How Oxygen levels affect cellular metabolism

 

                Animals need oxygen for the conversion of food into useful energy. The fundamental importance of oxygen has been understood for centuries, but 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.

The seminal discoveries by 2019 Nobel Laureates revealed the mechanism for one of life’s most essential adaptive processes. They established the basis for our understanding of how oxygen levels affect cellular metabolism and physiological function. Their discoveries have also paved the way for promising new strategies to fight anemia, cancer and many other diseases.

Oxygen at center stage

                        Oxygen, with the formula O₂, makes up about one fifth of Earth’s atmosphere. Oxygen is essential for animal life: it is used by the mitochondria present in virtually all 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.

During evolution, mechanisms developed to ensure a sufficient supply of oxygen to tissues and cells. The carotid body, adjacent to large blood vessels on both sides of the neck, contains specialized cells that sense the blood’s oxygen levels. The 1938 Nobel Prize in Physiology or Medicine to Corneille Heymans awarded discoveries showing how blood oxygen sensing via the carotid body controls our respiratory rate by communicating directly with the brain.

HIF (Hypoxia Inducible Factor)

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

Gregg Semenza 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 also studied O₂-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 wished to identify the cellular components mediating this response. In cultured liver cells he discovered 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.

                                     Erythropoietin (EPO)

Erythropoietin (EPO) is a hormone produced primarily by the kidneys, with small amounts made by the liver. EPO plays a key role in the production of red blood cells (RBCs), which carry oxygen from the lungs to the rest of the body.

      The body uses a dynamic feedback system to help maintain sufficient oxygen levels and a relatively stable number of RBCs in the blood.

·         Erythropoietin is produced and released into the blood by the kidneys in response to low blood oxygen levels (hypoxemia). The amount of erythropoietin released depends on how low the oxygen level is and the ability of the kidneys to produce erythropoietin.

·         EPO is carried to the bone marrow, where it stimulates production of red blood cells. The hormone is active for a short period of time and then eliminated from the body in the urine.

·         As oxygen levels in the blood rise to normal or near normal levels, the kidneys slow production of EPO.

However, if your kidneys are damaged and do not produce enough erythropoietin, then too few RBCs are produced and you can becomes anemic. Similarly, if your bone marrow is unable to respond to the stimulation from EPO, then you may become anemic. This can occur with some bone marrow disorders or with chronic diseases, such as rheumatoid arthritis.

If you have a condition that affects the amount of oxygen you breathe in, such as a lung disease, you may produce more EPO to try to compensate for the low oxygen level. People who live at high altitudes may also have higher levels of EPO and so do chronic tobacco smokers.

If you produce too much erythropoietin, which can happen with some benign or malignant kidney tumors and with a variety of other cancers, you may produce too many RBCs (polycythemia or erythrocytosis). This can lead to an increase in the blood’s thickness (viscosity) and sometimes to high blood pressure (hypertension), blood clots (thrombosis), heart attack, or stroke. Rarely, polycythemia is caused by a bone marrow disorder called polycythemia vera, not by increased erythropoietin.

VHL: Von Hippel Lindau

When oxygen levels are high, cells contain very little HIF-1α. However, when oxygen levels are low, the amount of HIF-1α increases so that it can bind to and thus regulate the EPO gene as well as other genes with HIF-binding DNA segments (Figure 1). Several research groups showed that HIF-1α, which is normally rapidly degraded, is protected from degradation in hypoxia. At normal oxygen levels, a cellular machine called the proteasome, recognized by the 2004 Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose, degrades HIF-1α. Under such conditions a small peptide, ubiquitin, is added to the HIF-1α protein. Ubiquitin functions as a tag for proteins destined for degradation in the proteasome. How ubiquitin binds to HIF-1α in an oxygen-dependent manner remained a central question.

The answer came from an unexpected direction. At about the same time as Semenza and Ratcliffe were exploring the regulation of the EPO gene, cancer researcher William Kaelin, Jr. was researching an inherited syndrome, von Hippel-Lindau’s disease (VHL disease). This genetic disease leads to dramatically increased risk of certain cancers in families with inherited VHL mutations. Kaelin showed that the VHL gene encodes a protein that prevents the onset of cancer. Kaelin also showed that cancer cells lacking a functional VHL gene express abnormally high levels of hypoxia-regulated genes; but that when the VHL gene was reintroduced into cancer cells, normal levels were restored. This was an important clue showing that VHL was somehow involved in controlling responses to hypoxia. Additional clues came from several research groups showing that VHL is part of a complex that labels proteins with ubiquitin, marking them for degradation in the proteasome. Ratcliffe and his research group then made a key discovery: demonstrating that VHL can physically interact with HIF-1α and is required for its degradation at normal oxygen levels. This conclusively linked VHL to HIF-1α.

              Under normal oxygen levels, hydroxyl groups are added at two specific positions in HIF-1α . 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 (so-called prolyl hydroxylases). Further research by Ratcliffe and others 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 had now elucidated the oxygen sensing mechanism and had shown how it works.

Figure 1. 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).

Oxygen shapes physiology and pathology

Thanks to the groundbreaking work of these Nobel Laureates, we know much more about how different oxygen levels regulate fundamental physiological processes. Oxygen sensing allows cells to adapt their metabolism to low oxygen levels: for example, in our muscles during intense exercise. Other examples of adaptive processes controlled by oxygen sensing include the generation of new blood vessels and the production of red blood cells. Our immune system and many other physiological functions are also fine-tuned by the O₂-sensing machinery. Oxygen sensing has even been shown to be essential during fetal development for controlling normal blood vessel formation and placenta development.

Oxygen sensing is central to a large number of diseases . For example, patients with chronic renal failure often suffer from severe anemia due to decreased EPO expression. EPO is produced by cells in the kidney and is essential for controlling the formation of red blood cells, as explained above. 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. Intense ongoing efforts in academic laboratories and pharmaceutical companies are now focused on developing drugs that can interfere with different disease states by either activating, or blocking, the oxygen-sensing machinery.

Figure 2. 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.

 

Von Hippel-Lindau Syndrome

What is von Hippel-Lindau disease?

Von Hippel-Lindau syndrome (VHL) is a hereditary condition associated with tumors arising in multiple organs. VHL-related tumors include hemangioblastomas, which are blood vessel tumors of the brain, spinal cord, and retina. The retinal tumors are also called retinal angiomas, which can lead to blindness if not treated in a timely manner. People with VHL also have an increased risk of developing clear cell renal cell carcinoma (ccRCC), which is a specific type of kidney cancer, as well as a type of tumor in the pancreas known as pancreatic neuroendocrine tumor (pNET). Tumors of the adrenal gland or pheochromocytoma can also develop, with a small number becoming metastatic, meaning they spread to other parts of the body. 

Other features of VHL include: kidney cysts, which are closed sacs usually filled with fluid; pancreatic cysts, epididymal cystadenomas, which are tumors near a man’s testicles; broad ligament cystadenomas, which occur near the fallopian tubes in women; and endolymphatic sac tumors (ELST), which are tumors of the inner ear that may cause hearing loss.

What causes VHL?

VHL is a genetic condition. This means that the risk of developing certain types of tumors and other features of VHL can be passed from generation to generation. The gene associated with VHL is also called VHL. Inheriting a deletion or mutation (alteration) in the VHL gene gives a person an increased risk of developing any of the different signs of VHL explained above, called manifestations. Nearly everyone who has VHL syndrome has an identifiable VHL genetic mutation.

How is VHL inherited?

Normally, every cell has 2 copies of each gene: 1 inherited from the mother and 1 inherited from the father. VHL follows an autosomal dominant inheritance pattern, in which inheriting 1 copy of the altered gene will likely result in a mutation of the second (normal) copy of the gene. This puts the individual at risk for developing cancer.

 

The increase in body size of humans and other vertebrates requires a

 physiological infrastructure to provide adequate delivery of oxygen to tissues

 and cells to maintain oxygen homeostasis. The heart, lungs and the vasculature

 are all part of a highly regulated system that ensures the distribution of the

 precise amount of oxygen needed throughout the mammalian organism.

1. The role of HIF-1
α pathway in cellular adaptation to hypoxic stress

Mammalian cells need to maintain proper oxygen hemostasis in order to execute their aerobic metabolism and energy generation. In cancer, heart diseases, or chronic obstructive pulmonary disorders, the cellular oxygen balance is highly impaired, and cells become hypoxic (having low oxygen (O2) levels). Hypoxia is common in many types of solid tumors, where tumor cells proliferate rapidly and form large solid tumor masses, leading to obstruction and compression of the blood vessels surrounding these masses. These abnormal blood vessels often do not function properly and result in poor O2 supply to the center tumor regions2. Tumor cells in this hypoxic region begin to adapt these low oxygen tension conditions by activating several survival pathways. Activation of HIF-1 transcription factor is the most recognized pathway adopted by hypoxic cells in this harsh microenvironment

 

 

    2. Regulation of HIF-1α pathway

The activity and accumulation of HIF-1α protein were found to be regulated at different levels throughout its life cycle inside the cells. Independently from O2 levels, HIF-1α is constitutively transcribed and synthesized through a series of signaling events involving several growth factors and other signaling molecules. HIF-1α undergoes quick degradation under normoxic conditions and normally has a very short half-life (about 5 min). In contrast, under hypoxic conditions, several pathways have been shown to control HIF-1α stability and transcriptional activity via post-transnational modifications involving hydroxylation, acetylation, ubiquitination, and phosphorylation reactions