Epigallocatechin-3-Gallate Suppresses BMP-6-Mediated SMAD1/5/8 Transactivation of Hepcidin Gene by Inducing SMILE in Hepatocytes

Hepcidin, a major regulator of systemic iron homeostasis, is mainly induced in hepatocytes by activating bone morphogenetic protein 6 (BMP-6) signaling in response to changes in the iron status. Small heterodimer partner-interacting leucine zipper protein (SMILE), a polyphenol-inducible transcriptional co-repressor, regulates hepatic gluconeogenesis and lipogenesis. Here, we examine the epigallocatechin-3-gallate (EGCG) effect on BMP-6-mediated SMAD1/5/8 transactivation of the hepcidin gene. EGCG treatment significantly decreased BMP-6-induced hepcidin gene expression and secretion in hepatocytes, which, in turn, abated ferroportin degradation. SMILE overexpression significantly decreased BMP receptor-induced hepcidin promoter activity. SMILE overexpression also significantly suppressed BMP-6-mediated induction of hepcidin mRNA and its secretion in HepG2 and AML12 cells. EGCG treatment inhibited BMP-6-mediated hepcidin gene expression and secretion, which were significantly reversed by SMILE knockdown in hepatocytes. Interestingly, SMILE physically interacted with SMAD1 in the nucleus and significantly blocked DNA binding of the SMAD complex to the BMP-response element on the hepcidin gene promoter. Taken together, these findings suggest that SMILE is a novel transcriptional repressor of BMP-6-mediated hepcidin gene expression, thus contributing to the control of iron homeostasis.

Involvement of Hepcidin in Cognitive Damage Induced by Chronic Intermittent Hypoxia in Mice

Obstructive sleep apnea (OSA) patients exhibit different degrees of cognitive impairment, which is related to the activation of reactive oxygen species (ROS) production by chronic intermittent hypoxia (CIH) and the deposition of iron in the brain. As a central regulator of iron homeostasis, whether hepcidin is involved in OSA-induced cognitive impairment has not been clarified. In order to simulate OSA, we established the mouse model by reducing the percentage of inspired O2 (FiO2) from 21% to 5%, 20 times/h for 8 h/day. We found hepcidin was rising during CIH, along with increasing iron levels and neuron loss. Then, we constructed a mouse with astrocyte-specific knockdown hepcidin gene (shHamp). During CIH exposure, the shHamp mice showed a lower level of total iron and neuronal iron in the hippocampus, via stabilizing ferroportin 1 (FPN1) and decreasing L-ferritin (FTL) levels, when compared with wild-type (WT) mice. Furthermore, the shHamp mice showed a decrease of ROS by downregulating the elevated NADPH oxidase (NOX2) and 4-hydroxynonenal (4-HNE) levels mediated by CIH. In addition, the shHamp mice presented improved cognitive deficit by improving synaptic plasticity and BDNF expression in the hippocampus when subjected to CIH. Therefore, our data revealed that highly expressed hepcidin might promote the degradation of FPN1, resulting in neuronal iron deposition, oxidative stress damage, reduced synaptic plasticity, and impaired cognitive performance during CIH exposure.

Hepcidin Downregulation Correlates With Disease Aggressiveness And Immune Infiltration in Liver Cancers

BackgroundHepcidin is a polypeptide hormone mainly produced by hepatocytes to modulate systemic iron balance. A drastic downregulation of the hepcidin gene was found in liver cancers. However, there is a paucity of information about the clinical significance of hepcidin gene downregulation in liver cancers.MethodsHepcidin expression profiles were assessed using multiple public datasets via several bioinformatics platforms. Clinical and pathological information was utilized to stratify patients for comparison. Patient survival outcomes were evaluated using the Kaplan-Meier plotter, a meta-analysis tool. Tumor immune infiltration was analyzed using the single sample gene set enrichment analysis (ssGSEA) approach on the Cancer Genome Atlas (TCGA) dataset. Hepcidin antagonist Fursultiamine was used to treat liver cancer HepG2 and Huh7 cells together with Sorafenib.ResultsHepcidin gene was predominantly expressed in benign liver tissues but drastically decreased in liver cancer tissues. Hepcidin reduction in liver cancers correlated with risk factors like non-alcoholic fatty liver disease (NAFLD) and liver fibrosis, as well as cancer grade and tumor stage. Hepcidin downregulation was associated with a rapid cancer progression and worse disease-specific survival, especially in patients of the White race without alcohol consumption history. Hepcidin expression in liver cancer tissues positively correlated with the bone morphogenetic protein-6 (BPM6)/interleukin-6 (IL6) cytokines and cytotoxic immune infiltration. Blocking hepcidin action with its antagonist Fursultiamine moderately reduced Sorafenib-induced apoptotic cell death in HepG2 and Huh7 cells.ConclusionHepcidin downregulation in liver cancers correlated with liver cancer risk factors, cancer aggressiveness, cytotoxic immune cell infiltration, and patient survival outcomes. BMP6/IL6 pathway insufficiency is a potential cause of hepcidin downregulation in liver cancers.

Comparison of the effects of zinc oxide and zinc oxide nanoparticles on the expression of hepcidin gene in rat liver

Objectives Nanoparticles have special properties, such as increased intestinal absorption, permeability, and so on. Zinc oxide (ZnO) nanoparticles have medical applications such as using in drug production. Studies of ZnO nanoparticles have shown the role of these particles in reducing or increasing the genes expression. Given the important role of hepcidin in the development of anemia and iron overload diseases, this study investigated the effect of ZnO nanoparticles on the hepatic expression of the hepcidin gene to help find a way to treat these diseases. Methods In this experimental study, 24 male Westar rats were divided into three groups: control, ZnO treating group and ZnO nanoparticle treating group. Both ZnO and ZnO nanoparticles were injected with 50 mg/kg body weight for 14 days. At the end, serums were collected and iron, ferritin and IL-6 levels were measured. Expression of the hepcidin gene was done by Real Time PCR. Results ZnO and the ZnO nanoparticle significantly increased the expression of the hepcidin gene relative to the control group. The increase in expression of the hepcidin gene in ZnO nanoparticles was more significant than in the ZnO. Conclusion ZnO nanoparticles led to significant increase in expression of the hepcidin gene.

DISC-a, the First in a Novel Class of Potent and Selective Matriptase-2 Inhibitors for the Treatment of Hematologic Disorders Characterized By Low Hepcidin

Hepcidin is known as the master regulator of systemic iron homeostasis with reduction in synthesis leading to the development of iron overload. Hepcidin gene expression is negatively modulated by matriptase-2 (MT-2), a liver-specific type II transmembrane serine protease. MT-2 cleaves hemojuvelin (HJV), leading to the extracellular release of soluble HJV fragments and suppression of hepcidin expression. Loss-of-function of MT-2 leads to increased hepcidin expression, as has been established by human genetics (Finberg et al., 2008) and genetic mouse models (Du et al., 2008). Therefore, inhibition of MT-2 represents a potential therapeutic strategy for diseases caused by inappropriately low hepcidin leading to iron overload or where therapeutic iron restriction may be used to control excessive erythrocytosis. Here we describe the characteristics of DISC-A, a potent (low nM Ki) small molecule MT-2 inhibitor for treatment of low hepcidin disorders, with a favorable pharmacokinetics profile in rats (Clp 6.4 ml/min/kg, and t ½ 4.6 hr) and monkeys (Clp 8.1 ml/min/kg, and t ½ 2.8 hr) and drug-like properties. DISC-A inhibits proteolytic activity of MT-2 expressed on the surface of transfected HEK293 cells and prevents shedding of MT-2 from the membrane (autocleavage). In addition, in MT-2 and HJV co-transfected HEK293A cells, DISC-A shows a dose dependent inhibition of HJV cleavage. The efficacy of DISC-A was evaluated in a rat model of low hepcidin. In this model, when Sprague-Dawley rats who are fed a standard iron diet (45 ppm) reach 8 - 9 weeks of age, they are administered erythropoietin (EPO) at 30 IU/animal/day for 4-consecutive days, before dosing with DISC-A. Under the conditions of the model, the increased erythropoiesis leads to increased iron utilization and consequently suppressed hepcidin levels. We determine hepcidin changes by measuring the changes in the expression of liver HAMP (the gene that encodes hepcidin) mRNA expression. Circulating soluble HJV is assayed as a direct measure of MT-2 activity. In this model, a single subcutaneous administration of DISC-A at 20 mg/kg resulted in a 50% reduction in soluble HJV, 14-fold increase in liver HAMP expression and >50% reduction in serum iron and transferrin saturation (TSAT) at 2, 4, 6, and 8 hours. The pharmacokinetics/pharmacodynamics response was robust. In summary, we have identified DISC-A, a novel, potent inhibitor of MT-2. We have demonstrated that DISC-A inhibits MT-2 proteolytic activity, prevents cleavage of HJV, and modulates hepcidin gene expression and iron homeostasis in vitro and in vivo. The favorable pharmacokinetics suggest compounds from these chemical series have the potential for clinical therapeutic benefit. Disclosures Hong: Disc Medicine: Current Employment, Current equity holder in private company. Babu:Aurigene Discovery Technologies: Current Employment. Blaustein:Disc Medicine: Current Employment, Current equity holder in private company. Nguyen:Disc Medicine: Current Employment, Current equity holder in private company. Rao:Aurigene Discovery Technologies: Current Employment. Savage:Disc Medicine: Current Employment, Current equity holder in private company. MacDonald:Disc Medicine: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Beconi:Disc Medicine: Current Employment, Current equity holder in private company. Venkatraman:Disc Medicine: Current Employment, Current equity holder in private company.