Discovering HIF Regulation

In the complicated, occasionally counter-intuitive world of signal transduction pathways, sometimes events turn out to be much simpler than first supposed.

Eugene Russo
Apr 20, 2003

APPETITE FOR DESTRUCTION: Schematic representation shows the 15-residue portion of the HIF-1a destruction sequence, which is bound to the B domain of pVHL in the pVHL-ElonginB-ElonginC complex.
Reference Reprinted with permission from science, 296:1886-9, 2002

In the complicated, occasionally counter-intuitive world of signal transduction pathways, sometimes events turn out to be much simpler than first supposed. Such is the case with an important oxygen- sensing pathway, the essential features of which investigators Bill Kaelin at Dana-Farber Cancer Institute and Peter Ratcliffe at the University of Oxford independently describe in these Hot Papers.1,2

Understanding how cells sense oxygen has potentially important implications for research in cancer, diabetes, and many ischemic diseases. The appropriate delivery of oxygen by the lungs, heart, blood, circulation, and blood vessels to all cells is a delicate operation: Too little oxygen impairs metabolism; too much oxygen is toxic.

The search for the oxygen-sensing mechanism has prompted a variety of theories, many of them complicated, some of them contradictory.3 Though not necessarily the definitive oxygen sensor, the pathway that these teams describe has significance. "I think most people are coming around to the view that this is certainly a major component of the oxygen-sensing pathway," says Kaelin, who is also a Howard Hughes Medical Institute Investigator. "That's not to say there couldn't be other inputs."

Pediatrics professor Gregg L. Semenza, Johns Hopkins University School of Medicine, says that the groups quite simply put the first definitive explanations into the hands of researchers. "What these studies demonstrated was a molecular mechanism by which changes in cellular oxygen concentration can lead to changes in gene expression."

Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age.

P. Jaakkola et al., "Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolylhydroxylation," Science, 292:468-72, April 20, 2001. (Cited in 251 papers)

M. Ivan et al., "HIF alpha targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing," Science, 292:464-8, April 20, 2001. (Cited in 246 papers)

A MOLECULAR CROSSROAD  Kaelin and Ratcliffe's papers emerged from two distinct fields: "We sort of met in the middle," says Kaelin. His work centered on tumor biology; Ratcliffe's, on oxygen sensing. Once the von Hippel-Lindau tumor suppressor gene was cloned in 1993,4 Kaelin's lab began studying its protein. His team wanted to know how inactivation of the VHL protein caused tumors to develop.

A dysfunctional VHL tumor suppressor gene causes VHL syndrome, in which an abnormal growth of blood vessels develops capillary clusters called angiomas. Angiomas beget highly vascularized tumors, likely a result of angiogenic growth factor overproduction, and the hormone erythropoietin (EPO). The tumors recruit sustenance by increasing the amount of red blood cells carrying oxygen. This feature of VHL-related tumors suggested to Kaelin that the VHL protein might play some role in sensing changes in oxygen. "It seemed to me that the tumors that you see in VHL disease are behaving as if they're hypoxic all the time, essentially," says Kaelin.

Ratcliffe, a nephrologist who'd been working explicitly on the problem of oxygen sensing, approached it by studying erythropoietin. His group was interested in how hypoxia, or oxygen deprivation, stimulates EPO production. "That sensing process remained enigmatic for several decades," says Ratcliffe. This conundrum drew him into the field in the early 1990s.

Ratcliffe says that his team realized early on that this was not a special sensing property of particular cells, but a widespread phenomenon that regulates angiogenesis, metabolism, and a whole range of cellular systemic responses to hypoxia. One important element of that phenomenon was the VHL protein. Consequently, both groups wound up studying proteins that were not on their original to-do lists: Ratcliffe, with VHL, and Kaelin, with hypoxia inducible factor (HIF)--a protein that maintains oxygen homeostasis and regulates hypoxia-inducible genes.

Several key findings helped piece together the oxygen sensor. In 1996, Kaelin's group showed that cells lacking VHL overproduce hypoxia-inducible mRNAs.5 Using this information, Ratcliffe's group found that cells lacking VHL overproduce the transcription factor HIF.6 Kaelin's and Ratcliffe's groups and others went on to show that VHL protein polyubiquinates HIF in the presence of oxygen.7-9 

REGULATORY REACTION  The big questions then: How does the VHL protein detect the presence of oxygen? In particular, what sites on the HIF molecule confer oxygen-sensing behavior? Is it the HIF that is modified or is it the VHL?

Kaelin's and Ratcliffe's groups each suspected there might be a posttrans-lational modification involved. Indeed, as reported in the two Hot Papers, the groups discovered a crucial modification of conserved prolyl residues in HIF. VHL binds only to the alpha subunit of HIF (one of the HIF complexes' two subunits) when an oxygen-dependent, posttranslational modification occurs, namely hydroxylation at proline 564 by a then-unidentified HIF-a prolyl-hydroxylase or HPH. Identifying a role for the hydroxylation was highly significant; prolyl hydroxylases were known to use dioxygen in the hydroxylation reaction. This established a clear connection between the availability of molecular oxygen and the regulatory reaction.

"The link to oxygen turned out to be much simpler than it had seemed," says Ratcliffe. Fifteen years of research had yielded the identification of three major steps: HIF, hydroxylation, and VHL. The story would have been much more complicated if the pathway were composed of, say, multiple kinases.

Interestingly, hints at the role of prolyl hydroxylation could have been gleaned earlier if researchers had looked to the field of collagen research where, for more than two decades, prolyl hydroxylases have been known to use molecular oxygen and to be highly sensitive to iron chelators and cobalt. "Had we read that field, it would have been helpful," says Ratcliffe.

A few months after the April 2001 papers were published, two groups, including Ratcliffe's, reported isolating the HPH in Drosophila, Caenorhabditis elegans, and mammalian cells.10,11 HPH is thought to be a promising drug target. Enzyme inhibition increases HIF levels, enabling the protection of hypoxic cells, like those typical of ischemic conditions. Kaelin is working with the South San Francisco-based biotech company FibroGen to pursue inhibitors that might protect against stroke. Several pharmaceutical companies are doing likewise.

The cancer case is not as straightforward, however. Enhancing HPH should decrease the level of HIF, theoretically providing an antiangiogenic treatment approach that cuts off oxygen supply to tumors. But, according to Kaelin, there are some contradictory reports as to whether HIF is protumorigenic or antitumorigenic. He says that he believes it's usually the former. Ratcliffe notes that ascorbate, because it enhances the activity of HPH, might figure into an antiangiogenic treatment. Recent work from Kaelin's group has solidified the role of HIF in VHL-associated tumors in murine studies. They've shown in mice that HIF downregulation is necessary for tumor suppression by VHL.12

Other follow-up findings have added layers of complexity to the oxygen-sensing system. Researchers found that one of HIF's two transactivation domains, the C-terminal transactivation domain of HIF-1a (CAD), is critical to the hydroxylation of HIF; its action links HIF to the well-studied transcriptional coactivator p300.13 Two papers later reported the crystal structures of that HIF transactivation domain bound to p300 and transcriptional coactivator CBP.14

Researchers continue to investigate the prevalence of protein hydroxylation as a signaling mechanism and to look for targets other than HIF. "The role of reactive oxygen species in the regulation of HIF-1a expression remains enigmatic," says Semenza. "The complexity of the system is considerable, as might be expected for a protein that plays a critical role in many developmental and physiological processes."

Eugene Russo ( is a freelance writer in Takoma Park, Md. 

1. P. Jaakkola et al., "Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation," Science, 292:468-72, April 20, 2001. (Cited in 251 papers)

2. M. Ivan et al., "HIF alpha targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing," Science, 292:464-8, April 20, 2001. (Cited in 246 papers)

3. J. Perkel, "Seeking a cellular oxygen sensor," The Scientist, 15[10]:15, May 14, 2001.

4. F. Latif et al., "Identification of the von Hippel-Lindau disease tumor-suppressor gene," Science, 260:1317-20, 1993.

5. O. Iliopoulos et al., "Negative regulation of hypoxia-inducible genes by the von Hippel Lindau protein," Proc Natl Acad Sci, 93:10595-9, 1996.

6. P.H. Maxwell et al., "The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis," Nature, 399:271-5, 1999.

7. M. Ohh et al., "Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein," Nat Cell Biol, 2:423-7, 2000.

8. T. Kamura et al., "Activation of HIF1 alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex," Proc Natl Acad Sci, 97:10430-5, 2000.

9. K. Tanimoto et al., "Mechanism of regulation of the hypoxia-inducible factor-l alpha by the von Hippel- Lindau tumor suppressor protein," EMBO J, 19:4298-309, 2000.

10. A.C.R. Epstein et al., "C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation," Cell, 107:43-54, 2001.

11. R.K. Bruick and S.L. McKnight, "A conserved family of prolyl-4-hydroxylases that modify HIF," Science, 294:1337-40, 2001.

12. K. Kondo et al., "Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein," Cancer Cell, 1:237-46, 2002.

13. D. Lando et al., "Asparagine hydroxylation of the HIF transactivation domain: A hypoxic switch," Science, 295:858-61, 2002.

14. B.A. Maher, "Hypoxic response takes shape," The Scientist, 16[14]:40, July 8, 2002.