Molecular Regulation-Biological Heme in Hematopoiesis
Role of
N. G. Abraham S UMMA R Y. Heme synthesis and degradation play pivotal roles in the regulation of growth and differentiation of erythroid and non-erythroid cells. Heme synthesis in mammalian cells involves eight enzymes which are localized in mitochondrial and cytoplasmic compartments. These enzymes have been well-characterized and cDNAs for six of the enzymes has been cloned. Two enzymes in the enzymes of the heme biosynthetic pathway, Gaminolevulinic acid synthase (ALAS) and poqhobilinogen deaminase (PBGD) have special features and may have regulatory functions in heme synthesis by hematopoietic cells. ALAS exists as two isozymes which are encoded by non-erythroid and erythroid-specific genes, respectively. By contrast, PBGD, which also exists as two isozymes, arises from a single gene comprised of two overlapping transcriptional units, each with its own promoter. Transcription from one or the other of these promoters gives rise through differential splicing to two distinct mRNA species which encode the distinct nonerythroid and erythroid isoforms. On the other hand, heme catabolism is determined by the levels of the heme oxygenase system. The enzyme has been puri&d and the cDNA for heme oxygenase has been cloned. Repression of heme oxygenase in eqthroid progenitor cells may initiate differentiation. In addition, recent evidence has suggested that heme may have a broader role in hematopoiesis and in the network of cytokine production by adherent stromal cells.
The hematopoietic microenvironment consists of cells, extracellular matrices and growth factors. The cells and matrices provide attachment sites for hematopoietic cells and regulate their proliferation and maturation by secretion of stimulatory and inhibitory cytokines. An understanding of hematopoietic regulation requires a detailed study of the interaction of not only the stimulatory and inhibitory factors, but of other types of factors such as interferons, arachidonic acid (AA), prostaglandins, leukotrienes and heme as well. Substantial evidence indicates that heme interacts with growth factors to form a network which regulates hematopoiesis. The vital, ubiquitous role of heme in mammalian physiology is attested to by its function as the prosN. G. Abraham, PhD, Professor of Medicine-immunology, New York Medical College, Valhalla, NY 10595, USA. Blood Reviews (1991) 5, 019-028 Q 1991 Longman Group UK Ltd
thetic group in a variety of important hemoproteins that are essential for various cellular processes. The role of heme in the regulation of globin and nonglobin protein synthesis is amply documented.’ Heme is involved in oxygen transport as the prosthetic group of hemoglobin, in the prostaglandin synthesis as the prosthetic group of cyclooxygenase, in the enzymatic decomposition of H,02 as the prosthetic group of catalase and peroxidase, in the inactivation of oxygen molecules, as the prosthetic group of mitochondrial and microsomal cytochrome P450. The latter refers to a family of isozymes for which heme serves as the prosthetic group that oxidizes a wide variety of structurally unrelated compounds, inactivation of leukotrienes, LTB4 and metabolism of AA to bioactive metabolites, some of which are involved in the signal transduction process of hematopoietic growth factors. However, the effect of heme (iron protoporphyrin
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IX) on erythropoiesis appears to be distinct from its direct involvement as a prosthetic group.‘O The expression of specific heme metabolic enzymes determines the level of cellular heme which is necessary for proper erythropoiesis. This concept is supported by evidence that hereditary or experimental alterations in enzymatic or biosynthetic events are often accompanied by a disturbance in heme levels, and that treatment with drugs or inhibitors of heme synthesis may affect progenitor cells, resulting in altered growth and differentiation.’ It is becoming increasingly evident that the role of heme metabolic enzymes in the regulation of hematopoiesis has a dual nature, since metabolic enzymes appear to participate in the implementation of both stimulation and suppression of erythropoiesis.’ Enhancement of erythropoiesis obtained with the growth factors 11-3 and Epo also results in increased levels of 6-aminolevulinic acid synthase (ALAS) and porphobilinogen deaminase (PBG-D) the proposed rate-limiting enzymes in the heme biosynthetic pathway. Suppression of erythropoiesis may be observed by either insufficient levels of one of the heme biosynthetic enzymes, or by increased heme degradation. More recently, heme has been shown to provide protection for hematopoietic cells against cytotoxic drugs and antiviral agents.2 The present review will emphasize the genetic expression of heme biosynthesis and degradation of pathways (Fig. l), since both processes are important in regulating the supply of heme ncessary for important cell functions, transductional signal of growth factor network which regulates the hematopoietic microenvironment, as well as for the adherent stromal ability for hormonal biotransformation. Several potential regulatory phenomena associated with the enzymatic steps of the pathways in which certain erythroid-specific and non erythroid
genes (ALAS and PBG-D), abnormalities of heme metabolism in certain hematological disorders and the mechanism by which heme oxygenase is expressed. The significant role of heme oxygenase in normal erythropoiesis and in the control of hematopoietic cellular heme is just beginning to emerge. For example, repression of heme oxygenase is seen as a trigger of erythroid differentiation. The fluctuation of heme oxygenase activity in erythroid systems indicates that the enzyme’s activity plays an important role in the regulation of cellular heme levels to ensure the coordination of heme and globin biosynthesis. Abnormalities in this type of regulation may be exemplified by the aberrant erythropoiesis and myelopoiesis which occur in the sideroblastic anemias and leukemia. &aminolevulinic
Acid Synthase (ALAS)
All evidence to date is consistent with the concept that ALAS [EC 2.3.1.371 is the rate-limiting enzyme in hepatic heme synthesis and is inducible by certain chemicals.3*4 ALAS catalyzes the formation of ALA from succinyl-CoA and glycine in the presence of its essential cofactor pyridoxal-5phosphate (vitamin B& Induction of hepatic ALAS can be increased by as much as 500-fold by a variety of drugs. In addition, a number of diverse substances such as ethanol, cobalt1 and certain steroid(s) act as inducers of ALAS activity.’ The induction of the enzyme activity in various systems is further modulated by the metabolic and hormonal status of the animals.5,6 ALAS is found exclusively in the matrix surface of the inner mitochondrial membrane.’ Mitochondrial ALAS has a molecular weight of about 77 000 Da and cytosolic ALAS of about 178 000 Da. Paterniti and Beattie’ * Cyclooxygenose * Prostocyclin synthose *Thromboxone synthose * Cytochrome P450
UROGEN
III
APOPROTEINS COPROGEN
III
BILIRUBIN Fig. 1 Intracellular localization of enzymes and intermediates of the heme biosynthetic and degradative pathways (as described in
Fig. 1) and utilization of heme for various important hemoprotein(s).
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obtained a value of 120 000 Da for the mitochondrial enzyme from untreated rat liver. Hepatic and erythroid ALAS differ in molecular weight determination and immunological properties as well as catalytic activity, and this suggests that ALAS may be encoded by more than one gene.g*‘O Bothwick et al” cloned ALAS and the cDNA identity was confirmed by comparison with the amino acid sequence of the purified enzyme. The gene spans 6.9 Kb of DNA and contains 10 exons.13 The promoter region of this gene contains a TATA box and a CAAT box and four copies of the Spl factor binding site CCGCCC. Riddle et al,13 as well as others,14 provide clear evidence that there are in fact two ALAS mRNAs with sites 2.2 Kb and 1.8 Kb present in hepatic and erythroid, respectively. Unlike hepatic ALAS which is negatively controlled by heme, erythroid ALAS is under positive control by heme or heme as an iron carrier.‘*15 In mature erythroid cells, i.e. hemoglobinized cells such as reticulocytes, heme synthesis appears to be inhibited by heme,’ 5*1’ mainly by direct repression of ALAS activity. Levere and Granick 3,4 demonstrated that in chick blastoderm, heme synthesis is also regulated by the levels of ALAS and that the end product heme, acts as a negative regulator of ALAS. However, heme can stimulate expression of ALAS in early erythroid progenitors and pluripotent stem cells. 15*18,1gSimilar results were described in mouse (MEL),” and human (K562)21 erythroleukemia cells. The difference in response of ALAS to heme in hepatic and early erythroid progenitors is not surprising since hepatic cells are more differentiated and have less demand for heme.
GAminolevulinic Acid Dehydratase (ALAD) ALAD [EC 4.2.1.241 participates in porphyrin/heme biosynthesis by catalyzing the condensation and cyclization of two molecules of ALA to PBG. Possible roles for both sulfhydryl groups and zinc in the catalytic activity of the enzyme have been suggested (for review, see ref. 1). As a result of its reactive sulfhydryl group, ALAD is highly sensitive to lead. Its activity decreases rapidly after low lead exposure long before clinical lead intoxication develops. Mammalian ALAD has been purified and reported to be a multimeric/oligomeric enzyme consisting of eight 31 000 Da subunits. cDNA clones encoding rat2* and human liver23 ALAD have also been isolated by immunoscreening of cDNA expression libraries. Recently, a genetic deficiency of ALAD was reported in bone marrow cells. The enzyme deficiency which is inherited in an autosomal dominant pattern, does not lead to clinical symptoms in the heterozygous state, but provides the enzymatic and metabolic basis for an acute porphyria syndrome in homozygotes who have extremely low ALAD (near 1 to 2%) levels.
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Porphobilinogen Deaminase (PBG-D) Uroporphyrinogen III is the first tetrapyrrole found in the pathway, and its formation is mediated by the concerted action of two cytoplasmic enzymes, uroporphyrinogen-I-synthase, currently called PBG-D (porphobilinogen ammonialyase polymerizing or hydroxymethyl bilane synthase) [EC 4.3.1.81 and uroporphyrinogen III cosynthetase or hydroxymethyl bilane hydrolase cyclizing [EC 4.2.1.751. Human PBG-D has also been purified more than 42 OOOfold24 and the molecular weight of the purified enzyme is 38 000 Da. Human erythrocyte PBG-D is relatively thermostable and contains a sulfhydryl group at its active site; it is subject to inactivation by photo-oxidation, and is inhibited by several divalent cations. In acute intermittent porphyria, PBG-D activity is decreased to 50% of its normal value in erythrocytes and lymphocytes. However, PBG-D activity is increased in lymphoproliferative disorders, such as Hodgkin’s and non-Hodgkin’s lymphomas.*’ The first PBG probe isolated was an erythroid rat cDNA.~~ This probe was then used for isolating human cDNAs from both erythroid and nonerythroid sources. The single copy PBG-D gene has a unique organization and consists of two overlapping transcriptional units, each with its own promoter.” The upstream promoter is active in all cell types (N-PBGD), whereas the downstream promoter is erythroid-specific (E-PBGD).28 Transcription from one or the other of these promoters gives rise through differential splicing to two distinct mRNA species which encode distinct isoforms of the protein.2g*30 The erythroid-specific promoter of PBG-D contains no TATA box nor CAAT box, but does exhibit several binding sites for the recently cloned erythroidspecific transcription factor,31 both in human3* and mouse.33 Contrary to the S-globin promoter, the E-PBGD promoter is totally inactive in nonerythroid cells, even in the presence of an SV40 enhancer. Potential control regions have also been localized by mapping DNase I hypersensitive sites of the PBG-D locus. DNA sequences at these sites have been characterized by in vitro footprinting and gel retardation assay (Fig. 2). These studies have demonstrated the binding of several factors, some of which correspondto previously identified proteins such as Spl or GF- 1.3’ These characteristics make the erythroidspecific promoter of PBG-D a suitable model to study the effects of various cytokines on the regulation of expression of erythroid-specific genes (Fig. 2). The authors have shown in normal human bone marrow culture that Epo induces PBG-D” in a concentrationdependent manner. Furthermore, hemin synergized the Epo effect and resulted in a 2-fold increase in PBG-D and a significant increase in BFU-E growth. Others have shown that during DMSO-induced MEL differentiation, the steady-state level of the erythroidPBGD-specific message increases up to 5-fold when
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eP
CAC
CAC
00 VW
Fig. 2 Regulatory regions of the mouse PBG-D gene. Top: Localization of DNase I hypersensitive sites (HSS). Five HSS were mapped in the PBG-D locus; sites A and E found, respectively, upstream and downstream from the gene, as well as site B located shortly after the first ubiquitous exon were observed in all types of nuclei. Site C situated downstream from the first exon and site D present within the erv-throid promoter region, were found exclusively in ervthropoietic cells. NEP: nonerythroid promoter, EP: erythroid promoter (with permission of B Grandchamp).
analyzed by Northern blot hybridization using a probe specific for erythroid ‘exon 2’.34 The second enzyme involved in catalyzing the formation of uroporphyrinogen III, i.e. uroporphyrinogen III has recently been cloned.35 The predicted/ deduced amino acid sequences of the nonerythroid and erythroid forms were essentially the same. Uroporphyrinogen Decarboxylase Uroporphyrinogen decarboxylase [EC 4.1.371 is a cytoplasmic enzyme that catalyzes conversion of urogen III to coprogen III. de Verneuil has shown that in humans an autosomal-dominant mutation produces a decrease of the enzyme activity.36 Uroporphyrinogen decarboxylase was purified to homogeneity from human erythrocytes more than 20 OOO-fold. The purified enzyme has a molecular weight of 46 000 and was not inhibited by iron.36 Uroporphyrinogen decarboxylase cDNA clone from a rat erythroid library and human was obtained.37 Northern and Southern blot analysis of the human genomic DNA indicate that uroporphyrinogen decarboxylase mRNA is identical in different cell types and the uroporphyrinogen decarboxylase gene exists as a single cop~.~’ There are two starting sites for initiation of two transcription initiation sites which have been identified for uroporphyrinogen decarboxylase by primer-extension and RNase mismatch cleavage analyses. The two sites of initiation are separated by six bp, which suggests utilization of one particular promotor in erythroid cells, similar to PBG-D, gene transcription. The promotor region of the uroporphyrinogen decarboxylase gene contains a TATA box and Spl binding site. Inherited deficiencies of uroporphyrinogen decarboxylase in man cause two
types of porphyria, hepatoerythropoietic porphyria (HEP) and the more common porphyria, cutanea tarda (PCT). Both disorders are characterized by an autosomal dominant pattern of inheritance (for review, see ref. 1). Ferrochelatase Ferrochelatase, or heme synthase [EC 4.99.1.11, is the enzyme that catalyzes the last step in the heme biosynthetic pathway. Ferrochelatase catalyzes the insertion of ferric Fe++, not Fe+++, but it appears that zinc and cobalt can also be inserted into protoporphyrin IX to form the corresponding heme analogues. Camadro and Labbe have shown that a single enzyme catalyzes both reactions. No other metals have been shown to be inserted by ferrochelatase, in fact certain metals act as inhibitors of the enzyme activity. 4o Ferrochelatase from human bone marrow and rat liver share similar features. Hemin, at appropriate concentrations can inhibit ferrochelatase activity in both rat liver and marrow cells by approximately 50%. The level of ferrochelatase activity is dependent on oxygen levels due to changes in the lipid environment of the enzymes in the mitochondrial membrane.41g42 Ferrochelatase activity in human lymphocytes or bone marrow was found to be inhibited by 50% in the presence of 10 uM hemin. Epo treatment increases ferrochelatase activity in patients’ peripheral blood. This activity was prevented by aluminum. 44 Ferrochelatase has been purified 1600-fold with a 6.5% yield. Antibodies to the purified enzyme from bovine kidney cross-react with MEL ferrochelatase induced by DMS0.45 Rutherford4’j and Sassa have suggested that ferrochelatase
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C Fii. 3 Analysis of gene transcription in hepatocyte nuclei from rats treated with CoCI,. Plasmids bound to nitrocellulose were hybridized with 32P-labelled runoff transcripts from nuclei isolated at different times after administration. (a) The 32Plabelled transcripts were hybridized with filter-bound immobilized plasmids containing cDNAs for heme oxygenase (HO), nonerythroid porphobilinogen deaminase (PBG-D) and the cloning vector of Okayama and Berg (OB). A-32P-labelled transcripts obtained from hepatocyte nuclei of control rats; B-nuclei obtained from rats treated with CoCI, and sacrificed 3 h later; C-nuclei obtained from rat hepatocytes treated with CoCI, and sacrificed 3 h later, labelled with [u-~*P]UTP in the presence of 1 l.rgg/mla-amanitin. (b) Quantification of transcriptional activation as determined by densitometric scanning of autoradiograph in (a) after correction for background binding to the cloning vector of Okayama and Berg; (c) Time course study of transcription of three different genes: heme oxygenase (HO), porphobilinogen deaminase (PBG-D) and r-actin. Transcripts were obtained from hepatocyte nuclei from (A) control rats and rats treated with CoCI, and sacrificed after (B) 1 h and (C) 3 h. The autoradiogram was exposed for 1 week with intensifying screens.
B may be the rate-limiting enzyme in erythroid differentiation. Other potential regulatory enzymes have been suggested, 48*49 but ferrochelatase was not among them. cDNA clones of ferrochelatase obtained from yeast indicate a 2.9 Kb genome, DNA contain an open reading frame of 1179 nucleotides5’ The open reading frame encodes a precursor form of the protein containing a 31 amino acid presequence. The mature ferrochelatase contains 362 amino acid residues with a molecular weight of 40 900.63 Mouse ferrochelatase was also cloned and Northern blot analysis demonstrated a several-fold increase in mRNA after erythroid induced to differentiation by DMSO (Taketani, Personal Communication). Genetic deficiency of ferrochelatase has been reported in cultured skin fibroblasts or mitogen-stimulated lymphocytes from patients with congenital
erythropoietic protoporphyria (CEP) resulting in accumulation of protoporphyrin. The level of the enzyme deficiency has been controversial for a long time. Bloomers1 suggested that ferrochelatase is a multimer whose function can be greatly inactivated with the mutation of one of the subunits of the enzyme. The basic enzymatic defect underlying the bovine CEP disorder, that is, a deficiency of ferrochelatase activity, was also demonstrated in cultured skin fibroblasts from affected cattle. Human ferrochelatase deficiency is inherited as an autosomal dominant trait, whereas the cattle enzymatic defect is inherited as an autosomal recessive trait. Heme oxygenase
Microsomal heme oxygenase [EC 1.14.99.31 plays an essential role in the physiological degradation of heme
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to biliverdin IX (for review, see ref. 1,52). In mammals, the microsomal heme oxygenase system consists of three individual enzymes: heme oxygenase, NADPH cytochrome c (P450) reductase [EC 1.6.2.41 and biliverdin reductase [EC 1.3.1.241 and requires NADPH and molecular oxygen for bilirubin formation. In hematopoietic cells, heme oxygenase level may play a crucial role during stem cell proliferation and differentiation. It has been shown that in human in vitro erythroid colony development system, heme oxygenase activity was elevated in the early phases of erythroid culture, after which it progressively declined. A similar observation was made in murine erythroid cultures stimulated by Epo.’ Other inducers of differentiation such as heme caused a repression in heme oxygenase activity and concomitant appearance of benzine-positive stain in human KS62 cells. It has been suggested that a decrement in erythroid heme oxygenase activity may be an important feature of the differentiation process. This reduction is due to the presence of transcriptional factor(s) induced during the onset of differentiation, which suppressed heme oxygenase mRNA. In patients with both X-linked and idiopathic-acquired siderobtastic anemia, and in ineffective erythropoiesis, heme oxygenase activity was increased by 40-60% above the normal levels. Cell growth and differentiation in these patients may have been hindered by the absence of a specific initiation factor which may control heme oxygenase levels.” Heme oxygenase activity is highest in the spleen followed by the liver and bone marrow. Human hepatic heme oxygenase has been purified and characterized.s3 Comparison between the primary structures of human and rat heme oxygenase, which was deduced from the nucleotide sequence of the cloned cDNA, was found to be 8Ooh.s3 The regulation of heme oxygenase activity has been reviewed in detai1.s2 Shibahara et al isolated cDNA clones for heme oxygenase from a rat spleen cDNA library in TgtII. When monkey cells (COS7) were transfected with an expression vector carrying the rat splenic heme oxygenase cDNA in tandem with the SV-40 promoter, the recombinant enzyme was highly expressed in the endoplasmic reticulum of transfected cells. Blot hybridization analysis of rat liver DNA suggested that there is a single gene for heme oxygenase in the rat genome.s2*54*55 In the author’s laboratory, the effect of several environmental agents on expression of heme oxygenase mRNA was examined. It was demonstrated that treatment of macrophages or hepatocytes with heme and several heavy metals increased the steadystate level of heme oxygenase mRNA, principally by increasing its rate of transcription (Fig. 3). Muller et a155 isolated and sequenced the rat heme oxygenase gene. The gene consists of 6830 nucleotides and is organized in four introns and five exons. In the 5’-flanking region, several potential binding sites for different transcription factors were found, such as HSE and GCN4.54,55 These factors have been
reported to regulate amino acid synthesis in yeast upon starvation, expression of genes for heat-shock proteins and metabollothioneins, and therefore seem to represent a stress response. In addition, upstream stimulatory factor originally identified in HeLa cells, interacts with the upstream promoter sequence of adenovirus two major late promoter (Ad2MLP). These factors contribute to the transcription of the human heme oxygenase gene. The cis-acting element, identified on the heme oxygenase gene as CACGTGACCCG, is located 34 bp upstream from the transcription initiation site, and contains the core sequence of the upstream promoter sequence of Ad2MLP.56 In addition, oncogene jun encodes a protein of which the carboxyl terminus is homologous to the DNA-binding domain of GCN4, which corresponds to AP-1 known to be involved in the gene activation by 12-O-tetradecanoyl-phorbol- 13-acetate (TPA).” Molecular Regulation of Heme Metabolic Enzyme in Hematopoietic Cells Early studies concentrated on the mechanism by which heme biosynthesis is regulated in erythroid and nonerythroid cells. It is believed that in nonerythroid cells, heme is regulated at the levels of the first enzyme in heme synthesis, ALAS1 Levere and Granick3s4 have indicated that mature erythroid progenitor cells are also regulated at S-ALAS. This regulation is due to inhibition of the first rate-limiting enzyme, ALAS.16r17 Abraham et al have shown that in more mature erythroid cells such as reticulocytes, heme inhibits ALAS and ALAD, the first two enzymes in heme synthesis. l6 More recently, it has been shown that the role of heme in this regulation of erythropoiesis is not limited to its effect on its own synthesis, but extends to inhibition of iron uptake.58 Studies in MEL and K562 cells reveal that the role of cellular heme in expression of heme metabolic enzymes in erythropoiesis is more complicated than that in hepatic or nonerythroid cells. Heme acts as an inducer of its own synthesis at the levels of ALAS and other heme biosynthetic intermediates. The only heme analogue which mimics heme action is cobalt protoporphyrin. Addition of cobalt protoporphyrin resulted in stimulation of heme biosynthesis in MEL cells. In vivo studies have shown that administration of Epo to polycythemic mice resulted in an increase in haeme biosynthetic enzymes and Fe” uptake. These results clearly demonstrated that the early effect of Epo is the induction of heme synthetic enzymes, and that heme plays an important role in erythroid cell proliferation and differentiation. Other compelling evidence for the role of heme in erythropoiesis came from Levere et a13v4in which addition of exogenous ALA to blood islands of the chick blastoderm, which are devoid of myeloid and lymphoid cells, enhanced globin synthesis. 3*4The role of PBG-D in erythroid differentiation has been described by the elegant work
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of Beru and Goldwasser.49 They demonstrated that in suppressed rat bone marrow which undergoes differentiation by Epo, the activities of PBG-D increased by 5-fold. The increase in PBG-D was also seen in human bone marrow treated with Epo. It is clear that the regulatory step in heme synthesis during erythropoiesis has not been precisely determined.48 Furthermore, examination of heme metabolism in hematopoietic cells must take into account the level of differentiation within the individual cell of the entire hemopoietic system. Recently, Ponka et al have suggested the possibility that iron, rather than heme can regulate erythroid differentiation.58 These investigators have used the reticulocyte system and have demonstrated that heme regulates its own synthesis by inhibiting the uptake of iron from transferrin. Further, Hradilek et a15’ have shown that when the level of cellular heme is increased. the efficiency of iron released is decreased. Heme is a very efficient iron carrier. When cellular heme reaches high levels, it will result in induction of heme oxygenase with concomitant heme degradation and iron release. Iron release may coordinately regulate ferritin and transferrin levels through the iron-responsive element, IRE.60,62 IREs are regulatory sequences in the 3’-untranslated region of the transferrin receptor mRNA and the 5’-untranslated region of ferritin mRNA.62 IREs interact with a cytosolic binding protein which exists in either a low or high affinity state. Iron deprivation results in a switch from the lower to higher affinity state of the IRE-binding protein. High affinity binding of the protein to the 3’IRE increases the metabolic half-life of the transferrin receptor mRNA, thereby increasing its translation and enabling increased iron acquisition. In addition, binding of the protein to the SIRE inhibits ferritin translation and subsequent depletion of the chelatable intracellular pool of iron. Recently, depression of ferritin mRNA translation by hemin has been demonstrated in vitro.61 Sporadic reports have examined the molecular regulation of heme metabolic enzymes in nonerythroid cells. Schoenfeld et al25 have observed elevated levels of PBG-D in lymphocytes obtained from leukemic patients. Furthermore, heme and heme analogues have been shown to influence cytokine production and to act synergistically with 11-2 to augment secretion of tumor necrosis factor (TNF) by lymphocytes in culture.(j3 Biological Role of Heme in Hematopoiesis The binding of growth factors is mediated through specific cell surface receptors which initiate a cascade of biochemical events leading to proliferation and differentiation. In many instances, the action of hemopoietic growth factors has a close association with the expression of oncogenes and their products. Expression of src-related oncogenes is associated with tyrosine kinase activity and this activity is also associ-
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ated with growth factors binding to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. 64 The c-fms gene product appears to be identical to the cell membrane receptor for macrophage colony stimulating factor (CSF-1). HL60 cells induced to differentiate to macrophages with vitamin D, or TPA express enhanced c-fms mRNA levels and receptors. We have recently demonstrated that hemin upregulates the expression of Epo receptors in murine (MEL) and human (HEL) erythroleukemia cells. Treatment of cells with hemin (50 uM) for l-2 days produced a progressive increase in Epo receptor expression. DMSO also increased receptor expression, but to a lesser degree. Induction of receptor expression was prevented by incubating cells with succinylacetone, which is an inhibitor of heme synthesis. Therefore, Epo receptor expression can be modulated by the levels of heme and hence the cells’ response to Epo. More recently, Griffin et aYj9 have shown that heme’s signal for differentiation may be coupled with specific changes in expression of c-myc and c-myb. Using HEL cells, we have shown that hemin induced a transient increase in c-myc mRNA after l-3 hours, which precedes that of PBG-D and globin mRNA levels. The time pattern of changes in c-myc expression differs from the pattern of expression of heme oxygenase, i.e., c-myc transcript levels rapidly increase, then return to control levels, whereas heme oxygenase expression continues to decrease for another 2-3 hours. Results also demonstrated that heme oxygenase was initially expressed at high levels, followed by a steady decrease during a latent period to approximately 25% of control value, while c-myc was gradually increased. We hypothesize that the rapid decrease in expression of heme oxygenase after exposure to hemin may be directly dependent on the earlier transient increase of c-myc. Moreover, we found that in K562 cells induced to differentiate with hemin, there was a drop in heme oxygenase activity before either ALAS or PBG-D was expressed (Fig. 4). This may suggest that the increase in ALAS and PBG-D is directly related to this decrease, and may indirectly depend on the transient increase in c-myc. Thus, there may be a cascade phenomenon in place, where a protooncogene causes a decrease in heme oxygenase which in turn is responsible for the increase in PBG-D and ALAS. Similarly, using Epo-treated Rauscher cells, it was found that there was a strong correlation between the magnitude of changes in c-myc and c-myb expression and the degree of Epo-induced hemoglobinization.65 Involvement of protooncogene and hemin in terminal differentiation was recently described.(j’ Prochownik showed that exposure of c-myc transfected cells to hemin allowed for induction of hemoglobin where c-myb transfected cells were refractory to hemin induction6’ More recently, it has been shown that in myeloid and erythroid cells, c-myc and c-myb
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0 HO l c-MYC m ALAS 0
0
6
3 HOURS
9
PBGD
12
15
AFTER STIMULATION
Fia. 4 Schematic oresentation of the changes in oncogene and heme metabolizing enzyme gene expression in hemin-stimulated K562 cells.
protein declines with differentiation well before proliferation ceases. Several studies have demonstrated that the expression of oncogenes, especially c-myc and c-myb is induced in cells stimulated to proliferate, c-myc in the transition from GO to Gr and c-myb in the transition from the G, to the S phase of the cell cycle. The expression of c-myc and c-myb mRNA diminishes when hemopoietic cell lines are induced to differentiate terminally; a progressive monotonic decline during macrophage differentiation. Stenzel’s group has reported that hemin induces mitogenesis in human T-lymphocytes. More recently, they found that heme and heme analogues, Snprotoporphyrin, an inhibitor of heme oxygenase, synergize with 11-2 in stimulating thymidine incorporation by lymphocytes. 63 Heme and Sn-protoporphyrin stimulate TNF-a and IFN-y production by peripheral blood macrophages in the presence of T-lymphocytes. 63 I- 1, TNF and IFN have previously been shown to synergize with 11-2 in the generation of killer cells. The author has studied the direct effect of heme and inhibitors of heme synthesis on the ability of adherent stromal cells to produce stimulatory and/or inhibitory factors. Stromal cells were incubated with and without inhibitors of heme synthesis and then the conditioned media (CM) were recovered and tested on BFU-E and CFU-GM growth. Results indicated that CM from adherent stromal cells exposed to inhibitors of heme had a significant inhibitory effect on BFU-E and CFU-GM growth. Formation of these inhibitory factors was prevented by supplementation of adherent stromal cells with heme. We suggested that within the adherent cell complex (macrophages, stromal cells), heme may provide the central network for the elaboration of growth-promoting substance(s) which directly promotes hemopoietic colony formation and possibly the expression of growth factors and/or receptors. A defect in heme synthesis may be accompanied by an
increase in negative regulation and suppression of pluripotent stem cells. For example, adherent cell macrophages deficient in heme may promote the release of excess E-type prostaglandin or other factors which counterbalance stimulatory factors. Adequate heme, on the other hand, may stimulate the synthesis and/or release of growth factor(s) from macrophages such as interleukins, which in turn signal the release of CSF(s) from stromal cells. These observations may lend support to the therapeutic potential of heme therapy for hematological disorders in which defective heme biosynthesis is frequent, e.g. in myelodysplastic syndrome and in sideroblastic syndromes.68 In conclusion, studies summarized here provide evidence that the expression of heme metabolic enzymes and the generation of specific gene products have pronounced effects on hematopoietic growth and differentiation within the bone marrow microenvironment. The interaction within the microenvironment and expression of oncogenes contribute to the complex interplay of heme metabolism and differentiation. Further elucidation of the molecular regulation of nonerythroid and erythroid heme metabolic enzymes is necessary to understand more fully such diverse entities as the role of heme and cytochrome P450 in endogenous and exogenous hormones and drugs in normal and aplastic anemia, chronic refractory anemia, chemotherapeutic marrow suppression, leukemia and various idiosyncratic drug reactions.
Acknowledgements I would like to express my sincere gratitude to Dr Richard D. Levere for his comments, encouragement and support. I would also like to acknowledge and thank my colleague Dr John D. Lutton for his collaborative assistance in our study, Dr Richard B. Levene for his time and effort spent on editing, and Mrs Joyce Eshet for her secretarial assistance. I owe enormous debts of gratitude to Dr Bernard Grandchamp for providing us with PBGD cDNA, and Dr Ron Hoffman for his previous collaborative
BLOOD REVIEWS studies on sideroblastic anemia patients. This manuscript was supported in part by US Public Health Service. Grant #AM29742.
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