RAB3A Regulates Melanin Exocytosis and Transfer Induced by Keratinocyte-Conditioned Medium

Funding: We would like to thank our group for the critical reading of the manuscript and the NMS microscopy and cell culture facilities, as well as José Belo’s group, for the kind gift of mouse embryonic fibroblasts. This study was supported by Fundação para a Ciência e a Tecnologia (Portugal) through grant PTDC/BIA-CEL/29765/2017 and PhD fellowships to LCC, MVN, LBL and AF (2020.08812.BD, PD/BD/137442/2018, SFRH/BD/131938/2017 and PD/BD/ 135506/2018, respectively). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, cofinanced by Fundação para a Cieˆncia e a Tecnologia (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). This article was supported by the LYSOCIL project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 811087. This work was also supported by iNOVA4Health e UIDB/04462/2020 and UIDP/04462/2020 and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (Portugal).Skin pigmentation is imparted by melanin and is crucial for photoprotection against UVR. Melanin is synthesized and packaged into melanosomes within melanocytes and is then transferred to keratinocytes (KCs). Although the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain unclear. Previously, our group proposed that coupled exocytosis/phagocytosis is the predominant mechanism of melanin transfer in human skin and showed an essential role for RAB11B and the exocyst tethering complex in this process. In this study, we show that soluble factors present in KC-conditioned medium stimulate melanin exocytosis from melanocytes and transfer to KCs. Moreover, we found that these factors are released by differentiated KCs but not by basal layer KCs. Furthermore, we found that RAB3A regulates melanin exocytosis and transfer stimulated by KC-conditioned medium. Indeed, KC-conditioned medium enhances the recruitment of RAB3A to melanosomes in melanocyte dendrites. Therefore, our results suggest the existence of two distinct routes of melanin exocytosis: a basal route controlled by RAB11B and a RAB3A-dependent route, stimulated by KC-conditioned medium. Thus, this study provides evidence that soluble factors released by differentiated KCs control skin pigmentation by promoting the accumulation of RAB3A-positive melanosomes in melanocyte dendrites and their release and subsequent transfer to KCs.publishersversionpublishe

The Dark Side of Melanin Secretion in Cutaneous Melanoma Aggressiveness

Funding Information: LC is funded by an FCT PhD fellowship (2020.08812.BD). AT is funded by an FCT PhD fellowship (2021.06204.BD). MP is funded by Liga Portuguesa Contra o Cancro – Núcleo Regional do Sul (LPCC-NRS). DB lab is supported by Fundação para a Ciência e a Tecnologia (FCT) through grant PTDC/BIA-CEL/29765/2017. This publication was funded by the iNOVA4Health - UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by FCT/Ministério da Educação e Ciência through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. Publisher Copyright: Copyright © 2022 Cabaço, Tomás, Pojo and Barral.Skin cancers are among the most common cancers worldwide and are increasingly prevalent. Cutaneous melanoma (CM) is characterized by the malignant transformation of melanocytes in the epidermis. Although CM shows lower incidence than other skin cancers, it is the most aggressive and responsible for the vast majority of skin cancer-related deaths. Indeed, 75% of patients present with invasive or metastatic tumors, even after surgical excision. In CM, the photoprotective pigment melanin, which is produced by melanocytes, plays a central role in the pathology of the disease. Melanin absorbs ultraviolet radiation and scavenges reactive oxygen/nitrogen species (ROS/RNS) resulting from the radiation exposure. However, the scavenged ROS/RNS modify melanin and lead to the induction of signature DNA damage in CM cells, namely cyclobutane pyrimidine dimers, which are known to promote CM immortalization and carcinogenesis. Despite triggering the malignant transformation of melanocytes and promoting initial tumor growth, the presence of melanin inside CM cells is described to negatively regulate their invasiveness by increasing cell stiffness and reducing elasticity. Emerging evidence also indicates that melanin secreted from CM cells is required for the immunomodulation of tumor microenvironment. Indeed, melanin transforms dermal fibroblasts in cancer-associated fibroblasts, suppresses the immune system and promotes tumor angiogenesis, thus sustaining CM progression and metastasis. Here, we review the current knowledge on the role of melanin secretion in CM aggressiveness and the molecular machinery involved, as well as the impact in tumor microenvironment and immune responses. A better understanding of this role and the molecular players involved could enable the modulation of melanin secretion to become a therapeutic strategy to impair CM invasion and metastasis and, hence, reduce the burden of CM-associated deaths.publishersversionpublishe

Melanin’s Journey from Melanocytes to Keratinocytes: Uncovering the Molecular Mechanisms of Melanin Transfer and Processing

Funding This research was funded by Fundação para a Ciência e a Tecnologia (Portugal) grant number PTDC/BIA-CEL/29765/2017 and Ph.D. fellowships to L.B.-L., L.C.C., M.V.N. and J.C. (numbers SFRH/BD/131938/2017, 2020.08812.BD, PD/BD/137442/2018, and PD/BD/136905/2018, respectively). This work was also supported by iNOVA4Health—UIDB/04462/2020 and UIDP/04462/2020—and by the Associated Laboratory LS4FUTURE (LA/P/0087/2020), two programs financially supported by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (Portugal).Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding keratinocytes. While the molecular mechanisms of melanin synthesis and transport in melanocytes are now well characterized, much less is known about melanin transfer and processing within keratinocytes. Over the past few decades, distinct models have been proposed to explain how melanin transfer occurs at the cellular and molecular levels. However, this remains a debated topic, as up to four different models have been proposed, with evidence presented supporting each. Here, we review the current knowledge on the regulation of melanin exocytosis, internalization, processing, and polarization. Regarding the different transfer models, we discuss how these might co-exist to regulate skin pigmentation under different conditions, i.e., constitutive and facultative skin pigmentation or physiological and pathological conditions. Moreover, we discuss recent evidence that sheds light on the regulation of melanin exocytosis by melanocytes and internalization by keratinocytes, as well as how melanin is stored within these cells in a compartment that we propose be named the melanokerasome. Finally, we review the state of the art on the molecular mechanisms that lead to melanokerasome positioning above the nuclei of keratinocytes, forming supranuclear caps that shield the nuclear DNA from UV radiation. Thus, we provide a comprehensive overview of the current knowledge on the molecular mechanisms regulating skin pigmentation, from melanin exocytosis by melanocytes and internalization by keratinocytes to processing and polarization within keratinocytes. A better knowledge of these molecular mechanisms will clarify long-lasting questions in the field that are crucial for the understanding of skin pigmentation and can shed light on fundamental aspects of organelle biology. Ultimately, this knowledge can lead to novel therapeutic strategies to treat hypo- or hyper-pigmentation disorders, which have a high socio-economic burden on patients and healthcare systems worldwide, as well as cosmetic applications.publishersversionpublishe

Melanocore uptake by keratinocytes occurs through phagocytosis and involves Protease-activated receptor-2 internalization

Funding: The authors would like to thank the scientific and technical assistance from the CEDOC Cell Culture, Flow Cytometry and Microscopy facilities. We also thank the Electron Microscopy Facility of Instituto Gulbenkian de Ciência for technical assistance. The authors also thank Dorothy Bennett and Elena Sviderskaya (St. George's University of London, UK), Susana Lopes (CEDOC, NOVA Medical School) and Paulo Matos (National Health Institute Doutor Ricardo Jorge, Portugal) for the kind gift of reagents and Paulo Matos also for expert advice. This article was supported by the LYSOCIL project. This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 811087. This article was also supported by Fundaçao para a Ciência e a Tecnologia ˜ (FCT), Portugal through grant PTDC/BIA-CEL/29765/2017, PhD fellowships to HM, MVN, LBL and LCC (PD/BD/114118/2015, PD/BD/137442/2018, SFRH/BD/131938/2017 and 2020.8812.BD, respectively), the FCT Investigator Program to DCB (IF/00501/2014/ CP1252/CT0001), and FCT Unit iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a programme financially supported by FCT / Ministério da Ciência, Tecnologia e Ensino Superior, through national funds.In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred - either in a membrane-bound melanosome or as a melanosome core, i.e., melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1, CtBP1/BARS, Cdc42 or RhoA, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct internalization routes of melanin internalization. Furthermore, we show that melanocore uptake induces Protease-activated receptor-2 (PAR-2) internalization by keratinocytes to a higher extent than melanosomes. Since skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore-based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.publishersversionpublishe