Analysis of the Effect of Soluble Transferrin Receptor 1 (sTfR1) on Iron Metabolism in Mice.

Transferrin receptor 1 (TfR1) is a cell surface-expressed protein that mediates cellular uptake of iron. The TfR1 molecule may be shed from cells by proteolytic cleavage at Arg₁₀₀ - Leu₁₀₁, resulting in a cleaved form called soluble transferrin receptor 1 (sTfR1), which circulates in the blood as a 74 kDa monomer bound to transferrin. Measurement of soluble transferrin receptor has been widely used for the diagnosis of iron deficiency, but the levels of the receptor are also increased in patients with ineffective erythropoiesis, because the amount of circulating sTfR1 is directly proportional to the total amount of cell-associated TfR1. The key factors that stimulate increased sTfR1 levels are the integrated effects of reduced iron availability and increased erythropoietic stimulation. It is notable that both in iron deficiency and in diseases such as thalassemia, in which there is active ineffective erthropoiesis, iron absorption is increased This raises the question of whether sTfR1 is merely an intermediate in the degradation of the protein or whether it is a physiological regulator of iron absorption. To investigate this potential signaling function of sTfR1, a hydrodynamic gene transfer technique was established to express transfected plasmid constructs of human sTfR1 (hsTfR1, aa89–759) and murine sTfR1 (MsTfR1, 86–763) from the livers of C57Bl6 mice. To assess the effect that hsTfR1 and MsTfR1 might have on iron metabolism, iron absorption, serum iron and gene expression of hepcidin was measured in hydrodynamically transfected mice. The efficacy of the hydrodynamic technique was demonstrated by sustained expression of hsTfR1 in mice, at a level six fold higher than the normal level of hsTfR1 in humans (Fig. 1). Iron absorption was determined in transfected mice by measuring the retention of gavage fed ⁵⁹Fe in a carrier iron solution, using a whole animal counting technique. Repeated experiments showed neither hsTfR1 nor MsTfR1 had any effect on the amount of iron absorbed. In agreement with these data, hepcidin levels were found by real time PCR to be unaltered in the same transfected mice. When serum iron levels were measured, expression of either hsTfR1 or MsTfR1 in mice resulted in significantly higher serum iron levels versus control mice (P=0.0141 and P=0.0192 respectively by t-test). We conclude that despite its attractiveness as a potential modifier of iron absorption, sTfR1 does not have any effect on hepcidin expression or iron absorption in mice. Mice transfected with hsTfR1 or MsTfR1 do have significantly higher serum iron levels, which may reflect an increased capability of these mice to retain transferrin in the serum. We have found that hydrodynamic transfection is a useful method to increase the levels of putative biologically active compounds in mice; this technique can be valuable in investigating the roles of serum or liver specific proteins in mice.

Fig. 1

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Sustained Expression of hsTfR1 in Mice

Fig. 1

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Sustained Expression of hsTfR1 in Mice

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