Hypoxia-inducible factors

Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment, specifically, to decreases in oxygen, or hypoxia.

Structure
Most, if not all, oxygen-breathing species express the highly-conserved transcriptional complex HIF-1, which is a heterodimer composed of an alpha and a beta subunit, the latter being a constitutively-expressed aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1 belongs to the PER-ARNT-SIM (PAS) subfamily of the basic-helix-loop-helix (bHLH) family of transcription factors. The alpha and beta subunit are similar in structure and both contain the following domains:


 * N-terminus – a bHLH domain for DNA binding
 * central region – Per-ARNT-Sim (PAS) domain which facilitates heterodimerization
 * C-terminus – recruits transcriptional coregulatory proteins

Members
The following are members of the human HIF family:

Role
The HIF signaling cascade mediates the effects of hypoxia, the state of low oxygen concentration, on the cell. Hypoxia often keeps cells from differentiating. However, hypoxia promotes the formation of blood vessels, and is important for the formation of a vascular system in embryos, and cancer tumors. The hypoxia in wounds also promotes the migration of keratinocytes and the restoration of the epithelium.

In general, HIFs are vital to development. In mammals, deletion of the HIF-1 genes results in perinatal death. HIF-1 has been shown to be vital to chondrocyte survival, allowing the cells to adapt to low-oxygen conditions within the growth plates of bones. HIF plays a central role in the regulation of human metabolism.

Mechanism
The alpha subunits of HIF are hydroxylated at conserved proline residues by HIF prolyl-hydroxylases, allowing their recognition and ubiquitination by the VHL E3 ubiquitin ligase, which labels them for rapid degradation by the proteasome. This occurs only in normoxic conditions. In hypoxic conditions, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a cosubstrate.

Hypoxia also results in a buildup of succinate, due to inhibition of the electron transport chain in the mitochondria. The buildup of succinate further inhibits HIF prolyl-hydroxylase action, since it is an end-product of HIF hydroxylation. In a similar manner, inhibition of electron transfer in the succinate dehydrogenase complex due to mutations in the SDHB or SDHD genes can cause a build-up of succinate that inhibits HIF prolyl-hydroxylase, stabilizing HIF-1α. This is termed pseudohypoxia.

HIF-1, when stabilized by hypoxic conditions, upregulates several genes to promote survival in low-oxygen conditions. These include glycolysis enzymes, which allow ATP synthesis in an oxygen-independent manner, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. HIF-1 acts by binding to HIF-responsive elements (HREs) in promoters that contain the sequence NCGTG.

It has been shown that muscle A kinase–anchoring protein (mAKAP) organized E3 ubiquitin ligases, affecting stability and positioning of HIF-1 inside its action site in the nucleus. Depletion of mAKAP or disruption of its targeting to the perinuclear (in cardiomyocytes) region altered the stability of HIF-1 and transcriptional activation of genes associated with hypoxia. Thus, "compartmentalization" of oxygen-sensitive signaling components may influence the hypoxic response.

The advanced knowledge of the molecular regulatory mechanisms of HIF1 activity under hypoxic conditions contrast sharply with the paucity of information on the mechanistic and functional aspects governing NF-κB mediated HIF1 regulation under normoxic conditions. However, HIF-1α stabilization is also found in non-hypoxic conditions through an, until recently, unknown mechanism. It was shown that NF-κB (nuclear factor κB) is a direct modulator of HIF-1α expression in the presence of normal oxygen pressure. siRNA (small interfering RNA) studies for individual NF-κB members revealed differential effects on HIF-1α mRNA levels, indicating that NF-κB can regulate basal HIF-1α expression. Finally, it was shown that when endogenous NF-κB is induced by TNFα (tumour necrosis factor α) treatment, HIF-1α levels also change in an NF-κB-dependent manner.

Anemia
Recently several drugs have been developed which act as selective HIF prolyl-hydroxylase inhibitors. The most notable of these include FibroGen's compounds FG-2216 and FG-4592, both are intended as orally acting drugs for use in the treatment of forms of anemia. By inhibiting HIF prolyl-hydroxylase, the activity of HIF-1α in the bloodstream is prolonged, which results in an increase in endogenous production of erythropoetin. Both of these drugs made it through to phase II clinical trials, but these were suspended temporarily in May 2007 following the death of a trial participant from fulminant hepatitis. However it is unclear whether this death was caused by FG-2216. The hold has been lifted in early 2008 as FDA has reviewed and approved a thorough response from FibroGen.

Inflammation, and cancer
In other scenarios and in contrast to the therapy outlined above, recent research suggests that HIF induction in normoxia is likely to have serious consequences in disease settings with a chronic inflammatory component. It has also been shown that chronic inflammation is self-perpetuating and that it distorts the microenvironment as a result of aberrantly active transcription factors. Consequently, alterations in growth factor, chemokine, cytokine and ROS balance occur within the cellular milieu that in turn provide the axis of growth and survival needed for de novo development of cancer and metastasis. The results of a recently published study have numerous implications for a number of pathologies where NF-κB and HIF-1 are deregulated, including rheumatoid arthritis and cancer. Therefore, it is thought that understanding the cross talk between these two key transcription factors, NF-κB and HIF, will greatly enhance the process of drug development.

HIF activity is involved in angiogenesis required for cancer tumor growth, so HIF inhibitors are under investigation for anti-cancer effects, (e.g. phenethyl isothiocyanate (PEITC) ).