Role of RAB27A in Skin Color Determination Figure 5. Knockdown of RAB27A expression in melanocytes by a lentivirus-driven shRNA significantly diminishes melanosome transfer into keratinocytes in co-culture. (A) Western blotting analysis of RAB27A in melanocytes treated with SCR or with RAB27A shRNA- expressing lentivirus (shRNA) following blasticidin-tolerant selection. Bright-field (upper panels) and differential interference contrast images (bottom panels) of melanocytes treated with SCR (B) or with shRNA (C). Scale bars = 10 mm. Immunohistochemistry for gp100 (green) in co-culture with melanocytes treated with SCR (D) or with shRNA (E) is demonstrated as described in Figure 3. Scale bars = 10 mm. (F) The ratios of keratinocytes harboring transferred melanosomes in co-cultures with melanocytes treated with SCR or with shRNA. The values represent means 6 SD. Statistical significance was evaluated by the Student’s t-test, and a p value of ,0.05 is considered statistically significant. doi:10.1371/journal.pone.0041160.g005 regulated both directly by MITF and indirectly via cAMP [39,44]. Consistent with those findings, the data presented here also suggest that MITF expression correlates with RAB27A expression. Additionally, MITF expression has been documented to be enhanced during UV-induced skin tanning accompanied with a remarkable increase in melanosome distribution throughout the epidermis in contrast to skin before UV exposure which contains melanosomes predominantly localized in BKCs [45]. Accompa- nied by the stimulated MITF expression, the enhancement of RAB27A expression would be harmonized with the increased melanin synthesis in melanocytes in order to effectively distribute melanosomes to keratinocytes and consequently to cover the epidermis for cutaneous photoprotection. In contrast, RAB27A mutations and gene knockdown have been reported not to affect melanin synthesis in melanocytes [46]. In Xenopus laevis, decreased expression of Rab27a and melanosome aggregation were observed when a dominant-negative Mitf was injected into their embryos, suggesting the involvement of Rab27a in melanosome transport in spite of the absence of subsequent melanosome transfer [47]. Since some species of amphibians, fish and reptiles can rapidly change their skin colors through mechanisms that translocate pigments and reorient reflective plates within chromatophores (pigment- containing and light-reflecting cells) which are largely responsible for modulating their skin color without changes in pigment production, RAB27A is a sort of vestigial machinery that is evolutionarily conserved to regulate skin color over the species. It is also of interest to explore the various experimental systems to uncover the detailed molecular mechanisms underlying skin and hair homeostasis. We have previously established several efficient cutaneous and hair follicle gene transfer systems in vivo using vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vec- tors [48–50]. However it remained a challenge to regulate the expression of specific genes only in targeted tissues and/or cells at will to examine long-term skin properties after grafting a cell mixture onto immunodeficient animals. In this study, we associated the in vivo HSS system with lentivirus-mediated gene knockdown for the first time and succeeded in developing HSSs containing melanocytes with continuously reduced expression levels of the gene of interest. This improved HSS technique could be a strong analytical tool to elucidate detailed molecular mechanisms of human skin disorders. Furthermore, the combina- tion of epidermal and dermal cell preparations from newborn and PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41160