Alk3, a BMP Type I Receptor Is Required for the Induction of Hepatic Hepcidin Gene Expression by Interleukin-6

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 686-686 ◽  
Author(s):  
Andrea U. Steinbicker ◽  
Claire Mayeur ◽  
Lisa K. Lohmeyer ◽  
Patricio Leyton ◽  
Sonya M. Kao ◽  
...  
Keyword(s):  
Gene Expression ◽  
General Hospital ◽  
Small Molecule ◽  
Iron Metabolism ◽  
Serum Iron ◽  
Small Molecule Inhibitors ◽  
Massachusetts General Hospital ◽  
Bmp Signaling ◽  
Type I ◽  
Hepcidin Gene

Abstract Abstract 686 Introduction: Anemia of chronic disease (ACD), the second most prevalent form of anemia, is commonly associated with chronic inflammatory, infectious, or neoplastic conditions. ACD is characterized by high hepcidin levels that decrease serum iron levels by inducing degradation of the iron exporter ferroportin. In contrast, a relative deficiency of hepcidin leads to ferroportin overexpression and iron overload. Hepcidin is transcriptionally regulated by interleukin-6 (IL-6) and bone morphogenic protein (BMP) signaling. Binding of BMP ligands to type II and type I BMP receptors induces the type II receptor to phosphorylate and activate one of four type I receptors. We sought to identify the type I BMP receptor that participates in the ability of IL-6 to induce hepatic hepcidin gene expression. Methods: The four type I BMP receptors are Alk1, Alk2, Alk3, and Alk6. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver. In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. Global deficiency of Alk2 or Alk3 is embryonic lethal. To selectively delete Alk2 or Alk3 in hepatocytes, we studied mice homozygous for Alk2 or Alk3 sequences flanked by loxP sites (Alk2fl/fl and Alk3fl/fl, respectively) that also carried a transgene specifying Cre recombinase under the control of the albumin gene promoter (Alb-Cre). Eight- to 12-week-old male mice (Alk2fl/fl, Alk2fl/fl; Alb-Cre, Alk3fl/fl, Alk3fl/fl; Alb-Cre) on a standard, iron-replete diet were injected via the tail vein with an adenovirus specifying IL-6 (Ad.IL-6) or an adenovirus specifying green fluorescent protein (GFP; Ad.GFP), as a control (1010 particles per ml for both). Seventy-two hours later, mice were euthanized, and blood was obtained for measurement of serum iron levels and transferrin saturations. Livers were harvested, and RNA was extracted. Hepatic levels of mRNAs encoding Alk2, Alk3, hepcidin, heme oxygenase-1 (HO-1, a transcriptional target of IL-6), and Id-1 (a BMP gene target) were measured by qRT-PCR. Hepatic STAT3 phosphorylation (a marker of IL-6 receptor activation) was measured using immunoblot techniques. Results: Liver-specific deletion of Alk2 or Alk3 caused mild and severe iron overload, respectively. Injection of Ad.IL-6, but not Ad.GFP, decreased serum iron levels and transferrin saturations in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. In contrast, infection of Alk3fl/fl; Alb-Cre mice with Ad.IL-6 did not alter serum iron levels and only modestly reduced transferrin saturations. Infection with Ad.IL-6 induced of hepatic hepcidin gene expression in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. Hepatic hepcidin mRNA levels were markedly reduced in Ad.GFP-infected Alk3fl/fl; Alb-Cre mice, and infection with Ad.IL-6 failed to increase hepcidin mRNA levels in this genotype. Ad.IL-6 infection induced hepatic Id-1 mRNA levels in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice, but not in Alk3fl/fl; Alb-Cre mice. Infection with Ad.IL-6 induced hepatic STAT-3 phosphorylation and HO-1 gene expression in all 4 genotypes of mice. Conclusions: Taken together, these results demonstrate that the response to IL-6, as reflected by STAT-3 phosphorylation and induction of HO-1 gene expression, does not require Alk2 or Alk3. In contrast, BMP signaling, predominantly via Alk3, is essential for the induction of hepcidin gene expression by IL-6. These results suggest that selective inhibition of Alk3 may represent a novel therapeutic approach to the treatment of ACD. Disclosures: Peterson: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties.

scholarly journals Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice

Blood ◽  
2011 ◽  
Vol 118 (15) ◽  
pp. 4224-4230 ◽  
Author(s):  
Andrea U. Steinbicker ◽  
Thomas B. Bartnikas ◽  
Lisa K. Lohmeyer ◽  
Patricio Leyton ◽  
Claire Mayeur ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Overload ◽  
Hereditary Hemochromatosis ◽  
Bmp Signaling ◽  
Type I ◽  
Anemia Of Chronic Disease ◽  
Hepcidin Expression ◽  
Hepatic Expression ◽  
Bmp Receptors ◽  
Hepcidin Gene

Abstract Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

Oral Administration Of a BMP Type I Receptor Inhibitor Prevents The Development Of Anemia Of Inflammation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2195-2195
Author(s):  
Claire Mayeur ◽  
Starsha A Kolodziej ◽  
Amy Wang ◽  
Xin Xu ◽  
Arthur Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oral Administration ◽  
Kinase Inhibitor ◽  
Bmp Signaling ◽  
Type I ◽  
Receptor Kinase ◽  
Citrate Buffer ◽  
Orally Bioavailable ◽  
Hepcidin Gene ◽  
Anemia Of Inflammation

Abstract Introduction Anemia of inflammation (AI) is a frequent complication of chronic infection, inflammatory diseases, heart failure, kidney disease, and cancer. Current therapies are directed at the treatment of the underlying disease, as well as parenteral iron supplementation and administration of erythropoiesis-stimulating agents. In AI, inflammatory cytokines, including interleukin 6 (IL6), induce hepatic synthesis of hepcidin, a hormone that reduces serum iron levels. Induction of hepatic hepcidin gene expression by IL6 also requires bone morphogenetic protein (BMP) signaling. Parenteral administration of BMP signaling inhibitors, including LDN-193189 (LDN, a small molecule inhibitor of BMP type I receptor kinases) can increase hemoglobin levels in rodent AI models. Although LDN is orally bioavailable, the ability of enterally-administered LDN to inhibit BMP signaling and ameliorate AI is not known. Methods We studied 10 week old C57bl/6 mice, fed a regular diet. To characterize the pharmacokinetic profile of LDN after oral administration, LDN (1 to 10 mg/kg in citrate buffer, pH 3.1) or vehicle was administered to mice by gavage. Additional mice received a single intraperitoneal injection of LDN (3 mg/kg in citrate buffer, pH 3.1). One and two hours later, mice were sacrificed, and plasma and liver were harvested. Plasma and hepatic LDN levels were measured by chromatography-tandem mass spectrometry. The ability of orally-administered LDN to inhibit hepatic BMP signaling was assessed by measuring phosphorylation of BMP-responsive Smad proteins (Smads 1 and 5) using immunoblot techniques and expression of genes encoding Id-1 (a BMP target gene) and hepcidin using qRT-PCR. To determine whether orally administered LDN can increase hemoglobin (Hb) levels in a mouse AI model, we induced chronic inflammation in mice by subcutaneously administering turpentine (5 mL/kg) once a week for three weeks. As controls, mice received weekly subcutaneous injections of saline. Turpentine-challenged mice were treated daily with LDN (1 mg/kg) or vehicle by gavage. One week after the third turpentine injection, blood was collected for measurement of complete blood counts. Results One hour after an oral administration of LDN (1, 3, and 10 mg/kg), plasma LDN levels were 133±27, 187±46, and 496±105 ng/mL, respectively, and hepatic LDN levels were 267±94, 259±98, and 610±22 ng/g wet tissue weight. Similar plasma and hepatic LDN levels were detected 2 hours after oral administration. One hour after intraperitoneal administration of LDN (3 mg/kg), plasma and liver LDN concentrations were 176±179 ng/mL and 178±80 ng/g, respectively. All three doses of orally-administered LDN markedly reduced hepatic levels of phosphorylated Smads 1 and 5 at one and two hours. Similarly, at one and two hours after oral administration of all three doses of LDN, hepatic Id-1 and hepcidin mRNA levels were decreased by at least 80% and 50%, respectively. Intraperitoneal and oral LDN administration were similarly effective at reducing hepatic Smads 1 and 5 phosphorylation and Id-1 and hepcidin gene expression. In response to turpentine injections, mice developed sterile abscesses associated with a systemic inflammation. Hb levels were less in turpentine-challenged mice that were treated with vehicle than in saline-challenged mice (12.7±0.7 vs 14.9±0.7 g/dL; p<0.0001) and were associated with a reduction in mean corpuscular volume (MCV, 43.2±0.5 vs 45.2±0.8 fl; p<0.0001). Hb levels and MCVs were greater in turpentine-challenged mice that were treated with orally-administered LDN (13.6±0.5 g/dL and 44.1±0.5 fl, respectively) than in turpentine-challenged mice that were treated with vehicle (p<0.005 and p<0.006, respectively). Conclusion LDN is an orally bioavailable BMP type I receptor kinase inhibitor. Oral administration of LDN inhibits hepatic BMP signaling and hepcidin gene expression and can increase hemoglobin levels in a mouse AI model. LDN may represent a novel therapeutic approach to the treatment of AI that does not require parenteral administration. Disclosures: Off Label Use: LDN-193189, BMP type I receptor kinase inhibitor. Yu:MGH: Patents & Royalties. Bloch:MGH: Patents & Royalties.

Heat-Etching with a Bullivant Type II Simple Freeze-Cleave Device

1970 ◽  
Vol 28 ◽  
pp. 106-107 ◽  
Author(s):  
Ronald S. Weinstein ◽  
N. Scott McNutt
Keyword(s):  
New Zealand ◽  
General Hospital ◽  
Massachusetts General Hospital ◽  
Type I ◽  
Type Ii ◽  
Collaborative Effort ◽  
Temperature And Pressure ◽  
The University

The Type I simple cold block device was described by Bullivant and Ames in 1966 and represented the product of the first successful effort to simplify the equipment required to do sophisticated freeze-cleave techniques. Bullivant, Weinstein and Someda described the Type II device which is a modification of the Type I device and was developed as a collaborative effort at the Massachusetts General Hospital and the University of Auckland, New Zealand. The modifications reduced specimen contamination and provided controlled specimen warming for heat-etching of fracture faces. We have now tested the Mass. General Hospital version of the Type II device (called the “Type II-MGH device”) on a wide variety of biological specimens and have established temperature and pressure curves for routine heat-etching with the device.

scholarly journals Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

Immunology ◽  
2019 ◽  
Vol 159 (3) ◽  
pp. 309-321
Author(s):  
Yukang Yuan ◽  
Ying Miao ◽  
Chenhua Zeng ◽  
Jin Liu ◽  
Xiangjie Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Type I Interferon ◽  
Type I ◽  
Antiviral Efficacy ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Role of BMP Signaling In the Anemia of Chronic Disease

Blood ◽  
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.

scholarly journals Changes in Serum Iron and Leukocyte mRNA Levels of Genes Involved in Iron Metabolism in Amateur Marathon Runners—Effect of the Running Pace

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 460 ◽  
Author(s):  
Agata Grzybkowska ◽  
Katarzyna Anczykowska ◽  
Wojciech Ratkowski ◽  
Piotr Aschenbrenner ◽  
Jędrzej Antosiewicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Metabolism ◽  
Serum Iron ◽  
Molecular Mechanisms ◽  
Mrna Levels ◽  
Marathon Runners ◽  
Time Points ◽  
Protein Levels ◽  
Reactive Protein ◽  
Blood Morphology

Iron is essential for physical activity due to its role in energy production pathways and oxygen transportation via hemoglobin and myoglobin. Changes in iron-related biochemical parameters after physical exercise in athletes are of substantial research interest, but molecular mechanisms such as gene expression are still rarely tested in sports. In this paper, we evaluated the mRNA levels of genes related to iron metabolism (PCBP1, PCBP2, FTL, FTH, and TFRC) in leukocytes of 24 amateur runners at four time points: before, immediately after, 3 h after, and 24 h after a marathon. We measured blood morphology as well as serum concentrations of iron, ferritin, and C-reactive protein (CRP). Our results showed significant changes in gene expression (except for TFRC), serum iron, CRP, and morphology after the marathon. However, the alterations in mRNA and protein levels occurred at different time points (immediately and 3 h post-run, respectively). The levels of circulating ferritin remained stable, whereas the number of transcripts in leukocytes differed significantly. We also showed that running pace might influence mRNA expression. Our results indicated that changes in the mRNA of genes involved in iron metabolism occurred independently of serum iron and ferritin concentrations.

Hydroxyurea Induces Hepcidin Expression In Monocytes In Sickle Cell Anemia Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2653-2653
Author(s):  
Kleber Yotsumoto Fertrin ◽  
Carolina Lanaro ◽  
Carla Fernanda Franco-Penteado ◽  
Dulcinéia Martins Albuquerque ◽  
Mariana R. B. Mello ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Iron Metabolism ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Serum Iron ◽  
Iron Status ◽  
Blood Transfusions ◽  
Hepcidin Expression

Abstract Abstract 2653 The antimicrobial peptide hormone hepcidin is a key regulator of iron metabolism. Although mainly produced in the liver, hepcidin is also known to be synthesized by monocytic-macrophagic cells. We have previously shown that hepcidin is overexpressed in mononuclear cells in patients with sickle cell anemia (SCA), but whether monocyte-derived hepcidin production is related to iron metabolism remains unknown. To gather further insight into the role of hepcidin in monocytes, we collected peripheral blood samples from adult SCA patients (n=54) and normal age- and race-matched controls (CTRL, n=13) for assessment of hematological parameters, biochemical markers of iron status (serum iron and transferrin saturation - TfSat), hemolysis (lactate dehydrogenase-LDH) and inflammation (C reactive protein - CRP), and separation of peripheral blood monocytes by Ficoll-Hypaque and Percoll gradients for gene expression analyses of genes involved in iron metabolism signaling (HAMP encoding hepcidin, and three genes belonging to different pathways known to influence hepcidin expression, STAT3, SMAD4 and TLR4). Plasma GDF-15 levels were also measured, as this protein has been shown to be a potent downregulator of hepcidin. SCA patients were further stratified according to the number of previous blood transfusions and to treatment with hydroxyurea (HU). All patients were in steady-state, had no history of iron chelation treatment and were not enrolled in a regular transfusion program. 18 patients were receiving HU and 15 from the non-HU group had received over 20 blood transfusions during their lifetime. As expected, SCA patients had elevated LDH levels, but no differences were found between control and SCA groups regarding serum iron, TfSat or CRP levels. Except for a higher red cell mean corpuscular volume, patients on HU did not differ significantly from patients not using HU. Plasma GDF-15 levels were higher in SCA patients (2146±506.4pg/mL) than in control individuals (228.5±21.0pg/mL, p<0.0001). Among the genes studied, HAMP expression was significantly increased in the SCA group as a whole compared to the CTRL group, but SCA patients on HU had higher levels of monocytic hepcidin expression when compared to the remaining individuals (CTRL 0.043±0.030, SCA on HU 1.240±0.426, remaining SCA 0.332±0.093, p=0.0196). There were no significant correlations between monocytic hepcidin expression and hemoglobin levels, hematocrit, leucocyte or reticulocyte counts, serum iron, LDH or CRP levels, TfSat or transfusion history. STAT3, SMAD4 and TLR4 gene expressions did not differ significantly, suggesting that a possible alternative cause for hepcidin upregulation unrelated to known mechanisms by IL-6, BMP6, LPS or GDF-15 could be an unexpected effect of hydroxyurea. To further investigate if HU was able to modulate hepcidin expression, we performed experiments with THP-1, a human monocytic lineage, since in vitro analysis would allow us to exclude the influence of circulating cytokines elevated in SCA patients. THP-1 cells were cultured in RPMI medium enriched with 10% fetal bovine serum at 37°C and 5% CO2 atmosphere, and were submitted to treatment with water as control, or HU dissolved in water in increasing concentrations (100μM, 400μM and 1600μM) for 6 hours (n≥4). Cell viability was not affected by treatment (>90% viable cells at all experiments), and HAMP gene expression was increased up to four times in the cell cultures exposed to HU (p=0.03), while STAT3 and SMAD4 expressions remained unchanged. We have shown that hepcidin expression is upregulated in monocytes in SCA patients, particularly in those receiving HU, and that HU is capable of inducing this expression in an in vitro model, independently from inflammatory cytokine-mediated stimulation. Our data suggest that, although liver-derived hepcidin has been shown to have a major role in iron metabolism, its monocyte-derived counterpart does not seem to be directly influenced by iron status and may have other functions. Some studies have demonstrated that hepcidin in other species has anti-inflammatory effects in vitro, and that patients with SCA on HU shift to a lower inflammatory status. Thus, monocytic hepcidin overexpression might be a response against the chronic inflammatory state in SCA, and HU treatment may enhance this response. This is the first description of monocyte-derived hepcidin in SCA and the influence of HU on its expression. Disclosures: No relevant conflicts of interest to declare.

TGFβ Signaling Regulates Hepcidin Gene Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 992-992
Author(s):  
Claire Mayeur ◽  
Patricio A Leyton ◽  
Starsha A Kolodziej ◽  
Kenneth D. Bloch
Keyword(s):  
Gene Expression ◽  
Iron Metabolism ◽  
Hepg2 Cells ◽  
Target Gene ◽  
Mrna Levels ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Hepcidin Gene ◽  
Anemia Of Inflammation ◽  
Pai 1

Abstract Abstract 992 Introduction: Hepcidin regulates iron metabolism by reducing duodenal iron absorption and iron release from macrophages and hepatocytes. In inflammatory states, including infection, neoplasia, and heart failure, cytokines induce hepcidin synthesis leading to the development of anemia of inflammation. The regulation of hepcidin gene expression by bone morphogenetic proteins (BMPs), members of the TGFβ family of growth factors, has been extensively investigated. In contrast, less is known about the regulation of hepcidin gene expression by other stimuli, including TGFβ itself. Although TGFβ expression is increased in inflammatory states, the role of TGFβ in the induction of hepcidin gene expression is controversial. To further elucidate the role TGFβ in iron metabolism, we investigated the regulation of hepcidin gene expression in the hepatoma cell line, HepG2. Methods: HepG2 cells were incubated with TGFβ (0.1, 0.5, 1, 2.5, and 5 ng/ml) for varying durations. RNA was extracted for measurement of levels of mRNAs encoding hepcidin, PAI-1 (a TGFβ-target gene), and Id-1 (a BMP-target gene). Cellular proteins were extracted to measure levels of phosphorylated TGFβ-responsive SMADs (using antibodies directed against phosphorylated SMAD2 or SMAD3) and levels of phosphorylated BMP-responsive SMADs (using antibodies directed to phosphorylated SMADs 1 and 5, SMAD1/5). The mechanisms by which TGFβ regulates hepcidin were investigated by pretreating cells with cycloheximide, an inhibitor of protein synthesis (50 μg/mL); Noggin (250 ng/mL) or LDN-193189 (100 nM), inhibitors of BMP signaling; or SB-431542 (5 μM), an inhibitor of the TGFβ type 1 receptor, Alk5. In additional experiments, HepG2 cells were transfected with an siRNA directed against Alk5, 72 hours before exposure to TGFβ. Results: In HepG2 cells, TGFβ induced hepcidin gene expression in a time- and dose-dependent manner: hepcidin mRNA levels were maximal at 2 hours after stimulation with TGFβ (1 ng/ml) and declined thereafter. Incubation of HepG2 cells increased PAI-1 and Id-1 mRNA levels, although increased PAI-1 mRNA levels persisted for at least 8 hours whereas Id-1 mRNA levels peaked at 2 hours. Cycloheximide did not block the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, or Id-1. TGFβ induced phosphorylation of SMADs 2 and 3, as well as SMAD1/5. Pretreatment of HepG2 cells with LDN-193189 (at concentrations that inhibit all four BMP type I receptors, as well as Alk1 which is a target of both BMPs and TGFβ) did not block the ability of TGFβ to induce hepcidin or Id-1 gene expression or phosphorylation of SMADs 2, 3, or 1/5. Pretreatment with Noggin gave similar results. Inhibition of Alk5 with SB-421542 blocked the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, and Id-1, as well as phosphorylation of SMADs 2, 3, or 1/5. TGFβ-stimulated hepcidin gene expression was inhibited by siRNA-mediated knockdown of Alk5. Conclusion: In HepG2 cells, TGFβ induces hepcidin gene expression via a mechanism which requires Alk5. Although, in addition to phosphorylation of SMADs 2 and 3, TGFβ induces phosphorylation of BMP-responsive SMADs, the failure of cycloheximide to inhibit the induction of hepcidin gene expression by TGFβ suggests that synthesis of BMPs is not required. Moreover, the inability of LDN-193189 to inhibit TGFβ-stimulated hepcidin gene expression suggests against a role for activation of Alk1 by TGFβ. Taken together our findings suggest that TGFβ stimulates hepcidin gene expression via a mechanism that requires Alk5 and may be mediated by signaling either via SMADs 2 and 3 or SMAD1/5. Targeting the regulation of hepcidin gene expression by TGFβ may offer a novel therapeutic approach to the anemia of inflammation. Disclosures: No relevant conflicts of interest to declare.

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