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 oxidative phosphorylation system is one of the best-characterized metabolic pathways. In mammals, the protein components and X-ray structures are defined for all complexes except complex I. Here, we show that NDUFA4, formerly considered a constituent of NADH Dehydrogenase (CI), is instead a component of the cytochrome c oxidase (CIV). Deletion of NDUFA4 does not perturb CI. Rather, proteomic, genetic, evolutionary, and biochemical analyses reveal that NDUFA4 plays a role in CIV function and biogenesis. The change in the attribution of the NDUFA4 protein requires renaming of the gene and reconsideration of the structure of CIV. Furthermore, NDUFA4 should be considered a candidate gene for CIV rather than CI deficiencies in humans.

When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

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 is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3-4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.

In this report, we describe a novel activation antigen that appears very early after T cell activation and is absent in resting lymphocytes, through which agonistic proliferative signals can be triggered by mAb binding. It has been designated as activation inducer molecule (AIM) and is a disulphide-linked heterodimeric structure containing two polypeptide chains of Mr 33,000 and 27,000. The expression of AIM can be induced by different activation stimuli such as PMA, PHA, or anti-CD3 mAb, but not by the Ca2+ ionophore A23187, and it precedes the expression of other activation molecules such as 4F2 or the IL-2-R. Once AIM antigens are expressed on lymphocytes after stimulation with submitogenic doses of PMA, the binding of anti-AIM mAbs triggers a strong proliferative response. Furthermore, a comitogenic effect of the anti-AIM mAbs is exerted in the presence of either PHA or anti-CD3 mAb. The activation of lymphocytes through AIM antigens induces both IL-2 and IL-2-R receptor synthesis and is inhibited by anti-IL-2-R mAbs.

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.

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.