Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies

The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 μM Fe). Caco-2 cells were exposed to 0, 80 and 120 μM Fe and responded with increased hepcidin production at 120 μM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered.     

Effect of <Emphasis Type="Italic">Boswellia serrata</Emphasis> Resin Supplementation on Basic Chemical and Mineral Element Composition in the Muscles and Liver of Broiler Chickens

Hepcidin synthesis is reported to be inadequate according to the body iron store in patients with non-alcoholic fatty liver disease (NAFLD) undergoing hepatic iron overload (HIO). However, the underlying mechanisms remain unclear. We hypothesize that hepatocyte nuclear factor-4α (HNF-4α) may negatively regulate hepcidin expression and contribute to hepcidin deficiency in NAFLD patients. The effect of HNF-4α on hepcidin expression was observed by transfecting specific HNF-4α small interfering RNA (siRNA) or plasmids into HepG2 cells. Both direct and indirect mechanisms involved in the regulation of HNF-4α on hepcidin were detected by real-time PCR, Western blotting, chromatin immunoprecipitation (chIP), and reporter genes. It was found that HNF-4α suppressed hepcidin messenger RNA (mRNA) and protein expressions in HepG2 cells, and this suppressive effect was independent of the potential HNF-4α response elements. Phosphorylation of SMAD1 but not STAT3 was inactivated by HNF-4α, and the SMAD4 response element was found essential to HNF-4α-induced hepcidin reduction. Neither inhibitory SMADs, SMAD6, and SMAD7 nor BMPR ligands, BMP2, BMP4, BMP6, and BMP7 were regulated by HNF-4α in HepG2 cells. BMPR1A, but not BMPR1B, BMPR2, ActR2A, ActR2B, or HJV, was decreased by HNF-4α, and HNF4α-knockdown-induced stimulation of hepcidin could be entirely blocked when BMPR1A was interfered with at the same time. In conclusion, the present study suggests that HNF-4α has a suppressive effect on hepcidin expression by inactivating the BMP pathway, specifically via BMPR1A, in HepG2 cells.     

Hepcidin, an antimicrobial peptide is downregulated in ceruloplasmin-deficient mice

Hepcidin, a principle regulator of iron metabolism, is synthesized by the liver. Contradictory results have been reported on the regulation of hepcidin expression in response to serum transferrin saturation and liver iron content. In the present study, we explore the expression of murine hepcidin mRNA and further analyze the relationship between liver hepcidin mRNA expression, liver iron stores, and serum iron level utilizing ceruloplasmin gene knockout mice. We find that hepcidin expression correlates significantly with serum transferrin saturation, whereas there is a negative correlation of hepcidin expression with liver tissue iron level.     

BMPER protein is a negative regulator of hepcidin and is up-regulated in hypotransferrinemic mice

The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.     

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down‐regulated by alcohol in cell lines and animal models. This down‐regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real‐time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down‐regulation of hepcidin expression leading to up‐regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.     

Alkaloid fraction of Mirabilis jalapa Linn. flowers has low cytotoxicity and increases iron absorption through Erythropoietin-Matriptase-2-Hepcidin pathway in iron deficiency Hepatocarcinoma cell model

In this study, we investigated the effects of an alkaloid fraction of Mirabilis jalapa L. flowers in terms of cytotoxicity, Erythropoietin (EPO), hepcidin, and Matriptase-2 (MT-2) expression levels in iron deficiency Hepatocarcinoma (HepG2) cell model. The iron deficiency HepG2 cell model was generated by induction with Deferoxamine (DFO) and was then treated with standard therapy Ferric Ammonium Citrate (FAC) and different alkaloid fraction doses. Subsequently, the type II transmembrane serine proteases (TTSPs) activity and MT-2 expression were measured using a fluorometer and immunocytochemistry methods, while the EPO and hepcidin levels and total iron were examined using an ELISA kit and a colorimetric assay, respectively. The data were then analyzed using ANOVA with a significance level of 95 %. According to the UV–vis Spectrophotometry and HPLC results, the alkaloid fraction of M. jalapa flowers had 6.17- and 4-times higher Betaxanthin levels, respectively, compared to the ethanol extract of M. jalapa flower. Furthermore, LC-MS/MS analysis showed that the most dominant compound is Indicaxanthin. The ethanol extract and alkaloid fraction of M. jalapa flowers were not cytotoxic (IC₅₀ > 30 ppm). Furthermore, the alkaloid fraction containing Indicaxanthin, Miraxanthin-V, and Boeravinone F is capable of increasing EPO levels, membrane and soluble TTSPs activity and MT-2 expression, decreasing hepcidin levels, and increasing intracellular iron levels in iron deficiency HepG2 cell model. In conclusion, the obtained alkaloid fraction of M. jalapa flowers has low cytotoxicity and the later increases iron absorption via EPO-MT2-hepcidin pathway in iron deficiency HepG2 cell model.     

The hereditary hemochromatosis protein, HFE, inhibits iron uptake via down-regulation of Zip14 in HepG2 cells

Lack of functional hereditary hemochromatosis protein, HFE, causes iron overload predominantly in hepatocytes, the major site of HFE expression in the liver. In this study, we investigated the role of HFE in the regulation of both transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI) uptake in HepG2 cells, a human hepatoma cell line. Expression of HFE decreased both TBI and NTBI uptake. It also resulted in a decrease in the protein levels of Zip14 with no evident change in the mRNA level of Zip14. Zip14 (Slc39a14) is a metal transporter that mediates NTBI into cells (Liuzzi, J. P., Aydemir, F., Nam, H., Knutson, M. D., and Cousins, R. J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 13612-13617). Knockdown of Zip14 with siRNA abolished the effect of HFE on NTBI uptake. To determine if HFE had a similar effect on Zip14 in another cell line, HeLa cells expressing HFE under the tetracycline-repressible promoter were transfected with Zip14. As in HepG2 cells, HFE expression inhibited NTBI uptake by approximately 50% and decreased Zip14 protein levels. Further analysis of protein turnover indicated that the half-life of Zip14 is lower in cells that express HFE. These results suggest that HFE decreases the stability of Zip14 and therefore reduces the iron loading in HepG2 cells.     

Hemojuvelin: a new link between obesity and iron homeostasis

The adipose tissue may play an active role in systemic iron regulation and this role may be determinant in obese patients. Indeed, we reported previously that hepcidin, the iron-regulatory hormone, is expressed in adipose tissue and its messenger RNA (mRNA) expression is increased in adipose tissue of morbidly obese patients. The objectives of this study were to characterize the status of hemojuvelin (HJV), another iron-regulatory protein, within the adipose tissue of morbidly obese patients. Since cell-associated HJV acts as a coreceptor of bone morphogenetic protein (BMP) to enhance hepcidin expression in liver cells, we investigated the possible involvement of this pathway in adipose tissue in regulating hepcidin expression. HJV expression was studied in adipose tissue of morbidly obese patients. Soluble HJV blood concentrations were assessed. Hepcidin regulation through BMP pathway was investigated in cultured adipocytes. HJV was expressed both at mRNA and protein levels in adipose tissue. Moreover, its mRNA expression was highly increased in adipose tissue of obese patients and correlated with mRNA hepcidin expression levels. Interestingly, HJV expressed by adipose tissue may be effective since cultured adipocytes increased their hepcidin expression when challenged with BMP2 through Smad effectors. In addition, blood concentrations of soluble HJV were significantly increased. In conclusion, adipose tissue may influence iron homeostasis in obese patients by expressing major iron-regulatory proteins and the BMP signaling pathway could be involved in regulating hepcidin expression in this tissue.     

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.