HIF signalling pathway
HIFs belong to a group of basic-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins that function as transcription factors responding to oxygen and other stresses (Semenza, 2012; Wang, Jiang, Rue & Semenza, 1995). HIFs function as heterodimeric transcription factors comprising a constitutively expressed beta unit, HIF1β (also known as aryl hydrocarbon receptor nuclear translocator, ARNT), and an alpha subunit that is oxygen liable (Figure 1). Currently, three alpha subunits have been identified and include HIF1α, HIF2α, HIF3α. The α and β subunits share a high percentage of both amino acid and structural homology, the N-terminal region (bHLH) which enables DNA binding, two PAS domains (PAS-A and PAS-B) that contribute to heterodimer stability and an oxygen dependent degradation domain.
In an oxygen rich environment, HIFs are instantaneously inactivated (Jewell, Kvietikova, Scheid, Bauer, Wenger & Gassmann, 2001) by posttranscriptional hydroxylation of conserved proline amino-acid residues within the α subunit. Hydroxylation generates a high affinity binding site for pVHL, leading to polyubiquitination and degradation by the proteosome. HIF prolyl hydroxylation is mediated by prolyl hydroxylases (PHDs). PHD activity requires several co-factors including molecular oxygen; a KM greater than 250 µM is above the oxygen concentration typically found in arterial blood (185 µM), and so the intracellular PO2 will always fall below the KM for oxygen, allowing enzyme activity to be modulated by oxygen availability over the entire physiological range (Hirsila, Koivunen, Gunzler, Kivirikko & Myllyharju, 2003). Obviously, evolution has come up with a mechanism that constitutively synthesizes molecules that allow a very fast response to hypoxia. This seems to be a crucial response. The prize for this is that if low oxygen conditions do not occur, all alpha subnits of HIFs are degraded without any further use.
Under hypoxic conditions, the loss of hydroxylation leads to the accumulation of HIFα isoforms and the formation of heterodimers with the constitutively expressed HIF1β (Kaelin & Ratcliffe, 2008). This HIF complex, together with a histone-acetyltransferase (p300), binds to hypoxia-response elements (HRE) in DNA, initiating or enhancing transcription of target genes. There is, however, a second tier of HIF regulation through the action of an asparagine hydroxylase, known as factor inhibiting HIF (FIH). Originally found to be a negative regulator of HIF1α, it was later shown to be an asparaginyl hydroxylase capable of hydroxylating N803 in the C-terminal domain of HIF1α (Schofield & Ratcliffe, 2004). FIH has a KM for molecular oxygen between 90 to 200µM. FIH hydroxylates target proteins with a higher efficiency at lower oxygen tensions than the PHDs, consequently continuing to hydroxylate target proteins in oxygen tensions below 5% (68µM). The efficiency of FIH hydroxylation appears to be substrate dependent; hydroxylation of HIF1α occurs with a greater efficiency (90%) than HIF2α (<30%) in normal physiological oxygen (Bracken et al., 2006; Yan, Bartz, Mao, Li & Kaelin, 2007). However, unlike the PHDs, FIH has a greater number of HIF-independent targets; for example, the efficacy for NOTCH hydroxylation appears to be far greater than HIFα (Coleman et al., 2007).
Both HIF1α and HIF2α isoforms have been extensively studied in pulmonary hypertension. The first direct evidence came from mice hemizygous for either HIF1α or HIF2α (Brusselmans et al., 2003; Shimoda, Manalo, Sham, Semenza & Sylvester, 2001). Pulmonary disease progression following chronic hypoxia exposure was substantially delayed in these models. The aberrant stability of both HIF1α and HIF2α was initially reported in whole lung tissue from PAH patients, then subsequently in pulmonary endothelial and smooth muscle cells from this patient group (Ball et al., 2014; Barnes, Chen, Sedan & Cornfield, 2017; Bonnet et al., 2006). Murine studies identified a definitive tissue-specific HIFα expression profile in the pulmonary vasculature, where HIF2α was found to be highly expressed in the endothelium. Genetic ablation of pulmonary endothelial HIF2α prevents the initiation and development of pulmonary vascular remodelling associated with pulmonary hypertension (Cowburn et al., 2016). Several groups have now reported that endothelial-loss of PHD2 orEGLN1 in mice leads to the aberrant stability of HIF2α with the development of occlusive vascular lesions and severe pulmonary hypertension (Dai, Li, Wharton, Zhu & Zhao, 2016; Tang et al., 2018). The concomitant genetic ablation of endothelial PHD2 and HIF2α in this model also inhibited the phenotype, offering near complete protection from pulmonary hypertension (Dai, Li, Wharton, Zhu & Zhao, 2016; Kapitsinou et al., 2016).
While murine genetic manipulation studies established HIF2α as the predominant HIFα isoform driving pulmonary hypertension, additional support has been found in gain-of-function gene mutations. Patients or mice with a HIF2α-gain-of-function mutation develop pulmonary vascular disease (Formenti et al., 2011; Tan et al., 2013), while mutations in VHL leads to mice with “Chuvash polycythemia” and pulmonary hypertension, which is rescued by heterozygous deletion of HIF-2α (but not HIF-1α) (Hickey et al., 2010).