Download Copper and iron interactions in a placental cell line (BeWo)

Biochemical Society Transactions (1998) 26
16
Copper and iron interactions in a placental cell
line (BeWo)
Ruth Danzeisen and Hany J. McArdle, The Rowett Research
Institute, Greenburn Road, Bucksburn, Aberdeen AB21 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. [l]). Thc important form of copper seemed
to be the copper protein, Cp, since direct injection of Cp into the
circulation could mimic the copper response. Ceruloplasmin (Cp)
is a glycoprotein, with a molecular weight of 131.OOO and each
molecule carries six copper atoms. It has ferroxidase activity, and
it is this property which is thought to be important in iron
mobilisation. The hypothesis is that iron is released from cells as
Fe(II) and Cp oxidises it to Fe(II1) so that it can be incorporated
into sexum transferrin (reviewed in [2]).
Iron transport across the placenta is poorly understood.
Uptake is from transferrin via receptor mediated endocytosis [3]
but there are no data on release into the fetal circulation. This
paper examines a role for Cp or other copper complexes in iron
efflux, using BeWo cells. BeWo cells are a choriocarcinoma cell
line, which will differentiate to form syncytia. We considered it
important to examine a role for Cp in both differentiated and
undfferentiated cells and this report presents data on Fe efflux in
the latter.
Cells were grown to 80 8 confluence in Hams F12
supplemented with 10 8 fetal calf serum and antibiotics. They
were incubated overnight with 0.1 ~Ci/ml'%e as Fe-transfemn
The following day, the cells were washed and incubated in
balanced salt solution with the addition of metal complexes as
detailed in the results. After incubation, the medium was
removed for counting and the cells collected using Pronase
digestion [3]. The supernatant, representing surface bound label,
and the cells, representing intracellular label, were counted
separately.
Data were corrected for cell death or cell damage by
measuring LDH activity in the medium and the cells. In all
experiments, less than 1 % of LDH activity was found in the
medium, showing that cell damage was insignificant. DNA
content of the cetkwas estimated using Hoeschst 33258.
After overnigiit-iincubation with 'Ve-labelled transferrin, cells
were incubated with or without 10 pg/ml Cp for increasing times
in 'Ve-free media. There was no difference in the efflux pattern.
By 6 h, approximately 35 % of intracellular iron had been
released to the medium in both conditions. (Fig 1)
S99
Table 1tnon Cu c a t of Bewo cek
Cells were incubated as described, digested in nitric acid and Cu
content measured using carbon furnace atomic absorption
spectroscopy. Results are the mean SEM of 6 plates in each
treatment
Control
CuHis,
Cp
Diamsar
c u hnol
3.2 f
13.9
4.5 f
none
/pgDNA)
0.9
It
0.6
detectable
*
e n
We tested the effect of Cu status of the cells on Fe efflux by
adding Cp, CuHis, or a Cu chelator, diamsar, to the overnight
incubation medium. This h'eatment altered the Cu content of the
cells as shown in Table 1. Even though the Cu content changed
dramatically, it made no difference to subsequent Fe efflux.
The data presented in this abstract show clearly that Cu levels
in BeWo cells have no effect on Fe efflux from the cells and that
extracellularCu complexes also have no effect on Fe efflux. The
results are somewhat surprising and need to be considered
carefully.
There is no doubt that Cp plays a role in iron metabolism in
vivo and the genetic and experimeytal data all point towards it
being essential for iron efflux, especially from liver, brain and
spleen cells [4]. It would be expected that it should also be
important in placenta, since Fe efflux is clearly necessary to
release Fe to the fetal circulation.
This would appear not to be the case. There are several
possible explanations. Iron accumulation does not occur in all
tissues, so that perhaps the placenta is one which does not need
Cp for Fe efflux. In the developing fetus, however, Cp mRNA is
present in the liver [5], and presumably, therefore, the protein is
available to oxidise the Fe(I1) as it is released. Alternatively, the
efflux may not be dependent directly on Cp in the medium, but on
an interaction of Cp with the membrane. We have identified a Cp
binding protein in microvillar membrane vesicles, and perhaps it
is also found on the baso-lateral membrane, where it could hold
Cp order to facilitate oxidation of Fe(I1) [6]. It may be important
that these cells were not differentiated. Harris and colleagues
have shown that proteins of Cu metabolism are expressed in
differentiated but not undifferentiated BeWo cells [7], and it is
possible that the same is true for proteins of Fe metabolism. A
third possibility is that Cp has an indirect role to play, rather than
a direct one, and it donates Cu to an intracellular protein which is
a closer analogue to Fet3p. In relation to this, it is well
established that liver expresses a large Cp transcript as well as the
3.7 kb mRNA [8] and it is tempting to speculate that this is a
membrane bound form of the protein, which serves to oxidise the
Fe as it is released from the cell. Which of these explanations is
correct remains to be determined.
This work was supported by the Scottish Office Agricultural,
Environmental and Fisheries Department
1
1C
2C
I
0
60
120
180
240
300
Time (mln)
Fie 1 Extracellular'cedo~lasmindoes not stimulate Fe
efflux &om BeWo cells. The ceh were incubated for increasing
periods of time as shown. Results are the mean f SEM; n = 4
The same result was observed when increasing
concentrations (up to 100 pg/ml) of Cp were added. Again, the
protein had no effect on Fe efflux. We considered the possibility
that low molecular weight complexes may have an effect on efflux
and incubated cells which had been loaded overnight with 'Ve as
Fe-transferrin with 2 pM CuHis,. Once again, there was no
effect on Fe efflux.
Lahey M. E., Gubler C. J., Chase M. S., Cartwright G.
E. and Wintrobe M. (1952) Blood 7, 1053-1074
Harris E. D. (1996) Nut. Rev. 53, 170-173
McArdle H. J., Douglas A. J. and Morgan E. H. (1984)
J. Cell. Physiol. 122, 405-409
Kaplan J. and OHalloran T. V. (1996) Science 271,
1510-1512
Linder M. C. (1991) Biochemistry of Copper, Elsevier,
New York
Hilton M., Spenser D. C., Ross P., Ramsey A. and
McArdle H. J. (1995) Biochim. Biophys. Acta 1245,
153-160
Qian Y., Majundar S., Reddy M. C. and Harris E. D.
(1996) Am. J. Physiol. 270, C188O-Cl884
Fleming R. E. and Gitlin J. D. (1990) J. Biol. Chem.
265, 7701-7707