Ferritin molecules play an important role in the control of intracellular iron distribution and in the constitution of long term iron stores. In vitro studies on recombinant ferritin subunits have shown that the ferroxidase activity associated with the H subunit is necessary for iron uptake by the ferritin molecule, whereas the L subunit facilitates iron core formation inside the protein shell. However, plant and bacterial ferritins have only a single type of subunit which probably fulfills both functions. To assess the biological significance of the ferroxidase activity associated with the H subunit, we disrupted the H ferritin gene (Fth) in mice by homologous recombination. Fth ؉/؊ mice are healthy, fertile, and do not differ significantly from their control littermates. However, Fth ؊/؊ embryos die between 3.5 and 9.5 days of development, suggesting that there is no functional redundancy between the two ferritin subunits and that, in the absence of H subunits, L ferritin homopolymers are not able to maintain iron in a bioavailable and nontoxic form. The pattern of expression of the wild type Fth gene in 9.5-day embryos is suggestive of an important function of the H ferritin gene in the heart.Iron is essential to all living organisms, but to prevent its toxicity it must be associated to specialized molecules. Of those, ferritins (Fts) 1 play special roles because of their ubiquitous distribution in all tissues, to the tight iron-dependent gene expression, and to their capacity to store large amounts of iron (up to 4,000 Fe atoms per molecule) inside a large protein shell, in a nontoxic and bioavailable form (reviewed in Ref.1). Mammalian ferritins are heteropolymers made of two different subunit types named H and L. The H chain carries a ferroxidase center which appears to be essential for iron incorporation (2), whereas the L chain facilitates iron mineralization inside the cavity (3). In prokaryotes and plants, ferritins are made of 24 identical subunits which all carry the ferroxidase activity. In mammals, multiple transcriptional regulations operate which modify H ferritin mRNA levels in response to cytokines (4), heme (5, 6), oncogenes (7), or to cell proliferation or differentiation (reviewed in Ref 8). In addition, ferritin mRNAs have unique features which allow efficient (9) and tissue-specific (10) translational regulation according to the iron status of the cell. Therefore, the conservation of the ferritin ferroxidase activity throughout evolution as well as the very complex genetic regulation of ferritin expression suggest that this catalytic activity is essential for ferritin biological function.We disrupted the H ferritin (Fth) gene in mice and found that H subunit-associated ferroxidase is necessary for early embryonic development because no Fth Ϫ/Ϫ embryos were found after 3.5 days post coitus. Our data also demonstrate that L ferritin gene product cannot substitute for the H subunit. In contrast, heterozygous Fth ϩ/Ϫ were healthy and indistinguishable from their control littermates. EXPERIME...

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Ferritin, the iron-storing molecule, is made by the assembly of various proportions of 2 different H and L subunits into a 24-mer protein shell. These heteropolymers have distinct physicochemical properties, owing to the ferroxidase activity of the H subunit, which is necessary for iron uptake by the ferritin molecule, and the ability of the L subunit to facilitate iron core formation inside the protein shell. It has previously been shown that H ferritin is indispensable for normal development, since inactivation of the H ferritin gene by homologous recombination in mice is lethal at an early stage during embryonic development. Here the phenotypic analysis of the mice heterozygous for the H ferritin gene (Fth ؉/؊ mice) is reported, and differences in gene regulation between the 2 subunits are shown. The heterozygous Fth ؉/؊ mice were healthy and fertile and did not present any apparent abnormalities. Although they had iron-overloaded spleens at the adult stage, this is identical to what is observed in normal Fth ؉/؉ mice. However, these heterozygous mice had slightly elevated tissue L ferritin content and 7-to 10-fold more L ferritin in the serum than normal mice, but their serum iron remained unchanged.

We have previously shown that hepcidin transgenic embryos are severely anemic and die around birth. Here, we report that embryonic hepcidin transgene expression decreases transferrin receptor 1 (TfR1) mRNA level in placenta, as shown by cDNA microarray analysis and quantitative RT-PCR, by a mechanism which is independent of placenta iron content and iron responsive element/iron regulatory protein (IRE/IRP) activity. On the contrary, iron injections into pregnant mothers result in increased placenta iron and ferritin content, and reduced IRE binding activity of IRP1 leading to decreased TfR1 mRNA level. Taken together, these results suggest that hepcidin action on placenta is mostly through transcriptional downregulation of the iron uptake machinery.

The abnormal concentrations of steroid hormones and free fatty acids in the plasma of HIV-infected subjects are associated with qualitative and quantitative alterations in two of the major steroid hormones carrier proteins, sex steroid-binding protein (SBP) and corticosteroid-binding globulin (CBG). The properties of SBP and CBG in the sera of two age-matched groups of 67 men healthy blood donors (controls) and 64 HIV+subjects: 11 CDC group II and III (ASY), 6 CDC group IVA and 47 groups IV C1+D (AIDS) were analyzed. The HIV+patients had SBP concentrations 39-51% above those of controls. The sera of AIDS patients had higher SBP association constants (Ka) for testosterone than did those of the II, III and IVA groups and controls. In contrast, the CBG concentrations in all the HIV+subjects were similar to those of the controls. However, the binding properties of HIV+CBG were abnormal: the Ka's for cortisol and 17 alpha hydroxyprogesterone binding were 50% below normal, while the number of binding sites was significantly higher. Such changes in these carrier proteins could result from conformational transitions; they may cause abnormal transfer of hormonal information and/or steroid hormones metabolism, thus modifying the impact of steroids on the immune response in HIV+subjects.

The molecular basis for the recently described hereditary hyperferritinemia-cataract syndrome is the presence of a mutation in the iron-responsive element (IRE) of the L ferritin gene, located on chromosome 19q13.3-13.4. Two mutations have been reported so far, altering adjacent nucleotides in the IRE loop, in a region that has been extensively studied in vitro and shown to mediate high affinity interaction with the iron-responsive protein. In this report, we describe two families with a new mutation in the bulge of the IRE stem, and we show that this mutation alters the protein-binding affinity of the IRE in vitro to the same extent as the loop mutation. In addition, we present evidence that some variability in the age of onset of cataract can be associated with this genetic syndrome, probably because of additional genetic or environmental factors that modulate the penetrance of the L ferritin defect in the lens. We confirm that the patients do not have increased iron stores despite the persistence of elevated serum ferritin levels and that, accordingly, they do not tolerate well venesection therapy. Further studies will be necessary to elucidate the mechanism responsible for the onset of cataract.

In vitro studies have shown that FFA induce conformational changes in human corticosteroid-binding globulin (CBG). We increased the plasma FFA concentrations of adult male rats by injecting heparin to determine whether such changes in CBG binding and immunological properties also occur in vivo. The in vivo transient activation of lipase by heparin produced a large increase in plasma FFA at 10 and 20 min (P < 0.01), which was maximal at 60 min (P < 0.005) and remained elevated at 120 min (P < 0.01) postinjection. This rise in FFA was associated with a 2- to 3-fold increase in the binding indices (C values; liters per g) of corticosterone (B) and progesterone to CBG 60-120 min postinjection (P < 0.001). There was a good positive correlation (r = 0.85) between the increase in B binding and the rise in plasma FFA in heparin-treated rats. The enhanced B binding to CBG resulted from a 2-fold increase in the apparent number of binding sites, without any significant change in the affinity constant (Ka). FFA extracted from postheparin plasma and a standard FFA mixture induced similar changes in B binding to purified mature rat CBG. The immunological behavior of CBG was not significantly changed after heparin-induced lipolysis, but the immunoreactivity of CBG from heparin-treated rats was more reduced by incubation with exogenous FFA than that from controls. FFA extracted from the plasma of heparin-treated rats and a standard FFA mixture both produced a dose-dependent drop in the immunodetection of pure CBG. These binding and immunological studies indicate that FFA mediate conformational changes in rat CBG in vivo. Thus, FFA, in addition to their roles as metabolic energy sources and components of complex lipids, can be rapid potent endogenous modulators of steroid-protein interactions.

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