Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte‐specific cell–cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1‐silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1‐silenced keratinocytes. Dsg1‐silenced keratinocytes increased melanocyte‐stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1‐silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1‐silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1‐deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.     

Endothelin‐1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet‐induced melanocyte homeostasis

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV‐induced melanocyte homeostasis. Selective ablation of endothelin‐1 (EDN1) in murine epidermis (EDN1^ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non‐cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte‐derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV‐induced melanocyte activation and recapitulated the phenotype seen in EDN1^ep?/? mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV‐induced melanocyte homeostasis in vivo.     

Melanocyte–keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes

Physical contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer to occur, but cellular signals induced during or after contact are not fully understood. Herein, it is shown that interactions between melanocyte and keratinocyte plasma membranes induced a transient intracellular calcium signal in keratinocytes that was required for pigment transfer. This intracellular calcium signal occurred due to release of calcium from intracellular stores. Pigment transfer observed in melanocyte–keratinocyte co‐cultures was inhibited when intracellular calcium in keratinocytes was chelated. We propose that a ‘ligand‐receptor’ type interaction exists between melanocytes and keratinocytes that triggers intracellular calcium signalling in keratinocytes and mediates melanin transfer.     

Novel method for isolating human melanoblasts from keratinocyte culture

The characterization of melanoblasts is important for understanding their in vivo development, melanoma formation, and pigment‐related disorders. However, no methods have been reported for the isolation of melanoblasts from human skin. Using a ‘calcium‐pulse’ technique, involving the differentiation of human keratinocytes with high calcium and the subsequent spontaneous separation of the epidermal sheets, we effectively isolated human melanoblasts (keratinocyte‐adapted melanoblasts, KaMBs) from keratinocyte culture. The KaMBs expressed early melanogenesis‐related genes, including BRN2, which is a known melanoblast marker. Moreover, the KaMBs displayed much higher proliferative and growth capacities than the primary melanocytes. Considering that keratinocytes might provide an in vivo‐like environment for KaMBs during isolation and in vitro culture, the ‘calcium‐pulse’ technique offers an unprecedented, easy, and efficient method for the isolation of human melanoblasts, retaining the original characteristics of these cells.     

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well‐integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co‐cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down‐regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase‐related protein‐1 (TRP‐1) negative in confluent well‐integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP‐1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP‐1 expression.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

Up‐Regulation and Redistribution of Bax in Ultraviolet B‐Irradiated Melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0–30 mJ/cm²). The levels of p53, Bax, and Bcl‐2, determined by immunoblotting, revealed a dose‐dependent increase in p53 and Bax, but the level of Bcl‐2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl‐2. These data suggest that the high constitutional level of Bcl‐2 may protect melanocytes from UVB‐induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

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

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

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

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.     

Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes

The hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy-induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum-free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty-eight hour co-treatment with 1,25(OH)(2)D(3) enhanced KGFR-mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c-Jun N-terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)(2)D(3) increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)(2)D(3) increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand-induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)(2)D(3) on KGFR-mediated ERK activation. Our results support the notion that the paracrine KGF-KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury.     

Changes in the Proliferation and Differentiation of Neonatal Mouse Pink‐Eyed Dilution Melanocytes in the Presence of Excess Tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild‐type at the pink‐eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum‐free primary culture, with or without the addition of L‐Tyr. Incubation with added L‐Tyr inhibited the proliferation of P/P melanocytes in a concentration‐dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L‐Tyr stimulated the proliferation of p/p melanoblasts–melanocytes derived from 0.5‐day‐old mice, but inhibited their proliferation when derived from 3.5‐ or 7.5‐day‐old mice. L‐Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5‐, 3.5‐ and 7.5‐day‐old p/p mice, but L‐Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole‐2,3,5‐tricarboxylic acid as well as the pheomelanin marker, 4‐amino‐3‐hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6‐dihydroxyindole‐2‐carboxylic acid, as well as the contents of pheomelanin and its precursor, 5‐S‐cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L‐Tyr increased the content of eumelanin and pheomelanin two‐ to threefold in cultured cells and media derived from 0.5‐, 3.5‐ and 7.5‐day‐old mice. These results suggest that the proliferation of p/p melanoblasts–melanocytes is stimulated by L‐Tyr, and that the differentiation of melanocytes is induced by L‐Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

Differences in visible light‐induced pigmentation according to wavelengths: a clinical and histological study in comparison with UVB exposure

The visible light spectrum is wide, and it can be hypothesized that all the wavelengths between 400–700 nm do not induce the same photobiological effects on pigmentation. We assessed the potential pro‐pigmenting effects of two single wavelengths located at both extremities of the visible spectrum: the blue/violet line (λ = 415 nm) and the red line (λ = 630 nm). We made colorimetric, clinical, and histological assessments with increasing doses of those lights on healthy volunteers. Then, we compared these irradiations to non‐exposed and UVB‐exposed skin. Colorimetric and clinical assessments showed a clear dose effect with the 415‐nm irradiation, in both skin type III and IV subjects, whereas the 630 nm did not induce hyperpigmentation. When compared to UVB irradiation, the blue–violet light induced a significantly more pronounced hyperpigmentation that lasted up to 3 months. Histological examination showed a significant increase of keratinocyte necrosis and p53 with UVB, as compared to 415‐ and 630‐nm exposures.     

Effect of the Rho kinase inhibitor Y-27632 on the proteome of hearts with ischemia-reperfusion injury

Growing attention has been given to the role of the Rho kinase pathway in the development of heart disease and ischemia/reperfusion (I/R) injury. Y‐27632 is a Rho kinase inhibitor demonstrated to protect against I/R injury, but the exact mechanism by which it does so remains to be elucidated. The goal of this project was to determine new targets by which Y‐27632 can protect the heart against I/R injury. Isolated rat hearts were perfused under aerobic conditions or subjected to I/R in the presence or absence of Y‐27632. Administration of Y‐27632 (1 μM) before ischemia and during the first 10 min of reperfusion resulted in complete recovery of cardiac function. 2‐D electrophoresis followed by MS identified four proteins whose levels were affected by Y‐27632 treatment. Lactate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase were significantly increased in the Y‐27632 treated group, while creatine kinase was normalized to control levels. In addition, we found increased level of two different molecular fragments of ATP synthase, which were normalized by Y‐27632. This increase suggests that during ischemia ATP synthase is subjected to degradation. The changes in metabolic enzymes' levels and their regulation by Y‐27632 suggest that the cardioprotective effect of Y‐27632 involves increased energy production.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

Co‐Culture of Mouse Epidermal Cells for Studies of Pigmentation

Interactions between melanocytes and keratinocytes in the skin suggest bi‐directional interchanges between these two cell types. Thus, melanocytes cultured alone may not accurately reflect the physiology of the skin and the effects of physiological regulators in vivo, because they do not consider possible interactions with keratinocytes. As more and more pigment genes are identified and cloned, the characterization of their functions becomes more of a challenge, particularly with respect to their roles in the processing and transport of melanosomes and their transfer to keratinocytes. Immortalized melanocytes mutant at these loci are now being routinely generated from mice, but interestingly, successful co‐culture of murine melanocytes and keratinocytes is very difficult compared with their human counterparts. Thus, we have now optimized co‐culture conditions for murine melanocytes and keratinocytes so that pigmentation and the effects of specific mutations can be studied in a more physiologically relevant context.     

Endothelin‐1 and α‐melanocortin have redundant effects on global genome repair in UV‐irradiated human melanocytes despite distinct signaling pathways

Human melanocyte homeostasis is sustained by paracrine factors that reduce the genotoxic effects of ultraviolet radiation (UV), the major etiological factor for melanoma. The keratinocyte‐derived endothelin‐1 (End‐1) and α‐melanocyte‐stimulating hormone (α‐MSH) regulate human melanocyte function, proliferation and survival, and enhance repair of UV‐induced DNA photoproducts by binding to the G_q‐ and G_i‐protein‐coupled endothelin B receptor (EDNRB), and the G_s‐protein‐coupled melanocortin 1 receptor (MC1R), respectively. We hereby report that End‐1 and α‐MSH regulate common effectors of the DNA damage response to UV, despite distinct signaling pathways. Both factors activate the two DNA damage sensors ataxia telangiectasia and Rad3‐related and ataxia telangiectasia mutated, enhance DNA damage recognition by reducing soluble nuclear and chromatin‐bound DNA damage binding protein 2, and increase total and chromatin‐bound xeroderma pigmentosum (XP) C. Additionally, α‐MSH and End‐1 increase total levels and chromatin localization of the damage verification protein XPA, and the levels of γH2AX, which facilitates recruitment of DNA repair proteins to DNA lesions. Activation of EDNRB compensates for MC1R loss of function, thereby reducing the risk of malignant transformation of these vulnerable melanocytes. Therefore, MC1R and EDNRB signaling pathways represent redundant mechanisms that inhibit the genotoxic effects of UV and melanomagenesis.     

The potent pro‐oxidant activity of rhododendrol–eumelanin is enhanced by ultraviolet A radiation

RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some consumers. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products and RD–eumelanin exerts a potent pro‐oxidant activity. Cellular antioxidants were oxidized by RD–eumelanin with a concomitant production of H₂O₂. In this study, we examined whether this pro‐oxidant activity of RD–eumelanin is enhanced by ultraviolet A (UVA) radiation because most RD–induced leukoderma lesions are found in sun‐exposed areas. Exposure to a physiological level of UVA (3.5 mW/cm²) induced a two to fourfold increase in the rates of oxidation of GSH, cysteine, ascorbic acid, and NADH. This oxidation was oxygen‐dependent and was accompanied by the production of H₂O₂. These results suggest that RD–eumelanin is cytotoxic to melanocytes through its potent pro‐oxidant activity that is enhanced by UVA radiation.     

Keratinocytes Control the Proliferation and Differentiation of Cultured Epidermal Melanocytes from Ultraviolet Radiation B‐Induced Pigmented Spots in the Dorsal Skin of Hairless Mice

Long‐term exposure of ultraviolet radiation B (UVB)‐induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR‐1 X HR//De) F₁. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts//melanocytes from UVB‐induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts//melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts//melanocytes and keratinocytes were prepared and co‐cultured in combination with control and irradiated mice in a serum‐free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non‐irradiated melanoblasts//melanocytes more greatly than those from non‐irradiated mice. In contrast, both non‐irradiated and irradiated adultmelanocytes proliferated and differentiated similarly when they were co‐cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB‐induced pigmented spots are regulated by keratinocytes, rather than melanocytes.