Exosomes released by keratinocytes modulate melanocyte pigmentation

Nature Communications, Jun 2015

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.

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Exosomes released by keratinocytes modulate melanocyte pigmentation

Abstract Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. Introduction In the thin outermost layer of the skin, melanocytes and surrounding keratinocytes form the epidermal–melanin unit1. Solar irradiation activates signalling cascades that induce the secretion of molecules including hormones and growth factors that lead to increased melanin synthesis in melanocytes2,3,4,5. Skin pigmentation requires close intercellular communication and results in skin tanning but also constitutes an important defense mechanism for photoprotection against Ultraviolet B exposure. Cells communicate via either soluble, secreted factors or via membrane vesicles, commonly called extracellular vesicles (EVs)6,7. Exosomes are endosome-derived EVs and correspond to the intraluminal vesicles (ILVs) released into the extracellular environment on fusion of multivesicular bodies (MVBs) with the plasma membrane. Exosomes harbour membrane and cytosolic components such as proteins, lipids and RNAs8,9. In this study we show for the first time that, in addition to soluble factors1,10,11,12, normal human keratinocytes (NHK) release exosomes that play a role in the regulation of pigmentation. Exosomes carrying selected microRNAs (miRNAs) are targeted to melanocytes and modulate the pigmented status of melanocytes by altering gene expression and enzyme activity. Results MVBs polarize to intercellular contact sites Previous studies reported that keratinocytes secrete vesicles with exosome-like features corresponding to the ILVs of MVBs13. Therefore, MVBs destined for secretion would be found in close proximity to the keratinocyte plasma membrane, as observed in other cell systems14. To visualize MVBs, NHKs were transduced with a lentivirus vector encoding CD63-GFP, a tetraspanin highly enriched in MVBs of most cell types15. After 3 days of transduction, immunofluorescence microscopy (IFM) showed that CD63-GFP-labelled compartments were primarily distributed around the nucleus (Fig. 1a, left panel). Interestingly, when transduced NHKs were co-cultured with normal human melanocytes a large fraction of CD63-positive compartments redistributed in NHK towards the areas of contact with melanocytes (Fig. 1a, right panel) as quantified by the increased distance of CD63-positive compartments from nuclei and relative to the control (Fig. 1b; P<0.01, t-test). Polarization of MVBs to the areas of cell–cell contact has similarly been observed in other exosome-secreting cell systems such as B and T cells14. Such a polarization is specific to MVBs since the distribution of early endosomes (EEA1) or Golgi (TGN46) was not drastically modified (Supplementary Fig. 1). To get further insight into such MVB redistribution in skin models, we analysed cell–cell contacts in the reconstructed skin epidermis (human epidermal model consisting of melanocytes and keratinocytes; see Methods) using immunoelectron microscopy (IEM). At high resolution, these observations revealed CD63-positive MVBs (labelling is for endogenous CD63) close to keratinocyte plasma membranes in the areas of contact with melanocytes (Fig. 1c), suggesting that those could potentially correspond to secretory MVBs. Figure 1: MVB polarization in cell culture and reconstructed epidermis. (a) CD63-GFP-transduced NHKs (green) in mono- or co-culture (ratio 1:1, melanocytes incubated with the same number of keratinocytes) with melanocytes were stained for tubulin (red) and PMEL (melanocyte-specific protein; blue) and were analysed using IFM (scale bar, 10 μm). Asterisks show the nuclei of keratinocytes and the arrow shows the site of contact between melanocyte and keratinocyte. (b) The distance of CD63-positive compartments from the centre of the corresponding nucleus was quantified in CD63-GFP-transduced NHK in mono- or co-culture with melanocytes (n=11; ***P<0.01, t-test). (c) EM analysis on ultrathin cryosections of Caucasian-reconstructed epidermis immunogold-labelled for endogenous CD63 (PAG 10 nm; scale bar, 1 μm). On the right, an inset corresponding to the magnified area of the back-boxed region depicts an MVB apposed to the keratinocyte plasma membrane in close association with melanocyte. Full size image Keratinocytes secrete exosomes interacting with melanocytes We (...truncated)


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Alessandra Lo Cicero, Cédric Delevoye, Floriane Gilles-Marsens, Damarys Loew, Florent Dingli, Christelle Guéré, Nathalie André, Katell Vié, Guillaume van Niel, Graça Raposo. Exosomes released by keratinocytes modulate melanocyte pigmentation, Nature Communications, 2015, Issue: 6, DOI: 10.1038/ncomms8506