Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

of the VEGF gene.36 Subsequent co-transactivation studies in Hep3B cells using a 47 bp 5’ human VEGF gene fragment bearing the HIF-1 binding site and expression vectors encoding HIF-1α and HIF-1β showed a greater increase in reporter gene transcription in hypoxic cells in comparison to cells transfected with the reporter construct alone. Co-transfection with a dominant negative form of HIF-1α resulted in inhibition of activation of the reporter gene. These studies implicated HIF-1 in the activation of VEGF transcription in hypoxic cells.46 Further proof for the importance of the HIF- 1/VEGF interaction has been shown in studies of HIF-1α–null mouse embryonic stem cells. Basal expression of VEGF mRNA in these cells is low and does not increase in response to hypoxia.47-49

3.2Increase in VEGF mRNA stability

In vitro mRNA degradation assays have been performed using VEGF mRNA and cytoplasmic extracts from normoxic and hypoxic PC12 cells. These experiments led to the identification of cis regulatory regions in the 3’ untranslated region (UTR) of the VEGF mRNA that confer lability to the VEGF mRNA transcript in normoxia and to the identification of sequences that are critical for increased stability of the mRNA in response to hypoxia.50 Levy et al. discovered a 500 bp region in the 3’ UTR that is critical for stabilization of VEGF mRNA by hypoxic cytoplasmic extracts. This region contains two consensus sequences (5’UUAUUUA (U/A)(U/A)-3’) that have previously been shown to mediate rapid turnover of many cytokines and oncogenes by binding specific endonucleases, thereby promoting RNA degradation.51-53 Deletion of this adenylate-rich element (ARE) resulted in stabilization of the mRNA,50 whereas insertion of VEGF 3’ UTR sequences into stable mRNA resulted in destabilization.54 Transfection studies in an experimental glioma model using a lacZ reporter gene under control of VEGF regulatory sequences showed maximal levels of reporter gene activity with constructs that included both the HIF-1 binding site and 3’ untranslated sequences.55

Gel shift mobility analyses have identified three separate elements within the VEGF 3’ UTR that possibly bind a hypoxia-inducible protein complex.50 These correspond to VEGF nucleotides 1472-1510, 1508-1573, and 16321678. RNA affinity purification and UV crosslinking studies led to the identification of three proteins, 17 kDa, 28 kDa, and 32 kDa in size, which form an RNA-protein complex under hypoxic conditions. Under normoxic conditions, this RNA-protein complex and VEGF mRNA are elevated in 786-0 cells, a Von-Hippel Landau protein (VHL) mutant cell line. Introduction of the wild-type VHL gene, a tumor suppressor protein into these cells results in reduction of VEGF mRNA stability in normoxia, thus

supporting the role of this hypoxia-inducible complex in mediating hypoxic stabilization of VEGF mRNA.50

HuR, a 36 kDa RNA binding protein, has been identified as being important for post-transcriptional stabilization of VEGF mRNA.56,57 Inhibition of HuR by antisense oligonucleotides prevents the hypoxiamediated increase in VEGF mRNA stability. However, overexpression of HuR alone may not be sufficient for increasing the stability of VEGF mRNA in hypoxia.56 Under hypoxic conditions, HuR binds to a 40 bp RNA element in the VEGF 3’ UTR only 4 nucleotides 5’ to the nonameric ARE described above. Although there is much more to be learned about the role of HuR and the hypoxia-inducible RNA-protein complex in stabilization of VEGF mRNA, it has been proposed that HuR binding alters the structure of the ARE and protects it from degradation by endonucleases.58

3.3Increase in VEGF mRNA translation

Under hypoxic conditions, overall protein synthesis is inhibited.59 Also, the 5’ UTR of VEGF has several characteristics that render initiation of translation by ribosomal scanning difficult: the 5’ UTR is longer than most eukaryotic 5’ UTRs, it has a high GC content that can form secondary structures, and it contains a short open reading frame. The VEGF gene counteracts these inherent problems by using an internal ribosomal entry site (IRES) in the 5’ UTR for efficient ribosomal scanning under hypoxic conditions.45 Use of the IRES allows for efficient translation in a capindependent mode, which is especially advantageous in conditions of stress when components of the eukaryotic initiation factor (eIF4 complex) may become rate-limiting. However, it is not known if a specific translation initiation factor exists that recognizes the IRES in VEGF or if that factor’s expression is itself increased in response to hypoxia.

3.4VEGF protein transport and secretion

Oxygen regulated protein of 150 kDa (ORP150), a chaperone protein required for intracellular transport of protein from the endoplasmic reticulum to the Golgi apparatus prior to protein secretion, was initially identified in hypoxic astrocytic cell cultures.60 ORP150 mRNA production is induced in response to hypoxia. Studies by Ozawa et al. using human macrophages transfected with adenovirus coding for ORP150 in either the sense or antisense orientation showed that overexpression of ORP150 resulted in increased VEGF secretion in hypoxia. Expression of ORP150 antisense mRNA resulted in increased accumulation of VEGF within the endoplasmic reticulum. These studies suggest that, under hypoxic conditions, ORP150

functions as a molecular chaperone to facilitate VEGF protein transport and secretion.61

4.REGULATION OF HIF-1 BY HYPOXIA

HIF-1 was first identified in hypoxic extracts of Hep3B, a hepatoma cell line that produces erythropoietin in a regulated fashion.62 It is a transcription factor that regulates oxygen homeostasis and controls angiogenesis, erythropoiesis, and glycolysis by transcriptional activation of several target genes under hypoxia.63 HIF-1 is a heterodimeric protein consisting of HIF- 1α and HIF-1β subunits. Both subunits are basic helix-loop-helix (bHLH) proteins containing a conserved PAS domain.64 HIF-1α is an 826-amino-acid polypeptide whose expression is uniquely regulated by hypoxia, whereas HIF-1β is constitutively expressed and is identical to the product of the arylhydrocarbon receptor nuclear translocator (ARNT) gene.65 HIF-1α contains a DNA binding/heterodimerization region and a C-terminal region with one or more transactivation domains (Figure 1).66,67 In response to hypoxia, the transactivated HIF-1α protein translocates to the nucleus, dimerizes with HIF-1β, and activates transcription of several target genes, including VEGF, by binding to the cis-acting hypoxia-responsive element 5’ -A/(G)CGTG-3’. 68

Figure 9-1. HIF-1α subunit organization. bHLH—basic helix-loop-helix protein; PAS (derived from PER, ARNT, SIM proteins in which it was first described); TAD (N)—amino terminal transactivation domain; ID—inhibitory domain; TAD (C)—carboxyl terminal transactivation domain. Adapted from Genes and Dev 15, 2675 (2001).

The regulation of HIF-1α by oxygen tension occurs at the level of HIF- 1α protein stabilization and transactivation. It is thought that tissue oxygen levels are sensed by a family of prolyl hydroxylase domain enzymes, PHD1, PHD2, and PHD3, which have a distinct pattern of subcellular localization and have different affinities for the proline residues in HIF-1α.69-72 Depending on the level of tissue oxygenation, PHD proteins add or remove a hydroxyl group to or from the prolines of HIF-1, thereby affecting its rate of degradation by ubiquitination and subsequent proteolysis. These enzymes

require iron as a cofactor and dioxygen as a co-substrate, which might explain the increased stability of HIF-1 under hypoxic conditions or after treatment with agents that remove (e.g., deferroxamine) or compete with iron (e.g., cobalt). Several studies have indicated that changes in reactive oxygen species or direct metal-catalyzed oxidation reactions (e.g., the Fenton reaction) are also involved in oxygen sensing and signaling.73,74 However, the precise mode in which these mechanisms interact with the prolyl hydroxylases is unknown. Using cell compartment-specific dyes, Liu et al. detected both the Fenton reaction and the HIF-1α subunit in the endoplasmic reticulum during normoxia.75 Inhibition of the Fenton reaction by scavenging of hydroxyl radicals results in inhibition of prolyl hydroxylases and translocation of HIF-1α to the nucleus.76

Under normoxic conditions, proline hydroxylases use molecular oxygen to hydroxylate the proline residues at positions 402 and 564 in the basic domain of the HIF-1α protein.71,77-80 Hydroxylation of the proline residues is followed by increased binding of the VHL-associated complex (VHL, elongin B, C, cullin-2, and Rbx-1), which interacts with HIF-1α via the transactivation domain (TAD-N) and also binds to Factor inhibiting HIF-1 protein (FIH-1). The VHL complex is recognized by an E3 ubiquitin ligase,81-83 which ubiquitinates HIF-1α, targeting it for proteosomal degradation by the 26S proteosome pathway84-86 (Figure 2). Under hypoxic conditions hydroxylation of the proline sites is inhibited, resulting in increased HIF-1α protein stabilization (Figure 3).

The second level of regulation of HIF-1 by oxygen tension occurs by affecting HIF-1α transactivation.66,87 HIF-1α contains two transactivation domains, TAD-N (amino acid residues 531-575) and TAD-C (residues 786-826) (Figure 2). The intervening region between the two domains comprises an inhibitory domain (ID).66 For transactivation of the HIF-1α

protein, two co-activators, the CH1 domain of P300 and CREB-binding protein (CBP), need to bind to the TAD-C region of HIF-1α.88,89 These

co-activators facilitate linking of the various transcription factors to the transcription initiation complex of several downstream genes.90 Under normoxic conditions, Factor inhibiting HIF-1 (FIH-1), an asparaginyl hydroxylase, interacts with HIF-1α at residues 757–826, which includes a part of the ID and the TAD-C domain, and also interacts with VHL, thus forming a ternary complex between the 3 proteins (Figures 2 and 3). FIH-1 binding leads to hydroxylation of asparagine 803 in TAD-C, thus preventing binding of the P300 and CBP proteins to the transactivation domain. Under hypoxic conditions, FIH-1 is unable to hydroxylate the asparagine residue in the transactivation domain thus allowing interaction between P300 and TAD-C and subsequent transactivation.

Figure 9-2. Oxygen-dependent hydroxylation events that regulate HIF-1α protein stability and transcriptional activity—Normoxia. CUL2, Elongin B, C constitute the VHL-associated complex. HDAC—histone deacetylases; FIH-1—factor inhibiting HIF-1; P402, P564—prolyl residues at 402, 564 respectively. Adapted from Trends in Mol Medicine 7(8), 345-350 (2001).

Figure 9-3. Oxygen-dependent hydroxylation events that regulate HIF-1α protein stability and transcriptional activity—Hypoxia. Adapted from Proc Natl Acad Sci U S A 99(8), 11570-11572 (2002).

Phosphorylation also appears to be necessary for transactivation of HIF- 1α. The C terminal region of HIF-1α is phosphorylated at multiple sites. Inhibitors of tyrosine kinases, serine/threonine phosphatases, diacylglycerol kinase, and the phosphoinositol-3-kinase pathway have all been shown to inhibit HIF-1α induction in response to hypoxia. However, the exact role of these molecules in the hypoxia signal transduction pathway is unknown. 65,91-94

5.ROLE OF VAN HIPPEL LANDAU (VHL) PROTEIN

VHL disease is a hereditary cancer syndrome characterized by the development of highly vascular tumors that overproduce hypoxia-inducible mRNAs such as VEGF.79 The VHL protein is a tumor suppressor protein that is functionally inactivated in renal carcinoma and hemangioblastoma cell lines.95,96 Loss of this function results in constitutive expression of VEGF mRNA and HIF-1α under non-hypoxic conditions.87 Reintroduction of wild-type VHL into these cells restored their hypoxia-inducible profile.97 VHL plays a critical role in the regulation of the HIF-1α protein under normoxic conditions by two mechanisms. The first, as mentioned above, occurs by decreasing the stability of the protein by binding to the hydroxylated TAD-N and recruiting the E3 ubiquitin ligase complex containing elongin B, C. VHL also binds to FIH-1 and acts as a transcriptional co-repressor that inhibits HIF-1α transactivation by recruiting histone deacetylases (HDACs), which repress transactivation of various genes.98 Intraocular gene transfer of an adenovirus vector expressing a VHL construct has been shown to inhibit angiogenesis in a monkey model of branch retinal vein occlusion.99 Thus, VHL may possibly be a good target for anti-angiogenic therapy for patients with retinopathy.

6.HIF-1 DEGRADATION

IN PROLONGED HYPOXIA

Prolonged hypoxia has been shown to shorten the half-life of HIF-1,100 and HIF-1 levels decline after 4 hours of hypoxia.101 Under conditions of continued hypoxia, a feedback mechanism for limiting HIF-1 activity comes into play. This feedback mechanism occurs via hydroxylases PHD2 and

PHD3, both of which respond to prolonged hypoxia with an increase in mRNA and protein expression.102,103 Also, both PHD2 and PHD3 are target

genes for HIF-1α.104 In response to continued hypoxia, an increase in these

hydroxylases results in destabilization and degradation of the HIF-1α protein, particularly after reoxygenation. In contrast to VHL, the p53 tumor suppressor protein, which is induced by hypoxia, has been implicated in HIF-1α degradation under conditions of continued hypoxia. One mechanism by which p53 limits the HIF-1α response is by competing for P300, thereby decreasing the activity of HIF-1α.105 Another mechanism is by direct interaction of p53 and HIF-1α, leading to recruitment of the ubiquitinprotein ligase MDM2, which binds p53 and triggers VHL-independent HIF- 1α degradation.106

7.OTHER ROLES AND REGULATORS OF HIF-1

HIF-1 upregulates several hypoxia-inducible genes. Also, although VEGF is a key angiogenic molecule, other angiogenic factors play a role in retinal angiogenesis. In a study by Kelly et al., injection of AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Expression of genes encoding the angiogenic factors angiopoietin-1 and 2, PDGF-B, placental growth factor, and VEGF were all increased in injected eyes, thus indicating that HIF-1α controls the expression of multiple angiogenic factors.107

Most of the mechanisms discussed above with respect to the regulation of HIF-1 and VEGF have been described in mainly non-ocular cell lines and in many cases tumor cell lines. Although the specific mechanisms and consequences of ischemia differ in each tissue, the molecular mechanisms described for HIF-1 and VEGF are very likely involved in retinal neovascularization. Retinal hypoxia is the most likely stimulus for the cascade of events that results in retinal neovascularization in various disease states. However, it is important to mention that both HIF-1α and VEGF

expression are responsive to other stimuli as well. These stimuli include growth factors, hormones, or cytokines.108,109 The increase in HIF-1α in

response to stimuli besides hypoxia is mediated via the PI3K/Akt-dependent signaling pathway.109 The mitogen activated protein kinase (MAPK) pathway does not participate in hypoxia signaling, but in tumor cells MAPK is required for transactivation activity of HIF-1α through p300/CBP.110 However, phosphorylation of HIF-1α is not directly mediated by MAPK.111

HIF-1α overexpression has been implicated in tumor vasculogenesis and progression of tumor growth.112 Studies in animal models of tumorigenesis

suggest that inhibition of HIF activity may be therapeutically beneficial.113,114 Given the central role of HIF-1 in retinal neovascularization,

it is very possible that inhibition of HIF-1 may be beneficial in treatment of retinal disease as well.

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