Iron chelation rescues hemolytic anemia and skin photosensitivity in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is an inborn error of heme synthesis resulting from uroporphyrinogen III synthase (UROS) deficiency and the accumulation of nonphysiological porphyrin isomer I metabolites. Clinical features are heterogeneous among patients with CEP but usually combine skin photosensitivity and chronic hemolytic anemia, the severity of which is related to porphyrin overload. Therapeutic options include symptomatic strategies only and are unsatisfactory. One promising approach to treating CEP is to reduce the erythroid production of porphyrins through substrate reduction therapy by inhibiting 5-aminolevulinate synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthetic pathway. We efficiently reduced porphyrin accumulation after RNA interference-mediated downregulation of ALAS2 in human erythroid cellular models of CEP disease. Taking advantage of the physiological iron-dependent posttranscriptional regulation of ALAS2, we evaluated whether iron chelation with deferiprone could decrease ALAS2 expression and subsequent porphyrin production in vitro and in vivo in a CEP murine model. Treatment with deferiprone of UROS-deficient erythroid cell lines and peripheral blood CD34+-derived erythroid cultures from a patient with CEP inhibited iron-dependent protein ALAS2 and iron-responsive element-binding protein 2 expression and reduced porphyrin production. Furthermore, porphyrin accumulation progressively decreased in red blood cells and urine, and skin photosensitivity in CEP mice treated with deferiprone (1 or 3 mg/mL in drinking water) for 26 weeks was reversed. Hemolysis and iron overload improved upon iron chelation with full correction of anemia in CEP mice treated at the highest dose of deferiprone. Our findings highlight, in both mouse and human models, the therapeutic potential of iron restriction to modulate the phenotype in CEP.

Liver Transplantation for Acute Intermittent Porphyria: Biochemical and Pathologic Studies of the Explanted Liver.

Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.

In ferrochelatase-deficient protoporphyria patients, ALAS2 expression is enhanced and erythrocytic protoporphyrin concentration correlates with iron availability.

The activity of the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase (ALAS2), the first and rate-limiting enzyme in heme biosynthesis, is down-regulated during iron-deficiency. Ferrochelatase (FECH), the last enzyme of this pathway, inserts iron into protoporphyrin IX (PPIX) to form heme. Patients with erythropoietic protoporphyria (EPP), an inherited deficiency in FECH, often show signs of iron deficiency in addition to phototoxicity which is caused by PPIX accumulation. However, iron supplementation often leads to exacerbation of phototoxicity. We report three EPP patients who had reduced erythrocytic PPIX concentrations when they were iron-deficient and their microcytic and hypochromic anemia deteriorated. Additionally, we found a significant increase in the amount of ALAS2 mRNA and protein among EPP patients. To verify the connection between FECH deficiency and ALAS2 over-expression, we inhibited FECH in cultured cells and found a subsequent increase in ALAS2 mRNA. We conclude that the primary deficiency in ferrochelatase leads to a secondary increase in ALAS2 expression. The combined action of these two enzymes within the heme biosynthetic pathway contributes to the accumulation of PPIX. Furthermore, we hypothesize that EPP patients may benefit from a mild iron deficiency since it would limit PPIX production by restricting ALAS2 over-expression.

Lon peptidase 1 (LONP1)-dependent breakdown of mitochondrial 5-aminolevulinic acid synthase protein by heme in human liver cells.

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system.

Hypoxic induction of human erythroid-specific δ-aminolevulinate synthase mediated by hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric basic helix-loop-helix transcription factor that regulates many key genes. δ-Aminolevulinate synthase (ALAS) catalyzes the first and rate-limiting reaction in the heme biosynthetic pathway. In this study, we show that hypoxia-induced expression of erythroid-specific ALAS2 is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased ALAS2 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells. Enforced HIF1α expression increased the level of ALAS2 expression, while HIF1α knockdown by RNA interference decreased the level of ALAS2 expression. In silico analysis revealed three potential hypoxia-response elements (HREs) that are located 611, 621, and 741 bp downstream of the ALAS2 gene. The results from reporter gene and mutation analysis suggested that these elements are necessary for a maximal hypoxic response. Chromatin immunoprecipitation and polymerase chain reaction showed that the HREs could be recognized and bound by HIF1α in vivo. These results demonstrate that the upregulation of ALAS2 during hypoxia is directly mediated by HIF1. We hypothesize that HIF1-mediated ALAS2 upregulation promotes erythropoiesis to satisfy the needs of an organism under hypoxic conditions. This may be accomplished via increased heme levels and an interaction between ALAS2 and erythropoietin.

Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in ß-thalassemic erythropoiesis.

BACKGROUND: ß-thalassemic syndromes are inherited red cell disorders characterized by severe ineffective erythropoiesis and increased levels of reactive oxygen species whose contribution to ß-thalassemic anemia is only partially understood. DESIGN AND METHODS: We studied erythroid precursors from normal and ß-thalassemic peripheral CD34(+) cells in two-phase liquid culture by proteomic, reverse transcriptase polymerase chain reaction and immunoblot analyses. We measured intracellular reactive oxygen species, heme levels and the activity of δ-aminolevulinate-synthase-2. We exposed normal cells and K562 cells with silenced peroxiredoxin-2 to H(2)O(2) and generated a recombinant peroxiredoxin-2 for kinetic measurements in the presence of H(2)O(2) or hemin. RESULTS: In ß-thalassemia the increased production of reactive oxygen species was associated with down-regulation of heme oxygenase-1 and biliverdin reductase and up-regulation of peroxiredoxin-2. In agreement with these observations in ß-thalassemic cells we found decreased heme levels related to significantly reduced activity of the first enzyme of the heme pathway, δ-aminolevulinate synthase-2 without differences in its expression. We demonstrated that the activity of recombinant δ-aminolevulinate synthase-2 is inhibited by both reactive oxygen species and hemin as a protective mechanism in ß-thalassemic cells. We then addressed the question of the protective role of peroxiredoxin-2 in erythropoiesis by exposing normal cells to oxidative stress and silencing peroxiredoxin-2 in human erythroleukemia K562 cells. We found that peroxiredoxin-2 expression is up-regulated in response to oxidative stress and required for K562 cells to survive oxidative stress. We then showed that peroxiredoxin-2 binds heme in erythroid precursors with high affinity, suggesting a possible multifunctional cytoprotective role of peroxiredoxin-2 in ß-thalassemia. CONCLUSIONS: In ß-thalassemic erythroid cells the reduction of δ-aminolevulinate synthase-2 activity and the increased expression of peroxiredoxin-2 might represent two novel stress-response protective systems.

Fish oil feeding up-regulates the expression of 5-aminolevulinate synthase 2 mRNA in rat brain.

In this study, we investigated the effect of fish oil on gene expression in the cerebral cortex, and found that 5-aminolevulinate synthase 2 (ALAS2) mRNA expression was up-regulated by fish oil feeding. ALAS2 promoter activity was found to be regulated by retinoic acid. Our results suggest that fish oil modulates neuronal functions via heme synthesis.

A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis.

A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C(5)N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C(5)N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C(5)N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of approximately 10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C(5)N. C(5)N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C(56) polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C(5)N product was observed. This three enzyme pathway is likely the general route to the C(5)N ring system in other natural products, including the antibiotic moenomycin.

Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells.

The process of erythroid differentiation in mouse Friend leukemia virus transformed cells (T3-C1-2) was examined by following changes in several enzyme activities of the heme biosynthetic pathway and in heme concentration while the cells were undergoing erythroid differentiation after treatment with dimethylsulfoxide. Untreated cells on the one hand, have a limited capacity for spontaneous differentiation. On the other hand, dimethylsulfoxide(DMSO)-treated cells showed an increase in the activities of delta-aminolevulinic acid (ALA) synthetase, ALA dehydratase, uroporphyrinogen-I synthetase, ferrochelatase, and heme concentration by days 1, 1.5, 2, and 4, respectively. The increase of the heme pathway enzymes and heme concentration followed the order of these enzymes or products as they are arranged in the heme biosynthetic pathway. These changes induced by DMSO were effectively inhibited by treatment with actinomycin D, suggesting that continued RNA synthesis is required for the differentiation process. 5-bromo-2'-deoxyuridine (BrdU) (10(-5) M) inhibited the DMSO-induced changes of the heme pathway enzymes. BrdU was most effective when it was present during the first 2 days of cell culture. It gradually lost its inhibitory effect when added after the 3rd day or later. The BrdU-mediated inhibition was completely overcome by the addition of thymidine (7 x 10(-5) M), but not by uridine (7 x 10(-5) M). All these data suggest that a sequential induction of the heme pathway enzyme takes place during erythroid differentiation of Friend leukemia cells, and that the sequential induction of the enzymes may be due to a sequential activation of genes coding for these enzyme activities.

Enhancement of heme and globin synthesis in cultured human marrow by certain 5 -H steroid metabolites.

These studies demonstrate that certain sex steroid metabolites are capable of significantly stimulating the synthesis of both heme and globin in cultured human bone marrow cells. These compounds, which are maximally effective at a concentration of 3 x 10(-8)M, are steroids of the C(19) and C(21) neutral type; share a common 5beta-H (A:B cis) configuration; and are derived from the in vivo biotransformation of testosterone or progesterone, or their intermediates, in man. Since these steroid metabolites have been shown to be capable in other systems of inducing the synthesis of delta-aminolevulinic acid synthetase, the limiting enzyme in the heme biosynthetic pathway, it is hypothesized that their action on human erythroid precursor cells is directed similarly at this enzymatic step leading thereby to increased heme production with consequent stimulation of globin synthesis. This steroid action is independent of erythropoietin, and since these compounds are effective at extremely low concentrations, it is suggested that they may play a physiologic role in the regulation of human erythropoiesis.

hem6: an ENU-induced recessive hypochromic microcytic anemia mutation in the mouse.

Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive, inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses reveal a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Intercross matings show that hem6 can suppress the porphyric phenotype of mice with erythropoietic protoporphyria (EPP). Furthermore, iron uptake studies in hem6 reticulocytes demonstrate defective incorporation of iron into heme that can be partially corrected by the addition of porphyrin precursors. Gene expression and enzymatic assays indicate that erythroid 5-aminolevulinic acid synthase (Alas2) is decreased in hem6 animals, suggesting a mechanism that could account for the anemia. Overall, these data lead to the hypothesis that hem6 encodes a protein that directly or indirectly regulates the expression of Alas2.

Porphyrin induction: equivalent effects of 5alphaH and 5betaH steroids in chick embryo liver cells.

The 5alphaH (A : B trans) and 5betaH (A : B cis) steroids are equipotent in inducing delta-aminolevulinic acid synthetase and porphyrin accumulation in chick embryo liver cells maintained in serum-free culture medium. Thus there is no specific steric requirement for porphyrin-inducing activity in steroids.

Tin: a potent inducer of heme oxygenase in kidney.

Tin greatly enhances heme breakdown in kidney, thus impairing heme-dependent cellular functions, such as cytochrome P-450 mediated drug biotransformation. This novel action of the metal results from a potent induction effect on heme oxygenase, the enzyme that catalyzes heme oxidation in microsomes. The possible toxicological implications of this tin effect in the kidney merit further investigation.

Effects of peripheral administration of GH and IGF-I on gene expression in the hippocampus of hypophysectomised rats.

OBJECTIVE: Growth hormone (GH) increases insulin-like growth factor I (IGF-I) production and both hormones affect hippocampal plasticity. We have previously shown that Hbb and Alas2 in the rat hippocampus were robustly regulated by GH-infusions for six days, whereas other transcripts were weakly affected. Here, we explored the effects of prolonged GH administration on transcripts linked to neuroprotection and investigated whether serum IGF-I administration may exert similar effects. DESIGN: Hypophysectomised female rats were infused with GH or IGF-I for 19 days. Hbb, Alas2 and seven additional GH- and IGF-I-related transcripts were quantified by Q-RT-PCR in rat hippocampus. RESULTS: Three transcripts, Hbb, Alas2, and Alox15 were increased by both GH and IGF-I administration. The other transcripts were marginally affected. CONCLUSION: The 19-day GH-infusion induced similar effects as those reported after 6-day GH treatment, with the addition of the regulation of transcript Alox15. IGF-I induced altered gene expression in relation to its effect on weight gain. This study underlines that there is an entity of transcripts involved in neuroprotection and vascular tone that is regulated by both systemic GH and IGF-I. For other transcripts, the longer duration of this study did not significantly enhance the marginal effects of GH administration seen previously.

Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin.

5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60-70%, but those of the cytosolic precursor protein were up-regulated by 2-5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on heme oxygenase-1, biliverdin IXalpha reductase and delta-aminolevulinic acid synthetase 1 in rats with wild-type or variant AH receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its monoglucuronide in moderately TCDD-resistant line B rats, but not in highly TCDD-resistant line A rats. In the mammalian heme degradation process, heme is cleaved to biliverdin by the rate-limiting enzyme heme oxygenase-1 (HO-1). Subsequently, biliverdin IXalpha reductase (BVRA) catalyzes the reduction of biliverdin to bilirubin. In heme biosynthesis, the rate-limiting enzyme is delta-aminolevulinic acid synthetase 1 (ALAS1). The effect of TCDD on HO-1, BVRA and ALAS1 was studied at the levels of mRNA (all three enzymes), protein expression (HO-1), and enzymatic activity (BVRA, liver only) in order to determine whether the accumulation of biliverdin could be due to their altered expression. In both lines A and B, 300 microg/kg TCDD transiently repressed hepatic HO-1 mRNA on day 2 but induced HO-1 protein expression at later time-points; however, the impact emerged earlier (day 14 vs. day 35) in line B rats. In spleen, TCDD repressed HO-1 mRNA and protein expression in lines A and B through days 2-35, but did not affect its mRNA levels in TCDD-sensitive L-E rats (10 days after 100 microg/kg). In all rat strains/lines, there was a strong repression of ALAS1 and a moderate induction of BVRA mRNA in liver, but mostly not in spleen. Hepatic BVRA activity was increased in lines A and B on day 14. At 5 weeks, it was still elevated in line A but reduced to 51% of control in line B. The results suggest that hepatic heme degradation is induced by TCDD in rats; however, this does not alone explain the accumulation of biliverdin in line B rats. Other factors such as the late repression of BVRA found here and possibly oxidative stress may be important contributors to biliverdin accumulation in these rats.

Liaison between heme metabolism and bioenergetics pathways-a multimodal elucidation for early diagnosis of oral cancer.

Metabolic alterations of oral epithelial cells under oxidative stress are important signatures for early diagnosis of oral cancer. Amongst different metabolic alterations, non-invasive photo-diagnostic methods have been extensively used for determining cellular heme metabolism and accumulation of protoporphyrin IX (PpIX) under administration of suitable photosensitizer. In this study, we report these metabolic alterations by direct analysis of oral exfoliated cells obtained from individuals with prolonged smoking habit without the exogenous administration of any photosensitizer. The relative expression level of relevant biomolecules of study groups were compared with clinically diagnosed and histopathologically confirmed leukoplakia (OLPK) and oral squamous cell carcinoma (OSCC) patients. The energy imbalance and variation in 'redox ratio' were examined through spectroscopic studies which showed an increasing trend (p < 0.001) in smokers to OSCC groups in comparison to nonsmoker control. Gene expression of important intermediates of the heme metabolic pathway (viz. 5-aminolevulinate synthase 1 (ALAS1), Ferrochelatase (FECH), hemeoxygenase 1 (HO-1) and ATP binding cassette subfamily G member 2 (ABCG2)) which affect production of PpIX was assessed. Relative mRNA level of ALAS1 and HO1 was upregulated whereas mRNA level of other genes (viz. FECH and ABCG2) were found to be downregulated in smokers as well as in cancer groups. Outcome of different spectroscopic studies on exfoliated cells (viz. fluorescence, atomic absorption and Fourier transform infrared spectroscopy) corroborated with the expression of biomarkers related to cellular endogenous metabolism related to heme cycle. This study indicates significant alterations in endogenous metabolic products, and cellular functional groups in oral epithelial cells among the study groups. Our study reports a strong possibility of diagnosis of early cancer signatures amongst habitual smokers by direct and non-invasive assessment of metabolic status of oral epithelial cells without exogenous administration of photosensitizers. The knowledge accrued from the study may guide clinicians in precise detection of precancer trend in the susceptible population through a noninvasive rapid screening method.

Control of heme metabolism with synthetic metalloporphyrins.

Studies with synthetic metal-porphyrin complexes in which the central iron atom of heme is replaced by other elements indicate that those heme analogues that cannot be enzymatically degraded to bile pigments possess novel biological properties that may have considerable clinical as well as experimental value. Such studies have revealed the important role that the central metal atom plays in determining the physiological and pharmacological properties of metal-porphyrin complexes; and they have demonstrated that the form in which animals and humans are exposed to trace metals, i.e., inorganic, organified, porphyrin-chelated, etc., can be of great importance in determining the biological responses that such elements elicit, especially with respect to actions on heme synthesis and degradation and cytochrome P-450 formation and function. Study of the biological properties of synthetic metalloporphyrins represents a potentially fruitful area of research and the results may have significant value for basic as well as clinical disciplines.

Haeme-regulated degradation of delta-aminolevulinate synthase 1 in rat liver mitochondria.

Protein turnover, which occurs at various rates, is critical for the homeostasis of cellular protein levels. However, the proteolysis systems that determine the turnover rate of mitochondrial proteins are largely unknown. Delta-aminolevulinic acid synthase (ALAS) 1, a rate-limiting enzyme in the haeme biosynthesis, is one of the mitochondrial proteins that have a very short lifetime. In this study, to reveal the regulatory mechanisms for ALAS1 degradation, we examined the turnover rates of ALAS1 in rat liver under several conditions. In primary rat hepatocytes, the degradation of ALAS1 was stimulated by haeme, and suppressed by inhibition of haeme biosynthesis. Furthermore, the haeme-stimulated degradation of ALAS1 was observed in the isolated mitochondria. These results suggested that, in mitochondria, there exists an ALAS1 degradation system that is regulated by cellular haeme level and plays a crucial role in the regulation of haeme biosynthesis.

Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase.

During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.