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The production of prostaglandins with an erythropoietin generating factor(s)
BIOCHEMICAL
MEDICINE
23,
55-63 (1980)
The Production of Prostaglandins with an Erythropoietin Generating Factor(s) W. A. NEAL, J. P. LEWIS, F. A. GARVER, AND C. L. LUTCHER Department of Research, Veterans Administration, Medical Center, Augusta, Ga.. The Departments of Medicine and Cell and Molecular Biology, Medical College of Georgiu, 30904’ Received September 14. 1979
Dukes et al. find that the action of erythropoietin (EPO)” in viva in mice, and in rat bone marrow cultures, is potentiated by prostaglandins in an EPO dose dependent fashion (1). Reduced glutathione increases the rate of the enzymatic conversion of dihomogammalinolenic (a conversion product of linoleic acid) and arachidonic acids to prostaglandins E, and EP, respectively (2), and the disappearance of reduced glutathione is parallel to erythropoietin activity as measured in AKR mice (3). Nugteren and Christ-Hazelhof describe the enzymatic conversion of prostaglandin endoperoxide into PGD, or PGE, by two different isomerases (4), which is related to the bioconversion of fatty acids to prostaglandins as described by Daniels and Pike (5). Both enzymes have an absolute requirement for glutathione (GSH), indicating a common reaction mechanism. The pH optimum of the isomerase(s) is 7.5 and the molecular weight between 26,000 and 34,000. Fisher et al. indicate that prostaglandins released from ischemic kidneys activate renal adenylate cyclase and indirectly initiate the production of erythropoietin (6). The purpose of this report is to compare the production of prostaglandins as described by Nugteren and Christ-Hazelhof, with the production of EPO by our erythropoietin generating (EGF) factor. ’ Supported by Veterans Administration and Medical College of Georgia (NIH Grants FR-5365-0061). We wish to acknowledge the excellent assistance of Mrs. Roberta Leverett. * Abbreviations: Erythropoiesis regulatory factors (ERFs), erythropoietin (EPG), erythropoietin generating factor (EGF), erythropoiesis stimulating factor (ESF), paroxysmal nocturnal hemoglobinuria (PNH), multiple myeloma (MM), erythropoiesis inhibitory factor (EIF), reduced glutathione (GSH), oxidized glutathione (G-SS-G), thin-layer chromatography (tic), dithiothreitol (DTT), prostaglandins (PGs). 55 0006-2944/80/010055-09502.00/O Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
56
NEAL
MATERIALS
ET AI..
AND METHODS
Lyophilized erythropoiesis regulatory factors ( 100 mg ERFs), obtained by column chromatography of the urine from a PNH patient (7) and also a fraction containing an EPO-generating factor(s) (EGFs but no EPO) (8) from a patient with multiple myeloma (MM), were treated by a modified bioconversion technique (with and without the addition of linoleic and arachidonic acids)3 as described by Daniels and Pike (5). Sheep seminal vesicular tissue, which contains an enzyme for converting unsaturated fatty acids to prostaglandins in the presence of GSH was not included and neither was hydroquinone. The hydroquinone was indicated as probably not needed (5), but 0.5 mg GSH/lOO mg ERFs was necessary. All of the required reagents were reduced in quantity proportionately to the amount of starting material. Incubation was done for I hr at 37°C. in pH 10 ammonium chloride, 0.1 M. Benzene extracts of the fractions were dried by rotatory evaporation. Thin-layer chromatography (tic) of these extracts was done on silver nitrate, silica gel precoated glass plates, using an ethyl acetate-acetic acid-methanol-2, 2, 4-trimethyl pentane-water solvent system as described by Green and Samuelsson (9). The bioconversion extraction techniques only were also done on the ERF fractions, and dried benzene extracts of these fractions were examined by tic. Antisera to EIF were produced in New Zealand white rabbits by intramuscular injection of 1 mg followed at weekly intervals with seven subcutaneous injections, Carver et al. (10). The antigen was first emulsified in complete Freund’s adjuvant. Blood was collected 5-7 days after the last injection. The EIF antigen was obtained by column electrophoresis and selective membrane fractionation (11-12). The most antigenic EIF fraction was in the retentate after ultrafiltration through a membrane with a cutoff at a molecular weight of 10,000. A chromatographic fraction of urinary PNH-ERF(s), (0.2 mg per mouse), obtained as described in the previous experiments (7) was incubated with prostaglandins PGD,, E, , E,, and Fzu (20-80 pg per mouse) for 30 min at 37°C and then frozen until assay time. ERF(s) and prostaglandin controls were also included. Rabbit anti-EIF serum (0.1 ml) was incubated with PGF,, and ERFs. Strain AKR female mice made polycythemic by exposure to a simulated altitude of 7320 m were used for the bioassay for ESF activity, The mice were intermittently kept in glass enclosures in an atmosphere of 8% oxygen and 92% nitrogen (13). The packed cell volumes were determined by a microhematocrit technique. The mice had a mean packed cell volume 3 The linoleic acid was obtained from was obtained from the Tridom Chemical
Fisher Scientific Company.
Company.
The arachidonic
acid
ERYTHROPOIESIS
REGULATORY
FACTORS
57
of 57.5 + 0.3% @EM) at the conclusion of the assay. Mice injected with 0.9% saline were used as controls with all groups. The values for the control groups injected with saline solution were subtracted from the ESF activity values. The results were recorded as the mean of the percentage of the 59Fe incorporated into red blood cells and as international standard B4 units of ESF activity, which were determined from a log dose-log response curve. Ten mice were used for each assay. Standards were interspersed between the unknowns. RESULTS
The movements of the standards and the benzene extracts of ERF(s) during thin-layer chromatography on silver nitrate-silica gel plates can be seen in Fig. 1, which is a photograph of individual tic experiments. The benzene extracts applied to the silver nitrate-silica gel plates were obtained from ERF and EGFs separately before and after treatment by a modified bioconversion technique (5). The qualitative results were the same with or without the addition of linoleic or arachidonic acids. There were components in the benzene extracts that moved in positions similar to the positions of linoleic acid, prostaglandins EB, Feu , and A,. The potentiation and inhibition of ESF activity with prostaglandins can be seen in Table 1, along with the neutralization of an inhibitor of erythropoiesis with an anti-EIF serum. The cancellation of ESF potentiation and inhibition by prostaglandins can be seen in Table 2. (The prostaglandins were kindly donated by Dr. J. E. Pike, The Upjohn Co.). DISCUSSION
The data in Table 1 demonstrating the potentiation and inhibition of ESF activity with prostaglandins confirms and extends the work by Dukes et al. (1). Prostaglandins appear to be present during thin-layer chromatogmphy of the benzene extracts of ERF fractions after bioconversion (Fig. 1). There appears to be a system for the production of prostaglandins E2 and F,, within our ERF fraction, and either F, or EIF in sufficient quantities inactivates all EPO activity. The inhibition of PGEl and PGE, by PGF, could be competitive due to the similarities of the structures of prostaglandins (9). The EIF is possibly the same lipid isolated from rabbit kidney by Erslev, Kazal, and Miller (14). A similar EIF has also been demonstrated in sheep, goat, and rabbit plasma (12). PGD* does not potentiate or inhibit ESF activity but prevents inhibition as seen in Table 2, possibly by competing for an active site. ’ Obtained through the courtesy of Drs. D. R. Bangham and Mary Cotes, Medical Research Council, National Institute for Medical Research, London, England.
NEAL ET AL.
EIF LA Ew(s)
0
PGE2 PGA2 AA EGF(s) PGEl PGD2 PGFz.o EGF(s)
FIG. 1. Thin-layer chromatography on silver nitrate-silica gel plates. The solvent system was ethyl acetate-acetic acid-methanol-2,2,4 trimethyl pentane-water, 110:30:35: IO: loo(9). Benzene extracts of ERF(s) and EGF(s) were done after bioconversion (5). Extracts were of fractions obtained from normal urine, from patients with paroxysmal nocturnal hemoglobinuria (PNH), and multiple myeloma (MM). Tracks: 1, EIF (I00 pg of an erythropoiesis inhibitory factor fraction from normal urine); 2. LA (20 pg linoleic acid); 3, ERF(s) (20 /~g of erythropoiesis regulatory factors from PNH urine): 4. PGE, (20 pg prostaglandin E,); 5, PGA, 20 pg prostaglandin A,); 6, AA (20 pg arachidonic acid); 7, EGF(s)(l00~~gofanerythropoietingeneratingfactor(s)fractionfromMMu~ne;8,PGE, (2Opg prostaglandin E,); 9, PGD, (20 pg prostaglandin Dz; 10, PGF, (30 pg prostaglandin F*; II. EGF(s) (100 Fg of an erythropoietin generating factor(s) fraction from MM urine).
Dukes rt ul. (1) find that the prostaglandin binding protein in human urinary erythropoietin preparations is not identical with erythropoietin. Our data indicate that several prostaglandins are present in our erythropoietin preparations after bioconversion, but only indications of PGF,, can be observed before applying the bioconversion technique. The prostaglandin potentiating ERF seems similar to the steroid potentiating ERF observed previously in that both ERF preparations potentiate erythropoiesis after a period of incubation with certain prostaglandins or certain steroids (15). However, the movement of steroids during thinlayer chromatography is not similar to the movement of the lipid extracts from ERFs as are the movements of prostaglandins after application of the bioconversion technique (unpublished observation). The presence of PGA, in the urinary ESF preparation could be an artifact of the isolation
ERYTHROPOIESIS
REGULATORY
FACTORS
59
TABLE 1 POTENTIATION AND INHIBITION OF ERYTHROPOIESIS AND NEUTRALIZATION OF INHIBITION BY ANTI-EIF SERUM Percentage 59Fe incorporated in RBC + SEM
Materials and amounts per mouse 0.2 mg ERF(s)” 40.0 pg PGE,* 0.2 mg ERF(s) + 40 pg PGE, 40.0 eg PGE, 0.2 mg ERF(s)* + 40 pg PGE, 0.1 mg EIF’ 0.2 mg ERF(s) + 0.1 mg EIF 0.2 mg ERF(s) + 0.2 mg EIF
20.19 1.25 34.69 7.00 36.91
0.2 0.2 0.2 0.2 0.2
13.50 7.68
mg mg mg mg mg
ERF(s) ERF(s) ERF(s) ERF(s) ERF(s)
+ + + + +
20 40 80 40 80
pg pg pg pg pg
6.12
PGF,, PGF,, PGE,, PGF,, + 0.1 ml anti-EIF PGF,, + 0.1 ml anti-EIF
21.53 20.07
rt t c 2 + 0 r 0 0 t -t 0 t t
ESF activity IU + SEM
0.89 0.63 1.43 0.91 0.75
0.46 0.15 0.78 0.14 0.84
2.61
0.12
0.53 1.05
0.30 0.16
0.65 0.46
0.47 0.46
f 2 k + k 0 t 0 0 + 2 0 t 2
0.02 0.01 0.03 0.02 0.02 0.05 0.01 0.02 0.01 0.01
a ERF(s) = Erythropoiesis regulatory factors, a chromatographic urinary fraction from a patient with paroxysmal nocturnal hemoglobinuria. * PGE,, PGE,, PGE,, are prostaglandins. They were donated by Dr. John F. Pike, The Upjohn Company, Kalamazoo, Mich. f EIF = Erythropoiesis inhibitory factor obtained by column electrophoresis of a urinary fraction collected in an electrofractionator. The neutralizing antiserum to EIF was diluted 1- 100. Anti-EIF serum was produced in rabbits as described by Garver et al( 10). The anti-EIF serum was mixed with PGF,, and incubated with ERF(s).
TABLE 2 THE CANCELLATION OF POTENTIATION AND INHIBITION OF ERYTHROPOIESISBY PROSTAGLAND~NS Materials and amounts per Mouse (m at) 0.2 0.2 0.2 0.2
mg mg mg mg
ERF(s) ERF(s) + 40 Fg PGDZ ERF(s) + 40 pg PGD* + 40 pg PGF,, ERF(s) + 40 pg PGE, + 40 pg PGF,,
j9Fe Incorporated RBC -c SEM (%) 16.20 16.19 15.16 19.71
r + + *
0.43 0.36 0.45 0.30
in
ERF Activity + SEM WJ) 0.36 0.36 0.32 0.44
-c 0.01 f 0.01 2 0.01 Yh0.01
0 The erythropoiesis regulating factor(s), ERF(s), were from a patient with paroxysmal nocturnal hemoglobinuria (7). The prostaglandins were donated by Dr. John E. Pike, The Upjohn Company, Kalamazoo, Mich.
60
NEAI.
ET AL
procedure. Daniels and Pike describe the formation of PGA, by the degradation of PGE, (loss of a molecule of water) (5). The prostaglandins obtained by bioconversion with the fraction containing EGF, which has been indicated to contain proerythropoietin but no EPO (8) did not include PGF4,. The latter PC apparently comes by a different pathway, although arachidonic acid is a precursor for PGF4, and PGE, (16). We usually see indications of PGE, and not E, (indication of E, was seen two times out of seven experiments) during tic of ERFs, and arachidonic acid (but not linoleic) is probably in the proximal pathway for the production of EPO. A glutathione-like compound and an enzyme with the same pH-optimum as indicated for the E-isomerase, as reported by Nugteren and Christ-Hazelhof, are needed for the EGF production of erythropoietin and also for the bioconversion of fatty acids to prostaglandins (4,5,15). The prostaglandin formed by the action of an enzyme on arachidonic acid and expedited by glutathione, possibly similar to the enzyme found in sheep seminal vesicular tissue by Van Dorp et ul. and Bergstriim et ul. and characterized by Nugteren and Christ-Hazelhof, is believed to bind to a specific protein which also is apparently in our ERF preparations. In the EGF fraction the conversion of arachidonic acid to PGE, appears to take place much more readily than the conversion of linoleic acid to PGE,. This could explain why we usually detect linoleic but not arachidonic acid, since arachidonic acid appears to be converted instantly and completely to PGE,. We can detect arachidonic acid by tic when the exogenous amount is increased to 1 mg per 100 mg of ERF during the bioconversion technique. Linoleic is converted to arachidonic acid by way of a-linolenic and dihomogammalinolenic acid. Foley, Gross, Nelson, and Fisher produced EPO in exhypoxic polycythemic mice and the isolated perfused canine kidney with arachidonic acid (18). The PGs indicated in Fig. I are observed after bioconversion with or without exogenous fatty acids. The EGF fraction is indicated to contain proerythropoietin at a molecular weight (MW) of 45,000, an enzyme at 32,000 for the conversion of pro-EPO to EPO, and a small amount of a component at 40,000 (19,20). The enzyme of the EGF fraction appears to be similar to the bull vesicular enzyme in that it has the same pH-optimum, molecular weight, and requirement for glutathione as the E-isomerase prostaglandin producing enzyme (4,17,20). These PGs cannot be extracted from EGFs with organic solvents (acetone, n-hexane, and benzene) without first applying the modified bioconversion technique (5), suggesting concurrence with the work by Fisher et al. (6) in that proerythropoietin initiates the production of erythropoietin by way of prostaglandins. The ESF extractable with organic solvents does not move with PGs during tic: It is apparently attached to fragments of protein (- 4.4%) (21). The experiments in Fig. 1
ERYTHROPOIESIS
REGULATORY
FACTORS
61
represent observations following extractions of fractions containing ERFs subjected to the modified bioconversion technique. Although Dukes et al. now reject the postulate that EPO is a glycoprotein-prostaglandin complex (1), the close association of a PG-producing system with a fraction indicated previously to contain proerythropoietin is apparent (22). The fact that neutralizing anti-EPO serum inactivates EPO and the product of EGF, whereas neutralizing anti-EGF serum only prevents the production of EPO (19) would place these factors proximately. It now seems that the enzyme, E-isomerase, is probably the antigen for the neutralizing antiEGF serum (4). PGD* had no ESF or EIF activity but it did prevent PGF,, from inhibiting ESF activity produced by EPO or PGEz, possibly by competing for the site of EIF inhibition or production. When the bioconversion technique is applied to a fraction containing only EGFs, PGF,, is not demonstrable. Miller et al. in their study of the modulation of granulopoiesis, find antagonistic actions for prostaglandins E and F (23). Their in vitro observations for the stimulation of granulopoiesis by PGF,, and inhibition by PGE, are opposite to our in ~ivo observations for erythropoiesis. If prostaglandin E2 activates adenyl cyclase and indirectly initiates the production of erythropoietin, as indicated by Fisher et al. (6), possibly PGF,, initiates the production of a comparable granulopoiesis factor via guanidylate cyclase (6,23), and inhibits erythropoiesis indirectly. The neutralization of the PGF,, induced inhibition of erythropoiesis by anti-EIF serum (Table 1) confirms the close association observed between PGF,, and EIF (12); however, anti-PGF,, and anti-EIF do not cross-react with their respective antigens (unpublished data).” Possibly PGF,, initiates the production of EIF. About 71% of the ESF activity in our urinary ERFs has been attributed to EGF (19). Previously we observed a parallelism between erythropoietin activity and the disappearance of GSH (3). It is now known also that dithiothreitol (DTT) will prevent EGF activity possibly by keeping GSH in the reduced state or reducing G-SS-G. Our data suggest that a major part of EPO from our urinary ERFs is biosynthesized with the aid of erythrocytic GSH after injection of ERFs, which contain proerythropoietin (3). SUMMARY
Thin-layer chromatography of ERFs extracts indicates components in ERFs, after treatment by a bioconversion technique which converts linoleic and/or arachidonic acids to prostaglandins, with the same mobility as prostaglandins E, and A,. Linoleic acid can be detected in ERFs prior 5 Anti-PGF,,
was a gift from Dr. Kenneth T. Kirton, The Upjohn Co.
NEAL ET AL.
62
to bioconversion. PGE, and PGE, potentiate erythropoiesis to about
the
same extent as PGF.,, (found in erythropoiesis inhibitory fractions, EIF) inhibits erythropoiesis. The inhibition produced by PGF,, can be prevented with a neutralizing antiserum to EIF. Our data suggest that fatty acids and prostaglandins are part of the pathway for the production of erythropoietin by an enzyme and reduced glutathione. REFERENCES I. Dukes, P. P., Shore, N. A., Hammond, G. D., and Ortega, J. A., irr “Erythropoiesis, Proceedings of the Fourth International Conference on Erythropoiesis” (K. Nakao, J. Fisher, and F. Takaku, Eds.), p. 698. University Park Press, Baltimore, London, Tokyo, 1975. 2. Nugteren, D. H., Beerthuis, R. K.. and Van Dorp, D. A.. Rec. True. Chim. Pays-Bus Be/g. 3.
85, 405 (1966).
Lewis, J. P., Alford, D. A., Smith, J. E.. Horton, B. F., and Smith. L. L.. Brir. J. Haemotol.
14, 457 (1968).
Nugteren, D. H., and Christ-Hazelhof, E., in “Proceedings of the Fourth International Prostaglandin Conference,” p. 87. Washington, D. C., May, 1979. 5. Daniels, E. G., and Pike, J. E., in “Prostaglandin Symposium of Worcester Foundation for Experimental Biology” (P. W. Ramwell and J. E. Shaw, Eds.), pp. 379-387. Interscience, New York, 1968. 6. Fisher, J. W., Gross, D. M., Foley, J. E.. and Jubiz. W., in “Erythropoiesis, Proceedings of the Fourth International Conference on Erythropoiesis” (K. Nakao, J. W. Fisher, and F. Takaku, Eds.), p. 698. University Park Press, Baltimore, London, Tokyo. 1975. 7. Lewis, J. P., Alford, D. A., Rathjen, J. H., Jr., and Lange, R. D., J. Lab. Clin. Med. 66, 4.
987 ( 1965).
Lewis, J. P.. Neal, W. A., Welch, E. T., Lewis, W. G., III, DuBose, C. M.. Jr., Wright, C.-S., and Smith, L. L., Proc. Sot. Exp. Biol. Med. 142, 293 (1973). 9. Green, K., and Samuelsson. B., J. Lipid Res. 5, 117 (1964). 10. Gamer, F. A., Nash,W. A., Lewis,J. P., Lutcher, C. L., Ozawa, T., Moores, R. R.. and Wright, C. S. Manuscript in preparation. 1I. Lewis, J. P., Neal, W. A., Moores, R. R., Smith, L. L., Wright, C.-S., and Welch, 8.
E. T., J. Lab.
C’h.
Med.
74, 608 (1969).
12. Neal, W. A., Lewis,J. P., Welch, E. T.. Lutcher, C. L., Moores. R. R., and Wright, C.-S., Amer. J. Vet. Res. 40, 493 (1979). 13. Lewis, J. P.. Neal, W. A., Alford. D. A., Moores, R. R., Gardner. E., Jr., Welch, E. T., Wright, C.-S.. and McWhirter, J. D.. Amer. J. Vet. Res. 31, 891 (1970). 14. Erslev. A. J., Kazal. L. A., and Miller, 0. P., in “Proceedings of the International SYmPOSiUm Erythropoieticum”(T. Travnicek and J. Neuwirt, Eds.), pp. 15. Charles
University, Prague,Czechoslovakia. 15. Lewis. J. P.. Alford, D. A., Neal, W. A., Moores, R. R., Welch, E. T., Gardner, E., Jr., Wright, C.-S., and Smith. L. L., Stand. J. Huemur. 8, 200 (1971). 16. Hinman.J. W., Ann. Rev. Biochem. 41, 161 (1972). 17. Lewis,J. P.. Welch, E. T., Neal. W. A.. Moores, R. R., Lewis, W. G., III, Wright,
C.-S.. DuBose.C. M., Jr., andSmith,L.
L., Proc.
SOC.
Exp.
Biol.
(1971). 18. Foley, J. E., Gross, D. M., Nelson, P. K., and Fisher, J. W.. J. Pharmaco/.
Med.
138, 322 ~np.
Ther.
207, 402 ( 1978).
19. Lewis. J. P.. Welch, E. T., Neal. W. A., Wright, C.-S., Gardner, E., Jr., Smith, L. L., Lange, R. D.. Biochem. Med. 14, 399 (1975).
ERYTHROPOIESIS
REGULATORY
FACTORS
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20. Neal, W. A., Welch, E. T., Lewis, J. P., Lutcher, C. L., Wright, C.-S., and Smith, L. L., Biochem. Med. 20, 229 (1978). 21. Lewis, J. P., Welch, E. T., Neal, W. A., DuBose, C. M., Jr., Lewis, W. G., III, Wright, C.-S., and Smith, L. L., Biochem. Med. 10, 374 (1974). 22. DuBose, C. M., Jr., Welch, E. T., Lewis, J. P., Neal, W. A., Lutcher, C. L., Biochem. Med. 17, 310 (1977). 23. Miller, H. M., Russell, T. R., Gross, M. A., and Yunis, A. A.,J. Lab. C/in. Med. 92, 983 (1978).
MEDICINE
23,
55-63 (1980)
The Production of Prostaglandins with an Erythropoietin Generating Factor(s) W. A. NEAL, J. P. LEWIS, F. A. GARVER, AND C. L. LUTCHER Department of Research, Veterans Administration, Medical Center, Augusta, Ga.. The Departments of Medicine and Cell and Molecular Biology, Medical College of Georgiu, 30904’ Received September 14. 1979
Dukes et al. find that the action of erythropoietin (EPO)” in viva in mice, and in rat bone marrow cultures, is potentiated by prostaglandins in an EPO dose dependent fashion (1). Reduced glutathione increases the rate of the enzymatic conversion of dihomogammalinolenic (a conversion product of linoleic acid) and arachidonic acids to prostaglandins E, and EP, respectively (2), and the disappearance of reduced glutathione is parallel to erythropoietin activity as measured in AKR mice (3). Nugteren and Christ-Hazelhof describe the enzymatic conversion of prostaglandin endoperoxide into PGD, or PGE, by two different isomerases (4), which is related to the bioconversion of fatty acids to prostaglandins as described by Daniels and Pike (5). Both enzymes have an absolute requirement for glutathione (GSH), indicating a common reaction mechanism. The pH optimum of the isomerase(s) is 7.5 and the molecular weight between 26,000 and 34,000. Fisher et al. indicate that prostaglandins released from ischemic kidneys activate renal adenylate cyclase and indirectly initiate the production of erythropoietin (6). The purpose of this report is to compare the production of prostaglandins as described by Nugteren and Christ-Hazelhof, with the production of EPO by our erythropoietin generating (EGF) factor. ’ Supported by Veterans Administration and Medical College of Georgia (NIH Grants FR-5365-0061). We wish to acknowledge the excellent assistance of Mrs. Roberta Leverett. * Abbreviations: Erythropoiesis regulatory factors (ERFs), erythropoietin (EPG), erythropoietin generating factor (EGF), erythropoiesis stimulating factor (ESF), paroxysmal nocturnal hemoglobinuria (PNH), multiple myeloma (MM), erythropoiesis inhibitory factor (EIF), reduced glutathione (GSH), oxidized glutathione (G-SS-G), thin-layer chromatography (tic), dithiothreitol (DTT), prostaglandins (PGs). 55 0006-2944/80/010055-09502.00/O Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
56
NEAL
MATERIALS
ET AI..
AND METHODS
Lyophilized erythropoiesis regulatory factors ( 100 mg ERFs), obtained by column chromatography of the urine from a PNH patient (7) and also a fraction containing an EPO-generating factor(s) (EGFs but no EPO) (8) from a patient with multiple myeloma (MM), were treated by a modified bioconversion technique (with and without the addition of linoleic and arachidonic acids)3 as described by Daniels and Pike (5). Sheep seminal vesicular tissue, which contains an enzyme for converting unsaturated fatty acids to prostaglandins in the presence of GSH was not included and neither was hydroquinone. The hydroquinone was indicated as probably not needed (5), but 0.5 mg GSH/lOO mg ERFs was necessary. All of the required reagents were reduced in quantity proportionately to the amount of starting material. Incubation was done for I hr at 37°C. in pH 10 ammonium chloride, 0.1 M. Benzene extracts of the fractions were dried by rotatory evaporation. Thin-layer chromatography (tic) of these extracts was done on silver nitrate, silica gel precoated glass plates, using an ethyl acetate-acetic acid-methanol-2, 2, 4-trimethyl pentane-water solvent system as described by Green and Samuelsson (9). The bioconversion extraction techniques only were also done on the ERF fractions, and dried benzene extracts of these fractions were examined by tic. Antisera to EIF were produced in New Zealand white rabbits by intramuscular injection of 1 mg followed at weekly intervals with seven subcutaneous injections, Carver et al. (10). The antigen was first emulsified in complete Freund’s adjuvant. Blood was collected 5-7 days after the last injection. The EIF antigen was obtained by column electrophoresis and selective membrane fractionation (11-12). The most antigenic EIF fraction was in the retentate after ultrafiltration through a membrane with a cutoff at a molecular weight of 10,000. A chromatographic fraction of urinary PNH-ERF(s), (0.2 mg per mouse), obtained as described in the previous experiments (7) was incubated with prostaglandins PGD,, E, , E,, and Fzu (20-80 pg per mouse) for 30 min at 37°C and then frozen until assay time. ERF(s) and prostaglandin controls were also included. Rabbit anti-EIF serum (0.1 ml) was incubated with PGF,, and ERFs. Strain AKR female mice made polycythemic by exposure to a simulated altitude of 7320 m were used for the bioassay for ESF activity, The mice were intermittently kept in glass enclosures in an atmosphere of 8% oxygen and 92% nitrogen (13). The packed cell volumes were determined by a microhematocrit technique. The mice had a mean packed cell volume 3 The linoleic acid was obtained from was obtained from the Tridom Chemical
Fisher Scientific Company.
Company.
The arachidonic
acid
ERYTHROPOIESIS
REGULATORY
FACTORS
57
of 57.5 + 0.3% @EM) at the conclusion of the assay. Mice injected with 0.9% saline were used as controls with all groups. The values for the control groups injected with saline solution were subtracted from the ESF activity values. The results were recorded as the mean of the percentage of the 59Fe incorporated into red blood cells and as international standard B4 units of ESF activity, which were determined from a log dose-log response curve. Ten mice were used for each assay. Standards were interspersed between the unknowns. RESULTS
The movements of the standards and the benzene extracts of ERF(s) during thin-layer chromatography on silver nitrate-silica gel plates can be seen in Fig. 1, which is a photograph of individual tic experiments. The benzene extracts applied to the silver nitrate-silica gel plates were obtained from ERF and EGFs separately before and after treatment by a modified bioconversion technique (5). The qualitative results were the same with or without the addition of linoleic or arachidonic acids. There were components in the benzene extracts that moved in positions similar to the positions of linoleic acid, prostaglandins EB, Feu , and A,. The potentiation and inhibition of ESF activity with prostaglandins can be seen in Table 1, along with the neutralization of an inhibitor of erythropoiesis with an anti-EIF serum. The cancellation of ESF potentiation and inhibition by prostaglandins can be seen in Table 2. (The prostaglandins were kindly donated by Dr. J. E. Pike, The Upjohn Co.). DISCUSSION
The data in Table 1 demonstrating the potentiation and inhibition of ESF activity with prostaglandins confirms and extends the work by Dukes et al. (1). Prostaglandins appear to be present during thin-layer chromatogmphy of the benzene extracts of ERF fractions after bioconversion (Fig. 1). There appears to be a system for the production of prostaglandins E2 and F,, within our ERF fraction, and either F, or EIF in sufficient quantities inactivates all EPO activity. The inhibition of PGEl and PGE, by PGF, could be competitive due to the similarities of the structures of prostaglandins (9). The EIF is possibly the same lipid isolated from rabbit kidney by Erslev, Kazal, and Miller (14). A similar EIF has also been demonstrated in sheep, goat, and rabbit plasma (12). PGD* does not potentiate or inhibit ESF activity but prevents inhibition as seen in Table 2, possibly by competing for an active site. ’ Obtained through the courtesy of Drs. D. R. Bangham and Mary Cotes, Medical Research Council, National Institute for Medical Research, London, England.
NEAL ET AL.
EIF LA Ew(s)
0
PGE2 PGA2 AA EGF(s) PGEl PGD2 PGFz.o EGF(s)
FIG. 1. Thin-layer chromatography on silver nitrate-silica gel plates. The solvent system was ethyl acetate-acetic acid-methanol-2,2,4 trimethyl pentane-water, 110:30:35: IO: loo(9). Benzene extracts of ERF(s) and EGF(s) were done after bioconversion (5). Extracts were of fractions obtained from normal urine, from patients with paroxysmal nocturnal hemoglobinuria (PNH), and multiple myeloma (MM). Tracks: 1, EIF (I00 pg of an erythropoiesis inhibitory factor fraction from normal urine); 2. LA (20 pg linoleic acid); 3, ERF(s) (20 /~g of erythropoiesis regulatory factors from PNH urine): 4. PGE, (20 pg prostaglandin E,); 5, PGA, 20 pg prostaglandin A,); 6, AA (20 pg arachidonic acid); 7, EGF(s)(l00~~gofanerythropoietingeneratingfactor(s)fractionfromMMu~ne;8,PGE, (2Opg prostaglandin E,); 9, PGD, (20 pg prostaglandin Dz; 10, PGF, (30 pg prostaglandin F*; II. EGF(s) (100 Fg of an erythropoietin generating factor(s) fraction from MM urine).
Dukes rt ul. (1) find that the prostaglandin binding protein in human urinary erythropoietin preparations is not identical with erythropoietin. Our data indicate that several prostaglandins are present in our erythropoietin preparations after bioconversion, but only indications of PGF,, can be observed before applying the bioconversion technique. The prostaglandin potentiating ERF seems similar to the steroid potentiating ERF observed previously in that both ERF preparations potentiate erythropoiesis after a period of incubation with certain prostaglandins or certain steroids (15). However, the movement of steroids during thinlayer chromatography is not similar to the movement of the lipid extracts from ERFs as are the movements of prostaglandins after application of the bioconversion technique (unpublished observation). The presence of PGA, in the urinary ESF preparation could be an artifact of the isolation
ERYTHROPOIESIS
REGULATORY
FACTORS
59
TABLE 1 POTENTIATION AND INHIBITION OF ERYTHROPOIESIS AND NEUTRALIZATION OF INHIBITION BY ANTI-EIF SERUM Percentage 59Fe incorporated in RBC + SEM
Materials and amounts per mouse 0.2 mg ERF(s)” 40.0 pg PGE,* 0.2 mg ERF(s) + 40 pg PGE, 40.0 eg PGE, 0.2 mg ERF(s)* + 40 pg PGE, 0.1 mg EIF’ 0.2 mg ERF(s) + 0.1 mg EIF 0.2 mg ERF(s) + 0.2 mg EIF
20.19 1.25 34.69 7.00 36.91
0.2 0.2 0.2 0.2 0.2
13.50 7.68
mg mg mg mg mg
ERF(s) ERF(s) ERF(s) ERF(s) ERF(s)
+ + + + +
20 40 80 40 80
pg pg pg pg pg
6.12
PGF,, PGF,, PGE,, PGF,, + 0.1 ml anti-EIF PGF,, + 0.1 ml anti-EIF
21.53 20.07
rt t c 2 + 0 r 0 0 t -t 0 t t
ESF activity IU + SEM
0.89 0.63 1.43 0.91 0.75
0.46 0.15 0.78 0.14 0.84
2.61
0.12
0.53 1.05
0.30 0.16
0.65 0.46
0.47 0.46
f 2 k + k 0 t 0 0 + 2 0 t 2
0.02 0.01 0.03 0.02 0.02 0.05 0.01 0.02 0.01 0.01
a ERF(s) = Erythropoiesis regulatory factors, a chromatographic urinary fraction from a patient with paroxysmal nocturnal hemoglobinuria. * PGE,, PGE,, PGE,, are prostaglandins. They were donated by Dr. John F. Pike, The Upjohn Company, Kalamazoo, Mich. f EIF = Erythropoiesis inhibitory factor obtained by column electrophoresis of a urinary fraction collected in an electrofractionator. The neutralizing antiserum to EIF was diluted 1- 100. Anti-EIF serum was produced in rabbits as described by Garver et al( 10). The anti-EIF serum was mixed with PGF,, and incubated with ERF(s).
TABLE 2 THE CANCELLATION OF POTENTIATION AND INHIBITION OF ERYTHROPOIESISBY PROSTAGLAND~NS Materials and amounts per Mouse (m at) 0.2 0.2 0.2 0.2
mg mg mg mg
ERF(s) ERF(s) + 40 Fg PGDZ ERF(s) + 40 pg PGD* + 40 pg PGF,, ERF(s) + 40 pg PGE, + 40 pg PGF,,
j9Fe Incorporated RBC -c SEM (%) 16.20 16.19 15.16 19.71
r + + *
0.43 0.36 0.45 0.30
in
ERF Activity + SEM WJ) 0.36 0.36 0.32 0.44
-c 0.01 f 0.01 2 0.01 Yh0.01
0 The erythropoiesis regulating factor(s), ERF(s), were from a patient with paroxysmal nocturnal hemoglobinuria (7). The prostaglandins were donated by Dr. John E. Pike, The Upjohn Company, Kalamazoo, Mich.
60
NEAI.
ET AL
procedure. Daniels and Pike describe the formation of PGA, by the degradation of PGE, (loss of a molecule of water) (5). The prostaglandins obtained by bioconversion with the fraction containing EGF, which has been indicated to contain proerythropoietin but no EPO (8) did not include PGF4,. The latter PC apparently comes by a different pathway, although arachidonic acid is a precursor for PGF4, and PGE, (16). We usually see indications of PGE, and not E, (indication of E, was seen two times out of seven experiments) during tic of ERFs, and arachidonic acid (but not linoleic) is probably in the proximal pathway for the production of EPO. A glutathione-like compound and an enzyme with the same pH-optimum as indicated for the E-isomerase, as reported by Nugteren and Christ-Hazelhof, are needed for the EGF production of erythropoietin and also for the bioconversion of fatty acids to prostaglandins (4,5,15). The prostaglandin formed by the action of an enzyme on arachidonic acid and expedited by glutathione, possibly similar to the enzyme found in sheep seminal vesicular tissue by Van Dorp et ul. and Bergstriim et ul. and characterized by Nugteren and Christ-Hazelhof, is believed to bind to a specific protein which also is apparently in our ERF preparations. In the EGF fraction the conversion of arachidonic acid to PGE, appears to take place much more readily than the conversion of linoleic acid to PGE,. This could explain why we usually detect linoleic but not arachidonic acid, since arachidonic acid appears to be converted instantly and completely to PGE,. We can detect arachidonic acid by tic when the exogenous amount is increased to 1 mg per 100 mg of ERF during the bioconversion technique. Linoleic is converted to arachidonic acid by way of a-linolenic and dihomogammalinolenic acid. Foley, Gross, Nelson, and Fisher produced EPO in exhypoxic polycythemic mice and the isolated perfused canine kidney with arachidonic acid (18). The PGs indicated in Fig. I are observed after bioconversion with or without exogenous fatty acids. The EGF fraction is indicated to contain proerythropoietin at a molecular weight (MW) of 45,000, an enzyme at 32,000 for the conversion of pro-EPO to EPO, and a small amount of a component at 40,000 (19,20). The enzyme of the EGF fraction appears to be similar to the bull vesicular enzyme in that it has the same pH-optimum, molecular weight, and requirement for glutathione as the E-isomerase prostaglandin producing enzyme (4,17,20). These PGs cannot be extracted from EGFs with organic solvents (acetone, n-hexane, and benzene) without first applying the modified bioconversion technique (5), suggesting concurrence with the work by Fisher et al. (6) in that proerythropoietin initiates the production of erythropoietin by way of prostaglandins. The ESF extractable with organic solvents does not move with PGs during tic: It is apparently attached to fragments of protein (- 4.4%) (21). The experiments in Fig. 1
ERYTHROPOIESIS
REGULATORY
FACTORS
61
represent observations following extractions of fractions containing ERFs subjected to the modified bioconversion technique. Although Dukes et al. now reject the postulate that EPO is a glycoprotein-prostaglandin complex (1), the close association of a PG-producing system with a fraction indicated previously to contain proerythropoietin is apparent (22). The fact that neutralizing anti-EPO serum inactivates EPO and the product of EGF, whereas neutralizing anti-EGF serum only prevents the production of EPO (19) would place these factors proximately. It now seems that the enzyme, E-isomerase, is probably the antigen for the neutralizing antiEGF serum (4). PGD* had no ESF or EIF activity but it did prevent PGF,, from inhibiting ESF activity produced by EPO or PGEz, possibly by competing for the site of EIF inhibition or production. When the bioconversion technique is applied to a fraction containing only EGFs, PGF,, is not demonstrable. Miller et al. in their study of the modulation of granulopoiesis, find antagonistic actions for prostaglandins E and F (23). Their in vitro observations for the stimulation of granulopoiesis by PGF,, and inhibition by PGE, are opposite to our in ~ivo observations for erythropoiesis. If prostaglandin E2 activates adenyl cyclase and indirectly initiates the production of erythropoietin, as indicated by Fisher et al. (6), possibly PGF,, initiates the production of a comparable granulopoiesis factor via guanidylate cyclase (6,23), and inhibits erythropoiesis indirectly. The neutralization of the PGF,, induced inhibition of erythropoiesis by anti-EIF serum (Table 1) confirms the close association observed between PGF,, and EIF (12); however, anti-PGF,, and anti-EIF do not cross-react with their respective antigens (unpublished data).” Possibly PGF,, initiates the production of EIF. About 71% of the ESF activity in our urinary ERFs has been attributed to EGF (19). Previously we observed a parallelism between erythropoietin activity and the disappearance of GSH (3). It is now known also that dithiothreitol (DTT) will prevent EGF activity possibly by keeping GSH in the reduced state or reducing G-SS-G. Our data suggest that a major part of EPO from our urinary ERFs is biosynthesized with the aid of erythrocytic GSH after injection of ERFs, which contain proerythropoietin (3). SUMMARY
Thin-layer chromatography of ERFs extracts indicates components in ERFs, after treatment by a bioconversion technique which converts linoleic and/or arachidonic acids to prostaglandins, with the same mobility as prostaglandins E, and A,. Linoleic acid can be detected in ERFs prior 5 Anti-PGF,,
was a gift from Dr. Kenneth T. Kirton, The Upjohn Co.
NEAL ET AL.
62
to bioconversion. PGE, and PGE, potentiate erythropoiesis to about
the
same extent as PGF.,, (found in erythropoiesis inhibitory fractions, EIF) inhibits erythropoiesis. The inhibition produced by PGF,, can be prevented with a neutralizing antiserum to EIF. Our data suggest that fatty acids and prostaglandins are part of the pathway for the production of erythropoietin by an enzyme and reduced glutathione. REFERENCES I. Dukes, P. P., Shore, N. A., Hammond, G. D., and Ortega, J. A., irr “Erythropoiesis, Proceedings of the Fourth International Conference on Erythropoiesis” (K. Nakao, J. Fisher, and F. Takaku, Eds.), p. 698. University Park Press, Baltimore, London, Tokyo, 1975. 2. Nugteren, D. H., Beerthuis, R. K.. and Van Dorp, D. A.. Rec. True. Chim. Pays-Bus Be/g. 3.
85, 405 (1966).
Lewis, J. P., Alford, D. A., Smith, J. E.. Horton, B. F., and Smith. L. L.. Brir. J. Haemotol.
14, 457 (1968).
Nugteren, D. H., and Christ-Hazelhof, E., in “Proceedings of the Fourth International Prostaglandin Conference,” p. 87. Washington, D. C., May, 1979. 5. Daniels, E. G., and Pike, J. E., in “Prostaglandin Symposium of Worcester Foundation for Experimental Biology” (P. W. Ramwell and J. E. Shaw, Eds.), pp. 379-387. Interscience, New York, 1968. 6. Fisher, J. W., Gross, D. M., Foley, J. E.. and Jubiz. W., in “Erythropoiesis, Proceedings of the Fourth International Conference on Erythropoiesis” (K. Nakao, J. W. Fisher, and F. Takaku, Eds.), p. 698. University Park Press, Baltimore, London, Tokyo. 1975. 7. Lewis, J. P., Alford, D. A., Rathjen, J. H., Jr., and Lange, R. D., J. Lab. Clin. Med. 66, 4.
987 ( 1965).
Lewis, J. P.. Neal, W. A., Welch, E. T., Lewis, W. G., III, DuBose, C. M.. Jr., Wright, C.-S., and Smith, L. L., Proc. Sot. Exp. Biol. Med. 142, 293 (1973). 9. Green, K., and Samuelsson. B., J. Lipid Res. 5, 117 (1964). 10. Gamer, F. A., Nash,W. A., Lewis,J. P., Lutcher, C. L., Ozawa, T., Moores, R. R.. and Wright, C. S. Manuscript in preparation. 1I. Lewis, J. P., Neal, W. A., Moores, R. R., Smith, L. L., Wright, C.-S., and Welch, 8.
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12. Neal, W. A., Lewis,J. P., Welch, E. T.. Lutcher, C. L., Moores. R. R., and Wright, C.-S., Amer. J. Vet. Res. 40, 493 (1979). 13. Lewis, J. P.. Neal, W. A., Alford. D. A., Moores, R. R., Gardner. E., Jr., Welch, E. T., Wright, C.-S.. and McWhirter, J. D.. Amer. J. Vet. Res. 31, 891 (1970). 14. Erslev. A. J., Kazal. L. A., and Miller, 0. P., in “Proceedings of the International SYmPOSiUm Erythropoieticum”(T. Travnicek and J. Neuwirt, Eds.), pp. 15. Charles
University, Prague,Czechoslovakia. 15. Lewis. J. P.. Alford, D. A., Neal, W. A., Moores, R. R., Welch, E. T., Gardner, E., Jr., Wright, C.-S., and Smith. L. L., Stand. J. Huemur. 8, 200 (1971). 16. Hinman.J. W., Ann. Rev. Biochem. 41, 161 (1972). 17. Lewis,J. P.. Welch, E. T., Neal. W. A.. Moores, R. R., Lewis, W. G., III, Wright,
C.-S.. DuBose.C. M., Jr., andSmith,L.
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19. Lewis. J. P.. Welch, E. T., Neal. W. A., Wright, C.-S., Gardner, E., Jr., Smith, L. L., Lange, R. D.. Biochem. Med. 14, 399 (1975).
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20. Neal, W. A., Welch, E. T., Lewis, J. P., Lutcher, C. L., Wright, C.-S., and Smith, L. L., Biochem. Med. 20, 229 (1978). 21. Lewis, J. P., Welch, E. T., Neal, W. A., DuBose, C. M., Jr., Lewis, W. G., III, Wright, C.-S., and Smith, L. L., Biochem. Med. 10, 374 (1974). 22. DuBose, C. M., Jr., Welch, E. T., Lewis, J. P., Neal, W. A., Lutcher, C. L., Biochem. Med. 17, 310 (1977). 23. Miller, H. M., Russell, T. R., Gross, M. A., and Yunis, A. A.,J. Lab. C/in. Med. 92, 983 (1978).