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Keywords:

  • melanoblast;
  • melanocyte;
  • keratinocyte;
  • epidermis;
  • melanocyte-stimulating hormone;
  • endothelins

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References

Melanocytes are neural crest-derived cells that synthesize melanin pigments by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 (dopachrome tautomerase, DCT) (Hearing, 2000; Ito, 2003; Mayer, 1973; Rawles, 1947). Fully differentiated melanocytes characterized by the activities of tyrosinase, TRP-1 and TRP-2 as well as by numerous mature melanosomes and well-developed dendrites can be seen in hair bulbs of the skin, where they secrete mature melanosomes into surrounding keratinocytes, giving rise to melanized hairs (Hirobe, 1995; Mann, 1962; Peters et al., 2002; Slominski and Paus, 1993). The hair bulb melanocytes are derived from melanoblasts, undifferentiated precursors, as well as differentiating melanocytes located in the epidermis (Hirobe, 1992b). In the hairy skin of mice, epidermal melanocytes are found only during the early weeks after birth (Hirobe, 1984; Hirobe and Takeuchi, 1977; Quevedo et al., 1966; Takeuchi, 1968; Weiss and Zelikson, 1975). By contrast, in the glabrous skin of human body as well as of ear, nose and tail of mice, epidermal melanocytes are also found in the adult (Hirobe, 1991; Quevedo et al., 1969).

Tyrosinase initiates melanin synthesis by catalyzing the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (dopa) and oxidation of dopa to dopaquinone (Hearing and Tsukamoto, 1991). TRP-1 maps to the mouse brown locus (Jackson et al., 1990), and possesses 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-oxidase activity (Jimenez-Cervantes et al., 1994; Kobayashi et al., 1994). In contrast, TRP-2 maps to the mouse slaty locus (Jackson et al., 1992), and possesses DCT activity (Tsukamoto et al., 1992) that converts dopachrome to DHICA (Korner and Pawelek, 1980).

Melanin synthesis occurs in specialized organelles called melanosomes (Seiji et al., 1963). Melanosome maturation is categorized in four stages: namely stages I and II include unmelanized immature premelanosomes, while melanized melanosomes are classified as stages III and IV (Fitzpatrick et al., 1969). In mammals, skin colors or coat colors are regulated by melanosome transfer from melanocytes to neighboring keratinocytes and/or to cells of hair bulbs. Mammalian melanocytes produce two types of melanin: brownish-black eumelanin and reddish-yellow pheomelanin (Ito, 2003; Prota, 1980). Although differences exist in molecular size and general properties, these melanins arise from a common metabolic pathway in which dopaquinone is a key intermediate (Hearing and Tsukamoto, 1991; Ito, 2003; Prota, 1980). Melanosomes are produced in varying sizes, numbers and densities in melanocytes. The melanosomes of hair bulb melanocytes are passed on to the hair shaft, where the final distribution patterns of the pigment are terminated. This distribution plays an important role in determining coat color of mice (Silvers, 1979). It is important to understand the regulatory mechanisms of the function of epidermal melanoblasts/melanocytes, especially in proliferation and differentiation, because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes.

The proliferation and differentiation of mammalian epidermal melanocytes are regulated by numerous genes. In mice, numerous coat color genes more than 200 alleles at over 90 loci (Bennett and Lamoreux, 2003; Silvers, 1979) may be required for the regulation of the development of melanocytes. In addition to these genetic controls, the epigenetic controls by the surrounding tissue environment, especially by keratinocytes, are important for the regulation of the proliferation and differentiation of epidermal melanocytes (Hirobe, 1994; Kunisada et al., 1998, 2000). Numerous factors produced in and released from keratinocytes may be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes (Halaban, 2000; Hirobe and Abe, 1999; Imokawa, 2004). Recently, many candidates for the keratinocyte-derived factors have been suggested in human and animals. In this article, studies on the regulation by the keratinocyte-derived factors of the proliferation and differentiation of mammalian epidermal melanocytes including human, rat, gerbil, guinea-pig and mice in normal circumstances or in experimental conditions by external stimuli, such as ultraviolet (UV) radiation are reviewed and discussed.

Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes

  1. Top of page
  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References

In the human skin, studies on the interaction between keratinocytes and melanocytes have been performed by two- (Abdel-Naser, 1999; Gordon et al., 1989; Seiberg et al., 2000; Toyoda et al., 1999; Valyi-Nagy et al., 1990, 1993) or three-dimensional (skin equivalent model, Archambault et al., 1995; De Luca et al., 1988; Scott and Haake, 1991; Seiberg et al., 2000; Todd et al., 1993; Valyi-Nagy et al., 1990) co-culture systems. Pure keratinocytes or melanocytes in primary or serial culture are prepared and used for co-culture experiments. Keratinocytes have been reported to stimulate the proliferation (Abdel-Naser, 1999; De Luca et al., 1988; Gordon et al., 1989; Scott and Haake, 1991; Todd et al., 1993; Toyoda et al., 1999; Valyi-Nagy et al., 1993), melanogenesis (Abdel-Naser, 1999; Archambault et al., 1995; Gordon et al., 1989; Seiberg et al., 2000; Todd et al., 1993; Toyoda et al., 1999; Valyi-Nagy et al., 1993) or dendritogenesis (Archambault et al., 1995; Gordon et al., 1989; Todd et al., 1993; Toyoda et al., 1999; Valyi-Nagy et al., 1990, 1993) of epidermal melanocytes in normal skin (Abdel-Naser, 1999; De Luca et al., 1988; Gordon et al., 1989; Scott and Haake, 1991; Seiberg et al., 2000; Toyoda et al., 1999; Valyi-Nagy et al., 1990, 1993) and/or in UVB-irradiated skin (Archambault et al., 1995; Todd et al., 1993). Undifferentiated keratinocytes (Valyi-Nagy et al., 1993) or keratinocytes cultured in a low Ca2+ medium (Abdel-Naser, 1999) stimulate the proliferation and melanogenesis/dendritogenesis of human melanocytes. By contrast, differentiated keratinocytes (Valyi-Nagy et al., 1993) or keratinocytes cultured in a high Ca2+ medium (Abdel-Naser, 1999) stimulate melanogenesis only. Because the proliferation of human keratinocytes cultured in a low Ca2+ medium is much more active than in a high Ca2+ medium (Hennings et al., 1980), undifferentiated keratinocytes or keratinocytes cultured in a low Ca2+ medium produce and release much more mitogens than differentiated keratinocytes or keratinocytes cultured in a high Ca2+ medium. Moreover, fetal keratinocytes seem to possess a greater stimulative effect on the proliferation of human epidermal melanocytes than neonatal keratinocytes (Scott and Haake, 1991), because fetal keratinocytes are thought to produce and release much more mitogens toward melanocytes than newborn keratinocytes.

In the mouse, studies on the interaction between keratinocytes and melanocytes have been mainly performed using a two-dimensional serum-free primary culture system of epidermal cell suspensions of newborn mice (Hirobe, 1992a). In the initial stage of the primary culture, keratinocytes proliferate well and epidermal melanoblasts and melanocytes start to proliferate around the keratinocyte colony, and after 8–9 days keratinocytes gradually die, then pure cultures of melanoblasts or melanocytes are obtained after 14 days (Hirobe, 1992a). The differentiation of mouse epidermal melanocytes also starts around the keratinocyte colony after 3–4 days in primary culture. The proliferation of mouse epidermal melanoblasts is induced by dibutyryl adenosine 3′:5′-cyclic monophosphate (DBcAMP) at 0.5 mM and basic fibroblast growth factor (bFGF) at 2.5 ng/ml in the presence of keratinocytes, but not in the absence of keratinocytes (Hirobe, 1992a, 1994). The proliferation of mouse epidermal melanocytes is induced by DBcAMP at 0.5–1 mM in the presence of keratinocytes (Hirobe, 1992c). The differentiation and melanogenesis/dendritogenesis of mouse epidermal melanocytes are induced by cAMP-elevating agent such as α-melanocyte-stimulating hormone (α-MSH, Hirobe, 1992c), DBcAMP (0.1–1 mM, Hirobe, 1992c), 3-isobutyl-1-methylxanthine (IBMX, Hirobe, 1992c) or adrenocorticotrophic hormone (ACTH)/ACTH fragments (Hirobe and Abe, 2000) in the presence of keratinocytes. These results suggest that keratinocytes produce and release factors involved in regulating the proliferation, differentiation, melanogenesis or dendritogenesis of neonatal mouse epidermal melanoblasts/melanocytes.

In adult mice, keratinocytes are also involved in regulating the proliferation and differentiation of epidermal melanocytes. It is reported that in pigmented spots of hairless mice (Furuya et al., 2002; Naganuma et al., 2001) induced by UVB long after the cessation of the irradiation, the proliferation and differentiation of epidermal melanocytes are stimulated. The stimulation is elicited by keratinocytes rather than melanocytes (Hirobe et al., 2002). Keratinocytes derived from irradiated skin may produce greater amount of mitogens and melanogens toward melanocytes than those from non-irradiated skin.

In an immortalized cell line of melanocytes, melan-a (Bennett et al., 1987), the increase in melanogenesis (tyrosinase activity and melanin synthesis) is augmented by the presence of an immortalized cell line of keratinocytes, SP-1 in culture (Lei et al., 2002; Yoon and Hearing, 2003), suggesting that SP-1 cells produce and release melanogens toward melanocytes.

Candidates of keratinocyte-derived factors

  1. Top of page
  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References

To identify the keratinocyte-derived mitogens and melanogens toward melanocytes, the following three methods have been mainly utilized. (1) Candidates of the mitogens and melanogens have been tested for their mitogenic and melanogenic effects on melanocytes by adding them into culture media. (2) Antibodies raised against candidates have been tested whether they are capable of neutralizing mitogenic and melanogenic effects of keratinocytes on melanocytes by adding them into culture media. (3) Enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) of conditioned media of cultured keratinocytes has been performed on candidates of mitogens and melanogens.

In the human epidermis, α-MSH (Chakraborty et al., 1996; Schauer et al., 1994; Slominski et al., 2000; Thody et al., 1983; Wakamatsu et al., 1997), ACTH (Chakraborty et al., 1996; Slominski et al., 2000; Wakamatsu et al., 1997)/ACTH fragments (Slominski et al., 2000; Wakamatsu et al., 1997) and nerve growth factor (NGF, Yaar et al., 1991) are produced in and released from keratinocytes and are involved in regulating the melanogenesis and/or dendritogenesis of melanocytes in primary and/or serial culture (Table 1). Endothelin (ET)-1 (Hara et al., 1995; Imokawa et al., 1992; Yada et al., 1991; Yohn et al., 1993) and granulocyte-macrophage colony-stimulating factor (GM-CSF, Imokawa et al., 1996) are the keratinocyte-derived factors that regulate the proliferation and melanogenesis/dendritogenesis of melanocytes (Table 1) in UVB or UVA-irradiated skins. Prostaglandin (PG) E2 and PGF2α are known to be produced and released from human keratinocytes by the stimulation of proteinase-activated receptor 2 (PAR-2) and to stimulate the dendritogenesis of human epidermal melanocytes in culture (Scott et al., 2004). By contrast, bFGF (Halaban et al., 1988) is involved in regulating melanocyte proliferation only (Table 1). Steel factor (SLF) (stem cell factor, SCF) is also expressed in cultured keratinocytes (Hachiya et al., 2001). These factors are involved in regulating the proliferation and melanogenesis/dendritogenesis of human epidermal melanocytes in normal skin (Schauer et al., 1994; Thody et al., 1983; Wakamatsu et al., 1997; Yaar et al., 1991) and/or in UVA (Imokawa et al., 1996)/UVB (Chakraborty et al., 1996; Hachiya et al., 2001; Halaban et al., 1988; Hara et al., 1995; Imokawa et al., 1992; Yada et al., 1991)-irradiated skin.

Table 1.  Keratinocyte-derived factors involved in regulating the proliferation and melanogenesis/dendritogenesis of epidermal melanocytes derived from human, rat, gerbil or guinea-pig
SpeciesFactorsMethodConcUVProlMelanDendMKAntiReference
  1. Method: the presence of the keratinocyte-derived factor is suggested by the method of enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis (Northern), radioimmunoassay (RIA), neutralization by the antibody (Anti) or liquid chromatography-mass spectrometry (LC-MS). Conc, concentration in conditioned medium (pg/ml)/skin (ng/g skin)/keratinocytes (pg/106 cells) or *the most effective concentration/used concentration. UV, irradiated (+, UVA or UVB) or non-irradiated (–); Prol, proliferation of melanocytes; Melan, melanogenesis of melanocytes; Dend, dendritogenesis of melanocytes; M, source of melanocytes, Skin, total skin; SC, serial culture; PC, primary culture; B 16, B16 mouse melanoma cells; K, source of keratinocytes; Anti, whether the effects are inhibited by the antibody.

Humanα-MSHRIA0.8–2.9 ng/g    SkinSkin Thody et al. (1983)
bFGFELISA10 ng/ml*±(UVB)+  SCSC+Halaban et al. (1988)
NGFRT-PCR/Northern100 ng/ml*   +PCPC Yaar et al. (1991)
ET-1RT-PCR/Northern10 nM*±(UVB)+++SCSC Imokawa et al. (1992)
α-MSHRIA21–41 pg/ml  + B16PC/SC+Schauer et al. (1994)
ET-1ELISA1 nM*±(UVB)  +PC/SCPC/SC+Hara et al. (1995)
GM-CSFELISA57–114 pg/ml±(UVA)++ SCSC+Imokawa et al. (1996)
α-MSHRIA/RT-PCR11–45 pg/ml±(UVB)   SCSC Chakraborty et al. (1996)
ACTHRIA/RT-PCR8–34 pg/ml±(UVB)   SCSC Chakraborty et al. (1996)
α-MSHRIA9 pg/106  ++SCSC Wakamatsu et al. (1997)
ACTHRIA447 pg/106  ++SCSC Wakamatsu et al. (1997)
α-MSHLC-MS     SkinSkin Slominski et al. (2000)
ACTHLC-MS     SkinSkin Slominski et al. (2000)
SLFELISA/RT-PCR ±(UVB)   SCSC Hachiya et al. (2001)
PGE2/PGF2αELISA    +PC/SCPC/SC Scott et al. (2004)
Ratα-MSHRIA0.3–0.7 ng/g    SkinSkin Thody et al. (1983)
Gerbilα-MSHRIA0.8–3.2 ng/g    SkinSkin Thody et al. (1983)
Guinea-pigSLFAnti UVB++ SkinSkin+Hachiya et al. (2001)

In animals, α-MSH is also produced in the skin of rat (Thody et al., 1983) and gerbil (Thody et al., 1983) (Table 1). In guinea-pig, SLF stimulates the proliferation and melanogenesis of melanocytes in UVB-irradiated skin (Hachiya et al., 2001) (Table 1).

It is reported that in mice, α-MSH is produced in the skin of adult hairless mouse (Thody et al., 1983). Alpha-MSH is also produced and released from SP-1 keratinocyte cell line and stimulates the melanogenesis of melan-a cells (Virador et al., 2001). However, in a recent study, the gene for proopiomelanocortin (POMC) is not expressed in cultured keratinocytes of newborn mice as well as in the epidermis and dermis separated by trypsin from prenatal and postnatal mice (Hirobe et al., 2004d), suggesting that no keratinocyte in prenatal and neonatal mouse skin produces and releases α-MSH or ACTH (Hirobe et al., 2004d). In mice, the major source of α-MSH and/or ACTH may be derived from the pituitary through the blood stream, but not from epidermal keratinocytes at least in prenatal and postnatal stages (Hirobe et al., 2004d).

By the study using a serum-free primary culture of neonatal mouse epidermal cell suspensions (Hirobe, 1992a,c), ET-1 is known to induce the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes in keratinocyte-depleted cultures (14 days in primary culture of epidermal cell suspensions) in the presence of DBcAMP and/or bFGF (Hirobe, 2001). Moreover, an anti-ET-1 antibody inhibits the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes in the primary culture of epidermal cell suspensions (Hirobe, 2001). These results suggest that ET-1 is a keratinocyte-derived factor involved in regulating the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes (Table 2). By using the same culture system, the same assay methods and ELISA of the conditioned medium of cultured keratinocytes in some cases (Table 2), it has been shown that ET-2 (Hirobe, 2001), ET-3 (Hirobe, 2001), leukemia inhibitory factor (LIF, Hirobe, 2002), SLF (Hirobe et al., 2003) and GM-CSF (Hirobe et al., 2004c) are keratinocyte-derived factors involved in regulating the proliferation of neonatal mouse epidermal melanoblasts and melanocytes as well as the differentiation of melanocytes (Table 2). However, hepatocyte growth factor (HGF, Hirobe et al., 2004a) regulates the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes (Table 2). ET-1, ET-2, ET-3, LIF, SLF, GM-CSF and HGF increase the mitotic indices of melanoblasts and melanocytes as well as the percentage of melanoblasts and melanocytes in the S phase of the cell cycle in keratinocyte-depleted cultures (Hirobe, 2001, 2002; Hirobe et al., 2003, 2004a,c, Table 2). A slight difference exists in the intensity of the induction of the proliferation and/or differentiation of melanocytes (Table 2). The most effective concentration of these factors is also different (Table 2). ET-1 and HGF are most effective in the stimulation of melanoblast proliferation (Table 2). HGF is most effective in the stimulation of melanocyte proliferation (Table 2). However, while ET-1 is more efficient in the stimulation of melanocyte differentiation (Table 2), HGF is more effective in the stimulation of dendritogenesis (Table 2). Moreover, LIF is effective at a lower concentration, while SLF is effective at a higher concentration than ET-1, ET-2, ET-3, HGF or GM-CSF (Table 2). It remains to be investigated in a future study what mechanisms are involved in regulating the intensity of the induction of the proliferation and differentiation.

Table 2.  Mouse keratinocyte-derived factors involved in regulating the proliferation and differentiation/melanogenesis of mouse epidermal melanoblasts/melanocytes
FactorsMethodUVStrainMb/MKMelanDendMb prolM prolM diff (%)Inh Mb (%)Inh M (%)Inh Diff (%)MISReference
  1. Method: the presence of the keratinocyte-derived factor is suggested by the method of radioimmunoassay (RIA), neutralization by the antibody (Anti) or enzyme-linked immunosorbent assay (ELISA). UV, irradiated (UVB) or non-irradiated (−). Strain, strain of mice used. HR, Hairless; B10, C57BL/10JHir; HRF1, (HR/De x HR-1)F1. Mb/M, source of melanoblasts (Mb) or melanocytes (M); Skin, total skin; Melan, melan-a cells; PC, primary culture; K, source of keratinocytes, SP-1, SP-1 keratinocyte cell line; SC, secondary culture; Melan, melanogenesis of melanocytes. Dend, whether the dendritogenesis is stimulated (+) by the factor; +++, intensive. Mb prol, induction of melanoblast proliferation by the factor (the most effective concentration: nM or ng/ml). M prol, induction of melanocyte proliferation by the factor (the most effective concentration: nM or ng/ml). M diff, induction of melanocyte differentiation (increase in the percentage of melanocytes in the melanoblast-melanocyte population, the most effective concentration: nM or ng/ml). Inh Mb, inhibition of melanoblast proliferation by the antibody (the most effective concentration, ng/ml or μg/ml). Inh M, Inhibition of melanocyte proliferation by the antibody (the most effective concentration, ng/ml or μg/ml). MI, mitotic indices of melanoblasts or melanocytes, up (increased). S, percentage of melanoblasts or melanocytes in the S phase of the cell cycle by the Flow Cytometry analysis; up, increased; up*, increased (Hirobe, Osawa and Nishikawa, unpublished data).

α-MSHRIAHRSkinSkin          Thody et al. (1983)
α-MSHRIA Melan-aSP-1+         Virador et al. (2001)
ET-1AntiB10PCPC++×3.0 (10 nM)×2.4 (10 nM)+39 (10 nM)−58 (250 ng)−55 (250 ng)−14 (250 ng)UpUp*Hirobe (2001)
ET-2AntiB10PCPC++×2.5 (10 nM)×2.1 (10 nM)+35 (10 nM)−63 (250 ng)−48 (250 ng)−18 (250 ng)UpUp*Hirobe (2001)
ET-3AntiB10PCPC++×2.6 (10 nM)×2.4 (10 nM)+37 (10 nM)−60 (250 ng)−52 (250 ng)−26 (250 ng)UpUp*Hirobe (2001)
LIFAntiB10PCPC++×2.1 (0.1 ng)×1.7 (0.1 ng)+35 (1 ng)−55 (2.5 μg)−42 (2.5 μg)−41 (2.5 μg)UpUp*Hirobe (2002)
SLFAntiB10PCPC++×2.3 (50 ng)×2.2 (50 ng)+24 (50 ng)−55 (2.5 μg)−43 (2.5 μg)−16 (2.5 μg)UpUpHirobe et al. (2003)
HGFAntiB10PCPC++++×3.0 (10 ng)×3.2 (10 ng)−63 (2.5 μg)−39 (2.5 μg)UpUpHirobe et al. (2004a)
GM-CSFAnti/ELISAB10PCPC++×2.6 (10 ng)×2.4 (10 ng)+22 (10 ng)−46 (25 μg)−33 (25 μg)−13 (25 μg)UpUpHirobe et al. (2004c)
GM-CSFAnti/ELISAUVBHRF1PCPC/SC++ ×2.4 (0.1 ng)+42 (10 ng)−90 (2.5 μg)−81 (2.5 μg)−56 (2.5 μg)Up Hirobe et al. (2004b)

In adult hairless mice, GM-CSF is also known to induce the proliferation and differentiation of epidermal melanocytes in the pigmented spots induced by UVB irradiation (Hirobe et al., 2004b). GM-CSF seems to be an important keratinocyte-derived factor involved in regulating the proliferation and differentiation of epidermal melanocytes from adult skin exposed to UVB (Hirobe et al., 2004b) (Table 2). In adult hairless mice, the inhibition of the proliferation of melanoblasts and melanocytes as well as the inhibition of melanocyte differentiation by an anti-GM-CSF antibody is much greater than that in newborn mice (Table 2). It is possible that the anti-GM-CSF antibody fail to inhibit completely the proliferation and differentiation of epidermal melanocytes of newborn mice, because in newborn mice many kinds of keratinocyte-derived factors other than GM-CSF are present and involved in regulating the proliferation and differentiation of melanocytes.

Molecules of candidates of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes may possess common characteristics. Namely, these molecules are more or less related to hematopoiesis or blood vessel formation, or they are supplied to target tissues through the blood stream. Alpha-MSH and ACTH (Eberle, 1988) are supplied from the pituitary through the blood stream. ETs (Yanagisawa et al., 1988) and bFGF (Dell'Era et al., 1991) are produced by endothelial cells. SLF (Nocka et al., 1990), LIF (Tomida et al., 1984) and GM-CSF (Morstyn and Burgess, 1988) are functional in hematopoietic cells. HGF (Bussolino et al., 1992) is also related to angiogenesis by stimulating endothelial cells to proliferate and migrate. It is possible that melanoblasts and melanocytes are influenced by factors released from blood vessels during the migratory process in development (Manova and Bachvarova, 1991; Matsui et al., 1990; Steel et al., 1992). After colonizing in the epidermis, melanocytes may require these factors for their proliferation and differentiation supplied from surrounding keratinocytes. Keratinocytes might be required to supply these factors to melanocytes for stimulating the proliferation and differentiation of melanocytes. However, this hypothesis remains to be investigated in a future study.

Mechanism of action of keratinocyte-derived factors

  1. Top of page
  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References

Alpha-MSH and ACTH induce the differentiation of mouse epidermal melanocytes in vivo (Hirobe and Takeuchi, 1977) and in vitro (Hirobe, 1992c; Hirobe and Abe, 2000). Alpha-MSH induces the differentiation of mouse epidermal melanocytes in the presence of keratinocyte-derived factors such as ET-1 (Hirobe, 2001), ET-2 (Hirobe, 2001), ET-3 (Hirobe, 2001), LIF (Hirobe, 2002), SLF (Hirobe et al., 2003) or GM-CSF (Hirobe et al., 2004c). In human epidermal melanocytes, α-MSH and ACTH stimulate melanogenesis (Wakamatsu et al., 1997). Alpha-MSH and ACTH bind to its specific receptor, melanocortin receptor-1 (MC1-R) (Cone et al., 1996), activate adenylate cyclase through G protein and then elevate cAMP from adenosine triphosphate (Im et al., 1998). Cyclic AMP exerts its effect in part through protein kinase A (PKA) (Insel et al., 1975). PKA phosphorylates and activates the cAMP-response element binding protein (CREB) that binds to cAMP-response element (CRE) present in the M promoter of the microphthalmia-associated transcription factor (Mitf) gene (Busca and Ballotti, 2000; Tachibana, 2000). The resulting transient increase in Mitf-M expression leads to the up-regulations of tyrosinase, TRP-1 and TRP-2 (Busca and Ballotti, 2000; Tachibana, 2000), to allow melanin synthesis (Figure 1). Alpha-MSH and ACTH act as melanogens toward mammalian epidermal melanocytes through signaling pathway of PKA. However, PGE2 and PGF2α released from human keratinocytes stimulate the function of human epidermal melanocytes through EP1, EP3 and FP receptors, and the stimulation of dendritogenesis by PGE2 and PGF2α is cAMP-independent and might be mediated through phospholipase C (PLC) (Scott et al., 2004). It is also known that α-MSH signaling can activate the stress-activated kinase p38 signaling pathway (Smalley and Eisen, 2000) although how important p38 signaling by α-MSH is in vivo is unclear. Moreover, cAMP signaling in melanocytes can activate the MAP kinase (MK) pathway (Busca et al., 2000). Taken together, these observations suggest that α-MSH may exert its effects via multiple signaling pathways.

image

Figure 1. Hypothesis of the mechanism of action of keratinocyte-derived factors on the proliferation and differentiation of mammalian epidermal melanocytes. The cross-talk between the signaling pathway is important for the proliferation and differentiation of melanocytes. *Evidenced for human only; **evidenced for mouse only; no asterisk, evidenced for both human and mouse. PGE2, prostaglandin E2; PGF2α, prostaglandin F2α; α-MSH, α-melanocyte-stimulating hormone; ACTH, adrenocorticotrophic hormone; ET-1, endothelin-1; ET-2, endothelin-2; ET-3, endothelin-3; SLF, steel factor; bFGF, basic fibroblast growth factor; LIF, leukemia inhibitory factor; HGF, hepatocyte growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; EP1/EP3/FP, receptor for prostaglandins; MC1R, melanocortin receptor-1; ETBR, endothelin receptor B; c-Kit, receptor for SLF; FGFR-1/-2, FGF receptor-1/-2; gp130LIFRα, LIF receptor; c-Met, receptor for HGF; GMCSFR, receptor for GM-CSF; PLC, phospholipase C; ATP, adenosine triphosphate; cAMP, adenosine 3′:5′-cyclic monophosphate; PKA, protein kinase A; CREB, cAMP responsive element binding protein, Mitf, microphthalmia associated transcription factor; PKC, protein kinase C; MK, MAP kinase.

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ET-1, ET-2 and ET-3 stimulate the proliferation of epidermal melanoblasts and melanocytes of newborn mice in cooperation with cAMP elevators and bFGF (Hirobe, 2001). The number of mitotic melanoblasts/melanocytes as well as the proliferation of melanoblasts/melanocytes in the S phase of the cell cycle is increased by ET-1, ET-2 and ET-3 (T. Hirobe, M. Osawa and S.-I. Nishikawa, unpublished data). ET-1, ET-2 and ET-3 are also capable of inducing the differentiation of mouse epidermal melanocytes in primary culture in cooperation with α-MSH or DBcAMP (Hirobe, 2001). ET-1, ET-2 and ET-3 also stimulate the DNA synthesis of cultured human epidermal melanocytes in the presence of cAMP elevator and serum (Imokawa et al., 1992). ET-1, ET-2 and ET-3 bind to their specific receptor, ETBR (Sakurai et al., 1990), stimulate 1,4,5-inositol-triphosphate formation and activate PKC of human epidermal melanocytes (Yada et al., 1991). ET-1, ET-2 and ET-3 act as mitogens and melanogens toward mammalian epidermal melanocytes by up-regulation of proteins required for the proliferation as well as of tyrosinase, TRP-1 and TRP-2 through signaling pathway of PKC (Imokawa et al., 1997) (Figure 1).

SLF in the presence of DBcAMP (or α-MSH) and/or bFGF induced the proliferation and differentiation of melanoblasts/melanocytes in primary culture (Hirobe et al., 2003). Cell cycle analysis shows that SLF stimulates the proliferation of melanoblasts and melanocytes by accelerating the transitions from G0/G1 to S as well as from G2 to M phases of the cell cycle (Hirobe et al., 2003). Mouse SLF is also reported to stimulate the proliferation of mouse epidermal melanoblasts and melanocytes in primary culture in the presence of fetal bovine serum and cholera toxin (Sviderskaya et al., 1995). In human skin, SLF stimulates the proliferation (Funasaka et al., 1992; Halaban et al., 1993) and dendritogenesis (Grichnik et al., 1998) of epidermal melanocytes in the presence of DBcAMP. SLF binds to its specific receptor, c-kit (Geissler et al., 1988), activates MK and acts as a mitogen and melanogen toward mammalian epidermal melanocytes through up-regulation of proteins required for the proliferation as well as of tyrosinase, TRP-1 and TRP-2 (Figure 1).

Basic FGF stimulates the proliferation of undifferentiated melanoblasts of newborn mice (Hirobe, 1992a) in the presence of DBcAMP and keratinocyte-derived factors such as ET-1 (Hirobe, 2001), ET-2 (Hirobe, 2001) and ET-3 (Hirobe, 2001), SLF (Hirobe et al., 2003), LIF (Hirobe, 2002), HGF (Hirobe et al., 2004a) or GM-CSF (Hirobe et al., 2004c). In human skin, bFGF stimulates the proliferation of epidermal melanocytes in the presence of DBcAMP (Halaban et al., 1988). Basic FGF is unable to stimulate the differentiation of mouse epidermal melanocytes in primary culture (Hirobe, 1992a). Basic FGF binds to its specific receptors, FGFR-1/FGFR-2 (Johnson and Williams, 1993), activates MK (Imokawa et al., 1997) and elicits up-regulation of proteins required for proliferation.

LIF stimulates the proliferation and differentiation of mouse epidermal melanocytes in primary culture in cooperation with cAMP elevators and bFGF (Hirobe, 2002). Cell cycle analysis shows that LIF stimulates the proliferation of melanoblasts and melanocytes by accelerating the transitions from G0/G1 to S as well as from G2 to M phases of the cell cycle (Hirobe, Osawa and Nishikawa, unpublished data). In human skin, there is no report that LIF stimulates the proliferation and differentiation of epidermal melanocytes. By the study of mammalian cells other than melanocytes LIF is known to act on cells by binding to the heterodimeric LIF receptor consisting of the two transmembrane proteins, gp130 and the LIF receptor α (LIFRα). At the times of bindings, the receptors dimerize and recruit the nonreceptor tyrosine kinases Jak 1 or 2 and Tyk2, which phosphorylate the cytoplasmic domains of the receptors. This results in the recruitment and phosphorylation of the latent signal transducer and activator of transcription (STAT) by the Jaks. The phosphorylated STATs dimerize and translocate to the nucleus where they act to regulate gene expression (Auernhammer and Melmed, 2000). LIF may stimulate the synthesis of proteins required for the proliferation as well as the melanin synthesis via signal transduction pathway of MK (Coughlin et al., 1988).

HGF stimulates the proliferation of mouse epidermal melanoblasts and melanocytes in culture in cooperation with cAMP elevator and/or bFGF (Hirobe et al., 2004a). The number of mitotic melanoblasts and melanocytes as well as the proportion of melanoblasts and melanocytes in the S phase of the cell cycle is increased by HGF (Hirobe et al., 2004a). HGF is unable to induce the differentiation of mouse epidermal melanocytes in primary culture, but the factor markedly stimulates the dendritogenesis of mouse epidermal melanocytes (Hirobe et al., 2004a). In human melanocytes, HGF alone is sufficient for stimulating their proliferation (Halaban et al., 1993; Matsumoto et al., 1991). HGF binds to its specific receptor, c-Met (Bottaro et al., 1991), activates MK and elicits up-regulation of proteins required for the proliferation (Figure 1).

GM-CSF stimulates the proliferation of cultured neonatal mouse epidermal melanoblasts and melanocytes (Hirobe et al., 2004c) as well as cultured adult epidermal melanoblasts and melanocytes from pigmented spots of hairless mice induced by UVB (Hirobe et al., 2004b) in cooperation with cAMP elevators and bFGF. In cultured neonatal melanoblasts/melanocytes, the number of mitotic melanoblasts/melanocytes as well as the proportion of melanoblasts/melanocytes in the S phase of the cell cycle is increased by GM-CSF (Hirobe et al., 2004c). GM-CSF is also capable of inducing the differentiation of neonatal mouse epidermal melanocytes in primary culture (Hirobe et al., 2004c) and of adult epidermal melanocytes in primary culture derived from pigmented spots of UVB-irradiated mice (Hirobe et al., 2004b). GM-CSF also stimulates the proliferation of human epidermal melanocytes in the presence of serum (Imokawa et al., 1996). GM-CSF binds to its specific receptor, GMCSFR (Chiba et al., 1990), activates the signal transducer and activator of transcription (STAT-1, STAT-3 and STAT-5) (Mui et al., 1995; Wang et al., 1995) or MK (Okuda et al., 1992) and elicits up-regulation of proteins required for the proliferation as well as of tyrosinase, TRP-1 and TRP-2 (Figure 1).

There may be a complex network in signaling pathway of these keratinocyte-derived factors (Figure 1). The proliferation of mouse epidermal melanoblasts may require three signaling pathways: PKA by cAMP elevators, PKC by ET-1/ET-2/ET-3 and MK by SLF/bFGF/LIF/HGF/GM-CSF (Hirobe, 1992a, 2001, 2002; Hirobe et al., 2003, 2004a,b,c). The proliferation of mammalian epidermal melanocytes may require two signaling pathways: PKA by cAMP elevators and PKC by ET-1/ET-2/ET-3 or MK by SLF/bFGF/LIF/HGF/GM-CSF (Halaban et al., 1988; Hirobe, 1992a, 2001, 2002; Hirobe et al., 2003, 2004a,b,c; Yada et al., 1991). The differentiation or melanogenesis/dendritogenesis of mammalian epidermal melanocytes may require two signaling pathways: PKA by cAMP elevators and PKC by ET-1/ET-2/ET-3 or MK by SLF/LIF/HGF/GM-CSF (Hirobe, 1992c, 2001, 2002; Hirobe et al., 2003, 2004a,b,c; Yada et al., 1991). There may be a cross-talk in signaling pathway to support activation of the proliferation and differentiation or melanogenesis/dendritogenesis of mammalian epidermal melanocytes elicited by these keratinocyte-derived factors.

A question arises how the proliferation and differentiation of mammalian melanocytes may be regulated at the gene level by signaling molecules activated by the keratinocyte-derived factors, namely signaling flow from cytoplasm to nucleus. The ubiquitous basic helix-loop-helix-leucine zipper transcription factor, Usf-1 is important for the UV activation of the tyrosinase promotor (Galibert et al., 2001). Melanocytes derived from mice knocked out for Usf-1 do not respond to UV in the sense that they no longer induce the expression of POMC in response to UV irradiation, suggesting that Usf-1 which is known to be regulated by the stress-activated kinase p38 (Galibert et al., 2001) might play an important role in UV-induced pigmentation (M.-D. Galibert, personal communication). It is possible that keratinocyte-derived factors may signal via this pathway and regulate the Usf-1 transcription factor in melanocytes and consequently control differentiation of mammalian melanocytes. However, this hypothesis remains to be investigated in an animal model.

There is also increasing evidence (reviewed in Vance and Goding, 2004) that the proliferation and dendricity of melanocytes is controlled by Mitf that is known to be regulated by both MK and p38 signaling (Mansky et al., 2002; Wu et al., 2000; Xu et al., 2000). It is possible therefore that keratinocyte-derived factors signaling via MK or p38 regulate Mitf in melanocytes and consequently control the proliferation of mammalian epidermal melanocytes. Precisely how Mitf might act as a regulator of proliferation in response to keratinocyte-derived signals remains an area for future work.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References

This work was supported in part by Grant-in-Aid for Scientific Research (No. 15591204) from the Ministry of Education, Science, Sports and Culture of Japan.

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  2. Summary
  3. Introduction
  4. Keratinocytes are involved in regulating the proliferation and differentiation of melanocytes
  5. Candidates of keratinocyte-derived factors
  6. Mechanism of action of keratinocyte-derived factors
  7. Acknowledgements
  8. References
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