have been reported to produce neurotransmitters, neuropeptides and hormones such as MSH, catecholamines, serotonin and melatonin, suggesting its plausible role in the regulation of sensory nerve endings in the epidermis [11]. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. It has been reported that tyrosinase activity in darker (African- descent) skin is significantly higher than in lighter (Caucasian) skin [12,13] in spite of no difference in tyrosinase expression levels among them [14,15]. Given that treatment with ionophore stimulators increase tyrosinase activity [16] and that a relatively lower melanosomal pH is observed in melanocytes from Cauca- sian skin compared to African-descent skin [17], melanosomal pH may be an important regulatory factor controlling enzymatic activity. Additionally, sodium/hydrogen exchangers (NHEs), which regulate intracellular pH and vesicular ATPase (V-ATPase), a vesicular proton pump, have been suggested to play roles in producing the variations of ethnic skin complexion [17,18]. Differences in the amounts and distribution patterns of melanosomes in keratinocytes are also proposed to determine distinct ethnic skin colors. Architectural analysis showing numer- ous melanosomes throughout the epidermis in darker skin in contrast to only a few melanosomes in lighter epidermis has facilitated investigations on the differences in melanosome transfer [19]. It has been demonstrated that protease-activated receptor-2 (PAR-2), a seven transmembrane G-protein-coupled receptor, regulates phagocytosis in keratinocytes [20] and that darker skin exhibits a higher expression of PAR-2 compared to lighter skin [21]. Additionally, inhibition of PAR-2 has been reported to effectively lighten skin complexion [22–25], illustrating that PAR- 2-mediated melanosome phagocytosis contributes to ethnic skin color differences. Apart from the numbers of melanosomes, keratinocytes from darker skin contain melanosomes that are predominantly distributed individually over the cytosol in contrast to lighter skin-derived keratinocytes which have their melano- somes in clusters [26,27]. These different distribution patterns of melanosomes in the epidermis have been suggested to be regulated by keratinocytes not by melanocytes [28]. Coat color mutations in mice, such as dilute (Myo5ad), ashen (Rab27aash) and leaden (Mlphln), demonstrate impaired actin- associated melanosome transport which results in coat color dilution, indicating that melanosome transport within melanocytes is also necessary for the pigmentation of hair as well as skin [29]. Rab27a, the protein encoded at the ashen locus, is a small Ras-like GTPase belonging to the Rab family which contains about 60 members in charge of various types of membrane transport, such as vesicle fusion and docking of transport vesicles to specific target organelles and/or plasma membranes during secretory processes [30,31]. It has been reported that Rab27a anchors melanosomes to the plasma membrane in collaboration with myosin Va, an actin-dependent motor protein encoded by the dilute locus, Slac2- a/melanophilin, a melanocyte-specific Rab27a effector encoded by the leaden locus and another effector Slp-2a in melanocytes [32– 35]. On the other hand, mutations in the genes encoding MYO5A, RAB27A and MLPH are known to result in one of three subtypes of an autosomal recessive inherited human pigmentary diseases called Griscelli Syndrome (GS), which is mostly characterized as diluted pigmentation in skin and hair. A mutation in SLAC2-A exhibits a phenotypic change only in hypopigmentation [36], whereas mutations in the other two genes cause a wider range of phenotypes, such as neurological impairment (MYO5A) and Role of RAB27A in Skin Color Determination immunodeficiency (RAB27A) [37]. Given that the physical interaction among these three factors to form a protein complex is required for melanosome transport [32–34,38] and that the three subtypes of GS share a trait of hypopigmentation, it is reasonable to consider that the MYO5A-SLAC2-A-RAB27A complex plays a pivotal role in melanosome transport and in consequent cutaneous pigmentation in humans. However, it remains to be elucidated which melanosome transport-related factors substantially contribute to the determination of ethnic skin color differences. In this study, we have evaluated the expressional correlation of several melanogenic- and/or melanosome transfer-related factors with the melanogenic activities in melanocytes from different ethnic backgrounds and subsequently validated their roles to elucidate the mechanisms underlying the ethnic skin color variations. The impact of RAB27A, which was selected as a possible candidate factor distinguishing ethnic skin color, on the amounts of melanocytic and epidermal melanin syntheses, on the transfer of melanosomes from melanocytes to keratinocytes and consequently on constitutive skin color was assessed using HSS technologies in collaboration with lentiviral-mediated shRNA technique to suppress RAB27A expression. Results Significantly higher numbers of melanosomes are transferred to keratinocytes in African-descent skin compared with Caucasian skin The numbers of melanosomes transferred to basal keratinocytes (BKCs) between African-descent and Caucasian skins were compared to understand the ethnic differences in melanosome transfer. Breast and upper inner arm skins and abdominal and upper inner arm skins surgically prepared from two different African-descent subjects (Fig. 1A, 1B) and two different Caucasian patients (Fig. 1C, 1D), respectively, showed higher numbers of melanosomes within BKCs in the African-descent skin compared to the Caucasian skin, where transferred melanosomes in BKCs were hardly observed (Fig. 1C, 1D). The number of melanosomes per BKC in African-descent skin was found to be significantly higher compared to Caucasian skin (Fig. 1E). The expression of RAB27A mRNA and protein is significantly correlated with melanin content in melanocytes For the assessment of the contribution of well-known melano- genic- and/or intracellular melanosome transport-related factors in melanocytes to the ethnic skin color variations, the correlation of the enzymatic activity of tyrosinase and the gene expression of tyrosinase, RAB27A and SLAC2-A with melanin content were first examined using 19 commercially obtained cell lines of melanocytes with various melanin contents (Fig. 2A–D). Following the observation that tyrosinase enzymatic activity correlated with melanin content within melanocytes (Fig. 2A), the highest correlation between RAB27A mRNA expression level and melanin content in melanocytes was found (Fig. 2C). Consistently, Western blotting analysis also confirmed the correlation of RAB27A protein expression with melanin content using an identical set of cell lines (Fig. 2E, 2F). In addition, the expression level of RAB27A mRNA correlated with that of MITF mRNA in agreement with a previous finding (Fig. 2H) [39]. However, there was no correlation of melanin content with levels of tyrosinase protein expression (Fig. 2E) or RAB27A protein expression (Fig. 2G) in this study. PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41160