Molecular Mechanisms Of Hif Regulation

Several of the factors involved in the process of neovascularization are regulated by Hypoxia-inducible-factor 1 (HIF-1), a transcription factor known as the "master regulator" of the physiological response to hypoxia. HIF represents an alternative strategy for the induction of both angiogenesis and arteriogenesis. Following a decline in intracellular oxygen, HIF triggers a coordinated molecular response to hypoxia in most cell types. A therapeutic approach employing a constitutively-active HIF construct could result in the expression of not only a single growth factor such as the VEGF-A isoforms, but also a spectrum of pro-angiogenic factors that might contribute to physiological neovascularization. HIF-1 is a heterodimeric transcription factor composed of an oxygen-sensitive HIF-1 a subunit and its consti-tutively expressed dimerization partner HIF-1 p (also termed ARNT, for aryl hydrocarbon receptor nuclear translocator). A second isoform of the alpha subunit, HIF-2a also termed EPAS-1, and a third isoform, HIF-3a, also exist with a more limited tissue distribution (see [21] for review). The lack of redundancy among these isoforms is highlighted by the lethality in early development of mice with targeted deletion of HIF-1 a [22-24]. HIF-2a and ARNT are also required for normal mouse development [25, 26]. HIF-1 a is comprised of a DNA binding, basic helix-loop-helix (bHLH) domain, and two Per-ARNT-Sim (PAS) domains, the latter mediating interactions with other regulatory proteins, including dimerization with ARNT. In addition, HIF-1 a has two carboxyl-terminal transactivation domains (TAD-N and TAD-C), which recruit transcriptional coactivators and cofactors such as CBP, p300, steroid receptor co-activator (SRC-1), transcriptional intermediary factor-2 (TIF-2) and the HIF-1 a-interacting histone deacetylase (HDAC) 7, all of which play an essential and largely non-redundant role in the initiation of transcription [27-32].

Tissue oxygen concentrations regulate HIF-1 activity by influencing both the stability and transcriptional activity of the HIF-1a subunit (reviewed in [33]). Under normoxic conditions HIF-1 a protein stability is regulated by several prolyl hydroxylase domain containing proteins (e.g. PHD's 1, 2 and 3) via the hydroxylation of conserved prolines (Pro402 and Pro564) within the oxygen-dependent degradation (ODD) domain of the protein. These modified residues are subsequently bound by the von Hippel-Lindau (VHL) tumor suppressor protein, the recognition component of the E3-ubiquitin ligase complex, which targets HIF-1 a for degradation by the 26S proteosome [reviewed in 34, 35]. The three PHD's serve distinct functions, as they show differing specificities for the two prolyl hydroxylation sites within HIF-1 a as well as for the HIF-1 a and HIF-2a isoforms [36]. Notably, PHD2 has been shown to be primarily responsible for degradation of HIF-1 a under normoxic conditions [37]. Depending on the cell type, degradation can occur in either the nucleus and/or the cytoplasm of the cell, suggesting that HIF-1a protein levels may be affected by subcellular distribution [38]. In addition to O2, the PHD's also utilize

2-oxoglutarate as a substrate, and require ascorbate as a cofactor. Their activity is also dependent upon coordination of ferrous iron within the active site [39, 40]. Because the PHD's exhibit a Km for O2 that is just above its atmospheric concentration, enzyme activity is tightly regulated under physiological conditions, thus contributing to the ability of the PHD's to function as effective oxygen sensors [41].

Expression of the PHD's themselves (specifically PHD2 and PHD3) is also regulated by oxygen availability, via HIF-dependent induction of gene transcription [42, 43]. An elevated level of the PHD2 isoform with hypoxia appears to act as a negative feedback mechanism, as PHD2 is capable of down-regulating HIF-1 a transcriptional activity during hypoxia [44]. A similar feedback function has been attributed to CITED/p35srj, a hypoxia-inducible gene product that competes with HIF-1 a for binding to p300, thereby down-regulating HIF-mediated transac-tivation under hypoxic conditions [45]. Regulation of the PHD's by hypoxia in vivo has been proposed to play a role in setting a new oxygen threshold as well as adapting to varying oxygen concentrations in different tissues [46]. Stability of the PHD1 and PHD3 proteins is also regulated under hypoxic conditions through the action of Siah1a/2 [47]. The Siah (seven in absentia homologue) proteins, which possess ubiquitin ligase activity, target the PHD's for proteosome-mediated degradation, thereby positively regulating HIF activity when oxygen concentration is low. Evidence for a role of the Siah proteins in HIF regulation was derived from experiments performed in vitro with mouse embryonic fibroblasts lacking either Siah 2 alone or both Siah2 and Siah1a. Siah null fibroblasts showed an increase in PHD3 half-life together with reduced HIF-1 a protein abundance while the physiological response to hypoxia was defective in Siah null mice. Other cellular proteins such as OS-9 play a role in the regulation of HIF hydryoxylation via the formation of a multiprotein complex with HIF and the PHD's [48]. In addition, it has been proposed that reactive oxygen species (ROS) generated by the mitochon-drial respiratory chain during hypoxia may have the ability to inhibit PHD activity via intracellular signaling pathways thereby leading to redundant mechanisms for the stabilization of HIF-1a during sustained hypoxia [49-51].

In addition to the regulation of both protein stability and transcriptional activity, HIF-1 a is also directly regulated in an oxygen-dependent manner by hydrox-ylation at an asparagine residue (Asn803) in the C-terminal activation domain. This modification is carried out by another 2-oxoglutarate-dependent dioxygenase, FIH (Factor-Inhibiting HIF; [52-54]) and functions to regulate the interaction of HIF-1 and p300/CBP [27, 55]. Importantly, these oxygen-dependent pathways for regulation of HIF-1 activity are relevant to the acceleration of several pathological mechanisms. Tumor growth, for example, can result from inhibition of HIF degradation via mutational inactivation of VHL [56] as well as loss of function of the Krebs cycle enzymes fumarate hydratase (FH) or succinate hydroxylase (SH). The substrates of FH and SH, fumarate and succinate, respectively, inhibit PHD activity [57]. Alternatively, high levels of HIF-1 activity in cancer cells have been attributed to binding of the glycolytic metabolites pyruvate and oxaloacetate to the

2-oxoglutatrate site of the PHD's, presumably providing these cells with a selective survival advantage in a hypoxic tissue milieu [58].

In addition to the oxygen-dependent pathways for the regulation of HIF activity, multiple additional hypoxia-independent signaling pathways exist that impact HIF-1 a activity. These pathways are stimulated by growth factors, hormones and cytokines such as IGF-1, IGF-2, FGF-2, insulin, EGF, TNF-a, angiotensin II and IL-ip as well as oncogenes such as HER2neu (reviewed in [59, 60]). These activators signal through the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR, mitogen-activated protein (MAPK)/MEK and/or NF-kB pathways and ultimately enhance intracellular levels of HIF-1 a by regulating HIF-1 a gene transcription and/or mRNA translation. Also, nitric oxide congeners (and specifically the NO donor NOC18) have been shown to regulate HIF-1a protein synthesis in a similar manner via PI3K and MAPK-dependent signalling [61]. Others have reported that a different NO donor, GSNO, activates HIF by inhibiting prolyl hydroxylase activity [62]. It has also recently been reported that TGFp1 increases HIF-1 a protein stability through a different mechanism, by down-regulating PHD2 gene expression via signaling through Smad-mediated signal-transduction pathways [63]. Interestingly, HIF activity also appears to be regulated by pH, as acidosis triggers nucleolar sequestration and inactivation of the VHL protein leading to prolonged stability and activity of HIF-1 a even under normoxic conditions [64].

As noted above, in hypoxic cells or tissues, HIF-1 target genes play a variety of roles, both at the level of individual cells and systemically, including the induction of angiogenesis, glucose metabolism, erythropoiesis, regulation of vascular tone, and cell proliferation and survival [65]. HIF-1 regulates gene expression by binding to a cis-acting hypoxia response element (5'-RCGTG-3') in the promoter/enhancer region of HIF target genes.

The central role of HIF-1 a in the regulation of hypoxia-driven angiogenesis has been extensively described elsewhere [33, 66-70]. HIF is known to induce the expression of a range of angiogenic factors, including VEGF, PDGF-B, PlGF, and angiopoeitins 2 and 4, among others [71-73]. In endothelial cells themselves, HIF-1 enhances neovascularization by playing a role in an autocrine response to hypoxia. Exposure of primary human vascular endothelial cells to hypoxia or overexpression of a constitutively-active form of HIF-1 a results in the induction of cytokines, growth factors and receptors as well as biological responses such as endothelial tube formation, that are consistent with angiogenic activation [74]. HIF-dependent autocrine loops involving VEGF's [75] and bFGF [76] have been shown to promote endothelial cell survival and angiogenic properties both in vitro and in vivo. In cultured endothelial cells (e.g., HUVECs), HIF-1 a has also been shown to decrease apoptosis in response to ischemia-reperfusion injury [77]. In general, however, when there is substrate limitation, HIF is also known to play a role in promoting hypoxic cell death by up-regulating pro-apoptotic genes such as BNIP3 as well as through stabilization of p53 (reviewed in [78])

Furthermore, HIF-1 a may contribute to the process of neovascularization by inducing expression of factors that promote homing of circulating bone-marrow-derived cells to ischemic tissue. Endothelial progenitor cells, as well as cells of the hematopoietic lineage, have been shown to be mobilized from the bone marrow and recruited to peripheral sites, stimulating endothelial cell growth and sprouting, either directly via incorporation into newly formed vessels or by supporting the process in a paracrine manner (recently reviewed in [79]). Recruitment, homing and maintenance of bone-marrow derived cells at these sites are dependent upon production of VEGF as well as stromal-derived factor (SDF-1) in ischemic tissues [80, 81]. HIF is also known to up-regulate expression of SDF-1 as well as its receptor, CXCR4 [82-84] thereby mediating the homing of circulating progenitor cells to sites of injury. Mesenchymal stem cells (MSC's) can also be recruited from the bone marrow to the peripheral blood upon initiation of an ischemic stimulus and contribute to tissue repair. Binding of VEGF and PlGF by VEGFR1 plays a critical role in this process. Migration of MSC in response to these cytokines in vitro is stimulated by HIF-mediated induction of VEGFR1 expression [85]. These studies show that HIF-1 may play a role in neovascularization both by directly inducing expression of angiogenic cytokines in ischemic tissue as well as by promoting the recruitment of circulating bone-marrow derived cells that contribute to this process.

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