Ceruloplasmin alters intracellular iron regulated proteins and pathways: ferritin, transferrin receptor, glutamate and hypoxia-inducible factor-1α.

Ceruloplasmin (Cp) is a ferroxidase important to the regulation of both systemic and intracellular iron levels. Cp has a critical role in iron metabolism in the brain and retina as shown in patients with aceruloplasminemia and in Cp-/-hep-/y mice where iron accumulates and neural and retinal degeneration ensue. We have previously shown that cultured lens epithelial cells (LEC) secrete Cp. The purpose of the current study was to determine if cultured retinal pigmented epithelial cells (RPE) also secrete Cp. In addition, the effects of exogenously added Cp on iron regulated proteins and pathways, ferritin, transferrin receptor, glutamate secretion and levels of hypoxia-inducible factor-1α in the nucleus were determined. Like LEC, RPE secrete Cp. Cp was found diffusely distributed within both cultured LEC and RPE, but the cell membranes had more intense staining. Exogenously added Cp caused an increase in ferritin levels in both cell types and increased secretion of glutamate. The Cp-induced increase in glutamate secretion was inhibited by both the aconitase inhibitor oxalomalic acid as well as iron chelators. As predicted by the canonical view of the iron regulatory protein (IRP) as the predominant controller of cellular iron status these results indicate that there is an increase in available iron (called the labile iron pool (LIP)) in the cytoplasm. However, both transferrin receptor (TfR) and nuclear levels of HIF-1α were increased and these results point to a decrease in available iron. Such confounding results have been found in other systems and indicate that there is a much more complex regulation of intracellularly available iron (LIP) and its downstream effects on cell metabolism. Importantly, the Cp increased production and secretion of the neurotransmitter, glutamate, is a substantive finding of clinical relevance because of the neural and retinal degeneration found in aceruloplasminemia patients. This finding and Cp-induced nuclear translocation of the hypoxia-inducible factor-1 (HIF1) subunit HIF-1α adds novel information to the list of critical pathways impacted by Cp.

Iron metabolism in the eye: a review.

This review article covers all aspects of iron metabolism, which include studies of iron levels within the eye and the processes used to maintain normal levels of iron in ocular tissues. In addition, the involvement of iron in ocular pathology is explored. In each section there is a short introduction to a specific metabolic process responsible for iron homeostasis, which for the most part has been studied in non-ocular tissues. This is followed by a summary of our current knowledge of the process in ocular tissues.

Lens epithelial cells synthesize and secrete ceruloplasmin: effects of ceruloplasmin and transferrin on iron efflux and intracellular iron dynamics.

Although an essential nutrient, iron can catalyze damaging free radical reactions. Therefore elaborate mechanisms have evolved to carefully regulate iron metabolism. Ceruloplasmin, a protein with ferroxidase activity, and transferrin, an iron binding protein have important roles in maintaining iron homeostasis in cells. Since oxidative damage is a hallmark of cataractogenesis, it is essential to determine iron's role in lenticular physiology and pathology. In the current study of lens epithelial cells, the effects of ceruloplasmin and transferrin on intracellular distribution and efflux of iron were determined. Both ceruloplasmin and transferrin increased iron efflux from these cells and their effects were additive. Ceruloplasmin had significant effects on extracellular iron distribution only in cases of iron overload. Surprisingly, both transferrin and ceruloplasmin had significant effects on intracellular iron distribution. Under physiological conditions, ceruloplasmin increased iron incorporation into the storage protein, ferritin. Under conditions of iron overload, it decreased iron incorporation into ferritin, which is consistent with increased efflux of iron. Measurements of an intracellular chelatable iron pool indicated that both transferrin and ceruloplasmin increased the size of this pool at 24 h, but these increases had different downstream effects. Finally, lens epithelial cells made and secreted transferrin and ceruloplasmin. These results indicate an important role for these proteins in iron metabolism in the lens.

The effect of UVB irradiation on ferritin subunit synthesis, ferritin assembly and Fe metabolism in cultured canine lens epithelial cells.

Ferritin is a multimeric protein consisting of heavy and light chains assembled in different tissue-specific ratios, which can protect cells from oxidative stress by storing reactive iron (Fe). Because the lens is constantly exposed to UV irradiation, we studied its effects on ferritin synthesis and Fe metabolism in cultured lens epithelial cells with and without ascorbic acid (Asc). UVB caused a large increase in accumulation of newly synthesized ferritin chains; this increase was additive to that induced by Asc. In contrast to the Asc-induced increase in Fe storage, Fe storage in ferritin was unaltered by UVB. Although UVB increased accumulation of newly synthesized ferritin chains, total ferritin levels were unaltered. In contrast, Asc, which induced a quantitatively similar increase in accumulation of newly synthesized ferritin chains, doubled the total amount of ferritin. Because UVB did not change Fe storage in ferritin or the size of the labile Fe pool, it was hypothesized and then determined that these newly synthesized chains did not assemble into functional holoferritin. Numerous studies detail the effects of various treatments on de novo ferritin synthesis; however, this study provides a cautionary note regarding the conclusions of such studies in the absence of data indicating assembly of functional ferritin molecules.

The effect of ascorbic acid and ferric ammonium citrate on iron uptake and storage in lens epithelial cells.

Ferritin is the major intracellular iron storage protein which has been shown to protect cells against oxidative damage. Recent reports that an inherited abnormality in human ferritin synthesis is associated with early bilateral cataracts underscore the importance of understanding ferritin synthesis and iron storage in lens epithelial cells. We previously demonstrated that ascorbic acid greatly increases de novo synthesis of ferritin in lens epithelial cells. The objectives of the present study were to determine: (1) the effects of ascorbic acid and ferric ammonium citrate on iron uptake by canine lens epithelial cells from iron bound to transferrin and from ferric chloride and (2) the incorporation of this element into ferritin. Iron uptake by lens epithelial cells from 59ferric chloride was 20 times higher than from 59iron-transferrin and iron deposition into ferritin was 8-fold higher when 59ferric chloride was the source. Ascorbic acid had a stimulatory effect on iron uptake from transferrin and on incorporation of this element into ferritin. The ascorbic acid-induced increase of iron uptake required de novo protein synthesis but not specifically de novo ferritin biosynthesis. Although ferritin is not directly involved in iron uptake, the level of ferritin protein could control the pool of intracellular iron. The present results indicate that iron homeostasis in lens epithelial cells is affected mainly by changes in apoferritin synthesis, which is greatly stimulated by ascorbic acid, rather than by altering the rate of protein degradation, which is very slow in these cells under all circumstances. Ferric ammonium citrate activates iron uptake from transferrin in a wide range of cell lines by generation of free radicals. Ferric ammonium citrate also increased iron uptake from Tf in lens epithelial cells. Ferric ammonium citrate treated cells incorporated 5 times more iron and deposited 2 times more iron into ferritin than control cells. Increased incorporation of iron into ferritin was due to ferric ammonium citrate-induced stimulation of de novo ferritin synthesis rather than an increased rate of iron deposition into pre-existing ferritin. Ferric ammonium citrate had a different effect on iron uptake from ferric chloride; total iron uptake was not significantly increased while deposition into ferritin was significantly decreased. These results demonstrate that iron homeostasis in lens epithelial cells is regulated by ascorbic acid and by changes in the rate of de novo ferritin synthesis. In addition, the differences in iron uptake from transferrin and ferric chloride and its subsequent incorporation into ferritin suggests that the mechanisms by which iron is incorporated into ferritin are source dependent.

Transferrin in after-cataract and as a survival factor for lens epithelium.

The Fe-transport protein, transferrin (Tf), is synthesized and secreted by whole lenses and cultured lens epithelial cells. Because of Tf's central role in cell growth and proliferation, its participation in lens cell proliferation following cataract extraction was explored using a rabbit model of after-cataract. Varying amounts of the central anterior lens capsule were removed (0, 35, or 80%) following extraction of the lens. The Tf content of and secretion by after-cataract lens capsular sacs containing regenerated lens tissue was determined ex vivo at 0, 3, 5, 7 and 9 weeks post-surgery. In all cases Tf content of and secretion by the lens sacs was higher than that of their contralateral controls (whole lenses). Tf secretion was up to 5-fold higher and metabolic labeling studies indicated secretion of newly synthesized Tf. The sacs contained up to 10 times the concentration of Tf as the control lenses. Human lens after-cataract capsular bags also secreted Tf. The function of Tf as a survival factor was tested on cultured lens epithelial cells. Cells cultured in serum-free medium had a survival rate of only 20-34% if the medium was changed each day. If the medium was never changed during this period, the survival rate was 43-52%, suggesting secretion of essential growth factors by these cells. Addition of 200 microg ml-1 Tf to the medium during each daily change increased survival to levels attained when the medium was not changed. Addition of Tf antibodies to the culture medium during each daily change decreased cell survival to 14%. Apparently Tf acts as a survival factor for lens epithelia and its synthesis is up-regulated in after-cataract lens sacs. These factors suggest that Tf may play an important role in the pathogenesis of lens epithelial cell proliferation and after-cataract formation following cataract surgery.

Regulation of ferritin levels in cultured lens epithelial cells.

In most eukaryotic cells, synthesis of the iron storage protein, ferritin is regulated by iron levels and redox conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical reactions. Although iron-catalysed reactions are believed to contribute to oxidative damage and cataractogenesis, little is known about iron storage in the lens. In this study, ferritin concentration was measured in cultured canine lens epithelial cells. Baseline ferritin concentration ranged from 76-163 ng (mg protein)-1; cells cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration. The iron chelator, Desferal, significantly decreased ferritin concentration. The reducing agent dithiothreitol decreased the hemin-induced increase in ferritin levels, but not baseline levels. In contrast, ascorbic acid induced a large increase in ferritin content. Other studies have shown that induction of ferritin synthesis can protect against oxidative damage. Regulation of ferritin levels may represent a mechanism by which the lens epithelium is protected from oxidative damage. In vivo, epithelial cells are normally exposed to much lower iron concentrations than the cultured lens epithelial cells in this study. However, in pathological circumstances, the iron content and redox state of the aqueous humor is dramatically altered and may affect the steady state levels of ferritin within the lens. This remains to be determined.

Effect of rat age on serum levels of growth hormone and somatomedins.

The somatomedins are a family of hormones which appear to mediate many of the anabolic actions of growth hormone; these processes often exhibit an age-associated deterioration in intact animals. We have demonstrated the validity of a radioreceptor assay for the determination of somatomedin levels in rat serum. In this assay, we measure displacement of 125I-labeled Multiplication Stimulating Activity (MSA) from receptors prepared from human placental membranes. Results with this procedure confirm and extend a previous report from this laboratory indicating a significant decrease in somatomedin levels during the latter part of the lifespan. Data are presented to eliminate possible artifactual explanations for the observed age-related changes. Furthermore, we find that the decrease in somatomedin levels can not be a simple result of an age-related decrease in basal levels of growth hormone in serum. We conclude that the decrease with age in circulating levels of the somatomedins is most probably attributable to a decrease in the activity of responsiveness of the tissues (most probably liver) which secrete somatomedins in response to stimulation by growth hormone.