Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.

Hypoxia inducible factor 1-alpha (HIF-1 alpha) is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

Acute Vhl gene inactivation induces cardiac HIF-dependent erythropoietin gene expression.

Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquitous tamoxifen-inducible recombinase Cre-ER(T2). Here, we characterize a widespread reduction in Vhl gene expression in Vhl(floxed)-UBC-Cre-ER(T2) adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O(2) tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER(T2) mouse lines, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice.

Mitochondrial reprogramming through cardiac oxygen sensors in ischaemic heart disease.

Under hypoxic conditions, mitochondria can represent a threat to the cell because of their capacity to generate toxic reactive oxygen species (ROS). However, cardiomyocytes are equipped with an oxygen-sensing pathway that involves prolyl hydroxylase oxygen sensors and hypoxia-inducible factors (HIFs), which induces a tightly regulated programme to keep ischaemic mitochondrial activity under control. The aim of this review is to provide an update on the pathways leading to mitochondrial reprogramming, which occurs in the myocardium during ischaemia, with particular emphasis on those induced by HIF activation. We start by studying the mechanisms of mitochondrial damage during ischaemia and upon reperfusion, highlighting the importance of the formation of the mitochondrial permeability transition pore during reperfusion and its consequences for cardiomyocyte survival. Next, we analyse hypoxia-induced metabolic reprogramming through HIF and its important consequences for mitochondrial bioenergetics, as well as the phenomenon known as the hibernating myocardium. Subsequently, we examine the mechanisms underlying ischaemic preconditioning, focusing, in particular, on those that involve the HIF pathway, such as adenosine signalling, sub-lethal ROS generation, and nitric oxide production. Finally, the role of the mitochondrial uncoupling proteins in ischaemia tolerance is discussed.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1alpha degradation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) accumulate HIF-1alpha in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ(2) induced an over-accumulation of HIF-1alpha in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1alpha degradation as a target for 15d-PGJ(2) based on: (1) HIF-1alpha colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ(2) inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1alpha in 15d-PGJ(2)-treated cells. Therefore, expression of HIF-1alpha is also modulated by lysosomal degradation.

Macrophage oxygen sensing modulates antigen presentation and phagocytic functions involving IFN-gamma production through the HIF-1 alpha transcription factor.

Low oxygen tension areas are found in inflamed or diseased tissues where hypoxic cells induce survival pathways by regulating the hypoxia-inducible transcription factor (HIF). Macrophages are essential regulators of inflammation and, therefore, we have analyzed their response to hypoxia. Murine peritoneal elicited macrophages cultured under hypoxia produced higher levels of IFN-gamma and IL-12 mRNA and protein than those cultured under normoxia. A similar IFN-gamma increment was obtained with in vivo models using macrophages from mice exposed to atmospheric hypoxia. Our studies showed that IFN-gamma induction was mediated through HIF-1alpha binding to its promoter on a new functional hypoxia response element. The requirement of HIF-alpha in the IFN-gamma induction was confirmed in RAW264.7 cells, where HIF-1alpha was knocked down, as well as in resident HIF-1alpha null macrophages. Moreover, Ag presentation capacity was enhanced in hypoxia through the up-regulation of costimulatory and Ag-presenting receptor expression. Hypoxic macrophages generated productive immune synapses with CD8 T cells that were more efficient for activation of TCR/CD3epsilon, CD3zeta and linker for activation of T cell phosphorylation, and T cell cytokine production. In addition, hypoxic macrophages bound opsonized particles with a higher efficiency, increasing their phagocytic uptake, through the up-regulated expression of phagocytic receptors. These hypoxia-increased immune responses were markedly reduced in HIF-1alpha- and in IFN-gamma-silenced macrophages, indicating a link between HIF-1alpha and IFN-gamma in the functional responses of macrophages to hypoxia. Our data underscore an important role of hypoxia in the activation of macrophage cytokine production, Ag-presenting activity, and phagocytic activity due to an HIF-1alpha-mediated increase in IFN-gamma levels.

Inadequate activation of the GTPase RhoA contributes to the lack of fibronectin matrix assembly in von Hippel-Lindau protein-defective renal cancer cells.

The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL+ cells. We have previously reported that VHL(-) cells fail to form beta1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.

A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery.

BACKGROUND: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest. RESULTS: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIFalpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFalpha peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system. CONCLUSION: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.

Accumulation of hypoxia-inducible factor-1alpha through a novel electrophilic, thiol antioxidant-sensitive mechanism.

15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a peroxisome-activated proliferator receptor-gamma (PPARgamma) agonist which contains an alpha,beta-unsaturated electrophilic ketone involved in nucleophilic addition reactions to thiols. Here we studied its effect on hypoxia-inducible factor-1alpha (HIF-1alpha) in human proximal tubular cells HK-2. 15d-PGJ(2) induced stabilization of HIF-1alpha protein, without affecting HIF-1alpha mRNA levels or proteasome activity, leading to its nuclear accumulation and activation of HIF-induced transcription. Accumulation of HIF-1alpha was unaffected by selective PPARgamma blockade nor mimicked by the PPARgamma agonists ciglitazone and 9,10-dihydro-15d-PGJ(2). N-acetylcysteine, reduced glutathione (GSH) or dithiothreitol (i.e. agents that act as thiol reducing agents and/or increase the GSH content), but not reactive oxygen species (ROS) scavengers, prevented 15d-PGJ(2)-induced HIF-1alpha accumulation whereas the inhibitor of GSH synthesis buthionine sulfoximine cooperated with 15d-PGJ(2) to accumulate HIF-1alpha. Finally, HIF-1alpha expression was increased by the electrophilic alpha,beta-unsaturated compounds acrolein and PGA(2), but not by 9,10-dihydro-15d-PGJ(2), which lacks the electrophilic cyclopentenone moiety. Taken together, these results point out to a new mechanism to increase pharmacologically the cell levels of HIF-1alpha through the electrophilic reaction of alpha,beta-unsaturated ketones with thiol groups.

von Hippel-Lindau tumor suppressor protein regulates the assembly of intercellular junctions in renal cancer cells through hypoxia-inducible factor-independent mechanisms.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.

Activation of HIF-prolyl hydroxylases by R59949, an inhibitor of the diacylglycerol kinase.

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

Role of iron (II)-2-oxoglutarate-dependent dioxygenases in the generation of hypoxia-induced phosphatidic acid through HIF-1/2 and von Hippel-Lindau-independent mechanisms.

Hypoxia-inducible factors (HIF-1/HIF-2) govern the expression of critical genes for cellular adaptation to low oxygen tensions. We have previously reported that the intracellular level of phosphatidic acid (PA) rises in response to hypoxia (1% O(2)). In this report, we have explored whether components of the canonical HIF/von Hippel-Lindau (VHL) pathway are involved in the induction of PA. We found that hypoxia induces PA in a cell line constitutively expressing a stable version of HIF-1alpha. PA induction was also found in HIF-1alpha- and 2alpha-negative CHO Ka13 cells, as well as in HIF-beta-negative HepaC4 cells. These data indicate that HIF activity is neither sufficient nor necessary for oxygen-dependent PA accumulation. PA generation was also detected in cells deficient for the tumor suppressor VHL, indicating that the presence of VHL was not required for the induction of PA. Here we show that PA accumulation also occurs at moderate hypoxia (5% O(2)), although to a lesser extent to that seen at 1% O(2), revealing that PA is induced at the same hypoxia range required to activate HIF-1. Prolyl hydroxylases (PHD) and asparaginyl hydroxylase (FIH) belong to the iron (II) and 2-oxoglutarate-dependent dioxygenase family and have been proposed as oxygen sensors involved in the regulation of HIFs. Chemical inhibition of these activities by treatment with iron chelators or 2-oxoglutarate analogs also results in a marked PA accumulation similar to that observed in hypoxia. Together these data show that PA accumulation in response to hypoxia is both HIF-1/2- and VHL-independent and indicate a role of iron (II)-2-oxoglutarate-dependent dioxygenases in the oxygen-sensing mechanisms involved in hypoxia-driven phospholipid regulation.

Specific oncolytic effect of a new hypoxia-inducible factor-dependent replicative adenovirus on von Hippel-Lindau-defective renal cell carcinomas.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are responsible for a hereditary cancer syndrome characterized by high susceptibility to hemangioblastomas of the retina and central nervous system, pheochromocytomas, and renal cell carcinomas. In agreement with its role as a tumor suppressor, the vast majority of spontaneous clear cell carcinomas of the kidney present loss of heterozygosity at the VHL locus. Recently, it has been shown that VHL works as the substrate recognition component of an E3 ubiquitination complex that targets the hypoxia-inducible factor (HIF) for proteosomal degradation. Under normal oxygen tension, the half-life of HIF transcription factors is extremely short because of its high degradation rate by the proteasome, resulting in undetectable HIF activity in normal cells. However, in VHL-deficient tumor cells, the HIF transcriptional pathway is constitutively activated because of impaired ubiquitination of this transcription factor. To target VHL-deficient tumors, we have exploited this feature to develop a conditionally replicative adenovirus (Ad9xHRE1A), the replication of which is HIF dependent. In this new oncolytic adenovirus, the expression of the E1A gene is controlled by an optimized minimal promoter containing HIF recognition elements. Here, we show that the induction of the E1A gene, as well as the viral replication and cytolytic effect of Ad9xHRE1A, are dependent on HIF activity. As a consequence, this virus efficiently kills VHL-deficient cells both in vitro and in vivo, as well as cells growing under hypoxic conditions. These data suggest that Ad9xHRE1A could be used as a highly specific therapy for VHL-deficient cancers and probably many other tumors that show extensive hypoxic areas or increased HIF activity by genetic alterations other than VHL loss.

The von Hippel Lindau/hypoxia-inducible factor (HIF) pathway regulates the transcription of the HIF-proline hydroxylase genes in response to low oxygen.

Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIFalpha proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIFalpha is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalyze HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIFalpha escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIFalpha in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIFalpha resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIFalpha.

Down-regulation of hypoxia-inducible factor-2 in PC12 cells by nerve growth factor stimulation.

Cellular responses to low oxygen tension are mediated, at least in part, by the activation of the hypoxia-inducible factors (HIFs). In the presence of oxygen, specific HIF residues become hydroxylated by the action of a recently described group of dioxygenases. These post-translational modifications target HIF for proteosomal degradation and prevent its transcriptional activity. Despite these detailed studies, little is known about the regulation of HIF by stimuli other than hypoxia. Here we report that, in rat pheochromocytoma PC12 cells, nerve growth factor (NGF) stimulation results in a decrease of both basal and hypoxia-induced levels of HIF-2 alpha protein. NGF treatment did not increase HIF-hydroxylase gene expression or activity, and the reduction of the HIF-2 alpha protein level upon stimulation was observed even in the presence of HIF-hydroxylase inhibitors such as deferoxamine or dimethyloxoglutarate. Thus, in contrast to the response to hypoxia, the effect of NGF on HIF-2 alpha protein levels is not mediated by the HIF hydroxilases. Quantitative real time (RT)-PCR showed that NGF stimulation results in a decrease of the HIF-2 alpha mRNA level similar to that found at the protein level. Interestingly, NGF effect was specific for HIF-2 alpha mRNA because it did not affect HIF-1 alpha mRNA levels. NGF treatment reduced HIF-2 alpha mRNA levels even in the presence of actinomycin D, suggesting an effect on mRNA stability. Finally, the effect of NGF on HIF2 alpha correlates with reduction of both basal and hypoxia-induced vascular endothelial growth factor mRNA levels. Reporter assays suggest that the reduced expression of hypoxia-inducible genes upon NGF treatment is related, at least in part, to the reduction of HIF-2 alpha protein. Hence, in PC12 cells the level of HIF-2 alpha protein and its effect on gene expression can be down-regulated by stimuli other than oxygen.

Role of the von Hippel-Lindau tumor suppressor gene in the formation of beta1-integrin fibrillar adhesions.

The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHLcancer syndrome and in most sporadic renal cancers. VHL is requiredfor the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exogenous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL- cells and their wild-type VHL stably transfected counterparts. The levels of beta1 and alphav integrins were increased in VHL- cells when compared with VHL+ transfectants. Early after plating, both VHL+ and VHL- cells were capable of assembling classic "patch-like" alphav focal contacts. As the culture advanced and cells became confluent, alphav integrins partly relocated to the intercellular junctions in VHL+ transfectants, which then developed large beta1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL- cells were unable to assemble beta1 fibrillar adhesions, and alphav focal contacts remained unchanged at all stages of the culture. Exogenous activation of beta1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL- cells to assemble beta1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common beta1-integrin chain was delayed in VHL- cells when compared with VHL+ cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of beta1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL- renal cancer cells.

Lack of evidence for the involvement of the phosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension.

Hypoxia-inducible factors (HIF) belong to an evolutionary conserved family of transcription factors, the activity of which is tightly regulated by oxygen levels. We have recently demonstrated that hypoxia activates the phosphoinositide 3-kinase (PI3K)/Akt pathway in some cell types, and other works have suggested that this pathway is involved in the activation of HIF. In the present work we studied the role of this pathway in the induction of HIF by hypoxia. Under hypoxic conditions the PI3K/Akt pathway was activated in some (PC12 and HeLa) but not all cell types (HepG2) tested, whereas the HIF protein was induced by hypoxia in all cases. Kinetics analysis showed that, when observed, the activation of PI3K/Akt occurred after HIF induction. In addition, the chemical inhibition of PI3K had no significant effect on the induction of the HIF protein or its transcriptional activity but prevented Akt activation. Accordingly, transient overexpression of a dominant negative form of the regulatory subunit of PI3K in HEK293T cells did not interfere with the induction of the HIF-alpha protein by hypoxia or affect HIF-mediated transcription in any of the cell types tested. Moreover, forced activation of the PI3K/Akt pathway did not affect the transcriptional activity of HIF under normoxic or hypoxic conditions. Thus, our data suggest that the activation of PI3K/Akt by hypoxia is cell type-specific and, when observed, lies downstream of HIF activation or in a parallel pathway. Furthermore, the activity of the PI3K/Akt is not sufficient for the activation of HIF nor is it essential for its induction by hypoxia.

c-Jun and hypoxia-inducible factor 1 functionally cooperate in hypoxia-induced gene transcription.

Under low-oxygen conditions, cells develop an adaptive program that leads to the induction of several genes, which are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1). On the other hand, there are other factors which modulate the HIF-1-mediated induction of some genes by binding to cis-acting motifs present in their promoters. Here, we show that c-Jun functionally cooperates with HIF-1 transcriptional activity in different cell types. Interestingly, a dominant-negative mutant of c-Jun which lacks its transactivation domain partially inhibits HIF-1-mediated transcription. This cooperative effect is not due to an increase in the nuclear amount of the HIF-1alpha subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1alpha are able to associate in vivo but not in vitro, suggesting that this interaction involves the participation of additional proteins and/or a posttranslational modification of these factors. In this context, hypoxia induces phosphorylation of c-Jun at Ser(63) in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser(63) and Ser(73) impair its functional cooperation with HIF-1. The functional interplay between c-Jun and HIF-1 provides a novel insight into the regulation of some genes, such as the one for VEGF, which is a key regulator of tumor angiogenesis.