Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94.

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe(+4)) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl(2)). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.

Inactivation of prolyl hydroxylase domain (PHD) protein by epigallocatechin (EGCG) stabilizes hypoxia-inducible factor (HIF-1α) and induces hepcidin (Hamp) in rat kidney.

HIF-1α plays a key role in iron uptake and transport in the liver, whose activity is tightly linked to the repression of hepcidin (Hamp). Hamp prevents intestinal iron uptake and cellular efflux by negatively modulating ferroportin. Hamp is also expressed in the kidneys, where transcriptional control by HIF-1α remains poorly understood. We show that the administration of epigallocatechin gallate (EGCG) results in a considerable Hamp expression in rat kidneys. We also provide evidence to show that EGCG inhibited prolyl hydroxylase (PHD) activity, essential for HIF-1α degradation in vivo and in vitro. Rats that were dosed with EGCG (60 mg/kg, intraperitoneal) over a 7 day time course stabilized HIF-1α protein in kidney tissues. Interestingly, Hamp gene expression was induced, even after subjecting rats to a 4h hypoxia treatment (8% oxygen). Using Hep3B cells, we determined that EGCG conferred its inhibitory action by complexing with PHD, altering its catalytic iron center and thus preventing HIF-1α hydroxylation. These data demonstrate EGCG's therapeutic potential in modulating hepcidin expression in diseases associated with altered iron metabolism.

Acellular haemoglobin attenuates hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes in haemodiluted rats.

Hb (haemoglobin)-based blood substitutes represent a class of therapeutics designed to correct oxygen deficit under conditions of anaemia and traumatic blood loss. The influences of these agents on HIF-1alpha (hypoxia-inducible factor-1alpha) target genes involved in adaptation to hypoxia have so far not been studied. In the study presented here, rats underwent 80% ET (exchange transfusion) with either HS (hetastarch) or a polymerized Hb OG (Oxyglobin). HS induced dramatic EPO (erythropoietin) gene transcription, reaching a maximum at 4 h post-ET. In contrast, OG suppressed EPO transcription until approx. 24 h post-ET. Large plasma EPO levels that were observed post-ET with HS were significantly blunted in animals transfused with OG. OG, unlike HS, induced a sharp increase in HO-1 (haem oxygenase-1) transcription at 4 h, which declined rapidly within 24 h, whereas modest increases in iNOS [inducible (nitric oxide synthase)] and constitutive NOS [eNOS (endothelial NOS)] were detected over the control. Our results demonstrate for the first time that severe haemodilution-induced erythropoietic responses in kidneys were attenuated by a low-oxygen-affinity cell-free Hb and suggest that tissue-specific oxygen-sensing pathways can be influenced by allosterically modified Hbs.

Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury.

BACKGROUND: Preconditioning phenomena provide evidence for adaptive responses to ischemia that have important implications for treatment/prevention of myocardial infarction. Hypoxia-inducible factor 1 (HIF-1) mediates adaptive transcriptional responses to hypoxia/ischemia. METHODS AND RESULTS: Exposure of wild-type mice to intermittent hypoxia resulted in protection of isolated hearts against ischemia-reperfusion injury 24 hours later. Cardiac protection induced by intermittent hypoxia was lost in Hif1a+/- mice heterozygous for a knockout allele at the locus encoding HIF-1alpha. Erythropoietin (EPO) mRNA expression was induced in kidneys of wild-type mice subjected to intermittent hypoxia, resulting in increased plasma EPO levels. EPO mRNA expression was not induced in Hif1a+/- mice. EPO administration to rats increased functional recovery and decreased apoptosis in isolated hearts subjected to ischemia-reperfusion 24 hours later. CONCLUSIONS: Hearts isolated from rodents subjected to intermittent hypoxia or EPO administration are protected against postischemic injury. Cardiac protection induced by intermittent hypoxia is critically dependent on Hif1a gene dosage. Our data suggest that additional studies to evaluate therapeutic applications of EPO administration are warranted.

Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1.

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding angiogenic growth factors, which are secreted by hypoxic cells and stimulate endothelial cells, leading to angiogenesis. To determine whether HIF-1 also mediates cell-autonomous responses to hypoxia, we have compared gene expression profiles in arterial endothelial cells cultured under nonhypoxic versus hypoxic conditions and in nonhypoxic cells infected with adenovirus encoding beta-galactosidase versus a constitutively active form of HIF-1alpha (AdCA5). There were 245 gene probes that showed at least 1.5-fold increase in expression in response to hypoxia and in response to AdCA5; 325 gene probes showed at least 1.5-fold decrease in expression in response to hypoxia and in response to AdCA5. The largest category of genes down-regulated by both hypoxia and AdCA5 encoded proteins involved in cell growth/proliferation. Many genes up-regulated by both hypoxia and AdCA5 encoded cytokines/growth factors, receptors, and other signaling proteins. Transcription factors accounted for the largest group of HIF-1-regulated genes, indicating that HIF-1 controls a network of transcriptional responses to hypoxia in endothelial cells. Infection of endothelial cells with AdCA5 under nonhypoxic conditions was sufficient to induce increased basement membrane invasion and tube formation similar to the responses induced by hypoxia, indicating that HIF-1 mediates cell-autonomous activation of endothelial cells.

Redox-dependent regulation of the conformation and function of human heat shock factor 1.

We present here evidence that redox-dependent thiol-disulfide exchange can provide a mechanism regulating the conformation and activity of the human heat shock transcription factor 1 (HSF1). Diamide and dithiothreitol were reagents used respectively to promote and reverse disulfide cross-link, and the resolution and detection of redox conformers of HSF1 were done according to recently published methods [Manalo, D. J., and Liu, A. Y.-C. (2001) J. Biol. Chem. 276, 23554-23561]. We showed that preincubation of the latent HSF1 monomer with diamide inhibited the in vitro heat-induced activation and trimerization of HSF1 and caused the formation of ox-hHSF1, a compact, disulfide cross-linked HSF1 conformer detectable in immuno-Western blot assay. These effects of diamide were dose-dependent and were rapidly and quantitatively reversed by dithiothreitol. Cysteine site-specific mutants of HSF1 were constructed and used to determine which of the five cysteine residues may be engaged in disulfide cross-link. Analysis of the in vitro transcribed and translated HSF1 proteins showed that while mutation of C1 (amino acid 36) and C2 (amino acid 103) had no effect on the redox sensitivity of HSF1, the mutation of C3 (amino acid 153) or double mutation of C4 and C5 (amino acids 373 and 378, respectively) to serine rendered HSF1 insensitive to diamide and prevented its conversion to ox-HSF1. HSF1 with a single cysteine to serine mutation at either the C4 or C5 position gave different ox-HSF1 conformers in the presence of diamide, suggesting that C3 could be disulfide cross-linked to either C4 or C5. The possibility that HSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional response in higher mammalian cells is discussed.

Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1 alpha.

To investigate whether the transcriptional activator hypoxia-inducible factor 1 (HIF-1) is required for ventilatory responses to hypoxia, we analyzed mice that were either wild type or heterozygous for a loss-of-function (knockout) allele at the Hif1a locus, which encodes the O(2)-regulated HIF-1 alpha subunit. Although the ventilatory response to acute hypoxia was not impaired in Hif1a(+/-) mice, the response was primarily mediated via vagal afferents, whereas in wild-type mice, carotid body chemoreceptors played a predominant role. When carotid bodies isolated from wild-type mice were exposed to either cyanide or hypoxia, a marked increase in sinus nerve activity was recorded, whereas carotid bodies from Hif1a(+/-) mice responded to cyanide but not to hypoxia. Histologic analysis revealed no abnormalities of carotid body morphology in Hif1a(+/-) mice. Wild-type mice exposed to hypoxia for 3 days manifested an augmented ventilatory response to a subsequent acute hypoxic challenge. In contrast, prior chronic hypoxia resulted in a diminished ventilatory response to acute hypoxia in Hif1a(+/-) mice. Thus partial HIF-1 alpha deficiency has a dramatic effect on carotid body neural activity and ventilatory adaptation to chronic hypoxia.