Transferrin gene expression. Effects of nutritional iron deficiency.

Nutritional iron deficiency was produced experimentally by raising newly hatched chicks on an iron-deficient diet for several weeks. During this time, hematocrit and hemoglobin values declined, iron stores were depleted, and the circulating level of transferrin increased 2- to 4-fold. The increase in serum transferrin was related to a similar increase in the rate of transferrin synthesis in liver. In addition, the level of transferrin mRNA sequences, as determined by hybridization to a specific cDNA, increased 2- to 3-fold, and more than 80% of the transferrin mRNA was associated with polyribosomes in both control and iron-deficient liver. These results demonstrate that the induction of transferrin synthesis in iron-deficient chicks is regulated directly by an increase in transferrin mRNA. The iron-mediated effects on transferrin also appear to be gene-specific since the rate of synthesis of serum albumin, the major secretory product of liver, was unaffected by any of the experimental conditions. Furthermore, when iron stores were rapidly replenished by the administration of iron-saturated ferritin, both the rate of transferrin synthesis and the level of transferrin mRNA returned to control values with 2 to 3 days.

Role of plasma membrane phospholipids in the uptake and release of transferrin and its iron by reticulocytes.

The involvement of membrane phospholipids in the utilization of transferrin-bound iron by reticulocytes was investigated using [59Fe]- and [125I]-labelled transferrin and rabbit reticulocytes which had been incubated with phospholipase A. Transferrin and iron uptake and release were all inhibited by phospholipase A which produced a marked decrease in the relative abundance of phosphatidylcholine and phosphatidylethanolamine and equivalent increases in their lyso-compounds in the reticulocyte plasma membrane. There was a close correlation between the iron uptake rate and the rate and amount of transferrin uptake and the amount of the lysophospholipids in the membrane. Incubation of the cells with exogenous lysophosphatidylethanolamine or lysophosphatidylcholine also produced inhibition of iron and transferrin uptake. The reduced uptake produced by phospholipase A could be reversed if the lyso-compounds were removed by fatty acid-free bovine serum albumin or by reincubation in medium 199. Treatment with phospholipase A was shown to increase the amount of transferrin bound by specific receptors on the reticulocyte membrane but to inhibit the entry of transferrin into the cells. The present investigation provides evidence that the phospholipid composition of the cell membrane influences the interaction of transferrin with its receptors, the processes of endocytosis and exocytosis whereby transferrin enters and leaves the cells, and the mechanism by which iron is mobilized between its binding to transferrin and incorporation into heme. In addition, the results indicate that phosphatidylethanolamine is present in the outer half of the lipid bilayer of reticulocyte membrane.

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase.

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with 125I and 59Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of N-ethylmaleimide which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37 degrees C but nearly all that taken up 4 degrees C was released when the cells were subsequently incubated with trypsin plus N-ethylmaleimide, despite the fact that about 80% of the 59Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells. These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37 degrees C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.