Class III β-tubulin, a novel biomarker in the human melanocyte lineage Heiko Locher a,n , Karien E. de Rooij b,e , John C.M.J. de Groot a , Remco van Doorn c , Nelleke A. Gruis c , Clemens W.G.M. Löwik b , Susana M. Chuva de Sousa Lopes d , Johan H.M. Frijns a , Margriet A. Huisman a a Department of Otorhinolaryngology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands b Department of Radiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands c Department of Dermatology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands d Department of Anatomy and Embryology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands e Percuros B.V., Plesmanlaan 1, 2333 BZ Leiden, The Netherlands article info Article history: Received 7 September 2012 Received in revised form 16 May 2013 Accepted 23 May 2013 Keywords: Class III beta-tubulin Humans Melanocytes Melanoblasts Neurons abstract It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experi- ments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells. & 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. 1. Introduction Over the last several years, there has been a notable increase in interest in the culture of skin and skin stem cells, in part for the generation of skin substitutes, in part with view of understanding cutaneous pathologies. It is of great importance to be able to identify the different skin cell subtypes accurately in culture in order to fully understand their biology. A widely used biomarker in this context is class III β-tubulin (TUBB3), which is generally thought to be expressed only in cells of the neural lineage. Microtubules are one of the main classes of cytoskeletal filaments involved in cellular morphology, motility, division and intracellular transport. They are composed of assemblies of het- erodimers one α- and one β-tubulin subunit (Bryan and Wilson, 1971; Downing and Nogales, 1998). There are multiple β-tubulin isotypes and their expression is tissue-specific and depends on the developmental stage (Ludueña, 1998). Isotype TUBB3 was first described in avian and mammalian species (Sullivan and Cleveland, 1986). Antibodies directed against TUBB3 epitopes distinguished between neurons and glial cells (Geisert and Frankfurter, 1989; Moody et al., 1989), so that it became a standard biomarker for neuronal identification. TUBB3 expression is often used to identify neuronal cells in skin-derived and (epidermal) stem cell cultures. In vivo it is used to trace the dendritic processes of neurons in the dermis and epidermis, of which the cell bodies are located elsewhere. TUBB3 is not exclusively neuron-specific. It is, albeit weakly, expressed in testicular and a few other tissue types (Burgoyne et al., 1988; Ludueña, 1998). Recently, it was shown to be present in cultures of mesenchymal and epithelial cells (Jouhilahti et al., 2008). A variety of solid tumors such as ovarian tumors, lung tumors and malignant melanomas also express TUBB3 and it has been suggested that this tubulin isotype is associated with cellular drug resistance (Mariani et al., 2011). Tubb3 has also been found expressed in developing melanoblasts in mouse and chicken embryo's(Adameyko et al., 2012, 2009). Interestingly, in a recent study positive immunoreactivity for the TUBB3 protein was reported in cells in the basal layer of normal human adult epidermis (K. Akasaka et al., 2009). Although it is suggestive that these cells could be melanocytes, no confirmation has been Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/diff Differentiation 0301-4681/$ - see front matter & 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) http://dx.doi.org/10.1016/j.diff.2013.05.003 Abbreviations: TUBB3, class III beta-tubulin; DCT, dopachrome tautomerase; TYRP1, tyrosinase-related protein 1; MITF, microphthalmia-associated transcrip- tion factor; α-MSH, alpha-melanocyte-stimulating hormone n Corresponding author. Tel.: +31 71 526 1179; fax: +31 71 524 8201. E-mail addresses: h.locher@lumc.nl (H. Locher), K.E.de_Rooij@lumc.nl (K.E. de Rooij), J.C.M.de_Groot@lumc.nl (J.C.M.J. de Groot), R.van_Doorn@lumc.nl (R.van Doorn), N.A.Gruis@lumc.nl (N.A. Gruis), C.W.G.M. Lowik@lumc.nl (C.W.G.M. Löwik), S.M.Chuva_de_Sousa_Lopes@lumc.nl (S.M. Chuva de Sousa Lopes), J.H.M. Frijns@lumc.nl (J.H.M. Frijns), M.A.Huisman@lumc.nl (M.A. Huisman). Differentiation 85 (2013) 173–181