Human Skin Model Containing Melanocytes: Essential Role of Keratinocyte Growth Factor for Constitutive Pigmentation—Functional Response to α-Melanocyte Stimulating Hormone and Forskolin

Abstract

To study human skin pigmentation in a physiological in vitro model, we developed a pigmented reconstructed skin reproducing the three-dimensional architecture of the melanocyte environment and the interactions of melanocyte with its cellular partners, keratinocytes, and fibroblasts. Co-seeding melanocytes and keratinocytes onto a fibroblast-populated collagen matrix led to a correct integration of melanocytes within the epidermal basal layer, but melanocytes remained amelanotic even after supplementation with promelanogenic factors. Interestingly, normalization of keratinocyte differentiation using keratinocyte growth factor instead of epidermal growth factor finally allowed an active pigmentary system to develop, as shown by the expression of key melanogenic markers, the production, and transfer of melanosome-containing melanin into keratinocytes. Various degrees of constitutive pigmentation were reproduced using melanocytes from different skin phenotypes. Furthermore, induction of pigmentation was achieved by treatment with known propigmenting molecules, αMSH and forskolin, thus demonstrating the functionality of the pigmentary system. This pigmented full-thickness skin model therefore represents a highly relevant tool to study the role of cell–cell, cell–matrix, and mesenchymal–epithelial interactions in the control of skin pigmentation.

Introduction

Human skin color, the most striking polymorphic trait of humans, is mainly due to melanin pigments produced by melanocytes and transferred in neighboring keratinocytes via their dendrites. The association involving one melanocyte and about 40 keratinocytes is called the epidermal melanin unit (EMU). Constitutive pigmentation depends on the quantity and the quality (pheo-/eumelanin ratio) of the melanin produced, as well as the size, mode of transfer, distribution, and degradation of the melanosomes inside the keratinocytes, and not on the number of melanocytes, which is relatively constant.¹ In addition, controls found throughout the surrounding tissue environment are also crucial regulators of pigmentation. In fact, keratinocytes regulate the adhesion, proliferation, survival, and morphology of melanocytes. The crosstalk between melanocytes and keratinocytes within the EMU is mediated both by direct connections via the E-cadherins, which allow gap junctions to form, and by a paracrine effect through keratinocyte-derived soluble factors, including α-melanocyte stimulating hormone (α-MSH), endothelins 1 and 3 (ET1 and ET3), stem cell factor (SCF), basic fibroblast growth factor (bFGF), prostaglandin E2, prostaglandin F2α, hepatocyte growth factor (HGF), or granulocyte macrophage colony-stimulating factor.^2,3 Keratinocytes also modulate the transcription of melanogenic proteins and subsequently, the quantity and quality of melanin.^4,5 It has also been shown that keratinocytes are active pigment-transfer recruiters and modulators of the distribution pattern of transferred melanosomes (aggregated versus individual melanosomes).^6–8 Furthermore, keratinocytes, via the increase in numerous released cytokines, are strongly involved in the propigmenting response of melanocyte after UV exposure.^3,9–11

Even though the role of the dermal compartment on pigmentation is far less documented, there is now evidence that the mesenchymal compartment, including fibroblasts and fibroblast-derived extracellular matrix (ECM) proteins, influences melanocyte proliferation, apoptosis resistance, morphology, and melanogenic activity.^12–25 Melanocyte adhesion and location at the dermal epidermal junction also depend on the microenvironment. In fact, several ECM proteins such as laminin or collagen IV are known to play a role in attachment of melanocytes onto the basement membrane via the interaction with specific integrins.¹⁶ Dermal fibroblasts play a regulatory role in constitutive pigmentation through the secretion of soluble factors. Some of those are the same as those secreted by keratinocytes such as HGF or SCF, and can be released in even greater amounts by fibroblasts than by keratinocytes.^20,24 Some are specifically secreted by fibroblasts, as illustrated by Dickkopf-1 (DKK1), which is responsible for the very light color of the palms and soles via a suppressive effect on melanocyte growth and activity as well as melanin uptake by keratinocytes.^23,25 More recently, the propigmenting effect of Neuregulin-1 secreted by fibroblasts derived from dark skin suggests its involvement in determining the human skin color.¹⁵ Furthermore, paracrine cytokine regulation loops may also exist; for example, keratinocyte-produced cytokines, such as IL1α or TNFα, may stimulate fibroblasts, which in turn release melanocyte-stimulating factors such as HGF or SCF.^20,26,27

Deregulations of melanocyte homeostasis and/or melanogenesis are the cause of various hyper- or hypopigmented lesions. For a better understanding of the pigmentation mechanisms and their deregulations, in vitro systems that reproduce the physiology of the pigmentary system as close as possible are required. The development of in vitro skin-engineered substitutes for biological, photobiological, and pharmacological research purposes has made significant progress over the years.^28,29 These models reproduce key structures of native skin, especially a three-dimensional (3D) organization and a differentiated epidermis. The successful integration of normal melanocytes provided a significant improvement of pigmented models, since pigmentation could be thus studied in conditions reproducing the EMU.^9,30,31 To overcome the absence of fibroblasts in these reconstructed epidermis, more complex organotypic pigmented skin models reconstructed on a dermal equivalent containing fibroblasts have been designed. Different fibroblast-containing dermal substrates have been used such as recolonized de-epidermized dermis (DED),^14,19,21,32 a glycoaminoglycan–collagen sponge,³³ or a fibroblast-populated collagen gel.^34–36 However, some of these models can hardly be considered a human normal skin model, because they contain melanocytes from mouse origin grown in a growth medium containing TPA, known to induce phenotypic and functional cellular alterations.^35–37 Beside, some researchers have developed disease skin models using non-normal cells such as melanoma or congenital café au lait macules cells.^34,35,37 Even though the integration of normal human melanocytes (NHMs) was achieved in the skin substitutes, key pigmentation parameters that could demonstrate that the pigmentary system is physiological (presence of melanogenic proteins, melanin production, and stimulation of pigmentation) have not been checked. Even more, some drawbacks were noticed. Okazaki et al. and Souto et al. cited an insufficient differentiation of the epidermis with an absence of the stratum granulosum and corneum.^32,35 Other defects concern the pigmentary system with a limited survival or a deficient functionality of melanocytes^21,14 or to the unresponsiveness of the model to physiological propigmenting agents.¹⁹

To study human skin pigmentation in a highly physiological in vitro model, our objective was to reconstruct a pigmented skin model (including human normal melanocytes, keratinocytes, and fibroblasts endowed with a functional pigmentary system) that is able to develop a real constitutive pigmentation (melanin production and transfer) and able to respond to known stimulators.

We first took advantage of our experience of (1) reconstructing skin models with a fibroblast-embedded collagen gel³⁸ and (2) reconstructing pigmented epidermis^9,31 and (3) combining both technologies to succeed in integrating melanocytes in the complete reconstructed skin. To stimulate melanin synthesis, various culture conditions were tested, supplementation with promelanogenic growth factors or modification of the keratinocyte homeostasis, resulting in (1) a correct integration of melanocytes and (2) a physiological melanocyte differentiation that allowed us to reproduce various pigmentation phenotypes. Finally, the functionality of the pigmentary system was verified by stimulation with the known propigmenting agents, αMSH and forskolin.

Materials and Methods

Cell culture

Epidermal normal human keratinocytes (NHKs) were isolated from Caucasian breast skin obtained from plastic surgery procedures, cultured as described³⁹ on a feeder layer of Swiss 3T3 fibroblasts, and used at passage 2. Human dermal fibroblasts were isolated after spreading from Caucasian mammary skin explants, amplified in the Dulbecco's modified Eagle's medium+10% fetal calf serum (FCS), and used at passage 8. NHMs were obtained from young foreskins of four different donors selected according to their pigmentation phenotype. Three Caucasian donor skins were lightly, moderately, darkly pigmented, and one African donor skin was black. Isolated melanocytes were amplified in a defined melanocyte culture medium M2 (Promocell) containing no phorbol ester and used at passages 4–6.

Skin reconstruction

NHMs, NHKs, and fibroblasts were amplified separately in their respective growth medium.

The dermal equivalent was obtained after contraction of a mixture of bovine type I collagen and fibroblasts (10⁶ cells for 7 mL) as described previously.^38,40 NHKs and NHMs were co-seeded on the contracted lattice at a concentration of 33,000 cells/cm² each: for this, 50,000 melanocytes and 50,000 keratinocytes were seeded inside a 1.5-cm² steel ring laid on the dermal equivalent. The culture was then kept submerged for 7 days, allowing cells to form a monolayer. The culture was then raised to the air–liquid interface (emersion phase) and kept at least for 1 week to allow the keratinocytes to stratify and differentiate. The culture medium used for the immersion and emersion phases was composed of the Minimal Essential Medium (Invitrogen), 10% FCS (Sigma), 10 ng/mL epidermal growth factor (EGF) (Becton Dickinson), 10⁻¹⁰ M Cholera toxin (Biomol), and 0.4 μg/mL hydrocortisone (Sigma) and was supplemented with M2 melanocyte growth factors (0.625 mL/L). Growth factors were SCF (10 ng/mL; R&D Systems), ET3 (100 nM; Sigma), bFGF (5 ng/mL; Sigma), and keratinocyte growth factor (KGF, 10 μg/mL; R&D Systems).

Induction of pigmentation by propigmenting agents

Reconstructed skin containing melanocytes from Caucasian moderately pigmented skin was made to test two propigmenting references, α-MSH (Sigma) and forskolin (Sigma). α-MSH and forskolin were added to the culture medium at the final concentration of 50 nM and 40 μM, respectively. They were renewed every second day. The treatment started on the first day of emersion phase (ED0) and ended 18 days after emersion (ED18). Another experiment was performed by adding 40 μM forskolin as soon as from the seeding of the keratinocytes and melanocytes onto the dermal equivalent.

Melanocyte morphology and distribution

After separation of the reconstructed epidermis from the dermis using forceps, dihydroxyphenylalanine (DOPA) reactions were performed as previously described,⁴¹ to visualize the melanocyte morphology and distribution throughout the surface of the epidermis.

Histology and immunostaining

Samples were fixed in 10% neutral formalin and treated for histology. Five- micrometer paraffin sections were stained with hematoxylin, eosin, and saffron. Melanin was colored by Fontana-Masson (FM) staining.

For immunolabeling, samples were embedded in Tissue Tek (Miles) and frozen in liquid nitrogen, or embedded in paraffin. Five-micrometer cryosections were postfixed in methanol at −20°C. Paraffin sections were deparaffined after several xylene/ethanol baths, and epitope demasking was performed by heating at 95°C for 20 min in a citrate buffer (pH 9). To detect melanogenic proteins, primary monoclonal antibodies (MoAb) NK1-beteb specific for Pmel-17 (Monosan), T311 specific for human tyrosinase (Novocastra), Ta99 specific for human tyrosine related protein-1 (TRP-1) (Signet), D5 specific for MITF (Neomarkers), and a polyclonal antibody D18 specific for human TRP-2 (Santa Cruz) were used.

For fluorescent immunochemistry, NKI/beteb (dilution 1/5), which recognizes Pmel-17, and Ta99 (dilution 1/50), which recognizes TRP-1, were both applied 30 min at room temperature (RT) on cryosections. Then, Alexa-488-coupled secondary antibodies (Molecular Probes) were applied for 30 min at RT. Nuclear counterstaining was performed with propidium iodide (PI).

For the enzymatic detections of tyrosinase by T311 (1/10), TRP-2 by D18 (1/100) on cryosections, and MITF by D5 (1/40) on paraffin-embedded section, biotinylated species specific of the secondary antibody were applied overnight at 4°C and revealed using the streptavidin–biotin peroxidase complex (LSAB2; Dako). 3-amino-9-ethylcarbazole (AEC) (Dako) or Histogreen (Vector Lab.) were used as enzymatic substrates. Counterstaining was performed with Papanicolaou Blue for AEC–LSAB2 staining and with Nuclear fast red (Vector Lab.) for Histogreen–LSAB2 revelation. Histogreen sections were dried after being passed through ethanol and xylene baths. AEC- and Histogreen-revealed slides were respectively mounted with immunomount (Thermo) and Vectamount (Vector Lab.).

For fluorescent co-staining of fibroblasts and melanocytes on cryosections, fibroblasts were first labeled with a WM15 MoAb specific of the CD13 antigen (dilution 1/10; AbDSerotec), which was applied for 30 min at RT, and which was followed by Alexa-488-coupled secondary antibody (dilution 1/150, 30 min RT; Molecular Probes). Melanocytes were fixed with methanol before labeling with Ta99 (dilution 1/50) and Alexa-568-coupled secondary antibody (dilution 1/200; Molecular Probes), both applied for 30 min at RT. Finally, nuclear counterstaining was performed with Hoechst dye (dilution 1/2500; Invitrogen). Negative controls have been performed by omitting primary antibodies.

The percentage of melanocytes present in basal layer and the Melanocyte/Keratinocyte ratio were assessed at ED14 by determining the ratio of TRP-1-stained cells to the number of total basal cells on cryosections. The number of total cells was estimated by the count of PI-labeled nuclei. Measurements were taken from three to five different fields for each sample (magnification×100), and in three independent experiments, each of which were performed in duplicate. The value was expressed as mean with the standard error of the mean.

Transmission electron microscopy

Samples were fixed for 1 h in 4% glutaraldehyde, then washed in PBS, and postfixed for 90 min in 2% osmium tetroxide. Samples were dehydrated and embedded in a Spurr resin. The resin was polymerized at 60°C. Eighty-nanometer-ultrathin sections were counterstained with uranyl acetate and lead citrate and observed with a JEOL 1010 electron microscope.

Colorimetric measurements

A Mercury 2000 spectrocolorimeter (Datacolor) was used to measure the luminance (L*) of pigmented reconstructed skin samples (three to six samples in each experiment) in the CIE L*a*b* color space. The lower the L* value, the darker the color of the skin sample.

Melanin content

Melanin pigments revealed by FM staining on 5-μm paraffin sections were quantified by an image analysis using the Pathfinder™ Cellscan morphoscan platform associated to CellMarker software. Melanin content is expressed as the percentage of melanin surface/epidermis surface.

Statistical analysis

Luminance and melanin content values are expressed as mean±standard deviation calculated for three to six different samples in each experiment and analyzed using the two-tailed unpaired Student's t-test.

Results

Induction of constitutive pigmentation

Integration of melanocytes in the reconstructed skin model

To develop a human pigmented skin model, NHKs and NHMs from Caucasian moderately pigmented skin were firstly amplified independently and then were co-seeded onto a dermal equivalent composed of a contracted fibroblast-embedded collagen gel (lattice). NHKs and fibroblasts were both from Caucasian skins. The skin was reconstructed in an EGF-based keratinocyte growth medium as described in the Material and Methods section. Several cell concentrations (from 33,000 to 300,000 keratinocytes/cm² and from 6700 to 33,000 melanocytes/cm²) and different NHK/NHM ratios (from 1/10 to 1/1) were tested (data not shown). These preliminary trials allowed us to select the optimal seeding concentration for each cell type, eventually set at 33,000 keratinocytes/cm² and 33,000 melanocytes/cm². Using these conditions, a skin model was obtained 7 days after emersion (ED7) with (1) a good epidermal structure (Fig. 1a) and (2) a correct integration of melanocytes in the basal layer revealed by immunostaining of TRP-1, a specific melanosome marker (Fig. 1b). DOPA reaction performed on the epidermal sheet showed that melanocytes were homogeneously distributed and maintained with a mostly bipolar morphology (Fig. 1c). However, using FM staining, no melanin pigment was visible neither in the melanocytes nor in the neighboring keratinocytes, indicating that melanocytes were inactive (Fig. 1d).

FIG. 1.

Integration of melanocytes and melanin synthesis in reconstructed skin: effect of various melanogenic growth factors. (a–d) Standard epidermal growth factor (EGF)-based medium condition; (e–l) addition of melanogenic growth factors in the EGF-based medium: stem cell factor (SCF; 10 ng/mL) (e, i), endothelin-3 (ET3; 100 nM) (f, j), basic fibroblast growth factor (bFGF; 5 ng/mL) (g, k), and SCF+ET3 (10 ng/mL +100 nM) (h, l). (a) hematoxylin-eosin-saffron (HES), (b) immunolabeling of tyrosine related protein-1 (TRP-1) revealing specifically melanocytes (green); (c, e–h) dihydroxyphenylalanine (DOPA) reaction on epidermal sheet; (d, i–l) Fontana-Masson staining (FM). Note the presence and the normal location of melanocytes at the basal layer of the epidermis, but the absence of melanin on skin sections after FM staining: Scale bars (a, b, d, i–l) 25 μm, (c, e–h) 50 μm.

Effects of melanogenic growth factors on constitutive pigmentation

To stimulate the differentiation of melanocytes and melanin synthesis, various growth factors were used to supplement the skin growth medium. In the presence of SCF, ET3, and bFGF, largely known to influence melanocytes and melanogenesis, the density and/or morphology of melanocytes was modulated. While the melanocyte density was increased by ET3 and bFGF, it was decreased by SCF alone (Fig. 1e–g). When combined with ET3, SCF induced an increase in the number of melanocytes, but an adverse cytotoxic effect was observed as shown by the round-shape morphology (Fig. 1h). However, in all conditions tested, the melanin pigment was still not detectable in the epidermis as shown by FM staining (Fig. 1i–l).

Induction of melanin production in the reconstructed skin cultured with KGF

Since keratinocytes are recognized for promoting melanocyte stimulation, we tested a growth factor, KGF, which is known to act on keratinocyte differentiation. Replacement of EGF by KGF, 4 days after NHK-NHM seeding, did not modify the basic architecture of the skin model that displayed a correct epidermal differentiation process with horny layer formation (Fig. 2a). Interestingly, KGF induced a normalized or an increased expression of differentiation markers such as K10 and loricrin at an early time ED7, compared to those observed with EGF (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/tec). Dermal fibroblasts could be observed within the dermal equivalent, and melanocytes were still correctly located at the basal layer of the epidermis as it is seen in normal human skin (Fig. 2b, c). FM staining revealed striking differences with the use of KGF compared that of EGF: melanin pigment could be detected at ED7, mostly in the basal layer of the reconstructed epidermis (Fig. 2d). The amount of melanin was increased at day 14 of emersion (ED14), and melanin granules were detected throughout the epidermal layers, thus resemble very much the vivo situation (Fig. 2e, f).

FIG. 2.

Stimulation of melanin production in reconstructed skin cultured with keratinocyte growth factor (KGF). Pigmented reconstructed skin (PRS) was cultured in KGF (10 ng/mL) instead EGF (10 ng/mL) containing a medium from the 4th day after epidermal cell seeding to the 7th or 14th day after emersion (ED7 or ED14): HES staining (a), double immunostaining of fibroblasts with a CD13 antibody (green) and melanocytes with a TRP-1 antibody (red) (b), and FM staining (d, e). For comparison, CD13/TRP-1 double immunostaining and FM coloration have been performed in a Caucasian normal human skin (NHS) (c, f). All nuclei were counterstained with Hoechst dye (blue). Note that melanin could be observed in the reconstructed skin comprising human normal melanocytes, keratinocytes, and fibroblasts, cultured in the KGF-containing medium. Scale bar=25 μm.

Characterization of the pigmentary system in the reconstructed skin

The pigmentary system was further characterized on the reconstructed skin model cultured in a KGF-containing medium, at ED14. DOPA reaction on epidermal sheet showed that melanocytes were homogeneously distributed and developed a dendritic shape (Fig. 3a, b), compared to that of melanocytes in vivo. The percentage of melanocytes present in the basal layer was calculated by measuring the number of TRP-1 positively stained cells compared to the number of unstained cells (keratinocytes) (see Materials and Methods section). We found that 5.88%±0.39% of the cells present in the basal layer were melanocytes, which corresponds to a melanocyte/keratinocyte ratio of around 1/18.

FIG. 3.

Characterization of the pigmentary system in the reconstructed skin. The pigmentary system in the pigmented reconstructed skin (PRS) has been analyzed at ED14 using (a, b) DOPA reaction on epidermal sheet, (c, d) transmission electron microscopy, (e–n) immunohistochemical labeling of the melanogenic proteins MITF, Tyrosinase, Pmel-17, TRP-1, and TRP-2 in reconstructed skin containing melanocytes isolated from a Caucasian moderately pigmented skin (e, g, i, k, m). For comparison, the same immunolabeling has been performed on NHS from a Caucasian individual (f, h, j, l, n). Note the homogeneous distribution and the dendritic shape of melanocytes (a, b), the presence of mature melanosomes into the dendrites, and their transfer into keratinocytes (arrows) (c, d). The expression of the key melanogenic proteins was found to be similar in PRS compared to human skin (e–n). Scale bars (a) 100 μm, (b) 10 μm, (c) 200 nm, (d) 1 μm, and (e–n) 25 μm.

The transfer of melanosomes within keratinocytes was studied using transmission electron microscopy. Detected ultrastructurally in between keratinocytes, dendrites contained stage III and stage IV mature melanosomes, which correspond to melanosomes with melanin deposits along fibrous striations or full of melanin, respectively (Fig. 3c). Interestingly, individual or clusters of mature melanosomes were observed within the keratinocytes (Fig. 3d).

The pigmentary system was characterized using immunolabeling of melanogenic proteins, Pmel-17, Tyrosinase, TRP-1, TRP-2, and the key melanogenic transcription factor MITF.

They confirmed that melanocytes were located at the basal layer and revealed that the pigment cells expressed Pmel-17, Tyrosinase, TRP-1, TRP-2, and MITF in the same way as they do in vivo (Fig. 3e–n). To assess the life span of the model, several parameters were evaluated using skin samples left in the emersion phase until ED28 (Supplementary Fig. S2). Although cell proliferation revealed by Ki67 staining was maintained until ED28, we found that the morphological qualities of the epidermis—global histological architecture, organization, and thickness of the viable epidermis—did not rise a sufficient level later than ED18. Therefore, the melanocyte behavior was analyzed from ED7 to ED18. Actually, melanocyte density, location, and activity assessed, respectively, by DOPA reaction, TRP-1, and FM staining, were constant all over this period. Precisely, the percentage of melanocytes at the basal layer at ED18, equal to 6.20±0.49 (corresponding to a melanocyte/keratinocyte ratio of 1/16), was very close to the one calculated at ED14 (see above). Altogether, the optimal lifespan of the model has been estimated to be ED18.

Reconstruction of skins of different phenotypes

We addressed the issue of integrating melanocytes from various constitutive pigmentation donors into the skin model. Four melanocyte strains were isolated from light to black skins and amplified before being used for skin reconstruction. A single keratinocyte and fibroblast strain from Caucasian donors was used. Samples were analyzed at ED14. As illustrated in Figure 4, all melanocyte strains, from the lighter to the darker, were correctly integrated and located into the skin model, and produce melanin. In correlation with the original skin phenotype, the color of skin samples varied gradually from light to dark. Luminance measurements and quantification of the FM staining confirmed that the more pigmented the melanocytes, the darker the skin, and the higher the amount of melanin.

FIG. 4.

Reconstruction of skins of different phenotypes. Melanocyte strains (M03, M106, M166, and M504) isolated from increasing constitutive pigmentation skins (light to black) have been integrated into the reconstructed skin containing same keratinocytes and fibroblasts. For comparison, a reconstructed skin containing no melanocytes (NHMs) is shown. At ED14, the macroscopic pictures of the samples, the melanin content revealed by FM staining on histological section, and quantified by image analysis, as well as the Luminance value (L*) showed that the more pigmented the melanocytes, the darker the reconstructed skin and the higher the amount of melanin. Two tailed Student's t-test *p<0.05, **p<0.01. Scale bar=25 μm.

Induction of pigmentation by propigmenting agents

To check the functionality of the pigmentary system in the skin reconstructs containing melanocytes from moderately pigmented skin, two propigmenting agents, αMSH and Forskolin, were tested in a set of independent reconstructed samples. The culture medium was supplemented with 50 nM α-MSH or 40 μM forskolin, from ED0 to ED18. In both treatment conditions, a visible darkening of the reconstructed skin was noticed with a corresponding decrease in the luminance value (DL>6) and a significant increase in melanin production within the epidermis, as illustrated with αMSH in Figure 5A. In another protocol, 40 μM forskolin was added 7 days earlier, that is, when the cells were seeded onto the dermal equivalent. This resulted in an intense pigmentation—the skin becoming almost black—correlated to a significant higher melanin content within the epidermis and a 26-point reduction in the luminance value (Fig. 5B).

FIG. 5.

Induction of pigmentation in the reconstructed skin by propigmenting agents. The culture medium was supplemented with (A) 50 nM α-melanocyte stimulating hormone from ED0 to ED18, or (B) 40 μM forskolin from the epidermal cells seeding to ED18. The treatment with the compounds induced a very intense darkening of the skin (a, b) with a corresponding decrease in Luminance L* (e). The melanin content, revealed on the skin section stained by FM staining (c, d) and quantified by image analysis (f), was found to be significantly increased by both propigmenting agents (two-tailed Student's t-test *p<0.05, ***p<0,001). Scale bar=25 μm.

Discussion

To study cutaneous pigmentation in a more physiological context, we have developed a pigmented reconstructed skin that reproduces (1) the 3D architecture of the melanocyte environment; (2) the interactions between melanocytes and their active cellular partners, keratinocytes, and fibroblasts; and (3) the functionality of the pigmentary system. The first experiments were based on our know-how of human skin reconstruction on fibroblast-populated living dermis and production of pigmented reconstructed epidermis onto DED.^9,31,38 They allowed us to obtain the integration of melanocytes within the epidermis, using a combined EGF-based keratinocyte medium supplemented with melanocyte factors from the M2 medium (devoid of any phorbol ester). However, although the skin model displayed a good histological quality, melanocytes were mostly bipolar and were found to be in a quiescent status with no melanin production.

One of the major reasons for the observed absence of constitutive pigmentation may be the presence of dermal fibroblasts. The development of a functional pigmented reconstructed epidermis without a living dermis has largely been reported by ourselves and others,^{9,30,31,42,43} but the introduction of fibroblasts in 3D models seemed more complex with regard to constitutive pigmentation and melanin synthesis. Indeed, fibroblasts have been previously shown to play a regulating role on melanocytes and melanogenesis. Although fibroblasts and the ECM increased pigmentation in 2D co-cultures,^13,20 fibroblasts seemed to contribute to keeping melanocytes in an amelanotic state in 3D reconstructed skin models.^14,19,21 Indeed, Cario-Andre et al. found that the introduction of fibroblasts, whatever their phototype, decreased pigmentation in epidermis reconstructed on dead DED.¹⁴ In addition, dermal fibroblasts have been shown to exert a negative regulative effect on melanocyte adhesion and survival.^19,21

One hypothesis was based on the fact that fibroblasts may inhibit melanin synthesis and reduce the number of melanocytes as a result of their known stimulatory effect on keratinocyte proliferation.²¹

Actually, the effects of fibroblasts on pigmentation may be more complex, since the recent work of Choi et al. reported a stimulating effect on pigmentation of one secreted protein, Neuregulin-1, found highly expressed in fibroblasts from phototype VI skin.¹⁵ However, the present experiments have been performed with fibroblasts from Caucasian skin. Introduction of fibroblasts from light to black skin in this full-thickness skin model should be investigated to provide an insight into their differential influence on pigmentation.

To (1) induce melanin synthesis in the skin model containing Caucasian fibroblasts and melanocytes and to (2) obtain a level of pigmentation similar to the in vivo situation, we tested the effects of different growth factors, SCF, ET3, and bFGF, known to act on melanocytes through their specific membrane receptors.^44–47 Literature reports multiple effects for these factors, especially for SCF, whose role in the development and the survival of melanocytes is well known, but which has also been described as a melanogen for human melanocytes.^48–50

However, the results were disappointing, suggesting that direct stimulation of melanocytes by SCF, ET3, or bFGF was not sufficient to induce pigmentation. It can be argued that other natural melanocyte activators may have been tested such as ET1 or pro-opiomelanocortin-derived peptides such as α-MSH. Although ET1 is thought to enhance melanocyte differentiation more than ET3 (though they share the same membrane receptor), its effect as a potent downregulator of E-cadherin, a key protein for melanocyte maintenance at the basal location,⁵¹ possibly via the upregulation of MCAM,⁵² could negatively affect melanocyte integration in reconstructed skin. Regarding the addition of αMSH in the basal culture medium, we have chosen to use it for functionality studies only, to avoid a potential saturation of the α-MSH/MC1R pathway, which is largely involved in the signaling of UV-induced pigmentation.

Based on the well-documented involvement of keratinocytes in pigmentation via the release of numerous paracrine factors and the direct connections taking place between keratinocytes and melanocytes,^2,3,10 we tried to overcome the problem of lack of pigmentation through an original approach, that is, the modulation of keratinocyte homeostasis.

Indeed, in a standard EGF-based medium, keratinocytes are highly proliferative, leading to a rapid epidermal turnover. We tried to improve keratinocyte differentiation and turnover, assuming that this could promote melanin production. Previous studies showed that the replacement of EGF by KGF in the culture medium may be a solution.^53,54 KGF, a human epithelial growth factor, member of the family of FGFs (FGF-7), has been shown to be a mitogen as efficient as EGF in conventional low-calcium keratinocyte cultures and, in contrast to EGF, to be able to stimulate the expression of early and late markers of keratinocyte differentiation (keratin 1 and filaggrin).⁵⁴ In addition, Gibbs et al. showed that the replacement of EGF by KGF for the reconstruction of epidermis on a dead DED normalized (1) the number of living cell layers (6–8 versus 14–16), (2) the Ki67 proliferation index (15% versus 30%), and (3) the granular expression of transglutaminase 1 and involucrin, and repressed the expression of keratinocyte keratins 6, 16, and 17 as observed in vivo.⁵³

Concerning K16 expression, it could be detected in both EGF and KGF conditions in our reconstructed skin model (Supplementary Fig. S1), in agreement with previous results obtained in nonpigmented organotypic models containing fibroblasts.^55,56 Altogether, these data suggest that the culture conditions as well as the presence of fibroblasts contribute in keeping keratinocytes in a relatively activated status.

However, interestingly, in presence of KGF, but not EGF, normalization of the expression of some differentiation markers of the epidermis such as K10 or loricrin could be observed (Supplementary Fig. S1). At the same time, our data show that the switch from EGF to KGF yielded the expected results in terms of development of constitutive pigmentation with the synthesis of melanin by melanocytes.

Chen et al. have recently shown that cultured melanocytes exposed to KGF have an increased tyrosinase and KGFR expression, thus suggesting that a direct effect of KGF on melanocytes and melanogenesis occurred.⁵⁷ However, this experiment was conducted in a monolayer system clearly different from the 3D and tricellular condition we created, which is able to restore physiological expression of the KGF receptor on the keratinocyte membrane only. Indeed, it has been reported that KFG is able to act only on keratinocytes through its specific receptor, FGFR2-IIIb, expressed exclusively in the epithelial cell lineage.^58,59 Furthermore, several other arguments support the idea that an indirect action of KGF via keratinocytes could prevail on melanocytes and melanogenesis. KGF has been shown to upregulate melanosome transfer by acting primarily on the recipient keratinocytes.⁶⁰ Additionally, KGF has shown to induce expression by keratinocyte of the melanocyte-stimulating factor SCF.⁶¹

Finally, improvement of keratinocyte differentiation with KGF may be involved in the secretion of specific paracrine factors requested for melanogenesis, as illustrated by Grabbe et al. for SCF.⁶² The normalization of the differentiation induced by KGF can also contribute to promote melanosome transfer to the keratinocytes, as suggested by Belleudi et al.⁶³

Characterization of the pigmentary system revealed that melanocytes correctly resided in the basal layer, developed dendrites, contained melanosomes expressing major melanogenic proteins like Pmel-17, tyrosinase, TRP-1, and TRP-2. Furthermore, the master melanocyte transcription factor MITF was detected in the melanocytes. Finally, the transfer of melanosomes containing melanin to neighboring keratinocytes demonstrated the functionality of the EMU with many similarities to the in vivo situation.

The melanocyte/keratinocyte ratios within the basal epidermal layer were estimated to be 1/16 and 1/18, which are in the same range of those found in vivo (1/8–1/21).^43,64 The basal ratio does not seem to depend on the seeding NHM/NHK ratio, which in our condition was high (1/1), reflecting the low keratinocyte density used (33,000 NHKs/cm²). This implies that keratinocytes strongly proliferate to form a confluent epidermal monolayer during the immersion phase (7 days). In most of the pigmented models, keratinocytes are seeded at a higher density, but cultured for a shorter immersion phase.^{14,19,21,30,34,35} The seeding density of melanocytes (33,000 NHMs/cm²) is similar to previously published data.^9,21,31,35 Seeding melanocytes with few keratinocytes can give an advantage to the melanocytes in terms of space and time to attach to the support as previously suggested.^19,35 However, the most interesting finding is that the final basal melanocyte/keratinocyte ratio equilibrated to a physiological balance as checked at ED7 and ED18. This reinforced previous assumptions that natural homeostasis between both cell types was strongly governed by keratinocytes.^65,66

As we obtained melanin synthesis with Caucasian melanocytes, the capacity of the model to integrate different strains of melanocytes from various phenotypes was tested. The results revealed that despite difficulties to culture highly pigmented melanocytes—primary thawed cultures are less prone to growth in the M2 culture medium used—the reconstructed skin model could accept more or less pigmented melanocytes, leading to pigmentation that was similar to the original phenotype. This indicates that such a system allows the study of various constitutive pigmentations. Finally, the stimulation of the pigmentary system by propigmenting modulators α-MSH and forskolin revealed the functionality of the model. In parallel, its potential use to evaluate depigmenting agents was also checked with Rucinol, a known in vitro and in vivo lightening agent (see Supplementary Fig. S3).^67,68 The stimulation of melanin synthesis by α-MSH demonstrated the responsiveness of the α-MSH/MC1R pathway, in contrast to negative data previously reported on pigmented epidermis reconstructed on a fibroblast-repopulated DED.¹⁹ The propigmenting effect of forskolin, a direct cAMP inducer bypassing MC1R, was even more impressive in our reconstructed skin model and confirmed the high efficacy of this diterpene derivative to darken the skin as previously demonstrated by the Fisher's group in MC1R-deficient humanized mice.⁶⁹ In the present study, we focused on the constitutive pigmentation and the responsiveness of melanocytes to chemical pro- or depigmenting agents. Future experiments will be designed to study the functionally of the pigmented skin model face to UV radiation.

In conclusion, a functional and pigmented human skin model comprising normal epidermal keratinocytes, melanocytes, and dermal fibroblasts was developed and will allow us to study the role of cell–cell and cell–matrix interactions in the control of skin pigmentation. This model will represent a powerful tool for elucidating the role of mesenchymal–epithelial interactions involved in melanocyte homeostasis.

Footnotes

Acknowledgments

We would like to thank Pr. Meenhard Herlyn for fruitful discussion and advices, Xavier Marat for help in supplying the depigmenting reference, and Catherine Olivry for artwork.

Disclosure Statement

No competing financial interests exist.

References

Nordland

J.J.

, Ortonne

J.-P.

The normal color of human skin. Nordlund

J.J.

, Boissy

R.E.

, Hearing

V.J.

, King

R.A.

, Oetting

W.S.

, Ortonne

J.P.

The Pigmentary System. Oxford: Blackwell Publishing Ltd., 2006; 504–520.

Haass

N.K.

, Herlyn

Normal human melanocyte homeostasis as a paradigm for understanding melanoma. J Invest Dermatol Symp Proc, 10:153. 2005.

Hirobe

Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes. Pigment Cell Res, 18:2. 2005.

Duval

, Smit

N.P.

, Kolb

A.M.

, Régnier

, Pavel

, Schmidt

Keratinocytes control the pheo/eumelanin ratio in cultured normal human melanocytes. Pigment Cell Res, 15:440. 2002.

Kippenberger

, Loitsch

, Solano

, Bernd

, Kaufmann

Quantification of tyrosinase, TRP-1, and TRP-2 transcripts in human melanocytes by reverse transcriptase- competitive multiplex PCR- regulation by steroid hormones. J Invest Dermatol, 110:364. 1998.

Minwalla

et al. Keratinocytes play a role in regulating distribution patterns of recipient melanosomes in vitro. J Invest Dermatol, 117:341. 2001.

Sharlow

E.R.

, Paine

C.S.

, Babiarz

, Eisinger

, Shapiro

, Seiberg

The protease-activated receptor-2 upregulates keratinocyte phagocytosis. J Cell Sci, 113:3093. 2000.

Weiner

, Han

, Scicchitano

B.M.

, Li

, Hasegawa

, Grossi

, Lee

, Brissette

J.L.

Dedicated epithelial recipient cells determine pigmentation patterns. Cell, 130:932. 2007.

Duval

, Régnier

, Schmidt

Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure. Pigment Cell Res, 14:348. 2001.

10.

Imokawa

Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Res, 17:96. 2004.

11.

Kadekaro

A.L.

, Kavanagh

R.J.

, Wakamatsu

, Ito

, Pipitone

M.A.

, bdel-Malek

Z.A.

Cutaneous photobiology. The melanocyte vs. the sun: who will win the final round?

Pigment Cell Res, 16:434. 2003.

12.

Balafa

, Smith-Thomas

, Phillips

, Moustafa

, George

, Blount

, Nicol

, Westgate

, MacNeil

Dopa oxidase activity in the hair, skin and ocular melanocytes is increased in the presence of stressed fibroblasts. Exp Dermatol, 14:363. 2005.

13.

Buffey

J.A.

, Messenger

A.G.

, Taylor

, Ashcroft

A.T.

, Westgate

G.E.

, MacNeil

Extracellular matrix derived from hair and skin fibroblasts stimulates human skin melanocyte tyrosinase activity. Br J Dermatol, 131:836. 1994.

14.

Cario-Andre

, Pain

, Gauthier

, Casoli

, Taieb

In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation. Pigment Cell Res, 19:434. 2006.

15.

Choi

, Wolber

, Gerwat

, Mann

, Batzer

, Smuda

, Liu

, Kolbe

, Hearing

V.J

. The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin. J Cell Sci, 15:3102. 2010.

16.

Hara

, Yaar

, Tang

, Eller

M.S.

, Reenstra

, Gilchrest

B.A.

Role of integrins in melanocyte attachment and dendricity. J Cell Sci, 107:2739. 1994.

17.

Hedley

S.J.

, Gawkrodger

D.J.

, Weetman

A.P.

, Macneil

Investigation of the influence of extracellular matrix proteins on normal human melanocyte morphology and melanogenic activity. Br J Dermatol, 135:888. 1996.

18.

Hedley

S.J.

, Wagner

, Bielby

, Smith-Thomas

, Gawkrodger

D.J.

, Mac Neil

The influence of extracellular matrix proteins on cutaneous and uveal melanocytes. Pigment Cell Res, 10:54. 1997.

19.

Hedley

S.J.

, Layton

, Heaton

, Chakrabarty

K.H.

, Dawson

R.A.

, Gawkrodger

D.J.

, MacNeil

Fibroblasts play a regulatory role in the control of pigmentation in reconstructed human skin from skin types I and II. Pigment Cell Res, 15:49. 2002.

20.

Imokawa

, Yada

, Morisaki

, Kimura

Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes. Biochem J, 330:1235. 1998.

21.

Lee

D.Y.

, Lee

J.H.

, Lee

E.S.

, Cho

K.H.

, Yang

J.M.

Fibroblasts play a stimulatory role in keratinocyte proliferation but an inhibitory role in melanocyte growth and pigmentation in a skin equivalent system from skin type IV. Arch Dermatol Res, 294:444. 2003.

22.

Scott

, Cassidy

, Busacco

Fibronectin suppresses apoptosis in normal human melanocytes through an integrin-dependent mechanism. J Invest Dermatol, 108:147. 1997.

23.

Yamaguchi

, Passeron

, Hoashi

, Watabe

, Rouzaud

, Yasumoto

, Hara

, Tohyama

, Katayama

, Miki

, Hearing

V.J.

Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/beta-catenin signaling in keratinocytes. FASEB J, 22:1009. 2008.

24.

Okazaki

, Yoshimura

, Uchida

, Harii

Correlation between age and the secretions of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts. Br J Dermatol, 153,Suppl 2:23. 2005.

25.

Yamaguchi

, Itami

, Watabe

, Yasumoto

, bdel-Malek

Z.A.

, Kubo

, Rouzaud

, Tanemura

, Yoshikawa

, Hearing

V.J.

Mesenchymal-epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation. J Cell Biol, 165:275. 2004.

26.

Matsumoto

, Okazaki

, Nakamura

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts. Biochem Biophys Res Commun, 188:235. 1992.

27.

Mildner

, Mlitz

, Gruber

, Wojta

, Tschachler

Hepatocyte growth factor establishes autocrine and paracrine feedback loops for the protection of skin cells after UV irradiation. J Invest Dermatol, 127:2637. 2007.

28.

Brohem

C.A.

, da Silva Cardeal

L.B.

, Tiago

, Soengas

M.S

, de Moraes Barros

S.B.

, Maria-Engler

S.S.

Artificial skin in perspective: concepts and applications. Pigment Cell Melanoma Res, 24:35. 2010.

29.

Groeber

, Holeiter

, Hampel

, Hinderer

, Schenke-Layland

Skin tissue engineering -In vivo and in vitro applications. Adv Drug Deliver Rev, 128:352. 2011.

30.

Bessou-Touya

, Picardo

, Maresca

, Surlève-Bazeille

J.E.

, Pain

, Taïeb

Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection. J Invest Dermatol, 111:1103. 1998.

31.

Regnier

, Duval

, Galey

J.B.

, Philippe

, Lagrange

, Tuloup

, Schmidt

Keratinocyte-melanocyte co-cultures and pigmented reconstructed human epidermis: models to study modulation of melanogenesis. Cell Mol Biol, 45:969. 1999.

32.

Souto

L.R.

, Rehder

, Vassallo

, Cintra

M.L.

, Kraemer

M.H.

, Puzzi

M.B.

Model for human skin reconstructed in vitro composed of associated dermis and epidermis. Sao Paulo Med J, 124:71. 2006.

33.

Nakazawa

, Nakazawa

, Sahuc

, Lepavec

, Collombel

, Damour

Pigmented human skin equivalents: new methods of reconstitution by grafting an epithelial sheet onto a non-contractile dermal equivalent. Pigment Cell Res, 10:382. 1997.

34.

Meier

, Nesbit

, Hsu

M.-Y.

, Martin

, Van Belle

, Elder

D.E.

, Schaumburg-Lever

, Garbe

, Walz

T.M.

, Donatien

, Crombleholme

T.M.

, Herlyn

Human melanoma progression in skin reconstructs-biological signifiance of bFGF. Am J Pathol, 156:193. 2000.

35.

Okazaki

, Suzuki

, Yoshimura

, Harii

Construction of pigmented skin equivalent and its application to the study of congenital disorders of pigmentation. Scand J Plast Reconstr Surg Hand Surg, 39:339. 2005.

36.

Yoshimura

, Tsukamoto

, Okazaki

, Virador

V.M.

, Lei

T.C.

, Suzuki

, Uchida

, Kitano

, Harii

Effects of all-trans retinoic acid on melanogenesis in pigmented skin equivalents and monolayer culture of melanocytes. J Dermatol Sci, 27,Suppl 1:S68. 2001.

37.

Liu

, Suwa

, Wang

, Takemura

, Fang

Y.R.

, Li

, Zhao

, Jin

Reconstruction of a tissue-engineered skin containing melanocytes. Cell Biol Int, 31:985. 2007.

38.

Bernerd

, Asselineau

Successive alteration and recovery of epidermal differentiation and morphogenesis after specific UVB-damages in skin reconstructed in vitro. Dev Biol, 183:123. 1997.

39.

Rheinwald

J.G.

, Green

Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinocyte colonies from single cells. Cell, 6:331. 1975.

40.

Asselineau

, Bernhard

, Bailly

, Darmon

Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture of human keratinocytes at the liquid-air interface. Exp Cell Res, 159:536. 1985.

41.

Staricco

R.J.

, Pinkus

Quantitative and qualitative data on the pigment cells of adult human epidermis. J Invest Dermatol, 28:33. 1957.

42.

Duval

, Schmidt

, Régnier

, Facy

, Asselineau

, Bernerd

The use of reconstructed human skin to evaluate UV-induced modifications and sunscreen efficacy. Exp Dermatol, 12,Suppl 2:64. 2003.

43.

Gibbs

, Murli

, De Boer

, Mulder

, Mommaas

A.M.

, Ponec

Melanosome capping of keratinocytes in pigmented reconstructed epidermis- effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis. Pigment Cell Res, 13:458. 2001.

44.

Halaban

, Langdon

, Birchall

, Cuono

, Baird

, Scott

, Moellmann

, McGuire

Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol, 107:1611. 1988.

45.

Reid

, Turnley

A.M.

, Maxwell

G.D.

, Kurihara

, Bartlett

P.F.

, Murphy

Multiple roles for endothelin in melanocyte development: regulation of progenitor number and stimulation of differentiation. Development, 122:3911. 1996.

46.

Swope

V.B.

, Medrano

E.E.

, Smalara

, Abdel-Malek

Z.A.

Long-term proliferation of human melanocytes is supported by the physiologic mitogens α-melanotropin, endothelin-1, and basic fibroblast growth factor. Exp Cell Res, 217:453. 1995.

47.

Wehrle-Haller

The role of kit-ligand in melanocyte development and epidermal homeostasis. Pigment Cell Res, 16:287. 2003.

48.

Luo

, Chen

, Searles

, Jimbow

Coordinated mRNA expression of c-kit with tyrosinase and TRP-1 in melanin pigmentation of normal and malignant human melanocytes and transient activation of tyrosinase by Kit/SCF-R. Melanoma Res, 5:303. 1995.

49.

Hachiya

, Kobayashi

, Ohuchi

, Takema

, Imokawa

The paracrine role of stem cell factor/c-kit signaling in the activation of human melanocytes in ultraviolet-B-induced pigmentation. J Invest Dermatol, 116:578. 2001.

50.

Kasamatsu

, Hachiya

, Higushi

, Ohuchi

, Kitahara

, Boissy

Production of the soluble form of kit, s-kit, abolishes stem cell factor-induced melanogenesis in human melanocytes. J Invest Dermatol, 128:1763. 2008.

51.

Jamal

, Schneider

R.J.

UV-induction of keratinocyte endothelin-1 downregulates E-cadherin in melanocytes and melanoma cells. J Clin Invest, 110:443. 2002.

52.

Mangahas

C.R.

, dela Cruz

G.V.

, Schneider

R.J.

, Jamal

Endothelin-1 upregulates MCAM in melanocytes. J Invest Dermatol, 123:1135. 2004.

53.

Gibbs

, Silva Pinto

A.N.

, Murli

, Huber

, Hohl

, Ponec

Epidermal growth factor and keratinocyte growth factor differentially regulate epidermal migration, growth, and differentiation. Wound Repair Regen, 8:192. 2000.

54.

Marchese

, Rubin

, Ron

, Faggioni

, Torrisi

M.R.

, Messina

, Frati

, Aaronson

S.A.

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family. J Cell Physiol, 144:326. 1990.

55.

Smiley

A.K.

, Klingenberg

J.M.

, Boyce

S.T.

, Supp

D.M.

Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting. Burns, 32:135. 2006.

56.

Stark

H.-J.

, Baur

, Breitkreutz

, Mirancea

, Fusenig

N.E.

Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J Invest Dermatol, 112:681. 1999.

57.

Chen

, Hu

, Li

W.H.

, Eisinger

, Seiberg

, Lin

B.C.

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Exp Dermatol, 19:865. 2010.

58.

Miki

, Bottaro

D.P.

, Fleming

T.P.

, Smith

C.L.

, Burgess

W.H.

, Chan

A.M.

, Aaronson

S.A.

Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci U S A, 89:246. 1992.

59.

Ornitz

D.M.

, Xu

, Colvin

J.S.

, McEwen

D.G.

, MacArthur

C.A.

, Coulier

, Gao

, Goldfarb

Receptor specificity of the fibroblast growth factor family. J Biol Chem, 271:15292. 1996.

60.

Cardinalli

, Bolasco

, Aspite

, Lotti

L.V.

, Torrisi

M.R.

, Picardo

Melanosomes transfer promote by keratinocyte growth factor in light and dark skin derived keratinocytes. J Invest Dermatol, 128:558. 2008.

61.

Belleudi

, Cardinali

, Kovacs

, Picardo

, Torrisi

M.R.

KGF promotes paracrine activation of the SCF/c-KIT axis from human keratinocytes to melanoma cells. Transl Oncol, 3:80. 2010.

62.

Grabbe

, Welker

, Rosenbach

, Nürnberg

, Krüger-Krasagakes

, Artuc

, Fiebiger

, Henz

B.M.

Release of stem cell factor from a human keratinocyte line, HaCaT, is increased in differentiating versus proliferating cells. J Invest Dermatol, 107:219. 1996.

63.

Belleudi

, Purpura

, Scrofani

, Persechino

, Leone

, Torrisi

M.R.

Expression and signaling of the tyrosine kinase FGFR2b/KGFR regulates phagocytosis and melanosome uptake in human keratinocytes. FASEB J, 25:170. 2011.

64.

Rosen

C.F.

, Seki

, Farinelli

, Stern

R.S.

, Fitzpatrick

T.B.

, Pathak

M.A.

, Gange

R.W.

A comparison of the melanocyte response to narrow band UVA and UVB exposure in vivo. J Invest Dermatol, 88:774. 1987.

65.

De Luca

, Franzi

A.T.

, D'Anna

, Zicca

, Albanese

, Bondanza

, Cancedda

Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium. Eur J Cell Biol, 46:176. 1988.

66.

Haake

A.R.

, Scott

G.A.

Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents. J Invest Dermatol, 96:71. 1991.

67.

Kim

D.S.

, Park

S.H.

, Choi

Y.G.

, Kwon

S.B.

, Kim

M.K.

, Na

J.L.

, Youn

S.W.

, Park

K.C.

Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis. Biol Phamacol Bull, 28:2216. 2005.

68.

Khemis

, Kaiafa

, Queille-Roussel

, Duteil

, Ortonne

J.-P.

Evaluation of efficacy and safety of rucinol serum in patients with melasma: a randomized controlled trial. Br J Dermatol, 156:997. 2007.

69.

Cui

, Widlund

H.R.

, Feige

, Lin

J.Y.

, Wilensky

D.L.

, Igras

V.E.

, D'Orazio

, Fung

C.Y.

, Schanbacher

C.F.

, Granter

S.R.

, Fisher

D.E.

Central role of p53 in the suntan response and pathologic hyperpigmentation. Cell, 128:853. 2007.

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