Erik R. Anderson

Hypoxia-Inducible Factor-2α Mediates the Adaptive Increase of Intestinal Ferroportin During Iron Deficiency in Mice

BACKGROUND & AIMS Iron deficiency and iron overload affect over a billion people worldwide. Dietary iron absorption in the small intestine is required for systemic iron homeostasis. Ferroportin (FPN) is the only characterized, mammalian, basolateral iron exporter. Despite the importance of FPN in maintaining iron homeostasis, its in vivo mechanisms of regulation are unclear. METHODS Systemic iron homeostasis was assessed in mice with intestine-specific disruption of genes encoding the von Hippel-Lindau tumor suppressor protein (Vhl), hypoxia-inducible factor (HIF)-1α, HIF-2α, and aryl hydrocarbon nuclear translocator (ARNT). RESULTS We observed biphasic regulation of Fpn during iron deficiency. Fpn was rapidly induced under conditions of low iron, which required the transcription factor HIF-2α. Targeted disruption of HIF-2α in the intestine inhibited Fpn induction in mice with low iron, through loss of transcriptional activation. Analysis of the Fpn promoter and in vivo chromatin immunoprecipitation assays demonstrated that HIF-2α directly binds to the Fpn promoter and induces its expression, indicating a mechanism of transcriptional regulation of Fpn following changes in systemic levels of iron. During chronic iron deficiency, FPN protein levels also increased, via increased stability through a HIF-2α-independent pathway. CONCLUSIONS In mice, expression of the gene that encodes Fpn and its protein levels are regulated by distinct pathways to provide a rapid and sustained response to acute and chronic iron deficiency. Therapies that target FPN might be developed for patients with iron-related disorders.

Endothelial PAS Domain Protein 1 Activates the Inflammatory Response in the Intestinal Epithelium to Promote Colitis in Mice

BACKGROUND & AIMS Hypoxic inflammation (decreased oxygen tension at sites of inflammation) is a feature of inflammatory bowel disease (IBD). The hypoxia response is mediated by the transcription factors hypoxia-inducible factor (HIF) 1α and endothelial PAS domain protein 1 (EPAS1 or HIF2α), which are induced in intestinal tissues of patients with IBD. HIF1α limits intestinal barrier dysfunction, but the role of EPAS1 has not been assessed under conditions of hypoxic inflammation or in models of IBD. METHODS Acute colitis was induced by administration of Citrobacter rodentium or dextran sulfate sodium (DSS) to transgenic hypoxia reporter mice (oxygen-dependent degradation-luciferase), mice with conditional overexpression of Epas1 (Epas1(LSL/LSL)), mice with intestinal epithelium-specific deletion of Epas1 (Epas1(ΔIE) ), or wild-type littermates (controls). Colon tissues from these mice and from patients with ulcerative colitis or Crohns disease were assessed by histologic and immunoblot analyses, immunohistochemistry, and quantitative polymerase chain reaction. RESULTS Levels of hypoxia and EPAS1 were increased in colon tissues of mice after induction of colitis and patients with ulcerative colitis or Crohns disease compared with controls. Epas1(ΔIE) mice had attenuated colonic inflammation and were protected from DSS-induced colitis. Intestine-specific overexpression of EPAS1, but not HIF-1α, led to spontaneous colitis, increased susceptibility to induction of colitis by C rodentium or DSS, and reduced survival times compared with controls. Disruption of intestinal epithelial EPAS1 attenuated the inflammatory response after administration of DSS or C rodentium, and intestine-specific overexpression of EPAS1 increased this response. We found EPAS1 to be a positive regulator of tumor necrosis factor-α production by the intestinal epithelium. Blocking tumor necrosis factor-α completely reduced hypoxia-induced intestinal inflammation. CONCLUSIONS EPAS1 is a transcription factor that activates mediators of inflammation, such as tumor necrosis factor-α, in the intestinal epithelium and promotes development of colitis in mice.

Hypoxia-Inducible Factor-2α Activation Promotes Colorectal Cancer Progression by Dysregulating Iron Homeostasis

Hypoxia-inducible factor (HIF), a key modulator of the transcriptional response to hypoxia, is increased in colon cancer. However, the role of HIF in colon carcinogenesis in vivo remains unclear. In this study, we found that intestinal epithelium-specific disruption of the von Hippel-Lindau tumor suppressor protein (VHL) resulted in constitutive HIF signaling, and increased HIF expression augmented colon tumorigenesis in the Apc(min/+) intestinal tumor model. Intestine-specific disruption of Vhl increased colon tumor multiplicity and progression from adenomas to carcinomas. These effects were ameliorated in mice with double disruption of Vhl and HIF-2α. Activation of HIF signaling resulted in increased cell survival in normal colon tissue; however, tumor apoptosis was not affected. Interestingly, a robust activation of cyclin D1 was observed in tumors of Apc(min/+) mice in which HIF-2α was activated in the intestine. Consistent with this result, bromodeoxyuridine incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis showed that dysregulation of the intestinal iron absorption transporter divalent metal transporter-1 (DMT-1) was a critical event in HIF-2α-mediated colon carcinogenesis. These data provide a mechanistic basis for the widely reported link between iron accumulation and colon cancer risk. Together, our findings show that a chronic increase in HIF-2α in the colon initiates protumorigenic signaling, which may have important implications in developing preventive and therapeutic strategies for colon cancer.

Intestinal HIF2α promotes tissue-iron accumulation in disorders of iron overload with anemia

Significance Several distinct congenital disorders can lead to tissue-iron overload with anemia. Tissue-iron accumulation is the major cause of mortality in these patients. Intestinal hypoxia-inducible factor-2α (HIF2α) and its downstream target gene divalent metal transporter-1 (DMT1) are essential for iron absorption during times of increased iron demand. However, the role of the intestinal HIF2α/DMT1 signaling axis in iron overload disorders has not been assessed. We demonstrate that HIF2α and DMT1 in the small intestine are highly activated early in mouse models of anemic iron overload and that disruption of their expression can prevent and improve tissue-iron accumulation in these disorders. These results demonstrate that HIF2α and DMT1 are ideal therapeutic targets in iron-overload disorders. Several distinct congenital disorders can lead to tissue-iron overload with anemia. Repeated blood transfusions are one of the major causes of iron overload in several of these disorders, including β-thalassemia major, which is characterized by a defective β-globin gene. In this state, hyperabsorption of iron is also observed and can significantly contribute to iron overload. In β-thalassemia intermedia, which does not require blood transfusion for survival, hyperabsorption of iron is the leading cause of iron overload. The mechanism of increased iron absorption in β-thalassemia is unclear. We definitively demonstrate, using genetic mouse models, that intestinal hypoxia-inducible factor-2α (HIF2α) and divalent metal transporter-1 (DMT1) are activated early in the pathogenesis of β-thalassemia and are essential for excess iron accumulation in mouse models of β-thalassemia. Moreover, thalassemic mice with established iron overload had significant improvement in tissue-iron levels and anemia following disruption of intestinal HIF2α. In addition to repeated blood transfusions and increased iron absorption, chronic hemolysis is the major cause of tissue-iron accumulation in anemic iron-overload disorders caused by hemolytic anemia. Mechanistic studies in a hemolytic anemia mouse model demonstrated that loss of intestinal HIF2α/DMT1 signaling led to decreased tissue-iron accumulation in the liver without worsening the anemia. These data demonstrate that dysregulation of intestinal hypoxia and HIF2α signaling is critical for progressive iron overload in β-thalassemia and may be a novel therapeutic target in several anemic iron-overload disorders.

Intestinal Hypoxia-inducible Factor-2α (HIF-2α) Is Critical for Efficient Erythropoiesis

Erythropoiesis is a coordinated process by which RBCs are produced. Erythropoietin, a kidney-derived hormone, and iron are critical for the production of oxygen-carrying mature RBCs. To meet the high demands of iron during erythropoiesis, small intestinal iron absorption is increased through an undefined mechanism. In this study, erythropoietic induction of iron absorption was further investigated. Hypoxia-inducible factor-2α (HIF-2α) signaling was activated in the small intestine during erythropoiesis. Genetic disruption of HIF-2α in the intestine abolished the increase in iron absorption genes as assessed by quantitative real-time reverse transcription-PCR and Western blot analyses. Moreover, the increase in serum iron following induction of erythropoiesis was entirely dependent on intestinal HIF-2α expression. Complete blood count analysis demonstrated that disruption of intestinal HIF-2α inhibited efficient erythropoiesis; mice disrupted for HIF-2α demonstrated lower hematocrit, RBCs, and Hb compared with wild-type mice. These data further cement the essential role of HIF-2α in regulating iron absorption and also demonstrate that hypoxia sensing in the intestine, as well as in the kidney, is essential for regulation of erythropoiesis by HIF-2α.

Iron Homeostasis in the Liver

Iron is an essential nutrient that is tightly regulated. A principal function of the liver is the regulation of iron homeostasis. The liver senses changes in systemic iron requirements and can regulate iron concentrations in a robust and rapid manner. The last 10 years have led to the discovery of several regulatory mechanisms in the liver that control the production of iron regulatory genes, storage capacity, and iron mobilization. Dysregulation of these functions leads to an imbalance of iron, which is the primary cause of iron-related disorders. Anemia and iron overload are two of the most prevalent disorders worldwide and affect over a billion people. Several mutations in liver-derived genes have been identified, demonstrating the central role of the liver in iron homeostasis. During conditions of excess iron, the liver increases iron storage and protects other tissues, namely, the heart and pancreas from iron-induced cellular damage. However, a chronic increase in liver iron stores results in excess reactive oxygen species production and liver injury. Excess liver iron is one of the major mechanisms leading to increased steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma.

Decreased hepcidin expression in murine β-thalassemia is associated with suppression of Bmp/Smad signaling.

To the editor: β-thalassemia is a genetic disorder of hemoglobin production characterized by ineffective erythropoiesis and anemia.[1][1] Iron overload, a major source of morbidity, results from inappropriately low expression of the gene encoding hepcidin ( Hamp1 ).[1][1] Hamp1 controls plasma

Activation of hypoxia-inducible factor-1α in type 2 alveolar epithelial cell is a major driver of acute inflammation following lung contusion.

Objective:Lung contusion is a major risk factor for the development of acute respiratory distress syndrome. Hypoxia-inducible factor-1&agr; is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension. We set to determine if hypoxia-inducible factor-1&agr; plays a role in the pathogenesis of acute inflammatory response and injury in lung contusion. Design:Nonlethal closed-chest unilateral lung contusion was induced in a hypoxia reporter mouse model and type 2 cell–specific hypoxia-inducible factor-1&agr; conditional knockout mice. The mice were killed at 5-, 24-, 48-, and 72-hour time points, and the extent of systemic and tissue hypoxia was assessed. In addition, injury and inflammation were assessed by measuring bronchoalveolar lavage cells (flow cytometry and cytospin), albumin (permeability injury), and cytokines (inflammation). Isolated type 2 cells from the hypoxia-inducible factor-1&agr; conditional knockout mice were isolated and evaluated for proinflammatory cytokines following lung contusion. Finally, the role of nuclear factor-&kgr;B and interleukin-1&bgr; as intermediates in this interaction was studied. Results:Lung contusion induced profound global hypoxia rapidly. Increased expression of hypoxia-inducible factor-1&agr; from lung samples was observed as early as 60 minutes, following the insult. The extent of lung injury following lung contusion was significantly reduced in conditional knockout mice at all the time points, when compared with the wild-type littermate mice. Release of proinflammatory cytokines, such as interleukin-1&bgr;, interleukin-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant, was significantly lower in conditional knockout mice. These actions are in part mediated through nuclear factor-&kgr;B. Hypoxia-inducible factor-1&agr; in lung epithelial cells was shown to regulate interleukin-1&bgr; promoter activity. Conclusion:Activation of hypoxia-inducible factor-1&agr; in type 2 cell is a major driver of acute inflammation following lung contusion.

Maternal intestinal HIF-2α is necessary for sensing iron demands of lactation in mice

Significance The benefits of breast milk in neonatal development are well characterized. However, very little is known about the essential nutrient components in breast milk that are critical for neonatal development and how these nutrients are maintained at adequate levels in breast milk. The present work demonstrates that the intestine is an essential sensor of systemic iron demand during lactation. During high iron demand from lactation, hypoxia-inducible factor-2α–mediated increase in maternal intestinal iron absorption is essential to maintain milk iron levels. This work demonstrates the significant role of maternal intestinal iron absorption in postnatal iron homeostasis of newborns and provides a therapeutic target to maintain iron homeostasis during pregnancy and lactation in anemic patients who are refractory to iron supplementation. The mechanisms that are essential for the maintenance of nutrient status in breast milk are unclear. Our data demonstrate that the intestine via hypoxia-inducible factor (HIF)-2α is an essential regulatory mechanism for maintaining the quality of breast milk. During lactation, intestinal HIF-2α is highly increased, leading to an adaptive induction of apical and basolateral iron transport genes. Disruption of intestinal HIF-2α (but not HIF-1α) or the downstream target gene divalent metal transporter (DMT)-1 in lactating mothers did not alter systemic iron homeostasis in the mothers, but led to anemia, decreased growth, and truncal alopecia in pups which was restored following weaning. Moreover, pups born from mothers with a disruption of intestinal HIF-2α led to long-term cognitive defects. Cross-fostering experiments and micronutrient profiling of breast milk demonstrated that the defects observed were due to decreased maternal iron delivery via milk. Increasing intestinal iron absorption by activation of HIF-2α or parenteral administration of iron-dextran in HIF-2α knockout mothers ameliorated anemia and restored neonatal development and adult cognitive functions. The present work details the importance of breast milk iron in neonatal development and uncovers an unexpected molecular mechanism for the regulation of nutritional status of breast milk through intestinal HIF-2α.

Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer

Significance Inflammation is a major risk factor for many cancers and the role of metabolic reprogramming in the inflammatory progression of cancer is not clear. We used a quantitative proteomic approach to identify mitochondrial proteins that are altered early in intestinal inflammation. We show that mitochondrial iron dysregulation is an early event that initiates mitochondrial dysfunction. Through the proteomic analysis, we identified a mitochondrial iron reductase, six-transmembrane epithelial antigen of prostate 4 (STEAP4), as being highly elevated during inflammation. Using intestinal epithelial-specific STEAP4 mice, we show that an increase in STEAP4 is sufficient to alter mitochondrial iron homeostasis. Chronic increase in mitochondrial iron leads to tissue injury and potentiates colon cancer, whereas mitochondrial iron chelation is protective in colitis and colitis-associated colon cancer models. Inflammatory bowel disease (IBD) is a chronic inflammatory disorder and is a major risk factor for colorectal cancer (CRC). Hypoxia is a feature of IBD and modulates cellular and mitochondrial metabolism. However, the role of hypoxic metabolism in IBD is unclear. Because mitochondrial dysfunction is an early hallmark of hypoxia and inflammation, an unbiased proteomics approach was used to assess the mitochondria in a mouse model of colitis. Through this analysis, we identified a ferrireductase: six-transmembrane epithelial antigen of prostate 4 (STEAP4) was highly induced in mouse models of colitis and in IBD patients. STEAP4 was regulated in a hypoxia-dependent manner that led to a dysregulation in mitochondrial iron balance, enhanced reactive oxygen species production, and increased susceptibility to mouse models of colitis. Mitochondrial iron chelation therapy improved colitis and demonstrated an essential role of mitochondrial iron dysregulation in the pathogenesis of IBD. To address if mitochondrial iron dysregulation is a key mechanism by which inflammation impacts colon tumorigenesis, STEAP4 expression, function, and mitochondrial iron chelation were assessed in a colitis-associated colon cancer model (CAC). STEAP4 was increased in human CRC and predicted poor prognosis. STEAP4 and mitochondrial iron increased tumor number and burden in a CAC model. These studies demonstrate the importance of mitochondrial iron homeostasis in IBD and CRC.