Melanocytes contribute to the vasculature of the choroid.

Melanocytes develop from the vertebrate embryo-specific neural crest, migrate, and localize in various organs, including not only the skin but also several extracutaneous locations such as the heart, inner ear and choroid. Little is known about the functions of extracutaneous melanocytes except for cochlear melanocytes, which are essential for hearing ability. In this study, we focused on the structure of the choroid, in which melanocytes are abundant around the well-developed blood vascular system. By comparing structural differences in the choroid of wild-type and melanocyte-deficient Mitfmi-bw/Mitfmi-bw mutant mice, our observations suggest that choroidal melanocytes contribute to the morphogenesis and/or maintenance of the normal vasculature structure of that tissue.

Dysregulated Expression of MITF in Subsets of Hepatocellular Carcinoma and Cholangiocarcinoma.

Cholangiocarcinoma represents the second most common primary liver tumor after hepatocellular carcinoma. Mahanine, a carbazole alkaloid derived from Murraya koenigii (Linn.) Spreng, has been used as folk medicine in Thailand, where the liver fluke-associated cholangiocarcinoma is common. The expression of microphthalmia-associated transcription factor (MITF) is maintained at immunohistochemically undetectable levels in hepatocytes and cholangiocytes. To explore the regulation of MITF expression in the liver, we immunohistochemically analyzed the MITF expression using hepatocellular carcinoma and cholangiocarcinoma specimens of the human liver cancer tissue array. MITF immunoreactivity was detected in subsets of hepatocellular carcinoma (6 out of 38 specimens; 16%) and cholangiocarcinoma (2/7 specimens; 29%). Moreover, immunoreactivity for glioma-associated oncogene 1 (GLI1), a transcription factor of the Hedgehog signaling pathway, was detected in 55% of hepatocellular carcinoma (21/38 specimens) and 86% of cholangiocarcinoma (6/7 specimens). Importantly, MITF was detectable only in the GLI1-positive hepatocellular carcinoma and cholangiocarcinoma, and MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. Subsequently, the effect of mahanine was analyzed in HepG2 human hepatocellular carcinoma and HuCCT1 and KKU-100 human cholangiocarcinoma cells. Mahanine (25 µM) showed the potent cytotoxicity in these hepatic cancer cell lines, which was associated with increased expression levels of MITF, as judged by Western blot analysis. MITF is over-expressed in subsets of hepatocellular carcinoma and cholangiocarcinoma, and detectable MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. MITF expression levels may be determined in hepatic cancer cells by the balance between the Hedgehog signaling and the cellular stress.

Microphthalmia-associated transcription factor ensures the elongation of axons and dendrites in the mouse frontal cortex.

Long interspersed element-1 (LINE-1) is a mammalian transposable element, and its genomic insertion could cause neurological disorders in humans. Incidentally, LINE-1 is present in intron 3 of the microphthalmia-associated transcription factor (Mitf) gene of the black-eyed white mouse (Mitfmi-bw allele). Mice homozygous for the Mitfmi-bw allele show the white coat color with black eye and deafness. Here, we explored the functional consequences of the LINE-1 insertion in the Mitf gene using homozygous Mitfmi-bw mice on the C3H background (C3H-bw mice) or on the C57BL/6 background (bw mice). The open-field test showed that C3H-bw mice moved more irregularly in an unfamiliar environment during the 20-min period, compared to wild-type mice, suggesting the altered emotionality. Moreover, C3H-bw mice showed the lower serum creatinine levels, which may reflect the creatine deficiency. In fact, morphologically abnormal neurons and astrocytes were detected in the frontal cortex of bw mice. The immunohistochemical analysis of bw mouse tissues showed the lower intensity for expression of guanidinoacetate methyltransferase, a key enzyme in creatine synthesis, in neurons of the frontal cortex and in glomeruli and renal tubules. Thus, Mitf may ensure the elongation of axons and dendrites by maintaining creatine synthesis in the frontal cortex.

Tetrahydrocurcumin in combination with deferiprone attenuates hypertension, vascular dysfunction, baroreflex dysfunction, and oxidative stress in iron-overloaded mice.

Excessive iron can generate reactive oxygen species (ROS), leading to oxidative stress that is closely associated with cardiovascular dysfunction. Iron overload was induced in male ICR mice by injection of iron sucrose (10mg/kg/day) for eight weeks. Iron overload was evidenced by increased serum iron indices. The mice developed increased blood pressure, impaired vascular function and blunted response of the autonomic nervous system. These effects were accompanied by increased malondialdehyde levels in various tissues, increased nitric oxide metabolites in plasma and urine, and decreased blood glutathione. Tetrahydrocurcumin (THU, 50mg/kg/day), deferiprone (or L1, 50mg/kg/day) or both was orally administered throughout the period of iron sucrose injection. The treatments significantly alleviated the deleterious cardiovascular effects of iron overload, and were associated with modulation of nitric oxide levels. An imbalance between endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) expression in response to iron overload was normalized by THU, L1 or the combination treatment. Moreover, the treatment decreased the upregulated expression levels of gp91phox, p47phox and HO-1. The combination of THU and L1 exerted a greater effect than THU or L1 monotherapy. These results suggest beneficial effects of THU and L1 on iron-induced oxidative stress, hypertension, and vascular dysfunction.

Regional Fluctuation in the Functional Consequence of LINE-1 Insertion in the Mitf Gene: The Black Spotting Phenotype Arisen from the Mitfmi-bw Mouse Lacking Melanocytes.

Microphthalmia-associated transcription factor (Mitf) is a key regulator for differentiation of melanoblasts, precursors to melanocytes. The mouse homozygous for the black-eyed white (Mitfmi-bw) allele is characterized by the white-coat color and deafness with black eyes due to the lack of melanocytes. The Mitfmi-bw allele carries LINE-1, a retrotransposable element, which results in the Mitf deficiency. Here, we have established the black spotting mouse that was spontaneously arisen from the homozygous Mitfmi-bw mouse lacking melanocytes. The black spotting mouse shows multiple black patches on the white coat, with age-related graying. Importantly, each black patch also contains hair follicles lacking melanocytes, whereas the white-coat area completely lacks melanocytes. RT-PCR analyses of the pigmented patches confirmed that the LINE-1 insertion is retained in the Mitf gene of the black spotting mouse, thereby excluding the possibility of the somatic reversion of the Mitfmi-bw allele. The immunohistochemical analysis revealed that the staining intensity for beta-catenin was noticeably lower in hair follicles lacking melanocytes of the homozygous Mitfmi-bw mouse and the black spotting mouse, compared to the control mouse. In contrast, the staining intensity for beta-catenin and cyclin D1 was higher in keratinocytes of the black spotting mouse, compared to keratinocytes of the control mouse and the Mitfmi-bw mouse. Moreover, the keratinocyte layer appears thicker in the Mitfmi-bw mouse, with the overexpression of Ki-67, a marker for cell proliferation. We also show that the presumptive black spots are formed by embryonic day 15.5. Thus, the black spotting mouse provides the unique model to explore the molecular basis for the survival and death of developing melanoblasts and melanocyte stem cells in the epidermis. These results indicate that follicular melanocytes are responsible for maintaining the epidermal homeostasis; namely, the present study has provided evidence for the link between melanocyte development and the epidermal microenvironment.

Induction of MITF expression in human cholangiocarcinoma cells and hepatocellular carcinoma cells by cyclopamine, an inhibitor of the Hedgehog signaling.

Microphthalmia-associated transcription factor (MITF) is a key regulator of differentiation of melanocytes and retinal pigment epithelial cells, but it also has functions in non-pigment cells. MITF consists of multiple isoforms, including widely expressed MITF-A and MITF-H. In the present study, we explored the potential role played by the Hedgehog signaling on MITF expression in two common types of primary liver cancer, using human cholangiocarcinoma cell lines, the KKU-100 and HuCCT1, along with the HepG2 human hepatocellular carcinoma cell line. Importantly, cholangiocarcinoma is characterized by the activated Hedgehog signaling. Here we show that MITF-A mRNA is predominantly expressed in all three human liver cancer cell lines examined. Moreover, cyclopamine, an inhibitor of the Hedgehog signalling, increased the expression levels of MITF proteins in HuCCT1 and HepG2 cells, but not in KKU-100 cells, suggesting that MITF expression may be down-regulated in some liver cancer cases.

Microphthalmia-associated transcription factor is expressed in projection neurons of the mouse olfactory bulb.

Microphthalmia-associated transcription factor (Mitf) is a key regulator for differentiation of the neural crest-derived melanocytes. Mitf consists of multiple isoforms, including melanocyte-specific Mitf-M and widely expressed Mitf-A and Mitf-H. Mitf mRNAs are also expressed in the brain, although the identity of Mitf-expressing cells remains unknown. We therefore aimed to identify Mitf-expressing cells in the brain. By the immunohistochemical analysis, we detected Mitf-expressing cells only in the olfactory bulb of the C57BL/6 mouse. The Mitf-expressing cells were then identified as projection neurons, mitral cells and tufted cells, both of which receive the signal from the olfactory neurons and transmit the information to the olfactory cortex. Real-time RT-PCR analysis showed the expression level of Mitf-M mRNA was comparable to the expression levels of Mitf-A and Mitf-H mRNAs in the olfactory bulb. We then analyzed Mitf-expressing neurons in the olfactory bulb of the homozygous black-eyed white (Mitf(mi-bw)) mouse that is characterized by the lack of melanocytes. Mitf was expressed in mitral cells and tufted cells in the olfactory bulb of the Mitf(mi-bw) mouse, thereby excluding the contribution of melanocytes to the detected expression of Mitf-M. In conclusion, Mitf, including Mitf-M, is expressed in mitral cells and tufted cells of the olfactory bulb.

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.

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.

Insertion of long interspersed element-1 in the Mitf gene is associated with altered neurobehavior of the black-eyed white Mitf(mi-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 (Mitf(mi-bw) ) allele is characterized by white-coat color and deafness with black eye, due to the loss of melanoblasts during embryonic development. The Mitf(mi-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.

The Great East-Japan Earthquake and devastating tsunami: an update and lessons from the past Great Earthquakes in Japan since 1923.

Japan has a long history of fighting against great earthquakes that cause structural damage/collapses, fires and/or tsunami. On March 11, 2011 at 14:46 (Friday), the Great East-Japan Earthquake (magnitude 9.0) attacked the Tohoku region (northeastern Japan), which includes Sendai City. The earthquake generated a devastating tsunami, leading to unprecedented disasters (~18,500 victims) in coastal areas of Iwate, Miyagi and Fukushima prefectures, despite the fact that people living in the Tohoku region are well trained for tsunami-evacuation procedures, with the mindset of "Tsunami, ten-den-ko." This code means that each person should evacuate individually upon an earthquake. Sharing this rule, children and parents can escape separately from schools, houses or workplaces, without worrying about each other. The concept of ten-den-ko (individual evacuation) is helpful for people living in coastal areas of earthquake-prone zones around the world. It is also important to construct safe evacuation centers, because the March 11(th) tsunami killed people who had evacuated to evacuation sites. We summarize the current conditions of people living in the disaster-stricken areas, including the consequences of the Fukushima nuclear accident. We also describe the disaster responses as the publisher of the Tohoku Journal of Experimental Medicine (TJEM), located in Sendai, with online support from Tokyo. In 1923, the Great Kanto Earthquake (magnitude 7.9) evoked a massive fire that destroyed large areas of Tokyo (~105,000 victims), including the print company for TJEM, but the Wistar Institute printed three TJEM issues in 1923 in Philadelphia. Mutual aid relationships should be established between distant cities to survive future disasters.

Otx2 is involved in the regional specification of the developing retinal pigment epithelium by preventing the expression of sox2 and fgf8, factors that induce neural retina differentiation.

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.

Coordinated expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 and heme oxygenase 2: evidence for a regulatory link between glycolysis and heme catabolism.

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.

Impaired development of melanoblasts in the black-eyed white Mitf(mi-bw) mouse, a model for auditory-pigmentary disorders.

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.

Lipocalin-type prostaglandin D synthase as a marker for the proliferative potential of melanocyte-lineage cells in the human skin.

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 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.