Transcriptional Regulation Of Hepcidin Research Articles

Melatonin Regulates Iron Homeostasis by Inducing Hepcidin Expression in Hepatocytes.

The pineal hormone, melatonin, plays important roles in circadian rhythms and energy metabolism. The hepatic peptide hormone, hepcidin, regulates iron homeostasis by triggering the degradation of ferroportin (FPN), the protein that transfers cellular iron to the blood. However, the role of melatonin in the transcriptional regulation of hepcidin is largely unknown. Here, we showed that melatonin upregulates hepcidin gene expression by enhancing the melatonin receptor 1 (MT1)-mediated c-Jun N-terminal kinase (JNK) activation in hepatocytes. Interestingly, hepcidin gene expression was increased during the dark cycle in the liver of mice, whereas serum iron levels decreased following hepcidin expression. In addition, melatonin significantly induced hepcidin gene expression and secretion, as well as the subsequent FPN degradation in hepatocytes, which resulted in cellular iron accumulation. Melatonin-induced hepcidin expression was significantly decreased by the melatonin receptor antagonist, luzindole, and by the knockdown of MT1. Moreover, melatonin activated JNK signaling and upregulated hepcidin expression, both of which were significantly decreased by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation analysis showed that luzindole significantly blocked melatonin-induced c-Jun binding to the hepcidin promoter. Finally, melatonin induced hepcidin expression and secretion by activating the JNK-c-Jun pathway in mice, which were reversed by the luzindole treatment. These findings reveal a previously unrecognized role of melatonin in the circadian regulation of hepcidin expression and iron homeostasis.

Mo1772 Lipopolysaccharides Can Accelerate the Development of Nonalcoholic Fatty Liver Disease, Caused by the Up-Regulation of Lipogenic Gene Expressions

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1774 Sitagliptin Can Inhibit the Acceleration of the Development in NAFLD Caused by Lipopolysaccharides Through Inhibiting Expression of MYD88 and NfκB

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1771 Adiponectin Antagonises Leptin-Induced Hepcidin Expression in Human Liver Cells: New Insights Into Obesity-Associated Iron Deficiency

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Transcriptional Regulation of Hepcidin by Iron.

Transcriptional regulation by iron in mammalian systems is poorly understood. Hepcidin, a 25 amino acid peptide that plays a central role in iron homeostasis, is transcriptionally regulated by iron. A region of the murine hepcidin promoter 1.6 to 1.8 kb upstream from the start of translation was recently identified to be important in transcriptional regulation by iron (Truksa J, et al. The distal location of the iron responsive region of the hepcidin promoter. Blood DOI 10.1182/blood-2007-05-091108, 2007). In order to identify transcription factors that might be important in regulation by iron, transcription factor microarray analyses (Panomics TranSignal Protein/DNA Array) were performed with nuclear extracts from livers of mice made iron deficient or iron loaded for 4 weeks. The analyses revealed 43 transcription factors that were upregulated in iron loaded liver nuclear extracts and 39 transcription factors that were upregulated in iron deficient nuclear extracts. In the region of the promoter we had found essential for transcriptional regulation by iron, −1.6 to −1.8 kb, consensus motifs were identified by Genomatix MatInspector for 10 transcription factors that corresponded to transcription factors upregulated in high iron nuclear extracts by array analyses. Similarly, the consensus sequences for 5 transcription factors corresponded to transcription factors identified in iron deficient nuclear extracts. Electrophoretic mobility shift assays were performed with probes across this region of the murine hepcidin promoter. Several probes exhibited differential binding between deficient and high iron nuclear extracts. These include the probe encompassing the CCAAT box and MEL1 motif, a probe containing a HLH motif, and a probe containing a bZIP and COUP motif. The probe containing the CCAAT motif was supershifted with antibodies against CBF, but was not supershifted with antibodies against SMAD4, CEBPα, and COUP. The probe containing a bZIP and COUP motif can be supershifted with antibodies against COUP-Tf and HNF4α, but not with antibodies against SMAD4, CEBPα, and COUP. Our data suggest that CBFA, COUP, and HNF4α are involved in transcriptional regulation of hepcidin by iron.

Hepcidin, a key regulator of iron metabolism, is transcriptionally activated by p53

Hepcidin is an iron-regulatory protein that is upregulated in response to increased iron or inflammatory stimuli. Hepcidin reduces serum iron and induces iron sequestration in the reticuloendothelial macrophages - the hallmark of anaemia of inflammation. Iron deprivation is used as a defense mechanism against infection, and it also has a beneficial effect on the control of cancer. The tumour-suppressor p53 transcriptionally regulates genes involved in growth arrest, apoptosis and DNA repair, and perturbation of p53 pathways is a hallmark of the majority of human cancers. This study inspected a role of p53 in the transcriptional regulation of hepcidin. Based on preliminary bioinformatics analysis, we identified a putative p53 response-element (p53RE) contained in the hepcidin gene (HAMP) promoter. Chromatin immunoprecipitation (ChIP), reporter assays and a temperature sensitive p53 cell-line system were used to demonstrate p53 binding and activation of the hepcidin promoter. p53 bound to hepcidin p53RE in vivo, andthis p53RE could confer p53-dependent transcriptional activation. Activation of p53 increased hepcidin expression, while silencing of p53 resulted in decreased hepcidin expression in human hepatoma cells. Taken together, these results define HAMP as a novel transcriptional target of p53. We hypothesise that hepcidin upregulation by p53 is part of a defence mechanism against cancer, through iron deprivation. Hepcidin induction by p53 might be involved in the pathogenesis of anaemia accompanying cancer.