Essential Role of RAB27A in Determining Constitutive Human Skin Color

Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs) in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (ΔL* = 4.4) in the HSSs. These data reveal the intrinsically essential role of RAB27A in human ethnic skin color determination and provide new insights for the fundamental understanding of regulatory mechanisms underlying skin pigmentation.     

Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co-cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono-cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono- and co-cultures. Removing certain keratinocyte growth factors from the co-culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte-melanocyte co-cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB-induced pigmentation, (ii) UVA-induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV-induced pigmentation in vitro.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte-keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Keratinocyte Extracellular Matrix-Mediated Regulation of Normal Human Melanocyte Functions

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation-associated protein detected by monoclonal antibody, MEL-5, was not changed on the kECM, while it was increased in melanocytes in co-culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co-culture compared to that in a control culture. We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca⁺⁺) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte–keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Toll‐like receptors 2 and 3 enhance melanogenesis and melanosome transport in human melanocytes

Because little is known about how the innate immune response influences skin pigmentation, we examined whether Toll‐like receptor (TLR) agonists participate in melanogenesis and melanosome transportation. We observed that TLR2/2 agonist HKLM and TLR3 agonist Poly(I:C) increased the amount of extracellular melanin from primary human epidermal melanocytes. HKLM, but not Poly(I:C), increased the melanogenic genes such as tyrosinase and dopachrome tautomerase. Poly(I:C) increased the expression of Rab27A, a molecule that facilitates melanosome transport to perimembranous actin filament. UVB irradiation induced Rab27A and melanosome transportation in a similar manner of Poly(I:C). SiRNA for TLR3 or Rab27A suppressed the perimembranous accumulation of Gp100‐positive vesicles in melanocytes and decreased melanin transfer to neighboring keratinocytes induced by both Poly(I:C) and UVB. These results suggest that the microenvironment in the epidermis and innate immune stimuli, such as microbiome and ultraviolet represented here by TLR2 and TLR3 agonists, could affect the melanogenesis in human melanocytes.     

Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders

Recently melanogenic paracrine or autocrine cytokine networks have been discovered in vitro between melanocytes and other types of skin cells. These include endothelin (ET)-1, granulocyte macrophage colony stimulating factor, membrane-type stem cell factor (SCF) and growth-related oncogene-alpha for interactions between keratinocytes and melanocytes, and hepatocyte growth factor and soluble type SCF for interactions between fibroblasts and melanocytes. These networks are also associated with corresponding receptors expressed on melanocytes, including ET B receptor and the SCF receptor, c-KIT. Consistent with in vitro findings on the melanogenic paracrine or autocrine cytokine networks, we have found that the up- or down-regulation of such networks is intrinsically involved in vivo in the stimulation of melanocyte functions in several epidermal hyper- or hypo-pigmentary disorders. These are ET-1/ET B receptor as well as membrane type SCF/c-KIT for ultraviolet B-melanosis, granulocyte macrophage colony stimulating factor for ultraviolet A-melanosis, ET-1/ET B receptor as well as membrane type SCF for lentigo senilis, growth related oncogene-alpha for Riehl's melanosis, sphingosylphosphorylcholine for hyperpigmentation in atopic dermatitis, ET-1 for seborrhoeic keratosis, soluble type SCF as well as hepatocyte growth factor for dermatofibroma and café-au-lait macules, and c-KIT for vitiligo vulgaris. These unveiled regulatory mechanisms involved in the abnormal up- or down-regulated levels of lesional melanocyte function provide new insights into therapeutic tools utilizing blockage of responsible cytokine networks.     

Pigmentation effects of solar-simulated radiation as compared with UVA and UVB radiation

Different wavelengths of ultraviolet (UV) radiation elicit different responses in the skin. UVA induces immediate tanning and persistent pigment darkening through oxidation of pre-existing melanin or melanogenic precursors, while UVB induces delayed tanning which takes several days or longer to develop and requires activation of melanocytes. We compared the effects of a 2-week repetitive exposure of human skin to solar-simulated radiation (SSR), UVA or UVB at doses eliciting comparable levels of visible tanning and measured levels of melanins and melanin-related metabolites. Levels of eumelanin and pheomelanin were significantly higher in the order of SSR, UVB, UVA or unexposed control skin. Levels of free 5-S-cysteinyldopa (5SCD) were elevated about 4-fold in SSR- or UVB-exposed skin compared with UVA-exposed or control skin. Levels of protein-bound form of 5SCD tended to be higher in SSR- or UVB-exposed skin than in UVA-exposed or control skin. Total levels of 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6H5MI2C were higher in SSR- than in UVB-exposed or control skin. These results show that SSR is more effective in promoting delayed tanning than UVB radiation alone, suggesting a synergistic effect of UVA radiation. Furthermore, free 5SCD may serve as a good marker of the effect of SSR and UVB.     

Ganoderma lucidum polysaccharide reduces melanogenesis by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway

Our previous study found that Ganoderma lucidum polysaccharide (GLP), bioactive ingredients from Ganoderma lucidum, protected fibroblasts from photoaging. However, whether GLP can affect melanogenesis in melanocytes through regulating paracrine mediators that secreted by keratinocytes and fibroblasts is unclear. We aimed to investigate the efficacy and mechanisms of action of GLP in melanogenesis by regulating paracrine effects of keratinocytes and fibroblasts. The effect of GLP on cell viability affected by GLP was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After an immortal keratinocyte line (HaCaT) and primary fibroblasts (FB) were treated with GLP, the supernatants of HaCaT and FB cells were collected and cocultured with an immortalized melanocyte line (PIG1). The expression levels of melanogenesis-associated genes in PIG1 cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Furthermore, FRS-2, ERK, JNK, and p38 phosphorylation levels were measured. Then, major melanogenic paracrine mediators in HaCaT and FB cells treated with GLP were evaluated by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). In addition, the expression of IL-6 and STAT3 was examined in HaCaT and FB cells. GLP was not cytotoxic to HaCaT and FB cells. The supernatants of GLP-treated HaCaT and FB cells downregulated the expression levels of MITF, TYR, TYRP1, TYRP2, RAB27A, and FSCN1 genes and inhibited the phosphorylation of FRS-2, ERK, JNK, and p38 in PIG1 cells. GLP also decreased FGF2 secretion in HaCaT and FB cells. Moreover, GLP reduced IL-6 expression and STAT3 phosphorylation in HaCaT and FB cells. GLP reduced melanogenesis in melanocytes by inhibiting the paracrine effects of keratinocytes and fibroblasts via IL-6/STAT3/FGF2 pathway.     

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21^CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.