A 2D and 3D melanogenesis model with human primary cells induced by tyrosine
Main Article Content
Keywords
co-culture, keratinocyte, melanin, melanocyte, pigmentation
Abstract
Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH4Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH4Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation.
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References
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24. Swope VB, Abdel-Malek ZA (2016) Significance of the melanocortin 1 and endothelin B receptors in melanocyte homeostasis and prevention of sun-induced genotoxicity. Front Genet 6: 146.
25. Zhang C, Zhou L, Huang J, Mei X, Wu Z, Shi W (2018) A preliminary study of growth characteristics of melanocytes co-cultured with keratinocytes in vitro. J Cell Biochem 119: 6173-80.
26. Hirobe T, Wakamatsu K, Ito S, Abe H, Kawa Y, Mizoguchi M (2002) Stimulation of the proliferation and differentiation of mouse pink‐eyed dilution epidermal melanocytes by excess tyrosine in serum‐free primary culture. J Cell Physiol 191:162-72.
27. Hirobe T, Abe H, Wakamatsu K, Ito S, Kawa Y, Soma Y, et al. (2007) Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis. Eur J Cell Biol 86:315-30.
28. Halaban R, Cheng E, Svedine S, Aron R, Hebert DN (2001) Proper folding and endoplasmic reticulum to golgi transport of tyrosinase are induced by its substrates, DOPA and tyrosine. J Biol Chem 276:11933-8.
2. Yamaguchi Y, Hearing VJ (2009) Physiological factors that regulate skin pigmentation. Biofactors 35: 193-199. 19449448
3. Lo JA, Fisher DE (2014) The melanoma revolution: From UV carcinogenesis to a new era in therapeutics. Science 346: 945-949. 25414302
4. Niu C, Aisa HA (2017) Upregulation of melanogenesis and tyrosinase activity: potential agents for vitiligo. Molecules 22: 1303. 28777326
5. Koo JH, Kim HT, Yoon HY, Kwon KB, Choi IW, Jung SH, et al. (2008) Effect of xanthohumol on melanogenesis in B16 melanoma cells. Exp Mol Med 40: 313-319. 18587269
6. Shoag J, Haq R, Zhang M, Liu L, Rowe GC, Jiang A, et al. (2013) PGC-1 coactivators regulate MITF and the tanning response. Mol Cell 49: 145–157. 23201126
7. Hu S, Huang J, Pei S, Ouyang Y, Ding Y, Jiang L, et al. (2019) Ganoderma lucidum polysaccharide inhibits UVB‐induced melanogenesis by antagonizing cAMP/PKA and ROS/MAPK signaling pathways. J Cell Physiol 234: 7330-7340. 30362532
8. Gledhill K, Rhodes LE, Brownrigg M, Haylett AK, Masoodi M, Thody AJ, et al. (2010) Prostaglandin-E2 is produced by adult human epidermal melanocytes in response to UVB in a melanogenesis-independent manner. Pigment Cell Melanoma Res 23:394-403.
9. Cardinali G, Ceccarelli S, Kovacs D, Aspite N, Lotti LV, Torrisi MR, et al. (2005) Keratinocyte growth factor promotes melanosome transfer to keratinocytes. J Invest Dermatol 125:1190-9.
10. Duval C, Smit NP, Kolb AM, Regnier M, Pavel S, Schmidt R (2002) Keratinocytes control the pheo/eumelanin ratio in cultured normal human melanocytes. Pigment Cell Res. 15(6):440-6.
11. Slominski A, Jastreboff P, Pawelek, J (1989) L-Tyrosine stimulates induction of tyrosinase activity by MSH and reduces cooperative interactions between MSH receptors in hamster melanoma cells. Biosci Rep 9:579-86.
12. Kongshoj, B, Mikkelsen N D, Kobayasi T, Lerche CM, Wulf HC (2007) Ammonium chloride and L-tyrosine enhance melanogenesis in vitro but not in vivo even in combination with ultraviolet radiation. Photodermatol Photoimmunol Photomed 23:197-202.
13. Pennacchi PC, de Almeida ME, Gomes OL, Faiao-Flores F, de Araujo Crepaldi MC, Dos Santos MF, et al. (2015) Glycated reconstructed human skin as a platform to study the pathogenesis of skin aging. Tissue Eng Part A 21:2417- 25.
14. Chen N, Hu Y, Li WH, Eisinger M, Seiberg M, Lin CB (2010) The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Exp Dermatol 19:865- 72.
15. Lei TC, Virador VM, Vieira WD, Hearing VJ (2002) A melanocyte–keratinocyte coculture model to assess regulators of pigmentation in vitro. Anal Biochem 305:260–268.
16. Roberts DW, Newton RA, Leonard JH, Sturm RA (2008) Melanocytes expressing MC1R polymorphisms associated with red hair color have altered MSH-ligand activated pigmentary responses in coculture with keratinocytes. J Cell Physiol 215:344-55.
17. Seiberg M (2001) Keratinocyte-melanocyte interactions during melanosome transfer. Pigment Cell Res 14:236-42.
18. Sturm RA (1998) Human pigmentation genes and their response to solar UV radiation. Mutat Res 422:69-76.
19. Jablonski NG, Chaplin G (2010) Human skin pigmentation as an adaptation to UV radiation. Proc Natl Acad Sci U S A 107:8962-8
20. Gibbs S, Murli S, De Boer G, Mulder A, Mommaas AM, Ponec M (2000) Melanosome capping of keratinocytes in pigmented reconstructed epidermis-effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis. Pigment Cell Res 13:458-66.
21. Lee HD, Lee WH, Roh E, Seo CS, Son JK, Lee SH, et al. (2011) Manassantin A inhibits cAMP-induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes. Exp Dermatol 20:761-3.
22. Seo GY, Ha Y, Park AH, Kwon OW, Kim YJ (2019) Extract inhibits α-MSH-induced melanogenesis in B16F10 cells via down-regulation of CREB signaling pathway. Int J Mol Sci 20:536.
23. Pateraki I, Andersen-Ranberg J, Jensen NB, Wubshet SG, Heskes AM, Forman V, et al. (2017) Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. Elife 14: e23001.
24. Swope VB, Abdel-Malek ZA (2016) Significance of the melanocortin 1 and endothelin B receptors in melanocyte homeostasis and prevention of sun-induced genotoxicity. Front Genet 6: 146.
25. Zhang C, Zhou L, Huang J, Mei X, Wu Z, Shi W (2018) A preliminary study of growth characteristics of melanocytes co-cultured with keratinocytes in vitro. J Cell Biochem 119: 6173-80.
26. Hirobe T, Wakamatsu K, Ito S, Abe H, Kawa Y, Mizoguchi M (2002) Stimulation of the proliferation and differentiation of mouse pink‐eyed dilution epidermal melanocytes by excess tyrosine in serum‐free primary culture. J Cell Physiol 191:162-72.
27. Hirobe T, Abe H, Wakamatsu K, Ito S, Kawa Y, Soma Y, et al. (2007) Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis. Eur J Cell Biol 86:315-30.
28. Halaban R, Cheng E, Svedine S, Aron R, Hebert DN (2001) Proper folding and endoplasmic reticulum to golgi transport of tyrosinase are induced by its substrates, DOPA and tyrosine. J Biol Chem 276:11933-8.
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Published
Jul 6, 2020
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Branquinho MS, Silva MB, Silva JC, Sales MC, Barros SB, Maria-Engler SS, Campa A. A 2D and 3D melanogenesis model with human primary cells induced by tyrosine. J Biol Methods [Internet]. 2020 Jul. 6 [cited 2024 Apr. 19];7(3):e134. Available from: https://jbmethods.org/index.php/jbm/article/view/327
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