Role of RAB27A in Skin Color Determination Figure 6. Reduced RAB27A expression in melanocytes significantly decreases the melanin content in the epidermis, resulting in a lighter skin surface color of HSS. HSSs were reconstructed by adding SCR- (A) or shRNA-treated melanocytes (B) to a mixture of normal keratinocytes and fibroblasts. Scale bars = 5 mm. Immunohistochemistry against melanosome specific protein TRP1 (red) in the HSS epidermis composed of SCR- (C) or shRNA-administered melanocytes (D) is demonstrated. Scale bars = 10 mm. The expression of RAB27A mRNA normalized to that of DOPAchrome tautomerase (DCT) (E), the surface color (F) and melanin content (G) are assessed in the HSS epidermis. Melanin content of the HSS epidermis composed of SCR-treated melanocytes was relatively expressed to be 1.0 (G). The values reported represent means 6 SD. Statistical significance was evaluated by the Student’s t-test and a p value,0.05 is considered statistically significant. doi:10.1371/journal.pone.0041160.g006 from perinatal mice onto the backs of athymic nude mice has been applied to the identification of the cellular requirements for skin appendage formation [51]. The novel approach described here may also be useful for studies related to hair homeostasis. Overall, our data show for the first time that RAB27A plays an important role in regulating the total amount of melanin in the epidermis and consequently in the determination of human ethnic skin color by controlling melanosome transport within melano- cytes. The suppressed expression of RAB27A resulted in reduced melanin content in HSSs, indicating the impact of a molecule which is not involved in melanin synthesis but in vesicular transport in melanocytes, on the contribution to skin color formation in vivo. It has been reported that melanosome transfer is accelerated in regions of senile lentigos [52,53]. Our findings provide new insights for the fundamental understanding of regulatory mechanisms that underlie skin pigmentation and provide a basis to develop an efficient strategy to treat cutaneous pigmentation disorders such as melasma, senile lentigo, seborrheic keratosis and vitiligo. Materials and Methods Materials Normal human epidermal melanocytes (NHEMs), keratinocytes (NHEKs) and fibroblasts were obtained from Kurabo Co. (Osaka, Japan). Four to six week-old SCID mice were supplied by Oriental Bio-service Kanto Co. (Tsukuba, Japan). Abdominal and breast skins were received from a healthy 58-year-old Caucasian female undergoing abdominoplasty and from a healthy 40-year-old African-descent female undergoing breast reduction, respectively. Upper inner arm skin tissues were supplied from a healthy 32- year-old Caucasian female and a 36-year-old African-descent female by punch biopsy. Ethics statement The collection of human skins was conducted according to Declaration of Helsinki principles and was approved by the Institutional Review Board of IntegReview (Austin, TX). Written informed consent was obtained from the subjects prior to the procedure. All mice were handled according to the guidelines of the Ethical Committee for Animal Experiments at the Kao Corp. (Tochigi, Japan). The protocol was approved by the Ethical Committee for Animal Experiments at the Kao Corp (Permit Number: N2009-0071A, N2009-0073A). All animal surgery was PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41160