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ABSTRACT: The adrenal medulla and pheochromocytomas are known to secrete various neuropeptides and vasoactive peptides. On the other hand, the production and secretion of peptides by adrenocortical tumors have not been studied in detail. The study reported here therefore set out to examine these two functions for two vasoactive peptides, endothelin-1 (ET-1) and adrenomedullin (ADM) in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive ET (irET) and irADM were detected in the culture medium of SW-13 cells. Northern blot analysis showed the expression of ET-1 and ADM mRNAs in SW-13 cells. On the other hand, no significant amounts of calcitonin-gene-related peptide, corticotropin-releasing-hormone, neuropeptide Y or urocortin were secreted by SW-13 cells. This study has shown that ET-1 and ADM are the two unique vasoactive peptides that are produced and secreted by adrenocortical carcinoma cells.
(C) 2000 Lippincott Williams & Wilkins, Inc.
Journal of Cardiovascular Pharmacology 07/2012; 36. · 2.29 Impact Factor
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ABSTRACT: Microphthalmia-associated transcription factor (Mitf) is a regulator for differentiation of melanoblasts that are derived from the neural crest. The mouse homozygous for the black-eyed white (Mitf(mi-bw)) allele is characterized by the white coat color and deafness, with black eye that is associated with the lack of melanocytes in skin and inner ear. The Mitf(mi-bw) mutation is an insertion of the LINE1 retrotransposable element into intron 3 of the Mitf gene that causes the selective deficiency of the melanocyte-specific Mitf isoform, Mitf-M. Here, we show the expression of Mitf-M mRNA in the trunk region of the homozygous Mitf(mi-bw)(bw) mouse at embryonic days (E) 11.5 and E12.5, but Mitf-M mRNA is undetectable at E13.5. In addition, using bw mouse that carries the lacZ transgene under the control of a melanoblast-specific promoter, we show that the number of migrating melanoblasts in bw embryos was less than 10% of that in control embryos at E11.5 and E12.5, and melanoblasts disappear by E13.5. The loss of melanoblasts in bw embryos was probably caused by apoptosis. Finally, forced expression of Mitf-M in the cultured neural tube of bw embryos ensured the differentiation of melanoblasts. Therefore, the correct dose of Mitf-M is required for the normal development of melanoblasts.
Genes to Cells 05/2012; 17(6):494-508. · 2.68 Impact Factor
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ABSTRACT: Melanocytes in the human epidermis actively produce and secrete various substances, thereby contributing to the maintenance of the skin homeostasis. Lipocalin-type prostaglandin D synthase (L-PGDS) that catalyzes the formation of prostaglandin D(2) (PGD(2) ) may be one of such secreted molecules. Once secreted, L-PGDS functions as a transporter for lipophilic ligands, including all-trans retinoic acid (RA). L-PGDS, therefore, may possess pleiotropic functions in the skin through PGD(2) and RA. We aimed to identify the cell types that express L-PGDS in human skin and to explore the role of L-PGDS in the growth potential of melanocyte-lineage cells. Immunohistochemical analysis for L-PGDS expression was performed with the tissue sections that were prepared from five malignant melanomas, six nevus cell nevi and one Spitz nevus. Normal skin tissues adjacent to the excised melanoma tissues were also analyzed. L-PGDS is expressed in epidermal melanocytes but its expression is undetectable in keratinocytes. Moreover, L-PGDS is undetectable in most benign nevus cells, which may reflect the marginally accelerated proliferation of nevus cells. In contrast, L-PGDS is overexpressed in malignant melanomas, although the frequency of L-PGDS-positive cells was variable (15-50%), depending on the specimens. Lastly, RNA interference analysis against human L-PGDS was performed with short interfering RNA. Knockdown of L-PGDS expression with short interfering RNA in cultured cells suggests that L-PGDS may restrict cell proliferation through RA. In conclusion, L-PGDS expression may contribute to the restricted proliferation of epidermal melanocytes, but conversely its overexpression may reflect the dysregulated proliferation of melanoma cells.
The Journal of Dermatology 02/2012; 39(8):699-704. · 1.49 Impact Factor
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ABSTRACT: 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.
Experimental hematology 01/2012; 40(6):477-86.e1. · 3.11 Impact Factor
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ABSTRACT: The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate.
PLoS ONE 01/2012; 7(11):e48879. · 4.09 Impact Factor
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ABSTRACT: 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 Tohoku Journal of Experimental Medicine 01/2012; 228(1):27-41. · 1.24 Impact Factor
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ABSTRACT: Lipocalin-type prostaglandin D synthase (L-PGDS) catalyses the formation of prostaglandin D(2) (PGD(2)) and also functions as a transporter for lipophilic ligands, including all-trans retinoic acid (RA). Here, we show that human epidermal melanocytes produce and secrete L-PGDS and PGD(2) in culture medium, whereas L-PGDS is not expressed in human melanoma cell lines, HMV-II, SK-MEL-28, 624 mel and G361. Treatment with RA (1 or 10 microM) for 4 days decreased the proliferation of melanocytes (30% decrease), but not melanoma cells. We therefore isolated L-PGDS-expressing cell lines from 624 mel cells. Treatment with RA decreased the proliferation of L-PGDS-expressing cells by 20%, but not mock-transfected cell lines lacking L-PGDS expression. RA induced expression of a cyclin-dependent kinase inhibitor p21(Cip1) in L-PGDS-expressing cells, but not mock-transfected cells. Moreover, RA increased the transient expression of a reporter gene carrying the RA-responsive elements in L-PGDS-expressing cell lines (at least 5-fold activation), compared to the 2-fold activation in mock-transfected cell lines, suggesting that L-PGDS may increase the sensitivity to RA. Lastly, the knockdown of L-PGDS expression by RNA interference was associated with the restoration of the RA-mediated decrease in proliferation of human and mouse melanocytes. In conclusion, L-PGDS may fine-tune the RA signalling in melanocytes.
Journal of biochemistry 08/2010; 148(2):139-48. · 1.95 Impact Factor
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ABSTRACT: 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.
Journal of biochemistry 10/2009; 147(1):143-51. · 1.95 Impact Factor
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 07/2009; 385(3):449-53. · 2.48 Impact Factor
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ABSTRACT: 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.
Journal of Investigative Dermatology 07/2009; 129(11):2594-603. · 6.31 Impact Factor
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ABSTRACT: 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.
FEBS Journal 02/2009; 276(5):1370-82. · 3.79 Impact Factor
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ABSTRACT: Prostaglandin (PG) D(2) exerts multiple actions through interaction with distinct receptors, DP1 and DP2. We have shown that PGD(2) induces the expression of heme oxygenase-1 (HO-1) in the retinal pigment epithelium (RPE) that is essential for survival of photoreceptors. HO-1 is a key enzyme in physiological heme degradation. Here, we explored the mechanism for the PGD(2)-mediated induction of HO-1 expression using ARPE-19 human RPE cells. ARPE-19 cells secrete PGD(2) and express DP2 mRNA, but not DP1 mRNA. Treatment with a DP2 agonist, 15(R)-15-methyl-PGD(2) or DK-PGD(2), increased HO-1 mRNA expression, and pretreatment with a DP2 antagonist, CAY10471, decreased the magnitude of the PGD(2)-mediated HO-1 induction. By contrast, either DP1 agonist or antagonist caused only marginal influence on HO-1 expression. Moreover, transient expression assays showed the DP2 agonist activated the HO-1-gene promoter in the enhancer-dependent manner. Thus, PGD(2) induces HO-1 mRNA expression through DP2 receptor, linking the PGD(2)-DP2 signaling with heme homeostasis.
Biochemical and Biophysical Research Communications 01/2009; 377(3):878-83. · 2.48 Impact Factor
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ABSTRACT: 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% O(2)) 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 1alpha was accumulated. Thus, the decrease in the expression of ubiquitous HMBS and UROS mRNAs is associated with accumulation of hypoxia-inducible factor 1alpha 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.
FEBS Journal 01/2009; 275(23):5947-59. · 3.79 Impact Factor
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ABSTRACT: Mitf has been reported to play a crucial role in regulating the differentiation of pigment cells in homeothermal animals, i.e. the melanocytes and the retinal pigment epithelium (RPE). However, less is known about the functions of Mitf in the developing RPE. To elucidate such functions, we introduced wild-type and dominant-negative Mitf expression vectors into chick optic vesicles by electroporation. Over-expression of wild-type Mitf altered neural retina cells to become RPE-like and repressed the expression of neural retina markers in vivo. In contrast, dominant-negative Mitf inhibited pigmentation in the RPE. The percentage of BrdU-positive cells decreased during normal RPE development, which was followed by Mitf protein expression. The percentage of BrdU-positive cells decreased in the wild-type Mitf-transfected neural retina, but increased in the dominant-negative Mitf-transfected RPE. p27(kip1), one of the cyclin-dependent kinase inhibitors, begins to be expressed in the proximal region of the RPE at stage 16. Transfection of wild-type Mitf induced expression of p27(kip1), while transfection of dominant-negative Mitf inhibited p27(kip1) expression. We found that Mitf was associated with the endogenous p27(kip1) 5' flanking region. These results demonstrate for the first time "in vivo" that Mitf uniquely regulates both differentiation and cell proliferation in the developing RPE.
Developmental Biology 01/2009; 326(2):335-46. · 4.07 Impact Factor
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ABSTRACT: Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the elderly. Risk factors include old age, female gender, obesity, smoking, low dietary intakes of antioxidants and increased exposure to the toxic metal cadmium (Cd(2+)). Supplementation with high-dose zinc (80 mg) provides some protection, but the mechanism(s) underlying such protection has not been fully elucidated. The present study had a focus on the human retinal pigment epithelial (RPE) cell line ARPE-19 in an attempt to demonstrate a reduction in intracellular Cd(2+) effect associated with heme oxygenase-1 (HO-1) expression by co-exposure with zinc (Zn(2+)) or manganese (Mn(2+)), which is known to be a more potent inhibitor of Cd(2+) uptake than Zn(2+). Our results indicated that co-exposure of 10 microM Cd(2+) with 5 microM Mn(2+) reduced the intracellular Cd(2+) effect by 50-60%, possibly by limiting the amounts of Cd(2+) entering cells through Mn(2+) transporter protein (ZIP8). A similar reduction in a Cd(2+) effect was achieved by co-exposure with 20 microM Zn(2+) while co-exposure with 5 and 10 microM Zn(2+) ions was ineffective. Mn(2+) ions as low as 2.5 microM were found to cause an increase in HO-1 mRNA expression levels in ARPE-19 cells, demonstrating for the first time that Mn(2+) is an inducer of HO-1. Mn(2+) ions at 1 microM induced HO-1 mRNA expression in the HEK293 human embryonic kidney cells. In contrast, Zn(2+) in 5, 10 or 20 microM concentrations did not induce expression of HO-1 in ARPE-19 cells or any other cells tested. These data suggest the superiority of Mn(2+) over Zn(2+) in preventing Cd(2+) uptake and accumulation in RPE to toxic levels. Further, induction of HO-1 by Mn(2+) could provide RPE with some resistance to enhanced oxidative stress arising from Cd(2+) accumulation in RPE as HO-1 is one of the frontline cellular antioxidant defense mechanisms.
Experimental Eye Research 11/2008; 87(6):587-93. · 3.26 Impact Factor
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Shigeyuki Uehara,
Yoshiko Izumi,
Yuko Kubo,
Chi Chiu Wang,
Katsuhiko Mineta,
Kazuho Ikeo,
Takashi Gojobori,
Masayoshi Tachibana,
Toshihiko Kikuchi,
Toshimitsu Kobayashi, Shigeki Shibahara,
Choji Taya,
Hiromichi Yonekawa,
Toshihiko Shiroishi,
Hiroaki Yamamoto
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ABSTRACT: Mammalian pigment cells produce melanin as the main pigment. Melanocytes, one of the two types of mammalian pigment cells, differentiate from the neural crest and migrate to a variety of organs during development. Melanocytes exist not only in the skin but also in other sites such as the cochlea where they are essential for hearing. Mitf(mi-bw) is one of the known recessive alleles of the mouse microphthalmia-associated transcription factor (Mitf) locus, which is essential for the development of pigment cells. Homozygous Mitf(mi-bw)/Mitf(mi-bw) mice have a completely white coat with black eyes and are deaf due to the lack of melanocytes. By comparing gene expression profiles in cochleae of wild-type and Mitf(mi-bw)/Mitf(mi-bw) mice, we now demonstrate the specific expression of glutathione S-transferase alpha 4 (Gsta4) in the stria vascularis. Gsta4 encodes one of the cytosolic glutathione S-transferases (GSTs) which participate in detoxification processes of many tissues. This gene is specifically expressed in intermediate cells of the stria vascularis, suggesting a novel function for cochlear melanocytes. Moreover, among mammalian pigment cells, expression of Gsta4 was restricted to cochlear melanocytes, suggesting that melanocytes in various tissues differentiate from one another depending on their location.
Pigment Cell & Melanoma Research 11/2008; 22(1):111-9. · 5.06 Impact Factor
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ABSTRACT: Hypoxia-inducible factor (HIF)-1 is important for cellular homeostasis under hypoxia. Expression of haem oxygenase-1 (HO-1), an essential enzyme in haem catabolism, varies under hypoxia, depending on cell types. Here, we studied the role of HIF-1alpha, a component of HIF-1, in the regulation of HO-1 expression using three human cell lines: HeLa cervical cancer, and ARPE-19 and D407 retinal pigment epithelial cells. Under hypoxia (1% O(2)), the expression of HO-1 mRNA was decreased in HeLa cells, increased in D407 cells, and unchanged in ARPE-19 cells, while HIF-1alpha protein was accumulated in these cell lines. Thus, HIF-1alpha is unlikely to function as a key regulator for HO-1 expression under hypoxia. We then used ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl hydroxylases, to accumulate HIF-1alpha protein under normoxia. Treatment with EDHB (250-500 microM) increased HIF-1alpha protein levels in HeLa and D407 cells, but not in ARPE-19 cells, whereas EDHB at lower concentrations (50-100 microM) consistently induced HO-1 mRNA expression (about 20-fold) in these three cell lines. Moreover, EDHB increased the HO-1 gene promoter activity via the enhancer that lacks a HIF-1-binding site. In conclusion, the signals evoked by hypoxia and after EDHB treatment differentially regulate HO-1 mRNA expression through HIF-1alpha-independent mechanisms.
Journal of Biochemistry 10/2008; 144(5):643-54. · 2.37 Impact Factor
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ABSTRACT: The retinal pigment epithelium (RPE) constitutes the blood-retinal barrier, whose function is impaired in various pathological conditions, including cerebral malaria, a lethal complication of Plasmodium falciparum infection. Prostaglandin (PG) D(2) is abundantly produced in the brain to regulate sleep responses. Moreover, PGD(2) is a potential factor derived from intra-erythrocyte falciparum parasites. Heme oxygenase-1 (HO-1) is important for iron homeostasis via catalysis of heme degradation to release iron, carbon monoxide and biliverdin/bilirubin, and may influence iron supply to the intra-erythrocyte falciparum parasites. Here, we showed that treatment of human RPE cell lines, ARPE-19 and D407, with PGD(2) significantly increased the expression levels of HO-1 mRNA, in a dose- and time-dependent manner. Transient expression assays showed that PGD(2) treatment increased the HO-1-gene promoter activity through the enhancer sequence, containing a Maf-recognition element. Thus, PGD(2) may contribute to the maintenance of heme homeostasis in the brain by inducing HO-1 expression.
Biochemical and Biophysical Research Communications 04/2008; 367(2):413-9. · 2.48 Impact Factor
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ABSTRACT: Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (-70/-29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2alpha (AP-2alpha), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2alpha synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (-103/-29) with Sp1 and AP-2alpha expression plasmids showed that Sp1 and AP-2alpha synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2alpha alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (-70/-29), whereas AP-2alpha bound to a more upstream promoter region (-103/-71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2alpha may be mediated by the binding of Sp1 to the promoter region (-70/-29) and the interaction with AP-2alpha, which binds to the promoter region (-103/-71).
Peptides 04/2008; 29(3):465-72. · 2.43 Impact Factor
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ABSTRACT: Heme is an essential molecule for life, as it is involved in sensing and using oxygen. Heme must be synthesized and degraded within an individual nucleated cell. Physiologic heme degradation is catalyzed by two functional isozymes of heme oxygenase, heme oxygenase-1 (HO-1) and HO-2, yielding carbon monoxide, iron, and biliverdin, an immediate precursor to bilirubin. HO-1 is an inducible enzyme, but the expression level of HO-2 is maintained in a narrow range. Characteristically, human HO-1 contains no Cys residue, whereas human HO-2 contains three Cys residues, each of which might be involved in heme binding. These features suggest separate physiologic roles of HO-1 and HO-2. Recent studies have shown that the expression levels of HO-1 and HO-2 are reduced under hypoxia, depending on the cell types. Moreover, we have proposed HO-2 as a potential O(2) sensor, because HO-2-deficient mice show hypoxemia and a blunted hypoxic ventilatory response with normal hypercapnic ventilatory response. HO-2-deficient mice also show hypertrophy of the pulmonary venous myocardium and enlargement of the carotid body. These morphometric changes are attributable to chronic hypoxemia. Here, we update the understanding of the regulation of HO-1 and HO-2 expression and summarize the regulatory role of HO-2 in the intercellular communication.
Antioxidants and Redox Signaling 01/2008; 9(12):2209-25. · 8.46 Impact Factor
