(Received for publication, October 27, 1994)
From the
Diphenylene iodonium chloride suppresses the cobaltous chloride-induced expression of erythropoietin by Hep3B cells to about 50% at a concentration of 30 nM. At that concentration, it has no effect on the response to low oxygen. The related compound iodonium diphenyl chloride acts similarly but is a much less effective inhibitor.
If, as reported, diphenylene iodonium chloride is a
specific inhibitor of cytochrome b, it follows that the
response to CoCl is dependent on that enzyme but the
response to hypoxia is not.
In 1988, Goldberg et al.(1) reported on a very
interesting and convincing model of the nature of the hypoxia-sensing
mechanism by which a hepatoma cell line regulates expression of the
erythropoietin (epo) ()gene. One of the attractive features
of this model is that it provides a common mechanism for the response
to hypoxia and to cobaltous chloride which also induces epo formation in vivo(2) as well as in vitro(3) .
The oxygen sensor has properties which are consistent with it being a
heme protein. Several other systems in which hypoxia induces specific
responses have been described (4, 5, 6) most
recently by Youngson et al.(7) who showed that fetal
rabbit pulmonary neuroepithelial cells respond to hypoxia by decreased
voltage-regulated, outward, potassium currents with no change in inward
currents. In this system, the response was inhibited by the compound,
diphenylene iodonium chloride (DPI) which has been characterized as an
inhibitor of superoxide-generating NADPH oxidase linked to cytochrome b(8) . A similar conclusion had previously been
reported for a possible oxygen sensor in the carotid
body(9, 10) . These observations again indicate that
the sensor may contain a heme protein, perhaps identical with
cytochrome b.
We report here on studies of the response of hepatoma cells (Hep3B) to both hypoxia and cobaltous chloride with respect to epo formation and show that responses to the two stimuli are differentially inhibited by DPI and by a similar compound iodonium diphenyl chloride (IDP).
Hep3B cells were maintained in T-25 flasks (Nunc) in 5 ml of
a medium consisting of 90% -minimal Eagle's medium (Sigma),
10% fetal bovine serum (Hyclone Laboratories, Inc.) with 5 units of
penicillin and 5 µg of streptomycin per ml. Cultures were split 1:5
once a week. Five days after a split, the cultures were split 1:2, and
fresh medium was added. At that time, CoCl
, DPI, or IDP was
added at the indicated concentrations. Incubations were at 37 °C in
5% CO
, 95% air or 5% CO
, 2% oxygen, and 93%
nitrogen for 24 h, after which medium was decanted and centrifuged at 4
°C for 10 min at 1600-1800 rpm, and the supernatant was
stored at -20 °C until put into the assay. We purchased DPI
from Cookson Chemical, Ltd. (Southhampton, UK) and IDP from Aldrich.
Culture supernatants were assayed at three different concentrations by the radioimmunoassay as described previously (11) using recombinant human epo (Amgen, Inc.) as standard. Iodination of the tracer epo was done by the method of Fraker and Speck(12) . The standard curve was fit, and titers of unknown samples were calculated using the SYSTAT version 5.2 statistical package for the Macintosh IIci computer.
The effects of varying concentrations of DPI on cobaltinduced
formation by Hep3B cells are shown in Fig. 1. The inhibitor is
extremely effective in suppressing the effect of 75 µM CoCl on epo production with 50% inhibition at a
concentration of about 30 nM. In contrast, the response to 2%
oxygen is not affected at all by DPI at 20 nM and is only
reduced by about 6% at 50 nM. At this latter concentration,
the response to cobaltous chloride is reduced by almost 70%. When IDP
was used as an inhibitor, at a concentration of 1 µM, we
once again found that the response to cobalt chloride was completely
abrogated whereas the response to 2% O
was unaffected (Table 1). In four other experiments with IDP at 4
µM, we found the response of Hep3B cells to CoCl
was 100% inhibited, whereas the response to 2% oxygen was
variable, ranging from 31% inhibition to 2-fold stimulation.
Figure 1:
Effect of diphenylene iodonium chloride
on induced erythropoietin expression. Data graphed are the means of
four to five experiments. Each experiment included a nonstimulated
control and a stimulated control (either CoCl or 2%
O
) to match each concentration of DPI used. The percent was
calculated from the increment over the nonstimulated control, due to
CoCl
(closed circles) or 2% O
(open squares) in the absence of inhibitor which was
taken as 100%. This system is very variable, with respect to the
response to CoCl
, and the coefficients of variation were as
follows: CoCl
alone, 0.52; CoCl
+ 10
nM DPI, 0.39; CoCl
+ 20 nM DPI,
0.65; CoCl
+ 50 nM DPI, 0.97; CoCl
+ 100 nM DPI, 0.93; 2% oxygen, 0.09; 2% 0
+ 20 nM DPI, 0.05; and 2% oxygen + 50 nM DPI, 0.02.
DPI is
considerably more toxic to Hep3B cells than is IDP, as judged by the
detachment of the cell monolayer. In our experiments, the possible
toxic effects of these compounds is controlled by our observation that
the response of the cells to 2% O is essentially the same
as that of control cells at concentrations of inhibitors that markedly
suppress the response to CoCl
. Our data may be interpreted
as showing that hypoxic conditions reduce the toxicity of DPI. To test
this possibility, we incubated Hep3B cells with both CoCl
and 2% O
at increasing concentrations of DPI. The
results of two experiments (Table 2) indicate that even at low
oxygen DPI inhibited the response to CoCl
. They also show
that when the cells are exposed to both stimuli, the response due to
CoCl
predominates.
The cobalt-induced expression of epo by Hep3B is very much more sensitive to DPI than is neutrophil NADPH oxidase. The latter is 50% inhibited by about 1 µM DPI(13) , about 30 times the concentration for 50% inhibition of epo induction.
Both DPI and IDP have been shown to
inhibit the cytochrome b-linked superoxide-generating NADPH
oxidase, with IDP being considerably less effective than
DPI(14) . Our observations indicate that DPI is a far more
potent inhibitor of the epo response to cobalt chloride than is IDP,
but that neither compound at the same concentration that suppressed
CoCl-induced epo formation has a significant effect on the
response to low oxygen. These results indicate that the mechanisms of
the cellular response to these two stimuli cannot be identical, and
that the role of cytochrome b, if it, in fact, is the only
site of inhibition by these compounds, involves the response to cobalt
chloride but not to low oxygen.
This dissociation of the Hep3B
response to CoCl and to hypoxia is also seen, on occasion,
when the cells spontaneously lose the ability to respond to CoCl
while retaining their response to low oxygen. When this happens,
we find, the cells still retain the ability to take up CoCl
(the cell pellet is purple).
The possible role for cytochrome
b in the regulation of epo formation is strengthened by the observation
of Görlach et al.(15) showing
that another hepatoma cell line, HepG2, has a hypoxia-sensitive
cytochrome b detectable by changes in the absorption spectrum.
These cells respond to both hypoxia and CoCl by increased
epo synthesis(16) .
While we do not yet know the detailed
mechanism of action of either of the stimuli on the expression of the
epo gene, our data suggest that the response to CoCl must
involve at least one more factor, possibly cytochrome b, than
does the response to hypoxia. This factor may be a heme protein, and it
is possible that the more proximal factor that responds to low oxygen
may be the non-heme protein associated with K
channel
activity as described for central neurons(14) .