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.

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.

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-␥ and IL-12 mRNA and protein than those cultured under normoxia. A similar IFN-␥ increment was obtained with in vivo models using macrophages from mice exposed to atmospheric hypoxia. Our studies showed that IFN-␥ induction was mediated through HIF-1␣ binding to its promoter on a new functional hypoxia response element. The requirement of HIF-␣ in the IFN-␥ induction was confirmed in RAW264.7 cells, where HIF-1␣ was knocked down, as well as in resident HIF-1␣ 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/CD3, CD3 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 upregulated expression of phagocytic receptors. These hypoxia-increased immune responses were markedly reduced in HIF-1␣-and in IFN-␥-silenced macrophages, indicating a link between HIF-1␣ and IFN-␥ 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-1␣-mediated increase in IFN-␥ levels.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output.

Hypoxia-inducible factors (HIF) are heterodimeric (␣/␤) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-␣ 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-␣ for degradation. In the absence of oxygen, HIF-␣ hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-␣ 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-1/2␣ protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-␣ in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-␣ was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O 2 as well as at 1% O 2 . 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.

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Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension.We found that the changes in transcription rates induced by hypoxia are the major determinant of RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 hours, to 5.7 hours in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect was mimicked by forced activation of the Hypoxia Inducible Factor pathway and prevented by its interference. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter..