Transferrin receptor expression is controlled differently by transferrin-bound and non-transferrin iron in human cells

We studied the effects of iron supplied as transferrin-bound iron and iron supplied as non-transferrin iron on transferrin receptor expression by human cell lines. Defined conditions of iron supply were represented by (i) 5 microg/ml of iron-saturated transferrin (transferrin medium) and by (ii) 500 microM ferric citrate (ferric citrate medium). Transferrin receptor expression of studied cell lines (HeLa, K562, Jiyoye) grown as long-term cultures in transferrin medium was somewhat higher (up to 137% of the mean fluorescence intensity) than in ferric citrate medium. The receptor expression corresponded with cellular iron regulatory protein (IRP) activity (ratio activated/total), which was also higher in transferrin medium (0.69-0.84) than in ferric citrate medium (0.33-0.60). However, unexpectedly much higher (about 65-135-fold) cellular iron levels were found in ferric citrate medium (13.9-14.9 nmol/10(6) cells) than in transferrin medium (0.11-0.21 nmol/10(6) cells). In contrast to the iron levels, cellular ferritin levels of the cells in ferric citrate medium (38.3-130 ng/10(6) cells) were only about 2-7-fold higher than in transferrin medium (6.8-61.5 ng/10(6) cells). We suggest that iron supplied as non-transferrin iron (ferric citrate) is apparently less available for the control of transferrin receptor expression via IRP activity than iron supplied as transferrin.

Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages

Lukas Kühn

The intracellular iron level exerts a negative feedback on transferrin receptor (TfR) expression in cells requiring iron for their proliferation, in contrast to the positive feedback observed in monocytes-macrophages. It has been suggested recently that modulation of TfR and ferritin synthesis by iron is mediated through a cytoplasmic protein(s) (iron regulatory element-binding protein(s) (IRE-BP)), which interacts with ferritin and TfR mRNA at the level of hairpin structures (IRE), thus leading to inhibition of transferrin mRNA degradation and repression of ferritin mRNA translation. In the present study we have evaluated in parallel the level of TfR expression, ferritin, and IRE-BP in cultures of: (i) circulating human lymphocytes stimulated to proliferate by phytohemagglutinin (PHA) and (ii) circulating human monocytes maturing in vitro to macrophages. The cells were grown in either standard or iron-supplemented culture. TfR and ferritin expression was evaluated at both the protein and mRNA level. IRE-BP activity was measured by gel retardation assay in the absence or presence of beta-mercaptoethanol (spontaneous or total IRE-BP activity, respectively). Spontaneous IRE-BP activity, already present at low level in quiescent T lymphocytes, shows a gradual and marked increase in PHA-stimulated T cells from day 1 of culture onward. This increase is directly and strictly correlated with the initiation and gradual rise of TfR expression, which is in turn associated with a decrease of ferritin content. Both the rise of TfR and spontaneous IRE-BP activity are completely inhibited in iron-supplemented T cell cultures. In contrast, the total IRE-BP level is similar in both quiescent and PHA-stimulated lymphocytes, grown in cultures supplemented or not with iron salts. Monocytes maturing in vitro to macrophages show a sharp increase of spontaneous and, to a lesser extent, total IRE-BP; the addition of iron moderately stimulates the spontaneous IRE-BP activity but not the total one. Here again, the rise of spontaneous IRE-BP from very low to high activity is strictly related to the parallel increase of TfR expression and, suprisingly, also with a very pronounced rise of ferritin expression observed at both the mRNA and protein level. It is noteworthy the effect of beta-mercaptoethanol is cell specific, i.e. the ratio of total versus spontaneous IRE-BP activity is different in activated lymphocytes and maturing monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

1991

Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron

Lukas Kühn

The cell-surface receptor for transferrin mediates cellular uptake of iron from serum. Transferrin receptor protein and mRNA levels are increased in cells treated with iron chelating agents, and are decreased by treatment with iron salts or hemin. Here we report that expression of human transferrin receptor cDNA constructions in stably transfected mouse fibroblasts is regulated both by the iron chelator, desferrioxamine, and by hemin. We found that sequences within the 3' noncoding region are required for the iron-dependent feed-back regulation of receptor expression, whereas the presence of the transferrin receptor promoter region is not necessary. Regulation by iron is observed when transcription is initiated at either the SV-40 early promoter or the transferrin receptor promoter, but deletion of a 2.3 kb fragment within the 2.6 kb 3' noncoding region of the cDNA abolishes regulation and increases the constitutive level of receptor expression. Furthermore, the 3' deletion does not affect the decrease in receptors which is observed in response to growth arrest, indicating that transferrin receptor expression is controlled by at least two distinct mechanisms.

1987

Molecular regulation of iron proteins

Lukas Kühn

Cellular iron metabolism comprises pathways of iron-protein synthesis and degradation, iron uptake via transferrin receptor (TfR) or release to the extracellular space, as well as iron deposition into ferritin and remobilization from such stores. Different cell types, depending on their rate of proliferation and/or specific functions, show strong variations in these pathways and have to control their iron metabolism to cope with individual functions. Studies with cultured cells have revealed a specific cytoplasmic protein, called 'iron regulatory protein' (IRP) (previously known as IRE-BP or IRF), that plays a key role in iron homoeostasis by regulating coordinately the synthesis of TfR, ferritin, and erythroid 5-aminolevulinate synthase (eALAS). Present in all tissues analysed, IRP is identical with the [4Fe-4S] cluster containing cytoplasmic aconitase. Under conditions of iron chelation, IRP is an apo-protein which binds with high affinity to specific RNA stem-loop elements (IREs) located 5' of the initiation codon in ferritin and eALAS mRNA, and 3' in the untranslated region of TfR mRNA. At 5' sites IRF blocks mRNA translation, whereas 3' it inhibits TfR mRNA degradation. Both effects compensate for low intracellular iron concentrations. Under high iron conditions, IRP is converted to the holo-protein and dissociates from mRNA. This reverses the control towards less iron uptake and more iron storage. Iron can therefore be considered as a feedback regulator of its own metabolism. It has recently become evident that nitric oxide, produced by macrophages and other cell types in response to interferon-gamma, induces the IRE-binding activity of IRF. Moreover measurements of the RNA-binding activity of IRP in tissue extracts may provide valuable information on iron availability

1994

Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages

Lukas Kühn

1991