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)
Testa
U
U
Department of Hematology-Oncology, Istituto Superiore di Sanità, Rome, Italy.
Kühn
L
L
Petrini
M
M
Quaranta
M T
MT
Pelosi
E
E
Peschle
C
C
D000328:Adult; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005293:Ferritins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism; D035925:Iron-Regulatory Proteins; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D035925:Iron-Regulatory Proteins; D010835:Phytohemagglutinins; D011990:Receptors, Transferrin; D005293:Ferritins; D007501:Iron