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Multiple Regulatory Steps in Erythroid Heme Biosynthesis
Archives of Biochemistry and Biophysics Vol. 384, No. 2, December 15, pp. 375–378, 2000 doi:10.1006/abbi.2000.2069, available online at http://www.idealibrary.com on
Multiple Regulatory Steps in Erythroid Heme Biosynthesis Steven I. Woodard* and Harry A. Dailey† ,1 *Department of Microbiology and †Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602
Received June 15, 2000, and in revised form August 16, 2000
Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent downstream rate-limiting steps. © 2000 Academic Press Key Words: heme synthesis; porphyrin; aminolevulinate synthase; coproporphyrinogen oxidase.
The regulation of heme biosynthesis differs significantly between erythroid precursor and nonerythroid cell types (1–3). Separate erythroid-specific and nonerythroid, or housekeeping, genes exist for the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), 2 and during erythroid differentiation, the housekeeping ALAS is downregulated and unable to satisfy cellular demands for ALA production (4, 5). For the remaining enzymes of the pathway, there exist erythroid-specific promoter elements that allow for induction of mRNAs of these enzymes during erythroid differentiation (see 2). Murine erythroleukemia (MEL) cells have frequently been employed to study various 1 To whom correspondence should be addressed. Fax: (706) 5427567. E-mail: [email protected]. 2 Abbreviations used: ALAS, 5-aminolevulinate synthase; MEL, murine erythroleukemia; DMSO, dimethyl sulfoxide.
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
aspects of this process. They are a convenient model since they are erythroid precursors whose erythroid program is arrested just prior to the induction of heme synthesis, and this arrest can be relieved by treatment of the cells with a variety of compounds, including dimethyl sulfoxide (DMSO) or butyric acid (6). A number of studies have been published that followed the induction of mRNA for enzymes of heme biosynthesis during erythroid differentiation (see 2, 7). On the basis of these data, it has been proposed that ALAS is the rate-limiting enzyme for heme biosynthesis during erythroid differentiation and that all downstream enzymes are present in excess of what is required, except in some porphyrias (1–3). However, in vitro measurement of enzyme activities in extracts from differentiating erythroid cells suggests that other sites in the pathway may be transiently limiting (7–9). From current data it is clear that, for at least ALAS, posttranscriptional regulation also occurs, so that mRNA levels do not always accurately reflect in situ enzyme activity (1, 3). Additionally, increases in enzyme activity for coproporphyrinogen oxidase, the antepenultimate enzyme in the pathway, do not appear to correspond with temporal increases in mRNA levels for this protein (8, 10). In the present work, we have approached the question of in situ enzyme activity by following the levels of pathway intermediates. We have made the assumption that any rate-limiting steps in situ would result in porphyrin accumulation and/or excretion behind that point. The rationale for this assumption is that if ALAS alone is rate limiting, then no intermediates should accumulate, but if a secondary enzyme activity is limiting, then an accumulation of intermediates before that site should occur. Thus, the pattern of porphyrin excretion might give an indication of limiting steps in cells. Our data presented below show that coproporphyrin and to a lesser extent protoporphyrin are produced at significant levels and excreted into the me375
376
WOODARD AND DAILEY
dium by differentiating MEL cells, indicating that in situ coproporphyrinogen oxidase is a rate-limiting step. METHODS MEL cell strain DS-19 was cultured at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS). Cells were induced with 2% (vol/vol) DMSO as described previously (8). In some experiments, effectors were added to cultured cells 24 h prior to induction. For the determination of porphyrin production, cultures were sampled at 24-h intervals, counted, and harvested at room temperature by centrifugation at 2500g for 10 min. Thus, the reported porphyrin excretion data represent amounts per 24 h and are not cumulative. Five milliliters of the cleared culture fluid was taken for porphyrin determinations, and the harvested cells were suspended in warm fresh medium containing 2% DMSO and effectors (where appropriate) and allowed to continue incubation. Porphyrin excretion. Porphyrins were extracted and concentrated by the method of Kennedy et al. (11). Prior to extraction of culture media samples for porphyrin determination, mesoporphyrin was added as an internal standard at a concentration of 50 nM. Any porphyrinogens present were then photooxidized in atmospheric air for 1 h at room temperature. This solution was loaded onto a C 18 Sep-Pak cartridge (Waters) that had been flushed successively with 10 ml of methanol and 15 ml of water. The cartridge was then washed with 50 ml of 10% (vol/vol) methanol. Porphyrins were eluted with 3 ml of 100% methanol, and this solution was evaporated at 60°C under N 2. The dried porphyrins were then dissolved in 10 l of Proto Solv (Porphyrin Products), and 990 l of 1 N HCl was added. After thorough mixing, the solution was filtered through 0.2-m Acrodiscs (Gelman) and stored in amber vials prior to HPLC separation. HPLC analysis. Porphyrins were analyzed using a Spheri 5 C 18 column (100 ⫻ 4.6 mm) (Pierce Chemical Co.) in a Beckman 344 HPLC system. The ammonium acetate buffer system of Lim and Peters (12) was used. Buffers A and B consisted of 1 M ammonium acetate/acetonitrile (9:1), pH 5.16, and methanol/acetonitrile (9:1), respectively. The flow rate was 1 ml/min. Following injection of 100 l, the program started with 20% B and 80% A. After 1 min, % B increased to 35% over 1.5 min and was held at 35% for 2 min. Then % B was increased to 70% over 0.5 min and maintained at that concentration for 4.5 min before being increased to 95% over 2.5 min. This concentration was maintained for 1.5 min. At 12 min, % B was recycled to 20% over 0.5 min. Eluted porphyrins were monitored by fluorescence detection using an Applied Biosystems 980 programmable fluorescence detector. The excitation wavelength was 395 nm and a 550-nm filter was used.
RESULTS AND DISCUSSION
Porphyrin production by induced and uninduced MEL cells. In the present study, we have examined the pattern of porphyrin excretion by MEL cells in an effort to see what steps in the heme biosynthetic pathway may be limiting during erythroid differentiation in MEL cells. It has been reported by others that protoporphyrin accumulates in the medium of differentiating MEL cells (13), and this observation was cited as support for the hypothesis that ferrochelatase was rate limiting for heme synthesis during MEL cell differentiation. We have previously shown that 48 h after induction the amount of heme that may be produced by MEL cells is correlated to the amount of exogenous
FIG. 1. Porphyrin excretion pattern by DMSO-induced MEL cells. Experimental details are given in the text. The four porphyrins detected are the amount of porphyrin produced in a 24-h period by 10 7 cells and the legend for these is as follows: coproporphyrin III (diagonal hatched bar), protoporphyrin IX (open box), uroporphyrin I (gray shade), and uroporphyrin III (black box). (A) DMSO-induced MEL cells; (B) DMSO-induced MEL cells in the presence of the inhibitor of heme synthesis, succinylacetone.
protoporphyrin added to the culture, which suggested that by 48 h after induction ferrochelatase activity is not rate limiting for heme production (9). Noninduced MEL cells produce and excrete small amounts of coproporphyrin, which must derive from chemically oxidized coproporphyrinogen, and protoporphyrin, which may be derived from protoporphyrin and/or protoporphyrinogen (Fig. 1A). DMSO induction results in a 10- to 100-fold increase in the amount of porphyrins excreted per day (Fig. 1A). To determine if cellular porphyrin synthesis results in the intracellular accumulation of free porphyrins, cells from 0, 24, 48, 72, and 96 h post DMSO induction were harvested, extracted, and analyzed for porphyrins. The data obtained did not show significant amounts of any free cellular porphyrins even at 96 h (data not shown), whereas the medium collected from these same cultures was routinely found to contain over 1 nmol of porphyrin per 10 7 cells produced per day in DMSOinduced cultures. When the cumulative excretion per 24 h was compared with total accumulation over the course of the experiment, it was found that the rates were additive so that the accumulation by 96 h was equal to the sum of the individual rates for all 4 days (data not shown). During this same period of time, cellular heme content also increased and accounted for over 90% of total cellular tetrapyrrole production (Fig. 1A). Effect of succinylacetone, SIH-iron, and ALA on porphyrin patterns. Succinylacetone inhibits the second step in the pathway, ALA dehydratase (PBG synthase). Addition of 500 M succinylacetone to the medium along with DMSO resulted in greatly reduced total porphyrin accumulation in the medium, with only
REGULATION OF HEME SYNTHESIS
FIG. 2. Porphyrin excretion pattern in DMSO-induced MEL cells in the presence of SIH-iron (A) or exogenous ALA (B). The legend for these figures is identical to that of Fig. 1. (A) SIH-iron treated cells; (B) ALA (100 M) added to DMSO-induced cultures.
small amounts of coproporphyrin being detected in these cultures (Fig. 1B). Under these conditions, cellular heme content did not increase, demonstrating that succinylacetone treatment prevented normal erythroid differentiation. Since one possible reason that free porphyrins are produced and excreted by cells is that there is insufficient iron available for heme formation by ferrochelatase, iron in the form of SIH-iron was added to the cultures. Since SIH-iron allows cells to load iron independent of the normal transferrin uptake system (14), the observations made were not dependent upon cellular iron transport machinery. Previously, it has been shown that addition of SIH-iron to differentiating MEL cell cultures increases cellular heme content about threefold over cultures supplied with only transferrin iron (14). In the current study, similar increases were found in DMSO-induced MEL cultures supplemented with 100 M SIH-iron and an overall decrease in porphyrin excretion was noted, with coproporphyrin being the major porphyrin detected (Fig. 2A). The increase in cellular heme seen in cells treated with SIH-iron cannot be accounted for solely by conversion of the porphyrins detected in DMSO-induced cultures without SIHiron. This is consistent with the proposed role for cellular iron stores in ALAS-2 activity induction during erythroid differentiation (3). However, the 10-fold decrease in coproporphyrin excretion in cultures supplemented with SIH-iron does suggest that some of the porphyrin excretion observed may be attributable to inadequate iron for ferrochelatase-mediated iron insertion. The lack of detectable uroporphyrin in cultures supplemented with SIH-iron may reflect the overall decreased level of culture porphyrins. Since production of ALA is thought to be a key regulatory step, cultures were grown with added ALA and porphyrin excretion was monitored. The data in Fig.
377
2B show that addition of 100 M ALA to DMSO-induced cultures results in large increases in all porphyrins, including uroporphyrins I and III, relative to induced controls. Heme content of these cultures at 96 h is not significantly higher than what is found in cultures without added ALA (data not shown), which is consistent with earlier observations by Laskey et al. (14). Interestingly, the total amount of tetrapyrroles produced by these supplemented cultures can account for only a few percent of the potential tetrapyrroles that could result if all of the ALA added to the cultures were converted to porphyrin. This is similar to what was noted with protoporphyrin supplementation of DMSO-induced MEL cell cultures (9). CONCLUSIONS
Induction of MEL cells with DMSO results in a 100fold increase in the excretion of coproporphyrin and protoporphyrin by day 4 postinduction. The possibility that the excretion of porphyrin was due entirely to decreased iron insertion attributable to low cellular iron concentrations appears not to be the case since supplementation of differentiating cultures with SIHiron, which does result in an increase in cellular heme production by differentiating MEL cells (14), decreased, but did not eliminate, culture porphyrin excretion. The addition of 100 M ALA to the culture medium of the induced or uninduced cells results in greatly increased porphyrin excretion, as one would expect if ALAS is a principal rate-limiting point. However, the presence of excreted coproporphyrin under all conditions is consistent with a role for coproporphyrinogen oxidase as a downstream regulatory point as has been suggested before from enzyme activity studies (7, 8). One additional possibility is that if transport of coproporphyrinogen from the cytoplasm, its site of synthesis, into the intramembrane space of the mitochondria where coproporphyrinogen oxidase is located is mediated by a specific transporter, then this transport system could be rate limiting. While an explanation for the need for such a regulatory site may not be immediately apparent, it should be remembered that coproporphyrin(ogen) is considerably more water soluble and less reactive than protoporphyrin or heme. Therefore, under conditions where ALAS may be producing more porphyrin precursors than can be converted into heme, it would be more advantageous for the cell to not convert this into free protoporphyrin, but instead excrete coproporphyrin, which is readily cleared by the body. The source of the protoporphyrin found in culture medium is unclear, but it is attractive to speculate that it may be derived from protoporphyrinogen (and, therefore, caused by limiting protoporphyrinogen oxidase), which has been shown in vitro to
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feedback inhibit porphobilinogen deaminase, the third pathway enzyme (15). This would then add an additional site of fine-tuned regulation to the pathway. ACKNOWLEDGMENT This work was supported by Grant DK 35898 from NIDDKD to H.A.D.
REFERENCES 1. May, B. K., Bashker, R., Bawden, M. J., and Cox, T. C. (1990) Mol. Biol. Med. 7, 405– 421. 2. Kappas, A., Sassa, S., Galbraith, R. A., and Nordmann, Y. (1995) in The Metabolic and Molecular Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), pp. 2103–2159, McGraw–Hill, New York. 3. Ponka, P. (1997) Blood 89, 1–25. 4. Yin, X., and Dailey, H. A. (1998) Blood Cells Mol. Dis. 24, 41–53. 5. Harigae, H., Suwabe, N., Weinstock, P. H., Nagai, M., Fujita, H., Yamamoto, M., and Sassa, S. (1998) Blood 91, 798 – 805.
6. Marks, P., and Rifkind, R. A. (1978) Annu. Rev. Biochem. 47, 419 – 448. 7. Lake-Bullock, H., and Dailey, H. A. (1993) Mol. Cell. Biol. 13, 7122–7132. 8. Conder, L. H., Woodard, S. I., and Dailey, H. A. (1991) Biochem. J. 275, 321–326. 9. Fadigan, A. B., and Dailey, H. A. (1987) Biochem. J. 243, 419 – 424. 10. Takahashi, S., Taketani, S., Akasaka, J.-e., Kobayashi, A., Hayashi, N., Yamamoto, M., and Nagi, T. (1998) Blood 92, 3436 – 3444. 11. Kennedy, S. W., Wingfield, D. C., and Fox, G. A. (1986) Anal. Biochem. 157, 1–7. 12. Lim, C. K., and Peters, T. J. (1984) Clin. Chim. Acta 139, 55– 63. 13. Rutherford, T., Thompson, G. C., and Moore, M. R. (1979) Proc. Natl. Acad. Sci. USA 76, 833– 836. 14. Laskey, J. D., Ponka, P., and Schulman, H. M. (1986) J. Cell Physiol. 129, 185–192. 15. Meissner, P. N., Adams, P., and Kirsch, R. (1993) J. Clin. Invest. 91, 1436 –1444.
Multiple Regulatory Steps in Erythroid Heme Biosynthesis Steven I. Woodard* and Harry A. Dailey† ,1 *Department of Microbiology and †Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602
Received June 15, 2000, and in revised form August 16, 2000
Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent downstream rate-limiting steps. © 2000 Academic Press Key Words: heme synthesis; porphyrin; aminolevulinate synthase; coproporphyrinogen oxidase.
The regulation of heme biosynthesis differs significantly between erythroid precursor and nonerythroid cell types (1–3). Separate erythroid-specific and nonerythroid, or housekeeping, genes exist for the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), 2 and during erythroid differentiation, the housekeeping ALAS is downregulated and unable to satisfy cellular demands for ALA production (4, 5). For the remaining enzymes of the pathway, there exist erythroid-specific promoter elements that allow for induction of mRNAs of these enzymes during erythroid differentiation (see 2). Murine erythroleukemia (MEL) cells have frequently been employed to study various 1 To whom correspondence should be addressed. Fax: (706) 5427567. E-mail: [email protected]. 2 Abbreviations used: ALAS, 5-aminolevulinate synthase; MEL, murine erythroleukemia; DMSO, dimethyl sulfoxide.
0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
aspects of this process. They are a convenient model since they are erythroid precursors whose erythroid program is arrested just prior to the induction of heme synthesis, and this arrest can be relieved by treatment of the cells with a variety of compounds, including dimethyl sulfoxide (DMSO) or butyric acid (6). A number of studies have been published that followed the induction of mRNA for enzymes of heme biosynthesis during erythroid differentiation (see 2, 7). On the basis of these data, it has been proposed that ALAS is the rate-limiting enzyme for heme biosynthesis during erythroid differentiation and that all downstream enzymes are present in excess of what is required, except in some porphyrias (1–3). However, in vitro measurement of enzyme activities in extracts from differentiating erythroid cells suggests that other sites in the pathway may be transiently limiting (7–9). From current data it is clear that, for at least ALAS, posttranscriptional regulation also occurs, so that mRNA levels do not always accurately reflect in situ enzyme activity (1, 3). Additionally, increases in enzyme activity for coproporphyrinogen oxidase, the antepenultimate enzyme in the pathway, do not appear to correspond with temporal increases in mRNA levels for this protein (8, 10). In the present work, we have approached the question of in situ enzyme activity by following the levels of pathway intermediates. We have made the assumption that any rate-limiting steps in situ would result in porphyrin accumulation and/or excretion behind that point. The rationale for this assumption is that if ALAS alone is rate limiting, then no intermediates should accumulate, but if a secondary enzyme activity is limiting, then an accumulation of intermediates before that site should occur. Thus, the pattern of porphyrin excretion might give an indication of limiting steps in cells. Our data presented below show that coproporphyrin and to a lesser extent protoporphyrin are produced at significant levels and excreted into the me375
376
WOODARD AND DAILEY
dium by differentiating MEL cells, indicating that in situ coproporphyrinogen oxidase is a rate-limiting step. METHODS MEL cell strain DS-19 was cultured at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS). Cells were induced with 2% (vol/vol) DMSO as described previously (8). In some experiments, effectors were added to cultured cells 24 h prior to induction. For the determination of porphyrin production, cultures were sampled at 24-h intervals, counted, and harvested at room temperature by centrifugation at 2500g for 10 min. Thus, the reported porphyrin excretion data represent amounts per 24 h and are not cumulative. Five milliliters of the cleared culture fluid was taken for porphyrin determinations, and the harvested cells were suspended in warm fresh medium containing 2% DMSO and effectors (where appropriate) and allowed to continue incubation. Porphyrin excretion. Porphyrins were extracted and concentrated by the method of Kennedy et al. (11). Prior to extraction of culture media samples for porphyrin determination, mesoporphyrin was added as an internal standard at a concentration of 50 nM. Any porphyrinogens present were then photooxidized in atmospheric air for 1 h at room temperature. This solution was loaded onto a C 18 Sep-Pak cartridge (Waters) that had been flushed successively with 10 ml of methanol and 15 ml of water. The cartridge was then washed with 50 ml of 10% (vol/vol) methanol. Porphyrins were eluted with 3 ml of 100% methanol, and this solution was evaporated at 60°C under N 2. The dried porphyrins were then dissolved in 10 l of Proto Solv (Porphyrin Products), and 990 l of 1 N HCl was added. After thorough mixing, the solution was filtered through 0.2-m Acrodiscs (Gelman) and stored in amber vials prior to HPLC separation. HPLC analysis. Porphyrins were analyzed using a Spheri 5 C 18 column (100 ⫻ 4.6 mm) (Pierce Chemical Co.) in a Beckman 344 HPLC system. The ammonium acetate buffer system of Lim and Peters (12) was used. Buffers A and B consisted of 1 M ammonium acetate/acetonitrile (9:1), pH 5.16, and methanol/acetonitrile (9:1), respectively. The flow rate was 1 ml/min. Following injection of 100 l, the program started with 20% B and 80% A. After 1 min, % B increased to 35% over 1.5 min and was held at 35% for 2 min. Then % B was increased to 70% over 0.5 min and maintained at that concentration for 4.5 min before being increased to 95% over 2.5 min. This concentration was maintained for 1.5 min. At 12 min, % B was recycled to 20% over 0.5 min. Eluted porphyrins were monitored by fluorescence detection using an Applied Biosystems 980 programmable fluorescence detector. The excitation wavelength was 395 nm and a 550-nm filter was used.
RESULTS AND DISCUSSION
Porphyrin production by induced and uninduced MEL cells. In the present study, we have examined the pattern of porphyrin excretion by MEL cells in an effort to see what steps in the heme biosynthetic pathway may be limiting during erythroid differentiation in MEL cells. It has been reported by others that protoporphyrin accumulates in the medium of differentiating MEL cells (13), and this observation was cited as support for the hypothesis that ferrochelatase was rate limiting for heme synthesis during MEL cell differentiation. We have previously shown that 48 h after induction the amount of heme that may be produced by MEL cells is correlated to the amount of exogenous
FIG. 1. Porphyrin excretion pattern by DMSO-induced MEL cells. Experimental details are given in the text. The four porphyrins detected are the amount of porphyrin produced in a 24-h period by 10 7 cells and the legend for these is as follows: coproporphyrin III (diagonal hatched bar), protoporphyrin IX (open box), uroporphyrin I (gray shade), and uroporphyrin III (black box). (A) DMSO-induced MEL cells; (B) DMSO-induced MEL cells in the presence of the inhibitor of heme synthesis, succinylacetone.
protoporphyrin added to the culture, which suggested that by 48 h after induction ferrochelatase activity is not rate limiting for heme production (9). Noninduced MEL cells produce and excrete small amounts of coproporphyrin, which must derive from chemically oxidized coproporphyrinogen, and protoporphyrin, which may be derived from protoporphyrin and/or protoporphyrinogen (Fig. 1A). DMSO induction results in a 10- to 100-fold increase in the amount of porphyrins excreted per day (Fig. 1A). To determine if cellular porphyrin synthesis results in the intracellular accumulation of free porphyrins, cells from 0, 24, 48, 72, and 96 h post DMSO induction were harvested, extracted, and analyzed for porphyrins. The data obtained did not show significant amounts of any free cellular porphyrins even at 96 h (data not shown), whereas the medium collected from these same cultures was routinely found to contain over 1 nmol of porphyrin per 10 7 cells produced per day in DMSOinduced cultures. When the cumulative excretion per 24 h was compared with total accumulation over the course of the experiment, it was found that the rates were additive so that the accumulation by 96 h was equal to the sum of the individual rates for all 4 days (data not shown). During this same period of time, cellular heme content also increased and accounted for over 90% of total cellular tetrapyrrole production (Fig. 1A). Effect of succinylacetone, SIH-iron, and ALA on porphyrin patterns. Succinylacetone inhibits the second step in the pathway, ALA dehydratase (PBG synthase). Addition of 500 M succinylacetone to the medium along with DMSO resulted in greatly reduced total porphyrin accumulation in the medium, with only
REGULATION OF HEME SYNTHESIS
FIG. 2. Porphyrin excretion pattern in DMSO-induced MEL cells in the presence of SIH-iron (A) or exogenous ALA (B). The legend for these figures is identical to that of Fig. 1. (A) SIH-iron treated cells; (B) ALA (100 M) added to DMSO-induced cultures.
small amounts of coproporphyrin being detected in these cultures (Fig. 1B). Under these conditions, cellular heme content did not increase, demonstrating that succinylacetone treatment prevented normal erythroid differentiation. Since one possible reason that free porphyrins are produced and excreted by cells is that there is insufficient iron available for heme formation by ferrochelatase, iron in the form of SIH-iron was added to the cultures. Since SIH-iron allows cells to load iron independent of the normal transferrin uptake system (14), the observations made were not dependent upon cellular iron transport machinery. Previously, it has been shown that addition of SIH-iron to differentiating MEL cell cultures increases cellular heme content about threefold over cultures supplied with only transferrin iron (14). In the current study, similar increases were found in DMSO-induced MEL cultures supplemented with 100 M SIH-iron and an overall decrease in porphyrin excretion was noted, with coproporphyrin being the major porphyrin detected (Fig. 2A). The increase in cellular heme seen in cells treated with SIH-iron cannot be accounted for solely by conversion of the porphyrins detected in DMSO-induced cultures without SIHiron. This is consistent with the proposed role for cellular iron stores in ALAS-2 activity induction during erythroid differentiation (3). However, the 10-fold decrease in coproporphyrin excretion in cultures supplemented with SIH-iron does suggest that some of the porphyrin excretion observed may be attributable to inadequate iron for ferrochelatase-mediated iron insertion. The lack of detectable uroporphyrin in cultures supplemented with SIH-iron may reflect the overall decreased level of culture porphyrins. Since production of ALA is thought to be a key regulatory step, cultures were grown with added ALA and porphyrin excretion was monitored. The data in Fig.
377
2B show that addition of 100 M ALA to DMSO-induced cultures results in large increases in all porphyrins, including uroporphyrins I and III, relative to induced controls. Heme content of these cultures at 96 h is not significantly higher than what is found in cultures without added ALA (data not shown), which is consistent with earlier observations by Laskey et al. (14). Interestingly, the total amount of tetrapyrroles produced by these supplemented cultures can account for only a few percent of the potential tetrapyrroles that could result if all of the ALA added to the cultures were converted to porphyrin. This is similar to what was noted with protoporphyrin supplementation of DMSO-induced MEL cell cultures (9). CONCLUSIONS
Induction of MEL cells with DMSO results in a 100fold increase in the excretion of coproporphyrin and protoporphyrin by day 4 postinduction. The possibility that the excretion of porphyrin was due entirely to decreased iron insertion attributable to low cellular iron concentrations appears not to be the case since supplementation of differentiating cultures with SIHiron, which does result in an increase in cellular heme production by differentiating MEL cells (14), decreased, but did not eliminate, culture porphyrin excretion. The addition of 100 M ALA to the culture medium of the induced or uninduced cells results in greatly increased porphyrin excretion, as one would expect if ALAS is a principal rate-limiting point. However, the presence of excreted coproporphyrin under all conditions is consistent with a role for coproporphyrinogen oxidase as a downstream regulatory point as has been suggested before from enzyme activity studies (7, 8). One additional possibility is that if transport of coproporphyrinogen from the cytoplasm, its site of synthesis, into the intramembrane space of the mitochondria where coproporphyrinogen oxidase is located is mediated by a specific transporter, then this transport system could be rate limiting. While an explanation for the need for such a regulatory site may not be immediately apparent, it should be remembered that coproporphyrin(ogen) is considerably more water soluble and less reactive than protoporphyrin or heme. Therefore, under conditions where ALAS may be producing more porphyrin precursors than can be converted into heme, it would be more advantageous for the cell to not convert this into free protoporphyrin, but instead excrete coproporphyrin, which is readily cleared by the body. The source of the protoporphyrin found in culture medium is unclear, but it is attractive to speculate that it may be derived from protoporphyrinogen (and, therefore, caused by limiting protoporphyrinogen oxidase), which has been shown in vitro to
378
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feedback inhibit porphobilinogen deaminase, the third pathway enzyme (15). This would then add an additional site of fine-tuned regulation to the pathway. ACKNOWLEDGMENT This work was supported by Grant DK 35898 from NIDDKD to H.A.D.
REFERENCES 1. May, B. K., Bashker, R., Bawden, M. J., and Cox, T. C. (1990) Mol. Biol. Med. 7, 405– 421. 2. Kappas, A., Sassa, S., Galbraith, R. A., and Nordmann, Y. (1995) in The Metabolic and Molecular Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), pp. 2103–2159, McGraw–Hill, New York. 3. Ponka, P. (1997) Blood 89, 1–25. 4. Yin, X., and Dailey, H. A. (1998) Blood Cells Mol. Dis. 24, 41–53. 5. Harigae, H., Suwabe, N., Weinstock, P. H., Nagai, M., Fujita, H., Yamamoto, M., and Sassa, S. (1998) Blood 91, 798 – 805.
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