Download Copper and iron homeostasis in mammalian cells and cell lines

Biochemical Society Transactions (1998) 26
135
Copper and iron homeostasis in mammalian cells
and cell lines
Roberta J. Ward', Maria-Laura Scarino', Armro Leone,
R. Crichton' and 'Hany J. McArdle
Robert
Unit6 de Biochimie, 'Universitt Catholique de Louvain B-1348
Louvain la Neuve Belgium, 'Istituto Nazionale della Nutrizione,
Rome, Italy, Dept of Pharmaceutical Sciences, University of
Salerno, 84080 Salerno, Italy, 'The Rowett Research Institute,
Aberdeen AB2 1 9SB Scotland.
It has been known for many years that the metabolism of
copper and iron are inter-related. Early data showed that when
pigs were made copper deficient, iron levels in specific tissues
rose . When copper was administered, the iron was released into
the plasma (see e.g. [I]). The inverse also seems to be the case,
in that iron deficiency results in increased Cu while iron overload
gives a decrease in Cu [2]. The molecular basis for the changes
are unclear, but recent studies in yeast have clarified the
interaction between these two metals and the degree of homology
between yeast and mammalian cells is strilung .
For example, prior to uptake, iron is reduced from Fe(II1) to
Fe(I1) and Cu(I1) to Cu(1) by Frelp [3]. This enzyme is similar
to the NADH oxidase described as involved in Cu(1I) reduction
by ourselves in liver [4]. Iron is re-oxidised by Fet3p [5], which
shows strong similarities to ceruloplasmin, and ceruloplasmin is
important in iron mobilisation in mammalian organisms. Ccc2p is
a Cu-ATPase (61 showing strong homology to ATWA and
ATP7B. the genes which are altered in Menkes' and Wilson
disease, respectively.
Despite the similarities. however, it is simplistic to presume
interactions are identical in each tissue and in each system. For
example, it is clear that in placenta, most of the iron is taken up
from transferrin and not from ionic complexes [7], as is the case
in yeast. In contrast, in the gut, it is likely that the mechanisms
are more closely related. In mammalian liver, the importance of
maintaining homeostasis of both copper and iron is likely to give
rise to a series of regulatory mechanisms which may have a yeast
homologue, but cannot a priori be assumed to do so.
As part of an initiative sponsored by the European Union, we
are examining the interactions between Cu and Fe in a variety of
different cells, both in vivo and in vitro, comparing the data
obtained and determining whether the mechanisms are identical or
whether there are important physiological, biochemical and
molecular differences. In this paper, we present some of the
preliminary data obtained from a series of in vivo studies, from
hepatocytes and macrophages in primary culture and from
placental and gut immortalised cells.
In the first series of experiments, male Wistar rats were either
made Fe deficient or overloaded using a Fe deficient diet (0.09 pg
Felday) or by i.p. injection with Fe-dextran (total administered
120 mg Fe). The effect on liver Cu concentration is given in
Table 1.
Similar results are obtained in vitro. Primary cultures of
hepatocytes were loaded with Fe using Fe-transferrin overnight
and Cu transport kmetics measured the following day. Iron
loading led to a decrease in the Vmax for Cu uptake, without any
change in Km. suggesting. as in yeast, a down-regulation of
transporter number. Copper loading the cells increased iron
uptake, while depleting the cells of Cu using a chelator made little
difference to Fe uptake. The changes in Cu uptake and
processing seem, from preliminary evidence, to be mediated by
changes in activity of the metalloreductase on the hepatocyte
membrane [4].
Table 1 The effect of changing Fe status on Cu content in rat
liver 'p <0.05 vs control, mean-? SEM n = 3 or 4
Control
Deficient
Overloaded
Cu (pg/g) 4.5k0.4
6.5?0. I '
3.74f0.6
S191
Iron levels in macrophages isolated from the Fe loaded or be
deficient rats changed in an unexpected manner. Iron loading
gave an increase in the amount of Fe of about two fold, but
showed little change in the Fe deficient animals. After stimulation
of these macrophages (LPS and interferon y), there was a
significant decrease in the release of NO from the cells, while iron
deficient cells showed the contrary. These observations are
particularly interesting given the lack of changes in iron levels and
indicate that iron homeostasis alone within the macrophage may
not be the prime determinant of its interaction with NO synthesis.
At present, we do not know how Cu levels are altered or how
proteins of Cu metabolism are changed.
In placental cells, there is no evidence for an interaction
between Cu and Fe. We loaded BeWo cells with Fe or with Cu,
presented as CuHis or as Cp, and measured the effect on Fe or
Cu uptake. In contrast to the liver, there was no effect. This is
somewhat surprising, given that the placenta has the
responsibility for transferring iron into the fetal circulation and
iron release is thought to be dependent on plasma ceruloplasmin.
In gut cells. we measured the effect of Cu and Fe on
parameters associated with the formation of tight junctions as well
as transportation. Caco-2 cells were grown on filters for periods
up to 18 D and used when the trans-epithelial electrical resistance
(TEER) had rcached steady state levels.
Adding low
concentrations o l Cu to the baso-lateral side of the cells resulted in
a dose-dependenc decrease in TEER. This did not achieve
significance at 10 pM Cu, but by SO kM,values had decreased to
about 10 70. Adding ascorbate alone had no effect on TEER but
when Fe ascorbate was added. the resistance decreased markedly.
All these changes were reversible. Interestingly the effect of Cu
on TEER was much decreased in cells which over-express the
metal binding protein, metallothionein.
In summary. the data presented show that Cu and Fe
interactions are not the same i n every cell, that the mechanisms
may not be as simple as those identified in ycast, but also show
that there are common pathways which will allow us to clarify
how these two e$\ential, yet toxic, elements, interact with each
other.
This work wah supported by SOAEFD, COST D8/0006/97
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