２２回日本研究皮膚科学会総会・抄録

２２回日本研究皮膚科学会総会・抄録

-150- Melanocyte(3) 9/5 Room-C
THE ROLES OF SCF AND ET-3 DURING MELANOCYTE DEVELOPMENT.
Hirotake Ono1,2, Mari Asano1, Yoko Kawa1, Yasuo Kubota1, Masako Mizoguchi1.
1Department of Dermatology, St. Marianna University School of Medicine, Kawasaki, 2Department of Biology, Keio University, Yokohama, Japan.
It has been known that stem cell factor (SCF) and endothelin-3 (ET-3) play important roles in the development of melanocytes from neural crest cells (NCCs). Previously, we reported that the addition of SCF to NCC cultures of wild type mice increases c-KIT+ cells and DOPA+ melanoblasts. In this study, we examined the effect of ET-3 on the proliferation and differentiation of cultured NCCs into melanocytes in the wild type and 'Steel' mutant mice. In the NCC culture of the wild type, the addition of ET-3 resulted in the marked differentiation of mature melanocytes. On the other hand, mature melanocytes, c-KIT+ cells and DOPA+ cells were not observed in the 'Steel' NCC culture. Also in such 'Steel' NCC culture, melanocytes appered when SCF was added with ET-3. These results suggest that there are SCF sources in the wild type explants, and that the survival of c-KIT+ melanoblasts supported by SCF is essential to the acceleration of melanization by ET-3.

-151- Melanocyte(3) 9/5 Room-C
EFFECTS OF EXTRACELLULAR MATRIX (ECM) ON MELANOCYTE DIFFERENTIATION IN CULTURED MOUSE NEURAL CREST CELLS (NCCs) WITH STEM CELL FACTOR (SCF).
Nagako Takano1,Yasuo Kubota1, Yoko Kawa1, Masaru Ito1, Mari Asano1, Hirotake Ono2, Masako Mizoguchi1.
1Department of Dermatology, St.Marianna University School of Medicine, Kanagawa, 2Department of Biology, Keio University, Tokyo, Japan.
SCF and ECM have been individually reported to affect melanocyte differentiation and proliferation. To examine the combined effects of ECM and SCF, we cultured NCCs in each well coated with fibronectin, collagen I, tenascin, chodroitin sulfate, or dermatan sulfate. Dopa (+) cells and c-kit (+) cells were counted at various time points. In the presence of SCF, there were more dopa (+) cells on all ECM-coated wells than on non-coated wells. However in the absence of SCF, there were few dopa (+) cells on both ECM-coated and non-coated wells. These findings suggest that ECM plays an important role in melanocyte differentiation in concert with SCF.

-152- Melanocyte(3) 9/5 Room-C
CELL MOTILITY IS ENHANCED IN H-RAS-TRANSFECTED MURINE MELANOCYTE CELL LINE MELAN-A.
Tatsuya Horikawa1, Keishi Araki1, Kenta Tsuru1, Hiroshi Nagai1, Masato Ueda1, Katsuhiko Tsukamoto2, Masamitsu Ichihashi1.
1Department of Dermatology, Kobe University School of Medicine, Kobe, 2Department of Dermatology, Yamanashi Medical Colledge, Yamanashi, Japan.
Melanocyte movement plays an critical role in the repigmenting phase of vitiligo. Clarification of regulating mechanisms of melanocyte movement will provide a good strategy for the treatment of vitiligo. Ras is a small GTP-binding protein of which activation results in intracellular signalling for various types of cellular response, especially cell proliferation. Recent studies have shown that ras activation may induce cell locomotion. In order to study if ras activation is related to melanocyte locomotion, we investigated the motility of H-ras tranfected murine melanocyte cell line melan-A. Using Boyden chamber invasion assay, we found that H-ras tranfected murine melanocytes show higher incidence of migration than parental cells. ET-1 and bFGF stimulated cell motility of parental melan-A but not a H-ras transfectant which shows higher motility without stimulation. These results suggest that H-ras plays a pivotal role in the induction of melanocyte locomotion, and that bFGF and ET-1 no longer can stimulate movement of H-ras-infected melanocytes.

-153- Melanocyte(3) 9/5 Room-C
UTILITY OF ADENOVIRUSES AS GENE EXPRESSION VECTORS IN MELANOCYTIC CELLS.
Masahiro Oka1,2, Mark Nesbit2, Kapaettu Satyamoorthy2, Mei-Yu Hsu2, Brian S. Glatt2, Masamitsu Ichihashi1, Meenhard Herlyn2.
1Department of Dermatology, Kobe University School of Medicine, Kobe, Japan. 2The Wistar Institute, Philadelphia, USA.
Melanoma progression occurs in distinct stages such as dysplastic nevus, radial and vertical growth phase primary melanoma and metastatic melanoma, based on their specific histopathological, biochemical and molecular characteristics. In melanoma cells, these characteristics are profoundly affected by the autocrine functions of growth factors such as basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), and interleukin-8(IL-8). However, dissection of the role of the individual growth factors has been previously hindered by the lack of a suitable experimental system to express these proteins in a controlled manner. We have now developed a system for efficient overexpression of genes in melanocytic cells using adenoviruses. Greater than 98% of melanocytes and melanoma cells can be infected with adenovirus/lacZ and a majority of the cells exhibit β-galactosidase activity over a period of 45 days.　　

-154- Melanocyte(3) 9/5 Room-C
KERATIN EXPRESION IN CULTURED HUMAN MELANOCYTES.
Yohtaro Katagata, Masakazu Kawaguchi, Shigeo Kondo.
Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan.
Human melanocytes are developmentally derived from neural crest cells and are localized in the basal cell layer of the epidermis. The main functions of melanocytes are to produce melanin and to supply the melanin to the epidermis as a protection from ultraviolet irradiation. In this paper, we studied whether keratin expresses in melanocytes, or not. Cultured melanocytes were grownusing Morinaga HFM cell kit and were incorporated with 35S-Met (50 mCi/ml) at 37℃　for 2h. Using an aqueous solution for keratin extrction, followed by 2D-PAGE and immunological techniques. From the results of the above experiments, we confirmed that at least four kinds of keratin subunits (K5, 8, 10, 14) and vimentin were expressed in cultured melanocytes. The differences of keratin expression between melanocytes and melanoma cells and absence of MRP-1 were suggested that some particular keratin and MRP-1 were related to change melanocytes into melanoma cells.