**6. Oxygen-dependent regulation of HIF by pVHL**

The importance of pVHL in the regulation of the HIF transcription factors, and the cellular transcriptional response to altered levels of oxygen, has provided tremendous insights into the mechanisms of cellular oxygen sensing. HIF was first discovered in the quest for transcriptional regulators of the erythropoietin gene (EPO), encoding the master regulator of red blood cell production [67]. It later emerged there were three HIF isoforms, HIF-1, HIF-2 [68, 69], and HIF-3 [70], each composed of a common, constitutive β-subunit (HIF-1β, also known as ARNT – aryl hydrocarbon receptor nuclear translocator) and a regulated alpha-subunit (HIF-1α, HIF-2α and HIF-3α respectively). HIF-1α is ubiquitously expressed at the mRNA level, thus HIF-1α protein is capable of being stabilized in all tissue types. HIF-1α is thought to drive core, canonical cellular responses to low oxygen levels (hypoxia) [71], including the metabolic switch to anaerobic glycolysis. The expression of HIF-2α mRNA and HIF-3α mRNA is more cell-type-specific and thus these transcription factors are thought to drive more specialized responses to hypoxia [69, 70, 72].

HIF-2α expression is generally more restricted to particular mesenchymal cell types, including endothelial cells in which it was first identified, hence its alias endothelial PAS domain-containing protein 1 (EPAS1) [69]. However, HIF-2α is also expressed in some epithelial malignancies, including ccRCC. HIF-3α expression is restricted to a select few cell types and can be alternatively spliced to yield several transcript variants [70]. The biological functions of HIF-3α have not been well-explored, although it is thought to antagonize the transcriptional responses of HIF-1α and HIF-2α [73–75].

HIF isoforms are all basic helix–loop–helix/Per-ARNT-SIM (bHLH–PAS) transcription factors, belonging to a much larger family that includes the oncogenic MYC proteins [76]. Each possess an N-terminal bHLH DNA-binding domain and two protein–protein interaction PAS domains responsible for dimerization. In addition, the three HIF-α isoforms each contain oxygen-dependent degradation domains (ODDDs), responsible for regulating protein abundance [77]. However, only HIF-1α and HIF-2α possess the C-terminal transactivation domains (C-TAD) [78].

In the presence of oxygen, HIF-α subunits are hydroxylated on two residues in the ODDD domains by a family of prolyl hydroxylase enzymes (PHD1, PHD2 and PHD3) [44, 45, 79]. These hydroxylated residues are recognized and bound by pVHL (in a complex with elongin B, elongin C and cullin 2) leading to its rapid ubiquitination and proteasomal degradation (**Figure 2**). Thus, when oxygen is abundant, HIF-α levels are low. However, since oxygen is a rate-limiting substrate for this reaction, HIF-α is stabilized in hypoxia. Inactivation of VHL in ccRCC cells will also block HIF from being degraded, leading to constitutive activation of HIF and its target genes, even in cells that are well-oxygenated. Accordingly, activation of both HIF and HIF target genes are hallmarks of ccRCC.

#### **Figure 2.**

*Regulation of HIF by PHD enzymes and pVHL E3 ligase. (A) In normal oxygen conditions (normoxia), the oxygen-dependent PHD enzymes (PHD1, PHD2 and PHD3) hydroxylate both HIF-1α and HIF-2α transcription factor isoforms. This causes HIF proteins to be recognized and ubiquitinated by the pVHL E3 ubiquitin ligase complex, which targets them for rapid degradation via the proteasome. (B) In low oxygen conditions (hypoxia), PHD enzymes are inactive due to the lack of their oxygen substrate. Therefore, HIF-1α and HIF-2α are not hydroxylated and are not targeted for degradation by pVHL. Due to their stabilization, they are able to dimerize with their obligate binding partner HIF-1β. This allows them to bind to DNA and upregulate their target genes. (C) When the VHL gene is inactivated (as is the case in ccRCC and some other cancers), pVHL is either not expressed or is dysfunctional. Therefore, pVHL is unable to recognize HIF-1α and HIF-2α, even in the presence of oxygen when they are hydroxylated by PHD enzymes. This causes inappropriate stabilization of HIF-1α and HIF-2α, which then dimerize with HIF-1β and upregulate their target genes, regardless of oxygen levels.*

#### *HIF Pathways in Clear Cell Renal Cancer DOI: http://dx.doi.org/10.5772/intechopen.96539*

In addition, HIF-1α and HIF-2α can be further modified at an additional site in the C-terminal TAD by an asparaginyl hydroxylase, termed factor inhibiting HIF 1 (FIH-1) [80, 81]. Similar to the PHD enzymes, FIH-1 activity is oxygen-dependent, but asparagine hydroxylation does not prompt recognition by pVHL. Instead, asparaginyl hydroxylated HIF is unable to bind to the transcriptional co-factor, CREB binding protein (CBP)/p300, which facilitates transcriptional activation at a subset of HIF-target genes [80–82]. Therefore, two distinct mechanisms act to control HIF activity and expression in an oxygen-dependent manner, one of which is blocked by VHL inactivation. In the context of ccRCC, this has two consequences. Firstly, FIH-1 may facilitate residual hypoxic regulation of HIF despite constitutive HIF stabilization [83]. Secondly, the transcriptional response to VHL inactivation in normoxic cells may not precisely mimic the transcriptional response to hypoxia.
