Letter to the Editor
aging (14). It was important to distinguish previously described increase in EOTAXIN in the advanced CTCL patients (17) from normal aging. We found EOTAXIN and MIP-1B to be significantly higher across all age groups in patients with MF/SzS, suggestive of their relevance to the disease pathogenesis (Fig. S1). In conclusion, we demonstrate for the first time that the immune profiles of MF/SzS cluster closer to HIV and then to normal controls and demonstrate unique, cancer-specific and age-related immunological changes. It underscores an immunological dysbalance and immune-compromised state of patients with MF/SzS.
References 1 Criscione V D, Weinstock M A. Arch Dermatol 2007: 143: 854–859. 2 Berger C L, Wang N, Christensen I et al. J Invest Dermatol 1996: 107: 392–397. 3 Axelrod P I, Lorber B, Vonderheid E C. JAMA 1992: 267: 1354–1358. 4 Greene M H, Dalager N A, Lamberg S I et al. Cancer Treat Rep 1979: 63: 597–606. 5 Korgavkar K, Xiong M, Weinstock M. JAMA Dermatol 2013: 149: 1295–1299. 6 Yurkovetsky Z R, Kirkwood J M, Edington H D et al. Clin Cancer Res 2007: 13: 2422–2428. 7 Little J A. Chromatographia 2004: 59: S177– S181.
Acknowledgements We thank Sue McCann, MRN for technical support with blood collection. This work was supported by SPORE NIH 5P50CA121973-03, Project 5 (to LJG) and UL1 RR024153 from the National Center for Research Resources (NCRR).
Author contributions LG and OA designed the study, analysed the data and wrote the paper. AL performed the Luminex assay. YL performed the statistical analysis. AL participated in the design of the work and the critical review of the paper.
Conflict of interest The authors state no conflict of interest.
8 Yoo E K, Cassin M, Lessin S R et al. J Am Acad Dermatol 2001: 45: 208–216. 9 Seo N, Tokura Y, Matsumoto K et al. Clin Exp Immunol 1998: 112: 403–409. 10 Hoppe R T, Medeiros L J, Warnke R A et al. J Am Acad Dermatol 1995: 32: 448–453. 11 Ginaldi L, De Martinis M, D’Ostilio A et al. Immunol Res 1999: 20: 117–126. 12 Litvinov I V, Jones D A, Sasseville D et al. Clin Cancer Res 2010: 16: 2106–2114. 13 Krejsgaard T, Litvinov I V, Wang Y et al. Blood 2013: 122: 943–950. 14 Shurin G V, Yurkovetsky Z R, Chatta G S et al. Cytokine 2007: 39: 123–129.
DOI: 10.1111/exd.12462 www.wileyonlinelibrary.com/journal/EXD
15 Tensen C P, Vermeer M H, van der Stoop P M et al. J Invest Dermatol 1998: 111: 222–226. 16 Rottman J B, Smith T L, Ganley K G et al. Lab Invest 2001: 81: 335–347. 17 Miyagaki T, Sugaya M, Fujita H et al. J Invest Dermatol 2010: 130: 2304–2311.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Age dependent biomarker expression in patient with MF/SzS and age matched controls. Table S1. Comparative analysis of soluble proteins.
Letter to the Editor
Leucine-rich glioma inactivated 3 is a melanogenic cytokine in human skin Hyo-Soon Jeong1, Yun-Mi Jeong1, Jandi Kim1, Seung Hoon Lee1, Hye-Ryung Choi2, Kyoung-Chan Park2, Beom Joon Kim3, Kwang Jin Baek1, Nyoun Soo Kwon1, Hye-Young Yun1 and Dong-Seok Kim1 1 Department of Biochemistry, Chung-Ang University College of Medicine, Seoul, Korea; 2Department of Dermatology, Seoul National University Bundang Hospital, Seongnam-si, Korea; 3Department of Dermatology, Chung-Ang University College of Medicine, Seoul, Korea Correspondence: Dong-Seok Kim, PhD, Department of Biochemistry, Chung-Ang University College of Medicine, 84 Heukseok-ro Dongjak-gu, Seoul 156-756, Korea, Tel.: +82-2-820-5768, Fax: +82-2-820-5768, e-mail: [email protected]
Abstract: Recently, we demonstrated that leucine-rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the effects of LGI3 on melanocytes remain unknown. The present study demonstrated that LGI3 can serve to stimulate melanogenesis without affecting cell viability. To determine the effects of LGI3 on melanin synthesis, normal human melanocytes and Mel-Ab cells were treated with recombinant LGI3 and melanin content was measured. Our results showed that LGI3 promoted melanin synthesis in both cell types. Moreover, upregulation of microphthalmia-associated transcription factor (MITF) and tyrosinase was observed at both the mRNA and protein levels via RT-PCR and Western blotting, respectively. Furthermore, immunohistochemical staining showed that the expression of LGI3
600
increased in the basal layer of melasma skin samples, whereas it decreased slightly in vitiligo samples. These results suggest that LGI3 may play a role as a melanogenic cytokine in human skin. Abbreviations: bFGF, basic fibroblast growth factor; CT, cholera toxin; ET-1, endothelin-1; GM-CSF, granulocyte–macrophage colony-stimulating factor; HGF, hepatocyte growth factor; LGI3, leucine-rich glioma inactivated 3; MITF, microphthalmia-associated transcription factor; SCF, stem cell factor; TPA, tetradecanoylphorbol-13-acetate; a-MSH, a-melanocyte-stimulating hormone. Key words: LGI3 – melanin – melanogenesis – MITF – tyrosinase
Accepted for publication 3 June 2014
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 596–605
Letter to the Editor
Questions addressed The present study focuses on the effects of LGI3 on melanin synthesis in melanocytes. We investigated whether LGI3 could affect melanin synthesis in human melanocytes and Mel-Ab cells using LGI3 recombinant protein. In addition, we also examined the effects of LGI3 on the expression of melanogenic proteins.
Experimental design For Data S1, see supporting information.
Results Previously, we showed that LGI3 protein was expressed in human skin tissue (6). We reported that an LGI3 antibody detected 60and 35-kDa proteins in skin, whereas it did not detect LGI3 in other tissues such as lung and heart (6). We suggested that the 60-kDa protein is full-length LGI3, because bacterially-expressed LGI3 also has a molecular mass of 60-kDa (3). Thus, the 35-kDa
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 596–605
protein may be a product from an alternative translation start site or a product of RNA splicing variants. In the present study, we used three different human skin cell types to examine LGI3 expression. Normal human keratinocytes generated LGI3 predominantly as 60-kDa proteins, whereas normal human fibroblasts and melanocytes produced LGI3 as 35-kDa proteins (Fig. S1). LGI3 is a secreted protein (14) and was shown to be secreted by transfected 293T cells (15). The most abundant form of secreted LGI3 was the 60-kDa protein in 3T3-L1 pre-adipocytes (5). Therefore, 35-kDa LGI3 may not be secreted from fibroblasts or melanocytes. We also reported that UVB irradiation induced the secretion of LGI3 from epidermal keratinocytes (6). Therefore, we hypothesised that LGI3 may participate in melanogenesis as a keratinocyte-derived cytokine. To assess whether LGI3 affects melanogenesis, normal human melanocytes were treated with LGI3 at 0–10 ng/ml for 4 days. As shown in Fig. 1a, LGI3 increased melanin production at concentrations of more than 1 ng/ml. To confirm the results with another cell type, Mel-Ab cells were treated with LGI3 at 0–10 ng/ml for 5 days. Thereafter, Mel-Ab cells were photographed under a phase contrast microscope (Fig. 1c). LGI3-treated Mel-Ab cells showed higher melanin
(a)
(b)
180
*
160 140
*
*
120
*
Melanin content(%)
Leucine-rich glioma inactivated 3 (LGI3) is a secreted leucine-rich repeat protein that has mainly been investigated in the brain due to its high expression level in that organ (1). LGI3 has been implicated in neuronal exocytosis, neuritogenesis and neuronal differentiation in the murine brain (2,3). LGI3 was also detected in aged monkey brains and was determined to be involved in amyloid peptide uptake by astrocytes (4). Outside of the brain, we have found that LGI3 is also expressed in adipose tissues and 3T3-L1 pre-adipocytes and regulates adipogenesis as a candidate adipokine (5). Recently, we reported that LGI3 is highly expressed in human skin and secreted from UVB-irradiated epidermal keratinocytes (6,7). However, the physiological roles of LGI3 in skin cells are not fully understood. Melanocytes are located in the bottom layer of the epidermis (8). Because melanocytes are derived from the neural crest, they may have properties of neuronal cells such as dendrites (8). In addition, LGI3 is the most abundantly expressed isoform of the LGI family (which includes LGI1, 2, 3 and 4) in melanoma cell lines (9). Therefore, it is possible that LGI3 could have an influence on melanocytes. Microphthalmia-associated transcription factor (MITF) plays a crucial role in melanocyte development and is mutated in patients with Waardenburg syndrome 2A (10). MITF binds and regulates melanogenic gene promoters; therefore, activation of MITF results in increased melanin synthesis (10). The activation of MITF enhances the expression of tyrosinase, which catalyses the conversion of tyrosine to melanin pigments (8). Many researchers are interested in the relationship between melanocytes and keratinocytes in the epidermis during melanogenesis (11–13). A variety of studies have suggested that the physiological functions of keratinocyte-derived cytokines, such as basic fibroblast growth factor (bFGF), stem cell factor (SCF/steel factor), hepatocyte growth factor (HGF), granulocyte–macrophage colony-stimulating factor (GM-CSF), nerve growth factor (NGF), a-melanocyte-stimulating hormone (a-MSH), adrenocorticotropic hormone, endorphins, endothelin-1 (ET-1), prostaglandin E2/F2a and leukaemia inhibitory factor (8), are all playing key roles in melanogenesis. For example, bFGF secreted by keratinocytes in vitro regulates melanocyte growth, survival and melanogenesis (8). However, the effects of LGI3 secreted by UVB-irradiated keratinocytes on melanocytes have not been investigated.
Melanin content(%)
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Actin
Figure 1. Effects of LGI3 on melanin synthesis. (a) Human melanocytes were treated with LGI3 at various concentrations (0–10 ng/ml) for 4 days. (b) Mel-Ab cells were treated with LGI3 at various concentrations (0–10 ng/ml) for 5 days. Melanin content was measured as described in ‘Materials and Methods’. Data represent the mean SD of triplicate assays expressed as percentages of the control. *P < 0.05 compared to the untreated control. (c) Phase contrast photographs were taken using a digital video camera. (d) Human melanocytes were treated with LGI3 (0–5 ng/ml). Total RNA was isolated from the cells, and cDNA was prepared. Equivalent amounts of cDNA were amplified with primers specific for MITF and tyrosinase. A GAPDH primer was used as a control to confirm loading of the target cDNA. The resulting PCR products were analysed by agarose gel electrophoresis. (e) After serum starvation, Mel-Ab cells were treated with LGI3 (5 ng/ml). Cells were harvested at the indicated time points and then subjected to Western blot analysis with antibodies against MITF and tyrosinase. Equal protein loading was confirmed by probing with an anti-actin antibody. Fold increases over the level of the control were determined by densitometric analysis and are shown below each lane.
601
Letter to the Editor
(a)
Normal skin F&M
(b)
LGI3
F&M
Normal skin F&M
LGI3
Vitiligo
Melasma LGI3
F&M
LGI3
F&M
Vitiligo
Melasma F&M
LGI3
LGI3
MITF and tyrosinase protein expression in Mel-Ab cells (Fig. 1e). Overall, these results indicate that LGI3 plays an important role in the induction of melanin synthesis. To further examine the expression and distribution of LGI3 in various skin tissues, we performed Fontana–Masson and immunohistochemical staining using normal, hyperpigmentary (melasma) and hypopigmentary (vitiligo) human skin samples. LGI3 was mostly expressed in the granular and cornified layers of normal human epidermis (Fig. 2). Differences in LGI3 expression between the basal layer of melasma and viltiligo epidermis are novel, interesting discoveries. In comparison with normal skin, melasma skin showed higher expression of LGI3 and vitiligo skin exhibited lower expression of LGI3 at the basal layer of the epidermis (Fig. 2). These results indicate that LGI3 may be a potent activator of melanin synthesis in the epidermis of the skin.
Conclusions Figure 2. Expression of LGI3 protein in various skin tissues. Fontana–Masson (F & M) and immunohistochemical staining for LGI3 were performed using normal, melasma and vitiligo skin samples (n = 2) as described in the Materials and Methods. (a) Original magnification, 9400. (b) Original magnification, 91000.
levels than the untreated control cells. As indicated by quantification of melanin content, LGI3-treated Mel-Ab cells demonstrated significantly increased pigmentation in a dose-dependent fashion, compared with the untreated control (Fig. 1b). To determine the effect of LGI3 on melanocyte viability, human melanocytes and Mel-Ab cells were treated with LGI3 at 0–10 ng/ml for 24 h. Crystal violet staining indicated that LGI3 did not exhibit cytotoxic effects on either cell type (data not shown). We also examined whether LGI3-induced melanogenesis involves the expression of MITF and tyrosinase. RT-PCR analysis showed that mRNA expression levels of MITF and tyrosinase in human melanocytes were upregulated when treated with concentrations of more than 0.1 ng/ml LGI3 (Fig. 1d). Moreover, LGI3 treatment increased
Our findings demonstrate for the first time that LGI3 stimulates melanin synthesis and triggers the expression of MITF and tyrosinase in melanocytes. Therefore, LGI3 may be an essential paracrine factor secreted by keratinocytes in the regulation of skin pigmentation. Based on these data, we suggest that LGI3 is a potent melanogenic cytokine in melanocytes in response to UVB irradiation.
Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 20100021335). H.S.J. and Y.M.J. performed the research and wrote this manuscript. J.K. and S.H.L. performed the research. H.R.C., K.C.P., B.J.K., K.J.B., N.S.K. and H.Y.Y. participated in the study design and data interpretation. D.S.K. contributed to the experimental design, data interpretation, writing of the manuscript, editing and submission of this manuscript. All authors read and approved the final manuscript.
Conflict of interests The authors have no conflicting interests to declare.
References
1 Lee S E, Lee A Y, Park W J et al. Gene 2006: 372: 8–17. 2 Park W J, Lim Y Y, Kwon N S et al. Neurochem Res 2010: 35: 789–796. 3 Park W J, Lee S E, Kwon N S et al. Neurosci Lett 2008: 444: 240–244. 4 Okabayashi S, Kimura N. NeuroReport 2008: 19: 1175–1179. 5 Kim H A, Park W J, Jeong H S et al. Biochim Biophys Acta 2012: 1821: 914–922. 6 Lee S H, Jeong Y M, Kim S Y et al. Exp Dermatol 2012: 21: 716–718. 7 Jeong Y M, Park W J, Kim M K et al. Wound Repair Regen 2013: 21: 634–640.
602
8 Costin G E, Hearing V J. Faseb J 2007: 21: 976– 994. 9 Rossi M R, Huntoon K, Cowell J K. Gene 2005: 356: 85–90. 10 Busca R, Ballotti R. Pigment Cell Res 2000: 13: 60–69. 11 Feinmesser M, Tsabari C, Fichman S et al. Am J Dermatopathol 2003: 25: 300–307. 12 Joachim R A, Handjiski B, Blois S M et al. Am J Pathol 2008: 173: 1379–1388. 13 Paterson E K, Ho H, Kapadia R et al. Exp Dermatol 2013: 22: 202–209. 14 Kegel L, Aunin E, Meijer D et al. ASN Neuro 2013: 5: 167–181.
15 Senechal K R, Thaller C, Noebels J L. Hum Mol Genet 2005: 14: 1613–1620.
Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Materials and methods. Figure S1. Expression of LGI3 protein in various skin cells.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 596–605
aging (14). It was important to distinguish previously described increase in EOTAXIN in the advanced CTCL patients (17) from normal aging. We found EOTAXIN and MIP-1B to be significantly higher across all age groups in patients with MF/SzS, suggestive of their relevance to the disease pathogenesis (Fig. S1). In conclusion, we demonstrate for the first time that the immune profiles of MF/SzS cluster closer to HIV and then to normal controls and demonstrate unique, cancer-specific and age-related immunological changes. It underscores an immunological dysbalance and immune-compromised state of patients with MF/SzS.
References 1 Criscione V D, Weinstock M A. Arch Dermatol 2007: 143: 854–859. 2 Berger C L, Wang N, Christensen I et al. J Invest Dermatol 1996: 107: 392–397. 3 Axelrod P I, Lorber B, Vonderheid E C. JAMA 1992: 267: 1354–1358. 4 Greene M H, Dalager N A, Lamberg S I et al. Cancer Treat Rep 1979: 63: 597–606. 5 Korgavkar K, Xiong M, Weinstock M. JAMA Dermatol 2013: 149: 1295–1299. 6 Yurkovetsky Z R, Kirkwood J M, Edington H D et al. Clin Cancer Res 2007: 13: 2422–2428. 7 Little J A. Chromatographia 2004: 59: S177– S181.
Acknowledgements We thank Sue McCann, MRN for technical support with blood collection. This work was supported by SPORE NIH 5P50CA121973-03, Project 5 (to LJG) and UL1 RR024153 from the National Center for Research Resources (NCRR).
Author contributions LG and OA designed the study, analysed the data and wrote the paper. AL performed the Luminex assay. YL performed the statistical analysis. AL participated in the design of the work and the critical review of the paper.
Conflict of interest The authors state no conflict of interest.
8 Yoo E K, Cassin M, Lessin S R et al. J Am Acad Dermatol 2001: 45: 208–216. 9 Seo N, Tokura Y, Matsumoto K et al. Clin Exp Immunol 1998: 112: 403–409. 10 Hoppe R T, Medeiros L J, Warnke R A et al. J Am Acad Dermatol 1995: 32: 448–453. 11 Ginaldi L, De Martinis M, D’Ostilio A et al. Immunol Res 1999: 20: 117–126. 12 Litvinov I V, Jones D A, Sasseville D et al. Clin Cancer Res 2010: 16: 2106–2114. 13 Krejsgaard T, Litvinov I V, Wang Y et al. Blood 2013: 122: 943–950. 14 Shurin G V, Yurkovetsky Z R, Chatta G S et al. Cytokine 2007: 39: 123–129.
DOI: 10.1111/exd.12462 www.wileyonlinelibrary.com/journal/EXD
15 Tensen C P, Vermeer M H, van der Stoop P M et al. J Invest Dermatol 1998: 111: 222–226. 16 Rottman J B, Smith T L, Ganley K G et al. Lab Invest 2001: 81: 335–347. 17 Miyagaki T, Sugaya M, Fujita H et al. J Invest Dermatol 2010: 130: 2304–2311.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Age dependent biomarker expression in patient with MF/SzS and age matched controls. Table S1. Comparative analysis of soluble proteins.
Letter to the Editor
Leucine-rich glioma inactivated 3 is a melanogenic cytokine in human skin Hyo-Soon Jeong1, Yun-Mi Jeong1, Jandi Kim1, Seung Hoon Lee1, Hye-Ryung Choi2, Kyoung-Chan Park2, Beom Joon Kim3, Kwang Jin Baek1, Nyoun Soo Kwon1, Hye-Young Yun1 and Dong-Seok Kim1 1 Department of Biochemistry, Chung-Ang University College of Medicine, Seoul, Korea; 2Department of Dermatology, Seoul National University Bundang Hospital, Seongnam-si, Korea; 3Department of Dermatology, Chung-Ang University College of Medicine, Seoul, Korea Correspondence: Dong-Seok Kim, PhD, Department of Biochemistry, Chung-Ang University College of Medicine, 84 Heukseok-ro Dongjak-gu, Seoul 156-756, Korea, Tel.: +82-2-820-5768, Fax: +82-2-820-5768, e-mail: [email protected]
Abstract: Recently, we demonstrated that leucine-rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the effects of LGI3 on melanocytes remain unknown. The present study demonstrated that LGI3 can serve to stimulate melanogenesis without affecting cell viability. To determine the effects of LGI3 on melanin synthesis, normal human melanocytes and Mel-Ab cells were treated with recombinant LGI3 and melanin content was measured. Our results showed that LGI3 promoted melanin synthesis in both cell types. Moreover, upregulation of microphthalmia-associated transcription factor (MITF) and tyrosinase was observed at both the mRNA and protein levels via RT-PCR and Western blotting, respectively. Furthermore, immunohistochemical staining showed that the expression of LGI3
600
increased in the basal layer of melasma skin samples, whereas it decreased slightly in vitiligo samples. These results suggest that LGI3 may play a role as a melanogenic cytokine in human skin. Abbreviations: bFGF, basic fibroblast growth factor; CT, cholera toxin; ET-1, endothelin-1; GM-CSF, granulocyte–macrophage colony-stimulating factor; HGF, hepatocyte growth factor; LGI3, leucine-rich glioma inactivated 3; MITF, microphthalmia-associated transcription factor; SCF, stem cell factor; TPA, tetradecanoylphorbol-13-acetate; a-MSH, a-melanocyte-stimulating hormone. Key words: LGI3 – melanin – melanogenesis – MITF – tyrosinase
Accepted for publication 3 June 2014
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 596–605
Letter to the Editor
Questions addressed The present study focuses on the effects of LGI3 on melanin synthesis in melanocytes. We investigated whether LGI3 could affect melanin synthesis in human melanocytes and Mel-Ab cells using LGI3 recombinant protein. In addition, we also examined the effects of LGI3 on the expression of melanogenic proteins.
Experimental design For Data S1, see supporting information.
Results Previously, we showed that LGI3 protein was expressed in human skin tissue (6). We reported that an LGI3 antibody detected 60and 35-kDa proteins in skin, whereas it did not detect LGI3 in other tissues such as lung and heart (6). We suggested that the 60-kDa protein is full-length LGI3, because bacterially-expressed LGI3 also has a molecular mass of 60-kDa (3). Thus, the 35-kDa
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 596–605
protein may be a product from an alternative translation start site or a product of RNA splicing variants. In the present study, we used three different human skin cell types to examine LGI3 expression. Normal human keratinocytes generated LGI3 predominantly as 60-kDa proteins, whereas normal human fibroblasts and melanocytes produced LGI3 as 35-kDa proteins (Fig. S1). LGI3 is a secreted protein (14) and was shown to be secreted by transfected 293T cells (15). The most abundant form of secreted LGI3 was the 60-kDa protein in 3T3-L1 pre-adipocytes (5). Therefore, 35-kDa LGI3 may not be secreted from fibroblasts or melanocytes. We also reported that UVB irradiation induced the secretion of LGI3 from epidermal keratinocytes (6). Therefore, we hypothesised that LGI3 may participate in melanogenesis as a keratinocyte-derived cytokine. To assess whether LGI3 affects melanogenesis, normal human melanocytes were treated with LGI3 at 0–10 ng/ml for 4 days. As shown in Fig. 1a, LGI3 increased melanin production at concentrations of more than 1 ng/ml. To confirm the results with another cell type, Mel-Ab cells were treated with LGI3 at 0–10 ng/ml for 5 days. Thereafter, Mel-Ab cells were photographed under a phase contrast microscope (Fig. 1c). LGI3-treated Mel-Ab cells showed higher melanin
(a)
(b)
180
*
160 140
*
*
120
*
Melanin content(%)
Leucine-rich glioma inactivated 3 (LGI3) is a secreted leucine-rich repeat protein that has mainly been investigated in the brain due to its high expression level in that organ (1). LGI3 has been implicated in neuronal exocytosis, neuritogenesis and neuronal differentiation in the murine brain (2,3). LGI3 was also detected in aged monkey brains and was determined to be involved in amyloid peptide uptake by astrocytes (4). Outside of the brain, we have found that LGI3 is also expressed in adipose tissues and 3T3-L1 pre-adipocytes and regulates adipogenesis as a candidate adipokine (5). Recently, we reported that LGI3 is highly expressed in human skin and secreted from UVB-irradiated epidermal keratinocytes (6,7). However, the physiological roles of LGI3 in skin cells are not fully understood. Melanocytes are located in the bottom layer of the epidermis (8). Because melanocytes are derived from the neural crest, they may have properties of neuronal cells such as dendrites (8). In addition, LGI3 is the most abundantly expressed isoform of the LGI family (which includes LGI1, 2, 3 and 4) in melanoma cell lines (9). Therefore, it is possible that LGI3 could have an influence on melanocytes. Microphthalmia-associated transcription factor (MITF) plays a crucial role in melanocyte development and is mutated in patients with Waardenburg syndrome 2A (10). MITF binds and regulates melanogenic gene promoters; therefore, activation of MITF results in increased melanin synthesis (10). The activation of MITF enhances the expression of tyrosinase, which catalyses the conversion of tyrosine to melanin pigments (8). Many researchers are interested in the relationship between melanocytes and keratinocytes in the epidermis during melanogenesis (11–13). A variety of studies have suggested that the physiological functions of keratinocyte-derived cytokines, such as basic fibroblast growth factor (bFGF), stem cell factor (SCF/steel factor), hepatocyte growth factor (HGF), granulocyte–macrophage colony-stimulating factor (GM-CSF), nerve growth factor (NGF), a-melanocyte-stimulating hormone (a-MSH), adrenocorticotropic hormone, endorphins, endothelin-1 (ET-1), prostaglandin E2/F2a and leukaemia inhibitory factor (8), are all playing key roles in melanogenesis. For example, bFGF secreted by keratinocytes in vitro regulates melanocyte growth, survival and melanogenesis (8). However, the effects of LGI3 secreted by UVB-irradiated keratinocytes on melanocytes have not been investigated.
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Figure 1. Effects of LGI3 on melanin synthesis. (a) Human melanocytes were treated with LGI3 at various concentrations (0–10 ng/ml) for 4 days. (b) Mel-Ab cells were treated with LGI3 at various concentrations (0–10 ng/ml) for 5 days. Melanin content was measured as described in ‘Materials and Methods’. Data represent the mean SD of triplicate assays expressed as percentages of the control. *P < 0.05 compared to the untreated control. (c) Phase contrast photographs were taken using a digital video camera. (d) Human melanocytes were treated with LGI3 (0–5 ng/ml). Total RNA was isolated from the cells, and cDNA was prepared. Equivalent amounts of cDNA were amplified with primers specific for MITF and tyrosinase. A GAPDH primer was used as a control to confirm loading of the target cDNA. The resulting PCR products were analysed by agarose gel electrophoresis. (e) After serum starvation, Mel-Ab cells were treated with LGI3 (5 ng/ml). Cells were harvested at the indicated time points and then subjected to Western blot analysis with antibodies against MITF and tyrosinase. Equal protein loading was confirmed by probing with an anti-actin antibody. Fold increases over the level of the control were determined by densitometric analysis and are shown below each lane.
601
Letter to the Editor
(a)
Normal skin F&M
(b)
LGI3
F&M
Normal skin F&M
LGI3
Vitiligo
Melasma LGI3
F&M
LGI3
F&M
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Melasma F&M
LGI3
LGI3
MITF and tyrosinase protein expression in Mel-Ab cells (Fig. 1e). Overall, these results indicate that LGI3 plays an important role in the induction of melanin synthesis. To further examine the expression and distribution of LGI3 in various skin tissues, we performed Fontana–Masson and immunohistochemical staining using normal, hyperpigmentary (melasma) and hypopigmentary (vitiligo) human skin samples. LGI3 was mostly expressed in the granular and cornified layers of normal human epidermis (Fig. 2). Differences in LGI3 expression between the basal layer of melasma and viltiligo epidermis are novel, interesting discoveries. In comparison with normal skin, melasma skin showed higher expression of LGI3 and vitiligo skin exhibited lower expression of LGI3 at the basal layer of the epidermis (Fig. 2). These results indicate that LGI3 may be a potent activator of melanin synthesis in the epidermis of the skin.
Conclusions Figure 2. Expression of LGI3 protein in various skin tissues. Fontana–Masson (F & M) and immunohistochemical staining for LGI3 were performed using normal, melasma and vitiligo skin samples (n = 2) as described in the Materials and Methods. (a) Original magnification, 9400. (b) Original magnification, 91000.
levels than the untreated control cells. As indicated by quantification of melanin content, LGI3-treated Mel-Ab cells demonstrated significantly increased pigmentation in a dose-dependent fashion, compared with the untreated control (Fig. 1b). To determine the effect of LGI3 on melanocyte viability, human melanocytes and Mel-Ab cells were treated with LGI3 at 0–10 ng/ml for 24 h. Crystal violet staining indicated that LGI3 did not exhibit cytotoxic effects on either cell type (data not shown). We also examined whether LGI3-induced melanogenesis involves the expression of MITF and tyrosinase. RT-PCR analysis showed that mRNA expression levels of MITF and tyrosinase in human melanocytes were upregulated when treated with concentrations of more than 0.1 ng/ml LGI3 (Fig. 1d). Moreover, LGI3 treatment increased
Our findings demonstrate for the first time that LGI3 stimulates melanin synthesis and triggers the expression of MITF and tyrosinase in melanocytes. Therefore, LGI3 may be an essential paracrine factor secreted by keratinocytes in the regulation of skin pigmentation. Based on these data, we suggest that LGI3 is a potent melanogenic cytokine in melanocytes in response to UVB irradiation.
Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 20100021335). H.S.J. and Y.M.J. performed the research and wrote this manuscript. J.K. and S.H.L. performed the research. H.R.C., K.C.P., B.J.K., K.J.B., N.S.K. and H.Y.Y. participated in the study design and data interpretation. D.S.K. contributed to the experimental design, data interpretation, writing of the manuscript, editing and submission of this manuscript. All authors read and approved the final manuscript.
Conflict of interests The authors have no conflicting interests to declare.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Materials and methods. Figure S1. Expression of LGI3 protein in various skin cells.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 596–605
