Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation.

The severity of T cell-mediated gastrointestinal (GI) diseases such as graft-versus-host disease (GVHD) and inflammatory bowel diseases correlates with a decrease in the diversity of the host gut microbiome composition characterized by loss of obligate anaerobic commensals. The mechanisms underpinning these changes in the microbial structure remain unknown. Here, we show in multiple specific pathogen-free (SPF), gnotobiotic, and germ-free murine models of GI GVHD that the initiation of the intestinal damage by the pathogenic T cells altered ambient oxygen levels in the GI tract and caused dysbiosis. The change in oxygen levels contributed to the severity of intestinal pathology in a host intestinal HIF-1α- and a microbiome-dependent manner. Regulation of intestinal ambient oxygen levels with oral iron chelation mitigated dysbiosis and reduced the severity of the GI GVHD. Thus, targeting ambient intestinal oxygen levels may represent a novel, non-immunosuppressive strategy to mitigate T cell-driven intestinal diseases.

Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

More Metabolism!

To celebrate our Focus Issue, we asked a selection of researchers working on different aspects of metabolism what they are excited about and what is still to come. They discuss emerging concepts, unanswered questions, things to consider, and technologies that are enabling new discoveries, as well as developing and integrating approaches to drive the field forward.

Sulfide oxidation promotes hypoxic angiogenesis and neovascularization.

Angiogenic programming in the vascular endothelium is a tightly regulated process for maintaining tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Here, we report that hypoxic upregulation of ·NO in endothelial cells reprograms the transsulfuration pathway to increase biogenesis of hydrogen sulfide (H2S), a proangiogenic metabolite. However, decreased H2S oxidation due to sulfide quinone oxidoreductase (SQOR) deficiency synergizes with hypoxia, inducing a reductive shift and limiting endothelial proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body (WBCreSqorfl/fl) and endothelial-specific (VE-cadherinCre-ERT2Sqorfl/fl) Sqor-knockout mice exhibit lower mass and angiogenesis than control mice. WBCreSqorfl/fl mice also exhibit decreased muscle angiogenesis following femoral artery ligation compared to control mice. Collectively, our data reveal the molecular intersections between H2S, O2 and ·NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization.

Hepatic SEL1L-HRD1 ER-associated degradation regulates systemic iron homeostasis via ceruloplasmin.

Iron homeostasis is critical for cellular and organismal function and is tightly regulated to prevent toxicity or anemia due to iron excess or deficiency, respectively. However, subcellular regulatory mechanisms of iron remain largely unexplored. Here, we report that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) in hepatocytes controls systemic iron homeostasis in a ceruloplasmin (CP)-dependent, and ER stress-independent, manner. Mice with hepatocyte-specific Sel1L deficiency exhibit altered basal iron homeostasis and are sensitized to iron deficiency while resistant to iron overload. Proteomics screening for a factor linking ERAD deficiency to altered iron homeostasis identifies CP, a key ferroxidase involved in systemic iron distribution by catalyzing iron oxidation and efflux from tissues. Indeed, CP is highly unstable and a bona fide substrate of SEL1L-HRD1 ERAD. In the absence of ERAD, CP protein accumulates in the ER and is shunted to refolding, leading to elevated secretion. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD is responsible for the degradation of a subset of disease-causing CP mutants, thereby attenuating their pathogenicity. Together, this study uncovers the role of SEL1L-HRD1 ERAD in systemic iron homeostasis and provides insights into protein misfolding-associated proteotoxicity.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1ß protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool.

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Renal-specific loss of ferroportin disrupts iron homeostasis and attenuates recovery from acute kidney injury.

Chronic kidney disease is increasing at an alarming rate and correlates with the increase in diabetes, obesity, and hypertension that disproportionately impact socioeconomically disadvantaged communities. Iron plays essential roles in many biological processes including oxygen transport, mitochondrial function, cell proliferation, and regeneration. However, excess iron induces the generation and propagation of reactive oxygen species, which lead to oxidative stress, cellular damage, and ferroptosis. Iron homeostasis is regulated in part by the kidney through iron resorption from the glomerular filtrate and exports into the plasma by ferroportin (FPN). Yet, the impact of iron overload in the kidney has not been addressed. To test more directly whether excess iron accumulation is toxic to kidneys, we generated a kidney proximal tubule-specific knockout of FPN. Despite significant intracellular iron accumulation in FPN mutant tubules, basal kidney function was not measurably different from wild type kidneys. However, upon induction of acute kidney injury (AKI), FPN mutant kidneys exhibited significantly more damage and failed recovery, evidence for ferroptosis, and increased fibrosis. Thus, disruption of iron export in proximal tubules, leading to iron overload, can significantly impair recovery from AKI and can contribute to progressive renal damage indicative of chronic kidney disease. Understanding the mechanisms that regulate iron homeostasis in the kidney may provide new therapeutic strategies for progressive kidney disease and other ferroptosis-associated disorders.NEW & NOTEWORTHY Physiological iron homeostasis depends in part on renal resorption and export into the plasma. We show that specific deletion of iron exporters in the proximal tubules sensitizes cells to injury and inhibits recovery. This can promote a chronic kidney disease phenotype. Our paper demonstrates the need for iron balance in the proximal tubules to maintain and promote healthy recovery after acute kidney injury.

Disruption of hypoxia-inducible factor-2α in neutrophils decreases colitis-associated colon cancer.

Neutrophils are abundant immune cells in the colon tumor microenvironment. Studies have shown that neutrophils are recruited into hypoxic foci in colon cancer. However, the impact of hypoxia signaling on neutrophil function and its involvement in colon tumorigenesis remain unclear. To address this, we generated mice with a deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α in neutrophils driven by the MRP8Cre (HIF-1αΔNeu) or (HIF-2αΔNeu) and littermate controls. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colon cancer, the disruption of neutrophils-HIF-1α did not result in any significant changes in body weight, colon length, tumor size, proliferation, or burden. However, the disruption of HIF-2α in neutrophils led to a slight increase in body weight, a significant decrease in the number of tumors, and a reduction in tumor size and volume compared with their littermate controls. Histological analysis of colon tissue from mice with HIF-2α-deficient neutrophils revealed notable reductions in proliferation as compared with control mice. In addition, we observed reduced levels of proinflammatory cytokines, such as TNF-α and IL-1ß, in neutrophil-specific HIF-2α-deficient mice in both the tumor tissue as well as the neutrophils. Importantly, it is worth noting that the reduced tumorigenesis associated with HIF-2α deficiency in neutrophils was not evident in already established syngeneic tumors or a DSS-induced inflammation model, indicating a potential role of HIF-2α specifically in colon tumorigenesis. In conclusion, we found that the loss of neutrophil-specific HIF-2α slows colon tumor growth and progression by reducing the levels of inflammatory mediators.NEW & NOTEWORTHY Despite the importance of hypoxia and neutrophils in colorectal cancer (CRC), the contribution of neutrophil-specific HIFs to colon tumorigenesis is not known. We describe that neutrophil HIF-1α has no impact on colon cancer, whereas neutrophil HIF-2α loss reduces CRC growth by decreasing proinflammatory and immunosuppressive cytokines. Furthermore, neutrophil HIF-2α does not reduce preestablished tumor growth or inflammation-induced colitis. The present study offers novel potential of neutrophil HIF-2α as a therapeutic target in CRC.

Dysregulated Amino Acid Sensing Drives Colorectal Cancer Growth and Metabolic Reprogramming Leading to Chemoresistance.

BACKGROUND & AIMS: Colorectal cancer (CRC) is a devastating disease that is highly modulated by dietary nutrients. Mechanistic target of rapamycin complex 1 (mTORC1) contributes to tumor growth and limits therapy responses. Growth factor signaling is a major mechanism of mTORC1 activation. However, compensatory pathways exist to sustain mTORC1 activity after therapies that target oncogenic growth factor signaling. Amino acids potently activate mTORC1 via amino acid-sensing GTPase activity towards Rags (GATOR). The role of amino acid-sensing pathways in CRC is unclear. METHODS: Human colon cancer cell lines, preclinical intestinal epithelial-specific GATOR1 and GATOR2 knockout mice subjected to colitis-induced or sporadic colon tumor models, small interfering RNA screening targeting regulators of mTORC1, and tissues of patients with CRC were used to assess the role of amino acid sensing in CRC. RESULTS: We identified loss-of-function mutations of the GATOR1 complex in CRC and showed that altered expression of amino acid-sensing pathways predicted poor patient outcomes. We showed that dysregulated amino acid-sensing induced mTORC1 activation drives colon tumorigenesis in multiple mouse models. We found amino acid-sensing pathways to be essential in the cellular reprogramming of chemoresistance, and chemotherapeutic-resistant patients with colon cancer exhibited de-regulated amino acid sensing. Limiting amino acids in in vitro and in vivo models (low-protein diet) reverted drug resistance, revealing a metabolic vulnerability. CONCLUSIONS: Our findings suggest a critical role for amino acid-sensing pathways in driving CRC and highlight the translational implications of dietary protein intervention in CRC.

Modulation of the HIF2α-NCOA4 axis in enterocytes attenuates iron loading in a mouse model of hemochromatosis.

Intestinal iron absorption is activated during increased systemic demand for iron. The best-studied example is iron deficiency anemia, which increases intestinal iron absorption. Interestingly, the intestinal response to anemia is very similar to that of iron overload disorders, as both the conditions activate a transcriptional program that leads to a hyperabsorption of iron via the transcription factor hypoxia-inducible factor 2α (HIF2α). However, pathways for selective targeting of intestine-mediated iron overload remain unknown. Nuclear receptor coactivator 4 (NCOA4) is a critical cargo receptor for autophagic breakdown of ferritin and the subsequent release of iron, in a process termed ferritinophagy. Our work demonstrates that NCOA4-mediated intestinal ferritinophagy is integrated into systemic iron demand via HIF2α. To demonstrate the importance of the intestinal HIF2α/ferritinophagy axis in systemic iron homeostasis, whole-body and intestine-specific NCOA4-/- mouse lines were generated and assessed. The analyses revealed that the intestinal and systemic response to iron deficiency was not altered after disruption of intestinal NCOA4. However, in a mouse model of hemochromatosis, ablation of intestinal NCOA4 was protective against iron overload. Therefore, NCOA4 can be selectively targeted for the management of iron overload disorders without disrupting the physiological processes involved in the response to systemic iron deficiency.

Dietary Iron Is Necessary to Support Proliferative Regeneration after Intestinal Injury.

BACKGROUND: Tissue repair and regeneration in the gastrointestinal system are crucial for maintaining homeostasis, with the process relying on intricate cellular interactions and affected by micro- and macro-nutrients. Iron, essential for various biological functions, plays a dual role in tissue healing by potentially causing oxidative damage and participating in anti-inflammatory mechanisms, underscoring its complex relationship with inflammation and tissue repair. OBJECTIVE: The study aimed to elucidate the role of low dietary iron in gastrointestinal tissue repair. METHODS: We utilized quantitative iron measurements to assess iron levels in inflamed regions of patients with ulcerative colitis and Crohn's disease. In addition, 3 mouse models of gastrointestinal injury/repair (dextran sulfate sodium-induced colitis, radiation injury, and wound biopsy) were used to assess the effects of low dietary iron on tissue repair. RESULTS: We found that levels of iron in inflamed regions of both patients with ulcerative colitis and Crohn's disease are elevated. Similarly, during gastrointestinal repair, iron levels were found to be heightened, specifically in intestinal epithelial cells across the 3 injury/repair models. Mice on a low-iron diet showed compromised tissue repair with reduced proliferation. In standard diet, epithelial cells and the stem cell compartment maintain adequate iron stores. However, during a period of iron deficiency, epithelial cells exhaust their iron reserves, whereas the stem cell compartments maintain their iron pools. During injury, when the stem compartment is disrupted, low iron levels impair proliferation and compromise repair mechanisms. CONCLUSIONS: Low dietary iron impairs intestinal repair through compromising the ability of epithelial cells to aid in intestinal proliferation.

Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes.

Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure, and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study, we used a multiomics approach to vertically integrate unbiased data generated using an assay for transposase-accessible chromatin with high-throughput sequencing, RNA-Seq, and proteomics. We then horizontally integrated these data across different models of hyperammonemia, including myotubes and mouse and human muscle tissues. Changes in chromatin accessibility and/or expression of genes resulted in distinct clusters of temporal molecular changes including transient, persistent, and delayed responses during hyperammonemia in myotubes. Known responses to hyperammonemia, including mitochondrial and oxidative dysfunction, protein homeostasis disruption, and oxidative stress pathway activation, were enriched in our datasets. During hyperammonemia, pathways that impact skeletal muscle structure and function that were consistently enriched were those that contribute to mitochondrial dysfunction, oxidative stress, and senescence. We made several novel observations, including an enrichment in antiapoptotic B-cell leukemia/lymphoma 2 family protein expression, increased calcium flux, and increased protein glycosylation in myotubes and muscle tissue upon hyperammonemia. Critical molecules in these pathways were validated experimentally. Human skeletal muscle from patients with cirrhosis displayed similar responses, establishing translational relevance. These data demonstrate complex molecular interactions during adaptive and maladaptive responses during the cellular stress response to hyperammonemia.

Medullary thymic epithelial NF-kB-inducing kinase (NIK)/IKKα pathway shapes autoimmunity and liver and lung homeostasis in mice.

Aberrant T cell development is a pivotal risk factor for autoimmune disease; however, the underlying molecular mechanism of T cell overactivation is poorly understood. Here, we identified NF-κB-inducing kinase (NIK) and IkB kinase α (IKKα) in thymic epithelial cells (TECs) as essential regulators of T cell development. Mouse TEC-specific ablation of either NIK or IKKα resulted in severe T cell-mediated inflammation, injury, and fibrosis in the liver and lung, leading to premature death within 18 d of age. NIK or IKKα deficiency abrogated medullary TEC development, and led to breakdown of central tolerance, production of autoreactive T cells, and fatal autoimmune destruction in the liver and lung. TEC-specific ablation of NIK or IKKα also impaired thymic T cell development from the double-negative through the double-positive stages and inhibited peripheral B cell development. These results unravel a hitherto unrecognized essential role of TEC-intrinsic NIK and IKKα pathways in autoimmunity and T cell-instigated chronic liver and lung diseases.

Oxygen battle in the gut: Hypoxia and hypoxia-inducible factors in metabolic and inflammatory responses in the intestine.

The gastrointestinal tract is a highly proliferative and regenerative tissue. The intestine also harbors a large and diverse microbial population collectively called the gut microbiome (microbiota). The microbiome-intestine cross-talk includes a dynamic exchange of gaseous signaling mediators generated by bacterial and intestinal metabolisms. Moreover, the microbiome initiates and maintains the hypoxic environment of the intestine that is critical for nutrient absorption, intestinal barrier function, and innate and adaptive immune responses in the mucosal cells of the intestine. The response to hypoxia is mediated by hypoxia-inducible factors (HIFs). In hypoxic conditions, the HIF activation regulates the expression of a cohort of genes that promote adaptation to hypoxia. Physiologically, HIF-dependent genes contribute to the aforementioned maintenance of epithelial barrier function, nutrient absorption, and immune regulation. However, chronic HIF activation exacerbates disease conditions, leading to intestinal injury, inflammation, and colorectal cancer. In this review, we aim to outline the major roles of physiological and pathological hypoxic conditions in the maintenance of intestinal homeostasis and in the onset and progression of disease with a major focus on understanding the complex pathophysiology of the intestine.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Impact of dietary manganese on experimental colitis in mice.

Diet plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). A recent epidemiological study has shown an inverse relationship between nutritional manganese (Mn) status and IBD patients. Mn is an essential micronutrient required for normal cell function and physiological processes. To date, the roles of Mn in intestinal homeostasis remain unknown and the contribution of Mn to IBD has yet to be explored. Here, we provide evidence that Mn is critical for the maintenance of the intestinal barrier and that Mn deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. Specifically, when treated with DSS, Mn-deficient mice showed increased morbidity, weight loss, and colon injury, with a concomitant increase in inflammatory cytokine levels and oxidative and DNA damage. Even without DSS treatment, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In contrast, mice fed a Mn-supplemented diet showed slightly increased tolerance to DSS-induced experimental colitis, as judged by the colon length. Despite the well-appreciated roles of intestinal microbiota in driving inflammation in IBD, the gut microbiome composition was not altered by changes in dietary Mn. We conclude that Mn is necessary for proper maintenance of the intestinal barrier and provides protection against DSS-induced colon injury.

A genetic mouse model of severe iron deficiency anemia reveals tissue-specific transcriptional stress responses and cardiac remodeling.

Iron is a micronutrient fundamental for life. Iron homeostasis in mammals requires sustained postnatal intestinal iron absorption that maintains intracellular iron concentrations for central and systemic metabolism as well as for erythropoiesis and oxygen transport. More than 1 billion people worldwide suffer from iron deficiency anemia (IDA), a state of systemic iron insufficiency that limits the production of red blood cells and leads to tissue hypoxia and intracellular iron stress. Despite this tremendous public health concern, very few genetic models of IDA are available to study its progression. Here we developed and characterized a novel genetic mouse model of IDA. We found that tamoxifen-inducible deletion of the mammalian iron exporter ferroportin exclusively in intestinal epithelial cells leads to loss of intestinal iron absorption. Ferroportin ablation yielded a robust phenotype of progressive IDA that develops in as little as 3 months following disruption of intestinal iron absorption. We noted that, at end-stage IDA, tissue-specific transcriptional stress responses occur in which the heart shows little to no hypoxic and iron stress compared with other peripheral organs. However, morphometric and echocardiographic analysis revealed massive cardiac hypertrophy and chamber dilation, albeit with increased cardiac output at very low basal heart rates. We propose that our intestine-specific ferroportin knockout mouse model of end-stage IDA could be used in future studies to investigate IDA progression and cell-specific responses to hypoxic and iron stress.

SMAD family member 3 (SMAD3) and SMAD4 repress HIF2α-dependent iron-regulatory genes.

Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.