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

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)

Interleukin-2-dependent transcriptional and post-transcriptional regulation of transferrin receptor mRNA

Lukas Kühn

Interleukin-2 (IL-2) controls the proliferation of the murine T cell line B6.1 and induces transferrin receptor (TfR) mRNA steady-state levels 50-fold when added to arrested, IL-2-deprived cells. In addition, TfR mRNA is post-transcriptionally regulated by intracellular iron. Low iron levels activate a cytoplasmic RNA-binding protein, called iron regulatory factor (IRF) or iron-responsive element-binding protein, which coordinately stabilizes TfR mRNA and inhibits ferritin mRNA translation. Since ferritin expression is known to be modulated by cytokines, we decided to investigate the mechanism by which IL-2 activates TfR gene expression in B6.1 cells. Induction by IL-2 of both nuclear and cytoplasmic TfR RNA was compared with run-on transcription rates in isolated nuclei. The results revealed a 3-fold increase in TfR gene transcription and a 6-fold rise in nuclear TfR RNA reaching its steady-state level within 2 h. The main accumulation of mature mRNA in the cytoplasm occurred after 6 h in parallel with the activation of IRF. However, stimulation of IRF binding activity by the iron chelator desferrioxamine, in the absence of IL-2, failed to induce TfR mRNA. Moreover, deprivation of growing B6.1 cells of IL-2 resulted in cell arrest and a rapid decay of TfR mRNA, which was not prevented by the activation of IRF with desferrioxamine. TfR mRNA stabilization appears, therefore, to depend on IL-2. We conclude that TfR mRNA expression is controlled by at least three steps at the onset of cell proliferation: (i) the growth factor-dependent activation of transcription; (ii) mRNA stabilization by IRF in the cytoplasm; and (iii) an additional IL-2-dependent activity which prevents TfR mRNA degradation. Our results indicate that expression of TfR, like ferritin, is controlled by both iron and cytokines.

1993

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

Lukas Kühn

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.

1998

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