Shigeki Shibahara

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase‐1 gene

Heme oxygenase‐1 (HO‐1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho‐1. Here we show that a heme‐binding factor, Bach1, is a critical physiological repressor of ho‐1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho‐1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho‐1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho‐1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound‐deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho‐1. Thus, regulation of ho‐1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor–activator antagonism.

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin‐releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5′‐untranslated region of the mRNA. The segment corresponding to the protein‐coding and the 3′‐untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin‐releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin‐releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1

Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero‐oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA‐binding activity. Deletion studies demonstrated that a heme‐binding region of Bach1 is confined within its C‐terminal region that possesses four dipeptide cysteine–proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA‐binding activity of Bach1 as a MafK hetero‐oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MAREs.

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor.

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin‐releasing factor precursor (prepro‐CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro‐CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro‐CRF than is the cryptic portion.

HLF/HIF-2α is a key factor in retinopathy of prematurity in association with erythropoietin

An HLF (HIF‐1α‐like factor)/HIF‐2α‐knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF‐knockdown (HLFkd/kd) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLFkd/kd mice expressing 80–20% reduction, depending on the tissue, in wild‐type HLF mRNA were fertile and apparently normal. Hyperoxia–normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild‐type mice, but not in HLFkd/kd mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLFkd/kd mice due to poor vascularization. Cre‐mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLFkd/kd mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLFkd/kd mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Coordinated expression of messenger RNAs for nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in the rat hippocampus following transient forebrain ischemia

Changes in nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 messenger RNA expression in the rat hippocampus following 20 min of transient forebrain ischemia were evaluated using Northern blot analysis and in situ hybridization histochemistry. Twelve hours after the insult, the level of nerve growth factor messenger RNA increased markedly in the granular cell layer of the dentate gyrus and by day 2 returned to control levels. The level of brain-derived neurotrophic factor messenger RNA showed a persistent and moderate increase. The highest expression of brain-derived neurotrophic factor messenger RNA was seen in the dentate granule cells on day 2 after the insult, and then the expression returned to the control levels. At 2 days post-ischemia, contents of messenger RNAs for nerve growth factor and brain-derived neurotrophic factor were reduced in the CA1 region, which may represent delayed loss of vulnerable CA1 pyramidal neurons. In contrast to brain-derived neurotrophic factor and nerve growth factor messenger RNA expression, the level of neurotrophin-3 messenger RNA declined in the CA1, the CA2 and the dentate granular layer immediately after ischemic insult. In the CA1 region, the reduced expression persisted for at least seven days, but in the dentate gyrus, neurotrophin-3 messenger RNA expression returned to the control levels after two days of post-ischemic recovery. These results suggest that nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 genes are differentially regulated and that each of their gene products may play different roles in the central nervous system under pathophysiological conditions.

Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling

Wnt signals regulate differentiation of neural crest cells through the β‐catenin associated with a nuclear mediator of the lymphoid‐enhancing factor 1 (LEF‐1)/T‐cell factors (TCFs) family. Here we show the interaction between the basic helix–loop–helix and leucine‐zipper region of microphthalmia‐associated transcription factor (MITF) and LEF‐1. MITF is essential for melanocyte differentiation and its heterozygous mutations cause auditory–pigmentary syndromes. Functional cooperation of MITF with LEF‐1 results in synergistic transactivation of the dopachrome tautomerase (DCT) gene promoter, an early melanoblast marker. This activation depends on the separate cis‐acting elements, which are also responsible for the induction of the DCT promoter by lithium chloride that mimics Wnt signaling. β‐catenin is required for efficient transactivation, but dispensable for the interaction between MITF and LEF‐1. The interaction with MITF is unique to LEF‐1 and not detectable with TCF‐1. LEF‐1 also cooperates with the MITF‐related proteins, such as TFE3, to transactivate the DCT promoter. This study therefore suggests that the MITF/TFE3 family is a new class of nuclear modulators for LEF‐1, which may ensure efficient propagation of Wnt signals in many types of cells.

Induction of haem oxygenase-1 by nitric oxide and ischaemia in experimental solid tumours and implications for tumour growth

SummaryInduction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either Nω-nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO+) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6–20 mg kg–1). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity.

Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer

Angiogenesis is necessary for the continued growth of solid tumors, invasion and metastasis. Several studies clearly showed that heme oxygenase-1 (HO-1) plays an important role in angiogenesis. In this study, we used the vital microscope system, transparent skinfold model, lung colonization model and transduced pancreatic cancer cell line (Panc-1)/human heme oxygenase-1 (hHO-1) cells, to precisely analyze, for the first time, the effect of hHO-1 gene on tumor growth, angiogenesis and metastasis. Our results revealed that HO-1 stimulates angiogenesis of pancreatic carcinoma in severe combined immune deficient mice. Overexpression of human hHO-1 after its retroviral transfer into Panc-1 cells did not interfere with tumor growth in vitro. While in vivo the development of tumors was accelerated upon transfection with hHO-1. On the other hand, inhibition of heme oxygenase (HO) activity by stannous mesoporphyrin was able transiently to delay tumor growth in a dose dependent manner. Tumor angiogenesis was markedly increased in Panc-1/hHO-1 compared to mock transfected and wild type. Lectin staining and Ki-67 proliferation index confirmed these results. In addition hHO-1 stimulated in vitro tumor angiogenesis and increased endothelial cell survival. In a lung colonization model, overexpression of hHO-1 increased the occurrence of metastasis, while inhibition of HO activity by stannous mesoporphyrin completely inhibited the occurrence of metastasis. In conclusion, overexpression of HO-1 genes potentiates pancreatic cancer aggressiveness, by increasing tumor growth, angiogenesis and metastasis and that the inhibition of the HO system may be of useful benefit for the future treatment of the disease.

Microsatellite polymorphism in the human heme oxygenase-1 gene promoter and its application in association studies with Alzheimer and Parkinson disease

Abstract Oxidative stress has been suggested to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease (PD). Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, also functions as an antioxidant enzyme. Here, we show that a (GT)n repeat in the human HO-1 gene promoter region is highly polymorphic, although no particular alleles are associated with AD or PD. This newly identified genetic marker should allow us to study the possible involvement of HO-1 in certain human diseases.