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Blood, 15 January 2006, Vol. 107, No. 2, pp. 419-420.
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HEMOSTASIS
Comment on Licht et al, page 584
EC does it with HIF
Gregg L. Semenza
THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE
Expression of a dominant-negative form of hypoxia-inducible factor 2 (HIF-2) in endothelial cells (ECs) disrupts cardiovascular development in mouse embryos, providing further evidence that HIFs exert both non–cell-autonomous and cell-autonomous control over ECs.
The developmental and physiologic processes that regulate O2 homeostasis in metazoans are directed by a family of hypoxia-inducible factors (HIFs), the prototype of which (HIF-1) was identified in 1992 as a mediator of erythropoietin gene transcription in hypoxic cells.1 Biochemical and molecular analyses revealed that HIF-1 was a heterodimer of constitutively expressed HIF-1 and O2-regulated HIF-1 subunits. Database searches identified HIF-2, which dimerizes with HIF-1 and transactivates an overlapping but distinct set of genes from those regulated by HIF-1.2 In contrast to the ubiquitous expression of HIF-1 and HIF-1, the expression of HIF-2 is tissue specific, most notably within vascular endothelial cells (ECs). HIF-3 is another family member that dimerizes with HIF-1. HIF-1 and HIF-2, but not HIF-3, contain transactivation domains that interact with the coactivator proteins CBP and p300 (see figure). The half-life of HIF-1, HIF-2, and HIF-3 is regulated by O2-dependent hydroxylation of prolyl residues, which is necessary for binding of the von Hippel-Lindau protein, the recognition component of an E3-ubiquitin ligase that targets the proteins for proteasomal degradation.3 In addition, hydroxylation of an asparaginyl residue in HIF-1 and HIF-2 blocks their interaction with coactivators. Because O2 is a rate-limiting substrate for HIF asparaginyl and prolyl hydroxylases, changes in oxygenation are transduced into changes in transcription.
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Mammalian hypoxia-inducible factors. HIF-1, HIF-2, HIF-3, and HIF-1 are each encoded by a distinct gene, with alternative mRNA splicing generating multiple isoforms (not shown) of HIF-1, HIF-3, and HIF-1. HIF-1, HIF-2, and HIF-3 dimerize with HIF-1 to form the functional proteins HIF-1, HIF-2, and HIF-3, respectively. The basic-helix-loop-helix (bHLH)–PAS domains mediate dimerization and DNA binding. All known HIF-1 binding sites contain the core DNA sequence 5'-RCGTG-3' (R, A, or G). Hydroxylation of prolyl residues (402 and 564 in human HIF-1) is required for the binding of the von Hippel-Lindau protein (VHL), which targets the proteins for ubiquitination and proteasomal degradation. Hydroxylation of an asparagine residue (803 in human HIF-1) blocks the binding of coactivators p300 and CBP. The HIF prolyl and asparaginyl hydroxylases use O2 and -ketoglutarate and generate CO2 and succinate as by-products.
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HIF-1 regulates genes encoding angiogenic cytokines, including vascular endothelial growth factor (VEGF), placental growth factor, angiopoietins 1 and 2, and stromal-derived factor 1, providing a mechanism by which cells are assured of adequate perfusion, as hypoxia-induced VEGF stimulates angiogenesis. Thus, HIF-1 was viewed as an extrinsic (non–cell-autonomous) regulator of ECs and their progenitors, which express VEGFR2 (Flk-1), VEGFR1 (Flt-1), Tie2, and CXCR4, the cell-surface receptors for VEGF, PLGF, angiopoietins, and SDF-1, respectively. HIF-2 was thought to function as an intrinsic (cell-autonomous) regulator of ECs. However, HIF-1 regulates the expression of hundreds of genes in ECs,4 and tumor angiogenesis is significantly impaired in mice with EC-specific loss of HIF-1.5 Mice completely lacking HIF-2 expression have normal cardiovascular development in certain genetic backgrounds,6 whereas complete HIF-1 deficiency results in major defects in cardiovascular development.1
A logical conclusion is that HIF-1 and HIF-2 play critical and partially overlapping roles in ECs. To test this hypothesis, Licht and colleagues generated transgenic mice with EC-specific expression of a dominant-negative form of HIF-2 (HIFdn) directed by Flk1 gene promoter/enhancer elements. Overexpressed HIFdn competes with HIF-1, HIF-2, and HIF-3 for dimerization with HIF-1, but the dimers cannot bind DNA or activate transcription. HIFdn transgenic embryos manifested cardiovascular defects similar to those of Tie2-deficient mice and the embryos completely lacked Tie2 expression. Thus, whereas loss of HIF-1 or HIF-2 activity in ECs is compatible with normal cardiovascular development, the combined loss of HIF-1 and HIF-2 activity is not. Overexpression of HIFdn may have confounding effects, such as competitive binding to proteins that interact with HIF-2 at residues not deleted in HIFdn, resulting in their cytoplasmic sequestration. It will be interesting to determine whether EC-specific HIF-1 deficiency phenocopies HIFdn and whether combined loss of HIF-1 and HIF-2 at earlier developmental stages (ie, prior to Flk1 expression) reveals essential roles for these proteins in hemangiopoiesis or vasculogenesis. Nevertheless, the results of Licht et al provide strong evidence that HIF-1 and HIF-2 play critical intrinsic roles in ECs. Because of their dual (extrinsic and intrinsic) effects, targeting these factors may represent a powerful approach to inhibiting angiogenesis in cancer and other disorders that are dependent upon neovascularization. 
References
- Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda). 2004;19: 176-182.
- Raval RR, Lau KW, Tran MG, et al. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol. 2005;25: 5675-5686.[Abstract/Free Full Text]
- Kaelin WG Jr. The von Hippel-Lindau protein, HIF hydroxylation, and oxygen sensing. Biochem Biophys Res Commun. 2005;338: 627-638.[CrossRef][Medline]
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- Manalo DJ, Rowan A, Lavoie T, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood. 2005;105: 659-669.[Abstract/Free Full Text]
- Tang N, Wang L, Esko J, et al. Loss of HIF-1 in endothelial cells disrupts a hypoxia-driven VEGF autocrine loop necessary for tumorigenesis. Cancer Cell. 2004;6: 485-495.[CrossRef][Medline]
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- Scortegagna M, Ding K, Oktay Y, et al. Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1–/– mice. Nat Genet. 2003;35: 331-340.[CrossRef][Medline]
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