Patrick H. Maxwell

Contrasting Properties of Hypoxia-Inducible Factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-Associated Renal Cell Carcinoma

ABSTRACT Defective function of the von Hippel-Lindau (VHL) tumor suppressor ablates proteolytic regulation of hypoxia-inducible factor α subunits (HIF-1α and HIF-2α), leading to constitutive activation of hypoxia pathways in renal cell carcinoma (RCC). Here we report a comparative analysis of the functions of HIF-1α and HIF-2α in RCC and non-RCC cells. We demonstrate common patterns of HIF-α isoform transcriptional selectivity in VHL-defective RCC that show consistent and striking differences from patterns in other cell types. We also show that HIF-α isoforms display unexpected suppressive interactions in RCC cells, with enhanced expression of HIF-2α suppressing HIF-1α 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-2α and that the proapoptotic gene encoding BNip3 responds positively to HIF-1α and negatively to HIF-2α, indicating that HIF-1α and HIF-2α have contrasting properties in the biology of RCC. In keeping with this, HIF-α isoform-specific transcriptional selectivity was matched by differential effects on the growth of RCC as tumor xenografts, with HIF-1α retarding and HIF-2α 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.

Structural basis for the recognition of hydroxyproline in HIF-1|[alpha]| by pVHL

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1α is the oxygen-regulated subunit of HIF-1, an αβ heterodimeric complex. HIF-1α is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel–Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1α by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1α. The crystal structure of a hydroxylated HIF-1α peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

Expression of Hypoxia-Inducible Factor-1α and -2α in Hypoxic and Ischemic Rat Kidneys

Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes. A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis. In vitro, the oxygen-regulated subunits HIF-1alpha and -2alpha are expressed in inverse relationship to oxygen tensions in every cell line investigated to date. The characteristics and functional significance of the HIF response in vivo are largely unknown. High-amplification immunohistochemical analyses were used to study the expression of HIF-1alpha and -2alpha in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (0.1% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia). These treatments led to marked nuclear accumulation of HIF-1alpha and -2alpha in different renal cell populations. HIF-1alpha was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli. Staining for HIF-1alpha colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1. HIF-2alpha was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts. The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1alpha and HIF-2alpha is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles. The expression of HIF-2alpha in peritubular fibroblasts suggests a role in erythropoietin regulation.

Regulation of E-cadherin Expression by VHL and Hypoxia-Inducible Factor

Mutations in von Hippel-Lindau tumor suppressor gene (VHL) underlie the VHL hereditary cancer syndrome and also occur in most sporadic clear cell renal cell cancers (CCRCC). Currently, the mechanism(s) by which VHL loss of function promotes tumor development in the kidney are not fully elucidated. Here, we show that VHL inactivation in precancerous lesions in kidneys from patients with VHL disease correlates with marked down-regulation of the intercellular adhesion molecule E-cadherin. Moreover, in VHL-defective cell lines (RCC4 and RCC10) derived from sporadic CCRCC, reexpression of VHL was found to restore E-cadherin expression. The product of the VHL gene has multiple reported functions, the best characterized of which is its role as the recognition component of an ubiquitin E3 ligase complex responsible for mediating oxygen-dependent destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits. We show that HIF activation is necessary and sufficient to suppress E-cadherin in renal cancer cells. Given the fundamental role of E-cadherin in controlling epithelial behavior, our findings give insight into how VHL inactivation/HIF activation may lead to kidney cancer and also indicate a mechanism by which reduced oxygenation could alter E-cadherin expression in other cancers and influence normal homeostasis in other epithelia.

Heterozygous deficiency of hypoxia-inducible factor–2α protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia

Chronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2alpha is a homologue of HIF-1alpha and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2alpha-deficient (Hif2alpha(+/-)) mice after exposure to 10% O(2) for 4 weeks. In contrast to WT mice, Hif2alpha(+/-) mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2alpha in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2alpha(+/-) mice after hypoxia, suggesting that HIF-2alpha-mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling.

The macrophage – a novel system to deliver gene therapy to pathological hypoxia

The use of activated macrophages in the treatment of cancer has been largely ineffectual. By ‘arming’ these cells with the ability to express a therapeutic gene we demonstrate significant advances in the efficacy of this approach. We have used a hypoxia-regulated adenoviral vector to transduce human macrophages with either a reporter or a therapeutic gene encoding human cytochrome P4502B6 (CYP2B6). Infiltration of transduced macrophages into a tumour spheroid results in induction of gene expression. We demonstrate significant tumour cell killing only in the presence of cyclophosphamide via activation by P4502B6 and show that this can be further targeted to tumours through hypoxia regulated gene expression.

Inhibition of Hypoxia Inducible Factor Hydroxylases Protects Against Renal Ischemia-Reperfusion Injury

Acute renal failure resulting from hypoperfusion and hypoxia is a significant clinical problem. Hypoxia activates the heterodimeric transcription factor hypoxia inducible factor (HIF), leading to changes in gene expression that promote tissue adaptation and survival. To determine whether HIF may protect the kidney from ischemia-reperfusion injury, we subjected hif1a(+/-) and hif2a(+/-) mice to renal ischemia-reperfusion injury. Injury was substantially more severe in hif(+/-) than in littermate controls, consistent with a protective role for HIF. Because wild-type mice exhibited submaximal HIF accumulation in response to no-flow ischemia, we tested compounds that might augment the protective HIF response following ischemia-reperfusion in these animals. We found that l-mimosine and dimethyloxalylglycine, two small molecules that activate HIF by inhibiting HIF hydroxylases, protected mouse kidneys from ischemia-reperfusion injury. Therefore, pharmacological activation of HIF may offer an effective strategy to protect the kidney from ischemic injury.

Xenon Preconditioning Protects against Renal Ischemic-Reperfusion Injury via HIF-1 alpha Activation

The mortality rate from acute kidney injury after major cardiovascular operations can be as high as 60%, and no therapies have been proved to prevent acute kidney injury in this setting. Here, we show that preconditioning with the anesthetic gas xenon activates hypoxia-inducible factor 1alpha (HIF-1alpha) and its downstream effectors erythropoietin and vascular endothelial growth factor in a time-dependent manner in the kidneys of adult mice. Xenon increased the efficiency of HIF-1alpha translation via modulation of the mammalian target of rapamycin pathway. In a model of renal ischemia-reperfusion injury, xenon provided morphologic and functional renoprotection; hydrodynamic injection of HIF-1alpha small interfering RNA demonstrated that this protection is HIF-1alpha dependent. These results suggest that xenon preconditioning is a natural inducer of HIF-1alpha and that administration of xenon before renal ischemia can prevent acute renal failure. If these data are confirmed in the clinical setting, then preconditioning with xenon may be beneficial before procedures that temporarily interrupt renal perfusion.

Abnormal sympathoadrenal development and systemic hypotension in PHD3–/– mice.

ABSTRACT 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-2α but not HIF-1α, supporting the nonredundant involvement of a PHD3-HIF-2α 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.

Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia.

A striking feature of Ewing sarcoma is the presence of blood lakes lined by tumor cells. The significance of these structures, if any, is unknown. Here, we report that the extent of blood lakes correlates with poor clinical outcomes, whereas variables of angiogenesis do not. We also show that Ewing sarcoma cells form vessel-like tubes in vitro and express genes associated with vasculogenic mimicry. In tumor models, we show that there is blood flow through the blood lakes, suggesting that these structures in Ewing sarcoma contribute to the circulation. Furthermore, we present evidence that reduced oxygen tension may be instrumental in tube formation by plastic tumor cells. The abundant presence of these vasculogenic structures, in contrast to other tumor types, makes Ewing sarcoma the ideal model system to study these phenomena. The results suggest that optimal tumor treatment may require targeting of these structures in combination with prevention of angiogenesis.