Miki Yoshizawa

Induction of heme oxygenase-1 as a response in sensing the signals evoked by distinct nitric oxide donors.

To gain insights into the cellular responses evoked by nitric oxide (NO), we have studied the effects of NO donors with distinct chemistries on the expression of heme oxygenase-1 mRNA by northern blot analysis. The expression levels of heme oxygenase-1 mRNA were increased significantly in DLD-1 human colorectal adenocarcinoma cells by treatment with each of three NO donors: sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO), and 3-morpholinosydnonimine (SIN-1). A combination of SIN-1 plus SNP or GSNO additively increased heme oxygenase-1 mRNA expression, whereas synergistic induction was seen with SNP plus GSNO. The SNP-mediated induction was not affected noticeably by extracellular superoxide dismutase, catalase, or mannitol, while the induction by SIN-1 was attenuated by superoxide dismutase. Thus, the SNP-mediated induction of heme oxygenase-1 mRNA expression may be independent of reactive oxygen species, and the induction by SIN-1 is mediated partly by peroxynitrite, which is generated by immediate reaction of NO and superoxide anion. Transient transfection assays suggested that treatment with SNP, but not with GSNO or SIN-1, increased the expression of a reporter gene through a cis-acting element, including the cadmium-responsive element, of the human heme oxygenase-1 gene. These results suggest that SNP induces heme oxygenase-1 mRNA expression through a mechanism different from that for GSNO or SIN-1. We therefore propose that induction of heme oxygenase-1 represents a common cellular response in sensing the signals evoked by distinct NO donors.

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.

Insertion of long interspersed element‐1 in the Mitf gene is associated with altered neurobehavior of the black‐eyed white Mitfmi‐bw mouse

Microphthalmia‐associated transcription factor (Mitf) is required for the differentiation of melanoblasts of the neural crest origin. The mouse homozygous for the black‐eyed white (Mitfmi‐bw) allele is characterized by white‐coat color and deafness with black eye, due to the loss of melanoblasts during embryonic development. The Mitfmi‐bw allele carries an insertion of long interspersed element‐1 (L1) in intron 3 of the Mitf gene, which may cause the deficiency of melanocyte‐specific Mitf‐M. Here, we show that the L1 insertion results in the generation of alternatively spliced Mitf‐M mRNA species, such as Mitf‐M mRNA lacking exon 3, exon 4 or both exons 3 and 4, each of which encodes Mitf‐M protein with an internal deletion. Transient expression assays showed the loss of or reduction in function of each aberrant Mitf‐M protein and the dominant negative effect of Mitf‐M lacking exon 4 that encodes an activation domain. Thus, the L1 insertion may decrease the expression level of functional Mitf‐M. Importantly, Mitf‐M mRNA is expressed in the wild‐type mouse brain, with the highest expression level in the hypothalamus. Likewise, aberrant Mitf‐M mRNAs are expressed in the bw mouse brain. The bw mice show the altered neurobehavior under a stressful environment, suggesting the role of Mitf‐M in sensory perception.

Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.

Lipocalin-type prostaglandin D synthase as a regulator of the retinoic acid signalling in melanocytes

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.

Transcriptional Control of the Human Heme Oxygenase-1 Gene by Stress

Heme oxygenase-1 is an essential enzyme in heme catabolism, releasing carbon monoxide, iron, and biliverdin. By transient expression analysis of the fusion genes, containing the firefly luciferase gene as a reporter under the human heme oxygenase-1 gene promoter, we have identified a 10-bp sequence, TGCTAGATTT, that confers the cadmium-mediated induction on the fusion gene, located about 4 kb upstream from the transcriptional initiation site (J. Biol. Chem. 269: 22858–67, 1994). This cadmium-responsive element (CdRE) is different from the consensus sequence of the metal-regulatory element of the metallothionein genes. Both heme oxygenase-1 and metallothionein are considered to be involved in the defense system against metal toxicity. Here we showed that the CdRE of the human heme oxygenase-1 gene is not responsive to zinc, whereas a metal-regulatory element (MRE) of the human metallothionein IIA gene is able to respond to both cadmium and zinc. These results suggest that the metal-selective activation of each gene promoter is mediated by a separate mechanism.