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).