Goldberg-Cohen et al: Regulation of vascular endothelial growth factor by hypoxia One of the key factors, which controls VEGF expression, is oxygen tension. A growing mass such as an embryo or a tumor is in need of oxygen when it can no longer rely on diffusion to sustain itself. The lack of oxygen, termed hypoxia, induces a cascade of events, which increase VEGF expression and ultimately the growth of new blood vessels.
abolished the destabilizing properties of the entire AU rich element (Akashi et al, 1994; Chen et al, 1994). The degradation of mRNAs containing AU rich elements in their 3´ UTR is facilitated by the binding of trans-acting factors which may promote exonuclease as well as site-specific endonucleolytic events. Tristetraproline (TTP) and AUF1 are two such trans-acting RNA binding proteins that bind AU rich elements and destabilize the mRNAs carrying these sequences (Brewer, 1991; Carballo et al, 1998; Lai and Blackshear, 2001). While AU rich elements allow for the rapid degradation of mRNAs they also appear to be able to bind trans-acting factors that act to increase mRNA stability under certain circumstances as discussed below for VEGF mRNA. Like GM-CSF, the 3´UTR of VEGF mRNA consists of multiple AU rich elements that render it vulnerable to rapid degradation. However, under hypoxic conditions, RNA binding proteins recognize and bind to their cognate AU rich sites on the 3´UTR of VEGF mRNA, increasing its stability and thus its expression several fold.
III. Hypoxic regulation of VEGF Hypoxia increases VEGF expression by several mechanisms which act at the level of mRNA transcription, stabilization and translation.
A. Upregulation of VEGF mRNA transcription VEGF transcription, as well as that of several other hypoxia inducible genes such as the glycolytic enzymes and erythropoietin, is increased with hypoxia. Most of these genes have Hypoxia Response Elements (HREs) that bind a heterodimeric helix-loop-helix transcription factor called Hypoxia Inducible Factor 1 (HIF-1) (Wang and Semenza, 1995; Semenza et al, 1996). HIF-1 binds to its recognition site on VEGF 5´ promoter and together with other trans acting factors mediates the increase in VEGF transcription with hypoxia. Several other transcription factors such as AP-1 and CREB also appear to influence the hypoxic induction of VEGF transcription most likely via direct interaction with HIF-1 (Abate et al, 1990).
IV. HuR A prominent member of the ARE binding protein family that acts to increase mRNA stability with hypoxia is HuR. This RNA binding protein belongs to the Embryonic Letal Abnormal Visual (ELAV) protein family first described in Drosophila (Robinow et al, 1988). The founding member, ELAV, is expressed immediately following neuroblast differentiation into neurons and is involved in the subsequent neuronal differentiation and maintenance (Robinow and White, 1991; Campos et al, 1985). Further studies identified four human homologues that were characterized as tumor antigens (Szabo et al, 1991). Three of the human ELAV-like proteins are expressed solely in terminal differentiation of neurons and neuroendocrine tumors (King et al, 1994; Barami et al, 1995; Jain et al, 1997) while the fourth, termed HuR, is found in proliferating cells and in tumors throughout the body (Ma et al, 1996). Classification as tumor antigens gave rise to extensive research into the essence of their RNA binding properties and resulted in the identification of three highly conserved RNA recognition motifs. Two of the RNA recognition motifs are in tandem separated from the third by a basic segment (Kenan et al, 1991). Subsequent studies confirmed that the ELAV-like proteins are prone to bind AU rich elements present in the 3´UTRs of mRNAs as well as to their polyA tails, which may contribute to their ability to protect mRNAs from ribonuclease degradation (Ma et al, 1997). As discussed above, HuR, the only ELAV family member not restricted to the nervous system but rather expressed throughout the body, is involved in increasing VEGF mRNA stability with hypoxia by binding to an AU rich recognition site on the VEGF mRNA 3´UTR. A study investigating the binding of HuR to c-fos mRNA identified a high affinity site containing three AU rich motifs AUUUA, AUUUUA, and AUUUUUA, all of which are critical for maximal binding (Ma et al, 1996). The requirement for a nonspecific number of U residues in
B. Hypoxic regulation of VEGF mRNA translation VEGF mRNA has an unusually long 5´ untranslated region (5´ UTR) containing stable secondary structures and a short in-frame initiation and termination codons. This significantly inhibits initiation of protein synthesis by the classical model of the cap-dependant ribosome scanning. VEGF mRNA can also be translated in a capindependent manner through an Internal Ribosome Entry Site (IRES). Under hypoxic conditions, and other conditions of stress, cap dependant translation is reduced. The presence of an IRES site allows the translation of VEGF and other IRES containing mRNAs to continue (Akiri et al, 1998; Stein et al, 1998).
C. Hypoxic stabilization of VEGF mRNA The half life of VEGF mRNA, like that of several other cytokine and oncogene mRNAs, is very short. Increased stability of a mRNA renders it more accessible to the translational machinery and thus increases the amount of its gene product. Shaw and Kamen (1986) reported a considerable decrease in the stability of "globin mRNA when an AU-rich element (ARE) from the 3´UTR of GM-CSF was introduced 3´ to the "-globin gene (Shaw and Kamen, 1986). Further studies indicated that the pentameric sequence AUUUA is necessary but not sufficient to induce degradation of mRNAs and mutations that specifically interrupted this pentameric sequence
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