Oxygen Sensing Gets a Second Wind

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Science  01 Feb 2002:
Vol. 295, Issue 5556, pp. 807-808
DOI: 10.1126/science.1069825

M ammalian cells are able to sense prolonged decreases in oxygen concentration (hypoxia)[HN1] through a conserved hypoxic response pathway. This pathway facilitates adaptation to hypoxia-induced physiological stress by regulating changes in gene expression, and is also critical for the execution of many physiological events, including formation of blood vessels during embryogenesis, and pathophysiological processes such as tumorigenesis[HN2]. A family of hypoxia-inducible transcription factors (HIFs) lies at the heart of this adaptive pathway. HIF proteins are activated by a decrease in the concentration of molecular oxygen (O2), which results in the induced expression of downstream target genes that mediate adaptation and survival of cells and the whole organism[HN3].

Until recently, the means by which cells sense alterations in oxygen tension and subsequently induce changes in HIF activity remained obscure. The first inkling of an oxygen sensing pathway in higher organisms came last year with the discovery of a family of oxygen-dependent enzymes responsible for the modulation of HIF stability[HN4] (1-4). A report by Lando et al.[HN5] (5) on page 858 of this issue now identifies a second, oxygen-dependent, posttranslational modification of HIF that regulates the ability of HIF to recruit stimulatory transcriptional cofactors to its target genes.

The HIF transcription factors are composed of two subunits: the hypoxia-regulated α subunit, HIF-1α (or its paralogs HIF-2α and HIF-3α), and the oxygen-insensitive HIF-1β subunit (also known as the arylhydrocarbon receptor nuclear translocator, or ARNT)[HN6]. Under normal oxygen conditions (normoxia), the HIF-1α subunit, although expressed, is rapidly degraded such that almost no HIF protein accumulates (see the figure). Under hypoxic conditions, degradation of the α subunit is blocked, allowing HIF-1α to accumulate within the nucleus where, upon binding to HIF-1β, it recognizes HIF-responsive elements (HREs) within the promoters of hypoxia-responsive target genes. Degradation of HIF-1α under normoxic conditions is triggered by posttranslational hydroxylation[HN7] of conserved proline[HN8] residues within a polypeptide segment known as the oxygen-dependent degradation domain (ODD). The hydroxylated proline residues in this sequence are recognized by the product of the von Hippel-Lindau tumor suppressor gene (pVHL)[HN9], a component of a ubiquitin ligase complex that tags the α subunit for degradation by the proteasome[HN10] (see the figure) (6, 7). This critical regulatory event is carried out by a family of iron (II)-dependent prolyl hydroxylase enzymes (3, 4) that use O2 as a substrate to catalyze hydroxylation of the target proline residues. Because O2 appears to be rate limiting for prolyl hydroxylase activity (3), these enzymes may represent bona fide oxygen sensors that provide a direct link between O2 concentration and components of the hypoxic response pathway.

Regulation of the HIF-1 transcription factor.

Under normoxic conditions, the ODD of HIF-1α is modified by a HIF-prolyl hydroxylase, triggering HIF-1α recognition by pVHL and subsequent degradation by the proteasome. Similarly, an asparaginyl hydroxylase modifies the C-TAD of HIF-1α, blocking its interaction with the transcriptional coactivator p300. Hypoxia blocks both prolyl hydroxylation and asparaginyl hydroxylation, allowing HIF-1α to accumulate and bind to p300, thereby promoting the transcription of downstream HIF-1 target genes, thus enabling cells and the whole organism to adapt to hypoxia.

Modulation of protein stability is just one means by which HIF activity is induced by hypoxia. In addition to the ODD domain, the α subunits of all three HIF isoforms contain two transactivation domains responsible for recruiting transcriptional coactivators essential for gene expression. One of the HIF transactivation domains overlaps the ODD, and regulation of its activity is likely to be a by-product of protein stability. The second carboxyl-terminal transactivation domain (C-TAD) operates independently of the ODD and is able to recruit coactivator complexes such as p300/CBP[HN11] only under hypoxic conditions (8-10). Remarkably, the regulatory switch controlling the activity of the C-TAD also involves an oxygen-dependent hydroxylation event, in this case targeted to a conserved asparagine[HN12] residue.

To examine oxygen-dependent regulation of the C-TAD, Lando and colleagues removed this domain from the α subunit, decoupling it from regulation of protein stability mediated through the ODD. When linked to the Gal4 DNA-binding domain[HN13], C-TAD stability is unaffected by hypoxia, yet remains able to stimulate transcription in response to hypoxia (8, 9, 11-13). Mass spectrometry[HN14] revealed that under normoxic conditions, the inactive C-TAD had a mass 16 daltons greater than predicted, reminiscent of ODD hydroxylation. Following a shift to hypoxic conditions, the mass increase was lost, coinciding with increased C-TAD activity. Parsimony would have predicted that the HIF prolyl hydroxylase enzymes would execute this regulatory step. However, mass spectrometry analysis revealed that a conserved asparagine residue, rather than a proline residue, is the target for hydroxylation in the C-TAD. Mutation of the key asparagine residue to alanine[HN15] resulted in loss of oxygen-dependent hydroxylation and led to constitutive C-TAD activity. Together, these data nicely fit a model in which the asparagine residue within the C-TAD is hydroxylated under normoxic conditions by a putative asparagine hydroxylase (see the figure). Furthermore, Lando and colleagues provide clear evidence that hydroxylation of the conserved asparagine residue blocks interaction of the C-TAD with the p300/CBP transcriptional coactivators. Under hypoxic conditions, asparagine hydroxylation is blocked, thereby derepressing the system and facilitating coactivator recruitment.

To date, the enzyme responsible for hydroxylation of the conserved C-TAD asparagine residue has not been identified. Well-characterized prolyl and lysyl hydroxylases are iron-binding enzymes that use 2-oxoglutarate as a cosubstrate. Lando and colleagues report that asparagine hydroxylation of the C-TAD can be abrogated by addition of iron chelators or competitive inhibitors of 2-oxoglutarate, indicating that this enzyme may also fall within the larger family of 2-oxoglutarate-dependent dioxygenases. Although it is tempting to believe that both the prolyl and asparaginyl hydroxylases will serve as direct oxygen sensors, further studies are required to assess whether these enzymes are sensitive to changes in O2 concentration capable of inducing a hypoxic response in vivo.

Discovery of this second oxygen-dependent switch raises the question of why HIF activity is subject to multiple independent levels of regulation. The work described by Lando and co-workers demonstrates that both hydroxylase switches must be flipped to fully induce HIF. It is possible that multiple levels of regulation allow for graded responses to subtle changes in O2 concentration. Alternatively, dependence upon two independent regulatory events may help to ensure that the hypoxic response pathway is tightly controlled. The products of well-characterized HIF target genes are known to promote increased vascularization and glycolytic metabolism, both of which are essential for solid tumor formation. Indeed, constitutive activation of HIF has been correlated with the progression of a variety of human tumors (14). Likewise, prolonged HIF induction leads to the expression of genes affecting the balance between cell death and survival and is required for promoting cell death pending failure to adapt to a hypoxic environment (15).

Do these two hydroxylase enzymes complete the story of oxygen-dependent HIF regulation, or might there be additional levels of HIF transcription factor modulation? By themselves, these prolyl and asparaginyl hydroxylase enzymes may not account for the effects of some hypoxia “mimics” such as carbon monoxide. Moreover, the secondary consequences of changes in oxygen concentration, such as alteration of intracellular redox potentials or the amount of reactive oxygen species, may influence HIF induction (16). These additional signals might regulate the prolyl and asparaginyl hydroxylase enzymes directly or influence HIF through unique pathways. Regardless of these potential complexities, the past year has been mighty good to HIF. The time is ripe for detailed enzymological studies of the relevant prolyl and asparaginyl hydroxylases, and the door now has been opened to the discovery of small-molecule agonists and antagonists of the hypoxic response pathway.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

Dictionaries and Glossaries

The On-line Medical Dictionary is provided by CancerWEB.

The BioTech Life Sciences Dictionary is made available by the Ellington Lab, University of Texas.

D. Glick's Glossary of Biochemistry and Molecular Biology is provided on the Web by Portland Press.

The Companion Web site for the third edition of Biochemistry by Mathews, van Holde, and Ahern offers brief introductions to concepts, molecules, and enzymes.

Web Collections, References, and Resource Lists

The Google Web Directory provides links to Internet resources in biochemistry, molecular biology, and cell biology.

P. Gannon's Cell & Molecular Biology Online is a collection of annotated links to Internet resources.

BioChemLinks provides links to educational Internet resources in biology, chemistry, and biochemistry.

K. House's Biology Web References for Students and Teachers includes sections of Internet resources related to biochemistry and molecular genetics.

GeneCards, provided by the Weizmann Institute of Science, is a database of human genes, their products, and their involvement in diseases, with links provided to other genome databases and resources.

Online Mendelian Inheritance in Man (OMIM) is a catalog of human genes and genetic disorders made available on the Web by the National Center for Biotechnology Information (NCBI). The database is also available on NCBI's Entrez system.

Online Texts and Lecture Notes

DNA-RNA-Protein is an educational presentation (available in basic and advanced versions) of the Nobel e-Museum. A presentation on transcription is included.

Molecular Genetics is a tutorial provided by U. Melcher, Department of Biochemistry and Molecular Biology, Oklahoma State University.

J. Kimball provides Kimball's Biology Pages, an online textbook and glossary. An introduction to transcription is included.

The Online Biology Book, made available by M. Farabee, Estrella Mountain Community College, Avondale, AZ, has a section on protein synthesis.

F. Lux, Division of Biological and Physical Sciences, Lander University, Greenwood, SC, provides lecture notes for a course on molecular biology.

The Department of Biological Sciences, Carnegie-Mellon University, makes available lecture notes by W. McClure for a biochemistry course, as well as lecture notes by G. Rule. Molecular Models for Biochemistry is a resource maintained by W. McClure, Department of Biological Sciences, Carnegie-Mellon University.

G. Gray, Department of Chemistry and Physics, Southwest Baptist University, Bolivar, MO, offers lecture notes for a biochemistry course. A presentation on protein structure and function is included.

W. Terzaghi, Department of Biology, Wilkes University, Wilkes-Barre, PA, offers lecture notes on transcription for a course on cell and molecular biology.

M. Hewlett, Department of Molecular and Cellular Biology, University of Arizona, offers lecture notes for a molecular biology course.

M. Mulligan, Biochemistry Department, Memorial University of Newfoundland, Canada, offers lecture notes for a biochemistry course on nucleic acid biochemistry and molecular biology.

General Reports and Articles

The 14 May 2001 issue of The Scientist had an article by J. Perkel titled “Seeking a cellular oxygen sensor.”

The 15 April 2000 issue of the Journal of Experimental Biology had a review article (full-text available in Adobe Acrobat format) by R. Wenger titled “Mammalian oxygen sensing, signalling and gene regulation.”

The 15 August 2000 issue of Genes & Development had a review article by G. Semenza titled “HIF-1 and human disease: One highly involved factor.”

Numbered Hypernotes

1. Hypoxia is defined in the On-line Medical Dictionary. The MSDS Hyperglossary has an entry for hypoxia. R. Hodgkiss, Gray Cancer Institute, Northwood, UK, provides an introduction to hypoxia. The summer 1999 issue of the magazine of the Physiological Society had an article about hypoxia by D. Millhorn titled “Regulation of signal transduction and gene expression by reduced oxygen.” R. Burn-Murdoch, Division of Physiology, King's College London, offers information on the causes and effects of hypoxia in humans.

2. The hypoxic response. The National Cancer Institute's Center for Cancer Research offers a research presentation titled “Molecular mechanisms of hypoxic response.” The 29 April 2000 issue of the Pharmaceutical Journal had an article by R. Airley, J. Monaghan, and I. Stratford titled “Hypoxia and disease: Opportunities for novel diagnostic and therapeutic prodrug strategies.” The 3 September 1996 issue of the Proceedings of the National Academy of Sciences had an article by P. Hochachka et al. titled “Unifying theory of hypoxia tolerance: Molecular/metabolic defense and rescue mechanisms for surviving oxygen lack”; the July 1997 issue had an article by P. Maxwell et al. titled “Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth.” The April 2000 issue of the Journal of Applied Physiology had a review article by G. Semenza titled “HIF-1: Mediator of physiological and pathophysiological responses to hypoxia.” The October 2000 issue of the Journal of Clinical Investigation had a perspective article about HIF-1 by G. Semenz titled “Surviving ischemia: Adaptive responses mediated by hypoxia-inducible factor 1.”

3. Transcription and transcription factors. Transcription and transcription factors are defined in xrefer's Dictionary of Biology. The Biochemistry companion Web site includes an introduction to transcription and transcription factors. U. Melcher's Molecular Genetics offers an introduction to transcription. A molecular biology tutorial on transcription is provided by the Interactive Genetics Web site of the University of California, Los Angeles. P. McClean, Department of Plant Sciences, North Dakota State University, includes a presentation on transcription and transcription factors in the lecture notes for a molecular genetics course. R. Keates, Department of Chemistry and Biochemistry, University of Guelph, Canada, provides lecture notes on the regulation of transcription for a course on regulation in biological systems.

4. Discovery of oxygen-dependent enzymes. The 20 April 2001 issue of Science had a Perspective by H. Zhu and H. F. Bunn titled “How do cells sense oxygen?” about the research article by M. Ivan et al. titled “HIF-alpha targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing” (1) and the research article by P. Jaakkola et al. titled “Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation” (2). The Howard Hughes Medical Institute offers a 20 April 2001 news article about the research titled “Researchers identify how cells respond to oxygen starvation.” The 9 November 2001 issue of Science had a report by R. Bruick and S. McKnight titled “A conserved family of prolyl-4-hydroxylases that modify HIF” (4).

5. D. Lando, D. Peet, and M. Whitelaw are in the Department of Molecular Biosciences, Adelaide University, Australia. D. Whelan and J. Gorman are in the CSIRO Health Sciences and Nutrition Division, Parkville, Victoria, Australia.

6. HIF-1 and subunits. The 20 January 1995 issue of the Journal of Biological Chemistry had an article by G. Wang and G. Semenza titled “Purification and characterization of hypoxia-inducible factor 1.” The June 1995 issue of the Proceedings of the National Academy of Sciences had an article by G. Wang et al. titled “Hypoxia-inducible factor 1 is a basic helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.” GeneTex provides catalog information about HIF-1 alpha and HIF-1 beta. The GeneCards database has an entry for HIF-1A and ARNT (HIF-1beta). OMIM has entries for hypoxia-inducible factor 1, alpha subunit; HIF1a and aryl hydrocarbon receptor nuclear translocator; ARNT (HIF1-beta).

7. Hydroxylation, hydroxyl group, and hydroxylase are defined in the On-line Medical Dictionary.

8. An introduction to proline is provided by the Biochemistry companion Web site. The Amino Acid Properties Web page, presented by the Section of Molecular and Cellular Biology, University of California, Davis, provides information about proline.

9. VHL (von Hippel-Lindau) tumor suppressor gene and protein. NCBI offers a presentation on Von Hippel-Lindau disease. The VHL Family Alliance provides an article by W. Kaelin titled “Recent insights into the functions of the von Hippel-Lindau protein.” The GeneCards database has an entry for VHL. The SWISS-PROT protein database has an entry for VHL. OMIM has an entry for von Hippel-Lindau syndrome; VHL. The online journal Expert Reviews in Molecular Medicine makes available a 19 March 2001 article by F. Richards titled “Molecular pathology of von Hippel-Lindau disease and the VHL tumour suppressor gene.” M. Czyzyk-Krzeska, Department of Molecular and Cellular Physiology, School of Medicine, University of Cincinnati, provides a research presentation on Von Hippel-Lindau tumor suppressor protein (pVHL) and gene expression. P. Jaakkola, Welcome Trust Cardiovascular Research Initiative, University of Oxford, provides a research presentation on the role of iron in the HIF-1a - pVHL interaction. O. Iliopooulos, Massachusetts General Hospital Cancer Center, provides a research presentation about the pVHL tumor suppressor protein. The 30 April 2001 issue of The Scientist had an article by J. Fisher Wilson titled “Understanding the VHL tumor suppressor complex.”

10. Proteasome is defined in xrefer's Dictionary of Biology. Kimball's Biology Pages include a presentation on the proteasome. The Highlights of Biochemistry Web site, provided by R Bergmann, Institute of General Botany, University of Hamburg, Germany, includes an illustrated presentation on proteasomes.

11. Transcriptional coactivators p300/CBP. S. Bhattacharya, Wellcome Trust Centre for Human Genetics, University of Oxford, provides an introduction to CBP and p300. The GeneCards database has entries for p300 and CREBBP (CBP, CREB-binding protein). GENATLAS has entries for p300 and CREBBP. Infobiogen's Atlas of Genetics and Cytogenetics in Oncology and Haematology has entries for p300 and CBP. The November 1996 issue of the Proceedings of the National Academy of Sciences had an article by Z. Arany et al. titled “An essential role for p300/CBP in the cellular response to hypoxia.” The 2 February 2001 issue of the Journal of Biological Chemistry had an article by J. Gu, J. Milligan, and L. E. Huang titled “Molecular mechanism of hypoxia-inducible factor 1alpha -p300 interaction” (9).

12. An introduction to asparagine is offered by the Biochemistry companion Web site. The Amino Acid Properties Web page provides information about asparagine.

13. An illustrated introduction to the GAL4 DNA-binding domain is provided in the Protein NMR Structure Gallery of the Protein NMR Spectroscopy Laboratory, Center for Advanced Biotechnology and Medicine, Rutgers University. U. Melcher's Molecular Genetics includes information about Gal4.

14. Mass spectrometry. The American Society for Mass Spectrometry offers a tutorial on mass spectrometry. The Mass Spectrometry Research Center, Vanderbilt University, provides a mass spectrometry tutorial. offers a primer on mass spectrometry in proteomics as well as a presentation by T. Mallet on molecular mass. W. McClure, Department of Biological Sciences, Carnegie Mellon University, offers an illustrated tutorial on molecular mass scales. The Cambridge Healthtech Institute offers a mass spectrometry glossary. The Mass Spectrometry Interest Group, National Cancer Institute at Frederick, MD, provides links to Web resources on mass spectrometry.

15. An introduction to alanine is provided by the Biochemistry companion Web site. The Amino Acid Properties Web page provides information about alanine.

16. R. K. Bruick and S. L. McKnight are in the Department of Biochemistry, University of Texas Southwestern Medical Center.


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