H. Franklin Bunn

Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1α protein. Both HIF-1α and HIF-1β mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1α protein was barely detectable in normoxic cells, even when HIF-1α was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1β protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1α protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1α protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1α protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1α protein.

Further identification of the nature and linkage of the carbohydrate in hemoglobin A1c.

We have found that Hb A1c contains neutral sugars which are only partially hydrolyzed from the N-termini of β chains. In both normal and diabetic Hb A1c, 0.2–0.3 equivalents of hexose were released, composed primarily of glucose and mannose in a 3:1 ratio. When Hb A1c was reduced with 3HNa BH4 and then treated with periodic acid, most of the radioactivity was recovered as 3H-formic acid with much lesser amounts of 3H-formaldehyde. From these results, we propose that in the red cell, glucose binds to the α-amino position of hemoglobin β-chains (valine) in an aldimine (Schiff base) linkage. This aldimine can then partially rearrange in a reversible manner to form a ketoamine linkage which is stable to acid hydrolysis. This Amadori-type rearrangement accounts for the formation of mannose, the C-2 epimer of glucose, as well as the inability to demonstrate 3HNa BH4 reduction at the C-1 position.

The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel

Abstract A small-scale technique for isoelectric focusing in polyacrylamide gels was used to separate hemoglobins in human and animal hemolysates. Chromatographically purified hemoglobins were used as standards to identify components in human hemolysates. Our results indicate that this technique offers a significant advance for high resolution separations of hemoglobins and a means for characterizing hemoglobin variants. The technique is suitable for routine analysis of multiple samples and may also be adapted for small-scale preparative purposes.

Inhibition of Hypoxia-inducible Factor 1 Activation by Carbon Monoxide and Nitric Oxide IMPLICATIONS FOR OXYGEN SENSING AND SIGNALING

It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dose-dependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, ∼0.5). Parallel experiments were done with 100 μm sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced accumulation of HIF-1α protein. Moreover, both NO and CO specifically targeted the internal oxygen-dependent degradation domain of HIF-1α, and also repressed the C-terminal transactivation domain of HIF-1α. Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream.

Continuous Subcutaneous Administration of Deferoxamine in Patients with Iron Overload

Since deferoxamine B, when administered as a single daily intramuscular injection of 0.75 g, is unable to promote sufficient urinary iron excretion to achieve net negative iron balance in siderosis, we evaluated its administration as a constant infusion over 24 hours. We compared intravenous and subcutaneous routes in 24 siderotic patients who had excreted 420 to 630 mg (mean, 480 mg) of iron per month on intramuscular therapy. With the intravenous route urinary iron excretions increased to 570 to 3690 mg (mean, 1595 mg) per month. Constant subcutaneous delivery was 90 per cent as effective as intravenous administration on a dose-for-dose basis. Noteworthy net cumulative urinary iron excretions (urinary iron excretions minus transfused iron), often in excess of 1 g per month, have been maintained in all patients. Constant subcutaneous deferoxamine administration may prove to be an effective and practical means of eliminating large quantities of iron in siderosis.

Clinical Consequences of Acquired Transfusional Iron Overload in Adults

We assessed the clinical sequelae of transfusional iron overload in 15 nonthalassemic adults (40 to 71 years of age) with anemias requiring transfusions. Iron loading had been present for less than four years in 14 patients. The number of units of blood transfused ranged from 60 to 210 (mean, 120). Liver-biopsy specimens in 10 patients contained seven to 26 times the normal amount of iron and typically showed focal portal fibrosis. Left ventricular cardiac function was impaired in only the most heavily transfused patients or in those with coexisting coronary-artery disease, All patients had glucose intolerance associated with significantly reduced insulin output, compared with controls (P < 0.01). Pituitary reserve of ACTH was limited in 10 of 12 patients, and that of gonadotropin in five of 13. We conclude that widespread subclinical organ dysfunction can result from transfusional iron overload developing in adulthood. The pattern of organ involvement resembles that encountered in idiopathic hemochromatosis.

Differences in the Interaction of 2,3-Diphosphoglycerate with Certain Mammalian Hemoglobins

The hemoglobins of man, horse, dog, rabbit, guinea pig, and rat all have relatively high (nonphysiologic) oxygen affinity when stripped of organic phosphates, and a strong reactivity with 2,3-diphosphoglycerate (2,3-DPG). Appropriately, their red cells contain high levels of 2,3-DPG. In contrast, the sheep, goat, cow, and cat have low oxygen affinity hemoglobins which interact weakly with 2,3-DPG, and low concentrations of red cell 2,3-DPG. These hemoglobins have structural differences at the NH2-terminus of the β chain, a site where 2,3-DPG is thought to bind.

Hemoglobin function in stored blood

Inorganic phosphate which is known to stimulate red cell glycolysis is present in one of the preservatives for storing whole blood, citrate-phosphate-dextrose (CPD), but not the other, acid-citrate-dextrose (ACD). Both of these preservatives for liquid storage were developed before 2,3-diphosphoglycerate (2,3-DPG) was found to be necessary for normal hemoglobin function. In a recent study we have shown that very high concentrations of phosphate (10, 15, and 20 mM) were deleterious for maintaining 2,3-DPG. In the present study a lower range of phosphate concentrations (2, 4, 6, and 8 mM) was studied for maintenance of 2,3-DPG and ATP during storage under blood banking conditions. The lowest concentration, 2 mM, which corresponds to CPD was found to be the best concentration for maintaining 2,3-DPG and thus hemoglobin function. Four mM phosphate was not quite as good but better than no phosphate. Six and 8 mM phosphate were considerably worse.

Pulmonary hypertension and nitric oxide depletion in sickle cell disease.

During the past decade a large body of experimental and clinical studies has focused on the hypothesis that nitric oxide (NO) depletion by plasma hemoglobin in the microcirculation plays a central role in the pathogenesis of many manifestations of sickle cell disease (SCD), particularly pulmonary hypertension. We have carefully examined those studies and believe that the conclusions drawn from them are not adequately supported by the data. We agree that NO depletion may well play a role in the pathophysiology of other hemolytic states such as paroxysmal nocturnal hemoglobinuria, in which plasma hemoglobin concentrations are often at least an order of magnitude greater than in SCD. Accordingly, we conclude that clinical trials in SCD designed to increase the bioavailability of NO or association studies in which SCD clinical manifestations are related to plasma hemoglobin via its surrogates should be viewed with caution.

Nonenzymatic glycosylation of protein: relevance to diabetes.

Glucose can react nonenzymatically with proteins to form stable covalent linkages. The most abundant minor hemoglobin component in human red cells is hemoglobin AIc: glucose is attached to the N-terminal amino group of the beta chain by a ketoamine linkage. Hemoglobin AIc is increased two to three-fold in the red cells of diabetic patients. It is formed slowly and continuously throughout the 120-day lifespan of the red cell. Measurement of hemoglobin AIc provides an index of average blood glucose levels over the preceding two or three months. Thus, hemoglobin AIc has proved to be useful in assessing diabetic control and, perhaps, in screening people for diabetes. Many other proteins, such as lens crystallins, collagen and proteins in serum and in red cell membrane, are modified by nonenzymatic glycosylation. This structural alteration may lead to impaired protein function and, perhaps, contribute to the long-term complications of diabetes.