Structural Biochemistry/Protein function/HIF switch

Introduction
Hypoxia refers to the condition of which inadequate oxygen is supplied to tissues and cells. The hypoxic response is facilitated by the hypoxia-inducible transcription factors, HIF-1 and HIF-2. HIF target gene activation is very specific and not indicative of which HIF alpha isoform is active.

Hypoxia and the HIFs in human physiology and disease
The oxygen tension in tissues is a lot lower than the ambient oxygen tensions due to the dramatic decrease in blood oxygen content as oxygen is released in the cells. Low oxygen or hypoxia acts as a stimulus for proper embryogenesis and wound healing and maintains the pluripotency of stem cells. Pathological hypoxia could be a result of high altitude or localized ischemia due to disruption of blood flow to a given area. Solid tumours also have hypoxic regions due to the severe structural abnormality of tumour microvessels. As a response to hypoxia, HIF transcription factors transactivate many genes including those that trigger angiogenesis, anaerobic metabolism and resistance to apoptosis. Structurally, HIFs are heterodimers that consist of one of three major oxygen labile HIF alpha subunits (1alpha, 2alpha, 3alpha) and a constitutive HIF1 beta subunit that combine to form the HIF-1, HIF-2, HIF-3 transcriptional complexes. Majority of the studied has been done on HIF-1alpha and HIF-2alpha. HIF-3alpha has similar basic helix-loop-helix and Per-Amt-SIM (PAS) domains as HIF-1alpha and HIF-2alpha, but it does not have the C-terminal transactivation domain. HIF-1alpha and HIF-2alpha are non-redundant, and they have distinct target genes and mechanisms of regulation. In some circumstances, HIF-1 drives the initial response to hypoxia, but after long exposure, it is HIF-2 that drives the hypoxic response.

HIF regulation
Under aerobic condition, both HIF 1 and 2 alpha are hydroxylated by specific prolyl hydroxylases at two conserved proline residues positions in the oxygen-dependent degradation domain. This reaction requires oxygen, 2-oxoglutarate, ascorbate, and iron (Fe2+) as a factor. Von Hippel-Lindau protein forms the substrate recognition module of an E3 ubiquitin ligase complex that directs HIF – 1 and 2 alpha polyubiquitylation and proteasomal degradation. Under hypoxic conditions, prolyl hydroxylase acitivity is inhibited, von Hippel-Lindau binding is abrogated and HIF-1 and 2 alpha are stabilized. Under normoxic conditions, HIF-1 and 2alpha cannot activate transcription due to oxygen-regulated enzyme, factor inhibiting HIF-1. Asn hydroxylation is also inhibited, which allows the p300/CBP complex to bind to HIF 1 and 2alpha, which results in HIF transactivation.

Outcomes of HIF-1 vs. HIF-2 activation
HIF-1alpha is known to be the master regulator of the hypoxic response and the important node that ensures the survival during hypoxic stress. HIF-2alpha was known as the endothelial PAS domain protein, an endothelium specific HIF-alpha isoform, which was thought to be more specialized that HIF-1alpha. Since HIF-2alpha is expressed in tissues of brain, heart, lung, kidney, liver, pancreas, and intestine, it suggests that it has roles in the hypoxia response. Recent studies show that both HIF-1 and HIF-2 participate in hypoxia-dependent gene regulation through complex and even antagonistic interactions. Post-DNA binding mechanism may be required for transactivation, because studies show that DNA binding does not have to correspond to increased transcriptional activity. Recent research confirms that endogenous HIF-2alpha is the main driver of EPO production. HIF-1 produces genes that encode glycolytic enzymes, enzymes that are involved in pH regulation, enzymes that promote apoptosis. HIF-2 produces genes that are involved in invasion and is proved to regulate enzymes in the glycolytic pathway without HIF-1. Interestingly, HIF-1 and HIF-2 are sometimes able to substitute for the isoform-specific functions of the other, meaning that their ability to activate specific target genes depends on specific context.

Different temporal and functional roles of HIF-1 vs. HIF-2
Multiple mechanisms converge to suggest context-dependent, HIF-alpha isoform-specific activation in response to variations in hypoxic intensities and duration. The balance between HIF-1 and HIF-2 activation allows the coordination regulation of the complex hypoxia-dependent processes that takes place in physiology.

HIFs in vascular development
During early embryonic development, the physiological hypoxic environment actives HIFs with the help from other non-hypoxic stimuli such as the renin-angiotensin system, growth factors, and immunogenic cytokines, which all regulates placental development and maturation. Embryonic blood vessels are generated through vasculogenesis, where cells are differentiated into endothelial cells. More blood vessels are made using both sprouting and non-sprouting angiogenesis, which can be remodeled into an adult circulatory system. The differential requirement for HIF-1 and HIF-2 activation during vessel formation and mutation is shown through studies done on mouse. HIF-1alpha knockout mice shows impaired erythropoiesis and finds cephalic vascularization in neural fold formation and the cardiovascular system. With various backgrounds, the HIF-2alpha mice can die either by E12.5 with muscular defects or months after birth due to multi-organ pathology and metabolic abnormalities. Loss of either HIF-1alpha or HIF-2alpha inhibits tumour angiogenesis in adult mouse, which suggests that HIF-1 drives vasculogenesis and early stages of angiogenesis. The formation of complete vasculature requires a smooth transition from the largely HIF-1-dependent transcription, through the period in which both HIF-1 and HIF-2 drive overlapping functions, to the HIF-2 dependent stage of vascular maturation.

HIFs in bone development
Bone can be formed through the mechanisms intramembranous and endochondrial ossification. Intramembranous ossification happens when the flat skull bones are formed and also the mesenchymal cells are differentiated into osteoblasts. Endochondrial ossification happens when other bones are developing; it has a two-staged mechanism. The mesenchymal cells change to chondrocytes, the primary cell type of cartilage, which forms an avascular and highly hypoxic matrix template or growth plate. As a permanent stress, hypoxia influences general chondrocyte metabolism and tissue-specific production of cartilage matrix proteins such as type two collage. The cartilaginous matrix was then replaced by highly vascularized bone tissue through degradation of the matrix and blood vessel invasion. Endochondral ossification requires both the hypertrophic differentiation of chondrocytes and the conversion of avascular cartilage tissue into highly vascularized bone tissue by degradation of the cartilage matrix, and vascular invasion, mainly through the activation of VEGF. Recent studies show that the HIF pathway is participating in membranous ossification and in both stages of endochondral ossification by binding angiogenesis to osteogenesis and regulating the spatiotemporal onset of angiogenesis in the growth plate. Both HIF-1alpha and HIF-2alpha are expressed in growth plate chondrocytes, HIF-1alpha is expressed in similar levels during all stages of chondrocyte differentiation, with its activity enhanced by hypoxia. HIF-2alpha is independent of oxygen-dependent hydroxylation as its levels increase with chondrocyte differentiation. HIF-1 functions as a survival factor in hypoxic chondrocytes by increasing anaerobic glycolysis and hindering apoptosis. It also promotes autophagy, which could extend the lifespan of chondrocytes. HIF-1 is also crucial in extracellular matrix synthesis, which involves the expression of important components required by proliferating chondrocytes in the proliferating zone. HIF-2 is a potent transactivator of many genes, such as type X collagen. Increased levels of HIF-2alpha have been correlated with the development of osteoarthritis. This suggests that HIF-1 is important in the process of hypoxia-dependent cartilage formation and maintenance. HIF-2 alpha participates in endochondral ossification and cartilage destruction, which may be less hypoxia-dependent. Both HIF-1 and HIF-2 are required for developing skeletal vascularity. HIF-1 is important for early stages during severely hypoxic conditions, whereas HIF-2 is more important for later stages.

HIFs in stem cells and cancer
Tumour hypoxia promotes tumour regression and resistance to therapy. It promotes the survival of tumour cells by shifting cells towards anaerobic metabolism, neovascularization and resistance to apoptosis. Hypoxia triggers increased genetic instability, invasion, metastasis and de-differentiation, which lead the tumour aggressiveness. Increased levels of tumour HIF-1alpha is associated with poor patient prognosis in multiple tumour types.

(a) This shows the oxygen gradient generated by the lack of oxygen within solid tumours. In (i), vessel occlusion or rapid tumour growth causes acute hypoxia that activates HIF-1alpha and HIF-2alpha. In (ii), only HIF-1alpha is activated to promote acute hypoxia respons, which could lead to angiogenesis or reperfusion or cell dealth shown on (iii). (iv) shows that chronic hypoxia can increase hypoxia-associated factor and HIF-2alpha levels by mediating a switch to HIF-2-dependent transcription that triggers tumour adaptation, proliferation and progression. (b) uses the blue line to show temporal regulation of HIF-1alpha, green line to show HIF-2alpha and red line to show HAF in response to chronic hypoxic exposure. The dashed lines show where the switch from HIF-1alpha to HIF-2alpha occurs.

Tumour HIF-1 provides an immediate response to acute or transient hypoxia due to rapid induction and negative feedback regulation. HIF-2alpha seems to be favoured by chronic hypoxic exposure. The HIF switch is clearly observed during development of RCC, where there is a slow shift from HIF-1alpha to HIF-2alpha expression with increasing tumour grade.

Stem cells have the ability for self-renewal, multilineage differentiation potential, and long-term viability. Embryotic stem cells can be extracted from the inner cell mass of blastocysis. Adult stem cells are found in tissues such as blood, bone marrow and adipose tissue. Both normal and malignant stem cells are situated in specialized areas where factors such as low oxygen play a crucial role in maintaining pluripotency and viability. Tumour cells have been shown to go through de-differentiation under hypoxic conditions. Thus, HIF-1 and HIF-2 both trigger the hypoxia-induced undifferentiated phenotype by activating the Notch pathway and activating the transcription of other stem-cell-specific factors. HIF-1 is the main driver for hypoxia-induced transcription in non-neoplastic embryonic stem cells. It is required for maintenance of the undifferentiated phenotype in GBM stem cells under hypoxic conditions. HIF-2 seems to be nonfunctional for this part. However, it is required for the proliferation of both stem and non-stem GBM cells; it is especially required for the survival of GBM stem cells. Both HIF1 and HIF2 can have hypoxia-independent functions in CSC maintenance. It seems that the HIF2 has similar functions as HIF1; however, it differs from HIF1 in that it has a unique role in stem cell maintenance under physiological oxygen tension, independently of hypoxia.

Conclusion
Studies have been conducted in understanding the complex regulation of HIFs in both physiological and pathophysiological processes. HIF-1 plays an important role in early vascular and bone development. The HIF switch is also seen in solid tumours where HIF-1 triggers the initial response to hypoxia and then HIF-2 triggers the hypoxic response during chronic hypoxia exposure. Thus, it is crucial for cells to switch from HIF-1 to HIF-2 whenever needed.