Iron excess upregulates SPNS2 mRNA levels but reduces sphingosine-1-phosphate export in human osteoblastic MG-63 cells.

We aimed to study the mechanisms involved in bone-related iron impairment by using the osteoblast-like MG-63 cell line. Our results indicate that iron impact the S1P/S1PR signalizing axis and suggest that iron can affect the S1P process and favor the occurrence of osteoporosis during chronic iron overload. INTRODUCTION: Systemic iron excess favors the development of osteoporosis, especially during genetic hemochromatosis. The cellular mechanisms involved are still unclear despite numerous data supporting a direct effect of iron on bone biology. Therefore, the aim of this study was to characterize mechanisms involved in the iron-related osteoblast impairment. METHODS: We studied, by using the MG-63 cell lines, the effect of iron excess on SPNS2 gene expression which was previously identified by us as potentially iron-regulated. Cell-type specificity was investigated with hepatoma HepG2 and enterocyte-like Caco-2 cell lines as well as in iron-overloaded mouse liver. The SPNS2-associated function was also investigated in MG-63 cells by fluxomic strategy which led us to determinate the S1P efflux in iron excess condition. RESULTS: We showed in MG-63 cells that iron exposure strongly increased the mRNA level of the SPNS2 gene. This was not observed in HepG2, in Caco-2 cells, and in mouse livers. Fluxomic study performed concomitantly on MG-63 cells revealed an unexpected decrease in the cellular capacity to export S1P. Iron excess did not modulate SPHK1, SPHK2, SGPL1, or SGPP1 gene expression, but decreased COL1A1 and S1PR1 mRNA levels, suggesting a functional implication of low extracellular S1P concentration on the S1P/S1PR signalizing axis. CONCLUSIONS: Our results indicate that iron impacts the S1P/S1PR signalizing axis in the MG-63 cell line and suggest that iron can affect the bone-associated S1P pathway and favor the occurrence of osteoporosis during chronic iron overload.

Modulation of ethanol effect on hepatocyte proliferation by polyamines.

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.

Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells.

UNLABELLED: In order to understand mechanisms involved in osteoporosis observed during iron overload diseases, we analyzed the impact of iron on a human osteoblast-like cell line. Iron exposure decreases osteoblast phenotype. HHIPL-2 is an iron-modulated gene which could contribute to these alterations. Our results suggest osteoblast impairment in iron-related osteoporosis. INTRODUCTION: Iron overload may cause osteoporosis. An iron-related decrease in osteoblast activity has been suggested. METHODS: We investigated the effect of iron exposure on human osteoblast cells (MG-63) by analyzing the impact of ferric ammonium citrate (FAC) and iron citrate (FeCi) on the expression of genes involved in iron metabolism or associated with osteoblast phenotype. A transcriptomic analysis was performed to identify iron-modulated genes. RESULTS: FAC and FeCi exposure modulated cellular iron status with a decrease in TFRC mRNA level and an increase in intracellular ferritin level. FAC increased ROS level and caspase 3 activity. Ferroportin, HFE and TFR2 mRNAs were expressed in MG-63 cells under basal conditions. The level of ferroportin mRNA was increased by iron, whereas HFE mRNA level was decreased. The level of mRNA alpha 1 collagen type I chain, osteocalcin and the transcriptional factor RUNX2 were decreased by iron. Transcriptomic analysis revealed that the mRNA level of HedgeHog Interacting Protein Like-2 (HHIPL-2) gene, encoding an inhibitor of the hedgehog signaling pathway, was decreased in the presence of FAC. Specific inhibition of HHIPL-2 expression decreased osteoblast marker mRNA levels. Purmorphamine, hedgehog pathway activator, increased the mRNA level of GLI1, a target gene for the hedgehog pathway, and decreased osteoblast marker levels. GLI1 mRNA level was increased under iron exposure. CONCLUSION: We showed that in human MG-63 cells, iron exposure impacts iron metabolism and osteoblast gene expression. HHIPL-2 gene expression modulation may contribute to these alterations. Our results support a role of osteoblast impairment in iron-related osteoporosis.

Bone status in a mouse model of genetic hemochromatosis.

UNLABELLED: Genetic hemochromatosis is a cause of osteoporosis; mechanisms leading to iron-related bone loss are not fully characterized. We assessed the bone phenotype of HFE (-/-) male mice, a mouse model of hemochromatosis. They had a phenotype of osteoporosis with low bone mass and alteration of the bone microarchitecture. INTRODUCTION: Genetic hemochromatosis is a cause of osteoporosis. However, the mechanisms leading to iron-related bone loss are not fully characterized. Recent human data have not supported the hypothesis of hypogonadism involvement. The direct role of iron on bone metabolism has been suggested. METHODS: Our aim was to assess the bone phenotype of HFE (-/-) male mice, a mouse model of human hemochromatosis, by using microcomputed tomography and histomorphometry. HFE (-/-) animals were sacrificed at 6 and 12 months and compared to controls. RESULTS: There was a significant increase in hepatic iron concentration and bone iron content in HFE (-/-) mice. No detectable Perls' staining was found in the controls' trabeculae. Trabecular bone volume (BV/TV) was significantly lower in HFE (-/-) mice at 6 and 12 months compared to the corresponding wild-type mice: 9.88 ± 0.82% vs 12.82 ± 0.61% (p = 0.009) and 7.18 ± 0.68% vs 10.4 ± 0.86% (p = 0.015), respectively. In addition, there was an impairment of the bone microarchitecture in HFE (-/-) mice. Finally, we found a significant increase in the osteoclast number in HFE (-/-) mice: 382.5 ± 36.75 vs 273.4 ± 20.95 ¢/mm(2) (p = 0.004) at 6 months and 363.6 ± 22.35 vs 230.8 ± 18.7 ¢/mm(2) (p = 0.001) at 12 months in HFE (-/-) mice vs controls. CONCLUSION: Our data show that HFE (-/-) male mice develop a phenotype of osteoporosis with low bone mass and alteration of the microarchitecture. They suggest that there is a relationship between bone iron overload and the increase of the osteoclast number in these mice. These findings are in accordance with clinical observations in humans exhibiting genetic hemochromatosis and support a role of excess iron in relation to genetic hemochromatosis in the development of osteoporosis in humans.

[Hereditary iron overload]. / Surcharges héréditaires en fer.

The field of hereditary iron overload has known, in the recent period, deep changes mainly related to major advances in molecular biology. It encompasses now a series of genetic entities. The mechanistic understanding of iron overload development and iron toxicity has greatly improved. The diagnostic approach has become essentially noninvasive with a major role for biological tests. From the therapeutic viewpoint, the phlebotomy treatment is now enriched by the possibility of resorting to oral chelation and by innovative perspectives directly linked to our improvement in the molecular understanding of these diseases.

Bacteroides fragilis prevents Salmonella Heidelberg translocation in co-culture model mimicking intestinal epithelium.

Salmonella Heidelberg is one of the most common serovar causing foodborne illnesses. To limit the development of digestive bacterial infection, food supplements containing probiotic bacteria can be proposed. Commensal non-toxigenic Bacteroides fragilis has recently been suggested as a next-generation probiotic candidate. By using an original triple co-culture model including Caco-2 cells (representing human enterocytes), HT29-MTX (representing mucus-secreting goblet cells), and M cells differentiated from Caco-2 by addition of Raji B lymphocytes, bacterial translocation was evaluated. The data showed that S. Heidelberg could translocate in the triple co-culture model with high efficiency, whereas for B. fragilis a weak translocation was obtained. When cells were exposed to both bacteria, S. Heidelberg translocation was inhibited. The cell-free supernatant of B. fragilis also inhibited S. Heidelberg translocation without impacting epithelial barrier integrity. This supernatant did not affect the growth of S. Heidelberg. The non-toxigenic B. fragilis confers health benefits to the host by reducting bacterial translocation. These results suggested that the multicellular model provides an efficient in vitro model to evaluate the translocation of pathogens and to screen for probiotics that have a potential inhibitory effect on this translocation.

Indicators of iron status are correlated with adiponectin expression in adipose tissue of patients with morbid obesity.

AIM: The aim of this study was to assess interactions between glucose and iron homoeostasis in the adipose tissue (AT) of obese subjects. METHODS: A total of 46 obese patients eligible for bariatric surgery were recruited into the study. Anthropometric and biochemical characteristics were assessed, and biopsies of subcutaneous (SCAT) and visceral adipose tissue (VAT) performed. The mRNA levels of genes involved in iron and glucose homoeostasis were measured in their AT and compared with a pool of control samples. RESULTS: Gene expression of hepcidin (HAMP) was significantly increased in the SCAT and VAT of obese patients, while transferrin receptor (TFRC) expression was reduced, compared with non-obese controls, suggesting a higher iron load in obese patients. Also, mRNA levels of adiponectin (ADIPOQ) were decreased in both SCAT and VAT in obese patients, and correlated negatively with hepcidin expression, while adiponectin expression was positively correlated with TFRC expression in both SCAT and VAT. Interestingly, TFRC expression in VAT correlated negatively with several metabolic parameters, such as fasting blood glucose and LDL cholesterol. CONCLUSION: Iron content appears to be increased in the SCAT and VAT of obese patients, and negatively correlated with adiponectin expression, which could be contributing to insulin resistance and the metabolic complications of obesity.

Stearoyl coenzyme A desaturase 1 expression and activity are increased in the liver during iron overload.

In humans, hepatic iron overload can lead to hepatocellular carcinoma development. Iron related dysregulation of hepatic genes could play a role in this phenomenon. We previously found that the carbonyl-iron overloaded mouse was a useful model to study the mechanisms involved in the development of hepatic lesions related to iron excess. The aim of the present study was to identify hepatic genes overexpressed in conditions of iron overload by using this model. A suppressive subtractive hybridization was performed between hepatic mRNAs extracted from control and 3% carbonyl-iron overloaded mice during 8 months. This methodology allowed us to identify stearoyl coenzyme A desaturase 1 (SCD1) mRNA overexpression in the liver of iron loaded mice. The corresponding enzymatic activity was also found to be significantly increased. In addition, we demonstrated that both SCD1 mRNA expression and activity were increased in another iron overload model in mice obtained by a single iron-dextran subcutaneous injection. Moreover, we found, in both models, that SCD1 mRNA was not only influenced by the quantity of iron in the liver but also by the duration of iron overload since SCD1 mRNA upregulation was not detected in earlier stages of iron overload. In addition, we found that cellular repartition likely influenced SCD1 mRNA expression. In conclusion, we demonstrated that iron excess in the liver induced both the expression of SCD1 mRNA and its corresponding enzymatic activity. The level and duration of iron overload, as well as cellular repartition of iron excess in the liver likely play a role in this induction. The fact that the expression and activity of SCD1, an enzyme adding a double bound into saturated fatty acids, are induced in two models of iron overload in mice leads to the conclusion that iron excess in the liver may enhance the biosynthesis of unsaturated fatty acids.

[HFE hemochromatosis: pathogenic and diagnostic approach]. / Hémochromatose HFE: approche pathogénique et diagnostique.

HFE hemochromatosis is the most frequent genetic iron overload disease. It is linked to the C282Y mutation of the HFE protein, protein encoded by the HFE gene, which is located on chromosome 6. The mechanisms accounting for iron excess are not only digestive hyperabsorption of iron but also excessive recycling of macrophagic iron coming from erythrophagocytosis and secreted into the blood. Both mechanisms are linked to an HFE-related hepatic failure in producing hepcidin, a key hormone of body iron regulation. The marked phenotypic variability of C282Y homozygosity expression is likely related to both genetic and environmental factors. The HFE gene discovery has rendered non invasive the positive diagnostic of HFE hemochromatosis, which is now based first on an increased level of plasma transferrin saturation leading to the request of the HFE mutation. Then, hepatic MRI is a reliable method to quantify iron overload. The HFE gene discovery has also paved the road of an enlarged field of differential diagnoses corresponding to novel entities of non-HFE related genetic iron overload syndromes.

Differential expression of laminin chains in hepatic lipocytes.

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B1 and B2 chains, but not A, were found in medium from 5-day-old lipocyte primary cultures by immunoblotting and immunoprecipitation of 35S-labeled proteins after reducing SDS-polyacrylamide gel electrophoresis. An additional polypeptide of Mr = 380,000 was identified by immunoprecipitation. Under non-reducing conditions only one Mr = 900,000 band was revealed. High levels of B1 and B2 mRNAs were also demonstrated in 5-day-old cultured lipocytes while at the time of seeding, only B2 chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the Mr = 380,000 polypeptide could be unrelated to the A chain of laminin.

Enterocytic differentiation of the human Caco-2 cell line is correlated with down-regulation of fibronectin and laminin.

Human intestinal Caco-2 cells were used to examine the expression of fibronectin (FN) and laminin (LN) during enterocytic differentiation. Combination of immunoprecipitation, Western and Northern blotting revealed that Caco-2 cells expressed a classical FN and a variant form of LN: besides B1 and B2 chains, LN contained a 350-kDa heavy chain instead of the 400-kDa A chain. Throughout Caco-2 cell differentiation, FN and LN synthesis decreased at both mRNA and protein levels. These data indicate that enterocytic differentiation involves both transcriptional and/or post-transcriptional down-regulation of FN and LN gene expression.

A reappraisal of hepatic siderosis in patients with end-stage cirrhosis: practical implications for the diagnosis of hemochromatosis.

The aim of this study was to describe the histologic pattern of iron distribution in end-stage cirrhosis due to various causes and to test the reliability of the hepatic iron index (equal to hepatic iron concentration divided by age) in excluding or confirming associated hemochromatosis in such a condition. Large slices of the resected livers of 30 patients transplanted for alcoholic and/or viral end-stage cirrhosis were assessed histologically for iron distribution and biochemically for hepatic iron concentration in the least and the most iron-overloaded nodules of each case. HLA-A3 was used as the marker for the hemochromatosis gene in the population studied. Intranodular parenchymal siderosis was found in 23 cases (12 spotty, 11 diffuse) with diffuse intrabiliary iron deposits apparent in only two cases. Although in 14 patients the hepatic iron index was significantly high (> 1.9) so as to suggest hemochromatosis, these cases did not correspond to homozygous hemochromatosis with respect to the prevalence of HLA-A3 antigen. End-stage cirrhosis arising from different causes is frequently complicated by parenchymal siderosis that may mimic hemochromatosis, including a hepatic iron index greater than 1.9. The diagnosis of hemochromatosis in patients with end-stage cirrhosis, even those with a hepatic iron index greater than 1.9, should rely mainly on clinical and histologic data.

Scintigraphic assessment of indium-111-labeled granulocyte splenic pooling: a new approach to inflammatory bowel disease activity.

We have conducted a prospective study into the sensitivity and the specificity of the fall in splenic activity (FSA) as an index of activity in inflammatory bowel disease (IBD). FSA was measured on scintiscans obtained at 3 and 24 hr postinjection of indium-111-labeled granulocytes. One hundred and twenty-two scans were acquired in 96 patients who were divided into six groups: Gr. I = normal volunteers (n = 10); Gr. II = inflammatory rheumatism (n = 10); Gr. III = abscesses (n = 17); Gr. IV = ulcerative colitis (UC: n = 23); Gr. V = colonic Crohn's disease (CCD: n = 22); and Gr. VI = ileal Crohn's disease (ICD: n = 14). FSA for Groups I and II was constantly below 10%, but it was increased in the other four groups (abscesses: 39% +/- 12%; UC: 35% +/- 13.5%; CCD: 23.7% +/- 14.7%; ICD: 21.5% +/- 11.7%). There was a significant correlation between fecal excretion of 111In (FEI) and FSA in patients with IBD (UC: r = 0.71, p less than 0.001; CCD: r = 0.74, p less than 0.001; ICD: r = 0.43, p less than 0.001). FSA was followed in 16 patients with IBD after medical treatment and there was a significant correlation between variations in FSA and in FEI (r = 0.879, p less than 0.001). FSA is a very sensitive although nonspecific index of disease activity in IBD and may replace FEI in the assessment of IBD activity.

Antiproliferative effect of deferiprone on the Hep G2 cell line.

Iron is an essential element in cellular metabolism and the growth of all living species, and is involved in DNA replication. The risk of hepatocellular carcinoma development is associated with an increase in iron availability. The aim of the present work was to investigate the effect of an oral iron chelator, deferiprone (CP20), on HepG2 cell-line proliferation in culture. HepG2 cell cultures were maintained in the absence of fetal calf serum (FCS) and in the presence or not (control cultures) of CP20 at the concentrations of 50 or 100 microM; deferoxamine (DFO) was used as an iron chelator reference. Cell proliferation was investigated by the analysis of DNA synthesis using [3H] methyl-thymidine incorporation and of the cell cycle by flow cytometry. Iron chelation efficiency in the culture model was studied by analyzing the effect of CP20 on radioactive iron uptake, intracellular ferritin level, and transferrin receptor expression. CP20, at the concentration of 50 or 100 microM, inhibited DNA synthesis after 48 hr of incubation and induced an accumulation of the cells in the S phase of the cell cycle. Iron chelators inhibited cellular iron uptake, decreased intracellular ferritin level, and increased transferrin receptor protein and mRNA levels. Our results show that CP20 as well as deferoxamine inhibit HepG2 cell proliferation and block cell cycle in the S phase.

Metabolic imaging of tissues by infrared fiber-optic spectroscopy: an efficient tool for medical diagnosis.

Infrared fingerprints of molecules in biology contain much information on cells metabolism allowing one to distinguish between healthy and altered tissues. Here, to collect infrared signatures, we used evanescent wave spectroscopy based on an original infrared transmitting tapered glass fiber. A strict control of the fiber diameter in the tapered sensing zone allows high sensitivity and wide spectral range exploration from 800 to 3000 cm(-1). Then, merely in depositing the mouse liver biopsies on the fiber, this device has enable us to differentiate between tumorous and healthy tissues.

[Diffuse pseudopolyposis of the colon: developmental form or cicatricial of hemorrhagic rectocolitis?]. / La pseudopolypose diffuse du côlon: forme évolutive ou cicatricielle de la rectocolite hémorragique?

The authors report the case of a patient with generalized polyposis associated with ulcerative colitis. The diagnosis of polyposis was made 20 years after the onset of colitis. The patient presented with the unusual clinical manifestations, ie poor general condition and severe denutrition, following a severe relapse of colitis. The mucosal surface of the entire colon, except the rectum, was covered by innumerable polyps, without any macroscopic or microscopic evidence of ulceration. Intestinal protein loss, as assessed by alpha-1-antitrypsin clearance, was very high (470 ml/d). Acute mucosal inflammation, as assessed by histologic study and by 111-Indium-labelled-leukocyte scintigraphy, was also present. The patient responded dramatically to total colectomy with ileorectal anastomosis. Histologically, the polyps were filiform, with a central core, containing vessels and smooth muscle fibers. This observation demonstrates that generalized polyposis, generally considered to be an asymptomatic sequela of ulcerative colitis, can also be associated with severe attacks of colitis.

[Histochemical detection of hepatic iron. A comparative study of four stains]. / Détection histochimique du fer hépatique. Etude comparative de quatre colorations.

Due to its specificity and easiness, Perls' stain is widely used in the histochemical assessment of liver iron content. However, it can underestimate slight iron overload, which can hamper screening for genetic hemochromatosis, especially in young people. The aim for the present study was to compare Perls' stain to three other specific iron stains (Tirmann-Schmeltzer (TRM); Hukill and Putt (HPT); Perls with Diaminobenzidine (DAB)), biochemical liver iron concentration (LIC) being used as the reference. 1) There is a significant difference between number of stained cells with TRM or DAB, compare with Perls' stain (p < 0.05). 2) Correlation (r) between histological assessment (T) and LIC was 0.39 for Perls' stain, 0.64 for TRM 0.53 for HPT and 0.64 for DAB. These data suggest that Perls' stain is not the most sensitive method for the assessment of slight iron overload. Tirmann-Schmeltzer's stain and Perls plus dAB should be preferred, especially in the screening of early liver siderosis.

[Current data on iron metabolism]. / Données actuelles sur le métabolisme du fer.

Iron is required for cellular life. However, abnormalities of its metabolism may lead to iron deficiency or iron overload, both conditions which are deleterious. Therefore, stock and distribution of iron in the body must be very stable. Classically, four major proteins are involved in iron metabolism: (a) transferrin which is implicated in its plasmatic transport, (b) transferrin receptor which regulates iron-transferrin uptake, (c) ferritin, the major iron storage protein, and (d) IRP (Iron Regulatory Protein) which regulates both the entry and storage of iron by linking to the IRE (Iron Responsive Element), a nucleotidic sequence found on transferrin receptor and ferritin mRNA. Thus, IRP adapts gene expression to the iron cellular status. Recent data give informations about new proteins involved in iron metabolism: HFE whose gene is mutated in genetic hemochromatosis, ceruloplasmin which permits cellular iron egress and frataxin which is implicated in the exit of iron from mitochondria.

[Hepatic fibrogenesis]. / Fibrogenèse hépatique.

The hepatic extracellular matrix is involved in both the stability of liver architecture and the hepatic function. Fibrogenesis occurs during various chronic liver diseases. It is the consequence of an imbalance between synthesis, deposition and degradation of extracellular matrix components leading to fibrosis and, then, cirrhosis. Hepatic stellate cells are the main source of extracellular matrix components in fibrogenesis. Among the factors involved in fibrogenesis, transforming growth factor beta 1 plays a central role. The vascular and cellular consequences of liver fibrogenesis require new specific diagnostic and therapeutic strategies.

[Genetics and physiopathology of hemochromatosis]. / Génétique et physiopathologie de l'hémochromatose.

Thanks to the discovery of the HFE gene and of its mutations, it is now established that the most frequent form of hemochromatosis is related to homozygosity for the mutation C282Y, and that other types of hemochromatosis, unrelated to HFE mutations, do exist such as the juvenile hemochromatosis. From a pathophysiological standpoint, the C282Y mutation impairs HFE protein expression at the surface of the membrane and disturbs the cellular entry of iron (carried by circulating transferrin) into the cryptic duodenal cell. This, in turn, is likely to lead to an aberrant programmation of the degree of iron influx from the digestive lumen into the apical duodenal cells. The resulting hyperabsorption, which forms the basis of iron overload in hemochromatosis, is likely to implicate an overexpression of the transmembrane iron carrier DMT1. It is remarkable to observe that these major improvements in the knowledge of hemochomatosis have been accompanied by similar improvements in the understanding of normal iron metabolism.