JCDSA  Vol.5 No.4 , December 2015
Melanin Uptake Reduces Cell Proliferation of Human Epidermal Keratinocytes
Abstract: Melanin, synthesized by melanocyte, is transferred to neighboring keratinocyte and finally accumulates in perinuclear site. Except functioning as an internal sunscreen to protect from UV damage, the potential effect of melanin on modulating the bioactivity of keratinocyte has not yet been fully investigated. In this study, we added melanin directly to the culture of human epidermal keratinocytes and the uptake of melanin was found to be dose- and time-dependent as determined by spectrophotometric method. The uptaken melanin accumulated perinuclearly in keratinocytes that is similar to the pattern observed in human solar lentigo tissue by microscopic examination. Pretreatment of keratinocytes with either niacinamide or trypsin inhibitor reduced the uptake of melanin dose-dependently, indicating a PAR-2-dependent pathway involved. Melanin uptake by keratinocytes inhibited cell proliferation as demonstrated both by the decrease of cell number and nuclear Ki-67 expression. Inhibited Ki-67 expression in melanin-containing keratinocyte was also found in human lentigo tissue. The cell cycle arrested at G1 phase in melanin-uptaken keratinocytes was confirmed by flow cytometric method. The protein expressions of cyclin-dependent kinase 1 (CDK1), CDK2, cyclin E, cyclin A and cyclin B were significantly reduced by melanin treatment. Microarray analysis, RT/real-time PCR and western blot demonstrated the inhibited expression of DKK1, a protein known to reduce skin pigmentation, in melanin-uptaken keratinocytes. Together, the direct incubation of keratinocyte with melanin might serve as a useful model to study the potential mechanisms involved in melanin uptake and pigmentation process.
Cite this paper: Yan, X. , Wang, T. , Ming, Y. , Yeh, Y. , Chen, T. and Pang, J. (2015) Melanin Uptake Reduces Cell Proliferation of Human Epidermal Keratinocytes. Journal of Cosmetics, Dermatological Sciences and Applications, 5, 300-310. doi: 10.4236/jcdsa.2015.54037.

[1]   Jordens, I., Westbroek, W., Marsman, M., et al. (2006) Rab7 and Rab27a Control Two Motor Protein Activities Involved in Melanosomal Transport. Pigment Cell Research, 19, 412-423.

[2]   Park, H.Y., Kosmadaki, M., Yaar, M. and Gilchrest, B.A. (2009) Cellular Mechanisms Regulating Human Melanogenesis. Cellular and Molecular Life Sciences, 66, 1493-1506.

[3]   Boissy, R.E. (2003) Melanosome Transfer to and Translocation in the Keratinocyte. Experimental Dermatology, 12, 5-12.

[4]   Reish, O., Townsend, D., Berry, S.A., Tsai, M.Y. and King, R.A. (1995) Tyrosinase Inhibition Due to Interaction of Homocyst(e)ine with Copper: The Mechanism for Reversible Hypopigmentation in Homocystinuria Due to Cystathionine Beta-Synthase Deficiency. The American Journal of Human Genetics, 57, 127-132.

[5]   Solano, F., Briganti, S., Picardo, M. and Ghanem, G. (2006) Hypopigmenting Agents: An Updated Review on Biological, Chemical and Clinical Aspects. Pigment Cell Research, 19, 550-571.

[6]   Kim, H., Choi, H.R., Kim, D.S., et al. (2012) Topical Hypopigmenting Agents for Pigmentary Disorders and Their Mechanisms of Action. Annals of Dermatology, 24, 1-6.

[7]   Singh, S.K., Kurfurst, R., Nizard, C., et al. (2010) Melanin Transfer in Human Skin Cells Is Mediated by Filopodia—A Model for Homotypic and Heterotypic Lysosome-Related Organelle Transfer. FASEB Journal, 24, 3756-3769.

[8]   Ando, H., Niki, Y., Yoshida, M., et al. (2010) Keratinocytes in Culture Accumulate Phagocytosed Melanosomes in the Perinuclear Area. Pigment Cell Melanoma Res, 23, 129-133.

[9]   Chakraborty, A.K., Funasaka, Y., Araki, K., et al. (2003) Evidence That the Small GTPase Rab8 Is Involved in Melanosome Traffic and Dendrite Extension in B16 Melanoma Cells. Cell and Tissue Research, 314, 381-388.

[10]   Ando, H., Niki, Y., Yoshida, M., et al. (2011) Involvement of Pigment Globules Containing Multiple Melanosomes in the Transfer of Melanosomes from Melanocytes to Keratinocytes. Cellular Logistics, 1, 12-20.

[11]   Tarafder, A.K., Bolasco, G., Correia, M.S., et al. (2014) Rab11b Mediates Melanin Transfer between Donor Melanocytes and Acceptor Keratinocytes via Coupled Exo/Endocytosis. Journal of Investigative Dermatology, 134, 1056-1066.

[12]   Byers, H.R., Maheshwary, S., Amodeo, D.M., et al. (2003) Role of Cytoplasmic Dynein in Perinuclear Aggregation of Phagocytosed Melanosomes and Supranuclear Melanin Cap Formation in Human Keratinocytes. Journal of Investigative Dermatology, 121, 813-820.

[13]   Yamaguchi, Y., Takahashi, K., Zmudzka, B.Z., et al. (2006) Human Skin Responses to UV Radiation: Pigment in the Upper Epidermis Protects against DNA Damage in the Lower Epidermis and Facilitates Apoptosis. FASEB Journal, 20, 1486-1488.

[14]   Takeuchi, S., Zhang, W., Wakamatsu, K., et al. (2004) Melanin Acts as a Potent UVB Photosensitizer to Cause an Atypical Mode of Cell Death in Murine Skin. Proceedings of the National Academy of Sciences of the United States of America, 101, 15076-15081.

[15]   Timares, L., Katiyar, S.K. and Elmets, C.A. (2008) DNA Damage, Apoptosis and Langerhans Cells—Activators of UV-Induced Immune Tolerance. Photochemistry and Photobiology, 84, 422-436.

[16]   Hsieh, W.L., Lin, Y.K., Tsai, C.N., et al. (2012) Indirubin, an Acting Component of Indigo Naturalis, Inhibits EGFR Activation and EGF-Induced CDC25B Gene Expression in Epidermal Keratinocytes. Journal of Dermatological Science, 67, 140-146.

[17]   Choi, H.-I., Sohn, K.-C., Hong, D.-K., et al. (2013) Melanosome Uptake Is Associated with the Proliferation and Differentiation of Keratinocytes. Archives of Dermatological Research. [Epub ahead of print]

[18]   Denicol, A.C., Dobbs, K.B., McLean, K.M., et al. (2013) Canonical WNT Signaling Regulates Development of Bovine Embryos to the Blastocyst Stage. Scientific Reports, 3, 1266.

[19]   Daoussis, D. and Andonopoulos, A.P. (2011) The Emerging role of Dickkopf-1 in Bone Biology: Is It the Main Switch Controlling Bone and Joint Remodeling? Seminars in Arthritis & Rheumatism, 41, 170-177.

[20]   Zhang, R., Oyajobi, B.O., Harris, S.E., et al. (2013) Wnt/β-Catenin Signaling Activates Bone Morphogenetic Protein 2 Expression in Osteoblasts. Bone, 52, 145-156.

[21]   Miao, C.G., Yang, Y.Y., He, X., et al. (2013) Wnt Signaling Pathway in Rheumatoid Arthritis, with Special Emphasis on the Different Roles in Synovial Inflammation and Bone Remodeling. Cellular Signalling, 25, 2069-2078.

[22]   Kocemba, K.A., Groen, R.W., van Andel, H., et al. (2012) Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma. PLoS One, 7, e30359.

[23]   Yamaguchi, Y., Itami, S., Watabe, H., et al. (2004) Mesenchymal-Epithelial Interactions in the Skin: Increased Expression of Dickkopf1 by Palmoplantar Fibroblasts Inhibits Melanocyte Growth and Differentiation. Journal of Cell Biology, 165, 275-285.

[24]   Yamaguchi, Y., Passeron, T., Watabe, H., et al. (2007) The Effects of Dickkopf 1 on Gene Expression and Wnt Signaling by Melanocytes: Mechanisms Underlying Its Suppression of Melanocyte Function and Proliferation. Journal of Investigative Dermatology, 127, 1217-1225.

[25]   Yamaguchi, Y., Passeron, T., Hoashi, T., et al. (2008) Dickkopf 1 (DKK1) Regulates Skin Pigmentation and Thickness by Affecting Wnt/Beta-Catenin Signaling in Keratinocytes. FASEB Journal, 22, 1009-1020.

[26]   Yamaguchi, Y., Morita, A., Maeda, A., et al. (2009) Regulation of Skin Pigmentation and Thickness by Dickkopf 1 (DKK1). Journal of Investigative Dermatology Symposium Proceedings, 14, 73-75.