A novel hypoxia-response element in the lactate dehydrogenase-B gene of the killifish Fundulus heteroclitus.

Previous studies have suggested that the lactate dehydrogenase-B gene (Ldh-B) of the Atlantic killifish, Fundulus heteroclitus, is a hypoxia-responsive gene. Here, we demonstrate that the F. heteroclitus Ldh-B promoter confers hypoxia-dependence upon reporter gene expression in transiently transfected mammalian (Hep3B) and fish (RTG-2 and RTH-149) cells in culture. Mutation and deletion analyses identified a putative hypoxia-response element (HRE) between 109 and 90 nucleotides upstream of the major start site. This HRE is characterized by the sequence 5'-GATGTG-3' spaced by 8 nucleotides from a perfect inverted repeat, and both sites are necessary for hypoxic induction of reporter gene expression in mammalian and fish cells. This HRE differs from the canonical sequence at one nucleotide position that is invariant among HREs from a wide range of hypoxia-sensitive genes. In fish cells, maximal induction of reporter gene expression driven by this HRE occurred at the lowest oxygen level tested (0.5%), took 48 h to 96 h, and was independent of glucose concentration (between 5.6 and 25 mM). Under all conditions tested, hypoxic induction of gene expression was lower in RTH-149 cells than in RTG-2, suggesting a potential defect in hypoxia signaling in RTH-149 cells. These results demonstrate that the F. heteroclitus Ldh-B promoter contains a novel HRE that is capable of driving reporter gene expression in a sequence-specific and oxygen-, time-, and cell line-dependent manner.

Erythropoietin, a hypoxia-regulated factor, elicits a pro-angiogenic program in human mesenchymal stem cells.

OBJECTIVE: The ability of erythropoietin (EPO) to elicit a pro-angiogenic effect on human mesenchymal stem cells (hMSC) was tested. hMSC are currently under study as therapeutic delivery agents that target tumor vessels. Hypoxia favors the differentiation of hMSC towards a pro-angiogenic program. However, the classical angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, are not fully capable of restoring this effect. The hypoxia-regulated factor, EPO, induces angiogenesis in endothelial cells. Here, EPO's pro-angiogenic effect on hMSC was analyzed. METHODS: hMSC were tested for EPO receptor expression by western blot, immunofluorescence, and flow cytometry assays. Downstream receptor signaling components JAK and STAT were measured by standard assays. Pro-angiogenesis effects mediated by EPO treatment of hMSC were measured by proliferation, cytokine, or pro-angiogenesis factor secretion, metalloprotease activation, migration, invasion, wound healing, and tubule formation assays. RESULTS: hMSC express the cognate EPO receptor and are capable of promoting angiogenesis following EPO treatment in all the angiogenesis assays tested. EPO-treated hMSC proliferate and secrete pro-angiogenesis factors more readily than untreated hMSC. EPO leads to increased hMSC chemotaxis, migration, and activation of matrix metalloprotease-2. This treatment causes greater recruitment of vessels as measured in an in vivo angiogenesis assay. CONCLUSION: EPO is capable of eliciting a pro-angiogenesis program in hMSC that instigates secretion of angiogenic factors and the subsequent recruitment of endothelium. This study defines a novel mechanism for tumor cell recruitment of blood vessels that is important to consider in the design of stem cell-based therapies.

Integrin-linked kinase: a hypoxia-induced anti-apoptotic factor exploited by cancer cells.

Based on cDNA microarray results, integrin-linked kinase (ILK) emerged as an interesting candidate in hypoxia-mediated survival mechanisms employed by cancer cells. This notion was confirmed here by the following observations: the 5' promoter region of the ilk gene contains hypoxia responsive elements (HRE) that bind hypoxia-inducible factor (HIF) transcription factor complexes and drive HRE-luciferase gene expression in reporter assays; ILK protein and kinase activity are induced following hypoxia; downstream targets of ILK signaling are induced following hypoxia treatment; inhibition of ILK leads to increased apoptosis; and HIF and ILK are co-localized within human cancer tissues. The identification of ILK as a player in hypoxia survival signaling employed by cancer cells further validates ILK as a unique target for cancer therapy.

NF-kappaB plays a key role in hypoxia-inducible factor-1-regulated erythropoietin gene expression.

OBJECTIVE: The aim of this study was to further define the signal transduction pathways leading to hypoxia-inducible factor-1 (HIF-1) erythropoietin (EPO) gene expression. MATERIALS AND METHODS: Human hepatocellular carcinoma cells (Hep3B) were exposed to hypoxia (1% oxygen) and examined for mRNA expression, as well as gene transactivation with RT-PCR and luciferase reporter gene assays, respectively. RESULTS: Treatment with LY294002 (a selective pharmacological inhibitor of phosphatidylinositol 3-kinase) significantly inhibited EPO protein and mRNA expression in Hep3B cells exposed to hypoxia for 24 hours, while treatment with PD098059 or SB203580 (selective pharmacological inhibitors of the MEK and p38 mitogen-activated protein kinase pathways, respectively) had no significant effects. The activity of AKT, a downstream target of PI3K, was increased by hypoxia and was also inhibited by LY294002. Genetic inhibition of AKT resulted in significant inhibition of NF-kappaB and HIF-1-mediated transactivation, as well as EPO gene expression, in response to hypoxia. Overexpression of constitutively active AKT resulted in increased NF-kappaB and HIF-1 transactivation. The selective inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), significantly blocked HIF-1 protein expression. Inhibition of NF-kappaB with a superrepressor dominant negative IkappaBalpha genetic construct also significantly blocked NF-kappaB and HIF-1 transactivation, as well as EPO gene expression. CONCLUSION: We propose a key role for NF-kappaB in EPO gene regulation in response to hypoxia.