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VEGFR-2
Most of the responses to VEGF have been attributed to VEGFR-2 signal transduction, as VEGFR-2 is considered the main receptor to mediate VEGF responses in endothelial cells [64]. Generally, upon activation, several specific tyrosine residues of the receptor are phosphorylated, inducing downstream signaling. In the VEGFR, this signaling is mediated by Src homology-2 (SH-2) domains and phosphotyrosine-binding domains [65]. The phosphorylation of different tyrosine residues activates different signal transduction pathways, which induce cytoskeletal reorganization, cell migration, or cellular permeability, respectively. Mitogenic function of VEGFR-2 signaling is mediated via PLC-g1, PKC, and ERK1/2 kinases, while survival signaling is mediated by PI3 kinase and Akt phosphorylation [30]. Activation of eNOS, either by PLC-g1 or by PI3K pathways, increases cellular permeability as does activation of p38, which also activates Hsp27, resulting in actin remodeling and cell migration [66].
Neuropilin
Neuropilin-1 and neuropilin-2 are transmembrane glycoproteins that share 44% homology [67]. As they lack an enzymatic intracellular activity, they are considered coreceptors of VEGFR, modulating VEGFR activity. There are, however, VEGF induced functions of NP-1 that are independent of VEGFR-2, challenging the notion of a pure coreceptor [68–70]. NP mediated VEGF responses may be connected to internalization of NP-1 after VEGF binding [71].
Heparan Sulfate Proteoglycan
Heparin-like molecules, HS and heparin, are negatively charged linear polysaccharides, of which heparan sulfates are synthesized by all mammalian cells. They are expressed on the cellular surface and in the ECM in a tissue specific manner. VEGF165 binds to heparin-like molecules via its heparin binding domain [15]. VEGF binding to VEGFR has been shown to be dependent on or enhanced by cellular heparin-like molecules in a concentration dependent manner [72–75] and they mediate VEGF165 interaction with NPs [76]. Binding to heparan sulfate prolongs the extracellular half-life of VEGF, as the former protects the latter from inactivation and degradation [12, 77]. Chaperone activity has also been suggested for these molecules [78]. The difference of heparan sulfate affinity of the isoforms is vital for the concentration gradient, which in turn is needed for vascular development [79].
13.2Regulation of VEGF Expression
The expression of VEGF is induced by a wide variety of extracellular signals, initiating signaling cascades and inducing enhanced expression of the VEGF gene. This expression is regulated at various checkpoints. Transcription of mRNA is regulated
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by a plethora of transcription factors and translation of the mRNA is regulated by mRNA stability. Posttranscriptional regulation of VEGF involves ER chaperones. An integrated overview of VEGF regulation is depicted in Fig. 13.1.
13.2.1Transcriptional Regulation
The 5¢ UTR region of VEGF is extremely long and contains a wide variety of promoter binding sites, such as consensus sites for Sp1/Sp3, AP-1, AP-2, Egr-1, Stat3, NFkB, or HIF-1 [6, 80, 81]. Additionally, it contains a WT1 binding site which has been connected to the responsiveness of VEGF to androgens, as the VEGF promoter lacks an androgen or estrogen receptor binding site [82]. The promoter region of VEGF does not contain a TATA box, but a GC-rich core region, which initiates transcription. The 5¢ flanking region of VEGF-A contains a hypoxia response element which is the binding region of HIF-1a/HIF1b [83] (discussed below). A repressive regulation of VEGF transcription has been shown for the transcription factor E2F1, which downregulates VEGF promoter activity in a p53-dependent manner under hypoxic conditions, binding in close proximity to Sp1 binding sites [25]. Additionally, a second internal promoter is responsible for the transcription of a truncated mRNA [80]. Phosphorylation by the MAPK ERK1/2 is important for the transactivation activity of both Sp1 and Sp3 [84, 85]. Sp1, but not Sp3, exhibits enhanced transactivation capacity after oxidative stress [86]. Participation of Sp1 or Sp3 has not been shown in the retina so far.
13.2.2Translational Regulation
In eukaryotic cells, the 5¢UTR carries a 7-methylguanosine cap, which is recognized and unwound by eukaryotic initiation factor (eIF), so the ribosome can reach the AUG start codon [87]. The IRES, originally discovered in viruses [88], which do not have a 7-methylguanosine cap, enables ribosomes to translate independently of the 7-methylguanosine cap and eIF proteins, ensuring translation when the canonical pathway is compromised, as it is in hypoxia [89]. IRES mediated translation is controlled by different factors, adding to the complexity of VEGF regulation [90]. In the retina, hyperglycemia induces VEGF production in Müller cells via IRES mediated translation which is dependent on 4E-BP1/2 noncanonical translation [46]. In addition to the classical AUG initiation codon, the 5¢UTR contains an alternate start codon, CUG [91]. The IRES can initiate at both initiation codons [92]. The 3¢ UTR is also involved in VEGF expression control. It contains a 3¢ UTR RNA instability site which consists of AU-rich elements (ARE), discussed in more detail below.
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Fig. 13.1 VEGF secretion can be controlled at three levels. (1) Transcriptional level, (2) translational level, and (3) posttranslational level. At transcriptional level, several transcription factors induce VEGF mRNA production. VEGF mRNA is shuttled out of the nucleus by HuR. VEGF mRNA translation is controlled by VEGF mRNA half-life which is prolonged by HuR at the ARE. Also, mRNA translation is enhanced by IRES. At posttranscriptional level, VEGF protein is protected by chaperones in the endoplasmic reticulum and its transportation to the Golgi apparatus is enhanced by ORP150
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13.2.3Hypoxia Induced VEGF Regulation
One of the main inducers of VEGF expression is hypoxia. Hypoxic VEGF upregulation is mediated by HIF-1a. In order to induce transcription, HIF-1a heterodimerizes with constitutively expressed HIF-1b. Under normoxic conditions, HIF-1a is bound to the von Hippel–Lindau tumor suppressor protein (VHL) and is rapidly degraded by the ubiquitin-proteasome pathway [93]. Under hypoxic condition, however, VHL releases HIF-1a, thereby no longer targeting it for degradation, and HIF-1a can dimerize with its partner. The heterodimer translocates to the nucleus, where it binds to the hypoxia responsive element (HRE) in the 5¢ UTR of the VEGF gene and induces translation [83, 94, 95]. Phosphorylation of HIF-1a by ERK1/2 enhances its transcriptional activity [96]. The small G-protein Rac1 is also involved this pathway [97]. Additionally, hypoxia induces the activation of the MAPK JNK1, which in turns phosphorylates c-Jun. Phospho-c-jun binds to the VEGF promoter and induces VEGF transcription. The expression of VEGF in the RPE, however, was not affected by JNK [98]. Inhibitory regulation also exists, as the transcription factor E2F1 mediates repressive promoter activity in a p53 dependent manner under hypoxic conditions [25].
VEGF regulation under hypoxia is also conducted posttranscriptionally by the regulation of the steady state level of the mRNA [99]. The half-life of VEGF mRNA is extremely short (less than 1 h [100]), but can be significantly enhanced by hypoxia. In VEGF mRNA, two cis-acting instability elements have been described in the 3¢ UTR [101]. These ARE predispose mRNA to degradation by endonucleases. Under hypoxic conditions, VEGF mRNA is stabilized by the binding of the RNA-binding protein HuR [99]. It protects VEGF mRNA from degradation by these nucleases, either by directly blocking the ARE sites or by inducing a conformational change, rendering the sites unavailable [87]. Additionally, HuR shuttles VEGF mRNA out of the nucleus, providing continuous protection [102]. In the diabetic retina, a role of HuR in VEGF regulation has been shown for pericytes which is dependent on PKC activity [52, 103]. In the RPE, VEGF expression by glucose deprivation has been shown to be mediated by the 3¢ UTR, but not 5¢ UTR. A direct involvement of HuR has been suggested [104].
13.2.4Posttranslational Regulation
VEGF is a secreted protein that is processed through the ER and the Golgi apparatus. Control of VEGF secretion can take place at this level. The ER chaperone ORP150 has been shown to be involved in VEGF secretion under hypoxia in tumor cells. It binds to VEGF and when overexpressed, VEGF secretion is enhanced, while inhibition of ORP150 retains VEGF in the ER [105]. The authors suggest an involvement of ORP150 in the transit of VEGF from the ER to the Golgi apparatus [105]. Even though this has not been shown for the retina so far, it has been hypothesized to play