Nobel Prize in Physiology or Medicine 2019: How do Cells Sense and Adapt to Availability of Oxygen

Nobel Prize Medal standing on a platform. Red and black background.

Oxygen makes up about one fifth of Earth’s atmosphere and is essential for human and animal life: it is used by the mitochondria present in virtually all human and animal cells in order to convert food into useful energy.

Otto Warburg, the recipient of the 1931 Nobel Prize in Physiology or Medicine, revealed that this conversion is an enzymatic process. And while the fundamental importance of the need oxygen to sustain life has been understood for centuries, how cells adapt to changes in levels of oxygen has long been unknown.

William G. Kaelin Jr., Sir Peter J. Ratcliffe and Gregg L. Semenza discovered how cells can sense and adapt to changing oxygen availability. They identified molecular machinery that regulates the activity of genes in response to varying levels of oxygen.

For their explanation of the molecular mechanisms underlying how cells adapt to variations in oxygen supply the Nobel Assembly at the Karolinska Institutet awarded the Nobel Prize in Physiology or Medicine 2019 to the 3 scientists.

Transcription factor
These changes in gene expression alter cell metabolism, tissue re-modelling, and even organismal responses such as increases in heart rate and ventilation.

In addition to the carotid body-controlled rapid adaptation to low oxygen levels known as hypoxia, there are other fundamental physiological adaptations. One of the key physiological response to hypoxia is the rise in levels of the hormone erythropoietin (EPO), which leads to increased production of erythropoiesis (red blood cells).

The importance of hormonal control of erythropoiesis was already known at the beginning of the 20th century, but how this process was itself controlled by O2 remained a mystery.

Regulating EPO
Gregg Semenza, MD, Ph.D, studied the EPO gene and how it is regulated by varying oxygen levels. By using gene-modified mice, specific DNA segments located next to the EPO gene were shown to mediate the response to hypoxia.

Sir Peter Ratcliffe, FRS, FMedSci, also studied oxygen-dependent regulation of the EPO gene, and both research groups found that the oxygen sensing mechanism was present in virtually all tissues, not only in the kidney cells where EPO is normally produced. These were important findings showing that the mechanism was general and functional in many different cell types.

Semenza discovered that in cultured liver cells a protein complex that binds to the identified DNA segment in an oxygen-dependent manner. He called this complex the hypoxia-inducible factor (HIF) .

Extensive efforts to purify the HIF complex began, and in 1995, Semenza was able to publish some of his key findings, including identification of the genes encoding HIF.

HIF was found to consist of two different DNA-binding proteins, so called transcription factors, now named HIF-1α and ARNT. Now the researchers could begin solving the puzzle, allowing them to understand which additional components were involved and how the machinery works.

VHL: Hippel-Lindau’s disease 
Von Hippel-Lindau’s disease is a genetic disease that leads to increased risk of certain cancers in families with inherited VHL mutations.

In 1995 William Kaelin, Jr. MD, studying the von Hippel-Lindau tumor suppressor gene, and after isolation of the first full-length clone of the gene, showed that this gene could suppress tumor growth in VHL mutant tumorigenic cell lines. Kaelin showed that the VHL gene encodes a protein that prevents the onset of cancer.

He also showed that cancer cells lacking a functional VHL gene express abnormally high levels of hypoxia-regulated genes. Kaelin further demonstrated that when the VHL gene was reintroduced into cancer cells, normal levels were restored.

This observation revealed an important clue showing that VHL was somehow involved in controlling responses to hypoxia.

When oxygen levels are high, cells contain very little HIF-1α. However, observation revealed that when oxygen levels are low, the amount of HIF-1α increases, bind to, and as a result, regulate the EPO gene as well as other genes with HIF-binding DNA segments (Figure 1).

Additional clues came from several research groups showing that VHL is part of a complex that labels the HIF-1α protein with a small peptide called ubiquitin, marking them for degradation by a cellular machine called the proteasome. However, how ubiquitin binds to HIF-1α in an oxygen-dependent manner remained a central question.

When oxygen levels are low (hypoxia), HIF-1α is protected from degradation and accumulates in the nucleus, where it associates with ARNT and binds to specific DNA sequences (HRE) in hypoxia-regulated genes (1). At normal oxygen levels, HIF-1α is rapidly degraded by the proteasome (2). Oxygen regulates the degradation process by the addition of hydroxyl groups (OH) to HIF-1α (3). The VHL protein can then recognize and form a complex with HIF-1α leading to its degradation in an oxygen-dependent manner (4).
When oxygen levels are low (hypoxia), HIF-1α is protected from degradation and accumulates in the nucleus, where it associates with ARNT and binds to specific DNA sequences (HRE) in hypoxia-regulated genes (1). At normal oxygen levels, HIF-1α is rapidly degraded by the proteasome (2). Oxygen regulates the degradation process by the addition of hydroxyl groups (OH) to HIF-1α (3). The VHL protein can then recognize and form a complex with HIF-1α leading to its degradation in an oxygen-dependent manner (4).

Association between VHL and HIF-1α
Peter Ratcliffe, in 1999, demonstrated that there was an association between VHL and HIF-1α, and that VHL regulated HIF-1α post-translational and oxygen-sensitive degradation.

Research focused on a specific portion of the HIF-1α protein known to be important for VHL-dependent degradation.

In 2001 both Kaelin and Ratcliffe simultaneously showed that this regulation of HIF-1α by VHL depends on hydroxylation of HIF-1α, a covalent modification that is itself dependent on oxygen.

In two simultaneously published articles they demonstrated that under normal oxygen levels, hydroxyl groups are added at two specific positions in HIF-1α (Figure 1).

This protein modification, called prolyl hydroxylation, allows VHL to recognize and bind to HIF-1α and thus explained how normal oxygen levels control rapid HIF-1α degradation with the help of oxygen-sensitive enzymes, a process called prolyl hydroxylases.

Further research by Ratcliffe and others research groups identified the responsible prolyl hydroxylases.

It was also shown that the gene activating function of HIF-1α was regulated by oxygen-dependent hydroxylation. The Nobel Laureates could now clearly explain the oxygen sensing mechanism.

Combined work
Through the combined work of these three laureates it was thus demonstrated that the response by gene expression to changes in oxygen is directly coupled to oxygen levels in the human and animal cell, allowing immediate cellular responses to occur to oxygenation through the action of the HIF transcription factor.

A better understanding of oxygen sensing shows that this process is central to a large number of diseases, including cancer (Figure 2).

For example, patients with chronic renal failure often suffer from severe anemia due to decreased EPO expression.

As explained earlier, EPO is produced by cells in the kidney and is essential for controlling the formation of red blood cells.

Moreover, the oxygen-regulated machinery has an important role in cancer. In tumors, the oxygen-regulated machinery is utilized to stimulate blood vessel formation and reshape metabolism for effective proliferation of cancer cells.

Drug development
The discovery of the proline hydroxylases that regulate HIF-1α stability enabled a search for hydroxylase inhibitors to increase HIF levels. This understanding has now opened up new pathways for pharmacologic discovery.

This understanding now guides the efforts of both academic laboratories and pharmaceutical companies, focused on developing drugs that can interfere with different disease states by either activating, or blocking, the oxygen-sensing machinery.

For example, a number of potential drugs that increase HIF function by inhibiting the PHD enzymes are already far along in clinical trials, with a recent series of publications demonstrating their clinical efficacy in treatment of anemia.

Future applications to inhibit the HIF pathway are also on the horizon. These are envisioned as a mean to slow the progression of some cancers that are induced by VHL mutations.

One of these is a specific blocker of EPAS1 function that was recently described by Kaelin and colleagues as capable of slowing tumor growth of VHL mutant cells in animal models.

The awarded mechanism for oxygen sensing has fundamental importance in physiology, for example for our metabolism, immune response and ability to adapt to exercise. Many pathological processes are also affected. Intensive efforts are ongoing to develop new drugs that can either inhibit or activate the oxygen-regulated machinery for treatment of anemia, cancer and other diseases.
The awarded mechanism for oxygen sensing has fundamental importance in physiology, for example for our metabolism, immune response and ability to adapt to exercise. Many pathological processes are also affected. Intensive efforts are ongoing to develop new drugs that can either inhibit or activate the oxygen-regulated machinery for treatment of anemia, cancer and other diseases.

Pharmacologically increased HIF function may aid in the treatment of a wide range of diseases, as HIF has been shown to be essential for phenomena as diverse as immune function, cartilage formation, and wound healing.

Conversely, inhibition of HIF function could also have many applications: increased levels of HIF are seen in many cancers as well as in some cardiovascular diseases, including stroke, heart attack, and pulmonary hypertension.

The discoveries of the Nobel Prize laureates is only the first step to a better understanding – helping research scientists to find novel applications in the fight against cancer and other diseases.

Reference
[1] The Nobel Prize in Physiology or Medicine 2019. NobelPrize.org. Nobel Media AB 2019. Wed. 9 Oct 2019.
[2] Semenza, G.L, Nejfelt, M.K., Chi, S.M. & Antonarakis, S.E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci USA, 88, 5680-5684
[3] Wang, G.L., Jiang, B.-H., Rue, E.A. & Semenza, G.L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.  Proc Natl Acad Sci USA, 92, 5510-5514
[4] Maxwell, P.H., Wiesener, M.S., Chang, G.-W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R. & Ratcliffe, P.J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature, 399, 271-275
[5] Mircea, I., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S. & Kaelin Jr., W.G. (2001) HIFa targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing. Science, 292, 464-468
[6] Jaakkola, P., Mole, D.R., Tian, Y.-M., Wilson, M.I., Gielbert, J., Gaskell, S.J., von Kriegsheim, A., Heberstreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W. & Ratcliffe, P.J. (2001). Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science, 292, 468-472