Intra- and inter-specific variation in metabolic gene expression in relationship to environmental hypoxia

In this study, we explored how environmental oxygen levels affect the metabolic phenotype of sympatric sunfish known to differ in their hypoxia tolerance. We examined bluegill (Lepomis macrochirus) and pumpkinseed (Lepomis gibbosus), two species commonly found in the same water bodies, though pumpkinseed are considered more hypoxia tolerant, and survive in hypoxic lakes that exclude bluegill. Freshly caught Lake Opinicon pumpkinseed possessed significantly higher glycolytic enzyme activities (PGI, ALD, GAPDH, ENO, and LDH) than bluegill, but after holding the fish in an oxygenated environment for 7days, pumpkinseed glycolytic enzymes (PGI, ALD, and LDH) and mRNA (LDHA and HIF1α) declined to bluegill's levels. When glycolytic enzymes and mRNA were compared in pumpkinseed populations from seven lakes, only Penyck Lake pumpkinseed had significantly elevated glycolytic enzyme activity that did not diminish with normoxic holding. The levels of mRNA for LDHA and HIF1α did not differ between lakes and did not change in response to normoxic holding in the Penyck Lake fish. Collectively, these studies on sunfish show that hypoxia tolerance contributes to ecological niche specialization between species, and provides an example of a population that has adapted chronically elevated glycolytic enzyme activity independent of current dissolved oxygen in the water.     

Induction of the glucose-6-phosphate dehydrogenase gene expression by chronic hypoxia in PC12 cells

We studied the regulation of glucose-6-phosphate dehydrogenase (G6PD) gene expression by chronic hypoxia. G6PD mRNA level and activity were increased in PC12 cells by hypoxia in a dose- and time-dependent manner. Cobalt chloride and dimethyloxalylglycine, which can mimic hypoxia, also activated G6PD gene expression. Interestingly, hypoxia-induced G6PD expression followed a time course much slower than that of phosphoglycerate kinase 1 (PGK1), a hypoxia-inducible factor (HIF)-dependent glycolytic enzyme. Hypoxic-G6PD induction was almost negligible in non-excitable Buffalo rat liver cells, although in these cells PGK1 was strongly upregulated by low PO(2). Furthermore, G6PD but not PGK1 induction was blocked by the antioxidants glutathione and N-acetylcysteine. These results suggest the dependence of G6PD gene expression on HIF and intracellular redox status and the differential hypoxic regulation of glucose-metabolizing enzymes.     

Effect of hypoxia on the expression of iron regulatory proteins 1 and the mechanisms involved

Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Iron regulatory proteins (IRPs, IRP1 and IRP2) are master regulators of cellular iron metabolism. Hypoxia triggers a broad range of gene responses that are primarily mediated by hypoxia-inducible factor-1 (HIF-1). In this study, we have shown that hypoxia could not only upregulate the expression of hypoxia inducible factor-1 but also downregulate the expression of IRP1. However, the molecular mechanisms that govern the IRP1 response to hypoxia are not known. Herein we suggested that HIF/HRE system was an essential link between IRP1 and hypoxia. The HRE of IRP1 5'-regulation regions could combine with HIF-1 in vitro. Dual-luciferase reporter assay showed that IRP1 was directly downregulated by HIF/HRE system.     

Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo

When oxygen becomes limiting, cells shift primarily to a glycolytic mode for generation of energy. A key regulator of glycolytic flux is fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric regulator of 6-phosphofructo-1-kinase (PFK-1). The levels of F-2,6-BP are maintained by a family of bifunctional enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB or PFK-2), which have both kinase and phosphatase activities. Each member of the enzyme family is characterized by their phosphatase:kinase activity ratio (K:B) and their tissue-specific expression. Previous work demonstrated that one of the PFK-2 isozyme genes, PFKFB-3, was induced by hypoxia through the hypoxia-inducible factor-1 (HIF-1) pathway. In this study we examined the basal and hypoxic expression of three members of this family in different organs of mice. Our findings indicate that all four isozymes (PFKFB-1-4) are responsive to hypoxia in vivo. However, their basal level of expression and hypoxia responsiveness varies in the different organs studied. Particularly, PFKFB-1 is highly expressed in liver, heart and skeletal muscle, with the highest response to hypoxia found in the testis. PFKFB-2 is mainly expressed in the lungs, brain and heart. However, the highest hypoxia responses are found only in liver and testis. PFKFB-3 has a variable low basal level of expression in all organs, except skeletal muscle, where it is highly expressed. Most importantly, its hypoxia responsiveness is the most ample of all three genes, being strongly induced in the lungs, liver, kidney, brain, heart and testis. Further studies showed that PFKFB-1 and PFKFB-2 were highly responsive to hypoxia mimics such as transition metals, iron chelators and inhibitors of HIF hydroxylases, suggesting that the hypoxia responsiveness of these genes is also regulated by HIF proteins. In summary, our data demonstrate that PFK-2 genes are responsive to hypoxia in vivo, indicating a physiological role in the adaptation of the organism to environmental or localized hypoxia/ischemia.     

Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferrioxamine

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.     

von Hippel-Lindau β-domain-luciferase fusion protein as a bioluminescent hydroxyproline sensor for a hypoxia-inducible factor prolyl hydroxylase assay

Hypoxia-inducible factor prolyl hydroxylases (HPHs) are responsible for hydroxylation of proline residues in hypoxia-inducible factor-α (HIF-α), resulting in von Hippel-Lindau (VHL)-mediated proteasome degradation of the hydroxylated proteins. Pharmacological inhibition of the enzyme leads to stabilization of HIF-α proteins and consequent activation of HIF, which provides therapeutic benefit for a variety of tissues undergoing ischemic stress. In an effort to develop a new assay for measuring HPH activity, we designed a fusion protein, VHL β-domain-luciferase. Recombinant fusion protein with a glutathione S-transferase (GST) tag was purified from Escherichia coli. GST-VHL β-domain-luciferase with C-terminal deletion (GVbL-CD) was obtained as a major product and found to have luciferase activity. In a GVbL-CD capture assay using HIF peptide-bound beads, at least a 13-fold increase in luciferase activity was elicited for HIF peptide with hydroxyproline compared with unhydroxylated HIF peptide. HPH inhibitory activities of known HPH inhibitors or HIF-1α inducers were assessed using this assay, whose results were in good agreement with those obtained from conventional methods. The competitive effect of 2-ketoglutarate on dimethyloxalylglycine-mediated HPH inhibition was assessed very well in the new assay. Taken together, the VHL β-domain protein with luciferase activity is of use for HPH activity assay.     

Nickel carcinogenesis: epigenetics and hypoxia signaling

Both water soluble and insoluble nickel compounds have been implicated in the etiology of human lung and nasal cancers. Water insoluble nickel compounds have been shown to enter cells by phagocytosis and are contained in cytoplasmic vacuoles, which are acidified thus accelerating the dissolution of soluble nickel from the particles. Using Newport Green, a dye that fluoresces when ionic nickel is bound, we have shown that following exposure (48-72 h) of human lung (A549) cells to NiS particles, most of the nickel is contained in the nucleus, while cells exposed to soluble NiCl2 exhibit most of the ions localized in the cytoplasm. This effect is consistent with previously published reports showing that short-term exposure of cells to crystalline nickel particles (1-3 days) is able to epigenetically silence target genes placed near heterochromatin, while similar short-term exposure to soluble nickel compounds are not able to induce silencing of genes placed near heterochromatin. However, a 3 week exposure of cells to soluble NiCl2 is also able to induce gene silencing. A similar effect was found in yeast cells where nickel was able to silence the URA-3 gene placed near (1.3 kb) a telomere silencing element, but not when the gene was placed farther away from the silencing element (2.0 kb). In addition to epigenetic effects, nickel compounds activate hypoxia signaling pathways. The mechanism of this effect involves the ability of either soluble or insoluble nickel compounds to block iron uptake leading to cellular iron depletion, directly affect iron containing enzymes, or both. This results in the inhibition of a variety of iron-dependent enzymes, such as aconitase and the HIF proline hydroxylases (PHD1-3). The inhibition of the HIF proline hydroxylases stabilizes the HIF protein and activates hypoxic signaling. Additional studies have shown that nickel and hypoxia decrease histone acetylation and increase the methylation of H3 lysine 9. These events are involved in gene silencing and hypoxia can also cause these effects in human cells. It is hypothesised that the state of hypoxia either by low oxygen tension or as a result of agents that signal hypoxia under normal oxygen tension (iron chelation, nickel and cobalt) results in low levels of acetyl CoA, which is a substrate for histone and other protein acetylation. This effect may in part be responsible for the gene silencing following nickel exposure and during hypoxia.