Christina Warnecke

Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs.

Cellular responses to oxygen are increasingly recognized as critical in normal development and physiology, and are implicated in pathological processes. Many of these responses are mediated by the transcription factors HIF‐1 and HIF‐2. Their regulation occurs through oxygen‐dependent proteolysis of the alpha subunits HIF‐1α and HIF‐2α, respectively. Both are stabilized in cell lines exposed to hypoxia, and recently HIF‐1α was reported to be widely expressed in vivo. In contrast, regulation and sites of HIF‐2α expression in vivo are unknown, although a specific role in endothelium was suggested. We therefore analyzed HIF‐2α expression in control and hypoxic rats. Although HIF‐2α was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, including brain, heart, lung, kidney, liver, pancreas, and intestine. Time course and amplitude of induction varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non‐parenchymal in some organs (kidney, pancreas, and brain), predominately parenchymal in others (liver and intestine) or equally distributed (myocardium). These data indicate that HIF‐2 plays an important role in the transcriptional response to hypoxia in vivo, which is not confined to the vasculature and is complementary to rather than redundant with HIF‐1.

Differentiating the functional role of hypoxia-inducible factor (HIF)-1α and HIF-2α (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2α target gene in Hep3B and Kelly cells

Activation of the hypoxia‐inducible factor α‐subunits, HIF‐1α and HIF‐2α, seems to be subject to similar regulatory mechanisms, and transgene approaches suggested partial functional redundancy. Here, we used RNA interference to determine the contribution of HIF‐1α vs. HIF‐2α to the hypoxic gene induction. Surprisingly, most genes tested were responsive only to the HIF‐1α siRNA, showing no effect by HIF‐2α knock‐down. The same was found for the activation of reporter genes driven by hypoxia‐responsive elements (HREs) from the erythropoietin (EPO), vascular endothelial growth factor, or phosphoglycerate kinase gene. Interestingly, EPO was the only gene investigated that showed responsiveness only to HIF‐2α knock‐down, as observed in Hep3B and Kelly cells. In contrast to the EPO‐HRE reporter, the complete EPO enhancer displayed dependency on HIF‐2α regulation, indicating that additional cis‐acting elements confer HIF‐2α specificity within this region. In 786‐0 cells lacking HIF‐1α protein, the identified HIF‐1α target genes were regulated by HIF‐2α. Overexpression of the HIFα subunits in different cell lines also led to a loss of target gene specificity. In conclusion, we found a remarkably restricted target gene specificity of the HIFα subunits, which can be overcome in cells with perturbations in the pVHL/HIF system and under forced expression.

Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors

Hypoxia‐inducible transcription factors (HIF) mediate complex adaptations to reduced oxygen supply, including neoangiogenesis. Regulation of HIF occurs mainly through oxygen‐dependent destruction of its α subunit. In the presence of oxygen, two HIFα prolyl residues undergo enzymatic hydroxylation, which is required for its proteasomal degradation. We therefore tested whether pharmacological activation of HIFα by hydroxylase inhibitors may provide a novel therapeutic strategy for the treatment of ischemic diseases. Three distinct prolyl 4‐hydroxylase inhibitors—l‐mimosine (l‐Mim), ethyl 3,4‐dihydroxybenzoate (3,4‐DHB), and 6‐chlor‐3‐hydroxychinolin‐2‐carbonic acid‐N‐carboxymethylamid (S956711)—demonstrated similar effects to hypoxia (0.5% O2) by inducing HIFα protein in human and rodent cells. l‐Mim, S956711, and, less effectively, 3,4‐DHB also induced HIF target genes in cultured cells, including glucose transporter 1 and vascular endothelial growth factor, as well as HIF‐dependent reporter gene expression. Systemic administration of l‐Mim and S956711 in rats led to HIFα induction in the kidney. In a sponge model for angiogenesis, repeated local injection of the inhibitors strongly increased invasion of highly vascularized tissue into the sponge centers. In conclusion, structurally distinct inhibitors of prolyl hydroxylation are capable of inducing HIFα and HIF target genes in vitro and in vivo and induce adaptive responses to hypoxia, including angiogenesis.

Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function.

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1α (HIF-1α), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1α on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1α protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1α significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1α and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.

GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis.

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

The Lysyl Oxidases LOX and LOXL2 Are Necessary and Sufficient to Repress E-cadherin in Hypoxia INSIGHTS INTO CELLULAR TRANSFORMATION PROCESSES MEDIATED BY HIF-1

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

Persistent induction of HIF-1α and -2α in cardiomyocytes and stromal cells of ischemic myocardium

Hypoxia‐inducible factor (HIF)‐1α and ‐2α are key regulators of the transcriptional response to hypoxia and pivotal in mediating the consequences of many disease states. In the present work, we define their temporo‐spatial accumulation after myocardial infarction and systemic hypoxia. Rats were exposed to hypoxia or underwent coronary artery ligation. Immunohistochemistry was used for detection of HIF‐1α and ‐2α proteins and target genes, and mRNA levels were determined by RNase protection. Marked nuclear accumulation of HIF‐1α and ‐2α occurred after both systemic hypoxia and coronary ligation in cardiomyocytes as well as interstitial and endothelial cells (EC) without pronounced changes in HIF mRNA levels. While systemic hypoxia led to widespread induction of HIF, expression after coronary occlusion occurred primarily at the border of infarcted tissue. This expression persisted for 4 wk, included infiltrating macrophages, and colocalized with target gene expression. Subsets of cells simultaneously expressed both HIF‐α subunits, but EC more frequently induced HIF‐2α. A progressive increase of HIF‐2α but not HIF‐1α occurred in areas remote from the infarct, including the interventricular septum. Cardiomyocytes and cardiac stromal cells exhibit a marked potential for a prolonged transcriptional response to ischemia mediated by HIF. The induction of HIF‐1α and ‐2α appears to be complementary rather than solely redundant.

HIF Activation Protects From Acute Kidney Injury

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.

Donor treatment with a PHD-inhibitor activating HIFs prevents graft injury and prolongs survival in an allogenic kidney transplant model.

Long-term survival of renal allografts depends on the chronic immune response and is probably influenced by the initial injury caused by ischemia and reperfusion. Hypoxia-inducible transcription factors (HIFs) are essential for adaptation to low oxygen. Normoxic inactivation of HIFs is regulated by oxygen-dependent hydroxylation of specific prolyl-residues by prolyl-hydroxylases (PHDs). Pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of tissue-protective genes. We examined the effect of donor treatment with a specific PHD inhibitor (FG-4497) on graft function in the Fisher–Lewis rat model of allogenic kidney transplantation (KTx). Orthotopic transplantation of the left donor kidney was performed after 24 h of cold storage. The right kidney was removed at the time of KTx (acute model) or at day 10 (chronic model). Donor animals received a single dose of FG-4497 (40 mg/kg i.v.) or vehicle 6 h before donor nephrectomy. Recipients were followed up for 10 days (acute model) or 24 weeks (chronic model). Donor preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond cold storage. It reduced acute renal injury (serum creatinine at day 10: 0.66 ± 0.20 vs. 1.49 ± 1.36 mg/dL; P < 0.05) and early mortality in the acute model and improved long-term survival of recipient animals in the chronic model (mortality at 24 weeks: 3 of 16 vs. 7 of 13 vehicle-treated animals; P < 0.05). In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcomes after allogenic KTx. Inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves clinical evaluation.

Evidence for a lack of a direct transcriptional suppression of the iron regulatory peptide hepcidin by hypoxia-inducible factors.

Background Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation. Methodology/Principal Findings Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased. Conclusions/Significance Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.