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Mitochondrial fatty acid oxidation (β-oxidation) is an essential metabolic process for energy production in eukaryotic cells, but the regulatory mechanisms of this pathway are largely unknown. In the present study, we found that several enzymes involved in β-oxidation are associated with CLPX, the AAA + unfoldase that is a component of the mitochondrial matrix protease ClpXP. Deletion of the CLPX gene increased β-oxidation activity in human hepatic cells but did not change the protein levels of enzymes involved in β-oxidation. Moreover, glucagon treatment resulted in decreased CLPX levels and increased β-oxidation activity in these cells, while the expression levels of enzymes involved in β-oxidation were not altered. Coimmunoprecipitation experiments revealed that glucagon treatment induced the dissociation of several β-oxidation-related enzymes from CLPX, suggesting that CLPX regulates β-oxidation by modulating the protein complex formation of enzymes catalyzing β-oxidation.

The caseinolytic mitochondrial matrix peptidase chaperone subunit (ClpX) plays an important role in the heme-dependent regulation of 5-aminolevulinate synthase (ALAS1), a key enzyme in heme biosynthesis. However, the mechanisms underlying the role of ClpX in this process remain unclear. In this in vitro study, we confirmed the direct binding between ALAS1 and ClpX in a heme-dependent manner. The substitution of C108 P109 [CP motif 3 (CP3)] with A108 A109 in ALAS1 resulted in a loss of ability to bind ClpX. Computational disorder analyses revealed that CP3 was located in a potential intrinsically disordered protein region (IDPR). Thus, we propose that conditional disorder-to-order transitions in the IDPRs of ALAS1 may represent key mechanisms underlying the heme-dependent recognition of ALAS1 by ClpX.

In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.

Heme is degraded by heme oxygenase to form iron, carbon monoxide (CO), and biliverdin. However, information about the catabolism of heme in erythroid cells is limited. In this study, we showed the production and export of bilirubin in murine erythroleukemia (MEL) cells. The production of bilirubin by MEL cells was enhanced when heme synthesis was induced. When mouse bone marrow cells were induced with erythropoietin to differentiate into erythroid cells, the synthesis of bilirubin increased. The expression of β-globin was enhanced by CO at the transcriptional level. These results indicate that constant production of CO from heme regulates erythropoiesis.

Sideroblastic anemias are anemic disorders characterized by the presence of ring sideroblasts in a patient's bone marrow. These disorders are typically divided into two types, congenital or acquired sideroblastic anemia. Recently, several genes were reported as responsible for congenital sideroblastic anemia; however, the relationship between the function of the gene products and ring sideroblasts is largely unclear. In this review article, we will focus on the iron metabolism in erythroid cells as well as in patients with congenital sideroblastic anemia.

(Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, is expressed in erythroblastic cells. (P)RR has multiple biological actions: prorenin activation, stimulation of the intracellular signaling including extracellular signal-regulated kinases, and functional complex formation with vacuolar H+-ATPase (v-ATPase). However, the functional implication of (P)RR in erythroblast cells has not been clarified. The aim of the present study was to clarify changes of (P)RR expression during erythropoiesis and a role of (P)RR in the heme synthesis. (P)RR expression was studied during rapamycin-induced erythropoiesis in a human erythroleukemia cell line, K562. Treatment with rapamycin (100 nM) for 48 hours significantly increased %number of hemoglobin-producing cells, γ-globin mRNA levels, erythroid specific 5-aminolevulinate synthase (ALAS2) mRNA levels, and heme content in K562 cells. Both (P)RR protein and mRNA levels increased about 1.4-fold during rapamycin-induced erythropoiesis. Suppression of (P)RR expression by (P)RR-specific small interference RNA increased ALAS2 mRNA levels about 1.6-fold in K562 cells, compared to control using scramble RNA, suggesting that (P)RR may down-regulate ALAS2 expression. By contrast, treatment with bafilomycin A1, an inhibitor of v-ATPase, decreased greatly % number of hemoglobin-producing cells and heme content in K562 cells, indicating that the v-ATPase function is essential for hemoglobinization and erythropoiesis. Treatment with bafilomycin A1 increased (P)RR protein and mRNA levels. In conclusion, we propose that (P)RR has dual actions on erythropoiesis: the promotion of erythropoiesis via v-ATPase function and the down-regulation of ALAS2 mRNA expression. Thus, (P)RR may contribute to the homeostatic control of erythropoiesis.

Human erytholeukemia K562 cells are induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, sodium butyrate and 1-β-d-arabinofuranosylcytosine. We have investigated the induction of erythroid differentiation of K562 cells by glutamine depletion. When K562 cells were cultured in glutamine-minus medium, the induction of hemoglobin synthesis, accompanied by those of heme-biosynthetic enzymes and erythroid transcriptional factors, was observed. This induction was dependent on the temporally marked decrease of intracellular level of glutathione, followed by the marked activation of p38MAPK and SAPK/JNK, but not ERK. Under glutamine-deficient conditions, the treatment of K562 cells with sodium butyrate resulted in the marked enhancement of the induction of heme biosynthesis. Glutamine depletion also accelerated the expressions of erythroid-related factors including α-globin and heme-biosynthetic enzymes, GATA-1 and NF-E2, in sodium butyrate-induced K562 cells. The transcriptional activity of β-globin gene promoter-reporter was markedly enhanced by these treatments, indicating that glutamine deficiency in combination with sodium butyrate treatment gives high efficiency of chemical-induced differentiation in the hematopoiesis process.

δ-Aminolevulinic acid (ALA)-induced porphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy. To clarify the mechanisms of the tumor-preferential accumulation of protoporphyrin, we examined the effect of the expression of heme-biosynthetic and -degradative enzymes on the ALA-induced accumulation of protoporphyrin as well as photodamage. The transient expression of heme-biosynthetic enzymes in HeLa cells caused variations of the ALA-induced accumulation of protoporphyrin. When ALA-treated cells were exposed to white light, the extent of photodamage of the cells was dependent on the accumulation of protoporphyrin. The decrease of the accumulation of protoporphyrin was observed in the cells treated with inducers of heme oxygenase (HO)-1. The ALA-dependent accumulation of protoporphyrin was decreased in HeLa cells by transfection with HO-1 and HO-2 cDNA. Conversely, knockdown of HO-1/-2 with siRNAs enhanced the ALA-induced protoporphyrin accumulation and photodamage. The ALA effect was decreased with HeLa cells expressing mitoferrin-2, a mitochondrial iron transporter, whereas it was enhanced by the mitoferrin-2 siRNA transfection. These results indicated that not only the production of porphyrin intermediates but also the reuse of iron from heme and mitochondrial iron utilization control the ALA-induced accumulation of protoporphyrin in cancerous cells.

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.

Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. Ring sideroblasts are erythroblasts characterized by iron accumulation in perinuclear mitochondria due to impaired iron utilization. There are two forms of sideroblastic anemia, i.e., inherited and acquired sideroblastic anemia. Inherited sideroblastic anemia is a rare and heterogeneous disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, or Fe-S cluster transport, and mitochondrial metabolism. The most common inherited sideroblastic anemia is X-linked sideroblastic anemia (XLSA) caused by mutations of the erythroid-specific δ-aminolevulinate synthase gene (ALAS2), which is the first enzyme of heme biosynthesis in erythroid cells. Sideroblastic anemia due to SLC25A38 gene mutations, which is a mitochondrial transporter, is the next most common inherited sideroblastic anemia. Other forms of inherited sideroblastic anemia are very rare, and accompanied by impaired function of organs other than hematopoietic tissue, such as the nervous system, muscle, or exocrine glands due to impaired mitochondrial metabolism. Moreover, there are still significant numbers of cases with genetically undefined inherited sideroblastic anemia. Molecular analysis of these cases will contribute not only to the development of effective treatment, but also to the understanding of mitochondrial iron metabolism.

Although heme oxygenase-1 (HO-1) is induced in keratinocytes after UV radiation, HO-1 expression during normal epidermal differentiation has not yet been reported. We showed by real-time PCR, western blotting, and ELISA that HO-1 mRNA and protein expression by cultured normal human keratinocytes was upregulated during epidermal differentiation induced by a high-calcium medium. Immunohistochemical staining and in situ hybridization showed the graduated expression of HO-1 in the upper epidermis, which was accompanied by suprabasal HO-1 mRNA expression, and the accumulation of bilirubin (BR) in the stratum corneum. We examined the activation of nuclear factor E2-related factor 2 (Nrf2), which is a pivotal transcription factor for HO-1 expression, by western blotting and by examining the mRNA expression of Nrf2 target genes, and excluded its role in HO-1 expression in epidermal differentiation. Next, we examined the regulation of HO-1 expression by inflammatory cytokines. IL-4 and IL-22 significantly reduced HO-1 mRNA and protein expression, whereas IL-1beta, IL-17A, and tumor necrosis factor-alpha (TNF-alpha) increased it. Finally, immunohistochemical studies on psoriatic lesional skin showed that HO-1 expression was downregulated in the parakeratotic epidermis, whereas it was retained in the orthokeratotic epidermis. These studies demonstrate that HO-1 is functionally expressed by keratinocytes in parallel with epidermal differentiation and that its expression is independently affected by several cytokines.

Heme oxygenase (HO) catalyzes oxidative breakdown of heme, and constitutes two isozymes, HO-1 and HO-2. Here, we explored the tissue-specific regulation of expression of HO-1 and HO-2 under hypoxemia. There was no significant change in the overall expression levels of HO-1 and HO-2 mRNAs and proteins in the lung during adaptation of C57BL/6 mice to normobaric hypoxia (10% O(2)). However, immunohistochemical analysis revealed the increased expression of HO-1 and HO-2 proteins after 28 days of normobaric hypoxia in the pulmonary venous myocardium that is the extension of the left atrial myocardium into pulmonary venous walls. Moreover, the expression of HO-2 protein was increased in the sub-endocardial myocardium of ventricles under hypoxia, while HO-1 protein level was increased in the full-thickness walls. Thus, hypoxemia induces expression of both HO-1 and HO-2 proteins in the myocardium. Using C57BL/6 mice lacking HO-2 (HO-2(-/-)), which manifest chronic hypoxemia, we also showed that the HO-1 protein level in the lung was similar between HO-2(-/-) mice and wild-type mice. Unexpectedly, HO-1 protein level was lower by 35% in the HO-2(-/-) mouse liver than the wild-type liver. These results indicate that the expression of HO-1 protein is regulated in a tissue-specific manner under hypoxemia.

Hypoxia induces expansion of erythroid precursor cells through erythropoietin production. However, it has also been suggested that hypoxia could enhance hemoglobin production in erythroid cells directly. To identify the molecules that are involved in hemoglobin production under hypoxia, we examined the expression profile of mRNAs in YN-1 human erythroleukemia cells under hypoxia. DNA array analysis revealed that the expression of transforming growth factor (TGF)-β1 and mitoferrin, which is a mitochondrial iron transporter, was induced after 6 h under hypoxia in YN-1 cells, whereas the increased expression of erythroid-specific 5-aminolevulinate synthase (ALAS2) and γ-globin mRNAs was observed after 48 h. Further analysis revealed that hypoxia enhanced the accumulation of TGF-β1 in the culture medium of cells of the YN-1-0-A line, which was a clonal variant of YN-1 and could be maintained in serum-free medium. Moreover, exogenous TGF-β1 induced hemoglobinization and the expression of ALAS2 mRNA in YN-1-0-A cells, but not of γ-globin and mitoferrin mRNAs. Importantly, a specific inhibitor of intracellular TGF-β signaling markedly reduced the degree of the hypoxia-mediated increase in the expression of ALAS2 mRNA in YN-1-0-A cells. On the other hand, nonhypoxic inducer of hypoxia-inducible factor 1 increased the expression of mitoferrin mRNA but not of TGF-β1 mRNA in YN-1 cells under normoxia, suggesting that mitoferrin mRNA expression may be regulated by hypoxia-inducible factor 1. Thus, our data suggest that hypoxia induces the expression of TGF-β1 and mitoferrin mRNAs through separate mechanisms in erythroid cells. TGF-β1 subsequently induces ALAS2 expression, which may contribute to terminal differentiation of erythroid cells.

Heme is synthesized in all cell types in aerobic organisms. Hydroxymethylbilane synthase (HMBS) and uroporphyrinogen III synthase (UROS) catalyze two consecutive reactions in the heme biosynthetic pathway, generating the first linear and the first cyclic tetrapyrroles, respectively. Each of the HMBS and UROS genes contains the two separate promoters that generate ubiquitous and erythroid-specific mRNAs. Despite the functional significance of HMBS and UROS, regulation of their gene expression remains to be investigated. Here, we showed that hypoxia (1% O2) decreased the expression of ubiquitous mRNAs for HMBS and UROS by three- and twofold, respectively, in human hepatic cells (HepG2 and Hep3B), whereas the expression of ubiquitous and erythroid HMBS and UROS mRNAs remained unchanged in erythroid cells (YN-1 and K562). Unexpectedly, hypoxia did not decrease the half-life of HMBS mRNA (8.4 h under normoxia versus 9.1 h under hypoxia) or UROS mRNA (9.0 versus 10.4 h) in hepatic cells. It is therefore unlikely that a change in mRNA stability is responsible for the hypoxia-mediated decrease in the expression levels of these mRNAs. Furthermore, expression levels of HMBS and UROS mRNAs were decreased under normoxia by treatment with deferoxamine or cobalt chloride in hepatic cells, while hypoxia-inducible factor 1α was accumulated. Thus, the decrease in the expression of ubiquitous HMBS and UROS mRNAs is associated with accumulation of hypoxia-inducible factor 1α protein. In conclusion, the expression of HMBS and UROS mRNAs may be coordinately regulated, which represents a newly identified mechanism that is important for heme homeostasis.

Heme plays an important biomodulating role in various cell functions. In this study, we examined the effects of hemin on cellular sensitivity to imatinib and other anti-leukemia reagents. Hemin treatment of human BCR/ABL-positive KCL22 leukemia cells increased IC(50) values of imatinib, that is, the drug resistance, in a dose-dependent manner without any change in the BCR/ABL kinase activity. Imatinib-induced apoptosis was also suppressed by hemin treatment in KCL22 cells. Hemin treatment increased the activity of gamma-glutamylcysteine synthetase (gamma-GCS) light subunit gene promoter, which contains a Maf recognition element (MARE). Protein levels of gamma-GCS and heme oxygenase-1 (HO-1), two MARE-containing genes, were also increased after hemin treatment. Knockdown of Nrf2 expression by RNA interference largely abolished the effect of hemin on imatinib-treated cells, suggesting that Nrf2 recognition of MARE is essential for the hemin-mediated protective effect. Similar to hemin, treatment of cells with delta-aminolevulinic acid (delta-ALA), the obligatory heme precursor, also increased IC(50) values of imatinib. In contrast, inhibition of cellular heme synthesis by succinylacetone increased the sensitivity of cells to imatinib in two imatinib-resistant cell lines, KCL22/SR and KU812/SR. Hemin treatment also decreased the sensitivity of cells to four anthracyclins, daunorubicin, idarubicin, doxorubicin, and mitoxantrone, in BCR/ABL-negative leukemia U937 and THP-1 cells, as well as in KCL22 cells. These findings thus indicate that cellular heme level plays an important role in determining the sensitivity of cells to imatinib and certain other anti-leukemia drugs and that the effect of heme may be mediated via its ability to upregulate Nrf2 activity.

Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h). Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.

Contains fulltext : 49456.pdf (Publisher’s version ) (Closed access) Mutations of the erythroid-specific 5-aminolaevulinate synthase (ALAS2) gene are known to be responsible for X-linked sideroblastic anaemia (XLSA). An amino acid (AA) substitution for arginine at the 452 AA position of the ALAS2 protein is the most frequent mutation, which has been found in approximately one-quarter of patients with XLSA. Despite its high frequency, there has been no report on the enzymatic activity of Arg452 mutant proteins. In this study, we examined enzymatic activity in vitro of two Arg452 mutants, Arg452Cys and Arg452His, which were found in two new pedigrees of XLSA. While these mutations must be responsible for the clinical phenotype of XLSA in patients, the enzymatic activity and stability of these mutant proteins studied in vitro are indistinguishable from those of the wild type protein. These findings suggest that the Arg452 mutation of the ALAS2 gene by itself does not decrease the enzymatic activity or the stability in vitro, and that there may be an additional factor(s) in the bone marrow, which ensures the full ALAS2 activity in vivo.

Heme oxygenase (HO) catalyzes physiological heme degradation and consists of two structurally related isozymes, HO-1 and HO-2. Here we show that HO-2-deficient (HO-2−/−) mice exhibit hypoxemia and hypertrophy of the pulmonary venous myocardium associated with increased expression of HO-1. The hypertrophied venous myocardium may reflect adaptation to persistent hypoxemia. HO-2−/− mice also show attenuated ventilatory responses to hypoxia (10% O2) with normal responses to hypercapnia (10% CO2), suggesting the impaired oxygen sensing. Importantly, HO-2−/− mice exhibit normal breathing patterns with normal arterial CO2 tension and retain the intact alveolar architecture, thereby excluding hypoventilation and shunting as causes of hypoxemia. Instead, ventilation–perfusion mismatch is a likely cause of hypoxemia, which may be due to partial impairment of the lung chemoreception probably at pulmonary artery smooth muscle cells. We therefore propose that HO-2 is involved in oxygen sensing and responsible for the ventilation–perfusion matching that optimizes oxygenation of pulmonary blood.

The retinal pigment epithelium (RPE) is essential for maintenance of photoreceptors and normally functions under conditions enriched with reactive oxygen species. RPE therefore expresses various defense enzymes against oxidative stress, including heme oxygenase-1 (HO-1). HO-1 catalyzes heme breakdown to release iron, carbon monoxide, and biliverdin, which is reduced to bilirubin, a potent radical scavenger. HO-1 expression is induced by various environmental factors, which has been established as a defense mechanism. To explore the hypothesis that the expression level of HO-1 is reduced in those RPE cells under certain conditions, we analyzed the effects of interferon-gamma and hypoxia, each of which represses the expression of HO-1 mRNA in other types of human cells. Expression levels of HO-1 mRNA were reduced by interferon-gamma in two human RPE cell lines, D407 and ARPE-19, which was consistently associated with the induction of mRNA for Bach1, a transcriptional repressor for the HO-1 gene. On the other hand, HO-1 and Bach1 mRNAs were induced by hypoxia in D407 cells but remained unchanged in ARPE-19 cells, suggesting that Bach1 is not a sole regulator for HO-1 expression. The hypoxia-mediated induction of HO-1 mRNA in D407 cells depends on gene transcription and protein synthesis, as judged by the effects of their inhibitors. The half-life of HO-1 mRNA did not change during hypoxia. Thus, hypoxia may increase transcription of the HO-1 gene through a certain protein factor in RPE cells. These results indicate that RPE cells maintain retinal homeostasis by repressing or inducing the expression of HO-1, depending on the microenvironment.

DESIGN: It has recently been shown that deficiency of adrenomedullin (AM), a potent vasodilator peptide, leads to insulin resistance. We studied expression of AM in NIH 3T3-L1 adipocytes and compared it with expression of resistin, an adipocyte-derived peptide hormone that is proposed to cause insulin resistance. Moreover, we studied the effects of tumor necrosis factor-alpha (TNF-alpha), a known mediator of insulin resistance, on the expression of AM and resistin in 3T3-L1 adipocytes. METHODS: 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. Expression of AM mRNA and resistin mRNA was examined by Northern blot analysis. Immunoreactive AM in the medium was measured by RIA. RESULTS: AM mRNA was expressed in preadipocytes, but barely detectable in adipocytes. Immunoreactive AM was detected in the medium of both preadipocytes and adipocytes, with about 2.5 times higher levels found in preadipocytes. In contrast, resistin mRNA was expressed in adipocytes, whereas it was not detected in preadipocytes. Treatment with TNF-alpha increased AM expression in both adipocytes and preadipocytes, whereas it decreased resistin mRNA levels in adipocytes. CONCLUSIONS: The present study has shown that AM expression was down-regulated and resistin expression was up-regulated during adipocyte differentiation of 3T3-L1 cells. TNF-alpha acted as a potent negative regulator of resistin expression and a potent positive regulator of AM expression in adipocytes, raising the possibility that in addition to its known actions in causing insulin resistance, TNF-alpha may also have actions against insulin resistance through AM and resistin.

Heme oxygenase 1 (HO-1) catalyzes heme breakdown, eventually releasing iron, carbon monoxide, and bilirubin IXalpha. HO-1 is induced by its substrate heme and various environmental factors, which represents a protective response against oxidative stresses. Here we show that hypoxia represses HO-1 expression in three human cell types but induces it in rat, bovine, and monkey cells, indicating the inter-species difference in the hypoxic regulation of HO-1 expression. The hypoxia-mediated repression of HO-1 expression is consistently associated with the induction of Bach1, a heme-regulated transcriptional repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a heterodimer with a small Maf protein. Expression of HO-1 was also reduced in human cells when exposed to interferon-gamma or an iron chelator desferrioxamine, each of which induced Bach1 expression. In contrast, induction of HO-1 expression by CoCl(2) is associated with reduced expression of Bach1 mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have identified a Maf recognition element in the human HO-1 gene that is required for repression of a reporter gene by hypoxia and targeted by Bach1. Therefore, Bach1 functions as a hypoxia-inducible repressor for the HO-1 gene, thereby contributing to fine-tuning of oxygen homeostasis in human cells.

Alas2 encodes the erythroid-specific δ-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with theAlas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking theAlas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype ofAlas2-null definitive erythroblasts was examined.Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes inAlas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate thatAlas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron,Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.

The properties of 9 delta -aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and deITC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions In the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified In ADP and indicates the highly heterogeneous nature of mutations in this disorder. (Blood. 2001;97:2972-2978) (C) 2001 by The American Society of Hematology.

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with δ-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific δ-aminolevulinic acid synthase, as well as of α-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)β1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGFβ1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGFβ1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.

Coproporphyrinogen oxidase [CPO] gene promoter regulatory element (CPRE) plays an important role in CPO gene regulation. To isolate a CPRE binding protein, we performed Southwestern screening of K562 cDNA expression library using CPRE as a probe and isolated a cDNA clone which encoded a novel protein, Klp1 (K562 cell-derived leucine-zipper-like protein 1). Klp1 mRNA was highly expressed in K562 cells, HeLa cells, and brain as a single transcript (1.4 kb). Gel mobility shift assays revealed that Klp1 specifically binds to CPRE. Computational analysis revealed that Klp1 has a leucine-zipper-like structure, a Leu-X-X-Leu-Leu motif, and a putative nuclear localization signal in the basic amino acid rich region. Transfection of the Klp1 expression vector into THP-1 cells resulted in transcriptional activation of a reporter construct containing CPRE. These results indicate that Klp1 is a DNA sequence-specific transcription factor that regulates gene expression of genes that contain CPRE in their regulatory region.

Transcription levels of the non-specific δ-aminolevulinate synthase (ALAS-N) and heme oxygenase-1 (HO-1) in the placenta at the terminal stage of pregnancy were comparable to those in the female adult liver and in the spleen, respectively. Immunohistochemical studies demonstrated that both enzymes were exclusively expressed in the trophoblast. During gestation, transcript of ALAS-N slightly increased, while HO-1 mRNA significantly decreased. Induced acute fetal hypoxia resulted in an increase in ALAS-N mRNA and in a decrease in HO-1 mRNA. These findings indicate that placental heme metabolism is influenced by the oxygen supply.

The erythroid transcription factor NF-E2 is an obligate heterodimer composed of two different subunits (p45 and p18), each containing a basic region-leucine zipper DNA binding domain, and it plays a critical role in erythroid differentiation as an enhancer-binding protein for expression of the beta-globin gene. We show here that dimethyl sulfoxide treatment of wild-type murine erythroleukemia cells, but not a mutant clone of dimethyl sulfoxide-resistant cells, increases NF-E2 activity significantly, which involves both up-regulation of DNA binding and transactivation activities. Both activities were reduced markedly by treatment of cells with 2-aminopurine but not by genistein. Activation of the Ras-Raf-MAP kinase signaling cascade increased NF-E2 activity significantly, but this was suppressed when MafK was overexpressed. Domain analysis revealed an activation domain in the NH2-terminal region of p45 and a suppression domain in the basic region-leucine zipper of MafK. These findings indicate that induction of NF-E2 activity is essential for erythroid differentiation of murine erythroleukemia cells, and serine/threonine phosphorylation may be involved in this process. In addition, they also suggest that a MafK homodimer can suppress transcription, not only by competition for the DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active transcription repressor.

Polyclonal B lymphocytosis was found in four patients having clinical and hematologic features resembling those of hairy cell leukemia (HCL). All four patients were women between 37 and 67 years of age. Three patients had splenomegaly. Lymphadenopthy was absent or slight. Persistent lymphocytosis was seen in all the patients, and anemia and/or thrombopenia was observed in three of the patients. Abnormal lymphocytes have long microvilli and prominent membranous ruffles on their surfaces. Bone marrow aspirates and biopsy specimens showed increased numbers of abnormal lymphocytes with round nuclei and abundant pale cytoplasm. Although these findings were similar to those of HCL, studies of Ig gene rearrangements and expression showed the polyclonal proliferation of B cells. We called this new disease hairy B-cell lymphoproliferative disorder (HBLD). All four patients exhibited a polyclonal increase in serum IgG. The morphology of the cells in HBLD was more similar to that of leukemia cells of a variant form of HCL (HCL-Japanese variant) than to typical HCL cells. The surface IgG+, CD5−, CD11c+, CD22+, CD24−, CD25− phenotype and the weak tartrate-resistant acid phosphatase activity in the cells were identical to those of HCL cells of the Japanese variant. Our findings suggest that the B cells in HBLD are the nonmalignant counterpart of leukemic B cells in HCL-Japanese variant.

Heme is an essential requirement for cell survival. Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and consists of two isozymes, HO-1 and HO-2. To identify the protein that regulates the expression or function of HO-1 or HO-2, we searched for proteins that interact with both isozymes, using protein microarrays. We thus identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) that synthesizes or degrades fructose-2,6-bisphosphate, a key activator of glycolysis, depending on cellular microenvironments. Importantly, HO-2 and PFKFB4 are predominantly expressed in haploid spermatids. Here, we show a drastic reduction in expression levels of PFKFB4 mRNA and protein and HO-2 mRNA in HepG2 human hepatoma cells in responses to glucose deprivation (≤ 2.5 mM), which occurred concurrently with remarkable induction of HO-1 mRNA and protein. Knockdown of HO-2 expression in HepG2 cells, using small interfering RNA, caused PFKFB4 mRNA levels to decrease with a concurrent increase in HO-1 expression. Thus, in HepG2 cells, HO-1 expression was increased, when expression levels of HO-2 and PFKFB4 mRNAs were decreased. Conversely, overexpression of HO-2 in HepG2 cells caused the level of co-expressed PFKFB4 protein to increase. These results suggest a potential regulatory role for HO-2 in ensuring PFKFB4 expression. Moreover, in D407 human retinal pigment epithelial cells, glucose deprivation decreased the expression levels of PFKFB4, HO-1, and HO-2 mRNAs. Thus, glucose deprivation consistently down-regulated the expression of PFKFB4 and HO-2 mRNAs in both HepG2 cells and RPE cells. We therefore postulate that PFKFB4 and HO-2 are expressed in a coordinated manner to maintain glucose homeostasis.

The renin–angiotensin system is known to enhance erythropoiesis. (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, has recently been identified. However, expression of (P)RR in erythroid cells has not been studied. The aim of the present study is to clarify expression of (P)RR in erythroid cells, and the effects of erythropoietin, angiotensin II, transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ) and interleukin-1β (IL-1β) on its expression. Western blot analysis showed that (P)RR protein was expressed in human cultured erythroid cell lines, YN-1 and YN-1-0-A (a clonal variant cell line of YN-1). Erythropoietin (1 IU/ml) increased (P)RR mRNA expression levels in YN-1-0-A cells (1.7-fold increase compared with control), but angiotensin II did not. Treatment of YN-1-0-A cells with IFN-γ (10 ng/ml) for 48 h increased the expression levels of (P)RR protein significantly (1.4-fold increase compared with control), whereas it had no significant effects on expression levels of (P)RR mRNA. Treatment of YN-1-0-A cells with TGF-β1 or IL-1β for 24 or 48 h had no significant effects on expression levels of (P)RR. The present study has shown for the first time expression of (P)RR in erythroid cells, raising the possibility that (P)RR may have a role in erythropoiesis and the pathophysiology of certain types of anemia.

Two distinct patterns of regulating heme biosynthesis have been observed in animals: while heme negatively regulates the synthesis of δ-aminolevulinate (ALA) synthase in non-erythroid cells, the expression of the enzyme is regulated developmentally in red blood cells. This observation eventually led to the cloning of both a tissue-specific ALA synthase isozyme (ALAS-E) that is expressed in erythroid-lineage cells and is distinct from the housekeeping isozyme (ALAS-N). We originally isolated cDNA clones encoding chicken ALAS-E by the combined use of an anti-chicken ALAS-N antibody, which was partially cross-reactive to chicken ALAS-E, and a λgt11 expression library. ALAS-E was also purified to homogeneity from rat reticulocyte lysate using a papain digestion method. The papain-resistant core catalytic domain overlaps with the evolutionarily conserved segment that had been described by sequence alignment of ALA synthases from a variety of species, suggesting that the papain-resistant domain represents the ancestral core of the enzyme. Blot hybridization analysis of RNA isolated from various developmental stage rat livers and from chicken and mouse erythroleukemia cells demonstrated that ALAS-E is the key enzyme which supplies large quantities of heme for hemoglobin synthesis. We are currently investigating the mechanisms which confer erythroid-specific transcriptional activation of the ALAS-E gene through transient transfection assays in erythroid cells using human and chicken ALAS-E genes. These experiments have identified promoter elements which are required for high level, erythroid-specific transcription of the genes.

Hereditary coproporphyria (HCP) is an acute hepatic porphyria with autosomal dominant inheritance, but with a variable degree of clinical expression. Molecular cloning, sequencing and expression of the defective gene for coproporphyrinogen oxidase (CPO) in a patient with HCP were carried out. Enzyme assays revealed that CPO activity in EBV-transformed lymphoblastoid cells from the proband and one of her sisters was approximately 50% of normal. Nucleotide sequence analysis of CPO cDNAs isolated from the proband's cells demonstrated three base substitutions, and three accompanying amino acid substitutions. An A514-->C transition causing a Asn172-->His substitution occurred in one allele, while two other transitions, G265-->A and G580-->A, caused Gly89-->Ser and Val194-->Ile substitutions, respectively, in the other allele. The A514-->C and the G580-->A transitions are known genetic polymorphisms. Transfection of CPO cDNA into Escherichia coli demonstrated that cDNA with the G265-->A transition produced a protein with less than 5% of normal enzyme activity. These findings indicate that the G265-->A transition, involving the highly conserved glycine residue at the 89th position, is responsible for the CPO defect in the patient and accounts for the partial deficiency of CPO activity in this pedigree.

δ-Aminolevulinic acid (ALA)-induced porphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy. To clarify the mechanisms of the tumor-preferential accumulation of protoporphyrin, we examined the effect of the expression of heme-biosynthetic and -degradative enzymes on the ALA-induced accumulation of protoporphyrin as well as photodamage. The transient expression of heme-biosynthetic enzymes in HeLa cells caused variations of the ALA-induced accumulation of protoporphyrin. When ALA-treated cells were exposed to white light, the extent of photodamage of the cells was dependent on the accumulation of protoporphyrin. The decrease of the accumulation of protoporphyrin was observed in the cells treated with inducers of heme oxygenase (HO)-1. The ALA-dependent accumulation of protoporphyrin was decreased in HeLa cells by transfection with HO-1 and HO-2 cDNA. Conversely, knockdown of HO-1/-2 with siRNAs enhanced the ALA-induced protoporphyrin accumulation and photodamage. The ALA effect was decreased with HeLa cells expressing mitoferrin-2, a mitochondrial iron transporter, whereas it was enhanced by the mitoferrin-2 siRNA transfection. These results indicated that not only the production of porphyrin intermediates but also the reuse of iron from heme and mitochondrial iron utilization control the ALA-induced accumulation of protoporphyrin in cancerous cells.

We established two cell lines, YN-1 and Y-1K, from the peripheral blood of two chronic myelogenous leukaemia patients in blastic crisis. Characterization of the YN-1 and Y-1K cells revealed that these cells expressed erythroid lineage markers. However, there was a marked difference in the level of gamma-globin mRNA and haemoglobin in YN-1 and Y-1K cells. YN-1 contained approximately 1-5% benzidine-positive staining cells, whereas no benzidine-positive cells were observed in Y-1K cells. Haemoglobin production in YN-1 cells was markedly increased with various chemical inducers of erythroid differentiation, but was not in Y-1K cells. In contrast, Y-1K cells expressed CD34 stem cell antigen and CD41 megakaryocyte-specific antigen. These observations suggested that, although both cell lines were committed to the erythroid lineage, each cell line represented a distinct differentiation stage in the erythroid differentiation programme. Y-1K seemed to correspond to an early stage of cells in erythroid lineage, whereas YN-1 represented a more advanced stage in human erythropoiesis.

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia. Human ALAS2 protein consists of 587 amino acids and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in the pathogenesis of X-linked sideroblastic anemia. In vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with X-linked sideroblastic anemia. Val562Ala showed the higher catalytic activity in vitro, but a shorter half-life in vivo compared to those of wild-type ALAS2 (WT). In contrast, the in vitro activity of Met567Ile mutant was about 25% of WT, while its half-life was longer than that of WT. However, in vivo catalytic activity of each mutant was lower than that of WT. In addition, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in vitro and in vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.

Hypoxemia is a common manifestation of various disorders and generates pressure overload to the heart. Here we analyzed the expression of lipocalin-type prostaglandin D synthase (L-PGDS) in the heart of C57BL/6 mice kept under normobaric hypoxia (10% O2) that generates hemodynamic stress. Northern and Western blot analyses revealed that the expression levels of L-PGDS mRNA and protein were significantly increased (> twofold) after 14 days of hypoxia, compared to the mice kept under normoxia. Immunohistochemical analysis indicated that L-PGDS was increased in the myocardium of auricles and ventricles and the pulmonary venous myocardium at 28 days of hypoxia. Moreover, using C57BL/6 mice lacking heme oxygenase-2 (HO-2(-/-)), a model of chronic hypoxemia, we showed that the expression level of L-PGDS protein was twofold higher in the heart than that of wild-type mouse. L-PGDS expression is induced in the myocardium under hypoxemia, which may reflect the adaptation to the hemodynamic stress.

Hypoxia induces expansion of erythroid precursor cells through erythropoietin production. However, it has also been suggested that hypoxia could enhance hemoglobin production in erythroid cells directly. To identify the molecules that are involved in hemoglobin production under hypoxia, we examined the expression profile of mRNAs in YN-1 human erythroleukemia cells under hypoxia. DNA array analysis revealed that the expression of transforming growth factor (TGF)-beta1 and mitoferrin, which is a mitochondrial iron transporter, was induced after 6 h under hypoxia in YN-1 cells, whereas the increased expression of erythroid-specific 5-aminolevulinate synthase (ALAS2) and gamma-globin mRNAs was observed after 48 h. Further analysis revealed that hypoxia enhanced the accumulation of TGF-beta1 in the culture medium of cells of the YN-1-0-A line, which was a clonal variant of YN-1 and could be maintained in serum-free medium. Moreover, exogenous TGF-beta1 induced hemoglobinization and the expression of ALAS2 mRNA in YN-1-0-A cells, but not of gamma-globin and mitoferrin mRNAs. Importantly, a specific inhibitor of intracellular TGF-beta signaling markedly reduced the degree of the hypoxia-mediated increase in the expression of ALAS2 mRNA in YN-1-0-A cells. On the other hand, nonhypoxic inducer of hypoxia-inducible factor 1 increased the expression of mitoferrin mRNA but not of TGF-beta1 mRNA in YN-1 cells under normoxia, suggesting that mitoferrin mRNA expression may be regulated by hypoxia-inducible factor 1. Thus, our data suggest that hypoxia induces the expression of TGF-beta1 and mitoferrin mRNAs through separate mechanisms in erythroid cells. TGF-beta1 subsequently induces ALAS2 expression, which may contribute to terminal differentiation of erythroid cells.

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.

Heme is a prosthetic group of various types of proteins, such as hemoglobin, myoglobin, cytochrome c, cytochrome p450, catalase and peroxidase. In addition, heme is involved in a variety of biological events by modulating the function or the state of hemoproteins. For example, protein synthesis is inhibited in erythroid cells under heme deficiency, as the consequence of the activation of heme-regulated inhibitor (HRI). Iron concentration in the cell is sensed and regulated by the heme-mediated oxidization and subsequent degradation of iron regulatory protein 2 (IRP2). Heme also binds to certain types of potassium channels, thereby inhibiting transmembrane K(+) currents. Importantly, heme determines its own fate; namely, heme regulates its synthesis and degradation through the feedback mechanisms, by which intracellular heme level is precisely maintained. Heme reduces heme synthesis by suppressing the expression of non-specific 5-aminolevulinate synthase (ALAS1) and stimulates heme breakdown by inducing heme oxygenase (HO)-1 expression. ALAS1 and HO-1 are the rate limiting enzymes in heme biosynthesis and catabolism, respectively. Accordingly, under the heme-rich condition, heme binds to cysteine-proline (CP) motifs of ALAS1 and those of transcriptional repressor Bach1, thereby leading to repression of mitochondrial transport of ALAS1 and induction of HO-1 transcription, respectively. Moreover, chemosensing functions of HO-2 containing CP motifs, another isozyme of HO, have been unveiled recently. In this review article, we summarize and update the pleiotropic effects of heme on various biological events and the regulatory network of heme biosynthesis and catabolism.

To identify erythroid-specific heme-regulated genes, we performed differential expression analysis between wild-type and heme-deficient erythroblasts, which had been prepared from wild-type and erythroid-specific delta-aminolevulinate synthase-null mouse ES cells, respectively. Among 8737 clones on cDNA array, 40 cDNA clones, including 34 unknown ESTs, were first selected by their high expression profiles in wild-type erythroblasts, and evaluated further for their erythroid-lineage specificity, expression in hematopoietic tissues in vivo, and heme-dependent expression, which yielded 11, 4, and 4 genes, respectively. Because of the selection strategy employed, the final 4 were considered as the newly identified erythroid-specific heme-regulated genes. These 4 genes were uncoupling protein 2, nucleolar spindle-associated protein, cellular nucleic acid-binding protein, and a novel acetyltransferase-like protein. These findings thus suggest that heme may regulate a wide variety of hitherto unrecognized genes, and further analysis of these genes may clarify their role in erythroid cell differentiation.

Adrenomedullin (AM), a potent vasodilator peptide, has been suggested to act against cardiovascular complications and insulin resistance in the metabolic syndrome. We have already reported the AM gene repression in the early phase of adipocyte differentiation of NIH 3T3-L1 cells. Here we show adipocyte differentiation-related regulatory element for AM gene repression (ADRE-AR) in 36-bp region (-2135/-2100) of the AM gene. 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. On the third day of differentiation, the promoter function was analyzed using the reporter plasmids, which contain the promoter region of AM gene (-4616/+108) in pGL3-basic luciferase reporter vector. The promoter activity decreased to about 20% in 3T3-L1 adipocytes when compared with 3T3-L1 preadipocytes, and a 36-bp region (-2135 to -2100) upstream from the transcription initiation site of the AM gene was necessary for higher AM gene expression in preadipocytes. This 36-bp ADRE-AR contains three copies of G/AAAA sequence (5'-GAAATGAAAGTAAAA-3') (-2124/-2110), which are conserved between mouse and human, and the introduction of mutations in each copy of G/AAAA sequence decreased the promoter activity in preadipocytes and adipocytes. Electrophoretic mobility shift assay showed that the full-length ADRE-AR was specifically bound by a certain nuclear protein(s). The present study has raised the possibility that ADRE-AR may play important roles in the AM gene expression in preadipocytes, and that the AM gene may be repressed through the ADRE-AR in adipocytes.

Heme oxygenase cleaves heme to form biliverdin, carbon monoxide (CO), and iron, and consists of two structurally related isozymes, HO-1 and HO-2. HO-2 is also known as a potential oxygen sensor. Here we show that the relative CO content in arterial blood, which reflects the total amount of endogenous heme degradation, dynamically changes in mice during acclimatization to normobaric hypoxia (10% O 2 ), with the two peaks at 1 day and 21 days of hypoxia. The expression levels of HO-1 and HO-2 proteins were decreased by 20% and 40%, respectively, in the mouse liver at 7 days of hypoxia, which returned to the basal levels at 14 days. On the other hand, HO-1 and HO-2 proteins were increased 2-fold and 1.3-fold, respectively, in the heart at 28 days of hypoxia. Thus, hypoxia induces or represses the expression of HO-1 and HO-2 in vivo, depending on cellular microenvironments.

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.

Adrenomedullin (AM) is a potent vasodilator peptide which has an inhibitory action on insulin secretion. Resistin is a novel peptide specifically secreted from adipocytes, and implicated in insulin resistance. We studied the expression of AM and resistin in 3T3-L1 adipocytes and preadipocytes by Northern blot analysis and radioimmunoassay. Immunoreactive-AM was detected in the culture media of 3T3-L1 preadipocytes and adipocytes, with higher concentrations found in preadipocytes. Northern blot analysis showed that AM mRNA was expressed in 3T3-L1 preadipocytes but was undetectable in adipocytes. In contrast, resistin mRNA was expressed in 3T3-L1 adipocytes, whereas it was not detected in 3T3-L1 preadipocytes. The present study thus showed that AM expression was decreased, and resistin expression increased, during adipocyte-differentiation of 3T3-L1 cells. (Hypertens Res 2003; 26 (Suppl): S41-S44)