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

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.

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

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.

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

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 ß-thalassemia major, which is characterized by a defective ß-globin gene. In this state, hyperabsorption of iron is also observed and can significantly contribute to iron overload. In ß-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 ß-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 ß-thalassemia and are essential for excess iron accumulation in mouse models of ß-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 ß-thalassemia and may be a novel therapeutic target in several anemic iron-overload disorders.

Intestinal hypoxia-inducible factor-2alpha (HIF-2alpha) 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.

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.

Survival signal REG3α prevents crypt apoptosis to control acute gastrointestinal graft-versus-host disease.

Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract remains the major cause of morbidity and nonrelapse mortality after BM transplantation (BMT). The Paneth cell protein regenerating islet-derived 3α (REG3α) is a biomarker specific for GI GVHD. REG3α serum levels rose in the systematic circulation as GVHD progressively destroyed Paneth cells and reduced GI epithelial barrier function. Paradoxically, GVHD suppressed intestinal REG3γ (the mouse homolog of human REG3α), and the absence of REG3γ in BMT recipients intensified GVHD but did not change the composition of the microbiome. IL-22 administration restored REG3γ production and prevented apoptosis of both intestinal stem cells (ISCs) and Paneth cells, but this protection was completely abrogated in Reg3g-/- mice. In vitro, addition of REG3α reduced the apoptosis of colonic cell lines. Strategies that increase intestinal REG3α/γ to promote crypt regeneration may offer a novel, nonimmunosuppressive approach for GVHD and perhaps for other diseases involving the ISC niche, such as inflammatory bowel disease.

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.

The hypoxia-inducible factor-C/EBPα axis controls ethanol-mediated hepcidin repression.

Hepcidin is a liver-derived peptide hormone and the master regulator of systemic iron homeostasis. Decreased hepcidin expression is a common feature in alcoholic liver disease (ALD) and in mouse models of ethanol loading. Dysregulation of hepcidin signaling in ALD leads to liver iron deposition, which is a major contributing factor to liver injury. The mechanism by which hepcidin is regulated following ethanol treatment is unclear. An increase in liver hypoxia was observed in an acute ethanol-induced liver injury model. The hypoxic response is controlled by a family of hypoxia-inducible transcription factors (HIFs), which are composed of an oxygen-regulated alpha subunit (HIFα) and a constitutively present beta subunit, aryl hydrocarbon receptor nuclear translocator (HIFß/Arnt). Disruption of liver HIF function reversed the repression of hepcidin following ethanol loading. Mouse models of liver HIF overexpression demonstrated that both HIF-1α and HIF-2α contribute to hepcidin repression in vivo. Ethanol treatment led to a decrease in CCAAT-enhancer-binding protein alpha (C/EBPα) protein expression in a HIF-dependent manner. Importantly, adenoviral rescue of C/EBPα in vivo ablated the hepcidin repression in response to ethanol treatment or HIF overexpression. These data provide novel insight into the regulation of hepcidin by hypoxia and indicate that targeting HIFs in the liver could be therapeutic in ALD.

Metabolomics identifies an inflammatory cascade involved in dioxin- and diet-induced steatohepatitis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent environmentally toxic compounds. Serum metabolomics identified azelaic acid monoesters as significantly increased metabolites after TCDD treatment, due to downregulation of hepatic carboxylesterase 3 (CES3, also known as triglyceride hydrolase) expression in an arylhydrocarbon receptor (AhR)-dependent manner in mice. The decreased CES3 expression was accomplished by TCDD-stimulated TGFß-SMAD3 and IL6-STAT3 signaling, but not by direct AhR signaling. Methionine- and choline-deficient (MCD) diet-treated mice also showed enhanced serum azelaic acid monoester levels after attenuation of hepatic CES3 expression, while db/db mice did not, thus suggesting an association with steatohepatitis. Forced expression of CES3 reversed serum azelaic acid monoester/azelaic acid ratios and hepatic TGFß mRNA levels in TCDD- and MCD diet-treated mice and ameliorated steatohepatitis induced by MCD diet. These results support the view that azelaic acid monoesters are possible indicators of TCDD exposure and steatohepatitis and suggest a link between CES3, TGFß, and steatohepatitis.