Effects of cell proliferation on the uptake of transferrin-bound iron by human hepatoma cells.
ABSTRACT The effects of cellular proliferation on the uptake of transferrin-bound iron (Tf-Fe) and expression of transferrin receptor-1 (TfR1) and transferrin receptor-2 (TfR2) were investigated using a human hepatoma (HuH7) cell line stably transfected with TfR1 antisense RNA expression vector to suppress TfR1 expression. At transferrin (Tf) concentrations of 50 nmol/L and 5 micromol/L, when Tf-Fe uptake occurs by the TfR1- and TfR1-independent (NTfR1)-mediated process, respectively, the rate of Fe uptake by proliferating cells was approximately 250% that of stationary cells. The maximum rate of Fe uptake by the TfR1- and NTfR1-mediated process by proliferating cells was increased to 200% and 300% that of stationary cells, respectively. The maximum binding of Tf by both TfR1- and NTfR1-mediated processes by proliferating cells was increased significantly to 160% that of stationary cells. TfR1 and TfR2-alpha protein levels expressed by proliferating cells was observed to be approximately 300% and 200% greater than the stationary cells, respectively. During the proliferating growth phase, expression of TfR1 messenger RNA (mRNA) increased to 300% whereas TfR2-alpha mRNA decreased to 50% that of stationary cells. In conclusion, an increase in Tf-Fe uptake by TfR1-mediated pathway by proliferating cells was associated with increased TfR1 mRNA and protein expression. An increase in Tf-Fe uptake by NTfR1-mediated pathway was correlated with an increase in TfR2-alpha protein expression but not TfR2-alpha mRNA. In conclusion, TfR2-alpha protein is likely to have a role in the mediation of Tf-Fe uptake by the NTfR1 process by HuH7 hepatoma cell in proliferating and stationary stages of growth.
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ABSTRACT: Nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) modulate iron regulatory protein (IRP) activity and may, therefore, affect iron uptake through transferrin receptor expression. However, iron also enters the cell as nontransferrin-bound iron (NTBI), and the aim of this study was to evaluate the effects of NO donors on NTBI transport in HepG2 cells, a model of liver physiology. Incubation with SNP and SNAP led to a time- and concentration-dependent reduction in Fe3+ and Fe2+ uptake, thus indicating an effect on the transporter rather than on the reductase. In terms of Fe2+ uptake, no variations in the Michaelis-Menten constant (Km) and a reduction in maximum uptake (Vmax) (50, 33, and 16.6 fmol/microgram protein/min in control, SNP-, and SNAP-treated cells, respectively) were detected, which suggested a decrease in the number of putative NTBI transport protein(s). Gel shift assays showed that IRP activity was reduced by SNP and slightly increased by SNAP. Northern blot analysis of transferrin receptor messenger RNA (mRNA) levels showed variations similar to those observed for IRPs, but both NO donors increased L-ferritin mRNA levels and had no effect on the stimulator of Fe transport (SFT) mRNA. In conclusion, NO donors significantly reduce NTBI transport in HepG2 cells, an effect that seems to be IRP and SFT independent. Moreover, the reduction in NTBI uptake after NO treatment suggests that this form of iron may play a minor role in the increased hepatic iron stores observed in inflammation or that other liver cells are more involved in this pathological condition.Hepatology 03/1999; 29(2):464-70. · 12.00 Impact Factor
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ABSTRACT: Freshly isolated rat hepatocytes display abut 36700 high-affinity sites to which differic transferrin may bind with an apparent association constant of 1.62 . 10(7) l . mol-1. Uptake of iron from diferic transferrin by hepatocytes is linear with time and is accelerated at increased diferric transferrin concentrations. Apotransferrin is able to decrease net iron uptake by hepatocytes from diferric transferrin by a process not dependent on the apotransferrin concentrations used or on the rate at which the cells take up iron. Immunoprecipitation of the apotransferrin during these incubations indicate that iron is being released from the cells to apotransferrin at the same time as iron is being taken up from diferric transferrin. The simultaneous uptake and release of iron, and the insensitivity to apotransferrin concentration, suggest that the processes of iron uptake and release occur via separate mechanisms. The effect of apotransferrin on net retention of iron may be one way in which the in vivo distribution of iron between sites of storage and utilization is controlled.Biochimica et Biophysica Acta 01/1981; 633(2):145-53. · 4.66 Impact Factor
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ABSTRACT: To explore a mechanism of interleukin (IL)-6-induced hypoferremia in rats, iron metabolism was investigated both in vivo and in vitro. Recombinant IL-6 was intraperitoneally administered to male Wistar rats and the serial change of parameters related to iron metabolism was examined. After administration of IL-6, plasma IL-6 concentration increased rapidly, reached its maximum in 1 hr and thereafter decreased quickly. Plasma IL-6 3 hr after IL-6 injection (50 micrograms/kg) was 3 units/ml, which is a concentration capable of inducing hepatic 125I-labeled transferrin uptake in vitro using isolated hepatocytes. Plasma iron concentration and transferrin saturation had decreased to approximately one third of the initial level within 3 hr and then recovered. Total iron binding capacity remained unchanged for 6 hr, then began to decrease. Red blood cell count and hemoglobin concentration showed no remarkable changes during this period. By ferrokinetic study with plasma that contained iron 59-labeled transferrin, the plasma iron disappearance half time, calculated from the disappearance curve, was significantly shortened from 55 min to 22 min by IL-6 treatment (p < 0.01). The ferritin concentration in the liver was increased significantly after the administration of IL-6 (p < 0.001), but transiently decreased in the spleen. The plasma ferritin showed a gradual increase during the 6-hr period after IL-6 injection. The uptake of 125I-labeled diferric transferrin by isolated hepatocytes was increased by IL-6 treatment and this increment was inhibited by addition of 100-fold excess unlabeled transferrin. On the other hand, no significant increment of 125I-labeled diferric transferrin uptake was observed in Kupffer cells.(ABSTRACT TRUNCATED AT 250 WORDS)Hepatology 06/1994; 19(6):1468-75. · 12.00 Impact Factor