Julián Aragonés

Substrate Fate in Activated Macrophages: A Comparison between Innate, Classic, and Alternative Activation

Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli. In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-13C2]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-γ or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1α activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for Hif-1α, the switch of PFK2 isoenzymes still occurs in LPS/IFN-γ–activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1α-independent mechanisms.

Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease

Cells in the human body need oxygen to function and survive, and severe deprivation of oxygen, as occurs in ischaemic heart disease and stroke, is a major cause of mortality. Nevertheless, other organisms, such as the fossorial mole rat or diving seals, have acquired the ability to survive in conditions of limited oxygen supply. Hypoxia tolerance also allows the heart to survive chronic oxygen shortage, and ischaemic preconditioning protects tissues against lethal hypoxia. The recent discovery of a new family of oxygen sensors — including prolyl hydroxylase domain-containing proteins 1–3 (PHD1–3) — has yielded exciting novel insights into how cells sense oxygen and keep oxygen supply and consumption in balance. Advances in understanding of the role of these oxygen sensors in hypoxia tolerance, ischaemic preconditioning and inflammation are creating new opportunities for pharmacological interventions for ischaemic and inflammatory diseases.

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 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.

Activation of HIF-prolyl Hydroxylases by R59949, an Inhibitor of the Diacylglycerol Kinase

Hypoxia-inducible factors (HIF) are heterodimeric (α/β) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-α 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-α for degradation. In the absence of oxygen, HIF-α hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-α 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-1/2α protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-α in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-α 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.

EFNA3 long noncoding RNAs induced by hypoxia promote metastatic dissemination

The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.