Prof Peter J Ratcliffe FRS
|Research Area:||Cell and Molecular Biology|
|Technology Exchange:||Cell sorting, Immunohistochemistry, Mass spectrometry, Transcript profiling and Transgenesis|
|Scientific Themes:||Physiology, Cellular & Molecular Biology|
|Keywords:||cancer, metabolism, epigenetics, cellular biology, angiogenesis and hypoxia|
My laboratory works on understanding the mechanisms by which cells sense and signal hypoxia (low oxygen levels). Oxygen is of fundamental importance for most living organisms, and the maintenance of oxygen homeostasis is a central physiological challenge for all large animals. Hypoxia is an important component of many human diseases including cancer, heart disease, stroke, vascular disease, and anaemia.
Working initially on regulation of the haematopoietic growth factor erythropoietin (which shows strong transcriptional upregulation by hypoxia), the laboratory discovered that the underlying oxygen sensitive signal pathway is widely operative in mammalian cells, extends to invertebrates, and mediates a range of other transcriptional responses including those regulating angiogenesis and metabolism. The laboratory went to define the oxygen sensing and signalling pathways that link the essential transcription factor, hypoxia inducible factor (HIF) to the availability of oxygen.
The laboratory discovered that these links involve an unprecedented mode of cell signalling involving post-translational hydroxylations at specific prolyl and asparaginyl residues within HIF that are catalysed by a series of non-haem Fe(II) enzymes belonging to the 2-oxoglutarate (2-OG) dependent dioxygenase superfamily. The obligate requirement for molecular oxygen in the reaction confers oxygen dependence, though emerging evidence suggests the enzymes integrate other signals generated by redox and metabolic stresses.
Together with its collaborators, the laboratory operates an extensive range of programmes exploring the extent, mechanisms and biological functions of these and related 2-OG oxygenases.
These programmes range across, protein science, structural biology and enzymology, through cell biology, systems physiology, epigenetics and cancer biology, to translational programmes in ischaemia therapeutics and integrative human physiology.
|Prof Christopher Schofield||Chemistry||Oxford University||UK|
|Peter Carmeliet||Vesalius Research Center, VIB, Leuven||Belgium|
|Prof Patrick Maxwell||School of Clinical Medicine||University of Cambridge||UK|
|Pablo Wappner||Instituto Leloir, Buenos Aires||Argentina|
Cell culture studies have implicated the oxygen-sensitive hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 in the regulation of neuronal apoptosis. To better understand this function in vivo, we have created PHD3(-/-) mice and analyzed the neuronal phenotype. Reduced apoptosis in superior cervical ganglion (SCG) neurons cultured from PHD3(-/-) mice is associated with an increase in the number of cells in the SCG, as well as in the adrenal medulla and carotid body. Genetic analysis by intercrossing PHD3(-/-) mice with HIF-1a(+/-) and HIF-2a(+/-) mice demonstrated an interaction with HIF-2alpha but not HIF-1alpha, supporting the nonredundant involvement of a PHD3-HIF-2alpha pathway in the regulation of sympathoadrenal development. Despite the increased number of cells, the sympathoadrenal system appeared hypofunctional in PHD3(-/-) mice, with reduced target tissue innervation, adrenal medullary secretory capacity, sympathoadrenal responses, and systemic blood pressure. These observations suggest that the role of PHD3 in sympathoadrenal development extends beyond simple control of cell survival and organ mass, with functional PHD3 being required for proper anatomical and physiological integrity of the system. Perturbation of this interface between developmental and adaptive signaling by hypoxic, metabolic, or other stresses could have important effects on key sympathoadrenal functions, such as blood pressure regulation. Hide abstract
The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling. Hide abstract
Molecular genetic analysis of hereditary leiomyomatosis and renal cell cancer (HLRCC) unexpectedly revealed germline defects in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH), stimulating great interest in the underlying mechanism of oncogenesis. It has been proposed that the associated accumulation of fumarate competitively inhibits the 2-oxoglutarate-dependent dioxygenases that regulate hypoxia-inducible factor (HIF), thus activating oncogenic hypoxia pathways. In this issue of Cancer Cell, Pollard and colleagues describe a genetic mouse model of FH deficiency that recapitulates aspects of the human disease, including HIF activation and renal cysts, enabling further insights into this unusual cancer syndrome. Hide abstract
Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IkappaB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IkappaBalpha were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes. Hide abstract
Defective function of the von Hippel-Lindau (VHL) tumor suppressor ablates proteolytic regulation of hypoxia-inducible factor alpha subunits (HIF-1alpha and HIF-2alpha), leading to constitutive activation of hypoxia pathways in renal cell carcinoma (RCC). Here we report a comparative analysis of the functions of HIF-1alpha and HIF-2alpha in RCC and non-RCC cells. We demonstrate common patterns of HIF-alpha isoform transcriptional selectivity in VHL-defective RCC that show consistent and striking differences from patterns in other cell types. We also show that HIF-alpha isoforms display unexpected suppressive interactions in RCC cells, with enhanced expression of HIF-2alpha suppressing HIF-1alpha and vice-versa. In VHL-defective RCC cells, we demonstrate that the protumorigenic genes encoding cyclin D1, transforming growth factor alpha, and vascular endothelial growth factor respond specifically to HIF-2alpha and that the proapoptotic gene encoding BNip3 responds positively to HIF-1alpha and negatively to HIF-2alpha, indicating that HIF-1alpha and HIF-2alpha have contrasting properties in the biology of RCC. In keeping with this, HIF-alpha isoform-specific transcriptional selectivity was matched by differential effects on the growth of RCC as tumor xenografts, with HIF-1alpha retarding and HIF-2alpha enhancing tumor growth. These findings indicate that therapeutic approaches to targeting of the HIF system, at least in this setting, will need to take account of HIF isoform-specific functions. Hide abstract
The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated alpha subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1-independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants (dpy-18, let-268, gon-1, mig-17, and unc-6), with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1-independent function of VHL-1 that is connected with extracellular matrix function. Hide abstract
HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF. Hide abstract
Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor. Hide abstract
Characterization of MINA function in normal physiology and cancer
MINA (Myc-Induced Nuclear Antigen) is an enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase family. Members of this family have been shown to catalyze novel post-translational modifications including hydroxylation and demethylation of proteins and nucleic acids (1). MINA was originally cloned as a direct transcriptional target of the proto-oncogene c-Myc (2), and as a consequence MINA is found to be overexpressed in a variety of human tumours (3-5). In several cases MINA ...
Differential regulation of the HIF-1 versus HIF-2 response in kidney cancer
BackgroundKidney cancer is increasing in incidence, with more than 8,500 cases / year in the UK. Without surgical cure, prognosis remains poor, despite recent advances in medical management. The most common form of kidney cancer is clear cell renal carcinoma (CCRC), the majority of which bear mutations in the von Hippel-Lindau tumour suppressor (pVHL). A major function of pVHL is regulation of the transcriptional response to oxygen through targeted degradation of hypoxia inducible factor (HIF). ...
Distinguishing oncogenic HIF pathways in Renal Cancer
BackgroundKidney cancer is increasing in incidence, with more than 8,500 cases / year in the UK. Without surgical cure, prognosis remains poor, despite recent advances in medical management. The most common form of kidney cancer is clear cell renal carcinoma (CCRC), the majority of which bear mutations in the von Hippel-Lindau tumour suppressor (pVHL). A major function of pVHL is regulation of the transcriptional response to oxygen through targeted degradation of hypoxia inducible factor (HIF) ...
Epigenetic reprogramming of the HIF transcriptional response in kidney cancer
BackgroundKidney cancer is increasing in incidence, with more than 8,500 cases / year in the UK. Without surgical cure, prognosis remains poor, despite recent advances in medical management. The most common form is clear cell renal carcinoma (CCRC), the majority of which bear mutations in the von Hippel-Lindau tumour suppressor (pVHL). A major function of pVHL is regulation of the transcriptional response to oxygen through targeted degradation of hypoxia inducible factor (HIF). Inactivation of ...
Investigating hydroxylases that target the protein synthesis apparatus and their potential as cancer targets
We have recently shown that protein hydroxylases related to the HIF asparaginyl hydroxylase (FIH) target ribosomal proteins and translation factors. The expression of these enzymes is induced by mitogenic stimuli, deregulated downstream of oncogene signalling, and increased in tumours, suggesting they may play a causal role in tumourigenesis. Consistent with this, deregulated protein synthesis is considered a hallmark of cancer, with activation of ribosomal biogenesis and translation initiation ...