Katsuya Ikuta

Body iron metabolism and pathophysiology of iron overload

Iron is an essential metal for the body, while excess iron accumulation causes organ dysfunction through the production of reactive oxygen species. There is a sophisticated balance of body iron metabolism of storage and transport, which is regulated by several factors including the newly identified peptide hepcidin. As there is no passive excretory mechanism of iron, iron is easily accumulated when exogenous iron is loaded by hereditary factors, repeated transfusions, and other diseased conditions. The free irons, non-transferrin-bound iron, and labile plasma iron in the circulation, and the labile iron pool within the cells, are responsible for iron toxicity. The characteristic features of advanced iron overload are failure of vital organs such as liver and heart in addition to endocrine dysfunctions. For the estimation of body iron, there are direct and indirect methods available. Serum ferritin is the most convenient and widely available modality, even though its specificity is sometimes problematic. Recently, new physical detection methods using magnetic resonance imaging and superconducting quantum interference devices have become available to estimate iron concentration in liver and myocardium. The widely used application of iron chelators with high compliance will resolve the problems of organ dysfunction by excess iron and improve patient outcomes.

Proline-rich antimicrobial peptide, PR-39 gene transduction altered invasive activity and actin structure in human hepatocellular carcinoma cells

SummaryPR-39 is an endogenous proline-rich antimicrobial peptide which induces the synthesis of syndecan-1, a transmembrane heparan sulphate proteoglycan involved in cell-to-matrix interactions and wound healing. Previously, we revealed that the expression of syndecan-1 was reduced in human hepatocellular carcinomas with high metastatic potential and speculated that syndecan-1 played an important role in inhibition of invasion and metastasis. It is assumed that a modification of this process with PR-39 and syndecan-1 may result in a new strategy by which it can inhibit the invasion and metastasis. Therefore, we transduced a gene of PR-39 into human hepatocellular carcinoma cell line HLF, which shows a low expression of syndecan-1 and a high in vitro invasive activity, and examined whether this procedure could reduce the invasive activity of tumour cells. In two transfectants with PR-39 gene, the syndecan-1 expression was induced and the invasive activity in type I collagen-coated chamber was inhibited. Moreover, these transfectants showed the suppression of motile activity assayed by phagokinetic tracks in addition to the disorganization of actin filaments observed by a confocal imaging system. In contrast, five transfectants with syndecan-1 gene in the HLF cells revealed suppression of invasive activity but did not alter the motile activity and actin structures of the cell. These results suggest that PR-39 has functions involved in the suppression of motile activity and alteration of actin structure on human hepatocellular carcinoma cells in addition to the suppression of invasive activity which might result from the induction of syndecan-1 expression.

Immunohistochemical detection of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver diseases

4-Hydroxy-2-nonenal (HNE) is one of the major components of lipid peroxidation product and has been shown to react with proteins to form HNE-protein adducts. HNE-protein adducts are relatively stable and can be used as a marker of radical-mediated cellular damage. We report herein the immunohistochemical analysis of HNE-protein adducts in human alcoholic liver diseases using a specific monoclonal antibody HNEJ-2. Cytoplasm of hepatocytes and bile duct epithelia was positively stained for HNE-protein adducts, and the nucleus was negligibly stained. The immunohistochemical intensity of hepatocytes was classified into three groups: strong, moderate, and faint staining. Strong staining was found in 43% of alcoholic liver diseases and in 4% of viral liver diseases. Hepatocytes of alcoholic liver diseases contained a higher amount of HNE-protein adducts than those of viral liver diseases, and the difference was statistically significant (p = 0.005; chi2 test). Semiquantitative analysis of the histological intensities of HNE-protein adducts and iron indicated a significant positive correlation (p = 0.084; Spearmans rank correlation). The localization of HNE-protein adducts and iron in hepatocytes appeared to be identical. These data suggested the correlation between HNE-protein adducts and iron. Our results indicate that HNE-protein adducts, a marker of oxidative stress-induced damage, are increased in human alcoholic liver damage, and that hepatic siderosis may act on the production of free radicals.

Dysregulation of systemic iron metabolism in alcoholic liver diseases.

Alcoholic liver diseases (ALD) are frequently associated with iron overload. Until recently, the effects of ethanol in hepatic iron uptake and intestinal iron absorption have not been clarified in detail. Two possible mechanisms for iron overload are the uptake of iron into hepatocytes in a specific manner through the increased expression of transferrin receptor (TfR) 1; and increased intestinal iron absorption by the lowering of hepcidin. It is worthwhile to examine whether a similar mechanism is present in the development of steatosis and non‐alcoholic steatohepatitis (NASH). Hepatocytes have several iron uptake pathways. Ethanol increases transferrin (Tf)‐mediated uptake via a receptor‐dependent manner, but downregulates the non‐Tf‐bound iron uptake. According to immunohistochemical study, TfR1 was increased in hepatocytes in 80% of hepatic tissues of patients with ALD, but was not detected in normal hepatic tissues. In an experimental model, ethanol exposure to the primary cultured‐hepatocytes in the presence of iron increased TfR1 expression and 59Fe‐labeled Tf uptake. In patients with ALD, intestinal iron absorption is increased by oral iron uptake assay. The regulatory hormone for iron homeostasis, hepcidin is downregulated in ethanol‐loaded mice liver. As well as ALD, a similar mechanism was present in the mouse model fed with a high‐fat diet, a model of the initial phenomenon of steatosis. The common mechanism for hepatic iron deposition and the triggering role of iron may be present in the development of ALD and non‐alcoholic fatty liver disease/NASH.

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.

MicroRNA‐146b improves intestinal injury in mouse colitis by activating nuclear factor‐κB and improving epithelial barrier function

The precise role of microRNAs in inflammatory disease is not clear. The present study investigated the effect of microRNA (miR‐146b) with respect to improving intestinal inflammation.

microRNA-18a induces apoptosis in colon cancer cells via the autophagolysosomal degradation of oncogenic heterogeneous nuclear ribonucleoprotein A1.

It is well known that microRNAs (miRs) are abnormally expressed in various cancers and target the messenger RNAs (mRNAs) of cancer-associated genes. While (miRs) are abnormally expressed in various cancers, whether miRs directly target oncogenic proteins is unknown. The present study investigated the inhibitory effects of miR-18a on colon cancer progression, which was considered to be mediated through its direct binding and degradation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). An MTT assay and xenograft model demonstrated that the transfection of miR-18a induced apoptosis in SW620 cells. A binding assay revealed direct binding between miR-18a and hnRNP A1 in the cytoplasm of SW620 cells, which inhibited the oncogenic functions of hnRNP A1. A competitor RNA, which included the complementary sequence of the region of the miR-18a-hnRNP A1 binding site, repressed the effects of miR-18a on the induction of cancer cell apoptosis. In vitro single and in vivo double isotope assays demonstrated that miR-18a induced the degradation of hnRNP A1. An immunocytochemical study of hnRNP A1 and LC3-II and the inhibition of autophagy by 3-methyladenine and ATG7, p62 and BAG3 siRNA showed that miR-18a and hnRNP A1 formed a complex that was degraded through the autophagolysosomal pathway. This is the first report showing a novel function of a miR in the autophagolysosomal degradation of an oncogenic protein resulting from the creation of a complex consisting of the miR and a RNA-binding protein, which suppressed cancer progression.

Recycling, degradation and sensitivity to the synergistic anion of transferrin in the receptor-independent route of iron uptake by human hepatoma (HuH-7) cells.

To secure iron from transferrin, hepatocytes use two pathways, one dependent on transferrin receptor (TfR 1) and the other, of greater capacity but lower affinity, independent of TfR 1. To clarify further similarities and differences of the two pathways, we have suppressed TfR 1 by 75-80% in human hepatoma-derived HuH-7 cells co-transfected with vectors bearing full-length TfR 1 cDNA or its first 100 bases in antisense orientation. Suppression of TfR 1 does not lead to down regulation of TfR 2, a recently described second transferrin receptor of as yet uncertain function. Both pathways depend on acidification of the compartments in which iron release from transferrin takes place. Recycling of transferrin is a feature of both pathways, but is substantially more efficient in the receptor-dependent route. Degradation of transferrin occurs only in the receptor-independent route, in the first example of a specific catabolic pathway of transferrin. Linkage of cellular iron uptake to release of the synergistic anion (without which iron is not bound by transferrin) is particularly evident in the receptor-independent pathway. Although the relative importance of the two pathways in normal and deranged hepatic iron metabolism remains to be determined, the receptor-independent route is a substantial accessory for iron uptake to the better-known receptor-dependent track.

Augmented hepatic Toll-like receptors by fatty acids trigger the pro-inflammatory state of non-alcoholic fatty liver disease in mice.

There is considerable evidence that intestinal microbiota are involved in the development of metabolic syndromes and, consequently, with the development of non‐alcoholic fatty liver disease (NAFLD). Toll‐like receptors (TLRs) are essential for the recognition of microbiota. However, the induction mechanism of TLR signals through the gut‐liver axis for triggering the development of non‐alcoholic steatohepatitis (NASH) or NAFLD remains unclear. In this study, we investigated the role of palmitic acid (PA) in triggering the development of a pro‐inflammatory state of NAFLD.

Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis

Previous reports have suggested that some probiotics inhibit tumorigenesis and cancer progression. However, the molecules involved have not yet been identified. Here, we show that the culture supernatant of Lactobacillus casei ATCC334 has a strong tumour-suppressive effect on colon cancer cells. Using mass spectrometry, we identify ferrichrome as a tumour-suppressive molecule produced by L. casei ATCC334. The tumour-suppressive effect of ferrichrome is greater than that of cisplatin and 5-fluorouracil, and ferrichrome has less of an effect on non-cancerous intestinal cells than either of those agents. A transcriptome analysis reveals that ferrichrome treatment induces apoptosis, which is mediated by the activation of c-jun N-terminal kinase (JNK). Western blotting indicates that the induction of apoptosis by ferrichrome is reduced by the inhibition of the JNK signalling pathway. This we demonstrate that probiotic-derived ferrichrome exerts a tumour-suppressive effect via the JNK signalling pathway.