Cardiac Hepcidin Expression Associates with Injury Independent of Iron

Background: Hepcidin regulates systemic iron homeostasis by downregulating the iron exporter ferroportin. Circulating hepcidin is mainly derived from the liver but hepcidin is also produced in the heart. We studied the differential and local regulation of hepcidin gene expression in response to myocardial infarction (MI) and/or chronic kidney disease (CKD). We hypothesized that cardiac hepcidin gene expression is induced by and regulated to severity of cardiac injury, either through direct (MI) or remote (CKD) stimuli, as well as through increased local iron content. Methods: Nine weeks after subtotal nephrectomy (SNX) or sham surgery (CON), rats were subjected to coronary ligation (CL) or sham surgery to realize 4 groups: CON, SNX, CL and SNX + CL. In week 16, the gene expression of hepcidin, iron and damage markers in cardiac and liver tissues was assessed by quantitative polymerase chain reaction and ferritin protein expression was studied by immunohistochemistry. Results: Cardiac hepcidin messenger RNA (mRNA) expression was increased 2-fold in CL (p = 0.03) and 3-fold in SNX (p = 0.01). Cardiac ferritin staining was not different among groups. Cardiac hepcidin mRNA expression correlated with mRNA expression levels of brain natriuretic peptide (β = 0.734, p < 0.001) and connective tissue growth factor (β = 0.431, p = 0.02). In contrast, liver hepcidin expression was unaffected by SNX and CL alone, while it had decreased 50% in SNX + CL (p < 0.05). Hepatic ferritin immunostaining was not different among groups. Conclusions: Our data indicate differences in hepcidin regulation in liver and heart and suggest a role for injury rather than iron as the driving force for cardiac hepcidin expression in renocardiac failure.

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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

Blood ◽

10.1182/blood-2020-138509 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 41-42

Author(s):

Vu Hong ◽

Ravi Krishna Babu ◽

Cecile Blaustein ◽

Sophia Nguyen ◽

Venkateshwar Rao ◽

...

Keyword(s):

Gene Expression ◽

Iron Overload ◽

Proteolytic Activity ◽

Iron Homeostasis ◽

Transferrin Saturation ◽

Subcutaneous Administration ◽

Hek293 Cells ◽

Private Company ◽

Hepcidin Expression ◽

Hepcidin Gene

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.

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Estrogen Metabolism in Abdominal Subcutaneous and Visceral Adipose Tissue in Postmenopausal Women

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2017-01474 ◽

2017 ◽

Vol 102 (12) ◽

pp. 4588-4595 ◽

Cited By ~ 33

Author(s):

Natalia Hetemäki ◽

Hanna Savolainen-Peltonen ◽

Matti J Tikkanen ◽

Feng Wang ◽

Hanna Paatela ◽

...

Keyword(s):

Adipose Tissue ◽

Mrna Expression ◽

Postmenopausal Women ◽

Messenger Rna ◽

Metabolic Diseases ◽

Quantitative Polymerase Chain Reaction ◽

Subcutaneous Fat ◽

Estrogen Metabolism ◽

Serum Samples ◽

Estrogen Exposure

Abstract Context In postmenopausal women, adipose tissue (AT) levels of estrogens exceed circulating concentrations. Although increased visceral AT after menopause is related to metabolic diseases, little is known about differences in estrogen metabolism between different AT depots. Objective We compared concentrations of and metabolic pathways producing estrone and estradiol in abdominal subcutaneous and visceral AT in postmenopausal women. Design, Setting, Patients, and Interventions AT and serum samples were obtained from 37 postmenopausal women undergoing surgery for nonmalignant gynecological reasons. Serum and AT estrone, estradiol, and serum estrone sulfate (E1S) concentrations were quantitated using liquid chromatography-tandem mass spectrometry. Activity of steroid sulfatase and reductive 17β-hydroxysteroid dehydrogenase enzymes was measured using radiolabeled precursors. Messenger RNA (mRNA) expression of estrogen-converting enzymes was analyzed by real-time reverse transcription quantitative polymerase chain reaction. Results Estrone concentration was higher in visceral than subcutaneous AT (median, 928 vs 706 pmol/kg; P = 0.002) and correlated positively with body mass index (r = 0.46; P = 0.011). Both AT depots hydrolyzed E1S to estrone, and visceral AT estrone and estradiol concentrations correlated positively with serum E1S. Compared with visceral AT, subcutaneous AT produced more estradiol from estrone (median rate of estradiol production, 1.02 vs 0.57 nmol/kg AT/h; P = 0.004). In visceral AT, the conversion of estrone to estradiol increased with waist circumference (r = 0.65; P = 0.022), and estradiol concentration correlated positively with mRNA expression of HSD17B7 (r = 0.76; P = 0.005). Conclusions Both estrone and estradiol production in visceral AT increased with adiposity, but estradiol was produced more effectively in subcutaneous fat. Both AT depots produced estrone from E1S. Increasing visceral adiposity could increase overall estrogen exposure in postmenopausal women.

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A potential inflammatory role of IL-31 in psoriatic arthritis: A correlation with Th17 cytokine profile

International Journal of Immunopathology and Pharmacology ◽

10.1177/2058738420907186 ◽

2020 ◽

Vol 34 ◽

pp. 205873842090718

Author(s):

LA Bautista-Herrera ◽

U De la Cruz-Mosso ◽

IV Román-Fernández ◽

I Parra-Rojas ◽

JG Soñanez-Organis ◽

...

Keyword(s):

Gene Expression ◽

Psoriatic Arthritis ◽

Mrna Expression ◽

Close Association ◽

Quantitative Polymerase Chain Reaction ◽

Serum Levels ◽

Cytokine Profile ◽

Orphan Receptor ◽

Cross Sectional ◽

Th17 Cytokine

The goals of our study were to determine the possible association of interleukin (IL)-31 with Th17 cytokine profile in serum and to quantify retinoic acid-related orphan receptor C ( RORC) mRNA expression in psoriatic arthritis (PsA) patients. This cross-sectional study was conducted in 50 patients with PsA and 30 control subjects (CS) matched by age and gender. The cytokine serum levels were quantified by magnetic bead–based assay using the Bio-Plex MAGPIX system, and RORC mRNA expression was determined by quantitative polymerase chain reaction (qPCR). As a result, significant differences in IL-31 were observed between study groups (77.23 pg/mL in PsA vs 64.4 pg/mL in CS, P < 0.001) and Th17 cytokine profile serum levels (IL-17A: 6.36 pg/mL in PsA vs 2.97 pg/mL in CS, P = 0.02; IL-17F: 44.15 pg/mL in PsA vs 23.36 pg/mL in PsA, P = 0.01; IL-17E: 3.03 pg/mL in PsA vs 0.82 pg/mL in CS, P < 0.001; IL-21: 36.45 pg/mL in PsA vs 12.44 pg/mL in CS, P = 0.02); however, significant differences were not observed for IL-23 (31.2 pg/mL in PsA vs 53.26 pg/mL in CS, P = 0.58). Furthermore, positive correlations between IL-31 and Th17 cytokine profile serum levels were found (IL-17A: rs = 0.64, P < 0.001; IL-17F: rs = 0.73, P < 0.001; IL-17E: rs = 0.70, P < 0.001; IL-21: rs = 0.54, P = 0.002; IL-23: rs = 0.5, P < 0.01). Regarding RORC gene expression, the PsA group showed an increase of 6.85-fold compared to the CS group. We did not find any association between the serum levels of cytokines and RORC gene expression. In conclusion, in PsA, there are increased serum levels of IL-31, IL-17A, IL-17F, IL-17E, and IL-21, but not IL-23. Moreover, there was a positive correlation of IL-31 with the Th17 cytokine profile and a high RORC gene expression. Altogether, these findings suggest a proinflammatory contribution of IL-31 in close association with the Th17 cytokine profile in PsA.

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Increase of E3 ubiquitin ligase NEDD4 expression leads to degradation of its target proteins PTEN/IGF1R during the formation of goose fatty liver

Journal of Animal Science ◽

10.1093/jas/skaa270 ◽

2020 ◽

Vol 98 (9) ◽

Author(s):

Chunchi Yan ◽

Minmeng Zhao ◽

Shuo Li ◽

Tongjun Liu ◽

Cheng Xu ◽

...

Keyword(s):

Fatty Liver ◽

Mrna Expression ◽

Messenger Rna ◽

Quantitative Polymerase Chain Reaction ◽

Normal Liver ◽

Protective Mechanism ◽

Nonalcoholic Fatty Liver ◽

Target Proteins ◽

Protein Levels ◽

Gene 4

Abstract Goose fatty liver may have a unique protective mechanism as it does not show a pathological injury even in the case of severe steatosis. Although neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) participates in repair and regeneration of injured liver through its target proteins, its role in nonalcoholic fatty liver disease remains unknown. Using quantitative polymerase chain reaction (PCR) and immunoblot analyses, here, we found that the messenger RNA (mRNA) and protein expressions of NEDD4 were induced in goose fatty liver compared with normal liver. The mRNA expression of the gene of phosphate and tension homology deleted on chromosome ten (PTEN) and insulin-like growth factor 1 receptor (IGF1R) was also induced in goose fatty liver; however, their protein expression was or tended to be suppressed. Moreover, the co-immunoprecipitation analysis indicated that there was a physical association between NEDD4 and PTEN in goose liver, which was consistent with the ubiquitination of PTEN in goose fatty liver. Furthermore, NEDD4 overexpression in goose primary hepatocytes suppressed the PTEN and IGF1R protein levels without a significant effect on their mRNA expression. In conclusion, the increased expression of NEDD4 leads to the degradation of PTEN and IGF1R proteins through ubiquitination in goose fatty liver, suggesting that NEDD4 may protect goose fatty liver from severe steatosis-associated injury via its target proteins during the development of goose fatty liver.

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Effect of High Levels of Growth Differentiation Factor 15 (GDF15) On Hepcidin Expression in Monocytes of β-Thalassemia Intermedia Patients.

Blood ◽

10.1182/blood.v114.22.4061.4061 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4061-4061

Author(s):

Kleber Yotsumoto Fertrin ◽

Carolina Lanaro ◽

Carla Fernanda Franco-Penteado ◽

Dulcinéia M Albuquerque ◽

Mariana R. B. Mello ◽

...

Keyword(s):

Gene Expression ◽

Mononuclear Cells ◽

Positive Control ◽

Beta Thalassemia ◽

Iron Loading ◽

Thalassemia Intermedia ◽

Control Groups ◽

Hepcidin Expression ◽

Healthy Control ◽

Hepcidin Gene

Abstract Abstract 4061 Poster Board III-996 Ineffective erythropoiesis in thalassemia has been associated with inappropriate suppression of hepatic synthesis of the key iron regulatory peptide hepcidin, leading to spontaneous iron overload. Hepcidin mRNA was found to be suppressed in hepatocyte cultures by high levels of growth differentiation factor 15 (GDF15) detected in sera from patients with thalassemic syndromes. GDF15 may inhibit hepcidin production by antagonizing positive regulatory cytokines such as bone morphogenic protein 6 (BMP6), shown to stimulate hepatic hepcidin expression in mouse models. Although mainly produced in the liver, human hepcidin production occurs to a lesser extent in circulating monocytes. Studies with monocytes in patients with anemia of chronic disease showed increased hepcidin expression and iron retention, but to the best of our knowledge, monocyte-derived hepcidin has not yet been characterized in iron-loading anemias such as beta-thalassemia intermedia (TI). We evaluated GDF15 plasmatic levels and correlated these to hemoglobin (Hb) levels, reticulocyte counts and gene expressions in monocytes from transfusion-independent, non-chelated TI patients, homozygous for the IVS-I-6 T→C mutation (n=18), healthy, age-matched controls with no iron deficiency or overload (n=10) and transfusion-independent sickle cell anemia (SCA) patients in steady state (n=5), as a positive control group in which hyperexpression of hepcidin in mononuclear cells has been previously demonstrated. Total RNA was extracted from monocytes isolated from peripheral blood mononuclear cells and determination of BMP6 and hepcidin gene expression was performed by Real Time Polymerase Chain Reaction. Plasma GDF15 levels were determined by ELISA. Mean TI patient Hb and serum ferritin levels were 7.05±0.21g/dL and 1846±346.4μg/L, respectively. Mean absolute reticulocyte count in TI was 163.9±21.5×103/mm3. Mean GDF15 plasma levels differences were statistically significant among TI, SCA and healthy control groups (8390±827, 1780±460 and 196±21pg/mL, p<0.0001, respectively). Hepcidin gene expression did not differ significantly between TI and healthy control groups (0.007±0.006 vs. 0.05±0.03, p>0.05, respectively) but was elevated in our positive control SCA patient group (0.56±0.20; p=0.04). BMP6 gene expression was significantly decreased in TI patients compared to healthy controls (1.17±0.15 vs. 0.51±0.11, p=0.01, respectively). GDF15 concentrations correlated positively with reticulocyte counts (r=0.47; p=0.007) and negatively with hemoglobin levels (r=-0.74; p<0.0001) and BMP6 gene expression (r=-0.62; p=0.006). Our data show very high GDF15 plasma levels in a relatively homogenous population of patients with iron overload secondary to beta-thalassemia intermedia. Correlation of GDF15 with hematimetric parameters reinforces its relation to the degree of erythropoietic activity in beta thalassemia due to ineffective erythropoiesis. In addition, our study demonstrates that monocyte-derived hepcidin, like its hepatic counterpart, is inappropriately suppressed in iron-overloaded beta thalassemia intermedia patients in the presence of increased GDF15 production, correlated to decreased levels of BMP6 expression. This supports the possibility that GDF15/BMP interaction regulates hepcidin production in monocytes and hepatocytes in a similar manner, and further studies of monocyte-derived hepcidin regulation may prove to be a suitable, non-invasive tool for the investigation of human liver-derived hepcidin pathways in thalassemia and other iron-loading anemias. Disclosures: No relevant conflicts of interest to declare.

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Role of BMP Signaling In the Anemia of Chronic Disease

Blood ◽

10.1182/blood.v116.21.2043.2043 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2043-2043

Author(s):

Andrea U. Steinbicker ◽

Ashley J. Vonner ◽

Chetana Sachidanandan ◽

Lisa Lohmeyer ◽

David T. Scadden ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Research Funding ◽

Critical Role ◽

Bmp Signaling ◽

Mrna Levels ◽

Anemia Of Chronic Disease ◽

Hepcidin Expression ◽

Hepcidin Gene ◽

Hepcidin Mrna

Abstract Abstract 2043 Introduction: Anemia of chronic disease (ACD) describes anemia associated with diverse chronic inflammatory, infectious, or neoplastic processes. These conditions are frequently associated with increased circulating levels of inflammatory cytokines such as interleukin 6 (IL-6). IL-6 regulates expression of the hormone hepcidin, which inhibits the release of iron from hepatocytes, macrophages, and enterocytes into the circulation. In addition to IL-6, hepcidin gene expression is known to be transcriptionally regulated by bone morphogenetic protein (BMP) signaling. Hypothesis: We hypothesized that BMP signaling is required for the induction of hepcidin gene expression by IL-6 and plays a critical role in the pathogenesis of ACD. Methods: We used a turpentine-dependent model of ACD in mice. Mice were challenged with weekly subcutaneous injections of turpentine, which induces anemia in an IL-6 dependent manner. This model was studied to determine hepcidin gene expression and rescue ACD using BMP inhibition. Moreover, we examined hepcidin gene expression in zebrafish injected with Pseudomonas aeruginosa, and in transgenic zebrafish overexpressing human IL-6. The regulation of hepcidin gene expression was also studied in the human hepatocarcinoma cell line (HepG2). Results: Injections of mice with IL-6 (0.8 μg/g ip) increased hepatic hepcidin mRNA levels expression at 24 hours and decreased serum iron concentrations. Both effects were prevented by a small molecule BMP type I receptor kinase inhibitor, LDN-193189, or protein BMP antagonists. Weekly turpentine injections induced microcytic anemia after 3 weeks with a decrease in hemoglobin levels from 12.8±0.3 to 9.7±1.7 g/dL (*p<0.01). Concurrent treatment with LDN-193189 prevented turpentine-induced anemia and microcytosis (*p<0.01 for both). In mice challenged with turpentine for 6 weeks, treatment with LDN-193189, beginning after anemia was established at week 3, led to an increase in hemoglobin levels at week 6 (10.9±0.1 vs 9.5±0.2 g/dL, LDN193189 vs vehicle, respectively; *p<0.05). In zebrafish, microinjection with Pseudomonas aeruginosa or overexpression of human IL-6 induced hepatic hepcidin expression, an effect which was blocked by LDN-193189. Incubation of HepG2 cells with IL-6 (100 ng/ml) increased hepcidin mRNA levels 2 to 5 fold. Pretreatment with LDN-193189, or recombinant protein BMP antagonists such as noggin, abrogated the induction of hepcidin expression by IL-6. Incubation of HepG2 cells with BMP6 (2.5 to 10 ng/ml) modestly increased hepcidin mRNA levels. However, the combination of IL-6 and BMP6 synergistically increased hepcidin gene expression (*p<0.05). Conclusion: BMP signaling appears to play a critical role in the pathogenesis of anemia in a mouse ACD model. Our findings support the concept that BMP signaling is required for the induction of hepcidin gene expression by IL-6 in vitro and in vivo. Moreover, manipulation of BMP signaling represents a potentially novel therapeutic approach to the treatment of anemia associated with inflammation. Disclosures: Steinbicker: Deutsche Forschungsgemeinschaft DFG: Research Funding. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Peterson:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Bloch:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Yu:Harvard Stem Cell Institute Seed Grant and the Howard Hughes Medical Institute Early Career Physician-Scientist Award: Honoraria, Research Funding; NHLBI 5K08HL079943: Research Funding.

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Paternal High Fat Diet and Exercise Regulate Sperm miRNA to Alter Placental Inflammation and Nutrient Transporter Expression in a Sex-Dependent Manner

Current Developments in Nutrition ◽

10.1093/cdn/nzaa054_096 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1024-1024

Author(s):

Kate Larson ◽

Amy Bundy ◽

James Roemmich

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Mirna Expression ◽

Placental Tissue ◽

Tissue Growth ◽

Tnf Alpha ◽

Dependent Manner ◽

High Fat ◽

Diet And Exercise ◽

Nutrient Transporter

Abstract Objectives We have shown that male offspring (F1) of fathers (F0) fed a high fat (HF) diet and exercised had greater skeletal muscle insulin signaling and reduced T2DM risk compared to fathers fed HF diet and remain sedentary. The current study extends this work by hypothesizing that F0 HF diet and exercise regulate F1 T2DM risk by early alterations in epigenetics of placental tissue growth via changes in sperm miRNA expression. Methods To test these hypotheses, three-week old male C57BL/6 mice were fed a normal-fat (NF) diet (16% fat) or a HF diet (45% fat) and assigned to either voluntary wheel running exercise or cage activity for 3 months prior to mating with NF diet fed dams. F0 sperm and placental tissue samples were collected to determine changes in placental and fetal weights, placental gene expression, and F0 sperm miRNA expression. Results F0 sperm miRNA 193b expression was decreased while miRNA 204 was increased by paternal exercise. Protein expression of di-methylated histone 3 lysine 9 was decreased with F0 HF diet. Placental and fetal tissue weights were decreased by F0 HF diet in F1 males while no changes in the F1 females. Placental proinflammatory cytokine mRNA expression, including IL-1 beta and TNF-alpha, was reduced by paternal exercise while nutrient transporter mRNA expression was decreased by paternal HF diet only in the placentae of F1 females. Treatment of primary placental cell with miRNA 193 inhibited TNF-alpha mRNA expression. In addition, treatment of the same cells with TNF-alpha increased SLC6a19. Moreover, paternal exercise increased body weight at weaning in a female offspring. Conclusions These results demonstrate that placental tissue weight, placental nutrient transporter gene expression and fetal weights are altered by paternal exercise while placental inflammatory gene expression are influenced by paternal exercise in offspring in a sex-specific manner. Funding Sources This work was supported by USDA ARS Project #3062–51,000-054–00D.

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Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

AJP Cell Physiology ◽

10.1152/ajpcell.00121.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. C888-C895 ◽

Cited By ~ 33

Author(s):

Timothy B. Chaston ◽

Pavle Matak ◽

Katayoun Pourvali ◽

Surjit K. Srai ◽

Andrew T. McKie ◽

...

Keyword(s):

Protein Expression ◽

Mrna Expression ◽

Promoter Activity ◽

Quantitative Polymerase Chain Reaction ◽

Hepatoma Cells ◽

Luciferase Reporter ◽

Smad Signaling ◽

Hepcidin Expression ◽

Huh7 Cells ◽

Hepcidin Mrna

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H2O2, hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

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Chronic ethanol feeding enhances miR-21 induction during liver regeneration while inhibiting proliferation in rats

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00019.2012 ◽

2012 ◽

Vol 303 (6) ◽

pp. G733-G743 ◽

Cited By ~ 33

Author(s):

Rachael P. Dippold ◽

Rajanikanth Vadigepalli ◽

Gregory E. Gonye ◽

Jan B. Hoek

Keyword(s):

Gene Expression ◽

Liver Regeneration ◽

Messenger Rna ◽

Expression Profiles ◽

Chronic Ethanol ◽

Sham Surgery ◽

Chronic Ethanol Consumption ◽

Mirna Expression Profiles ◽

Ethanol Feeding ◽

And Control

Liver regeneration is an important repair response to liver injury. Chronic ethanol consumption inhibits and delays liver regeneration in experimental animals. We studied the effects of chronic ethanol treatment on messenger RNA (mRNA) and microRNA (miRNA) expression profiles during the first 24 h after two-thirds partial hepatectomy (PHx) and found an increase in hepatic miR-21 expression in both ethanol-fed and pair-fed control rats after PHx. We demonstrate that the increase of miR-21 expression during liver regeneration is more robust in ethanol-fed rats. Peak miR-21 expression occurs at 24 h after PHx in both ethanol-fed and control rats, corresponding to the peak of hepatocyte S phase in control rats, but not in ethanol-exposed livers in which cell cycle is delayed. The induction of miR-21 24 h after PHx in control rats is not greater than the increase in expression of miR-21 due to sham surgery. However, in the ethanol-fed rat, miR-21 is induced to a greater extent by PHx than by sham surgery. To elucidate the implications of increased miR-21 expression during liver regeneration, we employed unbiased global target analysis using gene expression data compiled by our group. Our analyses suggest that miR-21 may play a greater role in regulating gene expression during regeneration in the ethanol-fed rat than in the control rat. Our analysis of potential targets of miR-21 suggests that miR-21 affects a broad range of target processes and may have a widespread regulatory role under conditions of suppressed liver regeneration in ethanol-treated animals.

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Transferrin receptor 1 controls systemic iron homeostasis by fine-tuning hepcidin expression to hepatocellular iron load

Blood ◽

10.1182/blood-2018-05-850404 ◽

2019 ◽

Vol 133 (4) ◽

pp. 344-355 ◽

Cited By ~ 20

Author(s):

Carine Fillebeen ◽

Edouard Charlebois ◽

John Wagner ◽

Angeliki Katsarou ◽

Jeannie Mui ◽

...

Keyword(s):

Transferrin Receptor ◽

Messenger Rna ◽

Iron Homeostasis ◽

Fine Tuning ◽

Deficiency Anemia ◽

Liver Iron ◽

Regulatory Pathways ◽

Hepcidin Expression ◽

Iron Load ◽

Transferrin Receptor 1

Abstract Transferrin receptor 1 (Tfr1) mediates uptake of circulating transferrin-bound iron to developing erythroid cells and other cell types. Its critical physiological function is highlighted by the embryonic lethal phenotype of Tfr1-knockout (Tfrc−/−) mice and the pathologies of several tissue-specific knockouts. We generated TfrcAlb-Cre mice bearing hepatocyte-specific ablation of Tfr1 to explore implications in hepatocellular and systemic iron homeostasis. TfrcAlb-Cre mice are viable and do not display any apparent liver pathology. Nevertheless, their liver iron content (LIC) is lower compared with that of control Tfrcfl/fl littermates as a result of the reduced capacity of Tfr1-deficient hepatocytes to internalize iron from transferrin. Even though liver Hamp messenger RNA (mRNA) and serum hepcidin levels do not differ between TfrcAlb-Cre and Tfrcfl/fl mice, Hamp/LIC and hepcidin/LIC ratios are significantly higher in the former. Importantly, this is accompanied by modest hypoferremia and microcytosis, and it predisposes TfrcAlb-Cre mice to iron-deficiency anemia. TfrcAlb-Cre mice appropriately regulate Hamp expression following dietary iron manipulations or holo-transferrin injection. Holo-transferrin also triggers proper induction of Hamp mRNA, ferritin, and Tfr2 in primary TfrcAlb-Cre hepatocytes. We further show that these cells can acquire 59Fe from 59Fe-transferrin, presumably via Tfr2. We conclude that Tfr1 is redundant for basal hepatocellular iron supply but essential for fine-tuning hepcidin responses according to the iron load of hepatocytes. Our data are consistent with an inhibitory function of Tfr1 on iron signaling to hepcidin via its interaction with Hfe. Moreover, they highlight hepatocellular Tfr1 as a link between cellular and systemic iron-regulatory pathways.

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