(Received for publication, April 26, 1995)
From the
Characterization of non-transferrin (non-Tf) iron transport by
K562 cells has revealed unique properties relative to iron uptake
mechanisms present in other cell types (Inman, R. S., and
Wessling-Resnick, M.(1993) J. Biol. Chem. 268,
8521-8528). Since treatment of K562 cells with phorbol esters
promotes stable megakaryocytic differentiation, we examined the uptake
of non-Tf iron in response to protein kinase C activation. Treatment of
K562 cells with phorbol esters increased the cellular uptake of Fe 4-6-fold compared with untreated cells. The
phorbol ester-induced stimulation of
Fe uptake was time-
and dose-dependent, with significantly enhanced transport observed only
after prolonged administration of 50 nM phorbol
12,13-dibutyrate (>8 h). These effects can be attributed to an
increased V
of transport (14.0 ± 5 versus 0.6 ± 0.2 pmol/min/10
cells) as well
as a 6-fold increase in the apparent K
(1.2 ± 0.4 versus 0.2 ± 0.06
µM). It is thought that the reduction of Fe
to Fe
is required as a first step in the uptake
mechanism, and the associated ferrireductase activity of K562 cells is
also enhanced with phorbol ester treatment by 5-10-fold (337
± 53 versus 43 ± 3 pmol/min/10
cells). Bryostatin-1, a protein kinase C activator that fails to
induce differentiation of K562 cells, did not promote this effect,
indicating that the enhanced transport activity is dependent on the
differentiation response. The idea that synthesis of a new class of
transporters is responsible for this effect is supported by the
observation that actinomycin D blocks up-regulation of non-Tf iron
transport. The increased transport and ferrireductase activity induced
upon differentiation also correlate with the appearance of saturable
iron-binding sites on the surface of K562 cells with K
0.4 µM. These
results indicate that non-Tf iron transport activity and the expression
of cell-surface iron-binding proteins can be controlled by
environmental factors that promote megakaryocytic differentiation of
K562 cells.
Although the major pathway for the cellular delivery of iron is
via receptor-mediated endocytosis of transferrin (Tf), ()considerable evidence for alternative pathways of non-Tf
iron uptake has been established. Properties defined for Tf-independent
iron transport systems for many different cell types suggest that there
may be two classes: ``high'' K
(5-30 µM) transport has been described in
studies of fibroblasts(1, 2) , HeLa
cells(1, 2) , Chinese hamster ovary cells(1) ,
hepatocytes(3) , HepG2 cells(4) , and L1210
cells(5) , while ``low'' K
(0.3-0.5 µM) iron transport has been
observed for reticulocytes (6, 7) and K562
cells(8) . Our knowledge about the factors that regulate the
appearance of these alternative pathways for iron acquisition is
limited, but it has been reported that non-Tf iron uptake by
fibroblasts can be up-regulated upon exposure to heavy metals (2) , suggesting that environmental factors can influence iron
assimilation through Tf-independent mechanisms. In contrast, modulation
of Tf receptor activity is well documented and, in particular, has been
found to be associated with cancer cell differentiation in Friend, M1,
HL-60, and K562 cell
leukemias(9, 10, 11, 12, 13, 14, 15) .
Among other chemical inducing agents, administration of tumor-promoting
phorbol esters is known to promote a rapid down-regulation of surface
Tf receptors, with an associated decline in receptor synthesis upon
prolonged exposure (>12 h)(16, 17) . For K562
cells, such alterations are accompanied by morphological and functional
changes due to megakaryocytic differentiation, including the expression
of glycoprotein IIIa (a platelet-specific antigen), the loss of
glycophorin A (an erythrocyte-specific lineage antigen), increased
platelet peroxidase activity, and secretion of granulocyte-macrophage
colony-stimulating factor and interleukin-6(18) . In this
report, we examine whether or not phorbol esters can also influence the
properties of the non-Tf iron transport system of K562 erythroleukemia
cells under these conditions. Our results indicate that while
Tf-mediated delivery is down-regulated when K562 cells are induced to
differentiate, non-Tf iron delivery is up-regulated. Thus, these two
different iron uptake mechanisms may be coordinately regulated.
To assess the effects of phorbol esters on non-Tf iron
uptake, K562 cells were treated with the concentrations of PDBu
indicated in Fig. 1for 16 h. The ability of the cells to
transport non-Tf iron was then measured as described under
``Materials and Methods.'' As shown in Fig. 1, the
rate of Fe uptake was significantly increased upon
exposure to 25 nM PDBu, with the maximum increase in non-Tf
transport activity induced by 50 nM PDBu. Treatment with PDBu
promoted a maximal stimulation of transport 3-5-fold over that
observed for control (untreated) K562 cells in a dose-dependent manner.
Figure 1:
Dose response of PDBu in the
stimulation of non-Tf iron uptake by K562 cells. K562 cells were
treated with the indicated concentrations of PDBu overnight. Fe uptake was then assayed as described under
``Materials and Methods.'' Briefly, 30 µl of 6 µM
FeNTA was added to 270 µl of PBS-washed K562
cells in uptake buffer (4.7-6.1
10
cells/ml).
After a 5-min incubation, the 250-µl reaction mixture was quenched
with 750 µl of ice-cold buffer containing 1 mM unlabeled
FeNTA to displace any nonspecific surface-bound radioactivity. The
cell-associated radioactivity measured in the same manner at 4 °C
was subtracted from that measured at 37 °C to obtain the specific
rate of
Fe transport. Shown are the averaged values from
triplicate transport measurements (±S.E.) as a function of
[PDBu] for a single experiment. These data are representative
of similar results obtained on three separate
occasions.
Effects of protein kinase C activation by phorbol esters can be
manifested within several minutes after administration; however,
treatment of K562 cells with phorbol esters is known to induce cellular
differentiation, which requires several hours to days before observable
changes occur(18) . To establish the earliest time point for
induction of the increase in non-Tf iron uptake, K562 cells were
treated with 50 nM PDBu for up to 16 h and then assayed for Fe transport activity. The results presented in Fig. 2demonstrate that increased transport rates are not
observed until after exposure to PDBu for >8 h.
Figure 2:
Time course of induction of
PDBu-stimulated Fe uptake. K562 cells were incubated with
50 nM PDBu at 37 °C for 0, 2, 4, 8, and 16 h and then
collected by centrifugation, washed with PBS, and resuspended in uptake
buffer. Transport activity was assayed exactly as described for Fig. 1with 5.0-6.6
10
K562 cells/ml.
Shown are the averaged values (±S.E.) for
Fe
transport rates determined in triplicate for K562 cells incubated with
PDBu for the indicated times. Data are from a single experiment that
was repeated three times.***, significantly different from control (p < 0.001).
The increase in Fe uptake could result from changes in the V
of uptake, the apparent K
, or both of these kinetic parameters.
We therefore determined these values for treated and control K562 cells
as described previously(8) . Double-reciprocal plots of initial
transport rate as a function of [FeNTA] were employed to
establish the V
and apparent K
. As shown by data presented in Fig. 3, both kinetic parameters were altered upon overnight
treatment with PDBu. Table 1presents a summary of the results
from these experiments. While control cells display a V
comparable to previously reported
activity(8) , PDBu-treated cells have a V
that is
20-fold greater (Table 1). The K
of transport was also increased
4-6-fold compared with control K562 cells.
Figure 3:
Double-reciprocal plots of transport rates
for PDBu-treated and control K562 cells. K562 cells were treated for 16
h with () or without (
) 50 nM PDBu and then assayed
for transport activity exactly as described for Fig. 1, except
that transport rates were determined as a function of
FeNTA concentration (75 nM to 1 µM).
Lineweaver-Burk plots of 1/rate versus 1/[FeNTA]
were constructed to determine K
and V
values from the slopes and intercepts of these
lines. The kinetic parameters determined for the experiment shown are
as follows: control cells, K
=
0.36 µM and V
= 1.03
pmol/min/10
cells; and PDBu-treated cells, K
= 2.37 µM and V
= 22.7 pmol/min/10
cells.
Experiments such as the one shown were performed on three separate
occasions with identical results.
In contrast to the
observed changes induced by phorbol esters, treatment of K562 cells
with bryostatin-1 fails to induce any significant alteration in the
properties of non-Tf iron uptake (Table 1). Like phorbol esters,
bryostatin-1 is an activator of protein kinase C; however, the latter
agent does not induce megakaryocytic differentiation of K562
cells(21) . Thus, our observations suggest that up-regulation
of non-Tf iron transport activity is specifically associated with
phorbol ester-mediated differentiation of K562 cells and the
concomitant expression of phenotypic markers of megakaryocytic lineage.
Since actinomycin D, an inhibitor of mRNA synthesis, blocks phorbol
ester-stimulated transport activity (Fig. 4), we conclude that
transcriptional activation associated with megakaryocytic
differentiation must be involved in the observed increase in the V and K
of non-Tf
iron transport. The simplest interpretation of these results is that
synthesis of a new class of transporters is induced under these
conditions, although it is not possible to rule out other
post-translational effects that may regulate the activity of the
existing iron uptake system.
Figure 4: Actinomycin D blocks phorbol ester-induced non-Tf iron transport by K562 cells. K562 cells were incubated with or without 50 nM PDBu and 1 µg/ml actinomycin D for 16 h. At the end of the incubation period, the cells were collected by centrifugation, and non-Tf iron uptake was measured exactly as described for Fig. 1. PMA, phorbol 12-myristate 13-acetate;***, significantly different from control (p < 0.001).
It is interesting to note that the
higher K of iron transport by phorbol
ester-treated K562 cells correlates well with apparent K
values reported for several other cell
types. This may suggest that upon differentiation, K562 cells express
an uptake mechanism akin to that observed for hepatocytes, fibroblasts,
and Chinese hamster ovary and HeLa
cells(1, 2, 3) . In the latter studies,
supraphysiological concentrations of calcium have been shown to promote
significant increases in non-Tf iron uptake. Moreover, depletion of
intracellular Ca
also correlates with decreased
transport activity. To establish whether the changes in transport
induced by PDBu may reflect an altered response of non-Tf iron uptake
to intra- or extracellular Ca
levels, treated and
control K562 cells were washed with 1 mM EGTA to deplete
intracellular Ca
or exposed to extracellular
Ca
(1 mM). As shown by the results presented
in Table 2, neither treatment had a significant effect on the
transport activity measured for PDBu-treated or control cells. These
observations are consistent with previous data from our laboratory
indicating that the non-Tf iron uptake system in K562 cells is
calcium-independent(8) ; most important, the results
demonstrate that stimulation of transport activity by PDBu does not
reflect an altered Ca
response of the non-Tf iron
uptake mechanism. The specificity of the non-Tf iron transporter in
K562 cells with respect to other transition metals is also distinct
from other uptake systems that have been characterized. Whereas the
transporter in K562 cells may be specific only for iron and
cadmium(8) , the non-Tf iron transporter in HeLa cells may be
able to transport cadmium, manganese, copper, and zinc as
well(1, 2) . Since the evidence presented above
indicates fundamental changes in transport characteristics, we further
examined the effects of 200 µM Cd
,
Co
, Cu
, Ni
, and
Mn
in the uptake assay. As shown by the results
presented in Table 2, only Cd
and
Cu
significantly inhibited non-Tf iron uptake by
control K562 cells, consistent with previous observations(8) .
None of the other cations were potent inhibitors of transport, unlike
observations made for non-Tf iron uptake by HeLa cells, Chinese hamster
ovary cells, and fibroblasts(1) . The stimulated transport
activity of PDBu-treated K562 cells was also resistant to inhibitory
effects of these metals; moreover, the efficacy of Cd
and Cu
to inhibit uptake was reduced (Table 2). This result may reflect the increase in apparent K
of transport upon phorbol ester
treatment. Thus, the specificity of non-Tf iron transport by K562 cells
does not appear to be altered upon differentiation.
A cell-surface
ferrireductase activity has been associated with non-Tf iron uptake by
K562 cells(8) . Reduction of Fe to
Fe
is thought to be the first step in the uptake
mechanism, and inhibition of the ferrireductase activity corresponds to
a block in non-Tf iron uptake(22) . Thus, the observed increase
in non-Tf iron uptake could be associated with a parallel stimulation
of the ferrireductase activity. To test this hypothesis, cell-surface
ferrireductase activity was measured in PDBu-treated and control K562
cells, with the results shown in Fig. 5. Extracellular reduction
of ferricyanide by PDBu-treated cells was stimulated 5-10-fold
compared with control K562 cells, with measured activities of 337
± 53 and 43 ± 3 pmol/min/10
cells,
respectively (n = 4). The enhanced ferrireductase
activity correlates well with the observed increase in non-Tf iron
uptake after overnight PDBu treatment and is compatible with the idea
that this function is closely associated with the uptake system. Since
reduction of Fe
to Fe
is presumably
a prerequisite for non-Tf iron transport(22, 23) , it
is possible that the observed stimulation of uptake is a functional
consequence of enhanced ferrireductase activity, although direct
evidence that the latter step is rate-limiting for transport is
lacking.
Figure 5:
Ferricyanide reductase activity is
stimulated in PDBu-treated K562 cells. K562 cells were treated with 50
nM PDBu overnight (16 h) and then assayed for cell-surface
ferrireductase activity as described under ``Materials and
Methods.'' Briefly, the capacity of cells to reduce
membrane-impermeant ferricyanide was measured by the production of
ferrocyanide upon incubation at 37 °C. Background activity was
measured at 4 °C in parallel reactions, and this value was
subtracted to yield specific cell-associated ferricyanide reductase
activity. Data are the average activity ± S.E. from four
independent experiments expressed as pmol/min/10 cells.
To identify proteins potentially involved in the phorbol
ester-induced transport activity, iron binding studies were performed.
As indicated by the results of Fig. 6, K562 cells do not display
a significant number of Fe-binding sites under basal
conditions. However, upon megakaryocytic differentiation, saturable
binding of
Fe to the surface of phorbol ester-treated K562
cells can be measured. Scatchard analysis of binding data reveals a K
for
Fe binding of 0.37
± 0.06 nM, with a single class of
5.4
10
binding sites/cell. Although the functional relationship
with the observed stimulation of non-Tf iron uptake remains unknown, it
is possible that the iron-binding proteins are components of a new
class of transporters expressed by K562 cells upon megakaryocytic
differentiation.
Figure 6:
Phorbol esters induce expression of
cell-surface iron-binding proteins. K562 cells treated with () or
without (
) 50 nM phorbol 12-myristate 13-acetate for 16 h
were assayed for cell-surface iron binding activity as described under
``Materials and Methods.'' Briefly, 0.5
10
cells were incubated with the indicated concentrations of
FeNTA for 1 h at 4 °C. Nonspecific binding, measured
in the presence of 5 mM unlabeled FeNTA, was subtracted to
yield specific iron binding activity (pmol of
Fe bound).
Shown are data from a single experiment representative of results
obtained on four separate occasions.
Tf-mediated iron delivery is known to be modulated by iron-induced changes in the half-life of receptor mRNA, as well as by regulation of the rate of transcription(24) . In contrast, non-Tf iron uptake does not appear to be influenced by intracellular iron levels(8) , indicating fundamental differences in the regulation of these two pathways for iron assimilation. Regulation of Tf receptor activity has also been correlated with leukemia cell differentiation and the cessation of cellular proliferation(9, 10, 11, 12, 13, 14, 15) . When human erythroleukemia K562 cells are exposed to phorbol esters, a rapid down-regulation of surface Tf receptors to intracellular compartments is observed(14, 15) , and prolonged exposure will diminish receptor synthesis(17) . In contrast, our results show that the non-Tf iron transport system is up-regulated by phorbol esters. Combined, these observations suggest that the cellular expression of these two different iron uptake mechanisms may be coordinately regulated. It is interesting to note that despite the down-regulation of the K562 cell Tf receptors under these conditions, the Tf-mediated iron delivery is only slightly suppressed(14, 25) , supporting the idea that the coordinate regulation between Tf-dependent and -independent pathways does not reflect changes in intracellular iron content.
Phorbol esters promote other changes in the phenotype of K562 cells as they cease to divide and begin to differentiate, expressing megakaryocytic markers(18) . The fact that actinomycin D blocks the PDBu-induced stimulation of Tf-independent transport indicates that mRNA and most likely protein synthesis are both required for this effect. Thus, altered transport activity appears to be the consequence of changes in cellular function due to the differentiation program. In fact, bryostatin-1, which activates protein kinase C but does not induce K562 cell differentiation(21) , fails to stimulate non-Tf iron transport activity. Two potential explanations can account for stimulation of non-Tf iron transport. 1) Expression of regulatory factors may modify the activity of existing plasma membrane iron transporters, and/or 2) synthesis of a new class of non-Tf transporters is induced to increase iron uptake. We favor the latter hypothesis due to the appearance of cell-surface iron-binding sites upon phorbol ester treatment of K562 cells; this activity may reflect properties of the new class of transport molecules.
Conrad et al.(26) have proposed that -integrin is
involved in non-Tf iron transport by K562 cells, although stringent
functional criteria to support a role for this extracellular matrix
adhesion factor in iron uptake are lacking. Since, upon megakaryocytic
differentiation of K562 cells, expression of glycoprotein IIIa or
-integrin is up-regulated(18) , it is possible
that the binding activity we observe is due to association of
Fe with
-integrin. The latter does indeed
bind cations; in fact, bound cation is displaced upon the binding of
-integrin by its ligand, typically fibrinogen or
another adhesion molecule that contains the RGD amino acid
motif(27) . We have examined the ability of RGD-containing
peptides to block both non-Tf iron transport and cell-surface iron
binding in basal and stimulated K562 cells. Since the RGD peptides do
not compete for uptake or binding, we conclude that neither of these
activities involves the ligand-displaceable cation-binding site of
-integrin. (
)Clearly, further investigation
is necessary to define the function of the cell-surface iron-binding
proteins that are induced by phorbol esters. As indicated above, we
speculate that these binding sites are functionally related to the
enhanced non-Tf iron system observed in K562 cells upon megakaryocytic
differentiation. Future efforts will not only help to identify the
molecules involved in iron assimilation, but may also explain why the
Tf-independent transport system is up-regulated during
megakaryocytopoiesis.