©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Inhibition by Iodonium Compounds of Induced Erythropoietin Expression (*)

(Received for publication, October 27, 1994)

Eugene Goldwasser (§) Petrit Alibali Annette Gardner

From the Department of Biochemistry and Molecular Biology, the University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(2) is dependent on that enzyme but the response to hypoxia is not.


INTRODUCTION

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) (^1)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).


MATERIALS AND METHODS

Hep3B cells were maintained in T-25 flasks (Nunc) in 5 ml of a medium consisting of 90% alpha-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(2), DPI, or IDP was added at the indicated concentrations. Incubations were at 37 °C in 5% CO(2), 95% air or 5% CO(2), 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.


RESULTS AND DISCUSSION

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(2) 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(2) was unaffected (Table 1). In four other experiments with IDP at 4 µM, we found the response of Hep3B cells to CoCl(2) 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(2) or 2% O(2)) to match each concentration of DPI used. The percent was calculated from the increment over the nonstimulated control, due to CoCl(2) (closed circles) or 2% O(2) (open squares) in the absence of inhibitor which was taken as 100%. This system is very variable, with respect to the response to CoCl(2), and the coefficients of variation were as follows: CoCl(2) alone, 0.52; CoCl(2) + 10 nM DPI, 0.39; CoCl(2) + 20 nM DPI, 0.65; CoCl(2) + 50 nM DPI, 0.97; CoCl(2) + 100 nM DPI, 0.93; 2% oxygen, 0.09; 2% 0(2) + 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(2) is essentially the same as that of control cells at concentrations of inhibitors that markedly suppress the response to CoCl(2). 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(2) and 2% O(2) at increasing concentrations of DPI. The results of two experiments (Table 2) indicate that even at low oxygen DPI inhibited the response to CoCl(2). They also show that when the cells are exposed to both stimuli, the response due to CoCl(2) 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(2)-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(2) and to hypoxia is also seen, on occasion, when the cells spontaneously lose the ability to respond to CoCl(2) while retaining their response to low oxygen. When this happens, we find, the cells still retain the ability to take up CoCl(2) (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(2) 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(2) 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) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL21676 and HL30121. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: 920 East 58th St., Chicago, IL 60637. Tel.: 312-702-1348; Fax: 312-702-0439.

(^1)
The abbreviations used are: epo, erythropoietin; DPI, diphenylene iodonium chloride; IDP, iodonium diphenyl chloride.


REFERENCES

  1. Goldberg, M. A., Dunning, S. P., and Bunn, H. F. (1988) Science 242, 1412-1415 [Medline] [Order article via Infotrieve]
  2. Goldwasser, E., Jacobson, L. O., Fried, W., and Plzak, L. (1957) Science 125, 1085 [Medline] [Order article via Infotrieve]
  3. Beru, N., McDonald, J., Lacombe, C., and Goldwasser, E. (1986) Mol. Cell. Biol. 6, 2571-2575 [Medline] [Order article via Infotrieve]
  4. Fishman, M. C., Greene, W. L., and Platika, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1448-1450 [Abstract]
  5. Delpiano, M. A., and Acker, H. (1989) Brain Res. 482, 235-246 [Medline] [Order article via Infotrieve]
  6. Kourembanas, S., Hannan, R. L., and Faller, D. V. (1990) J. Clin. Invest. 86, 670-674 [Medline] [Order article via Infotrieve]
  7. Youngson, C., Nurse, C., Yeger, H., and Cutz, E. (1993) Nature 365, 153-155 [CrossRef][Medline] [Order article via Infotrieve]
  8. Cross, A. R. (1987) Biochem. Pharm. 36, 489-493 [CrossRef][Medline] [Order article via Infotrieve]
  9. Acker, H., Dufau, E., Huber, J., and Sylvester, D. (1989) FEBS Lett. 256, 75-78 [CrossRef][Medline] [Order article via Infotrieve]
  10. Cross, A. R., Henderson, L., Jones, O. T. G., Delpiano, M. A., Hentschel, J., and Acker, H. (1990) Biochem. J. 272, 743-747 [Medline] [Order article via Infotrieve]
  11. Sherwood, J. B., and Goldwasser, E. (1979) Blood 54, 885-893 [Abstract]
  12. Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80, 849-857 [Medline] [Order article via Infotrieve]
  13. Jiang, C., and Haddad, G. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7198-7201 [Abstract]
  14. Ellis, J. A., Mayer, S. J., and Jones, O. T. G. (1988) Biochem. J. 251, 887-891 [Medline] [Order article via Infotrieve]
  15. Görlach, A., Holtermann, G., Jelkmann, W., Hancock, J. T., Jones, S. A., Jones, O. T. G., and Acker, H. (1993) Biochem. J. 290, 771-776 [Medline] [Order article via Infotrieve]
  16. Goldberg, M. A., Glass, G. A., Cunningham, J. M., and Bunn, H. F. (1982) Proc. Natl. Acad. Sci. U. S. A. 84, 7972-7976

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.