Cu loading alters expression of non-IRE regulated, but not IRE regulated, Fe dependent proteins in HepG2 cells.

This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using (125)I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein - iron responsive element regulation mechanism.

Regulation of the cysteine desulfurase Nfs1 and the scaffold protein IscU in macrophages stimulated with interferon-gamma and lipopolysaccharide.

Biogenesis of iron-sulfur (Fe-S) clusters in mammals involves a complex mitochondrial machinery that provides inorganic sulfide and iron for their assembly and insertion into apo-proteins. Mechanisms of Fe-S cluster assembly are just being unraveled, and regulation of the genes of this machinery remains unknown. In this study, we report that expression of two essential components of the Fe-S machinery, the cysteine desulfurase Nfs1 and its scaffold protein partner IscU, is down-regulated at both mRNA and protein levels when murine macrophages are physiologically stimulated with IFN-gamma and LPS. Regulation did not rely on cluster disassembly or NO production because exposure of cells to exogenous sources of NO did not alter Nfs1 expression, while it converted cytosolic Fe-S aconitase into its apo-form and because macrophages from NOS2 deficient mice displayed Nfs1 down-regulation. While IFN-gamma alone induced Nfs1 protein instability, LPS triggered a delayed decline of Nfs1, rather involving transcriptional events or mRNA instability. Also, the expression of IscU was down-regulated in IFN-gamma- and/or LPS-stimulated macrophages independently of NO, pointing to a general mechanism for marshalling the regulation of the Fe-S cluster assembly machinery in macrophages exposed to inflammatory stimuli.

RNA silencing of mitochondrial m-Nfs1 reduces Fe-S enzyme activity both in mitochondria and cytosol of mammalian cells.

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.

Interrelations between ceruloplasmin and Fe status during human pregnancy.

It is well established that Fe and ceruloplasmin interact in animals and in in vitro models. However, Fe-mediated regulation of ceruloplasmin has never been investigated in humans. In an observational study, 53 pregnant women aged 19-39 yr (29.8 +/- 0.7 yr, mean +/- SEM) were recruited at the Aberdeen Antenatal Clinic, Aberdeen Maternity Hospital, UK. All requirements for local ethical committees were followed. Venous blood samples were taken from each woman at 34 wk gestation for measurement of Fe status and ceruloplasmin. Various parameters were used to test for Fe status. The most sensitive one appeared to be soluble transferrin receptor, which increased with parity. In the population studied, there was no relationship between hemoglobin or ferritin and serum ceruloplasmin. However, using soluble transferrin receptor (sTfR) levels, we were able to demonstrate an inverse linear relationship (r = 0.37, p = 0.021, n = 41) between Fe status and ceruloplasmin. Fe supplementation, number of previous pregnancies, and smoking habits did not affect this relationship. Our data support in vitro results showing regulation of ceruloplasmin by Fe and also suggest that the interactions between Fe and ceruloplasmin should be considered when Fe supplementation is given.

Iron and copper interactions in development and the effect on pregnancy outcome.

During pregnancy, nutrients are transferred from mother to fetus across the placenta. The mechanisms whereby this occurs, and the adaptations that occur in response to deficiency or overload of iron (Fe) and copper (Cu) are examined in this review. Fe deficiency during pregnancy is common and has serious consequences both in the short and the long term such as fetal growth retardation and cardiovascular problems in the adult offspring. Similarly, Cu deficiency, although not so common, also has deleterious effects. The placenta minimizes the effect of the deficiency by up-regulating the proteins involved in Fe transfer. For example, transferrin receptor levels increase inversely to maternal Fe levels. Divalent metal transporter 1 (DMT1) mRNA in the iron-responsive element (IRE) regulated, but not the non-IRE regulated form is increased, as is the placenta Cu oxidase. Conversely, iron-regulated gene 1 (IREG1) expression is not affected. Fe deficiency increases Cu levels in maternal liver, serum and placenta, but has much less effect in the fetal serum and liver. Apart from maternal ceruloplasmin, mRNA levels of Cu-related proteins are not changed. The Cu oxidase, which we suggest fulfils the function of hephaestin in placenta, is regulated by Cu as well as by Fe. Fe deficiency also has marked effects on cytokine levels in the placenta. Tumor necrosis factor alpha (TNFalpha) and TNFalpha receptor 1 (TNFalphaR1) levels both increase. The data show that altering Fe status has a marked effect on metabolism of other metals and of other important mediators of cell function. This is particularly important during pregnancy, when the developing fetus is very vulnerable to inappropriate micronutrient status.

Placental ceruloplasmin homolog is regulated by iron and copper and is implicated in iron metabolism.

We previously reported an endogenous, membrane-bound Cu oxidase with homology to ceruloplasmin in BeWo cells, a placental choriocarcinoma cell line. In this previous study, ceruloplasmin immunoreactivity was localized to the perinuclear region and non-brush-border membranes. Here, we show that azide-sensitive oxidase activity is enriched in the same fractions, correlating subcellular localization of enzyme activity with ceruloplasmin immunoreactivity. Expression of the placental Cu oxidase is inversely proportional to Fe status and directly proportional to Cu status at enzyme and protein levels. To identify a role for the Cu oxidase, cells were exposed to (59)Fe-transferrin for 18 h in an environment of 20% O(2) or 5% O(2). At 5% O(2), Cu-deficient cells retain significantly more (59)Fe than control cells. This excess in (59)Fe accumulation is caused by a significant decrease in (59)Fe release. These results indicate that downregulation of the placental Cu oxidase in BeWo cells impairs Fe release. This effect is only apparent in an environment of limited O(2).