* Address reprint requests to Dr Genji Imokawa, Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga, Tochigi 321-34 Japan. E-mail: firstname.lastname@example.org
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-α 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-α 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.
Evidence that melanogenic paracrine cytokine networks (Fig. 1) exist between melanocytes and other types of skin cells, including keratinocytes and fibroblasts, which regulate melanocyte function, have recently been discovered mainly by our groups (1–5). Although these studies have used mainly cultured skin cells, they facilitate research directed toward regulatory mechanisms involved in melanocyte activation, as seen in several experimentally induced hyperpigmentation and hyperpigmentary disorders. In vivo studies directed toward identifying intrinsic paracrine cytokines involved in hyperpigmentary disorders have been difficult in the absence of information about whether the up or down-regulation of some cytokines or chemokines in the lesional skin is responsible for the constitutive activation or inactivation of lesional melanocytes. In part this is due to the wide variety of cytokines or chemokines not directly related to melanocyte stimulation that are highly expressed in hyperpigmentary disorders due to the concomitant presence of other types of abnormal epidermal cells in addition to melanocytes. Thus, during our research identifying paracrine cytokines intrinsically involved in experimentally induced hyperpigmentation or those responsible for the stimulatory effects of conditioned medium from cultured keratinocytes or fibroblasts, we used six criteria to determine if a cytokine is an intrinsic factor involved in epidermal hyperpigmentation, as follows: (i) The cytokine(s) should be highly expressed in the surrounding cells in response to several stimuli. (ii) The cytokine(s) should exist in supernatants of cultures or in the epidermis at concentrations sufficient to stimulate melanocytes. (iii) The stimulatory effect of the culture supernatants on melanocytes should be neutralized by an antibody to the cytokine, if it is secretable. (iv) The cytokine(s) should have the potential to activate melanocytes at physiological concentrations in vitro. (v) The induced hyperpigmentation should be suppressed by the corresponding receptor antibody or by receptor antagonists in vivo. (vi) Mutations in the gene encoding the cytokine or its receptor should produce an aberrant phenotype. In this review, based upon the above six criteria, melanocyte activation or inactivation mechanisms are described in several epidermal hyper- or hypo-pigmentary disorders including ultraviolet (UV) or chemical-induced hyperpigmentation (summarized in Table 1).
As UVB irradiation is well known as a potent stimulator for epidermal pigmentation, many researchers tried to identify melanogenic stimulatory factors responsible for UVB-induced pigmentation. Because cultured human melanocytes exposed to UVB tend to increase melanization but to be rather down-regulated in their proliferative activity, the melanocyte features for both stimulated proliferation and melanogenesis in UVB-exposed epidermis led us to assume the possibility that there are some paracrine factors produced and secreted by UVB-exposed keratinocytes, which after the interaction with melanocytes, leads in turn to the accentuated function of melanocytes and then to stimulated melanin production.
Basic fibroblast growth factor (bFGF) is a first cytokine discovered responsible for such keratinocyte-derived melanogenic stimulation (6). UVB-exposed human keratinocytes are stimulated to produce bFGF at both gene and protein levels, and keratinocyte extracts (not conditioned medium) have a potent ability to stimulate the proliferation of melanocytes in culture. However, whether bFGF is a constitutive mitogen or melanogen for human melanocytes in UVB-induced pigmentation remains to be clarified for the following reasons: (i) a secretory form of bFGF by human keratinocytes has not been found, (ii) other growth factors, such as c-AMP raising factors (6) are required for the stimulation of melanocytes by bFGF.
With an idea that the conditioning medium from human keratinocytes contains some melanogenic factors, the Gilchrest group had detected the potent ability of the conditioned medium from non-exposed keratinocytes to stimulate the proliferation and melanogenesis of human melanocytes in culture (7). In 1992, we found that the conditioned medium from UVB-exposed human keratinocytes has a potent ability to stimulate the proliferation and melanogenesis of cultured human melanocytes (2). A neutralizing antibody to endothelin (ET)-1 almost completely abolished the stimulatory effect of the conditioned medium on cultured melanocytes and exogenous ET-1 was found to be a potent mitogen and melanogen for human melanocytes (1). In a parallel study (2), we found that UVB-exposed human keratinocytes secrete vasoconstrictive peptides, termed ETs, which were first discovered in the conditioned medium of cultured endothelial cells (8). This increased secretion of ET-1 was accompanied by an increased transcription of ET-1 in UVB-exposed human keratinocytes in culture. Together with the discovery for the existence of the ET receptor in human melanocytes (1), these findings proposed a notion that ET-1 is an intrinsic melanogen and mitogen for human melanocytes.
The physiologic significance of ETs in UVB-induced epidermal pigmentation remained unclear in the absence of in vivo evidence of increased ET production by surrounding keratinocytes in the epidermis. No intrinsic melanogenic factor associated with UVB-induced epidermal pigmentation had so far been characterized in vivo, specifically with regard to the expression of melanogenesis-related gene transcripts. RT-PCR of RNA isolated from the epidermis of UVB-exposed human skin revealed that, whereas in non-exposed sites, ET-1, interleukin (IL)-1α and tyrosinase mRNA signals were scarcely detectable, there was a distinct increase in the expression of the IL-1α, ET-1 and tyrosinase genes 5 d after irradiation with UVB (9). This showed that the ET-1 gene is expressed in human epidermis and that UVB radiation increases it, as well as those of tyrosinase and IL-1α. The concurrent gene expression of IL-1α and ET-1 in UVB-exposed human epidermis was consistent with the previous evidence (2) that IL-1α is an essential cytokine for the production of ET-1 in cultured human keratinocytes in an autocrine fashion. Therefore, together with the evidence that ET-1 is a powerful melanogen, these findings suggest that UVB radiation stimulates the secretion of IL-1α by keratinocytes to increase ET-1 gene expression in an autocrine fashion. The up-regulated ET-1 secretion in keratinocytes results in the increased expression of the tyrosinase gene, together with the expression of growth-related genes in melanocytes, which leads to cutaneous pigmentation in vivo. The involvement of ET-1 in UVB-pigmentation was also corroborated by experiments in which extracts of Matricaria chamomilla L, which can act as an antagonist for ET-receptor binding-mediated signaling but has no inhibitory effect on tyrosinase activity in culture, have a significant inhibitory effect on UVB-induced pigmentation in vivo when applied daily immediately after UVB exposure of human skin (10). These findings suggest that ET-1 is an important mediator in the epidermis for UVB-induced pigmentation in vivo.
Stem cell factor (SCF) has been reported to stimulate the proliferation of human melanocytes in culture (11) and in xenografts of normal human skin (12). Therefore, it is of particular interest to characterize whether SCF/c-KIT signaling is also involved in the mechanism of melanocyte activation during UVB-induced pigmentation, perhaps accompanied by an increased expression of SCF and/or SCF receptor expression in keratinocytes and melanocytes, respectively. When human keratinocytes and melanocytes in culture were exposed to UVB radiation, transcripts of SCF and c-KIT (as assessed by reverse transcription polymerase chain reaction) and expression of those proteins (by enzyme-linked immunosorbent assay and Western blotting), increased significantly and peaked at a dose of 20–40 mJ/cm2 (13). In UVB-exposed human epidermis, SCF transcripts and protein expression were also markedly enhanced compared with the non-exposed epidermis. Immunohistochemistry with antibodies to SCF revealed an increased staining in the UVB-exposed epidermis, which was accompanied by a slight epidermal hyperplasia. In the course of UVB-induced pigmentation of brownish guinea pig skin, which is an ideal model for such a study as functional melanocytes exist in the epidermis and those respond well to UVB irradiation to increase pigmentation (14), the subepidermal injection of c-kit inhibitory antibodies completely abolished the induction of pigmentation in the UVB-exposed area, and there was no increase in the number of dihydroxyphenylalanine-positive melanocytes. These findings indicate that SCF/c-KIT signaling is critically involved in the biological mechanism of UVB-induced pigmentation. Therefore, we proposed that in addition to the ET-1/ETB receptor signaling, SCF/c-KIT signaling is also involved in the biologic mechanism of UVB pigmentation as a mitogen and as a melanogen for human melanocytes (Fig. 2).
Especially in relation to mechanisms of UVB-induced hyperpigmentation, the cytokines defined according to the above requirements described in the ‘Introduction’ section have so far been restricted to ET-1 and SCF for associations between melanocytes and keratinocytes. Other cytokines, such as α-melanocyte stimulating hormone (αMSH), did not satisfactorily fit the above requirements although the stimulatory effects on melanocyte function as well as their production within and secretion by human keratinocytes in response to stimuli such as UVB irradiation have already been well established (15, 16). However, whether αMSH is a constitutive mitogen or melanogens for human melanocytes in UVB-induced pigmentation remains to be clarified for the following reasons: (i) there is a relatively weak mitogenic effect of αMSH on human melanocytes among known melanogenic growth factors (15, 17); (ii) there is no evidence that antibodies to αMSH can abrogate the stimulation of proliferation and melanization in cultured human melanocytes elicited by conditioned medium obtained from UVB-exposed keratinocytes; (iii) other growth factors, such as bFGF are required for the stimulation of melanocytes by αMSH (15); (iv) while the secretion of αMSH requires a converting enzyme which cleaves the big precursor, proopiomelanocortin, to yield αMSH, there is no established biochemical identification of these enzymes in human keratinocytes despite the fact that immunostaining was observed in human keratinocytes for the prohormone convertases, PC1 and PC2 (18) in contrast to the biochemical identification of ET converting enzyme in human keratinocytes (19).
Unlike the effects of UVB radiation, little is known about how UVA exposure affects autocrine or paracrine linkage of cytokines between epidermal cells. UVA has been documented to elicit completely different biological and histological responses of epidermal cells and skin tissue despite the fact that UVA-induced pigmentation includes melanocyte responses identical, although to a smaller extent, with those evoked by UVB with regards to accentuated proliferation and melanization (14). Therefore, it is of considerable interest to clarify the cellular mechanisms involved in cutaneous pigmentation induced by UVA: if UVA-exposed human keratinocytes produce soluble factors that can stimuate DNA synthesis or melanogenesis of cultured human melanocytes, it is worthwhile to determine the nature of the cytokines directly associated with melanocyte activation. We found that UVA-induced melanosis is associated with other keratinocyte-derived growth factors, secretion of which are specifically stimulated after exposure of human keratinocytes to UVA (3). Thus, medium conditioned by UVA-exposed human keratinocytes elicited a significant increase in DNA synthesis by cultured human melanocytes in a UVA dose-dependent manner. Analysis of ET-1 and IL-1α in the conditioned medium by ELISA, both of which are major keratinocyte-drived cytokines involved in UVB-associated melanocyte activation, revealed that UVA exposure did not cause human keratinocytes to stimulate the secretion of the two cytokines. In contrast, the levels of several other cytokines such as IL-6, IL-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were significantly increased in the conditioned medium of human keratinocytes after exposure to UVA at a dose of 1.0 J/cm2. The gel chromatographic profile of UVA-exposed keratinocyte-conditioned medium demonstrated that there were two factors (P-1 and P-2) with molecular masses of approximately 20 and 1 kDa, respectively, that stimulate DNA synthesis in human melanocytes, and the larger species (P-1) also increased melanization as assessed by 14C-thiouracil incorporation. Quantitative analysis of cytokines in chromatographic fractions by ELISA revealed the P-1 fraction to be consistent with the molecular mass profile of GM-CSF. Furthermore, the stimulatory effect of the P-1 fraction on DNA synthesis in human melanocytes was neutralized by antibodies to GM-CSF, but not to bFGF or SCF, and binding and proliferation assay with recombinant GM-CSF demonstrated that human melanocytes possess specific binding sites for GM-CSF (Kd 2.11 nM; binding sites, 2.5–3.5 × 104per cell), and recombinant GM-CSF at concentrations of more than 10 nM significantly stimulated DNA synthesis and melanization. Although in vivo study is needed to verify the role of GM-CSF in UVA-induced pigmentation, these findings suggested that GM-CSF secreted by keratinocytes plays an essential role in the maintenance of melanocyte proliferation and UVA-induced pigmentation in the epidermis (Fig. 2).
Pigmentation in Lentigo Senilis
Lentigo senilis (LS) is the skin condition of common aging spots with accentuated epidermal pigmentation. Based upon the observation that large numbers of melanin-producing melanocytes are located in the vicinity of highly proliferating keratinocytes, as seen in hair follicles, we hypothesized that the proliferating keratinocytes in LS trigger the activation of neighboring melanocytes by secreting melanocyte-stimulating cytokines. As among the known keratinocyte-derived cytokines produced in response to their proliferation, ET-1 is the only cytokine, which has dual stimulatory effects on DNA synthesis and melanization of human melanocytes (1, 10, 11), it is of considerable interest to clarify whether ET production and secretion is accentuated in the epidermis of LS. Further, ET secretion was known to be regulated by ET converting enzyme (ECE-1α), which converts the propeptide, big-ET, to its active form, ET-1 (20). In addition to ET produced and secreted by keratinocytes, ET action on melanocytes is mediated via the ETB receptor which is a G protein-coupled transmembrane receptor (21). Therefore, we used immunohistochemistry and RT-PCR analysis to determine whether the epidermal ET cascade, consisting of ET production, ECE-1α (19), ET secretion and ETB receptor-mediated signaling (22, 23), is accentuated in the LS epidermis.
In LS lesional skin, the number of tyrosinase immuno-positive melanocytes was increased twofold over the perilesional epidermis (24). Immunohistochemistry using antibodies to ET-1 demonstrated relatively stronger staining in the lesional epidermis than in the perilesional epidermis. RT-PCR analysis concomitantly demonstrated accentuated expression of transcripts for ET-1 and for the ETB receptor in LS lesional skin, which was accompanied by a similar accentuated expression of tyrosinase mRNA compared with the perilesional control. The ET-1-inducible cytokine, transforming necrosis factor (TNF)α was consistently up-regulated in the LS lesional epidermis as determined at the transcriptional level and by immunostaining, while IL-1α was down-regulated. In contrast, ECE-1αmRNA was not substantially increased in the lesional epidermis. These findings suggest that an accentuation of the epidermal ET cascade, especially with respect to expression of ET and the ETB receptor, plays an important role in the mechanism involved in the hyperpigmentation of LS.
Subsequently, we demonstrated that in addition to the ET cascade, SCF and its receptor KIT protein is also constitutively associated with UVB-induced hyperpigmentation of the epidermis (13). This prompted us to determine whether SCF and its receptor c-KIT are also involved in the mechanism of epidermal hyperpigmentation of LS lesions (25).
In the epidermis of LS lesions, SCF expression is markedly accentuated. Thus, the LS lesional epidermis expresses increased levels of SCF mRNA transcripts concomitantly with the increased levels of SCF protein compared with non-lesional controls. In contrast, another melanogenic paracrine cytokine [e.g. growth-related oncogene-α (GROα) or bFGF)] or the SCF receptor occurs at similar levels of mRNA expression or immunostaining in the LS lesional and the non-lesional skin. By immuno-histochemistry, SCF is markedly increased in staining throughout the LS lesional epidermis which is corroborated by Western blotting using an antibody to SCF. In contrast, the SCF receptor KIT protein is not substantially stimulated in expression on melanocytes in the LS lesional epidermis compared with those in non-lesional skin. Based on the previous in vitro evidence on the association of the SCF/SCF receptor cascade with activation of melanocytes (11), these findings strongly suggest that the stimulated melanogenesis in the LS lesional epidermis is involved in the accentuated expression of SCF in LS lesional keratinocytes.
In epidermal keratinocytes, SCF is expressed as a membrane-bound form, not in a secretory or soluble cytokine form such as ET-1 (13). Even in stimulated conditions such as following UVB exposure, only the membrane-bound form of SCF is accentuated without the appearance of soluble SCF (13). In contrast, dermal fibroblasts can secrete soluble SCF, probably because of the action of proteolytic enzymes capable of cleaving membrane-bound SCF to release soluble SCF (5). In epidermal hyperpigmentation of dermatofibroma (DF) (26) or cafe-áu-lait macules (27), soluble SCF secreted by dermal fibroblasts plays an important role in activating epidermal melanocytes via a dispersion pathway through the basement membrane from the dermis toward the epidermis. In cases where soluble SCF is involved in the activation of epidermal melanocytes, dermal mast cells also accumulate or increase in the upper dermis, and SCF is also known as mast cell growth factor. Consistent with this, Longley et al. (28) demonstrated that abnormalities in the proteolytic processing of SCF tend to produce soluble SCF in the epidermis, which leads to the phenotype of mastocytosis. Further, Kunisada et al. (29) reported that the over-expression of soluble SCF in murine epidermis induces the accumulation of mast cells in the dermis in addition to increasing the number of melanocytes in the epidermis. In our study, as the number of mast cells is not increased in the lesional dermis of LS and as soluble SCF is not detected in the lesional epidermis by Western blotting, indicative of no mutation in the gene for the SCF in LS, it is likely that the accentuated production of membrane-bound SCF plays an essential role in the increased proliferation and melanogenesis of melanocytes, leading to the epidermal hyperpigmentation in LS. This is consistent with Kunisada's report (29) using mouse skin in which the expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis.
Thus, we suppose that an accentuation of two epidermal cascades, consisting of ET-1 and its receptor ETBR and SCF/SCF receptor, play important roles in the hyperpigmentation mechanism of LS. The concomitant accentuated expression of both linkages is also confirmed for ET-1, ETBR and tyrosinase by RT-PCR analysis, and for ET-1 and tyrosinase by immunohistochemistry in the same donor as tested for SCF. The preference for enhanced expression of ET-1 and SCF in the LS lesional epidermis and the melanogenic stimulation of melanocytes is corroborated by the existence of the synergistic effects of SCF and ET-1 in prompting melanogenesis and mitogenesis by cultured melanocytes via transactivation of SCF-stimulated KIT protein because of protein kinase C (PKC) activated by ET-1 (23). These marked synergistic effects are restricted to the cross-talk type of interaction only between ET-1 and SCF among the cytokines tested. Thus, it is likely that although there are multiple and complex mechanisms involved in the accentuated pigmentation of LS lesions, the coordinated and synergistic roles of SCF and ET-1 in the intracellular signaling mechanisms leading to melanogenesis (23) may support the rationale for such ligand specificity in the melanogenic stimulation of LS lesions. Our findings collectively suggest that in addition to the stimulated ET cascade, the accentuated SCF/SCF receptor cascade also contributes to the epidermal hyperpigmentation seen in LS lesions (Fig. 2).
Pigmentation in Phenyazonaphthol Induced-allergy (Riehl's Melanosis)
Phenyazonaphthol (PAN) allergy-induced late appearing hyperpigmentation is known as pigmented cosmetic dermatitis or Riehl's melanosis (30). We demostrated previously that specific melanogenic factors produced after a time course of hyperpigmentation because of PAN allergy in brownish guinea pig skin are associated with the induction of epidermal hyperpigmentation by the allergen (30). Biological evidence of signal transduction profiles suggest that melanogenic stimulation induced by PAN allergy is mediated through PKC activation within melanocytes by soluble factors secreted during biologic processing subsequent to allergic reaction (31). PAN allergy-induced hyperpigmentation clearly differs in its time course from ET or GM-CSF-associated pigmentation such as UVB or UVA melanosis, respectively (14). Further, there is no similarity in the chromatographic properties between the relevant cytokines in that the allergy-evoked soluble factor exhibits an apparent molecular mass of about 8–9 kDa estimated by gel filtration, which is different from that of ET-1 (2.3–2.7 kDa) or GM-CSF (14.5–15.5 kDa) (31).
Aside from studies focusing on the biological effects of exogenously added cytokines on human melanocytes, studies examining the paracrine linkage of cytokines between keratinocytes and melanocytes would provide new insight into intrinsic cellular mechanisms that eventually take part in allergy-induced melanosis. Therefore, we purified and characterized the PAN-induced melanogenic stimualting factor (PIMSF) that occurs in allergy-associated hyperpigmented skin (4).
PIMSF was purified from hyperpigmented skin extract to apparent homogeneity and showed a single band with an apparent molecular mass of 7.9 kDa upon SDS-PAGE. The PIMSF was similar in molecular profile to rat GROα (molecular mass of 7.9 kDa) upon SDS-PAGE. Immunoblotting analysis revealed consistently that the purified PIMSF cross-reacted with an anti-GROα antibody and that the bioactivities of PIMSF could be abrogated by the addition of the anti-GROα antibody. These findings suggested that purified PIMSF is identical to GRO family proteins or is very closely related to them. The similarity of the purified PIMSF to the GRO protein is also corroborated by their comparable cellular effects, including a marked stimulation of DNA synthesis in cultured guinea pig melanocytes at similar concentrations, accompanied by the release of inositol trisphosphate and intracellular calcium mobilization, which was in turn followed by PKC translocation. Therefore, it seems likely that melanogenic stimulation induced by PAN allergy is mediated by GROα secreted during biological processes subsequent to cutaneous allergic reaction.
GRO was identified originally as melanoma growth-stimulating activity for its ability to induce proliferation of human melanoma Hs294T cells (32); it is a member of a gene family encoding secretory proteins associated with the inflammatory response (33, 34). Structurally, GRO is related to a group of inflammatory proteins which includes IL-8, platelet factor 4, and β-thromboglobulin (35). In addition to its ability to stimulate the proliferation of melanoma cells, GRO has a variety of other biological effects including the ability to act as a neutrophil chemotatic factor (36) and to induce calcium mobilization in a variety of different target cells (37). These biological relationships suggest that GRO may also have a function in the inflammatory response. We demonstrated for the first time the ability of GROα to stimulate proliferation and melanogenesis of cultured melanocytes. This provides in vivo validation that keratinocytes, through GROα production and secretion, may play an important role in epidermal pigmentation. It has been reported that GROα mRNA is expressed in cultured human keratinocytes and that its expression is stimulated in response to UVB exposure (38) or to IL-1α treatment (39). In inflammatory and hyperproliferative skin diseases such as psoriasis, GROα mRNA is over-expressed selectively in the epidermis as a keratinocyte response to activated T cells (39). However, human foreskin fibroblasts express a 10-fold elevation in their steady-state levels of GRO mRNA in response to serum or PMA stimulation (40). It is interesting to note that IL-1 induces at least a 100-fold elevation of GROα mRNA expression without changing in c-myc or c-fos gene expression (40).
ET, which we have recently reported as a novel keratinocyte-derived mitogen and melanogen for human melanocytes in UVB-induced epidermal pigmentation (1, 9) is very similar in its mode of action to purified PIMSF. ET-1 is stimulated in production and secretion from keratinocytes by the action of IL-1 in an autocrine fashion and activates the signal transduction pathway, including Ins(1,4,5)P3 formation, intracellular Ca2+ mobilization, and PKC in human melanocytes. In UVB-exposed keratinocytes, ET-1 is secreted with a lag time of a few days following UVB irradiation (2). Significant differences between ET and GROα other than their molecular masses include the time course of their secretion in culture or in epidermal tissue. The increased secretion of ET-1 in cultured human keratinocytes require autocrine stimulation by IL-1α, which is released following UVB irradiation and which takes approximately 2–3 d to maximize (2). This time course of secretion is corroborated by the profile of ET-1 mRNA expression in cultured human keratinocytes and in UVB-exposed human skin. However, the production or secretion of PIMSF in guinea pig epidermis, estimated by melanogenic assay, was slight by day 20 postchallenge but became well defined by day 28 postchallenge. In our unpublished data, IL-1-activating factor activity, measured by fibroblast assay in epidermal homogenates during the late appearing pigmentation after PAN allergen challenge, was comparable in time course with PIMSF with a peak on day 28 postchallenge. Because several lines of evidence associate the initial process of GRO production by keratinocytes with the activation of the importance primary cytokine, IL-1α cascade, including keratinocyte type 1 receptor (39, 41), it is possible that the production or secretion of GROα in allergy-induced epidermis is triggered by the release of IL-1α. However, it remains unclear why a similar autocrine mechanism initiated by IL-1α secretion which leads to the secretion of GROα and ET-1 occurs in a different time course. Taken together, it seems likely that PAN allergy provides a new mechanism of hyperpigmentation in which biophysical factors, such as members of the GROα superfamily, synthesized within the epidermis, stimulate melanocytes through the activation of the PKC-related signal transduction system (Fig. 2).
Pigmentation in Atopic Dermatitis
Recently, we demonstrated that the skin of patients with atopic dermatitis (AD) contains a novel enzyme, sphingomyelin (SM) deacylase, which cleaves the N-acyl linkage of sphingomyelin (SM) (42–44). In the skin of patients with AD, SM deacylase is up-regulated and produces sphingosylphosphorylcholine (SPC) rather than ceramide (Cer) which would normally be generated by the action of sphingomyelinase (SMase). This results in a Cer deficiency, which is an etiologic factor in the barrier-disrupted dry skin of patients with AD (45). Consistent with the enhanced expression of SM deacylase in AD skin, the metabolic intermediates of SPC accumulate to a greater extent in the stratum corneum of AD patients compared with healthy controls (46). Available evidence demonstrates that SPC has potent biological activities in a variety of different cell types such as fibroblasts (47), keratinocytes (48, 49), and endothelial cells (50) in relation to in vivo altered tissue reactions attributable to the stimulation of wound healing, cutaneous inflammation and epidermal differentiation, and morphogenesis, respectively. In particular, their abilities to up-regulate the secretion of inflammatory cytokines and intercellular adhesion molecule-1 (ICAM-1) by human keratinocytes may exacerbate the dermatitis in the skin of patients with AD because the SM deacylase is localized in the upper epidermis, and the SPC would stimulate neighboring epidermal cells. As the epidermis is composed of keratinocytes, Langerhans cells and melanocytes, it is of considerable interest to determine how these lysosphingolipids would affect the function of human melanocytes with special reference to mechanisms involved in the increased pigmentation seen frequently in AD skin. Based upon available evidence that lysosphingolipids have multiple bio-modulator functions in various types of cells, we measured the biological effects of SPC on cultured human melanocytes to determine whether these lysosphingolipids have the potential to activate human melanocytes (51). The addition of SPC at 5–20 μM concentration significantly stimulated DNA ([3H] thymidine incorporation) and melanin synthesis ([14C] thiouracil incorporation and 3H2O release) in human melanocytes. Reverse transcription–polymerase chain reaction (RT-PCR) of RNA isolated from human melanocytes exposed to SPC revealed an accentuated expression of transcripts for tyrosinase, microphthalmia-associated transcription factor (MITF)-M, ET B receptor (ETBR) and the SCF receptor, c-KIT. An increased expression of tyrosinase protein was also consistently demonstrated by western blot analysis. This melanogenic stimulation by SPC was associated with a marked increase in the phosphorylation of mitogen-activated protein (MAP) kinase.
In some patients with chronic atopic eczema, a distinctive type of hyperpigmentation is found, particularly on the neck. Colver et al. have called this pathology the ‘Dirty Neck’ (52), and histological and ultrastructural studies of this ripple pigmentary area reveal an increase in melanocyte number and melanin incontinence in the dermis (53). However, little is known about the mechanism(s) underlying the distinguished pigmentation seen in AD. Inflammation frequently accompanies hyperpigmentation by secreting cytokines, chemokines or chemical mediators which are potent stimulants for melanogenesis by melanocytes. Some endogenous melanogenic factors, such as ET-1, SCF, and GROα, are constitutively involved in UVB-induced pigmentation or hyperpigmentation of several pigmentary disorders (2, 13, 26, 31). Although some of these factors may be responsible for the high incidence of pigmentation in AD skin, there is no available data on their secretion and distribution in AD, hampering the clarification of such a hyperpigmentation mechanism. The most likely explanation for the high incidence of pigmentation in AD would be that histamine secreted from mast cells accumulates in the AD skin and stimulates melanocytes to produce increased amounts of pigment because histamine is secreted in the dermis of AD skin (54) and increases tyrosinase activity in cultured human melanocytes (55). In contrast to the distribution of histamine secretion which is mainly located in the vicinity of mast cells in the inflamed dermis, SPC is produced presumably at concentrations as high as 10 μM in the upper epidermis [estimated from the concentration in the stratum corneum (46)] and is distributed in more abundance than histamine in both the non-inflamed and in the inflamed epidermis. Thus, the site of SPC production and its concentration (43) in the epidermis (46) physiologically favors its action on melanocytes compared with histamine. Thus collectively, these results suggest that increased levels of SPC play a role in the predisposition toward increased pigmentation in the epidermis of skin diseases such as AD wherein SM deacylase is over-expressed.
Pigmentation in Seborrhoeic Keratosis
Seborrhoeic keratosis (SK) is a common benign tumor with accentuated epidermal pigmentation. Based upon the observation that high-level melanin-producing melanocytes are located in the vicinity of highly proliferating keratinocytes as seen in hair follicles, we hypothesized that the proliferating keratinocytes in SK trigger the activation of neighbouring melanocytes by secreting melanocyte-stimulating cytokines. Thus, it seems reasonable to suppose that the accentuated melanization observed in SK is associated with increased production of ETs by the highly proliferating keratinocytes. Therefore, using immunohistochemistry and RT-PCR, we determined whether the production of ET-1 is accentuated in SK of acanthotic and deeply pigmented types (56, 57). Immunohistochemical analysis in SK revealed marked immunostaining wit anti-ET-1 in almost all basaloid normal controls. In parallel, RT-PCR of ET-1 mRNA demonstrated accentuated expression of ET-1 transcripts in SK in comparison with that in the perilesional normal control, accompanied by a similarly accentuated expression of tyrosinase mRNA. These findings suggest that ET-1 plays a part in the hyperpigmentation seen in SK.
The present study has provided, for the first time, insight into the role of ETs as a constitutive stimulator of human melanocytes in non-UV-associated hyperpigmentary disorders. The potential of keratinocytes located in SK to produce ET-1 was found to be significantly higher than that in perilesional normal controls. In contrast, in tissues other than epidermis, there were no significant differences between lesional skin and perilesional normal skin in the distribution or the intensity of ET-1 immunostaining. The increased production and localization of ET-1 as assessed in SK by immunostaining were paralleled by those of tyrosinase activity revealed by the dopa reaction, suggesting that melanocyte activation, including accentuated tyrosinase function, occurred in SK concomitantly with the stimulation of ET-1 production by surrounding keratinocytes.
There may be several mechanisms other than the above ET-1 paracrine pathway to account for the activation of melanocytes in SK. One possibility is that there are other cytokines, secretion of which might be enhanced in the epidermis, leading to the stimulated proliferation and melanization of human melanocytes. These may include bFGF, SCF, GM-CSF and hepatocyte growth factor (HGF), because of their known action on human melanocytes (3, 6, 11, 58). There has been no report concerning the production of GM-CSF, SCF or HGF in SK. Even if there are conditions under which these cytokines are highly expressed in SK, their contribution to the hyperpigmentation seen in SK may be low. These cytokines were found to have no significant potential for stimulating melanization in cultured human melanocytes (3, 11, 58), which is not consistent with the biological situation where both stimulation of proliferation and melanization occur in melanocytes in SK. ET-1 is the only cytokine reported, to date, which can stimulate both proliferation and melanization of human melanocytes at concentrations as low as 1 nM (1,11). Thus, it seems likely that the accentuated secretion of ET-1 in SK is responsible for the hyperpigmentary status of this benign neoplasm (Fig. 2).
Pigmentation in Dermatofibroma
Dermatofibroma (DF) is a benign dermal tumor of fibroblast-like cells (59); it is also known as histiocytoma, benign fibrous histiocytoma, and sclerosing hemangioma. It has been reported that some cases of multiple DF are associated with auto-immune diseases such as systemic lupus erythematosus (60, 61). In lesional DF skin, there is an abnormal accumulation of proliferating fibroblast-like cells and histiocytes surrounded by mature collagen and by increased capillaries. Of particular interest is the fact that the epidermis overlying the DF tumor is markedly hyperpigmented with a slight acanthosis (61), although the mechanism behind the pathogenesis of that epidermal hyperpigmentation remains to be clarified. Consistently, the number of tyrosinase immuno-positive melanocytes in the pigmented DF epidermis was significantly increased by twofold compared with non-lesional normal epidermis.
The characteristic features of DF, including fibroblastic tumors in the dermis and hyperpigmentation in the overlying epidermis, allow us to speculate that paracrine cytokines produced by highly proliferative fibroblast-like cells in the dermis might be primarily responsible for the melanogenic stimulation of melanocytes in the epidermis. In this connection, we have already found that conditioned culture medium from proliferating human fibroblasts has a potent ability to stimulate the proliferation of human melanocytes in culture (5, 11). Those stimulatory activities were associated with SCF and HGF which are exclusively secreted in abundance by human fibroblasts derived from aged persons.
Although there is little evidence for their paracrine role among skin cells, SCF and HGF have also been implicated to be mitogens for human melanocytes in vitro and in vivo (23, 62, 63). Treatment with HGF at a concentration of 10 nM has been reported to stimulate the proliferation of cultured human melanocytes (58). Using an in vivo injection protocol, Grichnik et al. (62) and Costa et al. (63) demonstrated a potent capacity of SCF to induce hyperpigmentation in the epidermis, which was accompanied by an increased proliferation of epidermal melanocytes. Consistent with its stimulatory potential, as SCF is also known as mast cell growth factor, increases in the numbers of mast cells and their degranulation were also observed concomitant with melanocyte activation. Thus, based upon the available evidence on the stimulatory effects of several skin cell-derived cytokines or chemokines on human melanocytes, it is most likely that SCF or HGF derived from the fibroblastic tumors is responsible for the stimulation of epidermal melanocytes, which leads to the hyperpigmentation restricted to the epidermis overlying the fibroblastic tumors in DF.
Despite the lack of a significant difference in the expression of SCF and HGF transcripts in lesional and in non-lesional DF epidermis, there was an increased expression of those transcripts in the lesional DF dermis relative to the non-lesional dermis (26). In contrast, transcripts of other melanogenic cytokines, such as GROα, bFGF and ET-1, are expressed similarly in lesional and in non-lesional DF dermis and epidermis. Because the gene expression is essentially expressed per cell (as standardized by G3PDH), the increased expression of SCF and HGF transcripts implied that cells of the fibroblastic DF tumors produce more of those proteins than do fibroblasts in the non-lesional dermis. In support of this, immunostaining with SCF or HGF antibodies in fibroblasts of the non-lesional DF dermis was substantially negative, indicating that non-proliferating normal fibroblasts produce insignificant amounts of SCF or HGF. Based upon this assumption, it is conceivable that there are copious amounts of SCF and HGF secreted throughout the lesional DF dermis, being sufficient to reach and activate melanocytes in the overlying epidermis. In contrast, the other melanogenic cytokines tested (including GROα, bFGF and ET-1) might also be secreted by fibroblastic tumors, but at concentrations insufficient to contact and activate melanocytes. The protein expression of SCF and HGF, as evidenced by immunohistochemistry, was markedly elevated in the lesional DF dermis compared with the non-lesional dermis, while for the other melanogenic cytokines tested, the transcripts and protein levels of GROα, bFGF and ET-1 were expressed at similar levels between the lesional and non-lesional DF dermis and epidermis. Taken together, our evidence suggests that in the lesional DF dermis, SCF and HGF are selectively produced and secreted by fibroblastic tumors at concentrations sufficient to activate melanocytes located in the adjacent epidermis, which results in the hyperpigmentation of the overlying skin.
A similar type of epidermal hyperpigmentation associated with SCF has been reported in mastocytosis in which the abnormal and excessive conversion of the membrane bound to the soluble form of SCF in the epidermis plays an essential role in stimulating epidermal melanocytes and proliferating dermal mast cells, respectively (64). Thus, it is conceivable that the threshold concentrations of SCF needed to stimulate melanocytes and mast cells are similar. If mast cells are activated beneath the epidermis by SCF, it seems reasonable to assume that melanocytes are also activated by the same SCF as SCF is also known as mast cell growth factor. Our finding that the numbers of mast cells are increased 5-fold in DF skin compared with non-lesional skin was an interesting indication that the stimulated status of epidermal melanocytes in DF can be ascribed to the action of SCF, which is largely secreted as a soluble isoform by fibroblastic tumors, but not by lesional epidermal cells, as supported by a significant increase in SCF gene and protein expression in the lesional dermis but not in the lesional epidermis compared with each non-lesional control. This is concluded because of the existence of soluble SCF which is released into the medium by cultured human fibroblasts at a level detectable by ELISA, but not by cultured human keratinocytes (17), and which diffuses to the nearby epidermis. Thus, it is unlikely that the expression of soluble SCF in the lesional epidermis is stimulated by unknown factors derived from the fibroblastic tumor, which results in the stimulation of melanocytes and mast cells as seen for mastocytosis. Unfortunately, as HGF is known just as a stimulator for hepatocytes and melanocytes (58) and as there are no other types of skin cells which are known to respond to HGF, it is difficult to define the role of HGF in the hyperpigmentation seen in DF. However, the high expression of HGF in the dermis at both the gene and the protein levels would support a distinct role for HGF in stimulating epidermal melanocytes in DF. Taken together, the above findings support the pathogenic mechanism of DF in which the proliferating fibroblast-like cells have a potent ability to secrete SCF and HGF which then diffuse toward the epidermis (Fig. 2). This would then result in the stimulation of melanocytes located at the basal layer of the epidermis, which would lead in turn to hyperpigmentation limited to the overlying epidermis, presumably accompanied by a concomitant accumulation of mast cells.
Pigmentation in Café-au-lait Macules
Café-au-lait macules (CALMs) are light to dark brown, well-circumscribed cutaneous macular areas with no hair. CALMs are the best-known cutaneous sign of Neurofibromatosis type-1 (NF1; von Recklinghausen's disease) and they are present in almost 100% of NF1 patients (65). Histological studies of CALMs in NF1 skin show increased epidermal melanization and an increased number of melanocytes with normal amounts of tyrosinase activity (66–68). The NF1 gene is localized to chromosome 17 (69), and encodes neurofibromin, a tumor-suppressor protein (70–72). It has been suggested that reduced neurofibromin levels in the epidermis of NF1 patients is responsible for the elevated melanogenesis and the increased density of melanocytes (73). However, this is not the complete mechanism by which melanization is locally stimulated in CALMs of NF1 skin, because the reduction in neurofibromin levels is systemic, not localized.
Although HGF, SCF, and bFGF had been shown to stimulate melanocyte proliferation in vitro (5, 6, 11, 17, 58, 74, 75) and in vivo (62, 64), keratinocytes do not secrete them at concentrations sufficient to stimulate melanocyte proliferation, even following exposure to various stimuli (17). In contrast to keratinocytes, human fibroblasts do secrete several melanogenic cytokines, such as bFGF, HGF and SCF, when they are rapidly growing or during inflammation (11, 17), which suggests the possibility that over-expression of these cytokines by dermal fibroblasts may activate melanocytes in the overlying epidermis. In DF, where the epidermis overlying the fibroblastic tumor is highly pigmented, rapidly growing fibroblasts are stimulated to secrete SCF and HGF, which suggests an important involvement of these cytokines in the accentuated epidermal pigmentation (26). Based upon the paracrine cytokine network now known to function within the skin to regulate epidermal pigmentation, it is intriguing to examine the patterns of cytokine secretion in cells located in CALMs of NF1 skin.
Histological studies of CALMs skin in NF1 patients showed an increased number of melanocytes with normal morphology (70–72). As NF1 is a congenital skin disease and as CALMs of NF1 skin have an accentuated melanization in their epidermis due to an increased number of epidermal melanocytes undergoing stimulated melanogenesis, we first examined the possibility that keratinocytes in the lesional epidermis secrete larger amounts of melanogenic cytokines than do keratinocytes in the non-lesional or healthy control epidermis (27). SCF and HGF proteins were not detectable in the lesional keratinocyte-conditioned medium. Further, comparison of secreted amounts of ET-1 and GM-CSF using ELISA revealed that there is no significant difference in the levels of ET-1 and GM-CSF secreted by keratinocytes isolated from CALMs of NF1 skin, the non-lesional NF1 skin and the healthy controls. This suggests that lesional keratinocytes have no significant alteration in their potential to secrete melanogenic cytokines and may not be involved in the hyperpigmentary mechanism of CALMs in NF1, although an alternative possibility that lesional melanocytes have increased sensitivity to cytokines through receptors remains to be clarified.
As epidermal melanogenic factors derived from dermal fibroblasts are known mainly to be SCF, HGF and/or bFGF in culture (11, 17), we next compared the secretion of SCF, HGF and bFGF as intrinsic cytokines leading to epidermal pigmentation by fibroblasts derived from CALM, from non-CALM and from healthy control skin. As for known cases where dermal fibroblasts produce and secrete larger amounts of melanogenic cytokines that lead to accentuated pigmentation in epidermis overlying fibroblasts in the dermis, we have recently found that such a mechanism occurs in vivo in DF, which is associated with the secretion of HGF and SCF by fibroblasts as melanogenic paracrine cytokines (26). Determination of cytokine levels demonstrated that in patients with NF1, the potential of dermal fibroblasts localized in CALMs to secrete HGF and SCF was significantly higher than that of fibroblasts derived from non-CALM skin or from normal control skin. These increases were accompanied by increased expression of mRNAs encoding HGF and SCF by cultured fibroblasts of CALMs from NF1 skin compared with healthy control skin. In contrast, the secretion of bFGF was not increased in fibroblasts derived from CALMs of NF1 skin compared with non-CALM skin and healthy control skin. These findings suggest the hypothesis that high levels of HGF and SCF secretion by dermal fibroblasts located in CALMs correlates with the stimulated melanogenesis in epidermal melanocytes.
The NF1 gene was cloned in 1990 (70–72), and belongs to the family of tumor suppressor genes. Neurofibromin is encoded by the NF-1 gene and is a major negative regulator of the Ras pathway, a key signal transduction pathway in cells. Loss of neurofibromin leads to increased levels of activated Ras, and thus increased downstream mitogenic signaling. It has been suggested that the reduction of neurofibromin in the epidermis of NF1 patients is responsible for the abnormal physiology such as the elevated melanogenesis and the increased density of melanocytes (73). However, this doesn't necessarily account for the localized hyperpigmented areas (CALMs) seen in NF1 because the reduction of neurofibromin is systemic, not localized. Our observation that fibroblasts localized beneath the CALMs, but not those in other areas, are stimulated to secrete SCF and HGF, suggests that the reduction of neurofibromin is not directly associated with the hyperpigmentation of CALM skin, but that additional unknown factors associated with neurofibromin cause fibroblasts to stimulate such cytokine secretion. Thus, the relation between reduced neurofibromin in patients with NF1 and the mechanisms of increased secretion of HGF and SCF by dermal fibroblasts remained unclear.
Available evidence indicates that there is a significant increase in mast cells in neurofibromas of NF1 and NF5 patients compared with the dermis of normal individuals, and that the density of mast cells in neurofibromas is independent of the NF type and the age of patients (76). An increase in the number of mast cells in CALMs skin was observed compared with non-CALM skin and with normal skin. Increased secretion of SCF by dermal fibroblasts derived from CALM skin of patients with NF1 may lead to the increased number of mast cells in neurofibromas of NF1. This was also corroborated by the fact that in DF, there are increased numbers of mast cells in the dermis overlying the fibroblastic tumors and those cells secrete a larger amounts of SCF than in non-lesional dermis (26). Thus, the fact that in addition to the increased numbers of activated melanocytes in the CALM epidermis, the number of mast cells located beneath the epidermis is increased over that in the non-lesional skin supported the suggestion that at least SCF is secreted in a soluble and diffusable form by fibroblasts at concentrations capable of directly influencing mast cells to proliferate. This strongly suggests that at least soluble SCF plays an important role in the epidermal hyperpigmentation of CALM skin (Fig. 2).
Hypopigmentation in Vitiligo Vulgaris
Vitiligo is an acquired idiopathic disorder of the skin (77). Although several hypotheses have been proposed for the loss of functional melanocytes in vitiligo skin, the etiology is still unknown. As discussed above, we found that paracrine cytokines (produced by keratinocytes) and their receptors (expressed on melanocytes), play important roles in the maintenance and activation of melanocyte function in the skin, leading to normal or to accentuated pigmentation. Based on the paracrine networks discovered so far, we hypothesize that some paracrine cytokines and their receptors may be involved in the melanocyte dysfunction or loss that occurs in vitiligo epidermis.
In the hypopigmented skin of vitiligo patients, it has been argued whether melanocytes remain but have lost their ability to synthesize melanin in melanosomes (77–79). However, whatever mechanisms underlie the loss of pigmentation in vitiligo lesions, it is quite conceivable that the deterioration of melanocyte functions, including melanogenesis, and their survival within the epidermis, are early events leading to the eventual complete loss of epidermal pigmentation. Several hypotheses have been proposed for the patho-physiological mechanism(s) involved in the dysfunction or degeneration of melanocytes in vitiligo skin. These include: (i) an autoimmune mechanism in which antibodies or cytotoxic T cells to melanocytes or its specific organelles (melanosomes) are produced in vitiligo patients and cause the death or apoptosis of melanocytes (80–82), (ii) an auto-cytotoxic mechanism in which superoxides, including hydroxyperoxide, are generated in abundance in the skin of vitiligo patients, which are toxic to melanocytes (83–87), and (iii) an abnormality in melanocytes or in surrounding keratinocytes in producing factors or their receptors, which are necessary for the survival or function of melanocytes within the epidermis (88). Of course, various combinations of those mechanisms may actually be involved in the pathogenesis of vitiligo.
In relation to the role of keratinocyte-derived cytokines that regulate melanocyte functions in the epidermis, pivotal roles for SCF and ET have been demonstrated in several hyperpigmentary disorders, including UVB-induced pigmentation (13, 23, 24, 26, 56, 57). Available evidence suggests that up-regulation of melanogenesis within melanocytes is mediated via increased levels of specific melanogenic cytokines or chemokines produced/released from keratinocytes as well as by the coordinated up-regulated expression of the respective receptors in melanocytes in response to several stimuli. However, many reports have demonstrated that the complete loss of epidermal pigmentation is generally mediated via point mutations of melanogenic ligand-specific receptors, such as c-kit (89, 90) or ETBR (91, 92), as well as of melanogenic cytokines, such as ET-3 (91) or SCF (93–95). Such defects are usually derived from a deficiency in the migration of neonatal melanoblasts from the neural crest to the epidermis or hair follicles during development. Collectively, the available evidence suggests an important and pivotal role of cytokine/receptor linkages in up-regulating or down-regulating melanocyte function. This prompted us to determine whether some cytokines and their receptors may be affected in vitiligo epidermis, which would result in melanocyte dysfunction.
In lesional vitiligo epidermis, there is an increased expression of ET-1 and SCF transcripts compared with non-lesional epidermis, and mSCF but not sSCF protein expression is increased in the lesional epidermis (96). Unexpectedly, this indicates an up-regulated cytokine production in the lesional epidermis, which suggests that there is no diminished ability of vitiligo keratinocytes to produce those melanogenic cytokines. That finding led us to determine whether the SCF receptor (KIT) or ETBR is altered in expression or in its ability to respond to their ligands which would result in a deficiency for inducing melanogenesis or maintaining melanocyte function in vitiligo skin. Following assessment of the distinct border between non-lesional and lesional skin by Fontana Masson staining, immunohistochemistry with antibodies to several melanocyte factors demonstrated that although there is a complete loss of immuno-reactive melanocytes in the center of the vitiligo lesional epidermis, there is an almost normal number of melanocytes expressing tyrosinase, ETBR and S100α protein at the edge of the lesional non-pigmented epidermis compared with the non-lesional pigmented epidermis. In contrast, there are very few melanocytes expressing KIT protein and/or MITF-M at the same edge. Quantitation of the number of melanocytes expressing such melanocytic factors reveals that the number of cells expressing KIT protein is significantly decreased at the edge of the lesional epidermis compared with the non-lesional epidermis, while there is only a slight decrease in the number of S100α, ETBR and tyrosinase immuno-positive cells at the same edge. Consistently, KIT protein expression is significantly decreased at the edge of vitiligo lesions compared with non-lesional epidermis. This suggests that a selective deficiency in KIT protein expression, among the melanocyte-associated molecules tested, occurs in melanocytes localized at the edge of the vitiligo lesional epidermis. A similar decrease in KIT protein expression in vitiligo skin has been reported using immunohistochemistry to show that the number of melanocytes expressing KIT protein begins to decrease at the edge of the vitiligo non-lesion but not in the lesion (although whether the lesional epidermis included the edge of the lesion remains unclarified) (97). Consistent with that study, we found that even at the edge of the non-lesional epidermis, there is a faint decrease in the number of melanocytes expressing KIT protein, tyrosinase and S100α as well as in the amount of c-kit protein.
MITF expression is regulated downstream of the SCF/SCF receptor linkage (98), and serves as a transcription factor regulating the expression of tyrosinase mRNA (99). In response to the activation of SCF/SCF receptor signaling, MITF is phosphorylated by activated MAP kinase (98), undergoes ubiquitination (100, 101) and is translocated to the nucleus to activate transcription of several genes, including tyrosinase. It has recently been reported that there is genetic cooperativity between MITF and Bcl2 in human melanocytes (102). Thus, a deficiency or loss of MITF expression in melanocytes is closely associated with their apoptosis as a result of the down-regulation of the suppressive apoptotic molecule, Bcl2 (102). Because of the close link between MITF and KIT protein expression via the E-box in the c-KIT promoter (103, 104) and between deficient KIT protein expression and a high susceptibility to apoptosis in subfertile human testes (105), it is of great interest to determine whether MITF expression is altered in melanocytes located at the border between the lesional and the non-lesional skin. Immunohistochemistry reveals that the number of cells expressing MITF-M is significantly decreased at the edge of the lesional epidermis compared with the non-lesional skin and that MITF-M protein expression is markedly decreased in the lesional as well as at the edge of the non-lesional epidermis compared with the non-lesional skin. This expression pattern of MITF-M seems to be consistent with the expression pattern of KIT protein. While the faint decrease in the expression of c-kit protein is nearly comparable with that of MITF-M protein at the edge of the non-lesional skin, there is a definite decrease in the expression both of c-kit and of MITF-M in the lesion. This observation strongly suggests that the observed deficiency of KIT protein expression in melanocytes localized at the edge of the vitiligo lesion results from the diminished expression of MITF-M. As a deficiency in MITF has been closely linked to the susceptibility of melanocytes to undergo apoptosis mediated via Bcl2 (102), our observations may indicate that melanocytes localized in the vitiligo lesional skin as well as at the edge of the non-lesional skin have a high predisposition toward apoptosis. In addition, because it seems likely that c-kit expressed on the melanocyte membrane plays an essential role in attracting SCF (which is produced in a membrane-bound form by epidermal keratinocytes), and serves a trafficking role by which melanocytes can stay within the epidermis, the KIT protein deficiency observed in vitiligo melanocytes may indicate that these melanocytes are less able to maintain their interactions with keratinocytes within the epidermis, thus allowing the melanocytes to detach from the epidermis. It should be noted that there is no report describing mutations in the gene for the c-KIT protein in lesional melanocytes of vitiligo epidermis.
In summary, the expression patterns observed for c-kit, MITF-M or tyrosinase in melanocytes located at the border between the lesional and the non-lesional vitiligo epidermis indicates that during the sequential process leading to the dysfunction or the loss of melanocytes, the initial event may result from a deficiency in MITF-M function within melanocytes in the non-lesional epidermis. Such a deficiency would lead to the decreased expression of its target molecule, tyrosinase, as well as to a decrease in c-kit expression via its transcriptional activation via the E-box in its promoter. The diminished signaling via the SCF/SCF receptor linkage and the distinct decrease in tyrosinase expression may underly the complete loss of melanocyte function at the border between the lesional and non-lesional vitiligo skin. Further, the probable increased susceptibility of melanocytes to apoptosis via the MITF/Bcl2 interaction (102) may also be involved in the loss of melanocytes in the lesional epidermis. Although biological mechanisms involved in the MITF-M deficiency observed in the vitiligo lesional melanocytes remain unclarified, recent evidence that oxidative stress such as H2O2 generation leads to the down-regulation of MITF expression (106) suggest that the auto-cytotoxic mechanism (83, 84) by superoxides may underlie the MITF-M deficiency in the vitiligo skin. In conclusion, we propose the hypothesis that the decreased expression of c-kit and its downstream events, including the expression of MITF-M, in melanocytes may be responsible for the dysfunction and/or the loss of melanocytes in vitiligo epidermis.
There are several difficulties in treating hyper- or hypo-pigmentary disorders in terms of down- or up-regulating the stimulated or inactivated melanocyte function toward the casual level of cellular activity because little has been known about the regulatory mechanisms involved in the abnormal elevated or diminished levels of lesional melanocyte function. Therefore, most treatments developed to date have been targeted at inhibitory or cytotoxic effects, such as the effects of ascorbic acid or hydroquinone on tyrosinase activity and on melanocyte viability, respectively. Based upon the newly discovered paracrine or autocrine mechanisms involved in hyper- or hypo-pigmentary disorders (Table 1), the development of more efficient regulation of pigmentary disorders is anticipated. Of special interest are the associated ligand receptors which are relatively manipulatable by cutaneous permeable chemicals with small molecules as an antagonist against specific receptors although an ET receptor antagonist has already been ultilized in the treatment of UVB-induced pigmentation. Furthermore, it would be worthwhile to determine if paracrine or autocrine regulation of melanocyte function is attributable to cancer genesis and the development of malignant melanoma.
Table 1. List of pigmentary disorders described in this chapter and associations with melanocyte proliferation, epidermal hyperplasia and intrinsic cytokine, chemokine or related receptor
Epidermal hyper- or hypo-pigmentary disorder
Intrinsic cytokine or chemokine or receptor
mSCF, membrane-bound type SCF; sSCF, soluble type SCF; SPC, sphingosylphosphorylcholine; PAN, phenyazonapthol.