CLPX regulates mitochondrial fatty acid ß-oxidation in liver cells.

Mitochondrial fatty acid oxidation (ß-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 ß-oxidation are associated with CLPX, the AAA+ unfoldase that is a component of the mitochondrial matrix protease ClpXP. The suppression of CLPX expression increased ß-oxidation activity in the HepG2 cell line and in primary human hepatocytes without glucagon treatment. However, the protein levels of enzymes involved in ß-oxidation did not significantly increase in CLPX-deleted HepG2 cells (CLPX-KO cells). Coimmunoprecipitation experiments revealed that the protein level in the immunoprecipitates of each antibody changed after the treatment of WT cells with glucagon, and a part of these changes was also observed in the comparison of WT and CLPX-KO cells without glucagon treatment. Although the exogenous expression of WT or ATP-hydrolysis mutant CLPX suppressed ß-oxidation activity in CLPX-KO cells, glucagon treatment induced ß-oxidation activity only in CLPX-KO cells expressing WT CLPX. These results suggest that the dissociation of CLPX from its target proteins is essential for the induction of ß-oxidation in HepG2 cells. Moreover, specific phosphorylation of AMP-activated protein kinase and a decrease in the expression of acetyl-CoA carboxylase 2 were observed in CLPX-KO cells, suggesting that CLPX might participate in the regulation of the cytosolic signaling pathway for ß-oxidation. The mechanism for AMP-activated protein kinase phosphorylation remains elusive; however, our results uncovered the hitherto unknown role of CLPX in mitochondrial ß-oxidation in human liver cells.

ATP6AP2 variant impairs CNS development and neuronal survival to cause fulminant neurodegeneration.

Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.

Establishment of a cell model of X-linked sideroblastic anemia using genome editing.

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, ß-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.

Iron metabolism in erythroid cells and patients with congenital sideroblastic anemia.

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.

Expression of (Pro)renin Receptor During Rapamycin-Induced Erythropoiesis in K562 Erythroleukemia Cells and Its Possible Dual Actions on Erythropoiesis.

(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.

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis.

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.

Ubiquitous expression and multiple functions of biologically active peptides.

Biologically active peptides are widely expressed throughout in human bodies. For example, endothelin-1 and adrenomedullin are expressed in almost all types of cells, including neurons, glial cells, fibroblasts, macrophages, cardiomyocytes, vascular endothelial cells, epithelial cells and cancer cells of various origins. Expression of both these peptides is induced by stimuli, such as hypoxia and inflammatory cytokines. They have a variety of biological functions, such as effects on brain function, hormone secretion, the cardiovascular system and cell proliferation. By contrast, orexins (hypocretins) and melanin-concentrating hormone (MCH) are specifically expressed in the hypothalamus, particularly in the lateral hypothalamus, although very low concentrations of these peptides are found in the peripheral tissues. Orexins and MCH play coordinated, but distinct physiological roles in the regulation of sleep-wake cycle, appetite, emotion and other brain functions. The cardiovascular system is regulated by cardiovascular peptides, such as natriuretic peptides, endothelins and angiotensin II. The renin-angiotensin system (RAS) is one of the most classical regulatory systems on blood pressure, electrolytes and kidney. (Pro)renin receptor is a novel member of the RAS and may be related to the pathophysiology of microvascular complications of hypertension and diabetes mellitus. Moreover, (pro)renin receptor forms a functional complex with vacuolar-type H(+)-ATPase, which plays an important physiological role in maintaining the acidic environment of intracellular compartments including secretory vesicles. Perhaps, the complex of (pro)renin receptor and vacuolar-type H(+)-ATPase may be important for the post-translational processing and secretion of many biologically active peptides.

Expression of (pro)renin receptor in breast cancers and its effect on cancercell proliferation.

(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the present study is to clarify expression of (P)RR and pathophysiological roles of (P)RR in human breast carcinomas. (P)RR expression was studied in 69 clinical cases of breast carcinoma by immunohistochemistry.Effects of (P)RR on cell proliferation were examined in cultured human breast carcinoma cells using (P)RR specific small interference RNA. Immunohistochemistry showed that(P)RR immunoreactivity was detected in the breast carcinoma cells in 50 of 69 cases of breast carcinoma (72%). The analysis on association between (P)RR immunoreactivity and clinicopathological parameters showed that the number of (P)RR positive cases was significantly greater in Ki-67 (a cell proliferation marker)≥10% group than in Ki-67<10% group (P=0.02). (P)RR was expressed in 4 types of human breast carcinoma cell lines. (P)RR specific small interference RNA inhibited proliferation of both MCF-7 (ERα positive) and SK-BR-3 (ERα negative) cells. The present study has shown, for the first time, the expression of (P)RR in human breast carcinoma tissues and cultured breast carcinoma cell lines. These findings have raised the possibility that the blockade of the (P)RR signaling may be a novel therapeutic strategy against breast carcinomas.

Identification of a novel erythroid-specific enhancer for the ALAS2 gene and its loss-of-function mutation which is associated with congenital sideroblastic anemia.

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the rate-limiting enzyme for heme biosynthesis in erythroid cells, and a missense mutation of the ALAS2 gene is associated with congenital sideroblastic anemia. However, the gene responsible for this form of anemia remains unclear in about 40% of patients. Here, we identify a novel erythroid-specific enhancer of 130 base pairs in the first intron of the ALAS2 gene. The newly identified enhancer contains a cis-acting element that is bound by the erythroid-specific transcription factor GATA1, as confirmed by chromatin immunoprecipitation analysis in vivo and by electrophoretic mobility shift assay in vitro. A promoter activity assay in K562 human erythroleukemia cells revealed that the presence of this 130-base pair region increased the promoter activity of the ALAS2 gene by 10-15-fold. Importantly, two mutations, each of which disrupts the GATA-binding site in the enhancer, were identified in unrelated male patients with congenital sideroblastic anemia, and the lower expression level of ALAS2 mRNA in bone marrow erythroblasts was confirmed in one of these patients. Moreover, GATA1 failed to bind to each mutant sequence at the GATA-binding site, and each mutation abolished the enhancer function on ALAS2 promoter activity in K562 cells. Thus, a mutation at the GATA-binding site in this enhancer may cause congenital sideroblastic anemia. These results suggest that the newly identified intronic enhancer is essential for the expression of the ALAS2 gene in erythroid cells. We propose that the 130-base pair enhancer region located in the first intron of the ALAS2 gene should be examined in patients with congenital sideroblastic anemia in whom the gene responsible is unknown.

Increased expression of (pro)renin receptor in aldosterone-producing adenomas.

(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the present study is to clarify expression and possible pathophysiological roles of (P)RR in aldosterone-producing adenomas (APAs) and other adrenal tumors. Expression of (P)RR was studied by immunocytochemistry, western blot analysis and real-time RT-PCR in adrenal tumor tissues obtained at surgery. Immunocytochemistry showed that (P)RR was expressed in normal adrenal glands and tumor tissues of adrenocortical tumors including APAs. In the normal adrenal glands, positive (P)RR immunostaining was observed in both adrenal cortex and medulla, with higher (P)RR immunostaining observed in zona glomerulosa and zona reticularis. Positive (P)RR immunostaining was also observed in the adrenocortical tumors, with elevated (P)RR immunostaining found in APAs, particularly in compact cells. By contrast, no apparent (P)RR immunostaining was observed in pheochromocytomas. Western blot analysis showed a band of (P)RR protein in normal adrenal glands and adrenocortical tumors at the position of 35 kDa. The relative expression levels of (P)RR protein were higher in tumor tissues of APAs than in attached non-neoplastic adrenal tissues of APAs. Real-time RT-PCR showed that expression levels of (P)RR mRNA were significantly increased in tumor tissues of APAs compared with other adrenal tumor tissues and attached non-neoplastic adrenal tissues of APAs. The present study has shown for the first time that expression of (P)RR is elevated in tumor tissues of APAs, raising the possibility that (P)RR may play pathophysiological roles in APAs, such as aldosterone secretion and cell proliferation.

Expression of (pro)renin receptor in human erythroid cell lines and its increased protein accumulation by interferon-γ.

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-ß1 (TGF-ß1), interferon-γ (IFN-γ) and interleukin-1ß (IL-1ß) 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 (1IU/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 (10ng/ml) for 48h 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-ß1 or IL-1ß for 24 or 48h 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.

The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability.

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.

Expression of kisspeptins and kisspeptin receptor in the kidney of chronic renal failure rats.

Kisspeptins are biologically active cleavage peptides of the KiSS-1 gene products with important roles in the suppression of tumor metastasis and in the reproduction. The aim of the present study is to clarify changes of the expression of kisspeptins and kisspeptin receptor in the kidney with and without chronic renal impairment. 5/6 nephrectomized rats were used as the rat model of chronic renal failure. Competitive quantitative RT-PCR showed that kisspeptin mRNA levels were decreased in the kidney of 5/6 nephrectomized rats at 56 days compared with sham-operated rats. In contrast, immunoreactive kisspeptin concentrations were increased in the kidney of 5/6 nephrectomized rats at 56 days. On the other hand, kisspeptin receptor mRNA levels were increased in the kidney of 5/6 nephrectomized rats at 14 and 56 days compared with sham-operated rats. Immunocytochemistry showed that kisspeptins and kisspeptin receptor were expressed in renal tubular cells, collecting duct cells, vascular smooth muscle cells in both rats. The intensity of kisspeptin receptor immunostaining was lower in 5/6 nephrectomized rats than in sham-operated rats. Western blot analysis confirmed that kisspeptin receptor protein levels were significantly decreased in the remnant kidney of 5/6 nephrectomized rats (about 23% of sham-operated rats), which is a good contrast to the kisspeptin receptor mRNA expression. The present study has shown that expression of kisspeptins and kisspeptin receptor are altered in the kidney tissues of chronic renal impairment, raising the possibility of their pathophysiological roles in chronic renal failure.

Expression of (pro)renin receptor in human kidneys with end-stage kidney disease due to diabetic nephropathy.

(Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, is a 350 amino-acid protein with a single transmembrane domain and may play important pathophysiological roles in diabetic nephropathy. The aim of the present study is to clarify the expression of (P)RR in the kidney with end-stage renal disease due to diabetic nephropathy. The kidney tissues were obtained at autopsy from patients with and without Type 2 diabetes mellitus (n=5 without diabetes mellitus; and n=8 with diabetes mellitus). Immunocytochemistry showed that (P)RR was mainly expressed in the tubular cells and collecting duct cells of the kidney without diabetic nephropathy. Cells in glomeruli were very weakly and sporadically immunostained for (P)RR. Vascular smooth muscle cells and endothelial cells were very weakly or were not immunostained for (P)RR. Adipocytes in the adipose tissue around the kidney were positively immunostained for (P)RR. Immunostaining pattern of (P)RR in the kidney with diabetic nephropathy was similar to that without diabetic nephropathy. However, most notably, (P)RR immunostaining in the tubular cells and collecting duct cells was clearly and frequently more strongly observed in the kidney with diabetic nephropathy up to the end-stage renal disease. The present study has raised the possibility that (P)RR expressed in the diabetic kidney may play a pathophysiological role in angiotensin I generation and renal fibrosis found in end-stage renal disease.

Presence of kisspeptin-like immunoreactivity in human adrenal glands and adrenal tumors.

Kisspeptins are neuropeptides which activate the hypothalamo-pituitary gonadal axis and are considered to play important physiological roles in the reproduction. Kisspeptins have also been reported to stimulate the aldosterone secretion from the adrenal cortex. However, the expression of kisspeptins in human adrenal glands and adrenal tumors has not been clarified yet. We, therefore, studied the presence of kisspeptin-like immunoreactivity (LI) in human adrenal glands and adrenal tumors (adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas) by radioimmunoassay and immunocytochemistry. Kisspeptin-LI was detected in all the tissues examined; normal portions of adrenal glands (3.0 +/- 2.3 pmol/g wet weight, n = 21, mean +/- SD), aldosterone-producing adenomas (4.6 +/- 3.3 pmol/g wet weight, n = 10), cortisol-producing adenomas (2.7 +/- 1.4 pmol/g wet weight, n = 14), adrenocortical carcinomas (1.7 +/- 0.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.8 +/- 0.8 pmol/g wet weight, n = 6). There was no significant difference in kisspeptin-LI levels among them. Immunocytochemistry showed positive kisspeptin-immunostaining in normal adrenal glands, with stronger immunostaining found in the medulla. Furthermore, positive kisspeptin-immunostaining was found in all types of adrenal tumors examined; adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas. The intensity of kisspeptin-immunostaining in these adrenal tumors was, however, not so strong as that in normal adrenal medulla. The present study has shown for the first time the presence of kisspeptin-LI in adrenal glands and adrenal tumors.

Hypoxia induces erythroid-specific 5-aminolevulinate synthase expression in human erythroid cells through transforming growth factor-beta signaling.

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.

Heme as a magnificent molecule with multiple missions: heme determines its own fate and governs cellular homeostasis.

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.

Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines. A possible strategy for the maintenance of intracellular heme level.

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.

Expression of heme oxygenase-1 is repressed by interferon-gamma and induced by hypoxia in human retinal pigment epithelial cells.

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.