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DOI: 10.1038/ncomms8506
OPEN
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.
1 Institut Curie, PSL Research University, UMR144, CNRS, F-75248 Paris, France. 2 Structure and Membrane Compartments, Centre National de la Recherche
Scientique, UMR144, Paris F-75248, France. 3 Cell and Tissue Imaging Facility, Infrastructures en Biologie Sante et Agronomie (IBiSA), Paris F-75248,
France. 4 Institut Curie, Centre de Recherche, Laboratoire de Spectrometrie de Masse Proteomique, Paris F-75248, France. 5 Laboratoires Clarins31
chaussee Jules Cesar, Pontoise 95300, France. Correspondence and requests for materials should be addressed to G.R. (email: graca.raposo@curie.fr).
ARTICLE
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, immunouorescence 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 quantied by the increased
*
*
TUBULIN
CD63-GFP
TUBULIN
CD63-GFP
PMEL
b
Distance from
nuclei (m)
***
5
4
3
2
1
0
Keratinocytes
Coculture
CD63-PAG10
CD63-PAG10
Melanocyte
Melanocyte
MVB
Keratinocyte
MVB
1 m
Keratinocyte
500 nm
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-specic protein;
blue) and were analysed using IFM (scale bar, 10 mm). 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 quantied in
CD63-GFP-transduced NHK in mono- or co-culture with melanocytes (n 11; ***Po0.01, t-test). (c) EM analysis on ultrathin cryosections of Caucasianreconstructed epidermis immunogold-labelled for endogenous CD63 (PAG 10 nm; scale bar, 1 mm). On the right, an inset corresponding to the magnied
area of the back-boxed region depicts an MVB apposed to the keratinocyte plasma membrane in close association with melanocyte.
2
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NHK
L
Exo
CD63-PAG10
96 kDa
Alix
75 kDa
CD63
37 kDa
TYRP1
PKH
DAPI
Alix
46 kDa
Tsg101
TYRP1
PKH
DAPI
Top view
PBS-PKH
CD63
50 kDa
46 kDa
Side view
96 kDa
50 kDa
Tsg101
100 nm
1.
07
5
1.
08
2
1.
10
1.
12
1.
13
1.
14
1.
15
1.
16
1.
17
1.
21
1.
06
1. 6
07
1. 2
09
1. 5
10
1. 7
11
1. 4
12
1. 3
13
1. 0
13
1. 5
13
1. 7
15
1
Front view
Exosomes NHK-PKH
e
+ Exosomes
FITC+
92.7
SSC
FITC+
4.21
FITC
Figure 2 | EV characterization and uptake. (a) EM analysis of exosomes from NHK immunogold-labelled for endogenous CD63 (PAG 10 nm, arrows).
(b) WB analysis of exosomes (Exo; 10 mg) and total cell lysate (L; 20 mg) from NHK or fractions recovered after OptiPrep gradient (c) using anti-Alix,
-CD63 and -Tsg101 antibodies. (d) Analysis by IFM of the interaction of PKH67-labelled (green) exosomes from NHK with melanocytes labelled for
TYRP1 (red) and DAPI (blue; scale bar, 10 mm). (e) FACS analysis of melanocytes incubated for 1 h at 37 C with FITC-labelled exosomes from NHK.
Melanocytes incubated with the last wash of the FITC-labelling were used as a control.
ARTICLE
1.4
**
***
Control
Caucasian
Caucasian UVB
Black
Exosomes
Control
Caucasian
Caucasian UVB
Black
0.8
0.6
0.4
0.2
0
Tyr
**
2
Control
Caucasian
Caucasian UVB
Black
1.5
1
0.5
Exosomes
***
2.5
Medium w/o
exosomes
1.2
MITF-M
Tyrosinase activity
(normalized to control)
e
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
*
Control
Caucasian
Caucasian UVB
Exosomes
Rab27a
Melanin content
(normalized to control)
in reconstructed epidermis
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Melanin content
(normalized to control)
in reconstructed epidermis
Melanin content
(normalized to control)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Control
Black
Exosomes
Figure 3 | Exosomal effects on the regulation of pigmentation. (a) Analysis of melanin content (optical density at 492 nm) in Caucasian melanocytes
incubated for 96 h with NHK exosomes (corresponding to 15 mg of protein) resuspended in PBS (left) and medium depleted of NHK exosomes (right;
Caucasian, Caucasian irradiated with ultraviolet B and Black; ratio 1:1, melanocytes incubated with exosomes or medium isolated from the same number of
keratinocytes). Melanocytes incubated without exosomes were used as a control. (b,c) Tyrosinase activity (b) or relative gene expression of Mitf-M,
Tyrosinase and Rab27a (c) were measured in Caucasian melanocytes incubated for 96 h with exosomes from NHK (Caucasian, Caucasian irradiated with
ultraviolet B and Black; ratio 1:1, 15 mg of exosomes). Melanocytes incubated without exosomes were used as a control. Intracellular melanin content
analysis of light phototype-reconstructed epidermis incubated with exosomes from Caucasian, ultraviolet B-irradiated Caucasian (d) or Black (e) NHK.
Reconstructed epidermis cultured only with medium and PBS were used as controls. Experiments in d,e were performed with different epidermis of the
same phototype using different melanocyte donors. The data are from three independent experiments. Values are means.d. (*Po0.05; **Po0.02;
***Po0.01, t-test, n 3).
ARTICLE
Fold change
2.5
b
**
2
1.5
Caucasian
Caucasian UVB
1
0.5
Melanin content
(normalized to control)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
miR-3196
e
***
NC Pre-miR-NC
Pre-miR-203
Melanin content
(normalized to control)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Caucasian
Black
1.4
1.2
1
0.4
0.2
0
Control
Caucasian UVB AntimiR-NC
Caucasian UVB AntimiR-3196
Exosomes
Pre-miR-NC
Pre-miR-3196
0.6
miR-203a
*** **
0.8
Melanin content
(normalized to control)
Fold change
Melanin conten
(normalized to control)
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
*
Control
Black Anti-miR-NC
Black Anti-miR-203
Exosomes
Figure 4 | miRNA and pigmentation. (a) Fold change of miR-3196 in exosomes from Black NHK and normalized to exosomes from Caucasian NHK.
(b) Fold change of miR-203-a in exosomes from Caucasian-irradiated NHK and normalized to exosomes from Caucasian NHK. (cf) Analysis of
intracellular melanin content in Caucasian melanocytes transfected with pre-miR-3196, (c) pre-miR-203 (d) or incubated for 96 h with exosomes from
ultraviolet B-irradiated Caucasian NHK transfected with anti-miR-3196 (e) or with exosomes from Black NHK transfected with anti-miR-203. (f) Values are
means.d. (*Po0.05; **Po0.02; ***Po0.01, t-test, n 3).
NATURE COMMUNICATIONS | 6:7506 | DOI: 10.1038/ncomms8506 | www.nature.com/naturecommunications
ARTICLE
ARTICLE
TYR activity assay. TYR activity was determined as previously described36 with
slight modications. Melanocytes were treated with exosomes from keratinocytes
(ratio 1:1, melanocytes incubated with exosomes from the same number of
keratinocytes) for 96 h and then suspended in phosphate buffer containing 1%
Triton X-100. After vortexing, cells were claried by centrifugation at 13,000 r.p.m.
for 10 min at 4 C. TYR (10 ml) substrate L-Dopa (15 mM) was incubated with 90 ml
of extract in a 96-well plate for 30 min at 37 C. The absorbance was read at
470 nm.
WB analysis. Cells were lysed on ice in lysis buffer (20 mM Tris, 150 mM NaCl,
0.1% Triton X-100, 1 mM EDTA, pH 7.2) with a protease inhibitor cocktail
(Roche). Lysates or exosomes were incubated in a sample buffer with or without
350 mM 2-mercaptoethanol (Sigma), boiled for 5 min and fractionated with
SDSPAGE using Nupage (412%) Bis-Tris gels (Invitrogen) and transferred to
nitrocellulose membranes (Millipore). The membranes were blocked in PBS/Tween
0.1% (PBS/T) with 5% non-fat dried milk, incubated with the indicated primary
antibody diluted in PBS/T, washed four times in blocking solution and incubated
with HRP-conjugated secondary antibody followed by washing in PBS/T.
Blots were developed using the ECL Plus Western blotting detection system
(GE Healthcare) according to the manufacturers instruction.
Quantitative real-time PCR. Total RNA was extracted from the control and
treated cells or exosomes using the RNeasy Mini kit (Qiagen) or mirVana miRNA
isolation kit (Ambion). The same amount of cDNA was synthesized using NCode
VILOmiRNA cDNA synthesis kit (Invitrogen) and random primers. Real-time
PCR was carried out with the LightCycler 480 (Roche), using the Syber green
uorescent probe, and normalized using the ribosomal gene S26 (for mRNA) or
RNU6 (for miRNA). MITF-M: Fw 50 -TCTACCGTCTCTCACTGGATTGG-30 ;
Rw 50 -GCTTTACCTGCTGCCGTTGG-30 . TYR: Fw 50 - TGCCAAGCATCCTAT
CTTCC-30 ; Rw 50 -CCATGTAGGATTCCCGGTTA-30 . S26: Fw 50 -CCGTGCCTC
CAAGATGACAA-30 ; Rw 50 -CGAATGACGAATTTCTTAATGGCCT-30 . Primers
for Rab27a, miR-203, miR-3196 and RNU6 were from QIAGEN.
Immunouorescence microscopy. Cells were grown on coverslips at 70% conuency and then rinsed in PBS and xed for 15 min in 4% paraformaldehyde/PBS
at room temperature. Fixed cells were washed in PBS and quenched for 10 min in
PBS/50 mM glycine, saturated in PBS containing 1 mg ml l BSA (blocking buffer)
and permeabilized in PBS/0.05% saponin/1 mg ml 1 BSA (incubation buffer, IB).
Cells were incubated for 1 h with the primary antibody diluted in IB, washed three
times in IB and incubated with the corresponding secondary antibodies diluted in
IB for 45 min. Coverslips were washed three times with IB and then mounted in
DABCO medium and examined on an Eclipse 80i Upright Microscope (Nikon)
equipped with a CoolSNAP HQ2 CCD Camera, a Piezo Flexure Objective Scanner
and 100 Plan Apo objective (1.4 numerical aperture CFI (chrome-free innity)).
Images are maximum-intensity z projections of three-dimensional image stacks
acquired every 0.2 mm using the Metamorph software (MDS Analytical Technologies, Sunnyvale, CA).
ARTICLE
found produces the best between-slide normalization to minimize the intensitydependent differences between the samples.
Data availability. The proteomics data set can be downloaded from
http://xfer.curie.fr/get/nil/quPNEagSx9J/raw.rar
References
1. Yamaguchi, Y., Brenner, M. & Hearing, V. J. The regulation of skin
pigmentation. J. Biol. Chem. 282, 2755727561 (2007).
2. Yamaguchi, Y. et al. Human skin responses to UV radiation: pigment in the
upper epidermis protects against DNA damage in the lower epidermis and
facilitates apoptosis. FASEB J. 20, 14861488 (2006).
3. Passeron, T. et al. SOX9 is a key player in ultraviolet B-induced melanocyte
differentiation and pigmentation. Proc. Natl Acad. Sci. USA 104, 1398413989
(2007).
4. Cardinali, G. et al. Melanosome transfer promoted by keratinocyte growth
factor in light and dark skin-derived keratinocytes. J. Invest. Dermatol. 128,
558567 (2008).
5. Raposo, G. & Marks, M. S. Melanosomes--dark organelles enlighten endosomal
membrane transport. Nat. Rev. Mol. Cell Biol. 8, 786797 (2007).
6. Gyorgy, B. et al. Membrane vesicles, current state-of-the-art: emerging role of
extracellular vesicles. Cell Mol. Life Sci. 68, 26672688 (2011).
7. Cocucci, E., Racchetti, G. & Meldolesi, J. Shedding microvesicles: artefacts no
more. Trends Cell Biol. 19, 4351 (2009).
8. Simons, M. & Raposo, G. Exosomes--vesicular carriers for intercellular
communication. Curr. Opin. Cell Biol. 21, 575581 (2009).
9. Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles,
and friends. J. Cell Biol. 200, 373383 (2013).
10. Hirobe, T. Role of keratinocyte-derived factors involved in regulating the
proliferation and differentiation of mammalian epidermal melanocytes.
Pigment Cell Res. 18, 212 (2005).
11. Hirobe, T. How are proliferation and differentiation of melanocytes regulated?
Pigment Cell Melanoma Res. 24, 462478 (2011).
12. Cui, R. et al. Central role of p53 in the suntan response and pathologic
hyperpigmentation. Cell 128, 853864 (2007).
13. Chavez-Munoz, C., Morse, J., Kilani, R. & Ghahary, A. Primary human
keratinocytes externalize stratin protein via exosomes. J. Cell Biochem. 104,
21652173 (2008).
14. Mittelbrunn, M. et al. Unidirectional transfer of microRNA-loaded exosomes
from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011).
15. Escola, J. M. et al. Selective enrichment of tetraspan proteins on the internal
vesicles of multivesicular endosomes and on exosomes secreted by human
B-lymphocytes. J. Biol. Chem. 273, 2012120127 (1998).
16. Thery, C. et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted
subcellular compartment distinct from apoptotic vesicles. J. Immunol. 166,
73097318 (2001).
17. Tauro, B. J. et al. Comparison of ultracentrifugation, density gradient
separation, and immunoafnity capture methods for isolating human colon
cancer cell line LIM1863-derived exosomes. Methods 56, 293304 (2012).
18. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a
novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654659
(2007).
19. Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that
promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10,
14701476 (2008).
20. Kraemer, A. et al. UVA and UVB irradiation differentially regulate
microRNA expression in human primary keratinocytes. PLoS ONE 8, e83392
(2011).
21. Fitzpatrick, T. B. The validity and practicality of sun-reactive skin types I
through VI. Arch. Dermatol. 124, 869871 (1988).
22. Choi, W. et al. Regulation of human skin pigmentation in situ by repetitive UV
exposure: molecular characterization of responses to UVA and/or UVB.
J. Invest. Dermatol. 130, 16851696 (2010).
23. Hearing, V. J. & Tsukamoto, K. Enzymatic control of pigmentation in
mammals. FASEB J. 5, 29022909 (1991).
24. Cheli, Y., Ohanna, M., Ballotti, R. & Bertolotto, C. Fifteen-year quest for
microphthalmia-associated transcription factor target genes. Pigment Cell
Melanoma Res. 23, 2740 (2010).
25. Shibahara, S. et al. Regulation of pigment cell-specic gene expression by MITF.
Pigment Cell Res. 13(Suppl 8): 98102 (2000).
26. Van Den Bossche, K., Naeyaert, J. M. & Lambert, J. The quest for the
mechanism of melanin transfer. Trafc 7, 769778 (2006).
Acknowledgements
We are grateful to Eric Gooris for insightful discussions and support during the course of
this work. We are grateful to Allison de Horsey (St Georges School, Newport, RI, USA)
for critical reading and editing of the manuscript. We are grateful to Matteo Gentili
for help with FACS analysis and to Joanna Kowal for the exchange of expertise on
techniques. We thank Lucie Sengmanivong and Vincent Fraisier from the PICT-IBiSA
Lhomond UMR144 Imaging facility of Institut Curie and the Nikon Imaging Centre
at Institut Curie-CNRS, members of the France-BioImaging national research
infrastructure. We acknowledge France-BioImaging infrastructure supported by the
French National Research Agency (ANR-10-INSB-04, )Investments for the future*).
This work was supported by Institut Curie, CNRS and Clarins.
Author contributions
A.L.C., G.R., G.v.N. and C.D. designed the study and analysed data; C.D. performed the
analysis on reconstructed epidermis; F.-G.M. performed the electron microscopy on the
reconstructed epidermis; D.L. and F.D. conducted mass spectrometry analysis; A.L.C.
carried out all the other experiments; C.G, N.A. and K.V. provided technical assistance
for the ultraviolet irradiation, participated in the scientic discussion and nanced the
project; A.L.C. and G.R. wrote the manuscript; C.D. and G.V.N. edited the manuscript;
G.R. directed the study.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing nancial interests: The authors declare no competing nancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Lo Cicero, A. et al. Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat. Commun. 6:7506 doi: 10.1038/ncomms8506 (2015).
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