Essential Role of RAB27A in Determining Constitutive Human Skin Color Yasuko Yoshida-Amano 1 , Akira Hachiya1*, Atsushi Ohuchi1, Gary P. Kobinger2, Takashi Kitahara1, Yoshinori Takema 3 , Mitsunori Fukuda4 1 Biological Science Laboratories, Kao Corporation, Haga, Tochigi, Japan, 2 Special Pathogens Program, National Microbiology Laboratory, Department of Medical Microbiology, Public Health Agency of Canada, University of Manitoba, Winnipeg, Manitoba, Canada, 3 Research and Development Global, Kao Corporation, Sumida-ku, Tokyo, Japan, 4 Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Abstract Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin- associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs) in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (DL* = 4.4) in the HSSs. These data reveal the intrinsically essential role of RAB27A in human ethnic skin color determination and provide new insights for the fundamental understanding of regulatory mechanisms underlying skin pigmentation. Citation: Yoshida-Amano Y, Hachiya A, Ohuchi A, Kobinger GP, Kitahara T, et al. (2012) Essential Role of RAB27A in Determining Constitutive Human Skin Color. PLoS ONE 7(7): e41160. doi:10.1371/journal.pone.0041160 Editor: Andrzej T. Slominski, University of Tennessee, United States of America Received March 30, 2012; Accepted June 18, 2012; Published July 23, 2012 Copyright: 2012 Yoshida-Amano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Competing Interests: The authors have read the journal’s policy and have the following conflicts: Yasuko Yoshida-Amano, Akira Hachiya, Atsushi Ohuchi, Takashi Kitahara, and Yoshinori Takema are employed by Kao Corporation, who were the funders of this study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: hachiya.akira@kao.co.jp Introduction Skin color is predominantly determined by the amount and types of melanin produced in melanosomes, melanocyte-specific organelles. In the process of mammal’s skin color formation, melanin synthesis is launched by the melanogenic enzymes, tyrosinase, dopachrome tautomerase and tyrosinase-related pro- tein-1, mutations in which cause hypopigmented or diluted color of skin [1,2]. In addition to those enzymes, L-tyrosine and L- dihydroxyphenylalanine have also been shown to positively regulate melanin synthesis through the stimulation of tyrosinase activity [3]. Pigmented melanosomes are then transported towards the cell membrane at the dendrite tips of melanocytes via the intracellular transport system, anchored to the plasma membrane, and then transferred to neighboring keratinocytes [4]. After their transfer to keratinocytes, melanosomes are distributed around the nuclei by a microtubule-associated motor protein, dynein, to form supranuclear melanin caps which protect keratinocytes from the harmful effects of ultraviolet exposure [5,6]. The substantial interaction between melanocytes and keratinocytes has been proposed by the anatomical finding that ,36 viable keratinocytes surround each melanocyte to form a specialized cell group called the epidermal melanin unit [7,8]. This ratio of the two cell types is consistently kept despite the extremely wide range of human skin color. Melanogenesis is controlled by a wide range of molecular-based mechanisms in melanocytes and keratinocytes [8], which result in the variety of skin and hair pigmentation. Among them, melanocyte stimulating hormone (MSH) and its receptor, MC1R, which regulate melanogenic signaling via cAMPs, have been actively investigated. Genetic and cellular studies have revealed that MC1R polymorphisms contribute to the differences in the ultraviolet sensitivity and in hair and skin color intensity in several ethnic groups [9,10]. Recently, melanocytes themselves PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41160