uring the 1990s, several labs in the United States and United Kingdom investigated the von Hippel-Lindau tumor suppressor (pVHL). Many important findings developed, including the belief that HIF (hypoxia-inducible factor) pathway activation could be central to renal cancer development. Interfering with this pathway could help in treating renal cancer and central nervous system hemangioblastoma. Despite the gains, many questions remain.
VHL is a cancer syndrome characterized by highly vascularized tumors, such as hemangioblastomas and clear cell carcinomas. Those with von Hippel-Lindau disease inherit one mutated copy of the VHL gene in the germ line. Their cells still behave normally, but every so often the gene's second copy mutates in somatic cells, leading to disease.
In 1993, National Institutes of Health researchers successfully identified and cloned the VHL tumor-suppressor gene.1 Subsequent work at the Oklahoma Medical Research Foundation and the Baylor College of Medicine in Houston, Texas, discovered Rbx1, a RING-H2 finger protein. As reported in the first featured paper and an accompanying paper, researchers learned that Rbx1 is not only a component of the VHL complex, but an essential component of a class of E3 ubiquitin ligases known as SCF (Skp1/Cul1(Cdc53)/F-box protein) ligases. These include the Elongin C-like protein Skp1 and Cullin family member Cul1.2 Furthermore, as reported in the second featured paper, work at the Wellcome Trust Centre in Oxford, U.K., discovered the HIF pathway activation. The mysteries of the VHL gene were beginning to unravel.
"Everything is building on everything else," says Joan Conaway, Howard Hughes Medical Institute associate investigator and a member of the molecular and cell biology research program, Oklahoma Medical Research Foundation, Oklahoma City. "Subsequent work carried out by several labs ... as well as our own, has provided firm biochemical evidence that the VHL ubiquitin ligase is directly responsible for ubiquitylation of hypoxia-inducible transcription factors."
In 1993, NIH scientists Marston Linehan, chief of the urologic oncology branch, Bert Zbar, chief of the laboratory of immunology of the National Cancer Institute, and Michael Lerman, the lab's principal investigator, identified and cloned the tumor-suppressor gene VHL. Unfortunately, the gene's amino acid sequence provided few clues to the protein's function.3
In 1995, in collaboration with NCI director Richard Klausner and Linehan, the Conaway group discovered that the VHL protein physically interacts with Elongin B and C proteins, which they had previously isolated as regulatory subunits of RNA polymerase II elongation factor Elongin.4 Then, in 1998 the group, in collaboration with the lab of Bill Kaelin at the Dana-Farber Cancer Institute in Boston demonstrated that the Cullin family member protein Cul2 is also a component of the endogenous VHL complex.5
The Conaway group then isolated an endogenous VHL complex from rat liver and found that more than 90 percent of the detectable VHL protein could be separated in a multiprotein complex that included Cul2 and the Elongin B and C proteins, which had all been shown to be capable of interacting with the VHL protein.4-6
In addition, the Oklahoma medical researchers learned that the VHL complex included a novel RING-H2 finger protein they named Rbx1, for Ring box protein 1. In collaboration with the labs of Wade Harper and Steve Elledge at Baylor College of Medicine, the Conaway group demonstrated that Rbx1 is not only a component of the VHL complex, which previously was unrecognized, but learned it was an essential part of the SCF ligases as well.2
"This work cemented the connection between VHL and ubiquitin ligase," says Joan Conaway, the senior author of this first featured hot paper. "The structural similarities between components of the VHL complex and SCF ubiquitin ligases, together with our finding that Rbx1 is present in both types of complexes, suggested strongly that the VHL complex was also a ubiquitin ligase. In addition, the finding that the Rbx1 RING-H2 finger domain is essential for activation of ubiquitylation of substrates fits nicely into what is becoming a new paradigm--that proteins containing RING finger domains are likely candidates to be ubiquitin ligases."
At about the same time, as reported in the second featured hot paper, Patrick Maxwell's group from the Wellcome Trust Centre provided a tantalizing clue as to the function of VHL, showing that the interaction of pVHL with HIF-1 is necessary for the oxygen-dependent degradation of HIF-alpha subunits. HIF-1 is a transcription factor responsible for oxygen-dependent regulation of genes implicated in blood vessel branching (angiogenesis). Thus, these findings suggest, at least in part, why patients with VHL disease develop vascular tumors that are riddled with blood vessels.
Maxwell and group co-leaders Peter Ratcliffe and Chris Pugh had been studying oxygen sensing, which led to their interest in the link between pVHL and oxygen regulation of gene expression. Studying the VHL tumor-suppressor gene product, they found that in VHL-defective cells, HIF alpha subunits are constitutively stabilized and HIF-1 is activated. However, if wild-type VHL is reintroduced into these cells, oxygen-dependent regulation is restored. Interestingly pVHL and HIF-alpha subunits co-immunoprecipitate; thus pVHL is part of the hypoxic HIF-1 DNA-binding complex.
"We were surprised by the lack of redundancy in the system," says Maxwell, the study's lead author and a nephrologist and consultant physician at Wellcome. "pVHL appears to be necessary for regulation of HIF, and patterns of gene expression usually induced by hypoxia are phenocopied [replicated] by VHL loss."
Previously, the group's long-standing research into oxygen-regulated gene expression regulation fueled the discovery that the HIF system has important effects on gene expression, angiogenesis, and growth of solid tumors.7 Extrapolating from this previous work, they hypothesized that in certain cancers, selection could occur for mutations activating the HIF system. Since the tumors of VHL are so highly angiogenic, pVHL was an attractive candidate. Additionally, other research groups had shown that certain hypoxia-regulated genes usually were constitutively active in cells lacking the VHL gene product.8,9
These findings, says The Wellcome researchers, have advanced understanding of tumor suppressor functions, of events leading to cancer and von Hippel-Lindau disease, of angiogenesis control, of how mammalian cells sense changes in oxygenation, and of how protein destruction is regulated.
Despite the gains, much remains unclear about pVHL's role in the body. Maxwell lists some unknowns: Which events occur between loss of the second pVHL allele and renal carcinoma? What are the genotype-phenotype relationships in VHL disease? What is the mechanism by which pVHL loss predisposes to pheochromocytoma? Why is VHL disease tissue restricted, given the widespread operation of the HIF system? What is the role of other pVHL functions, such as fibronectin assembly regulation, and do these interact with the HIF system? Do other ubiquitin E3 ligases regulate HIF in other cells?
The Conaway group continues investigating the function of the VHL ubiquitin ligase complex. Current efforts are aimed at defining the repertoire of cellular proteins regulated by the complex. Also, the group is researching the many other Elongin BC-interacting proteins. "We have hints that there may be a larger family of Elongin BC-based ubiquitin ligases, and we are now attempting to identify them," she says.
Since publication of the Nature paper, the Wellcome group has conducted display experiments involving proteins captured by VHL in cells with blocked protein destruction and identified only HIF-1 alpha and HIF-2 alpha.10 These experiments suggest that the alpha subunits of HIF-1 might be the only proteins targeted by pVHL, Maxwell says.
The group also undertook gene expression array analysis of cells lacking or re-expressing pVHL.11 They found that the expression of many known HIF targets is modulated by VHL, says Maxwell. Additionally, the tests showed that VHL-influenced surface membrane carbonic anhydrases are HIF-regulated.12
Currently, the group is focusing on clarifying cellular oxygen-sensing mechanisms. "We have made a lot of progress with the parameters that influence recognition of HIF-alpha to VHL," Maxwell comments. A paper with further information on how oxygen influences HIF is scheduled for publication later this year.
1. F. Latif et al., "Identification of the Von Hippel-Lindau disease tumor-suppressor gene," Science, 260:1317-20, May 28, 1993.
2. D. Skowyra et al., "Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1," Science, 284:662-5, 1999.
3. W.M. Linehan et al., "Genetic basis of renal cell cancer," Important Advances in Oncology, [review]:47-70, 1993.
4. D.R. Duan et al., "Inhibition of transcription elongation by the VHL tumor suppressor protein," Science, 269:1402-6, 1995.
5. K.M. Lonergan, et al., "Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2," Molecular and Cellular Biology, 18:732-41, 1998.
6. A. Pause et al., "The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins," Proceedings of the National Academy of Sciences, 94:2156-61, 1997.
7. P.H. Maxwell et al., "Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth," Proceedings of the National Academy of Sciences, 94(15):8104-9, 1997.
8. J.R. Gnarra et al., "Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene," Proceedings of the National Academy of Sciences, 93(20):10589-94, 1996.
9. O. Iliopoulos et al., "Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein," Proceedings of the National Academy of Sciences, 93:10595-99, 1996.
10. M.E. Cockman et al., "Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein," Journal of Biological Chemistry, 275:25733-41, Aug 18, 2000.
11. C.C. Wykoff et al., "Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling," Oncogene, 19:6297-305, Dec 14, 2000.
12. C.C. Wykoff et al., "Hypoxia-inducible expression of tumor-associated carbonic anhydrases," Cancer Research, 60:7075-83, Dec. 15, 2000.