ALAS2 mRNA Research Articles

Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol

5-Aminolevulinic acid synthase-1 (ALAS1) and heme oxygenase-1 (HO-1) are the rate-controlling enzymes for heme biosynthesis and degradation, respectively. Expression of these two genes showed tissue-specific expression pattern at both mRNA and protein levels in selected non-treated rat tissues. In the livers of rats receiving oral ethanol for 10 weeks, ALAS1 mRNA levels were increased by 65%, and the precursor and mature ALAS1 protein levels were increased by 1.8- and 2.3-fold, respectively, while no changes were observed in HO-1 mRNA and protein levels, compared with pair-fed controls. These results provide novel insights into the effects of chronic ethanol consumption on hepatic heme biosynthesis and porphyrias.

WITHDRAWN: Differential regulation of human ALAS1 mRNA and protein 3 levels by heme and cobalt protoporphyrin

WITHDRAWN: Differential regulation of human ALAS1 mRNA and protein 3 levels by heme and cobalt protoporphyrin

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.

PGC-1α: Looking behind the Sweet Treat for Porphyria

Porphyrias are a group of genetic disorders caused by mutations in enzymes of the heme biosynthesis pathway. Acute attacks of porphyria, reputedly the disease that incapacitated the British sovereign King George III (see this Cell cover), are precipitated by fasting and are treated by infusing heme or glucose, although the underlying molecular mechanisms remain unclear. In this issue of Cell, reveal that a transcriptional coactivator called PGC-1alpha is a key player in both induction of porphyria by fasting and amelioration of the symptoms by glucose treatment.

Increased Pulmonary Heme Oxygenase-1 and δ-Aminolevulinate Synthase Expression in Monocrotaline-Induced Pulmonary Hypertension

Monocrotaline (MCT), a pyrrolizidine alkaloid plant toxin, is known to cause pulmonary hypertension (PH) in rats. Recent findings suggest that pulmonary inflammation may play a significant role in the pathogenesis in MCT-induced PH. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, is known to be induced by various oxidative stresses, including inflammation and free heme, and its induction is thought essential in the protection against oxidative tissue injuries. In this study, we examined expression of HO-1 as well as non-specific delta-aminolevulinate synthase (ALAS1), the rate-limiting enzyme in heme catabolism and biosynthesis, respectively, in a rat model of PH produced by subcutaneous injection of MCT (60 mg/kg). MCT treatment caused infiltration of inflammatory cells, fibrosis of the interstitium, and pulmonary arterial wall thickening with marked elevation of right ventricular (RV) pressure, which are characteristics of MCT-induced PH. Gene expression of tumor necrosis factor-alpha (TNF-alpha) as well as DNA binding activity of nuclear factor-kappaB (NF-kappaB) increased at 1 week after MCT treatment, reached a maximum at 2 weeks, and then decreased to the pretreatment level at 3 weeks. HO-1 expression was markedly increased at 1 week, and continued to increase by 3 weeks following MCT treatment, both at transcriptional and protein levels in the mononuclear cells in the lung. ALAS1 mRNA levels in the lung also significantly increased at 2 weeks after MCT treatment. These findings suggest that pulmonary HO-1 expression was presumably induced by proinflammatory cytokine(s) in MCT-treated rats, resulting in the derepression of heme-repressible ALAS1 expression, and that HO-1 induction plays a significant role as an inflammatory factor in this condition.

Repression of 5-aminolevulinate synthase gene by the potent tumor promoter, TPA, involves multiple signal transduction pathways

The potent tumor promoter, 12- O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of α isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90 RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90 RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.

An alternatively-spliced exon in the 5′-UTR of human ALAS1 mRNA inhibits translation and renders it resistant to haem-mediated decay

Haem controls its own synthesis in non-erythroid cells primarily by regulation of ALAS1 mRNA stability. Alternative splicing of human ALAS1 generates two mRNAs with different 5′-UTRs: a major one, where exon 1B is omitted, and a minor form containing exon 1B. We show that, unlike the major ALAS1 mRNA, the minor form was resistant to haem-mediated decay. Furthermore, we demonstrate that the ALAS1 5′-UTR alone did not confer haem-mediated decay upon a heterologous mRNA and the inclusion of exon 1B inhibited translation. These data suggest that translation of ALAS1 mRNA itself might be required for destabilisation in response to haem.

Suppression of Erythroid-Specific 5-Aminolevulinate Synthase Using Short-Interfering RNA Alters Iron Metabolism and Inhibits Terminal Differentiation of Human Erythroleukemia Cells.

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the first and the rate limiting enzyme for heme biosynthesis in erythroid cells. ALAS2 plays a critical role in hemoglobin synthesis and erythrocyte maturation, since targeting the ALAS2 gene results in embryonic death in mice because of severe anemia. In humans, heritable mutations of the ALAS2 gene are responsible for X-linked sideroblastic anemia (XLSA). However, the effect of suppressed expression of ALAS2 on erythroid cell differentiation has not been examined in human cells. We therefore addressed this question, by stably suppressing ALAS2 mRNA with short-interfering RNA (siRNA) in a human erythroleukemia cell line, YN-1. After cloning of cells expressing low ALAS2 (ALAS2low cells), cells were induced to undergo erythroid differentiation by treatment with transforming growth factor beta1 (TGF-β1). Gene expression profiles of induced and uninduced cells were examined, including genes involved in globin synthesis and iron metabolism. Hemoglobin production, as judged by o-dianisidine staining, was significantly lower in ALAS2low cells than in control cells both before and after erythroid differentiation. Both alpha and gamma globin mRNA levels were also reduced in ALAS2low cells, compared with control cells. Decreased heme synthesis as well as reduced globin production in ALAS2low erythroid cells are consistent with our previous findings in murine erythroleukemia cells studied by antisense technology (Meguro K, et al. Blood 86:940–948, 1995), and extends our previous conclusion on the critical role of ALAS2 in heme and globin formation to human erythroid cells. Transferrin receptor (TFR) mRNA level was decreased in ALAS2low cells, and remained low following TGF-β1 treatment, whereas its level was increased in control cells during erythroid differentiation, which reflects enhanced iron uptake by differentiated control cells. Decreased TFR mRNA level in ALAS2low cells may suggest iron accumulation, since TFR mRNA is known to be unstable when intracellular iron level is increased. Notably, mitochondrial ferritin (MtF) mRNA level was decreased in control cells after differentiation, reflecting utilization of mitochondrial iron for heme synthesis, but it did not change in ALAS2low cells following TGF-β1 treatment. As accumulation of MtF protein is known to occur in iron-overloaded erythroid cells of patients with XLSA, our finding also suggests that there may be intramitochondrial iron accumulation in ALAS2low cells even after differentiation. In contrast to MtF mRNA, the level of cytosolic ferritin heavy chain mRNA was similar both in ALAS2low cells and control cells. These findings suggest that MtF levels, rather than cytosolic ferritin levels, may be a sensitive and specific indicator for iron accumulation in mitochondria. This study shows the critical role of ALAS2 not only in heme synthesis and hemoglobin formation, but also in iron metabolism in erythroid cells during their cell differentiation. An ALAS2low erythroid cell line, such as ALAS2-suppressed YN-1, will provide a good model for the study of relationship between heme biosynthesis and iron metabolism during terminal differentiation of human erythroid cells.

Iron Overload in an African American Woman with SS Hemoglobinopathy and a Promoter Mutation in the X-Linked Erythroid-Specific 5-Aminolevulinate Synthase (ALAS2) Gene.

Iron overload in African Americans unexplained by erythrocyte transfusion has been reported in a woman with sickle cell anemia (Castro et al., Blood ( 1998) 92 (suppl):13b) and in a woman with undefined mild anemia (Hitender et al., Am J Gastroenterol ( 2000) 95:2580). We evaluated a 41 year-old African American woman with sickle cell disease referred for management of congestive heart failure and recurrent stroke. She had received four units of erythrocyte transfusion over her lifetime, and had taken oral iron supplements intermittently for many years. Hemoglobin (Hb) was 7.6 g/dL, MCV 94 fL, MCH 20.7 pg, and RDW 23.3%. Erythrocyte MCV histogram revealed a small, distinct subpopulation of microcytes, but was also shifted right due to reticulocytosis (18.2%). Electrophoresis revealed 96.9% Hb S and 3.1% Hb A2. Transferrin saturation (TfSat) was 52%; serum ferritin (SF) was 1,362–3,065 ng/mL (4 measurements) without explanation other than iron overload. Serum transaminase levels were elevated. She declined liver biopsy. A desferrioxamine (DFO) urinary iron excretion test revealed 3,249 μg urinary Fe/24 h (reference range 100–300 μg urinary Fe/24 h). Thereafter, she took subcutaneous infusions of DFO five days weekly with fair compliance; other treatment included exchange transfusions (for management of strokes), hydroxyurea, and folic acid. Supplemental iron use was discontinued. Congestive heart failure improved; serum transaminase levels returned to normal. After 5.5 years of DFO infusions, SF was 561 ng/mL. We evaluated her iron-associated genes for pertinent mutations using denaturing high-performance liquid chromatography (dHPLC) and complete sequencing. She is heterozygous for a proximal promoter region mutation of ALAS2: C to G transversion at nucleotide −206 from the transcription start site, as defined by primer extension. HFE coding region mutations, including C282Y and H63D, were not detected; HFE alleles IVS4-44 C→T and IVS5-46 C→T were present. No single-nucleotide polymorphisms were detected in TFR2 (exons 2, 3; 5-13). This ALAS2 promoter mutation was first described in a Welsh family in which the heterozygous female proband had mild sideroblastic anemia, microcytosis, elevated TfSat and SF, and 87% reduction of ALAS2 mRNA in erythroid precursors, and had taken much supplemental iron (Bekri et al., Blood ( 2003) 102:698). Peto et al. reported that iron overload due to ineffective erythropoiesis in females heterozygous for X-linked sideroblastic anemia may be severe even when anemia is mild (Lancet ( 1983) 1:375). In women heterozygous for ALAS2 mutations, we propose that erythroid precursors without an ALAS2 mutation may sustain normal or near-normal levels of circulating erythrocytes and Hb, while erythroid precursors with an ALAS2 mutation stimulate iron absorption due to ineffective erythropoiesis and cause iron overload. In the present case, anemia due to the ALAS2 promoter mutation was partially masked by concurrent SS hemoglobinopathy, and iron overload was likely exacerbated by erythrocyte transfusion and iron supplements. We conclude that an ALAS2 promoter region mutation partly accounts for iron overload in the present patient with SS hemoglobinopathy, and that this or other ALAS2 mutations could explain the occurrence of non-transfusion iron overload in other African Americans with chronic anemia.

A promoter mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causes X-linked sideroblastic anemia

AbstractX-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). XLSA was diagnosed in a 32-year-old woman with a mild phenotype and moderately late onset. Pyridoxine therapy had no effect in the proband, but in her affected son engendered a modest increase in hemoglobin concentration and a 4-fold reduction in ferritin iron. Molecular analysis identified a C to G transversion at nucleotide -206 from the transcription start site, as defined by primer extension, in the proximal promoter region of ALAS2. No other mutations were found in the promoter region, the flanking intronic sequences, the exons, or the 3′ genomic region. The same mutation was found in her affected son but not in any other of her unaffected relatives. The mutation resulted in a 94% loss of activity relative to the wild-type sequence for a luciferase reporter construct containing the proximal 293 nucleotides (nt's) of the ALAS2 promoter when transfected into human erythroid K562 cells. Confirming the mutation's deleterious effect, the ALAS2 mRNA level in the proband's erythroid precursors was reduced 87%. The mutation occurred in or near 3 different putative transcription factor binding sites of unknown erythroid importance. The dramatic decreases in reporter activity and mRNA level suggest that the region of the mutation may bind a novel and important erythroid regulatory element.

Hypoxic up-regulation of erythroid 5-aminolevulinate synthase

AbstractThe erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

Drugs Mediate the Transcriptional Activation of the 5-Aminolevulinic Acid Synthase (ALAS1) Gene via the Chicken Xenobiotic-sensing Nuclear Receptor (CXR)

Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5'-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences.

Transcriptional Regulation of 5-Aminolevulinate Synthase by Phenobarbital and cAMP-Dependent Protein Kinase

5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5′-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.

Structure and Regulated Expression of the δ-Aminolevulinate Synthase Gene from Drosophila melanogaster

The structure of the single copy gene encoding the putative housekeeping isoform of Drosophila melanogaster delta-aminolevulinate synthase (ALAS) has been determined. Southern and immunoblot analyses suggest that only the housekeeping isoform of the enzyme exists in Drosophila. We have localized a critical region for promoter activity to a sequence of 121 base pairs that contains a motif that is potentially recognized by factors of the nuclear respiratory factor-1 (NRF-1)/P3A2 family, flanked by two AP4 sites. Heme inhibits the expression of the gene by blocking the interaction of putative regulatory proteins to its 5' proximal region, a mechanism different from those proposed for other hemin-regulated promoters. Northern and in situ RNA hybridization experiments show that maternal alas mRNA is stored in the egg; its steady-state level decreases rapidly during the first hours of development and increases again after gastrulation in a period where the synthesis of several mRNAs encoding metabolic enzymes is activated. In the syncytial blastoderm, the alas mRNA is ubiquitously distributed and decreases in abundance substantially through cellular blastoderm. Late in embryonic development alas shows a specific pattern of expression, with an elevated mRNA level in oenocytes, suggesting an important role of these cells in the biosynthesis of hemoproteins in Drosophila.

Cell biology of heme.

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. Heme serves as the prosthetic group of numerous hemoproteins (eg, hemoglobin, myoglobin, cytochromes, guanylate cyclase, and nitric oxide synthase) and plays an important role in controlling protein synthesis and cell differentiation. Cellular heme levels are tightly controlled; this is achieved by a fine balance between heme biosynthesis and catabolism by the enzyme heme oxygenase. On a per-cell basis, the rate of heme synthesis in the developing erythroid cells is at least 1 order of magnitude higher than in the liver, which is in turn the second most active heme producer in the organism. Differences in iron metabolism and in genes for 5-aminolevulinic acid synthase (ALA-S, the first enzyme in heme biosynthesis) are responsible for the differences in regulation and rates of heme synthesis in erythroid and nonerythroid cells. There are 2 different genes for ALA-S, one of which is expressed ubiquitously (ALA-S1), whereas the expression of the other (ALA-S2) is specific to erythroid cells. Because the 5'-untranslated region of the erythroid-specific ALA-S2 mRNA contains the iron-responsive element, a cis-acting sequence responsible for translational induction of erythroid ALA-S2 by iron, the availability of iron controls protoporphyrin IX levels in hemoglobin-synthesizing cells. In nonerythroid cells, the rate-limiting step of heme production is catalyzed by ALA-S1, whose synthesis is feedback-inhibited by heme. On the other hand, in erythroid cells, heme does not inhibit either the activity or the synthesis of ALA-S but does inhibit cellular iron acquisition from transferrin without affecting its utilization for heme synthesis. This negative feedback is likely to explain the mechanism by which the availability of transferrin iron limits heme synthesis rate. Moreover, in erythroid cells heme seems to enhance globin gene transcription, is essential for globin translation, and supplies the prosthetic group for hemoglobin assembly. Heme may also be involved in the expression of other erythroid-specific proteins. Furthermore, heme seems to play a role in regulating either transcription, translation, processing, assembly, or stability of hemoproteins in nonerythroid cells. Heme oxygenase, which catalyzes heme degradation, seems to be an important enzymatic antioxidant system, probably by providing biliverdin, which is an antioxidant agent.

Cell Biology of Heme

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. Heme serves as the prosthetic group of numerous hemoproteins (eg, hemoglobin, myoglobin, cytochromes, guanylate cyclase, and nitric oxide synthase) and plays an important role in controlling protein synthesis and cell differentiation. Cellular heme levels are tightly controlled; this is achieved by a fine balance between heme biosynthesis and catabolism by the enzyme heme oxygenase. On a per-cell basis, the rate of heme synthesis in the developing erythroid cells is at least 1 order of magnitude higher than in the liver, which is in turn the second most active heme producer in the organism. Differences in iron metabolism and in genes for 5-aminolevulinic acid synthase (ALA-S, the first enzyme in heme biosynthesis) are responsible for the differences in regulation and rates of heme synthesis in erythroid and nonerythroid cells. There are 2 different genes for ALA-S, one of which is expressed ubiquitously (ALA-S1), whereas the expression of the other (ALA-S2) is specific to erythroid cells. Because the 5’-untranslated region of the erythroid-specific ALA-S2 mRNA contains the iron-responsive element, a cis-acting sequence responsible for translational induction of erythroid ALA-S2 by iron, the availability of iron controls protoporphyrin IX levels in hemoglobin-synthesizing cells. In nonerythroid cells, the rate-limiting step of heme production is catalyzed by ALA-S1, whose synthesis is feedback-inhibited by heme. On the other hand, in erythroid cells, heme does not inhibit either the activity or the synthesis of ALA-S but does inhibit cellular iron acquisition from transferrin without affecting its utilization for heme synthesis. This negative feedback is likely to explain the mechanism by which the availability of transferrin iron limits heme synthesis rate. Moreover, in erythroid cells heme seems to enhance globin gene transcription, is essential for globin translation, and supplies the prosthetic group for hemoglobin assembly. Heme may also be involved in the expression of other erythroid-specific proteins. Furthermore, heme seems to play a role in regulating either transcription, translation, processing, assembly, or stability of hemoproteins in nonerythroid cells. Heme oxygenase, which catalyzes heme degradation, seems to be an important enzymatic antioxidant system, probably by providing biliverdin, which is an antioxidant agent.

Regulation of erythroid 5-aminolevulinate synthase expression during erythropoiesis

Erythroid tissue is the major site of heme production in the body. The synthesis of heme and globin chains is coordinated at both the transcriptional and post-transcriptional levels to ensure that virtually no free heme or globin protein accumulates. The key rate-controlling enzyme of the heme biosynthetic pathway is 5-aminolevulinate synthase (ALAS) and an erythroid-specific isoform (ALAS2) is up-regulated during erythropoiesis. Differentiation of embryonic stem cells with a disrupted ALAS2 gene has established that expression of this gene is critical for erythropoiesis and cannot be compensated by expression of the ubiquitous isoform of the enzyme (ALAS1). Interestingly, heme appears to be important for expression of globin and other late erythroid genes and for erythroid cell differentiation although the mechanism of this effect is not clear. Transcriptional control elements that regulate the human gene for ALAS2 have been identified both in the promoter and in intronic enhancer regions. Subsequent translation of the ALAS2 mRNA is dependent on an adequate iron supply. The mechanism by which transcription of the gene for ALAS2 is increased by erythropoietin late in erythropoiesis remains an interesting issue. Erythropoietin action may result in altered levels of critical erythroid transcription factors or modulate the phosphorylation/acetylation status of these factors. Defects in the coding region of the gene for ALAS2 underlie the disease state X-linked sideroblastic anemia. In this review, we focus on the regulation and function of erythroid-specific 5-aminolevulinate synthase during erythropoiesis and its role in the X-linked sideroblastic anemia.

Alteration of mRNA levels of δ-aminolevulinic acid synthase, ferrochelatase and heme oxygenase-1 in griseofulvin induced protoporphyria mice

Human erythropoietic protoporphria (EPP) is an inherited disorder of porphyrin metabolism and its experimental murine model can be produced by treatment with griseofulvin (GF). We investigated the alteration of mRNA expression in ferrochelatase (FeC), δ-aminolevulinic acid synthase (ALAS) and heme oxygenase-1 (HO-1) in liver, skin and peripheral blood cells of GF-treated mice. In liver, ALAS mRNA was enhanced dramatically by GF administration, in accord with thesis that the expression of ALAS is regulated by feedback mechanism. The expression of HO-1 mRNA increased most rapidly and drastically in liver, however its mechanism of regulation may be different from that of ALAS mRNA. The level of FeC mRNA in liver was less affected with GF treatment. Our results indicate that the inhibition of FeC by GF administration might occur primarily at post-transcriptional level. Similar effects were observed in the ALAS and HO-1 mRNA expression in peripheral blood cells, 2-fold increase in the ALAS mRNA and increase from undetectable level to detectable level in the HO-1 mRNA. In skin of GF-treated mice, average increases of 1.3-fold in the ALAS mRNA and 1.6-fold in the HO-1 mRNA were statistically insignificant. The FeC mRNA level was not altered in peripheral blood or in skin of GF-treated mice. The present study indicates that the molecular analysis is practicable in skin and peripheral blood. In further study, this model could contribute to investigate the pathogenesis of clinical manifestation including possibly cutaneous changes in EPP.

Insulin Inhibits δ-Aminolevulinate Synthase Gene Expression in Rat Hepatocytes and Human Hepatoma Cells

Insulin has been known to regulate intracellular metabolism by modifying the activity or location of many enzymes but it is only in the past few years that the regulation of gene expression is recognized to be a major action of this hormone. The present work provides evidences that insulin inhibits δ-aminolevulinate synthase (ALA-S) gene expression, the enzyme which governs the rate-limiting step in heme biosynthesis. The addition of 5 nM insulin to hepatocytes culture led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA in a dose-dependent manner, as measured by Northern and slot-blot analysis. Several clues as to how insulin regulates ALA-S transcription were determined. The inhibitory effect is achieved at physiological concentrations but much higher proinsulin doses are needed. Insulin's effect is rapid, quite specific, and protein synthesis is not required. Moreover, ALA-S mRNA half-life is not modified by the presence of the peptidic hormone. Our results demonstrate that the insulin effect is dominant; it overrides 8-CPT-cAMP plus phenobarbital-mediated induction. Also, insulin requires the activation of protein kinase C to exert its full effect. On the other hand, a 870-bp fragment of the ALA-S promoter region is able to sustain the inhibition of CAT expression in plasmid-transfected HepG2 cells. Thus, these results indicate that insulin plays an important role in regulating ALA-S expression by inhibiting its transcription.

Evidence That Protein Kinase C Is Involved in δ-Aminolevulinate Synthase Expression in Rat Hepatocytes

There are many factors that regulate the rate of synthesis of δ-aminolevulinate synthase (ALA-S), the enzyme which governs the rate-limiting step in heme biosynthesis. In rat hepatocytes, phenobarbital increases ALA-S gene transcription and dibutyryl cAMP potentiates this induction, whereas insulin and glucose have the opposite effect. The present report provides evidence that protein kinase C (PKC) activation negatively influences ALA-S mRNA levels, as measured by Northern and slot-blot analysis. The addition of 1,2-dioctanoyl-sn-glycerol (DOG) or 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator that mimics diacylglycerol function, to cultures led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA levels in a dose-dependent manner. This TPA effect depends on the specific activation of PKC because the analog 4α-phorbol 12,13-diacetate, a nonstimulatory PKC phorbol ester, is unable to inhibit ALA-S mRNA. Furthermore, the effect of TPA is blocked by the PKC inhibitors staurosporine and calphostin C. Desensitization of the PKC pathway by prolonged exposure to TPA abolished the subsequent action of the phorbol ester. On the other hand, neither TPA nor DOG modified the half-life of ALA-S mRNA. The study of the combinatorial action of TPA and cAMP revealed that the inhibitory effect of TPA overcomes dibutyryl cAMP induction. Thus, these results indicate that PKC plays an essential role in regulating ALA-S expression, probably at a transcriptional level.