable to ribonuclease digestion. The hypoxic regulation of HuR is not completely understood. Under normoxic conditions the bulk of HuR is sequestered in the nucleus. Under hypoxia, cytoplasmic HuR levels increase with no apparent increase in total HuR levels (Levy et al, 1998). This would suggest nucleocytoplasmic transport of HuR and indeed it was reported that HuR possess a shuttling signal termed HuR Nucleocytoplasmic Shuttling sequence (HNS) that may be significant to the process (Fan and Steitz, 1998). It remains to be investigated whether HuR binds VEGF mRNA in the nucleus and is transported to the cytoplasm as a complex or whether HuR is first transported to the cytoplasm where
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