The cellular response to O2 (oxygen) is a central process in animal cells and figures prominently in the pathophysiology of several diseases, including cancer, cardiovascular disease, and stroke. This process is coordinated by the HIF (Hypoxia-Inducible Factor) and its regulator, the pVHL (Von Hippel-Lindau tumor suppressor protein). HIF1 is a basic helix-loop-helix transcription factor that transactivates genes encoding proteins that participate in homeostatic responses to hypoxia. It induces expression of proteins controlling glucose metabolism, cell proliferation, and vascularization. Several genes involved in cellular differentiation are directly or indirectly regulated by hypoxia. These include Epo (Erythropoietin), LDHA (Lactate Dehydrogenase-A), ET1 (Endothelin-1), transferrin, transferrin receptor, VEGF (Vascular Endothelial Growth Factor), Flk1, FLT1 (Fms-Related Tyrosine Kinase-1), PDGF-Beta (Platelet-Derived Growth Factor-Beta), bFGF (basic Fibroblast Growth Factor), and others genes affecting glycolysis (Ref.1).
HIF1 consists of a heterodimer of two basic helix-loop-helix PAS (PerARNT-Sim) proteins, HIF1Alpha, and
HIF1Beta. HIF1Alpha accumulates under hypoxic conditions whereas
HIF1Beta is constitutively expressed. HIF1Alpha is an important mediator of the hypoxic response of tumor cells and controls the up-regulation of a number of factors important for solid tumor expansion including the angiogenic factor VEGF.
HIF1Beta is the ARNT (Aryl hydrocarbon Receptor Nuclear Translocator), an essential component of the xenobiotic response (Ref.2).
In the presence of O2, HIF is targeted for destruction by an E3 ubiquitin
ligase containing the
pVHL binds to a short HIF-derived peptide
when a conserved proline residue at the core of this peptide is hydroxylated.
The human genome contains EGL9 (Egg Laying Nine-9) homologues that are
EGLN2, and EGLN3 (also called PHD2,
PHD1, and PHD3 (Prolyl Hydroxylase
Domain-Containing Proteins) respectively). Prolyl hydroxylase post-translationally
modifies HIF1Alpha, allowing it to interact with the VHL
complex. Prolyl hydroxylase contains an iron moiety, so iron chelation
inhibits this activity. All three proteins of Prolyl hydroxylase can hydroxylate HIF1Alpha at one of
two proline sites within the ODD (Pro-402 and Pro-564). Analogous prolyl
residues are present in HIF2-Alpha and HIF3-Alpha. In the presence of
oxygen, the EGLN proteins are active and hydroxylate the ODD domain of
HIF1Alpha, which allows
pVHL to bind and polyubiquitinate HIF (Ref.3). VHL is part of a larger complex that includes Elongin-B,
Elongin-C, Cul2, RBX1 (Ring-Box 1) and a ubiquitin-conjugating enzyme (E2). This complex, together with a ubiquitin-activating enzyme
(E1), mediates the Ub (Ubiquitylation) of HIF1Alpha. The Ub modification
targets HIF1Alpha for
degradation, which can be blocked by proteasome inhibitors. Under hypoxic
conditions the HIF1Alpha subunits are not
pVHL, and they consequently accumulate and dimerize with
HIF1Beta and translocates to the nucleus, where they interacts with cofactors such as CBP (CREB Binding Protein)/p300 and the Pol II (DNA polymerase II) complex to bind to HREs (Hypoxia-Responsive Element) and activate transcription of target genes. HIF1Alpha-activated genes include VEGF, which promotes angiogenesis; GLUT1 (Glucose Transporter-1), which activates glucose transport; LDHA (Lactate Dehydrogenase), which is involved in the glycolytic pathway; and Epo, which induces erythropoiesis. HIF1Alpha also activates transcription of NOS (Nitric Oxide Synthase), which promotes angiogenesis and vasodilation. ARNT2 and MOP3 (Member of Pas superfamily-3) are other proteins that have been shown to heterodimerize with HIF1Alpha (Ref.4). HIF1Alpha can also be regulated by ERK2, which phosphorylate HIF1Alpha. HIF1Alpha also associates with the molecular chaperone HSP90 (Heat Shock Protein-90). HSP90 antagonists also inhibited HIF1Alpha transcriptional activity and dramatically reduced both hypoxia-induced accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. Recently, a factor inhibiting HIF1Alpha activation, FIH (Factor Inhibiting HIF1Alpha), has been described, representing a further level of HIF regulation.
Hypoxia also induces p53 protein accumulation. p53 directly interacts with HIF1Alpha and limits hypoxia-induced expression of HIF1Alpha by promoting MDM2-mediated ubiquitination and proteasomal degradation under hypoxic conditions. Furthermore, the degradation of HIF1Alpha by p53 in a hypoxic condition is inhibited by direct interaction with the JAB1 (Jun Activation domain Binding protein-1) and the ODD domain by blocking the interaction with p53. HIF1Alpha also associates with HNF4alpha2 (Hepatocyte Nuclear Factor-4-Alpha 2), which activates the Epo gene in concert with HIF1Alpha in response to hypoxic conditions. Hypoxia contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary diseases (Ref.5).