RESUMO
This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using (125)I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein - iron responsive element regulation mechanism.
Assuntos
Cobre/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas Reguladoras de Ferro/metabolismo , Ferro/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Cobre/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/genética , Humanos , Proteínas Reguladoras de Ferro/genética , Ligação Proteica/efeitos dos fármacos , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transferrina/genética , Transferrina/metabolismoRESUMO
It is well established that Fe and ceruloplasmin interact in animals and in in vitro models. However, Fe-mediated regulation of ceruloplasmin has never been investigated in humans. In an observational study, 53 pregnant women aged 19-39 yr (29.8 +/- 0.7 yr, mean +/- SEM) were recruited at the Aberdeen Antenatal Clinic, Aberdeen Maternity Hospital, UK. All requirements for local ethical committees were followed. Venous blood samples were taken from each woman at 34 wk gestation for measurement of Fe status and ceruloplasmin. Various parameters were used to test for Fe status. The most sensitive one appeared to be soluble transferrin receptor, which increased with parity. In the population studied, there was no relationship between hemoglobin or ferritin and serum ceruloplasmin. However, using soluble transferrin receptor (sTfR) levels, we were able to demonstrate an inverse linear relationship (r = 0.37, p = 0.021, n = 41) between Fe status and ceruloplasmin. Fe supplementation, number of previous pregnancies, and smoking habits did not affect this relationship. Our data support in vitro results showing regulation of ceruloplasmin by Fe and also suggest that the interactions between Fe and ceruloplasmin should be considered when Fe supplementation is given.