The 24th Annual Meeting of the Japanese Society for Pigment Cell Research (JSPCR-2012)


Thinking about pigment cells, at the margin of Lake Biwa, a much younger ancient lake

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November 24 & 25, 2012

Nagahama Institute of Bio-Science and Technology Nagahama, Shiga, Japan

President

Takahiro Kunisada

Secretary /  Treasurer

Chikako Nishigori

Local Advisory Committee

Hiroaki Yamamoto

Sponsors

CaHC

Kracie Home Products, Ltd

NIPPON MENARD COSMETIC CO., LTD.

NIHON L’OREAL

Olympus Medical Science Sales Corp.

POLA CHEMICAL INDUSTRIES, INC.

Shiseido Co.,Ltd.

TOKIWA Pharmaceutical Co. Ltd

Toray Industries, Inc.

WAKENYAKU CO. LTD

Important information on the JSPCR meetings, IFPCS council meeting and IFPCS workshop 2012

Date and Venue

November 23 (Fri) at Nagahama Institute of Bio-Science and Technology (http://www.nagahama-i-bio.ac.jp/english/index.html) in Japanese “Nagahama baio daigaku”

11 / 23 16:00 – JSPCR Board meeting at Nagahama Institute of Bio-Science and Technology

11 / 23 18:30 – JSPCR board & IFPCS council dinner at Nagahama Royal Hotel (http://www.daiwaresort.co.jp/english/14_nagah.html)

Accommodation for IFPCS council members will be at Nagahama Royal Hotel.

November 24 (Sat) at Nagahama Institute of Bio-Science and Technology

Shuttle Bus service will be provided from Nagahama Royal Hotel to Nagahama Institute of Bio-Science and Technology.

11 / 24 (Sat) IFPCS workshop & JSPCR annual meeting

 IFPCS WS session 1 Color pattern formation

 IFPCS WS session 2 Pigmentary disorders I

 IFPCS WS session 3 Tachibana memorial

11 / 24 (Sat) JSPCR council meeting

11 / 24 (Sat) IFPCS council meeting

11 / 24 (Sat) Social Gathering – IFPCS council members & JSPCR members (at Nagahama Royal Hotel)

Shuttle bus service will be provided from Nagahama Institute of Bio-Science and Technology to Nagahama Royal Hotel

11 / 25 (Sun) JSPCR General meeting at Nagahama Institute of Bio-Science and Technology

11 / 25 (Sun) IFPCS workshop & JSPCR annual meeting

Shuttle bus service will be provided from Nagahama Royal Hotel to Nagahama Institute of Bio-Science and Technology

 IFPCS WS session 4 Pigmentary disorders II

 IFPCS WS session 5 Melanoma science (Research)

 IFPCS WS session 6 Developmental biology of pigment cells

Brief Agenda

http://www.nagahama-i-bio.ac.jp/24thjspcr/en-program.html

More detailed information will be announced later.

Friday, November 23, 2012
16:00–17:30 JSPCR Board Meeting
Saturday, November 24, 2012
9:00–9:15 Opening Remarks
9:15–10:25 IFPCS Work Shop Session 1 Color pattern formation
IS-1 An update on mouse models of albinism and collaborative efforts for the systematic genetic diagnosis of all known forms of albinism
Lluis Montoliu
IS-2 Roles of Rab small GTPases in microtubule-dependent melanosome transport in melanocytes
Mitsunori Fukuda
OP-1 Innate immune regulation of melanogenesis in human melanocytes; TLR2 and TLR3 ligands participate in the synthesis and transportation of melanin
Saaya Koike, Kenshi Yamasaki, Setsuya Aiba
OP-2 Dermal fibroblasts uptake multiple melanosome-containing globules in a manner distinct from keratinocytes
Hideya Ando, Yusuke Higashi, Kaoru Akiyama, Masayuki Yagi, Masaaki Ito, Masamitsu Ichihashi
10:25–10:40 Break
10:40–12:15 Oral session I Melanins and Melanosomes
OP-3 Ultraviolet A (UVA)-induced oxidative degradation of melanin
Kazumasa Wakamatsu, Yukiko Nakanishi, Narimi Miyazaki, Ludger Kolbe, Shosuke Ito
OP-4 Thermally-induced cross-linking of dihydroxyindole moiety in fossil eumelanin
Shosuke Ito, Kazumasa Wakamatsu, Keely Glass, John D. Simon
OP-5 Conformational and vibrational spectroscopic assessment of dimers and trimers consisting of 5,6-dihydroxyindole-2-carboxylic acid
R. Hyogo, C. Natori, A. Nakamura, H. Okuda, M. Ojika, K. Wakamatsu, S. Ito, T. Sota
OP-6 Structural and vibrational analysis of dimers of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid from quantum chemical calculations
H. Okuda, R. Hyogo, K. Wakamatsu, S. Ito, T. Sota
OP-7 Structural analysis of 5,6-dihydroxyindole tetramers from quantum chemical calculations
Y. Masuda, H. Okuda, K. Koike, K. Monda, T. Nakamura, K. Wakamatsu, S. Ito, T. Sota
OP-8 Ultrastructural analysis of isolated melanosomes
Shuuichi Akazaki, Yujiro Nakano, Toshie Takahashi, Tomoki Nishida, Hirotarou Mori, Akio Takaoka2, Hitomi Aoki, Huayua Chen, Takahiro Kunisada, Kenzo Koike
12:15–13:30 Lunch/JSPCR Council Meeting
13:30–15:30 IFPCS Work Shop Session 2 Pigmentary disorders I
IS-3 α-MSH-mediated lipid signalling and vitiligo
V. Maresca, B. Bellei, M. Ottaviani, M.L. Dell’Anna, M. Picardo
IS-4 Vitiligo: new questions from clinical reseach
A. Taïeb, T. Jouary, K. Boniface, J. Seneschal, M. Cario-André, D. Mossalayi, K. Ezzedine
IS-5 New concept of melanocyte dysfunction in vitiligo vulgaris and tuberous sclerosis complex: role of Th17 cells and therapeutic implication
I. Katayama, Y. Kotobuki, D. Tsuruta, L. Yang, H. Murota, S. Itoi1, M. Nishioka, A. Tanaka, M. Tanaka, M. Wataya-Kaneda, A. Tanemura
OP-9 Morphological and ultrastructual assessment for lesional activation of dendritic cells in vitiligo vulgaris
Atsushi Tanemura, Saori Itoi, Yorihisa Kotobuki, Mari Wataya-Kaneda, Daisuke Tsuruta, Masamitsu Ishii, Ichiro Katayama
OP-10 A case of Langerhans cell histiocytosis with atypical skin manifestations of bulla, erythema, and depigmentation
Aya Takahashi, Mari Wataya-Kaneda, Hiroyuki Murota, Risa Matsumura, Hideaki Ota, Ichiro Katayama
OP-11 A case of hyperpigmentary mosaicism which localized at right leg, after several years some of macules spontaneously disappeared and others changed into hypopigmented macules with hypertrichosis and dyskeratosis
Takakazu Shibata
OP-12 Two cases of vitiligo universalis successfully treated with topical application of monobenzyl ether of hydroquinone
Katsuhiko Tsukamoto, Atsushi Osada, Toshio Oyama, Takayuki Minami3, Yoshio Kanemaru
15:30–15:45 Break
15:45–17:30 Oral session II Pigmentation
OP-13 Arenarol isolated from a marine sponge abrogates endothelin-1-stimulated expression of melanocyte-specific proteins by interrupting MEK phosphorylation in normal human melanocytes
Bong-keun Choi, Takeshi Fujiwara, Akihiko Kanamoto, Byung-yoon Cha, Je-tae Woo, Makoto Ojika, Genji Imokawa
OP-14 Low-molecular-weight polyphenol (Oligonol) suppresses production of melanin and reactive oxygen species in melanocytes
Kazuya Hagiwara, Masae Okura, Tokimasa Hida, Akihiro Yoneta, Yasuyuki Sumikawa, Toshiharu Yamashita
OP-15 Modulation of DKK1 as a novel approach to skin lightening
Sunghan Yim, Qi Hong, and Uma Santhanam
OP-16 Diacylglycerol kinase regulates tyrosinase expression and function in human melanocytes
Masakazu Kawaguchi, Julio C. Valencia, Takeshi Namiki, Tamio Suzuki, Vincent J. Hearing
OP-17 Paracrine interaction between UVB-exposed human keratinocytes and human melanocytes in co-culture system with cell insert leading to an increased expression of tyrosinase and its blockade by Wetherferin A
Takao Niwano, Hiroaki Nakajima, Genji Imokawa
OP-18 Effect of photo-aging on melanocyte motility
Masaki Yoshida and Nobuo Nagai
OP-19 Skin pigmentation and urinary 8-OHdG levels in humans
M. Iida, I. Yajima, N. Ohgami, M. Tamaki, M. Kato
17:30–17:45 Break
17:45–18:15 IFPCS Work Shop Session 3 Tachibana memorial
KL In memory of Dr. Masayoshi Tachibana: the Takeuchi Medalist of 2002
Kazuhisa Takeda
18:50 Get together for dinner
Sunday, November 25, 2012
8:30–9:00 JSPCR General meeting
9:05–11:05 IFPCS Work Shop Session 4 Pigmentary disorders II
IS-6 Regulation of interfollicular epidermal stem cells in the skin and possible implication in melasma
K.C. Park, H.R. Choi, S.H. Yang, H.S. Lee
IS-7 Lichen planus pigmentosus: where do we stand?
Davinder Parsad
IS-8 Recent proceedings in hereditary hypopigmentary disorders
Tamio Suzuki
OP-20 OCA2 polymorphisms are associated with skin color and risk of skin cancer in the Japanese population
Junko Yoshizawa, Yuko Abe, Yutaka Hozumi, Naoki Oiso, Tomohiko Narita, Akira Kawada, Kazuyoshi Fukai, Tomonori Motokawa, Kazumasa Wakamatsu, Shosuke Ito, Tomohiro Nakamura, Gen Tamiya, Tamio Suzuki
OP-21 A case report of Hermansky-Pudlak syndrome in Japan, harboring novel mutations in the HPS1 gene
Seiji Takeuchi, Yuko Abe, Taku amada, Seiji Kawano, Yutaka Hozumi, Tamio Suzuki, Chikako Nishigori
OP-22 A variant of linear atrophoderma of Moulin: hyper- and hypopigmented linear atrophoderma with peculiar area cutanea and lentiginosis following Blaschko’s lines
N. Oiso, A. Kawada (Dr. Oiso is a 2010 JSPCR Incentive Award Winner (one of two recipients))
OP-23 In vitro analysis of read-through effect of aminoglycosides to tyrosinase R278X nonsense mutation in melan-c cells
K. Fukai, H. Kunimoto, K. Nakajima, T. Suzuki, M. Ishii
11:05–11:20 Break
11:20–12:15 IFPCS Work Shop Session 5 Melanoma Research
IS-9 Lack of PTEN overcomes senescence barrier and promotes melanoma metastasis on a NRAS background
Lionel Larue
IS-10 Tyr705 phosphorylation and Ser727 phosphorylation in STAT3 have their own roles and regulation mechanisms in melanocytes and melanoma cells
M. Oka, M. Sakaguchi, T. Fukumoto, T. Iwasaki, Y. Fukami, C. Nishigori (Dr. Oka is a recipient of the JSPCR Research Fund 2010 (one of two recipients))
OP-24 Function of GNG2 in malignant melanoma
Ichiro Yajima, Mayuko Kumasaka, Nobutaka Ohgami and Masashi Kato
12:15–13:20 Lunch
13:20–14:50 IFPCS Work Shop Session 6 Genetics and Developmental Biology of Pigment Cells – 1
IS-11 Identification of melanocyte stem cells in eccrine glands
Takahiro Aoto, Natsuko Okamoto, Hisashi Uhara, Miyachi Yoshiki, Toshiaki Saida, Emi K. Nishimura
IS-12 Keratinocyte stem cells as a primary target for radiation-induced hair graying
H. Aoki, T. Kunisada (Dr. Aoki is a 2010 JSPCR Incentive Award Winner (one of two recipients))
IS-13 A rapid melanocyte-specific transgenesis system for phenotype-based gene function discovery
Masatake Osawa and Dongsoo Lee
OP-25 Endogenous retrovirus insertion in the Kit gene confers a hooded pattern of coat color to laboratory rats
T. Kuramoto, S. Nakanishi, B. Voigt, T. Serikawa
OP-26 Hyperpigmentation of internal organs (Fibromelanosis) in the Silky chicken is highly-influenced by EDNRB2, Mc1r and Id genes
Toyoko Akiyama, Ai Shinomiya, Keiji Kinoshita, Makoto Mizutani, and Yoichi Matsuda
14:50–15:05 Break
15:05–16:40 IFPCS Work Shop Session 6 Genetics and Developmental Biology of pigment cells – 2
OP-27 Melanocyte-specific Mitf-M is a key regulator for development of melanoblasts: lessons from the black-eyed white Mitf mi-bw mouse
Kazuhisa Takeda, Hiroki Hozumi, Yasuko Yoshida-Amano, Atushi Higashitani, Hiroaki Yamamoto, Shigeki Shibahara
OP-28 Mechanism of regulation of the proliferation and differentiation of melanocytes by a new mutation of mouse ruby-eye 2, ru2 d /Hps5 ru2-d
Tomohisa Hirobe (Dr. Hirobe is a recipient of the JSPCR Research Fund 2011 (one of two recipients))
OP-29 Effects of ionizing radiations on the development of mouse neural crest cells and melanocyte stem cells in the hair bulge
Tomohisa Hirobe and Kimihiko Sugaya
OP-30 Phenotypic characterization and genetic analysis of a novel spontaneous mutant mouse displaying age-related changes to the eye and coat colors
A. Ishikawa
OP-31 Analysis of endothelin receptor B expressed in spiral ganglion neurons
Nobutaka Ohgami, Machiko Iida, Ichiro Yajima, Masashi Kato
OP-32 Involvement of Mitf and its possible regulators in the retinal pigment epithelium development
D. Nishihara, A. Kawasaki-Nishihara, H. Nakamura, H. Yamamoto
16:40 Closing Remarks
Adjourn

KL

In memory of Dr. Masayoshi Tachibana: the Takeuchi Medalist of 2002

K. Takeda

Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi, Japan

Unfortunately, in May 2005, we lost a prominent researcher, Dr. Masayoshi Tachibana, who was an important contributor to clarify the function of microphthalmia-associated transcription factor (MITF), a master gene for the development of pigment cells, including melanocytes and retinal pigment epithelial cells. He was an otorhinolaryngologist and a researcher of the National Institute on Deafness and Other Communication Disorders in the National Institutes of Health (USA). He was therefore very interested in a mutant mouse, now well known as Mitfvga9, which showed a white coat color, deafness and microphthalmia (small eye). These phenotypes were almost the same as those of a well-known mutant mouse, microphthalmia (mi). Mitfvga9 mouse was one of the lines containing the transgene, β-galactosidase fused with the mouse vasopressin gene promoter, and under investigation by Dr. Heinz Arnheiter et al (National Institute of Neurological Disorders and Stroke). Dr. Tachibana showed the anatomical abnormalities of cochlea in Mitfvga9 mouse, in collaboration with Dr. Arnheiter et al. (1992). In 1993, Dr. Arnheiter et al identified the mouse mi gene, in which the transgene was inserted in the Mitfvga9 mouse. Then in 1994, Dr. Tachibana identified the human homologue of the mouse mi gene, and called it microphthalmia-associated transcription factor (MITF). Soon after that, Dr. Reed et al found that MITF is the responsible gene for Waardenburg syndrome (WS) type 2 (1994). Further, Dr. Tachibana intensively investigated the molecular characterization of MITF, and he showed that ectopic expression of MITF converts fibroblasts to cells with melanocyte characteristics (1996). Moreover, he focused on the molecular basis of the haploinsufficiency of WS2 (1996), MITF phosphorylation by glycogen synthase kinase 3 (2000), and transcriptional regulation of the MITF gene by PAX3, the responsible gene for WS1 (1998). Based on these findings, he was awarded the Takeuchi Medal in 2002. I worked together with him as a visiting fellow of the NIH in 1994–1997. He always had a passion toward a research. I regret his death heartily.

IS 1

An update on mouse models of albinism and collaborative efforts for the systematic genetic diagnosis of all known forms of albinism

A. Fernández1,2, E. Moltó1,2,3, M. Cantero2, M. Martínez2, L. Montoliu1,2

1CIBER de Enfermedades Raras, ISCIII, Madrid, Spain; 2Centro Nacional de Biotecnología-CSIC, Madrid, Spain; 3Universidad de Castilla-La Mancha, Toledo, Spain

Albinism is a rare genetic condition associated to a variable hypopigmentation phenotype, which can affect the pigmentation of only the eyes or both the eyes and the skin/hair, resulting in ocular (OA) or oculocutaneous albinism (OCA). Four forms of OCA and one of OA are known, associated to TYR (OCA1), OCA2 (OCA2), TYRP1 (OCA3), SLC45A2 (OCA4) and GPR143 (OA1) loci, respectively. Additional rarest syndromic forms of albinism, affecting other organs, can be grouped in the Hermansky-Pudlak syndrome (HP1-9) and the Chediak-Higashi syndrome (CHS1). In summary, a total of 15 genes are currently associated to various types of albinism. Murine counterparts for each and every of these 15 loci exist, and, hence, mouse mutant models of all these different types of albinism have been detected and/or produced and have served to our understanding of the albinism phenotype and the associated visual deficits, commonly associated in all types of albinism. In addition, for mouse models of OCA1, hearing deficits have been also reported. In this talk, several observed spontaneous and genetically-modified mouse mutant mice produced will be reviewed and discussed, focusing in those that have mostly contributed to our current knowledge of the albino condition.

In addition, and thanks to international collaborative efforts that have been instrumental in the validation phases of the experiments, we have initiated an approach to eventually attempt to genotype all known genetic mutations associated to albinism in any of the 15 loci mentioned above. Genetically diagnosed samples, from albino patients from France (in Bordeaux: Alain Taïeb, Benoît Arveiler; in Marseille: Robert Aquaron), Italy (in Milan: Vittoria Schiaffino), Spain (in Madrid: Carmen Ayuso) and Japan (in Yamagata: Tamio Suzuki), have served to define a set of mutations that can now be easily detected thanks to sophisticated innovative equipment, in collaboration with the laboratory of Angel Carracedo (University of Santiago de Compostela, Spain). Examples of these efforts will be presented and discussed during this lecture.

IS 2

Roles of Rab small GTPases in microtubule-dependent melanosome transport in melanocytes

M. Fukuda

Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi, Japan

Melanosomes are specialized organelles that synthesize and store melanin pigments in melanocytes. Melanosomes are formed and mature around the nucleus of the melanocyte and are transported to the cell periphery along two components of the cytoskeleton, microtubules and actin filaments. Mature melanosomes are first transported to the peripheral area of the cell by long-range, bidirectional, microtubule-dependent movements and are then transferred to actin filaments and carried by unidirectional, local movement at the cell periphery. These melanosome movements are thought to be essential for skin pigmentation in mammals, because defects in the transport of melanosomes have been shown to cause pigmentation disorders in mammals. Although the molecular mechanism of actin-dependent melanosome transport mediated by the Rab27A–Slac2-a/melanophilin–myosin Va complex has already been determined, almost nothing is known about the key factors involved in microtubule-dependent melanosome transport in melanocytes. In this study, we performed genome-wide screening for Rab proteins that are involved in microtubule-dependent melanosome transport by expressing 60 different constitutive active (and negative) mutants and succeeded in identifying Rab1A and Rab36 as novel regulators for melanosome transport on microtubules (J. Cell Sci., 2012; J. Biol. Chem., 2012; J. Cell Sci., in press). We discuss the possible mechanisms of microtubule-dependent melanosome transport mediated by Rab1A and Rab36 in mammalian skin melanocytes based on our findings.

IS 3

α-MSH-mediated lipid signalling and vitiligo

V. Maresca, B. Bellei, M. Ottaviani, M. L. Dell’Anna, M. Picardo

San Gallicano Dermatologic Institute, Rome, Italy

In association with the cAMP/PKA pathway, which is activated in response to stimulation with α-melanocyte stimulating hormone (α-MSH) and involving Microphthalmia-associated transcription factor (MITF), we have recently discovered a new α-MSH/Peroxisome proliferator activated receptor-γ (PPAR-γ) connection. We have observed that this link, exerts an influence on melanogeneis and proliferation and that the pathway which drive this connection is the PI(4,5)P2/PLCβ pathway. Vitiligo is a disease which is characterized by profound alterations of melanogenic activity and by a significant reduction in the proliferative potential of melanocytes. In primary cultures of melanocytes obtained from non lesional skin of vitiligo affected patients, compared to primary cultures of normal melanocytes, we have recently observed an altered proliferative behavior in response to α-MSH, that was not associated with alterations of the cAMP/PKA pathway and MITF response. These results suggest the investigation of new mechanisms of disease, and suggest that alterations in lipid transduction pathways may play a role in the pathogenesis of vitiligo.

IS 4

Vitiligo: new questions from clinical research

A. Taïeb 1,2,3, T. Jouary1,2,3, K. Boniface3, J. Seneschal1,2,3, M. Cario-André2,3, D. Mossalayi3, K. Ezzedine1,2,3

1Department of Dermatology and Pediatric Dermatology; 2National Reference Center for Rare Skin Disorders; 3Inserm U 1035, Université Bordeaux 2, France

In the last 5 years we have studied prospectively a large cohort of children and adults with vitiligo. Clinical and pathological observations made in this group of patients have been of major importance to direct our bench research. In particular, the relation of segmental to nonsegmental vitiligo has been questioned, new forms of vitiligo have been delineated, and the importance of factors affecting both immune responses (autoimmune and atopic diathesis) and melanocyte survival (premature hair graying) in patients and families have been underlined. Koebner's phenomenon and its role in primary melanocytorrhagy versus relation to inflammatory progression of disease are currently investigated. Better nomenclature and outcome measures to perform muticenter randomized trials are future goals of our group within the Vitiligo European task force, now joined by other international colleagues since last year Vitiligo Global Issues Consensus Conferences held in Seoul and Bordeaux. Based on a better delineation and understanding of human vitiligo, assays using in vitro human epidermal reconstructs and animal models may pave the way to much needed breakthrough therapies.

IS 5

New concept of melanocyte dysfunction in vitiligo vulgaris and tuberous sclerosis complex: role of Th17 cells and therapeutic implication

I. Katayama 1, Y. Kotobuki1, D. Tsuruta2, L. Yang1, H. Murota1, S. Itoi1, M. Nishioka1, A. Tanaka1, M. Tanaka1, M. Wataya-Kaneda1, A. Tanemura1

1Department of Dermatology Integrated Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; 2Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan

We have recently reported novel mechanisms of maintenance of melanocyte dysfunction in vitiligo vulgaris (VV) (Kotobuki et al. Pigment Cell Melanoma Res. 2012;25:219). Th17 cells significantly infiltrated vitiliginous skin associated with STAT3 activation in lesional keratinocytes and activated Langerhans cells located to the basal layer of the epidermis in VV. We also demonstrated the positive link between STAT3 activation and Th17 Cell infiltration to the lesional skin in VV (Tanemura et al. J Dermatol Sci. 2012;67:207). Th17-related cytokines significantly downregulated TRP, DCT and MITF expression and melanin production in vitro without apoptosis or necrosis of melanocytes. TNFα overexpressed in the lesional keratinocytes significantly induced IL17A in both keratinocytes and fibroblasts. These results suggest that epidermal and dermal cells contribute to melanocyte dysfunction through Th17 cell infiltration in VV although game players in the induction of TNFα in initiation and maintenance of melanocyte dysfunction in VV are not fully identified in the present study. Autoimmune mechanisms including CD8 related-melanocyte destruction or abnormal responses of skin resident cells to danger signals such as ultraviolet light, microorganisms or some chemical substances might trigger an abnormal innate immune response with TNFα overexpression in the skin.

In addition to VV, surprisingly, similar results were obtained in the hypomelanotic macules in tuberous sclerosis complex (TSC) (manuscript in submission). We have recently reported that rapamycin, a known mTOR inhibitor, significantly improved hypomelanotic macules in TSC (Wataya-Kaneda et al. Arch Dermatol 2012;148:138). Taken together, these results suggests that similar mechanisms are governed in the induction and maintenance of melanocyte dysfunctions in VV and TSC and that new therapeutic approaches using topical mTOR inhibitors might be promising not only in TSC but also in other hereditary or acquired hypopigmentary disorders.

IS 6

Regulation of interfollicular epidermal stem cells in the skin and possible implication in melasma

K. C. Park, H. R. Choi, S. H. Yang, H. S. Lee

Department of Dermatology, Seoul National University College of Medicine, Bundang Seoul National University Hospital, Gyeonggi-do, Korea

Defective barrier function is a hallmark of atopic dermatitis. But, it is also reported that lesional skin of melasma showed defective barrier function. In addition, pendulous melanocytes can be a characteristic feature of melasma which is related with reduced expression of type IV collagen along the basement membrane. Histologically, melasma is characterized by increased melanin in epidermal layer but melasma skin often has more melanin and melanophages in the dermis. All these findings suggest that melasma is not only a disease of melanocytes but also a disease of surrounding cells and microenvironment such as basement membrane abnormality.

It is believed that stem cells are under the control of surrounding environment. Because basement membrane will provide niches for the epidermal stem cells, changes in microenvironment will affect epidermal stem cells. Thus, strategies for manipulating cell to extracellular matrix interactions hold promise in preventing aging effects or controlling skin disease such as melasma. It is proposed that stem cell activation can be a new concept for the development of cosmetics. In this presentation, our study on epidermal stem cells including isolation technology, reconstruction of three dimensional skin models, and the effects of various factors on epidermal stem cells, will be presented. Our results clearly showed that stem cell activator can be useful in controlling skin aging and skin diseases including melasma.

IS 7

Lichen planus pigmentosus: where do we stand?

D. Parsad

Department of Dermatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Lichen planus pigmentosus (LPP) though a fairly common disorder of pigmentation in Indians, reports comprising a sizable number of patients are lacking in the literature. It is characterized by slate grey to brownish black pigmented macules distributed over the face and neck. The exact pathogenesis of this disorder is not known although lichenoid reaction to unknown agent/s or stimuli has been proposed.

Pigmentary disorders like ashy dermatosis, erythema dyschromicum perstans and lichen pigmentosus have been described with almost identical features in the literature. Erythema dyschromicum perstans (EDP) (ashy dermatosis) is a distinct and somewhat controversial cutaneous eruption closely linked with LPP. Distinguishing ashy dermatosis from LPP is not always easy and there are many overlapping features. Similarly, idiopathic eruptive macular pigmentation shares many clinical features with LPP

Thus LPP is disorder of great cosmetic concern and social stigma affecting quality of life and warrants further extensive study to demystify enigma surrounding LPP and closely related disorders like EDP or ashy dermatosis. There is an urgent need for consensus to define the nature and uniform label for this condition.

IS 8

Recent proceedings in hereditary hypopigmentary disorders

T. Suzuki

Department of Dermatology, Yamagata University Faculty of Medicine, Yamagata, Japan

Many specific gene products are sequentially synthesized and utilized by the melanocyte as it emigrates from its embryonic origin, migrates into specific target sites, synthesizes melanin(s) within a specialized organelle, melanosome, transfers pigment granules to neighboring cells, and responds to various exogenous cues. A mutation in many of the respective encoding genes can disrupt this process of melanogenesis and can result in hypopigmentary disorders. In other words, congenital pigmentary disorders are due to mutation(s) in various genes that cause defects in melanin synthesis, formation of the melanosomes, their transfer within melanocytes, as well as melanocyte maldevelopment. Many genes responsible for congenital pigmentary diseases have been reported.

Here, we summarize recent proceedings focusing on oculocutaneous albinism (OCA) and dyschromatosis symmetrica hereditaria (DSH). (i) OCA can be classified into two groups. One is the non-syndromic type, which is usually classified into four responsible genes. Another type is the syndromic type, which reveals not only OCA but also other symptoms, e.g. bleeding tendency, immunodeficiency. We reveal some topics found in our study, including a patient recently diagnosed as OCA3 which is very rare in non-African populations. (ii) The gene responsible for DSH has been identified as adenosine deaminase acting on RNA1 (ADAR1). The ADAR1 protein catalyzes the transformation of adenosine to inosine in double-stranded RNA substrates (so-called A-to-I editing) and is involved in various activities, such as viral inactivation, structural change of the protein, and the resultant cell survival. Some complications of DSH have been reported, and, intriguingly, several patients have been reported to develop neurological symptoms. We show new findings on ADAR1.

IS 9

Lack of PTEN overcomes senescence barrier and promotes melanoma metastasis on a NRAS background

A. Conde Perez1, C. Longvert1, G. Gros1, I. Puig1, F. Beermann2, L. Van Kempen3, L. Larue1

1Institut Curie, Centre de Recherche, CNRS UMR3347-INSERM U1021, Developmental Genetics of Melanocytes, Orsay, France; 2EPFL, Lausanne, Switzerland; 3Departments of Pathology and Oncology, Segal Cancer Centre/Jewish General Hospital, McGill University, Montreal, QC, Canada

Melanocyte transformation is a combinatorial multi-step, multi-etiological event that results in one of the most lethal types of skin cancer, melanoma, with the average 5 year survival prognosis of patients with metastatic melanoma <5%. Recent evidence has solidified the involvement of several signaling pathways with altered functions in melanoma, MAPK (NRAS and BRAF) and most notably PI3K (PTEN). Although BRAF and NRAS have been implicated in the proliferation status, PTEN`s exact role during melanoma initiation and progression remains uncertain. We aim to evaluate PTEN`s role in melanomagenesis and assert the possible cross-talk between both pathways, notably through NRAS and PTEN, in initiation/progression. Two human melanoma libraries were screened for NRAS and BRAF alterations, with concomitant loss of PTEN. Interestingly, it appears that both activation of NRAS with simultaneous loss of PTEN coexist, suggesting non-redundant functions. To further assess any functional cooperative effect, we generated mice displaying an active form of NRAS while lacking PTEN using the Cre-Lox system, with directed expression to the melanocyte lineage. Mice with both genetic aberrations had a dramatic increase in melanoma formation, and lung metastasis, with decreased latency period. These results suggest that PTEN loss and hyperactivation of NRAS cooperate, and that PTEN loss enhances melanoma initiation by bypassing the senescence barrier. Altogether, lack of PTEN with activated NRAS promotes melanoma initiation and progression.

IS 10

Tyr705 phosphorylation and Ser727 phosphorylation in STAT3 have their own roles and regulation mechanisms in melanocytes and melanoma cells

M. Oka 1, M. Sakaguchi1, T. Fukumoto1, T. Iwasaki2, Y. Fukami2, C. Nishigori1

1Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan; 2Research Center for Environmental Genomics, Organization of Advanced Science and Technology, Kobe University, Nada-ku, Kobe, Japan

The transcription factor signal transducer and activator of transcription 3 (STAT3) is activated in response to various growth factors, hormones, and cytokines and has an important role in their signaling. STAT3 has two important phosphorylation sites, Tyr705 and Ser727, for its activation. It has been generally thought that Tyr705 phosphorylation is a characteristic of STAT3 activation and that Ser727 phosphorylation is a secondary event after Tyr705 phosphorylation required for the maximal transcriptional activity of STAT3. In normal cells, the duration of STAT3 activation is temporary, usually lasting from a few minutes to several hours. However, constitutive activation of STAT3, as demonstrated by detection of Tyr705 phosphorylation, has been observed in melanomas. In addition, we have found that Ser727 is constitutively activated in normal melanocytes and melanoma cells irrespective of Tyr705 phosphorylation. These findings indicate that Ser727 phosphorylation on STAT3 is not necessarily a secondary event after Tyr705 phosphorylation and suggest that constitutive STAT3 phosphorylation at Tyr705 and Ser727 plays an important role in these cells. We will present our recent results on the role and regulation of constitutive STAT3 phosphorylation at Tyr705 and Ser727 in melanocytes and melanoma cells.

IS 11

Identification of melanocyte stem cells in eccrine glands

T. Aoto1*, N. Okamoto2*, H. Uhara3, M. Yoshiki2, T. Saida3, E. K. Nishimura1

1Department of Stem Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; 2Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan; 3Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan

Acral volar skin is unique in that it contains abundant eccrine glands instead of hair follicles as the main skin appendage and is minimally exposed to UV sunlight. Acral melanoma is the most prevalent subtype of melanoma in the non-Caucasian population. The preferential proliferation of early acral melanoma cells along and around epidermal eccrine ducts has indicated a close association between early acral melanoma in situ and eccrine glands. However, neither the presence of melanocytic cells in the eccrine glands nor the precise origin of these melanoma cells have been identified to date. Here, we report the identification of eccrine gland melanocyte stem cells in mouse acral skin. We found that unpigmented melanocytic cells reside in the secretary portion of eccrine glands using lineage-tagged H2B-GFP reporter mice and have characterized the development and maintenance of this population in adult mouse skin. These melanoblasts are normally kept in an immature and slow-cycling state but are able to self-renew and provide transit amplifying progeny which migrates upward toward the epidermis concurrently with the synthesis and deposition of melanin pigment in response to stresses including ionizing irradiation. These findings indicate that this population possesses several features consistent with adult stem cells. The existence of an acral melanocyte stem cell population within the eccrine gland might explain the preferential proliferation of early acral melanoma cells around eccrine glands during melanomagenesis.

IS 12

Keratinocyte stem cells as a primary target for radiation-induced hair graying

H. Aoki, T. Kunisada

Department of Tissue and Organ Development, Regeneration, and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan

A niche function for adult MSCs is exerted by hair follicle keratinocyte stem cells (KSCs) located in the bulge region. Radiation-induced hair graying is accompanied by an ectopic differentiation of follicular melanocyte stem cells (MSCs) in their niche located at the bulge region (Inomata et al. 2009). In post-IR mouse skin, we first found that IR-induced genotoxic stress detected by immunohistochemical staining of γH2AX as a genotoxicity marker was observed not only in c-Kit positive MSCs but also in CD34 positive follicular KSCs as well as inter-follicular keratinocytes. Only KSCs prepared from irradiated mice but not MSCs were functionally affected by IR as indicated by their reduced colony forming activity in culture and the delayed hair cycle in vivo. The effect of IR on KSCs was temporary and recovered to the normal level within a few days after irradiation both in vivo and in vitro. We further tested the potentials of MSCs after irradiation to proliferate and differentiate to melanocytes using a co-culture system on ST2 stromal cells as an alternative niche for MSCs and found that the stemness of MSCs was persistently maintained. Finally, the growth and differentiation of melanocytes (including MSCs) were suppressed with a co-culture of irradiated keratinocytes (including KSCs) in comparison with that without irradiation. These observations led us to conclude that the primary target of IR sufficient to induce hair graying is not MSCs but follicular KSCs.

IS 13

A rapid melanocyte-specific transgenesis system for phenotype-based gene function discovery

M. Osawa, D. Lee

Graduate School of Medicine, Gifu University, Gifu, Japan

The melanocyte affords an advantageous model for studying gene function through phenotype-based analysis, since alterations in genes involved in melanocyte regulation (i.e. in survival, proliferation, differentiation, cell migration, organelle transport, or stem cell controls) are easily identifiable as pigmentary phenotypes in the mouse. Traditionally, such gene alterations have been achieved by spontaneous or induced mutagenesis or gene engineering using gene targeting or transgenic technology. However, despite a number of recent methodological advances, the generation of genetically altered mice is still laborious and time-consuming, which largely hampers in vivo gene function assignment. Here, we have developed a melanocyte-specific transgenesis system, by which rapid gene functional assessment is allowed in mice. To establish this system, the following three technological features were combined: (i) derivation of an ES cell line from Tyr-Cre;K14-SCF transgenic mice, enabling Cre-regulatable melanocyte-specific genetic modification and resulting phenotype-based assessment in humanized skin pigmentation model mice; (ii) application of the ‘ES-mouse’ generation methodology, by which mice fully derived from genetically modified ES cells are generated in the F0 generation, permitting immediate assessment of the pigmentary phenotype resulting from the genetic alteration; (iii) use of a recombinase-mediated cassette exchange (RMCE)-based approach in ES cells to integrate a Cre-regulatable transgene cassette into the trans-criptionally permissive Rosa26 locus, ensuring stable and melanocyte-specific gene silencing or overexpression. As a proof-of-concept study, we generated transgenic lines carrying shRNA targeting the Tyr, Mitf, Bcl2 or RBP-J genes and found that each transgenic mice faithfully recapitulated the pigmentary phenotype of the corresponding gene knockout mice. Hence, these results clearly demonstrate the robustness of the transgenesis system in delineating gene function in the melanocyte lineage in vivo. Overall, our study suggests the broad applicability of the transgenesis system for understanding the molecular basis of melanocyte-related diverse biological phenomena, such as cell survival, proliferation, migration, stem cell regulation, and cancerous transformation to malignant melanomas.

OP 1

Innate immune regulation of melanogenesis in human melanocytes; TLR2 and TLR3 ligands participate in the synthesis and transportation of melanin

S. Koike, K. Yamasaki, S. Aiba

Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan

In human skin, melanocytes localize in the basal layer of the epidermis where innate immune stimuli such as microbe molecules affect the fate of the cells. However, little knowledge is available about innate immune functions for melanocytes and melanogenesis. To investigate the influence of innate immunity on melanogenesis, we stimulated normal human melanocytes with Toll-like receptor (TLR) ligands and measured extracellular and intracellular melanin and genes related to the melanogenesis. The TLR2 ligand HKLM increased the amount of extracellular melanin. Consistent with the increase in melanin release, TLR2 ligands increased genes related to melanogenesis such as tyrosinase and Dct. Interestingly, TLR3 ligands Poly (I:C) and Poly (I:C)-LMW increased the amount of extracellular melanin whereas TLR3 ligands little affected or decreased the amount of intracellular melanin content, tyrosinase activity, and genes related to melanogenesis. Co-culture of melanocytes with keratinocytes showed that both TLR2 and TLR3 ligands increase melanin contents in keratinocytes, indicating that the released melanin from TLRs-treated melanocytes was transported to keratinocytes. We then measured mRNA expression of Rac1, Rho A, and Rab27a, which are involved in the expansion and contraction of dendrites of melanocytes and melanosome transportation, and we confirmed that TLR3 ligands increased Rab27a gene expression in human melanocytes. These results suggested that TLR2 ligands increase the release of melanin by inducing the transcription of genes related to melanogenesis, and TLR3 ligands increase melanin release and have little affect on melanogenesis. Thus innate immune stimuli affect melanogenesis and melanin transportation from melanocytes to keratinocytes. Since not only microbe molecules but also host molecules such as heat shock proteins stimulate TLRs signaling, further study will reveal how microenvironments affect melanocyte biology.

OP 2

Dermal fibroblasts uptake multiple melanosome-containing globules in a manner distinct from keratinocytes

H. Ando 1, Y. Higashi1, K. Akiyama2, M. Yagi3, M. Ito4, M. Ichihashi5

1Department of Applied Chemistry and Biotechnology, Okayama University of Science, Okayama, Japan; 2Hanaichi Ultrastructure Research Institute Co., Okazaki, Japan; 3Rosette Co., Tokyo, Japan; 4Department of Dermatology, Niigata University, Niigata, Japan; 5SAISEI MIRAI Clinic Kobe, Kobe, Japan

We recently found that normal human melanocytes have a potency to produce multiple melanosome-containing globules from various areas of their dendrites, especially from the dendrites penetrating through a microporous membrane filter. Here we developed a method consisting of normal human melanocytes and fibroblasts separated by a microporous membrane filter that enabled multiple melanosome-containing globules to be transferred from melanocytes to fibroblasts. In a similar co-culture method consisting of normal human melanocytes and keratinocytes, efficient transfer of multiple melanosome-containing globules from melanocytes to keratinocytes was also observed, however, the inhibition of protease activated receptor-2 by soybean trypsin inhibitor decreased the phagocytic activity of keratinocytes efficiently, while it scarcely decreased the activity of fibroblasts. In addition, keratinocytes incorporated both isolated individual melanosomes and multiple melanosome-containing globules, while fibroblasts preferred to ingest multiple melanosome-containing globules. Furthermore, the engulfing figure of multiple melanosome-containing globules into fibroblasts observed by scanning electron microscopy was different from that of keratinocytes. These results suggested that dermal fibroblasts uptake multiple melanosome-containing globules in a manner distinct from keratinocytes.

OP 3

Ultraviolet A (UVA)-induced oxidative degradation of melanin

K. Wakamatsu 1, Y. Nakanishi1, N. Miyazaki1, L. Kolbe2, S. Ito1

1Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan; 2Beiersdorf AG, R&D, Skin Research Center, Hamburg, Germany

It is known that eumelanin is photoprotective while pheomelanin is phototoxic to pigmented tissues. UVA-induced tanning seems to result from the photooxidation of pre-existing melanin and essentially contributes no photoprotection. However, data available for melanin biodegradation remain limited. In this study, we first examined the photodegradation of eumelanin and pheomelanin in human black hairs. The UVA-induced oxidative degradation of eumelanin and pheomelanin can be estimated by the Free (formed by peroxidation in situ) to Total (after hydrogen peroxide oxidation) pyrole-2,3,5-tricarboxylic acid (PTCA) and thiazole-2,4,5-tricarboxylic acid (TTCA) to 4-amino-3-hydroxyphenylalanine (4-AHP) ratio, respectively. In black hairs, the ratio of Free to Total PTCA increases with hair aging, indicating fission of the dihydroxyindole moiety. In red hairs, the ratio of TTCA to 4-AHP increases with aging, indicating the conversion from benzothiazine to benzothiazole moiety. These photodegradation of melanins were confirmed by UVA (not UVB) irradiation of melanins from mice and human hairs and synthetic eumelanin and pheomelanin. These results show that both eumelanin and pheomelanin degrade by UVA and that Free/Total PTCA and TTCA/4-AHP ratios serve as sensitive indicators of photodegradation. It is likely that the UVA irradiation actually reduces photoprotection by melanins.

OP 4

Thermally-induced cross-linking of dihydroxyindole moiety in fossil eumelanin

S. Ito 1, K. Wakamatsu1, K. Glass2, J. D. Simon2,3

1Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan; 2Department of Chemistry, Duke University, Durham, NC, USA; 3Department of Chemistry, University of Virginia, Charlottesville, VA, USA

Eumelanin pigments consist of various ratios of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). Upon alkaline peroxide oxidation, these indole moieties give rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), respectively. In a recent study we detected considerable amounts of other pyrrole acids, pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) and pyrrole-2,3,4-tricarboxylic acid (isoPTCA), among the oxidation products of fossil ink sacs >160 million years old (K. Glass et al., Proc. Nat. Acad. Sci. 109: 10218–10223, 2012). PTeCA and isoPTCA arise from the cross-linking of the DHI moiety of eumelanin at the C2 and/or C3 positions. We mimicked the process of cross-linking by heating synthetic eumelanins prepared from various ratios of DHICA and DHI at 100°C for 18 days (or at 40°C for 180 days). The heated eumelanins were analyzed after alkaline peroxide oxidation as PTCA, PDCA, PTeCA, and isoPTCA by HPLC with ultraviolet detection. Upon heating, PTCA decreased rapidly due to decarboxylation while PDCA decreased gradually. Concurrently, PTeCA increased gradually to levels close to PTCA. IsoPTCA also increased gradually at lower levels. Similar changes were observed at 40°C at a much slower rate. These findings suggest that the PTeCA/PTCA ratio may serve as a good indicator of aging (cross-linking) of eumelanin.

OP 5

Conformational and vibrational spectroscopic assessment of dimers and trimers consisting of 5,6-dihydroxyindole-2-carboxylic acid

R. Hyogo 1, C. Natori2, A. Nakamura3, H. Okuda3, M. Ojika4, K. Wakamatsu2, S. Ito2, T. Sota1,3

1Department of Electrical Engineering and Bioscience, Waseda University; 2Department of Chemistry, Fujita Health University School of Health Sciences; 3Research Institute for Science and Engineering, Waseda University; 4Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University

Eumelanin is a macromolecule produced via oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). To verify the physical and chemical properties of DHI and/or DHICA related oligomers step by step is one way to approach resolving its structure. In this lecture, we report a new methodology synthesizing underivatized DHICA dimers and trimers and their conformational and vibrational spectroscopic properties.

We synthesized and isolated 4,4′- and 4,7′-DHICA dimers, and two atropoisomers of 4,4′:7′,4″-DHICA trimer (RR (SS), RS (SR)). This is the first report of the synthesis and isolation of underivatized trimers. The chemical structure of each product was identified or reconfirmed by mass spectrometry and 1H-NMR spectroscopy. It was found from FTIR spectroscopy that all the dimers and trimers have characteristic absorption bands in the wave number region where the DHICA monomer has no absorption peak. The wave numbers of the bands depend only on the degree of polymerization. Concretely speaking, the wave number of trimers are higher than those of dimers. In order to identify the origins of these absorption bands, quantum chemical calculations were carried out by Gaussian 03 with the B3LYP/6-31++G(d,p) level of theory. The origin of each band is identified as the vibrational mode including the stretching motion of C–C bonds between the adjacent DHICA units. Conformational information in vacuo was also obtained in the course of calculations. For each dimer, the most stable structure has no hydrogen bond between the adjacent DHICA units. For two atropoisomers of the trimer, the RS (SR) configuration is more stable than the RR (SS) configuration.

OP 6

Structural and vibrational analysis of dimers of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid from quantum chemical calculations

H. Okuda 1, R. Hyogo2, K. Wakamatsu3, S. Ito3, T. Sota1,2

1Research Institute for Science and Engineering, Waseda University; 2Department of Electrical Engineering and Bioscience, Waseda University; 3Department of Chemistry, Fujita Health University School of Health Sciences

Eumelanin is a macromolecule built from 5,6-dihydroxyindole (DHI) and its 2-carboxy derivative (DHICA). In general, the quantum chemical calculation is a powerful tool to assess the three-dimensional (3D) structures of organic molecules. As an aim at understanding the eumelanin structure, in this lecture, we report the 3D structures, stabilities, and parameters, which can be compared with experimental results, of DHI dimers, DHICA dimers, and DHI-DHICA heterodimers from quantum chemical calculations.

All quantum chemical calculations have been carried out by Gaussian 03 with the B3LYP/6-31++G(d,p) level of theory. The considered molecules are 2,4′- and 2,7′-DHI dimers, 4,4′-, 4,7′-, and 7,7′-DHICA dimers, and 2,4′- and 2,7′-DHI-DHICA heterodimers.

The stabilities estimated from Gibbs free energies of all dimers are consistent with the reported isolation yields. The stabilities of DHI dimers are consistent also with the chemical degradation. All dimers are twisted by the linkage between indoles and, thus, are non-planar. The torsion angles depend on the internal indole units’ hydrogen bond and range widely, which may support the highly heterogeneous 3D structures of eumelanin. The vibrational modes including the stretching motion of C–C bonds between adjacent indole units are IR active in each dimer and cause absorption bands in the wavenumber region of 975–1015 per centimeter. The corresponding wavenumbers become larger in the order of DHI dimers, DHI-DHICA heterodimers, and DHICA dimers.

OP 7

Structural analysis of 5,6-dihydroxyindole tetramers from quantum chemical calculations

Y. Masuda 1, H. Okuda2, K. Koike3, K. Monda3, T. Nakamura3, K. Wakamatsu4, S. Ito4, T. Sota1,2

1Department of Electrical Engineering and Bioscience, Waseda University; 2Research Institute for Science and Engineering, Waseda University; 3Beauty Research Center, KAO Corp; 4Department of Chemistry, Fujita Health University School of Health Sciences

5,6-Dihydroxyindole (DHI) is one of the precursor molecules of eumelanin. However, neither the three-dimensional (3D) structure of eumelanin nor that in each stage of the polymerization process is fully understood. The quantum chemical calculation is one of the most promising tools to study this issue. In this lecture, we report the 3D structures of DHI tetramers in the course of our program to unveil the structure of eumelanin.

All calculations have been carried out by Gaussian 03 and Gaussian 09. The HF/6-31G(d) have been used for optimizing structures and the B3LYP/6-31++G(d,p) for electronic energy calculations of the corresponding structures. Since all tetramers isolated experimentally are linear polyindolyl oligomers, we have assumed the tetramers linked through 2,4′ or 2,7′ linkages in the calculations. The initial chemical formulas have been identified according to the Gibbs free energies of DHI related tautomers. We have also considered five oxidative states and searched the most stable structure in each state.

Each stable tetramer linked through 2,4′ linkages has a structure like a pitch of helix independent of the degree of oxidation. It should be noted that NH groups are located outside of each tetramer. Tetramers linked through 2,7′ linkages have such a structure like the 2,4′ linked tetramers in the higher oxidative states because the inner NH...N hydrogen bonds are allowed. However, Coulomb repulsion between protons of NH groups leads to a spatially spread structure in the lower oxidative states. The present results suggest that possible structures of DHI oligomers are not planar ones but helical ones.

OP 8

Ultrastructural analysis of isolated melanosomes

S. Akazaki 1, Y. Nakano1, T. Takahashi1, T. Nishida2, H. Mori2, A. Takaoka2, H. Aoki3, H. Chen4, T. Kunisada3, K. Koike1

1Kao Corp. BRC; 2Osaka University UHVEM; 3Gifu University Department of Tissue and Organ Development Regeneration; 4Gifu University Department of Anatomy

Melanosomes are specialized organelles in pigment cells controlling melanin biosynthesis and storage. Matured melanosomes are transferred from pigment cells to neighboring cells such as keratinocytes. Melanin contributes to the determination of hair and skin color, the biological defense against ultraviolet rays, etc. in humans. As a part of investigating the characteristics of melanosomes themselves and their role in controlling the pigment system, we purified melanosomes and successively examined their ultrastructure by scanning electron microscopy imaging and transmission electron microscopy imaging. Since the details of the molecular structure of the melanin itself are unknown, little information about the three-dimensional ultrastructure of melanosomes is available. Some reports previously have described the three-dimensional ultrastructure of melanosomes from iridal stroma or retinal pigment epithelium. In our first attempt we focused on B16 melanoma cells derived from mouse. B16 cells were cultured, homogenized and melanosome containing fraction was isolated by density gradient centrifugation method. We demonstrate results about the three-dimensional ultrastructural imaging of extracted and isolated melanosomes.

OP 9

Morphological and ultrastructual assessment for lesional activation of dendritic cells in vitiligo vulgaris

A. Tanemura, S. Itoi, Y. Kotobuki, M. Wataya-Kaneda, D. Tsuruta, M. Ishii, I. Katayama

Department of Dermatology, Osaka University Graduate School of Medicine, Osaka, Japan

Although the autoimmune theory is the most widely-held consideration cited for the existence of HLA-DR + CLA + CD8 + cell and anti-melanocyte antibodies in vitiligo vulgaris patients, this theory does not fully explain the pathogenesis for autoimmune vitiligo. We had presented the changes of dendritic cells and immune-competent cells infiltrating vitiligo skin in the last IPCC meeting. In order to hunt the cue of Langerhans cells activation we performed an electron microscopic examination of lesional and nonlesional skin and consecutively investigated the more detailed morphological changes of Langerhans cells which are representative of antigen presentation and innate immune action to pathogens in the epidermis.

To investigate the comprehensive cellular immunity including Langerhans cells involved in vitiligo vulgaris.

We performed immunohistochemical analysis for the detection of immune competent cells with the following markers; CD4, CD8, Foxp3, IL-17A, HLA-DR, CD68, CD163, and E-cadherin in vitiligo lesions by utilizing the skin tissue of 20 cases from generalized vitiligo patients. Moreover, in order to observe detailed morphological changes and the interactions of melanocytes and Langerhans cells we performed an electron microscopic analysis of six cases.

We showed higher numbers of Th17 cells and macrophages in addition to cytotoxic T cells in vitiligo lesions compared to uninvolved skin as shown in the last meeting. We also found an exclusive localization of Langerhans cells beside a disfigured melanocyte without mature melanosomes so called α-dendritic cell on the basement membrane and the elongation of their dendrites in vitiligo skin. Activated Langerhans cells with elongated dendrites overexpressed one adhesion molecule, E-cadherin.

We showed a panel of immune competent cells infiltrating vitiligo skin and propose subsequent cellular immunological events triggered by Langerhans cells activation in autoimmune vitiligo.

OP 10

A case of Langerhans cell histiocytosis with atypical skin manifestations of bulla, erythema, and depigmentation

A. Takahashi 1, M. Wataya-Kaneda1, H. Murota1, R. Matsumura2, H. Ota2, I. Katayama1

1Department of Dermatology, Integrated Medicine Graduate School of Medicine, Osaka University; 2Department of Pediatrics, Integrated Medicine Graduate School of Medicine, Osaka University

Langerhans cell histiocytosis (LCH) is a reactive condition in which a clonal population of Langerhans cells accumulate in various tissues and cause damage. The characteristic skin presentations of LCH are yellowish brown scaly papules with purpura, ultimately to become crusted nodules or erosions. We report a case of LCH with atypical skin symptoms. She was a 3 months-year-old baby, and her skin features were depigmentations with bulla and erythema like the features of incontinentia pigmenti. We made a hypothesis about the pathogenesis of depigmentation in LCH based on the previous literature.

OP 11

A case of hyperpigmentary mosaicism which localized at right leg, after several years some of macules spontaneously disappeared and others changed into hypopigmented macules with hypertrichosis and dyskeratosis

T. Shibata

Shibata Clinic of Dermatology

I`ll report a case of hyperpigmentary mosaicism which localized on the right leg, and after several years some macules spontaneously disappeared and others changed into hypopigmented macules with hypertrichosis and dyskeratosis. This patient congenitally has philloid type hyperpigmentary macules only on her right leg. According to her growth, some of these macules spontaneously disappeared and others slowly changed into hypopigmented macules. Recently, these macules present hypertrichosis and dyskeratosis with surrounding hyperpigmentation and follicular pigmentation without using a steroid ointment. This rare case cannot be described by the stereotype concept of pigmentary mosaicism. Therapies for this case are irradiation of an excimer lamp (VTRAC), applying activated vitamin D3 ointment and prostaglandin ointment and suction blister epidermal grafting therapy (SBT). I’ll consider the possible course and future therapies of this pigmentary disorder and other vitiligo vulgaris.

OP 12

Two cases of vitiligo universalis successfully treated with topical application of monobenzyl ether of hydroquinone

K. Tsukamoto 1, A. Osada1, T. Oyama2, T. Minami3, Y. Kanemaru3

1Department of Dermatology, Yamanashi Prefectural Central Hospital, Yamanashi, Japan; 2Department of Pathology, Yamanashi Prefectural Central Hospital, Yamanashi, Japan; 3Department of Pharmacy, Yamanashi Prefectural Central Hospital, Yamanashi, Japan

Vitiligo is a common depigmentation disorder occurring in about 1% of the population worldwide. The disease causes the appearance of white patches on the skin. If vitiligo involves most of the body, it might be easier to depigment the normal remaining skin rather than to attempt repigmentation. For depigmentation therapy, monobenzyl ether of hydroquinone (MBEH) remains the only drug that the Food and Drug Administration approved for depigmentation therapy within the United States. The mechanism of toxicity of MBEH has remained little understood. Recently, Hariharan et al. reported that MBEH can induce cell death because of the disintegration of cellular membranes without activating the caspase cascade or DNA fragmentation. Therefore, the death pathway may be non-apoptotic.

We treated two severe vitiligo patients with 5–20% MBEH ointment. We checked their clinical effects and discuss its mechanism with histopathological findings of treated skin.

Treatment of two patients was started with 5% MBEH ointment and the concentration was elevated stepwise to 5–20%. We checked their clinical findings once a month, and when the normal skin was bleached, a skin biopsy was taken.

Depigmentation was obtained after 3 months, and almost complete bleaching was achieved in 6 months. The findings of histopathology were loss of melanocytes and deposition of melanophages in the dermis. Also the nuclear degeneration of melanocytes was seen.

Two severe vitiligo patients were successfully treated with MBEH. Histological findings showed that the melanocyte death pathway mediated by MBEH might be not apoptotic but necrotic.

OP 13

Arenarol isolated from a marine sponge abrogates endothelin-1-stimulated expression of melanocyte-specific proteins by interrupting MEK phosphorylation in normal human melanocytes

B.-K. Choi 1, T. Fujiwara2, A. Kanamoto2, B.-Y. Cha3, J.-T. Woo3, M. Ojika1, G. Imokawa3

1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan; 2OP Bio Factory Co., Ltd., Okinawa, Japan; 3Research Institute for Biological Functions, Chubu University, Kasugai, Japan

Using the re-pigmenting process of glucosamine-induced amelanotic B16 melanoma cells, we have found that a marine sponge extract has a potent anti-pigmenting effect and finally identified arenarol as its major active compound. In human melanocytes, arenarol significantly abrogated endothelin-1 [EDN-1 (10 nM)]-stimulated expression of tyrosinase (TYR), TRP1 and DCT at the translational and enzymatic activity (only for TYR) levels, which was accompanied by the attenuation of the increased expression level of MITF protein. Analysis of EDN-1 signaling demonstrated that arenarol (3 μM) definitely suppressed EDN-1-induced phosphorylation of MEK, ERK, MITF and CREB but not of Raf-1.

In contrast, forskolin (100 μM)-induced phosphorylation of CREB was not down-regulated by arenarol (3 μM). As for the mode of action of the suppressed phosphorylation of MEK, Raf-1 activity was not inhibited by arenarol (3 μM) in vitro and a protein phosphatase inhibitor (okadaic acid) did not affect the down-regulated phosphorylation of MEK induced by arenarol in human melanocytes. The sum of these findings suggests that arenarol abrogates endothelin-1-stimulated expression of melanocyte-specific proteins by interrupting MEK phosphorylation in as yet unknown mechanisms.

OP 14

Low-molecular-weight polyphenol (Oligonol) suppresses production of melanin and reactive oxygen species in melanocytes

K. Hagiwara, M. Okura, T. Hida, A. Yoneta, Y. Sumikawa, T. Yamashita

Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan

The fruit polyphenol resveratrol is known to have an affinity to tyrosinase and inhibits its biochemical activity. To examine anti-aging and anti-melanogenesis activities of Oligonol, a lychee-derived low-molecular-weight polyphenol, we analyzed the biochemical effects of Oligonol on tyrosinase (DOPA oxidase) activity, melanogenesis and production of reactive oxygen species (ROS) in human epidermal melanocytes and melanoma cells. Melanocytes were cultured in the presence of 10–30 μg/ml arbutin, Oligonol or LFP (high-molecular weight polyphenol), and then processed for tyrosinase assay and measurement of melanin. The results indicated that, Oligonol as well as arbutin suppressed tyrosinase activity in a dose-dependent manner. Tyrosinase suppression was observed at a lower concentration of Oligonol (IC50: 194.4 μM) than of arbutin (IC50: 2252.8 μM) or vitamin C (IC50: 381.0 μM). In addition, melanocytes cultured with Oligonol contained approximately 60% less melanin than those without Oligonol. Western blot analysis detected a decrease of tyrosinase in melanocytes cultured in the presence of 10–50 μg/ml Oligonol. Moreover, ROS generation was suppressed by a low concentration (1–5 μg/ml) of Oligonol in melanocytes and SK-mel-24 melanoma cells. Suppression of ROS generation by Oligonol was not disturbed by nicotinamide (a SIRT1 inhibitor), suggesting that Oligonol suppresses ROS generation without activating SIRT1. The results obtained suggest that through anti-melanogenesis and anti-ROS generation activities, Oligonol protects epidermal melanocytes from deterioration.

OP 15

Modulation of DKK1 as a novel approach to skin lightening

S. Yim, Q. Hong, U. Santhanam

Global R&D, Avon Products, Inc., Suffern, New York

Skin lightening is a rapidly growing segment of the global beauty industry. Development of skin lightening products often requires technologies that can inhibit melanin production. Melanin synthesis in skin is regulated by paracrine factors, e.g. Endothelin-3 from keratinocytes which control key melanogenic proteins such as tyrosinase. Current skin lightening products typically target tyrosinase or paracrine factors from keratinocytes. However, paracrine signaling from the dermis has not been explored. DKK1 is a protein that is highly expressed in palmoplantar fibroblasts. It is an inhibitor of the Wnt/β-catenin/MITF signaling pathway, suppressing the growth and function of melanocytes. It has been shown that the higher level of DKK1 expression in palmoplantar dermal fibroblasts contributes to the paler appearance of palmoplantar skin, suggesting that stimulating DKK1 expression may be a novel approach to inhibiting melanin synthesis. In the present study, we identified a natural extract that induces DKK1 protein expression in human fibroblasts. In order to evaluate the potential of this extract to influence melanin production, a combination of cosmetic skin lighteners with or without this extract was tested in the Melanoderm FT model. The blend containing this extract inhibited tyrosinase better than the blend without the extract, indicating that the natural extract, which stimulated DKK1, has the potential to lighten skin. A prototype skin care formulation containing the active blend was topically applied to the Melanoderm FT model for a period of time. The results indicated that melanin levels in the treated tissues were significantly inhibited. In addition, the whitening efficacy of this formulation was compared to select commercial skin lightening products using the Melanoderm FT model. The results demonstrated that our prototype formulation was significantly more efficacious compared to other products, as measured by the inhibition of melanin and by the appearance of the tissues. These findings collectively indicate that a natural extract that stimulates DKK1 has the potential of boosting the efficacy of existing skin lightening technologies and stimulation of DKK1 is a novel and viable approach to skin lightening.

OP 16

Diacylglycerol kinase regulates tyrosinase expression and function in human melanocytes

M. Kawaguchi 1,2, J. C. Valencia1, T. Namiki1, T. Suzuki2, V. J. Hearing1

1Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan

Diacylglycerol increases the melanin content of human melanocytes in vitro and increases the pigmentation of guinea pig skin in vivo, but the mechanisms underlying those effects remain unknown. In this study, we characterized the role of diacylglycerol kinase (DGK), which phosphorylates diacylglycerol to generate phosphatidic acid, in the regulation of pigmentation. Melanin content, tyrosinase activity and tyrosinase protein levels were significantly reduced by a DGK inhibitor, but tyrosinase and MITF mRNA levels were not changed by that inhibition, and there were no effects on the expression of other melanogenesis-related proteins. Glycosidase digestion revealed that inhibition of DGK reduced only the mature form of tyrosinase and the decrease of tyrosinase resulting from DGK inhibition could be blocked partially by protease inhibitors. These results suggest that DGK regulates melanogenesis via modulation of the post-translational processing of tyrosinase, which may be related with the protein degradation machinery.

OP 17

Paracrine interaction between UVB-exposed human keratinocytes and human melanocytes in co-culture system with cell insert leading to an increased expression of tyrosinase and its blockade by Wetherferin A

T. Niwano 1,2, H. Nakajima2, G. Imokawa3

1Tsuno Rice Fine Chemicals; 2School of Bioscience and Biotechnology, Tokyo University of Technology; 3Research Institute for Biological Functions, Chubu University

In this study, using a membrane-separated co-culture system with a cell culture insert, we have established a paracrine interaction model between UVB-exposed human primary keratinocytes (HPKs) (in the insert) and normal human melanocytes (NHMs) (in the well plate) leading to an increased expression of tyrosinase (TYR). The exposure of cultured HPKs to UVB radiation significantly stimulated the gene expression of MITF, TYR and tyrosinase-related protein 1 and the enzymatic activity of TYR in a dose-dependent manner in cultured NHMs at 24–36 and 72 h post-irradiation, respectively. In this system, UVB radiation significantly stimulated the secretion of IL-1α, IL-6/8, EDN1 but not αMSH and the addition of anti-EDN1 significantly neutralized the increased activity of TYR at 24 h post-irradiation addition timing. We used Wetherferin A (WFA) to examine a blocking effect on the paracrine interaction leading to the up-regulation of TYR activity. The addition of WFA (12.5-50 nM) at 0 h post-irradiation to the co-culture system significantly abolished the UVB-induced up-regulation of TYR activity after 72 h of culture. Treatment with WFA (50 nM) significantly suppressed the increased levels of IL-1α, IL-6/8 and EDN1 in the co-culture and also significantly abrogated the EDN1-stimulated activity of TYR in culture of NHMs alone. Intracellular signaling analysis using the culture with NHMs alone revealed that WFA (at 12.5, 25 or 50 nM) significantly abolished the EDN1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB. In contrast, intracellular calcium mobilization by EDN1 stimulation was not interrupted by WFA (50 nM). Taken together, these findings suggest that the co-culture system is a useful tool for selecting blockers for melanogenic paracrine interactions and it is anticipated that WFA is a potent signaling-interruptible anti-melanogenic agent.

OP 18

Effect of photo-aging on melanocyte motility

M. Yoshida, N. Nagai

Department of Animal Bio-Science, Nagahama Institute of Bio-Science and Technology

Photo-aging is a skin-specific aging process evoked by repetitive ultraviolet exposure via generation of reactive oxygen species and the concomitant accumulation of DNA damage. Various changes comprise photo-aging markers, such as the ruptures in elastin fibers, a decrease in collagen content, abnormal epidermal keratinization, and pigmentation. Pigmentation, in particular, is one of the most conspicuous signs of photo-aging and is also referred to as age spots. Age spots show untypical defined darkened areas that do not revert to the original color at least for several years. It is known that an abnormal accumulation of melanin causes this change, but what leads to this melanin accumulation is not fully understood.

A previous histological approach has shown an increase in the number of melanocytes in age spot areas. These melanocytes have been found to express p16, a marker of melanocyte aging, suggesting that the increase in prematurely aged melanocytes is involved in age spot formation.

To examine melanocyte characteristics, we produced photo-aged melanocytes by exposing cultured human melanocytes to UVA at 10 J/cm2 thrice a week, as previously reported for the production of photo-aged fibroblasts. Melanocyte proliferation was arrested after a 4-week exposure. Both melanin content and the population of dendriform cells were found to have increased after the treatment. These melanocytes showed up-regulation of p16, p19 and p21, markers of general aging as well as melanocyte aging, indicating that these photo-aged cells have characteristics similar to those of aged melanocytes. In a cell migration assay, these photo-aged cells showed less motility than normal melanocytes.

Our findings suggest that photo-aged melanocytes have low motility, and uneven distribution of them might induce pigmentation in age spot areas.

OP 19

Skin pigmentation and urinary 8-OHdG levels in humans

M. Iida, I. Yajima, N. Ohgami, M. Tamaki, M. Kato

Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi, Japan

Sunlight exposure-mediated health damages including skin cancer are serious global problems especially in Caucasians. Ultraviolet (UV) radiation is a major causal factor in the sunlight-mediated cutaneous diseases. UV works as an oxidative stressor via the promotion of free radical production. Experimental studies have revealed that oxidative stress is involved in the development of UV-induced skin diseases including skin cancer. However, there has been no study reported in which the influence of sunlight exposure in human daily life on 8-OHdG (oxidative DNA damage) levels. To reveal the influence of the sunlight exposure on oxidative stress in daily life is important to take appropriate preventative measures against sunlight exposure-related skin diseases. Therefore, we performed an epidemiologic study to investigate the correlations among levels of oxidative DNA damage, skin pigmentation, and sunlight exposure in daily life. A total of 127 healthy subjects aged 20–24 years participated in this study. We gave a questionnaire regarding sunlight-exposure conditions and measured their skin pigmentation and urinary 8-OHdG levels. Binary logistic regression analysis showed that increased skin pigmentation level, but not the use of sunscreen, was correlated with reduction of urinary 8-OHdG levels in humans. There was a significant correlation between urinary 8-OHdG levels and sunlight intensity 2 days before the day of urine sample collection. Meanwhile, there was no significant correlation between urinary 8-OHdG levels and sunlight exposure time and sunlight-exposed skin area. Some of these findings in humans were reproduced in an animal experimental study using HL-mice with hyperpigmented skin. In conclusion, we showed that an increase in intensity of sunlight in human daily life increased levels of DNA damage. We also showed a protective effect of skin pigmentation on sunlight exposure–mediated DNA damage.

OP 20

OCA2 polymorphisms are associated with skin color and risk of skin cancer in Japanese population.

J. Yoshizawa 1, Y. Abe1, Y. Hozumi1, N. Oiso2, T. Narita2, A. Kawada2, K. Fukai3, T. Motokawa4, K. Wakamatsu5, S. Ito5, T. Nakamura6, G. Tamiya6, T. Suzuki1

1Department of Dermatology, Faculty of Medicine, Yamagata University, Yamagata, Japan; 2Department of Dermatology, Faculty of Medicine, Kinki University, Osaka-Sayama, Japan; 3Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan; 4Cutaneous Drug Research Department, POLA Chemical Industries Inc., Yokohama, Japan; 5Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Japan; 6Advanced Molecular Epidemiology Research Institute, Yamagata University School of Medicine, Yamagata, Japan

Although many molecular mechanisms involved in melanin pigmentation have been identified, relatively little is understood about the genetic components responsible for variations in skin color within/between human populations. There are apparently some relationships between melanin pigmentation and carcinogenesis in human skin. Since melanin plays an important role in shielding the body from ultraviolet radiation and may serve as a scavenger for reactive oxygen species.

We have reported that OCA2 p.A481T rs74653330 (p = 6.18e-8) and, OCA2 p.H615R rs1800414 (p = 5.72e-6) are strongly associated with the mean of the melanin index in the Japanese female population.

We have now investigated the association of the variants in pigmentation-related genes with the risk of skin cancer, using 198 genomic DNA from Japanese patients with skin cancers. A multiple logistic regression analysis showed that only the OCA2 p.H615R variant was associated with the risk of MM (odds ratio 2.611; 95% confidence interval 1-165-5.850; P = 0.020). No association was detected between other variants and the risk of skin cancer. Furthermore, we analyzed an independent sample of a Japanese skin cancer group, and found that the OCA2 p.A481T variant was associated with the risk of SCC (odds ratio 0.317; 95% confidence interval 0.142–0.709; P = 0.005). No significant association between variants in MC1R and any skin cancer was found in either group.

This is the first report on the association between the genetic variants in the pigmentation genes and the risk of skin cancer in the East Asian population.

OP 21

A case report of Hermansky-Pudlak syndrome in Japan, harboring novel mutations in the HPS1 gene

S. Takeuchi 1, Y. Abe2T. Amada3, S. Kawano4, Y. Hozumi2, T. Suzuki2, C. Nishigori1

1Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine; 2Department of Dermatology, Yamagata University Faculty of Medicine; 3Department of Dermatology, Himeji Red Cross Hospital; 4Division of Laboratory Medicine, Department of Internal Related, Kobe University

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder that is characterized by congenital oculocutaneous albinism (OCA) and platelet storage pool deficiency. Nine responsible genes have been recognized causing different subtypes, whose products are involved in the biogenesis and/or function of intracellular organelles. Here we present a Japanese patient with HPS who has a novel mutation in the HPS1 gene. The patient is a 2 years old female who has been diagnosed as congenital OCA based on the depigmentation of skin and hair by physical examination. There is no family history of albinism and hemorrhagic tendency. Parents had never noticed apparent symptoms or episodes caused by hemorrhagic diathesis or bruising. Her bleeding time was within the normal range. Since genetic diagnosis indicated that she is probably not non-symptomatic OCA, a platelet aggregation test was analyzed, indicating that her collagen-induced platelet aggregation was impaired. To assure the diagnosis, the mutation in the HPS1 gene was examined by SSCP + direct sequencing. We detected c.965insC, p.T322insC in ex11 and c.1787G>T, p.G596V in ex18, the latter was a novel mutation that was never previously reported.

OP 22

A variant of linear atrophoderma of Moulin: hyper- and hypopigmented linear atrophoderma with peculiar area cutanea and lentiginosis following Blaschko's lines

N. Oiso, A. Kawada

Departments of Dermatology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan

Linear atrophoderma of Moulin (LAM) is an uncommon self-limited clinical disorder occurring in childhood or adolescence. It is characterized by asymptomatic, often hyperpigmented, atrophoderma following the lines of Blaschko on the trunk and/or the extremities. An atypical case of LAM with lentiginosis and hypopigmented lesions was recently described. We report an 18-year-old Japanese man with asymptomatic pigment anomalies on the left trunk and extremities. The patient noticed the development of hyper- and hypopigmented streaks at the age of 12 and asymptomatic dark brownish hyperpigmented spots within the streaks at the age of fifteen. The patient had no congenital malformations or delays in mental or physical development. The family had no history of pigmentary disorders or parental consanguinity. A physical examination revealed asymptomatic, slightly atrophic hyper- and hypopigmented streaks on the left trunk and extremities following the lines of Blaschko. Some lesions were characterized by faintly atrophic scaly erythema. The asymptomatic dark brownish hyperpigmented spots were present in a segmental or a spotty distribution within the linear streaks. Dermoscopy showed area cutanea with various colors and structure only on the affected lesions. The slightly atrophic scaly erythema showed either unusual area cutanea as crista cutis surrounding the hyperpigmented areas, or area cutanea distributed as a textile, wavy, linear, or polymorphous distribution. Histopathologic specimens showed acanthosis with irregular elongated rete ridges and different levels of melanin synthesis, even inside the hypopigmented streak. This case would be characterized by unregulated epidermal structure and uncontrolled melanin synthesis without the involvement of other organs. Our case suggests the importance of genetically determined relationship between regulated keratinization and melanin synthesis.

OP 23

In vitro analysis of read-through effect of aminoglycosides to tyrosinase R278X nonsense mutation in melan-c cells

K. Fukai 1, H. Kunimoto2, K. Nakajima2, T. Suzuki3, M. Ishii1

1Department of Dermatology and 2Immunology, Osaka City University; 3Department of Dermatology, Yamagata University

Among numerous mutations in Japanese OCA1 patients, the R278X nonsense mutation is found in 9% of all Japanese OCA1, being the most prevalent nonsense mutation. Aminoglycosides have been demonstrated to have a read-through effect on nonsense mutations, and have been tried for the treatment of muscular dystrophy. Here, we examined the read-through effect of aminoglycosides on melan-c cells in which tyrosinase R278X and the wild type tyrosinase were stably expressed by a lenti-virus system.

OP 24

Function of GNG2 in malignant melanoma

I. Yajima, M. Kumasaka, N. Ohgami, M. Kato

Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, Japan

Melanomagenesis is initially triggered by environmental agents including ultraviolet (UV) radiation, which induces genetic/epigenetic alterations in the chromosomes of melanocytes, and the incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer in the world. Since malignant melanoma is the most serious skin cancer, malignant melanoma is a threat for human life. However, an effective prevention and therapy for malignant melanoma has not been fully established.

We have previously reported that expression of G protein γ2 subunit (Gng2/GNG2), one of subunits of the Gβγ-dimer composing heterotrimeric G protein with a Gα-subunit, is reduced in human and murine malignant melanomas (Yajima, et al., 2012). Heterotrimeric G protein has been reported to be involved in various biological activities including cell proliferation, differentiation, invasion and angiogenesis via several intracellular signaling factors including c-SRC, FAK and PI3 kinase/AKT. At present, however, there are no reports about an effect of Gng2/GNG2 alone on cancer biology. In this study, we analyzed GNG2 function in human malignant melanomas in vitro and in vivo. We analyzed the phenotype of GNG2-overexpressed and -depleted human malignant melanoma cells using generation of GNG2-overexpressed cell clones and siRNA knockdown of GNG2 in human malignant melanoma cell lines to address relationships between GNG2 function and melanomagenesis. In the in vivo experiment, GNG2-overexpressed cell clones were inoculated in nude mice. The results show that melanomagenesis was suppressed in GNG2-overexpressed cells whereas it was enhanced in GNG2-depleted cells. Additionally, depletion of GNG2 rescued melanomagenesis in GNG2-overexpressed malignant melanoma cells. These results suggest that expression of GNG2 is reduced in malignant melanomas to avoid GNG2-mediated suppression of melanomagenesis. In summary, GNG2 could be a novel molecular marker and a target for malignant melanoma prevention and therapy.

OP 25

Endogenous retrovirus insertion in the Kit gene confers a hooded pattern of coat color to laboratory rats

T. Kuramoto, S. Nakanishi, B. Voigt, T. Serikawa

Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University

The ‘hooded’ phenotype is one of the oldest coat color mutations in the rat. Hooded rats have a pattern in which the entire ventral surface is white. Dorsally, pigmentation is limited to the head and shoulders (the ‘hood’) and a mid-dorsal stripe extending back to the tip of the tail. In addition to this hooded (h) phenotype, the Irish (hi) mutation causes a white spot on the belly between and behind the front legs. Here, we identified the h and hi alleles using a positional cloning approach. Genetic mapping and haplotype analysis revealed that the H locus spans about 92 kb on chromosome 14 in which five exons (1–5) of the Kit gene and the 48.8 kb genomic region upstream of the Kit gene are contained. We found an endogenous retrovirus (ERV) element was inserted into the first intron of the Kit gene in the h allele. In addition, a solitary long terminal repeat (LTR) was found at the same position to the ERV insertion in the hi allele. The ERV and the solitary LTR insertions were completely associated with the hooded and Irish coat patterns, respectively, across all colored rat strains examined. These findings suggest that the ERV insertion in the hooded allele may provoke the dysregulation of Kit expression and thereby causes the specific hooded pattern. The solitary LTR in the Irish allele may have been generated by homologous recombination between the 5′ and 3′ LTRs of the ERV. The Irish allele is a partial revertant of the hooded allele due to the residual solitary LTR. Thus, the hooded/Irish model system can be used to investigate spatiotemporal regulation of expression of the Kit gene that plays an important role in the development of the pigment cell.

OP 26

Hyperpigmentation of internal organs(Fibromelanosis) in the Silky chicken is highly-influenced by EDNRB2, Mc1r and Id genes

T. Akiyama 1, A. Shinomiya1, K. Kinoshita2, M. Mizutani2, Y. Matsuda2

1Department of Biology, Keio University, Yokohama, Japan; 2Avian Bioscience Research Center, Nagoya University, Nagoya, Japan

The Silky chicken is known to have a huge number of melanocytes in its inner organs (Fibromelanosis; Fm). Commonly, melanocytes that originate from the neural crest migrate through dorsolateral routes and settle to the integument of the body. The Silky chicken shows exceptional melanocyte migration though the dorsovental route and ectopic differentiation in visceral organs. Recently, we have reported that the responsible gene for Fm was mapped and the duplicate region of 130 kb containing endothelin 3 (EDN3) was located in the area. And then, two-fold expression of EDN3 was detected (Genetics 190, No2, 627–638, 2012). To disclose the Fm expression mechanism completely, we investigated the effects of E (Extension; Mc1r), Id (Inhibitor of dermal melanocytes on Z chromosome, control of shank color) and EDNRB2 genes on the Fm phenotype. We have recently identified several EDNRB2 mutants (mottling white, mow) from white plumage chickens. The heterozygous mutants (Mo+/mow) were crossed with Silky, and we examined the melanization of inner organs in their offspring having the Fm/fm+ genotype. The hyperpigmentation was clearly suppressed in Mo+/mowin comparison with wild type (Mo+/Mo+). These results demonstrate that normal EDNRB2is essential for Fm, and suggest that the two-fold EDN3is a trigger of hyperpigmentation. We also obtained F1 offspring from crossing females of Road Island Red having Id allele (fm+/fm+, e+/e+, Id/-) with males of white Silky (WS) (Fm/Fm, eb/eb, id+/id+) or black Silky (BS) (Fm/Fm, E/E, id+/id+) and their genotypes and the Fm phenotypes were compared. Both F1 females (Fm/fm+, e+/eb, id+/-, and Fm/fm+, E/e+, id+/-) displayed the Fm phenotype. On the other hand, the Fm phenotypes of F1 males are different; it was strongly suppressed in males (Fm/fm+, e+/eb, Id/id+) from WS, but appeared in males (Fm/fm+, E/e+, Id/id+) from BS. These results show that Id suppresses Fm expressionbut the E gene overcomes the suppressive effect of Id.

OP 27

Melanocyte-specific Mitf-M is a key regulator for development of melanoblasts: Lessons from the black-eyed white Mitfmi-bw mouse

K. Takeda 1, H. Hozumi1,2, Y. Yoshida-Amano2, A. Higashitani2, H. Yamamoto3, S. Shibahara1

1Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine; 2Graduate School of Life Sciences, Tohoku University; 3Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology

Microphthalmia-associated transcription factor (Mitf) has been considered as a regulator for differentiation of melanocytes and retinal pigment epithelial cells, which are derived from the neural crest and optic cup, respectively. The mouse homozygous for the Mitfmi-bw allele is characterized by the selective deficiency of the melanocyte-specific Mitf isoform, Mitf-M, and shows the white-coat color and deafness with black eye due to the lack of melanocytes. The Mitfmi-bw mutation is the insertion of a LINE-1 retrotransposable element into intron 3 of the Mitf gene. However, the molecular basis for the melanocyte loss has remained unknown. We therefore generated thehomozygous Mitfmi-bw (bw) mouse that carries the dopachrome tautomerase (Dct)-lacZ reporter transgene to track melanoblasts, because Dct is an early melanoblast marker. Mitf-M mRNA was expressed in the trunk region of the bw mouse at embryonic day (E) 11.5 and E12.5, but it was undetectable at E13.5. At E11.5 and E12.5, melanoblasts with lacZ expression are detectable in bw embryos, but they disappear by E13.5 mainly due to apoptosis. In addition, the number of melanoblasts with Dct-lacZ expression in bw mouse embryos (E11.5 and E12.5) was <10% of the control mouse embryos. Further, the electroporation of Mitf-M expression vector into the cultured neural tube of bw embryos (E9.5) enhanced the migration of neural crest cells and rescued the differentiation of melanoblasts, as judged by DOPA reaction. In conclusion, the correct dose of Mitf-M is required for the development of melanoblasts.

OP 28

Mechanism of regulation of the proliferation and differentiation of melanocytes by a new mutation of mouse ruby-eye 2, ru2d/Hps5ru2-d

T. Hirobe

Fukushima Restoration Support Headquarters, National Institute of Radiological Sciences

In our laboratory, a single autosomal recessive mutation spontaneously occurred in siblings of C57BL/10JHir (+/+) mice in 2006. This novel mutation, named ruby eye 2d (ru2d/Hps5ru2-d), exhibited a frameshift by 997G deletion in the Hps5 gene. To clarify the mechanism of the hypopigmentation, the characteristics of the neonatal development of ru2d/ru2d melanocytes were investigated in detail with special reference to those of +/+ melanocytes. In ru2d/ru2d mice, there were fewer epidermal melanocytes than in +/+ mice, whereas there was no difference in the number of epidermal melanoblasts between +/+ and ru2d/ru2d mice. The proliferation of ru2d/ru2d melanoblasts in serum-free primary culture system did not differ from that of +/+ melanoblasts. However, the differentiation of ru2d/ru2d melanocytes from melanoblasts was markedly inhibited. Epidermal melanocytes with increased dopa-melanin deposition and dendritogenesis were increased by injecting subcutaneously L-tyrosine (Tyr) into newborn ru2d/ru2d mice. Excess L-Tyr added to culture media from the initiation of the primary culture rescued the reduced differentiation through an increase in tyrosinase activity, expression of tyrosinase, TRP1, TRP2 and Kit, eumelanin synthesis and stage IV melanosomes. The eumelanin content in the epidermis and dermis in postnatal ru2d/ru2d mice was much lower than in +/+ mice, whereas similar pheomelanin content was observed 5.5 or 7.5 days after birth. Moreover, the eumelanin content in hairs of 5-week-old ru2d/ru2d mice was much lower than in +/+ mice, whereas pheomelanin content were two to four times greater than in +/+ mice. These results suggest thatthe ru2dallele inhibits melanocyte differentiation and the impaired differentiation is rescued by excess L-Tyr. The ru2d allele suppresses the differentiation of melanocytes through the inhibition of eumelanin synthesis, but stimulates pheomelanin synthesis in melanocytes.

OP 29

Effects of ionizing radiations on the development of mouse neural crest cells and melanocyte stem cells in the hair bulge

T. Hirobe, K. Sugaya

Fukushima Restoration Support Headquarters, National Institute of Radiological Sciences

The effects of ionizing radiations on the development of neural crest cells and melanocyte stem cells in the hair bulge are not well studied. Pregnant females of C57BL/10JHir (B10) mice at 9 days of gestation were whole-body irradiated with a single acute dose of γ-rays, and the effects of γ-rays were studied by scoring changes in the cutaneous coats of their offspring as well as in the development of melanoblasts and melanocytes in 18-day-old embryos. White spots were found in the mid-ventrum of the offspring irradiated with γ-rays. There were no melanoblasts and melanocytes there. To understand the mechanism of the development of white spots, the distribution of melanoblasts and melanocytes in the epidermis and hair follicles of irradiated 18-day-old embryos was investigated in detail. The number of epidermal melanoblasts/melanocytes and hair bulb melanocytes was markedly decreased even in 0.1 Gy-treated embryos, and decreased further as the dose was increased. The hair follicles of weanlings at 22–24 days correspond to the telogen stage of the first hair growth cycle. Although melanocyte stem cells exist in the hair bulge at this stage, there are no proliferating or differentiating melanoblasts/melanocytes there. The irradiation of γ-rays to 22–24-day-old B10 mice produced white hairs in 33–35-day-old mice (the second hair growth cycle) that may be derived from cell death of melanocyte stem cells as well as hypopigmentation in hair bulb melanocytes that may be caused by their abnormal differentiation. The frequency of white hairs did not increase in a dose-dependent manner, whereas the frequency of hypopigmented hair bulb melanocytes increased in a dose-dependent manner. These results suggest that γ-rays induce cell death of neural crest cells or melanocyte stem cells in the hair bulge, or inhibit their normal differentiation.

OP 30

Phenotypic characterization and genetic analysis of a novel spontaneous mutant mouse displaying age-related changes to the eye and coat colors

A. Ishikawa

Laboratory of Animal Genetics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Oca2p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat-color mutant gene on mouse chromosome 7. It was spontaneously discovered from wild Mus musculus castaneus captured in Indonesia. Usually, mice homozygous for Oca2p-cashave pink eyes and grey coat hair, and this phenotype remained unchanged throughout life. During development of a congenic strain carrying this gene as a homozygous state on the genetic background of the C57BL/6J inbred strain, I discovered a novel spontaneous mutant mouse whose eyes and coat hair change color with age. Here, I carried out phenotypic characterization and genetic analysis of this novel mutant. Phenotypic observations by the naked eye revealed that the pink eyes and grey coat hair of young mutant mice at about 16 days after birth became darker in color until approximately 3 months of age. Light and transmission-electron microscopic observations revealed that the mutants increased in pigmentation in the coat hair, epidermis and choroid, compared to normal homozygotes with pink eyes and grey coat hair. There were no clear differences in pigmentation of retinal pigment epithelial cells between the two types of mice. Also the cDNA sequence of the Oca2p-cas gene did not differ between the two types of mice. Mating experiments showed the possibility that this mutant phenotype might not be inherited as a simple Mendelian character. These results suggest that the present mutant phenotype may be caused by an unknown gene other than Oca2p-cas or epigenetics that switches on melanin biosynthesis in melanocytes of coat hair, epidermis and choroid.

OP 31

Analysis of endothelin receptor B expressed in spiral ganglion neurons.

N. Ohgami, M. Iida, I. Yajima, M. Kato

Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Matsumoto, Kasugai, Aichi, Japan

Impairments of endothelin receptor B (Ednrb/EDNRB) cause Waardenburg-Shah syndrome with hypopigmentation, congenital hearing loss and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of the inner ear. In this study, we found that Ednrb protein was expressed in spiral ganglion neurons (SGNs) from WT-mice on postnatal day 19 (P19), while it was undetectable in SGNs from WT-mice on P3. Furthermore, Ednrb homozygously deleted mice [Ednrb(-/-)-mice] with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs in Ednrb(-/-)-mice was clearly restored by introducing an Ednrb transgene driven by the dopamine beta-hydroxylase promoter [Ednrb(-/-);DBH-Ednrb-mice] on P19. Defects of melanocytes and hypopigmentation in the SV and skin in Ednrb(-/-)-mice were not restored in the Ednrb(-/-);DBH-Ednrb-mice. Thus, the results of this study suggest that Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributes partially to postnatal hearing development in mice.

OP 32

Involvement of Mitf and its possible regulators in the retinal pigment epithelium development

D. Nishihara 1,2, A. Kawasaki-Nishihara1,2, H. Nakamura1, H. Yamamoto2

1Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; 2Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

The retinal pigment epithelium (RPE), one component of the vertebrate eye, consists of a monolayer of melanin-pigment cells. Although the RPE is known to be indispensable for adult visual function, little is known about the molecular mechanisms underlying its development. During eye development, the eye primordium changes its shape to form a bilayered cup-like structure, called the optic cup (OC). The RPE is formed in the outer layer of OC. Some transcription factors, such as Mitf and Otx, are expressed in the outer layer of the OC and are thought to be essential for RPE development, since loss of function of these factors severely impairs RPE differentiation. However, the detailed underlying mechanisms for the regionalization and characterization of the RPE involving these transcription factors remain to be elucidated. Although mutant analyses are useful for understanding the functions of transcription factors in vivo, mice mutant for transcription factors required for RPE development display severe eye abnormalities. In such cases, with so many complicated phenotypes, analyses focusing on RPE development are not easily carried out. Therefore, to elucidate how eye-related transcription factors function during RPE development in detail, we conducted gene transfection into the chick OC by electroporation. Using that method, the transfected areas could be limited (not the whole developing eye affected) and easily controlled. The resultant eye phenotypes are not too disrupted to analyze how RPE development is affected with the transfected molecules in specific areas. Here we show how several transcription factors contribute to RPE regionalization and characterization by controlling the expression and function of Mitf, also a key player for RPE development.

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