GTMB 7 - Gene Therapy & Molecular Biology

Goldberg-Cohen et al: Regulation of vascular endothelial growth factor by hypoxiaOne of the key factors, which controls VEGFexpression, is oxygen tension. A growing mass such as anembryo or a tumor is in need of oxygen when it can nolonger rely on diffusion to sustain itself. The lack ofoxygen, termed hypoxia, induces a cascade of events,which increase VEGF expression and ultimately thegrowth of new blood vessels.III. Hypoxic regulation of VEGFHypoxia increases VEGF expression by severalmechanisms which act at the level of mRNA transcription,stabilization and translation.A. Upregulation of VEGF mRNAtranscriptionVEGF transcription, as well as that of several otherhypoxia inducible genes such as the glycolytic enzymesand erythropoietin, is increased with hypoxia. Most ofthese genes have Hypoxia Response Elements (HREs) thatbind a heterodimeric helix-loop-helix transcription factorcalled Hypoxia Inducible Factor 1 (HIF-1) (Wang andSemenza, 1995; Semenza et al, 1996). HIF-1 binds to itsrecognition site on VEGF 5´ promoter and together withother trans acting factors mediates the increase in VEGFtranscription with hypoxia. Several other transcriptionfactors such as AP-1 and CREB also appear to influencethe hypoxic induction of VEGF transcription most likelyvia direct interaction with HIF-1 (Abate et al, 1990).B. Hypoxic regulation of VEGF mRNAtranslationVEGF mRNA has an unusually long 5´ untranslatedregion (5´ UTR) containing stable secondary structuresand a short in-frame initiation and termination codons.This significantly inhibits initiation of protein synthesis bythe classical model of the cap-dependant ribosomescanning. VEGF mRNA can also be translated in a capindependentmanner through an Internal Ribosome EntrySite (IRES). Under hypoxic conditions, and otherconditions of stress, cap dependant translation is reduced.The presence of an IRES site allows the translation ofVEGF and other IRES containing mRNAs to continue(Akiri et al, 1998; Stein et al, 1998).C. Hypoxic stabilization of VEGF mRNAThe half life of VEGF mRNA, like that of severalother cytokine and oncogene mRNAs, is very short.Increased stability of a mRNA renders it more accessibleto the translational machinery and thus increases theamount of its gene product. Shaw and Kamen (1986)reported a considerable decrease in the stability of β-globin mRNA when an AU-rich element (ARE) from the3´UTR of GM-CSF was introduced 3´ to the β-globin gene(Shaw and Kamen, 1986). Further studies indicated thatthe pentameric sequence AUUUA is necessary but notsufficient to induce degradation of mRNAs and mutationsthat specifically interrupted this pentameric sequenceabolished the destabilizing properties of the entire AU richelement (Akashi et al, 1994; Chen et al, 1994).The degradation of mRNAs containing AU richelements in their 3´ UTR is facilitated by the binding oftrans-acting factors which may promote exonuclease aswell as site-specific endonucleolytic events.Tristetraproline (TTP) and AUF1 are two such trans-actingRNA binding proteins that bind AU rich elements anddestabilize the mRNAs carrying these sequences (Brewer,1991; Carballo et al, 1998; Lai and Blackshear, 2001).While AU rich elements allow for the rapiddegradation of mRNAs they also appear to be able to bindtrans-acting factors that act to increase mRNA stabilityunder certain circumstances as discussed below for VEGFmRNA. Like GM-CSF, the 3´UTR of VEGF mRNAconsists of multiple AU rich elements that render itvulnerable to rapid degradation. However, under hypoxicconditions, RNA binding proteins recognize and bind totheir cognate AU rich sites on the 3´UTR of VEGFmRNA, increasing its stability and thus its expressionseveral fold.IV. HuRA prominent member of the ARE binding proteinfamily that acts to increase mRNA stability with hypoxiais HuR. This RNA binding protein belongs to theEmbryonic Letal Abnormal Visual (ELAV) protein familyfirst described in Drosophila (Robinow et al, 1988). Thefounding member, ELAV, is expressed immediatelyfollowing neuroblast differentiation into neurons and isinvolved in the subsequent neuronal differentiation andmaintenance (Robinow and White, 1991; Campos et al,1985). Further studies identified four human homologuesthat were characterized as tumor antigens (Szabo et al,1991). Three of the human ELAV-like proteins areexpressed solely in terminal differentiation of neurons andneuroendocrine tumors (King et al, 1994; Barami et al,1995; Jain et al, 1997) while the fourth, termed HuR, isfound in proliferating cells and in tumors throughout thebody (Ma et al, 1996). Classification as tumor antigensgave rise to extensive research into the essence of theirRNA binding properties and resulted in the identificationof three highly conserved RNA recognition motifs. Two ofthe RNA recognition motifs are in tandem separated fromthe third by a basic segment (Kenan et al, 1991).Subsequent studies confirmed that the ELAV-like proteinsare prone to bind AU rich elements present in the 3´UTRsof mRNAs as well as to their polyA tails, which maycontribute to their ability to protect mRNAs fromribonuclease degradation (Ma et al, 1997).As discussed above, HuR, the only ELAV familymember not restricted to the nervous system but ratherexpressed throughout the body, is involved in increasingVEGF mRNA stability with hypoxia by binding to an AUrich recognition site on the VEGF mRNA 3´UTR. A studyinvestigating the binding of HuR to c-fos mRNAidentified a high affinity site containing three AU richmotifs AUUUA, AUUUUA, and AUUUUUA, all ofwhich are critical for maximal binding (Ma et al, 1996).The requirement for a nonspecific number of U residues in70