STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 353-358 ◽  
Author(s):  
Maria Vittoria Verga Falzacappa ◽  
Maja Vujic Spasic ◽  
Regina Kessler ◽  
Jens Stolte ◽  
Matthias W. Hentze ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Culture Conditions ◽  
Control Culture ◽  
Binding Motif ◽  
Iron Release ◽  
Hepcidin Expression ◽  
Inflammatory Conditions ◽  
Human Liver Cell ◽  
Hepcidin Mrna

Abstract Hepcidin is a key iron-regulatory hormone produced by the liver. Inappropriately low hepcidin levels cause iron overload, while increased hepcidin expression plays an important role in the anemia of inflammation (AI) by restricting intestinal iron absorption and macrophage iron release. Its expression is modulated in response to body iron stores, hypoxia, and inflammatory and infectious stimuli involving at least in part cytokines secreted by macrophages. In this study we established and characterized IL6-mediated hepcidin activation in the human liver cell line Huh7. We show that the proximal 165 bp of the hepcidin promoter is critical for hepcidin activation in response to exogenously administered IL6 or to conditioned medium from the monocyte/macrophage cell line THP-1. Importantly, we show that hepcidin activation by these stimuli requires a STAT3 binding motif located at position –64/–72 of the promoter. The same STAT binding site is also required for high basal-level hepcidin mRNA expression under control culture conditions, and siRNA-mediated RNA knockdown of STAT3 strongly reduces hepcidin mRNA expression. These results identify a missing link in the acute-phase activation of hepcidin and establish STAT3 as a key effector of baseline hepcidin expression and during inflammatory conditions.

Inflammatory Cytokine IL-1β Upregulates Hepcidin Expression in Human Hepatoma Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3593-3593
Author(s):  
Katsuya Ikuta ◽  
Junki Inamura ◽  
Junko Jimbo ◽  
Takaaki Hosoki ◽  
Motohiro Shindo ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Inflammatory Cytokines ◽  
Iron Metabolism ◽  
Inflammatory Cytokine ◽  
Iron Absorption ◽  
Human Hepatoma ◽  
Hepcidin Expression ◽  
Mrna Induction ◽  
Hepcidin Mrna

Abstract The anemia of chronic disease (ACD) is commonly observed in patients with inflammatory disorders, malignancies, and chronic infections. ACD is characterized by low serum iron concentration and elevated serum ferritin concentration. A number of previous findings suggest that impaired mobilization of iron from the reticuloendotherial system (RES), such as macrophages, and an inhibition of iron absorption from intestine contribute to ACD. However, the precise mechanisms for generating the conditions of ACD have long been unclear despite extensive investigations until hepcidin was recently found. Hepcidin was found as one of antimicrobial peptides, but hepcidin is now thought to be a key regulator in iron metabolism. Hepcidin inhibits iron absorption from enterocytes and iron efflux from RES, which have suggested that hepcidin could account for the pathogenesis of ACD. Some cytokines have also been shown to modulate iron metabolism in the condition of inflammation. Interleukin-6 (IL-6), one of inflammatory cytokines, has been reported to induce hepcidin production not only in vitro but also in vivo. However, it is not clear whether other cytokines have such effect or not. We, therefore, investigated the possibility that other inflammatory cytokines might have a regulatory effect on hepcidin expression. Because hepcidin is produced mainly by hepatocytes, we used human hepatoma-derived cell line HuH-7 cells and hepatoblastoma-derived cell line HepG2 cells. Cells are incubated with or without inflammatory cytokines, such as IL-1β, IL-6, tumor necrosis factor α (TNFα), and then total RNA was extracted. Quantitative RT-PCR was then performed, revealing that IL-1β upregulate hepcidin mRNA expression as well as IL-6, although TNFα down regulates hepcidin expression in both cell lines. We next investigated the dose-response effect of IL-1β and IL-6 on hepcidin mRNA expression. Strong induction of hepcidin mRNA by IL-1β was observed when cells were incubated with low concentrations of IL-1β (0.2 ng/ml), although much higher concentrations of IL-6 were needed for hepcidin mRNA upregulation. It was likely that the concentrations of IL-1β that needed for hepcidin upregulation were more physiological than those of IL-6. Since it has been reported that IL-1β induce IL-6 production in hepatocytes, there was a possibility that the effect of IL-1β on hepcidin mRNA expression was not direct but come from IL-6 induced by IL-1β. However, the manners of IL-6 mRNA induction and hepcidin mRNA induction by IL-1β stimulation were quite different when cells were incubated with IL-1β at the concentrations ranging from 0.1 to 10 ng/ml. Moreover, antibody against IL-6 did not inhibit the induction of hepcidin mRNA by IL-1β stimulation. Therefore, it is likely that the effect of IL-1β on hepcidin mRNA expression is independent from that of IL-6. We conclude that inflammatory cytokine IL-1β can induce hepcidin expression and might be a key cytokine in the condition of ACD as well as IL-6, and IL-1β might be more important than IL-6 in physiological situations.

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

2011 ◽  
Vol 300 (4) ◽  
pp. C888-C895 ◽  
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.

Competitive regulation of hepcidin mRNA by soluble and cell-associated hemojuvelin

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2884-2889 ◽  
Author(s):  
Lan Lin ◽  
Y. Paul Goldberg ◽  
Tomas Ganz
Keyword(s):  
Mrna Expression ◽  
Iron Homeostasis ◽  
Iron Concentration ◽  
Protein Product ◽  
Dependent Manner ◽  
Hepcidin Expression ◽  
Juvenile Hemochromatosis ◽  
Repulsive Guidance Molecule ◽  
Hepcidin Mrna

Abstract Mutations in a recently identified gene HJV (also called HFE2, or repulsive guidance molecule C, RgmC) are the major cause of juvenile hemochromatosis (JH). The protein product of HJV, hemojuvelin, contains a C-terminal glycosylphosphatidylinositol anchor, suggesting that it can be present in either a soluble or a cell-associated form. Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. However, neither the specific role of hemojuvelin in maintaining iron homeostasis nor its relationship to hepcidin has been experimentally established. In this study we used hemojuvelin-specific siRNAs to vary hemojuvelin mRNA concentration and showed that cellular hemojuvelin positively regulated hepcidin mRNA expression, independently of the interleukin 6 pathway. We also showed that recombinant soluble hemojuvelin (rs-hemojuvelin) suppressed hepcidin mRNA expression in primary human hepatocytes in a log-linear dose-dependent manner, suggesting binding competition between soluble and cell-associated hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron concentrations. We propose that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin expression in response to changes in extracellular iron concentration. (Blood. 2005;106:2884-2889)

Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat

2004 ◽  
Vol 286 (3) ◽  
pp. G385-G394 ◽  
Author(s):  
Kwo-yih Yeh ◽  
Mary Yeh ◽  
Jonathan Glass
Keyword(s):  
Mrna Expression ◽  
Serum Iron ◽  
Protein Production ◽  
Iron Absorption ◽  
Iron Concentration ◽  
Iron Chelator ◽  
Hepcidin Expression ◽  
Ferroportin 1 ◽  
Initial Decrease ◽  
Hepcidin Mrna

Hepcidin has been implicated as the iron stores regulator: a hepatic signaling molecule that regulates intestinal iron absorption by undefined mechanisms. The possibility that hepcidin regulates the expression of ferroportin 1 (FPT1), the basolateral iron transporter, was examined in rats after administration of LPS, an iron chelator, or His-tagged recombinant hepcidin (His-rHepc). In the liver, LPS stimulated a biphasic increase of hepcidin mRNA with peaks of mRNA at 6 and 36 h. Concurrently, hepatic FPT1 mRNA expression decreased to minimal level at 6 h and then increased with a peak at 24–36 h. LPS also induced biphasic changes in intestinal FPT1 mRNA expression, with decreased levels at 6 h and increased expression at 48 h. Whereas the initial decrease of FPT1 coincides with an LPS-induced decrease in serum iron, both intestinal and hepatic FPT1 expression recovered, whereas serum iron concentration continued to decrease for at least 24 h. Dietary iron ingestion increased intestinal ferritin protein production but did not reduce intestinal FPT1 mRNA expression. The iron chelator pyrrolidinedithiocarbamate (PDTC) stimulated hepatic hepcidin without suppressing intestinal FPT1 expression. In PDTC-treated rats, LPS stimulated no additional hepatic hepcidin expression but did increase intestinal FPT1 expression. Administration of HisrHepc induced significant reduction of intestinal FPT1 expression. Taken together, these data suggest that hepcidin mediates LPS-induced downregulation of intestinal FPT1 expression and that the hepcidin signaling pathway involves a PDTC-sensitive step.

Autocrine formation of hepcidin induces iron retention in human monocytes

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2392-2399 ◽  
Author(s):  
Igor Theurl ◽  
Milan Theurl ◽  
Markus Seifert ◽  
Sabine Mair ◽  
Manfred Nairz ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Iron Homeostasis ◽  
Mrna Levels ◽  
Anemia Of Chronic Disease ◽  
Hepcidin Expression ◽  
Inflammatory Monocytes ◽  
Cellular Iron ◽  
Iron Sequestration ◽  
Iron Retention ◽  
Hepcidin Mrna

Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

2011 ◽  
Vol 18 (3) ◽  
pp. 438-446 ◽  
Author(s):  
José P Oliveira-Filho ◽  
Peres R Badial ◽  
Paulo HJ Cunha ◽  
Juliana R Peiró ◽  
João P Araújo ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Hepcidin Mrna

CYTOKINE mRNA EXPRESSION IN HUMAN PLATELETS AND A MEGAKARYOCYTIC CELL LINE AND CYTOKINE MODULATION OF PLATELET FUNCTION

Cytokine ◽  
1997 ◽  
Vol 9 (6) ◽  
pp. 405-411 ◽  
Author(s):  
Gerald Soslau ◽  
Doris A. Morgan ◽  
Jonathan S. Jaffe ◽  
Isadore Brodsky ◽  
Yihe Wang
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Platelet Function ◽  
Human Platelets ◽  
Cytokine Mrna ◽  
Cytokine Modulation ◽  
Cytokine Mrna Expression ◽  
Megakaryocytic Cell

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

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