Manuel O. Landázuri

Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene.

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.

Induction of the Mitochondrial NDUFA4L2 Protein by HIF-1α Decreases Oxygen Consumption by Inhibiting Complex I Activity

The fine regulation of mitochondrial function has proved to be an essential metabolic adaptation to fluctuations in oxygen availability. During hypoxia, cells activate an anaerobic switch that favors glycolysis and attenuates the mitochondrial activity. This switch involves the hypoxia-inducible transcription factor-1 (HIF-1). We have identified a HIF-1 target gene, the mitochondrial NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2). Our results, obtained employing NDUFA4L2-silenced cells and NDUFA4L2 knockout murine embryonic fibroblasts, indicate that hypoxia-induced NDUFA4L2 attenuates mitochondrial oxygen consumption involving inhibition of Complex I activity, which limits the intracellular ROS production under low-oxygen conditions. Thus, reducing mitochondrial Complex I activity via NDUFA4L2 appears to be an essential element in the mitochondrial reprogramming induced by HIF-1.

Molecular Pathways: Hypoxia Response in Immune Cells Fighting or Promoting Cancer

Both malignant and stromal components in tumors are influenced by the physiologic conditions of the microenvironment. Hypoxia is a prominent feature of solid tumors as a result of defective vascularization and intense metabolic activity. The gene-expression control mechanisms that adapt tissues to hypoxia are exploited by tumors to promote angiogenesis and vasculogenesis. The functions of infiltrating immune cells (macrophages and lymphocytes) and other stromal components are also influenced by a limited O2 supply. Hypoxia-inducible factors (HIF) are the main molecular transcriptional mediators in the hypoxia response. The degradation and activity of HIF-1α and HIF-2α are tightly controlled by the fine-tuned action of oxygen-sensing prolyl and asparaginyl hydroxylase enzymes. Recent evidence indicates that hypoxia can modulate the differentiation and function of T lymphocytes and myeloid cells, skewing their cytokine-production profiles and modifying the expression of costimulatory receptors. This conceivably includes tumor-infiltrating lymphocytes. Hypoxia not only directly affects tumor-infiltrating leukocytes but also exerts effects on tumor cells and vascular cells that indirectly cause selective chemokine-mediated recruitment of suppressive and proangiogenic T-cell subsets. This review focuses on changes induced by hypoxia in immune cells infiltrating solid malignancies. Such changes may either promote or fight cancer, and thus are important for immunotherapy. Clin Cancer Res; 18(5); 1207–13. ©2011 AACR.

c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription

ABSTRACT 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-1α subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1α 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 Ser63 in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser63 and Ser73 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.

The chemokine SDF‐1α triggers a chemotactic response and induces cell polarization in human B lymphocytes

We studied the expression and possible functional role of chemokine receptors CXCR3, CXCR4 and CCR5 in normal human B lymphocytes. B cells from both peripheral blood and tonsils expressed high levels of CXCR4 but not the other chemokine receptors tested. CXCR4 ligand, stromal cell‐derived factor (SDF)‐1α, elicited a potent chemotactic response and induced a polarized motile phenotype in B cells, resulting in redistribution of the adhesion molecule ICAM‐3 to a posterior appendage of the cell, termed uropod, and of CXCR4 receptor to the leading edge of migrating B cells. Time‐lapse videomicroscopy studies revealed that SDF‐1α‐treated cells recruited additional bystander B cells through the uropod. SDF‐1α induced levels of cellular recruitment comparable to those elicited by polarization‐inducing anti‐ICAM‐3 monoclonal antibody, in an LFA‐1/ICAM‐1, −3‐dependent fashion. Moreover, this chemokine increased intracellular Ca2+ levels in B lymphocytes, and induced a rapid CXCR4 receptor down‐regulation on the cell surface membrane. These results provide new insight into the important biological role of SDF‐1α in physiological processes in which B cells participate, and suggest a key role for chemokines in normal B cell trafficking and recirculation.

The HIF-1α Hypoxia Response in Tumor-Infiltrating T Lymphocytes Induces Functional CD137 (4-1BB) for Immunotherapy

UNLABELLED The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by positron emission tomographic (PET) imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) of transplanted colon carcinomas, melanomas, and spontaneous breast adenocarcinomas are CD137 (4-1BB)-positive, as opposed to their counterparts in tumor-draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Importantly, hypoxia does not upregulate CD137 in hypoxia-inducible factor (HIF)-1α-knockout T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating and co-transferred HIF-1α-sufficient T lymphocytes. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies to the tumor tissue. As a result, low-dose intratumoral injections avoid liver inflammation, achieve antitumor systemic effects, and permit synergistic therapeutic effects with PD-L1/B7-H1 blockade. SIGNIFICANCE CD137 (4-1BB) is an important molecular target to augment antitumor immunity. Hypoxia in the tumor microenvironment as sensed by the HIF-1α system increases expression of CD137 on tumor-infiltrating lymphocytes that thereby become selectively responsive to the immunotherapeutic effects of anti-CD137 agonist monoclonal antibodies as those used in ongoing clinical trials.

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.

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.

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.

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.