Molecular mechanisms of melanosome exocytosis

• Autor(a) principal: Cabaço, Luís C.
• Data de Publicação: 2025
• Idioma: eng
• Título da fonte: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Texto Completo: http://hdl.handle.net/10362/178940
• topic: melanosome; Ciências da Saúde

Skin pigmentation relies on the pigment melanin and ensures photoprotection against ultraviolet (UV) radiation, avoiding the onset of skin cancers. Melanin is synthesized by melanocytes and stored within organelles designated melanosomes, which are tethered to actin in melanocyte dendrites through Rab27a, and finally transferred to keratinocytes. While the molecular players involved in melanogenesis have been extensively studied, those underlying melanosome exocytosis and melanin transfer remain unclear. Previously, our group found that Rab11b regulates melanin secretion and transfer in human skin. Here, we demonstrated that soluble factors, but not extracellular vesicles, present in keratinocyte-conditioned medium (KCM) stimulate melanin secretion from melanocytes, and transfer to keratinocytes. Moreover, we found that these factors are released by differentiated keratinocytes, but not by undifferentiated ones. Importantly, we ruled out the possibility that KCM increases melanin secretion and transfer as a consequence of increased melanin synthesis. For this, we quantified intracellular melanin levels in melanocytes cultured with or without KCM. Additionally, we confirmed that KCM does not increase the expression of microphthalmia-associated transcription factor (MITF) and its downstream transcript which encodes for tyrosinase in melanocytes, both required for melanogenesis. We also demonstrated that Rab3a, but not Rab11b, regulates KCM-stimulated melanin secretion and transfer, and shows enhanced colocalization with melanosomes in melanocyte dendrites upon incubation with KCM. Therefore, our results suggest that soluble factors released by differentiated keratinocytes control skin pigmentation by promoting the accumulation of Rab3a-positive melanosomes in melanocyte dendrites, and their release and subsequent transfer to keratinocytes. Furthermore, we found that Rab27a and the Rab3a guanine nucleotide exchange factor (Rab3il1) regulate KCM-stimulated melanin secretion. We also observed that Ca2+ -dependent signaling enhances KCM-induced melanin secretion, which raises a possible role for the Ca2+ -dependent Munc13-2 and Munc18-3 priming proteins in this process. Interestingly, immunoprecipitation and immunofluorescence assays suggested that Rab3a interacts with Rab27a on melanosome membranes, in the dendrites of KCM-stimulated melanocytes. Besides the characterization of the molecular machinery regulating KCM-induced melanin secretion, we identified a soluble factor present in KCM responsible for the stimulatory effect. Indeed, our results showed that KCM soluble factor(s) with a molecular weight lower than 3 KDa (< 3 KDa) is (are) the main stimulatory molecules in melanin secretion. Curiously, NMS-23-01 was abundantly detected in KCM < 3 KDa fraction by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Moreover, melanocytes cultured with NMS-23-01 demonstrated a dose-dependent increase in secreted melanin levels and a decrease in intracellular melanin content. Furthermore, the incubation of NMS-23-01 in both two-dimensional melanocyte/keratinocyte co-cultures and three-dimensional reconstructed human pigmented epidermises enhanced melanin transfer and augmented epidermal pigmentation. Thus, our results identified NMS-23-01 as the first molecule that increases skin pigmentation by specifically enhancing melanin secretion and subsequent transfer to keratinocytes. Importantly, NMS-24-01 (a specific antagonist of NMS-23-01) impaired both NMS-23-01 and KCM-stimulated melanin secretion levels. Additionally, we found that NMS 23-01 and KCM share the same molecular pathway of stimulated melanin secretion, independent of Rab11b and dependent of Rab27a, Rab3a and Rab3il1. Overall, our results suggest that NMS-23- 01 is at least one of the KCM soluble factors that stimulate melanin secretion. Furthermore, our studies indicate that two distinct pathways of melanosome exocytosis exist in melanocytes: a basal pathway controlled by Rab11b and another route controlled by the Rab27a-Rab3il1-Rab3a cascade, and triggered upon stimulation with NMS-23-01 (and possibly other small keratinocyte-derived soluble factors). Thus, this study contributed to a better understanding of fundamental processes of skin pigmentation, namely the molecular players involved in the stimulation of melanin secretion. Moreover, NMS-23-01 and its antagonist NMS-24-01 were identified in our work as novel and specific compounds to target melanin secretion and transfer. Therefore, they exhibit the potential to modulate skin color and serve as the basis of novel therapeutic strategies for hypo/hyperpigmentation disorders, potentially also with cosmetic applications. Importantly, the knowledge about NMS-23-01 and the molecular mechanisms stimulating melanin secretion and transfer can also be applied to stimulate skin tanning and photoprotection against UV-induced DNA damage.