Hypoxia Response Elements in the Aldolase A, Enolase 1, and Lactate Dehydrogenase A Gene Promoters Contain Essential Binding Sites for Hypoxia-inducible Factor 1

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3' and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1

Macrophage Migration Inhibitory Factor Activates Hypoxia-Inducible Factor in a p53-Dependent Manner

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Collateral circulation: Past and present

Following an arterial occlusion outward remodeling of pre-existent inter-connecting arterioles occurs by proliferation of vascular smooth muscle and endothelial cells. This is initiated by deformation of the endothelial cells through increased pulsatile fluid shear stress (FSS) caused by the steep pressure gradient between the high pre-occlusive and the very low post-occlusive pressure regions that are interconnected by collateral vessels. Shear stress leads to the activation and expression of all NOS isoforms and NO production, followed by endothelial VEGF secretion, which induces MCP-1 synthesis in endothelium and in the smooth muscle of the media. This leads to attraction and activation of monocytes and T-cells into the adventitial space (peripheral collateral vessels) or attachment of these cells to the endothelium (coronary collaterals). Mononuclear cells produce proteases and growth factors to digest the extra-cellular scaffold and allow motility and provide space for the new cells. They also produce NO from iNOS, which is essential for arteriogenesis. The bulk of new tissue production is carried by the smooth muscles of the media, which transform their phenotype from a contractile into a synthetic and proliferative one. Important roles are played by actin binding proteins like ABRA, cofilin, and thymosin beta 4 which determine actin polymerization and maturation. Integrins and connexins are markedly up-regulated. A key role in this concerted action which leads to a 2-to-20 fold increase in vascular diameter, depending on species size (mouse versus human) are the transcription factors AP-1, egr-1, carp, ets, by the Rho pathway and by the Mitogen Activated Kinases ERK-1 and -2. In spite of the enormous increase in tissue mass (up to 50-fold) the degree of functional restoration of blood flow capacity is incomplete and ends at 30% of maximal conductance (coronary) and 40% in the vascular periphery. The process of arteriogenesis can be drastically stimulated by increases in FSS (arterio-venous fistulas) and can be completely blocked by inhibition of NO production, by pharmacological blockade of VEGF-A and by the inhibition of the Rho-pathway. Pharmacological stimulation of arteriogenesis, important for the treatment of arterial occlusive diseases, seems feasible with NO donors

Molecular Genetic Analysis of the Silent Carrier of Beta Thalassemia

The silent carrier of (beta) thalassemia has a decreased (beta)/(alpha) globin synthesis ratio, but normal Hb A(,2) and Hb F levels and erythrocytic indices. A molecular genetic analysis has been performed using as subjects an Albanian family in which the father is a silent carrier, the mother has high Hb A(,2) (beta)-thalassemia trait, and both children have (beta) thalassemia. The relative excess (alpha)-globin in this family is not due to an increase in (alpha)-globin gene number. The maternal and paternal (beta)-globin genes were cloned from the daughter\u27s genomic DNA and characterized. The maternal gene contains a splice junction mutation in IVS-1 which results in (beta)(\u270) thalassemia. Nucleotide sequence analysis of the paternal gene failed to reveal any base changes of functional significance. When introduced into HeLa cells the gene was expressed at normal levels with proper processing of RNA. Haplotype analysis revealed that the affected son and daughter inherited different (epsilon)(gamma)(delta)(beta)-globin gene clusters from the father. However, the father is homozygous for all polymorphic restriction sites downstream from a Taq I site approximately 3 kb 5\u27 to the (delta)-globin gene, suggesting that either recombination within primordial germ cells of the father had occurred downstream from the Taq I site or the paternal silent carrier allele is not linked to the (beta)-globin gene cluster. In either case, structural and functional analyses demonstrate that the silent carrier allele is not due to a mutation within the (beta)-globin structural gene or its immediate flanking regions and as such represents a novel form of (beta)(\u27+) thalassemia. In addition, during the course of these studies a novel Rsa I polymorphism was identified approximately 550 base pairs 5\u27 to the (beta)-globin cap site. Population screening demonstrated the presence of this site in DNA from individuals of northern European, Mediterranean, Middle Eastern, African, Southeast Asian and Asian Indian descent, who also carried (beta)A, (beta)E, (beta)S, and (beta)-thalassemia alleles at the (beta)-globin locus